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.

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.

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.

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.

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 novel mechanism for chaperone-mediated telomerase regulation during prostate cancer progression.

Telomerase activity has been detected in >85% of all malignant human cancers, including 90% of prostate carcinomas. Using a well-characterized experimental prostate cancer system, we have found that telomerase activity is notably increased (>10-fold) during tumorigenic conversion. Expression profiles of the telomerase components (hTR and hTERT) revealed no substantive changes, which suggests a nontranscriptional mechanism for increased activity. Because the hsp90 chaperone complex functionally associates with telomerase, we investigated that relationship and found that along with telomerase activity, a number of hsp90-related chaperones are markedly elevated during transformation, as well as in advanced prostate carcinomas. Using the nontumorigenic cell protein extract as the source of telomerase, addition of purified chaperone components enhanced reconstitution of telomerase activity, which suggests a novel mechanism of increased telomerase assembly via a hsp90 chaperoning process during prostate cancer progression.

Prostate short-chain dehydrogenase reductase 1 (PSDR1): a new member of the short-chain steroid dehydrogenase/reductase family highly expressed in normal and neoplastic prostate epithelium.

Genes regulated by androgenic hormones are of critical importance for the normal physiological function of the human prostate gland, and they contribute to the development and progression of prostate carcinoma. We used cDNA microarrays comprised of prostate-derived cDNAs to profile transcripts regulated by androgens in prostate cancer cells. This study identified a novel gene that we have designated prostate short-chain dehydrogenase/reductase 1 (PSDR1), that exhibits increased expression on exposure to androgens in the LNCaP prostate cancer cell line. Northern analysis demonstrated that PSDR1 is highly expressed in the prostate gland relative to other normal human tissues. The PSDR1 cDNA and putative protein exhibit homology to the family of short-chain dehydrogenase/reductase enzymes and thus identify a new member of this family. Cloning and analysis of the putative PSDR1 promoter region identified a potential androgen-response element. We used a radiation-hybrid panel to map the PSDR1 gene to chromosome 14q23-24.3. In situ hybridization localizes PSDR1 expression to normal and neoplastic prostate epithelium. These results identify a new gene involved in the androgen receptor-regulated gene network of the human prostate that may play a role in the pathogenesis of prostate carcinoma.

Hevin, an antiadhesive extracellular matrix protein, is down-regulated in metastatic prostate adenocarcinoma.

Hevin, a gene closely related to the extracellular matrix protein SPARC, is an acidic cysteine-rich glycoprotein shown to be important for the adhesion and trafficking of cells through the endothelium. Through the use of differential display and differential EST analysis, we identified Hevin as a gene whose transcription is down-regulated in transformed prostate epithelial cell lines and metastatic prostate adenocarcinoma. These results were confirmed by comparing expression levels between normal and neoplastic human prostate tissues using Northern analysis. In situ hybridization with an 35S-labeled antisense riboprobe demonstrated the loss of Hevin expression in metastatic prostate carcinoma. The expression pattern of Hevin in transformed and metastatic epithelium may provide further insights into the complex cell adhesion events involved in the metastatic progression of prostate carcinoma.

PART-1: a novel human prostate-specific, androgen-regulated gene that maps to chromosome 5q12.

Genes regulated by androgenic hormones are of critical importance for the normal physiological function of the human prostate gland, and they contribute to the development and progression of prostate carcinoma. We used cDNA microarrays containing 1500 prostate-derived cDNAs to profile transcripts regulated by androgens in prostate cancer cells. This study identified a novel gene that we have designated PART-1 (prostate androgen-regulated transcript 1), which exhibited increased expression upon exposure to androgens in the LNCaP prostate cancer cell line. Northern analysis demonstrated that PART-1 is highly expressed in the prostate gland relative to other normal human tissues and is expressed as different transcripts using at least three different polyadenylation signals. The PART-1 cDNA and putative protein are not significantly homologous to any sequences in the nonredundant public sequence databases. Cloning and analysis of the putative PART-1 promoter region identified a potential binding site for the homeobox gene PBX-la, but no consensus androgen response element or sterol-regulatory element binding sites were identified. We used a radiation hybrid panel and fluorescence in situ hybridization to map the PART-1 gene to chromosome 5q12, a region that has been suggested to harbor a prostate tumor suppressor gene. These results identify a new gene involved in the androgen receptor-regulated gene network of the human prostate that may play a role in the etiology of prostate carcinogenesis.

Prostate-localized and androgen-regulated expression of the membrane-bound serine protease TMPRSS2.

Genes regulated by androgenic hormones are of critical importance for the normal physiological function of the human prostate gland, and they contribute to the development and progression of prostate carcinoma. We used cDNA microarrays containing 1500 cDNAs to profile transcripts regulated by androgens in prostate cancer cells and identified the serine protease TMPRSS2 as a gene exhibiting increased expression upon exposure to androgens. The TMPRSS2 gene is located on chromosome 21 and contains four distinct domains, including a transmembrane region, indicating that it is expressed on the cell surface. Northern analysis demonstrated that TMPRSS2 is highly expressed in prostate epithelium relative to other normal human tissues. In situ hybridization of normal and malignant prostate tissues localizes TMMPRSS2 expression to prostate basal cells and to prostate carcinoma. These results suggest that TMPRSS2 may play a role in prostate carcinogenesis and should be investigated as a diagnostic or therapeutic target for the management of prostate cancers.

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.

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.

Toll receptors: an expanding role in our understanding of human disease.

Toll receptor proteins in Drosophila are involved in establishing the dorsal-ventral axis in embryogenesis as well as participating in the innate immune response to invading pathogens. The basic mediators of this response show striking similarities in plants, insects, and vertebrates. The cytoplasmic signaling cascade is exemplified by the human interleukin-1 receptor complex (IL-1R), resulting in transcriptional activation of effector proteins through nuclear factor-kappaB (NF-kappaB). Six mammalian/human Toll-like receptors (TLR) have been described to date. The TLRs share the IL-1R cytoplasmic signaling cascade but are distinguished by their extracellular leucine-rich repeat (LRR) structure. The LRR superfamily comprises a diverse group of proteins, including a cohort involved in transmembrane signaling. Two of the human TLRs (TLR2, TLR4) have been shown to be involved in the innate response to bacterial pathogens and appear to provide a link between the innate and adaptive immune response. A better understanding of this response may provide improved therapeutic modalities in the treatment of bacterial and fungal sepsis, which continues to be a significant source of morbidity and mortality worldwide. In addition, similar to Drosophila, Toll receptors and related proteins in the LRR superfamily may also be involved in human development, as well as in noninfectious human disease.

Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray.

The development of cancer is the result of a series of molecular changes occurring in the cell. These events lead to changes in the expression level of numerous genes that result in different phenotypic characteristics of tumors. In this report we describe the assembly and utilization of a 5766 member cDNA microarray to study the differences in gene expression between normal and neoplastic human ovarian tissues. Several genes that may have biological relevance in the process of ovarian carcinogenesis have been identified through this approach. Analyzing the results of microarray hybridizations may provides new leads for tumor diagnosis and intervention.

Comparative hybridization of an array of 21,500 ovarian cDNAs for the discovery of genes overexpressed in ovarian carcinomas.

Comparative hybridization of cDNA arrays is a powerful tool for the measurement of differences in gene expression between two or more tissues. We optimized this technique and employed it to discover genes with potential for the diagnosis of ovarian cancer. This cancer is rarely identified in time for a good prognosis after diagnosis. An array of 21,500 unknown ovarian cDNAs was hybridized with labeled first-strand cDNA from 10 ovarian tumors and six normal tissues. One hundred and thirty-four clones are overexpressed in at least five of the 10 tumors. These cDNAs were sequenced and compared to public sequence databases. One of these, the gene HE4, was found to be expressed primarily in some ovarian cancers, and is thus a potential marker of ovarian carcinoma.