Abstract 5204: Parameters associated with metastatic dissemination are differentially modulated by specific isotypes of the retinoic acid receptor
Authors: Flumian Carolina , Berardi Damián E. , Cirigliano Stefano M. , Díaz Bessone María I. , Bal de Kier Joffé Elisa D. , Urtreger Alejandro J. , Todaro Laura B. 
Published: August 2015

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. 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 determined that both lines express the different RAR isotypes with the exception of RARβ in 4T1 cell line. Cells were treated for 96h with AM580 (RARα agonist, 200nM), AC55649 (RARβ agonist, 2μM), BMS961 (RARγ agonist, 50nM) 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±24.3μm, AC55649: 114.1±12.8μm vs. vehicle: 186.9±31.0μm, p&lt;0.05). An inverse effect was observed in 4T1 cells which increased its migratory potential after the treatment with AM580 (378.9±32.0μm vs. vehicle 302.2±31.9μm, p&lt;0.05).

The same agonist treatments were used to obtain conditioned media in order to evaluate soluble MMPs activity by zymography. AM580, AC55649 and BMS961 pre-treatments decreased soluble MMP2 activity in LM38-LP cells (0.53±0.03au (arbitrary units), 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 au and 2.04±0.26 au respectively, fold change vs. control).

Regarding adhesion assay, after the above mentioned treatments cells were detached, incubated 2h at 37°C in order to recover cell surface proteins and finally seeded. After 2h 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 144h and then inoculated into the tail vein of syngeneic mice. Lungs were removed 21 days later and number of superficial lung metastasis was determined. AC55649 treatment induced an increase in the metastatic potential (Md [Rg]: 143, [86-314] vs. control Md [Rg]: [21.5, 2-73], p&lt;0.05).

In conclusion, the activation of RARα and RARβ isotypes leads to opposite responses in different cell lines. We hypothesized that the differences in RARβ expression between this two cell lines should be responsible of this effect. Surprisingly, the activation of RARβ increased the metastatic potential of LM38-LP cells in spite of the negative regulation of parameters associated with the metastatic process.

Citation Format: Carolina Flumian, Damián E. Berardi, Stefano M. Cirigliano, María I. Díaz Bessone, Elisa D. Bal de Kier Joffé, Alejandro J. Urtreger, Laura B. Todaro. Parameters associated with metastatic dissemination are differentially modulated by specific isotypes of the retinoic acid receptor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5204. doi:10.1158/1538-7445.AM2015-5204