Abstract
Pancreatic cancer has the worst prognosis among major malignancies, largely due to its highly invasive property and difficulty in early detection. Mechanistic insights into cancerous transformation and especially metastatic progression are imperative for developing novel treatment strategies. The actin-regulating protein CAP1 is implicated in human cancers, while the role still remains elusive. In this study, we investigated roles for CAP1 and its phosphor-regulation in pancreatic cancer cells. No evidence supports remarkable up-regulation of CAP1 in the panel of cancer cell lines examined. However, knockdown of CAP1 in cancer cells led to enhanced stress fibers, reduced cell motility and invasion into Matrigel. Phosphorylation of CAP1 at the S308/S310 tandem regulatory site was elevated in cancer cells, consistent with hyper-activated GSK3 reported in pancreatic cancer. Inhibition of GSK3, a kinase for S310, reduced cell motility and invasion. Moreover, phosphor mutants had defects in alleviating actin stress fibers and rescuing the reduced invasiveness in the CAP1-knockdown PANC-1 cells. These results suggest a required role for transient phosphorylation for CAP1 function in controlling cancer cell invasiveness. Depletion of CAP1 also reduced FAK activity and cell adhesion, but did not cause significant alterations in ERK or cell proliferation. CAP1 likely regulates cancer cell invasiveness through effects on both actin filament turnover and cell adhesion. Finally, the growth factor PDGF induced CAP1 dephosphorylation, suggesting CAP1 may mediate extracellular signals to control cancer cell invasiveness. These findings may ultimately help develop strategies targeting CAP1 or its regulatory signals for controlling the invasive cycle of the disease.
Highlights
Www.nature.com/scientificreports in regulating the actin cytoskeleton, subsequent studies have revealed far more versatile roles for the protein in facilitating all key steps in the cycle of actin filament turnover, through multiple mechanisms carried out by all three of its structural domains[7,9]
CAP1 protein levels were determined in Western blotting in a panel of commonly used pancreatic cancer cell lines {PANC-126, CFPAC-127, AsPC-128 and Mia PaCa-229}, and compared to that in the immortalized but untransformed pancreas cell line hTERT-HPNE30, which serves as a control
We found that the untransformed hTERT-HPNE cells expressed comparable levels of CAP1 to those in the cancer cell lines
Summary
Www.nature.com/scientificreports in regulating the actin cytoskeleton, subsequent studies have revealed far more versatile roles for the protein in facilitating all key steps in the cycle of actin filament turnover, through multiple mechanisms carried out by all three of its structural domains[7,9]. Activation of FAK caused by CAP1 depletion in HeLa and metastatic breast cancer cells is believed to have overcome the negative effect from the reduced actin filament turnover on cell motility; as a result, knockdown of CAP1 stimulates cell motility and invasiveness[11,12,18]. GSK3 (Glycogen Synthase Kinase 3), which we identified as a kinase for S309 (S310 for human CAP1)[24], was reported to be hyper-activated in pancreatic cancer[25] Both the phosphor-mimetic DD (S307D/S309D) or the unphosphorylatable AA (S307A/S309A) mutant that resists regulation through transient phosphorylation exhibited defects in alleviating the enhanced stress fibers when re-expressed in the CAP1-knockdown HeLa cells[24]. PDGF (Platelet-Derived Growth Factor) induced dephosphorylation of CAP1 at S307/S309, suggesting that CAP1 likely links extracellular signals to cancer cell invasiveness
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