Purpose: The major obstacle in the management of advanced prostate cancer is the occurrence of resistance to endocrine therapy. Although the androgen receptor (AR) has been linked to therapy failure, the underlying escape mechanisms have not been fully clarified. Being closely related to the AR, the glucocorticoid receptor (GR) has been suggested to play a role in enzalutamide and docetaxel resistance. Given that glucocorticoids are frequently applied to prostate cancer patients, it is essential to unravel the exact role of the GR in prostate cancer progression. Experimental Design: Assessment of GR expression and functional significance in tissues from 177 prostate cancer patients, including 14 lymph node metastases, as well as in several human prostate cancer models, including androgen-dependent, androgen-independent, and long-term antiandrogen-treated cell lines. Results: Although GR expression is reduced in primary prostate cancer tissue, it is restored in metastatic lesions. Relapse patients with high GR experience shortened progression-free survival. GR is significantly increased upon long-term abiraterone or enzalutamide treatment in the majority of preclinical models, thus identifying GR upregulation as an underlying mechanism for cells to bypass AR blockade. Importantly, GR inhibition by RNAi or chemical blockade results in impaired proliferation and 3D-spheroid formation in all tested cell lines. Conclusions: GR upregulation seems to be a common mechanism during antiandrogen treatment and supports the notion that targeting the GR pathway combined with antiandrogen medication may further improve prostate cancer therapy. Clin Cancer Res; 24(4); 927–38. ©2017 AACR.

Prostate cancer is heterogeneous and patients would benefit from methods that stratify those who are likely to respond to systemic therapy. Here, we employ single-cell assays for transposase-accessible chromatin (ATAC) and RNA sequencing in models of early treatment response and resistance to enzalutamide. In doing so, we identify pre-existing and treatment-persistent cell subpopulations that possess regenerative potential when subjected to treatment. We find distinct chromatin landscapes associated with enzalutamide treatment and resistance that are linked to alternative transcriptional programs. Transcriptional profiles characteristic of persistent cells are able to stratify the treatment response of patients. Ultimately, we show that defining changes in chromatin and gene expression in single-cell populations from pre-clinical models can reveal as yet unrecognized molecular predictors of treatment response. This suggests that the application of single-cell methods with high analytical resolution in pre-clinical models may powerfully inform clinical decision-making.

Interleukin-4 plays a critical role in the regulation of immune responses and has been detected at high levels in the tumour microenvironment of cancer patients, where concentrations correlate with the grade of malignancy. In prostate cancer, interleukin-4 has been associated with activation of the androgen receptor, increased proliferation and activation of survival pathways such as Akt and NF-κB. However, its role in therapy resistance has not yet been determined. Here we investigate the influence of interleukin-4 on primary epithelial cells from prostate cancer patients. Our data demonstrate an increase in the clonogenic potential of these cells when cultured in the presence of interleukin-4. In addition, a Phospho-Kinase Array revealed that in contrast to previously published work, signal transducer and activator of transcription6 (STAT6) is the only signalling molecule activated after interleukin-4 treatment. Using the STAT6-specific inhibitor AS1517499 we could confirm the role of STAT6 in increasing colony-forming frequency. However, clonogenic recovery assays revealed that interleukin-4 does not rescue the effects of either irradiation or docetaxel treatment. We therefore propose that although the interleukin-4/STAT6 axis does not appear to be involved in therapy resistance, it does play a crucial role in the colony-forming abilities of the basal cell population in prostate cancer. IL-4 may therefore contribute to disease relapse by providing a niche that is favourable for the clonogenic growth of prostate cancer stem cells.

Inhibition of the androgen receptor (AR) by second-generation anti-androgens is a standard treatment for metastatic castration resistant prostate cancer (mCRPC), but it inevitably leads to the development of resistance. Since the introduction of highly efficient AR signalling inhibitors, approximately 20% of mCRPC patients develop disease with AR independent resistance mechanisms. In this study, we generated two anti-androgen and castration resistant prostate cancer cell models that do not rely on AR activity for growth despite robust AR expression (AR indifferent). They are thus resistant against all modern AR signalling inhibitors. Both cell lines display cross-resistance against the chemotherapeutic drug docetaxel due to MCL1 upregulation but remain sensitive to the PARP inhibitor olaparib and the pan-BCL inhibitor obatoclax. RNA-seq analysis of the anti-androgen resistant cell lines identified hyper-activation of the E2F cell-cycle master regulator as driver of AR indifferent growth, which was caused by deregulation of cyclin D/E, E2F1, RB1, and increased Myc activity. Importantly, mCRPC tissue samples with low AR activity displayed the same alterations and increased E2F activity. In conclusion, we describe two cellular models that faithfully mimic the acquisition of a treatment induced AR independent phenotype that is cross-resistant against chemotherapy and driven by E2F hyper-activation.

Novel drugs like Abiraterone or Enzalutamide, which target androgen receptor (AR) signaling to improve androgen deprivation therapy (ADT), have been developed during the past years. However, the application of these drugs is limited because of occurrence of inherent or acquired therapy resistances during the treatment. Thus, identification of new molecular targets is urgently required to improve current therapeutic prostate cancer (PCa) treatment strategies. PIAS1 (protein inhibitor of activated STAT1 (signal transducer and activator of transcription-1)) is known to be an important cell cycle regulator and PIAS1-mediated SUMOylation is essential for DNA repair. In this context, elevated PIAS1 expression has already been associated with cancer initiation. Thus, in the present study, we addressed the question of whether PIAS1 targeting can be used as a basis for an improved PCa therapy in combination with anti-androgens. We show that PIAS1 significantly correlates with AR expression in PCa tissue and in cell lines and demonstrate that high PIAS1 levels predict shorter relapse-free survival. Our patient data are complemented by mechanistic and functional in vitro experiments that identify PIAS1 as an androgen-responsive gene and a crucial factor for AR signaling via prevention of AR degradation. Furthermore, PIAS1 knockdown is sufficient to decrease cell proliferation as well as cell viability. Strikingly, Abiraterone or Enzalutamide treatment in combination with PIAS1 depletion is even more effective than single-drug treatment in multiple PCa cell models, rendering PIAS1 as a promising target protein for a combined treatment approach to improve future PCa therapies.

IL6/STAT3 signaling is associated with endocrine therapy resistance in prostate cancer, but therapies targeting this pathway in prostate cancer were unsuccessful in clinical trials so far. The mechanistic explanation for this phenomenon is currently unclear; however, IL6 has pleiotropic effects on a number of signaling pathways, including the androgen receptor (AR). Therefore, we investigated IL6-mediated AR activation in prostate cancer cell lines and ex vivo primary prostate tissue cultures in order to gain a better understanding on how to inhibit this process for future clinical trials. IL6 significantly increased androgen-dependent AR activity in LNCaP cells but importantly did not influence AR activity at castrate androgen levels. To identify the underlying mechanism, we investigated several signaling pathways but only found IL6-dependent changes in STAT3 signaling. Biochemical inhibition of STAT3 with the small-molecule inhibitor galiellalactone significantly reduced AR activity in several prostate and breast cancer cell lines. We confirmed the efficacy of galiellalactone in primary tissue slice cultures from radical prostatectomy samples. Galiellalactone significantly reduced the expression of the AR target genes PSA (P < 0.001), TMPRSS2 (P < 0.001), and FKBP5 (P = 0.003) in benign tissue cultures (n = 24). However, a high heterogeneity in the response of the malignant samples was discovered, and only a subset of tissue samples (4 out of 10) had decreased PSA expression upon galiellalactone treatment. Taken together, this finding demonstrates that targeting the IL6/STAT3 pathway with galiellalactone is a viable option to decrease AR activity in prostate tissue that may be applied in a personalized medicine approach.