Increased glucocorticoid receptor activity and proliferation in metastatic colon cancer

Metastasis is regarded as the fatal hallmark for colon cancer, but molecular mechanisms responsible for it have remained poorly defined. Glucocorticoid receptor (GR) within the tumor microenvironment mediates the effects of stress hormones which are used in clinics for their inflammation-modulatory and immunosuppressive properties. Further, epigenetic activation of GR promotes tumor heterogeneity and metastasis. Here, we sought to investigate the correlation between GR activation and proliferation and invasion in metastatic colon cancer microenvironment. We used proliferation/invasion assays, western blot, RT-qPCR, immunofluorescence staining and quantitative methylation to study glucocorticoid-GR signaling, including the involvement of CDK1, in human colon adenocarcinoma cell lines HT29 and T84 (a representative metastatic cell line). Nuclear expression levels of GR were significantly upregulated in metastatic T84 cells, and glucocorticoid derivative, dexamethasone (DEX) treatment caused increased proliferation and invasion in T84 cell, compared to HT29 cell. DEX treatment induced CDK1 expression which was accompanied by reduced CDK1 methylation, indicating epigenetic regulation. Depletion of GR suppressed proliferation of metastatic colon carcinoma cells and depletion of CDK1 had similar suppressing effects on proliferation as well as invasion of metastatic cells. Our study suggests that glucocorticoid-GR-CDK1 signaling induces proliferation and invasion of colon cancer cells and therapies involving the use of glucocorticoids need to exercise caution and re-evaluation.

Western blot. We used nuclear extraction kit (Millipore, China) to prepare the nuclear and cytosolic cell extracts. 1× SDS loading buffer was added to the proteins, and SDS electrophoresis was performed by resolving samples on SDS-10% PAGE gels. This was followed by transfer onto PVDF membranes (Bio-Rad, China) and blocking of membranes using 5% nonfat milk powder in PBST, and finally overnight incubation with primary antibodies at 4 °C and an hour long incubation with appropriate secondary antibodies with extensive washings in between. We used β-actin as loading control. Immun-Star HRP Substrate (Bio-Rad, China) was used for the visualization of protein bands on autoradiograms.
Immunofluorescence (IF) staining. Cells were fixed using 4% paraformaldehyde in PBS, and permeabilized for 10 minutes, using 0.5% Triton X-100 in PBS. This was followed by washing and overnight incubation at 4 °C with appropriate antibodies; washing to remove excess antodies and incubation with fluorophore-conjugated secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG) for 1 hour at room temperature. Finally, cells were counterstained with 4′,6-diamidino-2-phenylindole DAPI (Invitrogen, China) and visualized using a fluorescence microscope (Olympus, Japan).
Preparation of total RNAs. Total RNA content of cells was extracted by directly adding Trizol-LS reagent (Invitrogen, China) to the plates and following manufacturer's protocol. We collected aqueous phase after the addition of chloroform and added ethanol, followed by centrifugation to precipitate RNAs out. RNA pellet was dissolved in 10 μL of nuclease-free water and the purity and concentration measured on a NanoDrop instrument (ThermoScientific, China).
Quantitative RT-PCR. For quantitative estimation of mRNAs, we used oligo-dT primers for total cDNA synthesis, using the Superscript II Reverse Transcription kit (Invitrogen, China), and used ABI7300 machine (Applied Biosystems, China). mRNA levels were determined with relative to GAPDH expression using the SYBR Green PCR kit (Qiagen, China), respectively. Fold change was determined by the method of cycle Chromatin Immunoprecipitation (ChIP). ChIP was performed, exactly as described previously 11 .

Methylation quantitation.
We determined the levels of methylation in colon carcinoma cells using FFPE Bisulfite conversion kit from Active Motif (Shanghai, China). Manufacturer's protocol was used without any modifications and primers were designed, using established methodology 12 .
Cell proliferation assay. MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbr-omide] -based assay was performed to study cell proliferation, as previously described 13,14 . Cells were seeded into 96-well plates (5,000 cells/well in 200 µL medium) for 24 hr prior to the assay. Once the assay was complete, 20 µL of 5 mg/ml MTT (Sigma, China) solution was added to every single well and the plates incubated for another 4 hr in the cell culture incubator. This was followed by replacement of liquid with 150 µL DMSO (Sigma-, China). Finally, OD was determined at 490 nm using multi-microplate test system (Victor3, MA, USA).
Cell invasion assay. Cell invasion was studied using cell invasion kit purchased from R&D Systems (Shanghai, China) which contained 96-well boyden chamber with a polyethylene terephthalate membrane pre-coated with Basement Membrane Extract (BME) at medium density, to investigate cell invasion in vitro. Treated or control cells were placed in the top chamber and those that invaded through the BME and filter pores to the bottom of filter were quantified using a cell dissociation / calcein AM solution. www.nature.com/scientificreports www.nature.com/scientificreports/ Statistical analysis. Data is expressed as mean ± standard deviation (SD). We used one-way ANOVA and Student's t-test to compare three and two groups, respectively. p < 0.05 was considered significantly different.

Results
GR is constitutively activated in metastatic colon carcinoma cell line. The transcription factor, GR, is expressed in almost all types of cells within the human body. Upon ligand glucocorticoid binding, the glucocorticoid-GR (hormone-receptor) complex translocates to nucleus and then acts on target gene promoter resulting in modulation of gene transcription. In order to determine whether GR is activated in colon cancer, we performed western blotting analysis on extracts (nuclear and cytosolic) prepared from HT29 or T84 cell. As shown in Fig. 1A, we did not notice any difference in GR in protein abundance between these cell lines. However, GR levels were remarkably increased in the nuclear extracts in T84 cells, compared to HT29 cells (Fig. 1B). Moreover, the increased nuclear localization in T84 cells was also visualized by immunofluorescence staining (Fig. 1C). Taken together, these results suggest that GR is constitutively activated in metastatic colon carcinoma cells at a higher rate than the non-metastatic colon carcinoma cells.
Glucocorticoid-GR signaling promotes metastatic colon carcinoma proliferation. HT29 or the metastatic cells T84 were exposed to DEX (1 μM) for 24 hr or the DMSO (control). MTT proliferation assay was then performed and the results show that DEX-treatment caused a significant increase in cancer cell proliferation in T84 cells, compared to HT29 cells ( Fig. 2A). Then, we performed invasion assay and observed that, similar to proliferation, DEX-treatment resulted in more invasion of T84 cells, compared to HT29 cells (Fig. 2B). To verify that these observed results were an outcome of glucocorticoid-GR interaction, we silenced GR in T84 cells, using siRNA against GR. The silencing was very efficient (Fig. 2C) and the silencing of GR, followed by DEX treatment, resulted in much reduced proliferation (Fig. 2D) verifying an important role of GR in proliferation of metastatic cells T84. These results suggested that glucocorticoids stimulate metastatic colon cancer cell proliferation by activating GR.

Glucocorticoid-GR signaling induces gene expression of CDK1 in metastatic colon carcinoma.
Cancer proliferation and invasion is predominantly caused by dysregulation of CDKs [15][16][17][18] . In order to evaluate whether glucocorticoid-GR signaling involves regulation through CDK1, particularly in metastatic colon cancer cells, we first used ChIP assay to determine the occupancy of GR at CDK1 gene promoter (Fig. 3A,B). p65 was used as a control. Furthermore, we found that DEX exposure of T84 cells resulted in an increase in CDK1 expression at protein (Fig. 3C) and mRNA levels (Fig. 3D). Epigenetic regulation, such as methylation, also plays a role in eventual expression of genes 19 . Therefore, we also checked for the methylation of CDK1 in T84 cells treated with control or DEX. DEX-treated cells had significantly reduced methylation and this can explain the increased expression of CDK1. These results suggest that glucocorticoid-GR signaling stimulates CDK1 expression in www.nature.com/scientificreports www.nature.com/scientificreports/ metastatic colon carcinoma. There seems to be epigenetic mechanisms involved as well, which need to be studied further.

CDK1 stimulates proliferation and invasion of metastatic colon carcinoma cells.
Human metastatic colon cell line T84 were transiently transfected with CDK1 siRNA for 48 hr with non-specific siR-NAs as the controls. The depletion of CDKI was confirmed by western blot (Fig. 4A). MTT proliferation assay showed that inhibition of CDK1 suppressed cancer cell proliferation (Fig. 4B) as well as invasion (Fig. 4C), in a time-dependent manner. These results suggest that glucocorticoid-GR signaling stimulated-proliferation and invasion is mediated by increased expression of CDK1 gene in metastatic colon carcinoma.

Discussion
CRC is one of the major human cancers that is responsible for numerous deaths through-out the world. Despite the research efforts over past several decades, there has been no major breakthrough to substantially improve the way CRC patients are treated in the hospitals. As a result, there is urgent need to doscover novel diagnostic and prognostic markers of CRC for the ultimate goal of improving the outcome of CRC patients. As a step in this direction, here we show that glucocorticoid-GR signaling plays an important role in proliferation of metastatic human colon carcinoma cells, at least in part, by inducing CDK1 gene expression (Fig. 5).
Cancer heterogeneity poses as one of the major blockades for treatment of a variety of metastatic cancers 20 including colon cancer 21 . It has been reported that levels of stress hormones are elevated in patients with metastatic cancer including breast cancer, compared to those with age-matched healthy counterparts or patients without metastases 22 . In addition, patients of advanced breast cancer with deregulated cortisol rhythms usually have shorter survival 23 . Very recently, a study by Obradovic et al. demonstrated that the increase of stress hormones during breast cancer progression leads to the activation of the glucocorticoid receptor (GR) at distant metastatic sites, increased colonization but decreased survival 24 . We found that the stress hormone pathway is an effective inducer of proliferation of metastatic colon carcinoma. Furthermore, our data have demonstrated that inhibition of GR abolished glucocorticoid-mediated upregulation of cancer cell proliferation and invasion. As a result, our results support that caution is needed when administering corticosteroids to cancer patients, as proposed 24 , and inhibition of GR may actually be helpful for chemotherapy of metastatic colon cancer.
CDKs regulate the cell cycle by interacting with cyclins. CDK1 is the principal CDK that functions to promote G2-M transition, and regulates G1 progression and the G1-S transition within the cell cycle 25,26 . Cell proliferation, one of the hallmarks of malignant tumors, is often promoted by dysregulation of CDK activity, and change in CDK expression and/or activity is frequently observed in many human cancers 27 . Other than cell-cycle regulation, CDK1 is involved in apoptosis by caspase phosphorylation including the inhibitory phosphorylation of caspase-8. To this end, Zhang and colleagues have demonstrated that targeting CDK1 is an effective strategy to enhance the efficacy of MEK inhibition in mutant BRAF colorectal cancer cell lines and tumor xenografts 28 , consistent with a previous report on the relevance of CDK1 as a therapeutic target in human colorectal cancers 29 . In the current study, we have shown that depletion of either GR or CDK1 halted proliferation of metastatic colon carcinoma. These results further support the high relevance of GR or CDK1 as a therapeutic target in human colorectal cancer, especially for distant metastasis.
In conclusion, we identify glucocorticoid-GR-CDK1 signaling as a novel mediator of proliferation in metastatic colorectal cancer cells. Taken together, our data support development of novel strategies to inhibit CDK1 and/or GR for the treatment of metastatic human colorectal cancers.

Data Availability
All the data collected and analyzed for this study can be found in the submitted results (Figures).