The combination of retinoic acid and protein kinase C modulators as a novel therapy for the growth inhibition of cancer stem cells in a triple negative murine mammary cancer model

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

Munich

Congreso; 23rd Biennial Congress of the European Association for Cancer Research (EACR); 2014

European Association for Cancer Research (EACR)

Tumor stem cells are considered responsible of chemoresistance and metastatic dissemination of many breast tumors. Previously we have reported that mammospheres from LM38-LP cell line (composed by luminal, myoepithelial and cancer stem cells CSC) were able to regenerate LM38 in vitro and in vivo. In this work, we used stem-cell-enriched mammospheres from LM38-LP, in order to study whether retinoic acid (ATRA) and different pharmacological PKC inhibitors are involved in the modulation of proliferation, pluripotent genes expression, self-renewal and differentiation. Methodology: cells were treatment or not with ATRA (1uM) and/or PKCa/d inhibitors (Go6976 0.5 uM / Rottlerin 1 uM resp.) by 96H. By RT-PCR we could determine that mammospheres express high levels of Nanog, Sox2, Slug and Sox9 as compared to the original cell line. Surprisingly, only the ATRA/Go6976 combined treatment decreased Nanog and Slug mRNA levels, while the treatment with the PKCd inhibitor induced their expression. The ATRA/PKCa-inhibitor combined treatment induced a synergistic reduction in LM38-LP mammospheres growth rate as compared to either treatment alone(Diameters: Control: 176±63um; ATRA: 129±10µm; PKCa inhibitor: 130±11µm ATRA/PKCa inhibitor: 103±25µm), while PKCd inhibitor treatment induced the opposite effect (217±78µm). Moreover, the ATRA/PKCa-inhibitor combination dramatically affected CSC self-renewal (Number of secondary mammospheres: Control: 313±19; ATRA/PKCa inhibitor: 69±21). Next we analyzed whether the pre treatment of mammospheres with ATRA or PKC inhibitors alters the ability to grow in 3D culture in Matrigel. While ATRA induced the formation of lumen organized structures, Go6976 induced the formation of small colonies with apoptotic cell death signs, Rottlerin treated mammospheres form irregular structures similar to the control. Our results confirm that LM38-LP cell line presents pluripotent cells and the retinoid treatment in combination with the PKCa inhibitor, would be a novel targeted therapy against tumor progenitor stem cells.