Pan-inhibition of the three H 2 S synthesizing enzymes restrains tumor progression and immunosuppression in breast cancer.

BACKGROUND: Hydrogen sulfide (H2 S) is a significant endogenous mediator that has been implicated in the progression of various forms of cancer including breast cancer (BC). Cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) are the three principal mammalian enzymes responsible for H2 S production. Overexpression of CBS, CSE and 3MST was found to be associated with poor prognosis of BC patients. Moreover, H2 S was linked to an immune-suppressive tumor microenvironment in BC. Recently it was observed that BC cells, in response to single or dual inhibition of H2 S synthesizing enzymes, develop an escape mechanism by overexpressing alternative sources of H2 S generation. Thus, the aim of this work is to escape the H2 S compensatory mechanism by pan repressing the three enzymes using microRNAs (miRNAs) and to investigate their impact on the oncogenic and immunogenic profile of BC cells.

METHODS: BC female patients (n = 25) were recruited. In-silico analysis was used to identify miRNAs targeting CBS, CSE, and 3MST. MDA-MB-231 cells were cultured and transfected using oligonucleotides. Total RNA was extracted using Biazol, reverse transcribed and quantified using qRT-PCR. H2 S levels were measured using AzMc assay. BC hallmarks were assessed using trans-well migration, wound healing, MTT, and colony forming assays.

RESULTS: miR-193a and miR-548c were validated by eight different bioinformatics software to simultaneously target CBS, CSE and 3MST. MiR-193a and miR-548c were significantly downregulated in BC tissues compared to their non-cancerous counterparts. Ectopic expression of miR-193a and miR-548c in MDA-MB-231 TNBC cells resulted in a marked repression of CBS, CSE, and 3MST transcript and protein levels, a significant decrease in H2 S levels, reduction in cellular viability, inhibition of migration and colony forming ability, repression of immune-suppressor proteins GAL3 GAL9, and CD155 and upregulation of the immunostimulatory MICA and MICB proteins.

CONCLUSION: This study sheds the light onto miR-193a and miR-548c as potential pan-repressors of the H2 S synthesizing enzymes. and identifies them as novel tumor suppressor and immunomodulatory miRNAs in TNBC.