Molecular Mechanism Of BEST4 Regulating PI3K-Akt Signal Transduction And Its Role In Colorectal Cancer

Research background and purposeThe morbidity and mortality rates of colorectal cancer continue to rise,posing a grave threat to the health of humans.PI3K/Akt signal plays an important role in the onset and progression of colorectal cancer,and its aberrant activation can trigger important pathological processes,such as tumor cell proliferation,tumor cell invasion,angiogenesis,etc.Screening,identifying,and regulating the molecule of the PI3K/Akt signal,as well as elucidating its regulation mechanism,are therefore crucial for the prevention and treatment of colorectal cancer.This investigation will yield a potential new molecular target for the diagnosis and treatment of colorectal cancer.BEST4,a member of the Bestrophin protein family and newly discovered ion channel protein can function as a Cl-channel,HCO3-channel,or voltage-gated Ca2+ channel.BEST4 is predominantly expressed in the epithelial tissue of the intestinal mucosa,but its role and regulation mechanism in the occurrence and progression of colorectal cancer is unknown.The purpose of this study is to investigate the role of BEST4 in colorectal cancer and to describe the molecular mechanism of BEST4 regulation of PI3K/Akt signal transduction in order to determine the role of the BEST4/PI3K/Akt new signal transduction mechanism in the occurrence and progression of colorectal cancer and its clinical significance.Research method1.Using immunohistochemistry,the relationship between BSET4 and p-Akt is investigated.2.Using the exp erimental techniques of co-immunoprecipitation,immunoblotting,cell transfection,and plasmid construction,the interaction between BEST4 and p85α and the identification of its interaction key structural domain are studied.3.Co-immunoprecipitation,immunoblotting,cell transfection,plasmid construction,PI3 K activity detection,etc.are utilized to analyze and compare whether the interaction between BEST4 and p85α affects the interaction between p85α and p110 and the activity of p110 kinase.4.Immunoblotting,co-immunoprecipitation,cell transfection,plasmid construction,PI3 K activity detection,and other techniques are used to investigate the role of BEST4 in the PI3K/Akt signal transduction of EGF mediation.5.The heterotransplantation model of null mice is used to investigate the role of BEST4 in the drug resistance of gefitinib-treated colorectal cancer cells.6.The immunohistochemical method and the Spearman correlation analysis are employed to investigate the relationship between the expression of BEST4 in colorectal cancer tissue and the expression of downstream target genes MYC and CCND1 of the PI3K/Akt signal.Research results1.BSET4 and p85α interact to activate the PI3K/Akt signa.There exists a significant positive correlation between BSET4 and p-Akt in clinical colorectal cancer tissue samples.The interaction between BSET4 and p85α is revealed by co-immunoprecipitation assays.Besides,the C-terminal structural domain(307-473 aa)of BEST4 and the SH3 structural domain(1-78 aa)of p85α is vital to the interaction between BEST4 and p85α;BSET4 can increase the formation of p85α/p110α heterodimer via the interaction with p85α,thus intensifying the activity of p110 kinase,which is the molecular basis for BEST4 to activate the PI3K/Akt signal transduction.2.BEST4 presents the positive correlation property with expression of MYC and CCND1.Immunohistochemical results indicate that there is a general high expression between MYC and CCND1 in the high expression sample of BEST4,whereas there is a general low expression between MYC and CCND1 in the low expression sample of BEST4.The Spearman correlation analysis confirms that a significant positive correlation exists between the expressions of BEST4 and MYC or CCND1.3.BEST4 joins the PI3K/Akt signal induced by EGF,and promotes the drug resistance of colorectal cancer cell against the gefitinib treatmen.The research findings indicate that BEST4 joined the EGF-induced PI3K/Akt signal.EGF-induced p-Akt levels can be significantly increased by overexpression of BEST4.In contrast,the expression of knock-down BEST4 can significantly inhibit the EGF-induced increase in p-Akt.Importantly,we also discover that the overexpression of BEST4 is one of the major factors contributing to gefitinib resistance.ConclusionBEST4 can promote the formation of p85α/p110α heterodimer and enhance the activity of p110 kinase in colorectal cancer by interacting with PI3 K regulatory subunit p85α.Such a molecular event will subsequently result in the phosphorylation of Akt and promote the expression of downstream target genes MYC and CCND1,which is regarded as the crucial molecular mechanism by which BEST4 regulates the PI3K/Akt signal.Importantly,BEST4 can also regulate the PI3K/Akt signal induced by EGF by interacting with p85α,resulting in colorectal cancer cell tolerance to gefitinib treatment.Therefore,inhibiting BEST4 expression manually can significantly increase the sensitivity of colorectal cancer to gefitinib.In conclusion,the research reveals the molecular pathological mechanism by which BEST4 regulates PI3K/Akt signal transduction,which will contribute to the development of novel strategies for the diagnosis and treatment of colorectal cancer.