The Mechanism Of Feedback Activation Of PI3K/AKT Axis By Inhibiting STAT3 Activity In Breast Cancer Cells And The Strategy For The Rational Combination Of Targeted Therapies

Objective:To reveal the phenomenon and the molecular mechanism of the feedback activation of PI3K/AKT axis by inhibiting STAT3 activity in a variety of breast cancer cell lines and explore the rationality and significance of STAT3 and PI3K/AKT/mTOR signaling pathway(PAM)as the combination target therapy for breast cancer.Method:1.A series of breast cancer cell lines were treated with the inhibitor of STAT3(Stattic).CRISPR/Cas9 system or short hairpin RNA were used to establish a cell line that knocked out or knocked down the expression of STAT3 and the expression of p-STAT3,p-AKT and p-S6 protein was detected by western-blot.2.A series of breast cancer cells were treated with Stattic and the inhibitor of PAM alone or in combination including PF-4708671(the inhibitors of S6K1),NVP-AEW541(the inhibitors of IGF-1R)and GDC-0941(the inhibitors of PI3K)and the expression of p-STAT3,p-AKT and p-S6 protein was detected by western-blot.3.CRISPR/Cas9 system or short hairpin RNA were used to establish a series of breast cancer line that knocked out or knocked down the protein expressions of AKT.Mutant and wide-type cell lines were treated with Stattic and the expression of p-STAT3,p-AKT,p-S6,c-PARP protein was detected by western-blot.The proliferation activity of the mutant and widetype cells was detected by Cell Counting Kit-8(CCK-8).Breast cancer cells were treated with NVP-BEZ235(PI3K/mTOR inhibitor)and Stattic.The expression of p-STAT3,p-AKT,p-S6,c-PARP protein was detected by western-blot.The clonal formation and proliferative activity of breast cancer lines were detected with plate clone formation assay and CCK-8 assay respectively.MCF-7 was used to establish a breast cancer tumor xenograft model and tumor growth was detected under single or combined administration of Stattic and NVP-BZE235.Results:1.In many types of breast cancer cells,the pharmacological and genotypic inhibition of STAT3 could significantly enhance the activity of AKT.The sensitivity of Stattic on PI3K/AKT activation was different in different types of tumor cells.2.Both PF-4708671 and Stattic could inhibit S6 activity and enhance the activity of AKT,and further enhance the inhibitory effect on the activity of S6 when used in combination.However,the addition of Stattic did not significantly increase the activity of AKT induced by PF-4708671.Both NVP-AEW541 and GDC-0941 could inhibit the activity of AKT and S6 and block the feedback activation of AKT induced by Stattic.3.Compared with the control,when Stattic treated AKT mutant cell line,the activity of c-PARP was significantly increased and the proliferation activity of the cells was significantly reduced.When NVPBEZ235 and Stattic combination treatment inhibited the activity of STAT3 and AKT at the same time,the expression of c-PRAP was significantly incresed and the cloning formation rate and proliferation activity were significantly decreased compared with the single drug treatment.In vivo assay,the tumor growth was inhibited signigicantly when we block the Stat3 and PI3K/AKT signaling pathway together.Conclusion:In many types of breast cancer cell lines,inhibition the STAT3 activity can enhance the activity of PI3K/AKT axis.The mechanism is due to the positive regulatory of STAT3 on S6 activity.Inhibition of the activity of STAT3 makes S6 to release the negative feedback inhibition and lead to the feedback activation of AKT.In addition,we found that STAT3 and AKT are mutually regulated.Inhibition of the activity of STAT3 and PAM can inhibit the growth of breast cancer cells synergistically.The results provides some guidance on the target therapy on breast cancer.