Cleavage and Polyadenylation-Specific Factor 4 (CPSF4) Expression Is Associated with Enhanced Prostate Cancer Cell Migration and Cell Cycle Dysregulation, In Vitro.

Potential oncogene cleavage and polyadenylation specific factor 4 (CPSF4) has been linked to several cancer types. However, little research has been conducted on its function in prostate cancer (PCa). In benign, incidental, advanced, and castrate resistant PCa (CRPCa) patient samples, protein expression of CPSF4 was examined on tissue microarray (TMAs) of 353 PCa patients using immunohistochemistry. Using the 'The Cancer Genome Atlas' Prostate Adenocarcinoma (TCGA PRAD) database, significant correlations were found between high CPSF4 expression and high-risk genomic abnormalities such as ERG-fusion, ETV1-fusion, and SPOP mutations. Gene Set Enrichment Analysis (GSEA) of CPSF4 revealed evidence for the increase in biological processes such as cellular proliferation and metastasis. We further examined the function of CPSF4 in vitro and confirmed CPSF4 clinical outcomes and its underlying mechanism. Our findings showed a substantial correlation between Gleason groups and CPSF4 protein expression. In vitro, CPSF4 knockdown reduced cell invasion and migration while also causing G1 and G2 arrest in PC3 cell lines. Our findings demonstrate that CPSF4 may be used as a possible biomarker in PCa and support its oncogenic function in cellular proliferation and metastasis.

Comprehensive analysis of CPSF4-related alternative splice genes in hepatocellular carcinoma.

BACKGROUND: An important stage in controlling gene expression is RNA alternative splicing (AS), and aberrant AS can trigger the development and spread of malignancies, including hepatocellular carcinoma (HCC). A crucial component of AS is cleavage and polyadenylation-specific factor 4 (CPSF4), a component of the CPSF complex, but it is unclear how CPSF4-related AS molecules describe immune cell infiltration in the total tumor microenvironment (TME). METHODS: Using RNA-sequencing data and clinical data from TCGA-LIHC from the Cancer Genome Atlas (TCGA) database, the AS genes with differential expression were found. The univariate Cox analysis, KM analysis, and Spearman analysis were used to identify the AS genes related to prognosis. Screening of key AS genes that are highly correlated with CPSF4. Key genes were screened using Cox regression analysis and stepwise regression analysis, and prognosis prediction models and the topography of TME cell infiltration were thoroughly analyzed. RESULTS: A model consisting of seven AS genes (STMN1, CLSPN, MDK, RNFT2, PRR11, RNF157, GHR) was constructed that was aimed to predict prognostic condition. The outcomes of the HCC samples in the high-risk group were considerably worse than those in the lower risk group (p < 0.0001), and different risk patient groups were formed. According to the calibration curves and the area under the ROC curve (AUC) values for survival at 1, 2, and 3 years, the clinical nomogram performs well in predicting survival in HCC patients. These values were 0.76, 0.70, and 0.69, respectively. Moreover, prognostic signature was markedly related to immune infiltration and immune checkpoint genes expression. CONCLUSION: By shedding light on the function of CPSF4 and the seven AS genes in the formation and progression of HCC, this research analysis contributes to the development of more useful prognostic, diagnostic, and possibly therapeutic biomarkers.

CPSF4 promotes tumor-initiating phenotype by enhancing VEGF/NRP2/TAZ signaling in lung cancer.

Lung cancer is the leading cause of malignant tumor-related deaths worldwide. The presence of tumor-initiating cells in lung cancer leads to tumor recurrence, metastasis, and resistance to conventional treatment. Cleavage and polyadenylation specificity factor 4 (CPSF4) activation in tumor cells contributes to the poor prognosis of lung cancer. However, the precise biological functions and molecular mechanisms of CPSF4 in the regulation of tumor-initiating cells remain unclear. We demonstrated that CPSF4 promotes tumor-initiating phenotype and confers chemoresistance to paclitaxel both in vitro and in vivo. Mechanistically, we showed that CPSF4 binds to the promoters of vascular endothelial growth factor (VEGF) and neuropilin-2 (NRP2) and activated their transcription. In addition, we showed that CPSF4/VEGF/NRP2-mediated tumor-initiating phenotype and chemoresistance through TAZ induction. Furthermore, analysis of clinical data revealed that lung cancer patients with high CPSF4 expression exhibit high expression levels of VEGF, NRP2, and TAZ and that expression of these proteins are positively correlated with poor prognosis. Importantly, selective inhibition of VEGF, NRP2, or TAZ markedly suppressed CPSF4-mediated tumor-initiating phenotype and chemoresistance. Our findings reveal the mechanism of CPSF4 modulating tumor-initiating phenotype and chemoresistance in lung cancer and indicate that the CPSF4-VEGF-NRP2-TAZ signaling pathway may be a prognosis marker and therapeutic target in lung cancer.

WTAP promotes oesophageal squamous cell carcinoma development by decreasing CPSF4 expression in an m6A-dependent manner.

m6A is a widespread RNA modification. However, the mechanism through which m6A regulated the progress of oesophageal squamous cell carcinoma (ESCC) remains undetermined. The levels and prognosis of WTAP were analysed using an ESCC tissue microarray (87 ESCC and 44 paracancerous tissues). TCGA and Oncolnc databases validate WTAP expression and prognosis. CCK8, colony formation (CF), wound healing, transwell cell invasion (CI), and migration (CM) assays were employed for the detection of the biological impacts of WTAP. Expression of tumour stemness-related genes was assessed via qRT-PCR and western blotting. The m6A RNA methylation (m6AMe) quantitative kit was employed for cellular methylation level detection. Arraystar m6A-mRNA and lncRNA epitranscriptomic microarray analyses were used to screen low methylation, high expression, and prognosis-related candidate gene CPSF4. KEGG enrichment analysis was used to screen the downstream signalling pathways of CPSF4. WTAP, a methyltransferase "writer", was markedly enhanced in ESCC and was strongly correlated with poor patient outcome. WTAP knockdown inhibited the cell proliferation (CP), CI, CM, and stemness of ESCC cells in vitro and reduced the overall m6A modification (m6AMo) percentage of ESCC cells. CPSF4 is a target of WTAP-based m6AMo. WTAP-based m6AMo of CPSF4 transcript reduced the stability of CPSF4 by relying on YTHDF2. We identified the significant role of WTAP-catalysed m6AMo in ESCC tumourigenesis, wherein it facilitates ESCC tumour growth and metastasis through decreasing CPSF4 expression in an m6A-dependent manner.

LncRNA NEAT1 Regulates 5-Fu Sensitivity, Apoptosis and Invasion in Colorectal Cancer Through the MiR-150-5p/CPSF4 Axis.

BACKGROUND: Colorectal cancer (CRC) is one of the most prevalent malignancies in the world. Long non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) is involved in the development of many cancers. However, its role and mechanism in CRC progression still need further exploration. METHODS: The expression levels of lnc-NEAT1, microRNA-150-5p (miR-150-5p) and cleavage and polyadenylation specific factor 4 (CPSF4) were determined by quantitative real-time PCR (qRT-PCR). The sensitivity of cells to 5-fluorouracil (5-Fu) was measured by 3-(4,5-dimethyl-2 thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Cell apoptosis and invasion were evaluated by flow cytometry and transwell assays, respectively. Western blot (WB) analysis was used to assess the levels of resistance-related proteins and CPSF4 protein. Besides, dual-luciferase reporter assay was used to verify the interactions among lnc-NEAT1, miR-150-5p and CPSF4. Also, mice xenograft models were used to determine the effect of lnc-NEAT1 on CRC tumor growth in vivo. RESULTS: In CRC, the expression of lnc-NEAT1 was upregulated and miR-150-5p was downregulated, and the expression of both was negatively correlated. Silencing of lnc-NEAT1 promoted the 5-Fu sensitivity, enhanced the apoptosis and suppressed the invasion of CRC cells. MiR-150-5p could be sponged by lnc-NEAT1, and its inhibitors could partially reverse the effect of lnc-NEAT1 silencing on CRC progression. Besides, CPSF4 could be targeted by miR-150-5p, and its overexpression also could invert the effect of lnc-NEAT1 knockdown on CRC progression. Further, CPSF4 expression was regulated by lnc-NEAT1 and miR-150-5p. In addition, interference of lnc-NEAT1 reduced tumor volume and improved the sensitivity of CRC to 5-Fu in vivo. CONCLUSION: Lnc-NEAT1 acted as an oncogene in CRC through regulating CPSF4 expression by sponging miR-150-5p. The discovery of lnc-NEAT1/miR-150-5p/CPSF4 axis provided a novel approach for CRC genomic therapy strategy.

Cleavage and polyadenylation specific factor 4 promotes colon cancer progression by transcriptionally activating hTERT.

CPSF4 was identified as a crucial tumorigenic factor in lung cancer development. However, its precise function and the underlying molecular mechanisms in colon cancer progression remain completely unknown. Here, we demonstrate CPSF4 was highly expressed in human colon cancer cells and tissues. Its knockdown inhibited colorectal cancer progression in vitro, including cell proliferation, migration, invasion and stemness maintenance. In contrast, the ectopic overexpression of CPSF4 had the opposite effects in vitro and in vivo. Further mechanistic studies demonstrated that CPSF4 facilitated colorectal tumorigenesis and development partially through transcriptionally regulating hTERT expression by cooperating with NF-kB1 and co-anchoring at hTERT promoter -321 to -234 fragment. In addition, clinical samples analysis indicated that CPSF4 expression was positively correlated with hTERT, and the simultaneously high expression of CPSF4 and hTERT predicted poor patient outcome. Overall, our findings established CPSF4 as a pro-tumorigenic factor in colorectal cancer progression, and suggested that targeting CPSF4-hTERT axis may represent a promising therapeutic strategy in colon cancer treatment.

MiR-4458 inhibits breast cancer cell growth, migration, and invasiveness by targeting CPSF4.

Numerous studies have reported that CPSF4 is over-expressed in a large percentage of human lung cancers, and CPSF4 has been identified as a potential oncogene of human lung tumor. Downregulation of CPSF4 inhibits the proliferation and promotes the apoptosis of lung adenocarcinoma cells. A previous study by our group also found overexpression of CPSF4 in breast cancer (BC), and was closely associated with a poor prognosis for the patient. This study investigates microRNAs (miRNAs) that target CPSF4 to modulate BC cell proliferation. We found that miR-4458 was noticeably reduced in BC tissues and cells. Using a miR-4458 mimic, we found that cell proliferation, migration, and invasiveness were suppressed by miR-4458 overexpression, and were enhanced by reducing the expression of miR-4458. Moreover, the results from bioinformatics analyses suggest a putative target site in the CPSF4 3'-UTR. Furthermore, using luciferase reporter assays and Western blotting, we verified that miR-4458 directly targets the 3'-UTR of CPSF4 and downregulates COX-2 and h-TERT, which are downstream target genes of CPSF4. Additionally, PI3K/AKT and ERK were shown to be inhibited by miR-4458 overexpression in BC cells. Moreover, miR-4458 suppresses BC cell growth in vivo. Consequently, these results suggest that the miR-4458-CPSF4-COX-2-hTERT axis might serve as a potential target for the treatment of BC patients.

Synergistic Antitumor Effect of BKM120 with Prima-1Met Via Inhibiting PI3K/AKT/mTOR and CPSF4/hTERT Signaling and Reactivating Mutant P53.

BACKGROUND/AIMS: PI3KCA and mutant p53 are associated with tumorigenesis and the development of cancers. NVP-BKM120, a selective pan-PI3K inhibitor, exerts the antitumor activity by suppressing the PI3K signaling pathway. Prima-1Met, a low molecular weight compound, can rescue the gain-of-function of mutant p53 by restoring its transcriptional function. In this study, we investigated whether PI3K inhibition combined with mutant p53 reactivation could enhance the antitumor effect in thyroid cancer cells. METHODS: The effects of BKM120 and Prima-1Met on the proliferation, apoptosis, migration and invasion of thyroid cancer cells were measured by MTT, colony formation, flow cytometry, wound-healing and transwell assays, respectively. Thyroid differentiation was assessed by detecting the expression levels of specific markers using RT-PCR and Western blot. The in vivo antitumor efficacy was analyzed in a mouse xenograft model. RESULTS: The combinational treatment of BKM120 and Prima-1Met significantly enhanced the inhibitions of cell viability, colony formation, migration and invasion, and the induction of apoptosis in thyroid cell lines, and synergistically suppressed tumor xenograft growth by inhibiting the PI3K/Akt/mTOR and EMT signaling pathways, up-regulating p53 targeted genes, and triggering the release of cytochrome c. Moreover, the combination of BKM120 and Prima-1Met suppressed the stemlike traits of thyroid cancer cells and promoted their differentiation by upregulating the expression of thyroid-specific differentiation markers and repressing the expression of cancer stem cell markers. Furthermore, the mechanism study demonstrated that the combinational treatment synergistically abrogated the binding of CPSF4 at the promoter of hTERT and thus suppressed hTERT expression. Consistently, overexpression of hTERT rescued the inhibitions of cell viability, invasion and stem-like traits mediated by the combination of BKM120 and Prima-1Met. CONCLUSION: Our results showed that the combination of BKM120 with Prima-1Met synergistically suppressed the growth of thyroid cancer cells and tumor xenografts via inhibiting PI3K/Akt/mTOR and CPSF4/hTERT signaling and reactivating mutant p53.

Cleavage and polyadenylation specific factor 4 targets NF-κB/cyclooxygenase-2 signaling to promote lung cancer growth and progression.

Overexpression of cyclooxygenase 2 (COX-2) is frequently found in early and advanced lung cancers. However, the precise regulatory mechanism of COX-2 in lung cancers remains unclear. Here we identified cleavage and polyadenylation specific factor 4 (CPSF4) as a new regulatory factor for COX-2 and demonstrated the role of the CPSF4/COX-2 signaling pathway in the regulation of lung cancer growth and progression. Overexpression or knockdown of CPSF4 up-regulated or suppressed the expression of COX-2 at mRNA and protein levels, and promoted or inhibited cell proliferation, migration and invasion in lung cancer cells. Inhibition or induction of COX-2 reversed the CPSF4-mediated regulation of lung cancer cell growth. Cancer cells with CPSF4 overexpression or knockdown exhibited increased or decreased expression of p-IKKα/ß and p-IκBα, the translocation of p50/p65 from the cytoplasm to the nucleus, and the binding of p65 on COX-2 promoter region. In addition, CPSF4 was found to bind to COX-2 promoter sequences directly and activate the transcription of COX-2. Silencing of NF-κB expression or blockade of NF-κB activity abrogated the binding of CPSF4 on COX-2 promoter, and thereby attenuated the CPSF4-mediated up-regulation of COX-2. Moreover, CPSF4 was found to promote lung tumor growth and progression by up-regulating COX-2 expression in a xenograft lung cancer mouse model. CPSF4 overexpression or knockdown promoted or inhibited tumor growth in mice, while such regulation of tumor growth mediated by CPSF4 could be rescued through the inhibition or activation of COX-2 signaling. Correspondingly, CPSF4 overexpression or knockdown also elevated or attenuated COX-2 expression in tumor tissues of mice, while treatment with a COX-2 inducer LPS or a NF-κB inhibitor reversed this elevation or attenuation. Furthermore, we showed that CPSF4 was positively correlated with COX-2 levels in tumor tissues of lung cancer patients. Simultaneous high expression of CPSF4 and COX-2 proteins predicted poor prognosis of patients with lung cancers. Our results therefore demonstrated a novel mechanism for the transcriptional regulation of COX-2 by CPSF4 in lung cancer, and also offer a potential therapeutic target for lung cancers bearing aberrant activation of CPSF4/COX-2 signaling.

CREB-binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway.

CBP (CREB-binding protein) is a transcriptional co-activator which possesses HAT (histone acetyltransferases) activity and participates in many biological processes, including embryonic development, growth control and homeostasis. However, its roles and the underlying mechanisms in the regulation of carcinogenesis and tumor development remain largely unknown. Here we investigated the molecular mechanisms and potential targets of CBP involved in tumor growth and survival in lung cancer cells. Elevated expression of CBP was detected in lung cancer cells and tumor tissues compared to the normal lung cells and tissues. Knockdown of CBP by siRNA or inhibition of its HAT activity using specific chemical inhibitor effectively suppressed cell proliferation, migration and colony formation and induced apoptosis in lung cancer cells by inhibiting MAPK and activating cytochrome C/caspase-dependent signaling pathways. Co-immunoprecipitation and immunofluorescence analyses revealed the co-localization and interaction between CBP and CPSF4 (cleavage and polyadenylation specific factor 4) proteins in lung cancer cells. Knockdown of CPSF4 inhibited hTERT transcription and cell growth induced by CBP, and vice versa, demonstrating the synergetic effect of CBP and CPSF4 in the regulation of lung cancer cell growth and survival. Moreover, we found that high expression of both CBP and CPSF4 predicted a poor prognosis in the patients with lung adenocarcinomas. Collectively, our results indicate that CBP regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathways.

CPSF4 activates telomerase reverse transcriptase and predicts poor prognosis in human lung adenocarcinomas.

The elevated expression and activation of human telomerase reverse transcriptase (hTERT) is associated with the unlimited proliferation of cancer cells. However, the excise mechanism of hTERT regulation during carcinogenesis is not well understood. In this study, we discovered cleavage and polyadenylation specific factor 4 (CPSF4) as a novel tumor-specific hTERT promoter-regulating protein in lung cancer cells and identified the roles of CPSF4 in regulating lung hTERT and lung cancer growth. The ectopic overexpression of CPSF4 upregulated the hTERT promoter-driven report gene expression and activated the endogenous hTERT mRNA and protein expression and the telomerase activity in lung cancer cells and normal lung cells. In contrast, the knockdown of CPSF4 by siRNA had the opposite effects. CPSF4 knockdown also significantly inhibited tumor cell growth in lung cancer cells in vitro and in a xenograft mouse model in vivo, and this inhibitory effect was partially mediated by decreasing the expression of hTERT. High expression of both CPSF4 and hTERT proteins were detected in lung adenocarcinoma cells by comparison with the normal lung cells. Tissue microarray immunohistochemical analysis of lung adenocarcinomas also revealed a strong positive correlation between the expression of CPSF4 and hTERT proteins. Moreover, Kaplan-Meier analysis showed that patients with high levels of CPSF4 and hTERT expression had a significantly shorter overall survival than those with low CPSF4 and hTERT expression levels. Collectively, these results demonstrate that CPSF4 plays a critical role in the regulation of hTERT expression and lung tumorigenesis and may be a new prognosis factor in lung adenocarcinomas.