AP-2α Protein Research Articles

Abstract 5647: Investigating the role of AP-2α in melanomagenesis

Abstract Introduction: Transcription factor AP-2α (encoded by the TFAP2A gene) is a protein involved in the early development of multiple tissues, particularly the neural crest. Melanocytes are derived from the neural crest and their development appears to rely on AP-2α and its paralogues, along with master regulator MITF. Prior studies have indicated that AP-2α is a crucial regulator of melanoma metastasis. However, it is unclear if AP-2α plays a role in melanomagenesis. We sought to understand the role of AP-2α in melanomagenesis, hypothesizing that knock out (KO) of TFAP2A would suppress malignant potential in vitro and downregulate genetic pathways linked to malignant transformation. Methods: TFAP2A KO was generated with a single-guide RNA CRISPR/Cas9 system targeting exon 3 of the gene in the human melanoma cell line A375. Tumorigenesis phenotype was assessed by evaluating contact-independent colony formation with the in vitro soft agar assay. To identify downstream regulatory programs controlled by TFAP2A, we then utilized RNA-seq and Gene Set Enrichment Analysis (GSEA, San Diego). RNA-seq was performed on four clones each for wildtype and TFAP2A KO samples and the resulting data was processed using GSEA to identify gene sets modified after TFAP2A KO. Results: Targeting of TFAP2A with CRISPR/Cas9 effectively knocked out AP-2α protein expression in the A375 melanoma cell line. Colony formation on soft agar assay was significantly decreased after TFAP2A KO (P < 0.01). RNA-seq revealed significant differential expression of 11,195 genes after TFAP2A KO. Gene sets with negative enrichment after TFAP2A KO included those overall higher in tumors versus normal tissue (FWER P = 0.003) and multiple signaling pathways related to malignant transformation such as TGF-β (FWER P = 0.003), MYC (FWER P = 0.009), KRAS (FWER P = 0.03), and Notch (FWER P = 0.043). Conclusions: Our data suggests that TFAP2A plays a role in cutaneous melanomagenesis. TFAP2A may represent a therapeutic target for melanoma, though further pre-clinical studies are needed. We plan to further test this hypothesis in a melanoma mouse model with conditional TFAP2A KO under the control of inducible Cre recombinase driven by the mouse tyrosinase gene promoter. Citation Format: Nicholas Bartschat, Jeremy Chang, Mohammed Suraju, Ryan Nagel, Zhijie Li, Jeffrey White, Colin Kenny, Ronald Weigel. Investigating the role of AP-2α in melanomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5647.

Epigenetic Up-Regulation of ADAMTS4 in Sympathetic Ganglia is Involved in the Maintenance of Neuropathic Pain Following Nerve Injury.

Sympathetic axonal sprouting into dorsal root ganglia is a major phenomenon implicated in neuropathic pain, and sympathetic ganglia blockage may relieve some intractable chronic pain in animal pain models and clinical conditions. These suggest that sympathetic ganglia participated in the maintenance of chronic pain. However, the molecular mechanism underlying sympathetic ganglia-mediated chronic pain is not clear. Here, we found that spared nerve injury treatment upregulated the expression of ADAMTS4 and AP-2α protein and mRNA in the noradrenergic neurons of sympathetic ganglia during neuropathic pain maintenance. Knockdown the ADAMTS4 or AP-2α by injecting specific retro scAAV-TH (Tyrosine Hydroxylase)-shRNA ameliorated the mechanical allodynia induced by spared nerve injury on day 21 and 28. Furthermore, chromatin immunoprecipitation and coimmunoprecipitation assays found that spared nerve injury increased the recruitment of AP-2α to the ADAMTS4 gene promoter, the interaction between AP-2α and histone acetyltransferase p300 and the histone H4 acetylation on day 28. Finally, knockdown the AP-2α reduced the acetylation of H4 on the promoter region of ADAMTS4 gene and suppressed the increase of ADAMTS4 expression induced by spared nerve injury. Together, these results suggested that the enhanced interaction between AP-2α and p300 mediated the epigenetic upregulation of ADAMTS4 in sympathetic ganglia noradrenergic neurons, which contributed to the maintenance of spared nerve injury induced neuropathic pain.

Impact of selective serotonin reuptake inhibitors on neural crest stem cell formation

The use of antidepressants in pregnant women is rising, with rates up to 7.5% in the United States. Selective serotonin reuptake inhibitors (SSRIs) are currently the most common antidepressant prescribed to pregnant women. The teratogenic effects of SSRI exposure are debated because of discrepancies in epidemiological studies. As an alternative to epidemiological and animal studies, human embryonic stem cell research (hESC) provides a human-based experimental model to examine the risks of prenatal SSRI exposure. Neural crest stem cells (NCSCs) play an important role in craniofacial and cardiac development as precursors to craniofacial bones and heart septa. This study examines the effects of paroxetine (Paxil) and sertraline (Zoloft) exposure on proliferation, migration, and AP-2α protein expression of NCSC in vitro. hESCs were exposed to paroxetine and sertraline at three concentrations while undergoing directed differentiation into NCSCs. Our results indicate exposure to paroxetine significantly increased proliferation, migration, and AP-2α protein expression in NCSCs. Exposure to sertraline significantly decreased proliferation and significantly increased AP-2α protein expression in NCSC. This evidence suggests paroxetine and sertraline alter normal NCSC behavior and may thereby disrupt cardiac and craniofacial development.

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

Abstract 3395: Role of the activating protein 2 transcription factor in regulating cell invasion and migration in malignant glioma

Abstract Background: Malignant gliomas (MG) or high-grade astrocytomas are highly infiltrative brain tumors. Our previous work has shown that brain fatty acid binding protein (B-FABP) regulates the migratory properties of MG cells. B-FABP expression can be controlled by transcription factors such as NF1 and AP2. AP2 is a family of transcription factors involved in the regulation of genes responsible for cellular growth and differentiation during early development. Five AP2 genes have been identified in mice and humans: AP2α, AP2β, AP2γ, AP2δ and AP2ε. AP2 transcription factors are predominantly localized in the nucleus where they bind as homodimers or heterodimers to the consensus sequence GCCNNNGGC. Recent studies have shown that AP2α expression is inversely correlated with astrocytoma grade and that there are higher levels of AP2α in the cytoplasm of high-grade astrocytomas compared to low-grade astrocytomas. Objective: To study the regulation of B-FABP expression in response to AP2 in MG. Methodology: A panel of 10 MG cell lines was used to examine AP2α protein levels. Five of these cell lines were B-FABP +ve and five were B-FABP-ve. The effect of AP2 expression on endogenous B-FABP was examined by transfecting AP2 expression constructs into the B-FABP +ve U251 MG cell line. B-FABP levels were examined by western blot analysis and the subcellular localization of AP2 was studied by immunofluorescence analysis. Results: Western blot analysis indicates that AP2α is expressed in four out of five B-FABP-ve MG cell lines but in only one of the five B-FABP +ve MG cell lines tested. AP2 overexpression studies indicate that B-FABP levels are significantly reduced upon overexpression of AP2α, AP2β or AP2ε. Immunofluorescence data suggest preferential localization of AP2β in the cytoplasm of B-FABP +ve U251 MG cells. Our preliminary result shows difference in affinity towards heterodimerization between AP2 family members. Conclusion and future directions: AP2 is a negative regulator of B-FABP expression. Exclusion of AP2 from the nucleus allows transcription of B-FABP in MG cells, which in turn leads to increased cell migration. Experiments will be carried out to see the effect of AP2 heterodimerization on regulation of B-FABP transcription. Phosphorylation studies will be carried out to see whether AP2 phosphorylation affects its subcellular localization. This study may lead to new approaches to reduce B-FABP expression in MG, thereby reducing or preventing cell migration and tumor infiltration. Citation Format: Saket Jain, Ho Yin Poon, Roseline Godbout. Role of the activating protein 2 transcription factor in regulating cell invasion and migration in malignant glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3395. doi:10.1158/1538-7445.AM2014-3395

Repression of integrin-linked kinase by antidiabetes drugs through cross-talk of PPARγ- and AMPKα-dependent signaling: Role of AP-2α and Sp1

Nasopharyngeal carcinoma (NPC) is one of the most common cancers of the head and neck, particularly in Southern China and Southeast Asia with high treatment failure due to the development of local recurrence and distant metastasis. The molecular mechanisms related to the progression of NPC have not been fully understood. In this study, we showed that antidiabetes drugs rosiglitazone and metformin inhibit NPC cell growth through reducing the expression of integrin-linked kinase (ILK). Blockade of PPARγ and AMPKα overcame the effects of rosiglitazone and metformin on ILK protein. Importantly, overexpression of ILK abrogated the effect of rosiglitazone and metformin on NPC cell growth. Furthermore, these agents reduced ILK promoter activity, which was not observed in AP-2α, but not Sp1 site mutation in ILK gene promoter. In addition, silencing of AP-2α or overexpression of Sp1 reversed the effect of these agents on ILK protein expression and cell growth. Chromatin immunoprecipitation (ChIP) assay showed that rosiglitazone induced AP-2α, while metformin reduced Sp1 protein binding to the DNA sequences in the ILK gene promoter. Intriguingly, overexpression of Sp1 abolished the effect of rosiglitazone on AP-2α protein expression. Collectively, we show that rosiglitazone and metformin inhibit ILK gene expression through PPARγ- and AMPKα-dependent signaling pathways that are involved in the regulation of AP-2α and Sp1 protein expressions. The effect of combination of rosiglitazone and metformin demonstrates greater extent than single agent alone. The cross-talk of PPARγ and AMPKα signaling enhances the synergistic effects of rosiglitazone and metformin. This study unveils novel mechanisms by which oral antidiabetes drugs inhibit the growth of human NPC cells.

Analysis of TFAP2A mutations in Branchio-Oculo-Facial Syndrome indicates functional complexity within the AP-2α DNA-binding domain

Multiple lines of evidence indicate that the AP-2 transcription factor family has an important regulatory function in human craniofacial development. Notably, mutations in TFAP2A, the gene encoding AP-2α, have been identified in patients with Branchio-Oculo-Facial Syndrome (BOFS). BOFS is an autosomal-dominant trait that commonly presents with facial clefting, eye defects and branchial skin anomalies. Examination of multiple cases has suggested either simple haploinsufficiency or more complex genetic causes for BOFS, especially as the clinical manifestations are variable, with no clear genotype-phenotype correlation. Mutations occur throughout TFAP2A, but mostly within conserved sequences within the DNA contact domain of AP-2α. However, the consequences of the various mutations for AP-2α protein function have not been evaluated. Therefore, it remains unclear if all BOFS mutations result in similar changes to the AP-2α protein or if they each produce specific alterations that underlie the spectrum of phenotypes. Here, we have investigated the molecular consequences of the mutations that localize to the DNA-binding region. We show that although individual mutations have different effects on DNA binding, they all demonstrate significantly reduced transcriptional activities. Moreover, all mutant derivatives have an altered nuclear:cytoplasmic distribution compared with the predominantly nuclear localization of wild-type AP-2α and several can exert a dominant-negative activity on the wild-type AP-2α protein. Overall, our data suggest that the individual TFAP2A BOFS mutations can generate null, hypomorphic or antimorphic alleles and that these differences in activity, combined with a role for AP-2α in epigenetic events, may influence the resultant pathology and the phenotypic variability.

A miR-200b/200c/429-binding site polymorphism in the 3' untranslated region of the AP-2α gene is associated with cisplatin resistance.

The transcription factor AP-2α functions as a tumor suppressor by regulating various genes that are involved in cell proliferation and apoptosis. Chemotherapeutic drugs including cisplatin induce post-transcriptionally endogenous AP-2α, which contributes to chemosensitivity by enhancing therapy-induced apoptosis. microRNAs (miRNAs) miR-200b, miR-200c and miR-429 (miR-200b/200c/429) are up-regulated in endometrial and esophageal cancers, and their overexpression correlates with resistance to cisplatin treatment. Using computational programs, we predicted that the 3′ untranslated region (UTR) of AP-2α gene contains a potential miRNA response element (MRE) for the miR-200b/200c/429 family, and the single nucleotide polymorphism (SNP) site rs1045385 (A or C allele) resided within the predicted MRE. Luciferase assays and Western blot analysis demonstrated that the miR-200b/200c/429 family recognized the MRE in the 3′ UTR of AP-2α gene and negatively regulated the expression of endogenous AP-2α proteins. SNP rs1045385 A>C variation enhanced AP-2α expression by disrupting the binding of the miR-200b/200c/429 family to the 3′ UTR of AP-2α. The effects of the two polymorphic variants on cisplatin sensitivity were determined by clonogenic assay. The overexpression of AP-2α with mutant 3′ UTR (C allele) in the endometrial cancer cell line HEC-1A, which has high levels of endogenous miR-200b/200c/429 and low levels of AP-2α protein, significantly increased cisplatin sensitivity, but overexpression of A allele of AP-2α has no significant effects, compared with mock transfection. We concluded that miR-200b/200c/429 induced cisplatin resistance by repressing AP-2α expression in endometrial cancer cells. The SNP (rs1045385) A>C variation decreased the binding of miR-200b/200c/429 to the 3′ UTR of AP-2α, which upregulated AP-2α protein expression and increased cisplatin sensitivity. Our results suggest that SNP (rs1045385) may be a potential prognostic marker for cisplatin treatment.

Aurora-A Interacts with AP-2α and Down Regulates Its Transcription Activity

Aurora-A is a serine/threonine protein kinase and plays an important role in the control of mitotic progression. Dysregulated expression of Aurora-A impairs centrosome separation and maturation, which lead to disrupted cell cycle progression and tumorigenesis. However, the molecular mechanism by which Aurora-A causes cell malignant transformation remains to be further defined. In this report, using transcription factors array and mRNA expression profiling array, we found that overexpression of Aurora-A suppressed transcription activity of AP-2α, a tumor suppressor that is often downregulated in variety of tumors, and inhibited expression of AP-2α-regulated downstream genes. These array-based observations were further confirmed by microwell colorimetric TF assay and luciferase reporter assay. Downregulated transcription activity of AP-2α by Aurora-A was found to be associated with reduced AP-2α protein stability, which appeared to be mediated by Aurora-A enhanced ubiquitin-dependent proteasomal degradation of AP-2α protein. Interestingly, Aurora-A-mediated AP-2α degradation was likely dependent Aurora-A kinase activity since inhibition of Aurora-A kinase activity was able to rescue Aurora-A-induced degradation of AP-2α. Moreover, we defined a physical interaction between Aurora-A and AP-2α, and such interaction might bridge the suppressive effect of Aurora-A on AP-2α protein stability. These findings provide new insights into molecular mechanism by which Aurora-A acts as an oncogenic molecule in tumor occurrence and malignant development.

Transcription factor AP2 regulates human inhibin subunit gene expression during in vitro trophoblast differentiation

During human trophoblast differentiation, inhibin α subunit mRNA expression and protein secretion are increased. To understand how inhibin α subunit gene was regulated during syncytialization, we firstly cloned the inhibin α promoter and found a region with transcriptional activity related to the differentiation state. In this paper, we identified this protein and defined its DNA-binding site. Protein purification and identification were done by DNA affinity chromatography followed by mass spectrometry and western blotting. In order to confirm the identity of the protein, characterize its DNA-binding properties and to measure its expression during in vitro trophoblast differentiation, gel retardation assays and real-time polymerase chain reaction were done. We found that the cytotrophoblastic protein interacting with the inhibin α promoter was the transcription factor activating protein 2 (AP2). Western blotting using specific antibodies against AP2α and AP2γ confirmed that AP2α was the main subtype present in trophoblast cells, while AP2γ was barely detectable. Supershift experiments indicated that these two factors were able to bind to the sequence 5'-GCCtcaAGC-3'. We also observed an increase in AP2α mRNA and protein during in vitro trophoblast differentiation correlated with an increase in inhibin α subunit gene expression. Furthermore, AP2α and AP2γ overexpression in these cells was responsible for an increase in inhibin α subunit mRNA expression. We conclude that AP2 regulates the inhibin α subunit gene expression during trophoblast differentiation and may be a key regulator of syncytialization.

AP-2α expression and cell apoptosis of the lung tissue of rats with COPD and ECV304 cells stimulated by cigarette smoke extract

An increasing body of evidence suggests that apoptosis of structural cells in the lung might be an important upstream event in the pathogenesis of chronic obstructive pulmonary disease (COPD). AP-2α is one of the important transcription factors involved in the modulation of apoptosis in carcinogenesis and idiopathic-dilated cardiomyopathy. The relationship between AP-2α and apoptosis in COPD remains to be elucidated. The aim of the present study was to investigate the expression of AP-2α in the lung tissues of rats with COPD induced by smoking and its possible protective effect on cigarette smoke extract (CSE) induced endothelial cell apoptosis. Sprague-Dawley rats (n = 24) were randomly assigned to normal and COPD groups. The COPD group was exposed to smoke from 20 commercial unfiltered cigarettes for 80 d before morphological assessment of the lung tissue was performed. The expression of AP-2α in lung tissues was measured by Western blotting. To demonstrate the relationship between apoptosis and AP-2α, in vitro cell experiments were carried out. Cells were treated with different concentrations of CSE before proliferation was measured by MTT. Apoptosis was then determined by Hoechst staining and the expression of cleaved caspase-3 and AP-2α by Western blotting over time following treatment with 5% CSE. Cells were then infected with an AP-2α adenovirus vector and the expression of cleaved caspase-3 and AP-2α was compared to the control groups by Western blotting. The COPD group showed larger air spaces and significant decrease of FEV0.3/FVC compared with the rats in the control group (P<0.05). The expression of AP-2α was significantly higher in the lung tissue of rats with COPD compared with those of controls (P<0.05). In the ECV304 cells, CSE induced apoptosis (P<0.01) and caspase-3 activation in a time-dependent manner and reduced the cell proliferation rate in a dose-dependent manner (P<0.005). Moreover, 5% CSE treatment increased endogenous AP-2α protein expression. AP-2α overexpression inhibited 5% CSE-induced cell apoptosis and activated caspase-3 expression (P<0.05) AP-2α protects ECV304 cells against CSE-induced apoptosis and may play an important role in smoking induced-apoptosis in COPD.

Abstract 2353: CREB regulates AP-2α transcription to determine the malignant phenotype of melanoma

Abstract The Activator protein-2 (AP-2) family of transcription factors consists of five different proteins which are involved in the regulation of cell proliferation, differentiation, apoptosis and carcinogenesis. Among these proteins, AP-2α has been suggested to function as a tumor suppressor in many cancers including melanoma. We have previously shown that in melanoma cell lines, acquisition of the metastatic phenotype is associated with loss of AP-2α expression and gain in expression of cAMP-responsive element (CRE)-binding protein (CREB). This inverse correlation is associated with increase of melanoma invasion and metastasis resulting in overexpression of the melanoma cell adhesion molecule MCAM/MUC18 and protease-activated receptor 1 (PAR-1). However, the molecular mechanism involved in AP-2α regulation during melanoma progression remains unknown. Analyses of AP-2α promoter for transcription factor binding sites showed two distinct CRE-like sites as well as four E2F1 sites. Herein, we propose that the inhibition of AP-2α during the transition from the Radial Growth Phase (RGP) to the Vertical Growth Phase (VGP) occurs at the transcriptional level and is regulated through binding of CREB and E2F1. Hence, AP-2α protein expression was found to be increased after utilizing small hairpin RNA (shRNA) targeting CREB in two human metastatic melanoma cell lines, A375SM and C8161. Interestingly, E2F1 expression level was found to be reduced by 50% after CREB silencing in both cell lines. We also found a 20-fold increase in the luciferase activity driven by the AP-2α promoter after silencing CREB in both cell lines. In addition, point mutations at CRE-like binding sites as well as E2F1 increased the AP-2α promoter activity by more then 5-fold. To further establish the involvement of E2F1 in AP-2α regulation, we transiently overexpressed E2F1 in a low-metastatic melanoma cell line, SB-2. The AP-2α protein level as well as the promoter activity was significantly inhibited as compared to control. Finally, using Chromatin Immunoprecipitation (ChIP) assays in both CREB silenced cell lines, we were able to confirm that CREB and E2F1 bind to the AP-2α promoter thereby inhibiting its expression. Taken together, our studies demonstrate that CREB regulates AP-2α expression, thus, contributing to the metastatic phenotype of melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2353.

Abstract 1230: Nicotine induces expression of integrin-linked kinase in human lung carcinoma cells through A7 nAChR-mediated inhibition of AP-2A

Abstract We previously demonstrated that nicotine stimulates non small cell lung carcinoma (NSCLC) cell proliferation through nicotinic acetylcholine receptor (nAChR)-mediated induction of fibronectin expression. However, the mechanisms by which this agent affects human lung cancer occurrence and progression remain incompletely elucidated. Here, we show that nicotine increases the expression of integrin-linked kinase (ILK), a unique intracellular adaptor and kinase that has been implicated in the regulation of cancer cell growth/survival. This effect was blocked by the α7 nAChR antagonist, α-bungarotoxin, and by α7 nAChR siRNA. Silencing of ILK blocked the stimulatory effect of nicotine on phosphorylation of the PI3-K downstream target Akt and on cell growth. Furthermore, nicotine increased ILK gene promoter activity through inhibition of transcription factor AP-2α protein expression. Silencing of AP-2α enhanced, while exogenous expression of AP-2α attenuated, the stimulatory effect of nicotine on ILK expression. These findings appear to be relevant in vivo since we found that nicotine treatment increased tumor growth in animal models. Collectively, our results demonstrate that nicotine increases ILK gene expression through α7 nAChR-mediated inhibition of AP-2α followed by stimulation of PI3-K/Akt signaling. In turn, this leads to increased NSCLC cell growth. This study unveils a novel mechanism by which nicotine promotes human lung carcinoma cell growth/survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1230.

Nicotine Stimulates PPARβ/δ Expression in Human Lung Carcinoma Cells through Activation of PI3K/mTOR and Suppression of AP-2α

We previously showed that nicotine stimulates non-small cell lung carcinoma (NSCLC) cell proliferation through nicotinic acetylcholine receptor (nAChR)-mediated signals. Activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) has also been shown to induce NSCLC cell growth. Here, we explore the potential link between nicotine and PPARbeta/delta and report that nicotine increases the expression of PPARbeta/delta protein; this effect was blocked by an alpha7 nAChR antagonist (alpha-bungarotoxin), by alpha7 nAChR short interfering RNA, and by inhibitors of phosphatidylinositol 3-kinase (PI3K; wortmannin and LY294002) and mammalian target of rapamycin (mTOR; rapamycin). In contrast, this effect was enhanced by PUN282987, an alpha7 nAChR agonist. Silencing of PPARbeta/delta attenuated the stimulatory effect of nicotine on cell growth, which was overcome by transfection of an exogenous PPARbeta/delta expression vector. Of note, nicotine induced complex formation between alpha7 nAChR and PPARbeta/delta protein and increased PPARbeta/delta gene promoter activity through inhibition of AP-2alpha as shown by reduced AP-2alpha binding using electrophoretic gel mobility shift and chromatin immunoprecipitation assays. In addition, silencing of Sp1 attenuated the effect of nicotine on PPARbeta/delta. Collectively, our results show that nicotine increases PPARbeta/delta gene expression through alpha7 nAChR-mediated activation of PI3K/mTOR signals that inhibit AP-2alpha protein expression and DNA binding activity to the PPARbeta/delta gene promoter. Sp1 seems to modulate this process. This study unveils a novel mechanism by which nicotine promotes human lung carcinoma cell growth.

Overexpression of transcription factor AP-2 stimulates the P A promoter of the human uracil-DNA glycosylase (UNG) gene through a mechanism involving derepression

The P A promoter in the human uracil-DNA glycosylase gene ( UNG) directs expression of the nuclear form (UNG2) of UNG proteins. Using a combination of promoter deletion and mutation analyses, and transient transfection of HeLa cells, we show that repressor and derepressor activities are contained within the region of DNA marked by P A. Footprinting analysis and electrophoretic mobility shift assays of P A and putative AP-2 binding regions with HeLa cell nuclear extract and recombinant AP-2α protein indicate that AP-2 transcription factors are central in the regulated expression of UNG2 mRNA. Chromatin immunoprecipitation with AP-2 antibody demonstrated that endogenous AP-2 binds to the P A promoter in vivo. Overexpression of AP-2α, -β or -γ all stimulated expression from a P A-luciferase reporter gene construct approximately 3- to 4-fold. Interestingly, an N-terminally truncated AP-2α, lacking the activation domain but retaining the DNA binding and dimerization domains, stimulated P A to a level approaching that of full-length AP-2, suggesting that AP-2 overexpression stimulates P A activity by a mechanism involving derepression rather than activation, possibly by neutralizing an inhibitory effect of endogenous AP-2 or AP-2-like factors.

Fish Oil Inhibits Human Lung Carcinoma Cell Growth by Suppressing Integrin-Linked Kinase

We previously showed that synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) ligands inhibit non-small cell lung carcinoma (NSCLC) cell growth through multiple signaling pathways. Here, we show that dietary compounds, such as fish oil (which contains certain kinds of fatty acids like omega3 and omega6 polyunsaturated fatty acids), also inhibit NSCLC cell growth by affecting PPARgamma and by inhibiting the expression of integrin-linked kinase (ILK). Exogenous expression of ILK overcame, whereas silencing ILK enhanced the inhibitory effect of fish oil on cell growth. The inhibitor of p38 mitogen-activated protein kinase, SB239023, abrogated the inhibitory effect of fish oil on ILK expression, whereas the inhibitor of extracellular signal-regulated kinase, PD98059, had no effect. Transient transfection experiments showed that fish oil reduced ILK promoter activity, and this effect was abolished by AP-2alpha small interfering RNA and SB239023 and by deletion of a specific portion of the ILK gene promoter. Western blot analysis and gel mobility shift assay showed that fish oil significantly induced AP-2alpha protein expression and AP-2 DNA-binding activity in the ILK gene promoter and that this was dependent on PPARgamma activation. Blockade of AP-2alpha abrogated the effect of fish oil on ILK expression and on cell growth, whereas exogenous expression of AP-2alpha enhanced cell growth in the setting of fish oil exposure. Taken together, these findings show that fish oil inhibits ILK expression through activation of PPARgamma-mediated and p38 mitogen-activated protein kinase-mediated induction of AP-2alpha. In turn, this leads to inhibition of NSCLC cell proliferation. This study unveils a novel mechanism by which fish oil inhibits human lung cancer cell growth.

RETRACTED: Chronic Administration of Carbamazepine Down-regulates AP-2 DNA-binding Activity and AP-2α Protein Expression in Rat Frontal Cortex

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of author Stanley Rapoport, with approval from Biological Psychiatry Editor, John H. Krystal, MD. The National Institutes of Health has found that Dr. Jagadeesh S. Rao engaged in research misconduct by falsifying data in Figures 1, 3, and 5 of the aforementioned manuscript. No other authors were implicated in the data falsification

Interactions between c‐Myc/AP2 α regulate cell growth and survival

c-Myc and AP2α are both important transcription factors that are involved in cell proliferation, differentiation, carcinogenesis and apoptosis. c-Myc binds to a central CAC(G/A)TG sequence while AP2α binds to GCCN3-5GGC. It has been shown that AP2α can inhibit c-Myc DNA binding activity through either competing for the DNA binding site, or through protein/protein interactions. Since the interaction sites are the DNA binding domains of both proteins, we hypothesized conversely that c-Myc would block AP2α DNA binding activity. DNA binding activity of recombinant AP2α protein was determined by electrophoresis mobility shift assay (EMSA) in the presence and absence of recombinant c-Myc protein. Our EMSA showed that c-Myc blocked AP2α DNA binding activity stoichiometrically. However, if DNA probe was incubated with AP2α protein prior to adding c-Myc protein, such inhibitory effect was greatly alleviated. In addition to wild type AP2, a C-terminal truncated AP2 protein, AP2S420X was also tested, and displayed similar behavior. Studies to examine the effect of the c-Myc:AP2α interaction in vivo were conducted in two cell lines: HaCaT cells, which have abundant active AP2α, and MB-MDA-231 cells, which does not express AP2α. Both cell lines were infected with adenoviral expression constructs as follows: control, AP2α, c-Myc and AP2α+c-Myc, respectively. The clonogenic survival rate was found to be significantly lower in cells infected with c-Myc versus control or AP2α only group. Conversely, coinfection with AP2α+c-Myc was able to double cell survival and increase growth rate compared to cells infected with c-Myc alone. We conclude that c-Myc is an inhibitor of AP2α DNA binding, and AP2α counteracts the cytotoxic effect of c-Myc. Supported by NIH CA 66081

Regulation of KiSS-1 Metastasis Suppressor Gene Expression in Breast Cancer Cells by Direct Interaction of Transcription Factors Activator Protein-2α and Specificity Protein-1

KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2alpha (AP-2alpha) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2alpha binding elements, AP-2alpha-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2alpha into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2alpha wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2alpha regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2alpha directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2alpha and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2alpha in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis.

Activator Protein 2α Status Determines the Chemosensitivity of Cancer Cells: Implications in Cancer Chemotherapy

Cancer chemotherapeutic drugs induce apoptosis by several pathways. Inactivation of proapoptotic genes, or activation of survival signaling, leads to chemoresistance. Activator protein 2alpha (AP-2alpha), a developmentally regulated sequence-specific DNA-binding transcription factor, has been shown to function like a tumor suppressor. Here, we show that controlled expression of AP-2alpha, using tetracycline-inducible system, increased the chemosensitivity of cancer cells by severalfold by sensitizing cells to undergo apoptosis upon chemotherapy. Under these conditions, neither AP-2alpha expression nor drug treatment resulted in apoptosis induction, whereas in combination the cancer cells underwent massive apoptosis. We found that endogenous AP-2alpha protein is induced posttranscriptionally by various chemotherapeutic drugs. Blocking the endogenous AP-2alpha by small interfering RNA in human cancer cells lead to decreased apoptosis, increased colony formation, and chemoresistance irrespective of their p53 status upon chemotherapy. We further show that 5-aza-2'-deoxycytidine induced reexpression of AP-2alpha in MDA-MB-231 breast cancer cells (wherein AP-2alpha expression is silenced by hypermethylation), resulted in massive apoptosis induction, increased chemosensitivity, decreased colony formation, and loss of tumorigenesis upon chemotherapy. However, in MDA-MB-231 cells transfected with AP-2alpha small interfering RNA, 5-aza-2'-deoxycytidine treatment failed to increase apoptosis and chemosensitivity. The treatment also resulted in increased colony formation and efficient tumor formation upon chemotherapy. These results establish an important role for AP-2alpha in cancer cell chemosensitivity and provide new insights for modifying the chemosensitivity of cancer cells by activating apoptotic pathways.