Abstract

Evolutionarily conserved polarization and morphogenic pathways are critically important for human cancer outcomes. The tumour suppressor PTEN regulates the cdc42/PAR/aPKC polarity complex. PTEN deficiency is associated with impaired glandular morphogenesis that confers poor outlook in human colorectal cancer (CRC). We have investigated PTEN dependent glandular morphogenesis in a 3D organotypic Caco – 2 colorectal model system. PTEN mediated morphogenesis is dependent on coupling of cdc42 to spatial cues at the apical membrane (AM). Parental Caco – 2 cells formed regular hollow gland – like structures (glands) with polarized epithelial cells lined by a uniform AM interface around a single central lumen, in 3D culture. PTEN knockdown influenced expression or localization of cdc42 guanine nucleotide exchange factors (GEFs), inhibited cdc42 activation, disrupted AM integrity and induced a multilumen Caco – 2 glandular phenotype evocative of high grade cancer. While PTEN has lipid phosphatase-dependent and – independent functions, Caco – 2 morphogenesis was unaffected by oncogenic phosphatidylinositol 3 – kinase (PI3K) signalling or PI3K targeted treatment. To investigate effects of PTEN functional domains, we assessed effects of catalytically-active or – inactive PTEN mutants on cdc42 activity and/or AM integrity during 3D Caco – 2 morphogenesis. Here we show that PTEN mutants containing an intact C2 domain enhanced cdc42 activity in PTEN deficient colorectal epithelium. Transfection of PTEN deficient Caco – 2 (Caco – 2 ShPTEN) cultures with the PTEN C2 domain rescued AM integrity and defective 3D morphogenesis. Conversely, a C2 domain construct mutated at its CBR3 lipid-binding motif was ineffective. Fundamental attributes of the model system viz associations between AM integrity and gland morphology were conserved and had prognostic significance in human CRC . Taken together, these data show PTEN influences AM dynamics and gland formation in a predictive CRC morphogenesis model system, substantively through its catalytically inert membrane-targeting C2 domain. Dissection of PTEN C2 -cdc42 regulatory pathways of AM integrity may identify molecular targets for novel therapy, aimed at high grade CRC .