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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 ß-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.
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Triple-negative breast cancer (TNBC) represents an aggressive breast cancer subtype. Among young females, TNBC is the leading cause of cancer-related mortalities. Recently, long noncoding RNAs (lncRNAs) are representing a promising pool of regulators for tuning the aggressiveness of several solid malignancies. However, this still needs further investigations in TNBC. The main aim of this study is to unravel the expression pattern of sONE lncRNA and its mechanistic role in TNBC. Results showed that sONE is restrictedly expressed in TNBC patients; its expression level is inversely correlated with the aggressiveness of the disease. sONE acts as a posttranscriptional regulator to endothelial nitric oxide synthase (eNOS) and thus affecting eNOS-induced nitric oxide (NO) production from TNBC cells measured by Greiss reagent. Mechanistically, sONE is a potential tumor suppressor lncRNA in TNBC cells; repressing cellular viability, proliferation, colony-forming ability, migration, and invasion capacities of MDA-MB-231. Furthermore, sONE effects were found to be extended to affect the maestro tumor suppressor TP53 and the oncogenic transcription factor c-Myc. Knocking down of sONE resulted in a marked decrease in TP53 and increase in c-Myc and consequently altering the expression status of their downstream tumor suppressor microRNAs (miRNAs) such as miR-34a, miR-15, miR-16, and let-7a. In conclusion, this study highlights sONE as a downregulated tumor suppressor lncRNA in TNBC cells acting through repressing eNOS-induced NO production, affecting TP53 and c-Myc proteins levels and finally altering the levels of a panel of tumor suppressor miRNAs downstream TP53/c-Myc proteins.