J. R. Zatarain1, C. Phillips1, P. Johnson1, S. Widen1, T. G. Wood1, N. Druzhyna1, B. Szczesny1, C. Szabo1, C. Chao1, M. R. Hellmich1 1University Of Texas Medical Branch,Galveston, TX, USA
Introduction: Our laboratories have recently reported, cystathionine-β-synthase (CBS) is selectively upregulated in colorectal cancer (CRC), compared to patient-matched normal colonic mucosa. CBS produces the gaseous transmitter hydrogen sulfide (H2S), which we showed promotes CRC growth and metastasis by stimulating tumor cell bioenergetics (i.e., product of ATP for cell proliferation), and creating a localized H2S-rich microenvironment that stimulates tumor angiogenesis and local vasodilatation to feed and disseminate the growing cancer cells. However, the role of CBS/H2S in the progression of disease from adenoma to adenocarcinoma is unknown. The purpose of this study was to determine whether forced overexpression of CBS in a non-malignant colonic cell line (NCM356) would induce a malignant phenotype.
Methods: NCM356 (parental) cells express a very low level of endogenous CBS, similar to normal colonic mucosa, and are non-tumorigenic when xenografted into immune compromised mice. CBS overexpressing (NCMCBS) and control (NCMvec) cells where produced by transducing parental cells with a lentiviral vector containing a CBS cDNA or the empty vector, respectively. Western blotting was used to assess CBS protein levels. Levels of H2S were visualized in intact cells using the H2S-specific fluorescent probe, AzMC (7-azido-4-methylcoumarin). The amount of H2S was quantified by measuring the conversion of CBS substrates L-cysteine and L-homocysteine in protein extracts using AzMC. Cell growth rates where determined with a Coulter Counter. aminooxyacetic acid (AOAA) was used to inhibit CBS activity. Cell migration and invasion assay were performed in Boyden chambers with NIH3T3 conditioned media as a chemo-attractant. Anchorage-independent growth was assessed by soft-agar assay. Statistical significance (p≤0.05) was calculated using ANOVA or non-parametric Student t-test.
Results:NCMCBS cells showed a marked increase in H2S levels by microscopy and a 4-fold increased in NCM-CBS protein extracts, compared to either NCMvec or the parental cell line. CBS overexpression enhanced proliferation rate (p<0.03 NCMCBS vs. NCMvec or parental), cell migration, invasion through matrigel (p<0.01), and colony formation in soft agar (p<0.01). The stimulatory effects of CBS overexpression on NCM cell migration and invasion were inhibited by AOAA (p<0.01). Exosome-wide sequence analyses of the parental cells identified inactivating mutations in the APC and TP53 tumor suppressor genes, and an activating mutation in KRAS.
Conclusion:Colorectal carcinogenesis requires the accumulation of multiple mutations and the derangement of normal cellular metabolism. Our data demonstrate that forced overexpression of CBS can induce a malignant (i.e. invasive) phenotype in a cell line that is normally non-invasive, suggesting that CBS may play a role in adenoma to adenocarcinoma progression during colorectal carcinogenesis.