Regulation of polarized morphogenesis by protein kinase C iota in oncogenic epithelial spheroids

TY - JOUR

T1 - Regulation of polarized morphogenesis by protein kinase C iota in oncogenic epithelial spheroids

AU - Linch, Mark

AU - Sanz-Garcia, Marta

AU - Rosse, Carine

AU - Riou, Philippe

AU - Peel, Nick

AU - Madsen, Chris D.

AU - Sahai, Erik

AU - Downward, Julian

AU - Khwaja, Asim

AU - Dillon, Christian

AU - Roffey, Jon

AU - Cameron, Angus J. M.

AU - Parker, Peter J.

PY - 2014/2

Y1 - 2014/2

N2 - Protein kinase C iota (PKC), a serine/threonine kinase required for cell polarity, proliferation and migration, is commonly up- or downregulated in cancer. PKC is a human oncogene but whether this is related to its role in cell polarity and what repertoire of oncogenes acts in concert with PKC is not known. We developed a panel of candidate oncogene expressing MadinDarby canine kidney (MDCK) cells and demonstrated that H-Ras, ErbB2 and phosphatidylinositol 3-kinase transformation led to non-polar spheroid morphogenesis (dysplasia), whereas MDCK spheroids expressing c-Raf or v-Src were largely polarized. We show that small interfering RNA (siRNA)-targeting PKC decreased the size of all spheroids tested and partially reversed the aberrant polarity phenotype in H-Ras and ErbB2 spheroids only. This indicates distinct requirements for PKC and moreover that different thresholds of PKC activity are required for these phenotypes. By manipulating PKC function using mutant constructs, siRNA depletion or chemical inhibition, we have demonstrated that PKC is required for polarization of parental MDCK epithelial cysts in a 3D matrix and that there is a threshold of PKC activity above and below which, disorganized epithelial morphogenesis results. Furthermore, treatment with a novel PKC inhibitor, CRT0066854, was able to restore polarized morphogenesis in the dysplastic H-Ras spheroids. These results show that tightly regulated PKC is required for normal-polarized morphogenesis in mammalian cells and that H-Ras and ErbB2 cooperate with PKC for loss of polarization and dysplasia. The identification of a PKC inhibitor that can restore polarized morphogenesis has implications for the treatment of Ras and ErbB2 driven malignancies.

AB - Protein kinase C iota (PKC), a serine/threonine kinase required for cell polarity, proliferation and migration, is commonly up- or downregulated in cancer. PKC is a human oncogene but whether this is related to its role in cell polarity and what repertoire of oncogenes acts in concert with PKC is not known. We developed a panel of candidate oncogene expressing MadinDarby canine kidney (MDCK) cells and demonstrated that H-Ras, ErbB2 and phosphatidylinositol 3-kinase transformation led to non-polar spheroid morphogenesis (dysplasia), whereas MDCK spheroids expressing c-Raf or v-Src were largely polarized. We show that small interfering RNA (siRNA)-targeting PKC decreased the size of all spheroids tested and partially reversed the aberrant polarity phenotype in H-Ras and ErbB2 spheroids only. This indicates distinct requirements for PKC and moreover that different thresholds of PKC activity are required for these phenotypes. By manipulating PKC function using mutant constructs, siRNA depletion or chemical inhibition, we have demonstrated that PKC is required for polarization of parental MDCK epithelial cysts in a 3D matrix and that there is a threshold of PKC activity above and below which, disorganized epithelial morphogenesis results. Furthermore, treatment with a novel PKC inhibitor, CRT0066854, was able to restore polarized morphogenesis in the dysplastic H-Ras spheroids. These results show that tightly regulated PKC is required for normal-polarized morphogenesis in mammalian cells and that H-Ras and ErbB2 cooperate with PKC for loss of polarization and dysplasia. The identification of a PKC inhibitor that can restore polarized morphogenesis has implications for the treatment of Ras and ErbB2 driven malignancies.

KW - PROSTATE-CANCER CELLS

KW - ATYPICAL PKC

KW - RAS TRANSFORMATION

KW - IN-VIVO

KW - CAENORHABDITIS-ELEGANS

KW - PROLIFERATION CONTROL

KW - PHOSPHOLIPASE-C

KW - TIGHT JUNCTION

KW - BREAST-CANCER

KW - TUMOR-GROWTH

U2 - 10.1093/carcin/bgt313

DO - 10.1093/carcin/bgt313

M3 - Article

SN - 0143-3334

VL - 35

SP - 396

EP - 406

JO - Carcinogenesis

JF - Carcinogenesis

IS - 2

ER -