Reversal by RARα agonist Am580 of c-Myc-induced imbalance in RARα/RARγ expression during MMTV-Myc tumorigenesis.

INTRODUCTION: Retinoic acid signaling plays key roles in embryonic development and in maintaining the differentiated status of adult tissues. Recently, the nuclear retinoic acid receptor (RAR) isotypes α, ß and γ were found to play specific functions in the expansion and differentiation of the stem compartments of various tissues. For instance, RARγ appears to be involved in stem cell compartment expansion, while RARα and RARß are implicated in the subsequent cell differentiation. We found that over-expressing c-Myc in normal mouse mammary epithelium and in a c-Myc-driven transgenic model of mammary cancer, disrupts the balance between RARγ and RARα/ß in favor of RARγ. METHODS: The effects of c-Myc on RAR isotype expression were evaluated in normal mouse mammary epithelium, mammary tumor cells obtained from the MMTV-Myc transgenic mouse model as well as human normal immortalized breast epithelial and breast cancer cell lines. The in vivo effect of the RARα-selective agonist 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid (Am580) was examined in the MMTV-Myc mouse model of mammary tumorigenesis. RESULTS: Modulation of the RARα/ß to RARγ expression in mammary glands of normal mice, oncomice, and human mammary cell lines through the alteration of RAR-target gene expression affected cell proliferation, survival and tumor growth. Treatment of MMTV-Myc mice with the RARα-selective agonist Am580 led to significant inhibition of mammary tumor growth (~90%, P<0.001), lung metastasis (P<0.01) and extended tumor latency in 63% of mice. Immunocytochemical analysis showed that in these mice, RARα responsive genes such as Cyp26A1, E-cadherin, cellular retinol-binding protein 1 (CRBP1) and p27, were up-regulated. In contrast, the mammary gland tumors of mice that responded poorly to Am580 treatment (37%) expressed significantly higher levels of RARγ. In vitro experiments indicated that the rise in RARγ was functionally linked to promotion of tumor growth and inhibition of differentiation. Thus, activation of the RARα pathway is linked to tumor growth inhibition, differentiation and cell death. CONCLUSIONS: The functional consequence of the interplay between c-Myc oncogene expression and the RARγ to RARα/ß balance suggests that prevalence of RARγ over-RARα/ß expression levels in breast cancer accompanied by c-Myc amplification or over-expression in breast cancer should be predictive of response to treatment with RARα-isotype-specific agonists and warrant monitoring during clinical trials.

Adipocyte derived paracrine mediators of mammary ductal morphogenesis controlled by retinoic acid receptors.

We generated a transgenic (Tg)-mouse model expressing a dominant negative-(DN)-RARα, (RARαG303E) under adipocytes-specific promoter to explore the paracrine role of adipocyte retinoic acid receptors (RARs) in mammary morphogenesis. Transgenic adipocytes had reduced level of RARα, ß and γ, which coincided with a severely underdeveloped pubertal and mature ductal tree with profoundly decreased epithelial cell proliferation. Transplantation experiments of mammary epithelium and of whole mammary glands implicated a fat-pad dependent paracrine mechanism in the stunted phenotype of the epithelial ductal tree. Co-cultures of primary adipocytes, or in vitro differentiated adipocyte cell line, with mammary epithelium showed that when activated, adipocyte-RARs contribute to generation of secreted proliferative and pro-migratory factors. Gene expression microarrays revealed a large number of genes regulated by adipocyte-RARs. Among them, pleiotrophin (PTN) was identified as the paracrine effectors of epithelial cell migration. Its expression was found to be strongly inhibited by DN-RARα, an inhibition relieved by pharmacological doses of all-trans retinoic acid (atRA) in culture and in vivo. Moreover, adipocyte-PTHR, another atRA responsive gene, was found to be an up-stream regulator of PTN. Overall, these results support the existence of a novel paracrine loop controlled by adipocyte-RAR that regulates the mammary ductal tree morphogenesis.