Project description:In the majority of human tumors, expression of the c-MYC oncogene becomes constitutive. Here, we report that c-MYC directly regulates the expression of AP4 via CACGTG motifs in the first intron of the AP4 gene. Induction of AP4 was required for c-MYC-mediated cell cycle reentry of anti-estrogen arrested breast cancer cells and mitogen-mediated repression of the CDK inhibitor p21. AP4 directly repressed p21 by occupying four CAGCTG motifs in the p21 promoter via its basic region. AP4 levels declined after DNA damage, and ectopic AP4 interfered with p53-mediated cell cycle arrest and sensitized cells to apoptosis induced by DNA damaging agents. AP4 expression blocked induction of p21 by TGF-beta in human keratinocytes and interfered with up-regulation of p21 and cell cycle arrest during monoblast differentiation. Notably, AP4 is specifically expressed in colonic progenitor and colorectal carcinoma cells. In conclusion, our results indicate that c-MYC employs AP4 to maintain cells in a proliferative, progenitor-like state.
Project description:The gene encoding the transcription factor TFAP4/AP4 represents a direct target of the c-MYC oncoprotein. Here, we deleted Ap4 in ApcMin mice, a preclinical model of inherited colorectal cancer. Ap4 deficiency extends their average survival by 110 days and decreases the formation of intestinal adenomas and tumor-derived organoids. The effects of Ap4 deletion are presumably due to the reduced number of functional intestinal stem cells (ISCs) amenable to adenoma-initiating mutational events. Deletion of Ap4 also decreases the number of colonic stem cells and increases the number of Paneth cells. Expression profiling revealed that ISC signatures, as well as the Wnt/β-catenin and Notch signaling pathways are downregulated in Ap4-deficient adenomas and intestinal organoids. AP4-associated signatures are conserved between murine adenomas and human colorectal cancer samples. Our results establish Ap4 as rate-limiting mediator of adenoma initiation, as well as regulator of intestinal and colonic stem cell and Paneth cell homeostasis.
Project description:The basic helix-loop-helix transcription factor AP4/TFAP4/AP-4 is encoded by a c-MYC target gene and displays up-regulation concomitantly with c-MYC in colorectal cancer (CRC) and numerous other tumor types. Here a genome-wide characterization of AP4 DNA binding and mRNA expression was performed using a combination of microarray, genome-wide chromatin immunoprecipitation, next-generation sequencing, and bioinformatic analyses. Thereby, hundreds of induced and repressed AP4 target genes were identified. Besides many genes involved in the control of proliferation, the AP4 target genes included markers of stemness (LGR5 and CD44) and epithelial-mesenchymal transition (EMT) such as SNAIL, E-cadherin/CDH1, OCLN, VIM, FN1, and the Claudins 1, 4, and 7. Accordingly, activation of AP4 induced EMT and enhanced migration and invasion of CRC cells. Conversely, down-regulation of AP4 resulted in mesenchymal-epithelial transition and inhibited migration and invasion. In addition, AP4 induction was required for EMT, migration, and invasion caused by ectopic expression of c-MYC. Inhibition of AP4 in CRC cells resulted in decreased lung metastasis in mice. Elevated AP4 expression in primary CRC significantly correlated with liver metastasis and poor patient survival. These findings imply AP4 as a new regulator of EMT that contributes to metastatic processes in CRC and presumably other carcinomas.
Project description:Drug resistance makes treatment difficult in cancers. The present study identifies and analyzes drug resistance-related miRNA in colorectal cancer. We established 4 types of 5-fluorouracil (5-FU)-resistant colon cancer cell lines in vitro and in vivo. We then analyzed the miRNA expression profile by miRNA array in these 4 cell lines, and identified the drug resistance-related miRNAs. We examined the expression levels of the identified miRNA in 112 colorectal tumor samples from the patients. We identified 12 possible miRNAs involved in 5-FU resistance by miRNA arrays. We then examined the relationship between miR-31, which was the most promising among them, and drug resistance. The ectopic expression of mimic miR-31 showed significant 5-FU resistance in the parental DLD-1 cells, while anti-miR-31 caused significant growth inhibition in DLD/F cells; that is, 5-FU-resistant colon cancer cell line DLD-1 under exposure to 5-FU. When we exposed high doses of 5-FU to parent or 5-FU-resistant cells, the expression levels of miR-31 were raised higher than those of controls. Notably, the expression levels of miR-31 were positively correlated with the grade of clinical stages of colorectal tumors. The protein expression levels of factors inhibiting hypoxia-inducible factor 1 were downregulated by transfection of mimic miR-31 into DLD-1 cells. This study provides evidence supporting the association of miR-31 with 5-FU drug resistance and clinical stages of colorectal tumors.
Project description:In order to comprehensively identify genes directly regulated by AP4, a genome-wide chromatin-immunoprecipitation analysis (ChIP) followed by next generation sequencing (ChIP-seq) was performed after activation of a conditional AP4 allele in DLD-1 cells. One DLD-1 Sample was sequenced.
Project description:The c-Kit receptor tyrosine kinase is commonly over-expressed in different types of cancer. p53 activation is known to result in the down-regulation of c-Kit. However, the underlying mechanism has remained unknown. Here, we show that the p53-induced miR-34 microRNA family mediates repression of c-Kit by p53 via a conserved seed-matching sequence in the c-Kit 3'-UTR. Ectopic miR-34a resulted in a decrease in Erk signaling and transformation, which was dependent on the down-regulation of c-Kit expression. Furthermore, ectopic expression of c-Kit conferred resistance of colorectal cancer (CRC) cells to treatment with 5-fluorouracil (5-FU), whereas ectopic miR-34a sensitized the cells to 5-FU. After stimulation with c-Kit ligand/stem cell factor (SCF) Colo320 CRC cells displayed increased migration/invasion, whereas ectopic miR-34a inhibited SCF-induced migration/invasion. Activation of a conditional c-Kit allele induced several stemness markers in DLD-1 CRC cells. In primary CRC samples elevated c-Kit expression also showed a positive correlation with markers of stemness, such as Lgr5, CD44, OLFM4, BMI-1 and ?-catenin. On the contrary, activation of a conditional miR-34a allele in DLD-1 cells diminished the expression of c-Kit and several stemness markers (CD44, Lgr5 and BMI-1) and suppressed sphere formation. MiR-34a also suppressed enhanced sphere-formation after exposure to SCF. Taken together, our data establish c-Kit as a new direct target of miR-34 and demonstrate that this regulation interferes with several c-Kit-mediated effects on cancer cells. Therefore, this regulation may be potentially relevant for future diagnostic and therapeutic approaches.
Project description:To characterize the transcriptome of the transcription factor AP4 DLD-1 cells were infected with AP4 coding viruses for different periods of time. Adenovirus amplification and purification was performed as previously described (He et al., 1998). The minimal amount of virus needed to reach more than 90% infection efficiency was determined by monitoring GFP signals with fluorescence microscopy. DLD-1 cells were infected in serum-free medium with adenovirus for 3 hours. After removal an equal amount of medium containing 20% FBS was added. This study contains 17 Samples with different conditions/samples: Triplicates of non-infected cells as a control, triplicates of infections with an AP4 coding viruses harvested after 12, 24 and 48 hours and, as an additional control, a triplicate of cells infected with a GFP expressing virus harvested after 24 hours and unicates harvested after 12 and 48 hours.
Project description:BACKGROUND:Cellular senescence is a state of irreversible cell growth arrest and senescence cells permanently lose proliferation potential. Induction of cellular senescence might be a novel therapy for cancer cells. TRIB2 has been reported to participate in regulating proliferation and drug resistance of various cancer cells. However, the role of TRIB2 in cellular senescence of colorectal cancer (CRC) and its molecular mechanism remains unclear. METHODS:The expression of TRIB2 in colorectal cancer tissues and adjacent tissues was detected by immunohistochemistry and RT-PCR. The growth, cell cycle distribution and cellular senescence of colorectal cancer cells were evaluated by Cell Counting Kit-8 (CCK8) assay, flow cytometry detection and senescence-associated ?-galactosidase staining, respectively. Western blot, RT-PCR and luciferase assay were performed to determine how TRIB2 regulates p21. Immunoprecipitation (IP) and chromatin-immunoprecipitation (ChIP) were used to investigate the molecular mechanisms. RESULTS:We found that TRIB2 expression was elevated in CRC tissues compared to normal adjacent tissues and high TRIB2 expression indicated poor prognosis of CRC patients. Functionally, depletion of TRIB2 inhibited cancer cells proliferation, induced cell cycle arrest and promoted cellular senescence, whereas overexpression of TRIB2 accelerated cell growth, cell cycle progression and blocked cellular senescence. Further studies showed that TRIB2 physically interacted with AP4 and inhibited p21 expression through enhancing transcription activities of AP4. The rescue experiments indicated that silencing of AP4 abrogated the inhibition of cellular senescence induced by TRIB2 overexpression. CONCLUSION:These data demonstrate that TRIB2 suppresses cellular senescence through interaction with AP4 to down-regulate p21 expression. Therefore, TRIB2 could be a potential target for CRC treatment.
Project description:Ubiquitin specific peptidase 22 (USP22), a putative cancer stem cell marker, is overexpressed in liver metastases of colorectal cancer (CRC). However, the mechanism by which USP22 promotes CRC metastasis remains largely unknown. Here, we report that USP22 and AP4 are simultaneously overexpressed during TGF-?1-induced CRC cell epithelial-mesenchymal transition (EMT). USP22 up-regulation enhances CRC cell migration and invasion and EMT-related marker and AP4 expression, but these effects are partly blocked by AP4 knockdown. In addition, USP22 binds to the promoter region of AP4 to activate its transcription. In vivo, elevated USP22 expression promotes CRC cell metastasis to the lungs in nude mice, as evidenced by the fact that CRC metastatic nodules stain deeply positive for USP22 and AP4. In human CRC tissues, the genes encoding USP22 and AP4 are overexpressed in metastatic liver lesions compared with primary cancer tissues, and their overexpression is significantly associated with poor CRC patient survival. These findings indicate that USP22 and AP4 may serve as prognostic markers for predicting the risk of developing distant metastases in CRC.