Down-regulation of UDP-glucose dehydrogenase affects Epirubicin response and extracellular matrix components in MDA-MB-231 breast cancer cells.

DAIANA VITALE; ILARIA CAON; DAVIDE VIGETTI; MANUELA VIOLA; EVGENIA KAROUSOU; ELENA CARAVÀ; PAOLA MORETTO; ALBERTO PASSI; LAURA ALANIZ

Boston

Seminario; Gordon Research Seminar. 2018 Proteog lycans (GRS); 2018

Gordon Research Conference

Anthracyclines are antitumoral drugs used for treating several human malignancies, including breast cancer resistant to hormonal therapy. Epirubicin (EPI) is less cardiotoxic compared with other anthracyclines (e.g., doxorubicin). This has been attributed to its unique glucuronidation detoxification in liver. In this sense, an UDP-glucuronosyltransferase enzyme (UGT2B7) transfer a molecule of UDP-glucuronic acid (UDP-GlcUA) to EPI, transforming it into a glucuronide form. In addition to take part in EPI glucuronidation, UDP-GlcUA is a precursor of several glycosaminoglycans synthesis, such as hyaluronan (HA). UDP-GlcUA is formed by oxidation of UDP-glucose through catalytic action of UDP-glucose dehydrogenase enzyme (UGDH). Although association of extracellular matrix (ECM) with tumor microenvironment has been well documented, the importance of glucuronidation reaction and the role of UGDH in breast cancer treatment has not been studied yet. Furthermore, a possible modulation of these mechanisms by EPI in tumor cells would be clinically relevant. The aim of present study was to evaluate the effect of silencing UGDH gene on EPI response using an aggressive breast cancer cell line. MDA-MB-231 cells were transfected with 30 nM of UGDH or negative control siRNA using Nucleofector? Kit. After 24h of incubation, 1 µM EPI was added to complete 48h of incubation after transfection. Apoptosis and EPI accumulation assays were performed by flow cytometry. Cell viability was studied through a MTT assay. Tumor cell migration during transfection and EPI treatment was analyzed by wound healing assay. UGDH, UGT2B7, p53, ABCC1, ABCC2, ABCG2, HAS-2 and HAS-3 mRNA expression were determined by RT-qPCR, and results were normalized using β-actin as housekeeping gene. ECM variations during treatment were analyzed through a particle exclusion assay. After 48h post-transfection, a reduction of 90% of UGDH mRNA was obtained. When EPI accumulation was analyzed, we observed higher intracellular accumulation in cells that had been transfected with UGDH siRNA. As was expected, an increase in p53 mRNA expression was found in response to EPI treatment. However, apoptosis induction after silencing UGDH gene and EPI treatment was lower compared with non-transfected cells. In agreement with previous data, we found similar results when cell viability was studied. EPI treatment alone decreased cell viability in a 50%, nevertheless when cells were treated with EPI after silencing UGDH, cell viability was higher. These results indicates us that cells are eluding higher accumulation of this antitumor drug in any way unstudied yet. To understand possible mechanisms by cells are evading higher EPI accumulation, first we analyzed the expression of different genes related with drug elimination. UGT2B7 mRNA expression was enhanced after EPI treatment. We also observed even more increased levels when cells were transfected with UGDH siRNA before to treat them with EPI. Furthermore, is well known that high expression of drug efflux pumps is directly related to drug resistance. To determine if tumor cells are trying to favor expulsion of EPI, we analyzed the expression of different ABC drug transporter genes. In all cases, we found a notorious increase in mRNA levels, not only in response to EPI treatment, but also in combination with silencing of UGDH gene. When tumor cell migration was analyzed, we observed that in response to silencing UGDH, cells increased their migration compared with basal conditions. Same effect have been observed when we analyzed together silencing of UGDH and posterior EPI treatment. All these data together could explain the fact of obtaining less cell death and higher viability when cells were exposed to both conditions although MDA-MB-231 cells contained higher EPI inside. Finally, we studied the effect of both conditions on ECM components. Surprisingly, after silencing UGDH gene any variation was observed in pericellular area comparing with basal conditions. Furthermore, when tumor cells were treated with EPI after silencing UGDH, even more enhanced effect was observed. These data indicates us that although tumor cells lack UGDH enzyme, they are capable of expressing and even more favoring the expression of ECM components in that conditions. To determine a possible relationship with modulation of HA synthesis, in which UDP-GlcUA is involved, HAS-2 and HAS-3 mRNA expression were studied. In both cases we found an increase in mRNA levels in response to silencing UGDH and EPI treatment. In conclusion, silencing of UGDH gene allowed increasing EPI availability in breast cancer cells. Nevertheless, we found an opposite effect in response to higher EPI accumulation. This fact is related to mechanisms that involve drug efflux, as well as tumor aggressiveness, 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, such as HA, which is highly related to aggressive tumor phenotypes and drug resistance.