Cell Cycle-related And Expression-elevated Protein In Tumor Research Articles

Elevated CREPT Expression Enhances the Progression of Salivary Gland Adenoid Cystic Carcinoma

The elevated expression of Cell cycle-Related and Expression-elevated Protein in Tumor (CREPT) is reported to promote the growth of several tumors by enhancing Wnt/β-catenin signaling and cell cycle. However, the relevance of CREPT to the malignancy of salivary gland adenoid cystic carcinoma (SACC) remains unclear. The samples from 51 SACC patients were exploited in this study. We found that SACC samples exhibited a noticeably robuster CREPT expression than the para-cancerous tissues. Statistical analysis suggested that CREPT expression was significantly correlated with the T classification of SACC. To up- or down-regulating CREPT expression, the specific shRNA or full length of CREPT was delivered into SACC cell lines to examine the cell proliferation, migration, colony formation and implanted xenograft survival. Western blot assay and immunohistochemistry were applied to evaluate the expression of CREPT, cyclin D1, c-Myc and CDK4. Up-regulated CREPT in the low metastatic SACC line significantly promoted proliferation and colony formation, as well as cyclin D1, c-Myc and CDK4 expression. While knocking down of CREPT in the high metastatic SACC line remarkably reduced above effects. Furthermore, the SACC xenograft in mice confirmed that down-regulation of CREPT inhibited the in vivo tumor growth. Our study indicated that the elevated CREPT expression promoted the cell proliferation and tumor size of SACC by enhancing the expression of cyclin D1, c-Myc and CDK4, suggesting that CREPT contributed to SACC progression by stimulating cell proliferation, and might act as a potential target in future SACC therapy.

CREPT serves as a biomarker of poor survival in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human malignancies. Cell-cycle-related and expression-elevated protein in tumor (CREPT) plays an important role in the phosphorylation of RNA Pol II, and has been implicated in the development of several types of cancer. As yet, however, there have been no reports on its role in PDAC. Here, we aimed to explore the value of CREPT as a prognostic biomarker in PDAC. CREPT expression was assessed by immunohistochemistry (IHC) on a tissue microarray containing samples from 375 PDAC patients. Kaplan-Meier and Cox regression analyses were performed to explore the independent prognostic value of CREPT expression for the disease-free survival (DFS) and overall survival (OS) of PDAC patients. A Cell Counting Kit-8 (CCK8) assay was used to determine the growth rates and gemcitabine sensitivities of PDAC cells, while a Transwell assay was used to determine the migration and invasion abilities of PDAC cells. Subcutaneous xenografts were used to explore the effect of CREPT expression on tumor growth in vivo. We found that CREPT is highly expressed in tumor tissues and may serve as an independent prognostic biomarker for DFS and OS of PDAC patients. In vitro assays revealed that CREPT expression promotes the proliferation, migration, invasion and gemcitabine resistance of PDAC cells, and in vivo assays showed that CREPT expression knockdown led to inhibition of PDAC tumor growth. We conclude that high CREPT expression enhances the proliferation, migration, invasion and gemcitabine resistance of PDAC cells. In addition, we conclude that CREPT may serve as an independent prognostic biomarker and therapeutic target for PDAC patients.

Overexpression of cell-cycle related and expression-elevated protein in tumor (CREPT) in malignant cervical cancer.

ObjectiveTo explore the expression of cell-cycle related and expression-elevated protein in tumor (CREPT), cyclin D1, and transcription factor 4 (TCF4) in patients with cervical carcinoma.MethodsThis study enrolled 20 patients with cervical cancer and 10 control patients diagnosed with benign cervical lesions undergoing resection at the People’s Liberation Army General Hospital from January 2016 to December 2017. Cervical tissues were collected from all patients and their clinical characteristics were recorded. Protein and mRNA levels of CREPT, cyclin D1, and TCF4 were measured in tissue samples by immunohistochemistry, western blotting, and real-time polymerase chain reaction (PCR) and compared between the two groups.ResultProtein and mRNA expression levels of CREPT, cyclin D1, and TCF4 were all significantly higher in the cervical cancer compared with the control group, according to western blot and PCR, respectively. CREPT expression was also significantly higher in the cervical cancer group according to immunohistochemistry.ConclusionLevels of CREPT, cyclin D1, and TCF4 were significantly elevated in cervical carcinoma tissues, and their expression levels were positively correlated, suggesting that these factors might play important roles during the development of cervical carcinoma.

CREPT Promotes Melanoma Progression Through Accelerated Proliferation and Enhanced Migration by RhoA-Mediated Actin Filaments and Focal Adhesion Formation.

Melanoma is one of the most aggressive cancers, and patients with distant metastases have dire outcomes. We observed previously that melanoma progression is driven by a high migratory potential of melanoma cells, which survive and proliferate under harsh environmental conditions. In this study, we report that CREPT (cell-cycle related and expression-elevated protein in tumor), an oncoprotein highly expressed in other cancers, is overexpressed in melanoma cells but not melanocytes. Overexpression of CREPT stimulates cell proliferation, migration, and invasion in several melanoma cell lines. Further, we show that CREPT enhances melanoma progression through upregulating and activating Ras homolog family member A (RhoA)-induced actin organization and focal adhesion assembly. Our study reveals a novel role of CREPT in promoting melanoma progression. Targeting CREPT may be a promising strategy for melanoma treatment.

CREPT promotes glioma cell proliferation and invasion by activating Wnt/β-catenin pathway and is a novel target of microRNA-596

Cell cycle-related and expression elevated protein in tumor (CREPT) is emerging as a novel cancer-related gene that is dysregulated in many kinds of malignancies. However, the expression and biological role of CREPT in glioma remains unclear. In the present study, we aimed to explore the potential function and regulation mechanism of CREPT in glioma. Results showed that CRETP expression was significantly upregulated in glioma cell lines. Depletion of CREPT by siRNA-mediated gene silencing markedly decreased the proliferative and invasive capabilities of glioma cells. Bioinformatics analysis predicted CREPT as a target gene of microRNA-596 (miR-596), which was further verified by real-time quantitative polymerase chain reaction and Western blot analysis. miR-596 was significantly decreased in glioma tissues and cell lines, and inversely correlated with CREPT expression in clinical specimens. Knockdown of CREPT or overexpression of miR-596 significantly restricted the activation of Wnt/β-catenin signaling in glioma cells. Moreover, overexpression of CREPT partially reversed the miR-596-mediated inhibitory effect on proliferation, invasion and Wnt/β-catenin signaling in glioma cells. Overall, these results demonstrate that CREPT exerts an oncogenic role in glioma and its expression is regulated by miR-596. Our study highlights the important role miR-596/CREPT/Wnt/β-catenin signaling axis may play in glioma.

MicroRNA-449b-5p suppresses the growth and invasion of breast cancer cells via inhibiting CREPT-mediated Wnt/β-catenin signaling

Accumulating evidence has suggested that microRNA-449b-5p (miR-449b-5p) plays an important role in the development and progression of multiple cancers. However, little is known about the role of miR-449b-5p in breast cancer. In this study, we aimed to investigate the expression level, biological function and underlying mechanism of miR-449b-5p in breast cancer. Our results showed that miR-449b-5p expression was frequently down-regulated in breast cancer cell lines and tissues. The overexpression of miR-449b-5p significantly inhibited growth and invasion, and induced the cell cycle arrest of breast cancer cells. In contrast, the inhibition of miR-449b-5p showed the opposite effect. Interestingly, bioinformatic analysis predicted that cell cycle-related and expression-elevated protein in tumor (CREPT), an important oncogene in breast cancer, was a potential target gene of miR-449b-5p. The overexpression of miR-449b-5p decreased CREPT expression while miR-449b-5p inhibition promoted CREPT expression in breast cancer cells. Restoration of CREPT expression in miR-449b-5p mimics transfected cells partially reversed the suppressive effect of miR-449b-5p on breast cancer cell growth and invasion. Notably, our results showed that miR-449b-5p overexpression decreased the expression of β-catenin and suppressed the activation of Wnt/β-catenin/TCF-4 signaling via targeting CREPT. In addition, blocking Wnt/β-catenin partially reversed the promotion effect of miR-449b-5p inhibition on breast cancer cell growth and invasion. Overall, these results reveal a tumor suppressive role of miR-449b-5p that restricts the growth and invasion of breast cancer cells through targeting CREPT and inhibiting CREPT-mediated activation of Wnt/β-catenin signaling. Our study suggests that the miR-449b-5p/CREPT/Wnt/β-catenin axis may play an important role in the pathogenesis of breast cancer and miR-449–5p may serve as a potential therapeutic target for breast cancer.

MicroRNA-501–3p restricts prostate cancer growth through regulating cell cycle-related and expression-elevated protein in tumor/cyclin D1 signaling

MicroRNA-501–3p (miR-501–3p) has been reported as a novel cancer-related miRNA in many types of cancer. However, the precise biological function of miR-501–3p in prostate cancer remains unknown. In this study, we aimed to investigate the regulatory effect and mechanism of miR-501–3p on cell growth of prostate cancer cells. We found that miR-501–3p expression was significantly downregulated in prostate cancer tissues and cell lines. Gain-of-function experiments showed that upregulation of miR-501–3p expression significantly decreased cell proliferation and colony formation, and induced cell cycle arrest in the G0/G1 phase. Bioinformatics analysis predicted that cell cycle-related and expression-elevated protein in tumor (CREPT) was a potential target gene of miR-501–3p., and the results of our luciferase reporter assay confirmed that miR-501–3p bound to the 3′-untranslated region of CREPT at the predicted binding site. Moreover, miR-501–3p was shown to negatively regulate CREPT expression in prostate cancer cells. Correlation analysis showed that miR-501–3p was inversely correlated with CREPT expression in prostate cancer tissues. Knockdown studies revealed that miR-501–3p regulated the expression of cyclin D1 by targeting CREPT. Additionally, the inhibitory effect of miR-501–3p on prostate cancer cell growth was partially reversed by CREPT overexpression. Overall, these results suggest that miR-501–3p restricts prostate cancer cell growth by targeting CREPT to inhibit the expression of cyclin D1. These findings indicate that the miR-501–3p/CREPT/cyclin D1 axis plays a crucial role in the progression of prostate cancer and may serve as potential therapeutic target.

MicroRNA-383 acts as a tumor suppressor in colorectal cancer by modulating CREPT/RPRD1B expression.

CREPT (Cell-cycle-related and expression-elevated protein in tumor)/RPRD1B, a novel protein that enhances the transcription of Cyclin D1 to promote cell proliferation during tumorigenesis, was demonstrated highly expressed in most of tumors. However, it remains unclear how CREPT is regulated in colorectal cancers. In this study, we report that miR-383 negatively regulates CREPT expression. We observed that CREPT was up-regulated but the expression of miR-383 was down regulated in both colon cancer cell lines and colon tumor tissues. Intriguingly, we found that enforced expression of miR-383 inhibited the expression of CREPT at both the mRNA and protein level. Using a luciferase reporter, we showed that miR-383 targeted the 3'-UTR of CREPT mRNA directly. Consistently we observed that over expression of miR-383 shortened the half-life of CREPT mRNA in varieties of colorectal cancer cells. Furthermore, restoration of miR-383 inhibited cell growth and colony formation of colon cancer cells accompanied by inhibition of expression of CREPT and related downstream genes. Finally, we demonstrated that stable over expression of miR-383 in colon cancer cells decreased the growth of the tumors. Our results revealed that the abundant expression of CREPT in colorectal cancers is attributed to the decreased level of miR-383. This study shed a new light on the potential therapeutic therapy strategy for colorectal cancers using introduced miRNA.

Riboflavin Depletion Promotes Tumorigenesis in HEK293T and NIH3T3 Cells by Sustaining Cell Proliferation and Regulating Cell Cycle–Related Gene Transcription

Riboflavin is an essential component of the human diet and its derivative cofactors play an established role in oxidative metabolism. Riboflavin deficiency has been linked with various human diseases. The objective of this study was to identify whether riboflavin depletion promotes tumorigenesis. HEK293T and NIH3T3 cells were cultured in riboflavin-deficient or riboflavin-sufficient medium and passaged every 48 h. Cells were collected every 5 generations and plate colony formation assays were performed to observe cell proliferation. Subcutaneous tumorigenicity assays in NU/NU mice were used to observe tumorigenicity of riboflavin-depleted HEK293T cells. Mechanistically, gene expression profiling and gene ontology analysis were used to identify abnormally expressed genes induced by riboflavin depletion. Western blot analyses, cell cycle analyses, and chromatin immunoprecipitation were used to validate the expression of cell cycle-related genes. Plate colony formation of NIH3T3 and HEK293T cell lines was enhanced >2-fold when cultured in riboflavin-deficient medium for 10-20 generations. Moreover, we observed enhanced subcutaneous tumorigenicity in NU/NU mice following injection of riboflavin-depleted compared with normal HEK293T cells (55.6% compared with 0.0% tumor formation, respectively). Gene expression profiling and gene ontology analysis revealed that riboflavin depletion induced the expression of cell cycle-related genes. Validation experiments also found that riboflavin depletion decreased p21 and p27 protein levels by ∼20%, and increased cell cycle-related and expression-elevated protein in tumor (CREPT) protein expression >2-fold, resulting in cyclin D1 and CDK4 levels being increased ∼1.5-fold, and cell cycle acceleration. We also observed that riboflavin depletion decreased intracellular riboflavin levels by 20% and upregulated expression of riboflavin transporter genes, particularly SLC52A3, and that the changes in CREPT and SLC52A3 correlated with specific epigenetic changes in their promoters in riboflavin-depleted HEK293T cells. Riboflavin depletion contributes to HEK293T and NIH3T3 cell tumorigenesis and may be a risk factor for tumor development.

Cell cycle‐related and expression‐elevated protein in tumor overexpression is associated with proliferation behaviors and poor prognosis in non‐small‐cell lung cancer

The cell cycle‐related and expression‐elevated protein in tumor (CREPT) is overexpressed in several human malignancies. However, the clinical relevance of CREPT expression and its biological role in non‐small‐cell lung cancer (NSCLC) remains unclear. In this study, we detected the expression of CREPT in both NSCLC tissues and cell lines by immunohistochemistry, Western blot analysis, and RT‐PCR. The correlation between CREPT expression and clinicopathologic features was analyzed in 271 NSCLC patients. The prognostic value of CREPT expression was evaluated by Kaplan–Meier analysis and Cox regression analysis. CREPT was overexpressed in Calu‐1 cell lines by using plasmid vector and its biological function was explored both in vitro and in vivo. We found that CREPT was significantly overexpressed in NSCLC compared with paired adjacent non‐tumor tissues, and the expression level of CREPT was correlated with tumor differentiation, lymph node metastasis, and clinical stage. Kaplan–Meier analysis showed that the recurrence‐free survival and overall survival of high CREPT expression groups were significantly shorter than those of the low CREPT expression group. Multivariate analysis identified that CREPT might be an independent biomarker for the prediction of NSCLC prognosis. Overexpression of CREPT increased cell proliferation and enhanced the migration and invasion ability of Calu‐1 cells (a human NSCLC cell line with relative low CRPET expression) in vitro. Moreover, CREPT overexpression promoted tumor growth in a nude mice model. These results suggest that CREPT is closely relevant to the proliferation of NSCLC cells and it might be a potential prognostic marker in NSCLC patients.

Inhibition of CREPT restrains gastric cancer growth by regulation of cycle arrest, migration and apoptosis via ROS-regulated p53 pathway

CREPT (cell-cycle related and expression-elevated protein in tumor) was reported to be associated with growth of several human cancers; however, its clinical significance and regulatory mechanism still remain unclear in human gastric cancer. In the present study, we found CREPT was significantly increased in gastric cancer tissues compared to the matched adjacent normal tissues. CREPT silence inhibited the proliferation of gastric cancer cells through inducing G0/G1 phase cell cycle arrest, which was linked to the reduction of Cyclin D1 and Cyclin D-dependent kinase 4 (CDK4), and the elevation of p53 and p21. In addition, CREPT knockdown (KD) decreased migration of gastric cancer cells through up-regulating E-cadherin and down-regulating vimentin, N-cadherin and matrix metalloproteinase 1 (MMP-1) expressions. Further, CREPT KD induced apoptosis in gastric cancer cells, as evidenced by the increase of cleaved Caspase-3 and poly (ADP-ribose) polymerase (PARP). Intriguingly, suppressing p53 expressions significantly abolished CREPT silence-induced apoptosis, and reduction of cell viability. Moreover, CREPT KD caused reactive oxygen species (ROS) generation using discounted cash flow (DCF) analysis, which was reversed by ROS scavenger, N-acetyl-l-cysteine (NAC), pretreatment. Of note, NAC pretreatment abrogated apoptotic cell death in CREPT KD gastric cancer cells. In vivo, suppressing CREPT reduced the gastric tumor growth in gastric cancer xenograft models. Altogether, our results provided a novel insight into CREPT in regulating gastric cancer progression through apoptosis regulated by ROS/p53 pathways.

Overexpression of CREPT confers colorectal cancer sensitivity to fluorouracil.

AIMTo investigate expression of cell cycle-related and expression-elevated protein in tumor (CREPT) in colorectal cancer (CRC) and determine its prognostic value in response to 5-fluorouracil (5-FU).METHODSThe relative expression of CREPT in CRC tumor samples was determined using immunohistochemistry. The protein content in cell lines was analyzed by immunoblotting. Cell viability was measured with the CCK-8 assay. Cell cycle and apoptosis analyses were performed with flow cytometry.RESULTSCREPT was overexpressed in CRC tissues and correlated with histological grade. Clinicopathological analysis indicated that CREPT was positively related to tumor progression. Exogenous expression of CREPT stimulated cell proliferation and accelerated the cell cycle. More importantly, high expression of CREPT sensitized CRC cells to 5-FU treatment. Furthermore, we demonstrated that 5-FU elicited significant apoptosis in CREPT-positive cells.CONCLUSIONAberrant overexpression of CREPT contributes to tumorigenesis of CRC by promoting cell proliferation and accelerating the cell cycle, and confers sensitivity to 5-FU. CREPT is a potential prognostic biomarker for 5-FU in CRC.

Knocking-down of CREPT prohibits the progression of oral squamous cell carcinoma and suppresses cyclin D1 and c-Myc expression.

BackgroundAs a regulator essential for many cell cycle-related proteins, the robust expression of Cell cycle-Related and Expression-elevated Protein in Tumor (CREPT) implicates a poor diagnosis of endoderm and mesoderm-derived tumors. Whether CREPT plays the same role in the tumorigenesis derived from ectodermal tissues remains elusive.MethodsTo explore the role of CREPT in ectoderm-derived tumors, cells from 7oral squamous cell carcinoma (OSCC) lines and 84clinical OSCC samples were exploited in this study. Quantitative PCR, Western blot assay and immunohistochemistry were applied in the evaluation of CREPT, cyclin D1 and c-Myc expression. Knocking-down of CREPT was performed by lentivirus delivering specific shRNA of CREPT. The effects of CREPT on OSCC were examined by cell proliferation, colony formation, apoptosis, cell migration and xenograft implantation experiments.ResultsCompared with human normal oral keratinocytes, OSCC cell lines showed a significantly elevated expression of CREPT in both mRNA and protein levels. Consistently, samples from OSCC patients also exhibited a noticeably stronger CREPT expression than the noncancerous samples. In contrast, knocking down of CREPT in OSCC cell lines significantly reduced proliferation, colony formation and migration as well as the expression of cyclin D1 and c-Myc, but promoted apoptosis. Statistical analysis also suggested that CREPT expression was significantly correlated with the T and N classification of OSCC. Furthermore, CAL27 mouse xenograft model confirmed that down-regulation of CREPT prohibited cyclin D1 and c-Myc expression, through which decreased the in vivo tumor growth, but increased the survival ratio of hosts.ConclusionIn OSCC cell lines, up-regulated CREPT expression enhanced cell proliferation, migration and cell cycle as well as promoted cyclin D1 and c-Myc expression as it did in endoderm and mesoderm-origin tumors. Our study strongly suggests that CREPT could be used as a marker for the OSCC prognosis and might work as a potential target in future OSCC therapy.

High expression of CREPT promotes tumor growth and is correlated with poor prognosis in colorectal cancer

CREPT (cell cycle-related and expression elevated protein in tumor) is highly expressed in many kinds of cancer, and has been shown to be prognostic in certain cancers. However, the clinical significance of CREPT in colorectal cancer (CRC) has not been sufficiently investigated. In this study, we examined the CREPT expression in 225 clinical CRC tissues and paired adjacent normal tissues, and analyzed the correlation between CREPT expression and other clinicopathological features. We also evaluated the biological function of CREPT both in vitro and in vivo using knockdown or overexpressing CRC cells. Our results showed that CREPT expressed in 175 of 225 (77.8%) CRC patients and the CREPT expression was significantly associated with tumor differentiation (P = 0.000), Dukes' stages (P = 0.013) and metastasis (P = 0.038). Patients with high CREPT expression tended to have shorter survival time. Multivariate analysis showed that positive CREPT expression can be used as an independent predictor for CRC prognosis. CREPT knockdown cells showed inhibited cell proliferation and arrested cell cycle, while CREPT overexpressing cells showed increased proliferation and promoted cell cycle. In addition, CREPT overexpression significantly promoted tumor growth in vivo. Mechanism study showed that CREPT may regulate cell proliferation and cell cycle through the regulation on cyclin D3, CDK4 and CDK6.

Characterization of a monoclonal antibody against CREPT, a novel protein highly expressed in tumors.

CREPT (cell-cycle related and expression-elevated protein in tumor), a novel gene also called RPRD1B and C20ORF77, was recently identified to promote tumorigenesis through up-regulation of the expression of genes related to cell cycle. The previous study demonstrated that CREPT is highly expressed in a variety of tumors and enhances the expression of Cyclin D1 by promoting the formation of a chromatin loop. To study the correlation of CREPT expression with clinical factors in different tumors, we generated a monoclonal antibody (3E10) using purified recombinant human GST-CREPT protein as an antigen. In this study, we characterized the specificity of the monoclonal antibody and cloned the gene encoding the antibody for preparation of industrial production. Our results showed that the monoclonal antibody 3E10 was sensitive and specific to recognize human endogenous CREPT protein. We have mapped the epitope of the antibody and cloned the variable region sequence of the gene encoding the antibody. We confirmed that the cloned gene produced an equivalent antibody as that produced by the original hybridoma. This study provided a basis for large-scale production of the CREPT antibody, which will be useful for the study of the role of CREPT in different tumors.

CREPT/RPRD1B, a Recently Identified Novel Protein Highly Expressed in Tumors, Enhances the β-Catenin·TCF4 Transcriptional Activity in Response to Wnt Signaling

CREPT (cell cycle-related and expression elevated protein in tumor)/RPRD1B (regulation of nuclear pre-mRNA domain-containing protein 1B), highly expressed during tumorigenesis, was shown to enhance transcription of CCND1 and to promote cell proliferation by interacting with RNA polymerase II. However, which signaling pathway is involved in CREPT-mediated activation of gene transcription remains unclear. In this study, we reveal that CREPT participates in transcription of the Wnt/β-catenin signaling activated genes through the β-catenin and the TCF4 complex. Our results demonstrate that CREPT interacts with both β-catenin and TCF4, and enhances the association of β-catenin with TCF4, in response to Wnt stimulation. Furthermore, CREPT was shown to occupy at TCF4 binding sites (TBS) of the promoters of Wnt-targeted genes under Wnt stimulation. Interestingly, depletion of CREPT resulted in decreased occupancy of β-catenin on TBS, and over-expression of CREPT enhances the activity of the β-catenin·TCF4 complex to initiate transcription of Wnt target genes, which results in up-regulated cell proliferation and invasion. Our study suggests that CREPT acts as an activator to promote transcriptional activity of the β-catenin·TCF4 complex in response to Wnt signaling.