Pharmacologic reversion of epigenetic silencing of the PRKD1promoter blocks breast tumor cell invasion and metastasis

Abstract

IntroductionDNA methylation-induced silencing of genes encoding tumor suppressors is common in many types of cancer, but little is known about how such epigenetic silencing can contribute to tumor metastasis. The PRKD1 gene encodes protein kinase D1 (PKD1), a serine/threonine kinase that is expressed in cells of the normal mammary gland, where it maintains the epithelial phenotype by preventing epithelial-to-mesenchymal transition.MethodsThe status of PRKD1 promoter methylation was analyzed by reduced representation bisulfite deep sequencing, methylation-specific PCR (MSP-PCR) and in situ MSP-PCR in invasive and noninvasive breast cancer lines, as well as in humans in 34 cases of “normal” tissue, 22 cases of ductal carcinoma in situ, 22 cases of estrogen receptor positive, HER2-negative (ER+/HER2-) invasive lobular carcinoma, 43 cases of ER+/HER2- invasive ductal carcinoma (IDC), 93 cases of HER2+ IDC and 96 cases of triple-negative IDC. A reexpression strategy using the DNA methyltransferase inhibitor decitabine was used in vitro in MDA-MB-231 cells as well as in vivo in a tumor xenograft model and measured by RT-PCR, immunoblotting and immunohistochemistry. The effect of PKD1 reexpression on cell invasion was analyzed in vitro by transwell invasion assay. Tumor growth and metastasis were monitored in vivo using the IVIS Spectrum Pre-clinical In Vivo Imaging System.ResultsHerein we show that the gene promoter of PRKD1 is aberrantly methylated and silenced in its expression in invasive breast cancer cells and during breast tumor progression, increasing with the aggressiveness of tumors. Using an animal model, we show that reversion of PRKD1 promoter methylation with the DNA methyltransferase inhibitor decitabine restores PKD1 expression and blocks tumor spread and metastasis to the lung in a PKD1-dependent fashion.ConclusionsOur data suggest that the status of epigenetic regulation of the PRKD1 promoter can provide valid information on the invasiveness of breast tumors and therefore could serve as an early diagnostic marker. Moreover, targeted upregulation of PKD1 expression may be used as a therapeutic approach to reverse the invasive phenotype of breast cancer cells.

The AR/NCOA1 axis regulates prostate cancer migration by involvement of PRKD1

“…The low expression of PRKD1 in PC3 compared with AR-positive cell lines may be the explanation for its high motility. Epigenetic silencing of the PRKD1 promoter in PC3 cells could be a reason for the low expression, which was also shown for highly invasive breast cancer cell lines (Borges et al 2013). An inhibitory effect of PRKD1 on cellular proliferation could be explained by stimulation of secretion of matrix metalloproteinases-2 and -9 .…”

Molecular aspects of breast cancer resistance to drugs (Review)

“…Curcumin suppresses multiple signaling pathways (58), such as caspase activation, induction of cell death receptors, aggregation of Fas receptors, induction of p53, and p21 pathways (57), release of apoptosis-inducing factor (57,60), regulation of the cell cycle (58,61), inhibition of PI3K-AKT activation, mTOR inhibition, downregulation of androgen receptors (57), inhibition of AMP-activated protein kinase, inhibition of COX2 and LOX 5, inhibition of STAT3 activation (57), c-Jun kinase activation, induction of DNA fragmentation, depletion of intracellular Ca 2 , mitochondrial activation, direct damage to the DNA, anti-apoptotic protein suppression, autophagy, antioxidant mechanisms, proteasome activation, NF-κβ inhibition, among others (58,61). Thus, many studies have focused on improving its clinical effectiveness by modifying its delivery system through nanotechnology (62,63).…”

“…b) Epigenetic changes. Epigenetic silencing of tumor suppressor genes such as TP53 or PKD1 that can influence the tumor microenvironment and promote the progression of a breast tumor to an aggressive metastatic phenotype (2,28,39). c) miRNA.…”

“…It is the end result of successive genetic changes, altering regulation processes, producing tumor cells with important advantages of survival and growth. Breast cancer is one of the most common cancers in the USA and one of the main causes of cancer-related death in women worldwide (1,2). In the last 25 years in both North America and Europe, mortality from breast cancer has fallen as a result of early detection with screening program like mammography and the advent of more efficacious adjuvant systemic therapy (3).…”

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

“…13 Various studies have identified several genes that are frequently hypermethylated in OOSCC, 14,15 such as CDH1, CDKN2A, O-6-methylguanine-DNA methyltransferase (MGMT), death-associated protein kinase 1 (DAPK1), RARB, and RASSF1, but only few of those have been associated with metastasis. 16,17 In other cancers, various methylation markers have been associated with cell migration and invasion in vitro 18,19 and the presence of nodal metastasis. 19,20 In this study, we set out to identify novel methylation markers that are associated with the presence of lymph node metastases in patients with OOSCC.…”