Ezrin interacts with cortactin to form podosomal rosettes in pancreatic cancer cells.
OBJECTIVE
Pancreatic cancer is a highly invasive malignancy. Ezrin, a plasma membrane-cytoskeletal linker protein, is associated with the invasive behaviour of cancers. The purpose of this study was to elucidate a possible molecular mechanism for the invasive phenotype.


METHODS
Using a combination of techniques, such as western blotting, co-immunoprecipitation, confocal and light microscopy, invasion and adhesion assays, organotypic cultures and human samples as well as RNA interference (RNAi) and expression of various mutant ezrin constructs, the dynamic molecular nature of podosomes in pancreatic cancer was dissected out.


RESULTS
Podosome and podosomal rosette formation in pancreatic carcinoma (PaCa3) cells is ezrin dependent and associated with adhesion to fibronectin with subsequent digestion of this substrate. Ezrin binds to increasing amounts of cortactin during formation of the podosomal rosette, with the C-terminal region, specifically the actin-binding domain, mediating this molecular linkage. Further, it is shown that phosphorylation of Tyr353 and Thr567 sites on ezrin (conventionally shown to translocate ezrin to the plasma membrane) is not required for podosome formation. The podosomal rosette is revealed to be a highly dynamic and transient structure, which can metamorphose into other cellular processes, such as filopodia or lamellipodia, and thereby enable epithelial cancer cells to "palpate" the underlying substrate and modify their cytoskeletal behaviour accordingly. In human tumour tissues and organotypic cultures, specific subcellular expression of ezrin (basal membranous; cellular processes invading stroma) in pancreatic cancer cells can be correlated with tumour progression and disease-free survival (log-rank test (Mantel-Cox), p = 0.019).


CONCLUSIONS
Podosomes and their rosettes are driven by ezrin-cortactin interaction and this plays a role in pancreatic cancer invasion.
Centre for Tumour Biology, Institute of Cancer and CR-UK Clinical Centre, Barts & The London School of Medicine & Dentistry, John Vane Science Centre, London, UK. hemant.kocher@cancer.org.uk
H M
HM
Kocher
J
J
Sandle
T A
TA
Mirza
N F
NF
Li
I R
IR
Hart
D000328:Adult; D000368:Aged; D015153:Blotting, Western; D002277:Carcinoma / Q000378:metabolism* / Q000473:pathology; D002448:Cell Adhesion / Q000502:physiology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D051356:Cortactin / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D009361:Neoplasm Invasiveness; D010190:Pancreatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D017434:Protein Structure, Tertiary; D011554:Pseudopodia / Q000502:physiology / Q000648:ultrastructure; D034741:RNA, Small Interfering / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D000099149:Ezrin
D051356:Cortactin; D003598:Cytoskeletal Proteins; D005353:Fibronectins; D034741:RNA, Small Interfering; D000099149:Ezrin; C495868:CTTN protein, human