Studies On The Interaction Of CK2? And YBX1 And Its Functions In Cancer Progression

The PI3K/AKT pathway plays important roles in the pathogenesis and progression of tumors.Protein kinase CK2 alpha(CK2?),a highly conserved cytokinase with multiple substrates,is involved in the growth and migration of multiple malignancies.The overexpression of Y-box binding protein 1(YBX1)is related to tumor proliferation,drug resistance and poor prognosis.Studies have demonstrated that both CK2? and YBX1 could regulate the PI3K/AKT pathway.In addition,through Human Protein Reference Database(HPRD)we predicted that CK2 might be the upstream kinase of YBX1 and CK2 could phosphorylate YBX1 on S165 to exert its kinase function.Herein,we hypothesize that CK2 may interact with YBX1 and together regulate the PI3K/AKT signaling pathway.In this study,we assessed the expression of CK2? and YBX1 in several cancer cells.We explored the effects of CK2? on the expression of YBX1 and examined the regulation mechanisms of YBX1 by CK2?.We investigated the interaction of CK2? and YBX1 in different cancer cell lines.Furthermore,we evaluated the effects of combined inhibition of CK2? and YBX1 on cell viability,colony formation,cell-cycle arrest,wound healing,cell migration and chemotherapeutic drug resistance of cancer cell lines.The main results were as follows:(1)Overexpression of CK2? was associated with the expression of YBX1 in cancer cell lines.The expression of CK2? and YBX1 was measured by immunoblotting in various cancer cells,including GIST,ovarian,non-small cell lung,liver and gastric cancer cell lines.CK2? and YBX1 were expressed in all selected cell lines,without consideration of the type of cancer cell lines,indicating that expression of CK2? and YBX1 might be related in cancer cell lines.(2)CK2? could regulate the expression of YBX1.We assessed the effects of CK2? inhibitor CX4945 on YBX1 by immunoblotting in various cell lines,including GIST,ovarian,liver,and gastric cancer cell lines.CX4945 induced the degradation of YBX1 in all cell lines in a dose-dependent manner.We subsequently examined YBX1 expression after CK2? was knockdown by CK2? siRNA.Western blotting analysis showed that CK2? knockdown decreased the expression of YBX1 in SKOV3,ES2,HepG2,PC9 and OVCA429 cell lines;however,the deregulation of YBX1 had little influence on the expression of CK2?.As predicted,overexpression of CK2? induced an increased expression of YBX1;however,the K68M(kinase dead)mutant of CK2? had no effect on the expression of YBX1.These results suggested CK2? could regulate the expression of YBX1 which is dependent on its enzymatic activity.(3)CK2? regulated the expression of YBX1 at the transcriptional level.Expression of YBX1 was assessed by qRT-PCR after cells were transfected with WT-CK2? or K68M-CK2? plasmids.WT-CK2?,but not K68M-CK2?,resulted in the increase of YBX1 in 293 T cells.Next,YBX1 luciferase assay was carried out to determine the effects of CK2? on YBX1 transcriptional activation in 293 T cells.Overexpression of WT-CK2? activated YBX1,while overexpression of K68M-CK2? mutant showed no effects on YBX1 activation.Impressively,similar results were found in HeLa cells,suggesting that CK2? is an activator of YBX1,and K68 of CK2?(kinase activity site)plays a critical role in the regulation of YBX1 transcriptional activation.(4)CK2 interacted with YBX1.To investigate whether there is an interaction between CK2? and YBX1,we transfected 293 T with CK2? and YBX1 expression vectors and conducted CK2? or YBX1 immunoprecipitation(Co-IP).CK2? immunoprecipitation displayed an obvious YBX1 49 kDa band,while YBX1 immunoprecipitation displayed an obvious CK2? 42 kDa band.We subsequently evaluated CK2? and YBX1 interaction without extra transfection in GIST882 and SKOV3 cells.CK2? immunoprecipitation indicated a dominant YBX1 49 kDa band in GIST882 and SKOV3 cells;this result was confirmed by CK2? immunoblotting in YBX1 immunoprecipitation.Furthermore,the in vitro binding analysis indicated that CK2? directly bound to YBX1.These results demonstrated that CK2? could interact with YBX1.(5)Synergistic effects of combined inhibition of CK2? and YBX1.Additional inhibitory effects on PI3K/AKT pathway,cell viability,colony formation,cell-cycle arrest,wound-healing,Boyden chamber assay and drug resistance were obtained by the combined inhibition of CK2? and YBX1 in different cancer cell lines,which demonstrated decreased growth,migration and drug resistance.These results indicated that co-targeting CK2? and YBX1 achieved greater inhibition of the cell proliferation,migration and drug resistance ability.(6)Proliferation and migration induced by co-activation of CK2? and YBX1 was dependent on the PI3K/AKT pathway.We conducted cell viability and wound closure assays to assess the effects of PI3K/AKT pathway on cell phenotypes induced by co-activation of CK2? and YBX1.Overexpression of CK2? or YBX1 increased cell viability and wound closure ability.Co-overexpression of CK2? and YBX1 achieved greater promotion of cell viability and wound closure ability.Inhibition of PI3K/AKT pathway by LY294002 decreased cell viability and wound closure ability.Furthermore,after PI3K/AKT pathway was inhibited by LY294002,the induction of cell viability and wound closure induced by CK2? and YBX1 expression was abolished,indicating that the PI3K/AKT pathway plays an important role in the phenotypes of cancer cell lines induced by co-overexpression of CK2? and YBX1.Taken together,the results of this study explored the roles of CK2? and YBX1 in the genesis and progression of cancers,and identified the interaction between CK2? and YBX1,and uncovered the regulatory role of CK2? and YBX1 in the important signal pathways of cancers(PI3K/AKT/mTOR signaling pathway).These findings have significant implications for the understanding of how CK2? and YBX1 exert their carcinogenic roles in cancers,and co-targetting CK2? and YBX1 shed light on the development of novel anti-tumor drugs for cancer patients.