The integrin-linked kinase (ILK) suppresses anoikis

Disruption of integrin-extracellular matrix interactions in normal epithelial cells induces apoptosis, a process termed anoikis. Reduced sensitivity to anoikis appears to be an important hallmark of oncogenic transformation, particularly in the process of metastasis. Several pathways have been implicated in the suppression of anoikis, however, the events which take place proximal to the integrin receptors remain unclear. Integrin-linked kinase (ILK) is an integrin-interacting protein kinase which has been identified as a potential PDK-2, as it is capable of phosphorylating PKB/Akt on Ser-473, and stimulating its activity. Here, we show that ILK activity is stimulated upon adhesion of SCP2 mouse mammary epithelial cells to fibronectin, and inhibited in suspended cells. Overexpression of ILK in the anoikis-sensitive SCP2 cells results in a profound inhibition of anoikis, as determined by annexin V binding and activation of caspases 8 and 3. This effect is reversible by the transfection and expression of a dominant-negative, kinase deficient ILK (ILK KD), as well as by a dominant negative PKB/Akt (PKB AAA). On the other hand, transfection of a dominant negative form of FAK (FRNK) failed to reverse the suppression of anoikis by ILK. Furthermore, inhibition of ILK activity induced anoikis in two anoikis-resistant human breast cancer cell lines. These findings suggest that ILK plays a major role in the suppression of anoikis.     

The role of integrin-linked kinase (ILK) in cancer progression

Integrin-linked kinase (ILK) is an intracellular protein, which interacts with the cytoplasmic domains of integrin beta1 and beta3 subunits. ILK is a 59kDa protein containing a phosphoinositide phospholipid-binding domain flanked by an N-terminal ankyrin repeat domain and a C-terminal serine/threonine protein kinase domain. Genetic and biochemical evidence have established an essential role of ILK in connecting integrins to the actin cytoskeleton. Apart from integrins, ILK interacts with several adaptor and signaling proteins resulting in its activation and localization to focal adhesion plaques. The kinase activity of ILK is stimulated upon integrin engagement, as well as by growth factors and chemokines in a PI-3Kinase-dependent manner. ILK can mediate the phosphorylation of a variety of intracellular substrates, most notable of which are: protein kinase B (PKB/Akt), glycogen synthase kinase-3 (GSK-3) and myosin light chain. Gain and loss of function strategies have shown that overexpression, and/or constitutive activation of ILK results in oncogenic transformation and progression to invasive and metastatic phenotypes. In addition ILK expression and activity are upregulated in several types of cancers. In this review, we summarize the adaptor and signaling properties of ILK, and also progress in the identification of therapeutic strategies for inhibition of ILK activity.     

Cten signals through integrin-linked kinase (ILK) and may promote metastasis in colorectal cancer

CTEN/TNS4 is an oncogene in colorectal cancer (CRC), which can induce cell motility although its mechanistic basis of activity and the clinical implications of Cten expression are unknown. As Cten is in complex with integrins at focal adhesions, we hypothesised that it may interact with integrin-linked kinase (ILK). Through forced expression and knockdown of Cten in HCT116 and SW620 (respectively, showing low and high Cten expression), we showed that Cten could regulate ILK. However, inhibition of ILK after forced expression of Cten abrogated the motility-inducing effects of Cten, thereby demonstrating that the Cten-ILK interaction was functionally relevant. Combined knockdown of Cten and ILK had no additive effects on cell motility compared with knockdown of each individually. In order to investigate the clinical implications of Cten expression, a series of 462 CRCs were evaluated by immunohistochemistry. High expression of Cten was associated with advanced Dukes' stage (P<0.001), poor prognosis (P<0.001) and distant metastasis (P=0.008). The role of Cten in metastasis was tested by (a) intrasplenic injection of CRC cells stably transfected with a Cten expression vector into nude mice and (b) testing a series of primary human CRCs and their metastases by immunohistochemistry. Compared with controls, mice injected with cells expressing Cten developed larger tumours in the spleen (P<0.05) and liver (P<0.05). In the human cases, compared with primary tumours, the metastatic deposits had a significantly higher frequency of nuclear localisation of Cten (P=0.002). We conclude that Cten expression is of prognostic significance in CRC, and we delineate a Cten-ILK pathway controlling cell motility and possibly promoting metastasis.     

alpha(v) integrins regulate cell proliferation through integrin-linked kinase (ILK) in ovarian cancer cells

Integrins regulate both adhesion and signaling processes involved in proliferation and survival. alpha(v)beta(3) and alpha(v)beta(5) integrins have been shown to mediate cell adhesion and migration. Here we used human ovarian cancer cell lines (IGROV1, SKOV-3) that express alpha(v)beta(3) and alpha(v)beta(5) to study their role in cell proliferation and the signaling pathways involved. We found that alpha(v) integrins regulate cell proliferation through activation of integrin-linked kinase (ILK). An anti-alpha(v)-blocking antibody specifically inhibits the growth of IGROV1 and SKOV-3. The inhibition of cell proliferation involves alpha(v)beta(3) in IGROV1 cells, and both alpha(v)beta(3) and alpha(v)beta(5) in SKOV-3 cells. The reduced growth rate induced by alpha(v) integrin blockade is linked in both cell lines to G1/S cell cycle arrest. alpha(v) integrin blockade by neutralizing antibody as well as cyclic-RGD peptide caused an inhibition of ILK activity and phosphorylation of PKB/Akt on serine-473 but not on threonine-308, and was accompanied by an increase in p27(Kip1) expression. Overexpression of wild-type ILK rescued the phosphorylation of PKB/Akt on serine-473 in cells treated with anti-alpha(v) antibody. Inhibition of ILK by a pharmacological inhibitor results in inhibition of cell proliferation, PKB/Akt phosphorylation and increase of p27(Kip1). These results demonstrate that alpha(v) integrins regulate ovarian cancer cell proliferation through ILK.     

整合素连接激酶mRNA在非小细胞肺癌组织中的表达和意义

目的:比较整合素连接激酶(ILK)mRNA在非小细胞肺癌(NSCLC)组织及正常肺组织的表达差异,分析ILKmRNA的表达与NSCLC的恶性表型、侵袭程度、淋巴转移、远处转移、临床分期之间的关系。方法:取NSCLC患者及肺部良性病变手术切除标本,液氮及超低温冰箱保存,TRIzol法提取总RNA,RTPCR扩增ILKmRNA后对扩增产物进行电泳及图像分析。结果:35例肺癌、30例癌旁组织、29例正常肺及10例肺部良性病变组织ILKmRNA定性表达阳性率分别为82.9%(29/35)、76.7%(23/30)、55.2%(16/29)和60%(6/10),癌组织与正常肺组织之间ILKmRNA阳性率比较有显著性差异(P=0.027)。ILKmRNA定量表达为肺癌组1.67±0.349,癌旁组0.86±0.185,正常组0.43±0.102,肺部良性病变组0.78±0.201,除癌旁组织和肺部良性病变组织差别无统计学意义外,其余差异均有显著性(P<0.01)。NSCLC组织ILKmRNA定量表达的均值随细胞恶性程度、淋巴转移和远处转移及临床分期递增而随之递增(P<0.05)。结论:ILKmRNA在NSCLC组织中表达量高于癌旁组织、正常肺及肺部良性病变组织。高表达的ILKmRNA预示着NSCLC的高度恶性、高侵袭性、高转移性和预后不良,ILKmRNA可作为反映NSCLC恶性表型、转移和侵袭特性的分子标记物。     

Increased resistance of tumor cells to hyperthermia mediated by integrin-linked kinase.

PURPOSE: Integrin-linked kinase (ILK) is a serine-threonine kinase associated with anchorage-independent growth and tumorigenic transformation. Previous studies indicate that overexpression of ILK is common among several types of tumors, and it is involved in the regulation of tumor cell survival under stress. In this study, we examined the effects of ILK expression on tumor cellular response to hyperthermia. EXPERIMENTAL DESIGN: We used an adenovirus-mediated approach to overexpress the ILK gene in a prostate cancer cell line and examine its effects on heat stress-induced cell death. Clonogenic survival, as well as apoptosis, was evaluated in cells that overexpress ILK. In addition, the ability to form tumors in vivo was examined in syngeneic hosts. Finally, potential molecular mechanisms of ILK-mediated resistance to heat were examined by determining the status of a variety of signal transduction pathways. RESULTS: ILK overexpression made tumor cells significantly more resistant to the cell-killing effects of hyperthermia. This was correlated at the molecular level with the down-regulation of hyperthermia-induced activation of stress-activated protein kinase/c-Jun-NH(2)-terminal kinase, p38 mitogen-activated protein kinase activities, and caspase 9. The overexpression of ILK was also shown to induce a more rapid tumor growth in a murine prostate cancer cell line CONCLUSION: ILK plays an important role in tumor growth and tumor response to hyperthermia treatment.     

MicroRNA-mediated upregulation of integrin-linked kinase promotes Src-induced tumor progression

The tyrosine kinase c-Src is upregulated in various human cancers; however, the molecular mechanisms underlying c-Src-mediated tumor progression remain unclear. Here we show that downregulation of microRNA (miR)-542-3p is tightly associated with tumor progression via c-Src-related oncogenic pathways. In c-Src-transformed fibroblasts and human cancer cells that overexpress c-Src, miR-542-3p is substantially downregulated, and the ectopic expression of miR-542-3p suppresses tumor growth. We identified the integrin-linked kinase (ILK) as a conserved target of miR-542-3p. ILK upregulation promotes cell adhesion and invasion by activating the integrin-focal adhesion kinase (FAK)/c-Src pathway, and can also contribute to tumor growth via the AKT and glycogen synthase kinase 3β pathways. MiR-542-3p expression is downregulated by the activation of c-Src-related signaling molecules, including epidermal growth factor receptor, K-Ras and Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase. In human colon cancer tissues, downregulation of miR-542-3p is significantly correlated with the upregulation of c-Src and ILK. Our results suggest that the novel c-Src-miR-542-3p-ILK-FAK circuit plays a crucial role in controlling tumor progression.     

肿瘤血管生成调节与肿瘤治疗

肿瘤血管生成是一个非常复杂的过程，涉及到多种因子的调节。针对肿瘤血管生成的治疗对策在目前肿瘤治疗中具有十分重要的意义。本文就影响肿瘤血管生成的各种调节因子、抗血管生成的主要治疗靶标及研发现状作一综述，为肿瘤治疗的抗血管生成新靶标的设计研究提供信息。     

Inhibition of tumor angiogenesis by Brahma Rasayana (BR)

The current therapy for prostate cancer includes radical prostatectomy, radiation therapy and hormonal ablation. Chemotherapy also provides beneficial results for some patients with advanced prostate cancer but with several harmful side effects. Hence there is a need to identify and develop alternate therapies, which can reduce the disease progression with minimal or few side effects. Earlier studies from our laboratory have shown that a Polyherbal mixture, Brahma Rasayna (BR) rich in anti-oxidant principles has a potential to be an anti-tumor agent. BR treatment of MAT-LyLu cell inoculated Copenhagen rats resulted in a decrease of palpable tumor incidence, delay in tumor occurrence and lower mean tumor volumes. Also, a significant reduction in tumor weight and lung metastasis was observed in BR treated animals in comparison to untreated controls. In the present study, we focused to examine the effect of BR on angiogenesis and regulation of molecular markers involved in angiogenesis using in-vivo and in-vitro models. BR treatment showed a significant reduction in Factor VIII expression compared to control indicating reduced angiogenesis. BR treated tumor specimens showed a decrease in the pro-angiogenic factors like VEGF, MMP-9 and MMP-2. Methanolic extract of BR was found to inhibit the proliferation, tube formation, cell migration and attachment of HUVEC on matrigel in a dose dependant manner. These findings suggest the possible mechanism(s) of action of BR in the reduction of tumor growth and metastatic spread.     

Cell adhesion to the extracellular matrix protein fibronectin modulates radiation-dependent G2 phase arrest involving integrin-linked kinase (ILK) and glycogen synthase kinase-3beta (GSK-3beta) in vitro

Cell adhesion to extracellular matrix (ECM) is thought to confer resistance against cell-damaging agents, that is, drugs and radiation, in tumour and normal cells in vitro. The dependence of cell survival on beta1-integrin-linked kinase (ILK), protein kinase Balpha/Akt (PKBalpha/Akt) and glycogen synthase kinase-3beta (GSK-3beta) activity, which participate in beta1-integrin signalling and cell cycle progression was investigated as a function of radiation exposure. Colony-formation assays on polystyrene, fibronectin (FN), laminin (LA), bovine serum albumin (BSA) or poly-L-lysine (poly-L) (0-8 Gy), kinase assays, flow cytometric DNA and annexin-V analysis and immunoblotting were performed in nonirradiated and irradiated (2 or 6 Gy) A549 human lung cancer cells and CCD32 normal human lung fibroblasts. Cell contact to FN in contrast to polystyrene elevated basal ILK, PKBalpha/Akt and GSK-3beta kinase activities in A549 and CCD32 cells, as well as the basal amount of A549 G2 phase cells. Irradiation on FN or LA as compared to polystyrene, BSA or poly-L significantly improved cell survival. Following irradiation, kinase activities were stimulated strongly on polystyrene but showed to be less prominent on FN, which was because of the FN-related basal induction. Following irradiation, FN compared to polystyrene enlarged and prolonged G2 arrest in both the cell lines. For the analysis of phosphatidylinositol-3 kinase (PI3-K) dependence of protein kinases and cell cycle transition, the PI3-K inhibitors LY294002 and wortmannin were used showing decreased kinase activities, antiproliferative and radiation-dependent G2 accumulation-abrogating effects accompanied by downregulation of cyclin D1 and phospho-pRb in cells attached to polystyrene. Fibronectin partly abrogated these effects PI3-K-independently. These findings suggest a novel pathway that makes direct phosphorylation of GSK-3beta by ILK feasible after irradiation. Conclusively, the data indicate that ILK, PKBalpha/Akt and GSK-3beta are involved in modulations of the cell cycle after irradiation. These interactions are strictly dependent on ECM components in a cell line-specific manner. Our findings provide molecular insights into mechanisms likely to be important for ECM-dependent cell survival and cellular radioresistance as well as tumour growth.     

Cooperative signaling between Wnt1 and integrin-linked kinase induces accelerated breast tumor development

Introduction  

Breast cancer is genetically and clinically a heterogeneous disease. However, the exact contribution of different cell types and oncogenic mutations to this heterogeneity are not well understood. Recently, we discovered an interaction between Wnt and integrin-linked kinase (ILK) within the signaling cascade that regulates cell growth and survival. Interestingly, mammary-specific expression of either one of these proteins has been shown to promote mammary tumorigenesis. In light of our recent findings and to investigate the potential interaction between Wnt and ILK proteins during mammary tumor formation and progression, we established a transgenic mouse model that expresses both Wnt and ILK in mammary epithelial cells.     

The mitotic functions of integrin-linked kinase

The cytoskeleton is composed of three major constituents: actin filaments, intermediate filaments and microtubules. These are vital for numerous normal cellular processes including cell spreading and migration, intracellular organelle transport, mechanical strength, mitosis and cytokinesis. Deregulation of cytoskeletal components can lead to cells developing several oncogenic phenotypes; for example increased migration and invasiveness, defects in cellular morphogenesis and genetic instabilities due to errors in mitosis and cytokinesis. Integrin-linked kinase (ILK) is a protein with well established roles in regulating actin cytoskeletal reorganization, survival, proliferation, cell migration, invasion and epithelial to mesenchymal transition, and is therefore essential to normal cell physiology. In addition, ILK is overexpressed or deregulated in a number of human cancers and when experimentally overexpressed leads to the acquisition of a number of oncogenic phenotypes, some of which, such as increased cell migration, are actin-dependent. Here we shall focus on the recent finding that ILK also regulates the microtubule cytoskeleton and is involved in mitotic spindle organization. Therefore its deregulation may also lead to errors in cell division causing genomic instability, potentially further contributing to cancer development. In light of these findings, the therapeutic potential of the anti-mitotic effects of genetic or pharmacological inhibition of ILK will also be discussed.     

The MMAC1 tumor suppressor phosphatase inhibits phospholipase C and integrin-linked kinase activity

Loss of the tumor suppressor MMAC1 has been shown to be involved in breast, prostate and brain cancer. Consistent with its identification as a tumor suppressor, expression of MMAC1 has been demonstrated to reduce cell proliferation, tumorigenicity, and motility as well as affect cell-cell and cell-matrix interactions of malignant human glioma cells. Subsequently, MMAC1 was shown to have lipid phosphatase activity towards PIP3 and protein phosphatase activity against focal adhesion kinase (FAK). The lipid phosphatase activity of MMAC1 results in decreased activation of the PIP3-dependent, anti-apoptotic kinase, AKT. It is thought that this inhibition of AKT culminates with reduced glioma cell proliferation. In contrast, MMAC1's effects on cell motility, cell - cell and cell - matrix interactions are thought to be due to its protein phosphatase activity towards FAK. However, recent studies suggest that the lipid phosphatase activity of MMAC1 correlates with its ability to be a tumor suppressor. The high rate of mutation of MMAC1 in late stage metastatic tumors suggests that effects of MMAC1 on motility, cell - cell and cell - matrix interactions are due to its tumor suppressor activity. Therefore the lipid phosphatase activity of MMAC1 may affect PIP3 dependent signaling pathways and result in reduced motility and altered cell - cell and cell - matrix interactions. We demonstrate here that expression of MMAC1 in human glioma cells reduced intracellular levels of inositol trisphosphate and inhibited extracellular Ca2+ influx, suggesting that MMAC1 affects the phospholipase C signaling pathway. In addition, we show that MMAC1 expression inhibits integrin-linked kinase activity. Furthermore, we show that these effects require the catalytic activity of MMAC1. Our data thus provide a link of MMAC1 to PIP3 dependent signaling pathways that regulate cell - matrix and cell - cell interactions as well as motility. Lastly, we demonstrate that AKT3, an isoform of AKT highly expressed in the brain, is also a target for MMAC1 repression. These data suggest an important role for AKT3 in glioblastoma multiforme. We therefore propose that repression of multiple PIP3 dependent signaling pathways may be required for MMAC1 to act as a tumor suppressor.     

核糖核酸酶抑制因子与整合素连接激酶相互作用通过ILK/AKT/mTOR通路抑制膀胱癌体内外生长

背景与目的：蛋白质相互作用控制着细胞信号转导、跨膜转运、DNA合成及转录调控等生命过程,在生命活动中发挥重要作用。前期运用GST蛋白沉降技术与免疫共沉淀等已证明核糖核酸酶抑制因子(ribonuclease inhibitor,RI)与整合素连接激酶(intergrin-linked kinase,ILK)在体内外直接结合。该研究旨在探讨RI与ILK相互作用对ILK/AKT/mTOR通路与膀胱癌体内外生长的影响。方法：采用免疫荧光观察RI与ILK在膀胱癌EJ细胞中的共定位。采用荧光共振能量转移技术(fluorescence resonance energy transfer,FRET)验证RI与ILK的相互作用。构建过表达RI或ILK的EJ细胞系。采用蛋白［质］印迹法(Western blot)检测细胞中RI、ILK及ILK/AKT/mTOR通路相关蛋白的表达。采用细胞计数试剂盒(cell counting kit-8,CCK-8)与流式细胞术分析细胞增殖与细胞周期。构建膀胱癌裸鼠移植瘤模型,观察过表达RI或ILK对移植瘤生长的影响。采用免疫组织化学与免疫荧光分析瘤组织中RI、ILK及ILK/AKT/mTOR通路相关蛋白的表达。结果：EJ细胞中RI与ILK存在共定位的现象且RI与ILK存在相互作用。过表达RI抑制EJ细胞增殖并导致细胞周期阻滞于S期(P<0.05),阻碍膀胱癌移植瘤的生长(P<0.05),并抑制体内外ILK/AKT/mTOR通路的活化(P<0.05)。而过表达ILK促进细胞增殖与移植瘤的生长(P<0.05),促进体内外ILK/AKT/mTOR信号通路的激活(P<0.05)。结论：RI通过与ILK相互作用阻碍ILK/AKT/mTOR通路激活并抑制膀胱癌体内外的增殖生长。