Pharmacological Inhibition of PKCa and Retinoic Acid synergize to inhibit tumour progression and impairs selfrenewal of cancer stem cells through RARg2 modulation in a murine triple negative mammary cancer model

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

Natal

Congreso; 45th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology (SBBq); 2016

Brazilian Society for Biochemistry and Molecular Biology (SBBq)

INTRODUCTION Retinoids exert different effects on malignant phenotype reversion through the crosstalk with PKC pathway.OBJECTIVES We proposed to: A) Evaluate the combined effect of All Trans Retinoic Acid (ATRA) and PKCa inhibitor (Go6976) on: 1) cell proliferation, migration and cancer stem cells (CSC) self renewal and differentiation 2) Retinoic Acid Receptors (RARs) levels. B) Study the effect of ATRA/PKCa inhibitor treatment on tumour progression in vivo. MATERIALS AND METHODS We employed a murine mammary triple negative cell model (LM38LP), composed by luminal (LEP), myoepithelial (MEP) and CSC. We perform RTPCR to measure RARs. Drug interaction was analyzed by Chou-Talalays method. Migration potential was studied by wound healing assay. We employed mammoesphere assays to evaluated CSC. 3D culture in Matrigel to evaluate differentiation. LM38LP cells were inoculated orthotopically to get tumors in BALB/c mice, for in vivo studies. DISCUSSION AND RESULTS We found an inhibitory effect on cell proliferation exerted by ATRA/PKCa, inhibitor that was synergistic by Chou-Talalays method. Furthermore, ATRA/PKCa, inhibitor treatment reduced LM38LP migration more efficiently than each treatment alone, showing a synergic effect. In a 3D matrigel culture assay, ATRA pretreated CSC formed polarized colonies with presence of lumen. PKCa, inhibition impairs lumen formation induced by ATRA but led to smaller colonies. ATRA/PKCa, inhibitor treatment synergized to reduce mammospheres growth. Surprisingly, ATRA treatment induced CSC selfrenewal but the blockage of PKCa; activity impairs this effect. Interestingly, we found that ATRA treatment increased RARb2 and RARg2 in CSC. In addition, we observed that only the combined treatment induced a decrease of RARg2 in the migratory MEP cells and impairs RARg2 induction by ATRA in CSC. Finally, we observed that the combined treatment reduced LM38LP tumor growth and metastatic spread in a synergic manner. CONCLUSION Our findings suggest that ATRA and PKCa inhibition synergize to inhibit tumour progression in vitro and in vivo possibly through the decrease of RARg2.