HoxC6 regulation of early mammary epithelial growth and differentiation: implications for breast cancer potential.

Abstract

Abstract Abstract #2010 Background: Early menarche and breast development are increasingly common risk factors for breast cancer. Epigenetic mechanisms implicated include natural and anthropogenic steroids, such as estrogens, that disrupt endocrine signaling and dysregulate stem cell homeostasis. Animal models show that, although a proliferative response to estrogens in mammary epithelium is not observed until near puberty, in utero or neonatal exposure can recapitulate aberrant breast development. The molecular basis for the delay in proliferative response remains poorly understood and assessments of agent activity, potency and risk remain inaccurate. Here, we provide a candidate molecular mechanism involving the homeobox transcription factor HoxC6, through which more accurate assessments may be facilitated.
 Materials & Methods: Genetically and hormonally manipulated in vivo mouse models (HoxC6 targeted disruption, C6KO; and mammary epithelial-specific inducible HoxC6, MMTV-rtTA TRE-HoxC6-IRES-EGFP) and in vitro human breast cancer cell lines (MCF10A, MCF7 and MDA-MB-231; "231") were used to assess impacts on mammary epithelial growth, differentiation and expression of HoxC6 and its direct target genes IGFBP3, CD44 and FGFR2. Cell lines were manipulated using HoxC6 shRNA and cDNA lentivirus and activated Akt isoform retroviral expression vectors. Gene expression and signaling were measured by quantitative RT-qPCR, IHC, Western blot and using the BioPlex200 Multiplex System.
 Results: RT-qPCR analysis shows HoxC6 expression elevated in well-differentiated cell lines (MCF10A, 5.06X; MCF7, 5.16X) and decreased in more aggressive tumor cell lines (-1.95X, 231 cells; relative to A549 cells). HoxC6 knockdown in MCF10A cells results in an inhibition of cellular growth and decreased branching in 3D cultures and dysregulation of HoxC6 target genes. These results are consistent with in vivo results in which C6KO causes defective postnatal mammary epithelial growth/branching. Rescue of growth defects in knockdown MCF10A cells is achieved to varying extents with myristoylated Akt isoforms, however morphological defects persist and are associated with EMT. HoxC6 induction in stem cells is associated with altered self-renewal and differentiation. HoxC6 repression by estrogens was assessed in vivo under conditions of neonatal estrogen exposure and estrogen-free diets, showing alterations in HoxC6 and target gene expressions and other phenotypic changes.
 Discussion: Based on our results, we will present a model in which HoxC6 is estrogen-responsive and coordinates mammary stem/progenitor cell homeostatic pathways, including FGF and GH-IGF1 signaling in the control of pool size and branching morphogenesis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2010.