Investigation On The Function And Mechanism Of Positive Cofactor 4 In Cancer Development And Progression

Background:As one of the major diseases that seriously threaten healthy and life safety,cancer is rapidly increasing and presenting a younger trend.According to the“Global cancer statistics 2018”,there were 18.1 million new cancer patients worldwide in 2018,and about9.6 million died of cancer.The four most common cancers were lung cancer?11.6%?,breast cancer?11.6%?,prostate cancer?7.1%?and colorectal cancer?6.1%?.Despite advances in treatments,many patients still develop into metastatic disease,which is the leading cause of cancer death.Thus,there is an urgent need to characterize the underlying molecular mechanisms and identify novel therapeutic targets to improve the outcomes for cancer.Recent studies have shown that metabolic reprogramming is a hallmark of cancer cells and a key contributor to cancer progression.The Warburg effect,as known as aerobic glycolysis,is the best-characterized metabolic change in cancer cells,which facilitates cancer growth and progression by increasing glucose uptake,elevating lactate production,and supporting the energy demands.Emerging evidence has indicated the crucial role of the Warburg effect in cancer therapy as a novel target.Although a few transcription factors have been reported to regulate glycolysis,the transcriptional regulation of Warburg effect is still largely unknown,the development of anti-cancer drugs targeting the"Warburg effect"is also slow.The human positive cofactor 4?PC4?and its yeast ortholog SUB1?also named as coactivator p15?are initially identified as a coactivator of basal transcription.PC4 is located on chromosome 5p13 and encodes a 127-amino acid protein that has an important role in various cellular processes including transcription,DNA replication,DNA repair and chromatin organization.Our previous studies have shown that overexpression of PC4 is involved in the malignant transformation of normal dermal multipotent fibroblasts,indicating the crucial role of PC4 in tumorigenesis.Besides,PC4 is also found to be upregulated in lung cancer,astrocytoma and esophageal squamous cell carcinoma and positively related with tumor lymphatic metastasis and chemo-radiosensitivity.Although earlier studies showed the potential role of PC4 in cancer,the molecular mechanisms of PC4 in cancer development and progression are still unknown,especially in breast cancer.In this study,we first examined the expression of PC4 in 114 breast cancer specimens and analyzed the correlation between PC4 expression and metastasis and prognosis of patients.Through gene set enrichment analysis?GSEA?in breast cancer specimens and experimental verification,we explore the function of PC4 in breast cancer.Then,according to GSEA,we explore the mechanism of PC4 in breast cancer through detecting extracellular acidification rate?ECAR?,glucose intake,lactic acid production,ATP production and glycolysis related protein,and chromatin-immunoprecipitation?CHIP?experiment.At last,we examined the expression of PC4 in colorectal cancer and prostate cancer,and analyzed the correlation between PC4 expression level and clinicopathological characteristics and prognosis of patients.Methods and Results:1.PC4 is significantly upregulated in breast cancer and is closely correlated with metastasis and poor prognosis of patients.To explore the potential clinical significance of PC4,we firstly detected PC4expression level in 114 cases of breast cancer patients compared with their adjacent counterparts.In both invasive ductal carcinoma?IDC?and invasive lobular carcinoma?ILC?,aberrant PC4 overexpression was observed in carcinoma tissues from immunohistochemistry results,while weak positive signal was found in adjacent tissues and almost no positive signal in normal tissues.In addition,we analyzed PC4 expression in breast cancer with or without metastasis,and the intensity of PC4 in carcinoma with metastasis was apparently higher than that without metastasis.At last,the Kaplan-Meier analysis show that the higher PC4 expression group had poorer overall survival compared with lower PC4 expression group in breast cancer.2.PC4 promotes breast cancer growth and EMT mediated metastasis both in vivo and in vitro.To investigate the functional significance of increased PC4 expression in breast cancer,we conducted gene set enrichment analysis?GSEA?to compare the gene expression profiles of PC4^low and PC4^highigh breast cancer specimens.GSE9893 dataset containing 155cases of breast cancer patients was divided into PC4^low?n=78?and PC4^high?n=77?groups based on the median expression level of PC4.GSEA results demonstrated that the gene sets of Benporath_Proliferation and HALLMARK_Epithelial_Mesenchymal_Transition were obviously enriched in PC4^highigh breast cancer specimens.To verify the above results,MDA-MB-231 and MCF-7 cells were chosen for subsequent loss-of-function studies.The stable cell lines with PC4 knockdown were established by lentiviral infection?shPC4-1 and shPC4-2?.The CCK-8,colony formation assays and subcutaneous xenograft model show that knockdown of PC4 inhibit breast cancer growth.The wound healing assays,transwell assays and tumor metastasis model in mice show that knockdown of PC4 inhibit breast cancer metastasis.Quantitative PCR and Western blot analysis showed that downregulation of PC4 decreased the expression of Vimentin and N-cadherin and increased the expression of E-cadherin,indicating that PC4 could induce EMT.3.Glycolysis is critically involved in the oncogenic functions of PC4.To further illustrate the underlying mechanisms of PC4 mediated oncogenic advantages in breast cancer,GSEA in GSE9893 was conducted based on PC4 expression,and the gene sets of HALLMARKS_GLYCOLYSIS were evidently enriched in PC4^highigh breast cancer samples.Subsequently,extracellular acidification rate?ECAR?experiments show PC4 knockdown decreased overall glycolytic flux.PC4 knockdown also decreased glucose uptake,lactate production and ATP generation.Moreover,loss of PC4 inhibited the expression of GLUT1 and LDHA,the key enzymes of glycolysis.We next confirmed whether the oncogenic functions of PC4 were dependent on glycolysis.The glycolytic inhibitor 2-deoxy-D-glucose?2-DG?was used to block glycolysis in MDA-MB-231 cells,and the specific plasmid was used to overexpress PC4 before 2-DG treatment.Then,we found that overexpression of PC4 increased the proliferation,migration and invasion of breast cancer cells,which was inhibited by 2-DG.4.PC4 promotes glycolysis by regulating c-Myc transcription.To further illustrate the underlying mechanisms of PC4 mediated glycolysis advantages in breast cancer,GSEA in GSE9893 was conducted based on PC4 expression,and the gene sets of HALLMARKS_MYC_TARGETS_V1 were evidently enriched in PC4^high breast cancer samples.The qPCR and western blot results also showed that knockdown of PC4 observably inhibited the expression of c-Myc in breast cancer cells.To further demonstrate whether c-Myc was responsible for the oncogenic functions of PC4,we used the specific plasmid to overexpress of c-Myc in PC4-knockdown cells.As expected,c-Myc overexpression rescued the decreased glucose uptake,lactate production and ATP generation in PC4-knockdown MDA-MB-231 cells.Overexpression of c-Myc also reversed extracellular acidification rate?ECAR?loss in PC4-knockdown cells.Moreover,the inhibitory effects of PC4 knockdown on proliferation,migration and invasion were also rescued by c-Myc overexpression.At last,the downregulation of GLUT1,LDHA,Vimentin and upregulation of E-cadherin in PC4-knockdown cells were reversed by overexpression of c-Myc.Given the important role of PC4 in transcription regulation,we subsequently examined whether PC4 could directly activate c-Myc transcription to promote its expression.To confirm the relationship between PC4 and c-Myc,we performed a bioinformatic analysis for the presence of putative PC4-binding sites?BS?in the C-MYC promoter,and found two putative PC4 BS in a region encompassing 0.1 kb upstream the transcription start site?TSS?.Chromatin Immunoprecipitation?ChIP?showed that PC4binds to the C-MYC promoter in that region,indicating that C-MYC is a direct downstream target of PC4.5.PC4 is significantly upregulated in colorectal cancer and prostate cancer and is closely correlated with clinicopathological characteristics and poor prognosis of patients.To explore whether the overexpression of PC4 in cancers is universal,we selected colorectal cancer and prostate cancer as research samples.Through western blot,all CRC cells?SW480,SW620,LS174T,HCT116,LOVO,and HT29?demonstrated increased levels of PC4 compared with normal intestinal epithelial cells?CCD841?.Immunohistochemistry staining in CRC specimens revealed high levels of PC4 in carcinoma,medium levels in adjacent tissues,and almost undetectable levels in normal tissues.To confirm these results,we evaluated the PC4 protein levels in six fresh pairs of CRC samples and their matched adjacent tissues by western blotting and found upregulation of PC4 in carcinoma.In addition,PC4 expression in carcinoma was increased compared with adenoma.Regarding the staining score,the intensity of PC4 in well-diff erentiated tissue was reduced compared with that in moderately diff erentiated and poorly diff erentiated tissue.The T classification results showed that PC4 expression was increased in stage T3/T4 compared with stage T1/T2.Finally,the Kaplan-Meier analysis show that the higher PC4 expression group had poorer overall survival compared with lower PC4 expression group in colorectal cancer.The qPCR,western blot assays and immunofluorescent staining show that the expression level of PC4 were upregulated in prostate cancer cell lines?LAPC4,C4-2,PC3and DU145?compared with non-cancerous prostate epithelial cell lines?RWPE-1?.Moreover,the expression of PC4 was significantly elevated in androgen-independent prostate cancer?AIPC?cell lines?C4-2,PC3 and DU145?than that in androgen-dependent prostate cancer?ADPC?cell lines?LAPC4?.Immunohistochemistry staining in prostate cancer specimens show that high levels of PC4 were detected in carcinoma tissues,while almost undetectable levels in adjacent normal tissue.Then,we evaluated the possible correlation between PC4 expression and differentiation grade,and found that the intensity of PC4 in poorly differentiated tissue was significantly increased compared with that in well differentiated tissue.Moreover,PC4 expression in carcinoma with metastasis was obviously upregulated than that without metastasis.Finally,the Kaplan-Meier analysis show that the higher PC4 expression group had poorer overall survival compared with lower PC4 expression group in prostate cancer.Conclusion:1.PC4 is significantly upregulated in breast cancer and is closely correlated with metastasis and poor prognosis of patients.2.PC4 promotes breast cancer growth and EMT mediated metastasis both in vivo and in vitro.Moreover,glycolysis is critically involved in the oncogenic functions of PC4.3.PC4 promotes glycolysis by regulating c-Myc transcription.4.PC4 is significantly upregulated in colorectal cancer and prostate cancer and is closely correlated with clinicopathological characteristics and poor prognosis of patients.