Effect And Underlying Mechanism Of HORMAD1 On Docetaxel Resistance In Triple Negative Breast Cancer Cells

Objective:This study aims to explore the effect of HORMA domain containing 1(HORMAD1)on docetaxel resistance of Triple Negative Breast Cancer(TNBC)and its possible mechanism.Methods:Bioinformatic methods were used to check HORMAD1expression in breast cancer datasets.The clinicopathological information and tissue samples of TNBC patients undergoing neoadjuvant chemotherapy were collected to evaluate their pathological response,and the expression of HORMAD1 was analyzed by IHC staining.The relationship between HORMAD1 expression and clinicopathological characteristics and prognosis of patients was analyzed.In vitro experiments,the expression of HORMAD1 in cell lines was changed by overexpression plasmid or small interfering RNA(siRNA)/lentiviral vectors.The changes of related genes and proteins were detected by RT-PCR and western blot(WB).Cell Counting Kit?8(CCK-8)was used to verify the effects of HORMAD1 on cell proliferation and drug sensitivity.Flow cytometry was used to detect cell cycle,cell apoptosis,and intracellular reactive oxygen species.Caspase activity detection kits were used to detect the changes of caspase 3/8/9 induced by chemotherapeutic drugs.Protein expression and localization were detected by immunofluorescence.The ubiquitin binding of target protein was detected by immunoprecipitation method.Results:Bioinformatics and database analysis showed that HORMAD1 was only highly expressed in basal-like subtype and TNBC cell lines.IHC detection revealed that the expression of HORMAD1 in TNBC tissues with residual lesions after neoadjuvant chemotherapy was21.1%(23/109).In vitro experiments,cell proliferation,cell cycle,and apoptosis were not be affected by specific siRNA-HORMAD1 in TNBC cells.However,significant reduction of autophagy marker P62 and significant increase of Beclin1 were accompanied by HORMAD1 knockdown detected by WB.Overexpression of HORMAD1 was associated with a significant increase in P62 protein.Drug sensitivity of docetaxel could be enhanced in TNBC cells with knockdown expression of HORMAD1.Endogenous expression of HORMAD1 can be induced by DNA methyltransferase inhibitor5-aza-2-deoxycytidine in MDA-MB-231.Furthermore,the cytotoxicity of docetaxel to MDA-MB-231 cells was decreased.Apoptosis induced by docetaxel was significantly increased in the HORMAD1 knockdown group compared with control group detected by flow cytometry.WB detection found that anti-apoptotic protein BCL2 was significantly reduced in HORMAD1 knockdown group after docetaxel was exposed.Meanwhile,G₀/G₁-phase block and lower S-phase of TNBC cells was found in HORMAD1 knockdown group when exposed to docetaxel.After the addition of docetaxel,proliferation of HORMAD1 knockout group was significantly lower than that in control group.Meanwhile,more reactive oxygen species were found in the HORMAD1 knockout group,and the activity of caspase 3/8/9 were higher than that in the control group.Mechanism studies have found that early apoptosis induced by docetaxel was increased by HORMAD1 overexpression in MDA-MB-231cells,which was similar with the rate of autophagy inhibitor 3MA increasing the early apoptosis induced by docetaxel in MDA-MB-436 cells.Interestingly,although 3MA can enhance the decrease of cell activity induced by docetaxel,the sensitization effect was weakened with the increase of docetaxel concentration.Moreover,cell damage induced by docetaxel in the HORMAD1 knockdown group was not further increased with 3MA.In addition,the DNA damage marker,?H2AX,was significantly increased and the DNA damage repair marker,RAD51,was significantly decreased in the HORMAD1 knockdown group after docetaxel was added in TNBC cells.Moreover,Hoechst staining results showed that the proportion of nuclear fragmentation in HORMAD1knockout group was higher after docetaxel treatment.Further studies showed that cell proliferation was inhibited after continuous knockdown of HORMAD1 by lentiviral infection plus puromycin screening in TNBC cells.Also,cycle arrest occurs,and the proportion of senescent cells was increased.Meanwhile,significant increase in senescence related protein marker,such as p Rb,P27 detected by WB in HORMAD1 continuous knockdown group.At the same time,siRNA knockdown of HORMAD1 also found an increased proportion of senescent cells was also found in HORMAD1 transient knockdown group by siRNA.HORMAD1 knockdown did not affect P27 gene expression was not effected by HORMAD1 knockdown.However P27 protein expression were significantly increased after HORMAD1 knockdown,and vice versa.In rescue experiments,HORMAD1 knockdown combined with knockdown of P27 reduced the proportion of senescent cells compared with HORMAD1 knockdown alone.HORMAD1 knockdown in TNBC cells slowed down the degradation of P27 protein,and vice versa,according to the inhibition of protein synthesis by cyclohexane.The expression of HORMAD1 in TNBC cells inhibited the degradation of P27 protein by MG132.Protein IP assay confirmed that the expression of HORMAD1 in TNBC cells increased the ubiquitination of P27 protein.Conclusions:HORMAD1 was highly expressed only in the TNBC subtype.HORMAD1 negatively regulates P27 expression by promoting P27 ubiquitination degradation in TNBC cells.The inhibition of HORMAD1 on tumor cell senescence and the promotion of DNA damage tolerance may be the main reasons for its docetaxel resistance.