Transcription Factor AP-2 Gamma Research Articles

Transcriptional mechanism of E2F1/TFAP2C/NRF1 in regulating KANK2 gene in nephrotic syndrome

The mortality rate linked with nephrotic syndrome (NS) is quite high. The renal tubular injury influences the response of NS patients to steroid treatment. KN motif and ankyrin repeat domains 2 (KANK2) regulates actin polymerization, which is required for renal tubular cells to maintain their function. In this study, we found that the levels of KANK2 in patients with NS were considerably lower than those in healthy controls, especially in NS patients with acute kidney injury (AKI). To get a deeper understanding of the KANK2 transcriptional control mechanism, the core promoter region of the KANK2 gene was identified. KANK2 was further found to be positively regulated by E2F Transcription Factor 1 (E2F1), Transcription Factor AP-2 Gamma (TFAP2C), and Nuclear Respiratory Factor 1 (NRF1), both at mRNA and protein levels. Knocking down E2F1, TFAP2C, or NRF1 deformed the cytoskeleton of renal tubular cells and reduced F-actin content. EMSA and ChIP assays confirmed that all three transcription factors could bind to the upstream promoter transcription site of KANK2 to transactivate KANK2 in renal tubular epithelial cells. Our study suggests that E2F1, TFAP2C, and NRF1 play essential roles in regulating the KANK2 transcription, therefore shedding fresh light on the development of putative therapeutic options for the treatment of NS patients.

Transcription factor AP-2 gamma affects porcine early embryo development by regulating epigenetic modification

Research questionWhat is the role and mechanism of action of transcription factor AP-2 gamma (TFAP2C) in porcine early embryo development? DesignTFAP2C siRNA were injected into porcine oocytes, which subsequently underwent IVF. Different stages of embryos were collected for RNA sequencing, quantitative polymerase chain reaction, immunofluorescence staining to explore the affects in gene expression and epigenetic modification. Porcine fetal fibroblasts were transfected with siRNA, and cells were collected for chromatin immunoprecipitation and dual luciferase reporter assays. ResultsThe deficiency of TFAP2C led to disorders in early embryonic development; 1208 genes were downregulated and 792 genes were upregulated in TFAP2C knockdown (TFAP2C-KD) embryos. The expression of epigenetic modification enzymes KDM5B, SETD2 were significantly elevated in the TFAP2C-KD group (P < 0.001). Meanwhile, the modification levels of H3K4me3 and H3K4me2 were significantly decreased (P = 0.0021, P = 0.0029), and H3K36me3 and DNA methylation were significantly increased in TFAP2C-KD group (P = 0.0045, P = 0.0025). DNMT1 was mainly expressed in nuclei in the TFAP2C-KD group (P = 0.0103). In addition, TFAP2C could bind to the promoter region of SETD2, and the mutation of the TFAP2C binding site resulted in increased activity of SETD2 promoter (P < 0.001). ConclusionsThe knockdown of TFAP2C affects early embryonic development by regulating histone modification and DNA methylation.

Transcription factor AP-2 gamma/Krüppel-like factor 10 axis is involved in miR-3656-related dysfunction of endothelial cells in hypertension.

Dysfunction of endothelial cells links to microvascular rarefaction, reflecting the pathogenesis of hypertension. Our previous studies found that miR-3656 reduces nitric oxide generation and von Willebrand factor (vWF) cleavage, thereby retarding blood flow and potentially increasing blood pressure. In this paper, we investigated mechanism of transcription regulation contributing to miR-3656-damaged endothelial cells in hypertension. The effects of miR-3656 on function of endothelial cells were analyzed on the basis of proliferation, migration, tube formation, and apoptosis. The mRNA level and protein level of genes were examined using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Dual-luciferase reporter assay was performed to confirm the binding between miR-3656 and 3' untranslated region (UTR) of transcription factor AP-2 gamma ( TFAP2C ). The binding between TFAP2C and the promoter region of Krüppel-like factor 10 ( KLF10 ) was confirmed by chromatin immunoprecipitation-qPCR assay. miR-3656 impaired the cell proliferation, migration, tube formation, and apoptosis of endothelial cells. miR-3656 inhibited the expression of TFAP2C by directly targeting 3'UTR of TFAP2C ; moreover, miR-3656-induced injury of endothelial cells was rescued by TFAP2C overexpression. Furthermore, downregulated TFAP2C decreased KLF10 expression by binding to KLF10 promoter region, and upregulated KLF10 reversed the effects of silencing TFAP2C on endothelial cells. These inhibitory processes led to interference of miR-3656 to KLF10-promoted function of endothelial cells. TFAP2C/KLF10 axis is involved in miR-3656-related dysfunction of endothelial cells in hypertension. The 3'UTR of TFAP2C and KLF10 promoter region are the hubs of the TFAP2C/KLF10 axis.

TFAP2C Knockdown Sensitizes Bladder Cancer Cells to Cisplatin Treatment via Regulation of EGFR and NF-κB.

Simple SummaryBladder cancer (BCa) is considered one of the most common neoplasms of the urology system. Cisplatin-based chemotherapy has been the primary treatment for patients with advanced or metastatic BCa. Nevertheless, cisplatin resistance often limits the treatment of bladder cancer. We expect to find approaches to improve the therapeutic efficacy of cisplatin in bladder cancer. In recent years, many studies have shown that transcription factor AP-2 gamma (TFAP2C) acts as a key player in cancer development and and its expression level is closely related to the sensitivity of tumors to cisplatin. Our study investigated whether TFAP2C affects the sensitivity of BCa cells to cisplatin and the possible mechanisms. We found that TFAP2C expression was significantly upregulated in most BCa tissues compared to adjacent normal tissues. The present study confirmed that TFAP2C knockdown enhanced the anti-tumor effects of cisplatin by decreasing cisplatin-induced activation levels of epidermal growth factor receptor (EGFR) and nuclear factor kappaB (NF-κB). Specifically, this study provides a novel approach to improve the efficacy of cisplatin.Cisplatin is the first-line chemotherapy for advanced or metastatic bladder cancer. Nevertheless, approximately half of patients with BCa are insensitive to cisplatin therapy or develop cisplatin resistance during the treatment process. Therefore, it is especially crucial to investigate ways to enhance the sensitivity of tumor cells to cisplatin. Transcription factor AP-2 gamma (TFAP2C) is involved in cancer development and chemotherapy sensitivity. However, its relationship with chemotherapy has not been studied in BCa. In this study, we aimed to investigate the therapeutic potential of TFAP2C in human BCa. Results based on TCGA (The Cancer Genome Atlas), GTEx (The Genotype-Tissue Expression) and GEO (Gene Expression Omnibus) data showed that TFAP2C expression was upregulated in BCa tissues and that its high expression was associated with poor prognosis. Meanwhile, we demonstrated the overexpression of TFAP2C in BCa clinical specimens. Subsequently, in vitro, we knocked down TFAP2C in BCa cells and found that TFAP2C knockdown further increased cell cycle arrest and apoptosis caused by cisplatin. In addition, the inhibitory effect of cisplatin on BCa cell migration and invasion was enhanced by TFAP2C knockdown. Our data indicated that cisplatin increased epidermal growth factor receptor (EGFR) and nuclear factor-kappaB (NF-κB) activation levels, but TFAP2C knockdown suppressed this effect. Finally, in vivo data further validated these findings. Our study showed that TFAP2C knockdown affected the activation levels of EGFR and NF-κB and enhanced the anti-tumor effects of cisplatin in vivo and in vitro. This provides a new direction to improve the efficacy of traditional cisplatin chemotherapy.

LncRNA MILIP links YBX1 to translational activation of Snai1 and promotes metastasis in clear cell renal cell carcinoma

BackgroundDistant metastasis is the major cause of clear cell renal cell carcinoma (ccRCC)-associated mortality. However, molecular mechanisms involved in ccRCC metastasis remain to be fully understood. With the increasing appreciation of the role of long non-coding RNAs (lncRNAs) in cancer development, progression, and treatment resistance, the list of aberrantly expressed lncRNAs contributing to ccRCC pathogenesis is expanding rapidly.MethodsBioinformatics analysis was carried out to interrogate publicly available ccRCC datasets. In situ hybridization and qRT-PCR assays were used to test lncRNA expression in human ccRCC tissues and cell lines, respectively. Chromatin immunoprecipitation and luciferase reporter assays were used to examine transcriptional regulation of gene expression. Wound healing as well as transwell migration and invasion assays were employed to monitor ccRCC cell migration and invasion in vitro. ccRCC metastasis was also examined using mouse models in vivo. RNA pulldown and RNA immunoprecipitation were performed to test RNA–protein associations, whereas RNA-RNA interactions were tested using domain-specific chromatin isolation by RNA purification.ResultsMILIP expression was upregulated in metastatic compared with primary ccRCC tissues. The increased MILIP expression in metastatic ccRCC cells was driven by the transcription factor AP-2 gamma (TFAP2C). Knockdown of MILIP diminished the potential of ccRCC cell migration and invasion in vitro and reduced the formation of ccRCC metastatic lesions in vivo. The effect of MILIP on ccRCC cells was associated with alterations in the expression of epithelial-to-mesenchymal transition (EMT) hallmark genes. Mechanistically, MILIP formed an RNA-RNA duplex with the snail family transcriptional repressor 1 (Snai1) mRNA and bound to Y-box binding protein 1 (YBX1). This promoted the association between the YBX1 protein and the Snai1 mRNA, leading to increased translation of the latter. Snai1 in turn played an important role in MILIP-driven ccRCC metastasis.ConclusionsThe TFAP2C-responsive lncRNA MILIP drives ccRCC metastasis. Targeting MILIP may thus represent a potential avenue for ccRCC treatment.

TFAP2C-Mediated lncRNA PCAT1 Inhibits Ferroptosis in Docetaxel-Resistant Prostate Cancer Through c-Myc/miR-25-3p/SLC7A11 Signaling.

Recent evidence has shown that the induction of ferroptosis is a new therapeutic strategy for advanced prostate cancer (PCa) when used as a monotherapy or in combination with second-generation antiandrogens. However, whether ferroptosis inducers are effective against docetaxel-resistant PCa remains unclear. In addition, the biological role and intrinsic regulatory mechanisms of long noncoding RNAs (lncRNAs) in ferroptosis and chemoresistance are not well understood. In this study, we established two acquired docetaxel-resistant PCa cell lines and found that docetaxel-resistant PCa cells developed tolerance toward ferroptosis. In addition, dysregulated lncRNAs in drug-resistant and -sensitive PCa cells were identified by RNA sequencing, and we identified that prostate cancer-associated transcript 1 (PCAT1) was highly expressed in the docetaxel-resistant PCa cell lines and clinical samples. Overexpression of PCAT1 inhibited ferroptosis and increased docetaxel resistance, which could be attenuated by PCAT1 knockdown. Furthermore, we revealed that PCAT1 inhibited ferroptosis by activating solute carrier family 7-member 11 (SLC7A11) expression via reducing iron accumulation and subsequent oxidative damage. Mechanistically, we demonstrated that PCAT1 interacted with c-Myc and increased its protein stability using nucleotides 1093-1367 of PCAT1 and 151-202 amino acids of c-Myc protein, thereby transcriptionally promoting SLC7A11 expression. In addition, PCAT1 facilitated SLC7A11 expression by competing for microRNA-25-3p. Finally, transcription factor AP-2 gamma (TFAP2C) activated PCAT1 expression at the transcriptional level to reduce ferroptosis susceptibility and enhance chemoresistance. Collectively, our findings demonstrated that TFAP2C-induced PCAT1 promotes chemoresistance by blocking ferroptotic cell death through c-Myc/miR-25-3p/SLC7A11 signaling.

Interaction of transcription factor AP-2 gamma with proto-oncogene PELP1 promotes tumorigenesis by enhancing RET signaling.

A significant proportion of estrogen receptor-positive (ER+) breast cancer (BC) initially responds to endocrine therapy but eventually evolves into therapy-resistant BC. Transcription factor AP-2 gamma (TFAP2C) is a known regulator of ER activity, and high expression of TFAP2C is associated with a decreased response to endocrine therapies. PELP1 is a nuclear receptor coregulator, commonly overexpressed in BC, and its levels are correlated with poorer survival. In this study, we identified PELP1 as a novel interacting protein of TFAP2C. RNA-seq analysis of PELP1 knockdown BC cells followed by transcription factor motif prediction pointed to TFAP2C being enriched in PELP1-regulated genes. Gene set enrichment analysis (GSEA) revealed that the TFAP2C-PELP1 axis induced a subset of common genes. Reporter gene assays confirmed PELP1 functions as a coactivator of TFAP2C. Mechanistic studies showed that PELP1-mediated changes in histone methylation contributed to increased expression of the TFAP2C target gene RET. Furthermore, the TFAP2C-PELP1 axis promoted the activation of the RET signaling pathway, which contributed to downstream activation of AKT and ERK pathways in ER+ BC cells. Concomitantly, knockdown of PELP1 attenuated these effects mediated by TFAP2C. Overexpression of TFAP2C contributed to increased cell proliferation and therapy resistance in ER+ BC models, while knockdown of PELP1 mitigated these effects. Utilizing ZR75-TFAP2C xenografts with or without PELP1 knockdown, we provided genetic evidence that endogenous PELP1 is essential for TFAP2C-driven BC progression invivo. Collectively, our studies demonstrated that PELP1 plays a critical role in TFAP2C transcriptional and tumorigenic functions in BC and blocking the PELP1-TFAP2C axis could have utility for treating therapy resistance.

A novel xeno-organoid approach: exploring the crosstalk between human iPSC-derived PGC-like and rat testicular cells

Specification of germ cell-like cells from induced pluripotent stem cells has become a clinically relevant tool for research. Research on initial embryonic processes is often limited by the access to foetal tissue, and in humans, the molecular events resulting in primordial germ cell (PGC) specification and sex determination remain to be elucidated. A deeper understanding of the underlying processes is crucial to describe pathomechanisms leading to impaired reproductive function. Several protocols have been established for the specification of human pluripotent stem cell towards early PGC-like cells (PGCLC), currently representing the best model to mimic early human germline developmental processes in vitro. Further sex determination towards the male lineage depends on somatic gonadal cells providing the necessary molecular cues. By establishing a culture system characterized by the re-organization of somatic cells from postnatal rat testes into cord-like structures and optimizing efficient PGCLC specification protocols, we facilitated the co-culture of human germ cell-like cells within a surrogate testicular microenvironment. Specified conditions allowed the survival of rat somatic testicular and human PGCLCs for 14 days. Human cells maintained the characteristic expression of octamer-binding transcription factor 4, SRY-box transcription factor 17, and transcription factor AP-2 gamma and were recovered from the xeno-organoids by cell sorting. This novel xeno-organoid approach will allow the in vitro exploration of early sex determination of human PGCLCs.

Abstract 4371: PELP1-TFAP2C crosstalk promotes endocrine resistance in breast cancer cells

Abstract Background: A significant proportion of estrogen receptor positive (ER+) breast cancer (BC) initially respond to antiestrogens or aromatase inhibitors but become therapy resistant-BC. Development of effective therapies for endocrine therapy resistant BC represent the highest unmet need. PELP1 is a nuclear receptor coregulator, commonly overexpressed in BC, contributes to therapy resistance and correlate with poor survival. TFAP2C (transcription factor AP-2 gamma) is a known regulator of ER activity and high expression of TFAP2C is associated with a decreased response to Fulvestrant. The objective of this study is to examine the significance of PELP1-TFAP2C crosstalk in the development of breast cancer therapy resistance. Methods: We used yeast two-hybrid screen to identify PELP1 interacting transcription factors. Interaction of PELP1 and TFAP2C was confirmed by immunoprecipitation and GST pull down assays. Functional significance of the cross talk was tested using CellTiter-Glo, MTT and colony formation assays in the presence or absence of endocrine therapy. Mechanistic studies were conducted using shRNA, overexpression, Western blotting, reporter gene assays, RT-qPCR, ChIP-seq and RNA-seq analysis. Biological significance of PELP1 and TFAP2C in endocrine therapy resistance was examined using overexpression and under expression models of PELP1 and TFAP2C in multiple BC models including MCF7 and ZR75. Results: Yeast based screening of a mammary gland cDNA expression library using PELP1 as the bait identified TFAP2C as a novel interacting protein of PELP1. Immunoprecipitation assays using multiple BC cell lysates confirmed the interaction of PELP1 with TFAP2C. Using various deletions of PELP1, and by using GST pull down assays, we identified N-terminal 400-600aa region of PELP1 as the major interaction site for TFAP2C. Using RNA-seq of PELP1 knockdown BC model cells, we predicted TFAP2C as an enriched transcription factor in PELP1 regulated genes. The GSEA results from RNA-seq showed TFAP2C and PELP1 induce a subset of common genes. Reporter gene assays confirmed that PELP1 functions as a coactivator of TFAP2C. Mechanistic studies showed that TFAP2C activates both AKT and ERK pathways in ER+ cell lines, while knock down of PELP1 attenuated these effects. Overexpression of TFAP2C contributed to increased cell proliferation and endocrine therapy resistance in MCF7 and ZR75 models, while knock down of PELP1 attenuated these effects. Utilizing ZR75-TFAP2C xenograft models with WT PELP1 or PELP1 knock down, we provided genetic evidence that endogenous PELP1 is essential for TFAP2C drive n breast cancer progression in vivo. Conclusions: Collectively, our studies demonstrated that PELP1 functions as a coactivator of TFAP2C in modulating a set of ER target genes, TFAP2C functions as a transcription factor of PELP1 regulated genes and blocking the PELP1-TFAP2C axis could have therapeutic utility for treating therapy resistance. Supported by VA grant I01BX004545 (R.K. Vadlamudi) Citation Format: Junhao Liu, Suryavathi Viswanadhapalli, Mengxing Li, Uday P. Pratap, Weiwei Tang, Zexuan Liu, Yiliao Luo, Kristin A. Altwegg, Xiaonan Li, Gangadhara R. Sareddy, Rajeshwar R. Tekmal, Ratna K. Vadlamudi. PELP1-TFAP2C crosstalk promotes endocrine resistance in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4371.

MiR-29b-3p cooperates with miR-29c-3p to affect the malignant biological behaviors in T-cell acute lymphoblastic leukemia via TFAP2C/GPX1 axis

Mounting evidence has illustrated the tumor regulatory roles of microRNAs (miRNAs) in T-cell acute lymphoblastic leukemia (T-ALL), a malignant carcinoma originated from T-cell precursors. However, the possible regulation mechanisms underlying miR-29b/29c-3p in T-ALL have not been interrogated yet. The aim of our study was to probe the association and possible molecular mechanism of miR-29b/29c-3p and Glutathione Peroxidase 1 (GPX1), a predicted highly expressed gene in acute myeloid leukemia (LAML) tissues on the cancer genome atlas (TCGA) website. In our paper, it was observed that GPX1 was relatively overexpressed in T-ALL cells, compared with normal T cells. Loss-of-function assays demonstrated that GPX1 knockdown inhibited the proliferation and activated the apoptosis in T-ALL cells. Then miR-29b/29c-3p was confirmed to regulate GPX1 mRNA and protein expression via decreasing Transcription Factor AP-2 Gamma (TFAP2C) expression. In summary, miR-29b-3p and miR-29c-3p targeted TFAP2C so as to repress GPX1 transcription, thereafter inhibiting GPXA expression. In the end, rescue experiments proved the whole regulation mechanism of miR-29b/29c-3p in T-ALL. Overall, the miR-29b/29c-3p -TFAP2C-GPX1 axis helped us to have a better understanding of T-ALL pathogenesis.

TFAP2C-Activated MALAT1 Modulates the Chemoresistance of Docetaxel-Resistant Lung Adenocarcinoma Cells

Chemoresistance remains a great obstacle in effective lung adenocarcinoma (LUAD) treatment. Previously, we verified the role of microRNA-200b (miR-200b) in the formation of docetaxel (DTX)-resistant LUAD cells. This study aims to investigate the mechanism underlying the low level of miR-200b in DTX-resistant LUAD cells. The real-time reverse transcription (RT2) lncRNA PCR array system was applied to explore lncRNAs that potentially regulated miR-200b in DTX-resistant LUAD cells. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) contributed to the low miR-200b level in DTX-resistant LUAD cells. Functional assays were conducted to determine the role of MALAT1 in regulating the growth and metastasis of parental and DTX-resistant LUAD cells. Investigation revealed the mechanism of the competing endogenous RNA (ceRNA) pathway. MALAT1 regulated miR-200b by acting as a ceRNA. MALAT1 modulated the sensitivity of LUAD cells to DTX. E2F transcription factor 3 (E2F3) and zinc-finger E-box binding homeobox 1 (ZEB1) were two targets of miR-200b and mediated the function of MALAT1 in DTX-resistant LUAD cells. Transcription factor AP-2 gamma (TFAP2C) and ZEB1 activated the MALAT1 transcription. In conclusion, TFAP2C-activated MALAT1 modulated the chemoresistance of LUAD cells by sponging miR-200b to upregulate E2F3 and ZEB1. Our findings may provide novel therapeutic targets and perspectives for LUAD treatment.

TFAP2C promotes stemness and chemotherapeutic resistance in colorectal cancer via inactivating hippo signaling pathway

BackgroundAberrant expression of transcription Factor AP-2 Gamma (TFAP2C) has been reported to be implicated in malignant process of many cancers. The purpose of this study is to investigate the clinical significance and biological roles of TFAP2C in colorectal cancer (CRC).MethodsTFAP2C expression was evaluated by real-time PCR, Western blot and immunohistochemistry (IHC) respectively in clinical CRC tissues. Statistical analysis was performed to explore the correlation between TFAP2C expression and clinicopathological features, and overall and progression-free survival in CRC patients. In vitro and in vivo assays were performed to assess the biological roles of TFAP2C in CRC cells. Western blot, luciferase and Chromatin immunoprecipitation (ChIP) assays were used to identify the underlying pathway mediating the biological roles of TFAP2C in CRC.ResultsTFAP2C is robustly upregulated in CRC tissues and cells, and high expression of TFAP2C correlates with advanced clinicopathological features, poor prognosis and disease progression in CRC patients. Furthermore, upregulating TFAP2C enhances spheroids formation ability, the fraction of SP cells, expression of stem cell factors and the mitochondrial potential, and reduces the apoptosis induced by 5-fluorouracil in colorectal cancer cells in vitro, and promotes stemness and chemoresistance of CRC cells in vivo; while silencing TFAP2C yields an opposite effect. Importantly, downregulation of TFAP2C dramatically restores chemotherapeutic sensitivity of CRC cells to 5-FU in vivo. Our results further demonstrate that TFAP2C promotes stemness and chemoresistance of CRC cells to 5-FU by inhibiting Hippo signaling via transcriptionally upregulating ROCK1 and ROCK2 in CRC cells.ConclusionOur findings indicate that TFAP2C may serve as a novel prognostic factor in CRC patients, and a therapeutic target for the treatment of CRC, suggesting that silencing TFAP2C in combination with 5-FU may be an effective therapeutic strategy to improve survival in CRC patients.

S.12.05 - AP2gamma transcription factor as a modulator of hippocampal neurogenesis in an animal model of depression

The aim of this study was to determine the diagnostic performance of different diagnostic tests for histologic chorioamnionitis in patients at more than 20 weeks of gestation.A systematic search was carried out through MEDLINE, EMBASE, LILACS, CENTRAL and unpublished literature. Observational studies included with pregnant women (>20 weeks) with chorioamnionitis. The reference standard was the histopathological study of the placenta, umbilical cord and fetal membranes. Two independent researchers extracted data and performed a meta-analysis of diagnostic tests.Twenty-nine articles were included. The studies provided evidence in the form of maternal clinical and serological tests; tests of vaginal fluid, amniotic fluid or the umbilical cord; fetal monitoring and ultrasound tests. To assess the performance of maternal serum CRP, 13 studies were included, showing a combined sensitivity of 68.7% (95%CI 58%–77%) and a combined specificity of 77.1% (95%CI 67%–84%). Maternal leukocytosis was evaluated in four publications, showing a combined sensitivity of 51% (95%CI 40%–62%) and a combined specificity of 65% (95%CI 50%–78%).CRP and maternal leukocytosis, showed a low sensitivity and specificity. The sonographic evaluation of the fetal thymus is also more sensitive for the diagnosis of histologic chorioamnionitis than the fetal biophysical profile.

Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C

The epithelial-mesenchymal transition (EMT) regulator, Slug, plays multifaceted roles in controlling lung cancer progression, but its downstream targets and mechanisms in promoting lung cancer progression have not been well defined. In particular, the miRNAs downstream of Slug in non-small cell lung cancer (NSCLC) remain undetermined. Here, we report that miR-137 is downstream of the EMT regulator, Slug, in lung cancer cells. Slug binds directly to the E-box of the miR-137 promoter and up-regulates its expression in lung cancer cells. Knockdown of miR-137 abolished Slug-induced cancer invasion and migration, whereas upregulation of miR-137 was found to trigger lung cancer cell invasion and progression by direct suppressing TFAP2C (transcription factor AP-2 gamma). Clinical data showed that lung adenocarcinoma patients with low-level expression of Slug and miR-137 but high-level expression of TFAP2C experienced significantly better survival. miR-137 is a Slug-induced miRNA that relays the pro-metastatic effects of Slug by targeting TFAP2C. Our findings add new components to the Slug-mediated regulatory network in lung cancer, and suggest that Slug, miR-137, and TFAP2C may be useful prognostic markers in lung adenocarcinoma.

P.2.004 - The role of AP2gamma transcription factor in the modulation of adult glutamatergic neurogenesis in depression

P.2.004 - The role of AP2gamma transcription factor in the modulation of adult glutamatergic neurogenesis in depression

Mir-10a Regulates Autophagy and Cell Cycle in Normal Karyotype Acute Myeloid Leukaemia

Aim: Normal karyotype (NK) Acute Myeloid Leukemia (AML) accounts for approximately half of all AML cases. Almost two-thirds of these carry the Nucleophosmin 1(NPM1) gene mutation (NPM1c+). MicroRNAs (miRNA/miRs), short RNA molecules that act as post-transcriptional regulators of gene expression, have multiple roles in regulating key gene signalling networks in cancer, including in NK-AML. miR-10a is significantly overexpressed in NK-AML compared to normal bone marrow, particularly in those carrying the NPM1c+ mutation in which it is expressed at levels ~20-fold higher than in NPM1 wildtype (NPM1wt) NK-AML. We have previously shown that repression of miR-10a in the NPM1c+ AML cell line OCI-AML3 leads to inhibited proliferation and initiation of apoptosis. Here we aimed to further delineate the role of miR-10a in NK-AML.Results: miR-10a was shown to directly bind to and downregulate key members of the p53-mediated tumour suppressor gene network, including the CDKN1A (p21) inhibitor Transcription Factor AP2-gamma (TFAP2C) and Retinoblastoma (RB1) regulator Rb1-Inducible Coiled-Coil 1 (RB1C11). Inhibition of miR-10a resulted in significant repression of the cell cycle inhibitor p21, upregulation of RB1CC1, decrease in the expression of autophagy marker LC3-II, and the induction of apoptosis in serum-deprived cells. The individual modulation of these miR-10a target genes via siRNA (CDKN1A) and overexpression (RB1CC1) had similar effects, triggering apoptosis and also sensitizing OCI-AML3 cells to chemotherapy agents cytarabine and daunorubicin in transfected cells.Due to miR-10a’s potential role in regulating the central p53-p21-Rb1 tumour suppressor signalling axis in AML, miR-10a levels were quantified by QPCR in primary NK-AML patient samples and correlated to clinical outcome. Analysis of 61 patients from the German SAL AML2003 randomized clinical trial who received post-induction chemotherapy + allogeneic bone marrow transplantation (BMT) in a risk-adapted manner demonstrated that high miR-10a expression (> median expression) was associated with significantly poorer relapse-free survival (hazard ratio (HR) = 5.6, p < 0.001) compared those patient samples with low miR-10a expression. This is consistent with these patients being more resistant to post-induction chemotherapy and/or upfront BMT, and suggests that these patients may benefit from alternate therapy.To investigate the mechanism of miR-10a dysregulation in NPM1c+ AML cells, we assayed methylation of the miR-10a promoter in NPM1c+ (OCI-AML3) versus NPM1wt(HL60, MV4) cell lines. Consistent with the observed upregulation of miR-10a in NPM1c+ AML cells, hypomethylation of the miR-10a promoter was observed in OCI-AML3 cells (<5% CpG methylation) compared to the HL60 and MV4 (60% and 92% CpG methylation, respectively; p < 0.01), which was mirrored in NPM1c+ and NPM1WT primary patient samples. Exposure of the NPM1WT AML cells to hypomethylating agents decitabine and azacitidine resulted in reduced DNA methylation of the miR-10a promoter and a corresponding 20-100-fold upregulation of miR-10a (p < 0.001 for both lines). There was no significant association between miR-10a promoter hypomethylation with mutations of epigenetic modifiers such as DNA Methytransferanse 3A (DNMT3A; which are highly enriched in NPM1c+ AML) in primary patient specimens.Conclusion: Overexpression of the pro-survival miRNA miR-10a in NPM1c+ AML is epigenetically regulated, and results in modulation of the p53-p21-Rb1 tumour suppressor signalling axis in NK-AML. This may have clinical significance and identifies miR-10a as a potential novel biomarker and drug target for AML. DisclosuresNo relevant conflicts of interest to declare.

Stem cells derived from human first-trimester umbilical cord have the potential to differentiate into oocyte-like cells in vitro

Compared to stem cells derived from human term umbilical cord, stem cells derived from human first-trimester umbilical cord (hFTUC) exhibit a significantly greater proliferative potential, and more efficiency in terms of their in vitro differentiation. In the present study, we investigated whether hFTUC-derived stem cells are able to differentiate into germ cells. The hFTUC-derived stem cells were first isolated, expanded and then cultured in differentiation medium containing human follicular fluid, follicle-stimulating hormone (FSH)/luteinizing hormone (LH) and estradiol for 24 days. During the period of induction, a subpopulation of the cultured cells appeared that had a morphological resemblance to primordial germ cells (PGCs) and cumulus-oocyte complex (COC)-like cells, and oocyte-like cells (OLCs). The PGC-like cells expressed specific markers indicative of germ cell formation such as octamer-binding transcription factor 4 (OCT4), stage-specific embryonic antigen 1 (SSEA1), B lymphocyte-induced maturation protein-1 (BLIMP1), PR domain containing 14 (PRDM14), transcription factor AP-2 gamma (TFAP2C), VASA, STELLA, deleted in azoospermia-like (DAZL) and interferon-induced transmembrane protein 3 (IFITM3). The OLCs, which contained a single germinal vesicle, expressed oocyte-specific markers, such as synaptonemal complex protein 3 (SCP3), growth/differentiation factor-9 (GDF9), GDF9B and zona pellucida (ZP)1, ZP2 and ZP3. The COC-like cells secreted estradiol, vascular endothelial growth factor and leukemia inhibitory factor. Thus, our findings suggest that hFTUC-derived stem cells have an intrinsic ability to differentiate into OLCs, which may provide an in vitro model for the identification of factors involved in germ cell formation and differentiation.

TFAP2C (transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma))

Review on TFAP2C, with data on DNA/RNA, on the protein encoded and where the gene is implicated.

CITED2 mutation links congenital heart defects to dysregulation of the cardiac gene VEGF and PITX2C expression

CITED2, a cardiac transcription factor, plays an important role in cardiac development. CITED2 mutations lead to a constellation of cardiac defects, which include tetralogy of Fallot and outflow tract malformations. However, the mechanisms underlying these mutations are poorly understood. We investigated the function and mechanism of two missense mutations, G184S and S192G, responsible for tetralogy of Fallot and aortic stenosis, respectively. We found that CITED2 variants decreased its ability to mediate the expression of vascular endothelial growth factor (VEGF) and the expression of the paired-like homeodomain transcription factor 2-gamma (PITX2C), both of which are closely related to cardiac development. Luciferase reporter and mammalian two-hybrid assays showed that G184S and S192G in CITED2 restored the expression of VEGF, which was due to a reduction in its competitiveness with hypoxia inducible factor 1-alpha (HIF1-α) for binding to CBP/p300. In addition, we found that the G184S and S192G mutant decreased cooperation between CITED2 and transcription factor AP2-gamma (TFAP2C) in the transactivation of the PITX2C gene. These results provide important evidence that the mutation of CITED2 may play a role in the development of congenital heart disease (CHD) in humans.

UP‐REGULATION OF TFAP2C BY FGF DURING SHH INDUCTION IN LIMB BUDS

During development, limb outgrowth has been linked to a reciprocal feedback loop between Fibroblast growth factors (Fgfs) and Sonic Hedgehog (Shh). Fgfs from the apical ectodermal ridge (AER) at the distal tip of the limb bud promote secretion of Shh from the zone of polarizing activity (ZPA), a cluster of cells in the distal posterior aspect of limb bud mesoderm. In turn, Shh induces Fgf from the posterior AER. This reciprocal loop is also critical to limb regeneration, and previous studies have shown that Shh up‐regulation correlates with the capacity of Fgfs to accomplish regenerative limb outgrowth. Using gene arrays, we have identified a number of potential Fgf downstream targets in the Fgf‐Shh pathway. In this report, we examined the expression of Transcription factor AP‐2 gamma (Tfap2c).To determine the time course of Fgf‐induced Tfap2c up‐regulation, we implanted Fgf‐soaked beads into stage 23 chick wing buds and evaluated Tfap2c expression by in situ hybridization at several time points.Tfap2c was up‐regulated within 3 hours of Fgf exposure; however, treatment with the protein synthesis inhibitor cycloheximide abolished induction.We conclude that Tfap2c is a close, but indirect, downstream target of Fgf during limb development and regeneration. Further studies are needed to determine whether Tfap2c participates in the Fgf‐Shh pathway or plays an alternative role in limb outgrowth.Grant Funding Source: departmental