Dual targeting of H(2)S synthesizing enzymes; cystathionine β-synthase and cystathionine γ-lyase by miR-939-5p effectively curbs triple negative breast cancer

INTRODUCTION: Hydrogen sulfide (H(2)S) has been recently scrutinized for its critical role in aggravating breast cancer (BC) tumorigenicity. Several cancers aberrantly express H(2)S synthesizing enzymes; Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). However, their levels and interdependence in BC require further studies. OBJECTIVES: Firstly, this study aimed to demonstrate a comparative expression profile of H(2)S synthesizing enzymes in BC vs normal tissue. Moreover, to investigate the reciprocal relationship between CBS and CSE and highlight the importance of dual targeting. Finally, to search for a valid dual repressor of the H(2)S synthesizing enzymes that could cease H(2)S production and reduce TNBC pathogenicity. METHODS: Pairwise analysis of tumor vs. normal tissues of 40 BC patients was carried out. The TNBC cell line MDA-MB-231 was transfected with oligonucleotides to study the H(2)S mediated molecular mechanisms. In silico screening was performed to identify dual regulator(s) for CBS and CSE. Gene expression analysis was performed using qRT-PCR and was confirmed on protein level using Western blot. TNBC hallmarks were evaluated using MTT, migration, and clonogenicity assays. H(2)S levels were detected using a AzMc fluorescent probe. RESULTS: BC tissues exhibited elevated levels of both CBS and CSE. Interestingly, upon CBS knockdown, CSE levels increased compensating for H(2)S production in TNBC cells, underlining the importance of dually targeting both enzymes in TNBC. In silico screening suggested miR-939-5p as a regulator of both CBS and CSE with high binding scores. Low expression levels of miR-939-5p were found in BC tissues, especially the aggressive subtypes. Ectopic expression of miR-939-5p significantly repressed CBS and CSE transcript and protein levels, diminished H(2)S production and attenuated TNBC hallmarks. Moreover, it improved the immune surveillance potency of TNBC cells through up regulating the NKG2D ligands, MICB and ULBP2 and reducing the immune suppressive cytokine IL-10. CONCLUSION: This study sheds light on the reciprocal relationship between CBS and CSE and on the importance of their dual targeting, particularly in TNBC. It also postulates miR-939-5p as a potent dual repressor for CBS and CSE overcoming their redundancy in H(2)S production, a mechanism that can potentially attenuate TNBC oncogenicity and improves the immunogenic response.