Phosphoproteome Integration Reveals Patient-Specific Networks in Prostate Cancer.

We used clinical tissue from lethal metastatic castration-resistant prostate cancer (CRPC) patients obtained at rapid autopsy to evaluate diverse genomic, transcriptomic, and phosphoproteomic datasets for pathway analysis. Using Tied Diffusion through Interacting Events (TieDIE), we integrated differentially expressed master transcriptional regulators, functionally mutated genes, and differentially activated kinases in CRPC tissues to synthesize a robust signaling network consisting of druggable kinase pathways. Using MSigDB hallmark gene sets, six major signaling pathways with phosphorylation of several key residues were significantly enriched in CRPC tumors after incorporation of phosphoproteomic data. Individual autopsy profiles developed using these hallmarks revealed clinically relevant pathway information potentially suitable for patient stratification and targeted therapies in late stage prostate cancer. Here, we describe phosphorylation-based cancer hallmarks using integrated personalized signatures (pCHIPS) that shed light on the diversity of activated signaling pathways in metastatic CRPC while providing an integrative, pathway-based reference for drug prioritization in individual patients.

Targeting glutamine dependence with DRP-104 inhibits proliferation and tumor growth of castration-resistant prostate cancer.

BACKGROUND: Prostate cancer (PCa) continues to be one of the leading causes of cancer deaths in men. While androgen deprivation therapy is initially effective, castration-resistant PCa (CRPC) often recurs and has limited treatment options. Our previous study identified glutamine metabolism to be critical for CRPC growth. The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) blocks both carbon and nitrogen pathways but has dose-limiting toxicity. The prodrug DRP-104 is expected to be preferentially converted to DON in tumor cells to inhibit glutamine utilization with minimal toxicity. However, CRPC cells' susceptibility to DRP-104 remains unclear. METHODS: Human PCa cell lines (LNCaP, LAPC4, C4-2/MDVR, PC-3, 22RV1, NCI-H660) were treated with DRP-104, and effects on proliferation and cell death were assessed. Unbiased metabolic profiling and isotope tracing evaluated the effects of DRP-104 on glutamine pathways. Efficacy of DRP-104 in vivo was evaluated in a mouse xenograft model of neuroendocrine PCa, NCI-H660. RESULTS: DRP-104 inhibited proliferation and induced apoptosis in CRPC cell lines. Metabolite profiling showed decreases in the tricarboxylic acid cycle and nucleotide synthesis metabolites. Glutamine isotope tracing confirmed the blockade of both carbon pathway and nitrogen pathways. DRP-104 treated CRPC cells were rescued by the addition of nucleosides. DRP-104 inhibited neuroendocrine PCa xenograft growth without detectable toxicity. CONCLUSIONS: The prodrug DRP-104 blocks glutamine carbon and nitrogen utilization, thereby inhibiting CRPC growth and inducing apoptosis. Targeting glutamine metabolism pathways with DRP-104 represents a promising therapeutic strategy for CRPC.

Cas13d knockdown of lung protease Ctsl prevents and treats SARS-CoV-2 infection.

SARS-CoV-2 entry into cells requires specific host proteases; however, no successful in vivo applications of host protease inhibitors have yet been reported for treatment of SARS-CoV-2 pathogenesis. Here we describe a chemically engineered nanosystem encapsulating CRISPR-Cas13d, developed to specifically target lung protease cathepsin L (Ctsl) messenger RNA to block SARS-CoV-2 infection in mice. We show that this nanosystem decreases lung Ctsl expression in normal mice efficiently, specifically and safely. We further show that this approach extends survival of mice lethally infected with SARS-CoV-2, correlating with decreased lung virus burden, reduced expression of proinflammatory cytokines/chemokines and diminished severity of pulmonary interstitial inflammation. Postinfection treatment by this nanosystem dramatically lowers the lung virus burden and alleviates virus-induced pathological changes. Our results indicate that targeting lung protease mRNA by Cas13d nanosystem represents a unique strategy for controlling SARS-CoV-2 infection and demonstrate that CRISPR can be used as a potential treatment for SARS-CoV-2 infection.

Oncofetal protein glypican-3 is a biomarker and critical regulator of function for neuroendocrine cells in prostate cancer.

Neuroendocrine (NE) cells comprise ~1% of epithelial cells in benign prostate and prostatic adenocarcinoma (PCa). However, they become enriched in hormonally treated and castration-resistant PCa (CRPC). In addition, close to 20% of hormonally treated tumors recur as small cell NE carcinoma (SCNC), composed entirely of NE cells, which may be the result of clonal expansion or lineage plasticity. Since NE cells do not express androgen receptors (ARs), they are resistant to hormonal therapy and contribute to therapy failure. Here, we describe the identification of glypican-3 (GPC3) as an oncofetal cell surface protein specific to NE cells in prostate cancer. Functional studies revealed that GPC3 is critical to the viability of NE tumor cells and tumors displaying NE differentiation and that it regulates calcium homeostasis and signaling. Since our results demonstrate that GPC3 is specifically expressed by NE cells, patients with confirmed SCNC may qualify for GPC3-targeted therapy which has been developed in the context of liver cancer and displays minimal toxicity due to its tumor-specific expression. © 2023 The Pathological Society of Great Britain and Ireland.

Phosphorylated MED1 links transcription recycling and cancer growth.

Mediator activates RNA polymerase II (Pol II) function during transcription, but it remains unclear whether Mediator is able to travel with Pol II and regulate Pol II transcription beyond the initiation and early elongation steps. By using in vitro and in vivo transcription recycling assays, we find that human Mediator 1 (MED1), when phosphorylated at the mammal-specific threonine 1032 by cyclin-dependent kinase 9 (CDK9), dynamically moves along with Pol II throughout the transcribed genes to drive Pol II recycling after the initial round of transcription. Mechanistically, MED31 mediates the recycling of phosphorylated MED1 and Pol II, enhancing mRNA output during the transcription recycling process. Importantly, MED1 phosphorylation increases during prostate cancer progression to the lethal phase, and pharmacological inhibition of CDK9 decreases prostate tumor growth by decreasing MED1 phosphorylation and Pol II recycling. Our results reveal a novel role of MED1 in Pol II transcription and identify phosphorylated MED1 as a targetable driver of dysregulated Pol II recycling in cancer.

A glutaminase isoform switch drives therapeutic resistance and disease progression of prostate cancer.

Cellular metabolism in cancer is significantly altered to support the uncontrolled tumor growth. How metabolic alterations contribute to hormonal therapy resistance and disease progression in prostate cancer (PCa) remains poorly understood. Here we report a glutaminase isoform switch mechanism that mediates the initial therapeutic effect but eventual failure of hormonal therapy of PCa. Androgen deprivation therapy inhibits the expression of kidney-type glutaminase (KGA), a splicing isoform of glutaminase 1 (GLS1) up-regulated by androgen receptor (AR), to achieve therapeutic effect by suppressing glutaminolysis. Eventually the tumor cells switch to the expression of glutaminase C (GAC), an androgen-independent GLS1 isoform with more potent enzymatic activity, under the androgen-deprived condition. This switch leads to increased glutamine utilization, hyperproliferation, and aggressive behavior of tumor cells. Pharmacological inhibition or RNA interference of GAC shows better treatment effect for castration-resistant PCa than for hormone-sensitive PCa in vitro and in vivo. In summary, we have identified a metabolic function of AR action in PCa and discovered that the GLS1 isoform switch is one of the key mechanisms in therapeutic resistance and disease progression.

ACAA2 is a novel molecular indicator for cancers with neuroendocrine phenotype.

BACKGROUND: Neuroendocrine phenotype is commonly associated with therapy resistance and poor prognoses in small-cell neuroendocrine cancers (SCNCs), such as neuroendocrine prostate cancer (NEPC) and small-cell lung cancer (SCLC). Expression levels of current neuroendocrine markers exhibit high case-by-case variability, so multiple markers are used in combination to identify SCNCs. Here, we report that ACAA2 is elevated in SCNCs and is a potential molecular indicator for SCNCs. METHODS: ACAA2 expressions in tumour xenografts, tissue microarrays (TMAs), and patient tissues from prostate and lung cancers were analysed via immunohistochemistry. ACAA2 mRNA levels in lung and prostate cancer (PC) patients were assessed in published datasets. RESULTS: ACAA2 protein and mRNA levels were elevated in SCNCs relative to non-SCNCs. Medium/high ACAA2 intensity was observed in 78% of NEPC PDXs samples (N = 27) relative to 33% of adeno-CRPC (N = 86), 2% of localised PC (N = 50), and 0% of benign prostate specimens (N = 101). ACAA2 was also elevated in lung cancer patient tissues with neuroendocrine phenotype. 83% of lung carcinoid tissues (N = 12) and 90% of SCLC tissues (N = 10) exhibited medium/high intensity relative to 40% of lung adenocarcinoma (N = 15). CONCLUSION: ACAA2 expression is elevated in aggressive SCNCs such as NEPC and SCLC, suggesting it is a potential molecular indicator for SCNCs.

SPOP Promotes Ubiquitination and Degradation of the ERG Oncoprotein to Suppress Prostate Cancer Progression.

The ERG gene is fused to TMPRSS2 in approximately 50% of prostate cancers (PrCa), resulting in its overexpression. However, whether this is the sole mechanism underlying ERG elevation in PrCa is currently unclear. Here we report that ERG ubiquitination and degradation are governed by the Cullin 3-based ubiquitin ligase SPOP and that deficiency in this pathway leads to aberrant elevation of the ERG oncoprotein. Specifically, we find that truncated ERG (ΔERG), encoded by the ERG fusion gene, is stabilized by evading SPOP-mediated destruction, whereas prostate cancer-associated SPOP mutants are also deficient in promoting ERG ubiquitination. Furthermore, we show that the SPOP/ERG interaction is modulated by CKI-mediated phosphorylation. Importantly, we demonstrate that DNA damage drugs, topoisomerase inhibitors, can trigger CKI activation to restore the SPOP/ΔERG interaction and its consequent degradation. Therefore, SPOP functions as a tumor suppressor to negatively regulate the stability of the ERG oncoprotein in prostate cancer.

Cistrome analysis of YY1 uncovers a regulatory axis of YY1:BRD2/4-PFKP during tumorigenesis of advanced prostate cancer.

Castration-resistant prostate cancer (CRPC) is a terminal disease and the molecular underpinnings of CRPC development need to be better understood in order to improve its treatment. Here, we report that a transcription factor Yin Yang 1 (YY1) is significantly overexpressed during prostate cancer progression. Functional and cistrome studies of YY1 uncover its roles in promoting prostate oncogenesis in vitro and in vivo, as well as sustaining tumor metabolism including the Warburg effect and mitochondria respiration. Additionally, our integrated genomics and interactome profiling in prostate tumor show that YY1 and bromodomain-containing proteins (BRD2/4) co-occupy a majority of gene-regulatory elements, coactivating downstream targets. Via gene loss-of-function and rescue studies and mutagenesis of YY1-bound cis-elements, we unveil an oncogenic pathway in which YY1 directly binds and activates PFKP, a gene encoding the rate-limiting enzyme for glycolysis, significantly contributing to the YY1-enforced Warburg effect and malignant growth. Altogether, this study supports a master regulator role for YY1 in prostate tumorigenesis and reveals a YY1:BRD2/4-PFKP axis operating in advanced prostate cancer with implications for therapy.

A genetically defined disease model reveals that urothelial cells can initiate divergent bladder cancer phenotypes.

Small cell carcinoma of the bladder (SCCB) is a rare and lethal phenotype of bladder cancer. The pathogenesis and molecular features are unknown. Here, we established a genetically engineered SCCB model and a cohort of patient SCCB and urothelial carcinoma samples to characterize molecular similarities and differences between bladder cancer phenotypes. We demonstrate that SCCB shares a urothelial origin with other bladder cancer phenotypes by showing that urothelial cells driven by a set of defined oncogenic factors give rise to a mixture of tumor phenotypes, including small cell carcinoma, urothelial carcinoma, and squamous cell carcinoma. Tumor-derived single-cell clones also give rise to both SCCB and urothelial carcinoma in xenografts. Despite this shared urothelial origin, clinical SCCB samples have a distinct transcriptional profile and a unique transcriptional regulatory network. Using the transcriptional profile from our cohort, we identified cell surface proteins (CSPs) associated with the SCCB phenotype. We found that the majority of SCCB samples have PD-L1 expression in both tumor cells and tumor-infiltrating lymphocytes, suggesting that immune checkpoint inhibitors could be a treatment option for SCCB. We further demonstrate that our genetically engineered tumor model is a representative tool for investigating CSPs in SCCB by showing that it shares a similar a CSP profile with clinical samples and expresses SCCB-up-regulated CSPs at both the mRNA and protein levels. Our findings reveal distinct molecular features of SCCB and provide a transcriptional dataset and a preclinical model for further investigating SCCB biology.

Transcriptional profiling identifies an androgen receptor activity-low, stemness program associated with enzalutamide resistance.

The androgen receptor (AR) antagonist enzalutamide is one of the principal treatments for men with castration-resistant prostate cancer (CRPC). However, not all patients respond, and resistance mechanisms are largely unknown. We hypothesized that genomic and transcriptional features from metastatic CRPC biopsies prior to treatment would be predictive of de novo treatment resistance. To this end, we conducted a phase II trial of enzalutamide treatment (160 mg/d) in 36 men with metastatic CRPC. Thirty-four patients were evaluable for the primary end point of a prostate-specific antigen (PSA)50 response (PSA decline ≥50% at 12 wk vs. baseline). Nine patients were classified as nonresponders (PSA decline <50%), and 25 patients were classified as responders (PSA decline ≥50%). Failure to achieve a PSA50 was associated with shorter progression-free survival, time on treatment, and overall survival, demonstrating PSA50's utility. Targeted DNA-sequencing was performed on 26 of 36 biopsies, and RNA-sequencing was performed on 25 of 36 biopsies that contained sufficient material. Using computational methods, we measured AR transcriptional function and performed gene set enrichment analysis (GSEA) to identify pathways whose activity state correlated with de novo resistance. TP53 gene alterations were more common in nonresponders, although this did not reach statistical significance (P = 0.055). AR gene alterations and AR expression were similar between groups. Importantly, however, transcriptional measurements demonstrated that specific gene sets-including those linked to low AR transcriptional activity and a stemness program-were activated in nonresponders. Our results suggest that patients whose tumors harbor this program should be considered for clinical trials testing rational agents to overcome de novo enzalutamide resistance.

A pleiotropic ATM variant (rs1800057 C>G) is associated with risk of multiple cancers.

ATM (ataxia-telangiectasia mutated) is an important cell-cycle checkpoint kinase required for cellular response to DNA damage. Activated by DNA double strand breaks, ATM regulates the activities of many downstream proteins involved in various carcinogenic events. Therefore, ATM or its genetic variants may have a pleiotropic effect on cancer development. We conducted a pleiotropic analysis to evaluate associations between genetic variants of ATM and risk of multiple cancers. With genotyping data extracted from previously published genome-wide association studies of various cancers, we performed multivariate logistic regression analysis, followed by a meta-analysis for each cancer site, to identify cancer risk-associated single-nucleotide polymorphisms (SNPs). In the ASSET two-sided analysis, we found that two ATM SNPs were significantly associated with risk of multiple cancers. One tagging SNP (rs1800057 C>G) was associated with risk of multiple cancers (two-sided P = 5.27 × 10-7). Because ATM rs1800057 is a missense variant, we also explored the intermediate phenotypes through which this variant may confer risk of multiple cancers and identified a possible immune-mediated effect of this variant. Our findings indicate that genetic variants of ATM may have a pleiotropic effect on cancer risk and thus provide an important insight into common mechanisms of carcinogenesis.

PCK1 regulates neuroendocrine differentiation in a positive feedback loop of LIF/ZBTB46 signalling in castration-resistant prostate cancer.

BACKGROUND: Castration-resistant prostate cancer (CRPC) patients frequently develop neuroendocrine differentiation, with high mortality and no effective treatment. However, the regulatory mechanism that connects neuroendocrine differentiation and metabolic adaptation in response to therapeutic resistance of prostate cancer remain to be unravelled. METHODS: By unbiased cross-correlation between RNA-sequencing, database signatures, and ChIP analysis, combining in vitro cell lines and in vivo animal models, we identified that PCK1 is a pivotal regulator in therapy-induced neuroendocrine differentiation of prostate cancer through a LIF/ZBTB46-driven glucose metabolism pathway. RESULTS: Upregulation of PCK1 supports cell proliferation and reciprocally increases ZBTB46 levels to promote the expression of neuroendocrine markers that are conducive to the development of neuroendocrine characteristic CRPC. PCK1 and neuroendocrine marker expressions are regulated by the ZBTB46 transcription factor upon activation of LIF signalling. Targeting PCK1 can reduce the neuroendocrine phenotype and decrease the growth of prostate cancer cells in vitro and in vivo. CONCLUSION: Our study uncovers LIF/ZBTB46 signalling activation as a key mechanism for upregulating PCK1-driven glucose metabolism and neuroendocrine differentiation of CRPC, which may yield significant improvements in prostate cancer treatment after ADT using PCK1 inhibitors.

Tissue clearing techniques for three-dimensional optical imaging of intact human prostate and correlations with multi-parametric MRI.

BACKGROUND: Tissue clearing technologies have enabled remarkable advancements for in situ characterization of tissues and exploration of the three-dimensional (3D) relationships between cells, however, these studies have predominantly been performed in non-human tissues and correlative assessment with clinical imaging has yet to be explored. We sought to evaluate the feasibility of tissue clearing technologies for 3D imaging of intact human prostate and the mapping of structurally and molecularly preserved pathology data with multi-parametric volumetric MR imaging (mpMRI). METHODS: Whole-mount prostates were processed with either hydrogel-based CLARITY or solvent-based iDISCO. The samples were stained with a nuclear dye or fluorescently labeled with antibodies against androgen receptor, alpha-methylacyl coenzyme-A racemase, or p63, and then imaged with 3D confocal microscopy. The apparent diffusion coefficient and Ktrans maps were computed from preoperative mpMRI. RESULTS: Quantitative analysis of cleared normal and tumor prostate tissue volumes displayed differences in 3D tissue architecture, marker-specific cell staining, and cell densities that were significantly correlated with mpMRI measurements in this initial, pilot cohort. CONCLUSIONS: 3D imaging of human prostate volumes following tissue clearing is a feasible technique for quantitative radiology-pathology correlation analysis with mpMRI and provides an opportunity to explore functional relationships between cellular structures and cross-sectional clinical imaging.

Molecular determinants for enzalutamide-induced transcription in prostate cancer.

Enzalutamide, a second-generation androgen receptor (AR) antagonist, has demonstrated clinical benefit in men with prostate cancer. However, it only provides a temporary response and modest increase in survival, indicating a rapid evolution of resistance. Previous studies suggest that enzalutamide may function as a partial transcriptional agonist, but the underlying mechanisms for enzalutamide-induced transcription remain poorly understood. Here, we show that enzalutamide stimulates expression of a novel subset of genes distinct from androgen-responsive genes. Treatment of prostate cancer cells with enzalutamide enhances recruitment of pioneer factor GATA2, AR, Mediator subunits MED1 and MED14, and RNA Pol II to regulatory elements of enzalutamide-responsive genes. Mechanistically, GATA2 globally directs enzalutamide-induced transcription by facilitating AR, Mediator and Pol II loading to enzalutamide-responsive gene loci. Importantly, the GATA2 inhibitor K7174 inhibits enzalutamide-induced transcription by decreasing binding of the GATA2/AR/Mediator/Pol II transcriptional complex, contributing to sensitization of prostate cancer cells to enzalutamide treatment. Our findings provide mechanistic insight into the future combination of GATA2 inhibitors and enzalutamide for improved AR-targeted therapy.

Diverse AR-V7 cistromes in castration-resistant prostate cancer are governed by HoxB13.

The constitutively active androgen receptor (AR) splice variant 7 (AR-V7) plays an important role in the progression of castration-resistant prostate cancer (CRPC). Although biomarker studies established the role of AR-V7 in resistance to AR-targeting therapies, how AR-V7 mediates genomic functions in CRPC remains largely unknown. Using a ChIP-exo approach, we show AR-V7 binds to distinct genomic regions and recognizes a full-length androgen-responsive element in CRPC cells and patient tissues. Remarkably, we find dramatic differences in AR-V7 cistromes across diverse CRPC cells and patient tissues, regulating different target gene sets involved in CRPC progression. Surprisingly, we discover that HoxB13 is universally required for and colocalizes with AR-V7 binding to open chromatin across CRPC genomes. HoxB13 pioneers AR-V7 binding through direct physical interaction, and collaborates with AR-V7 to up-regulate target oncogenes. Transcriptional coregulation by HoxB13 and AR-V7 was further supported by their coexpression in tumors and circulating tumor cells from CRPC patients. Importantly, HoxB13 silencing significantly decreases CRPC growth through inhibition of AR-V7 oncogenic function. These results identify HoxB13 as a pivotal upstream regulator of AR-V7-driven transcriptomes that are often cell context-dependent in CRPC, suggesting that HoxB13 may serve as a therapeutic target for AR-V7-driven prostate tumors.

Systemic surfaceome profiling identifies target antigens for immune-based therapy in subtypes of advanced prostate cancer.

Prostate cancer is a heterogeneous disease composed of divergent molecular and histologic subtypes, including prostate adenocarcinoma (PrAd) and neuroendocrine prostate cancer (NEPC). While PrAd is the major histology in prostate cancer, NEPC can evolve from PrAd as a mechanism of treatment resistance that involves a transition from an epithelial to a neurosecretory cancer phenotype. Cell surface markers are often associated with specific cell lineages and differentiation states in normal development and cancer. Here, we show that PrAd and NEPC can be broadly discriminated by cell-surface profiles based on the analysis of prostate cancer gene expression datasets. To overcome a dependence on predictions of human cell-surface genes and an assumed correlation between mRNA levels and protein expression, we integrated transcriptomic and cell-surface proteomic data generated from a panel of prostate cancer cell lines to nominate cell-surface markers associated with these cancer subtypes. FXYD3 and CEACAM5 were validated as cell-surface antigens enriched in PrAd and NEPC, respectively. Given the lack of effective treatments for NEPC, CEACAM5 appeared to be a promising target for cell-based immunotherapy. As a proof of concept, engineered chimeric antigen receptor T cells targeting CEACAM5 induced antigen-specific cytotoxicity in NEPC cell lines. Our findings demonstrate that the surfaceomes of PrAd and NEPC reflect unique cancer differentiation states and broadly represent vulnerabilities amenable to therapeutic targeting.

Prostate Cancer Detection Rate of Freehand versus 3-Dimensional Template Mapping Biopsy Using a Magnetic Resonance Imaging-Ultrasound Fusion Device in Biopsy Naïve Men.

PURPOSE: Targeted prostate biopsy devices include a 3-dimensional digital template grid to guide systematic biopsy locations. Following a template could better ensure uniform and well distributed sampling of the prostate compared to the traditional freehand biopsy approach, possibly decreasing the chance of false-negative biopsy. Thus, we determined cancer detection rates obtained by conventional freehand systematic sampling vs template mapping sampling using a magnetic resonance imaging-ultrasound fusion device. MATERIALS AND METHODS: Men who underwent first line conventional or image guided prostate biopsy were identified retrospectively in an institutional review board approved protocol. Excluded from study were men with prior biopsy or treatment or fewer than 10 cores taken. Targeted cores obtained by image guided biopsy were censored from analysis to simulate systematic template biopsy. The resulting cancer detection rate was compared to that of conventional biopsy. RESULTS: We identified 1,582 patients between 2006 and 2014 who met the criteria for analysis, including 1,052 who underwent conventional biopsy and 530 who underwent template biopsy with a magnetic resonance imaging-ultrasound fusion device. Patient age, prostate specific antigen and the number of systematic cores were the same in the 2 groups. Template biopsy detected any prostate cancer in 257 of 530 men (48.5%) and clinically significant cancer in 196 (37.0%) while conventional biopsy detected any cancer in 432 of 1,052 (41.0%) (p=0.005) and clinically significant cancer in 308 (29.2%) (p=0.002). CONCLUSIONS: Template mapping systematic biopsy detected more prostate cancer than conventional sampling in biopsy naïve men. It is a promising cost-effective alternative to magnetic resonance imaging-ultrasound fusion biopsy as an upfront screening tool.

Regulated proteolysis of Trop2 drives epithelial hyperplasia and stem cell self-renewal via ß-catenin signaling.

The cell surface protein Trop2 is expressed on immature stem/progenitor-like cells and is overexpressed in many epithelial cancers. However the biological function of Trop2 in tissue maintenance and tumorigenesis remains unclear. In this study, we demonstrate that Trop2 is a regulator of self-renewal, proliferation, and transformation. Trop2 controls these processes through a mechanism of regulated intramembrane proteolysis that leads to cleavage of Trop2, creating two products: the extracellular domain and the intracellular domain. The intracellular domain of Trop2 is released from the membrane and accumulates in the nucleus. Heightened expression of the Trop2 intracellular domain promotes stem/progenitor self-renewal through signaling via ß-catenin and is sufficient to initiate precursor lesions to prostate cancer in vivo. Importantly, we demonstrate that loss of ß-catenin or Trop2 loss-of-function cleavage mutants abrogates Trop2-driven self-renewal and hyperplasia in the prostate. These findings suggest that heightened expression of Trop2 is selected for in epithelial cancers to enhance the stem-like properties of self-renewal and proliferation. Defining the mechanism of Trop2 function in self-renewal and transformation is essential to identify new therapeutic strategies to block Trop2 activation in cancer.

N-Myc promotes therapeutic resistance development of neuroendocrine prostate cancer by differentially regulating miR-421/ATM pathway.

BACKGROUND: MYCN amplification or N-Myc overexpression is found in approximately 40% NEPC and up to 20% CRPC patients. N-Myc has been demonstrated to drive disease progression and hormonal therapeutic resistance of NEPC/CRPC. Here, we aim to identify the molecular mechanisms underlying the N-Myc-driven therapeutic resistance and provide new therapeutic targets for those N-Myc overexpressed NEPC/CRPC. METHODS: N-Myc overexpressing stable cell lines for LNCaP and C4-2 were generated by lentivirus infection. ADT-induced senescence was measured by SA-ß-gal staining in LNCaP cells in vitro and in LNCaP xenograft tumors in vivo. Migration, cell proliferation and colony formation assays were used to measure the cellular response after overexpressing N-Myc or perturbing the miR-421/ATM pathway. CRISPR-Cas9 was used to knock out ATM in C4-2 cells and MTS cell viability assay was used to evaluate the drug sensitivity of N-Myc overexpressing C4-2 cells in response to Enzalutamide and ATM inhibitor Ku60019 respectively or in combination. RESULTS: N-Myc overexpression suppressed ATM expression through upregulating miR-421 in LNCaP cells. This suppression alleviated the ADT-induced senescence in vitro and in vivo. Surprisingly, N-Myc overexpression upregulated ATM expression in C4-2 cells and this upregulation promoted migration and invasion of prostate cancer cells. Further, the N-Myc-induced ATM upregulation in C4-2 cells rendered the cells resistance to Enzalutamide, and inhibition of ATM by CRISPR-Cas9 knockout or ATM inhibitor Ku60019 re-sensitized them to Enzalutamide. CONCLUSIONS: N-Myc differentially regulating miR-421/ATM pathway contributes to ADT resistance and Enzalutamide resistance development respectively. Combination treatment with ATM inhibitor re-sensitizes N-Myc overexpressed CRPC cells to Enzalutamide. Our findings would offer a potential combination therapeutic strategy using ATM kinase inhibitor and Enzalutamide for the treatment of a subset of mCRPC with N-Myc overexpression that accounts for up to 20% CRPC patients.