Abstract 3548: Hypoxia-induced Cystathionine-β-synthase (CBS) expression: impact on colonic epithelial cells metabolism, proliferation and migration

Abstract

Abstract Introduction: We have reported aberrant overexpression of CBS in human colorectal cancers, where its activity (H2S production) promotes tumor progression and metastasis. New data show that CBS expression increases in adenomatous polyps. The aims of this study were to identify possible mechanisms for CBS upregulation and determine the biological impact of its upregulation using a premalignant colonic epithelial cell line NCM356. Methods: Induction of CBS in response to LPS, TLR2&4 agonists, and hypoxia was assessed by immunoblotting. CBS overexpression was achieved using lentiviral expression vector (pReceiver-Lv103, GeneCopoeia™) in NCM356 cells. Global metabolic profiling (Metabolon, Durham) was performed on cellular extracts from NCM365 overexpressing cells (CBS-hi) and vector-transduced controls. High-resolution respirometry (Orobors Oxygraph-2k) assessed bioenergetics. H2S levels were measured using the fluorogenic probe 7-Azido-4-methylcoumarin. A Coulter counter was used to assess cell proliferation rates. Transwell assays were used to assess cell migration. CBS activity was inhibited with aminooxyacetic acid (AOAA). Results: CBS protein expression was increased by ~3-fold in pNCM after 2 and 24 hr of hypoxia (1% O2). Treatment with LPS but not the TLR agonists increased CBS expression 2-fold. Metabolic profiling of CBS-hi cells and controls identified 85 metabolites that were differentially expressed (65 increased and 20 decreased; p≤0.05 Welch’s 2-Sample t-Test). The metabolite lanthionine, which is produced by the CBS-dependent condensation of cysteine to produce H2S, showed the largest increase (12.3-fold increase, p=1.5E-06). A 4-fold increase in basal H2S production was measured in CBS-hi cells compared to control cultures. High-resolution respirometry showed that CBS-hi cells exhibited a significant increase in maximum respiration rate, reserve respiratory capacity and increased citrate synthase activity, suggesting increased cellular mitochondria mass. Treatment of CBS-hi cells either with pentose phosphate pathway inhibitor (oxythiamine) or lactate dehydrogenase inhibitor (XF-11) reducing growth by 90% and ~25% at day 5, respectively; the inhibitors had no effect on the vector control cells. Exposure of NCM356 cells to hypoxic conditions resulted in increased migration to conditioned media (p<0.001, NCM356 normoxia vs. hypoxia); inhibition of CBS using AOAA attenuated the hypoxia-induced migration. Conclusions: Hypoxia and LPS can induce CBS expression in a premalignant colonic epithelial cell line. Upregulation of CBS has a board impact on cellular metabolism including enhanced flux through the transsulfuration, glycolytic and pentose phosphate pathways, resulting in enhanced cellular bioenergetics, growth and migration. These data suggest that enhance CBS activity may be involved the adenoma to carcinoma sequence. Citation Format: Ches'Nique M. Phillips, John R. Zatarain, Michael E. Nicholls, Craig Porter, Steven Widen, Ketan Thanki, James W. Randall, Judith L. Hellmich, Manjit Maskey, Suimin Qiu, Thomas G. Wood, Nadiya Druzhyna, Bartosz Szczesny, Katalin Modis, Csaba Szabo, Celia Chao, Mark R. Hellmich. Hypoxia-induced Cystathionine-β-synthase (CBS) expression: impact on colonic epithelial cells metabolism, proliferation and migration [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 3548. doi:10.1158/1538-7445.AM2017-3548