Parameters associated with metastatic dissemination are differentially modulated by specific isotypes of the retinoic acid receptor

CAROLINA FLUMIAN; DAMIAN E. BERARDI; STÉFANO M. CIRIGLIANO; MARÍA INÉS DÍAZ BESSONE; ELISA D. BAL DE KIER JOFFE; ALEJANDRO J. URTREGER; LAURA B. TODARO

Filadelfia

Congreso; 106th Annual Meeting of the American Associaton for Cancer Research; 2015

American Associaton for Cancer Research (AACR)

Migration and adhesion are two critical processes highly related to metastasic dissemination and all trans retinoic acid (ATRA) is known to diminished migration and metalloprotease (MMP) secretion in breast cancer cell lines. In this work, our objective was to evaluate the effect of activating each retinoic acid receptor (RAR) isotype in adhesion, migration and MMPs secretion using two hormone-independent murine cell lines: LM38-LP y 4T1. First we could determine that both lines express the different RAR isotypes (with the exception of RARb in 4T1 cells). Cells were treated for 96h with AM580 (RARa agonist, 200 nM), AC55649 (RARb agonist, 2 µM), BMS961 (RARg agonist, 50 nM) or vehicle (DMSO). The migratory potential was evaluated by a wound healing assay. LM38-LP cell line reduced its migratory capacity in response to AM580 and AC55649 (AM580: 108.0±24.3 um, AC55649: 114.1±12.8 um vs. vehicle: 186.9±31.0 um). An inverse effect was observed in 4T1 cells in which increase the migratory potential was observed after the treatment with AM580 (378.9±32.0 um vs. vehicle 302.2±31.9 um). BMS961 pre-treatment did not modulate this biological parameter in neither of these cell lines. The same agonist treatments were used to obtain conditioned media in order to evaluate soluble MMPs activity of by zymography. AM580, AC55649 and BMS961 pre-treatments decreased soluble MMP2 activity in LM38-LP cells (0.53±0.03 au, 0.47±0.01 au, and 0.75±0.04 au respectively, fold change vs control). Conversely, AM580 pre-treatment increased MMP2 and MMP9 in 4T1 cells (1.57±0.10 and 2.04±0.26 respectively, fold change vs control). Regarding adhesion assay, after the above mentioned treatments cells were detached, incubated 2 h at 37°C in order to recover cell surface proteins and finally seeded. After 2 h incubation, non-adherent cells were washed out with PBS and adherent cells were fixed, stained and quantified by densitometry. We observed that AM580 and AC55649 diminished LM38-LP adhesive capacity (0.51±0.02 au, 0.83±0.00 au respectively, fold change vs control) while AC 55649 increased this parameter in 4T1 cells (1.28±0.06 au fold change vs control). Finally we performed an experimental lung metastasis assay using LM38-LP cells. Cells where treated with the different agonist during 144 h and then inoculated into the tail vein of syngeneic mice. Lungs were removed 21 days later and number of superficial lung metastasis was determined. Both, AC55649 and AM580, treatments induce a significant increase in the metastatic potential. In conclusion, the activation of RARa and RARb isotypes lead to opposite responses in different cell lines. We hypothesized that the differences in RARb expression between this two cell lines should be responsible of this effect. Surprisingly, the activation of RARa and RARb increased the metastatic potential of LM38-LP cells in spite of the negatively regulation of parameters associated with the metastatic process.