Transcription Factor Activating Enhancer-binding Protein 4 Research Articles

USP15, activated by TFAP4 transcriptionally, stabilizes SHC1 via deubiquitination and deteriorates renal cell carcinoma.

Ubiquitin-specific peptidase 15 (USP15), a critical deubiquitinating enzyme, has been demonstrated to improve substrate stabilization by hydrolyzing the bond between the substrate and ubiquitin, and is implicated in multiple carcinogenic processes. Prompted by the information cited from The Cancer Genome Atlas (TCGA) database and the Cancer Proteogenomic Data Analysis Site (cProSite), USP15 is selectively overexpressed in clear cell renal cell carcinoma (ccRCC) samples. We aimed to investigate the function of USP15 on ccRCC malignant features, which was emphasized in its deubiquitination of SHC adaptor protein 1 (SHC1). The overexpression of USP15 promoted the capacity of proliferation, migration, and invasion in ccRCC CAKI1 and 769-P cells, and these malignant biological properties were diminished by USP15 deletion in 786-O cells. USP15 accelerated tumor growth and lung metastasis invivo. In addition, deubiquitinase USP15 was further identified as a new protector for SHC1 from degradation by the ubiquitination pathway, the post-translational modification. In sequence, transcription factor activating enhancer binding protein 4 (TFAP4) was shown to be partly responsible for USP15 expression at the level of transcription, as manifested by the chromatin immunoprecipitation and pull-down assay. Based on the invitro and invivo data, we postulate that USP15 regulated by TFAP4 transcriptionally deteriorates ccRCC malignant biological properties via stabilizing SHC1 by deubiquitination.

Cucurbitacin E Triggers Cellular Senescence in Colon Cancer Cells via Regulating the miR-371b-5p/TFAP4 Signaling Pathway.

The induction of cellular senescence is considered as a potent strategy to suppress cancer progression. Cucurbitacin E (CE) belongs to the triterpenoids and has received substantial attention for its antineoplastic property. However, the function of CE on cellular senescence remained elusive. Herein, we revealed that CE significantly induced cellular senescence in colorectal cancer (CRC) cells. The CE effects on the cellular senescence in CRC cells were confirmed by observing the common features of the senescence, such as the enhanced activity of senescence-associated β-galactosidase, γ-H2AX positive staining, and upregulation of senescence-associated proteins including p53, p27, and p21. Moreover, CE exerted pro-senescent effects in CRC cells via attenuating the transcription factor activating enhancer-binding protein 4 (TFAP4) expression, and the ectopic expression of TFAP4 blocked the CE-induced senescence. Mechanistically, CE treatment caused a robust increase in miR-371b-5p, which markedly repressed TFAP4. In contrast, silencing of miR-371b-5p counteracted the percentages of CE-induced senescent cells from 37.49 ± 2.61 to 7.06 ± 0.91% in HCT-116 cells via derepressing TFAP4 to attenuate the expression of p53, p21, and p16. Altogether, these results demonstrated that dietary CE induces CRC cellular senescence via modulating the miR-371b-5p/TFAP4 axis and presents opportunities for potential therapeutic strategies against CRC.

Inhibitory effect of CC chemokine ligand 23 (CCL23)/ transcription factor activating enhancer binding protein 4 (TFAP4) on cell proliferation, invasion and angiogenesis in hepatocellular carcinoma

ABSTRACT Hepatocellular carcinoma (HCC) is a highly vascularized solid tumor with a fast growth rate. According to bioinformatics analysis, CC chemokine ligand 23 (CCL23) has clinical significance for survival and prognosis in HCC. The online databases TCGA and CCLE were used to analyze the expression level of CCL23, and its expression was also measured in HCC cell lines by RT-qPCR and Western blotting. The STRING database and co-immunoprecipitation were employed to evaluate the association between CCL23 and transcription factor activating enhancer binding protein 4 (TFAP4). Overexpression plasmids for CCL23 (Ov-CCL23) and TFAP4 (Ov-TFAP4) were transfected into Huh-7 cells to detect TFAP4 expression. Huh-7 cells injected with OV-negative control (NC)/Ov-CCL23 or OV-NC/Ov-CCL23 plus Ov-TFAP4 were utilized to study the function of CCL23/TFAP4. Cell proliferation, invasion and human umbilical vein endothelial cell tube formation assays were conducted. The database revealed decreased expression of CCL23 in HCC and that it was commonly downregulated in HCC cell lines. TFAP4 expression was negatively correlated with CCL23. The overexpression of CCL23 inhibited the proliferation and invasion of Huh-7 cells, whereas TFAP4 blocked these effects. Similarly, the supernatant of CCL23-upregulated cells exhibited significantly lower tube formation potential, and low vascular endothelial growth factor A (VEGFA), VEGFRs expression compared with those of non-transfected Huh-7 cells, while TFAP4 plasmid co-transfected markedly increased these. Taken together, the present study suggests that CCL23 is expressed at low levels in HCC; it inhibits HCC cell proliferation, invasion and angiogenesis in vitro; and its action is negatively associated with and can be blocked by TFAP4.

TFAP4 promotes the growth of prostate cancer cells by upregulating FOXK1.

Transcription factor activating enhancer binding protein 4 (TFAP4) has been indicated to be correlated with the progression of various human malignancies. However, the effect and regulatory mechanism of TFAP4 in prostate cancer (PC) remain unclear. The protein and mRNA expression were detected by western blotting and RT-qPCR. TFAP4 was overexpressed or knocked down in PC cells. The viability, invasion and migration of PC cells were analyzed by CCK-8, Transwell and wound healing assays. The colony formation was also determined. TFAP4 expression was upregulated in PC patients and cells; high TFAP4 expression predicted poor prognosis, and was associated with a range of clinicopathological features, including metastasis, clinical stage and Gleason score. Moreover, overexpression of TFAP4 promoted cell viability, migration, and invasion in vitro, whereas knockdown of TFAP4 revealed the opposite results. TFAP4 also positively regulated forkhead box K1 (FOXK1) expression. In addition, overexpression of FOXK1 reversed the effects of TFAP4 knockdown on PC cells. These findings clarified the biologic significance of TFAP4 in PC progression and revealed an association between TFAP4 and FOXK1, thus providing a new potential target for clinical therapy of PC.

Silencing of the Long Non-Coding RNA TTN-AS1 Attenuates the Malignant Progression of Osteosarcoma Cells by Regulating the miR-16-1-3p/TFAP4 Axis.

ObjectivesOsteosarcoma (OS) is a type of bone malignancy. This study attempted to explore the effect of long non-coding RNA TTN-AS1 (TTN-AS1) on OS and to determine its molecular mechanisms.MethodsThe expression of TTN-AS1, microRNA-16-1-3p (miR-16-1-3p), and transcription factor activating enhancer binding protein 4 (TFAP4) in OS was assessed using qRT-PCR. The OS cell proliferation, migration, and invasion were measured using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), wound-healing, and transwell assays. N-cadherin and MMP-2 protein level was determined with western blot. Interactions between TTN-AS1 and miR-16-1-3p or TFAP4 and miR-16-1-3p were confirmed using the dual-luciferase reporter assay. Additionally, an OS xenograft tumor model was constructed to assess the effect of TTN-AS1 on tumor growth.ResultsTTN-AS1 and TFAP4 expression was increased in OS, while miR-16-1-3p expression was decreased. TTN-AS1 silencing restrained OS cell proliferation, migration, invasion, N-cadherin and MMP-2 protein expression, and hindered tumor growth. MiR-16-1-3p overexpression retarded the malignant behavior of OS cells. TTN-AS1 played a carcinostatic role by down-regulating miR-16-1-3p in the OS cells. Moreover, miR-16-1-3p inhibition or TFAP4 elevation weakened the suppressive effect of TTN-AS1 silencing on OS cell tumor progression.ConclusionTTN-AS1 promoted the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of OS cells via mediating the miR-16-1-3p/TFAP4 axis. TTN-AS1 may be a critical target for improving OS.

MicroRNA-124 suppresses the invasion and proliferation of breast cancer cells by targeting TFAP4.

MicroRNA (miRNA/miR)-124 is widely accepted as the suppressor of different tumors. The present study aimed to improve understanding of the potential role of miR-124 in breast cancer. The gene expression profile change derived from the overexpression of miR-124 was investigated using RNA sequencing and bioinformatics analysis of the breast cancer cell line SKBR3. The results demonstrated that the gene expression profile of SKBR3 cells significantly changed. In addition, the transcription factor activating enhancer-binding protein 4 (TFAP4) gene was identified among the top 10 differentially expressed genes, and was identified as a novel target gene of miR-124 using a dual-luciferase reporter assay. TFAP4 knockdown in notably impaired SKBR3 cell migration and proliferation, which was consistent with decreasing migration and proliferation ability following overexpression of miR-124. Taken together, these results suggest that overexpression of miR-124 can suppress the migration and proliferation of SKBR3 cells by tarsgeting TFAP4. Thus, TFAP4 may act as a novel therapeutic target of breast cancer.

Transcription Factor AP4 Mediates Cell Fate Decisions: To Divide, Age, or Die.

Simple SummaryHere, we review the literature on Activating Enhancer-Binding Protein 4 (AP4)/transcription factor AP4 (TFAP4) function and regulation and its role in cancer. Elevated expression of AP4 was detected in tumors of various organs and is associated with poor patient survival. AP4 is encoded by a Myc target gene and mediates cell fate decisions by regulating multiple processes, such as cell proliferation, epithelial-mesenchymal transition, stemness, apoptosis, and cellular senescence. Thereby, AP4 may be critical for tumor initiation and progression. In this review article, we summarize published evidence showing how AP4 functions as a transcriptional activator and repressor of a plethora of direct target genes in various physiological and pathological conditions. We also highlight the complex interactions of AP4 with c-Myc, N-Myc, p53, lncRNAs, and miRNAs in feed-back loops, which control AP4 levels and mediate AP4 functions. In the future, a better understanding of AP4 may contribute to improved prognosis and therapy of cancer.Activating Enhancer-Binding Protein 4 (AP4)/transcription factor AP4 (TFAP4) is a basic-helix-loop-helix-leucine-zipper transcription factor that was first identified as a protein bound to SV40 promoters more than 30 years ago. Almost 15 years later, AP4 was characterized as a target of the c-Myc transcription factor, which is the product of a prototypic oncogene that is activated in the majority of tumors. Interestingly, AP4 seems to represent a central hub downstream of c-Myc and N-Myc that mediates some of their functions, such as proliferation and epithelial-mesenchymal transition (EMT). Elevated AP4 expression is associated with progression of cancer and poor patient prognosis in multiple tumor types. Deletion of AP4 in mice points to roles of AP4 in the control of stemness, tumor initiation and adaptive immunity. Interestingly, ex vivo AP4 inactivation results in increased DNA damage, senescence, and apoptosis, which may be caused by defective cell cycle progression. Here, we will summarize the roles of AP4 as a transcriptional repressor and activator of target genes and the contribution of protein and non-coding RNAs encoded by these genes, in regulating the above mentioned processes. In addition, proteins interacting with or regulating AP4 and the cellular signaling pathways altered after AP4 dysregulation in tumor cells will be discussed.

An integrated pan-cancer analysis of TFAP4 aberrations and the potential clinical implications for cancer immunity.

Studies have shown that transcription factor activating enhancer binding protein 4 (TFAP4) plays a vital role in multiple types of cancer; however, the TFAP4 expression profile is still unknown, as is its value within the human pan‐cancer analysis. The present study comprehensively analysed TFAP4 expression patterns from 33 types of malignancies, along with the significance of TFAP4 for prognosis prediction and cancer immunity. TFAP4 displayed inconsistent levels of gene expression across the diverse cancer cell lines, and displayed abnormal expression within most malignant tumours, which closely corresponded to overall survival. More importantly, the TFAP4 level was also significantly related to the degree of tumour infiltration. TFAP4 was correlated using gene markers in tumour‐infiltrating immune cells and immune scores. TFAP4 expression was correlated with tumour mutation burden and microsatellite instability in different cancer types, and enrichment analyses identified TFAP4‐associated terms and pathways. The present study comprehensively analysed the expression of TFAP4 across 33 distinct types of cancers, which revealed that TFAP4 may possibly play a vital role during cancer formation and development. TFAP4 is related to differing degrees of immune infiltration within cancers, which suggests the potential of TFAP4 as an immunotherapy target in cancers. Our study demonstrated that TFAP4 plays an important role in tumorigenesis as a prognostic biomarker, which highlights the possibility of developing new targeted treatments.

Expression of MiR-608 in Nonsmall Cell Lung Cancer and Molecular Mechanism of Apoptosis and Migration of A549 Cells.

Objective This Work is aimed at exploring the effect of microRNA (MiR)-608 on the function of nonsmall cell lung cancer (NSCLC) A549 cells and related mechanisms. Methods Blood samples of 106 NSCLC patients (experimental group) as well as 124 normal people (control group) were selected for relevant investigation. Polymerase chain reaction (PCR) as well as DNA sequencing was used to determine the genotyping of the MiR-608 rs4919510 polymorphism. MiR-608 expression in cells was detected by real-time PCR technology. Western blotting was used to detect changes in protein levels. NSCLC tissues as well as adjacent tissues were explored in 33 patients undergoing surgery. Results MiR-608 rs4919510 does not influence the incidence of NSCLC patients. In addition, MiR-608 expression was downregulated in the tumor tissue of NSCLC patients, while the transcription factor activating enhancer-binding protein 4 (TFAP4) expression was upregulated. MiR-608 promotes DOX- (Doxorubicin-) induced apoptosis by negatively regulating TFAP4 expression in NSCLC tissue. TFAP4 can significantly inhibit the migration of A549 cells. Conclusion The findings in this investigation can contribute to the effective treatment of NSCLC patients. Also, the investigation can provide some theoretical support for the application of new targets for NSCLC treatment.

MicroRNA-608 Promotes Apoptosis in Non-Small Cell Lung Cancer Cells Treated With Doxorubicin Through the Inhibition of TFAP4.

Lung cancer is the most commonly diagnosed type of cancer and the leading cause of cancer-associated death worldwide. MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules of ∼20–25 nucleotides in length. Single nucleotide polymorphisms are a class of genetic variation in the human genome, which when present in miRNA genes are associated with the risk of developing cancer. This study aimed to identify whether the miRNA (miR)-608 polymorphism rs4919510 influenced the incidence of lung cancer, and to explore the underlying mechanisms of miR-608 in the pathogenesis of the disease. A total of 37 patients with non-small cell lung cancer (NSCLC) were selected to determine the expression levels of miR-608; 96 NSCLC patients and 136 cancer-free healthy controls were recruited to determine the incidence of miR-608 rs4919510 in lung cancer patients. Additionally, the impact of miR-608 on the expression of predicted target genes, cell migration, viability, proliferation, and apoptosis was also assessed. We found that the presence of miR-608 rs4919510 did not affect the susceptibility of patients to NSCLC or the maturation of miR-608. miR-608 expression levels were found to be downregulated in NSCLC tissues. Furthermore, overexpression of miR-608 promoted doxorubicin-induced apoptosis in NSCLC cell lines A549 and HCC4006 by inhibiting the expression of transcription factor activating enhancer-binding protein 4 (TFAP4), and high expression levels of TFAP4 were observed in NSCLC tissues. Therefore, our results may provide valuable insights for the chemotherapeutical treatment of NSCLC.

TFAP4 Promotes Hepatocellular Carcinoma Invasion and Metastasis via Activating the PI3K/AKT Signaling Pathway.

Transcription factor activating enhancer binding protein 4 (TFAP4) is established as a regulator of human cancer genesis and progression. Overexpression of TFAP4 indicates poor prognosis in various malignancies. The current study was performed to quantify TFAP4 expression as well as to further determine its potential prognostic value and functional role in patients with hepatocellular carcinoma (HCC). We identified that the expression of TFAP4 mRNA in 369 tumor tissues was higher than that in 160 normal liver tissues. Upregulated TFAP4 expressions were discovered in HCC cell lines compared to the healthy liver cell line, and similarly, the levels of TFAP4 were higher in tumor tissues than its expression in paratumor tissues. High mRNA and protein expression of TFAP4 was associated with worse overall survival (OS) and disease-free survival (DFS). Additionally, TFAP4 expression emerged as a risk factor independently affecting both OS and DFS of HCC patients. Functional studies demonstrated that TFAP4 increased HCC cell migration and invasion. Further investigations found that TFAP4 promotes invasion and metastasis by inducing epithelial-mesenchymal transition (EMT) and regulating MMP-9 expression via activating the PI3K/AKT signaling pathway in HCC. In conclusion, our study demonstrated that TFAP4 is a valuable prognostic biomarker in determining the likelihood of tumor metastasis and recurrence, as well as the long-term survival rates of HCC patients. Exploring the regulatory mechanism of TFAP4 will also contribute to the development of new prevention and treatment strategies for HCC.

Elevated TFAP4 regulates lncRNA TRERNA1 to promote cell migration and invasion in gastric cancer.

Cancer cell invasion and metastasis are the leading causes of the high mortality rates in patients with malignant tumors. There is accumulating evidence to indicate that dysregulated long non‑coding RNAs (lncRNAs) may be involved in the progression of tumor invasion and metastasis. However, the regulatory mechanisms of the aberrant expression of lncRNAs remain largely unknown, although the roles of lncRNAs as drivers of tumor suppressive and oncogenic functions have appeared in prevalent cancer types in recent years. In the present study, we identified that the transcription factor, activating enhancer‑binding protein4 (TFAP4), acts as a key modulator of translation regulatory long non‑coding RNA 1(TRERNA1), which has been proven to promote the invasion and metastasis of gastric cancer (GC) cells. We revealed that TRERNA1 was upregulated in gastric carcinogenesis and promoted cell migration and invasion in GC. Using bioinformatics analysis, we observed that there were several potential binding sites of TFAP4 in the promoter region of TRERNA1. The knockdown of TFAP4 significantly reduced the expression level of TRERNA1, whereas the ectopic expression of TFAP4 significantly increased the expression level of TRERNA1 in GC cell lines. Dual luciferase reporter assay combined with chromatin immunoprecipitation (ChIP) revealed that TFAP4 specifically regulated the transcriptional activity of TRERNA1 by binding to the E‑box motifs in the TRERNA1 promoter. In addition, there was a positive correlation between the TFAP4 and TRERNA1 expression level in clinical GC cases, which also indicated that TFAP4 can directly modulate the expression of TRERNA1. In the present study, we provide a novel potential therapeutic target and strategy for GC.

Transcription factor AP-4 promotes tumorigenic capability and activates the Wnt/β-catenin pathway in hepatocellular carcinoma.

It has been reported that the transcription factor activating enhancer-binding protein 4 (TFAP4) is upregulated and associated with an aggressive phenotype in several cancers. However, the precise mechanisms underlying the oncogenic role of TFAP4 remain largely unknown.Methods: TFAP4 expression levels in hepatocellular carcinoma (HCC) cells and tissues were detected by quantitative real-time PCR (qPCR) and immunohistochemistry (IHC). In vitro and in vivo assays were performed to investigate the oncogenic function of TFAP4 in the tumor-initiating cell (TIC)-like phenotype and the tumorigenic capability of HCC cells. Luciferase reporter and chromatin immunoprecipitation (ChIP)-qPCR assays were performed to determine the underlying mechanism of TFAP4-mediated HCC aggressiveness.Results: TFAP4 was markedly upregulated in human HCC, and was associated with significantly poorer overall and relapse-free survival in patients with HCC. Furthermore, we found that overexpression of TFAP4 significantly enhanced, whereas silencing TFAP4 inhibited, the tumor sphere formation ability and proportion of side-population cells in HCC cells in vitro, and ectopic TFAP4 enhanced the tumorigenicity of HCC cells in vivo. Mechanistically, we demonstrated that TFAP4 played an important role in activating Wnt/β-catenin signaling by directly binding to the promoters of DVL1 (dishevelled segment polarity protein 1) and LEF1 (lymphoid enhancer binding factor 1).Conclusions: Our results provide new insight into the mechanisms underlying hyperactivation of the Wnt/β-catenin pathway in HCC, as well the oncogenic ability of TFAP4 to enhance the tumor-forming ability of HCC cells.

Overexpression of transcription factor activating enhancer binding protein 4 (TFAP4) predicts poor prognosis for colorectal cancer patients.

Transcription factor activating enhancer binding protein 4 (TFAP4) is an important regulator in the genesis and progression of human cancers. Overexpression of TFAP4 has been found to be correlated with several malignancies. The present study assessed the clinical importance of TFAP4 in colorectal cancer (CRC). First, immunohistochemistry was used to analyze TFAP4 expression and the association of TFAP4 expression with clinicopathological features on a tissue microarray containing 208 CRC patients. The results revealed that TFAP4 protein expression was significantly upregulated in CRC tissues compared with that in normal colon tissues (P<0.001). Of note, statistical analysis revealed that TFAP4 expression was significantly correlated with a high pathological grade (P=0.034), advanced clinical stage (P=0.024), enhanced tumor invasion (P=0.002) and lymph node metastasis (P=0.041). In addition, the Cancer Genome Atlas dataset further validated that TFAP4 mRNA levels were increased in CRC with advanced clinical stage (P=0.026), lymph node metastasis (P=0.018) and vascular invasion (P=0.046). Kaplan-Meier survival analysis demonstrated that CRC patients with high TFAP4 expression had shorter overall survival compared with those with low TFAP4 expression (P=0.011). Importantly, overexpression of TFAP4 was a valuable independent prognostic factor for CRC patients (hazard ratio, 8.200; 95% confidence interval, 1.838–36.591; P=0.006). In summary, TFAP4 may have an important role in CRC progression and upregulation of TFAP4 may be a predictor of poor prognosis for CRC patients.

MYCN promotes neuroblastoma malignancy by establishing a regulatory circuit with transcription factor AP4.

Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4) has been reported to be a direct transcriptional target of MYC. We show for the first time that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome. siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells led to inhibition of cell proliferation and migration. Chromatin immunoprecipitation assay demonstrated that TFAP4 expression is positively regulated by MYCN. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study suggests a regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying potential therapeutic targets for aggressive forms of this disease.

Abstract 2450: MYCN and TFAP4 promote neuroblastoma malignancy by cooperating in the regulation a subset of target genes involved in cancer cell growth and metastasis

Abstract Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4), which plays important roles in cancer progression, has been reported to be a direct transcriptional target of MYC. In this study, we have shown that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome and furthermore that siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells impaired migration and colony formation, and led to an increased proportion of cells in G1/S phase of the cell cycle. Chromatin immunoprecipitation and luciferase reporter assays demonstrated that TFAP4 expression is positively regulated by MYCN through direct promoter binding. In addition, when MYCN was overexpressed in neuroblastoma cells, TFAP4 was required for the observed increase in cell migration. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study unveils a complex regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying relevant therapeutic targets for aggressive forms of this disease. Citation Format: Chengyuan Xue, Denise M. Yu, Samuele Gherardi, Jessica Koach, Giorgio Milazzo, Laura Gamble, Bing Liu, Amanda Russell, Tao Liu, Belamy B. Cheung, Glenn M. Marshall, Giovanni Perini, Michelle Haber, Murray D. Norris. MYCN and TFAP4 promote neuroblastoma malignancy by cooperating in the regulation a subset of target genes involved in cancer cell growth and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2450.

Proteasome-dependent Degradation of Transcription Factor Activating Enhancer-binding Protein 4 (TFAP4) Controls Mitotic Division

TFAP4, a basic helix-loop-helix transcription factor that regulates the expression of a multitude of genes involved in the regulation of cellular proliferation, stemness, and epithelial-mesenchymal transition, is up-regulated in colorectal cancer and a number of other human malignancies. We have found that, during the G2 phase of the cell division cycle, TFAP4 is targeted for proteasome-dependent degradation by the SCF(βTrCP) ubiquitin ligase. This event requires phosphorylation of TFAP4 on a conserved degron. Expression of a stable TFAP4 mutant unable to interact with βTrCP results in a number of mitotic defects, including chromosome missegregation and multipolar spindles, which eventually lead to the activation of the DNA damage response. Our findings reveal that βTrCP-dependent degradation of TFAP4 is required for the fidelity of mitotic division.

Complementary Quantitative Proteomics Reveals that Transcription Factor AP-4 Mediates E-box-dependent Complex Formation for Transcriptional Repression of HDM2 >

Transcription factor activating enhancer-binding protein 4 (AP-4) is a basic helix-loop-helix protein that binds to E-box elements. AP-4 has received increasing attention for its regulatory role in cell growth and development, including transcriptional repression of the human homolog of murine double minute 2 (HDM2), an important oncoprotein controlling cell growth and survival, by an unknown mechanism. Here we demonstrate that AP-4 binds to an E-box located in the HDM2-P2 promoter and represses HDM2 transcription in a p53-independent manner. Incremental truncations of AP-4 revealed that the C-terminal Gln/Pro-rich domain was essential for transcriptional repression of HDM2. To further delineate the molecular mechanism(s) of AP-4 transcriptional control and its potential implications, we used DNA-affinity purification followed by complementary quantitative proteomics, cICAT and iTRAQ labeling methods, to identify a previously unknown E-box-bound AP-4 protein complex containing 75 putative components. The two labeling methods complementarily quantified differentially AP-4-enriched proteins, including the most significant recruitment of DNA damage response proteins, followed by transcription factors, transcriptional repressors/corepressors, and histone-modifying proteins. Specific interaction of AP-4 with CCCTC binding factor, stimulatory protein 1, and histone deacetylase 1 (an AP-4 corepressor) was validated using AP-4 truncation mutants. Importantly, inclusion of trichostatin A did not alleviate AP-4-mediated repression of HDM2 transcription, suggesting a previously unidentified histone deacetylase-independent repression mechanism. In contrast, the complementary quantitative proteomics study suggested that transcription repression occurs via coordination of AP-4 with other transcription factors, histone methyltransferases, and/or a nucleosome remodeling SWI.SNF complex. In addition to previously known functions of AP-4, our data suggest that AP-4 participates in a transcriptional-regulating complex at the HDM2-P2 promoter in response to DNA damage.