PTEN phosphatase-independent maintenance of apical membrane integrity during colorectal glandular morphogenesis


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Ravi Deevi1
1Queen’s University of Belfast, Belfast, UK

Background

Disruption of glandular morphogenesis (GM) is a hallmark of high grade, aggressive colorectal cancer (CRC) but causal mechanisms remain unclear. The tumour suppressor PTEN regulates three dimensional (3D) epithelial morphogenesis by coupling the GTPase cdc42 to spatial cues at the apical membrane (AM). PTEN knockdown disrupts AM integrity and induces a glandular phenotype evocative of high grade cancer in a Caco-2 colorectal organotypic model system. While PTEN has phosphatase-dependent and -independent functions, Caco-2 GM is unaffected by oncogenic phosphatidylinositol 3-kinase (PI3K) signalling. The aims of this study were to investigate effects of PTEN functional domains on 3D Caco-2 morphogenesis and to assess model fidelity to human CRC.

Method

GST PAK based cdc42 pull down, transfections, cell culture and three dimensional cultures of cells, confocal microscopy, western blotting and shRNA stable expression.

Results

Here we show that transient expression of either wild type PTEN or PTEN mutants containing an intact C2 domain enhanced cdc42 activity in PTEN-deficient colorectal epithelium cells. 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. Treatment of cells with sodium butyrate up regulates PTEN expression and also rescued morphogenesis defects in shCaco2 cells. Fundamental attributes of the model system viz associations between AM integrity and gland morphology were conserved and had prognostic significance in CRC. Taken together, these data show that the catalytically inert PTEN C2 domain influences AM dynamics and gland formation in a predictive CRC morphogenesis model system. Insights of PTEN C2-cdc42 pathways of AM integrity may identify molecular therapeutic targets for high grade CRC.

Conclusion

Overall, we conclude that PTEN/Cdc42 pathway regulates apical dynamics there by epithelial morphogenesis and this pathway may be useful as targets to treat colorectal cancer.