Upregulated LAMA3 modulates proliferation, adhesion, migration and epithelial­to­mesenchymal transition of cholangiocarcinoma cells.

A poor outcome for cholangiocarcinoma (CCA) patients is still a clinical challenge. CCA is typically recognized by the desmoplastic nature, which accounts for its malignancy. Among various extracellular matrix proteins, laminin is the most potent inducer for CCA migration. Herein, we accessed the expression profiles of laminin gene family and explored the significance of the key laminin subunit on CCA aggressiveness. Of all 11 laminin genes, LAMA3, LAMA5, LAMB3 and LAMC2 were concordantly upregulated based on the analysis of multiple public transcriptomic datasets and also overexpressed in Thai CCA cell lines and patient tissues in which LAMA3A upregulated in the highest frequency (97%) of the cases. Differential expression genes (DEGs) analysis of low and high laminin signature groups revealed LAMA3 as the sole common DEG in all investigated datasets. Restratifying CCA samples according to LAMA3 expression indicated the association of LAMA3 in the focal adhesion pathway. Silencing LAMA3 revealed that it plays important roles in CCA cell proliferation, adhesion, migration and epithelial-to-mesenchymal transition. Taken together, this research signifies the roles of dysregulated ECM homeostasis in CCA malignancy and highlights, for the first time, the potential usage of LAMA3 as the diagnostic biomarker and the therapeutic target to tackle the CCA stromal.

Role of laminin and cognate receptors in cholangiocarcinoma cell migration.

Extensive desmoplasia in cholangiocarcinoma (CCA) is associated with tumor aggressiveness, indicating a need for further understanding of CCA cell-matrix interaction. This study demonstrated laminin as the most potent attractant for CCA cell migration and the vast elevation of its receptor integrin ß4 (ITGB4) in CCA cell lines. Besides, their high expressions in CCA tissues were correlated with lymphatic invasion and the presence of ITGB4 was also associated with short survival time. ITGB4 silencing revealed it as the receptor for laminin-induced HuCCA-1 migration, but KKU-213 utilized 37/67-kDa laminin receptor (LAMR) instead. These findings highlight the role of ITGB4 and LAMR in transducing laminin induction of CCA cell migration and the potential of ITGB4 as diagnostic and prognostic biomarkers for CCA.

Blocking ERK1/2 signaling impairs TGF-ß1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells.

BACKGROUND: Transforming growth factor-ß (TGF-ß) plays a paradoxical role in cancer: it suppresses proliferation at early stages but promotes metastasis at late stages. This cytokine is upregulated in cholangiocarcinoma and is implicated in cholangiocarcinoma invasion and metastasis. Here we investigated the roles of non-Smad pathway (ERK1/2) and Smad in TGF-ß tumor promoting and suppressing activities in intrahepatic cholangiocarcinoma (ICC) cells. METHODS: TGF-ß1 effects on proliferation, invasion and migration of ICC cells, KKU-M213 and/or HuCCA-1, were investigated using MTT, colony formation, in vitro Transwell and wound healing assays. Levels of mRNAs and proteins/phospho-proteins were measured by quantitative (q)RT-PCR and Western blotting respectively. E-cadherin localization was examined by immunofluorescence and secreted MMP-9 activity was assayed by gelatin zymography. The role of ERK1/2 signaling was evaluated by treating cells with TGF-ß1 in combination with MEK1/2 inhibitor U0126, and that of Smad2/3 and Slug using siSmad2/3- and siSlug-transfected cells. RESULTS: h-TGF-ß1 enhanced KKU-M213 cell invasion and migration and induced epithelial-mesenchymal transition as shown by an increase in vimentin, Slug and secreted MMP-9 levels and by a change in E-cadherin localization from membrane to cytosol, while retaining the cytokine's ability to attenuate cell proliferation. h-TGF-ß1 stimulated Smad2/3 and ERK1/2 phosphorylation, and the MEK1/2 inhibitor U0126 attenuated TGF-ß1-induced KKU-M213 cell invasion and MMP-9 production but moderately enhanced the cytokine growth inhibitory activity. The latter effect was more noticeable in HuCCA-1 cells, which resisted TGF-ß-anti-proliferative activity. Smad2/3 knock-down suppressed TGF-ß1 ability to induce ERK1/2 phosphorylation, Slug expression and cell invasion, whereas Slug knock-down suppressed cell invasion and vimentin expression but marginally affected ERK1/2 activation and MMP-9 secretion. These results indicate that TGF-ß1 activated ERK1/2 through Smad2/3 but not Slug pathway, and that ERK1/2 enhanced TGF-ß1 tumor promoting but repressed its tumor suppressing functions. CONCLUSIONS: Inhibiting ERK1/2 activation attenuates TGF-ß1 tumor promoting effect (invasion) but retains its tumor suppressing role, thereby highlighting the importance of ERK1/2 in resolving the TGF-ß paradox switch.