Effect Of Intrabody Against CDK4 On Expression And Activity Of NPM1 In Breast Cancer Cells

Breast cancer is the most common cancer among women in all countries and has become the cancer with the highest incidence among Chinese women.Studies have shown that CDK4 and Cyclin D1 are overexpressed in about 50% of breast cancer,which is closely related to the occurrence,development and prognosis of breast cancer.Inhibition of CDK4 activity will contribute to the treatment of breast cancer.It is of great scientific significance to explore the the highly effective and specific strategies targeting CDK4 for breast cancer treatment and address their molecular mechanisms.In the previous study of our group,the anti-CDK4 intrabody with nuclear localization signal,NLS-AK3,was successfully prepared.NLS-AK3 can specifically bind endogenous CDK4 protein in cancer cells,and has good anti-tumor effects in vitro and in vivo.Differential proteomic analysis revealed that the expression of NLS-AK3 significantly down-regulated NPM1 protein levels in triple-negative breast cancer MDA-MB-231 cells.NPM1(nucleophosmin)is a multifunctional protein that is overexpressed in a variety of tumors and promotes the proliferation and metastasis of cancer cells.Studies have found that NPM1 can directly bind to downstream targets of Cyclin D1/CDK4 such as Rb,FOXM1,NF-?B/p65,STAT3,c-myc,etc and regulates the function of these proteins,which promote the proliferation and metastasis of tumor cells.In order to determine the effect of intrabody against CDK4(NLS-AK3)on expression and the interaction factors of NPM1 in triple negative breast cancer cells,this study obtained the following results through a series of experimental analyses including western blot,real-time PCR,GST pull downand immunoprecipitation:(1)the western blot and Q-PCR results showed that NLS-AK3 down-regulated NPM1 protein level and inhibited expression of NPM1 in MDA-MB-231 cells.(2)Predicted results from Gene MANIA website show that CDK4 may interact with NPM1,and then indirect immunofluorescence experiments and GST pull down assay were used to analyze the relationship between them.Indirect immunofluorescence experiments showed that CDK4 colocalized with NPM1 in the nucleus of MDA-MB-231 cells.GST-NPM1 and GST proteins were purified by affinity chromatography and conducted GST-pull down analysis for nuclear protein of MDA-MB-231 cells..GST-pull down analysis for nuclear protein of MDA-MB-231 cells by GST-NPM1 showed that NPM1 interacted with CDK4,Rb,phosphorylated Rb and NF-?B/p65 in MDA-MB-231 cells.(3)Western blot results showed that the expression of NLS-AK3 in MDA-MB-231 cells did not affect the total protein level of Rb,but decreased the phosphorylated Rb level and down-regulated the nuclear level of NF-?B/p65.The results of co-immunoprecipitation showed that NLS-AK3 inhibited the interaction between NPM1 and CDK4,Rb,phosphorylated Rb,NF-?B/p65 in the nucleus of MDA-MB-231 cells.(4)Q-PCR was used to detacet the effect of NLS-AK3 on the transcription of NF-?B/p65 downstream genes.It was found that NLS-AK3 down-regulated the m RNA levels of E2 F,CCNE1,CDK2,cyclin A,CDC2,PCNA,c-myc,Bcl-2 and up-regulated the m RNA levels of Bad.These results suggest that NLS-AK3 inhibits the expression of NPM1 and interferes the interaction of NPM1 with CDK4,phosphorylated Rb and NF-?B/p65,which may play an important role in inhibiting the proliferation of triple negative breast cancer cells.This study provides an experimental basis for exploring the molecular mechanism of intrabody against CDK4 mediated inhibition of breast cancer cell proliferation.