Role of a novel race-related tumor suppressor microRNA located in frequently deleted chromosomal locus 8p21 in prostate cancer progression.

The prostate cancer (PCa) genome is characterized by deletions of chromosome 8p21-22 region that increase significantly with tumor grade and are associated with poor prognosis. We proposed and validated a novel, paradigm-shifting hypothesis that this region is associated with a set of microRNA genes-miR-3622, miR-3622b, miR-383-that are lost in PCa and play important mechanistic roles in PCa progression and metastasis. Extending our hypothesis, in this study, we evaluated the role of a microRNA gene located in chromosome 8p-miR-4288-by employing clinical samples and cell lines. Our data suggests that (i) miR-4288 is widely downregulated in primary prostate tumors and cell lines; (ii) miR-4288 expression is lost in metastatic castration-resistant PCa; (ii) miR-4288 downregulation is race-related PCa alteration that is prevalent in Caucasian patients and not in African Americans; (iii) in Caucasians, miR-4288 was found to be associated with increasing tumor grade and high serum prostate-specific antigen, suggesting that miR-4288 downregulation/loss may be associated with tumor progression specifically in Caucasians; (iv) miR-4288 possess significant potential as a molecular biomarker to predict aggressiveness/metastasis; and (v) miR-4288 is anti-proliferative, is anti-invasive and inhibits epithelial-to-mesenchymal transition; and (vi) miR-4288 directly represses expression of metastasis/invasion-associated genes MMP16 and ROCK1. Thus, the present study demonstrates a tumor suppressor role for a novel miRNA located with a frequently lost region in PCa, strengthening our hypothesis that this locus is causally related to PCa disease progression via loss of microRNA genes. Our study suggests that miR-4288 may be a novel biomarker and therapeutic target, particularly in Caucasians.

MicroRNAs as Regulators of Prostate Cancer Metastasis.

Prostate cancer causes significant morbidity in men and metastatic disease is a major cause of cancer related deaths. Prostate metastasis is controlled by various cellular intrinsic and extrinsic factors, which are often under the regulatory control of various metastasis-associated genes. Given the dynamic nature of metastatic cancer cells, the various factors controlling this process are themselves regulated by microRNAs which are small non-coding RNAs. Significant research work has shown differential microRNA expression in primary and metastatic prostate cancer suggesting their importance in prostate pathogenesis. We will review the roles of different microRNAs in controlling the various steps in prostate metastasis.

Polyethylene Glycol 20k. Does It Fluoresce?

Polyethylene glycol (PEG) is a polyether compound commonly used in biological research and medicine because it is biologically inert. This simple polymer exists in variable chain lengths (and molecular weights). As they are devoid of any contiguous π-system, PEGs are expected to lack fluorescence properties. However, recent studies suggested the occurrence of fluorescence properties in non-traditional fluorophores like PEGs. Herein, a thorough investigation has been conducted to explore if PEG 20k fluoresces. Results of this combined experimental and computational study suggested that although PEG 20k could exhibit "through-space" delocalization of lone pairs of electrons in aggregates/clusters, formed via intermolecular and intramolecular interactions, the actual contributor of fluorescence between 300 and 400 nm is the stabilizer molecule, i.e., 3-tert-butyl-4-hydroxyanisole present in the commercially available PEG 20k. Therefore, the reported fluorescence properties of PEG should be taken with a grain of salt, warranting further investigation.

MicroRNA-4287 is a novel tumor suppressor microRNA controlling epithelial-to mesenchymal transition in prostate cancer.

Prostate cancer (PCa) is a significant cause of male morbidity in the United States. Despite recent advances in diagnosis and therapeutic interventions, significant fraction of cases still progress to an advanced stage. Various genetic/epigenetic elements that facilitate this progression are not yet completely known and the mechanism that favors advanced disease is an area of investigation. A characteristic feature associated with progressive disease is deletion of chromosome 8p (chr8p) region, that harbors tumor-suppressor NKX3.1. Previous studies from our group has shown that there are cluster of microRNAs (miRNAs) located within this region whose loss favors advanced, metastatic disease. miR-4287 is a novel miRNA located within this region that has not been studied before. In the present study, we analyzed the role of miR-4287 in PCa using clinical tissues and cell lines. We observed that miR-4287 is significantly downregulated in patient-derived tumor tissues. Receiver operating curve (ROC) analysis showed that miR-4287 distinguishes prostate cancer from normal with a specificity of 88.24% and with an Area under the curve (AUC) of 0.66. Further, we found that miR-4287 levels correlate inversely with patients' serum prostate-specific antigen levels. Ectopic over-expression of miR-4287 in PCa cell lines showed that miR-4287 plays a tumor suppressor role. miR-4287 led to an increase in G2/M phase of cell cycle in PCa cell lines. Further, ectopic miR-4287 inhibited PCa epithelial-to-mesenchymal transition (EMT) by directly repressing SLUG and stem cell marker CD44. Since miR-4287 specifically targets metastasis pathway mediators, miR-4287 has potential diagnostic and therapeutic significance in preventing advanced, metastatic disease.

MicroRNA determinants of neuroendocrine differentiation in metastatic castration-resistant prostate cancer.

Therapy-induced neuroendocrine prostate cancer (NEPC), an extremely aggressive variant of castration-resistant prostate cancer (CRPC), is increasing in incidence with the widespread use of highly potent androgen receptor (AR)-pathway inhibitors (APIs) such as Enzalutamide (ENZ) and Abiraterone and arises via a reversible trans-differentiation process, referred to as neuroendocrine differentiation (NED). The molecular basis of NED is not completely understood leading to a lack of effective molecular markers for its diagnosis. Here, we demonstrate for the first time, that lineage switching to NE states is accompanied by key miRNA alterations including downregulation of miR-106a~363 cluster and upregulation of miR-301a and miR-375. To systematically investigate the key miRNAs alterations driving therapy-induced NED, we performed small RNA-NGS in a retrospective cohort of human metastatic CRPC clinical samples + PDX models with adenocarcinoma features (CRPC-adeno) vs those with neuroendocrine features (CRPC-NE). Further, with the application of machine learning algorithms to sequencing data, we trained a 'miRNA classifier' that could robustly classify 'CRPC-NE' from 'CRPC-Adeno' cases. The performance of classifier was validated in an additional cohort of mCRPC patients and publicly available PCa cohorts. Importantly, we demonstrate that miR-106a~363 cluster pleiotropically regulate cardinal nodal proteins instrumental in driving NEPC including Aurora Kinase A, N-Myc, E2F1 and STAT3. Our study has important clinical implications and transformative potential as our 'miRNA classifier' can be used as a molecular tool to stratify mCRPC patients into those with/without NED and guide treatment decisions. Further, we identify novel miRNA NED drivers that can be exploited for NEPC therapeutic targeting.

BRN4 Is a Novel Driver of Neuroendocrine Differentiation in Castration-Resistant Prostate Cancer and Is Selectively Released in Extracellular Vesicles with BRN2.

PURPOSE: Neuroendocrine prostate cancer (NEPC), an aggressive variant of castration-resistant prostate cancer (CRPC), often emerges after androgen receptor-targeted therapies such as enzalutamide or de novo, via trans-differentiation process of neuroendocrine differentiation. The mechanistic basis of neuroendocrine differentiation is poorly understood, contributing to lack of effective predictive biomarkers and late disease recognition. The purpose of this study was to examine the role of novel proneural Pit-Oct-Unc-domain transcription factors (TF) in NEPC and examine their potential as noninvasive predictive biomarkers.Experimental Design: Prostate cancer patient-derived xenograft models, clinical samples, and cellular neuroendocrine differentiation models were employed to determine the expression of TFs BRN1 and BRN4. BRN4 levels were modulated in prostate cancer cell lines followed by functional assays. Furthermore, extracellular vesicles (EV) were isolated from patient samples and cell culture models, characterized by nanoparticle tracking analyses, Western blotting, and real-time PCR. RESULTS: We identify for the first time that: (i) BRN4 is amplified and overexpressed in NEPC clinical samples and that BRN4 overexpression drives neuroendocrine differentiation via its interplay with BRN2, a TF that was previously implicated in NEPC; (ii) BRN4 and BRN2 mRNA are actively released in prostate cancer EVs upon neuroendocrine differentiation induction; and (iii) enzalutamide treatment augments release of BRN4 and BRN2 in prostate cancer EVs, promoting neuroendocrine differentiation induction. CONCLUSIONS: Our study identifies a novel TF that drives NEPC and suggests that as adaptive mechanism to enzalutamide treatment, prostate cancer cells express and secrete BRN4 and BRN2 in EVs that drive oncogenic reprogramming of prostate cancer cells to NEPC. Importantly, EV-associated BRN4 and BRN2 are potential novel noninvasive biomarkers to predict neuroendocrine differentiation in CRPC.

microRNA-1246 Is an Exosomal Biomarker for Aggressive Prostate Cancer.

Because of high heterogeneity, molecular characterization of prostate cancer based on biopsy sampling is often challenging. Hence, a minimally invasive method to determine the molecular imprints of a patient's tumor for risk stratification would be advantageous. In this study, we employ a novel, digital amplification-free quantification method using the nCounter technology (NanoString Technologies) to profile exosomal serum miRNAs (ex-miRNA) from aggressive prostate cancer cases, benign prostatic hyperplasia, and disease-free controls. We identified several dysregulated miRNAs, one of which was the tumor suppressor miR-1246. miR-1246 was downregulated in prostate cancer clinical tissues and cell lines and was selectively released into exosomes. Overexpression of miR-1246 in a prostate cancer cell line significantly inhibited xenograft tumor growth in vivo and increased apoptosis and decreased proliferation, invasiveness, and migration in vitro miR-1246 inhibited N-cadherin and vimentin activities, thereby inhibiting epithelial-mesenchymal transition. Ex-miR-1246 expression correlated with increasing pathologic grade, positive metastasis, and poor prognosis. Our analyses suggest ex-miR-1246 as a promising prostate cancer biomarker with diagnostic potential that can predict disease aggressiveness.Significance: Dysregulation of exosomal miRNAs in aggressive prostate cancer leads to alteration of key signaling pathways associated with metastatic prostate cancer. Cancer Res; 78(7); 1833-44. ©2018 AACR.

A novel microRNA regulator of prostate cancer epithelial-mesenchymal transition.

The most frequent alteration in the prostate oncogenome is loss of chromosome (chr) 8p21 that has been associated with loss of NKX3.1 homeobox gene. Chr8p21 deletions increase significantly with tumor grade and are associated with poor prognosis in prostate cancer (PCa), suggesting critical involvement of this region in tumor progression. Recent studies suggest that apart from NKX3.1, this region harbors alternative tumor suppressors that are yet undefined. We proposed a novel, paradigm shifting hypothesis that this locus is associated with a miRNA gene cluster-miR-3622a/b- that plays a crucial suppressive role in PCa. Here we demonstrate the crucial role of miR-3622a in prostate cancer epithelial-to-mesenchymal transition (EMT). MicroRNA expression profiling in microdissected human PCa clinical tissues showed that miR-3622a expression is widely downregulated and is significantly correlated with poor survival outcome and tumor progression. To understand the functional significance of miR-3622a, knockdown and overexpression was performed using non-transformed prostate epithelial and PCa cell lines, respectively, followed by functional assays. Our data demonstrate that endogenous miR-3622a expression is vital to maintain the epithelial state of normal and untransformed prostate cells. miR-3622a expression inhibits EMT, progression and metastasis of PCa in vitro and in vivo. Further, we found that miR-3622a directly targets EMT effectors ZEB1 and SNAI2. In view of these data, we propose that frequent loss of miR-3622a at chr8p21 region leads to induction of EMT states that in turn, promotes PCa progression and metastasis. This study has potentially significant implications in the field of prostate cancer as it identifies an important miRNA component of a frequently lost chromosomal region with critical roles in prostate carcinogenesis which is a highly significant step towards understanding the mechanistic involvement of this locus. Also, our study indicates that miR-3622a is a novel PCa biomarker and potential drug target for developing therapeutic regimens against advanced PCa.

Significance of Proline Residue on Short Mucin Peptide Interactions with Mouse MUC1 Monoclonal Antibody Studied by Saturation Transfer Difference NMR Spectroscopy.

In this study we investigated to see whether or not a shortened MUC1 mucin peptide epitope with the sequence GVTSAPD containing a single prolyl residue would still bind specific monoclonal antibody as its native sequence (e.g., PDTRP), known to be the specific recognition site on the Variable Number Tandem Repeat (VNTR) region of MUC1 mucin by the immune system. The affinity of GVTSAPD peptide to a mouse Muc1 mucin specific monoclonal antibody (clone 6A4, IgG1 isotype) was investigated by Saturation Transfer Difference NMR spectroscopy (STD NMR). Results showed that the shortened mucin epitope GVTSAPD still retained affinity to Muc1 specific monoclonal antibody (mAb) while one that lacks the prolyl residue at position 6 lost its affinity, which suggests that P6 is necessay for antibody binding. The interactions observed by STD NMR occurred strongest at the P6 side chain 1H's (ßH and γH); the P6Hα showed lower degree of saturation transfer effect. Minor interactions also occurred at the methyl groups of V2' T3 and A5. Mucin peptides derived from the VNTR region have been the target of cancer vaccine research, thus properties associated with mucin peptide structure, conformation and antibody interaction are central to peptide design or engineering towards that end.