PTEN Mutant Research Articles

Abstract 374: In vitro and in vivo anti-tumor activity of ARQ 751, a potent and selective AKT inhibitor

Abstract Dysregulation of the PI3K-AKT signaling pathway is associated with a number of cancers. AKT can be activated through activated receptor tyrosine kinases, gain-of-function mutations of PIK3CA, PTEN deficiency, and AKT amplification or activating mutations such as AKT1-E17K. We present here the preclinical characterization of ARQ 751, which has distinct physico-chemical properties compared to our first generation inhibitor, ARQ 092. ARQ 751 has IC50 values of 0.55 nM, 0.81 nM and 1.31 nM for AKT1, 2 and 3, respectively, and does not inhibit any other kinase (out of the 245 tested) by greater than 50% at 5 μM, nor does it inhibit AKT lacking the PH domain. ARQ 751 strongly binds to wild-type AKT1 and mutant AKT1-E17K with Kd of 1.2 nM and 8.6 nM, respectively, and suppresses pAKT(S473) in 293T cells transiently transfected with AKT1-E17K. Additionally, membrane translocation of both wild-type and AKT1-E17K is inhibited in NIH 3T3 cells transiently transfected with wild-type AKT1 or mutant AKT1-E17K. Tests on 240 cancer cell lines (Oncopanel) showed the best antiproliferative effects on esophageal (100%, 3/3), breast (87.5%, 14/16), and head and neck cancer cells (67%, 4/6), with GI50 values GI50 < 1 μM. Cancer cell lines with PIK3CA/PIK3R1 mutations (73%; 33/45) are more sensitive to ARQ 751 (GI50<1μM) compared to wild-type (42%, 74/175). Interestingly, among PIK3CA/PIK3R1 wild type cell lines, the PTEN mutant (55%, 11/20) exhibited a similar sensitivity to PTEN wild-type cell lines (48%, 95/197). Of 17 tested breast cancer cell lines, all 8 with PIK3CA mutations are sensitive to ARQ 751. An in vivo efficacy study in AN3CA endometrial cancer xenograft model shows that ARQ 751 inhibits tumor growth (by up to 92% at the well tolerated continuous daily dose of 120 mg/kg) in a dose-dependent manner. Additionally, ARQ 751 exerted dose-dependent anti-tumor activity (by up to 98% at the 5 days on, 2 days off dose of 75mg/kg) in an AKT1-E17K mutant endometrial patient-derived xenograft (PDX) model. ARQ 751 causes significant pathway inhibition in vitro (at the concentrations of 3 nM on pAKT[S473] and 70 nM on pPRAS40 [T246]) and in vivo (on both pAKT[S473] and pPRAS40[T246] after 6 hours of dosing as low as 10 mg/kg) using AN3CA models. Pharmacokinetic data from repeat doses of ARQ 751 show a plasma half-life of 4 to 5 hours and no tissue accumulation. In conclusion, ARQ 751 is a potent and selective allosteric AKT inhibitor. PIK3CA/PIK3R1 and AKT mutations but not PTEN may predict ARQ 751 sensitivity. Our analysis suggests that endometrial, breast, esophageal, and head and neck cancers may represent viable indications for ARQ 751. Both PIK3CA/PIK3R1 and AKT1 mutations could be predictive biomarkers for patient selection, regardless of tumor type. Citation Format: Yi Yu, Ronald E. Savage, Sudharshan Eathiraj, Terence Hall, Brian Schwartz, Giovanni Abbadessa. In vitro and in vivo anti-tumor activity of ARQ 751, a potent and selective AKT inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 374.

Abstract 189: Protein kinase C alpha suppresses AKT activation in endometrial cancer

Abstract Endometrial cancer (EC) is the most common gynecological malignancy and the fourth leading cancer in women in the United States. Hyperactivation of the PI3K/AKT pathway is observed in ∼90% of EC cases and this pathway is the most prominent driver of endometrial carcinogenesis. Mutations in multiple PI3K/AKT pathway components (including PTEN, PIK3CA, PIK3R1, AKT) often coexist in EC, indicating that EC development requires full, unopposed activation of PI3K/AKT signaling. In this study, we identified PKCα as a negative regulator of AKT activation that suppresses endometrial carcinogenesis. Our analysis revealed that PKCα protein and mRNA are reduced or lost in 50% of EC cell lines and ∼60% of human ECs. PKCα deficiency was also observed in hyperplastic endometrial lesions in murine models with allelic knock-in of mutant Pten (ptenΔ4-5/+, ptenC124R/+ and ptenC129E/+) or endometrial-specific Pten deletion (ptenpr-/-, ptenltf-/-), indicating that loss of PKCα is an early event in endometrial carcinogenesis. Mechanistic analysis has further revealed that loss of PKCα is independent of changes in PI3K/AKT signaling; however, the enzyme acts as a potent negative regulator of the PI3K/AKT pathway in EC cells. PKC agonists reduced AKT phosphorylation/activity in EC cells that retain PKCα, via a PP2A-dependent mechanism. Conversely, PKCα knockdown led to increased AKT phosphorylation. Notably, while PKC agonists did not affect AKT in EC cells with loss of PKCα, exogenous expression of the enzyme in these cells restored the inhibitory effect of the agonists on PI3K/AKT signaling. Collectively these data indicate that PKCα has a restraining effect on PI3K/AKT signaling in EC cells, providing a basis for its loss during endometrial tumor progression. Physiological relevance of these findings was established using cell lines, patient samples and mouse models. PKCα restoration in human EC cells decreased expression of oncogenes such as cyclin D1 and Id1 and abolished their anchorage-independent growth, supporting a tumor suppressive role for the isozyme in this tissue. Analysis of PKCα expression in patient specimens determined that loss of PKCα expression correlates with high grade disease. Finally, in mice heterozygous for mutant Pten, PKCα knockout led to a 3-fold increase in endometrial tumor burden at three months, confirming a role of PKCα as a tumor suppressor in the endometrium. Taken together, our results provide evidence that PKCα 1) negatively regulates AKT signaling and the expression of important oncogenes in EC cells; and 2) functions as tumor suppressor in the endometrium whose loss is associated with disease progression. Thus, PKCα signaling represents a promising biomarker for risk stratification in early stage disease and may provide insight into therapeutic strategies for late stage disease. Supported by NIH grants CA036727, CA016056, and DK60632. Citation Format: Alice H. Hsu, Kathryn J. Curry, Kang-Sup Shim, Peter Frederick, Carl D. Morrison, Baojiang Chen, Subodh M. Lele, Takiko Daikoku, Sudhansu K. Dey, Gustavo Leone, Adrian R. Black, Jennifer D. Black. Protein kinase C alpha suppresses AKT activation in endometrial cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 189.

Abstract 1126: Importance of the REM (Ras exchange) motif for membrane interactions by RasGRP3

Abstract RasGRP comprises a family of guanine nucleotide exchange factors that activate small GTPases, regulating the dissociation of GDP from Ras GTPases to enhance the formation of the active GTP-bound form. RasGRP3 is a complex protein with REM (Ras exchange motif), GEF (catalytic domain), EF-hand, C1, SuPT, and PT (plasma membrane-targeting) domains, each of which can affect the cellular membranes to which RasGRP1/3 localizes. The C1 domain was the first membrane localizing domain identified in RasGRP1/3. It binds to the lipid second messenger diacylglycerol (DAG) and has the potential to mediate translocation of RasGRP1/3. The catalytic GEF domain and REM domain are both required for membrane localization of RasGRP1, affecting both endomembrane and plasma membrane targeting of RasGRP1. The objective of this study was to explore the importance of these two domains in translocation of RasGRP3. For these studies, we prepared full length RasGRP1/3, RasGRP1/3 C1 domains, and truncated RasGRP1/3 green fluorescent protein labeled constructs to transfect human prostate adenocarcinoma (LNCaP) cells. In the LNCaP cells, which are a PTEN mutant cell line with markedly elevated phosphoinositide levels, RasGRP1 translocates to the plasma membrane in response to PMA. In contrast, the full length RasGRP3 does not translocate to the plasma membrane even though it has a PT (plasma membrane-targeting) domain. In parallel, we examined the translocation of the C1 domain of RasGRP1/3 and of the truncated RasGRP1/3 constructs. The C1 domain of RasGRP3 was insufficient for targeting RasGRP3 to the plasma membrane. However, truncated RasGRP3s, which contain the PT domain and are missing the REM motif, showed stronger translocation. To better understand differences in the translocation pattern of these two proteins we compared RasGRP1 (“template”) and RasGRP3 (“target”) domains using Swiss-Model (homology or comparative modelling methods) and amino acid sequence alignment (NCBI/ BLAST). The percentage of identities showed remarkable differences in the structure of the REM and SuPT-PT domains. Based on these findings we prepared chimeras where we exchanged either the N or C terminus of RasGRP1/3. Exchange of the REM motif between RasGRP1 and RasGRP3 enhanced the translocation of RasGRP3, while it does not affect the translocation of RasGRP1. Exchange of C termini (SuPT-PT domain) of both proteins did not cause any changes in plasma membrane translocation. Our results clearly show that the N terminus (REM motif) of RasGRP3 is a critical element contributing to the difference between RasGRP1 and RasGRP3 in their ability to translocate to the plasma membrane. This difference in membrane translocation, in turn, should imply important differences in their regulation and, potentially, in their ligand selectivity in the living cell. Citation Format: Agnes Czikora, Noemi Kedei, Peter M. Blumberg. Importance of the REM (Ras exchange) motif for membrane interactions by RasGRP3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1126.

Fine-Tuning of Pten Localization and Phosphatase Activity Is Essential for Zebrafish Angiogenesis

The lipid- and protein phosphatase PTEN is an essential tumor suppressor that is highly conserved among all higher eukaryotes. As an antagonist of the PI3K/Akt cell survival and proliferation pathway, it exerts its most prominent function at the cell membrane, but (PIP3-independent) functions of nuclear PTEN have been discovered as well. PTEN subcellular localization is tightly controlled by its protein conformation. In the closed conformation, PTEN localizes predominantly to the cytoplasm. Opening up of the conformation of PTEN exposes N-terminal and C-terminal regions of the protein that are required for both interaction with the cell membrane and translocation to the nucleus. Lack of Pten leads to hyperbranching of the intersegmental vessels during zebrafish embryogenesis, which is rescued by expression of exogenous Pten. Here, we observed that expression of mutant PTEN with an open conformation rescued the hyperbranching phenotype in pten double homozygous embryos and suppressed the increased p-Akt levels that are characteristic for embryos lacking Pten. In addition, in pten mutant and wild type embryos alike, open conformation PTEN induced stalled intersegmental vessels, which fail to connect with the dorsal longitudinal anastomotic vessel. Functional hyperactivity of open conformation PTEN in comparison to wild type PTEN seems to result predominantly from its enhanced recruitment to the cell membrane. Enhanced recruitment of phosphatase inactive mutants to the membrane did not induce the stalled vessel phenotype nor did it rescue the hyperbranching phenotype in pten double homozygous embryos, indicating that PTEN phosphatase activity is indispensable for its regulatory function during angiogenesis. Taken together, our data suggest that PTEN phosphatase activity needs to be carefully fine-tuned for normal embryogenesis and that the control of its subcellular localization is a key mechanism in this process.

MP45-05 UROTHELIUM-SPECIFIC AND TEMPORALLY CONTROLLED BI-ALLELIC INACTIVATION OF BOTH PTEN AND P53 TRIGGERS BASAL-SUBTYPE MUSCLE-INVASIVE BLADDER CANCER

INTRODUCTION AND OBJECTIVES: PTEN is a tumor suppressor whose inhibitory activity towards phosphoinositide 3 kinase replies on membrane association, a process that is tightly regulated by electrostatic charges of PTEN tail domain. Precisely how and to what extent this domain affects PTEN’s activity remains unclear and is the subject of the present study. METHODS: Urothelial carcinoma cell lines without PTEN (UMUC3 and J82), or that expressing a mutated PTEN (T24), were transiently transfected with vector only, vector containing a full-length PTEN or vector containing a tailless PTEN, and their effects on cell proliferation, AKT activity, cell cycle and apoptosis were assessed under different culture conditions. To circumvent the extreme deleterious effects of the tailless PTEN, stable transfectants of UMUC3 were created that only expressed the tailless PTEN (or the full-length PTEN as a control) upon doxycycline exposure (e.g., stable transfection of CMV/rtTA and TRE-tailless PTEN (or TRE/full-length PTEN). The effects of their inducible expression were assessed in vitro and in xenograft mouse models. RESULTS: Introduction of full-length PTEN and tailless PTEN into PTEN-deficient UMUC3 and J82 cells inhibited proliferation at comparable levels in serum free and low serum (2%) media. However, in high serum (10%) medium, tailless PTEN was three times more inhibitory than full-length PTEN. This was due primarily to the phosphorylation of the serine/threonine clusters of the full-length PTEN’s tail domain, an effect absent in the tailless PTEN. Similar results were observed with transiently transfected T24 cells which expressed an endogenous mutant PTEN. UMUC3, J82 and T24 transfected with tailless PTEN could not sustain long-term culture because of its extreme deleterious effects, prompting us to establish doxycycline-inducible expression cell lines. UMUC3 inducibly expressing tailless PTEN exhibited significantly greater inhibition of proliferation, AKT phosphorylation, G1/S transition and significantly more apoptosis than UMUC3 inducibly expressing full-length PTEN. Finally, xenografted UMUC3 cells inducibly expressing full-length PTEN established tumors, albeit 30% smaller than the vector only controls. In contrast, those inducibly expressing tailless PTEN were devoid of any tumor in nude mice. CONCLUSIONS: Our in vitro and in vivo data are the first demonstration of the high potency of tailless PTEN as a tumor suppressor. They suggest that delivering tailless PTEN via viral vectors or other vehicles can be an effective approach to tackle advanced urothelial carcinomas with wild-type, mutated or deleted PTEN.

Regulatory Effects and Mechanism of Adenovirus-Mediated PTEN Gene on Hepatic Stellate Cells.

Tension homology deleted on chromosome ten (PTEN) is important in liver fibrosis. The purpose of this study was to evaluate the PTEN gene effects and mechanism of action on hepatic stellate cells (HSCs). The rat primary HSCs and human LX-2 cells were transfected by an adenovirus containing cDNA constructs encoding the wild-type PTEN (Ad-PTEN), the PTEN mutant G129E gene (Ad-G129E) and RNA interference targeting the PTEN sequence PTEN short hairpin RNA (PTEN shRNA), to up-regulate and down-regulate PTEN expression, respectively. The HSCs were assayed with a fluorescent microscope, real time PCR, Western blot, MTT, flow cytometry and Terminal-deoxynucleoitidyl transferase mediated nick end labeling. In addition, the CCl4 induced rat hepatic fibrosis model was also established to check the in vivo effects of the recombinant adenovirus with various levels of PTEN expression. The data have shown that the over-expressed PTEN gene led to reduced HSCs activation and viability, caspase-3 activity and cell cycle arrest in the G0/G1 and G2/M phases, as well as negative regulation of the PI3K/Akt and FAK/ERK signaling pathways in vitro. The over-expressed PTEN gene improved liver function, inhibited proliferation and promoted apoptosis of HSCs both in vitro and in vivo. These data have shown that gene therapy using the recombinant adenovirus encoding wild-type PTEN inhibits proliferation and induces apoptosis of HSCs, which is a potential treatment option for hepatic fibrosis.

C. elegans Methods to Study PTEN.

C. elegans encodes a PTEN homolog called DAF-18 and human PTEN can functionally replace DAF-18. Thus C. elegans provides a valuable model organism to study PTEN. This chapter provides methods to study DAF-18/PTEN function in C. elegans. We provide methods to genotype daf-18/Pten mutants, visualize and quantify DAF-18/PTEN in C. elegans, as well as to study physiological and developmental processes that will provide molecular insight on DAF-18/PTEN function.

Cytoplasm-predominant Pten associates with increased region-specific brain tyrosine hydroxylase and dopamine D2 receptors in mouse model with autistic traits.

BackgroundAutism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by impairment in social communication/interaction and inflexible/repetitive behavior. Several lines of evidence support genetic factors as a predominant cause of ASD. Among those autism susceptibility genes that have been identified, the PTEN tumor suppressor gene, initially identified as predisposing to Cowden heritable cancer syndrome, was found to be mutated in a subset of ASD patients with extreme macrocephaly. However, the ASD-relevant molecular mechanism mediating the effect of PTEN mutations remains elusive.MethodsWe developed a Pten knock-in murine model to study the effects of Pten germline mutations, specifically altering subcellular localization, in ASD. Proteins were isolated from the hemispheres of the male littermates, and Western blots were performed to determine protein expression levels of tyrosine hydroxylase (TH). Immunohistochemical stains were carried out to validate the localization of TH and dopamine D2 receptors (D2R). PC12 cells ectopically expressing either wild-type or missense mutant PTEN were then compared for the differences in TH expression.ResultsMice carrying Pten mutations have high TH and D2R in the striatum and prefrontal cortex. They also have increased phosphorylation of cAMP response element-binding protein (CREB) and TH. Mechanistically, PTEN downregulates TH production in PC12 cells via inhibiting the phosphoinositide 3-kinase (PI3K)/CREB signaling pathway, while PTEN reduces TH phosphorylation via suppressing MAPK pathway. Unlike wild-type PTEN but similar to the mouse knock-in mutant Pten, three naturally occurring missense mutations of PTEN that we previously identified in ASD patients, H93R, F241S, and D252G, were not able to suppress TH when overexpressed in PC12 cells. In addition, two other PTEN missense mutations, C124S (pan phosphatase dead) and G129E (lipid phosphatase dead), failed to suppress TH when ectopically expressed in PC12 cells.ConclusionsOur data reveal a non-canonical PTEN-TH pathway in the brain that may work as a core regulator of dopamine signaling, which when dysfunctional is pathogenic in ASD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13229-015-0056-6) contains supplementary material, which is available to authorized users.

Ezrin-radixin-moesin-binding phosphoprotein-50 regulates EGF-induced AKT activation through interaction with EGFR and PTEN.

Dysregulated epidermal growth factor receptor (EGFR) signaling, especially EGFR/AKT signaling, plays important roles in tumorigenesis and progression, the study on intracellular regulation of this signaling pathway has great clinical significance. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important antagonist of AKT activity. Its regulation of AKT activity can be enhanced by ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50)-mediated PTEN/EBP50/platelet-derived growth factor receptor (PDGFR) complex. EBP50 was reported to bind to EGFR, and that it may also mediate the formation of PTEN/EGFR complex to regulate EGFR/AKT signaling. In this study, experiments were performed to verify the hypothesis. Results showed that PTEN co-immunoprecipitated with EGFR, demonstrating PTEN/EGFR complex can form in tissue. Further studies showed that EBP50 knockdown decreased the amount of PTEN/EGFR complex by GST pull-down assay, and EBP50 overexpression increased the amount of PTEN/EGFR complex in a dose-dependent manner. While PTEN mutant (V403A), which can not bind with EBP50, only slightly mediated the formation of PTEN/EGFR complex, confirming that EBP50 specifically mediated the formation of the PTEN/EGFR complex. Both PTEN (V403A) and EGFR (L1043/1063F) mutants can not bind with EBP50. The expression of PTEN (V403A) or EGFR (L1043/1063F) mutant in cells resulted in higher AKT activation level than their respective wild-types by EGF stimulation, indicating that EBP50-mediated PTEN/EGFR complex can effectively inhibit EGF-induced AKT activation. EGF stimulation of siEBP50 cells induced higher AKT activation level compared with control cells, further confirming EBP50-mediated PTEN/EGFR complex can more effectively inhibit EGF-induced AKT activation. These results demonstrated the PTEN/EGFR complex formed under the mediation of EBP50, revealing a novel mechanism for negative regulation of EGF-induced AKT pathway, which may be an important molecular target for antineoplastic therapy.

Elevation of ω-3 Polyunsaturated Fatty Acids Attenuates PTEN-deficiency Induced Endometrial Cancer Development through Regulation of COX-2 and PGE2 Production.

Endometrial cancer is one of the most common gynecologic malignancies. Phosphatase and tensin homologue (PTEN)-mutation is frequently identified in endometrial cancer patients. Although high dietary intake of ω-3 polyunsaturated fatty acids (PUFAs) has been associated with reduced risk of endometrial cancer, the underlying mechanisms is still unknown. To this end, we evaluated the impact of ω-3 PUFAs using several endometrial cancer cellular and animal models. While ~27% and 40% of heterozygotic PTEN mutant mice developed endometrial cancer and atypical complex hyperplasia, respectively, none of the PTEN+/− mice developed cancer when we overexpressed an mfat-1 transgene, which allowed endogenous production of ω-3 PUFAs. Fish oil-enriched diet or expression of mfat-1 transgene significantly inhibited the growth of xenograft tumor derived from RL95-2 cells bearing a PTEN null mutation. At cellular level, ω-3 PUFAs treatment decreased the viability of RL95-2 cells, AKT phosphorylation, and cyclin D1 expression. These molecular events are primarily mediated through reduction of cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production. Exogenous PGE2 treatment completely blunted the impact of ω-3 PUFAs on endometrial cancer. Thus, we revealed the direct inhibitory effects of ω-3 PUFAs on endometrial cancer development and the underlying mechanisms involving reduction of COX-2 and PGE2.

Assessment of the In Vivo Activity of PI3K and MEK Inhibitors in Genetically Defined Models of Colorectal Cancer.

The objective of tailoring medicines for cancer patients according to the molecular profile of their disease holds great promise for the improvement of cancer therapy. Nevertheless, this approach has been limited, in part, due to the lack of predictive and informative preclinical studies. Herein, we describe an assessment of the therapeutic potential of targeting PI3K/mTOR and MAPK signaling in genetically defined mouse models of colorectal cancer mirroring disease subtypes targeted for novel therapy in the FOCUS4 trial. Our studies demonstrate that dual PI3K/mTOR inhibition is highly effective in invasive adenocarcinoma models characterized by combinatorial mutations in Apc and Pten; Apc and Kras; and Apc, Pten and Kras. MEK inhibition was effective in the combinatorial Apc and Kras setting, but had no impact in either Apc Pten mutants or in Apc Pten Kras triple mutants. Furthermore, we describe the importance of scheduling for combination studies and show that although no additional benefit is gained in Apc Pten mice, combination of PI3K/mTOR and MAPK inhibition leads to an additive benefit in survival in Apc Kras mice and a synergistic increase in survival in Apc Pten Kras mice. This is the first study using robust colorectal cancer genetically engineered mouse models to support the validity of PI3K/mTOR and MEK inhibitors as tailored therapies for colorectal cancer and highlight the potential importance of drug scheduling in the clinic.

Phosphorylation and inactivation of PTEN at residues Ser380/Thr382/383 induced by Helicobacter pylori promotes gastric epithelial cell survival through PI3K/Akt pathway.

Phosphorylation of PTEN at residues Ser380/Thr382/383 leads to loss of phosphatase activity and tumor suppressor function. Here, we found that phosphorylation of PTEN at residues Ser380/Thr382/383 was increased with gastric carcinogenesis, and more importantly, Helicobacter pylori was a trigger of this modification in chronic non-atrophic gastritis. H. pylori could phosphorylate and inactivate PTEN in vivo and in vitro, resulting in survival of gastric epithelial cells. Furthermore, stable expression of dominant-negative mutant PTEN or inhibition of Akt prevented the enhanced survival induced by H. pylori. These results indicate that PTEN phosphorylation at residues Ser380/Thr382/383 is a novel mechanism of PTEN inactivation in gastric carcinogenesis, and H. pylori triggers this modification, resulting in activation of the PI3K/Akt pathway and promotion of cell survival.

Abstract 1845: Chloroquine demonstrates limited effectiveness in an autochthonous preclinical model of prostate cancer

Abstract Chloroquine (CQ) is a well-known compound that is used for the treatment of malaria and certain autoimmune diseases. CQ can also block autophagy, an essential cellular process that is implicated with resistance to chemotherapy. Preclinical studies have demonstrated the anticancer effects of CQ and hydroxychloroquine when used in combination with other anticancer agents suggesting that blocking autophagy inhibits therapeutic resistance. However, recent data shows that CQ can have antitumor effects independent of autophagy. Furthermore, given the specific roles of autophagy during tumorigenesis, the therapeutic effects of CQ observed in xenograft studies may differ than those in an autochthonous tumor model. The aim of our study was to investigate the antitumor effects of CQ in an autochthonous tumor model and to determine if CQ could reverse therapeutic resistance through the induction of autophagy. To test this hypothesis we tested the therapeutic administration of CQ in a genetically engineered mouse model of prostate cancer driven by the conditional inactivation of PTEN. We also characterized the combinatorial effects of CQ in combination with additional molecular targeting that included mTOR (everolimus), AKT (AZD5363), MEK (Selumetinib; AZD6244/ARRY142886), 5α-reductase (dutasteride) inhibitors. Oral administration of CQ demonstrated antitumor activity and moderately, but significantly, inhibited growth PTEN-deficient prostate tumors in a drug intervention model. However, combination therapy with CQ did not enhance therapeutic efficacy over inhibitor monotherapy alone. Mice with PTEN-mutant castration-resistant prostate cancer (CRPC) are resistant to treatment with everolimus. Therefore, we tested whether blocking autophagy could restore sensitivity to everolimus in CRPC. Mice with mutant PTEN and ATG7, an autophagy regulatory gene, CRPC demonstrated increased sensitivity and activity to everolimus suggesting that defective autophagy restored resistance to everolimus. On, the contrary, the treatment the combination of CQ and everolimus failed to produce any treatment effects in PTEN-deficient/ATG7-wildtype CRPC. Our findings suggest that combinatorial strategies with CQ lack therapeutic efficacy in autochthonous tumors and might be ineffective in the clinical setting. Citation Format: Yurie Kura, Marco A. De Velasco, Naomi Ando, Emiko Fukushima, Barry R. Davies, Dennis Huzdar, Yutaka Yamamoto, Yuji Hatanaka, Takashi Oki, Nobutaka Shimizu, Kazuhiro Yoshimura, Masahiro Nozawa, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Chloroquine demonstrates limited effectiveness in an autochthonous preclinical model of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1845. doi:10.1158/1538-7445.AM2015-1845

A Fatty Acid Synthase Inhibitor Shows New Anticancer Mechanisms

The pharmacological modulation of proteins and molecules related to cancer is an important goal for translational science. Fatty acid synthase (FASN) is a complex dimeric protein that converts acetyl-CoA and malonyl-CoA into palmitic acid in a NADPH-dependent reaction in mammalian cells (Maier et al., 2006). Many cancers present a strong FASN expression and high enzymatic activity, and in recent years FASN has emerged as a relevant anticancer target. So far, several compounds have been reported to inhibit the enzymatic activity of FASN and to have an effect on the growth of malignant cells. Some of these compounds exhibit pharmacological limitations or induce weight loss, preventing their development as systemic drugs. In this issue of EBioMedicine, Ventura et al. report the characterization of the anti-tumor activity of TVB-3166, a potent and orally available FASN inhibitor that may provide a novel approach for cancer therapy (Ventura et al., 2015). The authors observed that FASN inhibition with TVB-3166 was able to induce apoptosis, to inhibit anchorage-independent cell growth under lipid-rich conditions, and to inhibit in vivo xenograft tumor growth in a dose-dependent manner without affecting non-cancer cells. FASN plays a critical role in a number of metabolic functions by catalyzing the terminal steps in the synthesis of long-chain saturated fatty acids. There is a strong FASN expression and high enzymatic activity in many cancers especially in carcinomas (Sebastiani et al., 2006), while FASN is expressed at low levels in most normal tissues, except the liver, adipose tissue, and lactating mammary gland (Sul and Wang, 1998), suggesting that cancer cells are more dependent on de novo palmitate synthesis catalyzed by FASN than normal cells (Menendez and Lupu, 2007). Palmitate and palmitate-derived lipids are essential components in cancer cell proliferation and survival as they provide energy metabolism and storage, membrane biosynthesis, and architecture and protein localization and activity. The well-documented upregulation of FASN in many human cancers as well as its association with poor clinical outcome (Witkiewicz et al., 2008) both strengthen the hypothesis that FASN is involved in the development, maintenance, and enhancement of the malignant phenotype. Interestingly, increased FASN expression has also been observed in some pre-neoplastic lesions and increases with tumor progression, supporting the hypothesis that the early up-regulation of FASN in precursor lesions might represent an obligatory metabolic acquisition that provides growth and survival advantages through multiple mechanisms. In the report by Ventura et al., the evidence for a role of FASN as a therapeutic target comes from both in vitro and in vivo models of human cancers. The authors found that pharmacological inhibition of FASN using TVB-3166 causes an increase in apoptosis in breast and prostate cancer cell lines. Interestingly, this response is not observed in non-cancer cells. These findings are consistent with previous reports that also used other FASN inhibitors (Brusselmans et al., 2003, Puig et al., 2009). The authors describe here unreported mechanisms of action including alteration on lipid raft architecture, palmitoylated protein localization disruption and inhibition of signal transduction through the PI3K-AKT-mTOR and s-catenin pathways. The effect of TVB-3166 on lipid raft architecture was examined using immuno-fluorescent confocal microscopy to check palmitoylated protein localization. The data reveal that FASN inhibition disrupts lipid raft distribution in the membranes, altering the localization of raft-associated proteins such as N-Ras, inhibits multiple signal transduction pathways including PI3K-AKT-mTPOR and B-catenin, and modulating the expression of several genes in metabolic, proliferation and apoptosis pathways. In addition to in vitro experiments, Ventura et al. also present important results using in vivo models. The authors show that FASN inhibition has anti-tumor activities in biologically diverse preclinical tumor models, including those expressing mutant K-Ras, ErbB2, c-Met, and PTEN. These results are globally in agreement with previous publication in which treatments with siRNA or chemical inhibitors of FASN have been shown to decrease tumor size in animal models of various types of cancers (De Schrijver et al., 2003, Puig et al., 2011). Finally, the authors performed both genetic and expression analyses and found that FASN inhibition modulates genes involved in lipid synthesis, signal transduction, cell cycle and apoptosis pathways. It may be very relevant for future development that those pathways were not affected in non-cancer cells. In conclusion, the results from this report show the potency, selectivity, reversible mechanism of action and in vivo availability of the FASN inhibitor TVB-3166, differentiating it from earlier FASN inhibitors such as C75 or cerulenin. The authors found that TVB-3166 modulates lipid synthesis, signal transduction, cell cycle and apoptosis pathways in both in vitro cell culture and in vivo tumor xenograft models. These results represent an important step in discovering the multiple mechanisms of action of FASN and pave the way for the identification of biomarkers with potential utility as predictive factors.

Abstract 5143: Rats heterozygous for Pten develop fibroadenoma and adenocarcinoma arising from the mammary gland

Abstract To further characterize the function of the tumor suppressor Pten in the role of tumorigenesis we have created a colony of Pten mutant Sprague Dawley (SD) rats. Utilizing zinc finger nuclease technology we introduced a 7bp deletion in Exon 7 of the Pten gene, creating a frameshift mutation and an early stop codon. Pten homozygous null animals are not viable so a colony of heterozygous animals is maintained. Spontaneous tumors of the mammary gland arise in these animals and were removed for staining and pathological analysis. We describe briefly the creation of this model and preliminary genotypic and proteomic characterization. Further we describe pathological observations made on tumor tissues removed from aging Pten heterozygous animals. We have confirmed a reduction in Pten expression by Western Blot utilizing a monoclonal antibody targeting a central locus in the protein. Heterozygous Pten animals develop spontaneous tumors of the mammary gland which were removed if animals demonstrated deteriorating health or any condition within our Humane Care and Use guidelines. Thirty six tissues from 16 animals were submitted for histopathological analysis. Eight of the animals were male, 8 of the animals were female and had a mean lifespan of 159 days (165 male, 153 female). Remaining consistent with human breast cancer incidence in women below 30 years of age, the most common lesion observed is a benign fibroadenoma (30/36 samples). The malignant adenocarcinoma was observed in 5/36 samples submitted. Fibroadenoma only lesions were observed in 12 of the 16 rats while both fibroadenoma and adenocarcinoma was observed in 4 of the 16 rats. Taken together we believe that a Pten heterozygous rat provides an additional tool for the elucidation of the role of Pten in human cancers. This model may also be useful in the study of neurological disorders such as Autism, Parkinson's disease, Alzheimer's disease, obesity, cardiovascular disease, and diabetes. Mutations in Pten strongly correlate with several human disorders including Cowden syndrome, Bannayan-Zonana syndrome, and Lhermitte-Duclos disease. These disorders result in a tumor pathologies including an increased risk for breast cancers. Citation Format: Aaron M. McCoy, Bala Manickam, Lara Reid, Edward Weinstein, Xiaoxia Cui. Rats heterozygous for Pten develop fibroadenoma and adenocarcinoma arising from the mammary gland. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5143. doi:10.1158/1538-7445.AM2015-5143

The PI3K/Akt Pathway Regulates Oxygen Metabolism via Pyruvate Dehydrogenase (PDH)-E1α Phosphorylation.

Inhibition of the PI3K/Akt pathway decreases hypoxia within SQ20B human head and neck cancer xenografts. We set out to understand the molecular mechanism underlying this observation. We measured oxygen consumption using both a Clark electrode and an extracellular flux analyzer. We made these measurements after various pharmacologic and genetic manipulations. Pharmacologic inhibition of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in vitro in SQ20B and other cell lines by 30% to 40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, which inhibits activity of this critical gatekeeper of mitochondrial respiration. Expressing wild-type PTEN in a doxycycline-inducible manner in a cell line with mutant PTEN led to an increase in PDH-E1α phosphorylation and a decrease in OCR. Pretreatment of SQ20B cells with dichloroacetate (DCA), which inhibits PDH-E1α phosphorylation by inhibiting dehydrogenase kinases (PDK), reversed the decrease in OCR in response to PI3K/Akt/mTOR inhibition. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects of PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway.

Abstract LB-063: PTEN function is controlled by recruitment to cytoplasmic vesicles

Abstract PTEN is thought to function at the plasma membrane where receptor tyrosine kinases activate PI 3-Kinases. Yet the majority of PTEN is located throughout the cytoplasm so that only a fraction of PTEN could be actively suppressing PI 3-K signaling at any time. Here we show that cytoplasmic PTEN is distributed along microtubules, tethered to vesicles via interaction with phosphatidylinositol 3- phosphate (PI(3)P), the signature lipid of endosomes. We demonstrate that the C2 domain of PTEN specifically binds PI(3)P via the CBR3-loop. Mutations that render the CBR3-loop incapable of PI(3)P binding abrogate PTEN function in cells but not in vitro. The loss-of-function in cells is rescued by fusion of the canonical PI(3)P vesicle targeting domain, FYVE, to CBR3-loop mutant PTEN, demonstrating the functional relevance of PTEN activity on endosomal membranes. These findings introduce an entirely unexpected site of action of the PTEN tumor suppressor. Furthermore, they introduce the concept of PI 3-K signal activation over the vast surface of the plasma membrane that is contrasted by PTEN-mediated signal termination on the discretely sized and much smaller surfaces of endocytic vesicles. Implications of these results for cancer signaling and growth control will be discussed. Note: This abstract was not presented at the meeting. Citation Format: Adam Naguib, Gylua Bencze, Hyejin Cho, Wu Zheng, Ante Tocilj, Elad Elkayam, Christopher R Faehnle, Nadia Jaber, Christopher Pratt, Muhan Chen, Wei-Xing Zong, Michael S Marks, Leemor Joshua-Tor, Darryl J Pappin, Lloyd C. Trotman. PTEN function is controlled by recruitment to cytoplasmic vesicles. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-063. doi:10.1158/1538-7445.AM2015-LB-063

Abstract 1140: PI3K/Akt inhibition decreases oxygen consumption In tumor cells by phosphorylating and inactivating pyruvate dehydrogenase PDH E1α subunit

Abstract The PI3K/mTOR pathway plays a central role in coupling metabolic processes to the cellular proliferative state. Pharmacologic inhibitors of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in transformed cell lines in vitro by 30-40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, an inhibitory modification of this critical gatekeeper of mitochondrial respiration. Expressing wild type PTEN in a doxycycline-inducible manner in a glioblastoma cell line with mutant PTEN led to an increase in PDH E1α phosphorylation and a decrease in OCR. The decrease in OCR with PI3K/Akt/mTOR inhibition was recapitulated by knocking down PDH E1α expression using siRNA. Pre-treatment with dichloroacetate (DCA), a known inhibitor of the pyruvate dehydrogenase kinases (PDKs) that phosphorylate PDH E1α, prevented the upregulation in PDH E1α phosphorylation, induced by inhibition of the pathway and also blunted the decrease in OCR. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway. Citation Format: George John Cerniglia, Souvik Day, Shannon M. Gallagher-Colombo, Natalie Daurio, Stephen Tuttle, Theresa M. Busch, Theresa M. Busch, Alexander Lin, Tatiana V. Esipova, Sergei A. Vinogradov, Constantinos Koumenis, Amit Maity. PI3K/Akt inhibition decreases oxygen consumption In tumor cells by phosphorylating and inactivating pyruvate dehydrogenase PDH E1α subunit. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1140. doi:10.1158/1538-7445.AM2015-1140

The tumor suppressor PTEN regulates motor responses to striatal dopamine in normal and Parkinsonian animals

Phosphatase and Tensin homolog deleted on chromosome 10 (PTEN) is a dual lipid–protein phosphatase known primarily as a growth preventing tumor suppressor. PTEN is also expressed in neurons, and pathways modulated by PTEN can influence neuronal function. Here we report a novel function of PTEN as a regulator of striatal dopamine signaling in a model of Parkinson's disease (PD). Blocking PTEN expression with an adeno-associated virus (AAV) vector expressing a small hairpin RNA (shRNA) resulted in reduced responses of cultured striatal neurons to dopamine, which appeared to be largely due to reduction in D2 receptor activation. Co-expression of shRNA-resistant wild-type and mutant forms of PTEN indicated that the lipid-phosphatase activity was essential for this effect. In both normal and Parkinsonian rats, inhibition of striatal PTEN in vivo resulted in motor dysfunction and impaired responses to dopamine, particularly D2 receptor agonists. Expression of PTEN mutants confirmed the lipid-phosphatase activity as critical, while co-expression of a dominant-negative form of Akt overcame the PTEN shRNA effect. These results identify PTEN as a key mediator of striatal responses to dopamine, and suggest that drugs designed to potentiate PTEN expression or activity, such as cancer chemotherapeutics, may also be useful for improving striatal responses to dopamine in conditions of dopamine depletion such as PD. This also suggests that strategies which increase Akt or decrease PTEN expression or function, such as growth factors to prevent neuronal death, may have a paradoxical effect on neurological functioning by inhibiting striatal responses to dopamine.

Identification of Androgen Receptor Splice Variants in the Pten Deficient Murine Prostate Cancer Model.

Androgen receptor (AR) variants are associated with resistance to anti androgen therapy both in human prostate cancer cell lines and clinical samples. These observations support the hypothesis that AR isoform accumulation is a consequence of selective therapeutic pressure on the full length AR. The Pten deficient prostate cancer model proceeds with well-defined kinetics including progression to castration resistant prostate cancer (CRPC). While surgical castration and enzalutamide treatments yield an initial therapeutic response, Pten-/-epithelia continue to proliferate yielding locally invasive primary tumor pathology. That most epithelium remains AR positive, but ligand independent, suggests the presence of oncogenic AR variants. To address this hypothesis, we have used a panel of recently described Pten-/- tumor cell lines derived from both from hormone intact (E4, E8) and castrated Pten mutants (cE1, cE2) followed by RACE PCR to identify and characterize three novel truncated, amino terminus containing AR variants (mAR-Va, b, c). Variants appear not only conserved throughout progression but are correlated with nearly complete loss of full length AR (AR-FL) at castrate androgen levels. The overexpression of variants leads to enhanced transcriptional activity of AR while knock down studies show reduced transcriptional output. Collectively, the identification of truncated AR variants in the conditional PTEN deletion model supports a role for maintaining the CRPC phenotype and provides further therapeutic applications of this preclinical model.