Mechanisms Of SKP2 Gene In Tumourigenesis And Radiation Tolerance By Targeting PDCD4

BackgroundBreast cancer is one of the most common malignant tumors in women and has been the leading cause of death in women with malignant tumors.At present,breast cancer treatment mainly takes surgery-based comprehensive treatment,including radiotherapy,chemotherapy and endocrine therapy.Radiotherapy is an important method of breast cancer treatment.The efficacy of radiotherapy depends on radiosensitivity,so it is important to improve radiotherapy sensitivity.SKP2(S-phase kinase-interacting protein),also known as p45 or FBXL1,is a member of the F-box family.F-box family proteins(Fbps)are proteins that contain at least one F-box domain.The F-box domain is a protein structural motif of approximately 50 amino acids that mediates the interaction between proteins,which was first identified in cyclin F.Fbps is one of the components of the SCF(SKP1-cullin-F-box)ubiquitin protein ligase complex,and in the SCF complex,SKP2 can be used as a substrate recognition factor.It regulates cell cycle,cell proliferation,and transcriptional processes by degrading cyclins,such as P27.P21,P53,and cyclin A.SKP2 also plays an important role in tumorigenesis and tumor development.SKP2 has been shown to be an oncogene and is overexpressed in tumors such as lymphoma,prostate cancer,breast cancer and non-small cell lung cancer.Overexpression of SKP2 is often observed in human tumor development and metastasis.SKP2 is carcinogenic in vitro and in vivo,and its overexpression promotes tumorigenesis in xenograft tumor models,while SKP2 inactivation strongly inhibits cancer development and progression by triggering strong cellular senescence and apoptotic responses.Recent studies have shown that SKP2 also plays an important role in DNA damage response and repair,SKP2 overexpressing cervical cancer cell lines showed more clone formation,higher cell viability and fewer DNA damage after radiotherapy.However,the role and mechanism of SKP2 in tumor radiation tolerance is still poorly understood.PDCD4(Programmed cell death protein 4),which was found in mouse cells in 1995,is a gene which promotes apoptosis and has a MA-3 domain.This gene was also found in human,chicken and rat cells,and was collectively named Programmed Cell Death Protein 4.The human PDCD4 gene is located on the 10q24 chromosome.PDCD4 interacts with the translation initiation factors eIF4A and eIF4G and inhibits the translation process.PDCD4 is a tumor suppressor gene that is down-regulated or deleted in a variety of tumors.PDCD4 overexpression inhibits tumor formation and progression.Down-regulation of PDCD4 is associated with poor prognosis in patients with lung cancer and colorectal cancer.PDCD4 can inhibit tumorigenesis and growth by up-regulating the expression levels of tumor suppressor genes such as p21 and p27,and promote apoptosis by down-regulating the expression levels of anti-apoptotic genes such as Bcl-xL and XIAP.PDCD4 plays a role in DNA damage response by interacting with eukaryotic initiation factor-4A(eIF4A)and P53.PDCD4 inhibits DNA damage responses by inhibiting P53 protein translation and phosphorylation.Previous studies have shown that SKP2 may interact with PDCD4,but the relationship between SKP2 and PDCD4 is still unclear.Chapter One Relationship and between SKP2 and PDCD4 and mechanisms of SKP2 on PDCD4ObjectiveThe SKP2 interacting protein was detected and validated.Study the correlation of expression level between SKP2 and PDCD4 and whether SKP2 can regulate PDCD4 expression.Then the molecular mechanisms of SKP2 on PDCD4 was studied from the aspects of phosphorylation and ubiquitination.Methods1.SDS-PAGE electrophoresis and mass spectrometry were used to detect and identificate the interacting proteins of SKP22.CO-IP assay was used to detect whether SKP2 interacts with PDCD4 in vivo.3.Western blot was used to detect whether SKP2 can combine with PDCD4 peptide or phosphorylated PDCD4 peptide in vitro.4.Immunohistochemistry and Western blot were used to detect SKP2 and PDCD4 protein expression levels in SKP2-/-and WT mouse MEFs and analyzed their correlation.5.Western blot was used to detect the expression levels of SKP2 and PDCD4 in four breast cancer cell lines,and the correlation was analyzed.6.Established SKP2 stably overexpressing MCF-7 breast cancer cell line and SKP2 stably knockout MDA-MB-231 breast cancer cell line.7.Western blot and QRT-PCR were used to detect the effect of SKP2 on the expression of PDCD4 protein and mRNA.8.Western blot was used to detect whether SKP2 promotes PDCD4 phosphorylation via the AKT signaling pathway.9.Western blot was used to detect effect of SKP2 on the PDCD4 ubiquitination level in vivo and in vitro.10.Western blot was used to detect the ubiquitination site of SKP2 on PDCD4.11.Western blot was used to detect the effect of SKP2 on the half-life of PDCD4 protein.12.Western blot was used to detect whether PDCD4 regulates reversely on SKP2.Results1.SKP2 interacted with PDCD4 in vitro and in vivo.The binding of SKP2 to PDCD4 requires the participation of the F-box domain and LRR domain of SKP2 and the serine phosphorylation of PDCD4 at position 67.2.The expression level of PDCD4 protein in SKP2-/-MEF cells was significantly up-regulated compared with WT MEF cells,and the expression of SKP2 and PDCD4 were negatively correlated in four breast cancer cells.3.Successfully established SKP2 stably overexpressing MCF-7 cell line and control MCF-7 cell line.4.Successfully established SKP2 stably knockout MDA-MB-231 cell line and control MDA-MB-231 cell line.5.SKP2 overexpression in MCF-7 cell line down-regulated PDCD4 protein expression level,but did not affect PDCD4 mRNA expression level.SKP2 down-regulation in MDA-MB-231 cell line up-regulated PDCD4 protein expression level but did not affect PDCD4 mRNA expression level.6.SKP2 can promote the phosphorylation of PDCD4 at position 67 by AKT signaling pathway,and reduce the stability of PDCD4.7.SKP2 can promote PDCD4 ubiquitination in vitro and in vivo,and SKP2 can mediate PDCD4 ubiquitination at K48 and degradation.8.The half-life of PDCD4 protein was significantly prolonged in SKP2-/-MEF cells compared with WT MEF cells.9.Downregulation of PDCD4 protein upregulated SKP2 protein expression levels in WT and SKP2-/-MEF cells.ConclusionSKP2 interacts with PDCD4.SKP2 expression level is negatively correlated with PDCD4 expression level in breast cancer cells.SKP2 negatively regulates PDCD4 protein expression level.SKP2 promotes PDCD4 phosphorylation at Ser67 via AKT signaling pathway and reduces the stability of PDCD4.PDCD4 is a ubiquitination substrate of SCF-SKP2 complex,and SCF-SKP2 complex can degrade PDCD4 by promoting PDCD4 ubiquitination at K48.Downregulation of SKP2 prolongs the half-life of PDCD4.PDCD4 can reversely regulate SKP2 expression level.Chapter Two SKP2 promotes breast cancer cell proliferation and radiation tolerance by regulating PDCD4ObjectiveThe results of the first chapter suggest that SKP2 can negatively regulates PDCD4 protein levels through PDCD4 ubiquitination and degradation.Since PDCD4 plays an important role in inhibiting tumor growth and DNA damage response.,this chapter focuses on whether SKP2 plays a role in cell proliferation,apoptosis,and DNA damage response through negative regulation of PDCD4 expression level.Methods1.Established SKP2 and PDCD4 stably overexpressing MCF-7 breast cancer cell line and the SKP2 and PDCD4 stably knockout MDA-MB-231 breast cancer cell line and the results were verified by western blot.2.Western blot was used to detect PCNA expression level and analyzed whether SKP2 regulates cell proliferation by inhibiting PDCD4.3.MTT and clone formation assays were used to detect whether SKP2 promotes cell proliferation by inhibiting PDCD4 in vitro.4.Nude mouse xenograft model and Ki-67 immunohistochemical staining were used to detect whether SKP2 promotes cell proliferation by inhibiting PDCD4 in vivo.5.Flow cytometry and Hoechest33342 staining were used to detect whether SKP2 regulates cell apoptosis through PDCD4.6.Western blot was used to detect Caspase-3 and Cleaved Caspase-3 expression levels and analyzed whether SKP2 regulates apoptosis through PDCD4.7.Western blot and immunofluorescence staining were used to determine the expression levels of SKP2 and PDCD4 before and after irradiation.8.Western blot was used to detect P53 and P53(Ser15)expression levels and analyzed whether SKP2 promotes DNA damage response by inhibiting PDCD4.9.Immunofluorescence was used to detect y-H2AX expression level and analyzed whether SKP2 promotes DNA damage response by inhibiting PDCD4.10.MTT and clone formation assays were used to detect whether SKP2 increases breast cancer cell survival after irradiation by inhibiting PDCD4 expression.Results1.Successfully established MCF-7-Con,MCF-7-SKP2 and MCF-7-SKP2-PDCD4 stable expression cell lines.Successfully established MDA-MB-231-Con,MDA-MB-231-sgSKP2 and MDA-MB-231-sgSKP2-shPDCD4 stable cell lines.2.SKP2 overexpression promoted PCNA protein expression in MCF-7 cell lines,while overexpression of PDCD4 reversed the effect of SKP2 overexpression.This effect was verified reversely in MDA-MB-231 cell lines.3.Compared with control cells,SKP2 overexpressing MCF-7 cell line showed faster cell proliferation rate and more clone formation,while PDCD4 overexpression significantly reversed the effect of SKP2-MCF-7 cell line.This effect was verified reversely in the MDA-MB-231 cell line.4.Compared to control cells,SKP2 overexpressing MCF-7 cells transplanted tumor in nude mice showed faster growth rates,greater volume,and heavier weight.Overexpression of PDCD4 significantly reversed the effect of SKP2-MCF-7 cells.This effect was verified reversely in MDA-MB-231 cell line.5.Ki-67 immunohistochemical staining showed that SKP2 overexpression promoted the proliferation of MCF-7 breast cancer cells in vivo,and this promotion can be reversed by PDCD4 overexpression.This effect was verified reversely in MDA-MB-231 cell line.6.Overexpression of SKP2 in MCF-7 cells reduced apoptosis after irradiation,whereas PDCD4 significantly reversed the effect of SKP2 overexpression.This effect was verified reversely in MDA-MB-231 cell line.7.The expression level of SKP2 was significantly increased after irradiation,and the expression level of PDCD4 was significantly decreased after irradiation.8.Downregulation of SKP2 in MDA-MB-231 cells increased the expression level of Cleaved Caspase-3 after irradiation,while down-regulation of PDCD4 reversed the effect of SKP2 down-regulation.9.Overexpression of SKP2 in MCF-7 cells down-regulated the expression level of y-H2AX after irradiation,while overexpression of PDCD4 reversed the effect of SKP2 overexpression.This effect was verified reversely in MDA-MB-231 cells.10.The expression level of P53(Seri 5)was significantly downregulated in SKP2-/-MEF cells than in WT MEF cells.11.Compared with control cells,SKP2 overexpressing MCF-7 cell line showed faster cell proliferation rate and more clone formation after irradiation,while PDCD4 overexpression significantly reversed the effect of SKP2 overexpression.This effect was verified reversely in MDA-MB-231 cell lines.ConclusionSKP2 promotes breast cancer cells proliferation in vitro and in vivo by negatively regulating PDCD4.SKP2 inhibits apoptosis in breast cancer cells by negatively regulating PDCD4.SKP2 promotes DNA damage response in breast cancer cells by negatively regulating PDCD4.SKP2 increases the survival rate of breast cancer cells after irradiation by negatively regulating PDCD4.Chapter Three Clinical correlation between SKP2 and PDCD4 in breast cancer and effect of radiotherapy combine with SKP2 inhibitor on breast cancerObjectiveThe results of the first chapter and the second chapter suggest that SKP2 may play a role in tumourigenesis in breast cancer patients as well as inhibit apoptosis and promote DNA damage response after irradiation by inhibiting PDCD4.these results suggest SKP2 targeted drugs may be used as sensitizers for breast cancer radiotherapy.We finally studied the clinical correlation between SKP2 and PDCD4 in breast cancer and the inhibitory effect of SKP2 inhibitor SMIP004 on breast cancer cell proliferation in vitro and in vivo.Methods1.Immunohistochemistry and Western blot were used to detect SKP2 and PDCD4 protein expression and correlation in breast cancer tissues and adjacent tissues.2.Reviewed the tumor bioinformatics database to detect the prognosis correlation and mutation sites of SKP2 and PDCD4 in breast cancer patients.3.Western blot,MTT and clone formation were used to detect the effect of radiotherapy combined with SKP2 inhibitor SMIP004 on breast cancer cells proliferation in vitro.4.Immunofluorescence was used detection of y-H2AX expression levels and the effect of SMIP004 on DNA damage response in vitro was analyzed.5.Nude mouse xenograft model was used to detect the effect of radiotherapy combined with SKP2 inhibitor SMIP004 on breast cancer cell proliferation in vivo.6.Caspase3 and γ-H2AX immunohistochemical staining were used to detect the effect of SMIP004 on apoptosis and DNA damage response in vivo.Results1.SKP2 is highly expressed in human breast cancer tissues and lowly expressed in para-carcinoma tissues.PDCD4 is lowly expressed in human breast cancer tissues and highly expressed in para-carcinoma tissues.There was a negative correlation between the expression of SKP2 and PDCD4 in breast cancer tissues and adjacent tissues.2.High expression of SKP2 in breast cancer patients is positively correlated with poor prognosis.High expression of PDCD4 is positively correlated with favourable prognosis.3.The common mutation site of SKP2 gene in breast cancer patients is the F-box domain,and the common mutation site of PDCD4 gene is the MA-3 domain.4.Radiotherapy combined with SKP2 inhibitor SMIP004 showed more significant inhibitory effect on breast cancer cell proliferation in vitro than radiotherapy alone.5.Radiotherapy combined with SKP2 inhibitor SMIP004 showed more significant inhibitory effect on breast cancer cell proliferation in vivo than radiotherapy alone.6.SKP2 inhibitor SMIP004 can promote breast cancer cell apoptosis and inhibit DNA damage response after radiation in breast cancer cells.ConclusionThere was a negative correlation between the expression of SKP2 and PDCD4 in human breast cancer tissues and para-carcinoma tissues.High expression of SKP2 in breast cancer patients is positively correlated with poor prognosis.High expression of PDCD4 is positively correlated with favourable prognosis.Radiotherapy combined with SKP2 inhibitor SMIP004 showed more significant inhibitory effect on breast cancer cell proliferation compared with radiotherapy alone in vitro and in vivo.SMIP004 can promote breast cancer cell apoptosis and inhibit DNA damage response after radiation in breast cancer cells.Radiotherapy combined with SKP2 targeted adjuvant therapy may prolong the survival of breast cancer patients.SKP2 inhibitors may be used as sensitizing drugs for radiotherapy.