ABSTRACT
Within the family of protein kinase C (PKC) molecules, the novel isoform PRKCE (PKCÉ) acts as a bona fide oncogene in in vitro and in vivo models of tumorigenesis. Previous studies have reported expression of PKCÉ in breast, prostate and lung tumors above that of normal adjacent tissue. Data from the cancer genome atlas suggest increased copy number of PRKCE in triple negative breast cancer (TNBC). We find that overexpression of PKCÉ in a non-tumorigenic breast epithelial cell line is sufficient to overcome contact inhibition and results in the formation of cellular foci. Correspondingly, inhibition of PKCÉ in a TNBC cell model results in growth defects in two-dimensional (2D) and three-dimensional (3D) culture conditions and orthotopic xenografts. Using stable isotope labeling of amino acids in a cell culture phosphoproteomic approach, we find that CTNND1/p120ctn phosphorylation at serine 268 (P-S268) occurs in a strictly PKCÉ-dependent manner, and that loss of PKCÉ signaling in TNBC cells leads to reversal of mesenchymal morphology and signaling. In a model of Ras activation, inhibition of PKCÉ is sufficient to block mesenchymal cell morphology. Finally, treatment with a PKCÉ ATP mimetic inhibitor, PF-5263555, recapitulates genetic loss of function experiments impairing p120ctn phosphorylation as well as compromising TNBC cell growth in vitro and in vivo. We demonstrate PKCÉ as a tractable therapeutic target for TNBC, where p120ctn phosphorylation may serve as a readout for monitoring patient response.
Subject(s)
Catenins/physiology , Crk-Associated Substrate Protein/metabolism , Protein Kinase C-epsilon/metabolism , Signal Transduction , ras Proteins/metabolism , Oncogenes , PhosphorylationABSTRACT
The synthesis and SAR of a series of 4-anilino-6, 7-dialkoxyquinoline-3-carbonitrile inhibitors of epidermal growth factor receptor (EGF-R) kinase are described. Condensation of 3, 4-dialkoxyanilines with ethyl (ethoxymethylene)cyanoacetate followed by thermal cyclization gave, regiospecifically, 6,7-dialkoxy-4-oxo-1, 4-dihydroquinoline-3-carbonitriles. Chlorination (POCl(3)) followed by the reaction with substituted anilines furnished the 4-anilino-6, 7-dialkoxyquinoline-3-carbonitrile inhibitors of EGF-R kinase. An alternate synthesis of these compounds starts with a methyl 3, 4-dialkoxybenzoate. Nitration followed by reduction (Fe, NH(4)Cl, MeOH-H(2)O) gave a methyl 2-amino-4,5-dialkoxybenzoate. Amidine formation using DMF-acetal followed by cyclization using LiCH(2)CN furnished a 6,7-dialkoxy-4-oxo-1,4-dihydroquinoline-3-carbonitrile, which was transformed as before. Compounds containing acid, ester, amide, carbinol, and aldehyde groups at the 3-position of the quinoline ring were also prepared for comparison, as were several 1-anilino-6,7-dimethoxyisoquinoline-4-carbonitriles. The compounds were evaluated for their ability to inhibit the autophosphorylation of the catalytic domain of EGF-R. The SAR of these inhibitors with respect to the nature of the 6,7-alkoxy groups, the aniline substituents, and the substituent at the 3-position was studied. The compounds were further evaluated for their ability to inhibit the growth of cell lines that overexpress EGF-R or HER-2. It was found that 4-anilinoquinoline-3-carbonitriles are effective inhibitors of EGF-R kinase with activity comparable to the 4-anilinoquinazoline-based inhibitors. A new homology model of EGF-R kinase was constructed based on the X-ray structures of Hck and FGF receptor-1 kinase. The model suggests that with the quinazoline-based inhibitors, the N3 atom is hydrogen-bonded to a water molecule which, in turn, interacts with Thr 830. It is proposed that the quinoline-3-carbonitriles bind in a similar manner where the water molecule is displaced by the cyano group which interacts with the same Thr residue.