title: PTEN phosphatase-independent maintenance of glandular morphology in a predictive colorectal cancer model system.
abstract: Organotypic models may provide mechanistic insight into colorectal cancer (CRC) morphology. Three-dimensional (3D) colorectal gland formation is regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN) coupling of cell division cycle 42 (cdc42) to atypical protein kinase C (aPKC). This study investigated PTEN phosphatase-dependent and phosphatase-independent morphogenic functions in 3D models and assessed translational relevance in human studies. Isogenic PTEN-expressing or PTEN-deficient 3D colorectal cultures were used. In translational studies, apical aPKC activity readout was assessed against apical membrane (AM) orientation and gland morphology in 3D models and human CRC. We found that catalytically active or inactive PTEN constructs containing an intact C2 domain enhanced cdc42 activity, whereas mutants of the C2 domain calcium binding region 3 membrane-binding loop (M-CBR3) were ineffective. The isolated PTEN C2 domain (C2) accumulated in membrane fractions, but C2 M-CBR3 remained in cytosol. Transfection of C2 but not C2 M-CBR3 rescued defective AM orientation and 3D morphogenesis of PTEN-deficient Caco-2 cultures. The signal intensity of apical phospho-aPKC correlated with that of Na(+)/H(+) exchanger regulatory factor-1 (NHERF-1) in the 3D model. Apical NHERF-1 intensity thus provided readout of apical aPKC activity and associated with glandular morphology in the model system and human colon. Low apical NHERF-1 intensity in CRC associated with disruption of glandular architecture, high cancer grade, and metastatic dissemination. We conclude that the membrane-binding function of the catalytically inert PTEN C2 domain influences cdc42/aPKC-dependent AM dynamics and gland formation in a highly relevant 3D CRC morphogenesis model system.
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University of Belfast, Belfast, United Kingdom.
authors.forename: Ishaan C
authors.initials: IC
authors.lastname: Jagan
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University of Belfast, Belfast, United Kingdom.
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Deevi
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University of Belfast, Belfast, United Kingdom.
authors.forename: Aliya
authors.initials: A
authors.lastname: Fatehullah
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University of Belfast, Belfast, United Kingdom.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Topley
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University of Belfast, Belfast, United Kingdom.
authors.forename: Joshua
authors.initials: J
authors.lastname: Eves
authors.affiliation: Centre for Public Health, Queen's University of Belfast, Belfast, United Kingdom.
authors.forename: Michael
authors.initials: M
authors.lastname: Stevenson
authors.affiliation: Department of Histopathology, Royal Victoria Hospital, Belfast, United Kingdom ; Northern Ireland Molecular Pathology Laboratory, Centre for Cancer Research and Cell Biology, Queen's University, Belfast, United Kingdom.
authors.forename: Maurice
authors.initials: M
authors.lastname: Loughrey
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University of Belfast, Belfast, United Kingdom ; Northern Ireland Molecular Pathology Laboratory, Centre for Cancer Research and Cell Biology, Queen's University, Belfast, United Kingdom.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Arthur
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University of Belfast, Belfast, United Kingdom.
authors.forename: Frederick Charles
authors.initials: FC
authors.lastname: Campbell
mesh_terms: D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D015179:Colorectal Neoplasms / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D045325:HCT116 Cells; D006801:Humans; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D014162:Transfection
chemical_list: D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; C115015:PKC-3 protein; D011493:Protein Kinase C; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human