KNOCKDOWN OF UDP-GLUCOSE DEHYDROGENASE FACILITATES EPIRUBICIN-RESISTANCE IN MDA-MB-231 BREAST CANCER CELLS BY REGULATION OF HYALURONAN SYNTHESIS.

VITALE, DAIANA L.; SPINELLI, FIORELLA M.; SEVIC, INA; CAON, ILARIA; DEMARCHI, GIANINA; VALLA, SOFÍA; CARAVÀ, ELENA; VIOLA, MANUELA; CRISTINA, CAROLINA; ALANIZ, LAURA

Cardiff

Conferencia; 11th International Conference on Hyaluronan; 2019

International Society of Hyaluronan Sciences

Epirubicin (EPI) is an antitumor drug used for treating several human malignancies. It has less toxicity due to a glucuronidation via a glucuronosyltransferase (UGT2B7) that binds EPI to UDP-glucuronic acid (UDP-GlcUA) 1. UDP-GlcUA is formed through the action of the UDP-glucose dehydrogenase (UGDH). Besides, UDP-GlcUA is a precursor of several glycosaminoglycans such as hyaluronan (HA) 2, but the importance of glucuronidation in breast cancer treatment has not been studied yet. The aim was to evaluate the effect of silencing UGDH gene on EPI response and its association with HA synthesis using an aggressive breast cancer cell line.MATERIALS & METHODS: MDA-MB-231 cells were transfected with UGDH or control siRNA using Nucleofector?. After 24h, 1µM EPI was added to complete 48h post-transfection. Apoptosis and EPI accumulation were detected by flow cytometry. Tumor cell migration was analyzed by wound healing assay. UGDH, UGT2B7, ABCC1-C2-G2, LC3, VEGF, EGF and HASes mRNAs expression were determined by RT-qPCR. ECM components were analyzed through a particle exclusion assay and levels of secreted HA were measured by ELISA. β-Catenin and p-Akt expression were determined by Western Blot and secreted VEGF through by ELISA. Lactate dehydrogenase (LDH) activity and pH levels were detected by colorimetric assays. RESULTS: After 48h post-transfection, a reduction of 90% of UGDH mRNA was obtained. Higher EPI accumulation was observed in cells transfected with UGDH siRNA. However, the induction of apoptosis in those conditions was significantly lower. We observed an increase in mRNA expression of ABC pumps, VEGF, EGF, UGT2B7 and LC3 after combined the silencing of UGDH with EPI treatment. Tumor cells increased their migration and ECM after silencing UGDH and EPI treatment comparing with basal conditions. In concordance, we observed an increase in HAS-3 mRNA levels; but we could not detected statistical differences in secreted HA. To study glycolytic metabolism, we analyzed the LDH activity in cell lysates and pH range in supernatants as result of our treatments. We observed a slight increase in LDH activity when cells were transfected. We found a significant decrease in pH of cell supernatants after all transfections (with UGDH or control siRNA). Akt/PI3K pathway was also analyzed and we observed an increase in p-Akt in response to both treatments. CONCLUSIONS: Knockdown of UGDH increased EPI availability in tumor cells. However, we found an opposite effect in response to higher EPI accumulation as consequence of mechanisms that involve drug efflux, migration and survival. Furthermore, in response to absence of UGDH, tumor cells were able to compensate this deficiency by up-regulation of expression of ECM components, and activate metabolic changes related to drug resistance.