Abstract 3914: Tcfap2c regulates growth and oncogenesis in neu-activated breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA TFAP2C and mouse homolog tcfap2c are transcription factors that play a critical role in mammary gland development and in maintaining the luminal breast cancer phenotype. Interestingly, several human HER2-positive breast cancer cell lines display genetic dependency on TFAP2C. However, the role of TFAP2C in carcinogenesis and progression in neu-activated breast cancer remains unknown. We examined the role of tcfap2c in mammary gland carcinogenesis by examining tumor formation in mice expressing an activated rat neu gene using MMTV-neu with and without mammary gland conditional knockout (KO) of tcfap2c using MMTV-Cre. The time until spontaneous palpable tumor formation was measured in mice harboring the neu oncogene with and without tcfap2c. Mice with homozygous conditional deletion of tcfap2c demonstrated a significant delay in tumor formation compared to littermate controls that retained tcfap2c expression (42 vs. 25 weeks, p<0.004). Immunohistochemistry of the tumors demonstrated a luminal phenotype; cytokeratin 8 (CK8), a luminal marker, was more strongly expressed compared to cytokeratin 5 (CK5), a basal marker, in tumors from both control (883 vs. 0.1 cells/HPF, p<0.0005) and KO animals (845 vs. 0, p<0.0005). Cleaved caspase-3 (CC3) showed that apoptosis was increased in tumors from KO compared to control animals (5 vs. 0.3 CC3-positive cells/HPF, p<0.0008). Ki-67 showed that proliferation was decreased in KO compared to control (348 vs. 142 cells/HPF, p<0.009). To validate the effects of tcfap2c, we established a cell line from a tumor obtained from an animal expressing activated neu with homozygous floxed tcfap2c lacking Cre expression. Paired cell lines with differential expression of tcfap2c were established by infecting the cells with Adenovirus-Cre (Ad-Cre) or Adenovirus-Empty (Ad-Empty). Proliferation was assessed by MTT and demonstrated that loss of tcfap2c expression resulted in a 30% decrease in proliferation rate (p<0.006). The difference in proliferation was confirmed in vivo through xenograft experiments of these paired cell lines inoculated into athymic (nude) mice. Mice xenografted with the paired cell lines demonstrated that KO of tcfap2c resulted in a significant delay in tumors reaching a 2 cm threshold requiring euthanasia (27.8 vs. 19.8 days post-inoculation, p<0.003). In order to identify a potential mechanism accounting for the differences in proliferation and delay in carcinogenesis, we identified tcfap2c target genes by comparing the binding sites of tcfap2c by chromatin-immunoprecipitation-sequencing (ChIP-seq) and differential expression by microarray/RT-PCR. We identified erbb3 and egfr as two tcfap2c target genes by ChIP-seq. KO of tcfap2c resulted in a 3-fold and 12-fold reduction in erbb3 and egfr expression, respectively (p<0.05). Taken together, these findings indicate that tcfap2c plays critical roles in oncogenesis and progression of HER2-positive mammary cancer. Citation Format: Jung M. Park, Tong Wu, George Woodfield, Anthony Cyr, James De Andrade, Weizhou Zhang, Frederick Domann, Ronald J. Weigel. Tcfap2c regulates growth and oncogenesis in neu-activated breast cancer. [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 3914. doi:10.1158/1538-7445.AM2014-3914