Integrin-Linked Kinase Overexpression and Its Oncogenic Role in Promoting Tumorigenicity of Hepatocellular Carcinoma

Background: Integrin-linked kinase (ILK) was first discovered as an integrin β1-subunit binding protein. It localizes at the focal adhesions and is involved in cytoskeleton remodeling. ILK overexpression and its dysregulated signaling cascades have been reported in many human cancers. Aberrant expression of ILK influenced a wide range of signaling pathways and cellular functions. Although ILK has been well characterized in many malignancies, its role in hepatocellular carcinoma (HCC) is still largely unknown. Methodology/Principal Findings: Quantitative PCR analysis was used to examine ILK mRNA expression in HCC clinical samples. It was shown that ILK was overexpressed in 36.9% (21/57) of HCC tissues when compared to the corresponding non-tumorous livers. The overall ILK expression level was significantly higher in tumorous tissues (P = 0.004), with a significant stepwise increase in expression level along tumor progression from tumor stage I to IV (P = 0.045). ILK knockdown stable clones were established in two HCC cell lines, BEL7402 and HLE, and were subjected to different functional assays. Knockdown of ILK significantly suppressed HCC cell growth, motility and invasion in vitro and inhibited tumorigenicity in vivo. Western blot analysis revealed a reduced phosphorylated-Akt (pAkt) at Serine-473 expression in ILK knockdown stable clones when compared to control clones. Conclusion/Significance: This study provides evidence about the clinical relevance of ILK in hepatocarcinogenesis. ILK was found to be progressively elevated along HCC progression. Here our findings also provide the first validation about the oncogenic capacity of ILK in vivo by suppressing its expression in HCC cells. The oncogenic role of ILK is implicated to be mediated by Akt pathway. © 2011 Chan et al.published_or_final_versio

ILK and cytoskeletal architecture: an important determinant of AQP2 recycling and subsequent entry into the exocytotic pathway

Within the past decade tremendous efforts have been made to understand the mechanism behind aquaporin-2 (AQP2) water channel trafficking and recycling, to open a path toward effective diabetes insipidus therapeutics. A recent study has shown that integrin-linked kinase (ILK) conditional-knockdown mice developed polyuria along with decreased AQP2 expression. To understand whether ILK also regulates AQP2 trafficking in kidney tubular cells, we performed in vitro analysis using LLCPK1 cells stably expressing rat AQP2 (LLC-AQP2 cells). Upon treatment of LLC-AQP2 cells with ILK inhibitor cpd22 and ILK-siRNA, we observed increased accumulation of AQP2 in the perinuclear region, without any significant increase in the rate of endocytosis. This perinuclear accumulation did not occur in cells expressing a serine-256-aspartic acid mutation that retains AQP2 in the plasma membrane. We then examined clathrin-mediated endocytosis after ILK inhibition using rhodamine-conjugated transferrin. Despite no differences in overall transferrin endocytosis, the endocytosed transferrin also accumulated in the perinuclear region where it colocalized with AQP2. These accumulated vesicles also contained the recycling endosome marker Rab11. In parallel, the usual vasopressin-induced AQP2 membrane accumulation was prevented after ILK inhibition; however, ILK inhibition did not measurably affect AQP2 phosphorylation at serine-256 or its dephosphorylation at serine-261. Instead, we found that inhibition of ILK increased F-actin polymerization. When F-actin was depolymerized with latrunculin, the perinuclear located AQP2 dispersed. We conclude that ILK is important in orchestrating dynamic cytoskeletal architecture during recycling of AQP2, which is necessary for its subsequent entry into the exocytotic pathway

The Expression of Genes Involved in Hepatocellular Carcinoma Chemoresistance Is Affected by Mitochondrial Genome Depletion

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Molecular Pharmaceutics, copyright © 2014 American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://pubs.acs.org/doi/10.1021/mp400732p.[EN] Deletions and mutations in mitochondrial DNA (mtDNA), which are frequent in human tumors, such as hepatocellular carcinoma (HCC), may contribute to enhancing their malignant phenotype. Here we have investigated the effect of mtDNA depletion in the expression of genes accounting for mechanisms of chemoresistance (MOC) in HCC. Using human HCC SK-Hep-1 cells depleted of mtDNA (Rho), changes in gene expression in response to antitumor drugs previously assayed in HCC treatment were analyzed. In Rho cells, a decreased sensitivity to doxorubicin-, SN-38-, cisplatin (CDDP)-, and sorafenib-induced cell death was found. Both constitutive and drug-induced reactive oxygen species generation were decreased. Owing to activation of the NRF2-mediated pathway, MDR1, MRP1, and MRP2 expression was higher in Rho than in wild-type cells. This difference was maintained after further upregulation induced by treatment with doxorubicin, SN-38, or CDDP. Topoisomerase-IIa expression was also enhanced in Rho cells before and after treatment with these drugs. Moreover, the ability of doxorubicin, SN-38 and CDDP to induce proapoptotic signals was weaker in Rho cells, as evidenced by survivin upregulation and reductions in Bax/Bcl-2 expression ratios. Changes in these genes seem to play a minor role in the enhanced resistance of Rho cells to sorafenib, which may be related to an enhanced intracellular ATP content together with the loss of expression of the specific target of sorafenib, tyrosine kinase receptor Kit. In conclusion, these results suggest that mtDNA depletion may activate MOC able to hinder the efficacy of chemotherapy against HCC