Michael A. Moses

Inhibition of the epidermal growth factor receptor by erlotinib prevents immortalization of human cervical cells by Human Papillomavirus type 16

The Human Papillomavirus type-16 (HPV-16) E6 and E7 oncogenes are selectively retained and expressed in cervical carcinomas, and expression of E6 and E7 is sufficient to immortalize human cervical epithelial cells. Expression of the epidermal growth factor receptor (EGFR) is often increased in cervical dysplasia and carcinoma, and HPV oncoproteins stimulate cell growth via the EGFR pathway. We found that erlotinib, a specific inhibitor of EGFR tyrosine kinase activity, prevented immortalization of cultured human cervical epithelial cells by the complete HPV-16 genome or the E6/E7 oncogenes. Erlotinib stimulated apoptosis in cells that expressed HPV-16 E6/E7 proteins and induced senescence in a subpopulation of cells that did not undergo apoptosis. Since immortalization by HPV E6/E7 is an important early event in cervical carcinogenesis, the EGFR is a potential target for chemoprevention or therapy in women who have a high risk for cervical cancer.

Heat shock protein 90: its inhibition and function

The molecular chaperone heat shock protein 90 (Hsp90) facilitates metastable protein maturation, stabilization of aggregation-prone proteins, quality control of misfolded proteins and assists in keeping proteins in activation-competent conformations. Proteins that rely on Hsp90 for function are delivered to Hsp90 utilizing a co-chaperone–assisted cycle. Co-chaperones play a role in client transfer to Hsp90, Hsp90 ATPase regulation and stabilization of various Hsp90 conformational states. Many of the proteins chaperoned by Hsp90 (Hsp90 clients) are essential for the progression of various diseases, including cancer, Alzheimers disease and other neurodegenerative diseases, as well as viral and bacterial infections. Given the importance of these clients in different diseases and their dynamic interplay with the chaperone machinery, it has been suggested that targeting Hsp90 and its respective co-chaperones may be an effective method for combating a large range of illnesses. This article is part of the theme issue ‘Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective’.

Phosphorylation induced cochaperone unfolding promotes kinase recruitment and client class-specific Hsp90 phosphorylation

During the Hsp90-mediated chaperoning of protein kinases, the core components of the machinery, Hsp90 and the cochaperone Cdc37, recycle between different phosphorylation states that regulate progression of the chaperone cycle. We show that Cdc37 phosphorylation at Y298 results in partial unfolding of the C-terminal domain and the population of folding intermediates. Unfolding facilitates Hsp90 phosphorylation at Y197 by unmasking a phosphopeptide sequence, which serves as a docking site to recruit non-receptor tyrosine kinases to the chaperone complex via their SH2 domains. In turn, Hsp90 phosphorylation at Y197 specifically regulates its interaction with Cdc37 and thus affects the chaperoning of only protein kinase clients. In summary, we find that by providing client class specificity, Hsp90 cochaperones such as Cdc37 do not merely assist in client recruitment but also shape the post-translational modification landscape of Hsp90 in a client class-specific manner.The Hsp90 chaperone cycle is influenced by multiple phosphorylation events but their regulatory functions are poorly understood. Here, the authors show that phosphorylation and unfolding of cochaperone Cdc37 tailors the Hsp90 chaperone cycle by recruiting kinases that promote distinct phosphorylation patterns.

Exploration of Benzothiazole-Rhodacyanines as Allosteric Inhibitors of Protein-Protein Interactions with Heat Shock Protein 70 (Hsp70)

Cancer cells rely on the chaperone heat shock protein 70 (Hsp70) for survival and proliferation. Recently, benzothiazole rhodacyanines have been shown to bind an allosteric site on Hsp70, interrupting its binding to nucleotide-exchange factors (NEFs) and promoting cell death in breast cancer cell lines. However, proof-of-concept molecules, such as JG-98, have relatively modest potency (EC50 ≈ 0.7-0.4 μM) and are rapidly metabolized in animals. Here, we explored this chemical series through structure- and property-based design of ∼300 analogs, showing that the most potent had >10-fold improved EC50 values (∼0.05 to 0.03 μM) against two breast cancer cells. Biomarkers and whole genome CRISPRi screens confirmed members of the Hsp70 family as cellular targets. On the basis of these results, JG-231 was found to reduce tumor burden in an MDA-MB-231 xenograft model (4 mg/kg, ip). Together, these studies support the hypothesis that Hsp70 may be a promising target for anticancer therapeutics.

Targeting the Hsp40/Hsp70 Chaperone Axis as a Novel Strategy to Treat Castration-Resistant Prostate Cancer

Castration-resistant prostate cancer (CRPC) is characterized by reactivation of androgen receptor (AR) signaling, in part by elevated expression of AR splice variants (ARv) including ARv7, a constitutively active, ligand binding domain (LBD)-deficient variant whose expression has been correlated with therapeutic resistance and poor prognosis. In a screen to identify small-molecule dual inhibitors of both androgen-dependent and androgen-independent AR gene signatures, we identified the chalcone C86. Binding studies using purified proteins and CRPC cell lysates revealed C86 to interact with Hsp40. Pull-down studies using biotinylated-C86 found Hsp40 present in a multiprotein complex with full-length (FL-) AR, ARv7, and Hsp70 in CRPC cells. Treatment of CRPC cells with C86 or the allosteric Hsp70 inhibitor JG98 resulted in rapid protein destabilization of both FL-AR and ARv, including ARv7, concomitant with reduced FL-AR- and ARv7-mediated transcriptional activity. The glucocorticoid receptor, whose elevated expression in a subset of CRPC also leads to androgen-independent AR target gene transcription, was also destabilized by inhibition of Hsp40 or Hsp70. In vivo, Hsp40 or Hsp70 inhibition demonstrated single-agent and combinatorial activity in a 22Rv1 CRPC xenograft model. These data reveal that, in addition to recognized roles of Hsp40 and Hsp70 in FL-AR LBD remodeling, ARv lacking the LBD remain dependent on molecular chaperones for stability and function. Our findings highlight the feasibility and potential benefit of targeting the Hsp40/Hsp70 chaperone axis to treat prostate cancer that has become resistant to standard antiandrogen therapy.Significance: These findings highlight the feasibility of targeting the Hsp40/Hsp70 chaperone axis to treat CRPC that has become resistant to standard antiandrogen therapy. Cancer Res; 78(14); 4022-35. ©2018 AACR.

Molecular Chaperone Inhibitors

Hsp70 and Hsp90 are molecular chaperones (heat shock proteins) that facilitate client protein maturation, stabilization of aggregation-prone proteins, quality control of misfolded proteins and maintenance of proteins in an activation-competent conformation. In general, these Hsps are part of the cellular proteostasis network that functions in normal and disease states to maintain protein homeostasis. Recent data suggest a role for certain components of the proteostasis network (e.g., the proteasome) in various aspects of immune responses, and lately molecular chaperones have also been suggested to play a role in immunity, although the exact nature of their function remains somewhat controversial. Given the growing importance of Hsp90 and Hsp70 in a number of different diseases, including cancer and neurodegenerative maladies, as well as their role in contributing to protein homeostasis in health and disease, pharmacologic targeting of Hsp70, Hsp90 and their respective co-chaperones remains an area of intense investigation, although the impact of Hsp inhibition on immune cells and systems remains poorly understood.

MP57-04 NOVEL THERAPIES TARGETING ANDROGEN RECEPTOR VARIANTS IN AN IN VITRO MODEL OF CASTRATE RESISTANT PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Prostate cancer (PCa) is the second leading cause of cancer deaths in men. AR signaling is known to play a critical role in androgen responsive prostate cancer cells. Androgen deprivation therapy (ADT) is a standard of care for patients when prostate cancer has spread beyond the prostate. Almost all the prostate cancer (PCa) deaths result from castration resistant prostate cancer (CRPC). With the development of newer anti-androgen Enzalutamide (ENZ) there has been a marked improvement in CRPC. However, almost all patients develop resistance to ENZ in part due to expression of ARv7. Thus, to date, no acceptable treatment options are available for ENZ resistant CRPC. In the present study we evaluated the effects of TET, a derivative of bis-benzyly isoquinoline, Tetrandrine on two enzalutamide resistant prostate cancer cell lines on sensitizing these cells to Enzalutamide. We also evaluated the effects of TET on AR and ARv7 levels. METHODS: Enz resistant Prostate cancer cell lines (22rv1 and LNCaP-abl) were used in the present study. Cells were grown in supplemented media and maintained at 370C in a 5%CO2 incubator (as described elsewhere). Where indicated cells were treated with Enzalutamide or TET alone or in combination. Cell viability was measured by crystal violet and MTT assays. Protein levels were measured by Western Blot assays. mRNA expression measured in RTPCR assays. RESULTS: Treatment with Enz had only a marginal effect on growth and viability of 22rv1 cells. TET inhibited growth and proliferation of enzalutamide resistant prostate cancer cells in both dose and time dependent manner with an IC50 in the range of 5-10uM at 72 hr. However TET treatment did not result in death of RWPE cells, a line of normal prostate cells. Moreover, combination of TET and Enz was more effective than either treatment alone. Treatment with TET resulted in decreased levels of full length AR as well as ARv7 within 24-48h. We also observed that TET treatment was associated with decreased cyclin D1 and increased CDK inhibitors p21 and p27. Over all Tet alone and in combination with Enz promoted cell growth arrest and cell death in ENZ resistant CRPC cells and sensitized these cells to Enz. CONCLUSIONS: This study shows that TET sensitizes CRPC cells to Enz in part by decreasing protein levels of AR and ARv7.

Abstract LB-110: Comparison of the effects of tubulin-targeting and DNA-damaging agents on the modulation and distribution of HSP90, pHSP90αT5/7, and tubulin in cancer cells

Microtubules (MTs) are essential architectural components of cells whose dynamic structures are critical for multiple functions including cell division, intracellular signaling, and trafficking. Many proteins are associated with MTs or their motors, including molecular chaperones such as heat shock protein 90 (HSP90), a key component of the cellular proteostatic machinery. The coordinated interplay of these processes is crucial for maintaining normal or malignant cell phenotypes. Alterations in protein levels, their localization and/or post-translational modifications (PTMs), such as phosphorylation or acetylation, are recognized as targets for disruption and may impact cell viability. To that end, we investigated the levels of HSP90 and pHSP90αT5/7 (a unique PTM previously associated with DNA damage) in both prostate (22RV1, PC3), and colorectal (HCT116wt, HCT116 DNA-PK -/-) carcinoma cell lines after treatment with microtubule-targeting agents (MTAs) or DNA-damaging agents (DDAs). We observed a dose dependent increase in the level of pHSP90αT5/7 protein in each of the cell lines treated with an MTA (paclitaxel (PTX), vincristine) or a DDA (gemcitabine, oxaliplatin) compared to the untreated control, whereas the levels of HSP90 remained monotonous throughout. The increases in pHSP90αT5/7 were independent of DNA-PK status for the HCT116 cells. These observations were accompanied by increasing levels of γ-H2AX and acetylated tubulin, the latter prominent following treatment with PTX. When cell lysates were separated into cytoplasmic (C) and nuclear (N) fractions, HSP90 and pHSP90αT5/7 were located predominantly in the C fractions while acetylated tubulin was surprisingly distributed between the C and N fractions as was γ-H2AX. Markers of cytoplasmic localization, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the voltage-dependent anion channel (VDAC), and of nuclear localization, including phospho-Histone H3 and acetylated-Histone H3, were each segregated to their appropriate cellular compartments. HSP90α was previously shown to be a substrate and chaperone of the nuclear kinase DNA-PK and its DNA-PK dependent phosphorylation in the nucleus at T5 and T7 was necessary for the apoptotic response to TRAIL (Solier S, et al PNAS, 105:32, 2012). In contrast, our data demonstrate that HSP90αT5/7 phosphorylation also occurs in response to MTA, but is localized in cytosol and is independent of DNA-PK. Meanwhile, others have reported that tubulin acetylation favors HSP90 recruitment to MTs, stimulating the signaling function of HSP90 clients (Giustiniani, J, et al Cell Sig, 21, 2009). The unique effects of MTAs and DDAs on pHSP90αT5/7 and tubulin acetylation have implications for malignant cell survival and drug resistance mechanisms. Experiments are underway to determine the significance of cytosolic pHsp90αT5/7 and nuclear acetylated tubulin in these cellular tumor models. Citation Format: Marianne S. Poruchynsky, Michael Moses, Len Neckers. Comparison of the effects of tubulin-targeting and DNA-damaging agents on the modulation and distribution of HSP90, pHSP90αT5/7, and tubulin in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-110. doi:10.1158/1538-7445.AM2017-LB-110

Abstract 1180: Targeting the HSP40/HSP70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) is frequently characterized by elevated expression of nuclear receptors able to at least partially maintain the androgen receptor (AR) transcriptional program. Elevated expression of a number of constitutively active AR splice variants lacking the ligand binding domain (LBD) (e.g., ARv7, which is ligand-independent and correlates with poor prognosis, reduced survival, and resistance to existing LBD-targeted standard of care therapy) is a frequent occurrence in CRPC. Thus, alternative approaches to disrupt AR signaling in CRPC are of great clinical importance, and a single strategy able to target AR and ARv7 remains a critical unmet need. As a steroid hormone nuclear receptor, the AR exists in an interactive and dynamic cycle with the molecular chaperones (heat shock proteins, HSPs) HSP40/HSP70/HSP90 for proper folding and remodeling of the AR LBD to bind ligand. Notably, HSP90 inhibitors promote AR degradation and display efficacy in prostate cancer xenograft models. Although it has been shown that ARv7 functions independently of HSP90, additional chaperone requirements of LBD-deficient ARv7 are not known. Thus, we tested the hypothesis that both AR and ARv7 are dependent on HSP40/HSP70 and that targeting these chaperones with specific inhibitors (C86 and JG98, respectively) will lead to AR/ARv7 destabilization and loss of transcriptional activity in models of CRPC. To determine if AR proteins associate with HSP40/HSP70, 22Rv1 CRPC cells (expressing endogenous AR and ARv7) were first transfected with FLAG-HSP40 or FLAG-HSP70. Immunoprecipitation with FLAG beads revealed AR and ARv7 associated with both chaperones, indicating potential functional dependence of these nuclear receptors on HSP40/HSP70. To further characterize these interactions, 22Rv1 lysate was probed with biotinylated-C86 and subjected to IP with streptavidin beads. C86 bound a significant fraction of HSP40 complexed with HSP70, AR, and ARv7. Excess unlabeled C86 or JG98 effectively competed away binding of HSP40/HSP70 to biotinylated-C86 with concomitant loss of associated AR and ARv7. Treatment of 22Rv1 cells with C86 or JG98 led to a time and dose-dependent decrease in AR and ARv7 protein, concomitant with a significant loss of viability. We also observed that HSP40/HSP70 inhibition markedly reduced AR and ARv7 transcriptional activity, as indicated by decreased AR (KLK3, TMPRSS2) and ARv7 (UBE2C) target gene expression. Finally, treatment of mice bearing 22Rv1 xenografts with JG231 (an analog of JG98 with enhanced PK properties) led to significantly smaller tumors relative to vehicle treated mice. Together, these data confirm the continued dependence of AR and ARv7 on HSP40/HSP70 molecular chaperones and they demonstrate the feasibility of targeting the HSP40/HSP70 axis to abrogate sustained AR-mediated signaling in CRPC. Citation Format: Michael A. Moses, Yeong Sang Kim, Genesis Rivera-Marquez, Matthew J. Watson, Sunmin Lee, Andrea Kravats, Sue Wickner, Jason Gestwicki, Jane Trepel, Len Neckers. Targeting the HSP40/HSP70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1180. doi:10.1158/1538-7445.AM2017-1180

Abstract 1005: Mitochondrial inhibition decreases the malignant phenotype of esophageal adenocarcinoma cells through the induction of autophagy

OBJECTIVE Esophageal adenocarcinoma (EAC) is a lethal disease for which novel therapies are needed. Malignant cell metabolism often shifts from mitochondrial respiration to aerobic glycolysis (Warburg effect). However, glucose alone does not meet the metabolic requirements of cancer cells; therefore, glutamine becomes an essential nutrient. A critical step in glutamine metabolism is the conversion of glutamine to glutamate by the enzyme glutaminase-1 (Gls-1), a potential therapeutic target. The present study was undertaken to identify the effect of targeting glutamine utilization by the mitochondrion in EAC. METHODS Assays were performed in 3 EAC and 1 Barrett9s cell lines: Flo-1, NCI-SB-ESC2 (Esc2), OE33, and CP-C. qRT-PCR and immunoblot were used for Gls-1 expression. Cyquant® and Millipore Invasion Kit measured proliferation and invasion. Flow cytometry, SA-β-galactosidase assay, and immunoblot assessed cell cycle, apoptosis, senescence, and autophagy. Seahorse Extracellular Flux Analyzer was used to quantitate mitochondrial respiration and glycolytic capacity. Cells were treated with bis-2 [5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethylsulfide (BPTES), an inhibitor of Gls-1, and/or metformin, a mitochondrial electron transport chain inhibitor. Glycolysis was inhibited with 2-deoxyglucose (2-DG). Flo-1 and Esc2 were transduced with shRNA targeting Gls-1. RESULTS Glutamine withdrawal decreased proliferation of Flo-1, OE33, and CP-C more than Esc2. Similarly, BPTES caused a more significant decrease in proliferation in Flo-1, OE33, and CP-C than in Esc2. BPTES growth inhibition was reversed by α-ketoglutarate, a metabolite of glutamate. Unlike BPTES, metformin decreased proliferation in all cell lines which was augmented with 2-DG inhibition of glycolysis. Metabolic and knockdown experiments were performed in Flo-1 and Esc2. Glucose partially blocked mitochondrial spare capacity in Flo-1, but completely abrogated the spare capacity in Esc2 suggesting a higher glycolytic dependency in Esc2. Also, knockdown of Gls-1 blocked mitochondrial spare capacity and increased glycolysis in both cell lines. Knockdown of gls-1 decreased proliferation and invasion. Glutamine withdrawal induced autophagy as evidenced by increased LC3 and pAMPK and decreased p70 S6 Kinase in both cell lines. Apoptosis and senescence were not observed. To test glutamine9s effect on glycolysis, glutamine repressed the negative regulator of glycolysis, thioredoxin interacting protein (TXNIP), suggesting that glutamine can increase glycolysis while simultaneously fueling the mitochrondria. CONCLUSIONS Mitochondrial respiration is mediated by both glucose and glutamine in EAC cells. Glutamine exerts an effect on glycolysis through repression of TXNIP. Whereas EAC cells have unique metabolic profiles, targeting common metabolic steps such as Gls-1 may be novel strategies to treat EAC. Citation Format: Deborah R. Depew, Paul L. Feingold, Kate Brown, Yuan Xu, Mahadev Rao, Michael Moses, Leonard M. Neckers, David S. Schrump, R Taylor Ripley. Mitochondrial inhibition decreases the malignant phenotype of esophageal adenocarcinoma cells through the induction of autophagy. [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 1005.