P08 Cystathionine-β-synthase (CBS)-derived hydrogen sulfide (H2S) supports colorectal tumor growth and angiogenesis in vivo

Introduction In vitro data presented at the current Meeting demonstrate that the upregulation of the H 2 S-producing enzyme cystathionine-®-synthase (CBS) in colorectal cancer cells supports bioenergetics, proliferation and migration. Several prior studies have shown that H 2 S also functions as a pro-angiogenic factor. By using a combination of genetic and pharmacological approaches, we have investigated whether CBS activity in cancer cells plays a role in the tumor growth and angiogenesis in vivo . Methods To determine the effects of CBS and CSE gene silencing on HCT116 xenograft growth in vivo , cells were transduced with a lentiviral vector containing either an enzyme-specific shRNA sequence or a non-targeting control shRNA sequence. The resulting HCT116 cell lines, designated shCBS, shCSE and shNT, were then injected subcutaneously in the right and left dorsum or either Nu/nu Balb/C or athymic nude mice (8–10 week old females). Changes in tumor volume (i.e., growth) over time were assessed by transcutaneous measurement using a caliper and calculated with the formula V = ( π /6) hd 2 . After 3–4 weeks, tumor tissue and blood plasma were harvested and tumor wet weights, H 2 S production/mg tumor protein, H 2 S levels in plasma, and tumor micro-blood vessel density were quantified. Micro-blood vessels were visualized by immunostaining tumor tissue sections for the mouse endothelial cell marker CD31 (Promin1) and quantified using a Chakley reticle. In a second series of experiments, mice bearing HCT116 xenografts or low passage (P2 to P3) patient-derived tumor xenografts (PDTXs) were randomized and treated (via subcutaneous injection) either with vehicle [phosphate buffered saline (PBS)], the CBS inhibitor aminooxyacetic acid (AOAA, 9 mg/kg/day) or the CSE inhibitor propargylglycine (PAG, 50 mg/kg/day) 6 days/week for up to 2 weeks. In a final set of experiments, peritumoral microvascular blood flow was measured using a PeriFlux 5000 laser-Doppler flowmeter. Changes in the subcutaneous microcirculatory perfusion were detected following intratumoral injections of either L-cysteine (1 mg/kg) or AOAA (9 mg/kg). Each in vivo experiment was repeated at least twice and statistical analyses were performed using GraphPad Prism 5 software. Results Consistent with the in vitro findings, shRNA-mediated knockdown of CBS expression (shCBS) significantly reduced the growth rate and size (i.e. volume) of HCT116 tumor xenografts. In contrast, silencing of CSE (shCSE) did not affect tumor growth. As expected, silencing of CBS or CSE in the tumor cells did not alter circulating H 2 S levels in tumor-bearing mice. Similar to the lack of inhibition of tumor growth by CSE silencing, the CSE inhibitor PAG failed to affect tumor growth in vivo . CBS suppression caused a significant reduction in the density of CD31-positive blood vessels within the tumor tissue as well as the prevalence of larger blood vessels and the extent of vessel branching, consistent with the hypothesis that CBS-derived H 2 S acts locally in a paracrine manner to stimulate tumor angiogenesis. Since small-molecule inhibitors are potentially more amenable to therapeutic translation than shRNA-based strategies, we next tested the effects of AOAA on tumor growth in vivo . Similar to CBS knockdown, AOAA treatment significantly slowed the rate of HCT116 xenograft growth, resulting in tumors with reduced size and weight at the time of harvest. A reduction in the plasma concentration of H 2 S confirmed the inhibitory effect of AOAA on systemic H 2 S production. Also, in accordance with the role of H 2 S as a local vasodilator, direct injection of AOAA into the tumor parenchyma significantly reduced, while intra-tumor administration of the CBS substrate L-cysteine increased peritumor blood flow. Finally, we conducted a series of translational studies with PDTXs, an emerging preclinical model for anti-cancer drug development, which closely recapitulates both the genetic and cellular heterogeneity of the patient’s original tumor tissue. We evaluated the effects of AOAA treatment on mice bearing PDTXs derived from the primary adenocarcinoma of a patient with pathological stage III disease with an activating Kras mutation and with a marked upregulation of endogenous CBS. Following randomization, mice were treated daily either with AOAA or vehicle. AOAA-treated animals showed a reduced rate of PDTX growth. Importantly, the growth inhibitory effects of AOAA on PDTX growth were also observed using tumor tissue from two other patients with stage II disease that also possessed a mutant Kras allele. Summary data using the PDTXs from three different patients showed that AOAA significantly reduced the rate of PDTX growth over the time course of the treatment. Conclusions The current study supports the conclusion that colon cancer cells overexpress CBS, which produces H 2 S to drive cellular bioenergetics and increase cancer cell proliferation. In addition to these cell-autonomous effects, the enhanced H 2 S production by cancer cells exerts paracrine effects on the tumor microenvironment to promote angiogenesis and peritumoral vasorelaxation. These studies identify CBS-derived H 2 S as a tumor promoting factor and a potential future anticancer drug target.