Analyses of Pax9 functions in the developing and adult mouse mammary gland

Abstract

The development of the mammary gland is strictly directed by hormones, local signaling, epithelial-mesenchymal cross talk, as well as participation of innate immune cells. On the other hand, the dysregulation of this orchestration may initiate breast cancer and facilitate its progression. In our study Pax9 was found, for the first time, significantly reversely correlated with breast cancer malignancy, being reduced or absent in human DCISs (96%) and invasive breast cancers (78%), as well as in MMTV-Neu and MMTV-PyMT induced mouse mammary tumours, while it is expressed in normal human and mouse mammary epithelium. By a full-range expression investigation, using semi-quantitative RT PCR and immunohistochemistry, covering all developmental stages of mouse mammary gland, Pax9 was found to be expressed in the ductal epithelium with a strict spatial-temporal pattern, with an expression peak at puberty. Reduction or deletion of Pax9 expression, using Pax9 hypomorphs and mammary gland-specific knockout mouse models, resulted in ductal branching delay during puberty, alveolar formation at the wrong position during pregnancy, disrupted epithelial cell apoptosis and engulfment of excess milk fat globulin during postlactational involution. Mammary ductal epithelial cell detachment, basement membrane disruption and tumour-like structure expansion have been found in the mammary glands of parous mice. Taken together, we found that Pax9 functions in the process of mammary epithelial cell differentiation, basement membrane integrity, apoptosis and epithelial cell engulfment. 4 By immunohistochemistry and western blot analysis of the mammary gland during involution, we identified the Stat signaling pathway as a candidate downstream pathway affected by Pax9 deficiency in the mammary gland, which may be responsible for apoptosis delay. Expression microarray profiling of Pax9 deficient and control mammary glands showed the increase of insulin growth factor binding protein 5 (Igfbp5, an essential regulator of mammary gland involution), monocyte to macrophage differentiation-associated (Mmd, immune and inflammation associated genes), and MMP3 and MMP12 (metalloproteinase) genes, and the decrease of inhibitor of DNA binding 2 (Id-2, functioning in mammary cells with low proliferation and invasiveness). Furthermore, myosin-related genes were strikingly up-regulated, which may be a cellular stress response to the milk stasis from impaired involution in the Pax9 deficient mammary gland. All these phenotypes we discovered in the mutants and molecular changes suggested by immunohistochemistry and gene expression profiling during involution, provided us with candidate networks regulated by Pax9 in mammary gland development. Further elucidation of these clues may help us to understand the multiple pathways in which Pax9 takes part in normal mammary differentiation, and the underlying mechanisms how its dysregulation may promote breast cancer formation and progression.