Dual targeting of H2S synthesizing enzymes; cystathionine β-synthase and cystathionine γ-lyase by miR-939-5p effectively curbs triple negative breast cancer

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

doi:10.1016/j.heliyon.2023.e21063

Dual targeting of H₂S synthesizing enzymes; cystathionine β-synthase and cystathionine γ-lyase by miR-939-5p effectively curbs triple negative breast cancer

Heliyon. 2023 Oct 17;9(10):e21063. doi: 10.1016/j.heliyon.2023.e21063. eCollection 2023 Oct.

Authors

Heba Nafea¹, Rana A Youness^{2

3}, Alyaa Dawoud¹, Nour Khater¹, Tamer Manie⁴, Reham Abdel-Kader⁵, Carole Bourquin⁶, Csaba Szabo⁷, Mohamed Z Gad¹

Affiliations

¹ Biochemistry Department, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo, Egypt.
² Pharmaceutical Biology Department, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo, Egypt.
³ Biology and Biochemistry Department, School of Life and Medical Sciences, University of Hertfordshire Hosted by Global Academic Foundation, New Administrative Capital, Cairo, Egypt.
⁴ Breast Surgery Department, National Cancer Institute, Cairo University, Cairo, Egypt.
⁵ Pharmacology and Toxicology Department, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo, Egypt.
⁶ School of Pharmaceutical Sciences and Institute of Pharmaceutical Sciences of Western Switzerland and Department of Anesthesiology, Pharmacology, Intensive Care and Emergency Medicine, University of Geneva, Geneva 1211, Switzerland.
⁷ Chair of Pharmacology, Section of Medicine, University of Fribourg, Fribourg, Switzerland.

Abstract

Introduction: Hydrogen sulfide (H₂S) has been recently scrutinized for its critical role in aggravating breast cancer (BC) tumorigenicity. Several cancers aberrantly express H₂S 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 H₂S 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 H₂S synthesizing enzymes that could cease H₂S 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 H₂S 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. H₂S 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 H₂S 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 H₂S 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 H₂S production, a mechanism that can potentially attenuate TNBC oncogenicity and improves the immunogenic response.

Keywords: CBS; CSE; IL-10; NKG2D; TNBC; miR-939-5p.