Epigenetic silencing of CREB3L1 by DNA methylation is associated with high-grade metastatic breast cancers with poor prognosis and is prevalent in triple negative breast cancers

Abstract

BackgroundCREB3L1 (cAMP-responsive element-binding protein 3-like protein 1), a member of the unfolded protein response, has recently been identified as a metastasis suppressor in both breast and bladder cancer.MethodsQuantitative real time PCR (qPCR) and immunoblotting were used to determine the impact of histone deacetylation and DNA methylation inhibitors on CREB3L1 expression in breast cancer cell lines. Breast cancer cell lines and tumor samples were analyzed similarly, and CREB3L1 gene methylation was determined using sodium bisulfite conversion and DNA sequencing. Immunohistochemistry was used to determine nuclear versus cytoplasmic CREB3L1 protein. Large breast cancer database analyses were carried out to examine relationships between CREB3L1 gene methylation and mRNA expression in addition to CREB3L1 mRNA expression and prognosis.ResultsThis study demonstrates that the low CREB3L1 expression previously seen in highly metastatic breast cancer cell lines is caused in part by epigenetic silencing. Treatment of several highly metastatic breast cancer cell lines that had low CREB3L1 expression with DNA methyltransferase and histone deacetylase inhibitors induced expression of CREB3L1, both mRNA and protein. In human breast tumors, CREB3L1 mRNA expression was upregulated in low and medium-grade tumors, most frequently of the luminal and HER2 amplified subtypes. In contrast, CREB3L1 expression was repressed in high-grade tumors, and its loss was most frequently associated with triple negative breast cancers (TNBCs). Importantly, bioinformatics analyses of tumor databases support these findings, with methylation of the CREB3L1 gene associated with TNBCs, and strongly negatively correlated with CREB3L1 mRNA expression. Decreased CREB3L1 mRNA expression was associated with increased tumor grade and reduced progression-free survival. An immunohistochemistry analysis revealed that low-grade breast tumors frequently had nuclear CREB3L1 protein, in contrast to the high-grade breast tumors in which CREB3L1 was cytoplasmic, suggesting that differential localization may also regulate CREB3L1 effectiveness in metastasis suppression.ConclusionsOur data further strengthens the role for CREB3L1 as a metastasis suppressor in breast cancer and demonstrates that epigenetic silencing is a major regulator of the loss of CREB3L1 expression. We also highlight that CREB3L1 expression is frequently altered in many cancer types suggesting that it could have a broader role in cancer progression and metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0672-x) contains supplementary material, which is available to authorized users.

Quaking-5 suppresses aggressiveness of lung cancer cells through inhibiting β-catenin signaling pathway

“…Genomic DNA was extracted from LC tissues and cell lines using a DNA extraction kit (Promega, USA). After quantification, 2 μg of DNA was treated with sodium bisulfate as previously reported [ 28 ], and used as the template DNA for PCR. The MSP primers were designed with the MethPrimer ( http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi ) [ 29 ] and sequences were listed in Supplementary Table 2 .…”

Epigenetic Mechanisms of Tamoxifen Resistance in Luminal Breast Cancer

“…For example, the number of genes has been reported to be methylated and consequently silenced, including tumor suppressor genes, such as secreted frizzled-related protein ( SFRP ), RASSF1A, WNT inhibitory factory factor 1 ( WIF1 ) inter-alpha-trypsin inhibitor heavy chain family member 5 (ITIH5), Dickkopf WNT signaling pathway inhibitor 3 ( DKK3 ), ATM serine/threonine kinase ( ATM ), long interspersed nuclear element 1 ( LINE1 ), cyclin-dependent kinase inhibitors ( CDKN2 and CDKN1B ), G1/S-specific cyclin-D2 ( CCND2 ), breast cancer 1 ( BRCA1 ), mutL homology 1 ( MLH1 ), glutathione S-transferase P ( GSTP1 ), homeobox protein ( HOXA5 ), cadherin-1 ( CDH1 ), metalloproteinase inhibitor 3 ( TIMP3 ), cAMP-responsive element-binding protein 3-like protein 1 ( CREB3L1 ) and hormone receptors ( ESR1 and PGR ). These genes have been shown to be involved in cell cycle regulation, DNA repair, cell detoxification, apoptosis and cell adhesion and invasion [ 38 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ]. The phospholipase A2 receptor (PLA2R1) is a transmembrane protein that plays a role in the clearance of phospholipase A2.…”

The Novel Transcription Factor CREB3L4 Contributes to the Progression of Human Breast Carcinoma

“…CREB3L1 is frequently upregulated in luminal and HER2amplified breast cancer but not in TNBC [31]. However, recently, Bray Denard1 reported that TNBC tumours with high CREB3L1 expression have a high response rate to doxorubicin-based chemotherapy [32].…”