To Explore The Mechanism Of CDK4/6 Inhibitor Palbociclib Involved In(RB+/CCNE1-)ER+ Positive Breast Cancer Resistance Through Autophagy Activated By MAPK Pathway

Objective: To investigate the mechanism of CDK4/6 inhibitors palbociclib(PAL)in the regulation of autophagy in(RB+/CCNE1-)ER+ breast cancer and clarify the mechanism of CDK4/6 inhibitor involved in(RB+/CCNE1-)ER+ breast cancer resistance through autophagy activated by MAPK pathway.Methods: PAL single-use tumor inhibition experiment: Human ER-positive breast cancer cells MCF-7 cells were inoculated into nude mice subcutaneously to construct ER-positive breast cancer tumor nude mice model.After the model was successfully established,the tumor-bearing nude mice were given drug intervention by gavage with normal saline and different doses of PAL(50mg/Kg,100 mg/Kg,150 mg/Kg).During the drug intervention,the general conditions such as the mental state and food intake of the nude mice were monitored every day,the body weight of the experimental nude mice and the volume of the transplanted tumor were measured every 3 days,and the corresponding graphs of body weight changes and the growth curve of the transplanted tumor volume were drawn.After the drug intervention,the transplanted tumor tissues were collected,and Western blot was used to detect the expression of autophagy key protein LC3 and the expression of MAPK pathway-related proteins p-ERK,p-RAF,p-MEK,ERK,RAF,MEK in the transplanted tumor tissues,Image J The software analyzes the gray value of the protein bands and calculates the ratios of LC3II/I,p-ERK/ERK,p-RAF/RAF,and p-MEK/MEK.The expressions of LC3,p-ERK,p-RAF and p-MEK in the transplanted tumor tissues were detected by immunohistochemistry(IHC).PAL and GSK1120212(GSK)combined with tumor inhibition experiment:Human ER-positive breast cancer cells MCF-7 cells were inoculated subcutaneously in nude mice to construct ER-positive breast cancer tumor nude mice model.After the model was successfully constructed,normal saline(control group),100 mg/Kg PAL(PAL group),1 mg/Kg GSK(GSK group),and 100 mg/Kg PAL+1 mg/Kg GSK(combined)were administered by gavage.Administration group)drug intervention was performed on tumor-bearing nude mice.During the drug intervention,the general conditions such as the mental state and food intake of the nude mice were monitored every day,the body weight of the experimental nude mice and the volume of the transplanted tumor were measured every 3 days,and the corresponding graphs of body weight changes and the growth curve of the transplanted tumor volume were drawn.After the drug intervention,the transplanted tumor tissues were collected,and the morphological changes of tumor cells in the transplanted tumor tissues of each group were observed by hematoxylin-eosin(HE)staining.Western blot was used to detect the expression of autophagy key protein LC3 in the transplanted tumor tissues of each group.Image J software was used to analyze the gray value of the protein band,and the ratio of LC3II/I was calculated.The expression of Ki67,LC3,p-ERK,p-RAF and p-MEK in the transplanted tumor tissues of each group was analyzed by IHC.Results: PAL single-use tumor inhibition experiment:(1)The ER-positive breast cancer tumor-bearing nude mouse model was successfully established,and the tumor formation rate was 100%.(2)There was no significant difference in the body weight of experimental nude mice in each group during the drug intervention period.(3)Compared with the control group,PAL concentration-dependently inhibited the volume of the transplanted tumor in nude mice,and the high dose of PAL had the most obvious inhibitory effect on the growth of the transplanted tumor.(4)The results of Western blot experiments showed that compared with the control group,PAL could increase the expression of LC3 II protein and the ratio of LC3II/I in the transplanted tumor tissues;The results of IHC experiments showed that compared with the control group,PAL could significantly increase the proportion of LC3-positive tumor cells in the transplanted tumor tissue.(5)The results of Western blot showed that compared with the control group,PAL could significantly up-regulate the expression of key proteins p-RAF,p-MEK and p-ERK in the MAPK signaling pathway.Image J software gray value analysis showed that PAL significantly increased the ratio of p-RAF/RAF,p-MEK/MEK,p-ERK/ER;IHC results showed that compared with the control group ratio,PAL could significantly increase p-RAF,p-MEK and p-ERK positive tumor cells in transplanted tumor tissues.Indicating that PAL can upregulate the MAPK signaling pathway.PAL and GSK combined with tumor inhibition experiment:(1)The ER positive breast cancer tumor-bearing nude mouse model was successfully established,and the tumor formation rate was 100%.(2)During drug intervention,compared with the experimental nude mice in the control group and the PAL and GSK single-drug group,the experimental nude mice in the PAL and GSK co-administration group started to lose weight from the 6th day of administration.(3)Compared with the control group,PAL has a certain inhibitory effect on the growth of the transplanted tumor in the experimental nude mice.The average volume of the transplanted tumor in the combined administration group of PAL and GSK was significantly smaller than that in the control group and the single-drug group,indicating that the combination of PAL and GSK has a synergistic inhibitory effect.HE staining results showed that compared with the control group,the PAL group had less necrosis,decreased tumor cell density,and decreased cell atypia.There was no obvious necrosis in the tumor tissue of the PAL and GSK combined administration group,and the tumor cell density and atypia were the smallest in this group.The results of IHC staining showed that compared with the control group,the proportion of Ki67-positive tumor cells in the PAL group was reduced,and the proportion of Ki67-positive tumor cells in the PAL and GSK group was the lowest,indicating that PAL can reduce the proliferation index of xenografts,and when combined with GSK has a synergistic inhibitory effect on the proliferation of xenografts.(4)The results of Western blot showed that MEK inhibitor GSK could significantly reduce the expression of LC3 II protein and the ratio of LC3II/I in PAL-treated xenograft tissues.The results of IHC experiments showed that compared with the control group,GSK could significantly reduce the proportion of LC3,p-RAF1,p-MEK and p-ERK positive tumor cells in PAL-treated xenograft tissues.The above experimental results indicated that in nude mouse ER-positive breast cancer xenografts,PAL-activated autophagy could be inhibited by GSK,and PAL activated autophagy in nude mouse ER-positive breast cancer xenografts through the MAPK signaling pathway.Conclusions:(1)The CDK4/6 inhibitor PAL could inhibits the growth of ER-positive breast cancer,and it has a synergistic inhibitory effect on ER-positive breast cancer when combined with MEK inhibitor GSK.(2)CDK4/6 inhibitor PAL may activate autophagy by regulating MAPK signaling pathway.(3)CDK4/6 inhibitor PAL activates autophagy through MAPK signaling pathway in ER-positive breast cancer xenograft model,which may be one of the molecular mechanisms of its drug resistance.