Abstract
BackgroundHepatocellular carcinoma (HCC) is the most common form of liver cancer and the third most lethal cancer worldwide. The epithelial to mesenchymal transition (EMT) describes the transformation of well-differentiated epithelial cells to a de-differentiated phenotype and plays a central role in the invasion and intrahepatic metastasis of HCC cells. Modulation of the transforming growth factor-β (TGF-β) signaling is known to induce various tumor-promoting and EMT-inducing pathways in HCC. The meta-analysis of a panel of EMT gene expression studies revealed that neuropilin 2 (NRP2) is significantly upregulated in cells that have undergone EMT induced by TGF-β. In this study we assessed the functional role of NRP2 in epithelial and mesenchymal-like HCC cells and focused on the molecular interplay between NRP2 and TGF-β/Smad signaling.MethodsNRP2 expression was analyzed in human HCC cell lines and tissue arrays comprising 133 HCC samples. Cell migration was examined by wound healing and Transwell assays in the presence and absence of siRNA against NRP2. NRP2 and TGF-β signaling were analyzed by Western blotting and confocal immunofluorescence microscopy.ResultsWe show that NRP2 is particularly expressed in HCC cell lines with a dedifferentiated, mesenchymal-like phenotype. NRP2 expression is upregulated by the canonical TGF-β/Smad signaling while NRP2 expression has no impact on TGF-β signaling in HCC cells. Reduced expression of NRP2 by knock-down or inhibition of TGF-β signaling resulted in diminished cell migration independently of each other, suggesting that NRP2 fails to collaborate with TGF-β signaling in cell movement. In accordance with these data, elevated levels of NRP2 correlated with a higher tumor grade and less differentiation in a large collection of human HCC specimens.ConclusionsThese data suggest that NRP2 associates with a less differentiated, mesenchymal-like HCC phenotype and that NRP2 plays an important role in tumor cell migration upon TGF-β-dependent HCC progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1919-0) contains supplementary material, which is available to authorized users.
Highlights
Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the third most lethal cancer worldwide
neuropilin 2 (NRP2) was found distributed in a patchy fashion (Fig. 1b) and interestingly, NRP2 was exclusively expressed in those patient samples that showed transforming growth factor (TGF)-β1 expression (Additional file 1: Figure S1)
NRP2 expression was found in dedifferentiated mesenchymal-like HCC cells in vitro which supports the idea that NRP2 expression correlates with a de-differentiated phenotype
Summary
Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the third most lethal cancer worldwide. Modulation of the transforming growth factor-β (TGF-β) signaling is known to induce various tumor-promoting and EMT-inducing pathways in HCC. Various signaling cascades are known to induce EMT, such as the Wnt/β-catenin, PI3K/AKT/ mTOR, Hedgehog, Ras/Raf/MEK/ERK, Notch and NFκB pathways, as well as transforming growth factor (TGF)-β [12,13,14,15]. These signals mostly converge on EMTtranscription factors (EMT-TFs) such as Snail, ZEB1 or Twist1/2 which transcriptionally repress E-cadherin and other epithelial junctional proteins as well as activate a mesenchymal gene expression signature. In HCC, TGF-β signaling has been shown to activate EMT-TFs and to repress their negative feedback loops by the downregulation of miRNAs that antagonize EMT-TFs [16, 17]
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