Targeting hydrogen sulphide signaling in breast cancer

Abstract

Introduction: Hydrogen sulphide (H2S) has been established as a key member of the gasotransmitters family that recently showed a pivotal role in various pathological conditions including cancer. Objectives: This study investigated the role of H2S in breast cancer (BC) pathogenesis, on BC immune recognition capacity and the consequence of targeting H2S using non-coding RNAs. Methods: Eighty BC patients have been recruited for the study. BC cell lines were cultured and transfected using validated oligonucleotide delivery system. Gene and protein expression analysis was performed using qRT-PCR, western blot and flow-cytometry. In-vitro analysis for BC hallmarks was performed using MTT, BrdU, Modified Boyden chamber, migration and colony forming assays. H2S and nitric oxide (NO) levels were measured spectrophotometrically. Primary natural killer cells (NK cells) and T cell isolation and chimeric antigen receptor transduction (CAR T cells) were performed using appropriate kits. NK and T cells cytotoxicity was measured. Finally, computational target prediction analysis and binding confirmation analyses were performed using different software and dual luciferase assay kit, respectively. Results: The H2S synthesizing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), exhibited elevated levels in the clinical samples that correlated with tumor proliferation index. Knock-down of CBS and CSE in the HER2+ BC and triple negative BC (TNBC) cells resulted in significant attenuation of BC malignancy. In addition to increased susceptibility of HER2+ BC and TNBC to the cytotoxic activity of HER2 targeting CAR T cells and NK cells, respectively. Transcriptomic and phosphoprotein analysis revealed that H2S signaling is mediated through Akt in MCF7, STAT3 in MDA-MB-231 and miR-155/ NOS2/NO signaling in both cell lines. Lastly, miR-4317 was found to function as an upstream regulator of CBS and CSE synergistically abrogates the malignancy of BC cells. Conclusion: These findings demonstrate the potential role of H2S signaling in BC pathogenesis and the potential of its targeting for disease mitigation.

Keywords

• Breast cancer
• Hydrogen sulphide
• miR-155/NOS2/NO signaling pathway
• PI3K/AKT signaling pathway
• Nitric oxide
• miR-4317