Pharmacological inhibition of Protein Kinase C Alpha (PKCa) and All Trans Retinoic Acid (ATRA) synergize to inhibit the proliferation, migration and cancer stem-like properties of a triple negative mammary cancer model

DAMIAN E. BERARDI; MARIA I. DIAZ BESSONE; CAROLINA FLUMIAN; STEFANO M. CIRIGLIANO; ELISA D. BAL DE KIER JOFFE; ALEJANDRO J. URTREGER; LAURA B. TODARO

San Diego

Congreso; 105th Annual Meeting of the American Associaton for Cancer Research; 2014

American Associaton for Cancer Research (AACR)

To study the interaction between PKCa and retinoic acid pathways on the biology of breast cancer, we employed a murine mammary triple negative cell model (LM38-LP, composed by luminal (LEP), myoepithelial (MEP) and cancer stem cells (CSC). We proposed to: A) Study the effect of ATRA (1uM) on the expression of PKCa and Retinoic Acid Receptors (RARs) in LEP, MEP and CSC (mammospheres); B) Evaluate the combined effect of ATRA and a PKCa pharmacological inhibitor (1 uM Go6976) on cell proliferation employing a method to determine the interaction type; C) Analyze the effect of ATRA/PKCa inhibitor on migration and MMP activity on LM38-LP;. D) Analyze the effect of ATRA/PKCa inhibitor on proliferation, self-renewal and differentiation of CSC; E) Study the expression profile of pluripotent genes in CSC and their modulation by treatments. By RT-PCR we found that ATRA (48 h) induced a decrease in PKCa in LEP, MEP and CSC. The same treatment increased RARb2 and RARg2 in CSC, and only RARb2 in LEP. ATRA and PKCa inhibitor interaction results from the proliferation assay were analyzed by Chou-Talalay´s method. We found that the inhibitory effect exerted by ATRA/PKCá inhibitor was synergistic with CI=0.59 (Synergistic with CI=0.7). ATRA/PKCá inhibitor treatment reduced LM38-LP migration more efficiently than each treatment alone, showing a synergic effect (% migration: Control: 80,1±6,9, ATRA: 55,4±5,6, PKCa inhibitor: 37,3±9,5, ATRA/PKCa inhibitor: 20,0±1,7). Furthermore, MMP2 activity was strongly reduced by the combined treatment. Interestingly, we observed that only the combined treatment induced a decrease of RARg2 expression in the highly migratory MEP cells. ATRA/PKCa inhibitor treatment synergized to reduce mammospheres growth (Diameter in um at 96h: Control: 176±8, ATRA: 129±10; PKCa inhibitor: 130±11 ATRA/PKCa inhibitor: 103±5). Pre-treatment with ATRA/PKCa inhibitor for 96h dramatically affected CSC self-renewal (Number of secondary mammospheres: Control: 313±19; ATRA/PKCa inhibitor: 69±21). While in a 3D matrigel culture assay ATRA-pretreated CSC formed organized colonies with presence of lumen, the combined treatment led to the formation of small undifferentiated structures and evidence of cell death. By RT-PCR we determined that CSC expressed higher levels of pluripotent genes, such as Nanog, Sox2, Slug and Sox9, than the parental LM38-LP cell line. Besides, only the combined treatment for 96 h induced a decrease in the levels of Nanog and Slug in CSC. Our findings suggest that the pharmacological inhibition of PKCa and ATRA synergize to inhibit proliferation and migration possibly through the decrease of RARg and the inhibition of MMP2 activity. Furthermore, the blockage of CSC expansion by the combined treatment, possibly occurred through the down regulation of Nanog and Slug.