Pan-inhibition of the three H2S synthesizing enzymes restrains tumor progression and immunosuppression in breast cancer

Alyaa Dawoud; Rana A Youness; Heba Nafea; Tamer Manie; Carole Bourquin; Csaba Szabo; Reham M Abdel-Kader; Mohamed Z Gad

doi:10.1186/s12935-024-03317-1

Pan-inhibition of the three H₂S synthesizing enzymes restrains tumor progression and immunosuppression in breast cancer

Cancer Cell Int. 2024 Apr 16;24(1):136. doi: 10.1186/s12935-024-03317-1.

Authors

Alyaa Dawoud¹, Rana A Youness², Heba Nafea¹, Tamer Manie³, Carole Bourquin⁴, Csaba Szabo⁵, Reham M Abdel-Kader⁶, Mohamed Z Gad⁷

Affiliations

¹ Biochemistry Department, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo, Egypt.
² Molecular Genetics and Biochemistry Department, Faculty of Biotechnology, German International University (GIU), New Administrative Capital, Cairo, Egypt.
³ Department of Breast Surgery, National Cancer Institute, Cairo University, Cairo, Egypt.
⁴ School of Pharmaceutical Sciences, Institute of Pharmaceutical Sciences of Western Switzerland, Department of Anaesthesiology, Pharmacology, Intensive Care and Emergency Medicine, University of Geneva, Geneva, 1211, Switzerland.
⁵ Chair of Pharmacology, Section of Science and Medicine, University of Fribourg, Fribourg, 1700, Switzerland.
⁶ Pharmacology and Toxicology Department, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo, Egypt.
⁷ Biochemistry Department, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo, Egypt. [email protected].

Abstract

Background: Hydrogen sulfide (H₂S) 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 H₂S production. Overexpression of CBS, CSE and 3MST was found to be associated with poor prognosis of BC patients. Moreover, H₂S 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 H₂S synthesizing enzymes, develop an escape mechanism by overexpressing alternative sources of H₂S generation. Thus, the aim of this work is to escape the H₂S 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. H₂S 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 H₂S 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 H₂S synthesizing enzymes. and identifies them as novel tumor suppressor and immunomodulatory miRNAs in TNBC.

Abstract

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