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Infertility affects 10–15% of couples, with half attributed to male factors. An improved understanding of the cell‐type‐specific dysfunction contributing to male infertility is needed to improve available therapies; however, human testicular tissues are difficult to obtain for research purposes. To overcome this, researchers have begun to use human induced pluripotent stem cells (hiPSCs) to generate various testis‐specific cell types in vitro. Peritubular myoid cells (PTMs) are one such testicular cell type that serves a critical role in the human testis niche but, to date, have not been derived from hiPSCs. This study set forth to generate a molecular‐based differentiation method for deriving PTMs from hiPSCs, mirroring in vivo patterning factors. Whole transcriptome profiling and quantitative polymerase chain reaction (qPCR) show that this differentiation method is sufficient to derive cells with PTM‐like transcriptomes, including upregulation of hallmark PTM functional genes, secreted growth and matrix factors, smooth muscle, integrins, receptors, and antioxidants. Hierarchical clustering shows that they acquire transcriptomes similar to primary isolated PTMs, and immunostaining shows the acquisition of a smooth muscle phenotype. Overall, these hiPSC‐PTMs will allow in vitro study of patient‐specific PTM development and function in spermatogenesis and infertility. [ABSTRACT FROM AUTHOR]

Objectives: To investigate global changes in ureters at the transcriptional, translational and functional levels, both while stents are indwelling and after removal and recovery, and to study the effects of targeting pathways that play a potential role. Methods: Pig ureters were stented for varying amounts of time (48 h, 72 h, 14 days) and the impact on peristalsis, dilatation and hydronephrosis were assessed. RNAseq, proteomic, histological and smooth muscle (SM) function analyses were performed on ureteric and kidney tissues to assess changes induced by stenting and recovery. Pathway analysis was performed using Ingenuity Pathway Analysis software. To study the impact of possible interventions, the effects of erythropoeitin (EPO) and a Gli1 inhibitor were assessed. Results: Stenting triggers massive ureteric dilatation, aperistalsis and moderate hydronephrosis within 48 h. Pathways associated with obstruction, fibrosis and kidney injury were upregulated by stenting. Increased expression of GLI1, clusterin‐α (a kidney injury marker) and collagen 4A2 (a fibrosis marker) was found in stented vs contralateral unstented ureters. EPO did not improve peristalsis or contraction force but did decrease non‐purposeful spasming seen exclusively in stented ureters. Tamsulosin administration increased contractility but not rate of peristalsis in stented ureters. Conclusions: Ureters respond to stents similarly to how they respond to an obstruction, that is, with activation of pathways associated with hydronephrosis, fibrosis and kidney injury. This is driven by significant dilatation and associated ureteric SM dysfunction. EPO and tamsulosin induced mild favourable changes in SM physiology, suggesting that targeting specific pathways has potential to address stent‐induced complications. [ABSTRACT FROM AUTHOR]

Hip implants are a successful solution for osteoarthritis; however, some individuals with metal‐on‐metal (MoM) and metal‐on‐polyethylene (MoP) prosthetics develop adverse local tissue reactions (ALTRs). While MoM and MoP ALTRs are presumed to be delayed hypersensitivity reactions to corrosion products, MoM‐ and MoP‐associated ALTRs present with different histological characteristics. We compared MoM‐ and MoP‐associated ALTRs histopathology with cobalt and chromium levels in serum and synovial fluid. We analyzed the gene expression levels of leukocyte aggregates and synovial fluid chemokines/cytokines to resolve potential pathophysiologic differences. In addition, we classified ALTRs from 79 patients according to their leukocyte infiltrates as macrophage‐dominant, mixed, and lymphocyte‐dominant. Immune‐related transcript profiles from lymphocyte‐dominant MoM‐ and MoP‐associated ALTR patients with perivascular lymphocytic aggregates were similar. Cell signatures indicated predominantly macrophage, Th1 and Th2 lymphocytic infiltrate, with strong exhausted CD8+ signature, and low Th17 and B cell, relative to healthy lymph nodes. Lymphocyte‐dominant ALTR‐associated synovial fluid contained higher levels of induced protein 10 (IP‐10), interleukin‐1 receptor antagonist (IL‐1RN), IL‐8, IL‐6, IL‐16, macrophage inflammatory protein 1 (MIP‐1α), IL‐18, MCP‐2, and lower cell‐attracting chemokine levels, when compared with prosthetic revisions lacking ALTRs. In addition, the higher levels of IP‐10, IL‐8, IL‐6, MIP‐1α, and MCP‐2 were observed within the synovial fluid of the lymphocyte‐dominant ALTRs relative to the macrophage‐dominant ALTRs. Not all cytokines/chemokines were detected in the perivascular aggregate transcripts, suggesting the existence of other sources in the affected synovia. Our results support the hypothesis of common hypersensitivity pathogenesis in lymphocyte‐dominant MoM and MoP ALTRs. The exhausted lymphocyte signature indicates chronic processes and an impaired immune response, although the cause of the persistent T‐cell activation remains unclear. The cytokine/chemokine signature of lymphocyte‐dominant‐associated ATLRs may be of utility for diagnosing this more aggressive pathogenesis. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) progression is driven by androgen receptor (AR) signaling. Unfortunately, androgen‐deprivation therapy and the use of even more potent AR pathway inhibitors (ARPIs) cannot bring about a cure. ARPI resistance (ie, castration‐resistant PCa, CRPC) will inevitably develop. Previously, we demonstrated that GRB10 is an AR transcriptionally repressed gene that functionally contributes to CRPC development and ARPI resistance. GRB10 expression is elevated prior to CRPC development in our patient‐derived xenograft models and is significantly upregulated in clinical CRPC samples. Here, we analyzed transcriptomic data from GRB10 knockdown in PCa cells and found that AR signaling is downregulated. While the mRNA expression of AR target genes decreased upon GRB10 knockdown, AR expression was not affected at the mRNA or protein level. We further found that phosphorylation of AR serine 81 (S81), which is critical for AR transcriptional activity, is decreased by GRB10 knockdown and increased by its overexpression. Luciferase assay using GRB10‐knockdown cells also indicate reduced AR activity. Immunoprecipitation coupled with mass spectrometry revealed an interaction between GRB10 and the PP2A complex, which is a known phosphatase of AR. Further validations and analyses showed that GRB10 binds to the PP2Ac catalytic subunit with its PH domain. Mechanistically, GRB10 knockdown increased PP2Ac protein stability, which in turn decreased AR S81 phosphorylation and reduced AR activity. Our findings indicate a reciprocal feedback between GRB10 and AR signaling, implying the importance of GRB10 in PCa progression. What's new? Androgen receptor (AR) signaling is the dominant driver of AR‐dependent castration‐resistant prostate cancer (CRPC) development and progression, even with continuous androgen deprivation therapy. The mechanisms causing the reactivation of AR signaling in CRPC remain unclear. Here, the authors demonstrate that the adaptor protein GRB10 sustains AR activity in CRPC. Data from high‐throughput proteomics analysis indicate that the reported tumor suppressor PP2A is a mediator of AR modulation by GRB10. GRB10 interacts with PP2A and promotes its degradation, thereby protecting phosphorylated AR from dephosphorylation and sustaining active AR signaling. Inhibiting GRB10 therapeutically could potentially improve CRPC management. [ABSTRACT FROM AUTHOR]

A subset of basal cell carcinomas (BCCs) are directly derived from hair follicles (HFs). In some respects, HFs can be defined as 'ordered' skin appendage growths, while BCCs can be regarded as 'disordered' skin appendage growths. The aim of the present study was to examine HFs and BCCs to define the expression of common and unique signaling pathways in each skin appendage. Human nodular BCCs, along with HFs and non-follicular skin epithelium from normal individuals, were examined using microarrays, qPCR, and immunohistochemistry. Subsequently, BCC cells and root sheath keratinocyte cells from HFs were cultured and treated with Notch signaling peptide Jagged1 (JAG1). Gene expression, protein levels, and cell apoptosis susceptibility were assessed using qPCR, immunoblotting, and flow cytometry, respectively. Specific molecular mechanisms were found to be involved in the process of cell self-renewal in the HFs and BCCs, including Notch and Hedgehog signaling pathways. However, several key Notch signaling factors showed significant differential expression in BCCs compared with HFs. Stimulating Notch signaling with JAG1 induced apoptosis of BCC cells by increasing Fas ligand expression and downstream caspase-8 activation. The present study showed that Notch signaling pathway activity is suppressed in BCCs, and is highly expressed in HFs. Elements of the Notch pathway could, therefore, represent targets for the treatment of BCCs and potentially in hair follicle engineering. [ABSTRACT FROM AUTHOR]

Motivation: Successful development and application of precision oncology approaches require robust elucidation of the genomic landscape of a patient's cancer and, ideally, the ability to monitor therapy-induced genomic changes in the tumour in an inexpensive and minimally invasive manner. Thanks to recent advances in sequencing technologies, 'liquid biopsy', the sampling of patient's bodily fluids such as blood and urine, is considered as one of the most promising approaches to achieve this goal. In many cancer patients, and especially those with advanced metastatic disease, deep sequencing of circulating cell free DNA (cfDNA) obtained from patient's blood yields a mixture of reads originating from the normal DNA and from multiple tumour subclones--called circulating tumour DNA or ctDNA. The ctDNA/cfDNA ratio as well as the proportion of ctDNA originating from specific tumour subclones depend on multiple factors, making comprehensive detection of mutations difficult, especially at early stages of cancer. Furthermore, sensitive and accurate detection of single nucleotide variants (SNVs) and indels from cfDNA is constrained by several factors such as the sequencing errors and PCR artifacts, and mapping errors related to repeat regions within the genome. In this article, we introduce SiNVICT, a computational method that increases the sensitivity and specificity of SNV and indel detection at very low variant allele frequencies. SiNVICT has the capability to handle multiple sequencing platforms with different error properties; it minimizes false positives resulting from mapping errors and other technology specific artifacts including strand bias and low base quality at read ends. SiNVICT also has the capability to perform time-series analysis, where samples from a patient sequenced at multiple time points are jointly examined to report locations of interest where there is a possibility that certain clones were wiped out by some treatment while some subclones gained selective advantage. Results: We tested SiNVICT on simulated data as well as prostate cancer cell lines and cfDNA obtained from castration-resistant prostate cancer patients. On both simulated and biological data, SiNVICT was able to detect SNVs and indels with variant allele percentages as low as 0.5%. The lowest amounts of total DNA used for the biological data where SNVs and indels could be detected with very high sensitivity were 2.5 ng on the Ion Torrent platform and 10 ng on Illumina. With increased sequencing and mapping accuracy, SiNVICT might be utilized in clinical settings, making it possible to track the progress of point mutations and indels that are associated with resistance to cancer therapies and provide patients personalized treatment. We also compared SiNVICT with other popular SNV callers such as MuTect, VarScan2 and Freebayes. Our results show that SiNVICT performs better than these tools in most cases and allows further data exploration such as time-series analysis on cfDNA sequencing data. [ABSTRACT FROM AUTHOR]

We conducted a microarray study to discover gene expression patterns associated with a lack of melanogenesis in nonpigmented hair follicles (HF) by microarray. Pigmented and non-pigmented HFs were collected and micro-dissected into the hair bulb (HB) and the upper hair sheaths (HS) including the bulge region. In comparison to pigmented HS and HBs, nucleotide excision repair (NER) family genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, XPA, NTPBP, HCNP, DDB2 and POLH exhibited statistically significantly lower expression in non- pigmented HS and HBs. Quantitative PCR verified microarray data and identified ERCC3 as highly differentially expressed. Immunohistochemistry confirmed ERCC3 expression in HF melanocytes. A reduction in ERCC3 by siRNA interference in human melanocytes in vitro reduced their tyrosinase production ability. Our results suggest that loss of NER gene function is associated with a loss of melanin production capacity. This may be due to reduced gene transcription and/or reduced DNA repair in melanocytes which may eventually lead to cell death. These results provide novel information with regard to melanogenesis and its regulation. [ABSTRACT FROM AUTHOR]

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer. It develops mainly via NE transdifferentiation of prostate adenocarcinoma in response to androgen receptor (AR)-inhibition therapy. The study of NEPC development has been hampered by a lack of clinically relevant models. We previously established a unique and first-in-field patient-derived xenograft (PDX) model of adenocarcinoma (LTL331)-to-NEPC (LTL331R) transdifferentiation. In this study, we applied conditional reprogramming (CR) culture to establish a LTL331 PDX-derived cancer cell line named LTL331_CR_Cell. These cells retain the same genomic mutations as the LTL331 parental tumor. They can be continuously propagated in vitro and can be genetically manipulated. Androgen deprivation treatment on LTL331_CR_Cells had no effect on cell proliferation. Transcriptomic analyses comparing the LTL331_CR_Cell to its parental tumor revealed a profound downregulation of the androgen response pathway and an upregulation of stem and basal cell marker genes. The transcriptome of LTL331_CR_Cells partially resembles that of post-castrated LTL331 xenografts in mice. Notably, when grafted under the renal capsules of male NOD/SCID mice, LTL331_CR_Cells spontaneously gave rise to NEPC tumors. This is evidenced by the histological expression of the NE marker CD56 and the loss of adenocarcinoma markers such as PSA. Transcriptomic analyses of the newly developed NEPC tumors further demonstrate marked enrichment of NEPC signature genes and loss of AR signaling genes. This study provides a novel research tool derived from a unique PDX model. It allows for the investigation of mechanisms underlying NEPC development by enabling gene manipulations ex vivo and subsequent functional evaluations in vivo. [ABSTRACT FROM AUTHOR]

Well-differentiated papillary mesothelioma (WDPM) is an uncommon mesothelial proliferation that is most commonly encountered as an incidental finding in the peritoneal cavity. There is controversy in the literature about whether WDPM is a neoplasm or a reactive process and, if neoplastic, whether it is a variant or precursor of epithelial malignant mesothelioma or is a different entity. Using whole exome sequencing of five WDPMs of the peritoneum, we have identified distinct mutations in EHD1, ATM, FBXO10, SH2D2A, CDH5, MAGED1, and TP73 shared by WDPM cases but not reported in malignant mesotheliomas. Furthermore, we show that WDPM is strongly enriched with C > A transversion substitution mutations, a pattern that is also not found in malignant mesotheliomas. The WDPMs lacked the alterations involving BAP1, SETD2, NF2, CDKN2A/B, LASTS1/2, PBRM1, and SMARCC1 that are frequently found in malignant mesotheliomas. We conclude that WDPMs are neoplasms that are genetically distinct from malignant mesotheliomas and, based on observed mutations, do not appear to be precursors of malignant mesotheliomas. [ABSTRACT FROM AUTHOR]

Background: Malignant peritoneal mesothelioma (PeM) is a rare and fatal cancer that originates from the peritoneal lining of the abdomen. Standard treatment of PeM is limited to cytoreductive surgery and/or chemotherapy, and no effective targeted therapies for PeM exist. Some immune checkpoint inhibitor studies of mesothelioma have found positivity to be associated with a worse prognosis. Methods: To search for novel therapeutic targets for PeM, we performed a comprehensive integrative multi-omics analysis of the genome, transcriptome, and proteome of 19 treatment-naïve PeM, and in particular, we examined BAP1 mutation and copy number status and its relationship to immune checkpoint inhibitor activation. Results: We found that PeM could be divided into tumors with an inflammatory tumor microenvironment and those without and that this distinction correlated with haploinsufficiency of BAP1. To further investigate the role of BAP1, we used our recently developed cancer driver gene prioritization algorithm, HIT'nDRIVE, and observed that PeM with BAP1 haploinsufficiency form a distinct molecular subtype characterized by distinct gene expression patterns of chromatin remodeling, DNA repair pathways, and immune checkpoint receptor activation. We demonstrate that this subtype is correlated with an inflammatory tumor microenvironment and thus is a candidate for immune checkpoint blockade therapies. Conclusions: Our findings reveal BAP1 to be a potential, easily trackable prognostic and predictive biomarker for PeM immunotherapy that refines PeM disease classification. BAP1 stratification may improve drug response rates in ongoing phases I and II clinical trials exploring the use of immune checkpoint blockade therapies in PeM in which BAP1 status is not considered. This integrated molecular characterization provides a comprehensive foundation for improved management of a subset of PeM patients. [ABSTRACT FROM AUTHOR]

Standardized and reproducible preclinical models that recapitulate the dynamics of prostate cancer are urgently needed. We established a bank of transplantable patient-derived prostate cancer xenografts that capture the biologic and molecular heterogeneity currently confounding prognostication and therapy development. Xenografts preserved the histopathology, genome architecture, and global gene expression of donor tumors. Moreover, their aggressiveness matched patient observations, and their response to androgen withdrawal correlated with tumor subtype. The panel includes the first xenografts generated from needle biopsy tissue obtained at diagnosis. This advance was exploited to generate independent xenografts from different sites of a primary site, enabling functional dissection of tumor heterogeneity. Prolonged exposure of adenocarcinoma xenografts to androgen withdrawal led to castration-resistant prostate cancer, including the first-in-field model of complete transdifferentiation into lethal neuroendocrine prostate cancer. Further analysis of this model supports the hypothesis that neuroendocrine prostate cancer can evolve directly from adenocarcinoma via an adaptive response and yielded a set of genes potentially involved in neuroendocrine transdifferentiation. We predict that these next-generation models will be transformative for advancing mechanistic understanding of disease progression, response to therapy, and personalized oncology. [ABSTRACT FROM AUTHOR]

Background: Protein-truncating mutations in the titin gene are associated with increased risk of atrial fibrillation. However, little is known about the underlying pathophysiology., Methods: We identified a heterozygous titin truncating variant (TTNtv) in a patient with unexplained early onset atrial fibrillation and normal ventricular function. We generated patient-specific atrial- and ventricular-like induced pluripotent stem cell-derived cardiomyocytes and engineered heart tissue to evaluate the impact of the TTNtv on electrophysiology, sarcomere structure, contractility, and gene expression., Results: We demonstrate that the TTNtv increases susceptibility to pacing-induced arrhythmia, promotes sarcomere disorganization, and reduces contractile force in atrial induced pluripotent stem cell-derived cardiomyocytes compared with their CRISPR/Cas9-corrected isogenic controls. In ventricular induced pluripotent stem cell-derived cardiomyocytes, this variant was associated with abnormal electrophysiology and sarcomere organization without a reduction in contractile force compared with their isogenic controls. RNA-sequencing revealed an upregulation of cell adhesion and extracellular matrix genes in the presence of the TTNtv for both atrial and ventricular engineered heart tissues., Conclusions: In a patient with unexplained atrial fibrillation, induced pluripotent stem cell-derived cardiomyocytes with a TTNtv showed structural and electrophysiological abnormalities in both atrial and ventricular models, while only atrial engineered heart tissues demonstrated reduced contractility. The observed chamber-specific effect suggests that structural disorganization and reduced contractile function may be associated with atrial myopathy in the presence of truncated titin.

Biomolecular condensates organize cellular environments and regulate key processes such as transcription. We previously showed that full-length androgen receptor (AR-FL), a major oncogenic driver in prostate cancer (PCa), forms nuclear condensates upon androgen stimulation in androgen-sensitive PCa cells. Disrupting these condensates impairs AR-FL transcriptional activity, highlighting their functional importance. However, resistance to androgen deprivation therapy often leads to castration-resistant prostate cancer (CRPC), driven by constitutively active splice variants like AR variant 7 (AR-V7). The mechanisms underlying AR-V7's role in CRPC remain unclear. In this study, we characterized the condensate-forming ability of AR-V7 and compared its phase behavior with AR-FL across a spectrum of PCa models and in vitro conditions. Our findings indicate that cellular context can influence AR-V7's condensate-forming capacity. Unlike AR-FL, AR-V7 spontaneously forms condensates in the absence of androgen stimulation and functions independently of AR-FL in CRPC models. However, AR-V7 requires a higher concentration to form condensates, both in cellular contexts and in vitro . We further reveal that AR-V7 drives transcription via both condensate-dependent and condensate-independent mechanisms. Using an AR-V7 mutant incapable of forming condensates, while retaining nuclear localization and DNA-binding ability, we reveal that the condensate-dependent regime activates part of the oncogenic KRAS pathway in CRPC models. Genes under this condensate-dependent regime were found to harbor significantly higher numbers of AR-binding sites and exhibited boosted expression in response to AR-V7. These findings uncover a previously unrecognized role of AR-V7 condensate formation in driving oncogenic transcriptional programs and shed light on its unique contribution to CRPC progression., Highlights: AR-V7 condensates form independently of both androgens and AR-FL in CRPC models.AR-V7 mediates condensate-dependent and independent transcriptionCondensate-dependent transcription enables boosted expression of oncogenic KRAS genesCondensate-dependent genes exhibit an exponential increase in expression, with a higher number of AR binding sites potentially playing a key role in their reliance on condensate formation.

Treatment-induced tumor dormancy is a state in cancer progression where residual disease is present but remains asymptomatic. Dormant cancer cells are treatment-resistant and responsible for cancer recurrence and metastasis. Prostate cancer treated with androgen-deprivation therapy (ADT) often enters a dormant state. ADT-induced prostate cancer dormancy remains poorly understood due to the challenge in acquiring clinical dormant prostate cancer cells and the lack of representative models. In this study, we aimed to develop clinically relevant models for studying ADT-induced prostate cancer dormancy. Dormant prostate cancer models were established by castrating mice bearing patient-derived xenografts (PDX) of hormonal naïve or sensitive prostate cancer. Dormancy status and tumor relapse were monitored and evaluated. Paired pre- and postcastration (dormant) PDX tissues were subjected to morphologic and transcriptome profiling analyses. As a result, we established eleven ADT-induced dormant prostate cancer models that closely mimicked the clinical courses of ADT-treated prostate cancer. We identified two ADT-induced dormancy subtypes that differed in morphology, gene expression, and relapse rates. We discovered transcriptomic differences in precastration PDXs that predisposed the dormancy response to ADT. We further developed a dormancy subtype-based, predisposed gene signature that was significantly associated with ADT response in hormonal naïve prostate cancer and clinical outcome in castration-resistant prostate cancer treated with ADT or androgen-receptor pathway inhibitors., Implications: We have established highly clinically relevant PDXs of ADT-induced dormant prostate cancer and identified two dormancy subtypes, leading to the development of a novel predicative gene signature that allows robust risk stratification of patients with prostate cancer to ADT or androgen-receptor pathway inhibitors., (©2022 The Authors; Published by the American Association for Cancer Research.)

Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of whole-exome sequencing, RNA sequencing, and mass spectrometry-based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies. SIGNIFICANCE: These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets., (©2021 American Association for Cancer Research.)

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer arising mostly from adenocarcinoma via neuroendocrine transdifferentiation following androgen deprivation therapy. Mechanisms contributing to both NEPC development and its aggressiveness remain elusive. In light of the fact that hyperchromatic nuclei are a distinguishing histopathologic feature of NEPC, we utilized transcriptomic analyses of our patient-derived xenograft (PDX) models, multiple clinical cohorts, and genetically engineered mouse models to identify 36 heterochromatin-related genes that are significantly enriched in NEPC. Longitudinal analysis using our unique, first-in-field PDX model of adenocarcinoma-to-NEPC transdifferentiation revealed that, among those 36 heterochromatin-related genes, heterochromatin protein 1α (HP1α) expression increased early and steadily during NEPC development and remained elevated in the developed NEPC tumor. Its elevated expression was further confirmed in multiple PDX and clinical NEPC samples. HP1α knockdown in the NCI-H660 NEPC cell line inhibited proliferation, ablated colony formation, and induced apoptotic cell death, ultimately leading to tumor growth arrest. Its ectopic expression significantly promoted NE transdifferentiation in adenocarcinoma cells subjected to androgen deprivation treatment. Mechanistically, HP1α reduced expression of androgen receptor and RE1 silencing transcription factor and enriched the repressive trimethylated histone H3 at Lys9 mark on their respective gene promoters. These observations indicate a novel mechanism underlying NEPC development mediated by abnormally expressed heterochromatin genes, with HP1α as an early functional mediator and a potential therapeutic target for NEPC prevention and management. Significance: Heterochromatin proteins play a fundamental role in NEPC, illuminating new therapeutic targets for this aggressive disease. Cancer Res; 78(10); 2691-704. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Carcinoma of the prostate is the most common cancer in men. Treatment of aggressive prostate cancer involves a regiment of radical prostectomy, radiation therapy, chemotherapy and hormonal therapy. Despite significant improvements in the last decade, the treatment of prostate cancer remains unsatisfactory, because a significant fraction of prostate cancers develop resistance to multiple treatments and become incurable. This prompts an urgent need to investigate the molecular mechanisms underlying the evolution of therapy-induced resistance of prostate cancer either in the form of castration-resistant prostate cancer (CRPC) or transdifferentiated neuroendocrine prostate cancer (NEPC). By analyzing micro-RNA expression profiles in a set of patient-derived prostate cancer xenograft tumor lines, we identified miR-100-5p as one of the key molecular components in the initiation and evolution of androgen ablation therapy resistance in prostate cancer. In vitro results showed that miR-100-5p is required for hormone-independent survival and proliferation of prostate cancer cells post androgen ablation. In Silico target predictions revealed that miR-100-5p target genes are involved in key aspects of cancer progression, and are associated with clinical outcome. Our results suggest that mir-100-5p is a possible therapeutic target involved in prostate cancer progression and relapse post androgen ablation therapy.

To avoid over- or under-treatment of primary prostate tumours, there is a critical need for molecular signatures to discriminate indolent from aggressive, lethal disease. Reprogrammed energy metabolism is an important hallmark of cancer, and abnormal metabolic characteristics of cancers have been implicated as potential diagnostic/prognostic signatures. While genomic and transcriptomic heterogeneity of prostate cancer is well documented and associated with tumour progression, less is known about metabolic heterogeneity of the disease. Using a panel of high fidelity patient-derived xenograft (PDX) models derived from hormone-naïve prostate cancer, we demonstrated heterogeneity of expression of genes involved in cellular energetics and macromolecular biosynthesis. Such heterogeneity was also observed in clinical, treatment-naïve prostate cancers by analyzing the transcriptome sequencing data. Importantly, a metabolic gene signature of increased one-carbon metabolism or decreased proline degradation was identified to be associated with significantly decreased biochemical disease-free patient survival. These results suggest that metabolic heterogeneity of hormone-naïve prostate cancer is of biological and clinical importance and motivate further studies to determine the heterogeneity in metabolic flux in the disease that may lead to identification of new signatures for tumour/patient stratification and the development of new strategies and targets for therapy of prostate cancer.

Purpose: Enzalutamide resistance has emerged as a major problem in the management of castration-resistant prostate cancer (CRPC). Research on therapy resistance of CRPCs has primarily focused on the androgen receptor pathway. In contrast, there is limited information on antiapoptotic mechanisms that may facilitate the treatment resistance. The inhibitor of apoptosis proteins (IAP) family is well recognized for its role in promoting treatment resistance of cancers by inhibiting drug-induced apoptosis. Here, we examined whether BIRC6, an IAP family member, has a role in enzalutamide resistance of CRPCs and could provide a therapeutic target for enzalutamide-resistant CRPC. Experimental Design: Use of enzalutamide-resistant CRPC models: (i) the transplantable, first high-fidelity LTL-313BR patient-derived enzalutamide-resistant CRPC tissue xenograft line showing primary enzalutamide resistance, (ii) MR42D and MR49F CRPC cells/xenografts showing acquired enzalutamide resistance. Specific BIRC6 downregulation in these models was produced using a BIRC6 -targeting antisense oligonucleotide (ASO-6w2). Gene expression was determined by qRT-PCR and gene expression profiling. Molecular pathways associated with growth inhibition were assessed via gene enrichment analysis. Results: Of eight IAPs examined, BIRC6 was the only one showing elevated expression in both enzalutamide-resistant CRPC models. Treatment with ASO-6w2 markedly suppressed growth of LTL-313BR xenografts and increased tumor apoptosis without inducing major host toxicity. Pathway enrichment analysis indicated that GPCR and matrisome signaling were the most significantly altered pathways. Furthermore, ASO-6w2 inhibited expression of prosurvival genes that were upregulated in the LTL-313BR line. Conclusions: BIRC6 targeting inhibited the growth of enzalutamide-resistant CRPC models and may represent a new option for clinical treatment of advanced, enzalutamide-resistant prostate cancer. Clin Cancer Res; 23(6); 1542-51. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies., Competing Interests: The authors have no conflicts of interest to report.

Optimal animal models of muscle invasive bladder cancer (MIBC) are necessary to overcome the current lack of novel targeted therapies for this malignancy. Here we report on the establishment and characterization of patient-derived primary xenografts (PDX). Patient tumors were grafted under the renal capsule of mice and subsequently transplanted over multiple generations. Patient tumor and PDX were processed for analysis of copy number variations by aCGH, gene expression by microarray, and expression of target pathways by immunohistochemistry (IHC). One PDX harbouring an FGFR3 mutation was treated with an inhibitory monoclonal antibody targeting FGFR3. Five PDX were successfully established. Tumor doubling time ranged from 5 to 11 days. Array CGH revealed shared chromosomal aberrations in the patient tumors and PDX. Gene expression microarray and IHC confirmed that PDXs maintain similar patterns to the parental tumors. Tumor growth in the PDX with an FGFR3 mutation was inhibited by the FGFR3 inhibitor. PDXs recapitulate the tumor biology of the patients' primary tumors from which they are derived. Investigations related to tumor biology and drug testing in these models are therefore more likely to be relevant to the disease state in patients. They represent a valuable tool for developing precision therapy in MIBC.

Purpose: Although novel agents targeting the androgen-androgen receptor (AR) axis have altered the treatment paradigm of metastatic castration-resistant prostate cancer (mCRPC), development of therapeutic resistance is inevitable. In this study, we examined whether AR gene aberrations detectable in circulating cell-free DNA (cfDNA) are associated with resistance to abiraterone acetate and enzalutamide in mCRPC patients., Experimental Design: Plasma was collected from 62 mCRPC patients ceasing abiraterone acetate (n = 29), enzalutamide (n = 19), or other agents (n = 14) due to disease progression. DNA was extracted and subjected to array comparative genomic hybridization (aCGH) for chromosome copy number analysis, and Roche 454 targeted next-generation sequencing of exon 8 in the AR., Results: On aCGH, AR amplification was significantly more common in patients progressing on enzalutamide than on abiraterone or other agents (53% vs. 17% vs. 21%, P = 0.02, χ(2)). Missense AR exon 8 mutations were detected in 11 of 62 patients (18%), including the first reported case of an F876L mutation in an enzalutamide-resistant patient and H874Y and T877A mutations in 7 abiraterone-resistant patients. In patients switched onto enzalutamide after cfDNA collection (n = 39), an AR gene aberration (copy number increase and/or an exon 8 mutation) in pretreatment cfDNA was associated with adverse outcomes, including lower rates of PSA decline ≥ 30% (P = 0.013, χ(2)) and shorter time to radiographic/clinical progression (P = 0.010, Cox proportional hazards regression)., Conclusions: AR gene aberrations in cfDNA are associated with resistance to enzalutamide and abiraterone in mCRPC. Our data illustrate that genomic analysis of cfDNA is a minimally invasive method for interrogating mechanisms of therapeutic resistance in mCRPC., (©2015 American Association for Cancer Research.)

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer which does not respond to hormone therapy. Research of NEPC has been hampered by a lack of clinically relevant in vivo models. Recently, we developed a first-in-field patient tissue-derived xenograft model of complete neuroendocrine transdifferentiation of prostate adenocarcinoma. By comparing gene expression profiles of a transplantable adenocarcinoma line (LTL331) and its NEPC subline (LTL331R), we identified DEK as a potential biomarker and therapeutic target for NEPC. In the present study, elevated DEK protein expression was observed in all NEPC xenograft models and clinical NEPC cases, as opposed to their benign counterparts (0%), hormonal naïve prostate cancer (2.45%) and castration-resistant prostate cancer (29.55%). Elevated DEK expression was found to be an independent clinical risk factor, associated with shorter disease-free survival of hormonal naïve prostate cancer patients. DEK silencing in PC-3 cells led to a marked reduction in cell proliferation, cell migration and invasion. The results suggest that DEK plays an important role in the progression of prostate cancer, especially to NEPC, and provides a potential biomarker to aid risk stratification of prostate cancer and a novel target for therapy of NEPC.

Castrate-resistant prostate cancer (CRPC) and neuroendocrine carcinoma of the prostate are invariably fatal diseases for which only palliative therapies exist. As part of a prostate tumor sequencing program, a patient tumor was analyzed using Illumina genome sequencing and a matched renal capsule tumor xenograft was generated. Both tumor and xenograft had a homozygous 9p21 deletion spanning the MTAP, CDKN2, and ARF genes. It is rare for this deletion to occur in primary prostate tumors, yet approximately 10% express decreased levels of methylthioadenosine phosphorylase (MTAP) mRNA. Decreased MTAP expression is a prognosticator for poor outcome. Moreover, it seems that this deletion is more common in CRPC than in primary prostate cancer. We show for the first time that treatment with methylthioadenosine and high dose 6-thioguanine causes marked inhibition of a patient-derived neuroendocrine xenograft growth while protecting the host from 6-thioguanine toxicity. This therapeutic approach can be applied to other MTAP-deficient human cancers as deletion or hypermethylation of the MTAP gene occurs in a broad spectrum of tumors at high frequency. The combination of genome sequencing and patient-derived xenografts can identify candidate therapeutic agents and evaluate them for personalized oncology.