Abstract 4276: Cystathionine-β-synthase overexpression increases cell proliferation, migration, bioenergetics and tumorigenesis in a non-tumorigenic colorectal cancer (CRC) cell line
Authors: Zatarain John R. , Phillips Ches’Nique M. , Nicholls Michael E. , Johnson Paul , Widen Steven G. , Wood Thomas G. , Druzhyna Nadiya , Szczesny Bartosz , Porter Craig , Modis Katalin , Szabo Csaba , Chao Celia , Hellmich Mark R. 
Published: July 2016

Introduction: We recently described overexpression of the enzyme, cystathionine-β-synthase (CBS) in human CRC (but not normal colonic mucosa) produces endogenous hydrogen sulfide (H2S) increasing tumor bioenergetics, cell proliferation, invasion, migration and promotes tumor angiogenesis. Its role in the progression of a colorectal adenoma to carcinoma remains elusive. The purpose of this study was to determine whether CBS overexpression in a non-tumorigenic human CRC cell line (NCM356) is sufficient to increase cell proliferation, migration, tumorigenesis, and metastasis. Methods: NCM356-p (parental) are non-tumorigenic when xenografted into athymic nude mice and express low levels of endogenous CBS, similar to normal colonic mucosa. RNASeq analysis determined mutation status. NCM-p were transduced with a lentiviral vector containing a CBS cDNA (NCM-C) or vector (NCM-v). H2S production was visualized with a fluorescent probe, 7-azido-4-methylcoumarine (AzMC). Cell proliferation rates where determined with a Coulter Counter. Cell migration and invasion assay were performed in Boyden chambers with NIH3T3 conditioned media (CM) as a chemoattractant. Anchorage-independent growth was assessed by soft-agar assay. Cellular bioenergetics was assessed using Oxygraph-O2K respirometer chamber. Tumorigenesis and metastasis were assessed injecting cells subcutaneously and orthotopically into nude mice, respectively. Aminooxyacetic acid (AOAA) was used to inhibit CBS activity. Statistical significance (p≤0.05) set using ANOVA or non-parametric Student t-test. Results: CBS overepression and H2S production was verified by Western blot and AzMC fluorescence in the NCM-C cells. CBS overexpression demonstrated significantly increased proliferation rate (p&lt;0.03 NCM-C vs. NCM-v or NCM-p) over 4 days in culture, invasion through matrigel (p&lt;0.01), migration (p&lt;0.001) toward NIH3T3 CM and increased colony formation in soft agar (p&lt;0.01). CBS inhibition with AOAA (1mM) decreased invasion and migration (p&lt;0.01) in the NCM-C cells compared to NCM-v. In high-resolution respirometry, respiratory rate was significantly higher in the NCM-C compared to -p and -v. (141.8 ± 1.5 vs. 33.7 ± 0.8 vs. 72.2 ± 1.77 pmol·s−1·mg−1, -p v -C p &lt; 0.0001, -v v -C p &lt; 0.0005, -p v -v p &lt; 0.001). Exome-wide sequence analyses of the NCM-p identified inactivating mutations in the APC and TP53 tumor suppressor genes, and an activating mutation in KRAS. Although the NCM-C cells produced significantly larger tumors in mice compared with NCM-v or NCM-p (p&lt;0.001 2-w ANOVA), none of the cell lines caused liver metastasis in vivo. Conclusion: A non-tumorigenic cell line with some known mutations can progress to a tumorigenic, but not metastatic, phenotype with CBS overexpression and H2S production. Our data supports the importance of CBS/H2S axis in the adenoma to carcinoma sequence.

Citation Format: John R. Zatarain, Ches’Nique M. Phillips, Michael E. Nicholls, Paul Johnson, Steven G. Widen, Thomas G. Wood, Nadiya Druzhyna, Bartosz Szczesny, Craig Porter, Katalin Modis, Csaba Szabo, Celia Chao, Mark R. Hellmich. Cystathionine-β-synthase overexpression increases cell proliferation, migration, bioenergetics and tumorigenesis in a non-tumorigenic colorectal cancer (CRC) cell line. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4276.