Antiandrogen Resistance Research Articles

PTEN Loss Promotes Intratumoral Androgen Synthesis and Tumor Microenvironment Remodeling via Aberrant Activation of RUNX2 in Castration-Resistant Prostate Cancer.

Purpose: Intratumoral androgen synthesis (IAS) is a key mechanism promoting androgen receptor (AR) reactivation and antiandrogen resistance in castration-resistant prostate cancer (CRPC). However, signaling pathways driving aberrant IAS remain poorly understood.Experimental Design: The effect of components of the AKT-RUNX2-osteocalcin (OCN)-GPRC6A-CREB signaling axis on expression of steroidogenesis genes CYP11A1 and CYP17A1 and testosterone level were examined in PTEN-null human prostate cancer cell lines. Pten knockout mice were used to examine the effect of Runx2 heterozygous deletion or abiraterone acetate (ABA), a prodrug of the CYP17A1 inhibitor abiraterone on Cyp11a1 and Cyp17a1 expression, testosterone level and tumor microenvironment (TME) remodeling in vivoResults: We uncovered that activation of the AKT-RUNX2-OCN-GPRC6A-CREB signaling axis induced expression of CYP11A1 and CYP17A1 and testosterone production in PTEN-null prostate cancer cell lines in culture. Deletion of Runx2 in Pten homozygous knockout prostate tumors decreased Cyp11a1 and Cyp17a1 expression, testosterone level, and tumor growth in castrated mice. ABA treatment also inhibited testosterone synthesis and alleviated Pten loss-induced tumorigenesis in vivoPten deletion induced TME remodeling, but Runx2 heterozygous deletion or ABA treatment reversed the effect of Pten loss by decreasing expression of the collagenase Mmp9.Conclusions: Abnormal RUNX2 activation plays a pivotal role in PTEN loss-induced IAS and TME remodeling, suggesting that the identified signaling cascade represents a viable target for effective treatment of PTEN-null prostate cancer, including CRPC. Clin Cancer Res; 24(4); 834-46. ©2017 AACR.

DBC1 promotes castration-resistant prostate cancer by positively regulating DNA binding and stability of AR-V7.

Constitutively active AR-V7, one of the major androgen receptor (AR) splice variants lacking the ligand-binding domain, plays a key role in the development of castration-resistant prostate cancer (CRPC) and anti-androgen resistance. However, our understanding of the regulatory mechanisms of AR-V7-driven transcription is limited. Here we report DBC1 as a key regulator of AR-V7 transcriptional activity and stability in CRPC cells. DBC1 functions as a coactivator for AR-V7 and is required for the expression of AR-V7 target genes including CDH2, a mesenchymal marker linked to CRPC progression. DBC1 is required for recruitment of AR-V7 to its target enhancers and for long-range chromatin looping between the CDH2 enhancer and promoter. Mechanistically, DBC1 enhances DNA-binding activity of AR-V7 by direct interaction and inhibits CHIP E3 ligase-mediated ubiquitination and degradation of AR-V7 by competing with CHIP for AR-V7 binding, thereby stabilizing and activating AR-V7. Importantly, DBC1 depletion suppresses the tumorigenic and metastatic properties of CRPC cells. Our results firmly establish DBC1 as a critical AR-V7 coactivator that plays a key role in the regulation of DNA binding and stability of AR-V7 and has an important physiological role in CRPC progression.

Regulation of the glucocorticoid receptor via a BET-dependent enhancer drives antiandrogen resistance in prostate cancer.

In prostate cancer, resistance to the antiandrogen enzalutamide (Enz) can occur through bypass of androgen receptor (AR) blockade by the glucocorticoid receptor (GR). In contrast to fixed genomic alterations, here we show that GR-mediated antiandrogen resistance is adaptive and reversible due to regulation of GR expression by a tissue-specific enhancer. GR expression is silenced in prostate cancer by a combination of AR binding and EZH2-mediated repression at the GR locus, but is restored in advanced prostate cancers upon reversion of both repressive signals. Remarkably, BET bromodomain inhibition resensitizes drug-resistant tumors to Enz by selectively impairing the GR signaling axis via this enhancer. In addition to revealing an underlying molecular mechanism of GR-driven drug resistance, these data suggest that inhibitors of broadly active chromatin-readers could have utility in nuanced clinical contexts of acquired drug resistance with a more favorable therapeutic index.

Abstract 670: Integrative genomic analysis of alterations driving anti-androgen treatment resistance in vitro

Abstract Prostate cancer is one of the most frequently diagnosed cancers in the world. It is the second most common type of cancer and the fifth leading cause of cancer death in American men. Enzalutamide (MDV) and apalutamide (ARN-509) are selective competitive antagonists of the androgen receptor (AR). Abiraterone acetate (ABI) is an androgen biosynthesis inhibitor which is used in combination with prednisone in metastatic castration-resistant prostate cancer. Although the anti-androgen and AR inhibitors provide breakthrough treatments of metastatic castration-resistant prostate cancer, approximately a third of patients fail to show PSA response (>50% decline from baseline after 12 weeks of treatment), while another third of patients progress after a few cycles of treatment. Recently, the acquired F876L mutation of the androgen receptor (AR) was identified in advanced prostate cancer cells with acquired resistant to enzalutamide and apalutamide. To systematically identify and understand the mechanisms of resistance, we evaluated 22RV1 and LNCaP prostate cancer cells which were resistant to the treatment of enzalutamide (MDV), apalutamide (ARN-509), and abiraterone acetate (ABI). We isolated the DNA and RNA from both resistant cells and their parent cells. We did RNAseq for gene expression analysis, whole exome sequencing to identify the DNA alterations, and array CGH for copy number change analysis. We confirmed the acquired F876L mutation of the AR identified in LNCaP cells resistant to apalutamide. We identified the novel G644R and R630Q mutations of AR, R213* mutation of TP53, N372H mutation of BRCA1, and E2621G mutation of ATM in LNCaP cells which were resistant to the treatment of enzalutamide and apalutamide. For copy number alterations, we identified acquired copy number gain on chromosome 3, 8, and 18 on 22RV1 resistant cells, and acquired copy number loss on LNCaP resistant cells. Besides acquired DNA alterations, we identified 16 up-regulated and 24 down-regulated genes which are common in both 22RV1 and LNCap cells resistant to enzalutamide and apalutamide. Through pathway enrichment analysis, we identified the Wnt/B-catenin, cAMP signaling, and TREM1 signaling pathways were up-regulated in 22RV1 and LNCap cells resistant to enzalutamide and apalutamide. Detection of acquired genetic alterations in cells resistant to anti-androgen treatment provides us with important insights into the mechanisms of their resistance. Further testing of clinical samples collected at disease progression after treatment with these agents is required to validate these findings. Citation Format: Dong Shen, Brendan Hodkinson, Deborah Ricci, Karin Verstraeten, Michael Schaffer, Michael Gormley, Shibu Thomas. Integrative genomic analysis of alterations driving anti-androgen treatment resistance in vitro [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 670. doi:10.1158/1538-7445.AM2017-670

Abstract 4165: SOX2 promotes lineage plasticity and antiandrogen resistance in TP53 and RB1 deficient prostate cancer

Abstract Some cancers evade targeted therapies through a mechanism known as lineage plasticity, whereby tumor cells acquire phenotypic characteristics of a cell lineage whose survival no longer depends on the drug target. Here we show, using in vitro and in vivo prostate cancer models, that these tumors can develop resistance to the antiandrogen drug enzalutamide by a phenotypic shift from androgen receptor (AR) dependent luminal epithelial cells to AR independent basal-like cells. This lineage plasticity is enabled by loss of TP53 and RB1 function, is mediated by increased expression of the reprogramming transcription factor SOX2 and can be reversed by restoring TP53 and RB1 function or by inhibiting SOX2 expression. Thus, mutations in tumor suppressor genes can create a state of increased cellular plasticity that, when challenged with antiandrogen therapy, promotes resistance through lineage switching. Citation Format: Ping Mu, Zeda Zhang, Matteo Benelli, Wouter Karthaus, Elizebeth Hoover, Chi-Chao Chen, John Wongvipat, Sheng-Yu Ku, Dong Gao, Zhen Cao, Neel Shah, Elizabeth Adams, Wassim Abida, Philip Watson, Davide Prandi, Chun-Hao Huang, Elisa de Stanchina, Scott Lowe, Leigh Ellis, Himisha Beltran, Mark Rubin, David Goodrich, Francesca Demichelis, Charles L. Sawyers. SOX2 promotes lineage plasticity and antiandrogen resistance in TP53 and RB1 deficient 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 4165. doi:10.1158/1538-7445.AM2017-4165

Abstract 316: Identification of SGK1 as a potential therapeutic target in castrate resistance prostate cancer

Abstract Introduction: Prostate cancer (PC) is the most common male cancer in the UK, with approximately 1 in 8 men developing the disease within their lifetime (Prostate Cancer UK). The androgen receptor (AR) has a crucial role in the proliferation and progression of prostate cancer. Patients respond to anti-androgen therapy in the early stage of the disease, however many will develop resistance, entering a “castrate-resistant” disease state (CRPC), carrying a very poor prognosis, posing a major clinical challenge (1). The development of second generation anti-androgen therapies, such as Enzalutamide and ARN-509, have shown promise in the treatment of CRPC patients, but response rates of just 50% and the development of resistance to these drugs have limited their success in the clinic (2,3). This study aims to interrogate the global gene expression consequences of anti-androgen resistance in an LNCaP prostate cancer cell line model, resistant to Enzalutamide. In our gene microarray, SGK1 demonstrates high expression in Enzalutamide resistant cells. Our subsequent experiments suggest SGK1 may serve as a biomarker of resistance or perhaps an exploitable target in CRPC. Methods: A gene microarray was used to determine the gene profile of parental LNCaP cells, sensitive to anti-androgen drugs, versus in-house generated LNCaP-Enzalutamide Resistant cells. QPCR to determine the mRNA level of SGK1 +/- Dihydrotestosterone (DHT), +/-Dexamethasone. Western blot used to detect the protein level of the SGK1. IncuCyte used to determine the proliferation. Wound Healing Assay to detect the direction of the cells toward the edges Results: These preliminary data demonstrate an increase in expression of SGK1 in the LNCaP-Enz-R cell line model, compared with parental LNCaP cell, at both the protein and mRNA levels. We have demonstrated that stimulating AR by DHT also increases SGK1 expression at both the protein and mRNA levels, in both cell lines. In addition, it is shown that activation of the glucocorticoid receptor (GR) by stimulation with Dexamethasone increases SGK1 expression at the mRNA level only in LNCaP cells, however high SGK1 expression is observed at the mRNA AND protein level in the LNCaP-Enz-R cell line. Inhibition of SGK1 using small molecular inhibitors significantly decreases proliferation and migration of the LNCaP-Enz-R cell line, whereas no significant difference is seen in the parental, androgen sensitive LNCaP cell line. Conclusion: Increased expression of SGK1 in Enzalutamide, Casodex and ARN509 resistant cell line models was observed, compared with parental LNCaP cells. AR regulates SGK1 in both parental LNCaP and LNCaP-Enz-R cell line models. GR regulates SGK1 in the LNCaP-Enz-R cell line alone. SGK1 has a vital role in the proliferation and migration of the LNCaP-Enz-R cell line model. SGK1 could represent either a future biomarker of Enzalutamide-resistance, or a potential therapeutic target in advanced disease. Citation Format: Massar I. Alsamraae, Urzsula McClurg, Craig N. Robson, Stuart McCracken. Identification of SGK1 as a potential therapeutic target in castrate resistance 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 316. doi:10.1158/1538-7445.AM2017-316

Cellular identity crisis: Antiandrogen resistance by lineage plasticity

ABSTRACTA recent publication in Science demonstrates the ability of prostate cancer cells to switch lineages from one that is dependent on androgen signaling to a cell type that is not. Known as lineage plasticity, this phenomenon is driven by the transcription factor SOX2 in RB1 and TP53-deficient prostate cancer. SOX2 is a potential prognostic marker and therapeutic target in castration resistant prostate cancer.

Androgen Receptor Regulation of Local Growth Hormone in Prostate Cancer Cells.

Prostate cancer (PCa) growth is mainly driven by androgen receptor (AR), and tumors that initially respond to androgen deprivation therapy (ADT) or AR inhibition usually relapse into a more aggressive, castration-resistant PCa (CRPC) stage. Circulating growth hormone (GH) has a permissive role in PCa development in animal models and in human PCa xenograft growth. As GH and GH receptor (GHR) are both expressed in PCa cells, we assessed whether prostatic GH production is linked to AR activity and whether GH contributes to the castration-resistant phenotype. Using online datasets, we found that GH is highly expressed in human CRPC. We observed increased GH expression in castration-resistant C4-2 compared with castration-sensitive LNCaP cells as well as in enzalutamide (MDV3100)-resistant (MDVR) C4-2B (C4-2B MDVR) cells compared with parental C4-2B. We describe a negative regulation of locally produced GH by androgens/AR in PCa cells following treatment with AR agonists (R1881) and antagonists (enzalutamide, bicalutamide). We also show that GH enhances invasive behavior of CRPC 22Rv1 cells, as reflected by increased migration, invasion, and anchorage-independent growth, as well as expression of matrix metalloproteases. Moreover, GH induces expression of the AR splice variant 7, which correlates with antiandrogen resistance, and also induces insulinlike growth factor 1, which is implicated in PCa progression and ligand-independent AR activation. In contrast, blockade of GH action with the GHR antagonist pegvisomant reverses these effects both in vitro and in vivo. GH induction following ADT or AR inhibition may contribute to CRPC progression by bypassing androgen growth requirements.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

CCN3-EZH2-AR feedback loop: new targets for enzalutamide and castration resistant prostate cancer.

The development of castration resistant prostate cancer and anti-androgen resistance remains one of the largest hurdles in the successful treatment of prostate cancer. Therefore, the identification of dysregulated pathways contributing to this resistance and determining ways to target these mechanisms is of utmost importance. In the recent publication in Cancer Research, Fong et al. identify a novel role for cytoplasmic CCN3 in prostate cancer progression and enzalutamide resistance. The authors demonstrate that CCN3 expression inhibits androgen receptor signaling and thereby suppresses enzalutamide-resistant prostate cancer cell proliferation, colony formation, and xenograft tumor growth. The data from this manuscript highlight an intriguing potential therapeutic target for the treatment of CRPC and are a critical step forwards towards treating enzalutamide resistant prostate cancer.

Evasion of targeted cancer therapy through stem-cell-like reprogramming

ABSTRACTProstate cancer variants expressing alternative lineage markers appear at relapse from antiandrogen therapy. We show that loss of the retinoblastoma (RB1) and tumor protein 53 (TP53) genes drives expression of stem cell reprogramming factors, lineage plasticity, and antiandrogen resistance. Epigenetic manipulation restores antiandrogen sensitivity—suggesting an approach for treating lethal prostate cancers.

Rb1 and Trp53 cooperate to suppress prostate cancer lineage plasticity, metastasis, and antiandrogen resistance.

Prostate cancer relapsing from antiandrogen therapies can exhibit variant histology with altered lineage marker expression, suggesting that lineage plasticity facilitates therapeutic resistance. The mechanisms underlying prostate cancer lineage plasticity are incompletely understood. Studying mouse models, we demonstrate that Rb1 loss facilitates lineage plasticity and metastasis of prostate adenocarcinoma initiated by Pten mutation. Additional loss of Trp53 causes resistance to antiandrogen therapy. Gene expression profiling indicates that mouse tumors resemble human prostate cancer neuroendocrine variants; both mouse and human tumors exhibit increased expression of epigenetic reprogramming factors such as Ezh2 and Sox2. Clinically relevant Ezh2 inhibitors restore androgen receptor expression and sensitivity to antiandrogen therapy. These findings uncover genetic mutations that enable prostate cancer progression; identify mouse models for studying prostate cancer lineage plasticity; and suggest an epigenetic approach for extending clinical responses to antiandrogen therapy.

Cancer-Associated Fibroblasts Modify the Response of Prostate Cancer Cells to Androgen and Anti-Androgens in Three-Dimensional Spheroid Culture.

Androgen receptor (AR) targeting remains the gold standard treatment for advanced prostate cancer (PCa); however, treatment resistance remains a major clinical problem. To study the therapeutic effects of clinically used anti-androgens we characterized herein a tissue-mimetic three-dimensional (3D) in vitro model whereby PCa cells were cultured alone or with PCa-associated fibroblasts (CAFs). Notably, the ratio of PCa cells to CAFs significantly increased in time in favor of the tumor cells within the spheroids strongly mimicking PCa in vivo. Despite this loss of CAFs, the stromal cells, which were not sensitive to androgen and even stimulated by the anti-androgens, significantly influenced the sensitivity of PCa cells to androgen and to the anti-androgens bicalutamide and enzalutamide. In particular, DuCaP cells lost sensitivity to enzalutamide when co-cultured with CAFs. In LAPC4/CAF and LNCaP/CAF co-culture spheroids the impact of the CAFs was less pronounced. In addition, 3D spheroids exhibited a significant increase in E-cadherin and substantial expression of vimentin in co-culture spheroids, whereas AR levels remained unchanged or even decreased. In LNCaP/CAF spheroids we further found increased Akt signaling that could be inhibited by the phosphatidyl-inositol 3 kinase (PI3K) inhibitor LY294002, thereby overcoming the anti-androgen resistance of the spheroids. Our data show that CAFs influence drug response of PCa cells with varying impact and further suggest this spheroid model is a valuable in vitro drug testing tool.

Abstract 5040: Genomic and proteomic characterization of anti-androgen resistant cell lines

Abstract Background: Prostate cancer is the most common cancer and second most frequent cause of cancer-related deaths for men in the United States. AR-axis inhibitors have been shown to improve both progression-free survival (PFS) and overall survival (OS) in castration resistant prostate cancer (CRPC) patients however an estimated 50% of patients will go on to develop resistance to these therapies. Some mechanisms of resistance observed in the clinic include Androgen Receptor (AR) dependent events such as increased AR copy number, activating point mutations in AR, or splice variants (ARV7) both of the latter leading to constitutively active versions of AR. Alternatively, AR-independent resistance mechanisms include up-regulation of other oncogenic signaling pathways such as FGFR and PI3/Akt and induction of the nuclear hormone Glucocorticoid Receptor (GR). Finally, an increase in the emergence of a neuroendocrine phenotype is observed in the clinic with CRPC patients and is thought to be associated with chronic AR-antagonist treatment. To better understand the specific proteomic and genomic changes that occur during chronic AR inhibitor treatment we developed two enzalutamide (ENZA) resistant cell lines and performed targeted proteomic and global gene expression analysis. Methods: We generated LnCaP and VCAP ENZA-resistant (ENZA-Res) prostate cancer cell lines. We performed gene expression analysis by using both cDNA microarray and RNA-SEQ techniques to identify novel or differential gene expression patterns between ENZA-Res and sensitive phenotypes. We also employed a targeted proteomic approach to identify changes in the relative expression levels of proteins that have historically been associated with anti-androgen resistance including, ARV7, GR and SGK-1. Results: We have shown that ENZA-Res cell lines can undergo a significant change in morphology that is associated with an elongated neuronal-like appearance. This phenotype is associated with an induction in the mRNA expression of neuroendocrine markers, CHGA and REST along with a strong induction of GR mRNA and protein expression. We have shown that the neuroendocrine derived prostate cancer cell line, H660, also demonstrates high GR mRNA and protein content suggesting that GR may have a role in the maintenance of neuroendocrine and/ or ENZA-Res phenotypes when AR is absent. In VCaP ENZA-Res cells a significant increase in ARV7 mRNA and protein content was observed compared to parental ENZA sensitive VCaP cells. Lastly, we employed a combination of cDNA microarray and RNA-SEQ techniques to identify a molecular signature associated with anti-androgen resistance. We have identified a unique subset of genes that are differentially up-regulated in ENZA-Res cell lines compared to ENZA-sensitive cell lines. This novel molecular signature identifies several candidate genes as potential therapeutic targets that may be important in mediating anti-androgen resistance. Citation Format: Jennifer Hertzog, Mike Russell, Michael Quigley, Marco Gottardis, Theresa McDevitt. Genomic and proteomic characterization of anti-androgen resistant cell lines. [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 5040.

Abstract 238: Androgen insensitivity syndrome germline loss-of-function mutations in the androgen receptor that acquire somatic gain-of-function in prostate cancer

Abstract Genomic alterations in the androgen receptor (AR) commonly occur in patients with advanced prostate cancer resistant to androgen deprivation therapies. While still retaining androgen sensitivity, recurring AR mutations become drivers of antiandrogen resistance by causing alterations in the ligand binding pocket that enables one specific antiandrogen to bind that particular mutant receptor in an agonist conformation, thereby aberrantly activating AR transcriptional activity. We chronically treated antiandrogen-sensitive preclinical prostate cancer models (LNCaP-AR and CWR22Pc) with antiandrogens followed by high-throughput sequencing of the AR exons 2-8 to search for novel mutations that may be associated with antiandrogen resistance. The mutation A597T in the DNA binding domain was found in four LNCaP-AR xenograft tumors from mice receiving enzalutamide or ARN-509, while the ligand binding domain mutation P893S was uncovered in CWR22Pc cells treated in vitro with combined androgen depletion and bicalutamide. Recent whole exome sequencing studies of clinical prostate cancer identified single patients with each of these mutations at high allele frequencies and without concurrent AR amplification (www.cBioPortal.org). A597T occurred in a Gleason 6 primary tumor, whereas P893S was found in a patient with metastatic castration resistant prostate cancer who had received bicalutamide during the course of treatment. Remarkably, both A597T and P893S were earlier reported as germline loss-of-function mutations in patients with androgen insensitivity syndrome. Using an AR reporter assay in AR-null cells coupled with overexpression of these AR mutants, we confirmed that P893S is not activated by the androgen dihydrotestosterone (DHT), but nevertheless is potently stimulated by bicalutamide. In silico modeling showed that in contrast to the wild-type AR, cofactor-recruiting helix 12 of the ligand binding domain of P893S substantially drifted away from an agonist conformation when bound to DHT but was able to adopt the active conformation when simulated with bicalutamide. Importantly, overexpression of AR-P893S in CWR22Pc cells conferred bicalutamide resistance in vivo. These findings highlight an unexpected contextual element to the function of AR mutants, and suggest that rare or private AR mutations may nonetheless act as drivers of clinical progression. Citation Format: Philip A. Watson, Minna D. Balbas, Zeda Zhang, Taslima F. Ishmael, Kayla E. Lawrence, John Wongvipat, Sophie D. Sawyers, Yi-Mi Wu, Dan Robinson, Yang Shen, Arul M. Chinnaiyan, Charles L. Sawyers. Androgen insensitivity syndrome germline loss-of-function mutations in the androgen receptor that acquire somatic gain-of-function in prostate 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 238.

Abstract 3571: Targeted suppression of inhibitor of apoptosis proteins amplifies apoptosis and improves response to enzalutamide in prostate cancer

Abstract Prostate cancer is the most common malignancy and the 2nd leading cause of cancer death in men worldwide. First-line treatment includes androgen deprivation therapy in addition to androgen receptor (AR) antagonists that work to block androgen signaling. Although most patients respond initially to hormone deprivation therapy, responses are not durable and the disease progresses to a lethal stage, termed castrate resistant prostate cancer (CRPC). Interestingly, most CRPCs are still dependent on AR signaling for growth and survival, therefore more potent AR-antagonists such as enzalutamide have recently been FDA approved for treatment of CRPC. However, response rates to enzalutamide can be limited by emergence of acquired resistance or intrinsic resistance. A universal mechanism of resistance to cytotoxic therapies is upregulation of anti-apoptotic programs that function to override death signals and permit survival of the tumor cell. In prostate cancer, overexpression of the Inhibitor of Apoptosis (IAP) proteins cIAP1, cIAP2, and XIAP has been demonstrated in progression to CRPC. Additionally, aberrant expression of these proteins is reported to contribute to anti-androgen resistance. Therefore, to achieve more robust responses to AR inhibition with enzalutamide, we targeted IAP proteins in order to amplify the apoptotic signal and increase cell death in prostate cancer cells. Using a small-molecule IAP inhibitor, we evaluated response to enzalutamide upon IAP inhibition in prostate cancer cell lines LNCAP and C4-2. Here we demonstrate greater proliferation inhibition, reduced cell survival and synergistic induction of apoptosis signaling in response to combined AR antagonism and IAP inhibition. Furthermore, we demonstrate depletion of cIAP1 protein expression upon enzalutamide treatment indicating a possible role for AR in regulating anti-apoptotic pathways. Taken together these results demonstrate that IAP proteins may be a critical survival pathway in CRPC and antagonism of these proteins through small-molecule inhibition can amplify apoptosis and increase cell death in response to enzalutamide. Targeting a critical survival pathway can lower the apoptotic threshold providing a possible therapeutic opportunity to treat and prevent resistance to enzalutamide and increase benefit to patients with this lethal form of prostate cancer. Citation Format: Amanda Pilling, Ok Hwang, Clara Hwang. Targeted suppression of inhibitor of apoptosis proteins amplifies apoptosis and improves response to enzalutamide in prostate 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 3571.

Functional analysis of androgen receptor mutations that confer anti-androgen resistance identified in circulating cell-free DNA from prostate cancer patients.

BackgroundThe androgen receptor (AR) is a pivotal drug target for the treatment of prostate cancer, including its lethal castration-resistant (CRPC) form. All current non-steroidal AR antagonists, such as hydroxyflutamide, bicalutamide, and enzalutamide, target the androgen binding site of the receptor, competing with endogenous androgenic steroids. Several AR mutations in this binding site have been associated with poor prognosis and resistance to conventional prostate cancer drugs. In order to develop an effective CRPC therapy, it is crucial to understand the effects of these mutations on the functionality of the AR and its ability to interact with endogenous steroids and conventional AR inhibitors.ResultsWe previously utilized circulating cell-free DNA (cfDNA) sequencing technology to examine the AR gene for the presence of mutations in CRPC patients. By modifying our sequencing and data analysis approaches, we identify four additional single AR mutations and five mutation combinations associated with CRPC. Importantly, we conduct experimental functionalization of all the AR mutations identified by the current and previous cfDNA sequencing to reveal novel gain-of-function scenarios. Finally, we evaluate the effect of a novel class of AR inhibitors targeting the binding function 3 (BF3) site on the activity of CRPC-associated AR mutants.ConclusionsThis work demonstrates the feasibility of a prognostic and/or diagnostic platform combining the direct identification of AR mutants from patients’ serum, and the functional characterization of these mutants in order to provide personalized recommendations regarding the best future therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0864-1) contains supplementary material, which is available to authorized users.

Pilot study on the detection of antiandrogen resistance using serial diffusion‐weighted imaging of bone metastases in prostate cancer

To evaluate serial apparent diffusion coefficient (ADC) measurements of bone metastases in prostate cancer to determine whether antiandrogen resistance can be detected and time to progression estimated. Diffusion-weighted imaging (DWI) was performed at 1.5T in nine patients with treatment-naïve metastatic prostate cancer (20 lesions) before antiandrogen treatment, after 1, 2, and 3 months of treatment, and thereafter every 4 months over 31 months or until antiandrogen resistance was detected. Tumor volumes were stable over time. Time courses of the ADCs when averaged over entire lesions and on functional diffusion maps (fDMs) were analyzed using marginal linear model (MLM) analysis. Starting at 1 month, MLM analysis revealed decreasing mean ADCs (P = 0.001) over time. Simultaneously, the percentage of voxels with significantly higher ADCs decreased (P = 0.004), whereas the percentage of voxels with significantly lower ADCs increased (P < 0.001) on fDMs. Both mean ADCs (P = 0.042) and percentages of voxels with significantly higher ADCs on fDMs (P = 0.039) decreased more rapidly over time in patients with a shorter progression-free interval (PFI). Likewise, higher (P = 0.001) and more rapidly increasing (P = 0.002) percentages of voxels with significantly lower ADCs on fDMs were associated with a shorter PFI. The results of our pilot study suggest that the evolution of ADCs over time may permit early identification of antiandrogen resistance in bone metastases. J. Magn. Reson. Imaging 2016;43:1407-1416.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Single-Cell Analysis of Prostate CTCs Highlights Resistance Mechanisms

Abstract Activation of noncanonical WNT signaling promotes antiandrogen resistance in prostate cancer.