Metastatic prostate cancer (mPC) comprises a spectrum of diverse phenotypes. However, the extent of inter- and intra-tumor heterogeneity is not established. Here we use digital spatial profiling ...(DSP) technology to quantitate transcript and protein abundance in spatially-distinct regions of mPCs. By assessing multiple discrete areas across multiple metastases, we find a high level of intra-patient homogeneity with respect to tumor phenotype. However, there are notable exceptions including tumors comprised of regions with high and low androgen receptor (AR) and neuroendocrine activity. While the vast majority of metastases examined are devoid of significant inflammatory infiltrates and lack PD1, PD-L1 and CTLA4, the B7-H3/CD276 immune checkpoint protein is highly expressed, particularly in mPCs with high AR activity. Our results demonstrate the utility of DSP for accurately classifying tumor phenotype, assessing tumor heterogeneity, and identifying aspects of tumor biology involving the immunological composition of metastases.
Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. However, current light-sheet microscopes ...have imposed constraints on the size, shape, number of specimens, and compatibility with various clearing protocols. Here we present a multi-immersion open-top light-sheet microscope that enables simple mounting of multiple specimens processed with a variety of clearing protocols, which will facilitate wide adoption by preclinical researchers and clinical laboratories. In particular, the open-top geometry provides unsurpassed versatility to interface with a wide range of accessory technologies in the future.
TMPRSS2 is an androgen-regulated cell-surface serine protease expressed predominantly in prostate epithelium. TMPRSS2 is expressed highly in localized high-grade prostate cancers and in the majority ...of human prostate cancer metastases. Through the generation of mouse models with a targeted deletion of Tmprss2, we demonstrate that the activity of this protease regulates cancer cell invasion and metastasis to distant organs. By screening combinatorial peptide libraries, we identified a spectrum of TMPRSS2 substrates that include pro-hepatocyte growth factor (HGF). HGF activated by TMPRSS2 promoted c-MET receptor tyrosine kinase signaling, and initiated a proinvasive epithelial-to-mesenchymal transition phenotype. Chemical library screens identified a potent bioavailable TMPRSS2 inhibitor that suppressed prostate cancer metastasis in vivo. Together, these findings provide a mechanistic link between androgen-regulated signaling programs and prostate cancer metastasis that operate via context-dependent interactions with extracellular constituents of the tumor microenvironment.
The vast majority of prostate cancer deaths are due to metastasis. Loss of TMPRSS2 activity dramatically attenuated the metastatic phenotype through mechanisms involving the HGF-c-MET axis. Therapeutic approaches directed toward inhibiting TMPRSS2 may reduce the incidence or progression of metastasis in patients with prostate cancer.
The significance of ERG in human prostate cancer is unclear because mouse prostate is resistant to ERG-mediated transformation. We determined that ERG activates the transcriptional program regulated ...by YAP1 of the Hippo signaling pathway and found that prostate-specific activation of either ERG or YAP1 in mice induces similar transcriptional changes and results in age-related prostate tumors. ERG binds to chromatin regions occupied by TEAD/YAP1 and transactivates Hippo target genes. In addition, in human luminal-type prostate cancer cells, ERG binds to the promoter of YAP1 and is necessary for YAP1 expression. These results provide direct genetic evidence of a causal role for ERG in prostate cancer and reveal a connection between ERG and the Hippo signaling pathway.
•Upregulation of ERG is sufficient to cause age-dependent prostate tumors in mice•ERG transcriptional output is similar to the output of YAP1 of the Hippo pathway•ERG is necessary for YAP1 expression in luminal-type human prostate cancer cells•Activation of YAP1 is sufficient to cause age-dependent prostate tumors in mice
Nguyen et al. show that ERG activates YAP1-regulated transcriptional output and that activation of ERG or YAP1 in mouse prostate induces similar transcriptional changes and age-related prostate tumors. Moreover, pharmacological inhibition of YAP1 suppresses growth of ERG-positive human prostate tumor xenografts.
Therapy for advanced prostate cancer centers on suppressing systemic androgens and blocking activation of the androgen receptor (AR). Despite anorchid serum androgen levels, nearly all patients ...develop castration-resistant disease. We hypothesized that ongoing steroidogenesis within prostate tumors and the maintenance of intratumoral androgens may contribute to castration-resistant growth. Using mass spectrometry and quantitative reverse transcription-PCR, we evaluated androgen levels and transcripts encoding steroidogenic enzymes in benign prostate tissue, untreated primary prostate cancer, metastases from patients with castration-resistant prostate cancer, and xenografts derived from castration-resistant metastases. Testosterone levels within metastases from anorchid men 0.74 ng/g; 95% confidence interval (95% CI), 0.59-0.89 were significantly higher than levels within primary prostate cancers from untreated eugonadal men (0.23 ng/g; 95% CI, 0.03-0.44; P < 0.0001). Compared with primary prostate tumors, castration-resistant metastases displayed alterations in genes encoding steroidogenic enzymes, including up-regulated expression of FASN, CYP17A1, HSD3B1, HSD17B3, CYP19A1, and UGT2B17 and down-regulated expression of SRD5A2 (P < 0.001 for all). Prostate cancer xenografts derived from castration-resistant tumors maintained similar intratumoral androgen levels when passaged in castrate compared with eugonadal animals. Metastatic prostate cancers from anorchid men express transcripts encoding androgen-synthesizing enzymes and maintain intratumoral androgens at concentrations capable of activating AR target genes and maintaining tumor cell survival. We conclude that intracrine steroidogenesis may permit tumors to circumvent low levels of circulating androgens. Maximal therapeutic efficacy in the treatment of castration-resistant prostate cancer will require novel agents capable of inhibiting intracrine steroidogenic pathways within the prostate tumor microenvironment.
Although androgens are depleted in castration resistant prostate cancer (CRPC), metastases still express nuclear androgen receptor (AR) and androgen regulated genes. We recently reported that ...C-terminal truncated constitutively active AR splice variants contribute to CRPC development. Since specific antibodies detecting all C-terminal truncated AR variants are not available, our aim was to develop an approach to assess the prevalence and function of AR variants in prostate cancer (PCa).
Using 2 antibodies against different regions of AR protein (N- or C-terminus), we successfully showed the existence of AR variant in the LuCaP 86.2 xenograft. To evaluate the prevalence of AR variants in human PCa tissue, we used this method on tissue microarrays including 50 primary PCa and 162 metastatic CRPC tissues. RT-PCR was used to confirm AR variants. We observed a significant decrease in nuclear C-terminal AR staining in CRPC but no difference between N- and C-terminal AR nuclear staining in primary PCa. The expression of the AR regulated proteins PSA and PSMA were marginally affected by the decrease in C-terminal staining in CRPC samples. These data suggest that there is an increase in the prevalence of AR variants in CRPC based on our ability to differentiate nuclear AR expression using N- and C-terminal AR antibodies. These findings were validated using RT-PCR. Importantly, the loss of C-terminal immunoreactivity and the identification of AR variants were different depending on the site of metastasis in the same patient.
We successfully developed a novel immunohistochemical approach which was used to ascertain the prevalence of AR variants in a large number of primary PCa and metastatic CRPC. Our results showed a snapshot of overall high frequency of C-terminal truncated AR splice variants and site specific AR loss in CRPC, which could have utility in stratifying patients for AR targeted therapeutics.
The neuroendocrine phenotype is associated with the development of metastatic castration-resistant prostate cancer (CRPC). Our objective was to characterize the molecular features of the ...neuroendocrine phenotype in CRPC.
Expression of chromogranin A (CHGA), synaptophysin (SYP), androgen receptor (AR), and prostate-specific antigen (PSA) was analyzed by IHC in 155 CRPC metastases from 50 patients and in 24 LuCaP prostate cancer patient-derived xenografts (PDX). Seventy-one of 155 metastases and the 24 LuCaP xenograft lines were analyzed by whole-genome microarrays. REST splicing was verified by PCR.
Coexpression of CHGA and SYP in >30% of cells was observed in 22 of 155 metastases (9 patients); 11 of the 22 metastases were AR(+)/PSA(+) (6 patients), 11/22 were AR-/PSA- (4 patients), and 4/24 LuCaP PDXs were AR(-)/PSA(-). By IHC, of the 71 metastases analyzed by whole-genome microarrays, 5 metastases were CHGA(+)/SYP(+)/AR(-), and 5 were CHGA(+)/SYP(+)/AR(+). Only CHGA(+)/SYP(+) metastases had a neuroendocrine transcript signature. The neuronal transcriptional regulator SRRM4 transcript was associated with the neuroendocrine signature in CHGA(+)/SYP(+) metastases and all CHGA(+)/SYP(+) LuCaP xenografts. In addition, expression of SRRM4 in LuCaP neuroendocrine xenografts correlated with a splice variant of REST that lacks the transcriptional repressor domain.
(i) Metastatic neuroendocrine status can be heterogeneous in the same patient, (ii) the CRPC neuroendocrine molecular phenotype can be defined by CHGA(+)/SYP(+) dual positivity, (iii) the neuroendocrine phenotype is not necessarily associated with the loss of AR activity, and (iv) the splicing of REST by SRRM4 could promote the neuroendocrine phenotype in CRPC.
The CYP17A1 inhibitor abiraterone markedly reduces androgen precursors and is thereby effective in castration-resistant prostate cancer (CRPC). However, abiraterone increases progesterone, which can ...activate certain mutant androgen receptors (AR) identified previously in flutamide-resistant tumors. Therefore, we sought to determine if CYP17A1 inhibitor treatment selects for progesterone-activated mutant ARs.
AR was examined by targeted sequencing in metastatic tumor biopsies from 18 patients with CRPC who were progressing on a CYP17A1 inhibitor (17 on abiraterone, 1 on ketoconazole), alone or in combination with dutasteride, and by whole-exome sequencing in residual tumor in one patient treated with neoadjuvant leuprolide plus abiraterone.
The progesterone-activated T878A-mutant AR was present at high allele frequency in 3 of the 18 CRPC cases. It was also present in one focus of resistant tumor in the neoadjuvant-treated patient, but not in a second clonally related resistant focus that instead had lost one copy of PTEN and both copies of CHD1. The T878A mutation appeared to be less common in the subset of patients with CRPC treated with abiraterone plus dutasteride, and transfection studies showed that dutasteride was a more potent direct antagonist of the T878A versus the wild-type AR.
These findings indicate that selection for tumor cells expressing progesterone-activated mutant ARs is a mechanism of resistance to CYP17A1 inhibition.
PDF
