AP-2α Expression Research Articles

AP-2α decreases TMZ resistance of recurrent GBM by downregulating MGMT expression and improving DNA damage

AimsThe incidence of recurrent gliomas is high, exerting low survival rates and poor prognoses. Transcription factor AP-2α has been reported to regulate the progression of primary glioblastoma (GBM). However, the function of AP-2α in recurrent gliomas is largely unclear. MethodsThe expression of AP-2α and O6-methylguanine DNA-methyltransferase (MGMT) was detected in recurrent glioma tissues and cell lines by Western blots, the regulation mechanisms between AP-2α/MGMT promoter and RA/AP-2α promoter were studied by luciferase reporter assays, EMSA, and chIP assays. The effects of AP-2α and TMZ/RA treatment on cell viability in vitro and in vivo were investigated by MTT assays, γH2AX staining, comet assays and intracranial injection. Key findingsAP-2α expression negatively correlates with the expression of MGMT in glioma samples. AP-2α could directly bind with the promoter of the MGMT gene, suppresses transcriptional levels of MGMT and downregulate MGMT expression in TMZ-resistant U87MG-R and T98G cells, but TMZ treatment decreases AP-2α expression and increases MGMT expression. The extended TMZ treatment and increased TMZ concentrations reversed these effects. Moreover, AP-2α overexpression combines with TMZ to decrease cell viability, concurrently with improved DNA damage marker γH2AX. Furthermore, retinoic acid (RA) activates RAR/RXR heterodimers, which bind to RA-responsive elements (RAREs) of the AP-2α promoter, and activates AP-2α expression in recurrent glioma cells. Finally, in intracranial relapsed glioma mouse model, both RA and TMZ could retard tumor development and prolong the mouse survival. SignificanceAP-2α activation by gene overexpression or RA treatment reveals the suppressive effects on glioma relapse, providing a novel therapeutic strategy against malignant refractory gliomas.

AP-2α gene deregulation is associated with renal cell carcinoma patient survival

BackgroundRenal cell carcinoma (RCC), one of the most fatal urologic tumors, accounts for approximately 3% of all adult cancers and exhibits a high metastatic index at diagnosis and a high rate of relapse. Radical or partial nephrectomy is a curative option for nonmetastatic RCCs. Targeted therapy has been shown to improve the survival of patients with metastatic RCCs. However, the underlying cellular and molecular events associated with RCC pathogenesis are not well known.MethodsTo investigate the clinical role of the transcription factor activator protein (AP)-2α in RCC, methylated CpG island recovery assays and microarray analysis were employed. COBRA and RT‒qPCR assays were performed to assess AP-2α expression in RCC.ResultsA negative correlation was noted between AP-2α mRNA expression levels and methylation status. Multivariate analyses showed that AP-2α mRNA was a major risk factor not only for overall and disease-free survival in RCC but also for disease-free survival in clear cell RCC.ConclusionsOur results indicated that AP-2α expression was deregulated in RCC and associated with overall patient survival and disease-free survival. Such findings suggest that AP-2α might play an important role in the pathogenesis of RCC.

Epigenetically modified AP-2α by DNA methyltransferase facilitates glioma immune evasion by upregulating PD-L1 expression

Programmed death-ligand 1 (PD-L1) ensures that tumor cells escape T-cell-mediated tumor immune surveillance. However, gliomas are characteristic of the low immune response and high-resistance therapy, it is necessary to understand molecular regulatory mechanisms in glioblastoma, especially the limited regulation of PD-L1 expression. Herein, we show that low expression of AP-2α is correlated with high expression of PD-L1 in high-grade glioma tissues. AP-2α binds directly to the promoter of the CD274 gene, not only inhibits the transcriptional activity of PD-L1 but enhances endocytosis and degradation of PD-L1 proteins. Overexpression of AP-2α in gliomas enhances CD8+ T cell-mediated proliferation, effector cytokine secretion, and cytotoxicity in vitro. Tfap2a could increase the cytotoxic effect of Cd8+ T cells in CT26, B16F10, and GL261 tumor-immune models, improve anti-tumor immunity, and promote the efficacy of anti-PD-1 therapy. Finally, the EZH2/H3K27Me3/DNMT1 complex mediates the methylation modification of AP-2α gene and maintains low expression of AP-2α in gliomas. 5-Aza-dC (Decitabine) treatment combines with anti-PD-1 immunotherapy to efficiently suppress the progression of GL261 gliomas. Overall, these data support a mechanism of epigenetic modification of AP-2α that contributes to tumor immune evasion, and reactivation of AP-2α synergizes with anti-PD-1 antibodies to increase antitumor efficacy, which may be a broadly applicable strategy in solid tumors.

TWIST1 interacts with β/δ-catenins during neural tube development and regulates fate transition in cranial neural crest cells.

Cell fate determination is a necessary and tightly regulated process for producing different cell types and structures during development. Cranial neural crest cells (CNCCs) are unique to vertebrate embryos and emerge from the neural plate borders into multiple cell lineages that differentiate into bone, cartilage, neurons and glial cells. We have previously reported that Irf6 genetically interacts with Twist1 during CNCC-derived tissue formation. Here, we have investigated the mechanistic role of Twist1 and Irf6 at early stages of craniofacial development. Our data indicate that TWIST1 is expressed in endocytic vesicles at the apical surface and interacts with β/δ-catenins during neural tube closure, and Irf6 is involved in defining neural fold borders by restricting AP2α expression. Twist1 suppresses Irf6 and other epithelial genes in CNCCs during the epithelial-to-mesenchymal transition (EMT) process and cell migration. Conversely, a loss of Twist1 leads to a sustained expression of epithelial and cell adhesion markers in migratory CNCCs. Disruption of TWIST1 phosphorylation in vivo leads to epidermal blebbing, edema, neural tube defects and CNCC-derived structural abnormalities. Altogether, this study describes a previously uncharacterized function of mammalian Twist1 and Irf6 in the neural tube and CNCCs, and provides new target genes for Twist1 that are involved in cytoskeletal remodeling.

VIPR1 promoter methylation promotes transcription factor AP-2α binding to inhibit VIPR1 expression and promote hepatocellular carcinoma cell growth in vitro

To explore the transcriptional regulation mechanism and biological function of low expression of vasoactive intestinal peptide receptor 1 (VIPR1) in hepatocellular carcinoma (HCC). We constructed plasmids carrying wild-type VIPR1 promoter or two mutant VIPR1 promoter sequences for transfection of the HCC cell lines Hep3B and Huh7, and examined the effect of AP-2α expression on VIPR1 promoter activity using dual-luciferase reporter assay. Pyrosequencing was performed to detect the changes in VIPR1 promoter methylation level in HCC cells treated with a DNA methyltransferase inhibitor (DAC). Chromatin immunoprecipitation was used to evaluate the binding ability of AP-2α to VIPR1 promoter. Western blotting was used to assess the effect of AP-2α knockdown on VIPR1 expression and examine the differential expression of VIPR1 in the two cell lines. The effects of VIPR1 overexpression and knockdown on the proliferation, cell cycle and apoptosis of HCC cells were analyzed using CCK8 assay and flow cytometry. We also observed the growth of HCC xenograft with lentivirus-mediated over-expression of VIPR1 in nude mice. Compared with the wild-type VIPR1 promoter group, co-transfection with the vector carrying two promoter mutations and the AP-2α-over-expressing plasmid obviously restored the luciferase activity in HCC cells (P < 0.05). DAC treatment of the cells significantly decreased the methylation level of VIPR1 promoter and inhibited the binding of AP-2α to VIPR1 promoter (P < 0.01). The HCC cells with AP-2α knockdown showed increased VIPR1 expression, which was lower in Huh7 cells than in Hep3B cells. VIPR1 overexpression in HCC cells caused significant cell cycle arrest in G2/M phase (P < 0.01), promoted cell apoptosis (P < 0.001), and inhibited cell proliferation (P < 0.001), while VIPR1 knockdown produced the opposite effects. In the tumor-bearing nude mice, VIPR1 overexpression in the HCC cells significantly suppressed the increase of tumor volume (P < 0.001) and weight (P < 0.05). VIPR1 promoter methylation in HCC promotes the binding of AP-2α and inhibits VIPR1 expression, while VIPR1 overexpression causes cell cycle arrest, promotes cell apoptosis, and inhibits cell proliferation and tumor growth.

AP-2α-Mediated Activation of E2F and EZH2 Drives Melanoma Metastasis.

In melanoma metastasis, the role of the AP-2α transcription factor, which is encoded by TFAP2A, is controversial as some findings have suggested tumor suppressor activity while other studies have shown high TFAP2A expression in node-positive melanoma associated with poor prognosis. Here we demonstrate that AP-2α facilitates melanoma metastasis through transcriptional activation of genes within the E2F pathway including EZH2. A BioID screen found that AP-2α interacts with members of the nucleosome remodeling and deacetylase (NuRD) complex. Loss of AP-2α removed activating chromatin marks in the promoters of EZH2 and other E2F target genes through activation of the NuRD repression complex. In melanoma cells, treatment with tazemetostat, an FDA-approved and highly specific EZH2 inhibitor, substantially reduced anchorage-independent colony formation and demonstrated heritable antimetastatic effects, which were dependent on AP-2α. Single-cell RNA sequencing analysis of a metastatic melanoma mouse model revealed hyperexpansion of Tfap2a High/E2F-activated cell populations in transformed melanoma relative to progenitor melanocyte stem cells. These findings demonstrate that melanoma metastasis is driven by the AP-2α/EZH2 pathway and suggest that AP-2α expression can be used as a biomarker to predict responsiveness to EZH2 inhibitors for the treatment of advanced melanomas. SIGNIFICANCE: AP-2α drives melanoma metastasis by upregulating E2F pathway genes including EZH2 through inhibition of the NuRD repression complex, serving as a biomarker to predict responsiveness to EZH2 inhibitors.

Role of AP-2α/TGF-β1/Smad3 axis in rats with intervertebral disc degeneration

ObjectiveStudies have proposed the role of AP-2α in human disease. However, few have focused on its effects on intervertebral disc degeneration (IDD). This study intends to discuss the role of AP-2α in IDD by regulating TGF-β1 and Smad3 expression. MethodsThe AP-2α and TGF-β1 expression in IDD NP clinical samples was detected. Rat models of IDD were established by acupuncture. The rats were injected with AP-2α low expression adeno-associated virus or TGF-β1 high expression adeno-associated virus to observe their effects on pathological damages, NP cell apoptosis, matrix metalloproteinase-2 (MMP-2), MMP-9, Smad3, Aggrecan and collagen (Col)-2 expression in NP tissues. The NP cells were isolated and transfected with silenced AP-2α or overexpressed TGF-β1 vector to figure out their functions in growth, senescence and apoptosis. ResultsAP-2α and TGF-β1 were upregulated in NP tissues of patients and rats with IDD. AP-2α silencing limited the activation of TGF-β1 signaling pathway. Reduced AP-2α ameliorated pathological changes, declined MMP-2, MMP-9 and Smad3 expression and elevated Aggrecan and Col-2 expression in NP tissues of rats with IDD, and speeded up the growth and depressed senescence and apoptosis of NP cells of rats with IDD. Up-regulating TGF-β1 weakened the effect of down-regulated AP-2α on NP tissues and cells in IDD. ConclusionCollectively, our study demonstrates that knockdown of AP-2α restricts TGF-β1 and Smad3 expression to promote proliferation and depress senescence and apoptosis of NP cells in rats with IDD.

Early Embryonic Expression of AP-2α Is Critical for Cardiovascular Development

Congenital cardiovascular malformation is a common birth defect incorporating abnormalities of the outflow tract and aortic arch arteries, and mice deficient in the transcription factor AP-2α (Tcfap2a) present with complex defects affecting these structures. AP-2α is expressed in the pharyngeal surface ectoderm and neural crest at mid-embryogenesis in the mouse, but the precise tissue compartment in which AP-2α is required for cardiovascular development has not been identified. In this study we describe the fully penetrant AP-2α deficient cardiovascular phenotype on a C57Bl/6J genetic background and show that this is associated with increased apoptosis in the pharyngeal ectoderm. Neural crest cell migration into the pharyngeal arches was not affected. Cre-expressing transgenic mice were used in conjunction with an AP-2α conditional allele to examine the effect of deleting AP-2α from the pharyngeal surface ectoderm and the neural crest, either individually or in combination, as well as the second heart field. This, surprisingly, was unable to fully recapitulate the global AP-2α deficient cardiovascular phenotype. The outflow tract and arch artery phenotype was, however, recapitulated through early embryonic Cre-mediated recombination. These findings indicate that AP-2α has a complex influence on cardiovascular development either being required very early in embryogenesis and/or having a redundant function in many tissue layers.

The miR-26a/AP-2α/Nanog signaling axis mediates stem cell self-renewal and temozolomide resistance in glioma.

Aberrant expression of transcription factor AP-2α has been functionally associated with various cancers, but its clinical significance and molecular mechanisms in human glioma are largely elusive.Methods: AP-2α expression was analyzed in human glioma tissues by immunohistochemistry (IHC) and in glioma cell lines by Western blot. The effects of AP-2α on glioma cell proliferation, migration, invasion and tumor formation were evaluated by the 3-(4,5-dimethyNCthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) and transwell assays in vitro and in nude mouse models in vivo. The influence of AP-2α on glioma cell stemness was analyzed by sphere-formation, self-renewal and limiting dilution assays in vitro and in intracranial mouse models in vivo. The effects of AP-2α on temozolomide (TMZ) resistance were detected by the MTT assay, cell apoptosis, real-time PCR analysis, western blotting and mouse experiments. The correlation between AP-2α expression and the expression of miR-26a, Nanog was determined by luciferase reporter assays, electrophoretic mobility shift assay (EMSA) and expression analysis.Results: AP-2α expression was downregulated in 58.5% of glioma tissues and in 4 glioma cell lines. AP-2α overexpression not only reduced the proliferation, migration and invasion of glioma cell lines but also suppressed the sphere-formation and self-renewal abilities of glioma stem cells in vitro. Moreover, AP-2α overexpression inhibited subcutaneous and intracranial xenograft tumor growth in vivo. Furthermore, AP-2α enhanced the sensitivity of glioma cells to TMZ. Finally, AP-2α directly bound to the regulatory region of the Nanog gene, reduced Nanog, Sox2 and CD133 expression. Meanwhile, AP-2α indirectly downregulated Nanog expression by inhibiting the interleukin 6/janus kinase 2/signal transducer and activator of transcription 3 (IL6/JAK2/STAT3) signaling pathway, consequently decreasing O6-methylguanine methyltransferase (MGMT) and programmed death-ligand 1 (PD-L1) expression. In addition, miR-26a decreased AP-2α expression by binding to the 3' untranslated region (UTR) of AP-2α and reversed the tumor suppressive role of AP-2α in glioma, which was rescued by a miR-26a inhibitor. TMZ and the miR-26a inhibitor synergistically suppressed intracranial GSC growth.Conclusion: These results suggest that AP-2α reduces the stemness and TMZ resistance of glioma by inhibiting the Nanog/Sox2/CD133 axis and IL6/STAT3 signaling pathways. Therefore, AP-2α and miR-26a inhibition might represent a new target for developing new therapeutic strategies in TMZ resistance and recurrent glioma patients.

AP-2α expression in papillary thyroid carcinoma predicts tumor progression and poor prognosis.

BackgroundThe activator protein (AP)-2α is involved in a wide variety of biologic processes in tumor. However, little is known about the role of AP-2α in human papillary thyroid carcinoma (PTC).MethodsThe immunohistochemical method was used to detect AP-2α expression in 63 PTC cases. Western blotting was carried out to assess the change in expression of certain proteins. The bioinformatics analysis of 496 PTC samples comes from The Cancer Genome Atlas (TCGA). The Gene Set Enrichment Analysis (GSEA) was performed using TCGA data set. Cell transfection was used to induce related protein expression or to repress it by RNA interference procedures.ResultsOur results demonstrated that AP-2α expression was higher in tumor tissues than the corresponding adjacent nontumor tissues, the positive substances of AP-2α were observed mainly in the cytoplasm of PTC, and AP-2α was positively correlated with histologic type (P=0.026) of PTC patients. The high expression of AP-2α mRNA was associated significantly with tumor stages (P=0.011), histologic type (P=0.019), and independently predicted shorter overall survival (P=0.005) based on TCGA analysis. Patients with high AP-2α mRNA expression have shorter overall survival compared to those with low AP-2α mRNA expression, particularly in advanced tumor stages (III and IV) of PTC patients (P=0.011). Multivariate analysis suggested that AP-2α mRNA expression might be an independent prognostic indicator for the survival of patients with PTC (P=0.037). Moreover, the association between enhanced AP-2α expression and two pathways (notch signaling and focal adhesion) was revealed by GSEA, and then confirmed by cellular experiments.ConclusionTaken together, our findings suggest that AP-2α may be a potential prognostic molecular marker and therapeutic target for PTC patients.

Long non-coding RNA CCAL promotes hepatocellular carcinoma progression by regulating AP-2α and Wnt/β-catenin pathway

ObjectiveLong non-coding RNAs are emerging as key molecules in cancer progression. LncRNA-CCAL has shown to be highly expressed and important in regulating CRC and osteosarcoma development. Nevertheless, the expression and mechanism of CCAL in HCC is still not well understood. MethodsqRT-PCR and ISH were used to evaluate CCAL expression in HCC tissues and cell lines. Histone H3 methylation and acetylation levels across CCAL promoter region were examined by chromatin immunoprecipitation assays. Transfection of Lv-CCAL-shRNAs into HCC cell lines was used to evaluate cellular invasion and proliferation. The influence of CCAL depletion on AP-2α expression and Wnt/β-catenin pathway was analyzed by qRT-PCR, western blot and immunofluorescence. ResultsHigher expression of CCAL was found in HCC tumor tissues compared with normal tissues, and was associated with tumor metastasis and TNM stage. Furthermore, the decreased histone H3 methylation and increased histone H3 acetylation across CCAL promoter region contributed to the upregulation of CCAL in HCC. Moreover, the depletion of CCAL inhibited HCC cellular invasion and proliferation, and promoted cell apoptosis. In addition, CCAL depletion up-regulated AP-2α expression and inhibited Wnt/β-catenin pathway activation. ConclusionsCCAL has an important role in hepatic carcinogenesis and may serve as a new target for HCC diagnosis and treatment.

Mllt10 knockout mouse model reveals critical role of Af10-dependent H3K79 methylation in midfacial development

Epigenetic regulation is required to ensure the precise spatial and temporal pattern of gene expression that is necessary for embryonic development. Although the roles of some epigenetic modifications in embryonic development have been investigated in depth, the role of methylation at lysine 79 (H3K79me) is poorly understood. Dot1L, a unique methyltransferase for H3K79, forms complexes with distinct sets of co-factors. To further understand the role of H3K79me in embryogenesis, we generated a mouse knockout of Mllt10, the gene encoding Af10, one Dot1L complex co-factor. We find homozygous Mllt10 knockout mutants (Mllt10-KO) exhibit midline facial cleft. The midfacial defects of Mllt10-KO embryos correspond to hyperterolism and are associated with reduced proliferation of mesenchyme in developing nasal processes and adjacent tissue. We demonstrate that H3K79me level is significantly decreased in nasal processes of Mllt10-KO embryos. Importantly, we find that expression of AP2α, a gene critical for midfacial development, is directly regulated by Af10-dependent H3K79me, and expression AP2α is reduced specifically in nasal processes of Mllt10-KO embryos. Suppression of H3K79me completely mimicked the Mllt10-KO phenotype. Together these data are the first to demonstrate that Af10-dependent H3K79me is essential for development of nasal processes and adjacent tissues, and consequent midfacial formation.

Alterted SLIT2/ROBO1 signalling is linked to impaired placentation of missed and threatened miscarriage in early pregnancy.

Two-thirds of early pregnancy failures present with reduced trophoblast invasion, and SLIT2/ROBO1 signalling is considered to play an important role in trophoblast function during pregnancy. We investigated SLIT2/ROBO1 signalling associated with missed and threatened miscarriage during early gestation. Human placenta samples were collected from women with missed miscarriage (n = 25), threatened miscarriage (n = 22) and termination of pregnancy controls (n = 32). Corresponding decreases in beta human chorionic gonadotrophin (β-hCG) levels and shallow trophoblast invasion were observed in patients with missed and threatened miscarriage, immunohistological staining revealed abnormal Slit2 and Robo1, as well as E-cadherin and activating protein-2 alpha (AP-2α) expression in villi and extravillous trophoblasts, and the expression of these proteins were confirmed in villi and decidua of miscarriage material by Western blotting. Using HTR8/SVneo cells, blocking SLIT2/ROBO1 signalling promoted cell migration, proliferation and suppressed differentiation. Moreover, blocking SLIT2/ROBO1 signalling in HTR8/SVneo cells altered trophoblast differentiation-related and angiogenesis-related gene mRNA expression, which also occurred in the tissues of missed and threatened miscarriage. SLIT2/ROBO1 signalling may regulate trophoblast differentiation and invasion causing restricting β-hCG production, shallow trophoblast invasion and inhibiting placental angiogenesis in missed and threatened miscarriage during the first trimester.

AP-2α reverses vincristine-induced multidrug resistance of SGC7901 gastric cancer cells by inhibiting the Notch pathway.

Multidrug resistance (MDR) remains a major clinical obstacle in the treatment of gastric cancer (GC) since it causes tumor recurrence and metastasis. The transcription factor activator protein-2α (AP-2α) has been implicated in drug-resistance in breast cancer; however, its effects on MDR of gastric cancer are far from understood. In this study, we aimed to explore the effects of AP-2α on the MDR in gastric cancer cells selected by vincristine (VCR). Decreased AP-2α levels were markedly detected by RT-PCR and Western blot in gastric cancer cell lines (BGC-823, SGC-7901, AGS, MKN-45) compared with that in the gastric epithelial cell line (GES-1). Furthermore, we found that the expression of AP-2α in SGC7901/VCR or SGC7901/adriamycin (ADR) cells was lower than in SGC7901 cells. Thus, a vector overexpressing AP-2α was constructed and used to perform AP-2α gain-of-function studies in SGC7901/VCR cells. The decreased IC50 values of the anti-cancer drugs in sensitive and resistant cells after transfect with pcDNA3.1/AP-2α were determined in SGC7901/VCR cells by MTT assay. Moreover, flow cytometry analysis indicated that overexpressed AP-2α induced cell cycle arrest in the G0/G1 phase and promoted cell apoptosis of VCR-selected SGC7901/VCR cells. RT-PCR and Western blot demonstrated that overexpressed AP-2α can significantly induce the down-regulation of Notch1, Hes-1, P-gp and MRP1 in SGC7901/VCR cells. Similar effects can be observed when Numb (Notch inhibitor) was introduced. In addition, the intracellular ADR accumulation was markedly detected in AP-2α overexpressed or Numb cells. In conclusion, our results indicate that AP-2α can reverse the MDR of gastric cancer cells, which may be realized by inhibiting the Notch signaling pathway.

Soluble TNF Regulates TACE via AP-2α Transcription Factor in Mouse Dendritic Cells.

Dendritic cells (DCs), the essential immunoregulatory and APCs, are major producers of the central mediator of inflammation, soluble TNF-α (sTNF). sTNF is generated by TNF-α converting enzyme (TACE) proteolytic release of the transmembrane TNF (tmTNF) ectodomain. The mechanisms of TACE and sTNF regulation in DCs remain elusive. This study newly defines that sTNF regulates TACE in mouse DCs by engaging the AP-2α transcription factor. We found that the expression of AP-2α was higher, whereas the expression and activity of TACE were lower, in wild-type DCs (wtDCs) than in TNF knockout (TNFko) DCs. Exogenous sTNF rapidly and simultaneously induced increases of AP-2α expression and decreases of TACE expression and activity in wtDCs and TNFko DCs, indicating that AP-2α and TACE are inversely dependent on sTNF and are functionally associated. To define this functional association, we identified an AP-2α binding site in TACE promoter and demonstrated, using EMSAs and chromatin immunoprecipitation assays, that AP-2α could bind to TACE promoter in a TNF-dependent manner. Additionally, sTNF simultaneously enhanced AP-2α expression and decreased TACE promoter luciferase activity in DCs. Similarly, transfection of AP-2α cDNA decreased TACE promoter luciferase activity, TACE expression, and TACE enzymatic activity in wtDCs or TNFko DCs. In contrast, transfection of AP-2α small interfering RNA increased TACE promoter luciferase activity, TACE expression, and TACE enzymatic activity in wtDCs. These results show that TACE is a target of, and is downregulated by, sTNF-induced AP-2α transcription factor in DCs.

AP-2α inhibits hepatocellular carcinoma cell growth and migration.

Transcription factor AP-2α is involved in many types of human cancers, but its role in hepatocellular carcinogenesis is largely unknown. In this study, we found that expression of AP-2α was low in 40% of human hepatocellular cancers compared with adjacent normal tissues by immunohistochemical analysis. Moreover, AP-2α expression was low or absent in hepatocellular cancer cell lines (HepG2, Hep3B, SMMC-7721 and MHHC 97-H). Human liver cancer cell lines SMMC-7721 and Hep3B stably overexpressing AP-2α were established by lentiviral infection and puromycin screening, and the ectopic expression of AP-2α was able to inhibit hepatocellular cancer cell growth and proliferation by cell viability, MTT assay and liquid colony formation in vitro and in vivo. Furthermore, AP-2α overexpression decreased liver cancer cell migration and invasion as assessed by wound healing and Transwell assays, increasing the sensitivity of liver cancer cells to cisplatin analyzed by MTT assays. Also AP-2α overexpression suppressed the sphere formation and renewed the ability of cancer stem cells. Finally, we found that AP-2α is epigenetically modified and modulates the levels of phosphorylated extracellular signal-regulated protein kinase (ERK), β-catenin, p53, EMT, and CD133 expression in liver cancer cell lines. These results suggested that AP-2α expression is low in human hepatocellular cancers by regulating multiple signaling to affect hepatocellular cancer cell growth and migration. Therefore, AP-2α might represent a novel potential target in human hepatocellular cancer therapy.

MiR-193a-5p Targets the Coding Region of AP-2α mRNA and Induces Cisplatin Resistance in Bladder Cancers.

Transcription factor AP-2 alpha (AP-2α or TFAP2A) is a newly identified prognostic marker of chemotherapy; its expression is positively correlated with chemosensitivity and survival of cancer patients. Using computational programs, we predicted that the coding region of AP-2α gene contains a potential miRNA response element (MRE) of miR-193a-5p, and the single nucleotide polymorphism (SNP) site (c.497A>G, rs111681798) resides within the predicted MRE. The results of luciferase assays and Western blot analysis demonstrated that miR-193a-5p negatively regulated the expression of AP-2α proteins, but have no influence on the mutant AP-2α (c.497A>G). Infection with lentiviral AP-2α gene or miR-193a-5p inhibitor in the bladder cancer cells decreased migration and cisplatin resistance, while knockdown of AP-2α gene or overexpression of miR-193a-5p in the urothelial cell line SV-HUC-1 increased migration and cisplatin resistances. We concluded that miR-193a-5p induced cisplatin resistance by repressing AP-2α expression in bladder cancer cells.

AP2α controls the dynamic balance between miR-126&amp;126* and miR-221&amp;222 during melanoma progression

Accumulating evidences have shown the association between aberrantly expressed microRNAs (miRs) and cancer, where these small regulatory RNAs appear to dictate the cell fate by regulating all the main biological processes. We demonstrated the responsibility of the circuitry connecting the oncomiR-221&222 with the tumor suppressors miR-126&126* in melanoma development and progression. According to the inverse correlation between endogenous miR-221&222 and miR-126&126*, respectively increasing or decreasing with malignancy, their enforced expression or silencing was sufficient for a reciprocal regulation. In line with the opposite roles of these miRs, protein analyses confirmed the reverse expression pattern of miR-126&126*-targeted genes that were induced by miR-221&222. Looking for a central player in this complex network, we revealed the dual regulation of AP2α, on one side directly targeted by miR-221&222 and on the other a transcriptional activator of miR-126&126*. We showed the chance of restoring miR-126&126* expression in metastatic melanoma to reduce the amount of mature intracellular heparin-binding EGF like growth factor, thus preventing promyelocytic leukemia zinc finger delocalization and maintaining its repression on miR-221&222 promoter. Thus, the low-residual quantity of these two miRs assures the release of AP2α expression, which in turn binds to and induces miR-126&126* transcription. All together these results point to an unbalanced ratio functional to melanoma malignancy between these two couples of miRs. During progression this balance gradually moves from miR-126&126* toward miR-221&222. This circuitry, besides confirming the central role of AP2α in orchestrating melanoma development and/or progression, further displays the significance of these miRs in cancer and the option of utilizing them for novel therapeutics.

RGD Peptides-Conjugated Pluronic Triblock Copolymers Encapsulated with AP-2α Expression Plasmid for Targeting Gastric Cancer Therapy in Vitro and in Vivo.

Gastric cancer, a high-risk malignancy, is a genetic disease developing from a cooperation of multiple gene mutations and a multistep process. Gene therapy is a novel treatment method for treating gastric cancer. Here, we developed a novel Arg-Gly-Asp (RGD) peptides conjugated copolymers nanoparticles-based gene delivery system in order to actively targeting inhibit the growth of gastric cancer cells. These transcription factor (AP-2α) expression plasmids were also encapsulated into pluronic triblock copolymers nanoparticles which was constituted of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEO-block-PPO-block-PEO, P123). The size, morphology and composition of prepared nanocomposites were further characterized by nuclear magnetic resonance (NMR), transmission electron microscopy (TEM) and dynamic light scattering (DLS). In MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) analysis, these nanocomposites have minor effects on the proliferation of GES-1 cells but significantly decreased the viability of MGC-803, suggesting they own low cytotoxicity but good antitumor activity. The following in vivo evaluation experiments confirmed that these nanocomposites could prevent the growth of gastric cancer cells in the tumor xenograft mice model. In conclusion, these unique RGD peptides conjugated P123 encapsulated AP-2α nanocomposites could selectively and continually kill gastric cancer cells by over-expression of AP-2α in vitro and in vivo; this exhibits huge promising applications in clinical gastric cancer therapy.

Targeted inhibition of microRNA-200c on expression of AP-2α to enhance the proliferation of colon cancer cells in vitro

Objective To investigate the impact of miR-200c overexpression on colon cancer cell proliferation ability and the related mechanism. Methods MicroRNAs which may combined with the transcription factor AP-2α were screened and forecasted by the bioinformatics database, while its eukaryotic expression plasmids and specific inhibitor were synthesized. Plasmids PEZX-miR-200c, PEZX-NC, pmir-GLO-AP-2α3'UTR, pmir-GLO and the specific inhibitors miR-67-inhibtor, miR-200c-inhibitor were transfected in vitro into colon cancer HCT-116 and SW480 cells and the HEK293T cell by Lipofectamine2000. The expression of AP-2α mRNA and protein in colon cancer cells was analyzed by qRT-PCR,Western blot and immunocytochemical staining. CCK-8 assay and flow cytometry were adopted to observe the effect of miR-200c on colon cancer cells proliferation and apoptosis. Dual-Luciferase assay experiments were performed to observe the relative luciferase activity induced by miR-200c. Results The proliferation activity was significantly decreased in anti-miR-200c /SW480 group, while in PEZX-miR-200c/HCT-116 group, it was higher than that in PEZX-NC/HCT-116 group. The apoptosis ability was significantly increased in anti-miR-200c/SW480 group [(78±0.7)% vs (66±1.1)%, P<0.05]. The expression of AP-2α both in mRNA and protein levels was decreased in PEZX-miR-200c/HCT-116 group, while the protein level was increased in Anti-miR-200c/SW480 group. The relative luciferase activity inhibited by miR-200c was decreased in HEK-293T cells transfected with PEZX-miR-200c and pmir-GLO-AP-2α3'UTR (0.51±0.09 vs 0.98±0.04, P<0.01). Conclusion MicroRNA-200c could promote cell proliferation ability by targeting transcriptional factor AP-2α in human colorectal cancer cells. Key words: Transcription factor AP-2α; microRNA-200c; Colonic neoplasms