Epigenetic Silencing of CREB3L1 by DNA Methylation Is Associated With

Ward et al. Breast Cancer Research (2016) 18:12 DOI 10.1186/s13058-016-0672-x RESEARCHARTICLE Open Access 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 Alison K. Ward1, Paul Mellor1, Shari E. Smith1, Stephanie Kendall1, Natasha A. Just1, Frederick S. Vizeacoumar1, Sabuj Sarker2, Zoe Phillips2, Riaz Alvi2, Anurag Saxena3, Franco J. Vizeacoumar1,4, Svein A. Carlsen4 and Deborah H. Anderson1,4* Abstract Background: CREB3L1 (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. Methods: Quantitative 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. Results: This 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. (Continued on next page) * Correspondence: [email protected] 1Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada 4Cancer Research, Saskatchewan Cancer Agency, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada Full list of author information is available at the end of the article © 2016 Ward et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ward et al. Breast Cancer Research (2016) 18:12 Page 2 of 21 (Continued from previous page) Conclusions: Our 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. Keywords: Prognostic marker, Triple negative breast cancer, CREB3L1, DNA methylation, Unfolded protein response Background both a bZIP domain and a DNA binding domain [1]. CREB3L1 is a member of the CREB/ATF family of CREB3L1 is located on chromosome 11, a chromo- transcription factors and functions as a transducer of the some that contains a number of loci that are fre- unfolded protein response (UPR) [1]. A large fraction of quently altered in breast cancer [7–9]. It is an proteins synthesized in the cell undergo folding and endoplasmic reticulum transmembrane protein and post-translational modification in the endoplasmic activated in a similar manner to ATF6, via Site-1- reticulum before being released to perform their desired protease (S1P) and S2P cleavage in the Golgi appar- function. This process can be disrupted by endoplasmic atus followed by translocation to the nucleus [10]. reticulum stress resulting from hypoxia, glucose or nu- This mature activated form is a transcription factor, trient depletion, change in calcium homeostasis, or ex- acting on both endoplasmic reticulum stress respon- pression of mutant or misfolded proteins, and can lead sive elements (ESRE) and cyclic AMP responsive ele- to the accumulation of unfolded proteins that if released ments (CRE) to increase expression of target genes from the endoplasmic reticulum can have detrimental such as GRP78 [11]. effects. The accumulation of unfolded proteins in the A number of studies have identified roles for the lumen of the endoplasmic reticulum initiates the UPR. members of the UPR in breast cancer development, pro- The UPR works to regain endoplasmic reticulum gression and resistance to therapy. PERK expression has homeostasis by reducing protein translocation into the been shown to be vital for the initiation and progression endoplasmic reticulum, increasing the protein-folding of breast cancers. Inhibition of PERK expression in ani- capacity of this organelle, decreasing translation initi- mal models results in an increase in reactive oxygen spe- ation, and increasing protein degradation [2]. Prolonged cies leading to increased DNA damage and a halting of activation of the UPR leads to apoptosis [3]. the cell cycle [12]. ATF4 activation was shown to confer There are three main transducers of the UPR, namely resistance to the chemotherapy agent taxol in hypoxic activating transcription factor-6 (ATF6), inositol requir- tumors [13]. A similar finding demonstrated that in- ing 1 (IRE1), and PRK-like endoplasmic reticulum kinase creased expression of GRP78 is associated with che- (PERK). Under non-stress conditions they are held in moresistance in breast cancer [14, 15]. XBP1 expression their inactive form by association with chaperone pro- has been linked to resistance to anti-estrogen therapies, teins, such as GRP78, bound to their endoplasmic including tamoxifen, which is especially problematic as reticulum luminal domain. As unfolded proteins accu- XBP1 is rapidly induced by estrogens [16–18]. Recently mulate in the endoplasmic reticulum, GRP78 disassoci- XBP1 has been shown to be important in driving TNBC ates from ATF6, IRE1, and PERK, and binds to the oncogenesis through the formation of transcriptional hydrophobic regions of unfolded proteins, which are complexes with hypoxia inducing factor 1α (HIF1α) [19]. subsequently either refolded, or ubiquitinated and de- Although not specific to breast cancer, CREB3L1, like graded [4]. Activated IRE1 cleaves the mRNA of X-box the other members of the UPR, has also been shown to binding protein 1 (XBP1). The spliced form of XBP1 is perform important roles in cancer. Epigenetic downreg- translated into a potent transcriptional activator that ulation of CREB3L1 mRNA expression by DNA methy- stimulates the transcription of UPR target genes. PERK lation is associated with increased tumor grade and phosphorylates eIF2α, which in turn causes a global re- aggressive phenotype in bladder cancer [20]. Also, duction in mRNA translation. Paradoxically, phosphory- CREB3L1 has been shown to be necessary for the che- lated eIF2α also selectively promotes the translation of motherapeutic drug doxorubicin to block cell prolifera- specific mRNAs, such as ATF4 [5], which activates the tion and may function as a biomarker in predicting transcription of genes involved in amino acid metabol- response to therapy [21, 22]. Doxorubicin increases cer- ism and apoptosis [6]. amide production, which in turn stimulates regulated CREB3L1, also termed OASIS (old astrocyte intramembrane proteolysis of CREB3L1 to its mature ac- specifically-induced substance) in mice, is the most tive form. CREB3L1 then activates expression of target recently identified member of the UPR containing genes, including p21, a cell cycle inhibitor [21, 23]. In Ward et al. Breast Cancer Research (2016) 18:12 Page 3 of 21 addition, CREB3L1 may also play a role in limiting the DNA, one for RNA and the other lysed for western blot spread of viral expression as CREB3L1 expression blocks analysis of CREB3L1 protein levels, as detailed below. In proliferation of virally infected Huh7 cells [24]. some instances, cells were treated with the proteasomal Our previous work showed that highly metastatic rat and inhibitor, MG132 (Sigma Aldrich, Oakville, ON, human breast cancer cell lines had reduced expression of Canada), at a concentration of 3 μM for the last 18 hours CREB3L1 compared to poorly metastatic breast cancer cell

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