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Recent studies in cancer patients and animal models demonstrate that intestinal microbiota influence the therapeutic efficacy of cancer treatments, including immune checkpoint inhibition. However, no studies to-date have investigated relationships between gastrointestinal microbiota composition and response to checkpoint inhibition in advanced metastatic castrate resistant prostate cancer (mCRPC). We performed 16S rRNA gene sequencing of fecal DNA from 23 individuals with mCRPC progressing on enzalutamide and just prior to treatment with anti-PD-1 (pembrolizumab) to determine whether certain features of the microbiome are associated with treatment response (defined as serum PSA decrease50% at any time on treatment or radiographic response per RECIST V.1.1). Global bacterial composition was similar between responders and non-responders, as assessed by multiple alpha and beta diversity metrics. However, certain bacterial taxa identified by sequencing across multiple 16S rRNA hypervariable regions were consistently associated with response, including the archetypal oral bacterium Streptococcus salivarius. Quantitative PCR (qPCR) of DNA extracts from fecal samples confirmed increased Streptococcus salivarius fecal levels in responders, whereas qPCR of oral swish DNA extracts showed no relationship between oral Streptococcus salivarius levels and response status. Contrary to previous reports in other cancer types, Akkermansia muciniphila levels were reduced in responder samples as assessed by both 16S rRNA sequencing and qPCR. We further analyzed our data in the context of a previously published "integrated index" describing bacteria associated with response and non-response to checkpoint inhibition. We found that the index was not reflective of response status in our cohort. Lastly, we demonstrate little change in the microbiome over time, and with pembrolizumab treatment. Our results suggest that the association between fecal microbiota and treatment response to immunotherapy may be unique to cancer type and/or previous treatment history.

Prostate cancer (PCa) remains a leading cause of cancer-related deaths in American men and treatment options for metastatic PCa are limited. There is a critical need to identify new mechanisms that contribute to PCa progression, that distinguish benign from lethal disease, and that have potential for therapeutic targeting. P2X4 belongs to the P2 purinergic receptor family that is commonly upregulated in cancer and is associated with poorer outcomes. We observed P2X4 protein expression primarily in epithelial cells of the prostate, a subset of CD66+ neutrophils, and most CD68+ macrophages. Our analysis of tissue microarrays representing 491 PCa cases demonstrated significantly elevated P2X4 expression in cancer- compared with benign-tissue spots, in prostatic intraepithelial neoplasia, and in PCa with ERG positivity or with PTEN loss. High-level P2X4 expression in benign tissues was likewise associated with the development of metastasis after radical prostatectomy. Treatment with the P2X4-specific agonist cytidine 5'-triphosphate (CTP) increased Transwell migration and invasion of PC3, DU145, and CWR22Rv1 PCa cells. The P2X4 antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) resulted in a dose-dependent decrease in viability of PC3, DU145, LNCaP, CWR22Rv1, TRAMP-C2, Myc-CaP, BMPC1, and BMPC2 cells and decreased DU145 cell migration and invasion. Knockdown of P2X4 attenuated growth, migration, and invasion of PCa cells. Finally, knockdown of P2X4 in Myc-CaP cells resulted in significantly attenuated subcutaneous allograft growth in FVB/NJ mice. Collectively, these data strongly support a role for the P2X4 purinergic receptor in PCa aggressiveness and identify P2X4 as a candidate for therapeutic targeting. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Prostate cancer (PCa) remains a leading cause of cancer-related deaths in American men and treatment options for metastatic PCa are limited. There is a critical need to identify new mechanisms that contribute to PCa progression, that distinguish benign from lethal disease, and that have potential for therapeutic targeting. P2X4 belongs to the P2 purinergic receptor family that is commonly upregulated in cancer and is associated with poorer outcomes. Herein, we report that the P2X4 purinergic receptor is overexpressed in PCa, associated with PCa metastasis, and a driver of tumor development in vivo. We observed P2X4 protein expression primarily in epithelial cells of the prostate, a subset of CD66+ neutrophils, and most CD68+ macrophages. Our analysis of tissue microarrays representing 491 PCa cases demonstrated significantly elevated P2X4 expression in cancer compared to benign tissue spots, in prostatic intraepithelial neoplasia, in cancer from White compared to Black men, and in PCa with ERG positivity or with PTEN loss. High P2X4 expression in benign tissues was likewise associated with the development of metastasis after radical prostatectomy. Treatment with P2X4-specific agonist CTP increased transwell migration and invasion of PC3, DU145, and CWR22Rv1 PCa cells. P2X4 antagonist 5-BDBD treatment resulted in a dose-dependent decrease in viability of PC3, DU145, LNCaP, CWR22Rv1, TRAMP-C2, Myc-CaP, BMPC1, and BMPC2 cells and decreased DU145 cell migration and invasion. Knockdown of P2X4 attenuated growth, migration, and invasion of PCa cells. Finally, knockdown of P2X4 in Myc-CaP cells resulted in significantly attenuated subcutaneous allograft growth in FVB/NJ mice. Collectively, these data strongly support a role for the P2X4 purinergic receptor in PCa aggressiveness and identifies P2X4 as a candidate for therapeutic targeting.

Background It is well known that the gastrointestinal (GI) microbiota can influence the metabolism, pharmacokinetics, and toxicity of cancer therapies. Conversely, the effect of cancer treatments on the composition of the GI microbiota is poorly understood. We hypothesized that oral androgen receptor axis-targeted therapies (ATT), including bicalutamide, enzalutamide, and abiraterone acetate, may be associated with compositional differences in the GI microbiota. Methods We profiled the fecal microbiota in a cross-sectional study of 30 patients that included healthy male volunteers and men with different clinical states of prostate cancer (i.e., localized, biochemically recurrent, and metastatic disease) using 16S rDNA amplicon sequencing. Functional inference of identified taxa was performed using PICRUSt. Results We report a significant difference in alpha diversity in GI microbiota among men with versus without a prostate cancer diagnosis. Further analysis identified significant compositional differences in the GI microbiota of men taking ATT, including a greater abundance of species previously linked to response to anti-PD-1 immunotherapy such as Akkermansia muciniphila and Ruminococcaceae spp. In functional analyses, we found an enriched representation of bacterial gene pathways involved in steroid biosynthesis and steroid hormone biosynthesis in the fecal microbiota of men taking oral ATT. Conclusions There are measurable differences in the GI microbiota of men receiving oral ATT. We speculate that oral hormonal therapies for prostate cancer may alter the GI microbiota, influence clinical responses to ATT, and/or potentially modulate the antitumor effects of future therapies including immunotherapy. Given our findings, larger, longitudinal studies are warranted.

The human microbiome may influence prostate cancer initiation and/or progression through both direct and indirect interactions. To date, the majority of studies have focused on direct interactions including the influence of prostate infections on prostate cancer risk and, more recently, on the composition of the urinary microbiome in relation to prostate cancer. Less well understood are indirect interactions of the microbiome with prostate cancer, such as the influence of the gastrointestinal or oral microbiota on pro- or anti-carcinogenic xenobiotic metabolism, and treatment response. We review the literature to date on direct and indirect interactions of the microbiome with prostate inflammation and prostate cancer. Emerging studies indicate that the microbiome can influence prostate inflammation in relation to benign prostate conditions such as prostatitis/chronic pelvic pain syndrome and benign prostatic hyperplasia, as well as in prostate cancer. We provide evidence that the human microbiome present at multiple anatomic sites (urinary tract, gastrointestinal tract, oral cavity, etc.) may play an important role in prostate health and disease. In health, the microbiome encourages homeostasis and helps educate the immune system. In dysbiosis, a systemic inflammatory state may be induced, predisposing remote anatomical sites to disease, including cancer. The microbiome’s ability to affect systemic hormone levels may also be important, particularly in a disease such as prostate cancer that is dually affected by estrogen and androgen levels. Due to the complexity of the potential interconnectedness between prostate cancer and the microbiome, it is vital to further explore and understand the relationships that are involved.

Mitochondria are critical regulators of energy production, metabolism and cell death. Warburg observed that in the presence of oxygen, many cancer cells take up abundant amounts of glucose, and excrete the carbon mainly as lactate. Warburg postulated that this aerobic glycolysis was essential and universal in cancer, and was caused by mitochondrial dysfunction. Yet, more recent findings suggest that aerobic glycolysis also occurs in rapidly dividing normal cells to support anabolic reactions needed for cell growth and replication. Furthermore, a number of metabolites generated by the tricarboxylic acid cycle and the electron transport chain are also used for macromolecular synthesis in proliferating cancer and normal cells. This suggests that an increase in mitochondrial activity, especially those functions related to anabolic metabolism, may be required for cancer development and progression. One mechanism to increase mitochondrial function is through mitochondrial biogenesis, which is partly regulated by regulating mitochondrial DNA (mtDNA) copy number. Alterations in mtDNA sequence and copy number have been implicated in various cancers. Since most prior measurements of mtDNA copy number have been performed using homogenates of complex tissues composed of multiple cell types, very little is known about cell type-specific mtDNA copy number heterogeneity. Here, we validated a quantitative in situ hybridization approach to study mtDNA levels at the single cell level in tissue sections. In hierarchically organized self-renewing tissues, higher levels of mtDNA were observed in stem/proliferative compartments than in differentiated compartments. Also, we found zonal heterogeneity of mtDNA levels in the liver that mirrors the known oxygen tension gradient. In the kidney, we observed markedly lower mtDNA levels in glomeruli and the collecting ducts compared to cortical proximal and distal tubules. This work provides visualization of mtDNA copy number in distinct cell populations in situ for the first time across normal mammalian tissues, and can serve as a foundation for quantifying cell type-specific mtDNA copy number dynamics in any relevant diseases, especially in cancers and their precursor lesions. Citation Format: JIAYU CHEN, Qizhi Zheng, Lauren B. Peiffer, Jessica L. Hicks, Michael C. Haffner, Avi Z. Rosenberg, Moshe Levi, Xiaoxin Wang, Busra Ozbek, Srinivasan Yegnasubramanian, Angelo M. De Marzo. An in situ hybridization assay reveals high mitochondrial DNA copy number in stem cell/proliferative cell compartments [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4790.