Androgen-dependent Prostate Cancer Research Articles

Interleukin-10 Induces Expression of Neuroendocrine Markers and PDL1 in Prostate Cancer Cells.

Interleukin-10 (IL10) is best studied for its inhibitory action on immune cells and ability to suppress an antitumour immune response. But IL10 also exerts direct effects on nonimmune cells such as prostate cancer epithelial cells. Elevated serum levels of IL10 observed in prostate and other cancer patients are associated with poor prognosis. After first-line androgen-deprivation therapy, prostate cancer patients are treated with androgen receptor antagonists such as enzalutamide to inhibit androgen-dependent prostate cancer cell growth. However, development of resistance inevitably occurs and this is associated with tumour differentiation to more aggressive forms such as a neuroendocrine phenotype characterized by expression of neuron specific enolase and synaptophysin. We found that treatment of prostate cancer cell lines in vitro with IL10 or enzalutamide induced markers of neuroendocrine differentiation and inhibited androgen receptor reporter activity. Both also upregulated the levels of PDL1, which could promote tumour survival in vivo through its interaction with the immune cell inhibitory receptor PD1 to suppress antitumour immunity. These findings suggest that IL10's direct action on prostate cancer cells could contribute to prostate cancer progression independent of IL10's suppression of host immune cells.

Chelerythrine suppresses proliferation and metastasis of human prostate cancer cells via modulating MMP/TIMP/NF-κB system.

Chelerythrine is a natural benzo[c]phenanthridine alkaloid found in many herbs and displays a wide range of antitumor activities. Here, the present study tested their effects on prostate cancer cells. The addition of chelerythrine can significantly inhibit the proliferation of androgen-independent prostate cancer DU145 and PC-3 cells at the concentration of 5 and 10μM, but not on androgen-dependent prostate cancer LNCaP cells as well as normal prostate epithelial cell line PrEC cells. Wound migration and transwell invasion assay showed the similar inhibitory effect of chelerythrine on the migration and invasion of DU145 and PC-3 cells in the same condition. Western blot analysis further confirmed that chelerythrine not only dramatically decreased MMP-2, MMP-9, and uPA protein expression, but also augmented the expression of their endogenous inhibitors (TIMP-1 and TIMP-2) and plasminogen activator inhibitors (PAI-1 and PAI-2) in both cancer cells. Meanwhile, NF-κB and AP-1 transcription factors were all suppressed as evidenced by the decline of p-p65, c-Fos, and c-Jun protein expression in both cells. Taken together, these findings suggested that chelerythrine could reduce the metastasis of androgen-independent prostate cancer cells via modulation of MMP/TIMP system and inactivation of NF-κB pathway.

Increased nuclear factor I/B expression in prostate cancer correlates with AR expression.

Most prostate cancers express androgen receptor (AR), and our previous studies have focused on identifying transcription factors that modify AR function. We have shown that nuclear factor I/B (NFIB) regulates AR activity in androgen-dependent prostate cancer cells in vitro. However, the status of NFIB in prostate cancer was unknown. We immunostained a tissue microarray including normal, hyperplastic, prostatic intraepithelial neoplasia, primary prostatic adenocarcinoma, and castration-resistant prostate cancer tissue samples for NFIB, AR, and synaptophysin, a marker of neuroendocrine differentiation. We interrogated publically available data sets in cBioPortal to correlate NFIB expression and AR and neuroendocrine prostate cancer (NEPCa) activity scores. We analyzed prostate cancer cell lines for NFIB expression via Western blot analysis and used nuclear and cytoplasmic fractionation to assess where NFIB is localized. We performed co-immunoprecipitation studies to determine if NFIB and AR interact. NFIB increased in the nucleus and cytoplasm of prostate cancer samples versus matched normal controls, independent of Gleason score. Similarly, cytoplasmic AR and synaptophysin increased in primary prostate cancer. We observed strong NFIB staining in primary small cell prostate cancer. The ratio of cytoplasmic-to-nuclear NFIB staining was predictive of earlier biochemical recurrence in prostate cancer, once adjusted for tumor margin status. Cytoplasmic AR was an independent predictor of biochemical recurrence. There was no statistically significant difference between NFIB and synaptophysin expression in primary and castration-resistant prostate cancer, but cytoplasmic AR expression was increased in castration-resistant samples. In primary prostate cancer, nuclear NFIB expression correlated with cytoplasmic NFIB and nuclear AR, while cytoplasmic NFIB correlated with synaptophysin, and nuclear and cytoplasmic AR. In castration-resistant prostate cancer samples, NFIB expression correlated positively with an AR activity score, and negatively with the NEPCa score. In prostate cancer cell lines, NFIB exists in several isoforms. We observed NFIB predominantly in the nuclear fraction of prostate cancer cells with increased cytoplasmic expression seen in castration-resistant cell lines. We observed an interaction between AR and NFIB through co-immunoprecipitation experiments. We have described the expression pattern of NFIB in primary and castration-resistant prostate cancer and its positive correlation with AR. We have also demonstrated AR interacts with NFIB.

Mechanisms of Androgen Receptor Agonist- and Antagonist-Mediated Cellular Senescence in Prostate Cancer.

The androgen receptor (AR) plays a leading role in the control of prostate cancer (PCa) growth. Interestingly, structurally different AR antagonists with distinct mechanisms of antagonism induce cell senescence, a mechanism that inhibits cell cycle progression, and thus seems to be a key cellular response for the treatment of PCa. Surprisingly, while physiological levels of androgens promote growth, supraphysiological androgen levels (SAL) inhibit PCa growth in an AR-dependent manner by inducing cell senescence in cancer cells. Thus, oppositional acting ligands, AR antagonists, and agonists are able to induce cellular senescence in PCa cells, as shown in cell culture model as well as ex vivo in patient tumor samples. This suggests a dual AR-signaling dependent on androgen levels that leads to the paradox of the rational to keep the AR constantly inactivated in order to treat PCa. These observations however opened the option to treat PCa patients with AR antagonists and/or with androgens at supraphysiological levels. The latter is currently used in clinical trials in so-called bipolar androgen therapy (BAT). Notably, cellular senescence is induced by AR antagonists or agonist in both androgen-dependent and castration-resistant PCa (CRPC). Pathway analysis suggests a crosstalk between AR and the non-receptor tyrosine kinase Src-Akt/PKB and the PI3K-mTOR-autophagy signaling in mediating AR-induced cellular senescence in PCa. In this review, we summarize the current knowledge of therapeutic induction and intracellular pathways of AR-mediated cellular senescence.

Stabilization of ADAM9 by N-\u03b1-acetyltransferase 10 protein contributes to promoting progression of androgen-independent prostate cancer

N-α-Acetyltransferase 10 protein (Naa10p) was reported to be an oncoprotein in androgen-dependent prostate cancer (PCa; ADPC) through binding and increasing transcriptional activity of the androgen receptor (AR). PCa usually progresses from an androgen-dependent to an androgen-independent stage, leading to an increase in the metastatic potential and an incurable malignancy. At present, the role of Naa10p in androgen-independent prostate cancer (AIPC) remains unclear. In this study, in silico and immunohistochemistry analyses showed that Naa10 transcripts or the Naa10p protein were more highly expressed in primary and metastatic PCa cancer tissues compared to adjacent normal tissues and non-metastatic cancer tissues, respectively. Knockdown and overexpression of Naa10p in AIPC cells (DU145 and PC-3M), respectively, led to decreased and increased cell clonogenic and invasive abilities in vitro as well as tumor growth and metastasis in AIPC xenografts. From the protease array screening, we identified a disintegrin and metalloprotease 9 (ADAM9) as a potential target of Naa10p, which was responsible for the Naa10p-induced invasion of AIPC cells. Naa10p can form a complex with ADAM9 to maintain ADAM9 protein stability and promote AIPC’s invasive ability which were independent of its acetyltransferase activity. In contrast to the Naa10p-ADAM9 axis, ADAM9 exerted positive feedback regulation on Naa10p to modulate progression of AIPC in vitro and in vivo. Taken together, for the first time, our results reveal a novel cross-talk between Naa10p and ADAM9 in regulating the progression of AIPC. Disruption of Naa10p–ADAM9 interactions may be a potential intervention for AIPC therapy.

3D-QSAR and Molecular Docking Studies on Design Anti-Prostate Cancer Curcumin Analogues.

Prostate cancer is one of the most common tumors in the world and the fifth leading cause of male cancer death. Although the treatment of localized androgen-dependent prostate cancer has been successful, the efficacy of androgen-independent metastatic disease is limited. Curcumin, a natural product, has been found to inhibit the proliferation of prostate cancer cells. To design curcumin analogs with higher biological activity and lower toxicity and side effects for the treatment of prostate cancer. In this study, the three dimensional-quantitative structure activity relationship (3DQSAR) and molecular docking studies were performed on 34 curcumin analogs as anti-prostate cancer compounds. We introduced OSIRIS Property Explorer to predict drug-related properties of newly designed compounds. The optimum CoMSIA model exhibited statistically significant results: the cross-validated correlation coefficient q2 is 0.540 and non-cross-validated R2 value is 0.984. The external predictive correlation coefficient Rext 2 is 0.792. The information of structure-activity relationship can be obtained from the CoMSIA contour maps. In addition, the molecular docking study of the compounds for 3ZK6 as the protein target revealed important interactions between active compounds and amino acids. Compound 28i may be a new type of anti-prostate cancer drug with higher biological activity and more promising development.

Role of nitric oxide-based immunotherapy in augmenting prostate cancer progression by targeting androgen receptor heterogeneity.

e17537 Background: A significant proportion of men with Prostate Cancer (PCa) develop castration resistant prostate cancer (CRPC) and do not respond to hormonal agents that decrease androgens. In trying to understand the causes of androgen resistance that develop in CRPC, it is considered most relevant to study the role of Androgen receptor (AR) in the development and progression of PCa from androgen dependent to androgen independent state. Recent studies have highlighted the significance of tumor microenvironment (TME) in regulation of PCa progression in addition to AR. A key molecule in the regulation of TME interactions is nitric oxide (NO). We have shown in our recent study, the critical association of NO with the TME in CRPC. However, the effects of NO to modulate the progression of PCa to CRPC with respect to AR still remains largely unexplored. Methods: 22RV1, LNCaP, LNCaPAPIPC(cells expressing no AR), and LNCaPshAR/pATK (cells expressing low AR), cells were used for the study. Cell proliferation was first assessed by MTT assay. The castrated SCID mice were grafted with 22RV1 cells and were treated with GSNO at the dosage of 10mg/kg/day IP. After treatment, animals were humanely sacrificing. Tumor RNA and proteins were analysed for markers that are important for PCa progression using qPCR, western blot and cytokine antibody array. Animal experiments were carried out in compliance with the IACUC of University of Miami. GraphPad Prism (GraphPad Software) was used for statistical analysis. Results: In addition to reducing the tumor burden, the expression of anti-inflammatory (M2) macrophages (CD206 and Arginase1) is decreased and that of the pro-inflammatory (M1) macrophage (iNOS) is increased in mice which received increased NO levels. Furthermore, to study the effects of NO on progression of PCa from androgen dependent to androgen independent stage, we characterized the LNCAP cell models with differential extent of AR knockdown (LNCaP, LNCaPshAR/pATK and LNCaPAPIPC) for the effects of increased NO levels. Results showed that NO had significant impact on cell proliferation on androgen dependent PCa cells however the effects were negligible in cells expressing low or no AR, suggesting that effects of NO on PCa cell proliferation are AR dependent. Conclusions: Our results suggest that during PCa progression, NO suppresses TAMs to target the TME in an AR dependent manner. Further studies are undergoing to establish the impacts of NO in PCa progression.

Zinc: The Wonder Drug for the Treatment of Carcinomas.

Evidence is evolving that support the relationship that all carcinomas exhibit the following important relationships: The malignant cells exhibit a significant decreased zinc compared to the normal cells. The higher zinc levels that exist in the normal cells are cytotoxic in the malignant cells. The decrease in zinc is due to the down regulation of the ZIP-family zinc uptake transporter. These cells are as "ZIP-deficient/decreased zinc" malignancies. This provides a target for a chemotherapy that can restore the high zinc levels that will manifest cytotoxic effects in the malignant cells. In order to achieve this, a vehicle that facilitates the uptake and accumulation of zinc in the ZIP-deficient cells is required. The zinc ionophore, clioquinol, exhibits the properties that will provide these requirements. This is demonstrated by the treatment of a patient with 3% Clioquinol Cream, which successfully suppressed the progression of androgen-dependent prostate cancer. This treatment should also be efficacious for pancreatic cancer, liver cancer, breast cancer, thyroid cancer, kidney cancer, stomach cancer, gall bladder cancer, and lung cancer; which are carcinomas that exhibit decreased zinc. Thus, it is appropriate to describe that "Zinc is the wonder drug for the treatment of carcinomas".

BAT1 Alters Migration, Invasion and Pro‐Inflammatory Cytokines in Prostate Cancer

Prostate cancer (PCa) is the most frequently diagnosed non‐cutaneous cancer and the second cause of cancer related deaths in men. Though PCa is often treated with radical prostatectomy and radiation therapy, 15%–30% of patients may undergo biochemical recurrence. The current biomarkers are inadequate to identify recurrence, thus there is a demand for the development of biomarkers that can identify recurrence. In preliminary data, HLA‐B associated transcript 1 (BAT1) was down‐regulated in patients with PCa recurrence when compared with non‐recurrent PCa patients. BAT1 has been identified as an anti‐inflammatory gene in other types of diseases. The role of BAT1 in PCa has not been identified. We hypothesize that BAT1 expression will affect cell migration, invasion, and inflammatory cytokine expression in vitro and in vivo. Our objective was to identify the role of BAT1 expression in PCa. We down‐regulated and upregulated BAT1 in androgen independent PC3 and androgen dependent 22RV1 prostate cancer cell lines using BAT1 small‐interfering RNA (siRNA) and complementary DNA (cDNA) lentiviral particle infections, respectively. In vitro assays were performed to measure BAT1 protein expression, migration, invasion and gene expression in prostate cancer cells. Western blot analysis showed that BAT1 protein expression was significantly down‐regulated in PC3 and 22RV1 siRNA transfected cells and overexpressed in PC3 and 22RV1 cDNA infected cells when compared to control. BAT1 down‐regulation significantly increased cell migration and invasion when compared to control. In contrast, BAT1 overexpression significantly decreased cell migration and invasion when compared to control. Real time PCR (RT‐PCR) showed changes in expression of pro‐inflammatory cytokines (TNF‐α and IL‐6) and genes that regulate cell adhesion and migration (MMP10, MMP13 and TIMPs) in BAT1 overexpressed cells when compared to BAT1 siRNA cells. In vivo studies were performed using severe combined immunodeficient (SCID) orthotopic mice injected with 22RV1 cells previously transfected with BAT1 short hairpin ribonucleic acid (shRNA) or BAT1 cDNA. The cells were injected directly into the prostate and allowed to form tumors for analysis. Immunohistochemistry analysis showed increases in the expression of TNF‐α, IL‐6 and MMP10 in tumors developed from BAT1 shRNA treated mice when compared to tumors developed from BAT1 cDNA treated mice. These results suggest that BAT1 expression alters inflammation through the modulation of TNF‐α and IL‐6 expression and can eventually promote migration and invasion through the expression of MMPs and TIMPs.Support or Funding InformationThis work is supported by the NIGMS‐RISE Program #R25GM061838

Isorhamnetin inhibited the proliferation and metastasis of androgen-independent prostate cancer cells by targeting the mitochondrion-dependent intrinsic apoptotic and PI3K/Akt/mTOR pathway.

The present study investigated the effects of Isorhamnetin on two types of prostate cancer cells (androgen-independent and androgen-dependent) and explored its possible mechanisms underlying such effects. Treatment with Isorhamnetin significantly inhibited cell growth and induced lactate dehydrogenase (LDH) release of androgen-independent DU145 and PC3 prostate cancer cells, but exhibited almost no toxicity effect on androgen-dependent LNCaP prostate cancer cell line or normal human prostate epithelial PrEC cells, which was achieved by the induction of apoptosis in a mitochondrion-dependent intrinsic apoptotic pathway. Furthermore, Isorhamnetin inhibited cell migration and invasion in concentration-dependent manners by enhancing mesenchymal−epithelial transition (MET) and inhibiting matrix metalloproteinase (MMP) 2 (MMP-2) and MMP-9 overexpression. In addition, Isorhamnetin also down-regulated the expression of phosphorylated PI3K (p-P13K), Akt (p-Akt), and mTOR (p-mTOR) proteins in both cancer cells, revealing Isorhamnetin to be a selective PI3K–Akt–mTOR pathway inhibitor. In summary, these findings propose that Isorhamnetin might be a novel therapeutic candidate for the treatment of androgen-independent prostate cancer.

Quantitative imaging of the receptor for advanced glycation end-products in prostate cancer

Current screening and monitoring of prostate cancer (PCa) is insufficient, producing inaccurate diagnoses. Presence of the receptor for advanced glycation end-products (RAGE) is associated with signature characteristics of PCa development such as cell proliferation, anchorage-independent growth, angiogenesis, migration, invasion, and poor patient survival. Therefore, we developed a preclinical multimodal imaging strategy targeted at RAGE to diagnose and monitor PCa. In this work, RAGE-targeted multimodal nanoparticles (64Cu-Cy5-G4-CML) were synthesized and rendered functional for nuclear and optical imaging using previously established methods. The probe's binding affinity and targeting specificity was assessed in androgen-dependent (LNCaP) and androgen-independent (DU145) prostate cancer cells using flow cytometry and confocal microscopy. In vivo PET-CT imaging was used to evaluate RAGE levels in DU145 and LNCaP xenograft models in mice. Then, tumors were excised post-imaging for histological staining and autoradiography to further assess RAGE levels and targeting efficiency of the tracer. Finally, RAGE levels from human PCa samples of varying Gleason Scores were evaluated using Western blot and immunohistochemical staining. PCa cell culture studies confirmed adequate RAGE-targeting with 64Cu-Cy5-G4-CML with KD between 360 and 540nM as measured by flow cytometry. In vivo PET-CT images of PCa xenografts revealed favorable kinetics, rapid blood clearance, and a non-homogenous, enhanced uptake in tumors, which varied based on cell type and tumor size with mean uptake between 0.5 and 1.4%ID/g. RAGE quantification of human samples confirmed increased RAGE uptake corresponding to increased Gleason scoring. Our study has shown that RAGE-targeted cancer imaging is feasible and could significantly impact PCa management.

REPRODUCTIVE FUNCTION AND ANTITUMOR ACTIVITY: DIFFERENT ROLES FOR THE HYPOTHALAMIC HORMONE GnRH

The decapeptide GnRH (Gonadotropin-Releasing Hormone), whose amino acidic sequence was discovered by Dr. A.V. Schally, was initially identified as the key hypothalamic hormone involved in the control of reproductive functions. GnRH, by binding to specific receptors (GnRH-R) at the pituitary level, stimulates the synthesis and secretion of the two gonadotropins (LH, luteinizing hormone and FSH, follicle stimulating hormone) and the downstream production of steroid hormones at the gonadal level. At present, these receptors represent the molecular targets of the standard pharmacological treatments for hormone-related tumors, such as androgen-dependent prostate cancer. Actually, chronic administration of synthetic GnRH agonists induces the desensitization of pituitary receptors and, subsequently, the suppression of testicular androgen production. The physiological role of GnRH in reproductive functions, and its regulation, represented a very important line of research for professor Martini and His colleagues. In the last three decades it has become increasingly clear that GnRH-R are expressed also in a wide range of tumors, both related and unrelated to the reproductive system; in particular GnRH-R are expressed in prostate cancers after development of resistance to androgen ablation therapy (castration resistant prostate cancer, CRPC), a tumor known to be refractory to standard chemotherapy. Activation of these receptors by means of GnRH agonists is associated with a significant antiproliferative/antimetastatic/antiangiogenic activity. These different biological effects at pituitary vs. prostate tissues are related to specific intracellular signal transduction pathways. Based on these observations, tumor GnRH-R are presently considered an effective molecular target for novel therapies (‘targeted’ therapies). In particular, GnRH-based bioconjugates, in which a standard cytotoxic drug is linked to a GnRH analog, have been developed. The rationale for this ‘targeted’ therapy is that the GnRH analog behaves as the targeting moiety by binding to GnRH-R in tumors, thus specifically delivering (targeting) the cytotoxic drug to tumor cells. At the level of tumor cells, the bioconjugate is internalized and degraded at the lysosomal level; in this way the anticancer drug is specifically released into the tumor cells to exert its cytotoxic effects, while sparing normal cells. In conclusion, GnRH-R are expressed not only at the pituitary level but also in a wide range of tumor tissues; these receptors are at present under investigation as an effective molecular target for the development of novel therapeutic strategies.

Mechanism of Anti-Cancer Activity of Curcumin on Androgen-Dependent and Androgen-Independent Prostate Cancer.

Prostate cancer (PCa) is a heterogeneous disease and ranked as the second leading cause of cancer-related deaths in males worldwide. The global burden of PCa keeps rising regardless of the emerging cutting-edge technologies for treatment and drug designation. There are a number of treatment options which are effectively treating localised and androgen-dependent PCa (ADPC) through hormonal and surgery treatments. However, over time, these cancerous cells progress to androgen-independent PCa (AIPC) which continuously grow despite hormone depletion. At this particular stage, androgen depletion therapy (ADT) is no longer effective as these cancerous cells are rendered hormone-insensitive and capable of growing in the absence of androgen. AIPC is a lethal type of disease which leads to poor prognosis and is a major contributor to PCa death rates. A natural product-derived compound, curcumin has been identified as a pleiotropic compound which capable of influencing and modulating a diverse range of molecular targets and signalling pathways in order to exhibit its medicinal properties. Due to such multi-targeted behaviour, its benefits are paramount in combating a wide range of diseases including inflammation and cancer disease. Curcumin exhibits anti-cancer properties by suppressing cancer cells growth and survival, inflammation, invasion, cell proliferation as well as possesses the ability to induce apoptosis in malignant cells. In this review, we investigate the mechanism of curcumin by modulating multiple signalling pathways such as androgen receptor (AR) signalling, activating protein-1 (AP-1), phosphatidylinositol 3-kinases/the serine/threonine kinase (PI3K/Akt/mTOR), wingless (Wnt)/ß-catenin signalling, and molecular targets including nuclear factor kappa-B (NF-κB), B-cell lymphoma 2 (Bcl-2) and cyclin D1 which are implicated in the development and progression of both types of PCa, ADPC and AIPC. In addition, the role of microRNAs and clinical trials on the anti-cancer effects of curcumin in PCa patients were also reviewed.

Stevia eupatoria and Stevia pilosa Extracts Inhibit the Proliferation and Migration of Prostate Cancer Cells.

Background and Objectives: Prostate cancer is the second most harmful disease in men worldwide and the number of cases is increasing. Therefore, new natural agents with anticancer potential should be examined and the response of existing therapeutic drugs must be enhanced. Stevia pilosa and Stevia eupatoria are two species that have been widely used in traditional medicine, but their effectiveness on cancer cells and their interaction with antineoplastic drugs have not been studied. The aim of this study was to evaluate the anticancer activity of Stevia pilosa methanolic root extract (SPME) and Stevia eupatoria methanolic root extract (SEME) and their effect, combined with enzalutamide, on prostate cancer cells. Materials and Methods: The study was conducted on a human fibroblast cell line, and on androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cell lines. The cell viability was evaluated using a Trypan Blue exclusion test for 48 h, and the migration by a wound-healing assay for 24, 48, and 72 h. Results: The results indicate that SPME and SEME were not cytotoxic at concentrations less than 1000 μg/mL in the human fibroblasts. SPME and SEME significantly reduced the viability and migration of prostate cancer cells in all concentrations evaluated. The antiproliferative effect of the Stevia extracts was higher in cancer cells than in normal cells. The enzalutamide decreased the cell viability in all concentrations tested (10–50 µM). The combination of the Stevia extracts and enzalutamide produced a greater effect on the inhibition of the proliferation and migration of cancer cells than the Stevia extracts alone, but not of the enzalutamide alone. Conclusion: The results indicate that SPME and SEME have an inhibitory effect on the viability and migration of prostate cancer cells and do not interfere with the enzalutamide anticancer effect. The data suggest that Stevia extracts may be a potential source of molecules for cancer treatment.

Targeting tumor-associated macrophage (TAM) mediated inhibition of T-cell migration in prostate cancer using epigenetic modifying agents.

166 Background: Cytotoxic T lymphocytes (CTLs) perform vital anti-tumor functions and are critical to the efficacy of many anticancer therapies. In prostate cancer, the characteristic paucity of activated CTLs within the tumor microenvironment (TME) may be a key factor in disease progression and likely underlies the limited role for immune checkpoint inhibitors (ICIs) in prostate cancer treatment. In this study, we utilized novel microfluidic technologies to evaluate whether TAMs may be driving the exclusion of T cells from the prostate TME and whether the immunosuppressive functions of TAMs could be modified by epigenetic modifying agents. Methods: Primary macrophages and autologous T cells were derived from peripheral blood samples of prostate cancer patients at the University of Wisconsin. Mono-, co-, and tri-culture systems of macrophages, T cells, and 22RV1 cells (androgen-dependent prostate cancer cell line) were cultured in 2D and 3D in microfluidic cell culture platforms. Culture systems were treated with the EZH2 inhibitors (EZH2i) DZNep or EPZ-6438 or left untreated. Macrophages were also treated with M1 (IFN-g) and M2 (IL-4) polarizing cytokines. Systems were analyzed for T cell migration as well as mRNA and protein expression in each cell population. Results: Autologous macrophages inhibited activated T cell migration towards tumor cells in a multi-cellular microscale TME. T cell migration was restored through treatment with EZH2i. Gene expression analysis identified that EZH2i altered macrophage gene expression in the unpolarized and M1/M2 polarized states. In particular, there was increased expression of genes involved in T cell recruitment/chemotaxis, including CXCL10, CXCL11, CXCL12, following EZH2i treatment. Conclusions: We used novel microfluidic technologies to model and analyze multicellular TMEs using primary, patient-derived cells. We demonstrate that TAM-mediated suppression of T cell migration is mediated, in part, through epigenetic pathways, which can be targeted with EZH2i. Treatment with EZH2i, alone or in combination other therapies such as ICIs, may enhance cytotoxic T cell migration and activity in primary prostate cancer.

MUC1-C regulates lineage plasticity driving progression to neuroendocrine prostate cancer

Neuroendocrine prostate cancer (NEPC) is an aggressive malignancy with no effective targeted therapies. The oncogenic MUC1-C protein is overexpressed in castration-resistant prostate cancer (CRPC) and NEPC, but its specific role is unknown. Here, we demonstrate that upregulation of MUC1-C in androgen-dependent PC cells suppresses androgen receptor (AR) axis signaling and induces the neural BRN2 transcription factor. MUC1-C activates a MYC→BRN2 pathway in association with induction of MYCN, EZH2 and NE differentiation markers (ASCL1, AURKA and SYP) linked to NEPC progression. Moreover, MUC1-C suppresses the p53 pathway, induces the Yamanaka pluripotency factors (OCT4, SOX2, KLF4 and MYC) and drives stemness. Targeting MUC1-C decreases PC self-renewal capacity and tumorigenicity, suggesting a potential therapeutic approach for CRPC and NEPC. In PC tissues, MUC1 expression associates with suppression of AR signaling and increases in BRN2 expression and NEPC score. These results highlight MUC1-C as a master effector of lineage plasticity driving progression to NEPC.

Pharmacological inhibition of androgen receptor expression induces cell death in prostate cancer cells.

The androgen receptor (AR) plays an important role in the pathogenesis and development of prostate cancer (PCa). Mostly, PCa progresses to androgen-independent PCa, which has activated AR signaling from androgen-dependent PCa. Thus, inhibition of AR signaling may be an important therapeutic target in androgen-dependent and castration-resistant PCa. In this study, we determined the anticancer effect of a newly found natural compound, sakurasosaponin (S-saponin), using androgen-dependent and castration-resistant PCa cell lines. S-saponin induces mitochondrial-mediated cell death in both androgen-dependent (LNCaP) and castration-resistant (22Rv1 and C4-2) PCa cells, via AR expression. S-saponin treatment induces a decrease in AR expression in a time- and dose-dependent manner and a potent decrease in the expression of its target genes, including prostate-specific antigen (PSA), transmembrane protease, serin 2 (TMPRSS2), and NK3 homeobox 1 (NKX3.1). Furthermore, S-saponin treatment decreases B-cell lymphoma-extra large (Bcl-xL) and mitochondrial membrane potential, thereby increasing the release of cytochrome c into the cytosol. Moreover, Bcl-xL inhibition and subsequent mitochondria-mediated cell death caused by S-saponin were reversed by Bcl-xL or AR overexpression. Interestingly, S-saponin-mediated cell death was significantly reduced by a reactive oxygen species (ROS) scavenger, N-acetylcystein. Animal xenograft experiments showed that S-saponin treatment significantly reduced tumor growth of AR-positive 22Rv1 xenografts but not AR-negative PC-3 xenografts. Taken together, for the first time, our results revealed that S-saponin induces mitochondrial-mediated cell death in androgen-dependent and castration-resistant cells through regulation of AR mechanisms, including downregulation of Bcl-xL expression and induction of ROS stress by decreasing mitochondrial membrane potential.

Oxidative stress and redox signaling in CRPC progression: therapeutic potential of clinically-tested Nrf2-activators.

Androgen deprivation therapy (ADT) is the mainstay regimen in patients with androgen-dependent prostate cancer (PCa). However, the selection of androgen-independent cancer cells leads to castrate resistant prostate cancer (CRPC). The aggressive phenotype of CRPC cells underscores the need to elucidate mechanisms and therapeutic strategies to suppress CRPC outgrowth. Despite ADT, the activation of androgen receptor (AR) transcription factor continues via crosstalk with parallel signaling pathways. Understanding of how these signaling cascades are initiated and amplified post-ADT is lacking. Hormone deprivation can increase oxidative stress and the resultant reactive oxygen species (ROS) may activate both AR and non-AR signaling. Moreover, ROS-induced inflammatory cytokines may further amplify these redox signaling pathways to augment AR function. However, clinical trials using ROS quenching small molecule antioxidants have not suppressed CRPC progression, suggesting that more potent and persistent suppression of redox signaling in CRPC cells will be needed. The transcription factor Nrf2 increases the expression of numerous antioxidant enzymes and downregulates the function of inflammatory transcription factors, e.g., nuclear factor kappa B. We documented that Nrf2 overexpression can suppress AR-mediated transcription in CRPC cell lines. Furthermore, two Nrf2 activating agents, sulforaphane (a phytochemical) and bardoxolone-methyl (a drug in clinical trial) suppress AR levels and sensitize CRPC cells to anti-androgens. These observations implicate the benefits of potent Nrf2-activators to suppress the lethal signaling cascades that lead to CRPC outgrowth. This review article will address the redox signaling networks that augment AR signaling during PCa progression to CRPC, and the possible utility of Nrf2-activating agents as an adjunct to ADT.

MicroRNA-337-3p suppresses cell viability, apoptosis, and autophagy by modulating PPARγ expression in androgen-dependent human prostate cancer

We proposed that autophagy activated through PPARγ in prostate cancer could protect cancer against androgen deprivation. Our MTT and western blotting assay results showed that mimics of miR-337-3p repressed autophagy flux under androgen deprivation, whereas inhibitors had the opposite effect. Neither mimics nor inhibitors influenced cell motility and invasion. miR-337-3p downregulated PPARγ expression in dual luciferase reporter assays and promoted AMPK phosphorylation. The overexpression of PPARγ or AMPK agonists partly diminished the adverse influence of mimics on proliferation and cell autophagy. In a mouse xenograft model, tumors transplanted with LNCap cells having a miR-337-3p deficiency had a significantly larger volume and lower rate of apoptosis than in controls after orchidectomy. Our findings showed that the negative effects of miR-337-3p on cell survival after androgen deprivation through PPARγ degeneration and modulated autophagy could serve as a novel therapeutic target for androgen-dependent prostate cancer and provide a strategy against resistance to anti-androgen therapy.

Bacterial steroid-17,20-desmolase is a taxonomically rare enzymatic pathway that converts prednisone to 1,4-androstanediene-3,11,17-trione, a metabolite that causes proliferation of prostate cancer cells

The adrenal gland has traditionally been viewed as a source of “weak androgens”; however, emerging evidence indicates 11-oxy-androgens of adrenal origin are metabolized in peripheral tissues to potent androgens. Also emerging is the role of gut bacteria in the conversion of C21 glucocorticoids to 11-oxygenated C19 androgens. Clostridium scindens ATCC 35,704 is a gut microbe capable of converting cortisol into 11-oxy-androgens by cleaving the side-chain. The desA and desB genes encode steroid-17,20-desmolase. Our prior study indicated that the urinary tract bacterium, Propionimicrobium lymphophilum ACS-093-V-SCH5 encodes desAB and converts cortisol to 11β-hydroxyandrostenedione. We wanted to determine how widespread this function occurs in the human microbiome. Phylogenetic and sequence similarity network analyses indicated that the steroid-17,20-desmolase pathway is taxonomically rare and located in gut and urogenital microbiomes. Two microbes from each of these niches, C. scindens and Propionimicrobium lymphophilum, respectively, were screened for activity against endogenous (cortisol, cortisone, and allotetrahydrocortisol) and exogenous (prednisone, prednisolone, dexamethasone, and 9-fluorocortisol) glucocorticoids. LC/MS analysis showed that both microbes were able to side-chain cleave all glucocorticoids, forming 11-oxy-androgens. Pure recombinant DesAB from C. scindens showed the highest activity against prednisone, a commonly prescribed glucocorticoid. In addition, 0.1 nM 1,4-androstadiene-3,11,17-trione, bacterial side-chain cleavage product of prednisone, showed significant proliferation relative to vehicle in androgen-dependent growth LNCaP prostate cancer cells after 24 h (2.3 fold; P < 0.01) and 72 h (1.6 fold; P < 0.01). Taken together, DesAB-expressing microbes may be an overlooked source of androgens in the body, potentially contributing to various disease states, such as prostate cancer.