Roles Of RAD21 In Regulating Triple-negative Breast Cancer Cells Chemosensitivity To Adriamycin And 5-FU

Background:Breast cancer is the most common malignancy in female worldwide Triple-negative breast cancer(TNBC)lacks the expression of estrogen receptor,progesterone receptor and HER2.It contributes to ’15%of breast cancers cases Patients with TNBC have an increased probability of distant metastases,recurrence and death compared with other types of breast cancer.The standard treatment for TNBC involves cytotoxic chemotherapy regimens.Neoadjuvant chemotherapy(NAC)used in TNBC usually includes anthracycline-based chemotherapy and paclitaxel-based chemotherapy.Among the anthracycline-based chemotherapy,the combination of Adriamycin,5-FU and cyclophosphamide is the most commonly used treatment.NAC increases survival of patients with TNBC,but more than 50%of TNBC patients with NAC treatment have residual cancer burden.The insensitivity of NAC is associated with metastases and recurrence after treatment.However,the molecular mechanism of TNBC chemoresistance is not completely understood.Cohesin is a large and highly conserved ring-shaped protein complex from fission yeast to human beings,consisted of SMC1,SMC3 and RAD21.RAD21 is the core submit of cohesin,which is the only physical correlation between SMC1/SMC3 heterodimer and STAG1/2 submits.What is more,RAD21 could also regulate the binding or dissociation of cohesion complex with chromatin,maintain the stability of the cell genorme and regulate the cell identity genes transcription.RAD21 is one of the driver genes in breast cancer.RAD21 gene amplification or overexpression are associated with poor outcome and chemoresistance in breast cancer.Recent studies indicate that RAD21 might determine drug response of certain drug regimens,such as 5-Fluorouracial,cyclophosphamide and Etopside.But the underlying mechanisms have not been fully identified yet.In this study,we aims to explore the molecular mechanism of RAD21 in regulation of drugs chemosensitivity in TNBC.It will be important to determine whether RAD21 can be a predictive marker of response to neoadjuvant chemotherapy and personalized therapy in breast cancer.Objective:To explore the molecular mechanism of RAD21 in regulation of chemosensitivity to Adriamycin and 5-FU in TNBC.It will be important to determine whether RAD21 can be a predictive marker of response to neoadjuvant chemotherapy and personalized therapy in breast cancer.Methods:Flow cytometry was used to detect reactive oxygen species(ROS)level in RAD21 depleted breast cancer cells.Western Blotting was performed to examine DNA damage by phosphorylation of H2AX at Ser 139.Western Blotting、RT-qPCR and Dual-luciferase reporter assay were performed to analyze the regulatory effect of RAD21 on the NRF2/ABCC3 pathway.Cell counting kit-8(CCK-8)proliferation assay and Western Blotting were used to examine the role of RAD21 in the regulation of the sensitivity to Adriamycin in TNBC cells.The effects of RAD21 on cellular Adriamycin uptake and retention were evaluated by fluorescent microscope experiments in TNBC cells.EdU proliferation assay was performed to examine the effect on cell proliferation in the RAD21 depleting breast cancer cells after 5-FU treatment.Colony formation assay and CCK-8 proliferation assay were performed to examine the regulation of RAD21 on the sensitivity of TNBC cells to 5-FU.Migration assay was used to detect the metastasis effect of RAD21 in triple-negative breast cancer cells after 5-FU treatment.Western Blotting was used to examine the expression level of ABHD5 in RAD21 depletion breast cancer cells.Results:1.RAD21 knockdown induces ROS accumulation and DNA damage in TNBC cellsIn the triple-negative breast cancer cell lines MDA-MB-231 and BT549,RAD21 knockdown increased intracellular ROS levels and the protein level of RAD50,while the protein level of RAD51 was decreased.DNA damage was examined by phosphorylation of y-H2AX in RAD21 depletion cells2.RAD21 depletion activates the NRF2/ABCC3 signaling pathway in TNBC cellsIn RAD21 knocking-down MDA-MB-231 cells,the protein level of NRF2 was increased while mRNA level of NRF2 did not alter.The up-regulation of NRF2 protein was probably due to an increase in its stabilization by elevated ROS level.In MDA-MB-231 cells,RAD21 depletion selectively activated NRF2 partial target genes such as ABCC3 and FTH1,but not other target genes such as GSTP1 and ABCG2.3.RAD21 knockdown reduces chemosensitivity to Adriamycin in TNBC cells by activating NRF2/ABCC3 signaling pathway.0.5μM Adriamycin treatment for different durations resulted in a decrease in the expression of RAD21 and increases of NRF2 and ABCC3 in MDA-MB-231 and BT549 cells.RAD21 depleted breast cancer cells were more resistant to Adriamycin compared with control cells.RAD21 overexpressed cells were more sensitive to Adriamycin compared with control cells.In RAD21-knockdown MDA-MB-231 cells apoptosis-related protein cleaved-caspase3 was significantly reduced compared with control cells,which indicated that RAD21 depletion lead to a decrease of apoptosis in breast cancer cells.Compared with control cells,the cellular uptake of Adriamycin was reduced and the efflux of Adriamucin was enhanced in RAD21 depleted breast cancer cells.The inhibition of NRF2 by ML-385 significantly attenuated the increase of Adriamycin efflux in RAD21 depletion MDA-MB-231 cells4、RAD21 depletion increases the chemosensitivity to 5-FU in TNBC cells.In RAD21-knockdown MDA-MB-231 cells,the sensitivity to 5-FU was significantly increase compared with control cells.RAD21 depletion resulted in the reductions of colony formation efficiency and migration ability induced by 5-FU in MDA-MB-231 cells.In MDA-MB-231 and BT549 cells treated with 5-FU,the protein levels of DPYD,UCK1 and TYMP were declined.There were no obvious differentiation of DP YD and UCK1 protein levels in RAD21 knockdown TNBC cells compared with control cells.It suggested that RAD21 depletion induced increase of chemosensitity to 5-FU in TNBC cells could not mediated by DP YD and UCK15、RAD21 depletion may inhibit ABHD5 up-regulation and promotes the chemosensitivity to 5-FU in TNBC cells.In MDA-MB-231 cells exposured to 5-FU,the protein level and mRNA level of ABHD5 were increased in a dose-dependent manner and time-dependent manner.In RAD21-knockdown MDA-MB-231 cells,the mRNA level and protein level of ABHD5 were not changed.5-FU induced an increase of ABHD5 protein in control cells,but it failed to promote the ABHD5 protein accumulation in RAD21 depleted breast cancer cells.Conclusions:1.RAD21 maintains intracellular ROS homeostasis and genome integrity in TNBC cells.2.RAD21 knockdown reduces chemosensitivity to Adriamycin in TNBC cells by activating NRF2/ABCC3 signaling pathway.3.RAD21 depletion results in an increase of chemosensitivity to 5-FU in TNBC cells by inhibiting ABHD5 up-regulation.