CLEC3A Promotes Tamoxifen Resistance Via PI3K/AKT Signaling In ER+ Breast Cancer

Breast cancer is the most common malignancy and the second leading cause of cancer-related deaths in women,newly diagnosed breast cancer cases are increasing annually,and it has become the top killer of women’s health.The tumorigenesis and development of breast cancer are closely related to sex hormone level due to its estrogen-dependent pattern.Pathologically,patients with positive estrogen receptor account for about70%,so endocrine therapy is extremely important for these patients.Tamoxifen is a selective ER modulator antagonizing the effect of estrogen on breast cancer cells by competing with ER.In the past 30 years,tamoxifen has become the most prescribed drug for endocrine therapy of premenopausal breast cancer.It can significantly improve overall survival and disease-free survival.However,there are still 30-50% of patients who cannot benefit from tamoxifen treatment,which is mainly due to primary and secondary tamoxifen resistance.Therefore,it is of great significance to study the molecular mechanism related to tamoxifen resistance.Objectives1.To explore the differential expression of CLEC3 A in tamoxifenresistant breast cancer cells and pathological samples,and investigate the relationship between CLEC3 A expression and tamoxifen sensitivity.2.To study the molecular function of CLEC3 A in tamoxifen resistant breast cancer cells.3.To uncover the mechanism of CLEC3 A in ER+ breast cancer tamoxifen resistant cells.Methods1.We used 4-hydroxytamoxifen(the active metabolite of tamoxifen)to continuously induce MCF7 and T47 D cells to generate tamoxifenresistant MCF7 R and T47 DR cells.Morphological difference between these cell lines before and after induction was observed with an inverted phase contrast microscope,and the half maximal inhibitory concentration(IC50)of 4OHT on MCF7,MCF7 R,T47D and T47 DR cells was detected by CCK-8 assay.2.Real-time quantitative PCR and western blot were applied to detect the differential expression of CLEC3 A m RNA and protein in tamoxifensensitive cells and tamoxifen-resistant cells,respectively.Meanwhile,we also collected 7 pairs of primary and recurrent breast cancer specimens from patients who received tamoxifen treatment,and performed immunohistochemistry(IHC)to evaluate the expression of CLEC3 A in primary and recurrent lesions.3.Small interfering RNAs(si RNAs)targeting CLEC3 A were transfected into in MCF7 R and T47 DR,and CLEC3 A overexpressing plasmids were introduced into MCF7 and T47 D to elevate the expression of CLEC3 A.Using CCK-8 assay and plate clone formation assay,the effect of CLEC3 A knockdown/overexpressing on cell proliferation was investigated.Flow cytometry and western blot were used to detect the effects of CLEC3 A knockdown/overexpressing on cell apoptosis and apoptosis-related protein expression.Finally,the wound healing experiment and Transwell assay were used to explore the effect of CLEC3 A on breast cancer cell migration and invasion.4.The mechanism of CLEC3 A in ER+ breast cancer is unknown.We used western blot to detect the effect of CLEC3 A knockdown/overexpressing on PI3K/AKT pathway activity.Next,rescue experiments were performed to verify the relationship between CLEC3 A and PI3K/AKT pathways.Results1.Tamoxifen-resistant MCF7R/T47 DR were morphologically different with their parental MCF7/T47 D cells.The tamoxifen-resistant cells were more likely to be round and spherical,while their parental cells were close to spindle.In addition,the IC50 of the 4OHT to drug-resistant cells is different from that of their parental cells.2.Real-time quantitative PCR and western blot results showed that compared with their parental cells MCF7/T47 D,the protein and m RNA levels of CLEC3 A in the tamoxifen-resistant MCF7R/T47 DR cells were significantly elevated.Moreover,the primary and recurrent tumor tissues of breast cancer patients who received tamoxifen treatment were evaluated by IHC,and the results showed that CLEC3 A expression was increased in the recurrent tumor tissue compared with the primary tumor.3.(1)The results of real-time quantitative PCR and western blot showed that compared with the control group,si RNA targeting CLEC3 A downregulated the expression of CLEC3 A in MCF7R/T47 DR.And,the CLEC3 A overexpressing plasmids significantly elevated the expression of CLEC3 A in MCF7/T47 D.(2)Compared with the control,the inhibition rate of 4OHT on cell proliferation significantly increased and the pro-apoptotic ability was significantly enhanced after CLEC3 A knockdown in the MCF7R/T47 DR,and the expression of apoptosis-related markers changed accordingly.Compared with the control group,after CLEC3 A overexpressing in MCF7/T47 D,the cell proliferation rate increased and the apoptosis decreased,and the expression levels of apoptosis-related markers changed accordingly.(3)The results of wound healing showed that CLEC3 A knockdown did not increase or decrease the healing speed in MCF7 R.In addition,the number of migrated cells in the CLEC3 A knockdown group was not significantly increased or decreased.The results of the Transwell assay showed that there was no significant difference in the number of invaded cells between CLEC3 A knockdown group and the control.4.The effect of CLEC3 A on proliferation and apoptosis in ERpositive breast cancer cells can be partially reversed by the inhibition of AKT by MK2206.Knockdown of CLEC3 A while applying AKT inhibitors could inhibit the survival of ER-positive breast cancer cells and increase apoptosis.Conclusions1.Compared with their parental cells,MCF7R/T47 DR cells were morphologically changed.In addition,MCF7 R and T47 DR cells successfully developed tamoxifen resistance.2.Compared with their parental cells,the m RNA and protein expression of CLEC3 A in MCF7R/T47 DR cells are significantly increased.In addition,compared with the primary tumor tissue,CLEC3 A protein expression in recurrent tumor tissues was also significantly increased,indicating that CLEC3 A was closely related to breast cancer tamoxifen resistance.3.CLEC3 A expression was knocked down in MCF7R/T47 DR cells with si RNAs targeting CLEC3 A.Moreover,CLEC3 A overexpressing plasmids were also transfected into MCF7/T47 D cells.With CCK-8experiment,plate clone formation experiment and flow cytometry,the proapoptotic ability and inhibitory ability of 4OHT on cell proliferation were enhanced in MCF7R/T47 DR.And,CLEC3 A promoted cell proliferation and inhibited apoptosis in MCF7/T47 D.Moreover,CLEC3 A had no effect on cell migration or invasion in tamoxifen-resistant cells.4.In MCF7/T47 D cells overexpressing CLEC3 A,CLEC3A did not change the expression of PI3K/AKT,but promoted their phosphorylation,thereby enhancing the activity of this pathway.The phenotypic changes caused by CLEC3 A overexpressing in MCF7/T47 D cells can be partially reversed by the AKT inhibitor MK2206.CLEC3 A regulated the proliferation,apoptosis and tamoxifen resistance of ER-positive breast cancer cells through the PI3K/AKT pathway.