The integrin-linked kinase gene up-regulated by hypoxia plays its pro-survival role in colorectal cancer cells

Abstract

Context: Colorectal cancer (CRC) is a leading cause of cancer death in recent years. It is believed that there are hypoxic regions in both early and advanced stage of tumor and hypoxia is able to reinforce the aggressiveness of tumor cells and accelerate the progression of cancer. Objective: Until now the mechanisms by which hypoxia promotes the progression of CRC are far from well understood. Integrin-linked kinase (ILK) is a crucial mediator and over-expressed in CRC patients. But whether ILK is involved in the process that hypoxia promotes CRC cells growth and silencing the ILK gene results in CRC cells apoptosis is not clear. Materials and methods: Lentivirus transfection, invasion assay, TUNEL assay, Bromodeoxyuridine incorporation and mitochondrial function assay were applied to demonstrate our hypothesis. Results: In this study, we found that hypoxia induced the expression of ILK in a time-dependent manner, and after knocking down ILK expression with ILK shRNA, the cells proliferation promoted by hypoxia was inhibited in HT29 cell line. Moreover, blocking the ILK pathway led to caspase-3 and caspase-9 activations, the decrease of mitochondrial membrane potential, and cells apoptosis. And the inhibitory effects of hypoxia on cells apoptosis were mediated by the ILK pathway. In addition, hypoxia promoted HT29 cells metastasis and invasion through the ILK pathway. Conclusions: Therefore, we conclude that the CRC cells survival and invasion enhanced by hypoxia are mediated by ILK, and ILK may be an important potential therapeutic target for CRC.     

Small interfering RNA targeting integrin-linked kinase inhibited the growth and induced apoptosis in human bladder cancer cells

Integrin-linked kinase (ILK), an intracellular serine/threonine kinase, is implicated in cell growth and survival, cell-cycle progression, tumor angiogenesis, and cell apoptosis. Recent studies showed that the expression and activity of ILK increased significantly in many types of solid tumors. However, the exact molecular mechanism of ILK underlie tumor has not been fully ascertained. The purpose of our study was to determine whether knockdown of ILK would inhibit cell growth and induce apoptosis in bladder cancer cells using a plasmid vector based small interfering RNA (siRNA). The experiments showed that knockdown of ILK could remarkably inhibit cell proliferation and growth, regulate cell cycle and induce apoptosis of bladder cancer BIU-87 and EJ cells. We demonstrated that knockdown of ILK inhibited phosphorylation of downstream signaling targets protein kinase B/Akt, glycogen synthase kinase 3-beta (GSK-3β), and reduced expression of β-catenin in BIU-87 as well as EJ cells by Western blot and Immunofluorescence analysis. In addition, down-regulation of ILK also could increase expression of Ribonuclease inhibitor (RI), an important acidic cytoplasmic protein with many functions. BALB/C nude mice injected with the BIU-87 cells transfected ILK siRNA showed a significant inhibition of the tumor growth with lighter tumor weight, lower microvessels density and higher apoptosis rate than those in the other two control groups. In conclusion, these results suggest that ILK might be involved in the development of bladder cancer, and could be served as a novel potential therapy target for human bladder cancer. Our study may be of biological and clinical importance.     

Downregulation of integrin-linked kinase inhibits epithelial-to-mesenchymal transition and metastasis in bladder cancer cells

Integrin-linked kinase (ILK) is a multifunctional serine/threonine kinase in cytoplasm. Recent studies showed that cancer patients with increased ILK expression had low survival, poor prognosis and increased metastasis. Although the causes of ILK overexpression remain to be fully elucidated, accumulating evidence suggests that its oncogenic capacity derives from its regulation of several downstream targets that provide cells with signals that promote proliferation, survival and migration. However, the mechanisms underlying tumor metastasis by ILK is still not fully understood. Epithelial–mesenchymal transition (EMT) is a critical event of cancer cells that triggers invasion and metastasis. We recently reported that knockdown of ILK inhibited the growth and induced apoptosis in human bladder cancer cells. Therefore, we postulate that ILK might involve in EMT. Here we further investigate the function of ILK with RNA interference in bladder cancer cells. Knockdown of ILK impeded an EMT with low Vimentin, Snail, Slug and Twist as well as high E-cadherin expression in vivo and vitro. In addition, we found that knockdown of ILK inhibited cell proliferation, migration and invasion as well as changed cell morphology, adhesion and rearranged cytoskeleton in vitro. We also demonstrated that ILK siRNA inhibited phosphorylation of downstream signaling targets Akt and GSK3β, increased expression of nm23-H1, as well as reduced expression of MMP-2 and MMP-9 in vivo and vitro. Furthermore, downregulation of ILK could increase expression of Ribonuclease inhibitor (RI), an important acidic cytoplasmic protein with many functions. Finally, the effects of ILK siRNA on bladder cancer cell phenotype and invasiveness translate into suppression for tumorigenesis and metastasis in vivo. Taken together, our findings highlight that ILK signaling pathway plays a novel role in the development of bladder cancer through regulating EMT. ILK could be a promising diagnostic marker and therapeutic target for bladder cancer.     

A growth-suppressive function for the c-fes protein-tyrosine kinase in colorectal cancer

The human c-fes locus encodes a non-receptor protein-tyrosine kinase implicated in myeloid, vascular endothelial, and neuronal cell differentiation. A recent analysis of the tyrosine kinome in colorectal cancer identified c-fes as one of only seven genes with consistent kinase domain mutations. Although four mutations were identified (M704V, R706Q, V743M, S759F), the consequences of these mutations on Fes kinase activity were not explored. To address this issue, Fes mutants with these substitutions were co-expressed with STAT3 in human 293T cells. Surprisingly, the M704V, R706Q, and V743M mutations substantially reduced Fes autophosphorylation and STAT3 Tyr-705 phosphorylation compared with wild-type Fes, whereas S759F had little effect. These mutations had a similar impact on Fes kinase activity in a yeast expression system, suggesting that they inhibit Fes by affecting kinase domain structure. We have also demonstrated for the first time that endogenous Fes is strongly expressed at the base of colonic crypts where it co-localizes with epithelial cells positive for the progenitor cell marker Musashi-1. In contrast to normal colonic epithelium, Fes expression was reduced or absent in colon tumor sections from most individuals. Fes protein levels were also low or absent in a panel of human colorectal cancer cell lines, including HT-29 and HCT 116 cells. Introduction of Fes into these lines with a recombinant retrovirus suppressed their growth in soft agar. Together, our findings strongly implicate the c-Fes protein-tyrosine kinase as a tumor suppressor rather than a dominant oncogene in colorectal cancer.     

PRAK suppresses oncogenic ras-induced hematopoietic cancer development by antagonizing the JNK pathway

The p38 mitogen-activated protein kinase (MAPK) pathway regulates multiple physiologic and pathologic processes, including cancer development. PRAK, a p38 substrate protein kinase, has previously been implicated in the suppression of skin carcinogenesis. In the current study, we show that PRAK deletion accelerates hematopoietic cancer development in a mouse model harboring an oncogenic ras allele, Eμ-N-Ras(G12D), specifically expressed in hematopoietic cells. Further investigation reveals that enhanced hematopoietic tumorigenesis by PRAK deficiency is associated with hyperactivation of the c-jun-NH(2)-kinase (JNK) pathway both in vivo and in primary hematopoietic cells isolated from spleens. In primary splenocytes, PRAK deficiency further enhanced oncogenic ras-induced cell proliferation and promoted ras-mediated colony formation on semisolid medium in a JNK-dependent manner. In addition, deletion of PRAK leads to abrogation of ras-induced accumulation of senescence markers. These findings indicate that PRAK suppresses hematopoietic cancer formation in this mouse model by antagonizing oncogenic ras-induced activation of the JNK pathway. Our results suggest that PRAK may function as a tumor suppressor in multiple types of cancers.     

Quercetin inhibits inducible ICAM-1 expression in human endothelial cells through the JNK pathway

The cell adhesion molecule intercellular adhesion molecule-1(ICAM-1) plays a pivotal role in inflammatory responses. Quercetin (3,3',4',5,7-pentahydroxyflavone), a naturally occurringdietary flavonol, has potent anti-inflammatory properties. The effect of quercetin on ICAM-1 expression induced by agonists phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor- (TNF-) in human endothelial cell line ECV304 (ECV) wasinvestigated. Quercetin treatment downregulated both PMA-and TNF--induced surface expression, as well as the ICAM-1 mRNAlevels, in ECV cells in a dose-dependent (10-50 µM) manner.Quercetin had no effect on PMA- or TNF--induced nuclear factor-B(NF-B) activation. However, under similar conditions a remarkabledose-dependent downregulation of activator protein-1 (AP-1) activationwas observed. This decrease in AP-1 activation was observed to beassociated with the inhibitory effects of quercetin on the c-JunNH₂-terminal kinase (JNK) pathway.These results suggest that quercetin downregulates both PMA- andTNF--induced ICAM-1 expression via inhibiting both AP-1 activationand the JNK pathway.

     

Stem-like and non-stem human pancreatic cancer cells distinguished by morphology and metastatic behavior

We report here that XPA1 human pancreatic cancer cells are dimorphic. After injection in the spleen, XPA1 cells isolated from the primary tumor in the spleen were predominantly round; while cells isolated from the resulting liver metastasis and ascites were comprised of both round- and spindle-shaped cell types. Cancer cells previously grown in the spleen and re-implanted in the spleen developed large primary tumors in the spleen only. Cancer cells isolated from liver metastasis and re-transplanted to the spleen resulted in a primary tumor in the spleen and liver metastasis. Cancer cells derived from ascites and re-transplanted to the spleen developed primary tumors in the spleen and distant metastasis in the liver, lung, and diaphragm in addition to ascites formation. Spindle and round cells were differentially labeled with fluorescent proteins of different colors. After co-injection of the two cell types in the spleen, cells were isolated from the primary tumors, liver metastasis, and ascites and analyzed by color-coded fluorescence microscopy and fluorescence-activated cell sorting (FACS). No significant differences between the percentages of spindle-shaped and round cancer cells in the primary tumor and the liver metastasis were observed. However, spindle-shaped cancer cells were enriched in the ascites. One hundred percent of the spindle-shaped and round cancer cells expressed CD44, suggesting that morphology and metastatic behavior rather than CD44 expression can distinguish the stem-like cells of the XPA1 pancreatic cancer cell line. The spindle-shaped cancer cells had the greater capability for distant metastasis and ascites formation, suggesting they are stem-like cells, which can be readily targeted for therapy.     

Silencing of the integrin-linked kinase gene suppresses the proliferation, migration and invasion of pancreatic cancer cells (Panc-1)

Integrin-linked kinase (ILK) is an ankyrin repeat-containing serine-threonine protein kinase that is involved in the regulation of integrin-mediated processes such as cancer cell proliferation, migration and invasion. In this study, we examined the effect of a lentivirus-mediated knockdown of ILK on the proliferation, migration and invasion of pancreatic cancer (Panc-1) cells. Immunohistochemical staining showed that ILK expression was enhanced in pancreatic cancer tissue. The silencing of ILK in human Panc-1 cells led to cell cycle arrest in the G0/G1 phase and delayed cell proliferation, in addition to down-regulating cell migration and invasion. The latter effects were mediated by up-regulating the expression of E-cadherin, a key protein in cell adhesion. These findings indicate that ILK may be a new diagnostic marker for pancreatic cancer and that silencing ILK could be a potentially useful therapeutic approach for treating pancreatic cancer.     

Modulation of integrin-linked kinase (ILK) expression in human oesophageal squamous cell carcinoma cell lines by the EGF and TGFβ1 growth factors

Background  

Integrin-linked kinase (ILK) is a ubiquitously expressed protein kinase that has emerged as one of the points of convergence between integrin- and growth factor-signalling pathways.     

G1 arrest and expression of cyclin-dependent kinase inhibitors in tamoxifen-treated MCF-7 human breast cancer cells

Treatment of exponentially growing MCF-7 human breast carcinoma cells with tamoxifen (TAM) inhibits cell growth in a dose-dependent manner. However, the molecular basis for the drug's activity and its relationship to the cell cycle have not yet been clearly established. In this study, we analyzed cell cycle-related proteins used for immunoblotting and flow cytometry in TAM-treated MCF-7 cells. In addition, the ratio of apoptosis in the cell was analyzed using labeling of DNA strand breaks (TdT assay). In flow-cytometric DNA distribution analysis, the S-phase fraction showed a marked decrease and a concomitant increase in G1- and G2-phase cells accompanying the inhibitory effect of TAM; these changes were time- and dose-dependent. Immunoblotting revealed that the levels of p53 and p21(WAF1/CIP1) in TAM-treated cells increased in a time- and dose-dependent manner, whereas those of p27(KIP1) and p16 slightly increased or remained unchanged. Furthermore, cyclin D3 and B showed sharp decreases, in contrast with p53 and p21(WAF1/CIP1) DNA-apoptosis dual analysis using flow cytometry revealed that the TAM-treated samples contained apoptotic cells, the majority of which were arrested in G1 or G2 and showed suppression of Bcl-2 protein. These results suggest that the tumorigenic effect of TAM on MCF-7 cells arises through antitumor effects that are due to the expression of cyclin-dependent kinase inhibitors, especially p21(WAF1/CIP1) and these are regulated by the decrease of wild-type p53. The proposed mechanism is similar to that underlying the cytotoxic effects of other agents and ionizing irradiation that cause DNA damage.     

Butyrate induces cell apoptosis through activation of JNK MAP kinase pathway in human colon cancer RKO cells

Butyrate has been shown to display anti-cancer activity through the induction of apoptosis in various cancer cells. However, the underlying mechanism involved in butyrate-induced apoptosis is still not fully understood. Here, we investigated the cytotoxicity mechanism of butyrate in human colon cancer RKO cells. The results showed that butyrate induced a strong growth inhibitory effect against RKO cells. Butyrate also effectively induced apoptosis in RKO cells, which was characterized by DNA fragmentation, nuclear staining of DAPI, and the activation of caspase-9 and caspase-3. The expression of anti-apoptotic protein Bcl-2 decreased, whereas the apoptotic protein Bax increased in a dose-dependent manner during butyrate-induced apoptosis. Moreover, treatment of RKO cells with butyrate induced a sustained activation of the phosphorylation of c-jun N-terminal kinase (JNK) in a dose- and time-dependent manner, and the pharmacological inhibition of JNK MAPK by SP600125 significantly abolished the butyrate-induced apoptosis in RKO cells. These results suggest that butyrate acts on RKO cells via the JNK but not the p38 pathway. Butyrate triggered the caspase apoptotic pathway, indicated by an enhanced Bax-to-Bcl-2 expression ratio and caspase cascade reaction, which was blocked by SP600125. Taken together, our data indicate that butyrate induces apoptosis through JNK MAPK activation in colon cancer RKO cells.     

A novel Jun N-terminal kinase (JNK)-binding protein that enhances the activation of JNK by MEK kinase 1 and TGF-beta-activated kinase 1.

We have identified a novel Jun N-terminal kinase (JNK)-binding protein, termed JNKBP1, and examined its binding affinity for JNK1, JNK2, JNK3, and extracellular signal-regulated kinase 2 (ERK2) in COS-7 cells. JNKBP1 preferentially interacted with the JNKs, but not with ERK2. Furthermore, we investigated the effect of overexpressing JNKBP1 on the JNK and ERK signaling pathways in COS-7 cells. JNKBP1 alone had only a marginal effect on JNK activity. However, the activation of JNK by MEK kinase 1 and TGF-beta-activated kinase 1 was significantly enhanced in the presence of JNKBP1. In contrast, JNKBP1 had no or very little effect on the ERK signaling pathway. These results suggest that JNKBP1 functions to facilitate the specific and efficient activation of the JNK signaling pathways.