George W. Woodfield

TFAP2C Controls Hormone Response in Breast Cancer Cells through Multiple Pathways of Estrogen Signaling

Breast cancers expressing estrogen receptor-alpha (ERalpha) are associated with a favorable biology and are more likely to respond to hormonal therapy. In addition to ERalpha, other pathways of estrogen response have been identified including ERbeta and GPR30, a membrane receptor for estrogen, and the key mechanisms regulating expression of ERs and hormone response remain controversial. Herein, we show that TFAP2C is the key regulator of hormone responsiveness in breast carcinoma cells through the control of multiple pathways of estrogen signaling. TFAP2C regulates the expression of ERalpha directly by binding to the ERalpha promoter and indirectly via regulation of FoxM1. In so doing, TFAP2C controls the expression of ERalpha target genes, including pS2, MYB, and RERG. Furthermore, TFAP2C controlled the expression of GPR30. In distinct contrast, TFAP2A, a related factor expressed in breast cancer, was not involved in estrogen-mediated pathways but regulated expression of genes controlling cell cycle arrest and apoptosis including p21(CIP1) and IGFBP-3. Knockdown of TFAP2C abrogated the mitogenic response to estrogen exposure and decreased hormone-responsive tumor growth of breast cancer xenografts. We conclude that TFAP2C is a central control gene of hormone response and is a novel therapeutic target in the design of new drug treatments for breast cancer.

Identification of Primary Gene Targets of TFAP2C in Hormone Responsive Breast Carcinoma Cells

The TFAP2C transcription factor is involved in mammary development, differentiation, and oncogenesis. Previous studies established a role for TFAP2C in the regulation of ESR1 (ERα) and ERBB2 (Her2) in breast carcinomas. However, the role of TFAP2C in different breast cancer phenotypes has not been examined in detail. To develop a more complete characterization of TFAP2C target genes, ChIP‐seq with anti‐TFAP2C antibody and expression arrays with TFAP2C knock down were analyzed in MCF‐7 breast carcinoma cells. Genomic sequences common to the ChIP‐seq data set defined the consensus sequence for TFAP2C chromatin binding as the nine base sequence SCCTSRGGS (S = G/C, r = A/G), which closely matches the previously defined optimal in vitro binding site. Comparing expression arrays before and after knock down of TFAP2C with ChIP‐seq data demonstrated a conservative estimate that 8% of genes altered by TFAP2C expression are primary target genes and includes genes that are both induced and repressed by TFAP2C. A set of 447 primary target genes of TFAP2C was identified, which included ESR1 (ERα), FREM2, RET, FOXA1, WWOX, GREB1, MYC, and members of the retinoic acid response pathway. The identification of ESR1, WWOX, GREB1, and FOXA1 as primary targets confirmed the role of TFAP2C in hormone response. TFAP2C plays a critical role in gene regulation in hormone responsive breast cancer and its target genes are different than for the Her2 breast cancer phenotype.

Sumoylation Pathway Is Required to Maintain the Basal Breast Cancer Subtype

The TFAP2C/AP-2γ transcription factor regulates luminal breast cancer genes, and loss of TFAP2C induces epithelial-mesenchymal transition. By contrast, the highly homologous family member, TFAP2A, lacks transcriptional activity at luminal gene promoters. A detailed structure-function analysis identified that sumoylation of TFAP2A blocks its ability to induce the expression of luminal genes. Disruption of the sumoylation pathway by knockdown of sumoylation enzymes, mutation of the SUMO-target lysine of TFAP2A, or treatment with sumoylation inhibitors induced a basal-to-luminal transition, which was dependent on TFAP2A. Sumoylation inhibitors cleared the CD44(+/hi)/CD24(-/low) cell population characterizing basal cancers and inhibited tumor outgrowth of basal cancer xenografts. These findings establish a critical role for sumoylation in regulating the transcriptional mechanisms that maintain the basal cancer phenotype.

Transcriptional regulation of the GPX1 gene by TFAP2C and aberrant CpG methylation in human breast cancer.

The complexity of gene regulation has created obstacles to defining mechanisms that establish the patterns of gene expression characteristic of the different clinical phenotypes of breast cancer. TFAP2C is a transcription factor that has a critical role in the regulation of both estrogen receptor-alpha (ERα) and c-ErbB2/HER2 (Her2). Herein, we performed chromatin immunoprecipitation and direct sequencing (ChIP-seq) for TFAP2C in four breast cancer cell lines. Comparing the genomic binding sites for TFAP2C, we identified that glutathione peroxidase (GPX1) is regulated by TFAP2C through an AP-2 regulatory region in the promoter of the GPX1 gene. Knockdown of TFAP2C, but not the related factor TFAP2A, resulted in an abrogation of GPX1 expression. Selenium-dependent GPX activity correlated with endogenous GPX1 expression and overexpression of exogenous GPX1 induced GPX activity and significantly increased resistance to tert-butyl hydroperoxide. Methylation of the CpG island encompassing the AP-2 regulatory region was identified in cell lines where TFAP2C failed to bind the GPX1 promoter and GPX1 expression was unresponsive to TFAP2C. Furthermore, in cell lines where GPX1 promoter methylation was associated with gene silencing, treatment with 5′-aza-2-deoxycytidine (5′-aza-dC) (an inhibitor of DNA methylation) allowed TFAP2C to bind to the GPX1 promoter resulting in the activation of GPX1 RNA and protein expression. Methylation of the GPX1 promoter was identified in ∼20% of primary breast cancers and a highly significant correlation between the TFAP2C and GPX1 expression was confirmed when considering only those tumors with an unmethylated promoter, whereas the related factor, TFAP2A, failed to demonstrate a correlation. The results demonstrate that TFAP2C regulates the expression of GPX1, which influences the redox state and sensitivity to oxidative stress induced by peroxides. Given the established role of GPX1 in breast cancer, the results provide an important mechanism for TFAP2C to further influence oncogenesis and progression of breast carcinoma cells.

TFAP2C governs the luminal epithelial phenotype in mammary development and carcinogenesis

Molecular subtypes of breast cancer are characterized by distinct patterns of gene expression that are predictive of outcome and response to therapy. The luminal breast cancer subtypes are defined by the expression of estrogen receptor-alpha (ERα)-associated genes, many of which are directly responsive to the transcription factor activator protein 2C (TFAP2C). TFAP2C participates in a gene regulatory network controlling cell growth and differentiation during ectodermal development and regulating ESR1/ERα and other luminal cell-associated genes in breast cancer. TFAP2C has been established as a prognostic factor in human breast cancer, however, its role in the establishment and maintenance of the luminal cell phenotype during carcinogenesis and mammary gland development have remained elusive. Herein, we demonstrate a critical role for TFAP2C in maintaining the luminal phenotype in human breast cancer and in influencing the luminal cell phenotype during normal mammary development. Knockdown of TFAP2C in luminal breast carcinoma cells induced epithelial–mesenchymal transition with morphological and phenotypic changes characterized by a loss of luminal-associated gene expression and a concomitant gain of basal-associated gene expression. Conditional knockout of the mouse homolog of TFAP2C, Tcfap2c, in mouse mammary epithelium driven by MMTV-Cre promoted aberrant growth of the mammary tree leading to a reduction in the CD24hi/CD49fmid luminal cell population and concomitant gain of the CD24mid/CD49fhi basal cell population at maturity. Our results establish TFAP2C as a key transcriptional regulator for maintaining the luminal phenotype in human breast carcinoma. Furthermore, Tcfap2c influences development of the luminal cell type during mammary development. The data suggest that TFAP2C has an important role in regulated luminal-specific genes and may be a viable therapeutic target in breast cancer.

Interaction of TFAP2C with the Estrogen Receptor-α Promoter Is Controlled by Chromatin Structure

Purpose: Transcriptional regulation of estrogen receptor-α (ERα) involves both epigenetic mechanisms and trans-active factors, such as TFAP2C, which induces ERα transcription through an AP-2 regulatory region in the ERα promoter. Attempts to induce endogenous ERα expression in ERα-negative breast carcinomas by forced overexpression of TFAP2C have not been successful. We hypothesize that epigenetic chromatin structure alters the activity of TFAP2C at the ERα promoter. Experimental Design: DNA methylation, histone acetylation, and chromatin accessibility were examined at the ERα promoter in a panel of breast carcinoma cell lines. TFAP2C and polymerase II binding were analyzed by chromatin immunoprecipitation. Epigenetic chromatin structure was altered using drug treatment with 5-aza-2′-deoxycytidine (AZA) and trichostatin A (TSA). Results: The ERα promoter in the ERα-negative lines MDA-MB-231, MCF10A, and MCF7-5C show CpG island methylation, histone 3 lysine 9 deacetylation, and decreased chromatin accessibility compared with ERα-positive cell lines MCF7 and T47-D. Treatment with AZA/TSA increased chromatin accessibility at the ERα promoter and allowed TFAP2C to induce ERα expression in ERα-negative cells. Chromatin immunoprecipitation analysis showed that binding of TFAP2C to the ERα promoter is blocked in ERα-negative cells but that treatment with AZA/TSA enabled TFAP2C and polymerase II binding. Conclusion: We conclude that the activity of TFAP2C at specific target genes depends upon epigenetic chromatin structure. Furthermore, the combination of increasing chromatin accessibility and inducing TFAP2C provides a more robust activation of the ERα gene in ERα-negative breast cancer cells.

AP2alpha alters the transcriptional activity and stability of p53.

AP2α and p53 form nuclear complexes that establish a functional partnership, which regulates the expression of certain genes involved in cell growth and metastasis. The growth effects of AP2α are mediated through p21WAF1/CIP1 and the ability for AP2α to coactivate p21 requires p53. Herein, we have localized the AP2-binding region of p53 to amino acids 305–375. Analysis of 26 distinct p53 alleles established a correlation between AP2α binding and transcriptional coactivation. The L350P point mutation was the only nonbinding allele that retained normal transcriptional activity by reporter assay. Although both wild-type and L350P alleles facilitated binding of AP2α to the p21 promoter, the L350P allele was significantly reduced in its ability to induce the endogenous p21 gene, demonstrating a striking difference in activity comparing reporter assays with activation of endogenous p53 target genes. Interestingly, expression of AP2 in the absence of radiation repressed p53-mediated induction of p21 and this effect was explained by a reduction in p53 stability induced by AP2α overexpression. We conclude that AP2α has competing effects on p53 activity through coactivation and decreased stability. These findings may provide a mechanism to account for the discrepancies reported for the association between AP2 and p21 expression in tumor tissue.

Distinct Pathways Regulated by RET and Estrogen Receptor in Luminal Breast Cancer Demonstrate the Biological Basis for Combination Therapy

Objective:We investigated directed therapy based on TFAP2C-regulated pathways to inform new therapeutic approaches for treatment of luminal breast cancer. Background:TFAP2C regulates the expression of genes characterizing the luminal phenotype including ESR1 and RET, but pathway cross talk and potential for distinct elements have not been characterized. Methods:Activation of extracellular signal-regulated kinases (ERK) and AKT was assessed using phosphorylation-specific Western blot. Cell proliferation was measured with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] after siRNA (small interfering RNA) gene knockdown or drug treatment. Cell cycle, Ki-67, and cleaved caspase 3 were measured by fluorescence-activated cell sorting. Tumorigenesis was assessed in mice xenografts. Results:Knockdown of TFAP2C or RET inhibited GDNF (glial cell line–derived neurotrophic factor)–mediated activation of ERK and AKT in MCF-7 cells. Similarly, sunitinib, a small-molecule inhibitor of RET, blocked GDNF-mediated activation of ERK and AKT. Inhibition of RET either by gene knockdown or by treatment with sunitinib or vandetanib reduced RET-dependent growth of luminal breast cancer cells. Interestingly, knockdown of TFAP2C, which controls both ER (estrogen receptor) and RET, demonstrated a greater effect on cell growth than either RET or ER alone. Parallel experiments using treatment with tamoxifen and sunitinib confirmed the increased effectiveness of dual inhibition of the ER and RET pathways in regulating cell growth. Whereas targeting the ER pathway altered cell proliferation, as measured by Ki-67 and S-phase, anti-RET primarily increased apoptosis, as demonstrated by cleaved caspase 3 and increased TUNEL (terminal deoxyneucleotidyl transferase dUTP nick end labeling) expression in xenografts. Conclusions:ER and RET primarily function through distinct pathways regulating proliferation and cell survival, respectively. The findings inform a therapeutic approach based on combination therapy with antiestrogen and anti-RET in luminal breast cancer.

Inhibition of RET increases the efficacy of antiestrogen and is a novel treatment strategy for luminal breast cancer.

Purpose: Recent findings suggest that combination treatment with antiestrogen and anti-RET may offer a novel treatment strategy in a subset of patients with breast cancer. We investigated the role of RET in potentiating the effects of antiestrogen response and examined whether RET expression predicted the ability for tyrosine kinase inhibitor (TKI) to affect extracellular signal–regulated kinase 1/2 (ERK1/2) activation in primary breast cancer. Experimental Design: Growth response, ERK1/2 activation, Ki-67, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling were assessed in breast cancer cell lines in vitro and in xenografts with vandetanib and/or tamoxifen. Thirty tumors with matched normal breast tissue were evaluated for RET expression and response to TKI treatment. Results: Vandetanib potentiated the inhibitory effect of tamoxifen in hormone responsive (P = 0.01) and hormone insensitive (P < 0.001) estrogen receptor α (ERα)-positive breast cancer cells. Vandetanib significantly repressed tumorigenesis of MCF-7 xenografts (P < 0.001), which displayed decreased activation of ERK1/2 and AKT. Vandetanib and tamoxifen reduced the growth of established tumors with a greater effect of dual therapy compared with single agent (P = 0.003), with tamoxifen-reducing proliferative index and vandetanib-inducing apoptosis. In primary breast cancers, RET expression correlated with the ERα-positive subtype. Relative decrease in ERK1/2 phosphorylation with TKI treatment was 42% (P < 0.001) in RET-positive tumors versus 14% (P = ns) in RET-negative tumors. Conclusions: Vandetanib potentiated the antigrowth effects of tamoxifen in breast cancer, which was mediated through RET activation. RET predicted response to TKI therapy with minimal effects on ERK1/2 activation in RET-negative tumors. The preclinical data support evaluation of antiestrogen in combination with TKI as a potential treatment strategy for RET-positive luminal breast cancer. Clin Cancer Res; 20(8); 2115–25. ©2014 AACR.

Discovery of SMAD4 promoters, transcription factor binding sites and deletions in juvenile polyposis patients.

Inactivation of SMAD4 has been linked to several cancers and germline mutations cause juvenile polyposis (JP). We set out to identify the promoter(s) of SMAD4, evaluate their activity in cell lines and define possible transcription factor binding sites (TFBS). 5′-rapid amplification of cDNA ends (5′-RACE) and computational analyses were used to identify candidate promoters and corresponding TFBS and the activity of each was assessed by luciferase vectors in different cell lines. TFBS were disrupted by site-directed mutagenesis (SDM) to evaluate the effect on promoter activity. Four promoters were identified, two of which had significant activity in several cell lines, while two others had minimal activity. In silico analysis revealed multiple potentially important TFBS for each promoter. One promoter was deleted in the germline of two JP patients and SDM of several sites led to significant reduction in promoter activity. No mutations were found by sequencing this promoter in 65 JP probands. The predicted TFBS profiles for each of the four promoters shared few transcription factors in common, but were conserved across several species. The elucidation of these promoters and identification of TFBS has important implications for future studies in sporadic tumors from multiple sites, and in JP patients.