Stem Cell Marker Nestin Is Critical for TGF-β1-Mediated Tumor Progression in Pancreatic Cancer

Abstract

The stem cell marker nestin is an intermediate filament protein that plays an important role in cell integrity, migration, and differentiation. Nestin expression occurs in approximately one third of pancreatic ductal adenocarcinoma (PDAC), and its expression strongly correlates with tumor staging and metastasis. Little is known about the mechanisms by which nestin influences PDAC progression. Here, nestin overexpression in PDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymal transition (EMT) in vitro; conversely, knockdown of endogenous nestin expression reduced the migration rate and reverted cells to a more epithelial phenotype. Mouse xenograft studies showed that knockdown of nestin significantly reduced tumor incidence and volume. Nestin protein expression was associated with Smad4 status in PDAC cells; hence, nestin expression might be regulated by the TGF-b1/Smad4 pathway in PDAC. We examined nestin expression after TGF-b1 treatment in human pancreatic cancer PANC-1 and PANC-1 shSmad4 cells. The TGF-b1/Smad4 pathway induced nestin protein expression in PDAC cells in a Smad4-dependent manner. Moreover, increased nestin expression caused a positive feedback regulator of the TGF-b1 signaling system. In addition, hypoxia was shown to induce nestin expression in PDAC cells, and the hypoxiainduced expression of nestin is mediated by the TGF-b1/Smad4 pathway. Finally, the antimicrotubule inhibitors, cytochalasin D and withaferin A, exhibited anti-nestin activity; these inhibitors might be potential antimetastatic drugs. Our findings uncovered a novel role of nestin in regulating TGF-b1-induced EMT. Antinestin therapeutics may serve as a potential treatment for PDAC metastasis. Mol Cancer Res; 11(7); 768-79. Ó2013 AACR. IntroductionThe incidence of pancreatic ductal adenocarcinoma (PDAC) is rising to the fifth leading cause of cancerrelated mortality. The condition is associated with the poorest prognosis among all gastrointestinal cancers, with a 5-year survival rate less than 4% (1, 2). A main reason for this extremely poor prognosis is the cancer's tendency to invade adjacent tissues and metastasize to regional lymph at a relatively early stage. Patients with PDAC frequently

Utilization of Liquid Chromatography Mass Spectrometry Analyses to Identify LKB1–APC Interaction in Modulating Wnt/β-Catenin Pathway of Lung Cancer Cells

“…The pLKO.1-shRNA scrambled control vector was pLKO_TRC025. Lipofectamine 2000 reagent (Invitrogen) was used for lentiviral production in 293T cells with a packaging construct (pCMV-DR8.91), an envelope construct (pMD.G), and different shRNA constructs as previously described (22,23).…”

“…Western blotting was performed as previously described (23,24). Primary antibodies used were anti-smooth muscle actin (SMA, ab5698), anti-N-cadherin (ab12221; Abcam), anti-E-cadherin (sc-8426), anti-vimentin (sc-32322), anti-APC (sc-54), anti-CD44 (sc-18849), anti-LKB1 (sc-5638 and sc-32245), anti-COX2 (sc-0736), anti-survivin (sc-752), anti-VEGF (sc-152), anti-cyclin D1 (sc-718), antic-Myc (9E10; sc-40), and total b-catenin (sc-1496; Santa Cruz Biotechnology), anti-GSK3b (#9315), anti-p-GSK3b (#9336, Cell Signal), anti-active-b-catenin (Millipore), and mouse anti-b-actin (Sigma Aldrich).…”

“…Transient transfections and luciferase reporter assays were conducted using the Dual-Glo Luciferase Assay System from Promega (Cat. #E2920), as previously described (22,23,25).…”

Stem Cells in Pancreatic Cancer

“…In pancreatic carcinoma, one third of tumor cells present nestin expression which is correlated with tumor staging and metastasis. Nestin-expressing cells are involved in epithelial-to-mesenchymal transition (EMT) and seem to be the origin of pancreatic intraepithelial neoplasia lesions [19]. Recently, presence of Nestin in various types of malignancy was associated with tumoral angiogenesis and was proposed as an angiogenic marker [20].…”

Pancreatic Cancer Stem Cells

“…Their study, in some way, illuminated our way to detect pancreatic cancer at PanIN stage and to prevent the progression of PanIN, and offered new targets for treatments. Additionally, there are studies showing other CSC markers such as the nestin [23,24], aldehyde dehydrogenase (ALDH-1) [23][24][25][26], ABCG2 and c-Met [24] pancreatic adenocarcinoma [25].…”