Introduction Disruption of colorectal gland formation characterises high grade, aggressive CRCs but causal mechanisms remain unclear. Glandular morphogenesis can be modelled in three-dimensional (3D) culture systems that enable investigation of specific oncogenic signals. We have shown that the tumour suppressor PTEN regulates 3D glandular morphogenesis in a Caco-2 colorectal organotypic model system through effects on the Rho-GTPase cdc42. Cdc42 is activated by specific guanine nucleotide exchange factors (GEFs) and influences gland lumen formation by regulation of apical membrane (AM) assembly. PTENknockdown inhibits cdc42, disrupts AM integrity and induces a vacuolar, multilumen glandular phenotype evocative of high grade CRC. While PTENhas catalytically -active or -inactive functional domains relevant to phosphatidylinositol 3-kinase (PI3K) activity, Caco-2 gland development was unaffected by PI3K signalling.
Methods We used wild type PTEN-expressing Caco-2 cells and isogenic stable PTENknockdown Caco-2 (KD) clones in two- (2D) and three-dimensional (3D) cultures as model systems. Cell membrane localization of specific cdc42 GEFs was investigated by cell fractionation and immunoblot. Effects of catalytically -active or -inactive PTENmutants on cdc42 activity and/or AM integrity during 3D morphogenesis were investigated by transfection and confocal microscopy. Apical membrane integrity was assessed in human CRCby semiquantitative score of the AM marker, NHERF-1. CRCgland morphology was assessed by a validated grading system.
Results PTENexpression enhanced cell membrane recruitment of cdc42 GEFs with a specific role in 3D morphogenesis (Tuba, ITSN2). PTENmutants containing an intact catalytically-inactive C2 domain enhanced cdc42 activity, restored AM integrity and rescued defective morphogenesis of 3D PTEN-KD Caco-2 cultures. 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.
Conclusion PTENdeficiency impairs GEF membrane recruitment, cdc42 activation, apical membrane assembly and CRCglandular morphogenesis in a predictive colorectal cancer model system.
PTEN-cdc42 regulatory pathways influence AM integrity and colorectal glandular morphogenesis. Dissection of these networks may identify molecular targets for novel therapy, aimed at high grade CRC.
Disclosure of Interest None Declared.