OP17 Cystathionine-β-synthase (CBS) is upregulated in colorectal cancer cells and promotes their proliferation

Introduction Although the pathophysiological roles of hydrogen sulfide (H2S) have been demonstrated or proposed in many diseases, the role of endogenously produced H2S in the pathogenesis of cancer has not yet been explored. Using surgical specimens from human colorectal cancer patients, and human colorectal cancer cell lines and corresponding control tissues and cells, we have investigated the potential changes in the expression of H2S-producing enzymes in colorectal cancer cells, and the potential role of endogenous H2S in the proliferation and invasion of colorectal cancer cells in vitro. Methods Freshly resected human colorectal tumor tissue, along with a specimen of patient-matched normal mucosa (wide margin), was collected under an IRB approved protocol and snap frozen in liquid N2. The colon cancer cells line HT-29 and the non-tumorigenic colon epithelial cell line derived from the normal margin of a rectal cancer specimen, NCM356 (NCM) were cultured in DMEM supplemented with 10% fetal bovine serum. HCT116 and LoVo cells were cultured in McCoy’s 5A and F-12, respectively. For detection of H 2S production in lysates of NCM or HCT116 cells, the amperometric H2S-specific sensor or the methylene blue assay were used. In addition, Western blotting for CSE, CBS and 3-MST was conducted in cell and tissue homogenates. For shRNA-mediated silencing of CBS and CSE, HCT116 cells were transduced with either a lentiviral vector containing shRNA sequences targeting CBS or CSE. A non-targeting control shRNA sequence (shNT) was used to control of off-target effects. To overexpress CBS in the normal colon mucosa cell line NCM, gene transfer was accomplished using an adenovirus at a MOI of 10, with a green fluorescent protein – expressing adenovirus used as a control. For cellular migration and invasion assays, 105cells were suspended in serum-free media containing 0.1% BSA and then added to 6.5 mm Transwell chambers (8 mm pore) uncoated (migration assay) or coated with a thin layer (45 μl) of growth factor reduced matrigel (cell invasion assay). Cells were allowed to migrate toward the bottom chamber containing fibroblast (NIH-3T3 cells) conditioned media at 37 °C for 6 or 24 h for cell migration and invasion, respectively. Migrated and invaded cells were then fixed in Carson’s fixative, stained in 0.33% toluidine blue and quantified by visual counting. For HCT116-EAhy926 co-culture experiments, the cancer cell line was grown to confluence on the bottom wells of the transwell chambers containing 600 μl of DMEM media. Endothelial cells were grown until 60–70% confluent and then trypsinized, suspended at a density of 105 cells/ml in DMEM and added to the upper chamber (100 μl of the cell suspension). Cell migration was then assessed. For assessment of cell proliferation, the xCELLigence system (Roche) was used. Results Comparison of human colon cancer specimens with patient-matched normal mucosa tissue revealed the selective upregulation of the H2S-producing enzyme cystathionine-β-synthase (CBS) in the cancers. In contrast, the expression of the other two H2S-producing enzymes, cystathionine-γ-lyase (CSE) and 3-MST remained unchanged. Similar to colon tumors, colon adenocarcinoma-derived cell lines (HCT116, HT-29, LoVo) exhibited the selective upregulation of CBS, when compared to NCM cells. Homogenates of the patient-derived colon tumor specimens, as well as homogenates of the colon cancer-derived cell lines exhibited increased rates of H2S production, which was blocked by the CBS inhibitor aminooxyacetic acid (AOAA), but not by the CSE inhibitor propargylglycine PAG. Gene specific shRNA sequences in lentiviral vectors were used to suppress the expression of either CBS or CSE in HCT116 cells. Densitometric analyses of Western blots revealed an approximately 50% decrease in CBS expression with comparable reductions in both cell proliferation and H2S production. In contrast to CBS, silencing of CSE or the pharmacological CSE inhibitor propargylglycine (PAG, 3 mM) did not significantly affect either HCT116 cell proliferation or H2S production. To further define the function of CBS in colon cancer cells, we treated both NCM (which express CSE, but only low levels of CBS), and HCT116 cells (which overexpress CBS) with the pharmacological CBS inhibitor, amino-oxyacetic acid (AOAA). Consistent with the CBS knockdown experiments, AOAA treatment inhibited the growth of HCT116 colon cancer cells, but did not affect the proliferation of the slower-growing non-malignant NCM356 cell line. Conversely, forced overexpression of CBS in NCM356 cells significantly increased their basal rate of proliferation. AOAA treatment also suppressed the migration and invasion of HCT116 cells, while the presence of the H2S donor NaHS (30 μM) in the lower chamber slightly enhanced HCT116 cell migration and invasion in serum-free media. Inhibition of CBS with AOAA reduced endothelial cell migration in colon cancer/endothelial cell co-cultures. Conclusions The H2S-producing enzyme CBS is selectively overexpressed in human colorectal cancer cells, and significantly contributes to their proliferation, migration and invasion. Together with additional in vitroand in vivo data presented at the current meeting, these data support the view that CBS-derived H2S serves as an endogenous cancer cell survival factor, and identify CBS as a potential future antitumor drug target.