Genomic Characterization of Preclinical Prostate Cancer Cell Line Models.

As we move into the era of precision medicine, the growing relevance of genetic alterations to prostate cancer (PCa) development and treatment demonstrates the importance of characterizing preclinical models at the genomic level. Our study investigated the genomic characterization of eight PCa cell lines to understand which models are clinically relevant. We designed a custom AmpliSeq DNA gene panel that encompassed key molecular pathways targeting AR signaling, apoptosis, DNA damage repair, and PI3K/AKT/PTEN, in addition to tumor suppressor genes. We examined the relationship between cell line genomic alterations and therapeutic response. In addition, using DepMap's Celligner tool, we identified which preclinical models are most representative of specific prostate cancer patient populations on cBioPortal. These data will help investigators understand the genetic differences in preclinical models of PCa and determine which ones are relevant for use in their translational research.

PARP Inhibitors and Prostate Cancer: To Infinity and Beyond BRCA.

The U.S. Food and Drug Administration recently approved two poly-adenosine diphosphate-ribose polymerase (PARP) inhibitors, olaparib and rucaparib, for treatment of biomarker-positive metastatic castrate resistant prostate cancer. The benefits of PARP inhibition have been well characterized in patients who have BRCA1 and BRCA2 mutations in several forms of cancer. BRCA1 and BRCA2 occupy key roles in DNA damage repair, which is comprised of several different pathways with numerous participants. Patients with mutations in other key genes within the DNA damage repair pathway may also respond to treatment with PARP inhibitors, and identification of these alterations could significantly increase the percentage of patients that may benefit from PARP inhibition. This review focuses on the potential for synthetically lethal interactions between PARP inhibitors and non-BRCA DNA damage repair genes. IMPLICATIONS FOR PRACTICE: The treatment potential of PARP inhibition has been well characterized in patients with BRCA1 and BRCA2 mutations, but there is compelling evidence for expanding the use of PARP inhibitors to mutations of other non-BRCA DNA damage repair (DDR) genes. This could increase the percentage of patients that may benefit from treatment with PARP inhibitors alone or in combination with other therapies. Understanding the significance of PARP inhibitor-sensitizing alterations in other common non-BRCA DDR genes will help guide clinical decisions to provide targeted treatment options to a wider population of patients.

Comparison of Eight Technologies to Determine Genotype at the UGT1A1 (TA)n Repeat Polymorphism: Potential Clinical Consequences of Genotyping Errors?

To ensure accuracy of UGT1A1 (TA)n (rs3064744) genotyping for use in pharmacogenomics-based irinotecan dosing, we tested the concordance of several commonly used genotyping technologies. Heuristic genotype groupings and principal component analysis demonstrated concordance for Illumina sequencing, fragment analysis, and fluorescent PCR. However, Illumina sequencing and fragment analysis returned a range of fragment sizes, likely arising due to PCR "slippage". Direct sequencing was accurate, but this method led to ambiguous electrophoregrams, hampering interpretation of heterozygotes. Gel sizing, pyrosequencing, and array-based technologies were less concordant. Pharmacoscan genotyping was concordant, but it does not ascertain (TA)8 genotypes that are common in African populations. Method-based genotyping differences were also observed in the publication record (p < 0.0046), although fragment analysis and direct sequencing were concordant (p = 0.11). Genotyping errors can have significant consequences in a clinical setting. At the present time, we recommend that all genotyping for this allele be conducted with fluorescent PCR (fPCR).

Association of androgen metabolism gene polymorphisms with prostate cancer risk and androgen concentrations: Results from the Prostate Cancer Prevention Trial.

BACKGROUND: Prostate cancer is highly influenced by androgens and genes. The authors investigated whether genetic polymorphisms along the androgen biosynthesis and metabolism pathways are associated with androgen concentrations or with the risk of prostate cancer or high-grade disease from finasteride treatment. METHODS: A nested case-control study from the Prostate Cancer Prevention Trial using data from men who had biopsy-proven prostate cancer (cases) and a group of biopsy-negative, frequency-matched controls was conducted to investigate the association of 51 single nucleotide polymorphisms (SNPs) in 12 genes of the androgen pathway with overall (total), low-grade, and high-grade prostate cancer incidence and serum hormone concentrations. RESULTS: There were significant associations of genetic polymorphisms in steroid 5α-reductase 1 (SRD5A1) (reference SNPs: rs3736316, rs3822430, rs1560149, rs248797, and rs472402) and SRD5A2 (rs2300700) with the risk of high-grade prostate cancer in the placebo arm of the Prostate Cancer Prevention Trial; 2 SNPs were significantly associated with an increased risk (SRD5A1 rs472402 [odds ratio, 1.70; 95% confidence interval, 1.05-2.75; Ptrend = .03] and SRD5A2 rs2300700 [odds ratio, 1.94; 95% confidence interval, 1.19-3.18; Ptrend = .01]). Eleven SNPs in SRD5A1, SRD5A2, cytochrome P450 family 1, subfamily B, polypeptide 1 (CYP1B1), and CYP3A4 were associated with modifying the mean concentrations of serum androgen and sex hormone-binding globulin; and 2 SNPs (SRD5A1 rs824811 and CYP1B1 rs10012; Ptrend < .05) consistently and significantly altered all androgen concentrations. Several SNPs (SRD5A1 rs3822430, SRD5A2 rs2300700, CYP3A43 rs800672, and CYP19 rs700519; Ptrend < .05) were significantly associated with both circulating hormone levels and prostate cancer risk. CONCLUSIONS: Germline genetic variations of androgen-related pathway genes are associated with serum androgen concentrations and the risk of prostate cancer. Further studies to examine the functional consequence of novel causal variants are warranted. Cancer 2016;122:2332-2340. © 2016 American Cancer Society.

Androgen receptor variation affects prostate cancer progression and drug resistance.

Significant therapeutic progress has been made in treating prostate cancer in recent years. Drugs such as enzalutamide, abiraterone, and cabazitaxel have expanded the treatment armamentarium, although it is not completely clear which of these drugs are the most-effective option for individual patients. Moreover, such advances have been tempered by the development of therapeutic resistance. The purpose of this review is to summarize the current literature pertaining to the biochemical effects of AR variants and their consequences on prostate cancer therapies at both the molecular level and in clinical treatment. We address how these AR splice variants and mutations affect tumor progression and therapeutic resistance and discuss potential novel therapeutic strategies under development. It is hoped that these therapies can be administered with increasing precision as tumor genotyping methods become more sophisticated, thereby lending clinicians a better understanding of the underlying biology of prostate tumors in individual patients.

Dual targeting of the androgen receptor and hypoxia-inducible factor 1α pathways synergistically inhibits castration-resistant prostate cancer cells.

Enzalutamide is a potent second-generation androgen receptor (AR) antagonist with activity in metastatic castrate-resistant prostate cancer (CRPC). Although enzalutamide is initially effective, disease progression inevitably ensues with the emergence of resistance. Intratumoral hypoxia is also associated with CRPC progression and treatment resistance. Given that both AR and hypoxia inducible factor-1 α (HIF-1α) are key regulators of these processes, dual targeting of both signaling axes represents an attractive therapeutic approach. Crosstalk of the AR and HIF-1α signaling pathways were examined in prostate cancer cell lines (LNCaP, 22Rv1) with assays measuring the effect of androgen and hypoxia on AR-dependent and hypoxia-inducible gene transcription, protein expression, cell proliferation, and apoptosis. HIF-1α inhibition was achieved by siRNA silencing HIF-1α or via chetomin, a disruptor of HIF-1α-p300 interactions. In prostate cancer cells, the gene expression of AR targets (KLK3, FKBP5, TMPRSS2) was repressed by HIF-signaling; conversely, specific HIF-1α target expression was induced by dihydrotestosterone-mediated AR signaling. Treatment of CRPC cells with enzalutamide or HIF-1α inhibition attenuated AR-regulated and HIF-1α-mediated gene transcription. The combination of enzalutamide and HIF-1α inhibition was more effective than either treatment alone. Similarly, the combination also reduced vascular endothelial growth factor protein levels. HIF-1α siRNA synergistically enhanced the inhibitory effect of enzalutamide on cell growth in LNCaP and enzalutamide-resistant 22Rv1 cells via increased enzalutamide-induced apoptosis. In conclusion, the combination of enzalutamide with HIF-1α inhibition resulted in synergistic inhibition of AR-dependent and gene-specific HIF-dependent expression and prostate cancer cell growth.

Genetic variation: effect on prostate cancer.

The crucial role of androgens in the development of prostate cancer is well established. The aim of this review is to examine the role of constitutional (germline) and tumor-specific (somatic) polymorphisms within important regulatory genes of prostate cancer. These include genes encoding enzymes of the androgen biosynthetic pathway, the androgen receptor gene, genes that encode proteins of the signal transduction pathways that may have a role in disease progression and survival, and genes involved in prostate cancer angiogenesis. Characterization of deregulated pathways critical to cancer cell growth have lead to the development of new treatments, including the CYP17 inhibitor abiraterone and clinical trials using novel drugs that are ongoing or recently completed [1]. The pharmacogenetics of the drugs used to treat prostate cancer will also be addressed. This review will define how germline polymorphisms are known affect a multitude of pathways, and therefore phenotypes, in prostate cancer etiology, progression, and treatment.

Epidithiodiketopiperazines (ETPs) exhibit in vitro antiangiogenic and in vivo antitumor activity by disrupting the HIF-1α/p300 complex in a preclinical model of prostate cancer.

The downstream targets of hypoxia inducible factor-1 alpha (HIF-1α) play an important role in tumor progression and angiogenesis. Therefore, inhibition of HIF-mediated transcription has potential in the treatment of cancer. One attractive strategy for inhibiting HIF activity is the disruption of the HIF-1α/p300 complex, as p300 is a crucial coactivator of hypoxia-inducible transcription. Several members of the epidithiodiketopiperazine (ETP) family of natural products have been shown to disrupt the HIF-1α/p300 complex in vitro; namely, gliotoxin, chaetocin, and chetomin. Here, we further characterized the molecular mechanisms underlying the antiangiogenic and antitumor effects of these ETPs using a preclinical model of prostate cancer. In the rat aortic ring angiogenesis assay, gliotoxin, chaetocin, and chetomin significantly inhibited microvessel outgrowth at a GI50 of 151, 8, and 20 nM, respectively. In vitro co-immunoprecipitation studies in prostate cancer cell extracts demonstrated that these compounds disrupted the HIF-1α/p300 complex. The downstream effects of inhibiting the HIF-1α/p300 interaction were evaluated by determining HIF-1α target gene expression at the mRNA and protein levels. Dose-dependent decreases in levels of secreted VEGF were detected by ELISA in the culture media of treated cells, and the subsequent downregulation of VEGFA, LDHA, and ENO1 HIF-1α target genes were confirmed by semi-quantitative real-time PCR. Finally, treatment with ETPs in mice bearing prostate tumor xenografts resulted in significant inhibition of tumor growth. These results suggest that directly targeting the HIF-1α/p300 complex with ETPs may be an effective approach for inhibiting angiogenesis and tumor growth.

Bridging Health Disparities: a Genomics and Transcriptomics Analysis by Race in Prostate Cancer.

As the era of cancer genomics expands, disproportionate rates of prostate cancer incidence and mortality by race have demonstrated increasing relevance in clinical settings. While Black men are most particularly affected, as data has historically shown, the opposite is observed for Asian men, thus creating a basis for exploring genomic pathways potentially involved in mediating these opposing trends. Studies on racial differences are limited by sample size, but recent expanding collaborations between research institutions may improve these imbalances to enhance investigations on health disparities from the genomics front. In this study, we performed a race genomics analysis using GENIE v11, released in January 2022, to investigate mutation and copy number frequencies of select genes in both primary and metastatic patient tumor samples. Further, we investigate the TCGA race cohort to conduct an ancestry analysis and to identify differentially expressed genes highly upregulated in one race and subsequently downregulated in another. Our findings highlight pathway-oriented genetic mutation frequencies characterized by race, and further, we identify candidate gene transcripts that have differential expression between Black and Asian men.

Detection of Extracellular Vesicle-Derived RNA as Potential Prostate Cancer Biomarkers: Role of Cancer-type SLCO1B3 and ABCC3.

Extracellular vesicles (EVs) provide a minimally invasive liquid biopsy source of tumor-specific markers for patients who have already undergone prostatectomies. Our laboratory has previously demonstrated enrichment of the cancer-type solute carrier organic anion transporter family 1B3 (ct-SLCO1B3) and the ATP Binding Cassette Subfamily Member C (ABCC3) in castration-resistant cell lines (CRPC). However, their expression in EVs has yet to be explored. Our study demonstrated that ct-SLCO1B3 and ABCC3 are highly detectable in CRPC cell line-derived EVs. We also showed that ct-SLCO1B3 and ABCC3 were detectable in a CRPC xenograft mouse model, both intratumorally and in plasma-derived EVs. Our results provide evidence for EV-contained ct-SLCO1B3 and ABCC3 as novel, EV-based tumor markers for prostate cancer progression.

Targeting the metastatic niche: Single-cell lineage tracing in prime time.

Identification of actionable drug targets remains a rate-limiting step of, and one of the most prominent barriers to successful drug development for metastatic cancers. CRISPR-Cas9, a tool for making targeted genomic edits, has given rise to various novel applications that have greatly accelerated discovery in developmental biology. Recent work has coupled a CRISPR-Cas9-based lineage tracing platform with single-cell transcriptomics in the unexplored context of cancer metastasis. In this perspective, we briefly reflect on the development of these distinct technological advances and the process by which they have become integrated. We also highlight the importance of single-cell lineage tracing in oncology drug development and suggest the profound capacity of a high-resolution, computational approach to reshape cancer drug discovery by enabling identification of novel metastasis-specific drug targets and mechanisms of resistance.

GNRH2 Polymorphism in Men With Prostate Cancer Treated With Androgen Deprivation Therapy.

BACKGROUND/AIM: Gonadotropin-releasing hormone 2 (GNRH2) is a poorly-studied peptide hormone that is widely distributed in the central nervous system and expressed in peripheral tissues of mammals. The non-synonymous rs6051545 variant in GNRH2 (A16V) has been linked to higher serum testosterone concentrations. This study investigated whether the A16V variant is associated with altered androgen-deprivation therapy (ADT) progression-free survival (PFS) and overall survival (OS). PATIENTS AND METHODS: We examined the expression of GNRH2 in prostate tissue microarrays comprising normal tissue, prostatic hyperplasia, and prostate cancer using immunofluorescence. We also evaluated the GNRH2 genotype in 131 patients with prostate cancer who received ADT and compared PFS and OS between the variant and wild-type genotypes. RESULTS: GNRH2 was detected in all prostate tissues, although expression did not vary with Gleason grade or disease stage (p=0.71). The GNRH2 A16V genotype was not associated with PFS or OS; however, univariate and multivariate analyses revealed Gleason score and definitive local therapy were each associated with PFS (p≤0.0074), whereas age and Gleason score were associated with OS (p≤0.0046). CONCLUSION: GNRH2 is expressed in normal, hyperplastic, and neoplastic prostate tissues; the A16V variant is not related to treatment outcome or survival.

Racial disparities in the association between variants on 8q24 and prostate cancer: a systematic review and meta-analysis.

Recent studies implicate single nucleotide polymorphisms (SNPs) within the 8q24 region as a risk factor for prostate cancer (PCa). New developments suggest that 8q24 encodes regulators of the nearby MYC gene, a known oncogene. In order to better understand the implications of SNPs in this region, we performed meta-analyses, stratified by race, of seven SNPs and one microsatellite marker previously identified as risk loci on the 8q24 region of the genome. In addition, we reviewed the literature examining the possible associations between these polymorphisms and clinicopathological features of PCa. The results of the meta-analyses indicate that rs6983267, rs1447295, rs6983561, rs7837688, rs16901979, and DG8S737 are significantly associated with a higher risk for PCa for at least one race, whereas the variants rs13254738 and rs7000448 are not. The degree of association and frequency of the causative allele varied among men of different races. Though several studies have demonstrated an association between certain 8q24 SNPs and clinicopathological features of the disease, review of this topic revealed conflicting results.

Drugging the undruggable: activity-based protein profiling offers opportunities for targeting the KLK activome.

The vast majority of the human proteome is yet to be functionally characterized thus hindering ongoing investigations on potential drug resistance mechanisms and advanced treatment options. Chemical proteomics is a powerful solution for enzyme profiling and the development of next generation cancer therapeutics previously deemed undruggable by small molecules. Within this field, activity-based protein profiling (ABPP) is a specialized technology capable of discriminating enzyme interactions that occur within complex, biological environments. In a recent publication by Lovell et al, the kallikrein-related peptidase (KLK) family of serine proteases that is highly implicated in the progression of prostate cancer (PCa) was subject to ABPP to elucidate enzymatic activities in the presence of enzalutamide. This is the first report of ABPP in PCa and of activity-based chemical probes selective for individual KLKs. Further, the study reveals androgen receptor-dependent activity among KLK proteins, particularly in mediating the invasion of the bone microenvironment.

PARP inhibitors on the move in prostate cancer: spotlight on Niraparib & update on PARP inhibitor combination trials.

PARP inhibitors were recently introduced as a novel targeted therapy for biomarker positive metastatic castration resistant prostate cancer (mCRPC) patients, a population that inevitably acquires resistance to existing standard care regimens. Olaparib and rucaparib are now FDA-approved for mCRPC, while talazoparib and niraparib are advancing through the clinical stage of development. We highlight the recent results of the GALAHAD trial testing the efficacy of niraparib in mCRPC patients with DNA damage repair gene defects and compare its performance to key PARP inhibitor trials (PROFOUND, olaparib; TRITON2, rucaparib; TALAPRO-1, talazoparib). Finally, we briefly discuss recent updates on emerging PARP inhibitor and androgen receptor targeting combination trials as a novel treatment strategy for upfront treatment of mCRPC and in earlier disease settings.

Serum markers, obesity and prostate cancer risk: results from the prostate cancer prevention trial.

Molecular mechanisms linking obesity to prostate cancer involve steroid hormone and insulin/insulin-like growth factor 1 (IGF1) pathways. We investigated the association of circulating serum markers (e.g. androgens and IGFs/IGFBPs) with BMI and in modifying the association of obesity with prostate cancer risk. Data and specimens for this nested case-control study are from the Prostate Cancer Prevention Trial, a randomized, placebo-controlled trial of finasteride for prostate cancer prevention. Presence or absence of cancer was determined by prostate biopsy. Serum samples were assayed for sex steroid hormone concentrations and IGF1 axis analytes. Logistic regression estimated odds ratio and 95% CIs for risk of overall, low-grade (Gleason 2-6), and high-grade (Gleason 7-10) cancers. We found significant associations between BMI with serum steroids and IGFs/IGFBPs; the IGF1 axis was significantly associated with several serum steroids. Serum steroid levels did not affect the association of BMI with prostate cancer risk; however, IGFBP2 and IGFs modified the association of obesity with low- and high-grade disease. While serum steroids and IGFs/IGFBPs are associated with BMI, only the IGF1 axis contributed to obesity-related prostate cancer risk. Understanding the biological mechanisms linking obesity to prostate cancer risk as it relates to circulating serum markers will aid in developing effective prostate cancer prevention strategies and treatments.

Serum estrogen levels and prostate cancer risk in the prostate cancer prevention trial: a nested case-control study.

OBJECTIVE: Finasteride reduces prostate cancer risk by blocking the conversion of testosterone to dihydrotestosterone. However, whether finasteride affects estrogens levels or change in estrogens affects prostate cancer risk is unknown. METHODS: These questions were investigated in a case-control study nested within the prostate cancer prevention trial (PCPT) with 1,798 biopsy-proven prostate cancer cases and 1,798 matched controls. RESULTS: Among men on placebo, no relationship of serum estrogens with risk of prostate cancer was found. Among those on finasteride, those in the highest quartile of baseline estrogen levels had a moderately increased risk of Gleason score < 7 prostate cancer (for estrone, odds ratio [OR] = 1.51, 95% confidence interval [CI] = 1.06-2.15; for estradiol, OR = 1.50, 95% CI = 1.03-2.18). Finasteride treatment increased serum estrogen concentrations; however, these changes were not associated with prostate cancer risk. CONCLUSION: Our findings confirm those from previous studies that there are no associations of serum estrogen with prostate cancer risk in untreated men. In addition, finasteride results in a modest increase in serum estrogen levels, which are not related to prostate cancer risk. Whether finasteride is less effective in men with high serum estrogens, or finasteride interacts with estrogen to increase cancer risk, is uncertain and warrants further investigation.

The ERCC1 N118N polymorphism does not change cellular ERCC1 protein expression or platinum sensitivity.

Genetic polymorphisms in ERCC1 are thought to contribute to altered sensitivity to platinum-based chemotherapy. Although ERCC1 N118N (500 C>T, rs11615) is the most studied polymorphism, the impact of this polymorphism on platinum-based chemotherapy remains unclear. This is the first study in which the functional impact of ERCC1 N118N on gene expression and platinum sensitivity was explored. The aim of this study is to investigate if the reduced codon usage frequency of AAT, which contains the variant allele of the silent mutation, has functional impact on ERCC1 in a well-controlled biological system. Specifically, the ERCC1 cDNA clone with either the C or T allele was introduced into an ERCC1 deficient cell line, UV20, and assayed for the effect of the two alleles on ERCC1 transcription, translation and platinum sensitivity. Both ERCC1 mRNA and protein expression levels increased upon cisplatin treatment, peaking at 4h post-treatment, however there were no differences between the two alleles (p>0.05). Cells complemented with ERCC1 showed significantly higher survival proportion than the parental cell line following platinum exposure (p<0.0001), although no differences were observed between the cells transfected with the wild type or the polymorphic allele. These data suggest that N118N itself is not related to the phenotypic differences in ERCC1 expression or function, but rather this polymorphism may be linked to other causative variants or haplotypes.

Diversity on demand: multi-ancestry meta-analysis improves genetic risk prediction in prostate cancer.

Several genome-wide association studies have been conducted to identify genetic risk factors associated with prostate cancer, but their ability to discover new genetic variants and their applicability across ancestry groups have been limited by their lack of genetic diversity, owing to an underrepresentation of non-European populations. A recent meta-analysis published in Nature Genetics by Conti et al. has used a multi-ancestry approach to identify 86 new genetic loci associated with prostate cancer risk, refine leads in known risk regions, and develop a genetic risk score that is transferable across population groups. The findings of this study represent a significant advancement in genetic risk prediction for prostate cancer and their incorporation into standard screening protocols may lead to significant improvements in clinical outcomes.

Antitumor Activity of NLG207 (Formerly CRLX101) in Combination with Enzalutamide in Preclinical Prostate Cancer Models.

Effective treatments for patients with metastatic castration-resistant prostate cancer following disease progression on enzalutamide are currently an unmet clinical need. Simultaneous inhibition of the hypoxia-inducible factor (HIF)-1α and androgen receptor (AR) pathways has been previously shown to overcome enzalutamide resistance in vitro Combination treatment with NLG207, a nanoparticle-drug conjugate of camptothecin and inhibitor of HIF-1α, and enzalutamide was evaluated in preclinical prostate cancer models of enzalutamide resistance. The effect of NLG207 and enzalutamide on average tumor volume and tumor re-growth after 3 weeks of treatment was evaluated in vivo using the subcutaneous 22Rv1 xenograft and castrated subcutaneous VCaP xenograft models. Correlative assessments of antitumor activity were evaluated in vitro using cell proliferation and qPCR assays. NLG207 8 mg/kg alone and in combination with enzalutamide reduced average tumor volume by 93% after 3 weeks of treatment (P < 0.05) in comparison with vehicle control in the subcutaneous 22Rv1 xenograft model. Notably, the addition of NLG207 also enhanced the efficacy of enzalutamide alone in the castrated subcutaneous VCaP xenograft model, decreasing the median rate of tumor growth by 51% (P = 0.0001) in comparison with enzalutamide alone. In vitro assessments of cell proliferation and gene expression further demonstrated antitumor activity via AR-HIF-1α crosstalk inhibition. Combination treatment with NLG207 and enzalutamide was shown to be effective in preclinical prostate cancer models of enzalutamide resistance. Clinical investigation of this treatment combination is ongoing (NCT03531827).