Study Of Drug Resistance Genes In CDK4/6 Inhibitor-resistant Luminal-type Breast Cancer

Background:Breast cancer is the most common cancer and the leading cause of cancer death among women worldwide.According to a recent report by the American Cancer Society,an estimated 281,550 new cases of invasive breast cancer and 49,290deaths are expected to occur among US women in 2022.The incidence rate of breast cancer varies greatly among countries,with higher rates in developed regions and lower rates in developing regions.However,the mortality rate of breast cancer is higher in less developed countries due to late diagnosis and limited access to treatment.Breast cancer can be classified into different subtypes based on molecular markers such as hormone receptors（HR）,human epidermal growth factor receptor 2（HER2）,and Ki-67.The most common subtype is luminal A,which is HR-positive,HER2-negative,and has low Ki-67 expression.Luminal A breast cancers tend to have a good prognosis and respond well to endocrine therapy.Luminal B breast cancers are also HR-positive but have either HER2-positive or high Ki-67 expression.They have a worse prognosis than luminal A cancers and may benefit from chemotherapy or anti-HER2 therapy.Other subtypes include HER2-enriched（HR-negative,HER2-positive）,basal-like（HR-negative,HER2-negative,CK5/6-positive or EGFR-positive）.The incidence and mortality of different breast cancer subtypes vary by race/ethnicity,age,and other factors.In general,Black women have a higher proportion of aggressive subtypes such as basal-like breast cancers than White women.They also have a higher mortality rate from breast cancer than White women overall（27.6 vs.19.7 deaths per 100,000 in 2016-2020）and two-fold higher among adult women younger than 50 years（12.1 vs.6.5 deaths per 100,000）.Asian/Pacific Islander women have a lower incidence and mortality rate of breast cancer than other racial/ethnic groups.However,they have a higher proportion of HER2-enriched subtype than White women.CDK4/6 inhibitors are a novel drug class that target cyclin-dependent kinases 4and 6（CDK4/6）,which are key regulators of cell cycle progression and proliferation.They have shown remarkable efficacy in combination with endocrine therapy for hormone receptor-positive（HR+）,human epidermal growth factor receptor 2-negative（HER2-）metastatic breast cancer（MBC）.However,resistance to CDK4/6 inhibitors is inevitable and limits their clinical benefit.The mechanisms of resistance can be classified into cell cycle-specific and cell cycle-non-specific categories.Cell cycle-specific resistance involves alterations in the CDK4/6-Rb-E2F pathway,such as mutations or amplifications of CDK4/6,loss of Rb function,or activation of E2F transcription factors.Cell cycle-non-specific resistance involves changes in signaling pathways that bypass CDK4/6 inhibition,such as PI3K/AKT/mTOR,ERBB2/3,FGFR1/2,or MAPK pathways.Additionally,tumor heterogeneity,epithelial-mesenchymal transition（EMT）,immune evasion,and pharmacokinetic factors may also contribute to resistance.Several strategies have been proposed to overcome or prevent CDK4/6 inhibitor resistance,such as optimizing endocrine therapy combinations,switching to other CDK inhibitors or targeted therapies,exploiting synthetic lethality interactions,or modulating the tumor microenvironment^[4].Objective:This study used bioinformatics methods to perform differential gene analysis on Palbociclib-resistant Luminal-type breast cancer cell line MCF7,and performed core gene screening,downstream regulatory network construction,and drug resistance prediction.Knock down the up-regulated genes in the resistant cell line,and determine the vitality of the breast cancer cell line after knocking down the genes to verify the close relationship between the gene and the drug-resistant phenotype.Methods:Use R language as the basic language for bioinformatics analysis.Select the sequencing samples of MCF7-Palbociclib-resistant cells and MCF7parental cells in three datasets GSE98987,GSE130437 and GSE140758 for analysis.Use DESeq2,edgeR,and limma differential analysis methods to obtain differential genes according to data type.Use Robust rank aggregation algorithm and WGCNA algorithm to perform dimensionality reduction processing on differential genes to obtain core genes.Input core genes into cytoscape to obtain gene interaction network.Select IFIT3 in the core genes for subsequent experiments,knock down the IFIT3gene in the MCF7 cell line,and perform qRT-PCR and Western Blot to verify the level of gene knockdown.CCK8 experiment was performed to verify cell viability.Results:After differential analysis and dimensionality reduction screening of the samples in the three datasets GSE98987,GSE130437,and GSE140758,a total of 15core genes related to drug resistance phenotype were obtained,namely IFIT3,CFB,IFITM1,OASL,MVP,S100P,IFIT1,DDX60,OAS1,H19,IFI44,STAT6,ALDH1A3,CLIC3,S100A9.STAT1 and STAT3 are the main transcription factors that regulate core genes.Among them,STAT1 acts on MVP and IFIT3,and STAT3 acts on CFB and OAS1.Propofol,propofol maleate,and phenfluorothiazine have significant effects on core genes in MCF7 cell lines.MCF7 cell lines with IFIT3 gene knockdown showed stronger sensitivity to palbociclib drugs.The IC50 values of palbociclib in MCF7 cells with IFIT3 knockdown,MCF7 parental cells,and MCF7empty virus-transfected cells were 12.66μM,17.02μM,and not reached（>20μM）,respectively.Conclusion:The 15 genes represented by IFIT3,CFB,MVP,IFITM1,OSA1,and H19 may be the core gene group that causes drug resistance in Luminal-type breast cancer.STAT1 and STAT3 are the key transcription factors that regulate these genes.MCF7 cell lines with IFIT3 gene knockdown showed strong sensitivity to Palbociclib.Overexpression of IFIT3 gene is one of the reasons for Palbociclib resistance.