Epigenetic silencing of miRNA-338-5p and miRNA-421 drives SPINK1-positive prostate cancer

Abstract

PurposeSerine Peptidase Inhibitor, Kazal type-1 (SPINK1) overexpression defines the second most recurrent and aggressive prostate cancer (PCa) subtype. However, the underlying molecular mechanism and pathobiology of SPINK1 in PCa remains largely unknown.Experimental DesignMicroRNA-prediction tools were employed to examine the SPINK1-3’UTR for miRNAs binding. Luciferase reporter assays were performed to confirm the SPINK1-3’UTR binding of shortlisted miR-338-5p/miR-421. Further, miR-338-5p/-421 overexpressing cancer cells (SPINK1-positive) were evaluated for oncogenic properties using cell-based functional assays and mice xenograft model. Global gene expression profiling was performed to unravel the biological pathways altered by miR-338-5p/-421. Immunohistochemistry and RNA in-situ hybridization was carried-out on PCa patients’ tissue microarray for SPINK1 and EZH2 expression respectively. Chromatin immunoprecipitation assay was performed to examine EZH2 occupancy on the miR-338-5p/-421 regulatory regions. Bisulfite sequencing and methylated DNA-immunoprecipitation was performed on PCa cell lines and patients’ specimens.ResultsWe established a critical role of miRNA-338-5p/-421 in post-transcriptional regulation of SPINK1. Ectopic expression of miRNA-338-5p/-421 in SPINK1-positive PCa cells abrogate oncogenic properties including cell-cycle progression, stemness and drug resistance, and show reduced tumor burden and distant metastases in mice model. Importantly, we show SPINK1-positive PCa patients exhibit increased EZH2 expression, suggesting its role in miRNA-338-5p/-421 epigenetic silencing. Furthermore, presence of CpG dinucleotide DNA methylation marks on the regulatory regions of miR-338-5p/-421 in SPINK1-positive PCa cells and patients’ specimens confirms epigenetic silencing.ConclusionOur findings revealed that miRNA-338-5p/-421 are epigenetically silenced in SPINK1-positive PCa, while restoring the expression of these miRNAs using epigenetic drugs or synthetic mimics could abrogate SPINK1-mediated oncogenesis.TRANSLATIONAL IMPACTWe establish a regulatory model involving the functional interplay between SPINK1, miRNA-338-5p/miRNA-421 and EZH2, thereby, revealing hitherto unknown mechanism of SPINK1 up-regulation in SPINK1-positive subtype. Our findings provide a strong rationale for the development of potential therapeutic strategies for SPINK1-positive malignancies. We demonstrate that restoring miRNA-338-5p/miRNA-421 expression using epigenetic drugs including DNMTs inhibitors in combination with HDACs or HKMTs inhibitors or miRNA synthetic mimics in SPINK1-positive prostate cancer abrogate SPINK1-mediated oncogenicity. The major findings of this study will not only advance the prostate cancer field, but will also be valuable for treatment and disease management of other SPINK1-positive malignancies.

Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer

“…Further, a bidirectional negative-feedback loop between AR and ZEB1 has been established, which drives EMT and stem cell-like features upon androgen deprivation in LuCaP35 tumor explants 16 . Recently, we have shown that SPINK1 expression positively correlates with EZH2, a member of Polycomb repressive complex 2, known to induce pluripotency and stemness 13 . Furthermore, SOX2 has been implicated as a key regulator in governing pluripotency and drives NEtransdifferentiation 44 .…”

“…Prostatosphere assay. 22RV1-shSCRM and -shSPINK1 cells (1 × 10 4 ) were plated in low adherence 6-well cell culture dishes in serum-free DMEM-F12 media (1:1, Invitrogen) supplemented with B27 (1×, Invitrogen), EGF (20 ng/ml, Invitrogen), FGF (20 ng/ml, Invitrogen) as previously described 13 . A small population of cells forming prostatospheres were collected by centrifugation and were mechanically dissociated into single cell suspension, followed by re-plating in fresh culture media.…”

“…SPINK1, also known as tumor-associated trypsin inhibitor (TATI) or pancreatic secretory trypsin inhibitor (PSTI) was previously discovered in the urine of ovarian cancer patients 10 . Under normal physiological condition, SPINK1 inhibits the premature activation of pancreatic proteases, however, multiple reports have observed elevated levels of SPINK1 in cancer tissues, and shown its role in cancer progression [11][12][13][14] . Moreover, SPINK1 acts as an autocrine/paracrine factor and imparts oncogenic traits via EGFR downstream signaling 11,15 .…”

New Hope in Prostate Cancer Precision Medicine? miRNA Replacement and Epigenetics

“…Many tumor cells will become castration resistant with mestatases (mCRPC), and approximately 10% of patients develop a molecular subtype with high expression of SPINK1. The study by Bhatia and colleagues (1) proposes that repressed levels of miRNA-338-5p/-421 are involved in the regulation of SPINK1 expression. Increased levels of miRNA-338-5p/-421 by epigenetic modulation or by miRNA replacement therapy will subsequently downregulate SPINK1 as a targeted therapy in this subgroup of patients with mCRPC.…”

Identification of Key Genes and Evaluation of Their Prognosis Potential in Hepatocellular Carcinoma by Integrated Bioinformatics Analysis

“…Low expression of TAT was found in HCC, and downregulation of TAT promoted tumor progression [31]. Several previous studies revealed that SPINK1 is associated with various cancers development [32][33][34]. In HCC, researchers found the expression of SPINK1 was increased in tumor tissues by micro-arrays analysis, high level of SPINK1 promoted the proliferation, migration and invasion ability of HCC cells [33].…”