Philip M. Spanheimer

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.

The response to neoadjuvant chemotherapy predicts clinical outcome and increases breast conservation in advanced breast cancer

BACKGROUND The aim of this study was to determine outcomes in patients with breast cancer treated with neoadjuvant chemotherapy. METHODS Seventy-two consecutive patients receiving neoadjuvant chemotherapy for breast cancer were enrolled. RESULTS Mastectomy was avoided in 46% of patients, and 42% converted to negative nodes after neoadjuvant chemotherapy. Thirteen patients (18%) achieved a pathologic complete response, which was associated with the estrogen receptor (ER)-negative/human epidermal growth factor receptor 2 (Her2)-negative subtype (58%) and was significantly less likely to occur in the ER+/Her2- subtype (2%) (P < .01). Patients with the ER+/Her2+ subtype were most likely to have no response or progression during chemotherapy, compared with those with the ER-/Her2- subtype (50% vs 0%, P = .01). Five-year survival for patients achieving a pathologic complete response was 100%, compared with 74% in the group with partial response and 48% in the group with no response or progression (P = .01). CONCLUSIONS Neoadjuvant chemotherapy for patients with advanced breast cancer provided prognostic information, allowed evaluation of response to chemotherapy, decreased the mastectomy rate, and potentially reduced the need for axillary lymph node dissection.

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.

Complications and survival associated with operative procedures in patients with unresectable pancreatic head adenocarcinoma.

Unresectable tumors of the pancreatic head are encountered in up to 20% of patients taken for resection. The objective of this study was to evaluate the complications and outcome associated with palliative surgical procedures to help guide management decisions in these patients.

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.

Differentiation of small bowel and pancreatic neuroendocrine tumors by gene-expression profiling.

BACKGROUND Between 10% and 20% of patients with neuroendocrine tumors (NETs) present with metastases of unknown primary site. Because knowledge of the primary site has important implications for treatment, we set out to define gene-expression profiles to differentiate between small-bowel NETs (SBNETs) and pancreatic NETs (PNETs). METHODS RNA was extracted from tumor and normal tissues in 11 patients with SBNETs and 15 patients with PNETs, and qPCR was performed for 367 GPCR genes. Differentially expressed genes were identified using the RT2 Profiler. Whole genome expression analysis was performed on 11 SBNETs, 5 PNETS, and corresponding normal tissues. Statistical significance was evaluated by the Student t test and ANOVA. RESULTS Whole-genome analysis revealed 173 significantly differentially expressed genes in SBNETs and normal tissues and in 52 in PNETs. GPCR arrays identified 28 genes in SBNETs and 18 in PNETs, with significant expression differences from normal tissues. In all SBNETs, 2 genes were significantly upregulated by more than fivefold: OXTR and GPR113. No PNETs shared this profile, whereas 73% had a greater than fivefold downregulation of ADORA1 and SCTR. These genes also allowed for determination of the primary site in 8 of 10 liver metastases. CONCLUSION Differential expression patterns using as few as 2 to 4 GPCR genes successfully discriminated primary sites in small bowel and pancreatic NETs.

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.

The role of Tcfap2c in tumorigenesis and cancer growth in an activated Neu model of mammary carcinogenesis

TFAP2C/AP-2γ influences development of the mammary gland and regulates patterns of gene expression in luminal and HER2-amplified breast cancer. The roles of TFAP2C in mammary gland tumorigenesis and in pathways critical to cancer progression remain poorly understood. To gain greater insight into oncogenic mechanisms regulated by TFAP2C, we examined mammary tumorigenesis in MMTV-Neu transgenic female mice with or without conditional knockout (KO) of Tcfap2c, the mouse homolog of TFAP2C. Loss of Tcfap2c increased the latency of tumorigenesis and tumors that formed demonstrated reduced proliferative index and increased apoptosis. In addition, tumors formed in Tcfap2c KO animals had a significant reduction in Egfr levels without a change in the expression of the Neu oncogene. The MMneu-flAP2C cell line was established from tumor tissue derived from MMTV-Neu/Tcfap2cL/L control animals and parallel cell lines with and without expression of Tcfap2c were created by transduction with adenovirus-empty and adenovirus-Cre, respectively. KO of Tcfap2c in vitro reduced activated phosphorylated-Erk, decreased cell viability, repressed tumor growth and was associated with attenuation of Egfr expression. Chromatin immunoprecipitation and direct sequencing and expression analysis confirmed that Egfr was a Tcfap2c target gene in murine, as well as human, mammary carcinoma cells. Furthermore, decreased viability of mammary cancer cells was directly related to Egfr functional blockade. We conclude that TFAP2C regulates tumorigenesis, cell growth and survival in HER2-amplified breast cancer through transcriptional regulation of EGFR. The findings have important implications for targeting the EGFR pathway in breast cancer.

EGFR Is Regulated by TFAP2C in Luminal Breast Cancer and Is a Target for Vandetanib

Expression of TFAP2C in luminal breast cancer is associated with reduced survival and hormone resistance, partially explained through regulation of RET. TFAP2C also regulates EGFR in HER2 breast cancer. We sought to elucidate the regulation and functional role of EGFR in luminal breast cancer. We used gene knockdown (KD) and treatment with a tyrosine kinase inhibitor (TKI) in cell lines and primary cancer isolates to determine the role of RET and EGFR in regulation of p-ERK and tumorigenesis. KD of TFAP2C decreased expression of EGFR in a panel of luminal breast cancers, and chromatin immunoprecipitation sequencing (ChIP-seq) confirmed that TFAP2C targets the EGFR gene. Stable KD of TFAP2C significantly decreased cell proliferation and tumor growth, mediated in part through EGFR. While KD of RET or EGFR reduced proliferation (31% and 34%, P < 0.01), combined KD reduced proliferation greater than either alone (52% reduction, P < 0.01). The effect of the TKI vandetanib on proliferation and tumor growth response of MCF-7 cells was dependent upon expression of TFAP2C, and dual KD of RET and EGFR eliminated the effects of vandetanib. The response of primary luminal breast cancers to TKIs assessed by ERK activation established a correlation with expression of RET and EGFR. We conclude that TFAP2C regulates EGFR in luminal breast cancer. Response to vandetanib was mediated through the TFAP2C target genes EGFR and RET. Vandetanib may provide a therapeutic effect in luminal breast cancer, and RET and EGFR can serve as molecular markers for response. Mol Cancer Ther; 15(3); 503–11. ©2016 AACR.