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The fact that the PI3K/mTOR pathway is deregulated in a large number of human malignancies, and its importance for different cellular responses, makes it an attractive drug target. Pharmacological PI3K inhibitors have played a very important role in studying cellular responses involving these enzymes. Currently, a wide range of selective PI3K inhibitors have been tested in preclinical studies and some have entered clinical trials in oncology. Rapamycin and its analogs targeting mTOR are effective in many preclinical cancer models. Although rapalogs are approved for the treatment of some cancers, their efficacy in clinical trials remains the subject of debate. Due to the complexity of the PI3K/mTOR signaling pathway, developing an effective anti-cancer therapy remains a challenge. The biggest challenge in curing cancer patients with various signaling pathway abnormalities is to target multiple components of different signal transduction pathways with mechanism-based combinatorial treatments. Frontiers in Oncology July 2014 | Targeting PI3K/mTOR signaling in cancer | 2 Table of Contents 04 Targeting PI3K/mTOR Signaling in Cancer Alexandre Arcaro 05 Genomic Determinants of PI3K Pathway Inhibitor Response in Cancer Britta Weigelt and Julian Downward 21 Abrogating Endocrine Resistance by Targeting ERα and PI3K in Breast Cancer Emily M. Fox, Carlos L. Arteaga and Todd W. Miller 27 Targeting PI3K in Cancer: Any Good News? Miriam Martini, Elisa Ciraolo, Federico Gulluni and Emilio Hirsch 36 S6K2: The Neglected S6 Kinase Family Member Olivier E. Pardo and Michael J. Seckl 47 Targeting PI3K/Akt/mTOR Signaling in Cancer Camillo Porta, Chiara Paglino and Alessandra Mosca 58 p110 δ PI3 Kinase Pathway: Emerging Roles in Cancer Niki Tzenaki and Evangelia A. Papakonstanti 74 Did We Get Pasteur, Warburg and Crabtree on a Right Note? Lakshmipathi Vadlakonda, Abhinandita Dash, Mukesh Pasupuleti, Kotha Anil Kumar and Pallu Reddanna 78 The Paradox of Akt -mTOR Interactions Lakshmipathi Vadlakonda, Abhinandita Dash, Mukesh Pasupuleti, Kotha Anil Kumar and Pallu Reddanna 87 Role of PI3K-AKT-mTOR and Wnt Signaling Pathways in Transition of G1-S Phase of Cell Cycle in Cancer Cells Lakshmipathi Vadlakonda, Mukesh Pasupuleti and Reddanna Pallu Frontiers in Oncology July 2014 | Targeting PI3K/mTOR signaling in cancer | 3 EDITORIAL published: 22 April 2014 doi: 10.3389/fonc.2014.00084 Targeting PI3K/mTOR signaling in cancer Alexandre Arcaro* Department of Clinical Research, Division of Pediatric Hematology/Oncology, University of Bern, Bern, Switzerland *Correspondence: alexandre.arcaro@dkf.unibe.ch Edited and reviewed by: Paolo Pinton, University of Ferrara, Italy Keywords: Akt, cancer, clinical trials, mTOR, phosphoinositide 3-kinase The phosphoinositide 3-kinase (PI3K)/mammalian target of REFERENCES rapamycin (mTOR) pathway is very frequently activated in human 1. 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Abrogating endocrine resistance by targeting review the genetic determinants of response to these targeted ER alpha and PI3K in breast cancer. Front Oncol (2012) 2:doi:10.3389/fonc. agents (10). Fox et al. discuss the potential of co-targeting PI3K 2012.00145 and the estrogen receptor (ER) in breast cancer (11). Conflict of Interest Statement: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed ACKNOWLEDGMENTS as a potential conflict of interest. Work in the author’s laboratory is supported by grants from the European Union FP7 (ASSET, project number: 259348 and Received: 19 March 2014; accepted: 05 April 2014; published online: 22 April 2014. Citation: Arcaro A (2014) Targeting PI3K/mTOR signaling in cancer. Front. Oncol. LUNGTARGET, project number: 259770), the Swiss National Sci- 4:84. doi: 10.3389/fonc.2014.00084 ence Foundation (Grant 31003A-146464), the Fondation FORCE, This article was submitted to Molecular and Cellular Oncology, a section of the journal the Novartis Stiftung für Medizinisch-Biologische Forschung, the Frontiers in Oncology. Jubiläumsstiftung der Schweizerischen Mobiliar Genossenschaft, Copyright © 2014 Arcaro. This is an open-access article distributed under the terms of the Stiftung zur Krebsbekämpfung, the Huggenberger-Bischoff the Creative Commons Attribution License (CC BY). The use, distribution or repro- duction in other forums is permitted, provided the original author(s) or licensor are Stiftung zur Krebsforschung, the UniBern Forschungsstiftung, the credited and that the original publication in this journal is cited, in accordance with Stiftung für klinisch-experimentelle Tumorforschung, Bern and accepted academic practice. No use, distribution or reproduction is permitted which the Berner Stiftung für krebskranke Kinder und Jugendliche. does not comply with these terms. www.frontiersin.org April 2014 | Volume 4 | Article 84 | 4 REVIEW ARTICLE published: 31 August 2012 doi: 10.3389/fonc.2012.00109 Genomic determinants of PI3K pathway inhibitor response in cancer Britta Weigelt 1 and Julian Downward 1,2 * 1 Signal Transduction Laboratory, Cancer Research UK London Research Institute, London, UK 2 Division of Cancer Biology, The Institute of Cancer Research, London, UK Edited by: The phosphoinositide 3-kinase (PI3K) pathway is frequently activated in cancer as a result Alexandre Arcaro, University of Bern, of genetic (e.g., amplifications, mutations, deletions) and epigenetic (e.g., methylation, Switzerland regulation by non-coding RNAs) aberrations targeting its key components. Several lines of Reviewed by: Edward Prochownik, University of evidence demonstrate that tumors from different anatomical sites depend on the continued Pittsburgh Medical Center, USA activation of this pathway for the maintenance of their malignant phenotype.The PI3K path- Hua Yan, New York University School way therefore is an attractive candidate for therapeutic intervention, and inhibitors targeting of Medicine, USA different components of this pathway are in various stages of clinical development. Bur- *Correspondence: geoning data suggest that the genomic features of a given tumor determine its response Julian Downward , Signal Transduction Laboratory, Cancer Research UK to targeted small molecule inhibitors. Importantly, alterations of different components of London Research Institute, 44 the PI3K pathway may result in distinct types of dependencies and response to specific Lincoln’s Inn Fields, London WC2A therapeutic agents. In this review, we will focus on the genomic determinants of response 3LY, UK. to PI3K, dual PI3K/mechanistic target of rapamycin (mTOR), mTOR, and AKT inhibitors in e-mail: julian.downward@ cancer.org.uk cancer identified in preclinical models and clinical trials to date, and the development of molecular tools for the stratification of cancer patients. Keywords: PI3K pathway inhibitors, drug response, genetic determinant, cancer INTRODUCTION either isoform specific [i.e., class I isoforms p110α, p110β, p110γ, The phosphoinositide 3-kinase (PI3K) signaling pathway regulates p110δ; (glossary box)] or pan-class I PI3K inhibitors, dual numerous processes in the normal cell such as growth, prolifera- PI3K/mechanistic target of rapamycin (mTOR) inhibitors, mTOR tion, survival, motility, and metabolism (Engelman et al., 2006). inhibitors, and AKT inhibitors, which are all currently in various In human cancer, the PI3K pathway is one of the most frequently stages of clinical development (Table 1). activated signal transduction pathways, and its prominent role Over the past years it has become apparent that irrespective is highlighted by the array of mechanisms targeting several of of the cancer type and small molecule inhibitor or antibody its key components (Figure 1). Mutations and/or amplifications used, kinase inhibitor response is limited to those tumors whose of genes encoding receptor tyrosine kinases (RTKs) upstream proliferation and survival are reliant on the activation of the tar- of class I PI3Ks (glossary box), including the human epidermal geted oncogenic kinase (Sharma and Settleman, 2007; Janne et al., growth factor receptors EGFR (ERBB1) and HER2 (ERBB2), of 2009). Bernard Weinstein coined the term “oncogene addiction” the PI3K catalytic subunits p110α (PIK3CA) and p110β (PIK3CB), to describe this phenomenon (Weinstein, 2002), which has impor- the PI3K regulatory subunits p85α (PIK3R1) and p85β (PIK3R2), tant implications for the targeting of kinases: given the incredibly the PI3K effector AKT (AKT1), and of the PI3K activator KRAS diverse repertoire of genetic and epigenetic aberrations observed are frequently observed in cancer [Catalog Of Somatic Mutations within a given cancer type, only the subset of tumors “addicted” In Cancer (COSMIC), http://www.sanger.ac.uk/cosmic; Forbes to the continued activation of the oncogenic kinase targeted et al., 2011], as is loss of function of the tumor suppressors will prove vulnerable to the therapeutic intervention. Consis- phosphatase and tensin homolog (PTEN) and inositol polyphos- tent with this “oncogene addiction” concept, strong associations phate 4-phosphatase-II (INPP4B), negative regulators of PI3K between a tumor’s genotype and its response to small mole- signaling, through mutations, deletions, or epigenetic mechanisms cule kinase inhibitors or antibodies targeting kinases have been (Gewinner et al., 2009; Fedele et al., 2010; Hollander et al., 2011). identified. For example, melanomas harboring BRAF V600E muta- Given that the PI3K pathway is frequently activated in can- tions are selectively sensitive to the BRAF inhibitor Vemurafenib cers, that tumorigenesis and/or maintenance of the malignant (Flaherty et al., 2010), non-small cell lung cancers (NSCLCs) phenotype of different tumor types is driven by its continued harboring EGFR mutations to the EGFR inhibitors Gefitinib or activation (Bader et al., 2005; Hollander et al., 2011), and that Erlotinib (Pallis et al., 2011), HER2 amplified breast and gastric kinases are amendable to pharmacological intervention, it is not cancers to the HER2 targeting agents Trastuzumab or Lapatinib surprising that there has been great interest in the development (Stern, 2012), and KIT and PDGFRA mutant gastrointestinal of allosteric and ATP-competitive small molecule inhibitors tar- stromal tumors to Imanitib Mesylate and other small molecule geting different components of this pathway downstream of RTKs inhibitors targeting mutant KIT and PDGFRα (Antonescu, 2011). (Liu et al., 2009). These targeted agents include PI3K inhibitors, Importantly, however, cancers harboring only wild-type copies of www.frontiersin.org August 2012 | Volume 2 | Article 109 | 5 Weigelt and Downward PI3K pathway inhibitor response FIGURE 1 | Class I PI3K signal transduction pathway. Components of tested in clinical trials (gray boxes). mTOR, mechanistic target of the class I PI3K signaling pathway (left) and of the mitogen-activated rapamycin; mTORC, mTOR complex; PI3K, phosphoinositide 3-kinase; protein kinase (MAPK) pathway (right) recurrently targeted by PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol genetic/epigenetic alterations in cancer are depicted with a red asterisk. (3,4,5)-triphosphate; PTEN, phosphatase and tensin homolog; RTK, Several PI3K pathway inhibitors downstream of RTKs are currently being receptor tyrosine kinase; TSC, tuberous sclerosis protein. the genes mentioned above seem not to be sensitive to the same GENOMIC DETERMINANTS OF PI3K PATHWAY INHIBITOR agents. RESPONSE IN PRECLINICAL MODELS As PI3K pathway inhibitors progress into trials focusing on their The ease of therapeutic intervention using in vitro cell culture clinical efficacy (Table 1), it is critical to identify their genomic and the wealth of data available on the mutational landscape of determinants of response and to select the patient population most known cancer genes in the most common cell lines obtainable likely to benefit from treatment. In fact, it has been suggested to from commercial repositories have made cancer cell line panels incorporate predictive biomarkers throughout the clinical drug the model of choice for the preclinical study of drug response. development process from phase I studies onward in order to Furthermore, with the advent of methods for massively parallel enrich trials with patients more likely to respond to a given targeted sequencing, it is now possible to identify the genomic determi- therapy and to increase the chances of drug registration (Carden nants of therapy response in in vitro models in a genome-wide et al., 2010). For the guidance and prioritization of predictive bio- fashion (Barretina et al., 2012; Garnett et al., 2012). In general, marker candidates in early clinical trials, results derived from the sensitivity or resistance of cancer cell lines to a given targeted study of preclinical models are of importance. agent are determined by short-term treatment ranging from 48 to In this review, we focus on the genomic determinants of 120 h of cells grown on tissue culture plastic using several dilu- response to PI3K pathway inhibitors in cancer identified in preclin- tions of the inhibitor. At the endpoint, cell number or cell viability ical models and clinical trials to date, and discuss the challenges for is assessed and drug response reported as half-maximal inhibitory the development of molecular tools for the stratification of cancer concentration (IC50 ), or the concentration needed to reduce the patients. growth of treated cells to half that of untreated or vehicle treated Frontiers in Oncology | Molecular and Cellular Oncology August 2012 | Volume 2 | Article 109 | 6 Weigelt and Downward PI3K pathway inhibitor response Table 1 | Open clinical trials testing PI3K pathway inhibitors in cancer*. Inhibitor name Company Target Clinical Cancer type trial phase PAN-CLASS I PI3K INHIBITORS BAY80-6946 Bayer Class I PI3K I Advanced solid cancers ZSTK474 Zenyaku Kogyo Class I PI3K I Advanced solid cancers GSK1059615 GlaxoSmithKline Class I PI3K I Terminated BKM120 Novartis Class I PI3K I and II (Advanced) solid cancers; NSCLC, endometrial, prostate, breast, colorectal, pancreatic, renal cell, GIST, melanoma, glioblastoma, leukemia, SCCHN, TCC GDC-0941 Roche/Genentech Class I PI3K I and II Solid cancers; breast, NSCLC, non-Hodgkin’s lymphoma PX866 Oncothyreon Class I PI3K I and II Prostate, NSCLC, SCCHN, colorectal, glioblastoma XL147 (SAR245408) Exelixis/Sanofi-Aventis Class I PI3K I and II Solid cancers; endometrial, ovarian, breast, NSCLC ISOFORM SPECIFIC PI3K INHIBITORS BYL719 Novartis p110α I Advanced solid cancers; SCCHN GDC-0032 Roche/Genentech p110α I Solid cancers INK-1117 Intellikine p110α I Advanced solid cancers GSK2636771 GlaxoSmithKline p110β I/IIa Advanced solid cancers (PTEN deficient) IPI-145 Infinity p110γ, p110δ I Advanced hematological malignancies AMG319 Amgen p110δ I Relapsed or refractory lymphoid malignancies CAL-101 (GS-1101) Gilead sciences p110δ I, II, and III Chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin’s lymphoma; mantle cell lymphoma, acute myeloid leukemia, multiple myeloma DUAL PI3K/mTOR INHIBITORS DS-7423 Daiichi Sankyo PI3K/mTOR I Advanced solid cancers; colorectal, endometrial GDC-0980 Roche/Genentech PI3K/mTOR I (Advanced) solid cancers; non-Hodgkin’s lymphoma, breast, prostate, endometrial, renal cell GSK2126458 GlaxoSmithKline PI3K/mTOR I Advanced solid cancers PWT33597 Pathway Therapeutics PI3K/mTOR I Advanced solid cancers or malignant lymphoma SF1126 Semafore PI3K/mTOR I Advanced solid cancers BEZ235 Novartis PI3K/mTOR I and II Advanced solid cancers; renal cell, breast BGT226 Novartis PI3K/mTOR I and II Completed (advanced solid cancers; breast) PF-04691502 Pfizer PI3K/mTOR I and II Advanced solid cancers; breast, endometrial PF-05212384 (PKI-587) Pfizer PI3K/mTOR I and II Advanced solid cancers; endometrial XL765 (SAR245409) Exelixis/Sanofi-Aventis PI3K/mTOR I and II Advanced breast, gliomas, glioblastoma multiforme mTOR KINASE INHIBITORS AZD2014 AstraZeneca mTOR I Advanced solid cancers; breast AZD8055 AstraZeneca mTOR I Recurrent glioma INK-128 Intellikine mTOR I Advanced solid cancers; multiple myeloma, Waldenstrom macroglobulinemia OSI-027 Astellas Pharma mTOR I Advanced solid cancers; lymphoma CC-223 Celgene Corporation mTOR I and II Advanced solid cancers; non-Hodgkin’s lymphoma, multiple myeloma, NSCLC ALLOSTERIC mTOR INHIBITORS (RAPAMYCIN ANALOGS) Sirolimus (Rapamycin) Wyeth/Pfizer mTOR I, II, and III Advanced solid cancers; breast, liver, rectum, NSCLC, leukemias, lymphomas, head and neck, pancreatic, ovarian, fallopian tube, glioblastoma, fibromatosis Everolimus** (RAD001) Novartis mTOR I, II, and III Solid cancers; leukemias, lymphomas, breast, bladder, head and neck, kidney/renal cell, liver, gastric, thyroid, neuroendocrine tumors, ovarian, fallopian tube, cervical, colorectal, brain and central nervous system, prostate, endometrial, esophageal, melanoma, NSCLC, SCLC, germ cell, soft tissue sarcoma, osteosarcoma, nasopharyngeal, glioma, Waldenstrom’s macroglobulinemia (Continued) www.frontiersin.org August 2012 | Volume 2 | Article 109 | 7 Weigelt and Downward PI3K pathway inhibitor response Table 1 | Continued Inhibitor name Company Target Clinical Cancer type trial phase Temsirolimus** (CCI-779) Wyeth/Pfizer mTOR I and II Advanced solid cancers; breast, endometrial, ovarian, prostate, liver, kidney/renal cell, SCCHN, NSCLC, melanoma, sarcoma, lymphomas, leukemia, brain and central nervous system, bladder, urethral Ridaforolimus (MK-8669) Merck/Ariad mTOR I Advanced solid cancers; endometrial, ovarian, breast, NSCLC, renal cell, soft tissue sarcoma AKT INHIBITORS (ATP-Competitive) ARQ 092 ArQule/Daiichi Sankyo AKT I Advanced solid cancers AZD5363 AstraZeneca AKT I Advanced solid cancers GSK2141795 GlaxoSmithKline AKT I Completed/not recruiting (advanced solid cancers; lymphoma) GDC-0068 Roche/Genentech AKT I and II Advanced solid cancers; prostate cancer GSK2110183 GlaxoSmithKline AKT I and II Solid cancers, hematological malignancies, multiple myeloma, Langerhans cell histiocytosis, chronic lymphocytic leukemia ALLOSTERIC AKT INHIBITORS MK-2206 Merck AKT I and II Advances solid cancers; breast, endometrial, ovarian, fallopian tube, peritoneal, gastric, gastroesophageal junction, colorectal, prostate, NSCLC, SCLC, melanoma, kidney, leukemias, lymphomas, biliary, head and neck, liver, thymic, nasopharyngeal *Data retrieved from http://clinicaltrials.gov and http://www.fda.gov/ (May 2012). **Temsirolimus: approved for the treatment of advanced renal cell carcinoma; Everolimus: approved for the treatment of progressive neuroendocrine tumors of pancreatic origin, for advanced renal cell carcinoma after failure of treatment with sunitinib or sorafenib, for renal angiomyolipoma and tuberous sclerosis complex, and for subependymal giant cell astrocytoma associated with tuberous sclerosis. GIST, gastrointestinal stromal tumor; NSCLC, non-small cell lung cancer; SCCHN, squamous cell carcinoma of the head and neck; SCLC, small cell lung cancer; TCC, transitional cell carcinoma of the urothelium. cells (GI50 ). In addition, xenograft studies in immunodeficient PIK3CA mutations as determinant of response to PI3K inhibi- mice injected with human cancer cell lines or human tumor tis- tion (O’Brien et al., 2010; Sanchez et al., 2011), dual PI3K/mTOR sues, as well as transgenic mouse models have been employed to inhibition (Serra et al., 2008; Brachmann et al., 2009; Lehmann assess anti-tumor activity of PI3K pathway inhibitors in vivo using et al., 2011; Sanchez et al., 2011), mTOR kinase inhibition (Weigelt tumor growth, proliferation, apoptosis, and/or levels of pathway et al., 2011), allosteric mTOR inhibition (Sanchez et al., 2011; activation state as read-out of treatment response. Weigelt et al., 2011), and AKT inhibition (She et al., 2008; Meuillet Using these preclinical approaches, several groups attempted et al., 2010; Table 2). In one report, however, which assessed seven to define genomic determinants of response to PI3K pathway estrogen receptor (ER)-positive breast cancer cell lines and their inhibitors. It should perhaps not come as a surprise that genetic response to the allosteric mTOR inhibitor Rapamycin (Sirolimus), alterations leading to PI3K pathway activation, including PIK3CA no correlation with PIK3CA mutation status but to some extent gain-of-function mutations and/or PTEN mutations/PTEN loss with a PIK3CA mutation associated gene signature was found of function and/or amplification of HER2, have been repeatedly (Loi et al., 2010). In vitro and xenograft models of breast can- identified as predictors of response to these agents (Table 2). cer have also demonstrated that cells harboring amplification of However, tumor type-specific differences have been observed. For the RTK HER2 are dependent on PI3K pathway activation and example, in ovarian cancer cells both PIK3CA mutations and sensitive to its inhibition through targeting of PI3K (O’Brien PTEN deficiency have been reported to predict PI3K pathway et al., 2010; Tanaka et al., 2011), dual PI3K/mTOR (Brachmann inhibitor response (Ihle et al., 2009; Di Nicolantonio et al., 2010; et al., 2009), AKT (She et al., 2008), and mTOR kinase (Weigelt Meuillet et al., 2010; Santiskulvong et al., 2011; Tanaka et al., 2011; et al., 2011). In fact, mTOR kinase inhibitors seem to lead to a Meric-Bernstam et al., 2012), whereas in breast cancer the associ- more effective decrease of PI3K pathway signaling than allosteric ations between PTEN loss of function and response are less clear mTOR inhibitors given that HER2 amplified breast cancer cells (She et al., 2008; Brachmann et al., 2009; Lehmann et al., 2011; in vitro have been found to be unresponsive to the rapamycin ana- Sanchez et al., 2011; Tanaka et al., 2011; Weigelt et al., 2011), which log (“rapalog”) Everolimus (RAD001; glossary box; Weigelt et al., will be discussed in greater detail below. 2011). Whereas PIK3CA mutations and HER2 amplification have Several studies provided evidence to suggest that cancer cells been identified in the majority of preclinical breast cancer studies harboring PIK3CA gain-of-function mutations are selectively sen- as determinant of sensitivity to PI3K pathway inhibition down- sitive to inhibitors of different components of the PI3K pathway. stream of RTKs, the correlation between PTEN deficiency and In breast cancer, cell culture, and/or xenograft models identified response is less clear. In some studies, results were inconclusive as Frontiers in Oncology | Molecular and Cellular Oncology August 2012 | Volume 2 | Article 109 | 8 Weigelt and Downward PI3K pathway inhibitor response Table 2 | Genomic determinants of response to PI3K pathway inhibitors identified in preclinical cancer models. Inhibitor (target) Cancer type Preclinical model Genomic determinant Reference of response GDC-0941 (Class I PI3K) Breast Cell lines PIK3CA mutation O’Brien et al. (2010) Cell line xenografts HER2 amplification BEZ235 (PI3K/mTOR) Breast Cell lines PIK3CA mutation Serra et al. (2008) Cell line xenografts BEZ235 (PI3K/mTOR) Breast Cell lines PIK3CA mutation Brachmann et al. (2009) Cell line xenografts HER2 amplification BEZ235 (PI3K/mTOR) Breast Cell lines PIK3CA mutation Lehmann et al. (2011) (PTEN deficiency) BKM120 (Class I PI3K), Breast Cell lines PIK3CA mutation Sanchez et al. (2011) BGT226 (PI3K/mTOR), Everolimus (mTOR) PP242 (mTOR kinase) Breast Cell lines PIK3CA mutation Weigelt et al. (2011) Everolimus (mTOR) HER2 amplification (only for PP242) Rapamycin (mTOR) Breast Cell lines None (not PIK3CA Loi et al. (2010) mutations) AKTi-1/2 (AKT) Breast Cell lines PIK3CA mutation She et al. (2008) Cell line xenografts HER2 amplification Everolimus (mTOR) Non-malignant breast Cell lines (isogenic) PIK3CA mutation Di Nicolantonio et al. (2010) (knock-in) Temsirolimus (mTOR) Multiple myeloma Cell lines PTEN deficiency Shi et al. (2002) Everolimus (mTOR) Glioblastoma multiforme Cell lines None (not PTEN Yang et al. (2008) Human tumor deficiency) xenografts BEZ235 (PI3K/mTOR) Ovarian Cell lines PIK3CA mutation Santiskulvong et al. (2011) PTEN deficiency WAY-175, WAY-176 Various (breast, prostate, melanoma, lung, Cell lines PIK3CA mutation Yu et al. (2008) (Class I PI3K) colon) PX866 (PI3K) Various (non-small cell lung cancer, colon, Cell line xenografts PIK3CA mutation Ihle et al. (2009) breast, pancreatic, prostate, ovarian, PTEN deficiency multiple myeloma) CH5132799 (PI3K) Various (breast, ovarian, prostate, Cell lines PIK3CA mutation Tanaka et al. (2011) endometrial) Cell line xenografts Temsirolimus (mTOR) Various (glioblastoma, prostate) Cell lines PTEN deficiency Neshat et al. (2001) Everolimus (mTOR) Various (prostate, glioblastoma, breast, Cell lines PIK3CA mutation Di Nicolantonio et al. (2010) ovarian, cervical) PTEN deficiency Rapamycin (mTOR) Various (neuroendocrine, cervical, Cell lines PIK3CA mutation Meric-Bernstam et al. (2012) hepatocellular, melanoma, ovarian, colon, PTEN deficiency breast, renal cell, glioblastoma, breast) PHT-427 (AKT/PDPK1) Various (pancreatic, prostate, ovarian, Cell line xenografts PIK3CA mutation Meuillet et al. (2010) breast, lung) 25 PI3K pathway Various (lung, colorectal, gastric, breast, Cell lines None (p-AKT levels) Dan et al. (2010) inhibitors (PI3K, ovarian, brain, renal, melanoma, prostate) PI3K/mTOR, AKT) A-443654 (AKT) Various (bladder, blood, bone, breast, Cell lines SMAD4 mutation Garnett et al. (2012); CNS, GI tract, kidney, lung, ovary, (http://www.cancerrxgene.org/; pancreas, skin, soft tissue, thyroid, upper Release 2, July 2012) aerodigestive, uterus) AKT inhibitor VIII (AKT) Various (bladder, blood, bone, breast, Cell lines PIK3CA mutation Garnett et al. (2012); CNS, GI tract, kidney, liver, lung, ovary, ERBB2 mutation (http://www.cancerrxgene.org/; pancreas, prostate, skin, soft tissue, Release 2, July 2012) thyroid, upper aerodigestive, uterus) (Continued) www.frontiersin.org August 2012 | Volume 2 | Article 109 | 9 Weigelt and Downward PI3K pathway inhibitor response Table 2 | Continued Inhibitor (target) Cancer type Preclinical model Genomic determinant Reference of response MK-2206 (AKT) Various (bladder, blood, bone, breast, Cell lines PTEN mutation Garnett et al. (2012); CNS, GI tract, kidney, liver, lung, ovary, (http://www.cancerrxgene.org/; pancreas, prostate, skin, soft tissue, Release 2, July 2012) thyroid, upper aerodigestive, uterus) AZD6482 (p110β) Various (bladder, blood, bone, breast, Cell lines PTEN mutation Garnett et al. (2012); CNS, GI tract, kidney, liver, lung, ovary, PIK3CA mutation (http://www.cancerrxgene.org/; pancreas, prostate, skin, soft tissue, Release 2, July 2012) thyroid, upper aerodigestive, uterus) BEZ235 (PI3K/mTOR) Various (bladder, blood, bone, breast, Cell lines CDKN2A mutation Garnett et al. (2012); CNS, GI tract, kidney, liver, lung, ovary, NRAS mutation (http://www.cancerrxgene.org/; pancreas, prostate, skin, soft tissue, Release 2, July 2012) thyroid, upper aerodigestive, uterus) Temsirolimus (mTOR) Various (bladder, blood, bone, breast, Cell lines PTEN mutation Garnett et al. (2012); CNS, GI tract, kidney, liver, lung, ovary, (http://www.cancerrxgene.org/; pancreas, prostate, skin, soft tissue, Release 2, July 2012) thyroid, upper aerodigestive, uterus) GDC-0941 (Class I Various (bladder, bone, breast, CNS, GI Cell lines None (TET2 mutations Garnett et al. (2012); PI3K), AZD8055 (mTOR tract, kidney, liver, lung, ovary, pancreas, associated with (http://www.cancerrxgene.org/; kinase), Rapamycin prostate, skin, soft tissue, thyroid, upper AZD8055 response, Release 2, July 2012) (mTOR), JW-7-52-1 aerodigestive, uterus) however only 3/554 cell (mTOR) lines were TET2 mutant) CONFIRMATORY STUDIES USING ANIMAL MODELS BEZ235 (PI3K/mTOR) Prostate and glioblastoma Cell line xenografts PTEN deficiency Maira et al. (2008) Rapamycin (mTOR) Breast and pancreatic Cell line xenografts PIK3CA mutation Meric-Bernstam et al. (2012) WYE-354 (mTOR Prostate and glioblastoma Cell line xenografts PTEN deficiency Yu et al. (2009) kinase) BEZ235 (PI3K/mTOR) Lung PIK3CA H1047R PIK3CA H1047R Engelman et al. (2008) mouse model mutation Rapamycin (mTOR), Ovarian endometrioid adenocarcinoma Apc flox/flox ; PTEN deficiency Wu et al. (2011) API-2 (AKT) Ptenflox/flox mouse model CNS, central nervous system; GI, gastrointestinal. only a subset of PTEN null breast cancer cell lines were sensitive preclinical models has suggested that PTEN deficient cancers may to PI3K pathway inhibition (She et al., 2008; Lehmann et al., 2011; depend on p110β rather than p110α signaling (Jia et al., 2008; Wee Sanchez et al., 2011), whilst others found PTEN deficient breast et al., 2008; Edgar et al., 2010; Ni et al., 2012), and a p110β isoform cancer cells to be preferentially resistant to treatment with PI3K specific inhibitor (GSK2636771) is currently being tested in a clin- (Tanaka et al., 2011), dual PI3K/mTOR (Brachmann et al., 2009), ical trial of PTEN deficient malignancies (NCT01458067). In fact, mTOR kinase, and allosteric mTOR inhibitors (Weigelt et al., as in different disease contexts selective targeting of specific p110 2011). These data are consistent with the notion that aberrations in isoforms may be more beneficial and less toxic than pan-PI3K the different components of the PI3K pathway are not necessarily inhibition (Jia et al., 2009; Vanhaesebroeck et al., 2010; Jamieson equivalent in their biological impact and their potential to activate et al., 2011; Tzenaki et al., 2012), also p110α, p110γ, and p110δ spe- the signaling pathway (Stemke-Hale et al., 2008; Vasudevan et al., cific inhibitors are being assessed in clinical trials (Table 1). The 2009; Dan et al., 2010). Moreover, these observations also sug- contribution of the p85 isoforms (glossary box) to PI3K inhibitor gest that sensitivity of PTEN deficient breast cancer cells to PI3K response is however not yet fully understood. There is evidence to pathway inhibitors may be dependent on epistatic interactions suggest that different cancer types express different levels of p110 between PI3K pathway genes and genes from other signaling path- and p85 isoforms (Cortes et al., 2012; Tzenaki et al., 2012), which ways such as the MAPK pathway, as well as the release of negative may lead to tumor type-specific combinations of catalytic and feedback loops and the node targeted by pharmacologic inhibition regulatory PI3K subunits. It remains to be determined whether (Efeyan and Sabatini, 2010; Zhang and Yu, 2010). Recent work in certain PI3K inhibitors show preferential activity against specific Frontiers in Oncology | Molecular and Cellular Oncology August 2012 | Volume 2 | Article 109 | 10 Weigelt and Downward PI3K pathway inhibitor response p110/p85 isoform combinations and whether distinct mutations Temsirolimus (CCI-779). Consistent with this result, the PTEN in the regulatory subunits PIK3R1 or PIK3R2 have an impact on null glioblastoma cell line U-87MG and the prostate cancer cell PI3K inhibitor response. line PC3 were found to be sensitive to Rapamycin in vitro (Di The general effect of PIK3CA gain-of-function mutations in Nicolantonio et al., 2010), and when grown as xenografts to the the sensitization to PI3K pathway inhibitors has been confirmed dual PI3K/mTOR inhibitor BEZ235 (Maira et al., 2008) and the in a mouse model with inducible expression of human onco- ATP-competitive mTOR inhibitor WYE-354 (Yu et al., 2009). At genic p110α (i.e., p110α H1047R), where treatment of the p110α variance with these findings, PTEN loss of function was shown H1047R driven lung adenocarcinomas with the dual PI3K/mTOR to be a poor predictor of Everolimus response in a panel of 17 inhibitor BEZ235 led to marked tumor regression (Engelman et al., glioblastoma multiforme cell lines, and in human glioblastoma 2008). In xenografts derived from the breast cancer cell line MCF7 xenograft models (Yang et al., 2008). and the pancreatic carcinoid cell line BON, both harboring an The data discussed above on the genomic determinants of PI3K activating PIK3CA mutation, treatment with the allosteric mTOR pathway inhibitor response identified in vitro are based on the inhibitor Rapamycin (Sirolimus) was associated with a signifi- analysis of up to 60 cancer cell lines, which were selected based cant decrease in tumor volume (Meric-Bernstam et al., 2012). on different criteria by independent investigators. Recently, two Moreover, using PIK3CA wild-type human breast immortalized large-scale studies subjected hundreds of cancer cell lines derived epithelial cells (hTERT-HME1) or non-malignant MCF10A breast from tumors stemming from different anatomical sites and tis- cells, knock-in of the E454K or H1047R PIK3CA mutant alle- sue types to transcriptomic profiling, copy number profiling, and les sensitized non-transformed human breast cells to the rapalog massively parallel sequencing. Owing to their unprecedented scale Everolimus (Di Nicolantonio et al., 2010). and approach employed, these studies unraveled several associa- Also when focusing on an array of tumor types rather than tions between genetic aberrations and response to specific targeted on a single disease entity, PIK3CA mutant cell lines, or cell line therapies (Barretina et al., 2012; Garnett et al., 2012). Garnett et al. derived xenografts were found to be selectively sensitive to PI3K (2012) tested up to 714 cell lines for their response to 138 anti- pathway inhibition (Table 2). For example, analysis of xenografts cancer agents including ten PI3K pathway inhibitors downstream derived from pancreatic, prostate, ovarian, NSCLC, and ovarian of RTKs (http://www.cancerrxgene.org/; Release 2, July 2012), and cancer cells revealed that those harboring PIK3CA mutations were observed that, in line with previous findings, cancer cells harbor- among the most sensitive to the AKT inhibitor PHT-427 (Meuil- ing mutations in PIK3CA and PTEN were sensitive to treatment let et al., 2010). This observation has been validated in a large with the AKT inhibitor VIII and MK-2206, respectively. Of note, panel of breast, ovarian, prostate, and endometrial cancer cells, also ERBB2 mutations were associated with AKT inhibitor VIII given that those with PIK3CA mutations were found to be signif- response. Sensitivity to the AKT inhibitor A-443654 and the dual icantly more sensitive to the PI3K inhibitor CH5132799 in vitro PI3K/mTOR inhibitor BEZ235, however, was not determined by than those without (Tanaka et al., 2011). Other studies assessing PI3K pathway aberrations but by the presence of SMAD4 and mixed tumor type cell line panels, however, have identified both CDKN2A mutations, respectively (Table 2). In Garnett et al. (2012) activating PIK3CA mutations and PTEN loss of function as deter- mutations in PTEN were associated with response to the mTOR minant of PI3K pathway inhibitor response. This was observed inhibitor Temsirolimus, and not only PTEN but also PIK3CA in a panel of breast, melanoma, lung, colon, prostate cancer cells mutations predicted response to the PI3K isoform specific p110β treated in vitro with the PI3K inhibitors WAY-175 and WAY-176 inhibitor AZD6482 (http://www.cancerrxgene.org/; Release 2). On (Yu et al., 2008), in a panel of human lung, colon, breast, pancre- the other hand, contrary to previous reports, no mutations predic- atic, ovarian, and multiple myeloma cell line derived xenografts tive of response to the PI3K inhibitor GDC-0941, the mTOR kinase treated with the PI3K inhibitor PX866 (Ihle et al., 2009), and in inhibitor AZD8055, and the mTOR inhibitors Rapamycin and JW- cell line panels of various tumor types treated in vitro with the 7-52-1 were identified (http://www.cancerrxgene.org/; Release 2; allosteric mTOR inhibitors Everolimus (Di Nicolantonio et al., Garnett et al., 2012; Table 2). 2010) or Rapamycin (Meric-Bernstam et al., 2012). In one study, Mutation analysis has already become part of the diagnostic the evaluation of the in vitro efficacy of 25 PI3K pathway inhibitors armamentarium for lung and colon cancers (Allegra et al., 2009; in a panel of 39 human cancer cell lines did not identify any genetic Keedy et al., 2011), and is also likely to be implemented in the man- determinant of sensitivity (Dan et al., 2010). agement of other tumor types. In fact, the potential determinants It is interesting to note that whilst loss of PTEN function of PI3K pathway inhibitor response identified in preclinical studies has been shown to be a strong activator of the PI3K pathway may provide a rationale for the guidance of predictive biomark- as determined by levels of AKT phosphorylation (Stemke-Hale ers to be assessed in early clinical trials. It should be noted here et al., 2008), only a few studies identified PTEN mutations/PTEN that in addition to genomic response predictors also non-genetic deficiency as a single genomic determinant of response to PI3K predictors of PI3K inhibitor response have been put forward, yet pathway inhibitors. Murine PTEN deficient ovarian endometri- none of them has been fully validated. In breast cancer, a gene oid adenocarcinomas arising in Apcflox/flox ; Ptenflox/flox mice have expression signature predictive of in vitro sensitivity to the PI3K been shown to be sensitive to Rapamycin and the AKT inhibitor inhibitor GDC-0941 (O’Brien et al., 2010), and a PIK3CA muta- API-2 (Wu et al., 2011). Also in a panel of multiple myeloma tion associated gene signature (PIK3CA-GS) derived from exon 20 (Shi et al., 2002), glioblastoma, and prostate cancer cell lines PIK3CA mutations able to predict PIK3CA mutation status in pri- (Neshat et al., 2001), PTEN deficiency was reported to be asso- mary breast cancers and predictive of Rapamycin response in vitro ciated with enhanced sensitivity to the allosteric mTOR inhibitor have recently been described (Loi et al., 2010). In addition, several www.frontiersin.org August 2012 | Volume 2 | Article 109 | 11 Weigelt and Downward PI3K pathway inhibitor response groups found increased phosphorylated (p)-AKT baseline levels clear cell renal cell carcinomas rarely harbor mutations in PI3K as a read-out for PI3K pathway activation to be associated with its pathway components (COSMIC), however commonly show loss therapeutic intervention (Noh et al., 2004; Yu et al., 2008; Dan et al., of function of the tumor suppressor genes PTEN (Brenner et al., 2010; Meric-Bernstam et al., 2012). Despite the potential utility of 2002; Velickovic et al., 2002) or von Hippel Lindau (VHL), a critical these approaches, it should be mentioned that gene expression sig- regulator of the hypoxic response (Kim and Kaelin, 2004; Linehan natures and immunohistochemical assessment of phosphorylated et al., 2010). Clear cell renal cell cancer has been suggested to be a proteins have proven challenging to implement in routine clinical cell metabolism, angiogenesis-dependent and hypoxia-driven dis- practice (Pinhel et al., 2010; Weigelt et al., 2012). ease, and its response to mTOR inhibition thought to stem from its Although predictive markers of sensitivity to PI3K pathway impact on proliferation and cell survival but also from the fact that inhibitors, such as PIK3CA mutations, are of importance for treat- the hypoxia-inducible-factor 1-α (HIF1-α) is under translational ment tailoring, markers predictive of resistance may be useful. control of the mTOR complex 1 (mTORC1; glossary box; Thomas In fact, tumors harboring a given therapeutic target not uncom- et al., 2006; Linehan et al., 2010). Exploratory subgroup analysis monly display primary (i.e., de novo) resistance or develop resis- of the 209 patients from the Temsirolimus single agent arm of the tance over time (van der Heijden and Bernards, 2010; Turner phase III global ARCC trial (Hudes et al., 2007) investigated PTEN and Reis-Filho, 2012). In several studies discussed here assess- and HIF1-α protein expression levels by immunohistochemistry ing determinants of single agent PI3K pathway inhibitor response, (IHC) on formalin fixed paraffin embedded nephrectomy or core KRAS mutations were found to be associated with resistance to biopsy derived tissues. Importantly, baseline PTEN or HIF1-α lev- these targeted agents (Engelman et al., 2008; Brachmann et al., els were shown not to be associated with single agent Temsirolimus 2009; Ihle et al., 2009; Dan et al., 2010; Meuillet et al., 2010; Gar- response (Figlin et al., 2009; Table 3). Furthermore, in a retrospec- nett et al., 2012), as were mutations in APC, BRAF, or MYCN tive subgroup analysis from a phase II clinical trial of ARCC (Atkins (http://www.cancerrxgene.org/; Garnett et al., 2012). et al., 2004) including 20 patients (Cho et al., 2007), carbonic anhy- Finally, in addition to genetic alterations of components of drase IX (CA9), p-AKT, and PTEN protein expression levels using the PI3K pathway germline polymorphisms may affect response IHC or VHL mutation status were shown not to be significantly of patients treated with targeted therapies. Ng et al. (2012) have associated with single agent Temsirolimus response. There was, recently identified a common intronic deletion polymorphism of however, a significant positive association between higher p-rpS6 the BIM gene that leads to the generation of an alternative spliced expression, a downstream effector of mTORC1 (Figure 1), and BIM isoform lacking the BH3 domain, which is required for tyro- clinical Temsirolimus response (Cho et al., 2007). It should be sine kinase inhibitor induced apoptosis. This polymorphism was noted that the analysis above was performed in a limited number shown to confer intrinsic resistance to RTK inhibitors in chronic of patients and their statistical power to reveal the associations myeloid leukemia and EGFR mutated NSCLC cell lines (Ng et al., should be taken into account. 2012). It is plausible that this and other germline polymorphisms The vast majority of completed to date trials testing PI3K may results in resistance to agents targeting the PI3K pathway. pathway inhibitors in tumor types other than renal cell carci- Taken together, preclinical studies focusing on breast can- noma also focused on rapalogs, but only few studies assessed cer only have repeatedly identified PIK3CA mutations and potential genomic predictors (Table 3). Based on the rationale HER2 amplifications as predictors of sensitivity to PI3K path- that PIK3CA mutations may predict response to PI3K pathway way inhibitors. In other tumor types, however, the genotype-drug inhibitors, breast, cervical, endometrial, and ovarian cancers were response associations are less defined and PIK3CA mutations, sequenced for the presence of activating PIK3CA mutations and PTEN loss of function or both, or CDKN2A mutations have been treated with different allosteric mTOR inhibitors (i.e., rapalogs) or reported as determinants of response. Furthermore, the in vitro the PI3K inhibitor PX866 either as single agent or combination in a and animal model studies revealed that in cancer cells other than prospective phase I clinical trial. A partial response was observed in breast cancer, where MAPK pathway mutations are rare (COS- 30% of the 23 patients with tumors harboring a PIK3CA mutation MIC), KRAS mutations may confer resistance to single agent PI3K in contrast to 10% of 70 patients whose tumors were PIK3CA wild- pathway inhibitor treatment, as do mutations in BRAF, APC and type (Janku et al., 2012), consistent with the preclinical observa- MYCN. tions. Interestingly, whilst in preclinical models mutations in KRAS have been found to confer resistance to PI3K pathway inhibition, as GENOMIC DETERMINANTS OF PI3K PATHWAY INHIBITOR discussed above, in this trial 2/7 ovarian cancer patients with coex- RESPONSE IN CLINICAL TRIALS isting PIK3CA and KRAS or BRAF mutations responded to the Rapamycin analogs (“rapalogs”; glossary box) were the first PI3K anti-PI3K pathway treatment (Janku et al., 2012). This finding may pathway inhibitors to be tested in clinical trials for the treatment of be tumor type-specific, given that the same group had previously cancer, and Everolimus and Temsirolimus have been approved by described that colorectal cancers harboring simultaneous PIK3CA the US Food and Drug Administration (FDA) for the treatment of and KRAS mutations were resistant to PI3K pathway inhibitor advanced renal cell carcinoma (ARCC), and Everolimus has also treatment (Janku et al., 2011). A similar, but not statistically sig- been approved for the treatment of progressive neuroendocrine nificant, trend was observed in a retrospective subgroup analysis tumors of pancreatic origin and non-malignant kidney and brain of 43 patients with different tumor types but most frequently tumors (Table 1). colorectal cancer from phase I/II clinical study of single agent The determinants of mTOR inhibition in renal cell carcino- Everolimus (Tabernero et al., 2008; Di Nicolantonio et al., 2010). mas may differ from those of other solid malignancies. In fact, Patients whose tumors harbored PIK3CA mutations or PTEN loss Frontiers in Oncology | Molecular and Cellular Oncology August 2012 | Volume 2 | Article 109 | 12 Table 3 | Genomic determinants of response to PI3K pathway inhibitors identified in clinical trials. Inhibitor name Cancer type Clinical trial Patients (n) Genomic Non-genomic Genomic Reference (target) determinant of determinant of determinant of Weigelt and Downward www.frontiersin.org sensitivity sensitivity resistance Temsirolimus (mTOR), single Renal cell carcinoma NCT00065468 209 (Temsirolimus arm; NA None (PTEN or HIF1-α NA Figlin et al. agent (retrospective subgroup ∼60% assessed) protein expression (2009) analysis; phase III) assessed) Temsirolimus (mTOR), single Renal cell carcinoma ND (retrospective 20 None (VHL mutation p-rpS6 (Ser235) NA Cho et al. agent subgroup analysis; assessed) (2007) phase II) Temsirolimus (mTOR), single Breast, cervical, NCT00761644, 140 (217) PIK3CA mutation NA Coexisting KRAS Janku et al. agent, or combined; endometrial, ovarian NCT00877773, mutation (2011, 2012) Rapamycin (mTOR), cancer (colorectal, head NCT01054313, (tissue-specific) combined; PX866 (PI3K), and neck) NCT00610493, single agent NCT00726583 (phases I/II) Everolimus (mTOR), single Colorectal, breast, ND (retrospective 43 PIK3CA mutation NA Coexisting KRAS Di agent melanoma, pancreas, subgroup analysis; PTEN loss of mutation Nicolantonio HNSCC phase I/II) function et al. (2010) Temsirolimus (mTOR), Ovarian, uterine, cervix, NCT00761644 (phase I) 74 PIK3CA mutation NA NA Moroney et al. combined breast cancer PTEN loss of (2011) function Everolimus (mTOR), Neuroendocrine NCT00113360 60 (17 assessed) NA p-AKT (Thr308; PSF) NA Meric- combined carcinoma (retrospective subgroup Bernstam analysis; phase II) et al. (2012) Everolimus (mTOR), single NSCLC NCT00124280 (phase I) 58 (40 assessed) NA p-AKT (Ser473, Thr308; NA Soria et al. agent PFS) (2009) Everolimus (mTOR), single SCLC NCT00374140 (phase II) 40 (22 assessed) NA S6K NA Tarhini et al. agent (2010) Temsirolimus (mTOR), single Glioblastoma NCT00016328 (phase II) 56 (43 assessed) NA p-S6K (Thr421/Ser424) NA Galanis et al. agent multiforme (2005) Deforolimus (Ridaforolimus; Sarcoma NCT00288431 20 NA p-rpS6 (Ser235/236) NA Iwenofu et al. mTOR), single agent, and NCT00093080 (phase (2008) combination I/II; retrospective subgroup analysis) Temsirolimus (mTOR), single Neuroendocrine NCT00093782 37 (35 assessed) NA p-mTOR (Ser2448) NA Duran et al. agent carcinoma (phase II) (2006) (Continued) August 2012 | Volume 2 | Article 109 | 13 PI3K pathway inhibitor response Weigelt and Downward PI3K pathway inhibitor response CA9, carbonic anhydrase IX; HNSCC, head and neck squamous cell carcinoma; NA, not assessed; ND, not defined; NSCLC, non-small cell lung cancer; PFS, progression-free survival; SCLC, small cell lung cancer. Behbakht et al. of function were more likely to benefit from Everolimus, except in Chawla et al. Ellard et al. presence of coexistent KRAS/BRAF mutations (Di Nicolantonio Reference et al., 2010). A high percentage of responders with PI3K path- (2009) (2012) (2011) way aberrations as determined by PIK3CA mutations or PTEN loss of function was also reported in a phase I trial of lipo- somal Doxorubicin, Bevacizumab, and Temsirolimus (Moroney determinant of et al., 2011). Taken together, these results suggest PI3KCA gain- resistance of-function mutations may predict sensitivity to PI3K pathway Genomic inhibitors, whereas KRAS and BRAF mutations may lead to resis- tance in some tumor types such as colorectal cancer. Importantly, NA NA NA however, the data available demonstrate that PIK3CA activating None (p27Kip1, IGF-1R, p-S6K, p-4E-BP1, cyclin mutations are neither required nor sufficient for a tumor to be eIF4E, p-AKT, FKBP12) None (p-AKT, p-mTOR, PTEN, p-S6K, 4E-PB1, None (PTEN, p-AKT, CA9, ER, PR, HER2 sensitive to PI3K pathway inhibitors, and that a substantial pro- portion of cases with PIK3CA activating mutations may be de novo determinant of Non-genomic resistant to these agents. sensitivity assessed) Not only genomic predictors but also PI3K pathway activation state as determined by expression levels of markers upstream of D1) mTORC1, such as p-AKT, or downstream of mTORC1, such as p-S6K (Figure 1), have been shown to correlate with sensitivity to allosteric mTOR inhibitors in breast cancer cell lines in vitro (Noh determinant of et al., 2004; Meric-Bernstam et al., 2012). In fact, several clini- cal trials testing rapalogs evaluated PI3K signaling biomarkers on sensitivity Genomic baseline tumor tissue by IHC rather than performing sequenc- ing analysis. In a phase II study, high p-AKT levels on baseline NA NA NA and on-treatment fine needle aspirations of tumors from patients with neuroendocrine carcinoma (n = 17) assessed by reverse phase protein arrays correlated with longer progression-free survival 212 (∼80 assessed) (PFS; Meric-Bernstam et al., 2012). Moreover, in NSCLC patients 49 (47 assessed) 54 (51 assessed) treated with Everolimus (n = 40), p-AKT levels at baseline deter- Patients (n) mined by IHC were reported to be independent predictors of PFS (Soria et al., 2009; Table 3). It should be noted, however, that the authors emphasized that tissue fixation had large effects on immunoreactivity when assessing phosphorylated proteins, which may compound the implementation of this IHC predictive test in NCT00255788 (phase clinical practice. In a phase II trial, p-S6K levels assessed by IHC in base- NCT00093080 NCT00429793 Clinical trial line glioblastoma multiforme samples were associated with sin- (phase II) (phase II) gle agent Temsirolimus response (n = 44; Galanis et al., 2005). Also in a small retrospective subgroup analysis of two phase I/II clinical trials p-rpS6, downstream of p-S6K, was correlated II) with early response of sarcomas to the rapalog Deforolimus (i.e., peritoneal malignancies Ridaforolimus) alone or in combination with doxorubicin (n = 20; Epithelial ovarian and Bone and soft tissue Iwenofu et al., 2008). Not only expression levels of activated (i.e., phosphorylated) S6K/rpS6 have been found to correlate with Breast cancer Cancer type response to allosteric mTOR inhibitors, but in phase II study, sarcoma total S6K expression in baseline SCLC tumor tissue defined by IHC was reported as a potential predictive biomarker for the ther- apeutic benefit of Everolimus (n = 22; Tarhini et al., 2010). In addition, higher baseline levels of p-mTOR itself assessed by IHC Ridaforolimus (mTOR), single Temsirolimus (mTOR), single Everolimus (mTOR), single predicted for a better response to Temsirolimus in patients with neuroendocrine carcinoma in a phase II study (n = 35; Duran Table 3 | Continued et al., 2006). Other, similarly powered phase II trials however did not identify Inhibitor name any correlates between potential biomarkers assessed in archival tumor material using IHC and treatment response (Table 3). In (target) agent agent agent metastatic breast cancers, no association between p-AKT, PTEN, CA9, ER, progesterone receptor or HER2 expression, and response Frontiers in Oncology | Molecular and Cellular Oncology August 2012 | Volume 2 | Article 109 | 14 Weigelt and Downward PI3K pathway inhibitor response to Everolimus was found (Ellard et al., 2009). In bone and soft response was found (Di Nicolantonio et al., 2010; Moroney et al., tissue sarcomas, an extended subgroup analysis (n ≈ 80; Chawla 2011) but not in others (Ellard et al., 2009; Figlin et al., 2009; et al., 2012) did not confirm the previously published analysis Chawla et al., 2012). A similar picture is seen when expression on 20 patients, which identified p-rpS6 levels as Ridaforolimus levels of phosphorylated proteins of the PI3K pathway are used response predictors (Iwenofu et al., 2008). In fact, neither the as a read-out of its activation state and determinant of response potential biomarkers upstream of mTORC1, including PTEN, (Table 3). IHC of phosphorylated proteins has proven challenging p-AKT, FKBP21, or IGF-1R, nor downstream of mTORC1, p- (Soria et al., 2009; Pinhel et al., 2010) and also PTEN staining is not S6K, 4E-BP1, eIF4E, or p27kip1, were predictive of clinical benefit routinely performed. Recent reports focused on the reproducibil- response to Ridaforolimus (Chawla et al., 2012). Furthermore, ity of PTEN staining protocols and scoring (Sakr et al., 2010; Garg p-AKT, p-mTOR, p-4E-BP1, and cyclin D1 expression levels in et al., 2012), however guidelines for accurate PTEN testing and its epithelial ovarian and peritoneal tumors were shown not to utility as predictive marker have yet to be established. be associated with partial/complete tumor responses to Tem- Early clinical trials often analyze archival tissue of the primary sirolimus, however cyclin D1 expression seemed to correlate with tumor for the presence of specific mutations and the response PFS ≥6 months (Behbakht et al., 2011). of the metastatic lesions are correlated with the mutational sta- The completed phase I/II clinical trials to date are to some tus. Recent analyses of paired primary tumors and metastases extent consistent with the preclinical observations in that tumors have revealed that there is a high level of discordance in PTEN harboring PIK3CA mutations may be more likely to respond to expression level and PIK3CA mutation status, which may influ- PI3K pathway inhibitors. It is important to note, however, that not ence patient selection and response to PI3K targeted therapies all patients with tumors harboring PIK3CA mutations are sensi- (Dupont Jensen et al., 2011; Gonzalez-Angulo et al., 2011). tive to PI3K pathway inhibitor treatment, and, on the other hand, Despite the interest in the development of biomarkers for that also subsets of patients with wild-type PIK3CA/PTEN cancers patient selection in clinical trials testing PI3K pathway inhibitors, are responsive. The results from studies analyzing the activation none of the biomarkers tested so far is supported by level I evi- state of PI3K pathway components by IHC are variable and no dence. Importantly, however, one of the most exciting results of consistent determinant of response has been identified to date. allosteric mTOR inhibitors in the context of a clinical study was the BOLERO-2 trial, where patients with ER-positive advanced DEVELOPMENT OF MOLECULAR TOOLS FOR THE breast cancers resistant to aromatase inhibitors were randomized STRATIFICATION OF CANCER PATIENTS to receive Exemestane (a non-steroidal aromatase inhibitor) plus For the development of molecular markers for patient stratifica- Everolimus or Exemestane plus Placebo (Baselga et al., 2012). The tion in clinical trials testing the efficacy of PI3K pathway inhibitors, rationale for this stemmed from preclinical observations that resis- it is crucial to take into account the observations that in some tance to endocrine therapy in breast cancer is associated with tumor types, either PIK3CA activating mutations or PTEN loss activation of the PI3K pathway (Miller et al., 2011). Despite the of function are predictors of sensitivity, whereas in other tumor lack of a patient stratification biomarker, this trial demonstrated types, both predict sensitivity to these agents (Table 2). These data that addition of Everolimus to Exemestane increased the median imply that the mutational repertoire and the epistatic interactions PFS from 4.1 to 10.6 months (Baselga et al., 2012). Although a between different components of the PI3K pathway may be distinct substantial proportion of patients included in this trial may har- in different tumor types, that genetic lesions in different compo- bor PIK3CA activating mutations, given that they are more likely nents of the pathway may not have the same functional effects in to occur in ER-positive postmenopausal patients (Kalinsky et al., different tumor types, and that a genetic determinant identified in 2009), other mechanisms resulting in PI3K pathway activation are one cancer type may not necessarily be applicable to another. This likely to play a role in resistance to endocrine therapy. The material is perhaps best exemplified by BRAF V600E mutations, which are from this trial will constitute a unique resource to determine the predictive of response to Vemurafenib in melanoma, however col- genomic and epigenomic determinants of sensitivity to concurrent orectal cancer patients harboring oncogenic BRAF V600E mutations mTOR inhibition and endocrine treatment in breast cancer. derive limited if any benefit from this drug due to increased EGFR expression (Prahallad et al., 2012). Likewise, the clinical trials dis- FUTURE PERSPECTIVES AND CHALLENGES cussed above provide evidence to suggest that ovarian cancers with Despite the critical role of the PI3K pathway in cancer, the intro- coexisting PIK3CA and MAPK pathway mutations may be sensi- duction of single-agent PI3K pathway inhibitors into the clinic tive to PI3K pathway inhibition, whereas colorectal cancers har- may be challenging. In fact, of all PI3K pathway inhibitors dis- boring the same repertoire of mutations affecting these genes may cussed here, one of the most exciting targeted agents is the p110δ be resistant (Di Nicolantonio et al., 2010; Janku et al., 2011, 2012). inhibitor CAL-101, which has shown remarkable clinical activity Results from preclinical studies performed have further sug- in certain hematological diseases including chronic lymphocytic gested that cancer cells harboring PIK3CA mutations might be leukemia. Inhibition of p110δ is though to target both the malig- among the most sensitive to single agent PI3K pathway inhibitors. nant B cells and the tumor microenvironment of chronic lymphoid These data were in part confirmed in the three clinical trials assess- leukemia (Fruman and Rommel, 2011). The clinical trials per- ing tumor PIK3CA mutational status (Di Nicolantonio et al., 2010; formed thus far using allosteric mTOR inhibitors as single agents Janku et al., 2011, 2012; Moroney et al., 2011). The predictive value have seen some stable diseases and partial responses, however of the PTEN status is however less clear, as in some clinical trials an by no means are these responses as dramatic as for example for association between PTEN deficiency and PI3K pathway inhibitor Vemurafenib in BRAF mutant melanoma (Flaherty et al., 2010). www.frontiersin.org August 2012 | Volume 2 | Article 109 | 15 Weigelt and Downward PI3K pathway inhibitor response Based in the preclinical data, kinase inhibitors seem to target the that some targeted agents may be effective in vivo due to targeting PI3K pathway more robustly and, in contrast to allosteric mTOR of tumor microenvironment interactions (Fruman and Rommel, inhibitors, also promote apoptotic effects in vitro and in vivo 2011), which are unlikely to be uncovered using conventional (Brachmann et al., 2009; O’Brien et al., 2010; Weigelt et al., 2011). in vitro cell culture models or by the genomic characterization Several feedback loops upon PI3K/AKT/mTOR inhibition have of tumor cells only. been described, which amongst others lead to activation of the With the advent of massively parallel sequencing technologies, MAPK signaling pathway or re-activation of the PI3K pathway several studies have documented intra-tumor genetic heterogene- (reviewed in Carracedo and Pandolfi, 2008; Efeyan and Sabatini, ity in solid cancers (reviewed in Turner and Reis-Filho, 2012; 2010; Chandarlapaty, 2012; Laplante and Sabatini, 2012). These Yap et al., 2012), and revealed that certain mutations, includ- feedbacks may play a role in the modest responses observed thus ing PIK3CA or PTEN mutations, may be only prevalent in a far using single agent rapalogs, and for the optimal activity of PI3K subset of tumor cells in a given cancer (Gerlinger et al., 2012; pathway inhibitors, co-administration with other agents may be Shah et al., 2012). This has not only consequences for can- required. cer drug resistance and the clinical utility of single agent tar- Numerous clinical trials are currently testing the safety and geted therapy, but also questions whether potential biomarkers efficacy of combination PI3K pathway and MEK inhibitors in assessed in a single biopsy will be representative of the entire advanced solid tumors to target both the driver and poten- tumor. tial “escape” pathways. As discussed above, such combinatorial Given the crucial role of the PI3K pathway in cancer, inhibitors approach has been successfully performed in breast cancer, where of its components are expected to be effective in subsets of many the combination of an aromatase inhibitor with Everolimus led different cancer types. Preclinical models have proven useful in to substantial improvement of PFS (Baselga et al., 2012). For the the identification of potential predictive biomarkers, however tis- identification of optimal combinations of PI3K pathway inhibitors sue collection and assessment of biomarkers even in early clinical with other agents, Drosophila models may provide an effective trials are crucial, as is the development of robust and accurate tool as these have been successfully employed for the identifica- companion diagnostics. With the number of ongoing clinical tri- tion of agents with optimized pharmacological profiles (Das and als currently testing a wide gamut of PI3K pathway inhibitors, Cagan, 2010; Dar et al., 2012). For combinatorial treatments it our community should expect a wealth of data, which will help will be crucial to understand the activation of negative feedback improve therapeutic strategies for cancer patients. loops in the PI3K pathway and the cross-talk with other pathway upon inhibition of its different components, and whether the feed- GLOSSARY back activation is dependent on specific epistatic interactions and PHOSPHOINOSITIDE 3-KINASE CLASSES distinct in tumors from different anatomical sites. According to their structures and substrate specificities, PI3Ks are Drug sensitivity and resistance are likely to constitute conver- divided into three classes, and class I PI3Ks are directly activated gent phenotypes, meaning that they may be driven by distinct by cell surface receptors (Liu et al., 2009). Class IA PI3Ks are genetic aberrations in the same tumor type (Gerlinger and Swan- heterodimeric lipid kinases composed of a p110 catalytic sub- ton, 2010; Turner and Reis-Filho, 2012; Weigelt et al., 2012; Yap unit (isoforms p110α, p110β, and p110δ, encoded by PIK3CA, et al., 2012). It has become apparent from in vitro studies that PIK3CB, and PIK3CD, respectively), and a regulatory subunit there are significant correlations between specific mutations and (p85α and its splice variants p55α and p50α), p85β, and p55γ, treatment response, however the negative predictive value of these encoded by PIK3R1, PIK3R2, and PIK3R3, respectively); the class mutations is often poor and not all sensitive cancers are identified IB PI3K is composed of the p110γ catalytic subunit, encoded by by single mutations/single gene panels. For example, O’Brien et al. PIK3CG, and the regulatory subunit p101, p84/p87 (Liu et al., (2010) showed that in their cell line panel tested, PIK3CA muta- 2009; Vanhaesebroeck et al., 2010). tions and HER2 amplification showed excellent specificity (100 RAPAMYCIN AND RAPAMYCIN ANALOGS (“RAPALOGS”) and 95%, respectively) and a high positive predictive value, but relatively low sensitivity (∼30%) and a poor negative predictive Mechanistic target of rapamycin is a serine/threonine kinase that value as single markers in predicting drug responsiveness in the interacts with several proteins to form two distinct signaling com- cell line panel analyzed. Additional biomarkers will therefore be plexes called mTORC1 and mTORC2 (Laplante and Sabatini, required to identify all patients likely to respond to PI3K path- 2012). Rapamycin and rapamycin analogs (“rapalogs”) bind the way inhibitors. To date, the majority of studies have focused on FK506-binding protein (FKBP12) and together target preferen- the analysis of PIK3CA mutations or PTEN deficiency as potential tially the mTORC1 by an allosteric mechanism, however pro- determinants of PI3K pathway inhibitor response. However, also longed treatment may also inhibit mTORC2 and disrupt its main activating mutations in other components of the pathway, such as substrate AKT, possibly in a tissue-specific manner (Sarbassov PIK3R1 (Jaiswal et al., 2009; Urick et al., 2011) or mTOR (Sato et al., et al., 2006; Lamming et al., 2012). 2010; Hardt et al., 2011), or loss of function of TSC1/2 (COSMIC; El-Hashemite et al., 2003; Sjodahl et al., 2011) or INPP4B (Gewin- ACKNOWLEDGMENTS ner et al., 2009; Fedele et al., 2010) may play a role in PI3K pathway We apologize to those authors whose work we could not cite inhibitor response. Furthermore, and as mentioned above, the directly due to space constraints. We would like to thank J. S. remarkable single agent activity of the PI3K isoform specific p110δ Reis-Filho (The Breakthrough Breast Cancer Centre, London) for inhibitor CAL-101 in chronic lymphocytic leukemia, a disease in advice and critical reading of the manuscript. Britta Weigelt is which PI3K pathway aberrations are relatively rare, emphasizes funded by a Cancer Research UK postdoctoral fellowship. Frontiers in Oncology | Molecular and Cellular Oncology August 2012 | Volume 2 | Article 109 | 16 Weigelt and Downward PI3K pathway inhibitor response REFERENCES in postmenopausal hormone- Cortes, I., Sanchez-Ruiz, J., Zuluaga, El-Hashemite, N., Zhang, H., Henske, Allegra, C. J., Jessup, J. M., Somer- receptor-positive advanced breast S., Calvanese, V., Marques, M., Her- E. P., and Kwiatkowski, D. J. field, M. R., Hamilton, S. R., Ham- cancer. N. Engl. J. Med. 366, nandez, C., Rivera, T., Kremer, L., (2003). Mutation in TSC2 and acti- mond, E. H., Hayes, D. F., Mcallister, 520–529. 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Oncol. 9, 58–64. cal, cellular, and in vivo activity of any third-party graphics etc. Frontiers in Oncology | Molecular and Cellular Oncology August 2012 | Volume 2 | Article 109 | 20 REVIEW ARTICLE published: 16 October 2012 doi: 10.3389/fonc.2012.00145 Abrogating endocrine resistance by targeting ERα and PI3K in breast cancer Emily M. Fox1 , Carlos L. Arteaga1,2,3 and Todd W. Miller4 * 1 Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN, USA 2 Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN, USA 3 Breast Cancer Research Program, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN, USA 4 Department of Pharmacology and Toxicology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA Edited by: Antiestrogen therapies targeting estrogen receptor α (ER) signaling are a mainstay for Alexandre Arcaro, University of Bern, patients with ER+ breast cancer. While many cancers exhibit resistance to antiestrogen Switzerland therapies, a large body of clinical and experimental evidence indicates that hyperactivation Reviewed by: Keisuke Ito, Harvard Medical School, of the phosphatidylinositol 3-kinase (PI3K) pathway promotes antiestrogen resistance. In USA addition, continued ligand-independent ER signaling in the setting of estrogen deprivation Clement Lee, Mount Sinai School of may contribute to resistance to endocrine therapy. PI3K activates several proteins which Medicine, USA promote cell cycle progression and survival. In ER+ breast cancer cells, PI3K promotes *Correspondence: ligand-dependent and -independent ER transcriptional activity. Models of antiestrogen- Todd W. Miller, Dartmouth–Hitchcock Medical Center, One Medical Center resistant breast cancer often remain sensitive to estrogen stimulation and PI3K inhibition, Drive, HB-7936, Lebanon, NH 03756, suggesting that clinical trials with combinations of drugs targeting both the PI3K and ER USA. pathways are warranted. Herein, we review recent findings on the roles of PI3K and ER e-mail: todd.w.miller@dartmouth.edu in antiestrogen resistance, and clinical trials testing drug combinations which target both pathways. We also discuss the need for clinical investigation of ER downregulators in combination with PI3K inhibitors. Keywords: PI3K, breast cancer, antiestrogen, aromatase, fulvestrant, tamoxifen, estrogen receptor INTRODUCTION testing of drug combinations targeting the ER and PI3K pathways, At least 75% of breast cancers express estrogen receptor α (ER) and the need to clinically address the potential for continued ER and/or progesterone receptor (PR), which are tumor biomark- signaling in patients treated with endocrine therapies. ers of estrogen dependence. Antiestrogen treatments for patients with ER+ or PR+ breast cancer inhibit ER by antagonizing estro- RATIONALE FOR COMBINED TARGETING OF THE ER AND gen ligand binding to ER (tamoxifen and other selective estrogen PI3K PATHWAYS receptor modulators, SERMs), inhibiting dimerization and down- We and others identified a requirement for PI3K in the estrogen- regulating ER (fulvestrant and other selective estrogen receptor independent growth of long-term estrogen-deprived (LTED) ER+ downregulators, SERDs), or blocking estrogen production (aro- breast cancer cells, which mirror clinical resistance to AIs (Sabnis matase inhibitors, AIs; letrozole, anastrozole, exemestane). While et al., 2007; Crowder et al., 2009; Miller et al., 2010). Proteomic such endocrine therapies have changed the natural history of ER+ profiling revealed amplification of PI3K signaling via the mTOR breast cancer, many tumors exhibit de novo or acquired drug substrates p70S6 kinase and p85S6 kinase, and the PI3K effec- resistance (Table 1). The only clinically validated mechanism of tor AKT in ER+ human breast cancer cells adapted to hormone resistance to endocrine therapy is overexpression or amplifica- deprivation. Treatment with the ATP-competitive PI3K/mTOR tion of the ERBB2 (HER2) protooncogene (Arpino et al., 2004; dual inhibitor BEZ235 (Maira et al., 2008) completely suppressed De Laurentiis et al., 2005; Ellis et al., 2006). However, only 10% the emergence of hormone-independent ER+ cells and induced of ER+ breast cancers exhibit HER2 overexpression, prompt- apoptosis in cell lines harboring activating mutations in PIK3CA ing the need for discovery of other mechanisms of antiestrogen (gene that encodes the p110α subunit of PI3K) or PTEN loss resistance. (PTEN antagonizes PI3K signaling). In contrast, the TORC1 A large body of experimental and clinical evidence suggests inhibitor everolimus (Schuler et al., 1997) had only a partial effect that hyperactivation of the phosphatidylinositol 3-kinase (PI3K) (Miller et al., 2010; Sanchez et al., 2011). This partial effect may pathway, the most frequently mutated pathway in breast cancer, be attributable to feedback activation of PI3K/AKT upon inhibi- promotes antiestrogen resistance. PI3K is commonly activated by tion of TORC1 (O’Reilly et al., 2006; Carracedo et al., 2008; Miller growth factor receptor tyrosine kinases and G-protein-coupled et al., 2009), suggesting that direct inhibitors of PI3K may be more receptors in breast cancer cells. The signaling cascades triggered by effective than rapalogs in this setting. PI3K, including PDK1, AKT, and SGK among others, promote cell In a siRNA screen against 779 kinases, we implicated insulin growth and survival. For detailed information, we refer the reader receptor (InsR) in the hormone-independent growth of MCF- to a recently published, comprehensive review of this material 7/LTED cells. InsR and its homolog IGF-1R dimerize and, upon (Miller et al., 2011a). Herein, we focus on updated findings, clinical ligand binding, potently activate PI3K. IGF-1R has also been www.frontiersin.org October 2012 | Volume 2 | Article 145 | 21 “fonc-02-00145” — 2012/10/15 — 10:50 — page 1 — #1 Fox et al. ER and PI3K in endocrine resistance Table 1 | Frequencies of breast cancer recurrence and resistance to anti-estrogen therapies in patients with ER+ breast cancer. Population Treatment Effect Trial/reference Follow-up: 5 years 10 years Early-stage Adjuvant anastrozole × 5 years Distant recurrence: 9.8% 19.7% ATAC (Cuzick et al., 2010) Post-menopausal Adjuvant tamoxifen × 5 years Distant recurrence: 12.5% 24% Follow-up: 5 years 8 years Early-stage Adjuvant letrozole × 5 years Recurrence: 14.5% 23.6% BIG 1-98 (Regan et al., 2011) Post-menopausal Adjuvant tamoxifen × 5 years Recurrence: 18% 28% Advanced Post-menopausal Anastrozole No clinical benefit: 33% FIRST (Robertson et al., 2009a) No prior Tx Fulvestrant (high-dose regimen) No clinical benefit: 27.5% Advanced Post-menopausal Exemestane No clinical benefit: 68.5% EFECT (Chia et al., 2008) Progressed on AI Fulvestrant (loading-dose regimen) No clinical benefit: 67.8% Median follow-up: 5.3 years Disease-free following Letrozole With disease: 2% MA.17 5 years of adjuvant tamoxifen Placebo With disease: 4.9% (Goss et al., 2008) Median follow-up: 2.5 years Disease-free following Exemestane With disease: 9% NSABP B-33 5 years of adjuvant tamoxifen Placebo With disease: 11% (Mamounas et al., 2008) shown to confer antiestrogen resistance in MCF-7 cells (Zhang can be shifted using PI3K and ER inhibitors in preclinical models et al., 2011). Treatment with the ATP-competitive IGF-1R/InsR (Figure 2; Creighton et al., 2010; Miller et al., 2010), suggesting inhibitor OSI-906 suppressed PI3K activation and hormone- that cells may defer to the other pathway when one is inhibited. independent ER+ cell growth (Fox et al., 2011). Network mapping Crosstalk between the PI3K and ER pathways has also been of the 42 kinases individually implicated in MCF-7/LTED cell suggested as a mechanism of endocrine resistance (Musgrove growth in this screen revealed that PI3K is a central hub in and Sutherland, 2009). PI3K activation was shown to induce these signaling pathways (Figure 1). Interestingly, a recent study ER phosphorylation at the putative AKT/p70S6K site Ser167 and showed that in ER+ breast cancer cells treated with BEZ235 or estrogen-independent transcriptional activity (Campbell et al., with PI3K siRNA, exogenous 17β-estradiol rescued the cells from 2001; Yamnik et al., 2009). However, treatment of such cells in drug- and siRNA-induced apoptosis (Crowder et al., 2009; Sanchez hormone-depleted conditions with everolimus or the pan-PI3K et al., 2011). This suggests that in ER+ cancers treated with PI3K inhibitor BKM120 (Maira et al., 2012) did not decrease ER phos- inhibitors, estrogen suppression should be maintained and, there- phorylation at Ser167 , ER-DNA binding, or ER transcriptional fore, combined inhibition of both PI3K and ER may be more reporter activity (Miller et al., 2011b). These data collectively effective than single-agent therapies. suggest that PI3K effectors do not modulate ER in the absence Clinical evidence further indicates that PI3K pathway activa- of estrogens. Analysis of the effects of BKM120 and fulves- tion is associated with antiestrogen resistance. Patients bearing trant on hormone-independent cell growth showed synergy in primary ER+ breast tumors which exhibit a protein expres- 6/8 ER+ lines. In mice bearing ER+ breast cancer xenografts, sion/phosphorylation signature of PI3K activation, as deter- single-agent treatment with BKM120 or fulvestrant slowed tumor mined using reverse-phase protein arrays (RPPA), have a shorter growth, while the combination induced tumor regression. Sim- recurrence-free survival (Miller et al., 2010). RPPA analysis of ER+ ilarly, treatment with the ATP-competitive IGF-1R/InsR dual primary breast tumors obtained from patients following 2–3 weeks inhibitor OSI-906, which blocks downstream activation of PI3K in of treatment with the AI letrozole showed that a protein signa- MCF-7 cells, slowed tumor growth and induced regression when ture of insulin signaling was associated with high post-AI tumor combined with fulvestrant (Fox et al., 2011). These data further cell proliferation (Fox et al., 2011). Overexpression of HER2 or imply that combined targeting of the ER and PI3K pathways is FGFR1, or loss of INPP4B, molecular lesions which activate the more effective than single-agent therapies. PI3K pathway, also confer antiestrogen resistance in patients with ER+ breast cancer (Arpino et al., 2004; De Laurentiis et al., 2005; CLINICAL TRIALS TESTING DRUG COMBINATIONS Ellis et al., 2006; Gewinner et al., 2009; Turner et al., 2010). Also TARGETING THE ER AND PI3K PATHWAYS noteworthy is the inverse correlation between levels of PI3K acti- Herein, we will review three recent clinical studies that evalu- vation and ER protein in human tumors. This ER/PI3K balance ated the benefit of adding the TORC1 inhibitor everolimus to Frontiers in Oncology | Molecular and Cellular Oncology October 2012 | Volume 2 | Article 145 | 22 “fonc-02-00145” — 2012/10/15 — 10:50 — page 2 — #2 Fox et al. ER and PI3K in endocrine resistance FIGURE 1 | Phosphatidylinositol 3-kinase is a central hub in signaling Fox et al., 2011). Ingenuity Pathways Analysis revealed that these 42 pathways required for estrogen-independent ER+ breast cancer kinases map to several protein networks that overlap with PI3K cell growth. MCF-7/LTED cells transiently transfected with a siRNA signaling (red box, enlarged in bottom panel). Proteins involved in these library targeting 779 kinases were reseeded in hormone-depleted networks are displayed as nodes. Solid lines indicate direct relationships medium. Cell viability was measured 4–5 days later by Alamar blue between proteins, and dotted lines indicate indirect interactions. Green assay. Median cell growth in four independent experiments was nodes represent the kinases identified in the screen, as well as others calculated for each kinase siRNA relative to non-silencing controls. whose knockdown was predicted by the Ingenuity software to negatively Individual knockdown of 42 kinases inhibited MCF-7/LTED cell growth affect cell growth. The various nodal shapes represent the functional class ≥33% (p ≤ 0.05) in at least three of four experiments (detailed in of the gene product. www.frontiersin.org October 2012 | Volume 2 | Article 145 | 23 “fonc-02-00145” — 2012/10/15 — 10:50 — page 3 — #3 Fox et al. ER and PI3K in endocrine resistance endocrine therapy. (1) In the first study, post-menopausal women with early-stage ER+ breast cancer were randomized to neoad- juvant therapy with the AI letrozole ± everolimus for 4 months. The addition of everolimus increased clinical response and sup- pression of tumor cell proliferation at 2 weeks (measured by Ki67 IHC) compared to letrozole alone (Baselga et al., 2009). (2) In the TAMRAD study, post-menopausal patients with metastatic, ER+, AI-resistant breast cancer were randomized to treatment with tamoxifen ± everolimus. The addition of everolimus improved clinical benefit rate, time-to-progression, and disease-free sur- vival compared to tamoxifen alone (Bachelot et al., 2010). (3) The phase III BOLERO-2 study included 724 post-menopausal women with metastatic, ER+, HER2-negative breast cancer. While 84% of patients exhibited sensitivity to prior endocrine therapy, all were resistant to non-steroidal AIs (letrozole, anastrozole) at the time of randomization to treatment with the steroidal AI exemestane ± everolimus. The addition of everolimus increased progression-free survival (PFS) from 4.1 months (exemestane alone) to 10.6 months (Baselga et al., 2012). While the addition of a TORC1 inhibitor prevents disease progression in patients with antiestrogen-resistant breast can- cer, inhibition of TORC1 relieves negative feedback on activators of PI3K (e.g., IGF-1R, IRS-1, HER3; O’Reilly et al., 2006; Car- racedo et al., 2008; Miller et al., 2009). These data suggest that direct inhibitors of PI3K may be more effective. Early clini- cal testing of PI3K inhibitors in combination with antiestrogens suggests that this strategy is feasible. In a phase Ib trial, post- menopausal patients with advanced ER+ disease are being treated with letrozole plus the PI3K inhibitor BKM120. This drug combination is safe and exhibits promising anti-tumor activity FIGURE 2 | Estrogen receptor inhibition with fulvestrant induces upregulation of PI3K signaling. Ovariectomized athymic mice were s.c. (Mayer et al., 2012). implanted with MCF-7 cells and a 10-day-release E2 pellet (0.12 mg). Twelve days later, mice were randomized to treatment with vehicle or fulvestrant RATIONALE FOR AN ER DOWNREGULATOR IN COMBINATION (5 mg/week, s.c., clinical formulation). Tumors were harvested after 3–4 WITH A PI3K INHIBITOR IN AI-RESISTANT BREAST CANCER weeks of treatment. Tumor lysates were analyzed by immunoblotting using the indicated antibodies; each lane contains equal amount of protein from A recent comparison of high-dose fulvestrant (an ER downreg- two to three tumors. Fulvestrant treatment decreased the levels of ER and ulator) to the AI anastrozole as first-line treatment for advanced ER-regulated genes products (PR, IGF-1R), but increased levels of breast cancer revealed that fulvestrant provided a longer time- P-AKT-T308 and P-AKT-S473, suggesting increased activation of PI3K. All to-progression (Robertson et al., 2009a). In other studies, ∼35% lanes were from the same membrane. of patients who progressed on an AI responded to second-line FIGURE 3 | Diagram of a clinical trial with a PI3K pathway inhibitor another AI plus a PI3K inhibitor, or fulvestrant plus the PI3K inhibitor. in AI-resistant breast cancer. Patients with breast cancer that progressed FDG-PET scans would be performed before and after 4 weeks of on AI therapy will be subjected to a biopsy to confirm ER+, HER2-negative, therapy to identify early metabolic changes. Patients will be treated PIK3CA-mutant status. Eligible patients would then be randomized to until progression. Frontiers in Oncology | Molecular and Cellular Oncology October 2012 | Volume 2 | Article 145 | 24 “fonc-02-00145” — 2012/10/15 — 10:50 — page 4 — #4 Fox et al. ER and PI3K in endocrine resistance fulvestrant (Ingle et al., 2006; Perey et al., 2007). This suggests These findings may be validated clinically in a phase II clini- that in some clinical situations, downregulation of ER may be cal trial where post-menopausal patients with AI-resistant, ER+, superior to estrogen deprivation (AI) therapy (Robertson et al., HER2-negative, PIK3CA-mutant breast cancer are randomized to 2009a). We recently reported that ER retains transcriptional activ- treatment with another AI plus a PI3K inhibitor vs. fulvestrant ity in estrogen-independent LTED cells and primary human breast plus a PI3K inhibitor (Figure 3). The novel agent in such a trial tumors (i.e., following AI therapy), and drives the estrogen- would be the PI3K inhibitor, but the comparison would be an AI independent growth of LTED cells (Miller et al., 2011b). These vs. fulvestrant. The primary endpoint would be PFS. Incorpora- data suggest that estrogen (ligand)-independent ER activity may tion of non-invasive imaging with [18 F]FDG-PET at baseline and promote resistance to AI therapy. While their side effect profiles after several weeks of treatment could identify metabolic changes are generally similar, AI therapy increases the risk of bone fractures indicative of a pharmacodynamic effect. This comparison would and joint disorders more so than fulvestrant (Howell et al., 2002, inform us whether (1) the addition of a PI3K inhibitor to an AI 2005; Osborne et al., 2002; Goss et al., 2003; Robertson et al., 2003; is beneficial, (2) downregulation of ER is superior to estrogen Howell and Sapunar, 2011). Fulvestrant, which is administered deprivation (AI) therapy in the context of PI3K inhibition, and intramuscularly, is associated with injection site pain, and only (3) metabolic inhibition at an early time point as reflected by induces partial ER downregulation in tumors (Robertson et al., FDG-PET is predictive of PFS. 2009b). Hence, the development of a more potent, orally avail- able ER downregulator/inhibitor may provide a convenient and ACKNOWLEDGMENTS effective treatment option for patients with ER+ breast cancer. This work was supported by the National Institutes of Health Cancer cells harboring activating mutations in PIK3CA exhibit K99CA142899, R00CA142899 (Todd W. Miller), Breast Can- increased sensitivity to PI3K inhibition (Miller et al., 2010; Sanchez cer Specialized Program of Research Excellence (SPORE) et al., 2011; Maira et al., 2012), suggesting that this class of drugs P50CA98131, Vanderbilt-Ingram Cancer Center Support Grant may be most effective against tumors with mutations in the PI3K P30CA68485; a grant from the Breast Cancer Research Foundation pathway. In mice bearing ER+, HER2-negative, PIK3CA-mutant (Carlos L. Arteaga); American Cancer Society Clinical Research MCF-7 breast cancer xenografts, treatment with the combina- Professorship Grant CRP-07-234 (Carlos L. Arteaga) and Post- tion of fulvestrant and BKM120 induced tumor regression (Miller doctoral Fellowship Grant PF-10-184-01-TBE (Emily M. Fox); the et al., 2011b). Using [18 F]FDG-PET imaging as an early biomarker Lee Jeans Translational Breast Cancer Research Program (Carlos L. of metabolic inhibition, treatment with BKM120 but not fulves- Arteaga); and Stand Up to Cancer/American Association for Can- trant decreased tumor FDG uptake. BKM120 increased tumor cell cer Research Dream Team Translational Cancer Research Grant apoptosis, while fulvestrant decreased tumor cell proliferation. SU2C-AACR-DT0209 (Carlos L. Arteaga). REFERENCES with estrogen receptor-positive breast reveals a link between the PI3K path- after neoadjuvant letrozole. J. Clin. Arpino, G., Green, S. J., Allred, D. C., cancer. J. Clin. Oncol. 27, 2630–2637. way and lower estrogen receptor (ER) Oncol. 24, 3019–3025. Lew, D., Martino, S., Osborne, C. Campbell, R. A., Bhat-Nakshatri, P., levels and activity in ER+ breast Fox, E. M., Miller, T. W., Balko, J. M., K., et al. (2004). HER-2 amplifica- Patel, N. M., Constantinidou, D., cancer. Breast Cancer Res. 12, R40. Kuba, M. 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B., Smith, menopausal women: a prospective 30 September 2012; published online: 16 acterization of NVP-BEZ235, a new D., et al. (2006). mTOR inhibition combined analysis of two multicenter October 2012. orally available dual phosphatidyli- induces upstream receptor tyrosine trials. Cancer 98, 229–238. Citation: Fox EM, Arteaga CL and Miller nositol 3-kinase/mammalian target kinase signaling and activates Akt. Sabnis, G., Goloubeva, O., Jelovac, D., TW (2012) Abrogating endocrine resis- of rapamycin inhibitor with potent in Cancer Res. 66, 1500–1508. Schayowitz, A., and Brodie, A. (2007). tance by targeting ERα and PI3K in vivo antitumor activity. Mol. Cancer Osborne, C. K., Pippen, J., Jones, S. Inhibition of the phosphatidylinos- breast cancer. Front. Oncol. 2:145. doi: Ther. 7, 1851–1863. E., Parker, L. M., Ellis, M., Come, itol 3-kinase/Akt pathway improves 10.3389/fonc.2012.00145 Mamounas, E. P., Jeong, J. H., Wick- S., et al. (2002). Double-blind, ran- response of long-term estrogen- This article was submitted to Frontiers erham, D. L., Smith, R. E., Ganz, P. domized trial comparing the efficacy deprived breast cancer xenografts to in Molecular and Cellular Oncology, a A., Land, S. R., et al. (2008). Benefit and tolerability of fulvestrant ver- antiestrogens. Clin. Cancer Res. 13, specialty of Frontiers in Oncology. from exemestane as extended adju- sus anastrozole in postmenopausal 2751–2757. Copyright © 2012 Fox, Arteaga and vant therapy after 5 years of adjuvant women with advanced breast cancer Sanchez, C. G., Ma, C. X., Crow- Miller. This is an open-access article dis- tamoxifen: intention-to-treat analy- progressing on prior endocrine ther- der, R. J., Guintoli, T., Phom- tributed under the terms of the Creative sis of the National Surgical Adjuvant apy: results of a North American trial. maly, C., Gao, F., et al. (2011). Commons Attribution License, which Breast And Bowel Project B-33 trial. J. Clin. Oncol. 20, 3386–3395. Preclinical modeling of combined permits use, distribution and reproduc- J. Clin. Oncol. 26, 1965–1971. Perey, L., Paridaens, R., Hawle, H., phosphatidylinositol-3-kinase inhi- tion in other forums, provided the origi- Mayer, I. A., Abramson, V. G., Balko, Zaman, K., Nole, F., Wildiers, H., bition with endocrine therapy for nal authors and source are credited and J. M., Isakoff, S. J., Kuba, M. et al. (2007). Clinical benefit of ful- estrogen receptor-positive breast can- subject to any copyright notices concern- G., Sanders, M., et al. (2012). vestrant in postmenopausal women cer. Breast Cancer Res. 13, R21. ing any third-party graphics etc. Frontiers in Oncology | Molecular and Cellular Oncology October 2012 | Volume 2 | Article 145 | 26 “fonc-02-00145” — 2012/10/15 — 10:50 — page 6 — #6 REVIEW ARTICLE published: 08 May 2013 doi: 10.3389/fonc.2013.00108 Targeting PI3K in cancer: any good news? Miriam Martini † , Elisa Ciraolo † , Federico Gulluni † and Emilio Hirsch* Molecular Biotechnology Center, University of Turin, Turin, Italy Edited by: The phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates several cellular Alexandre Arcaro, University of Bern, processes and it’s one of the most frequently deregulated pathway in human tumors. Switzerland Given its prominent role in cancer, there is great interest in the development of inhibitors Reviewed by: Agnès Noël, University of Liege, able to target several members of PI3K signaling pathway in clinical trials. These drug can- Belgium didates include PI3K inhibitors, both pan- and isoform-specific inhibitors, AKT, mTOR, and Clement M. Lee, Mount Sinai School dual PI3K/mTOR inhibitors. As novel compounds progress into clinical trials, it’s becoming of Medicine, USA urgent to identify and select patient population that most likely benefit from PI3K inhibition. *Correspondence: In this review we will discuss individual PIK3CA mutations as predictors of sensitivity and Emilio Hirsch, Molecular Biotechnology Center, Dipartimento resistance to targeted therapies, leading to use of novel PI3K/mTOR/AKT inhibitors to a di Biotecnologie Molecolari e Scienze more “personalized” treatment. per la Salute, Via Nizza 52, Turin Keywords: PI3K, cancer, therapeutics, genetic determinants, class II phosphatidylinositol 3-kinase 10126, Italy. e-mail: emilio.hirsch@unito.it † Miriam Martini , Elisa Ciraolo and Federico Gulluni have contributed equally to this work. INTRODUCTION KIT and PDGFRA mutant gastrointestinal stromal tumors (GIST) Over the past years, it has become widely accepted that can- to Imatinib (Antonescu, 2011). cer is a multistep genetic disease that arises by the activation of The phosphatidylinositol 3-kinase (PI3K) signaling pathway specific oncogenes, inactivation of tumor suppressor genes, and regulates several processes in normal cell such as survival, metab- stochastic accumulation of genetic alterations driving tumor pro- olism, and motility and it’s one of the most frequently deregulated gression (Vogelstein and Kinzler, 2004). Despite the genetic and pathway in human cancer (Cantley, 2002; Samuels et al., 2004; Liu epigenetic complexity observed in cancer, tumor growth and sur- et al., 2009a). Mutations and/or amplifications of the PI3K cat- vival can be impaired by the inactivation of a single oncogene. alytic subunits p110α (PIK3CA) and p110β (PIK3CB), the PI3K This phenomenon is called “oncogene addiction” (term coined regulatory subunits p85α (PIK3R1) and p85β (PIK3R2), the PI3K by Weinstein, 2000), and reveals a possible “Achilles heel” within effector AKT (AKT1) are often observed in cancer1 . Moreover, the cancer cell that can be therapeutically exploited (Weinstein, mutations, deletions, or epigenetic changes of negative regula- 2000, 2002). The hypothesis of oncogene addiction refers to the tors of PI3K axis (Gewinner et al., 2009; Hollander et al., 2011), observation that a tumor cell, despite several genetic alterations, is such as phosphatase and tensin homolog (PTEN) and inositol dependent on a single oncogenic pathway responsible for sustain- polyphosphate-4-phosphatase, type II (INPP4B), may alter sensi- ing the malignant phenotype. An important implication is that tivity to chemo- and targeted-therapies (Steelman et al., 2008; Kim switching off this crucial pathway upon which cancer cells are et al., 2012). dependent should have negative effects on cancer while sparing While class I is the most well characterized of the PI3K to normal cells. Therefore pharmacological inhibition of this crucial date, the family comprises other two groups; class II and class pathway cause an “addiction shock,” resulting in the blockade of III. Each class of PI3-kinase has unique preferences for phospho- cell growth or in cell death (Sharma and Settleman, 2007; Janne inositide substrates and produces specific lipid second messengers, et al., 2009). responding to a wide variety of signaling molecules. The “addiction paradigm” has been pharmacologically Class II PI3Ks consists of three members named PI3K-C2α, exploited and drugs designed to specifically inhibit mutated pro- PI3K-C2β, and PI3K-C2γ. Unlike class I, they lack a regulative teins have led to what is commonly known as “personalized subunit and appear to be monomers of high molecular weight, cancer medicine.” At present only a small subset of anticancer predominantly associated with intracellular membranes (Falasca therapies are administered based upon the genetic alterations et al., 2007). PI3K-C2α and PI3K-C2β have a broad tissue dis- present in individual tumors (Martini et al., 2012). For example, tribution and are almost ubiquitously expressed, while PI3K-C2γ breast cancer patients with amplification/overexpression of the displayed a very restricted expression pattern, limited to liver, pan- human epidermal growth factor receptor (EGFR) 2 (HER-2) are creas, and prostate (Kok et al., 2009). The precise nature of class II selectively sensitive to Trastuzumab and Lapatinib (Stern, 2012), substrates and lipid products is still debated. Class II members are melanomas harboring BRAF V600E mutations to Vemurafenib (Flaherty et al., 2010), non-small cells lung cancers (NSCLC) with mutated EGFR to Erlotinib and Gefitinib (Pallis et al., 2011), and 1 www.sanger.ac.uk/cosmic www.frontiersin.org May 2013 | Volume 3 | Article 108 | 27 Martini et al. PI3K as a pharmacological target in cancer thought to act similarly to class III mainly generating PtdIns(3)P selectivity for the ATP binding site, PI3K inhibitors have been in vitro and in vivo (Falasca and Maffucci, 2007), nonetheless, classified into different groups (Table 1). they can also produce PtdIns(3,4)P2 in vitro (Vanhaesebroeck The first group encompasses inhibitors able to bind all class I et al., 2010) and PI3K-C2α has been also reported to produce PI3Ks (pan inhibitors), and in particular PI3Kα, PI3Kβ, PI3Kγ, PtdIns(3,4,5)P3 in vitro (Gaidarov et al., 2001). Class II PI3Ks and PI3Kδ. Wortmannin and LY294002, the first two prototype are activated downstream of different receptor types including PI3K inhibitors, represented for a long time a useful tool in the RTKs (EGFR and PDGFR) (Brown et al., 1999; Arcaro et al., 2000, study of PI3K function in cellular processes, given their effective- 2002; Falasca and Maffucci, 2007) and GPCRs (Maffucci et al., ness at low concentration (nM). Nevertheless, given their poor 2005). Several stimuli promote PI3K-C2α activation such as hor- pharmacokinetic properties and lack of selectivity, these com- mones (insulin) (Brown et al., 1999), chemokines (Turner et al., pounds have limited their therapeutic potential. The availability 1998), and cytokines (TNFα and leptin) (Ktori et al., 2003). Simi- of the crystal structure of the p110 isoform-specific catalytic sub- larly PI3K-C2β is activated by growth factor (EGF) (Arcaro et al., units gave a boost to the development of new PI3K inhibitors 2002) and phospholipids (LPA) (Maffucci et al., 2005) while at the (Vadas et al., 2011). Therefore, several novel compounds have present there are no study investigating PI3K-C2γ upstream acti- been further developed in order to improve pharmacokinetic pro- vators. A recent study reported that PI3K-C2α has an essential role files, to increase target specificity and to minimize toxicity. At the in angiogenesis resulting in embryo lethality, impaired endothelial present, several promising pan-PI3K inhibitors are under devel- cell signaling, and RhoA activation (Yoshioka et al., 2012). opment and evaluation in clinical trials for cancer therapy. These The increasing understanding of the mechanisms underlying molecules predominantly display cytostatic effects with conse- the role of the PI3K pathway in tumorigenesis has encouraged quent G1 phase arrest in vitro and favorable anticancer effects many pharmaceutical companies and academic laboratories to in vivo. focus their efforts on the development of inhibitors targeting the Lately, a second group of PI3K inhibitors has been developed PI3K signaling pathway at different levels. to overcome the toxicity displayed by the treatment with pan- In this review, we will discuss the challenges for the develop- PI3K inhibitors. They are characterized by greater selective activ- ment of novel inhibitors to target the PI3K signaling pathway ity (isoform-specific) and several molecules are currently under and the binary relationship between PI3K mutations in cancer evaluation in preclinical and clinical studies. genotype and personalized medicine. On the other hand, since PI3K and mTOR share several struc- tural similarities, many chemical compounds, under evaluation in TARGETING PI3K SIGNALING PATHWAY IN CANCER clinical trials, are able to inhibit both catalytic subunits. This third Since PI3K/AKT/mTOR axis has been classified among the most group of inhibitors is termed “dual PI3K/mTOR” and they have frequently activated pathway in cancer, members of the cascade the advantage of inhibiting not only all class I isoforms but also represent an attractive target for cancer therapeutics (Miled et al., mTORC1 and mTORC2 thus having a strongest effectiveness in 2007). The activation of the PI3K signaling pathway contributes switching off the PI3K signaling pathway. to several aspects of tumorigenesis as tumor development, pro- Given the growing interest in inhibiting PI3K signaling path- gression, invasiveness, and metastasis formation. A number of way, the drug development landscape is becoming increasingly molecules targeting members of the PI3K axis have been devel- crowded and highly competitive, so that several pharmaceutical oped and evaluated in preclinical studies as well as in clinical trials companies, such as Novartis, Sanofi-Aventis, Roche/Genentech, (Figure 1). Based on pharmacokinetics properties and isoform Bayer, and GlaxoSmithKline, are currently in competition to FIGURE 1 | Schematic representation of PI3K pathway and sites of action of PI3K signaling pathway inhibitors in solid tumors and hematological malignancies. Frontiers in Oncology | Molecular and Cellular Oncology May 2013 | Volume 3 | Article 108 | 28 Martini et al. PI3K as a pharmacological target in cancer Table 1 | PI3K inhibitors tested in preclinical and clinical models, genetic determinants of response, and open clinical trials described for each compound. Group Selectivity Compound Cancer type Genetic determinant Clinical trial status of response I Pan-class I Class I PI3K GDC-0941 Breast HER-2 amplification I-II in breast, (Roche/Genentech) PIK3CA mutations non-Hodgkin’s lymphoma, NSCLC Melanomas, MM, – non-Hodgkin’s lymphoma, NSCLC, ovarian BKM120 Breast PIK3CA mutations I-II in breast CRC, (Novartis) endometrial, GIST, GBM, leukemia, melanoma, NSCLC, pancreatic, renal cell, SCCHN, TCC CRC, endometrial, GIST, – GBM, leukemia, melanoma, NSCLC, pancreatic prostate PX866 Breast, CRC, MM, PIK3CA mutation, PTEN I-II in CRC, GBM, (Oncothyreon) NSCLC, pancreatic loss NSCLC, SCCHN prostate, ovarian BAY 80-6946 Advanced solid cancers PIK3CA mutations I-II in advanced solid (Bayer) cancers II Isoform PI3Kα GDC-0032 Solid cancers – I-II in solid cancers specific (Roche/Genentech) PI3Kβ GSK2636771 Advanced solid cancers – I-IIa in advanced solid (GlaxoSmithKline) cancers with PTEN deficiency PI3Kγ and PI3Kδ IPI-145 (Infinity) Hematological – I-IIa in advanced malignancies hematological malignancies PI3Kδ CAL-101 (Gilead AML, CLL, Hodgkin’s – I-II-III in AML, CLL, Sciences) and non-Hodgkin’s Hodgkin’s and lymphoma, MCL non-Hodgkin’s lymphoma, MCL, MM III Dual PI3K and mTOR BEZ235 (Novartis) Breast PIK3C2a mutation, I-II in breast, renal cell PI3K/mTOR HER-2 amplification, PTEN loss Ovarian PIK3C2a mutation, PTEN loss bring their own inhibitors in the market. On the basis of these PI3K SIGNALING INHIBITION IN SOLID TUMORS considerations, we will describe and discuss results for the most Several PI3K inhibitors have progressed through early clinical important PI3K inhibitors currently in clinical trial. safety and dose-escalation studies to phase I clinical trials on www.frontiersin.org May 2013 | Volume 3 | Article 108 | 29 Martini et al. PI3K as a pharmacological target in cancer solid tumors. This allowed to define the treatment efficacy and to BKM120 (NOVARTIS) identify patient population that will benefit from PI3K inhibitor BKM120 is an oral pyrimidine-derived pan-PI3K inhibitor with administration. Furthermore, a few number of compounds are potent activity at nanomolar concentrations against all class I PI3K now progressing to phase Ib expansion cohort and phase II single isoform while didn’t show any activity against other classes of PI3K agent efficacy studies. as well as mTOR. In vitro preclinical models showed that BKM120 Several early reports were presented in the last 3 years revealing has a strong anti-proliferative activity in more than 400 cancer safety and some preliminary activity data about the use of class I cell lines. The antitumor effects of BKM120 was also described in PI3K and dual mTOR/PI3K inhibitors in patients. several xenograft models of lung cancer (Fruman and Rommel, 2011) and metastatic HER-2+ breast cancer (Nanni et al., 2012). GDC-0941 (GENENTECH-ROCHE) Clinical data indicate that it is unlikely that BKM120 will achieve GDC-0941 is a potent and selective oral inhibitor of class I PI3K exposures sufficient to significantly engage the off-target activ- with activity against DNA-PK and also mTOR but at high concen- ity at tolerated doses and schedules, however careful dose range trations (Folkes et al., 2008). GDC-0941 is currently under eval- selection is required to ensure specific targeting of PI3K signaling uation in several phase I clinical trials on patients with advanced pathway (Brachmann et al., 2012). Given its ability to penetrate the solid tumors, such as HER-2 positive metastatic breast cancer and blood-brain barrier, BKM120 may represent an attractive option advanced NSCLC2 . Several studies have documented the effects of for the treatment of glioblastoma multiforme (GBM), the most GDC-0941 on cell viability inhibition at submicromolar concen- common and aggressive malignant primary brain tumor (Koul tration in several tumor types including glioblastoma, breast, and et al., 2012). The first-in-human phase I dose-escalation study prostate cell lines carrying specific alteration in the PI3K signal- investigated the MTD, safety, preliminary activity, and pharmaco- ing pathway. GDC-0941 exhibited excellent inhibition on MCF7, dynamics of BKM120 (Bendell et al., 2012). The study reports that T-47D, and SK-BR-3 breast derived cancer cell lines as a single BKM120 was well tolerated with a dose-dependent safety profile agent while in combination with rapamycin promotes apopto- and it describes related side effects, such as hyperglycemia, rash, sis and down regulates cell cycle machinery components, such nausea, fatigue, and mood alterations. In particular hyperglycemia as cyclin D1 (Zheng et al., 2012). In addition, the combinatorial is consistent with inhibition of PI3K signaling and has been treatment of GDC-0941 and the Docetaxel in a panel of 25 breast observed with other PI3K/mTOR/Akt pathway inhibitors. Distur- tumor cell lines (HER-2+, luminal, and basal subtypes) increases bance of glucose homeostasis, as evidenced by hyperglycemia, was the rate of apoptosis and enhances sensitivity to Docetaxel (Wallin more common at higher doses and may be attributed to BKM120 et al., 2012). The efficacy of combined GDC-0941 and chemo- and inhibition of p110. Pharmacodynamics data demonstrate a dose- targeted-therapies has also been demonstrated for other agents related inhibition of the PI3K signaling with significant decrease such as Trastuzumab, Pertuzumab, and Docetaxel (Yao et al., 2009). in pS6 phosphorylation and decreased [18F]fluorodeoxyglucose In NSCLC GDC-0941 synergizes also with the MEK inhibitor uptake. In another study, 77 patients with CRC, breast, lung, and (U0126) by promoting G0-G1 arrest and cell apoptosis (Zou et al., endometrial cancers received oral BKM120 in monotherapy once 2012). Several clinical studies are currently evaluating the rela- daily (Call et al., 2010). PR were observed in two patients, a tive bioavailability, absorption, metabolism, excretion, and effects triple negative breast cancer with KRAS and p53 mutation and of GDC-0941 in patient with advanced or metastatic tumors, a ER+/HER− metastatic breast cancer carrying PIK3CA muta- alone or in combination with Paclitaxel, Erlotinib, Carboplatin, or tions. At the same time, 58% of patients showed stable disease Bevacizumab (see text footnote 2) GDC-0973 (MEK inhibitor). (SD) response. Safety data about GDC-0941 monotherapy regimen have been recently released. Phase I studies with a 3 + 3 dose-escalation BAY 80-6946 (BAYER HEALTHCARE) design showed that GDC-0941 is generally well tolerated at doses BAY 80-6946 is a potent pan-class I PI3K inhibitor with IC50 below 450 mg every day (QD) and twice a day (BID) in patient at sub-nanomolar concentration against PI3Kα (0.5 nM), PI3Kβ with advanced solid tumors. After treatment, signs of clinical activ- (3.7 nM), PI3Kδ (0.7 nM), and PI3Kγ (6.4 nM) while it is inac- ity have been reported, including a partial response (PR) by the tive against around 240 protein/lipid kinases and RTKs. BAY response evaluation criteria in solid tumors (RECIST) in a patients 80-6946 has shown antitumor activity against a panel of 140 with melanoma, ovarian, endocervical, and ER+/HER-breast can- tumor cell lines with an IC50 of 1–100 nM in about 60 tumor cer (Wagner et al., 2009; Moreno Garcia et al., 2011; Von Hoff et al., cell lines. BAY 80-6946 displayed a strong activity of the PI3K 2011). Several side effects have been described after administra- signaling pathway inhibiting AKT (Thr308 and Ser473) as well tion of GDC-0941 in about 10% of patients, in particular nausea, as PRAS40, 4EBP1, and FOXOs phosphorylation in tumor cells diarrhea, fatigue, dysgeusia, and decreased appetite. In two dif- carrying PIK3CA activating mutations. BAY 80-6946 has been ferent studies, the maximum tolerated dose (MTD) was enriched demonstrated to induce apoptosis in a subset of PIK3CA mutant at 450 mg with a dose limiting toxicities (DLT) of grade 3 (Gr) tumors at concentrations lower that 100 nM in preclinical stud- macular rash and asymptomatic T -wave inversion in ECG, Gr3 ies. The pharmacokinetics, pharmacodynamics, and MTD of BAY thrombocytopenia, and Gr4 hyperglycemia (Moreno Garcia et al., 80-6946 have been determined in a phase I escalation multicen- 2011; Von Hoff et al., 2011). ter study in patients with advanced solid tumors. Unlike other PI3K inhibitors, BAY 80-6946 is administered intravenously as 1-h infusion once weekly for 3 weeks every month. Data deriv- 2 www.clinicaltrials.gov ing from phase I study revealed a MTD at 0.8 mg/kg and several Frontiers in Oncology | Molecular and Cellular Oncology May 2013 | Volume 3 | Article 108 | 30 Martini et al. PI3K as a pharmacological target in cancer side effects including hyperglycemia, fatigue, nausea, alopecia, leukemia. In addition, in some cases leukemias cell display overex- diarrhea, mucositis, dysgeusia, and grade 2/3 anemia. Moreover pression of PI3Kα, PI3Kβ, and PI3Kγ. Conversely, the expression a phase I study assessed safety, pharmacokinetics, and clinical of PI3Kδ has been found up-regulated in acute myelogenous benefit in patients with advanced solid tumors, including breast, leukemia (AML) and in a subset of promyelocytic leukemia (APL). endometrial, gastric cancer. AML comprises a heterogeneous group of tumors characterized by uncontrolled proliferation of hematopoietic precursors thus NVP-BEZ235 (NOVARTIS) leading to accumulation of blast cells in the bone marrow. These NVP-BEZ235 is a reversible, orally available, and selective inhibitor blasts are blocked in their differentiation program at different of PI3K and TORC1/2. Several preclinical studies have already stage of maturation. This blast accumulation causes a progres- demonstrate its efficacy in a variety of solid tumors such as sive failure in the hematopoiesis process, which in turn leads to melanomas (Roper et al., 2011), breast (Brunner-Kubath et al., anemia, neutropenia, and thrombocytopenia (Smith et al., 2004). 2011), CRCs (Manara et al., 2010; Roper et al., 2011), and sarcomas The standard therapeutic approach for the treatment of AML is (Manara et al., 2010). This compound suppresses cell proliferation, based on high-dose chemotherapy, nonetheless the prognosis of induces G1 cell cycle arrest and promotes autophagy by inhibit- AML remains poor with a 5-year survival rate in a 15–30% of ing the activity of AKT, S6K, S6, and 4EBP1 target proteins (Serra patients. A recent evidence demonstrated that up-regulation of et al., 2008; Cerniglia et al., 2012). In addition, data on glioma the PI3K/AKT/mTOR axis is a common feature in AML. Con- xenograft demonstrate a reduction of the expression of the vascu- sequently, several pan-PI3K and dual PI3K/mTOR inhibitors, in lar endothelial growth factor (VEGF) on tumor vasculature, thus particular BKM120 and BEZ235, are undergoing phase I clini- suggesting an anti-angiogenetic effect for NVP-BEZ235 (Liu et al., cal development to assess safety, dose, and preliminary efficacy 2009b). Recently, NVP-BEZ235 also emerged as inhibitor of ATM in patients with advanced leukemias, relapsed or refractory acute and DNA-PK at low concentration (100 nM). In this context, treat- lymphoblastic and myelocytic leukemia (see text footnote 2). ment with NVP-BEZ235 may have significant radio sensitizing Several studies demonstrated that treatment with the PI3Kδ effects with important implications in the rational design of clini- selective inhibitor, EC87114, inhibits AML cell proliferation with- cal trials (Mukherjee et al., 2012). At present, this molecule is under out affecting the proliferation of normal hematopoietic progenitor evaluation in phase I/II clinical trials in patients with advanced cells (Sujobert et al., 2005; Billottet et al., 2006). In this context, solid malignancies, including GBM (Salkeni et al., 2012), breast, PI3Kδ inhibitors represent a promising therapeutic treatment for renal cell carcinoma (RCC), castration-resistant prostate cancer AML without producing undesirable side effects that are con- (CRPC), endometrial carcinoma, and pancreatic neuroendocrine versely expected for other pan-PI3K inhibitors (Di Nicolantonio tumors (see text footnote 2), alone or in combination with other et al., 2010). Additionally, since PI3Kδ is expressed in leukocytes drugs such as Paclitaxel, Trastuzumab, Everolimus, and MEK162. and plays a key role in B-cell signaling (Jou et al., 2002; Bilancio A 3 + 3 dose-escalation schedule with NVP-BEZ235 revealed tol- et al., 2006), it represents an interesting target in B-cells malig- erability at 600 mg BID dose and preliminary signs of clinical and nancies. Mice with deleted or kinase dead PI3Kδ exhibit B-cell pharmacodynamic activity (Arkenau et al., 2012). In a phase IB defects, such as lack of B1 lymphocytes, decreased number of dose-escalation study, NVP-BEZ235 was administered in combi- mature B-cell and impaired antibody production. The absence of nation with Trastuzumab in HER-2+ metastatic breast cancer (15 PI3Kδ in B-cell leads also to a reduction in AKT phosphorylation pts) with altered PI3K/PTEN status, showing an acceptable safety and decreased PtdIns(3,4,5)P3 levels (Okkenhaug and Vanhaese- profile and PR or SD in one and four patients respectively (Krop broeck, 2003). All these data support the use of PI3Kδ inhibitors in et al., 2012). NVP-BEZ235 was also tested as a single agent or B-cell malignancies including the chronic lymphocytic leukemia Trastuzumab-combined with a novel formulation based on a solid (CLL). dispersion system (SDS) sachet (Peyton et al., 2011). The MTD for Given the increasing interest in inhibiting PI3Kδ for the the new formulated NVP-BEZ235 was determined as 1600 mg/day, treatment of hematopoietic malignancies, several inhibitors are dose chosen for the ongoing phase II clinical trials. After treatment, currently under evaluation in preclinical and in clinical trials. in 28 patients with advanced solid tumors, a stable response has Presently, the most promising PI3Kδ inhibitor is represent by been evidence in a 40% of cases (Wen et al., 2012). Overall, these CAL-101 (Calistoga Pharmaceuticals/Gilead Sciences), an orally studies display that NVP-BEZ235 is generally well tolerated and available selective inhibitor with an IC50 of 2.5 nM for PI3Kδ and the most common side effects include nausea, diarrhea, AST/ALT 820, 565, 89 nM for PI3Kα, PI3Kβ, PI3Kγ respectively (Lannutti elevation, and headache. DLT include fatigue, asthenia, Gr3 throm- et al., 2011). At the present, CAL-101 is undergoing in preclinical bocytopenia, and Gr3 mucositis (Peyton et al., 2011; Arkenau et al., and clinical development in a variety of lymphoid malignan- 2012; Wen et al., 2012). cies thanks to its high selectivity. Phase I studies, on patients with relapsed or refractory hematologic malignancies, including PI3K SIGNALING INHIBITION IN HEMATOLOGICAL non-Hodgkin lymphoma (NHL), CLL, and AML, revealed a very MALIGNANCIES favorable toxicity profile and pharmacokinetics during a week of Although the majority of PI3K inhibitors are under development CAL-101 oral administration (Fruman and Rommel, 2011). The for the treatment of solid tumors, hematological malignancies also predominant toxicity, caused by high serum transaminases, was represent a therapeutic area of interest, especially for isoform- observed in 21% of patients at doses of 200 and 350 mg. However, selective PI3K inhibitors. Constitutive activation of class I PI3K this side effect resulted to be reversible disappearing with a tempo- isoform has been identified in high percentage of acute and chronic rary discontinuation of the drug (Fruman and Rommel, 2011). In www.frontiersin.org May 2013 | Volume 3 | Article 108 | 31 Martini et al. PI3K as a pharmacological target in cancer this study, 57 patients were treated with different dosages, corre- levels significantly correlates with resistance to Erlotinib in GBM sponding to 50, 100, 200, and 350 mg. Clinical responses were seen thus suggesting that targeting of PI3K-C2β in resistant GBM may at all dose levels resulting in 50% of reduction in lymphadenopa- represent a new therapeutic approach (Low et al., 2008). thy and around 6 months of stable disease. In a successive phase I To further support the hypothesis the role of PI3K-C2β in drug clinical trial CAL-101 was administered orally one or two times per resistance, a siRNA-based study reported that in a panel of kinases, day for a cycle of 28 day in 54 patients with CLL. After treatment down regulation PIK3C2B is one of the Tamoxifen sensitizing with CAL-101, 26% of patients achieved a PR with 80% of patients target in breast cancer cells (Iorns et al., 2009). Furthermore, over- showing reduced lymphadenopathy by ≥50% (Di Nicolantonio expression of PI3K-C2β significantly inhibited cisplatin-induced et al., 2010; Fruman and Rommel, 2011). apoptosis and cleavage of caspase-3 in esophageal squamous cell carcinoma (Liu et al., 2011). Altogether these data suggest that RELEVANCE FOR SELECTIVE INHIBITION OF CLASS II PI3Ks PI3K-C2β may have a role in promoting resistance to chemothera- Pharmacological inhibitors selectively targeting class II PI3Ks have peutic drugs and that interference with PI3K-C2β activity might be not been described yet. In particular, in vitro PI3K-C2α is refrac- a rational possibility for treatment of cisplatin-resistant esophageal tory to inhibition by LY294002 and Wortmannin (Virbasius et al., cancer patients. On the other hand, it has been recently showed that 1996; Domin et al., 1997), two very well-known PI3Ks inhibitors. down regulation of PI3K-C2β may specifically confer resistance However, emerging evidence suggests that class II PI3Ks may have to leukemia cells to chemotherapy (thioguanine and mercaptop- crucial role in different types of tumor independently from class I urine) (Diouf et al., 2011). Reduced levels of PI3K-C2β results in PI3K activity. increased degradation of MSH2, a DNA mismatch repair enzyme involved in genomic integrity maintenance and drug resistance. PI3K-C2α Although it’s becoming increasingly clear the involvement of PI3K- RNAi-based silencing of PI3K-C2α in a large set of cancer cell lines C2β in chemotherapy resistance, further studies are needed to showed that this enzyme is crucially required for cancer cell sur- clarify when the targeting of this enzyme could promote drug vival in vitro (Elis et al., 2008). A small but significantly difference sensitivity. in the DNA copy number and mRNA levels, was then reported A recent study also reported that PIK3C2B gene single- in vivo in 19 hepatitis B-positive hepatocellular carcinoma com- nucleotide polymorphisms (SNP) is associated to cancer risk sus- pared with non-tumor tissues counterparts (Ng et al., 2009). The ceptibility (Koutros et al., 2010). The authors examined the asso- PIK3C2A gene was also found to be up-regulated in breast cancer ciation between several SNPs in PI3Ks genes (PIK3CD, PIK3C2A, stem-like cells characterized by increased tumorigenicity com- PIK3R3, PIK3AP1, and PIK3C2B) and prostate cancer risk: among pared with the normal counterpart (Zhou et al., 2007). Moreover, it the five genes, only PIK3C2B showed a cluster of SNPs related to was reported that PI3K-C2α is directly down-modulated at a trans- prostate cancer risk. PIK3C2B was also found to be significantly lational level by miR-30e-3p in DLD1 CRC cells (Schepeler et al., mutated in a recent whole-exome sequencing screening in NSCLC 2012). In particular miR-30e-3p levels are significantly decreased (Liu et al., 2012). during early events of CRC carcinogenesis thus raising the possi- bility that PIK3C2A gene may be up-regulated in the initial steps of PI3K-C2γ CRC onset. On the other hand, Yoshioka et al. (2012) showed that At the present very little is known about the role of PI3K-C2γ PI3K-C2α has an essential role during angiogenesis process and in in cancer. The chromosomal region 12p12, containing the human vascular barrier function. After subcutaneous injection of Lewis PIK3C2G gene, is found significantly amplified in a subset of ovar- lung carcinoma (LLC) or B16-BL6 melanoma tumors, Pik3c2a ian cancer (Lambros et al., 2005) and in about 60% of pancreatic endothelial-restricted knock-out mice had reduced tumor vol- ductal adenocarcinoma (PDAC) (Harada et al., 2008). Recently umes/weights compared to control, suggesting that the in vivo bioinformatics analysis reported significative association between pro-angiogenetic function of PI3K-C2α is required for tumor PIK3C2G mutations and GBM related signaling pathways (Dong growth and maintenance. Further studies are needed to better et al., 2010). Another study also suggests a new mechanism through understand the role PI3K-C2α in tumor angiogenesis, however which Bcr-Abl induces abnormal homing of leukemia cells by designing of specific inhibitors targeting PI3K-C2α could repre- reducing PI3K-C2γ expression (Yu et al., 2010). In this way, Bcr- sent a promising new anti-angiogenetic approach to arrest tumor Abl is not only responsible for class I PI3K activation in cell growth. migration (Martelli et al., 2006) but also for PI3K-C2γ reduced expression (Yu et al., 2010). PI3K-C2β Altogether these data suggest that class II PI3Ks may exert addi- An increasing number of studies described the involvement of the tional or complementary role to class I PI3Ks in promoting cancer, gene PIK3C2B, encoding for PI3K-C2β, in cancer. Amplification being involved in specific kind of tumors. The development of of PIK3C2B gene at 1q32 was reported in 6 out of 103 GBM tumors selective inhibitors targeting class II PI3Ks may thus represent a and in 4 of these cases amplification correlates with PI3KC2B promising way of action, once the precise role of these enzymes in mRNA over-expression (Knobbe and Reifenberger, 2003). Ampli- cancer will be elucidated. fication of chromosomal region 1q32.1 (PIK3C2B\MDM4) was also reported in around 8% of GBM tumor samples (Rao et al., FUTURE PERSPECTIVES 2010) and in whole genome amplification analysis (Nobusawa As PI3K inhibitors progress into trials focusing on their clinical et al., 2010). On the other hand increased PI3K-C2β protein efficacy, it is critical to identify genomic determinants of response Frontiers in Oncology | Molecular and Cellular Oncology May 2013 | Volume 3 | Article 108 | 32 Martini et al. PI3K as a pharmacological target in cancer and to stratify the patient population that will most likely benefit to respond to a given targeted therapy and to increase the chances from the treatment (Weigelt and Downward, 2012). of drug registration (Carden et al., 2010). For the guidance and Historically rapalogs were the first PI3K pathway inhibitors prioritization of predictive biomarker candidates in early clinical tested in clinical trials for cancer therapy and currently preclini- trials, results derived from the study of preclinical models are of cal models and early clinical data suggested that PIK3CA muta- primary importance to develop accurate companion diagnostic tions may predict sensitivity to treatment with PI3K/AKT/mTOR tools. inhibitors (Engelman et al., 2008; Ihle et al., 2009). In particu- lar, it has been shown that patients with advanced malignancies CONCLUSION carrying PIK3CA pathway aberrations, showed high percent- In summary, although available dataset showed that a high per- age of response to rapalogs and/or PI3K pan-inhibitor (Janku centage of tumors harboring PIK3CA will likely benefit from inhi- et al., 2011b; Moroney et al., 2011). Moreover, PI3K pathway bition of the PI3K pathway, a substantial proportion of patients aberrations due to PIK3CA mutations or PTEN loss correlate with PIK3CA activating mutations may be de novo resistant to with increase response to Doxorubicin, Bevacizumab, and Tem- these agents. On the other hand, not all the patients with PIK3CA sirolimus in patients with advanced gynecologic and breast malig- mutations are sensitive to PI3K inhibitors and not all the patients nancies (Moroney et al., 2011). Likewise, ovarian cancers with with wt PIK3CA/PTEN tumors are responsive. Moreover, while coexisting PIK3CA and MAPK pathway mutations are sensitive to the majority of the studies have restricted the analysis on the PI3K inhibition, whereas CRC with the same repertoire of muta- PIK3CA and PTEN status, also occurring in other members of the tions are resistant (Di Nicolantonio et al., 2010; Janku et al., 2011a, pathway, such as mTOR activating mutations, or INPP4B loss of 2012). The identification of feedback loops leading to MAPK function, may play a role in the response to inhibitors. Therefore, activation, upon PI3K inhibition, underscores the potential of a the possibility to target PI3K signaling pathway in cancer requires combined therapeutic approach with PI3K and MAPK inhibitors deeper investigation, in order to identify additional biomarkers (Carracedo et al., 2008; Laplante and Sabatini, 2012). According to and to improve therapeutic strategies in the clinic. these findings, it has been proposed to incorporate predictive bio- markers during the clinical drug development process from phase ACKNOWLEDGMENTS I studies onward in order to enrich trials with patients more likely This work was supported by a grant from Fondazione Cariplo. REFERENCES of the p110delta isoform of PI3K cancer cells. Breast Cancer Res. Treat. Di Nicolantonio, F., Arena, S., Antonescu, C. R. (2011). The GIST in B-cell antigen and IL-4 recep- 129, 387–400. 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A., specialty of Frontiers in Oncology. orectal cancer. PLoS ONE 6:e25132. D., Ward, S. G., and Westwick, J. Ligon, K. L., and Alfred Yung, W. Copyright © 2013 Martini, Ciraolo, doi:10.1371/journal.pone.0025132 (1998). The CC chemokine mono- K. (2012). Current clinical devel- Gulluni and Hirsch. This is an open- Salkeni, M. A., Beg, M. S., Olowokure, cyte chemotactic peptide-1 activates opment of PI3K pathway inhibitors access article distributed under the terms O. O., Fathallah, H., Thomas, both the class I p85/p110 phos- in glioblastoma. Neuro-oncology 14, of the Creative Commons Attribution H., Mercer, C. A., et al. (2012). phatidylinositol 3-kinase and the 819–829. License, which permits use, distribution A dose escalation, single arm, class II PI3K-C2alpha. J. Biol. Chem. Yao, E., Zhou, W., Lee-Hoeflich, S. T., and reproduction in other forums, pro- phase Ib/II combination study 273, 25987–25995. Truong, T., Haverty, P. M., Eastham- vided the original authors and source of BEZ235 with everolimus to Vadas, O., Burke, J. E., Zhang, X., Berndt, Anderson, J., et al. (2009). Sup- are credited and subject to any copy- determine the safety, pharmacody- A., and Williams, R. L. (2011). pression of HER2/HER3-mediated right notices concerning any third-party namics, and pharmacokinetics in Structural basis for activation and growth of breast cancer cells with graphics etc. www.frontiersin.org May 2013 | Volume 3 | Article 108 | 35 REVIEW ARTICLE published: 24 July 2013 doi: 10.3389/fonc.2013.00191 S6K2: the neglected S6 kinase family member Olivier E. Pardo* and Michael J. Seckl * Division of Cancer, Department of Surgery and Cancer, Imperial College, Hammersmith Hospital, London, UK Edited by: S6 kinase 2 (S6K2) is a member of the AGC kinases super-family. Its closest homolog, Alexandre Arcaro, University of Bern, S6K1, has been extensively studied along the years. However, due to the belief in the Switzerland community that the high degree of identity between these two isoforms would translate Reviewed by: Enrico Vittorio Avvedimento, in essentially identical biological functions, S6K2 has been largely neglected. Neverthe- University Naples Federico II, Italy less, recent research has clearly highlighted that these two proteins significantly differ in Shailender Singh Kanwar, University their roles in vitro as well as in vivo. These findings are significant to our understanding of Michigan, USA of S6 kinase signaling and the development of therapeutic strategies for several diseases *Correspondence: including cancer. Here, we will focus on S6K2 and review the protein–protein interactions Olivier E. Pardo and Michael J. Seckl , Division of Cancer, Department of and specific substrates that determine the selective functions of this kinase. Surgery and Cancer, Imperial College, Keywords: S6K2, S6 kinase, selectivity, specificity, function, RPS6KB2, cancer 1st Floor ICTEM Building, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK e-mail: o.pardo@imperial.ac.uk; m.seckl@imperial.ac.uk INTRODUCTION (9). The presence in the C-terminus of S6K2 of a nuclear local- The ribosomal protein S6 kinases constitute a super-family of pro- ization sequence (NLS) means that this isoform is predominantly teins initially discovered based on their ability to phosphorylate a localized to the nuclei of quiescent cells (10). In addition, the long 40S ribosomal subunit component, the ribosomal S6 protein. The form of S6K2 contains in its 13 amino acid extension an additional p90 ribosomal S6 kinases (RSKs), comprising RSK1–4 (1), were putative NLS. This results in the different cellular distribution of first identified followed by the p70 ribosomal S6 kinase, S6K1 (2, these two isoforms as the two NLS motifs in p56 S6K2 confers 3). It took an additional 10 years for the p70 ribosomal S6 kinase constitutive nuclear localization to this variant, while p54 S6K2 homolog, S6K2, to be discovered (4–6). The high degree of homol- shuttles between the nucleus and the cytoplasm in response to ogy between S6K1 and S6K2 has for many years led researchers to growth factor signaling. The C-terminus of S6K2 also contains a assume that these were redundant kinases with essentially over- proline-rich region which has been proposed to promote interac- lapping functions. This introduced a bias toward S6K1-oriented tion with SH3 and WW domains putatively present in its binding research, as this isoform came to be considered the prototypical partners (4). While shorter isoforms of S6K1 have been shown to S6K. However, more recent research clearly indicates that these two be generated by alternate mRNA splicing (11), no such variants isoforms also have distinct biological functions the understand- have yet been reported for S6K2. However, the high degree of con- ing of which may have implications for therapeutic intervention. servation between the two proteins raises the possibility that simi- Therefore, while other publications exist that review S6Ks and their lar regulation may take place for the RPS6KB2 gene. Indeed, eight upstream pathways (7, 8), here we will focus specifically on S6K2 transcripts have been reported for S6K2 with a corresponding pro- and highlight the distinct biological function of this isoform. tein found for only one (ID ENST00000312629) of the four protein coding transcripts (ID ENST00000539188, ENST00000524934, STRUCTURE OF S6K2 ENST00000524814, ENST00000312629). This may have impor- Human S6K2 is encoded by the 15 exons of the RPS6KB2 tant functional consequences as, unlike its full length counterpart, gene on chromosome 11 (11q13). The S6K2 mRNA (ID an S6K1 splice variant, p31S6K1, was shown to have oncogenic ENST00000312629) gives rise to two protein products through potential (12). the use of alternative translational start sites: a long form (p56 S6K2) and a short form (p54 S6K2) that differ by the presence S6K2 ACTIVATION AND POST-TRANSLATIONAL or absence of an N-terminal 13 amino acid segment. The overall MODIFICATION structure of S6K2 is very close to that of S6K1 (Figure 1A). The S6K2 ACTIVATION kinase domain of S6K2 shares 83% amino acid identity with that Many of the residues that are required for kinase activation are of S6K1, a fact that has long justified the lack of interest in finding common between S6K1 and S6K2 as seven of the eight ser- isoform-specific substrates for these proteins. The kinase domain ine/threonine phosphorylation sites present on S6K1 are con- is followed toward the C-terminus by a kinase extension domain served in S6K2 (Thr-228, Ser-370, Thr-388, Ser-403, Ser-410, and a pseudo-substrate inhibitory region. The greatest degree of Ser-417, and Ser-423 on p54 S6K2) (4, 6, 10) (Figure 1A). The divergence between S6K1 and S6K2 lies in the C-and N-terminus, a activation of S6K2 occurs in a step-wise manner (Figure 1B). fact that has enabled the development of S6K2-specific antibodies An initial barrier to overcome is the repression exerted by the www.frontiersin.org July 2013 | Volume 3 | Article 191 | 36 Pardo and Seckl S6 kinase 2 in health and disease FIGURE 1 | Structure and activation of S6K2. (A) Domain organization of region (KE); C-terminal regulatory region (CR); pseudo-substrate domain (PS); S6K2, post-translational modifications together with involved enzymes, and turn motif (TM); hydrophobic motif (HM); pseudo-substrate region (PS); percentage homology with S6K1. Nuclear localizations sequences (NLS); phosphorylation (P), ubiquitination (Ub); acetylation (Ac). (B) Step-wise model N-terminal regulatory region (NR); kinase domain (KD); kinase extension of activation of S6K2. C-terminal autoinhibitory pseudo-substrate domain. This is dealt Despite S6K1 and S6K2 both lying downstream of mTOR with by phosphorylation of the three proline-directed serines in (Figure 2), there is evidence to indicate that they may be regulated the autoinhibitory domain, Ser-410, Ser-417, and Ser-423 down- through different pools of this upstream kinase. Indeed, both S6K stream of MEK/ERK signaling. We and others have found this first isoforms react differently to nutrient deprivation, a known modu- step to be crucial for S6K2 activation in various cell types (13, 14), lator of mTOR activity. For instance inhibition of protein synthesis as this domain exerts a far more repressive role on S6K2 activity by leucine deprivation in myotubes, results in dephosphorylation than it’s equivalent for S6K1 (15, 16). This event is presumed to of S6K1, without affecting S6K2 activity (21). The existence of two open the kinase conformation, exposing additional phosphoryla- separate pools of mTOR regulating the two S6K isoforms is further tion sites to activating kinases. In agreement with this, deletion suggested by the differential sensitivity of S6K1 and 2 kinase activ- of the autoinhibitory region increases basal activity of S6K2 and ity to the mTOR inhibitor, rapamycin. Indeed, the involvement sensitizes the kinase to activation by various agonists (15). Subse- of mTORC1 in the activation of S6K2, led several researchers to quent phosphorylation of Ser-370 then enables phosphorylation report the sensitivity of S6K2 to this inhibitor (14, 17, 22). How- of Thr-388 by the mTORC1 complex followed by that of Thr-228 ever, the majority of reports suggesting equivalent sensitivity of by PDK1 (17). The T388 site lies within a conserved sequence of S6K1 and 2 to rapamycin used concentrations of this drug that the kinase extension domain (F-X-X-F/Y-S/T-F/Y) known as the non-selectively inhibit the MEK/ERK pathway, therefore indirectly hydrophobic motif, a region found in many AGC kinases. Phos- targeting S6K2 independently of its effect on mTOR (13). Hence, phorylation of this site by mTOR is achieved following the binding when used at the minimal concentrations that fully inhibit S6K1 of the mTORC1 complex component Raptor to the TOR signaling activity, rapamycin often fails to significantly alter S6K2 activ- (TOS) motif present in both S6K1 and 2 (18, 19). Interestingly, ity in several cell systems [(13) and unpublished data from our despite the conservation of the hydrophobic motif, substitution lab]. These findings are consistent with the reported existence of a of Thr-388 by a glutamic acid (T388E) renders S6K2, but not rapamycin-resistant mTORC1 activity pool (23, 24) that can effi- S6K1, constitutively active. However, phosphorylation of both the ciently be targeted by mTOR ATP-competitive inhibitors (23, 25). Ser-370 and Thr-228 is crucial for S6K2 activity. Indeed, sub- stitution of the latter site for alanine renders the T388E mutant ADDITIONAL PHOSPHORYLATION EVENTS inactive while that of the first prevents Thr-388 phosphorylation. While expression of an mTOR variant targeted to the nucleus As S6K2 is mainly a nuclear protein and mTOR shuttles between increases activation of S6K2, nuclear localization of S6K2 is not the cytoplasm and the nucleus, it was shown that S6K2 activity indispensable for activation of this kinase. Indeed, S6K2, but not was increased by targeting mTOR expression to the nucleus (20). S6K1, is phosphorylated in vitro as well as in vivo by protein Frontiers in Oncology | Molecular and Cellular Oncology July 2013 | Volume 3 | Article 191 | 37 Pardo and Seckl S6 kinase 2 in health and disease FIGURE 2 | Signaling pathways upstream and downstream of S6K2 that regulate its activation, localization, expression, and functions. kinase C (PKC) (26). The site of phosphorylation was identified S6K1 was found to relocates to membrane ruffles where the acti- as S486 in p54 S6K2 (S486 in p56 S6K2), located within the C- vated RTKs are expected to reside. Although this pathway seems terminal NLS. While phosphorylation of this site did not affect shared between S6K1 and 2, it is worth noting that while SRC the activity of S6K2, it impaired the function of the NLS leading family members were equally able to phosphorylate both iso- to cytoplasmic accumulation of the kinase upon cell stimulation forms in vitro, S6K2, but not S6K1, was tyrosine phosphorylated with PKC agonists such as PMA. In contrast, S6K1 sub-cellular in response to FYN transgene expression in vivo. This may reflect localization was not modulated by this treatment, highlighting differential wiring of these isoforms to the SRC family members a specific mechanism regulating S6K2 nucleo-cytoplasmic shut- through alternate cellular multi-protein complexes. tling. All PKC isoforms were capable of phosphorylating S6K2, In addition to phosphorylation events, S6K2 is also the target with PKCδ appearing to be the most efficient in vitro. However, of ubiquitination (28, 29) and of acetylation on a lysine residue this specificity seemed to disappear in vivo with all PKCs being close to the C-terminal PDZ binding motif (30). The latter mod- equally potent. ification does not impact on S6K2 kinase activity or sub-cellular In addition to being serine/threonine phosphorylated, S6K2, localization but increases the stability of this kinase (see Control as well as S6K1, can be tyrosine phosphorylated downstream of of S6K2 Steady-State Levels). receptor tyrosine kinase activation (27). Both S6Ks were found to associate with the PDGFR, HGFR, and CSFR. Upon stimulation CELLULAR EXPRESSION AND LOCALIZATION OF S6K2 of these receptors, N-terminal tyrosine phosphorylation of S6Ks CONTROL OF S6K2 STEADY-STATE LEVELS occurred (Y39 on S6K1 and Y45 on S6K2) in a SRC-dependent There is currently little to no information on the transcriptional manner. This event did not result in modulation of S6Ks activity or translational regulation of S6K2 expression. However, much or the gross redistribution of these enzymes, although a fraction of more is known about the regulation of S6K2 stability. Steady-state www.frontiersin.org July 2013 | Volume 3 | Article 191 | 38 Pardo and Seckl S6 kinase 2 in health and disease levels of S6K2 and 1 are regulated through the opposing effects compared to those found in corresponding tumor samples [(42) of ubiquitination and acetylation. Indeed, degradation of S6K1 and see S6K2 Protein Levels in Cancer and Normal Corresponding and S6K2 is mediated by ubiquitination followed by proteosomal Tissues]. degradation (28, 29). This is promoted by growth factor signaling in cell lines although independently of phosphorylation/activation EXPRESSION OF S6K2 IN CANCER AND ITS SIGNIFICANCE of these kinases. Conversely, cell stress, such as that induced by UV S6K2 protein levels in cancer and normal corresponding tissues exposure, stabilizes both proteins. However, unlike for S6K1, the S6K2 has been shown to be expressed in the overwhelming major- molecular pathway regulating S6K2 ubiquitination is currently ity (88%) of cancer samples investigated and the level for this unknown. Indeed, while the ROC1 ubiquitin ligase was shown to kinase compared between several cancer types and corresponding specifically interact and ubiquitinate S6K1 (31), the corresponding normal tissue. These studies demonstrated that normal tissue usu- partner for S6K2 has not yet been identified. S6K1/2 degrada- ally express low levels of this kinase as compared to those found in tion is counteracted by the stabilization of this protein through tumor samples (42–45) and that overexpression of S6K2 was more a C-terminal lysine acetylation event. This occurs through inter- common than that of S6K1 (e.g., 80 versus 25% in breast and 18 action with the acetyltransferases p300 and p300/CBP-associated versus 8% for endometrial cancer). In addition to changes in the factor (PCAF) (Figure 2). Hence, overexpression of p300 or inhi- levels of expression of S6K2, investigators also identified changes bition of deacetylases leads to an increase in the levels of both in the sub-cellular localization of this kinase between normal and kinases (30). Interestingly, overexpression of p300 has been linked malignant tissues. For instance, Filonenko et al. demonstrated to decreased overall survival in patients suffering from a wide that nuclear accumulation of S6K2 was a distinguishing feature variety of malignancies (32–36) while that of PCAF has been asso- of breast cancer cell in situ, whereas this kinase was only found ciated with drug resistance (37–39). However, the role of S6K2 in in the cytoplasm of normal breast cells (43). Moreover, presence these backgrounds still remains to be established. S6K1 seems to be of S6K2 in the nucleus positively correlated with proliferating cell targeted by both HDAC and sirtuins for de-acetylation, but S6K2 nuclear antigen (PCNA) and Ki-67 staining in breast cancer tis- seems entirely dependent on HDAC showing differential regula- sues, demonstrating a link between the presence of S6K2 in this tion of these isoforms. The involvement of HDACs in this process compartment and cell proliferation (46). It is noteworthy that no may provide a further link between these kinases and the transcrip- such correlation existed in the case of S6K1. Interestingly, nuclear tional machinery (see Regulation of Transcription). Moreover, the localization of S6K2 was further increased in cells localized at the stabilization of S6K2 by acetylation together with the pro-survival periphery of the tumor where tumor cells are in contact with and drug resistance phenotypes associated with overexpression of healthy tissue. Hence, it is tempting to speculate that the nuclear this kinase (see Control of Cell Survival) may hinder the efficiency activity of S6K2 is somehow promoting tumorigenesis. The role of of HDAC inhibitors in the clinic. S6K2 in tumorigenesis is further suggested by the fact that expres- sion levels of S6K2 in various tissues seem to influence the role of SUB-CELLULAR LOCALIZATION OF S6K2 S6K1 in mediating PTEN haplo-insufficiency-driven tumorigenic- As mentioned above, S6K2 mainly resides in the nucleus of resting ity. Indeed, S6K1 downregulation impaired tumor development cells. Closer examination reveals that it is the long form of S6K2 downstream of mTORC1 hyperactivation in Pten ± mice only in that is predominantly nuclear by virtue of its two NLSs. In con- tissues where S6K2 expression levels were low (41). Furthermore, trast, the short form of S6K2 shuttles between the nucleus and the in endometrial cancer, increased nuclear localization of S6K2 cor- cytoplasm in response to growth factor signaling. In addition to its related with tumor grade (44), while in lung cancer, increased diffuse nuclear localization, a proportion of S6K2, but not S6K1, expression of S6K2 correlated with drug resistance (42). This has been shown to co-localize with CTR453 and γ-tubulin at the would further suggest that S6K2 expression is linked to cancer level of the centrosome. This localization, demonstrated in multi- progression. ple cell lines using both immunofluorescence and immunoblotting Interestingly, phosphorylation of the ribosomal S6 protein was of purified centrosome (40), was stable throughout the cell cycle. shown not to correlate with expression levels of either S6K1 or Finally, cytoplasmic S6K2 has been shown to have a speckled distri- S6K2 in endometrial and breast cancer (43, 44). This lack of cor- bution (40), although the structural components of these speckles relation is in contradiction with the results obtained from animal remains undetermined. models showing that S6K2 knockout mice, unlike their S6K1 coun- terpart, showed a dramatic reduction in the cellular levels of S6 TISSUE EXPRESSION OF S6K2 IN HEALTH AND DISEASE phosphorylation (47). Hence, the lack of correlation found in tis- PHYSIOLOGICAL EXPRESSION OF S6K2 sue samples may be an artifact generated by the modalities of S6K2 is expressed at various levels in different mouse and human tissue processing or the saturation of these phosphorylation events tissues, and its expression levels often inversely correlate with those beyond a certain threshold of S6K expression. However, conclu- of S6K1 (41). In Humans, S6K2 expression was found in all tissues sions on this issue are further complicated by results obtained with the exception of the neuropil, the peripheral nervous sys- in knock-in mice where the five phosphorylation sites in S6 are tem, and adipocytes. However, expression levels between organs replaced by alanine residues. Indeed, these animals show a pheno- vary considerably, with highest levels found in the gastrointesti- typic overlap with that of S6K1−/− mice including a cell growth nal tract, the central nervous system and the lung. In contrast, defect associated with reduction in cell size (48). This is somehow most mesenchymal cells stain weakly for S6K2. Although S6K2 is surprising considering the lack of impact on S6 phosphorylation detected in normal tissues, its expression levels are often very low of knocking out S6K1 and the previously mentioned privileged Frontiers in Oncology | Molecular and Cellular Oncology July 2013 | Volume 3 | Article 191 | 39
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