To study the interaction between PKCα 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 (1μM) on the expression of PKCα and Retinoic Acid Receptors (RARs) in LEP, MEP and CSC (mammospheres); B) Evaluate the combined effect of ATRA and a PKCα pharmacological inhibitor (1 uM Go6976) on cell proliferation employing a method to determine the interaction type; C) Analyze the effect of ATRA/PKCα inhibitor on migration and MMP activity on LM38-LP;. D) Analyze the effect of ATRA/PKCα 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 PKCα in LEP, MEP and CSC. The same treatment increased RARβ2 and RARγ2 in CSC, and only RARβ2 in LEP.

ATRA and PKCα inhibitor interaction results from the proliferation assay were analyzed by Chou-Talalay´s method. We found that the inhibitory effect exerted by ATRA/PKCα inhibitor was synergistic with CI=0.59 (Synergistic with CI<0.7).

ATRA/PKCα 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, PKCα inhibitor: 37,3±9,5, ATRA/PKCα inhibitor: 20,0±1,7; p≤0.05). Furthermore, MMP2 activity was strongly reduced by the combined treatment. Interestingly, we observed that only the combined treatment induced a decrease of RARγ2 expression in the highly migratory MEP cells.

ATRA/PKCα inhibitor treatment synergized to reduce mammospheres growth (Diameter in µm at 96h: Control: 176±8, ATRA: 129±10; PKCα inhibitor: 130±11 ATRA/PKCα inhibitor: 103±5 p≤0.05). Pre-treatment with ATRA/PKCα inhibitor for 96h dramatically affected CSC self-renewal (Number of secondary mammospheres: Control: 313±19; ATRA/PKCα inhibitor: 69±21; p≤0.05). 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 PKCα and ATRA synergize to inhibit proliferation and migration possibly through the decrease of RARγ 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.

Citation Format: Damian E. Berardi, Maria I. Diaz Bessone, Carolina Flumian, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Pharmacological inhibition of protein kinase C alpha (PKCα) and all trans retinoic acid (ATRA) synergize to inhibit the proliferation, migration and cancer stem-like properties of a triple-negative mammary cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 209. doi:10.1158/1538-7445.AM2014-209