The Role And Mechanism Of EPHA5 In The Trastuzumab Resistance Of HER2-positive Breast Cancer

BackgroundAs an effective targeted drug,trastuzumab has been widely used in the treatment of HER2-positive breast cancer patients,and has achieved great success in clinical practice.However,due to the congenital lack of sensitivity to Herceptin in some patients,only 12%-34%of patients are effective,and nearly 50%of patients are prone to drug resistance within one year,leading to treatment failure.Therefore,it is of great social value to further study the potential mechanism of Herceptin resistance to improve clinical efficacy and benefit more patients.EPHA5,as a receptor tyrosine kinase,has been proved to be involved in the occurrence and development of a variety of tumors,and is closely related to the effect of cell dormancy on the treatment of tumors.However,up to now,the clinical significance of EPHA5 in HER2-positive breast cancer and its role and mechanism in trastuzumab resistance remain unclear.ObjectiveThe purpose of this study was to explore whether EPHA5 may play a role in trastuzumab resistance by regulating breast cancer stem-like characteristics using vivo and vitro experiments.Methods1.To evaluate whether the expression of EPHA5 has an impact on the prognosis of HER2-positive breast cancer patients treated with trastuzumab.Paraffin masses of 88HER2-positive breast cancer patients treated with trastuzumab were collected for immunohistochemical analysis.The expression of EPHA5 in breast cancer tissues was detected.The expression of EPHA5 in HER2-positive breast cancer patients was analyzed with age,menopausal status,pathological grade,hormone receptor?HR?status,lymph node metastasis status and TNM score.To evaluate the correlation between the expression of EPHA5 and trastuzumab resistance.Blood samples were collected from 20 patients with HER2-positive breast cancer who were sensitive and tolerant to trastuzumab?the diagnosis of HER2-positive breast cancer was based on ASCO/CAP guidelines?.The expression of EPHA5 in plasma was detected by qRT-PCR in two groups?resistant and sensitive to trastuzumab?.2.The EPHA5-deficient SKBR3-KO cell line was constructed by CRISPR/Cas9technique in the trastuzumab-sensitive SKBR3 cell line,and the stable overexpression cell line JIMT-1-OE was constructed by lentiviral technique in the trastuzumab-resistant JIMT-1 cell line.The viability of SKBR3-KO cells treated with trastuzumab?no-load as control?and JIMT-1-OE cells treated with trastuzumab?no-load as control?were detected by CCK-8 assay.Flow cytometry was used to observe the effects of EPHA5 knockout and overexpression by trastuzumab on cell cycle and apoptosis.The breast cancer stem cells from SKBR3-KO and3.Breast cancer stem cells were isolated by cell surface marker CD44⁺/CD24^-/low.qRT-PCR and western blot were used to detect the regulation of EPHA5 on tumor stem cell markers.EPHA5 knockout of SKBR3 breast cancer cells or EPHA5over-expression of JIMT-1 cells?no-load as control?were involved in the maintenance of stemness identified by mammosphere formation assay in vitro.The stem cells separated from SKBR3-KO cells and the control group were injected into the breast pad of nude mice to establish a tumorigenic model.Trastuzumab was given one week later to observe the effect of EPHA5 deletion on tumorigenesis,metastasis and survival of nude mice.The effect of EPHA5 deletion on the therapeutic effect of trastuzumab in vivo was evaluated.4.The effects of EPHA5 on Notch1 and PI3K/AKT signaling pathways in HER2-positive breast cancer cells were observed by immunofluorescence staining and Western Blot.Notch1 inhibitor FLI-06 and PBS were used in SKBR3-KO cells and no-load control group respectively.Apoptosis test,cell cycle analysis and mammosphere formation assay were also used to verify whether Notch1 signaling pathway played a role in the resistance to trastuzumab through the rescue experiment.Results1.In 88 HER2-positive breast cancer specimens treated with trastuzumab,the expression of EPHA5 protein was significantly correlated with clinical stage?P=0.036?and lymph node metastasis?P=0.033?.There was no significant correlation with age?P=0.348?,state of extremity?P=0.087?,tumor grade?P=0.383?,ER?P=0.868?and P R?P=0.595?.Cox regression analysis showed that lymph node metastasis?P=0.006,HR=0.345,95%CI=0.217-0.549?and EPHA5 expression levels?P=0.032,HR=0.231,95%CI=0.149-0.359?were independent influencing factors of overall survival?OS?in 88 patients.Kaplan-Meier curve analysis revealed that there was a significant correlation between the low expression of EPHA5 and poor prognosis of breast cancer patients received trastuzumab treatment?log-rank test,P=0.0353?.The expression of EPHA5 in the plasma of 40 patients with trastuzumab resistant HER2-positive breast cancer was significantly lower than that of the control group.2.Western Blot results showed that EPHA5 expression was negative in SKBR3-KO cells and increased significantly in JIMT-1-OE cells?compared with the control group?.CCK8 test and apoptotic test showed that the sensitivity of EPHA5-deleted breast cancer cell SKBR3 to trastuzumab was significantly lower than that of control group,while the sensitivity of EPHA5-overexpressed breast cancer cell JIMT-1 to trastuzumab was significantly higher than that of control group.Flow cytometry showed that over-expression of EPHA5 could block cell cycle in S phase.3.The stem cell markers NANOG,CD133 and CD44 from SKBR3-KO CSCs were significantly higher than those from the control group,while E-cadherin and CD24was significantly lower than those from the control group.The stem cell markers NANOG,CD133 and CD44 from JIMT-1-OE CSCs were significantly lower than those from the control group,while E-cadherin and CD24 was significantly higher than those from the control group.Stem cell culture showed that the ability of CSCs from SKBR3-KO was significantly higher than that of control group,and the ability of CSCs from JIMT-1-OE was significantly lower than that of control group.In vivo experiments in nude mice showed that the lung and liver metastases of nude mice in SKBR3-KO group were significantly lower than those in control group,and the body weight and survival time of nude mice in SKBR3-KO group were lower than those in control group.4.Immunofluoresence cell staining and western blot results showed that EPHA5could regulate the changes of Notch 1 and PI3K/AKT signaling pathways related proteins NICD,Hes1,Hey1,PTEN and AKT in SKBR3-KO and JIMT-1-OE cell lines.In the rescue experiment,SKBR3-KO cells were treated with Notch1 inhibitor FLI-06 and PBS,respectively.The results showed that the cell viability,apoptosis,stem cell surface markers and the capacity of forming mammoshperes induced by EPHA5 could be reversed by FLI-06.ConclusionThe expression of EPHA5 in HER2-positive breast cancer patients with trastuzumab resistant was down-regulated.EPHA5 expression was closely related to lymph node metastasis,TNM status and poor prognosis.The deletion of EPHA5could significantly increase the resistance of HER2-positive breast cancer cell lines to trastuzumab through promoting the proliferation and DNA synthesis,inhibit the apoptosis.EPHA5 may affect stemness of breast cancer cells through Notch1 and PI3K/AKT pathways.The results of this study suggest that EPHA5 is expected to be a new target for reversing trastuzumab in HER2-positive breast cancer patients.