Abstract 4791: Cystathionine-β-Synthase (CBS) and the progression of colorectal carcinogenesis

Abstract

Abstract Introduction: We recently demonstrated that cystathionine-β-synthase (CBS) stimulates colon cancer bioenergetics, migration, invasion and angiogenesis and is increased in colorectal cancer compared to normal colonic mucosa. The role of CBS in carcinogenesis is still, however, unknown. Here we compare CBS protein levels and immunohistochemistry in human colonic biopsies at different stages of colorectal carcinogenesis. In addition, we used Azoxymethane (AOM) in CBS knockdown mice to study the development of aberrant crypt foci (ACF), a model of sporadic colorectal carcinogenesis. Methods: CBS protein expression was assessed in human biopsies of normal mucosa, tubular adenoma and carcinoma in situ by Western blotting. Immunohistochemical (IHC) staining was used to assess CBS levels in formalin-fixed/paraffin-embedded specimens of normal colonic mucosa, adenomatous polyps and colon adenocarcinoma. To assess the effect of CBS gene dosage on azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation CBS heterozygous mice (CBS+/-) and wild-type (CBS+/+) controls were treated with the mutagen AOM at 10mg/kg via intraperitoneal injection once per week for 5 weeks. At the end of 16 weeks, the colons were harvested and ACF visualized with methylene blue staining by 3 independently blinded observers. Significance (p ≤ 0.05) was determined using GraphPad Prism 7 software. Results: Consistent with our previously reported findings CBS levels were relatively low in biopsies of normal mucosa. By comparison, it was elevated in polyps exhibiting both low- and high-grade dysplasia. IHC staining of normal mucosa and hyperplastic polyps revealed CBS immunoreactivity in a small number of cells located along the basal lamina aspect of the colonic crypts in both normal and hyperplastic polyps. A sight increase in cytoplasmic CBS staining also was noted in the epithelial cells of hyperplastic polyps. In contrast, the epithelial cells of tubular adenoma specimens exhibited higher levels of diffuse cytoplasmic CBS staining with frequent focal areas of intense staining adjacent to mucin-containing vesicles. Sections of adenocarcinoma exhibited diffuse CBS staining throughout the cytoplasm of the cancer cells. AOM treatment induced significantly less ACF in the colons of CBS(+/-) mice compared to wild-type controls. The loss of one allele of CBS reduced the number of AOM-induced ACF by half. The mean number of ACF ± SD for CBS(+/+) and CBS(+/-) were 10.9 ± 5.2 and 5.6 ± 2.5, respectively (p < 0.05). Conclusion: CBS protein levels increase and the pattern of expression changes with progression from benign hyperplastic polyps to invasive adenocarcinoma, suggesting the enzyme may play a functional role in colorectal carcinogenesis. In addition, CBS single allele knockdown significantly reduces ACF formation. CBS, and its product H2S, may, therefore, be a driver of carcinogenesis. Citation Format: John R. Zatarain, Ketan Thanki, Michael E. Nicholls, Manjit Maskey, Muhammad U. Jawad, Ches'Nique Phillips, Katalin Modis, Csaba Szabo, Celia Chao, Mark R. Hellmich. Cystathionine-β-Synthase (CBS) and the progression of colorectal carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4791. doi:10.1158/1538-7445.AM2017-4791