IPO5 Induces Resistance To EGFR-Targeted Therapy Via Ras Pathway In Colorectal Cancer

Background and purpose of the study:Karyopherin nuclear transport receptors play essential roles in regulating the transport of proteins from the cytoplasm into the nucleus.Dysfunctional nuclear transport can lead to alterations in both expression levels and spatiotemporal distribution of tumor suppressor genes,proto-oncogenes,and other macromolecular proteins,subsequently resulting in oncogenesis and altered sensitivity of cancer cells to chemotherapeutic agents.IPO5,a member of the karyopherin nuclear transport receptor family,is highly expressed in colorectal cancer and can activate the Ras pathway,promoting colorectal cancer proliferation.However,whether it contributes to colorectal cancer resistance to chemotherapy drugs remains unreported.This study aims to explore the role and molecular mechanism of IPO5 in EGFR-targeted drug resistance,providing new insights and a theoretical basis for the treatment of colorectal cancer.Methods:1)Public database analysis of IPO5 expression in colorectal cancer,screen cell lines with different Ras gene mutations and assess their cetuximab sensitivity using the CCK-8 assay;2)Determine IPO5 expression levels in these colorectal cancer cell lines using Western Blot;3)Knockdown IPO5 expression in cell lines exhibiting high expression using siRNA interference fragments and overexpress IPO5 in cell lines with low expression using plasmid DNA;4)Investigate the toxic effects of cetuximab on colorectal cancer cell lines under varying IPO5 expression conditions using the CCK-8 assay;examine the impact of cetuximab on apoptosis in colorectal cancer cell lines under different IPO5 expression scenarios using flow cytometry;assess the effect of exogenous IPO5 expression intervention on Colorectal cancer cell proliferation under cetuximab treatment using plate clone formation assays;5)Validate the influence of IPO5 on cetuximab treatment in vivo by constructing a subcutaneous tumor model in nude mice and utilizing immunohistochemical(IHC)techniques;6)Establish cetuximab-resistant LoVo-CR cells using in vitro low-concentration gradient increment combined with high-dose intermittent shock therapy,and detect their resistance index(RI);compare protein expression levels to parental cells;observe the sensitivity and proliferation ability of LoVo-CR to cetuximab under different IPO5 expression conditions using CCK-8 assays and plate clone formation assays;7)Determine the expression of the Ras pathway and EGFR following IPO5 expression intervention using Western Blot,and analyze whether IPO5 can promote EGFR by transporting RASAL2 into the nucleus using nuclear-cytoplasmic fractionation experiments;8)Examine whether IPO5-mediated resistance to cetuximab treatment in colorectal cancer is related to NLS binding and nuclear translocation of RASAL2 using CCK-8 assays.Results:IPO5 is highly expressed in colorectal cancer,and the expression level of IPO5 in colorectal cancer cell lines is negatively correlated with its sensitivity to cetuximab treatment.Knockdown of IPO5 expression may enhance cetuximab sensitivity in colorectal cancer cells and increase the apoptosis rate and clonogenic ability following cetuximab treatment.Conversely,overexpression of IPO5 may enhance resistance to cetuximab and decrease the apoptosis rate and clonogenic ability after cetuximab treatment.Similar effects have been observed in cells that are already resistant to cetuximab.In the established cetuximab-resistant cell line LoVO-CR,the expression of Hras,IPO5,AKT,and EGFR is upregulated,and IPO5 knockdown enhances cetuximab sensitivity.Mechanistically,IPO5 transports RASAL2 into the nucleus by binding with NLS,subsequently activating the Ras pathway,increasing EGFR expression,and causing abnormal EGFR activation,which promotes resistance to EGFR-targeted chemotherapy.Conclusions:IPO5 expression in colorectal cancer cells demonstrates a negative correlation with cetuximab sensitivity.IPO5 may mediate cetuximab resistance in colorectal cancer by binding to NLS,facilitating RASAL2 transport into the nucleus,alleviating the inhibitory impact of RASAL2 within the cytoplasm and cell membrane,and promoting the activation of the Ras pathway and EGFR.