AP-2 Alpha Research Articles

Overcoming Cancer Persister Cells by Stabilizing the ATF4 Promoter G-quadruplex.

Persister cells (PS) selected for anticancer therapy have been recognized as a significant contributor to the development of treatment-resistant malignancies. It is found that imposing glutamine restriction induces the generation of PS, which paradoxically bestows heightened resistance to glutamine restriction treatment by activating the integrated stress response and initiating thegeneral control nonderepressible 2-activating transcription factor 4-alanine, serine, cysteine-preferring transporter 2 (GCN2-ATF4-ASCT2)axis. Central to this phenomenon is the stress-induced ATF4 translational reprogramming. Unfortunately, directly targeting ATF4 protein has proven to be a formidable challenge because of its flat surface. Nonetheless, a G-quadruplex structure located within the promoter region of ATF4 (ATF4-G4) is uncovered and resolved, which functions as a transcriptional regulator and can be targeted by small molecules. The investigation identifies the natural compound coptisine (COP) as a potent binder that interacts with and stabilizes ATF4-G4. For the first time, the high-resolution structure of the COP-ATF4-G4 complex is determined. The formation of this stable complex disrupts the interaction between transcription factor AP-2 alpha (TFAP2A) and ATF4-G4, resulting in a substantial reduction in intracellular ATF4 levels and the eventual death of cancer cells. These seminal findings underscore the potential of targeting the ATF4-G4 structure to yield significant therapeutic advantages within the realm of persister cancer cells induced by glutamine-restricted therapy.

IGF2BP3 suppresses ferroptosis in lung adenocarcinoma by m6A-dependent regulation of TFAP2A to transcriptionally activate SLC7A11/GPX4.

Ferroptosis is recently discovered as an important player in the initiation, proliferation, and progression of human tumors. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) has been reported as an oncogene in multiple types of cancers, including lung adenocarcinoma (LUAD). However, little research has been designed to investigate the regulation of IGF2BP3 on ferroptosis in LUAD. qRT-PCR and western blot were used to measure the mRNA and protein expression of IGF2BP3 and transcription factor AP-2 alpha (TFAP2A). CCK-8 assay was performed to determine cell viability. DCFH-DA and C11-BODIPY staining were used to detect the levels of intracellular reactive oxygen species (ROS) and lipid ROS. The corresponding assay kits were used to analyze the levels of malondialdehyde (MDA) and glutathione (GSH). SRAMP website and m6A RNA immunoprecipitation (Me-RIP) were used to predict and confirm the m6A modification of TFAP2A. RIP experiments were conducted to confirm the binding of IGF2BP3 and TFAP2A. RNA stability assay was performed using actinomycin D. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter experiments were performed to confirm the interaction between TFAP2A and cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11) or glutathione peroxidase 4 (GPX4). Mice xenotransplant model was also constructed to explore the effect of IGF2BP3 on LUAD tumor growth and ferroptosis. IGF2BP3 and TFAP2A were both highly expressed in LUAD. IGF2BP3 or TFAP2A knockdown induced ferroptosis by aggravating erastin-induced cell viability suppression, increasing the production of intracellular ROS, lipid ROS, and MDA, and decreasing GSH synthesis, GSH/GSSG ratio, and cystine uptake. Mechanistically, IGF2BP3 stabilized TFAP2A expression via m6A modification. Moreover, sh-IGF2BP3-mediated ferroptosis was significantly abated by TFAP2A overexpression. Furthermore, TFAP2A binds to the promoters of SLC7A11 and GPX4 to promote their transcription. Also, IGF2BP3 depletion suppressed LUAD tumor growth by inducing ferroptosis in mice. IGF2BP3 suppresses ferroptosis in LUAD by m6A-dependent regulation of TFAP2A to promote the transcription of SLC7A11 and GPX4. Our findings suggest that targeting IGF2BP3/TFAP2A/SLC7A11/GPX4 axis might be a potential therapeutic choice to increase ferroptosis sensitivity in LUAD.

Polymorphism rs2327430 in TCF21 predicts the risk and prognosis of gastric cancer by affecting the binding between TFAP2A and TCF21

BackgroundSingle nuclear polymorphisms (SNPs) have been published to be correlated with multiple diseases. Transcription Factor 21 (TCF21) is a critical transcription factor involved in various types of cancers. However, the association of TCF21 genetic polymorphisms with gastric cancer (GC) susceptibility and prognosis remains unclear.MethodsA case-control study comprising 890 patients diagnosed with GC and an equal number of cancer-free controls was conducted. After rigorous statistical analysis, molecular experiments were carried out to elucidate the functional significance of the SNPs in the context of GC.ResultsTCF21 rs2327430 (OR = 0.78, P = 0.026) provides protection against GC, while rs4896011 (OR = 1.39, P = 0.005) exhibit significant associations with GC risk. Furthermore, patients with the (TC + CC) genotype of rs2327430 demonstrate a relatively favorable prognosis (OR = 0.47, P = 0.012). Mechanistically, chromatin immunoprecipitation assay and luciferase reporter assay revealed that the C allele of rs2327430 disrupts the binding of Transcription Factor AP-2 Alpha (TFAP2A) to the promoter region of TCF21, resulting in increased expression of TCF21 and inhibition of malignant behaviors in GC cells.ConclusionOur findings highlight the significant role of TCF21 SNPs in both the risk and prognosis of GC and provide valuable insights into the underlying molecular mechanisms. Specifically, the disruptive effect of rs2327430 on TCF21 expression and its ability to modulate malignant cell behaviors suggest that rs2327430 may serve as a potential predictive marker for GC risk and prognosis.

Inhibition of Usp14 ameliorates renal ischemia-reperfusion injury by reducing Tfap2a stabilization and facilitating mitophagy

Mitochondrial dysfunction is recognized as a pivotal contributor to the pathogenesis of renal ischemia-reperfusion (IR) injury. Mitophagy, the process responsible for removing damaged protein aggregates, stands as a critical mechanism safeguarding cells against IR injury. Currently, the role of deubiquitination in regulating mitophagy still needs to be completely elucidated. This study aimed to evaluate the impact of ubiquitin-specific peptidase 14 (Usp14), a deubiquitinase, in IR injury by influencing mitophagy. Utilizing a murine model of renal IR injury, Usp14 silencing was found to ameliorate kidney injury, leading to decreased levels of serum creatinine and blood urea nitrogen, alongside diminished oxidative stress and inflammation. In renal epithelial cells subjected to hypoxia/reoxygenation (H/R), Usp14 knockdown increased cell viability and reduced apoptosis. Further mechanistic studies revealed that Usp14 interacted with and deubiquitinated transcription factor AP-2 alpha (Tfap2a), thereby suppressing its downstream target gene, TANK binding kinase 1 (Tbk1), to influence mitophagy. Tfap2a overexpression or Tbk1 inhibition reversed the protective effects of Usp14 silencing on renal tubular cell injury and its facilitation of mitophagy. In summary, our study demonstrated the renoprotective role of Usp14 knockdown in mitigating renal IR injury by promoting Tfap2a-mediated Tbk1 upregulation and mitophagy. These findings advocate for exploring Usp14 inhibition as a promising therapeutic avenue for mitigating IR injury, primarily by enhancing the clearance of damaged mitochondria through augmented mitophagy.

Tyrosine phosphorylation-mediated YAP1-TFAP2A interactions coordinate transcription and trastuzumab resistance in HER2+ breast cancer

Trastuzumab resistance in HER2+ breast cancer (BC) is the major reason leading to poor prognosis of BC patients. Oncogenic gene overexpression or aberrant activation of tyrosine kinase SRC is identified to be the key modulator of trastuzumab response. However, the detailed regulatory mechanisms underlying SRC activation-associated trastuzumab resistance remain poorly understood. In the present study, we discover that SRC-mediated YAP1 tyrosine phosphorylation facilitates its interaction with transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha, TFAP2A), which in turn promotes YAP1/TEAD-TFAP2A (YTT) complex-associated transcriptional outputs, thereby conferring trastuzumab resistance in HER2+ BC. Inhibition of SRC kinase activity or disruption of YTT complex sensitizes cells to trastuzumab treatment in vitro and in vivo. Additionally, we also identify YTT complex co-occupies the regulatory regions of a series of genes related to trastuzumab resistance and directly regulates their transcriptions, including EGFR, HER2, H19 and CTGF. Moreover, YTT-mediated transcriptional regulation is coordinated by SRC kinase activity. Taken together, our study reveals that SRC-mediated YTT complex formation and transcriptions are responsible for multiple mechanisms associated with trastuzumab resistance. Therefore, targeting HER2 signaling in combination with the inhibition of YTT-associated transcriptional outputs could serve as the treatment strategy to overcome trastuzumab resistance caused by SRC activation.

Fluconazole-Induced Protein Changes in Osteogenic and Immune Metabolic Pathways of Dental Pulp Mesenchymal Stem Cells of Osteopetrosis Patients.

Osteopetrosis is a rare inherited disease caused by osteoclast failure, resulting in increasing bone density in humans. Patients with osteopetrosis possess several dental and cranial complications. Since carbonic anhydrase II (CA-II) deficiency is a major cause of osteopetrosis, CA-II activators might be an attractive potential treatment option for osteopetrosis patients. We conducted comprehensive label-free quantitative proteomics analysis on Fluconazole-treated Dental Pulp Mesenchymal Stem/Stromal Cells from CA-II-Deficient Osteopetrosis Patients. We identified 251 distinct differentially expressed proteins between healthy subjects, as well as untreated and azole-treated derived cells from osteopetrosis patients. Twenty-six (26) of these proteins were closely associated with osteogenesis and osteopetrosis disease. Among them are ATP1A2, CPOX, Ap2 alpha, RAP1B and some members of the RAB protein family. Others include AnnexinA1, 5, PYGL, OSTF1 and PGAM4, all interacting with OSTM1 in the catalytic reactions of HCO3 and the Cl- channel via CAII regulation. In addition, the pro-inflammatory/osteoclast regulatory proteins RACK1, MTSE, STING1, S100A13, ECE1 and TRIM10 are involved. We have identified proteins involved in osteogenic and immune metabolic pathways, including ERK 1/2, phosphatase and ATPase, which opens the door for some CA activators to be used as an alternative drug therapy for osteopetrosis patients. These findings propose that fluconazole might be a potential treatment agent for CAII- deficient OP patients. Altogether, our findings provide a basis for further work to elucidate the clinical utility of azole, a CA activator, as a therapeutic for OP.

TFAP2A activates HMGA1 to promote glycolysis and lung adenocarcinoma progression

BackgroundLung cancer is the most common cancer in the world. High Mobility Group AT-Hook 1 (HMGA1) is found to be associated with the glycolytic pathway in a variety of cancers, and abnormal glycolysis function is one of the important characteristics of cancer cells. Therefore, this paper discusses the effect of HMGA1 on glycolysis of lung adenocarcinoma (LUAD) cells MethodsThe mRNA expression data were downloaded from TCGA-LUAD database. Groups were set according to the median expression of HMGA1, followed by GSEA enrichment analysis. The upstream transcriptional regulators of HMGA1 were predicted by bioinformatics. The correlation between HMGA1 and Transcription Factor AP-2 Alpha (TFAP2A) and their expression in LUAD tissues were analyzed as well. mRNA expression levels of HMGA1 and TFAP2A were detected by qRT-PCR. The binding of HMGA1 and TFAP2A was demonstrated by ChIP and dual luciferase reporter assays. Cell function experiments were utilized to assay proliferation, apoptosis, glycolysis ability of LUAD cells, and glycolysis-related protein expression in each treatment group. ResultsHMGA1 was highly expressed in LUAD patients’ tissues and enriched in the glycolytic pathway. Additionally, silencing HMGA1 markedly hampered cell proliferation and glycolysis, and promoted cell apoptosis. The upstream transcriptional regulator TFAP2A was predicted to be highly expressed in LUAD. ChIP and dual luciferase reporter assays confirmed the targeted relationship between HMGA1 and TFAP2A. Cell rescue assay confirmed that TFAP2A promoted glycolysis and LUAD progression by activating HMGA1. ConclusionTFAP2A promotes glycolysis, proliferation and hampers apoptosis of LUAD cells by stimulating HMGA1. Hence, TFAP2A/HMGA1 may be a feasible therapeutic target for LUAD. Availability of data and materialsAll the data within this manuscript could be gotten from corresponding author at reasonable request.

RELA promotes the progression of oral squamous cell carcinoma via TFAP2A-Wnt/β-catenin signaling.

Oral squamous cell carcinoma (OSCC) has emerged as the most prevailing oral malignancy worldwide, characterized by cervical solid lymph node metastasis and strong local invasiveness. Overexpression of Transcription Factor AP-2 alpha (TFAP2A) is observed in a significant proportion of OSCC cases. In this study, we aimed to elucidate the function of TFAP2A in the progression of OSCC and the related molecular signaling pathways. The role of RELA was predicted using bioinformatics analysis. The mRNA abundances of RELA, TFAP2A, and β-catenin were assessed by Western blot and quantitative real-timePCR. The relationship between RELA, TFAP2A, and β-catenin and their correlation with clinicopathological characteristics of OSCC was evaluated. The target of RELA and TFAP2A was identified by the chromatin immunoprecipitation as well as luciferase reporter assay. The colony formation assay and MTS assay were performed to determine the proliferative level of OSCC cells. OSCC cell motility was determined by Transwell assay and wound-healing assay. The protein expressions of epithelial-mesenchymal transition-associated factors were evaluated by Western blot. The expressions of RELA and TFAP2A were elevated in OSCC, and their expressions displayed a positive correlation. The expression levels of RELA and TFAP2A were found to be associated with TNM staging and lymphatic metastasis of OSCC patients. RELA upregulation promoted OSCC progression, as manifested by increased levels of proliferation, invasion, and migration of OSCC cells. We also demonstrated that RELA was directly bound to the promoter of TFAP2A transcription, which activated multiple malignant and metastatic phenotypes. Furthermore, TFAP2A activated the Wnt/β-catenin signaling by targeting the promoter regions of β-catenin. The study found that RELA is critical for promoting the progression of OSCC via the RELA-TFAP2A-Wnt/β-catenin signaling pathway. The RELA-TFAP2A-Wnt/β-catenin signaling pathway is a potential target for reducing the aggressiveness of OSCC.

Transcription factor AP-2α activates RNA polymerase III–directed transcription and tumor cell proliferation by controlling expression of c-MYC and p53

Deregulation of transcription factor AP2 alpha (TFAP2A) and RNA polymerase III (Pol III) products is associated with tumorigenesis. However, the mechanism underlying this event is not fully understood and the connection between TFAP2A and Pol III-directed transcription has not been investigated. Here, we report that TFAP2A functions as a positive factor in the regulation of Pol III-directed transcription and cell proliferation. We found TFAP2A is also required for the activation of Pol III transcription induced by the silencing of filamin A, a well-known cytoskeletal protein and an inhibitor in Pol III-dependent transcription identified previously. Using a chromatin immunoprecipitation technique, we showed TFAP2A positively modulates the assembly of Pol III transcription machinery factors at Pol III-transcribed gene loci. We found TFAP2A can activate the expression of Pol III transcription-related factors, including BRF1, GTF3C2, and c-MYC. Furthermore, we demonstrate TFAP2A enhances expression of MDM2, a negative regulator of tumor suppressor p53, and also inhibits p53 expression. Finally, we found MDM2 overexpression can rescue the inhibition of Pol III-directed transcription and cell proliferation caused by TFAP2A silencing. In summary, we identified that TFAP2A can activate Pol III-directed transcription by controlling multiple pathways, including general transcription factors, c-MYC and MDM2/p53. The findings from this study provide novel insights into the regulatory mechanisms of Pol III-dependent transcription and cancer cell proliferation.

UBE2T promotes breast cancer tumor growth by suppressing DNA replication stress.

Breast cancer is a leading cause of cancer-related deaths among women, and current therapies benefit only a subset of these patients. Here, we show that ubiquitin-conjugating enzyme E2T (UBE2T) is overexpressed in patient-derived breast cancer samples, and UBE2T overexpression predicts poor prognosis. We demonstrate that the transcription factor AP-2 alpha (TFAP2A) is necessary for the overexpression of UBE2T in breast cancer cells, and UBE2T inhibition suppresses breast cancer tumor growth in cell culture and in mice. RNA sequencing analysis identified interferon alpha-inducible protein 6 (IFI6) as a key downstream mediator of UBE2T function in breast cancer cells. Consistently, UBE2T inhibition downregulated IFI6 expression, promoting DNA replication stress, cell cycle arrest, and apoptosis and suppressing breast cancer cell growth. Breast cancer cells with IFI6 inhibition displayed similar phenotypes as those with UBE2T inhibition, and ectopic IFI6 expression in UBE2T-knockdown breast cancer cells prevented DNA replication stress and apoptosis and partly restored breast cancer cell growth. Furthermore, UBE2T inhibition enhanced the growth-suppressive effects of DNA replication stress inducers. Taken together, our study identifies UBE2T as a facilitator of breast cancer tumor growth and provide a rationale for targeting UBE2T for breast cancer therapies.

Long noncoding RNA CDKN2B-AS1 silencing protects against esophageal cancer cell invasion and migration by inactivating the TFAP2A/FSCN1 axis.

Esophageal cancer (EC) is the most aggressive malignancy in the gastrointestinal tract. Long noncoding RNA cyclin-dependent kinase inhibitor 2 B antisense RNA 1 (CDKN2B-AS1) is implicated in EC development. However, the specific mechanisms involved remain poorly defined. Therefore, this research aimed to explore the mechanism of action of CDKN2B-AS1 in EC. Quantitative real-time polymerase chain reaction was conducted to measure CDKN2B-AS1 expression in EC cells and western blotting was utilized to evaluate transcription factor AP-2 alpha (TFAP2A) and fascin actin-bundling protein 1 (FSCN1) expression. After gain-of-function and loss-of-function assays, cell proliferation, migration, invasion, apoptosis, and apoptosis-related protein expression were assessed using cell counting kit-8, scratch tests, Transwell assays, flow cytometry, and western blotting, respectively. The binding relationship between CDKN2B-AS1 and TFAP2A was assessed by RNA immunoprecipitation and RNA pull-down assays. The binding relationship between TFAP2A and FSCN1 was evaluated using dual-luciferase reporter and chromatin immunoprecipitation assays. Tumor xenografts from nude mice were used for in vivo verification. CDKN2B-AS1, TFAP2A, and FSCN1 were upregulated in EC cells. Mechanistically, CDKN2B-AS1 transcriptionally activated FSCN1 by recruiting TFAP2A to the FSCN1 promoter. Silencing CDKN2B-AS1 or TFAP2A suppressed EC cell proliferative, migrating, and invasive properties and augmented apoptosis. TFAP2A was bound to CDKN2B-AS1 and the FSCN1 promoter. Overexpression of TFAP2A or FSCN1 abolished the effects of CDKN2B-AS1-silencing on EC cell function. CDKN2B-AS1 silencing curtailed tumorigenesis in nude mice, which was nullified by the upregulation of TFAP2A or FSCN1. Our findings demonstrated the antioncogenic effects of silencing CDKN2B-AS1 in EC through inactivation of the TFAP2A/FSCN1 axis.

Circ_FURIN promotes trophoblast cell proliferation, migration and invasion in preeclampsia by regulating miR-34a-5p and TFAP2A.

Emerging evidence has shown that circular RNAs (circRNAs) play vital roles in the progression of diverse human diseases. However, the functions of circRNAs in preeclampsia (PE) are largely unknown. In this study, we aimed to explore the functions of the circRNA furin, paired basic amino acid cleaving enzyme (circ_FURIN) in PE development. qRT-PCR and western blot analyses were conducted to determine the levels of circ_FURIN, miR-34a-5p and transcription factor AP-2 alpha (TFAP2A). A Cell Counting Kit-8 (CCK-8) assay and a 5'-ethynyl-2'-deoxyuridine (EdU) incorporation assay were utilized to evaluate the cell proliferation ability. Transwell assays were adopted to estimate cell migration and invasion. A dual-luciferase reporter assay and an RNA pulldown assay were utilized to analyze the relationships among circ_FURIN, miR-34a-5p and TFAP2A. It was found that circ_FURIN was downregulated in PE placental tissues and hypoxia-treated placental trophoblast cells. Overexpression of circ_FURIN promoted trophoblast cell proliferation, migration and invasion under hypoxic conditions. Circ_FURIN functioned as the sponge for miR-34a-5p. MiR-34a-5p overexpression abrogated the effects of circ_FURIN on the proliferation, migration and invasion of trophoblast cells under hypoxic conditions. In addition, TFAP2A was demonstrated to be the target gene of miR-34a-5p. TFAP2A silencing ameliorated the promotive effects of miR-34a-5p inhibition on trophoblast cell proliferation, migration and invasion under hypoxic conditions. In conclusion, circ_FURIN enhanced trophoblast cell proliferation, migration and invasion under hypoxic conditions by elevating TFAP2A expression through sponging miR-34a-5p.

An integrated analysis of DNA promoter methylation, microRNA regulation, and gene expression in gastric adenocarcinoma.

BackgroundGastric adenocarcinoma (GAC), a common type of gastric cancer, poses a significant public health threat worldwide. This study aimed to determine the transcriptional regulatory mechanisms of GAC.MethodsHTSeq-FPKM raw data were obtained from The Cancer Genome Atlas Stomach Adenocarcinoma data collection. Subsequently, the limma package in R was used to identify differentially expressed genes (DEGs). Differentially methylated genes (DMGs), DEGs, and differentially expressed microRNAs (miRNAs) in normal, and tumor tissues of the same patients were screened and compared using R software tools. A functional enrichment analysis was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) for various DEGs, DMGs, promoter methylation, and miRNAs. DEG-specific methylation and transcription factors were analyzed using ENCODE ChIP-seq.ResultsDEGs were centrally modified by the histone trimethylation of lysine 27 on histone H3 (H3K27me3). Upstream transcription factors of DEGs were enriched in different ChIP-seq clusters, such as Forkhead Box M1, E2F Transcription Factor 4, and suppressor of zest 12. Integrated regulatory networks of DEGs, promoter methylation, and miRNAs were constructed. Two miRNAs (hsa-mir-1 and hsa-mir-133a) and four DEGs (A disintegrin and metalloproteinase domain 12, transcription factor AP-2 alpha, solute carrier family 5 member 7, and cadherin 19) separately played important roles in the integrated regulatory network. Therefore, these DEGs, DMGs, promoter methylation, and miRNAs may play an important role in GAC pathogenesis.ConclusionsIn summary, the present study results provide insights into the oncogenesis and progression of GAC, thus accelerating the development of novel targeted GAC therapies.

TFAP2A inhibits microRNA-126 expression at the transcriptional level and aggravates ischemic neuronal injury

Neuronal injury induced by cerebral ischemia poses a serious health risk globally, and there is no effective clinical therapy. This study was performed to investigate the role of transcription factor AP-2 alpha (TFAP2A) in cerebral ischemia, and the underlying mechanisms, using an in-vitro model (PC-12 cells) of oxygen–glucose deprivation (OGD), and an in-vivo model (rat) of transient global cerebral ischemia (tGCI). The results for CCK-8 and Hoechst staining showed that silencing of TFAP2A enhanced the viability and decreased the rate of apoptosis of PC12 cells subjected to OGD. ChIP assays were performed to evaluate the binding of TFAP2A to the promoter region of microRNA (miR)-126, and we found that TFAP2A inhibits the expression of miR-126. Further mechanistic investigation revealed that miR-126 targets polo like kinase 2 (PLK2), and that overexpression of PLK2 activates the IκBα–NF-κB signaling pathway and suppresses the growth of PC12 cells subjected to OGD. For our in-vivo assay, we used TTC staining to analyze the infarction area in the brain tissues of rats, and Nissl staining to evaluate the number of surviving brain neurons. The pathological conditions associated with neuronal injury in rat brain tissues were assessed by staining the tissues with hematoxylin–eosin. Our results indicate that TFAP2A downregulates miR-126, and thereby upregulates PLK2 and activates the IκBα–NF-κB pathway, which increased neuronal injury following cerebral ischemia.

MiR-204-5p/TFAP2A feedback loop positively regulates the proliferation, migration, invasion and EMT process in cervical cancer.

MicroRNAs (MiRNAs) have been clarified as crucial regulators of the pathological processes in various carcinomas in the past years. Interestingly, existing evidence has manifested that microRNA-204-5p (miR-204-5p) is engaged in the initiation and progression of multiple carcinomas. However, the potential of miR-204-5p in cervical cancer remains to be disentombed. This study focused on unraveling the detailed role of miR-204-5p in cervical cancer. MiR-204-5p exhibited a low level in cervical cancer cells. The functional assays demonstrated that miR-204-5p upregulation exerted suppressive impact on the functions of cervical cancer cells, including proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) process. Moreover, transcription factor AP-2 alpha (TFAP2A) was screened to be the most affected target gene by miR-204-5p, and TFAP2A was discovered to transcriptionally repress miR-204-5p in cervical cancer. The mutual regulation between TFAP2A and miR-204-5p was testified through molecular mechanism assays. Final rescued-function assays demonstrated that overexpression of TFAP2A could recover the suppressed cellular process caused by miR-204-5p upregulation. In conclusion, miR-204-5p/TFAP2A feedback loop promoted the proliferative and motorial capacities of cervical cancer cells. This finding suggested a novel modulatory loop of miR-204-5p/TFAP2A in cervical cancer, offering promising biomarkers for cervical cancer therapy.

TFAP2A is a novel regulator that modulates ferroptosis in gallbladder carcinoma cells via the Nrf2 signalling axis.

Ferroptosis is a recently identified form of controlled cell death generally associated with the accumulation of lipid-associated reactive oxygen species (ROS). However, the molecular mechanisms underlying ferroptosis have not been established. Microarray expression data for three human gallbladder carcinoma (GBC) and matched non-tumour specimens were downloaded from the Gene Expression Omnibus (GEO) repository. Candidate genes were filtered using bioinformatic analysis. After cell transfection, candidate gene impacts on cell proliferation, migration, invasion and ferroptosis (ferrous iron (Fe2+) and malondialdehyde (MDA) levels) were assessed. We screened 626 differentially expressed genes (DEGs) including 465 that were downregulated and 161 that were upregulated in the three tissue pairs. These DEGs were used to construct a protein-protein interaction (PPI) network. Functional enrichment analysis revealed the top three modules in the network and four hub genes. Transcription factor AP-2 alpha (TFAP2A) was screened and showed overexpression in The Cancer Genome Atlas (TCGA) digestive system tumour data and a relationship with clinical survival. In vitro, GBC exhibited upregulated expression of TFAP2A, whose inhibition reduced GBC cell proliferation, migration, and invasion. Fe2+ and MDA levels were elevated. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed TFAP2A enrichment in oxidative stress. Subsequent experiments demonstrated that TFAP2A silencing attenuated the expression of key genes associated with oxidative stress such as heme oxygenase 1 (HO-1), nuclear factor erythroid 2 like 2 (Nrf2), ferritin heavy chain 1 (FTH1) and NAD(P)H quinone dehydrogenase 1 (NQO1). Bioinformatic and experimental analyses reveal that TFAP2A plays a vital role in ferroptosis and hence is a potential therapeutic target for GBC treatment.

KMT2D Haploinsufficiency in Kabuki Syndrome Impairs Differentiation of Neural Crest Cells

ObjectiveKabuki Syndrome (KS), caused by haploinsufficiency in lysine methyltransferase KMT2D, is a multi‐system disease characterized by distinct facial dysmorphisms, mild‐to‐moderate intellectual disability, renal and cardiac abnormalities, short stature and humoral immune deficiency. Many of the tissues in which these abnormalities manifest are derived from or developmentally modulated by the neural crest cell (NCC) lineage. This study’s objective was to determine which features of the KS phenotype can be directly linked to genetic and epigenetic dysregulation in KMT2D haploinsufficient NCCs.MethodsPeripheral blood mononuclear cells from a human donor haploinsufficient for KMT2D (KS patient, KMT2D+/−) or wild type (WT) control were used to generate induced pluripotent stem (iPS) cells. WT and KS NCCs were generated from WT and KS iPS cells, respectively. Whole transcriptome RNA sequencing (RNAseq) was performed on iPS cells (WT and KS) and NCCs (WT and KS) to characterize differential gene expression. Chromatin immunoprecipitation sequencing (ChIPseq) of histone 3 lysine 4 trimethylation (H3K4Me3) and histone 3 lysine 27 acetylation (H3K27Ac) was performed on NCCs (WT and KS) to characterize differential histone modifications at gene regulatory elements. Analysis of RNAseq and ChIPseq data was performed using BioWardrobe platform.ResultsGene set enrichment analysis of transcriptome data indicates that, compared to WT NCCs, KMT2D haploinsufficient NCCs have ectopic expression of several genes critical to normal NCC organization, morphogenesis and tissue differentiation, including NCC transcriptional repressor Msh homeobox 2 (MSX2). Analysis also identified several genes with decreased expression in KMT2D+/− NCCs, including transcription factor AP‐2 alpha (TFAP2A), which serves as a master regulator of NCC lineage progression. ChIPseq data indicate differential enrichment of H3K4Me3 and H3K27Ac histone modifications at the TFAP2A locus in WT compared to KS NCCs, which suggests that TFAP2A may have a direct role in the impaired differentiation of NCCs in KS. Further studies to explore the roles of TFAP2A and other genes identified by these data are underway.ConclusionThese results show that KMT2D haploinsufficiency impairs differentiation of the NCC lineage cells through epigenetic and transcriptomic dysregulation, thus supporting the classification of KS as a neurocristopathy.Support or Funding InformationNIH T32 Training Grant 5T32AI060515‐14 (WJL), R35 DE027557 (SB), Center for Pediatric Genomics Cincinnati Research Foundation (AWL).

The comprehensive investigation of transcription factor AP-2 alpha in lung adenocarcinoma.

BackgroundTranscription factor AP-2 alpha (TFAP2A) has been reported to participate in various tumors. However, the transcriptional levels and prognostic values of TFAP2A remain elusive in lung adenocarcinoma (LUAD). The purpose of the present study was to investigate the impact of the TFAP2A in LUAD.MethodsThe transcriptional levels and prognostic effects of TFAP2A were explored in patients with LUAD using various online databases, including the GEPIA, Oncomine, and Kaplan-Meier plotter databases. Meanwhile, meta-analyses were performed to verify the expression levels and prognostic effects of TFAP2A in LUAD using the Lung Cancer Explorer (LCE) database. In addition, target genes of TFAP2A were identified in the Animal TFDB3.0 dataset.ResultsOur comprehensively study indicated the mRNA expression levels of TFAP2A in LUAD were significantly higher than that of normal controls. In the survival analyses, higher TFAP2A levels were related to the shortened survival time of patients with LUAD. Meanwhile, the meta-analyses based on the LCE database also suggested the overexpressed TFAP2A led to worsen prognosis of patients with LUAD. Finally, the cell division cycle 6 (CDC6) and aurora kinase A (AURKA) were regarded as two target genes of TFAP2A.ConclusionsWe have performed comprehensive analyses for TFAP2A in patients with LUAD. Patients with higher TFAP2A levels demonstrated worsen prognosis than those with lower TFAP2A levels. The CDC6 and AURKA might be two target genes of TFAP2A. Further molecular biological experiments were required to study the mechanisms of TFAP2A in LUAD.

LINC00160 Mediates Sunitinib Resistance <i>via</i> SAA1/STAT3 Signaling Pathways and ABCB1 Transmembrane Transport in Renal Cell Carcinoma

Patients with advanced renal cell carcinoma (RCC) who are resistant to sunitinib currently have limited clinical options for treatment. Therefore, it is necessary to explore the biological basis of sunitinib resistance and to uncover new targets for the intervention of sunitinib resistance. In this study, we identified that long non-coding RNA 160 (LINC00160) was associated with sunitinib resistance in RCC. Resistant RCC cells highly expressed LINC00160 to recruit transcriptional factor AP-2 alpha (TFAP2A), which bound to serum amyloid A1 (SAA1) promoter regions and activated its expression. On one hand, SAA1 linked to ATP binding cassette subfamily B member 1 (ABCB1) protein, which facilitated sunitinib cellular efflux and diminished drug accumulation. On the other hand, SAA1 stimulated JAK-STAT signaling pathways, which countered cellular survival inhibition from drug. All these regulatory networks were well organized and collaborated, thus promoting sunitinib resistance in renal cell carcinoma. LINC00160 mediates sunitinib resistance via SAA1/STAT3 signaling pathways and ABCB1 transmembrane transport in RCC, which offers an opportunity for targeted intervention and molecular therapies in the future. Funding Statement: Our study was supported by the Key Research and Development Plan in China (Grant No. 2017YFB1303100), the National Natural Science Foundation of China (Grant No. 81927807, 81874090, 81672528, 81773282 & 81972630) and Individual Innovative Research Funding of Union Hospital (Grant No. 02.03.2019-156). Declaration of Interests: The authors declared no conflicts of interest. Ethics Approval Statement: This study was approved by the Ethics Committees of Huazhong University of Science and Technology, and all aspects of the study comply with the criteria established by the Declaration of Helsinki. Animal experiments were approved by the Institutional Animal Use and Care Committee of Tongji Medical College, Huazhong University of Science and Technology (HUST).

Repression of transcription factor AP-2 alpha by PPAR\u03b3 reveals a novel transcriptional circuit in basal-squamous bladder cancer

The discovery of bladder cancer transcriptional subtypes provides an opportunity to identify high risk patients, and tailor disease management. Recent studies suggest tumor heterogeneity contributes to regional differences in molecular subtype within the tumor, as well as during progression and following treatment. Nonetheless, the transcriptional drivers of the aggressive basal-squamous subtype remain unidentified. As PPARɣ has been repeatedly implicated in the luminal subtype of bladder cancer, we hypothesized inactivation of this transcriptional master regulator during progression results in increased expression of basal-squamous specific transcription factors (TFs) which act to drive aggressive behavior. We initiated a pharmacologic and RNA-seq-based screen to identify PPARɣ-repressed, basal-squamous specific TFs. Hierarchical clustering of RNA-seq data following treatment of three human bladder cancer cells with a PPARɣ agonist identified a number of TFs regulated by PPARɣ activation, several of which are implicated in urothelial and squamous differentiation. One PPARɣ-repressed TF implicated in squamous differentiation identified is Transcription Factor Activating Protein 2 alpha (TFAP2A). We show TFAP2A and its paralog TFAP2C are overexpressed in basal-squamous bladder cancer and in squamous areas of cystectomy samples, and that overexpression is associated with increased lymph node metastasis and distant recurrence, respectively. Biochemical analysis confirmed the ability of PPARɣ activation to repress TFAP2A, while PPARɣ antagonist and PPARɣ siRNA knockdown studies indicate the requirement of a functional receptor. In vivo tissue recombination studies show TFAP2A and TFAP2C promote tumor growth in line with the aggressive nature of basal-squamous bladder cancer. Our findings suggest PPARɣ inactivation, as well as TFAP2A and TFAP2C overexpression cooperate with other TFs to promote the basal-squamous transition during tumor progression.