Mechanisms involved in the inhibitory effect of retinoids on a murine mammary cell line (LM38 LP) composed by luminal and myoepithelial subpopulations
Authors: Laura Todaro, Paola Campodonico, Maria Veloso, Lydia Puricelli, Eduardo Farias, Elisa Bal de Kier Joffe
Journal: Cancer Research (2008)

2665  Retinoids regulate growth, differentiation and apoptosis of many cell types by binding to RAR/RXR nuclear receptors. These receptors up regulate expression of target genes by binding to their retinoic acid response elements (RARE) in their promoters or by repressing other genes by inhibiting the action of transcription factors like AP1. Previously, we demonstrated that LM38 LP, composed by luminal (LEP) and myoepithelial (MEP) cells, express the main RAR/RXR, and that ATRA inhibited its growth and metastatic dissemination. Now we studied the mechanisms involved in retinoids inhibitory effect with the aim to analyze the interaction between LEP and MEP cells in this response.  The retinoic acid pathway activation was confirmed by RARE-Luc assays and by monitoring the increased levels of RAR target genes, RARb2 and CRBP by RT PCR / western blot (WB).  Retinoids inhibited LM38 LP in vitro cell growth, this effect was reversed by RARa antagonist Ro415253 (Ro).  Confocal microscopy of LM38 LP 3D cultures showed, that ATRA increased the expression of activated caspase 3 and promoted p27 nuclear translocation in the colony center, indicating that both arrest and apoptosis were taking place. In 2D control cultures, half of MEP cells were positive for p27 while LEP islets expressed P-RB. Three days after treatment with retinoids we observed an increase in the number of p27 stained arrested MEP while LEP islets became negative for P-RB acquiring p27 expression, except for a single cell layer around the islets that kept cycling. Cell cycle analysis by FACS of LM38 LP cells treated with ATRA showed G1 arrest. Similar results were observed in vivo, where immunohistochemistry of tumors grown in mice treated with ATRA twice a week for 15 days, showed increased staining for p27 and extense areas of apoptotic and necrotic cells with some activated caspase 3 positive cells. Besides arrest and apoptosis, retinoids induced senescence mainly in MEP cells (β Gal activity). Interestingly retinoids did not affect LM38 LP clonogenic capacity.  Cell cycle arrest and apoptosis were confirmed by WB showing a decrease in D1 / B1 cyclins, P-ERK and activated caspase 3, after 30 minutes of stress conditions. Both retinoids inhibited LM38 LP secreted MMPs and uPA, but contrarily to its effect on proliferation, Ro mimicked ATRA.  Conclusion: RARs activation by retinoids inhibit LM38 LP mammary tumors growth affecting in a differential fashion their LEP and MEP cellular compartments by induction of cell cycle arrest, senescence and apoptosis, suggesting the presence of a retinoid-resistant clonogenic subpopulation. Physical interaction between these cellular compartments seems to generate survival stimuli strong enough to overcome the retinoids antitumoral effects. The lack of modulation by Ro on the proteolytic activity is an indication that this antagonist is not involved in the transrepression RAR and AP1