LSD1 inhibition suppresses ASCL1 and de-represses YAP1 to drive potent activity against neuroendocrine prostate cancer.

Lysine-specific demethylase 1 (LSD1 or KDM1A ) has emerged as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Among mCRPC subtypes, neuroendocrine prostate cancer (NEPC) is an exceptionally aggressive variant driven by lineage plasticity, an adaptive resistance mechanism to androgen receptor axis-targeted therapies. Our study shows that LSD1 expression is elevated in NEPC and associated with unfavorable clinical outcomes. Using genetic approaches, we validated the on-target effects of LSD1 inhibition across various models. We investigated the therapeutic potential of bomedemstat, an orally bioavailable, irreversible LSD1 inhibitor with low nanomolar potency. Our findings demonstrate potent antitumor activity against CRPC models, including tumor regressions in NEPC patient-derived xenografts. Mechanistically, our study uncovers that LSD1 inhibition suppresses the neuronal transcriptional program by downregulating ASCL1 through disrupting LSD1:INSM1 interactions and de-repressing YAP1 silencing. Our data support the clinical development of LSD1 inhibitors for treating CRPC - especially the aggressive NE phenotype. Statement of Significance: Neuroendocrine prostate cancer presents a clinical challenge due to the lack of effective treatments. Our research demonstrates that bomedemstat, a potent and selective LSD1 inhibitor, effectively combats neuroendocrine prostate cancer by downregulating the ASCL1- dependent NE transcriptional program and re-expressing YAP1.

In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo.

Engineering chimeric antigen receptors (CAR) or T cell receptors (TCR) helps create disease-specific T cells for targeted therapy, but the cost and rigor associated with manufacturing engineered T cells ex vivo can be prohibitive, so programing T cells in vivo may be a viable alternative. Here we report an injectable nanocarrier that delivers in vitro-transcribed (IVT) CAR or TCR mRNA for transiently reprograming of circulating T cells to recognize disease-relevant antigens. In mouse models of human leukemia, prostate cancer and hepatitis B-induced hepatocellular carcinoma, repeated infusions of these polymer nanocarriers induce sufficient host T cells expressing tumor-specific CARs or virus-specific TCRs to cause disease regression at levels similar to bolus infusions of ex vivo engineered lymphocytes. Given their ease of manufacturing, distribution and administration, these nanocarriers, and the associated platforms, could become a therapeutic for a wide range of diseases.

Systematic and functional characterization of novel androgen receptor variants arising from alternative splicing in the ligand-binding domain.

The presence of intact ligand-binding domain (LBD) ensures the strict androgen-dependent regulation of androgen receptor (AR): binding of androgen induces structural reorganization of LBD resulting in release of AR from HSP90, suppression of nuclear export which otherwise dominates over import and nuclear translocation of AR as a transcription factor. Thus, loss or defects of the LBD abolish constraint from un-liganded LBD as exemplified by constitutively active AR variants (AR-Vs), which are associated with emerging resistance mechanism to anti-AR therapy in castration-resistant prostate cancer (mCRPC). Recent analysis of the AR splicing landscapes revealed mCRPC harboring multiple AR-Vs with diverse patterns of inclusion/exclusion of exons (exons 4-8) corresponding to LBD to produce namely exon-skipping variants. In silico construction for these AR-Vs revealed four novel AR-Vs having unique features: Exclusion of specified exons introduces a frameshift in variants v5es, v6es and v7es. ARv56es maintains the reading frame resulting in the inclusion of the C-terminal half of the LBD. We systematically characterized these AR-Vs regarding their subcellular localization, affinity for HSP90 and transactivation capability. Notably, ARv5es was free from HSP90, exclusively nuclear, and constitutively active similarly as previously reported for v567es. In contrast, v6es and v7es were similar in that they are cytoplasmic, transcriptionally inactive and bind HSP90, ARv56es was present in both nucleus and cytoplasm, does not bind HSP90 and is transcriptionally inactive. Converting these transcriptionally inactive AR-Vs into active forms, we identified the two separate elements that allosterically suppress otherwise constitutively active AR-Vs; one in exon 5 for v6es and v7es and the other in exon 8 for v56es. Our findings identify a novel constitutively active AR-V, ARv5es and establish a method to predict potential activities of AR-Vs carrying impaired LBD.

A diagnosis of prostate cancer and pursuit of active surveillance is not followed by weight loss: potential for a teachable moment.

BACKGROUND: Obesity is a risk factor for incident prostate cancer (PC) as well as risk of disease progression and mortality. We hypothesized that men diagnosed with lower-risk PC and who elected active surveillance (AS) for their cancer management would likely initiate lifestyle changes that lead to weight loss. METHODS: Patients were enrolled in the Prostate Active Surveillance Study (PASS), a multicenter prospective biomarker discovery and validation study of men who have chosen AS for their PC. Data from 442 men diagnosed with PC within 1 year of study entry who completed a standard of care 12-month follow-up visit were analyzed. We examined the change in weight and body mass index (BMI) over the first year of study participation. RESULTS: After 1 year on AS, 7.5% (33/442) of patients had lost 5% or more of their on-study weight. The proportion of men who lost 5% or more weight was similar across categories of baseline BMI: normal/underweight (8%), overweight (6%) and obese (10%, χ2 test P=0.44). The results were similar for patients enrolled in the study 1 year or 6 months after diagnosis. By contrast, after 1 year, 7.7% (34/442) of patients had gained >5% of their weight. CONCLUSIONS: Only 7.5% of men with low-risk PC enrolled in AS lost a modest (⩾5%) amount of weight after diagnosis. Given that obesity is related to PC progression and mortality, targeted lifestyle interventions may be effective at this 'teachable moment', as men begin AS for low-risk PC.

Prognostic value of Ki67 in localized prostate carcinoma: a multi-institutional study of >1000 prostatectomies.

BACKGROUND: Expanding interest in and use of active surveillance for early state prostate cancer (PC) has increased need for prognostic biomarkers. Using a multi-institutional tissue microarray resource including over 1000 radical prostatectomy samples, we sought to correlate Ki67 expression captured by an automated image analysis system with clinicopathological features and validate its utility as a clinical grade test in predicting cancer-specific outcomes. METHODS: After immunostaining, the Ki67 proliferation index (PI) of tumor areas of each core (three cancer cores/case) was analyzed using a nuclear quantification algorithm (Aperio). We assessed whether Ki67 PI was associated with clinicopathological factors and recurrence-free survival (RFS) including biochemical recurrence, metastasis or PC death (7-year median follow-up). RESULTS: In 1004 PCs (∼4000 tissue cores) Ki67 PI showed significantly higher inter-tumor (0.68) than intra-tumor variation (0.39). Ki67 PI was associated with stage (P<0.0001), seminal vesicle invasion (SVI, P=0.02), extracapsular extension (ECE, P<0.0001) and Gleason score (GS, P<0.0001). Ki67 PI as a continuous variable significantly correlated with recurrence-free, overall and disease-specific survival by multivariable Cox proportional hazard model (hazards ratio (HR)=1.04-1.1, P=0.02-0.0008). High Ki67 score (defined as ⩾5%) was significantly associated with worse RFS (HR=1.47, P=0.0007) and worse overall survival (HR=2.03, P=0.03). CONCLUSIONS: In localized PC treated by radical prostatectomy, higher Ki67 PI assessed using a clinical grade automated algorithm is strongly associated with a higher GS, stage, SVI and ECE and greater probability of recurrence.

HDAC inhibition impedes epithelial-mesenchymal plasticity and suppresses metastatic, castration-resistant prostate cancer.

PI3K (phosphoinositide 3-kinase)/AKT and RAS/MAPK (mitogen-activated protein kinase) pathway coactivation in the prostate epithelium promotes both epithelial-mesenchymal transition (EMT) and metastatic castration-resistant prostate cancer (mCRPC), which is currently incurable. To study the dynamic regulation of the EMT process, we developed novel genetically defined cellular and in vivo model systems from which epithelial, EMT and mesenchymal-like tumor cells with Pten deletion and Kras activation can be isolated. When cultured individually, each population has the capacity to regenerate all three tumor cell populations, indicative of epithelial-mesenchymal plasticity. Despite harboring the same genetic alterations, mesenchymal-like tumor cells are resistant to PI3K and MAPK pathway inhibitors, suggesting that epigenetic mechanisms may regulate the EMT process, as well as dictate the heterogeneous responses of cancer cells to therapy. Among differentially expressed epigenetic regulators, the chromatin remodeling protein HMGA2 is significantly upregulated in EMT and mesenchymal-like tumors cells, as well as in human mCRPC. Knockdown of HMGA2, or suppressing HMGA2 expression with the histone deacetylase inhibitor LBH589, inhibits epithelial-mesenchymal plasticity and stemness activities in vitro and markedly reduces tumor growth and metastasis in vivo through successful targeting of EMT and mesenchymal-like tumor cells. Importantly, LBH589 treatment in combination with castration prevents mCRPC development and significantly prolongs survival following castration by enhancing p53 and androgen receptor acetylation and in turn sensitizing castration-resistant mesenchymal-like tumor cells to androgen deprivation therapy. Taken together, these findings demonstrate that cellular plasticity is regulated epigenetically, and that mesenchymal-like tumor cell populations in mCRPC that are resistant to conventional and targeted therapies can be effectively treated with the epigenetic inhibitor LBH589.

Management of patients with advanced prostate cancer: recommendations of the St Gallen Advanced Prostate Cancer Consensus Conference (APCCC) 2015.

The first St Gallen Advanced Prostate Cancer Consensus Conference (APCCC) Expert Panel identified and reviewed the available evidence for the ten most important areas of controversy in advanced prostate cancer (APC) management. The successful registration of several drugs for castration-resistant prostate cancer and the recent studies of chemo-hormonal therapy in men with castration-naïve prostate cancer have led to considerable uncertainty as to the best treatment choices, sequence of treatment options and appropriate patient selection. Management recommendations based on expert opinion, and not based on a critical review of the available evidence, are presented. The various recommendations carried differing degrees of support, as reflected in the wording of the article text and in the detailed voting results recorded in supplementary Material, available at Annals of Oncology online. Detailed decisions on treatment as always will involve consideration of disease extent and location, prior treatments, host factors, patient preferences as well as logistical and economic constraints. Inclusion of men with APC in clinical trials should be encouraged.

Integrative molecular profiling of routine clinical prostate cancer specimens.

BACKGROUND: Comprehensive molecular profiling led to the recognition of multiple prostate cancer (PCa) molecular subtypes and driving alterations, but translating these findings to clinical practice is challenging. PATIENTS AND METHODS: We developed a formalin-fixed paraffin-embedded (FFPE) tissue compatible integrative assay for PCa molecular subtyping and interrogation of relevant genetic/transcriptomic alterations (MiPC). We applied MiPC, which combines capture-based next generation sequencing and quantitative reverse transcription PCR (qRT-PCR), to 53 FFPE PCa specimens representing cases not well represented in frozen tissue cohorts, including 8 paired primary tumor and lymph node metastases. Results were validated using multiplexed PCR based NGS and Sanger sequencing. RESULTS: We identified known and novel potential driving, somatic mutations and copy number alterations, including a novel BRAF T599_V600insHT mutation and CYP11B2 amplification in a patient treated with ketoconazole (a potent CYP11B2 inhibitor). qRT-PCR integration enabled comprehensive molecular subtyping and provided complementary information, such as androgen receptor (AR) target gene module assessment in advanced cases and SPINK1 over-expression. MiPC identified highly concordant profiles for all 8 tumor/lymph node metastasis pairs, consistent with limited heterogeneity amongst driving events. MiPC and exome sequencing were performed on separately isolated conventional acinar PCa and prostatic small cell carcinoma (SCC) components from the same FFPE resection specimen to enable direct comparison of histologically distinct components. While both components showed TMPRSS2:ERG fusions, the SCC component exclusively harbored complete TP53 inactivation (frameshift variant and copy loss) and two CREBBP mutations. CONCLUSIONS: Our results demonstrate the feasibility of integrative profiling of routine PCa specimens, which may have utility for understanding disease biology and enabling personalized medicine applications.

EPLIN downregulation promotes epithelial-mesenchymal transition in prostate cancer cells and correlates with clinical lymph node metastasis.

Epithelial-mesenchymal transition (EMT) is a crucial mechanism for the acquisition of migratory and invasive capabilities by epithelial cancer cells. By conducting quantitative proteomics in experimental models of human prostate cancer (PCa) metastasis, we observed strikingly decreased expression of EPLIN (epithelial protein lost in neoplasm; or LIM domain and actin binding 1, LIMA-1) upon EMT. Biochemical and functional analyses demonstrated that EPLIN is a negative regulator of EMT and invasiveness in PCa cells. EPLIN depletion resulted in the disassembly of adherens junctions, structurally distinct actin remodeling and activation of ß-catenin signaling. Microarray expression analysis identified a subset of putative EPLIN target genes associated with EMT, invasion and metastasis. By immunohistochemistry, EPLIN downregulation was also demonstrated in lymph node metastases of human solid tumors including PCa, breast cancer, colorectal cancer and squamous cell carcinoma of the head and neck. This study reveals a novel molecular mechanism for converting cancer cells into a highly invasive and malignant form, and has important implications in prognosis and treating metastasis at early stages.

IL-6 promotes prostate tumorigenesis and progression through autocrine cross-activation of IGF-IR.

As an established mediator of inflammation, interleukin-6 (IL-6) is implicated to facilitate prostate cancer progression to androgen independence through transactivation of the androgen receptor. However, whether IL-6 has a causative role in de novo prostate tumorigenesis was never investigated. We now provide the first evidence that IL-6 can induce tumorigenic conversion and further progression to an invasive phenotype of non-tumorigenic benign prostate epithelial cells. Moreover, we find that paracrine IL-6 stimulates the autocrine IL-6 loop and autocrine activation of insulin-like type I growth factor receptor (IGF-IR) to confer the tumorigenic property and also that activation of signal transducer and activator of transcription 3 (STAT3) is critical in these processes. Inhibition of STAT3 activation or IGF-IR signaling suppresses IL-6-mediated malignant conversion and the associated invasive phenotype. Inhibition of STAT3 activation suppresses IL-6-induced upregulation of IGF-IR and its ligands, namely IGF-I and IGF-II. These findings indicate that IL-6 signaling cooperates with IGF-IR signaling in the prostate microenvironment to promote prostate tumorigenesis and progression to aggressiveness. Our findings suggest that STAT3 and IGF-IR may represent potential effective targets for prevention or treatment of prostate cancer.

The androgen-regulated type II serine protease TMPRSS2 is differentially expressed and mislocalized in prostate adenocarcinoma.

Transmembrane serine protease 2 (TMPRSS2) is an androgen-regulated member of the type two transmembrane protease (TTSP) family. Two other members of the TTSP family, matriptase and hepsin, are over-expressed in prostate adenocarcinoma and mechanistically influence cancer cell invasion and metastasis. This study was performed to determine TMPRSS2 protein expression in primary and metastatic prostate cancers. We developed a monoclonal antibody capable of the sensitive and specific detection of TMPRSS2 protein. TMPRSS2 regulation by androgen and presence in seminal fluid was measured. TMPRSS2 localization and expression was evaluated in 415 cases of primary prostate cancer and 144 prostate cancer metastases by immunohistochemistry. We determined that TMPRSS2 protein expression is regulated by androgens and that TMPRSS2 is a component of the normal seminal fluid proteome. TMPRSS2 protein is abundantly expressed in the prostate, with low levels in the epithelia of the colon, stomach, epididymis and breast. Pancreatic acini, hepatic bile ducts, testicular Leydig cells and the kidney also express TMPRSS2. In the prostate, TMPRSS2 protein is specifically localized to the secretory epithelium, with enhanced expression in the plasma membrane orientated towards the ductal lumen. TMPRSS2 expression was significantly higher in both neoplastic prostate and in the epithelium of prostatic hyperplasia compared to normal epithelium (p < 0.01). TMPRSS2 expression was further elevated in higher Gleason grade cancers (patterns 4 and 5) compared to pattern 3 (p = 0.04). Furthermore, in most high-grade cancers, TMPRSS2 was mislocalized, being expressed in the cytoplasm as well as in the cell membrane. Prostate cancer metastases also generally expressed high levels of TMPRSS2. In summary, the TMPRSS2 protease is expressed highly in primary and metastatic prostate cancers and is associated with tumour cell differentiation. Based on studies with the related proteins matriptase and hepsin, TMPRSS2 should be investigated for causal roles in prostate carcinogenesis.

Profiling influences of senescent and aged fibroblasts on prostate carcinogenesis.

Experimental evidence suggests that ageing-associated alterations in the tissue microenvironment act to promote prostate carcinogenesis. In this review, we survey the cellular state of senescence, review its causes, and describe associations with ageing and cancer. We further discuss senescent stromal gene expression changes, which may mediate these effects, and that may serve as therapeutic targets.

Characterization of hK4 (prostase), a prostate-specific serine protease: activation of the precursor of prostate specific antigen (pro-PSA) and single-chain urokinase-type plasminogen activator and degradation of prostatic acid phosphatase.

hK4 (prostase, KLK4), a recently cloned prostate-specific serine protease and a member of the tissue kallikrein family, is a zymogen composed of 228 amino acid residues including an amino-terminal propiece, Ser-Cys-Ser-Gln-. A chimeric form of hK4 (ch-hK4) was constructed in which the propiece of hK4 was replaced by that of prostate-specific antigen (PSA) to create an activation site susceptible to trypsin-type proteases. ch-hK4 was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified with an overall yield of 25%. The zymogen was readily self-activated during the refolding process to generate an active form (21 kDa) of hK4 (rhK4). rhK4 cleaved the chromogenic substrates Val-Leu-Arg-pNA (S-2266), Pro-Phe-Arg-pNA (S-2302), Ile-Glu-Gly-Arg-pNA (S-2222), and Val-Leu-Lys-pNA (S-2251), indicating that rhK4 has a trypsin-type substrate specificity. The rhK4 was inhibited by aprotinin (6 kDa), forming an equimolar 27 kDa complex. rhK4 readily activated both the precursor of PSA (pro-PSA) and single chain urokinase-type plasminogen activator (scuPA, pro-uPA). rhK4 also completely degraded prostatic acid phosphatase but failed to cleave serum albumin, another protein purified from human seminal plasma. These results indicate that hK4 may have a role in the physiologic processing of seminal plasma proteins such as pro-PSA, as well as in the pathogenesis of prostate cancer through its activation of pro-uPA.

Project normal: defining normal variance in mouse gene expression.

The mouse has become an indispensable and versatile model organism for the study of development, genetics, behavior, and disease. The application of comprehensive gene expression profiling technologies to compare normal and diseased tissues or to assess molecular alterations resulting from various experimental interventions has the potential to provide highly detailed qualitative and quantitative descriptions of these processes. Ideally, to interpret experimental data, the magnitude and diversity of gene expression for the system under study should be well characterized, yet little is known about the normal variation of mouse gene expression in vivo. To assess natural differences in murine gene expression, we used a 5406-clone spotted cDNA microarray to quantitate transcript levels in the kidney, liver, and testis from each of 6 normal male C57BL6 mice. We used ANOVA to compare the variance across the six mice to the variance among four replicate experiments performed for each mouse tissue. For the 6 kidney samples, 102 of 3,088 genes (3.3%) exhibited a statistically significant mouse variance at a level of 0.05. In the testis, 62 of 3,252 genes (1.9%) showed statistically significant variance, and in the liver, there were 21 of 2,514 (0.8%) genes with significantly variable expression. Immune-modulated, stress-induced, and hormonally regulated genes were highly represented among the transcripts that were most variable. The expression levels of several genes varied significantly in more than one tissue. These studies help to define the baseline level of variability in mouse gene expression and emphasize the importance of replicate microarray experiments.

From genomics to proteomics: techniques and applications in cancer research.

New technologies designed to facilitate the comprehensive analyses of genomes, transcriptomes and proteomes in health and disease are poised to exert a dramatic change on the pace of cancer research and to impact significantly on the care of cancer patients. These approaches have already demonstrated the power of molecular medicine in discriminating among disease subtypes that are not recognizable using traditional pathological criteria and in identifying specific genetic events involved in cancer progression. This review outlines the current status of these technologies and highlights recent studies in which they have been applied in the context of carcinogenesis.