TFAP2C governs the luminal epithelial phenotype in mammary development and carcinogenesis

Abstract

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 CD24(hi)/CD49f(mid) luminal cell population and concomitant gain of the CD24(mid)/CD49f(hi) 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.

Figure 1. ChIP-SEQ Demonstrates TFAP2C Binds to Luminal Target Genes

A. Examples of ChIP-SEQ data from MCF-7 cells for select luminal target genes GATA3, FBP1, FOXA1, MYB, RET, ESR1, KRT8 and MUC1 showing that TFAP2C binds to the regulatory regions of the genes. Data also show that TFAP2C binds to the first intron of the CD44 gene. The y-axis represents normalized coverage (reads per million mapped). Full ChIP-SEQ dataset available under accession number GSE44257. B. Summary of analysis showing preference of TFAP2C binding to Luminal Differentiation and ERα-associated genes compared to Basal gene expression cluster.

Figure 2. Knockdown of TFAP2C in Luminal Cell Lines

TFAP2C was knocked down in MCF-7 and T47-D cells using siRNA compared to non-targeting (NT) siRNA. A. Relative RNA expression for genes shown normalized to expression with NT siRNA (line at 1.0) and demonstrated significant reduction in TFAP2C and luminal target genes and increase in CD44 expression. B. Western blot for protein expression shows reduced expression of TFAP2C and luminal genes with increase in CD44 expression. C. Relative RNA expression as in panel A performed with T47-D cells. D. Western blot for protein expression as in panel C performed with T47-D.

Figure 3. Stable knockdown of TFAP2C induced EMT

MCF-7 cell clone 45 derived by stable expression of shRNA targeting TFAP2C (sKD-C) compared to cell clone derived with non-targeting shRNA (sKD-NT). The clone used for all data in this figure was sKD-C-clone 45. A. Expression pattern of TFAP2A and TFAP2C by RT-PCR in stable cell clones. B. Cell morphology with using the brightfield microscopy 400x magnification. C. Section of expression array comparing sKD-NT vs. sKD-C cells demonstrates down-regulation of Luminal Differentiation Markers and up-regulation of Mesenchymal Markers. D. Western blot showing protein expression of AP-2 proteins and examples of luminal differentiation and mesenchymal markers. E. Relative expression of CD44 gene by RT-PCR for TFAP2C shows increased CD44 RNA in sKD-C cells, confirming array in A and protein in D. F. Distribution of CD44/CD24 subpopulations in stable cell clones of MCF7 by flow cytometry demonstrate an increase in the CD44^+/hi/CD24^−/low population.

Figure 4. Expression profile of sKD-NT and sKD-C stable cell clones

A. Heatmap of selected genes (p <0.01) for luminal associated genes for sKD-C, clone 45 compared to sKD-NT clone. B. Expression of RNA for luminal-marker genes normalized to expression in sKD-NT cells (line at 1.0) for sKD-C, clone 45 and clone 46. C. Western blots showing repression of protein expression of luminal target genes in sKD-C, clone 45 and clone 46 compared to sKD-NT cells. D. Heatmap of selected basal genes (p <0.01) for sKD-C clone 45. E. Expression of RNA for select basal genes normalized to expression in sKD-NT cells (line at 1.0) for sKD-C, clone 45 and clone 46. F. Western blot showing protein expression for selected basal genes for sKD-C, clone 45 and clone 46, compared to sKD-NT cells. Expression of TFAP2C confirmed to be abrogated in sKD-C, clone 46 by western blot (note: expression of TFAP2C in clone 45 presented in figure 3).

Figure 5. Whole Mounts of Mammary Gland-Specific Tcfap2c Knockout Mouse

A. Representative whole mounts of mammary gland 4–6-week old virgin animals. Inset demonstrates qualitative differences between overall gland structures, including more end buds and fewer branch points in KO glands. B. Quantitation of whole mounts revealed reduced distal migration of TEBs with reduced fat pad invasion in KO (−/−) animals compared to control (wt/wt), n=6 glands per group, statistics from two-tailed Student’s T Test. C. Quantitation of branch points and end buds, revealed an increase in end bud structures and a decrease in branch points in KO (−/−) compared to control (wt/wt) glands. N=6 glands per group, statistics from two-tailed Student’s T Test.

Figure 6. Immunohistochemical Analysis of Tcfap2c Knockout Mouse

A. Immunohistochemical analysis of mammary gland 3 from virgin control and KO mice at 6 weeks of age. KO (Tcfap2c^−/−) glands demonstrated loss of TCFAP2C reactivity in luminal cell populations, but not in myoepithelial basal cell populations. CK5 and CK8 staining of the same representative duct in control and KO animals demonstrate CK5 staining of myoepithelial layer and CK8 staining of luminal cells. Last panel notes CK5 staining in terminal end bud (TEB) structures with increased CK5-positive cells in KO animals. B. Examples of CK5 staining in luminal cells in single cell lined mammary ductal structures in KO animals. C. Quantification of CK5 staining demonstrated a statistical increase in KO animals. D. Quantification of CK5 staining in TEB structures demonstrates statistical increase in KO animals; n= 3 mice per group and data from 10 TEB/mouse; p=0.01, student’s t-test.

Figure 7. Flow Cytometric Analysis of Tcfap2c Null Mammary Glands

A/B. Representative images from individual flow cytometry runs from different litters. A. CD31-, CD45-, and Ter119-negative (Lin−) cells were derived from mammary gland 4 pooled from five control and five KO 10–12 week-old adult animals. B. Example of data from single mammary gland isolated from control and KO animal. For FACS analysis, cells were gated for CD31-, CD45-, and Ter119-negative (Lin−) cells. C–E. Quantitation of flow cytometric analysis based on all experiments including pooled and single mammary gland data. Total numbers of FACS flow data combined were five control (wt/wt) and five KO (−/−) samples (one FACS run from five pooled glands and four FACS runs from individual mammary glands). Cells from KO glands had a statistically significant reduction in identifiable luminal cells (C), a statistically significant increase in identifiable basal cells (D), and a statistically significant reduction in the luminal/basal ratio (E); statistical analysis using Student’s T-test.