CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) has been shown to upregulate gene transcription during tumorigenesis. However, how STAT3 initiates transcription remains to be exploited. This study is to reveal the role of CREPT (cell cycle-related and elevated-expression protein in tumours, or RPRD1B) in promoting STAT3 transcriptional activity. METHODS: BALB/c nude mice, CREPT overexpression or deletion cells were employed for the assay of tumour formation, chromatin immunoprecipitation, assay for transposase-accessible chromatin using sequencing. RESULTS: We demonstrate that CREPT, a recently identified oncoprotein, enhances STAT3 transcriptional activity to promote tumorigenesis. CREPT expression is positively correlated with activation of STAT3 signalling in tumours. Deletion of CREPT led to a decrease, but overexpression of CREPT resulted in an increase, in STAT3-initiated tumour cell proliferation, colony formation and tumour growth. Mechanistically, CREPT interacts with phosphorylated STAT3 (p-STAT3) and facilitates p-STAT3 to recruit p300 to occupy at the promoters of STAT3-targeted genes. Therefore, CREPT and STAT3 coordinately facilitate p300-mediated acetylation of histone 3 (H3K18ac and H3K27ac), further augmenting RNA polymerase II recruitment. Accordingly, depletion of p300 abolished CREPT-enhanced STAT3 transcriptional activity. CONCLUSIONS: We propose that CREPT is a co-activator of STAT3 for recruiting p300. Our study provides an alternative strategy for the therapy of cancers related to STAT3.

CREPT is required for murine stem cell maintenance during intestinal regeneration.

Intestinal stem cells (ISCs) residing in the crypts are critical for the continual self-renewal and rapid recovery of the intestinal epithelium. The regulatory mechanism of ISCs is not fully understood. Here we report that CREPT, a recently identified tumor-promoting protein, is required for the maintenance of murine ISCs. CREPT is preferably expressed in the crypts but not in the villi. Deletion of CREPT in the intestinal epithelium of mice (Vil-CREPTKO) results in lower body weight and slow migration of epithelial cells in the intestine. Vil-CREPTKO intestine fails to regenerate after X-ray irradiation and dextran sulfate sodium (DSS) treatment. Accordingly, the deletion of CREPT decreases the expression of genes related to the proliferation and differentiation of ISCs and reduces Lgr5+ cell numbers at homeostasis. We identify that CREPT deficiency downregulates Wnt signaling by impairing ß-catenin accumulation in the nucleus of the crypt cells during regeneration. Our study provides a previously undefined regulator of ISCs.

NOK associates with c-Src and promotes c-Src-induced STAT3 activation and cell proliferation.

Signal transducers and activators of transcription 3 (STAT3) is reported to regulate cell proliferation, survival, and differentiation, and thus plays a central role in development and carcinogenesis. Accumulating evidence demonstrated the involvement of cellular Src (c-Src) tyrosine kinase in the activation of STAT3. Additionally, novel oncogene with kinase-domain (NOK), a receptor protein tyrosine kinase that involves in cell transformation and tumorigenesis, was found to activate STAT3 signaling by a JAK2-dependent mechanism. However, whether the existence of the interaction between c-Src/STAT3 and NOK/STAT3 signals is still unknown. In this study, we showed that NOK formed a complex with c-Src and facilitated the interaction between c-Src and STAT3. In the complex, NOK greatly elevated the c-Src-mediated STAT3 activation by increasing the phosphorylation level of STAT3 on Tyr705. Truncated and mutation experiments further demonstrated that the kinase activity was responsible for the synergistic effect of NOK and c-Src on STAT3 activation. In addition, NOK and c-Src synergistically promoted cell proliferation and tumor growth in nude mice. Taken together, our results indicate that NOK associates with c-Src and promotes c-Src-induced STAT3 activation in a kinase-dependent manner. We proposed that the axis that NOK promoted c-Src-induced STAT3 activation is critical in cell proliferation and tumorigenesis.

CREPT/RPRD1B associates with Aurora B to regulate Cyclin B1 expression for accelerating the G2/M transition in gastric cancer.

Gastric cancer, like most of other cancers, has an uncontrolled cell cycle regulated by cyclins and cyclin-dependent kinases (CDKs). In this study, we reported that gastric cancer cells showed an accelerated G2/M transition promoted by CREPT/RPRD1B and Aurora kinase B (Aurora B). We found that CREPT/RPRD1B and Aurora B were coordinately expressed during the cell cycle in gastric cancer cells. Deletion of CREPT/RPRD1B disturbed the cell progression and extended the length of cell cycle, leading to a significant accumulation of mitotic cells. Mechanistically, we revealed that CREPT/RPRD1B interacted with Aurora B to regulate the expression of Cyclin B1 in gastric cancer cells. Interestingly, Aurora B phosphorylates S145 in a well-conserved motif of CREPT/RPRD1B. We proposed that phosphorylation of CREPT/RPRD1B by Aurora B is required for promoting the transcription of Cyclin B1, which is critical for the regulation of gastric tumorigenesis. Our study provides a mechanism by which gastric tumor cells maintain their high proliferation rate via coordination of Aurora B and CREPT/RPRD1B on the expression of Cyclin B1. Targeting the interaction of Aurora B and CREPT/RPRD1B might be a strategy for anti-gastric cancer therapy in the future.

Overexpression of CREPT confers colorectal cancer sensitivity to fluorouracil.

AIM: To 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). METHODS: The 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. RESULTS: CREPT 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. CONCLUSION: Aberrant 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.

Polo-like kinase 1 (PLK1)-dependent phosphorylation of methylenetetrahydrofolate reductase (MTHFR) regulates replication via histone methylation.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating the folate cycle and its genetic variations have been associated with various human diseases. Previously we identified that MTHFR is phosphorylated by cyclin-dependent kinase 1 (CDK1) at T34 and MTHFR underlies heterochromatin maintenance marked by H3K9me3 levels. Herein we demonstrate that pT34 creates a binding motif that docks MTHFR to the polo-binding domain (PBD) of polo-like kinase 1 (PLK1), a fundamental kinase that orchestrates many cell cycle events. We show that PLK1 phosphorylates MTHFR at T549 in vitro and in vivo. Further, we uncovered a role of MTHFR in replication. First, MTHFR depletion increased the fraction of cells in S phase. This defect could not be rescued by siRNA resistant plasmids harboring T549A, but could be restored by overproduction of Suv4-20H2, the H4K20 methyltransferase. Moreover, siMTHFR attenuated H4K20me3 levels, which could be rescued by Suv4-20H2 overproduction. More importantly, we also investigated MTHFR-E429A, the protein product of an MTHFR single nucleotide variant. MTHFR-E429A overexpression also increased S phase cells and decreased H4K20me3 levels, and it is linked to a poor glioma prognosis in the Chinese population. Collectively, we have unveiled a vital role of PLK1-dependent phosphorylation of MTHFR in replication via histone methylation, and implicate folate metabolism with glioma.

BCL10 regulates RNF8/RNF168-mediated ubiquitination in the DNA damage response.

Timely and proper cellular response to DNA damage is essential for maintenance of genome stability and integrity. B-cell lymphoma/leukemia 10 (BCL10) facilitates ubiquitination of NEMO in the cytosol, activating NFκB signaling. Translocation and/or point mutations of BCL10 associate with mucosa-associated lymphoid tissue lymphomas and other malignancies. However, the mechanisms by which the resulting aberrant expression of BCL10 leads to cellular oncogenesis are poorly understood. In this report, we found that BCL10 in the nucleus is enriched at the DNA damage sites in an ATM- and RNF8-dependent manner. ATM-dependent phosphorylation of BCL10 promotes its interaction with and presentation of UBC13 to RNF8, and RNF8-mediated ubiquitination of BCL10 enhances binding of BCL10 and UBC13 to RNF168. This allows mono-ubiquitination on H2AX by RNF168 and further poly-ubiquitination by the RNF8/RNF168-containing complex. Depletion of BCL10 compromised homology recombination-mediated DNA double-strand break (DSB) repair because of insufficient recruitment of BRCA1, RAD51, and the ubiquitinated DNA damage response factors. Taken together, our results demonstrate a novel function of BCL10 in delivering UBC13 to RNF8/RNF168 to regulate ubiquitination-mediated DSB signaling and repair.

MTHFR promotes heterochromatin maintenance.

Methylenetetrahydrofolate reductase (MTHFR), a key enzyme in the folate cycle, catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine. Methionine serves as the precursor of the active methyl donor S-adenosylmethionine, which provides methyl groups for many biological methylations. It has been reported that MTHFR is highly phosphorylated under unperturbed conditions and T34 is the priming phosphorylation site. In this report, we generated a phospho-specific antibody that recognized T34-phosphorylated form of MTHFR and revealed that MTHFR was phosphorylated at T34 in vivo and this phosphorylation peaked during mitosis. We further demonstrated that the cyclin-dependent kinase 1 (CDK1)/Cyclin B1 complex is the kinase that mediates MTHFR phosphorylation at T34 and the MTHFR immunocomplex purified from mitotic cells exhibited lower enzymatic activity. Inhibition of MTHFR expression resulted in a decrease of H3K9me3 levels, and an increase of transcription of the centromeric heterochromatin markers. Taken together, our results demonstrated that CDK1/Cyclin B1 phosphorylates MTHFR on T34 and MTHFR plays a role in the heterochromatin maintenance at the centromeric region.

Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination.

DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian cells. Interestingly, we show that Ago2 forms a complex with Rad51 and that the interaction is enhanced in cells treated with ionizing radiation. We demonstrate that Rad51 accumulation at DSB sites and HR repair depend on catalytic activity and small RNA-binding capability of Ago2. In contrast, DSB resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs to facilitate repair by HR.

CtIP is required for DNA damage-dependent induction of P21.

DNA endonuclease CtIP is involved in both DNA double-strand break (DSB) repair and transcriptional repression/activation. The cyclin-dependent kinase inhibitor P21, which is induced at transcription level in response to a variety of stresses, controls G1/S transition. In this report, we found that CtIP bound to the P21 promoter, and this binding was enhanced in response to DNA damage. Concomitantly, ectopic expression of CtIP increased P21 promoter activity, and this increment was enhanced upon camptothecin treatment. Conversely, DNA damage failed to induce P21 gene expression in CtIP-deficient cells. Taken together, our data demonstrate that CtIP is required for DNA damage-induced P21 induction.

Nampt is involved in DNA double-strand break repair.

DNA double-strand break (DSB) is the most severe form of DNA damage, which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer. Nicotinamide phosphoribosyltransferase (Nampt), which is involved in nicotinamide adenine dinucleotide metabolism, is overexpressed in a variety of tumors. In this report, we found that Nampt physically associated with CtIP and DNA-PKcs/Ku80, which are key factors in HR and NHEJ, respectively. Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair. Furthermore, the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased ß-galactosidase staining, indicating a delay in the onset of cellular senescence in normal human fibroblasts. Taken together, our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair, contributing to the acceleration of cellular senescence.

Protein phosphatase PP6 is required for homology-directed repair of DNA double-strand breaks.

DNA double-strand breaks (DSBs) are among the most lethal lesions associated with genome stability which, when destabilized, predisposes organs to cancers. DSBs are primarily fixed either with little fidelity by non-homologous end joining (NHEJ) repair or with high fidelity by homology-directed repair (HDR). The phosphorylated form of H2AX on serine 139 (γ-H2AX) is a marker of DSBs. In this study, we explored if the protein phosphatase PP6 is involved in DSB repair by depletion of its expression in human cancer cell lines, and determined PP6 expression in human breast cancer tissues by immunohistochemistry staining. We found that bacterially-produced PP6c (the catalytic subunit of PP6)-containing heterotrimeric combinations exhibit phosphatase activity against γ-H2AX in the in vitro phosphatase assays. Depletion of PP6c or PP6R2 led to persistent high levels of γ-H2AX after DNA damage and a defective HDR. Chromatin immunoprecipitation assays demonstrated that PP6c was recruited to the region adjacent to the DSB sites. Expression of PP6c, PP6R2, and PP6R3 in human breast tumors was significantly lower than those in benign breast diseases. Taken together, our results suggest that γ-H2AX is a physiological substrate of PP6, and PP6 is required for HDR and its expression may harbor a protective role during the development of breast cancer.