Background
CREB3L1 is a member of the CREB/ATF family of transcription factors and functions as a transducer of the unfolded protein response (UPR) [
1]. A large fraction of proteins synthesized in the cell undergo folding and post-translational modification in the endoplasmic reticulum before being released to perform their desired function. This process can be disrupted by endoplasmic reticulum stress resulting from hypoxia, glucose or nutrient depletion, change in calcium homeostasis, or expression of mutant or misfolded proteins, and can lead to the accumulation of unfolded proteins that if released from the endoplasmic reticulum can have detrimental effects. The accumulation of unfolded proteins in the lumen of the endoplasmic reticulum initiates the UPR. The UPR works to regain endoplasmic reticulum homeostasis by reducing protein translocation into the endoplasmic reticulum, increasing the protein-folding capacity of this organelle, decreasing translation initiation, and increasing protein degradation [
2]. Prolonged activation of the UPR leads to apoptosis [
3].
There are three main transducers of the UPR, namely activating transcription factor-6 (ATF6), inositol requiring 1 (IRE1), and PRK-like endoplasmic reticulum kinase (PERK). Under non-stress conditions they are held in their inactive form by association with chaperone proteins, such as GRP78, bound to their endoplasmic reticulum luminal domain. As unfolded proteins accumulate in the endoplasmic reticulum, GRP78 disassociates from ATF6, IRE1, and PERK, and binds to the hydrophobic regions of unfolded proteins, which are subsequently either refolded, or ubiquitinated and degraded [
4]. Activated IRE1 cleaves the mRNA of X-box binding protein 1 (XBP1). The spliced form of XBP1 is translated into a potent transcriptional activator that stimulates the transcription of UPR target genes. PERK phosphorylates eIF2α, which in turn causes a global reduction in mRNA translation. Paradoxically, phosphorylated eIF2α also selectively promotes the translation of specific mRNAs, such as ATF4 [
5], which activates the transcription of genes involved in amino acid metabolism and apoptosis [
6].
CREB3L1, also termed OASIS (old astrocyte specifically-induced substance) in mice, is the most recently identified member of the UPR containing both a bZIP domain and a DNA binding domain [
1].
CREB3L1 is located on chromosome 11, a chromosome that contains a number of loci that are frequently altered in breast cancer [
7‐
9]. It is an endoplasmic reticulum transmembrane protein and activated in a similar manner to ATF6, via Site-1-protease (S1P) and S2P cleavage in the Golgi apparatus followed by translocation to the nucleus [
10]. This mature activated form is a transcription factor, acting on both endoplasmic reticulum stress responsive elements (ESRE) and cyclic AMP responsive elements (CRE) to increase expression of target genes such as
GRP78 [
11].
A number of studies have identified roles for the members of the UPR in breast cancer development, progression and resistance to therapy. PERK expression has been shown to be vital for the initiation and progression of breast cancers. Inhibition of PERK expression in animal models results in an increase in reactive oxygen species leading to increased DNA damage and a halting of the cell cycle [
12]. ATF4 activation was shown to confer resistance to the chemotherapy agent taxol in hypoxic tumors [
13]. A similar finding demonstrated that increased expression of GRP78 is associated with chemoresistance in breast cancer [
14,
15]. XBP1 expression has been linked to resistance to anti-estrogen therapies, including tamoxifen, which is especially problematic as XBP1 is rapidly induced by estrogens [
16‐
18]. Recently XBP1 has been shown to be important in driving TNBC oncogenesis through the formation of transcriptional complexes with hypoxia inducing factor 1α (HIF1α) [
19].
Although not specific to breast cancer, CREB3L1, like the other members of the UPR, has also been shown to perform important roles in cancer. Epigenetic downregulation of CREB3L1 mRNA expression by DNA methylation is associated with increased tumor grade and aggressive phenotype in bladder cancer [
20]. Also, CREB3L1 has been shown to be necessary for the chemotherapeutic drug doxorubicin to block cell proliferation and may function as a biomarker in predicting response to therapy [
21,
22]. Doxorubicin increases ceramide production, which in turn stimulates regulated intramembrane proteolysis of CREB3L1 to its mature active form. CREB3L1 then activates expression of target genes, including
p21, a cell cycle inhibitor [
21,
23]. In addition, CREB3L1 may also play a role in limiting the spread of viral expression as CREB3L1 expression blocks proliferation of virally infected Huh7 cells [
24].
Our previous work showed that highly metastatic rat and human breast cancer cell lines had reduced expression of CREB3L1 compared to poorly metastatic breast cancer cell lines [
25]. We further showed that re-expression of CREB3L1 reduced the in vitro metastatic cell properties, including cell migration, invasion, survival under hypoxic conditions and anchorage-independent growth. In a rat model of breast cancer, CREB3L1-re-expressing cells initially formed large tumors (>0.5 cm
3), in which 70 % of them regressed to a nearly undetectable size. None of these rats had metastases as compared to a 90 % metastasis rate for the rats with the corresponding CREB3L1-deficient cells [
25]. These results suggest that CREB3L1 plays a key role in suppressing tumorigenesis and metastasis.
In this report, we characterize the expression of CREB3L1 in a large panel of breast cancer and non-cancer cell lines and determine whether epigenetic mechanisms regulate CREB3L1 expression in breast cancer. In addition, we characterize CREB3L1 mRNA expression, gene methylation and protein localization in a large number of human tumor samples. Finally, we expanded our analysis of tumor samples from the Cancer Genome Atlas with associated patient data to derive cancer-specific stage association and predict clinical outcome.
Methods
Cell culture
A panel containing 40 breast cancer cell lines (and 4 non-tumorigenic breast cell lines) was obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA 30-4500 K). Cells were cultured according to ATCC recommendations for fewer than 6 months from the time of resuscitation. All cell lines were authenticated by the supplier (
http://www.ATCC.org).
To examine the impact of DNA methylation and/or histone acetylation on CREB3L1 expression, the human breast cancer cell lines BT20, HCC1806 and MDA-MB-468 cells were treated with a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (DAC) (Sigma Aldrich, Oakville, ON, Canada), and/or a histone deacetylase inhibitor, trichostatin A (TSA) (Sigma Aldrich, Oakville, ON, Canada). Cells were grown to 60–70 % confluency and treated with DAC (1 μM) for 96 hours (changing to fresh DAC-containing media every 24 hours), with or without TSA (1 μM), for the last 18 hours as previously reported [
26‐
28]. Cytotoxicity measurements were carried out using a Cytotox Glo Cytotoxicity assay (Promega, Madison, WI, USA G9290) according to their instructions, and no cytotoxicity was observed at 1 μM TSA. Three independent experiments were performed with triplicate samples, with one set used to prepare DNA, one for RNA and the other lysed for western blot analysis of CREB3L1 protein levels, as detailed below. In some instances, cells were treated with the proteasomal inhibitor, MG132 (Sigma Aldrich, Oakville, ON, Canada), at a concentration of 3 μM for the last 18 hours prior to lysis, to prevent the degradation of CREB3L1 protein and better enable visualization on western blots.
Breast tumor samples
Sections from 216 human primary breast tumors and corresponding de-identified clinical data were obtained from the Manitoba Breast Tumor Bank (Winnipeg, MB, Canada). Four different tumor types were obtained including: infiltrating ductile, infiltrating colloid, infiltrating lobular and infiltrating papillary. For the purposes of assessing the possible correlation between low CREB3L1 expression and more advanced or aggressive tumor type, we have ordered these tumor types from least to most aggressive (colloid, lobular, ductile, papillary) based on several sources [
29‐
33]. Tumor samples were graded by pathologists at the time of diagnosis based on mitotic count, nuclear pleomorphisms and tubule formation, from low (grade 4) to high (grade 9) according to the Nottingham derivation of the Scarff Bloom Richardson system, in which grades 4 to 5 are low, 6 to 7 are medium, and 8 to 9 are high [
34]. The estrogen and progesterone receptor status of the tumors was also provided by the Manitoba Breast Tumor Bank. No data were available for the human epidermal growth factor receptor 2 (HER2) status of these samples. Each section consisted of 40–70 % invasive tissue, with the remainder of the tissue being composed of stroma and fat. Samples were accessed and handled according to approved ethics committee guidelines at both the University of Saskatchewan and the Manitoba Tumor Bank (ethics approval number 14-37).
Western blot analysis
CREB3L1 protein expression in breast cancer cell lines was quantified by western blot analysis as previously described [
35]. Briefly, SDS-PAGE was performed using 50 μg total protein, unless otherwise stated, as determined by Lowry (Sigma Aldrich, Oakville, ON, Canada TP0300). Samples were transferred to nitrocellulose and were probed with CREB3L1 (11235-2-AP from Protein Tech, Rosemont, IL, USA; rabbit, 1:500) or ß-actin (C-4 from Santa Cruz Biotechnology, Dallas, TX, USA; mouse, 1:500) primary antibodies, followed by infrared 680 nm or 800 nm dye-tagged secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA; 200 ng/ml). Blots were imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA), quantified, normalized to a ß-actin loading control, and reported relative to that in MDA-kB2 cells.
Methylation sequencing
DNA was extracted from breast tumor sections and cell cultures using the QIAamp DNA Mini Kit (Qiagen, Toronto, ON, Canada). Sodium bisulfite treatment was performed using the EpiTect Bisulfite kit (Qiagen, Toronto, ON, Canada) to convert unmethylated cytosine residues to uracil according to the supplier’s instructions. CpGenome Human Methylated DNA Standard, (Cedarlane, Burlington, ON, Canada) and Epitect Control unmethylated DNA (Qiagen, Toronto, ON, Canada) were used as positive and negative controls, respectively.
Primers were designed using Primer3 software [
36] to amplify (from bisulfite-treated DNA) two overlapping fragments of
CREB3L1 spanning base pair −492 to +290 relative to the transcription start site (Additional file
1: Table S1). Our preliminary data indicated that DNA methylation was concentrated within the beginning of the coding region, thus, a set of primers that amplify a fragment from −51 to +258 to target this methylation-rich region was used. PCR was performed with 100 ng bisulfite-treated DNA in a 50-μl reaction with the TaKaRa EpiTaq HS kit (Cedarlane, Burlington, ON, Canada) according to the supplier’s instructions. The thermocycling protocol consisted of 45 cycles of 10 seconds at 98 °C, 30 seconds at annealing temperature (Additional file
1: Table S1), and 1 minute at 72 °C. QIAquick PCR Purification Kit (Qiagen, Toronto, ON, Canada) was used to purify PCR products prior to sequencing. Sanger sequencing was performed by the Plant Biotechnology Institute (Saskatoon, SK, Canada) and results were visualized with MacVector version 12.5 software (MacVector, Inc., Apex, NC, USA). Only sequences containing efficient C to T conversions, indicative of effective sodium bisulfite conversion at the non-CpG sites (that would not be methylated) were used for methylation analyses. Sequences were assessed for the presence of methylated cytosine residues at CpG dinucleotide motifs by using a qualitative assessment at each possible methylated position. Methylation was scored as low, but present and given a value of 1, if the C peak was above background noise even if some T was also present. Methylation was scored as high and given a value of 2, if the C peak was the tallest peak observed at that position. Initially sequences were analyzed from −429 to +259, relative to the translational start site, a region that includes 60 CpG sites. As most methylations were concentrated between −15 and +259, subsequent analyses focused on these 24 CpG sites.
Quantitative real-time PCR
Total RNA was extracted from cell lines using the RNeasy kit (Qiagen, Toronto, ON, Canada) and from breast tumor sections using the PicoPure RNA Isolation kit (Life Technologies). RNA was reverse-transcribed to cDNA using Superscript II Reverse Transcriptase and oligo-dT primers (Invitrogen) according to the supplier’s instructions. CREB3L1 expression was measured by quantitative real-time PCR performed using TaqMan probes (assay ID Hs00999642_m1, Life Technologies, Waltham, MA, USA) and TaqMan Gene Expression Master Mix (Life Technologies, Waltham, MA, USA) according to manufacturer’s protocols. The sequence of these primers is proprietary, but it amplifies a 103-bp fragment at the junction of exons 8 and 9 such that it will only detect the full-length transcript. Relative expression was calculated using expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (assay ID Hs99999905_m1, Life Technologies, Waltham, MA, USA) as a reference gene. Samples were analyzed in triplicate per reaction using the StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Results are the mean of two independent reactions and reported as the relative change in expression compared to the MDA-kB2 cell line (for the cell line analysis), or control normal breast tissue sample (Manitoba Breast Tumor Bank, Winnipeg, MB, Canada) for the tumor sample analysis. Where samples were available, the HER2 status of the tumor samples was determined by qPCR as above, but using HER2-specific TaqMan probes (assay ID Hs1001580_m1, Life Technologies, Waltham, MA, USA), relative to a GAPDH reference gene. HER2 status was reported as compared to the MDA-kB2 cell line (HER2-positive, but not amplified) as follows (negative <1, positive = 1–9, amplified >9).
Copy number variation
CREB3L1 gene copy number was assessed using a digital droplet PCR assay [
37]. The number of target fragments in the original sample is calculated using a Poisson distribution and the copy number of the gene of interest is calculated by normalizing it to a reference gene (
AP3B1) to adjust for global ploidy changes [
38].
DNA extracted from human breast tumors was digested with HaeIII (New England Biolabs, Whitby, ON, Canada) prior to analysis, as per supplier’s instructions. To measure CREB3L1 gene copy number, custom-designed CREB3L1 primers and FAM-labeled TaqMan probe (forward 5′- GATCCAGCTTCCTGGACTTG-3′, reverse 5′-GTAAGATGAAGGGTCTCCGTTC-3′, probe 5′-ACGAGTCGGACTTCCTCAACAATGC-3′; Bio-Rad, Mississaugo, ON, Canada) were combined in a duplex reaction with pre-validated AP3B1 primers and HEX-labeled TaqMan probe (assay ID dHsaCP1000483, Bio-Rad, Mississaugo, ON, Canada) and ddPCR Supermix for Probes (Bio-Rad, Mississaugo, ON, Canada). Droplets were generated using the QX100 Droplet Generator (Bio-Rad, Mississaugo, ON, Canada) and thermocycled to the completion of the following protocol: 10 minutes at 95 °C; 40 cycles of 30 seconds at 94 °C and 1 minute at 60 °C; 10 minutes at 98 °C. Droplets were analyzed using the QX100 Droplet Reader (Bio-Rad, Mississaugo, ON, Canada) and copy numbers were computed with QuantaSoft software (Bio-Rad, Mississaugo, ON, Canada) after normalization to AP3B1. Results are reported as the average copy number of CREB3L1 genes per cell.
Immunohistochemistry
Immunohistochemical staining of CREB3L1 protein was performed on formalin-fixed paraffin-embedded human breast tumor samples obtained from the Manitoba Tumor Bank, according to the manufacturer’s instructions using Pierce Peroxidase Detection Kit (Thermo Scientific, Burlington, ON, Canada). Briefly, sections were de-paraffinized and heated in citrate buffer (pH = 6), followed by an overnight incubation at 4 °C with a rabbit polyclonal anti-CREB3L1 antibody (1:100; Protein Tech, Rosemont, IL, USA 11235-2-AP). Subsequently, slides were incubated with a horseradish peroxidase goat anti-rabbit secondary antibody (1:1000; Abcam, Toronto, ON, Canada ab6721) for 2 hours at room temperature and reacted with 3,3′-Diaminobenzidine (DAB) for 15 minutes. Sections were counterstained with hematoxylin. CREB3L1 staining was evaluated by a pathologist using two criteria, staining intensity (absent = 0, weak = 1, moderate = 2, strong = 3, very strong = 4) and % cells staining positive (0–5 % = 0, 6–49 % = 1, 50–69 % = 2, 70–89 % = 3, 90–100 % = 4). Scores were added together and described as little or no CREB3L1 staining (combined score 0–1), low (scores 2–3), medium (scores 4–5) and high (scores 6–8) CREB3L1 expression, analogous to the Allred system [
39]. In addition, the subcellular location of CREB3L1 was evaluated as nuclear or cytoplasmic.
In silico analysis
Publically available RNA-Seq Version 2 containing normalized gene expression datasets for 24 different cancer types were downloaded from the online database, The Cancer Genome Atlas (TCGA;
http://tcga-data.nci.nih.gov). These data contained expression profile, clinical information, tumor stage, and immunohistochemical (IHC) results for the determination of breast cancer subtype for each breast tissue sample. The RNA-seq by expectation-maximization (RSEM) algorithm-normalized gene expression profile was downloaded from TCGA. Although the microarray-based dataset is also available in this database, to exclude problems that could be caused by combining different platforms, measurement types, and normalization procedures, we used only the RNAseqV2 dataset for all our gene expression analyses. To identify different subtypes of breast cancer, we used the IHC annotations available within our downloaded TCGA dataset. This approach was further verified by an additional independent analysis, where the TNBC population was defined as the group of samples with the lowest 10 % of estrogen receptor (ER), progesterone receptor (PR) and HER2 expression. Analysis of these populations confirmed that the same patients matched the IHC category. Similarly, for stage-specific classification, the annotation of each patient from the downloaded TCGA dataset was used. The subtype classifications or stage-specific classifications were analyzed using python scripts with pylab and the scipy stat built-in libraries to generate the graphs showing relationships with methylation and expression. The non-parametric Mann–Whitney
U test was used to compare two groups. The methylation data were also downloaded from TCGA (Human Methylation 450 K data). Methylation probes were deconvolved for every single region across the promoter, and the intronic and exonic regions. Normalized values were downloaded onto Gene-E to generate the heatmap. Methylation data of either selected regions as identified in Li et al. [
40] or as pre-selected by cBioPortal were used to generate correlation analyses between methylation and gene expression.
The relationship between CREB3L1 methylation and expression (Fig.
6c) was analyzed using cBioPortal (
http://www.cbioportal.org/index.do) [
41,
42]. The dataset analyzed was the breast invasive carcinoma (TCGA Provisional) containing 737 cases, and was accessed on 12 February 2015. Kaplan–Meier survival analysis was carried out using KM-plotter (
http://kmplot.com/analysis/) [
43]. Gene expression data and relapse-free survival information were downloaded from Gene Expression Omnibus (GEO) (Affymetrix microarrays only), European Genome-phenome Archive (EGA) and TCGA. The database is handled by a PostgreSQL server, which integrates gene expression and clinical data simultaneously. To analyze the prognostic value of a particular gene, the patient samples were split into two groups at the median of the proposed biomarker. The two patient cohorts were compared using a Kaplan–Meier survival plot, and the hazard ratio with 95 % confidence intervals and log-rank
p value were calculated. The database was accessed on 22 February 2015.
Statistical analyses
Spearman correlations were determined using free online software [
44]. Statistical analyses were performed using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA) software. Significance was set at
p <0.05 and error reported as plus or minus the standard deviation (SD). The non-parametric Mann–Whitney
U test was used to compare two groups. The Kruskal–Wallis test was used to compare four or more groups of sample data, using SPSS Statistics 23. Provided that significant differences were detected by the Kruskal–Wallis test, a post-hoc test was performed using pairwise comparisons. Survival analysis was performed using the Kaplan–Meier estimator with the non-parametric log-rank test to measure the equity of strata.
Discussion
Previous investigations by our laboratory have implicated
CREB3L1 as a metastatic suppressor gene in breast cancer models in vitro and in vivo. Transfection of CREB3L1 into cells that normally express low levels of CREB3L1 reduced cell migration, invasion, anchorage-independent growth and tolerance of hypoxia [
25]. Consistent with these effects, knockdown of endogenous CREB3L1 in cells demonstrated that loss of CREB3L1 expression significantly increased migration, invasion, anchorage-independent growth, and tolerance of hypoxia. Further support for a role for CREB3L1 as a metastatic suppressor was provided by in vivo studies where rats were injected with CREB3L1-null or CREB3L1-expressing cells. The CREB3L1-null cells formed large primary tumors (29/30 animals) with frequent lymph node metastases (26/30 animals) [
25]. Strikingly, the CREB3L1-expressing cells failed to develop metastases (0/30 animals) and after initially forming large tumors (>0.5 cm
3), 70 % of these tumors (21/30) regressed to a nearly undetectable size [
25].
Here we have expanded upon our previous study to include 40 human breast cancer cell lines and over 200 human breast cancer tumor samples and investigated DNA methylation and its role in the regulation of CREB3L1 expression. We found that in luminal and HER2 amplified breast cell lines and tumors, CREB3L1 mRNA expression was frequently increased, whereas in TNBC cell lines and tumor samples, CREB3L1 expression was frequently low. As CREB3L1 has an important role in metastasis suppression, its low expression in TNBC may contribute to its more aggressive and metastatic phenotype.
CREB3L1 is activated in response to cellular stress as part of the endoplasmic reticulum stress response process [
53‐
55]. Viral infections and treatment with the chemotherapy agent, doxorubicin, have also been shown to induce cell stress and the activation of CREB3L1 [
21,
24]. Tumors form in a stressful cellular environment with low nutrients and low oxygen levels (hypoxia), suggesting that CREB3L1 may be activated during tumor development and progression.
A recent study demonstrated that CREB3L1 expression was required for the chemotherapeutic agent doxorubicin to block the proliferation of cancer cells [
21]. Doxorubicin, but not other chemotherapy agents that cause DNA damage (etoposide, bleomycin) or cell cycle arrest (paclitaxel), cause increased ceramide production that induces the trafficking of CREB3L1 from the endoplasmic reticulum to the Golgi complex [
21]. CREB3L1 has been shown to be proteolytically activated and the mature protein translocated into the nucleus where it could alter the transcription of genes important for cell proliferation [
21,
24]. As doxorubicin was only effective in blocking cell proliferation in CREB3L1-expressing cells, this suggests that only patients expressing CREB3L1 are likely to benefit from doxorubicin treatment. In support of this idea, a recent report showed that higher levels of CREB3L1 expression strongly inversely correlated with tumor volume upon doxorubicin treatment in renal cell carcinoma xenografts (
r = −0.891;
p = 0.017) [
22], leading to the suggestion that CREB3L1 could be a biomarker that predicts doxorubicin treatment outcome. These results also suggest that the loss of CREB3L1 may contribute to doxorubicin treatment resistance.
Other members of the UPR have been implicated in tumor development. GRP78 has been shown to protect tumor cells from cytotoxic T cell immune response and apoptosis following Ca
2+ depletion [
56]. Additionally, increased GRP78 expression has been associated with chemoresistance in breast cancer [
15]. PERK and XBP1 are both important factors in tolerance of hypoxia; loss of expression of either factor inhibits tumor growth and increased apoptosis following hypoxia [
57,
58]. Conversely, decreased expression of UPR transducers has been observed in the progression from normal to high-grade tumors in mouse models of prostate cancer [
53,
55]. It is thought that increased activation of the UPR may impart tolerance of hypoxia in some tumors, and downregulation may promote tumor progression by preventing activation of apoptosis [
55].
We have assessed human breast tumor samples of various grades for the expression of CREB3L1. Low-grade and medium-grade breast tumors had increased CREB3L1 expression, when compared to normal breast tissue samples. In contrast, high-grade (8 and 9) breast tumors had reduced CREB3L1 expression (
p = 0.001). Our results suggest that CREB3L1 expression is initially upregulated in response to the stressful conditions that exist within the tumor environment, as observed for other stress response proteins [
53‐
55]. In contrast to other stress response proteins, loss of CREB3L1 expression is prevalent in high-grade tumors and may be required to avoid apoptosis under prolonged stress conditions. This would allow the de-repression of genes necessary for angiogenesis and metastasis, which we have shown are negatively regulated by CREB3L1 [
25]. Overall, CREB3L1 was lost in 31 % (67/213) of the human breast tumor samples analyzed, but importantly CREB3L1 was lost from a much larger fraction of the high-grade (8 and 9) metastatic breast tumors (51 % of grade 8 and 73 % of grade 9 breast tumors;
p = 0.001). Thus, CREB3L1 may provide a cytoprotective effect early in tumor development and later decreased expression allows progression to high-grade tumors.
Our analysis also found that breast cancer patients with low CREB3L1 expression have a shorter relapse-free survival time specifically for the luminal A and TNBC subtypes. As similar results were not seen for luminal B and HER2 amplified breast cancers, the impact of low CREB3L1 expression may not be significant in the context of HER2 expression. This suggests that low CREB3L1 is a marker for poor prognosis in both luminal A breast cancer and TNBC.
Our data in human breast cancer cell lines suggest that epigenetic silencing of CREB3L1 contributes to reducing CREB3L1 mRNA expression for at least some cell lines, an effect that was reversed by the inhibition of histone acetylation with TSA and/or inhibition of DNA methylation with DAC. We also found that some cell lines had little or no
CREB3L1 DNA methylation and yet, still had little or no CREB3L1 mRNA. As methylation outside of the region tested could also impact
CREB3L1 transcription it is possible that other regions within the
CREB3L1 gene also have key roles in its regulation. In this regard the analysis of TCGA data for
CREB3L1 DNA methylation suggested there might be additional CpG sites outside of the 688 nucleotides tested that could influence CREB3L1 mRNA expression. For example, regions number 16, 19 and 20 (near the 3′ end of intron 1) all had increased methylation in breast tumor samples as compared to normal breast tissue (Additional file
9: Figure S5b), raising the possibility that methylation in these regions could influence CREB3L1 mRNA expression.
In contrast, some cell lines had significant levels of
CREB3L1 DNA methylation, yet expressed relatively high levels of CREB3L1 mRNA (e.g., MDA-MB-415, MDA-MB-231). This could also be due to changes in methylation within other regions of the
CREB3L1 gene that could impact transcription. In addition to DNA methylation, CREB3L1 mRNA levels can also be influenced by alterations in histone modifications, as well as by the regulation of RNA processing, transport and stability [
59,
60]. Further the high CREB3L1 mRNA levels and low protein expression observed in some breast cancer cell lines (e.g., HCC202) could be the result of translational deregulation or the rapid turnover of CREB3L1 protein. An E3 ubiquitin ligase, HRD1, has been reported to ubiquitinate CREB3L1 to induce proteasomal-mediated degradation in C6 glioma cells and mouse embryonic fibroblasts to maintain low levels of CREB3L1 protein [
52]. Other cell lines have an abundance of CREB3L1 protein, but very low levels of the corresponding mRNA (e.g., HCC1500), raising the possibility that the protein is not being turned over as rapidly in these cells, perhaps as a result of defects in HRD1-mediated ubiquitination and degradation.
Within the region tested (−429 to +259, numbering relative to the translational start site), we noted that for cell lines with less
CREB3L1 DNA methylation, the most frequently methylated CpG sites were 259 and 238. Cell lines with more methylation had an increased number of methylated CpG sites 5′ to these, suggesting that
CREB3L1 DNA methylation proceeds in a hierarchical fashion with some sites methylated preferentially. Although the human breast tumor samples typically had lower levels of
CREB3L1 DNA methylation, they also had similar preferential methylation at CpG sites 221, 238 and 259. The analysis of
CREB3L1 DNA methylation in breast tumors in the TCGA database, also with suggested CpG sites 238 and 259 (i.e., region number 2), were usually more methylated in tumors with low CREB3L1 mRNA expression (Additional file
11: Figure S7), but were generally less methylated in breast tumors than in normal breast tissue (Additional file
9: Figure S5a). Together these results suggest that
CREB3L1 methylation at these sites in particular may negatively regulate CREB3L1 expression.
Overall, CREB3L1 expression was negatively correlated with its gene methylation, suggesting that epigenetic silencing is one mechanism that contributes to decreased CREB3L1 levels. Increased
CREB3L1 gene methylation and low CREB3L1 mRNA expression were both correlated with more aggressive types of breast cancer [
61] and higher tumor grade (8 and 9). We therefore conclude that the loss of CREB3L1 expression in cells of high metastatic potential is due in some cases to methylation of a region of CpG sites near the CREB3L1 start site. In support of this, high levels of
CREB3L1 DNA methylation and low levels of CREB3L1 mRNA expression were more frequently observed in TNBC cell lines and breast tumor samples. This is consistent with previous reports showing more DNA hypermethylation in TNBC [
62], in some cases due to overexpression of DNA methyltransferase enzymes [
63].
The subcellular localization of CREB3L1 also changes with tumor progression. In low-grade breast tumors CREB3L1 is predominantly a nuclear protein, consistent with its processing into the mature form and translocation into the nucleus as part of the cellular stress response to activate CREB3L1. High-grade breast tumors that still express CREB3L1 typically have mainly cytoplasmic protein localization, which likely would prevent CREB3L1-mediated transcriptional repression of target genes that promote cell growth, survival, migration, invasion, angiogenesis and metastasis [
25]. Similar subcellular localization patterns of CREB3L1 are observed during bladder cancer progression, suggesting a common function for CREB3L1 in at least these two cancer types [
20].
Our results in TNBC and high-grade tumors are in agreement with a recent study by Rose et al
. who found that CREB3L1 gene expression was downregulated in bladder cancer and that the loss of expression was associated with DNA methylation of the gene [
20]. They also reported that DNA methylation was associated with invasive tumor subtypes of bladder cancer. In support of a wider role for CREB3L1 in multiple cancer types, we determined that CREB3L1 mRNA expression is altered in many types of cancer (Fig.
8). Cancers of the breast, prostate, kidney (papillary), stomach and pancreas all had generally increased CREB3L1 expression in tumors as compared to the corresponding normal tissue. Our work in breast cancer suggests that this should provide a protective effect. However, as we saw for breast cancers with reduced CREB3L1, like TNBC, the disease is typically more aggressive and advanced resulting in a poor prognosis within these groups if tumors have reduced CREB3L1. Importantly, we identified a large group of cancer types where low CREB3L1 expression is prevalent, including lung squamous cell carcinoma, melanoma and cancers of the kidney (clear cell), bladder, colon, liver, adrenal gland, rectum and cervix. This raises the possibility that loss of CREB3L1 could play a key role in cancer progression and metastasis across a broad group of cancer types.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AKW, PM, SES, SAC, and DHA were involved in the study concept and design. AKW, PM, SES, SK, and NAJ carried out experiments and acquired the data. AKW, PM, SES, SS, FSV, FJV, ZP, RA, AS, and DHA analyzed and interpreted the data. AKW, PM, and DHA wrote the manuscript. All authors have read and approved the final version of the manuscript.