John R. Zatarain

Inhibition of hydrogen sulfide biosynthesis sensitizes lung adenocarcinoma to chemotherapeutic drugs by inhibiting mitochondrial DNA repair and suppressing cellular bioenergetics

Therapeutic manipulation of the gasotransmitter hydrogen sulfide (H2S) has recently been proposed as a novel targeted anticancer approach. Here we show that human lung adenocarcinoma tissue expresses high levels of hydrogen sulfide (H2S) producing enzymes, namely, cystathionine beta-synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), in comparison to adjacent lung tissue. In cultured lung adenocarcinoma but not in normal lung epithelial cells elevated H2S stimulates mitochondrial DNA repair through sulfhydration of EXOG, which, in turn, promotes mitochondrial DNA repair complex assembly, thereby enhancing mitochondrial DNA repair capacity. In addition, inhibition of H2S-producing enzymes suppresses critical bioenergetics parameters in lung adenocarcinoma cells. Together, inhibition of H2S-producing enzymes sensitize lung adenocarcinoma cells to chemotherapeutic agents via induction of mitochondrial dysfunction as shown in in vitro and in vivo models, suggesting a novel mechanism to overcome tumor chemoresistance.

Screening of a composite library of clinically used drugs and well-characterized pharmacological compounds for cystathionine β-synthase inhibition identifies benserazide as a drug potentially suitable for repurposing for the experimental therapy of colon cancer

Abstract Cystathionine-β-synthase (CBS) has been recently identified as a drug target for several forms of cancer. Currently no potent and selective CBS inhibitors are available. Using a composite collection of 8871 clinically used drugs and well-annotated pharmacological compounds (including the LOPAC library, the FDA Approved Drug Library, the NIH Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the ‘Killer Plates’ collection and a small custom collection of PLP-dependent enzyme inhibitors), we conducted an in vitro screen in order to identify inhibitors for CBS using a primary 7-azido-4-methylcoumarin (AzMc) screen to detect CBS-derived hydrogen sulfide (H2S) production. Initial hits were subjected to counterscreens using the methylene blue assay (a secondary assay to measure H2S production) and were assessed for their ability to quench the H2S signal produced by the H2S donor compound GYY4137. Four compounds, hexachlorophene, tannic acid, aurintricarboxylic acid and benserazide showed concentration-dependent CBS inhibitory actions without scavenging H2S released from GYY4137, identifying them as direct CBS inhibitors. Hexachlorophene (IC50: ∼60μM), tannic acid (IC50: ∼40μM) and benserazide (IC50: ∼30μM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: ∼3μM) and NSC67078 (IC50: ∼1μM), while aurintricarboxylic acid (IC50: ∼3μM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: ∼1μM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50 of ∼6μM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: ∼6μM), tannic acid (IC50: ∼20μM), benserazide (IC50: ∼20μM), and NSC67078 (IC50: ∼0.3μM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: ∼300μM). In contrast, although a CBS inhibitor in the cell-free assay, aurintricarboxylic acid failed to inhibit HCT116 proliferation at lower concentrations, and stimulated cell proliferation at 300μM. Copper-containing compounds present in the libraries, were also found to be potent inhibitors of recombinant CBS; however this activity was due to the CBS inhibitory effect of copper ions themselves. However, copper ions, up to 300μM, did not inhibit HCT116 cell proliferation. Benserazide was only a weak inhibitor of the activity of the other H2S-generating enzymes CSE and 3-MST activity (16% and 35% inhibition at 100μM, respectively) in vitro. Benserazide suppressed HCT116 mitochondrial function and inhibited proliferation of the high CBS-expressing colon cancer cell line HT29, but not the low CBS-expressing line, LoVo. The major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity and suppressed HCT116 cell proliferation in vitro. In an in vivo study of nude mice bearing human colon cancer cell xenografts, benserazide (50mg/kg/days.q.) prevented tumor growth. In silico docking simulations showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.

Upregulation of cystathionine-β-synthase in colonic epithelia reprograms metabolism and promotes carcinogenesis

The trans-sulfuration enzyme cystathionine-β-synthase (CBS) and its product hydrogen sulfide (H2S) are aberrantly upregulated in colorectal cancers, where they contribute to tumor growth and progression by both autocrine and paracrine mechanisms. However, it is unknown whether the CBS/H2S axis plays a role in colorectal carcinogenesis. Here, we report upregulation of CBS in human biopsies of precancerous adenomatous polyps and show that forced upregulation of CBS in an adenoma-like colonic epithelial cell line is sufficient to induce metabolic and gene expression profiles characteristic of colorectal cancer cells. Differentially expressed metabolites (65 increased and 20 decreased) clustered into the glycolytic pathway, nucleotide sugars, intermediates of the pentose phosphate pathway, and lipogenesis, including primarily phospholipids, sphingolipids, and bile acids. CBS upregulation induced broad changes in the NCM356 cell transcriptome with over 350 differentially expressed genes. These genes overlapped significantly with gene sets related to glycolysis, hypoxia, and a colon cancer cell phenotype, including genes regulated by NF-κB, KRAS, p53, and Wnt signaling, genes downregulated after E-cadherin knockdown, and genes related to increased extracellular matrix, cell adhesion, and epithelial-to-mesenchymal transition. The CBS-induced switch to an anabolic metabolism was associated with increased NCM356 cell bioenergetics, proliferation, invasion through Matrigel, resistance to anoikis, and CBS-dependent tumorigenesis in immunocompromised mice. Genetic ablation of CBS in CBS heterozygous mice (CBS+/- ) reduced the number of mutagen-induced aberrant colonic crypt foci. Taken together, these results establish that activation of the CBS/H2S axis promotes colon carcinogenesis. Cancer Res; 77(21); 5741-54. ©2017 AACR.

Tu1975 H2S Inhibition of Cystathionine-β-Synthase (CBS) Using Novel Prodrug Decreases Colorectal Cancer Xenograft Growth With Less Toxicity Than Aminooxyacetic Acid (AOAA)

G A A b st ra ct s enhanced MEK/ERK activation coincided with those that inhibited mTORC2-mediated Akt phosphorylation on Ser473, suggesting a role of mTORC2. Knockdown of Rictor markedly increased baseline levels of ERK phosphorylation and treatment with NVP-BEZ235 did not produce further enhancement of ERK activation. These results imply that Rictor or mTORC2 exerts feedback inhibition of the MEK/ERK pathway in pancreatic cancer cells. Conclusion: We propose that dual PI3K/mTOR inhibitors suppress a novel negative feedback loop mediated by mTORC2 thereby leading to enhanced MEK/ERK pathway activity in pancreatic cancer cells.

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

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 Citation Format: John R. Zatarain, Ketan Thanki, Michael E. Nicholls, Manjit Maskey, Muhammad U. Jawad, Ches9Nique 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

Abstract 1857: Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Introduction: Ulcerative colitis (UC) is a highly morbid, chronic inflammatory disease characterized by mucosal ulceration of the colonic mucosa and is associated with the higher risk of colitis-associated cancer (CAC). The exact mechanism(s) causing UC progression to CAC is currently unknown. The balanced activity of transsulfuration pathway enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS), and their production of endogenous hydrogen sulfide gas, are critical to maintenance of the colonic homeostasis. CSE activity is suggested to be important for wound healing and mucosal protection. Consistent with this, we have previously showed a decrease CSE expression in human colonic mucosa obtained from patients with chronic UC, compared to normal colonic mucosa by immunocytochemistry. By contrast, increased CBS expression is implicated in the progression of sporadic colorectal carcinoma. However, the role of these enzymes in CAC is unknown. We hypothesize that the dysregulation in CSE/CBS expression and activity is important to the progression from UC to CAC. Methods: CSE null mice and wild type Sv129/B6 (control) mice were used in azoxymethane-dextran sodium sulfate (AOM-DSS) colon cancer model which mimics human CAC. The disease development was followed up to day 80. Confocal microscopy and Western blot was used to assess the gene expression during cancer development. Size, number, and time interval to tumor formation, as well inflammation were assessed. Results: Abrogation of CSE expression using CSE null animals in AOM-DSS model of CAC accelerated the time to tumor development and resulted in an increase in both tumor size (p<0.001) and number (p<0.001) compared to wild-type controls. CBS protein expression was increased within the colonic tumor when compared to the normal margin in AOM-DSS treated animals by Western blot analysis and tissue immunostaining. Interestingly, CAC liver metastases, an exceedingly rare finding in this mouse model, were identified. Conclusion: Taken together, our human and murine data suggest that dysregulation in the transsulfuration pathway enzymes CSE and CBS expression/activity may be critical contributor to the CAC development in UC and may serve as a potential biomarker for disease progression in the future. Citation Format: Paul Johnson, Ches’Que M. Phillips, Carl Grim, John R. Zatarain, Aakash H. Gajjar, Suimin Qiu, Rui Wang, Celia Chao, Iryna V. Pinchuk, Mark R. Hellmich. Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis. [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 1857.

Abstract 4276: Cystathionine-β-synthase overexpression increases cell proliferation, migration, bioenergetics and tumorigenesis in a non-tumorigenic colorectal cancer (CRC) cell line

Introduction: We recently described overexpression of the enzyme, cystathionine-β-synthase (CBS) in human CRC (but not normal colonic mucosa) produces endogenous hydrogen sulfide (H 2 S) 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). H 2 S 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 H 2 S production was verified by Western blot and AzMC fluorescence in the NCM-C cells. CBS overexpression demonstrated significantly increased proliferation rate (p −1 ·mg −1 , -p v -C p 2 S production. Our data supports the importance of CBS/H 2 S 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.