Analysis Of The Resistance Mechanism Of Breast Cancer Cells To Palbociclib And The Study Of The Anti-breast Cancer Effect Of Novel MTOR Inhibitor FP-208 In Cooperation With Palbociclib

Objective:To establish a breast cancer cell line T47D-Pal R with the characterization of palbociclib resistance,and explore the mechanism of acquired drug resistance.To further explore the anti-breast cancer effect of novel m TOR inhibitor FP-208 in conjunction with CDK4/6 inhibitor palbociclib.Methods:The ER-positive breast cancer cell line T47D was used as parent cells,and the in vitro palbociclib-resistant breast cancer cell line T47D-Pal R was induced by gradient increasing low concentrations of palbociclib.The drug resistance of T47D-Pal R cells was evaluated by Cell Titer-Glo Luminescent Cell Viability Assay（Promega）.The differential gene expression of T47D and T47D-Pal R cells at the transcriptome level was detected by RNA-seq.The m RNA and protein relative expression levels of CDK2,CDK4,CDK6,E2F1,Rb and other drug-resistant genes in T47D and T47D-PALR cells were detected by Real-time PCR and Western-Blot.The anti-breast cancer effect of novel m TOR inhibitor FP-208 in cooperation with palbociclib was detected by Cell Titer-Glo Luminescent Cell Viability Assay（Promega）and Western-Blot methods.Results:1.The T47D-Pal R resistant cell line was successfully established after 10 months of induction,which could grow stably in20μM palbociclib culture medium.The cell morphology of T47D-PALR cell has changed significantly compared with the parent cell T47D.The inhibitory effect of palbociclib against T47D and T47D-Pal R cells were tested using CTG assay with an IC₅₀of0.069±0.018μM and 4.281±0.167μM in vitro,and the resistance index（RI）is 62.Furthermore,the monoclonal cell of T47D-PALR 39#was established and tested with an IC₅₀more than 10μM,and the resistance indexs higher than 144.9,indicating high drug resistance.2.Compared with T47D,the abnormally high expression of CDK6 in T47D-Pal R cells was detected by RNA-seq.The relative m RNA expression of CDK2,CDK4,CDK6 and E2F1 in T47D-Pal R resistant cells were all higher than that of T47D cells（P<0.05）,while the m RNA relative expression levels of Rb had no significant difference（P>0.05）.The protein relative expressions of p-Rb,CDK4,CDK6,and E2F1 in T47D-Pal R resistant cells were all higher than those of T47D（P<0.05）,but there was no significant difference between CDK2 and total Rb protein expression levels（P>0.05）.3.Whether in the parent cells T47D or the drug-resistant cells T47D-Pal R,the combination of novel m TOR inhibitor FP-208 and palbociclib has a synergistic anti-breast cancer effect,with CI values less than 0.7.Compared with palbociclib and FP-208 alone,the combination of palbociclib and FP-208 significantly inhibited the relative expression of related proteins in the AKT-m TOR-S6 and CDK4/6-RB-E2F1 pathways.Conclusion:A stable and highly drug-resistant breast cancer cell line T47D-Pal R was established by by gradient increasing low concentrations of the inhibitor palbociclib.The resistance mechanism may be associated with the increase of m RNA and protein relative expression of CDK4,CDK6,E2F1 and other drug-resistant genes,as well as CDK2 amplification and other mechanisms.The combination of FP-208,a novel m TOR inhibitor,and palbociclib,a CDK4/6 inhibitor,has a synergistic anti-breast cancer effect,but the mechanism of action remains to be further explored and discovered.