Elevated focal adhesion kinase expression facilitates oral tumor cell invasion

BACKGROUND: Understanding the molecular mechanisms of metastasis is critical with respect to oral tumorigenesis. The focal adhesion kinase (FAK) is an intracellular tyrosine kinase associated with the regulation of cell growth, migration, and survival. The purpose of the current study was to determine whether elevated FAK expression in oral malignancies was associated with increased invasiveness and oral carcinoma. METHODS: Immunohistochemical analysis was used to assess levels of FAK expression in archived oral carcinoma tissue samples. Invasion assays after transfections were used to assess the effect of increased FAK expression on invasive potential of oral tumor cells. RESULTS: The human oral carcinoma cell line SCC25 was significantly more invasive (P < 0.05) and expressed higher levels of FAK compared with the less invasive human oral carcinoma cell line SCC15. FAK expression was 3.0-fold higher in the SCC15 cell line and 5.0-fold higher in the SCC25 cell line compared with normal epithelial cells. In the highly invasive SCC25 cell line, FAK expression was 1.5-fold higher compared with the less invasive SCC15 cell line. FAK immunostaining in oral tumors was significantly more intense compared with the immunostaining in surrounding normal epithelium or chronic mucositis. Overexpression of FAK in low-invading SCC15 cells resulted in a 4.5-fold increase in the rate of invasion compared with untransfected or neotransfected control SCC15 cell lines and a nearly 1.5-fold greater rate compared with the highly invasive untransfected SCC25 cell line. CONCLUSIONS: The current results suggest that enhanced expression of FAK in oral carcinoma cells may lead to a selective growth advantage and increased invasive potential of the primary oral tumor.     

Epidermal growth factor induces matrix metalloproteinase-1 (MMP-1) expression and invasion in glioma cell lines via the MAPK pathway

Glioblastoma multiforme (GBM) is an aggressive cancer with a poor survival rate. A key component that contributes to the poor prognosis is the capacity of glioma cells to invade local brain tissue in a diffuse manner. Among various proteases that aid in the process of invasion, matrix metalloproteinase-1 (MMP-1) has been identified as an important contributory factor in various cancers. Apart from its traditional role in cleaving its primary extracellular matrix (ECM) substrates, and like other members of the matrix metalloproteinase family, MMP-1 can activate latent forms of bio-active molecules initiating downstream pro-invasive and pro-oncogenic signaling mechanisms. MMP-1 expression is regulated by several growth factors including epidermal growth factor (EGF). Due to the fact that the epidermal growth factor receptor (EGFR) is aberrantly overexpressed in GBM, we wanted to examine in greater detail the signaling mechanisms by which MMP-1 expression and invasion is driven by EGF in GBM cells. T98G cells treated with EGF resulted in an induction of MMP-1 expression following EGFR activation. Inhibition of EGFR by both pharmacologic and genetic approaches abrogated this induction. Repression of the mitogen activated protein kinase (MAPK) signaling led to the inhibition of EGF-induced MMP-1 whereas the PI3-kinase/AKT signaling was not associated with EGFR-mediated MMP-1 induction. Inhibition of EGFR signaling also led to a decrease in T98G invasion. These data suggest that EGFR mediated MMP-1 regulation is mainly via the MAPK pathway in T98G cells and inhibition of EGFR and MMP-1 results in a decrease in T98G cell invasion.     

Activation of focal adhesion kinase enhances the adhesion and invasion of pancreatic cancer cells via extracellular signal-regulated kinase-1/2 signaling pathway activation

Background  

Interaction with integrin and focal adhesion kinase (FAK) regulates the cancer cell adhesion and invasion into extracellular matrix (ECM). In addition, phosphorylation of FAK correlates with the increase of cell motility and invasion. Adhesion and spreading of cancer cells on a variety of ECM proteins, including collagen type IV (Coll IV), leads to an increase in tyrosine phosphorylation and activation of FAK. In this study, we investigated the mechanism of activation of FAK and its downstream extracellular signal-regulated kinase (ERK)-1/2 signaling following stimulation by interleukin (IL)-1α and adhesion to ECM with subsequent enhancement of pancreatic cancer cell adhesion and invasion.     

Cannabinoids inhibit glioma cell invasion by down-regulating matrix metalloproteinase-2 expression

Cannabinoids, the active components of Cannabis sativa L. and their derivatives, inhibit tumor growth in laboratory animals by inducing apoptosis of tumor cells and impairing tumor angiogenesis. It has also been reported that these compounds inhibit tumor cell spreading, but the molecular targets of this cannabinoid action remain elusive. Here, we evaluated the effect of cannabinoids on matrix metalloproteinase (MMP) expression and its effect on tumor cell invasion. Local administration of Delta(9)-tetrahydrocannabinol (THC), the major active ingredient of cannabis, down-regulated MMP-2 expression in gliomas generated in mice, as determined by Western blot, immunofluorescence, and real-time quantitative PCR analyses. This cannabinoid-induced inhibition of MMP-2 expression in gliomas (a) was MMP-2-selective, as levels of other MMP family members were unaffected; (b) was mimicked by JWH-133, a CB(2) cannabinoid receptor-selective agonist that is devoid of psychoactive side effects; (c) was abrogated by fumonisin B1, a selective inhibitor of ceramide biosynthesis; and (d) was also evident in two patients with recurrent glioblastoma multiforme. THC inhibited MMP-2 expression and cell invasion in cultured glioma cells. Manipulation of MMP-2 expression by RNA interference and cDNA overexpression experiments proved that down-regulation of this MMP plays a critical role in THC-mediated inhibition of cell invasion. Cannabinoid-induced inhibition of MMP-2 expression and cell invasion was prevented by blocking ceramide biosynthesis and by knocking-down the expression of the stress protein p8. As MMP-2 up-regulation is associated with high progression and poor prognosis of gliomas and many other tumors, MMP-2 down-regulation constitutes a new hallmark of cannabinoid antitumoral activity.     

Stimulation of beta1 integrin down-regulates ICAM-1 expression and ICAM-1-dependent adhesion of lung cancer cells through focal adhesion kinase

Adhesion molecules are involved in intracellular signaling in various physiological and pathological processes including metastasis and growth of tumor cells. Tumor cells interact with various host cells as well as with extracellular matrices through certain adhesion molecules such as integrins. We here propose that stimulation of beta1 integrin reduces intercellular adhesion molecule (ICAM)-1-mediated interaction of lung cancer cells with CTLs. This concept is based on the following findings: (a) engagement of beta1 integrins on certain lung cancer cells by a specific antibody or by ligand matrices such as fibronectin and collagen markedly reduced ICAM-1 expression on the cell surface and induced sICAM-1; (b) down-regulation of ICAM-1 by stimulation of beta1 integrins was abrogated by tyrosine kinase inhibitors or by transfection of dominant negative truncations of focal adhesion kinase (FAK); (c) engagement of beta1 integrins also reduced ICAM-1-dependent adhesion of lung cancer cells to T cells, a process completely inhibited by tyrosine kinase inhibitors and by transfection of dominant negative forms of FAK; and (d) stimulation of beta1 integrins prevented killing of lung cancer cells by autologous CTLs. In malignant tumors, cancer cells, including lung cancer cells, are surrounded by extracellular matrix proteins such as fibronectin and collagen. This suggests that the engagement of beta1 integrins by matrix proteins potentially occurs in cancer cells in vivo and that continuous stimulation via beta1 integrins reduces ICAM-1-expression, ICAM-1-mediated adhesion of cancer cells to CTLs and their killing by CTLs. Our results suggest that such processes can lead to the escape of lung cancer cells in vivo from immunological surveillance.     

Prevention of irradiation-induced glioma cell invasion by temozolomide involves caspase 3 activity and cleavage of focal adhesion kinase

Sublethal doses of irradiation enhance the invasiveness of human malignantglioma cells. This can be inhibited by subtoxic concentrations of temozolomide (TMZ) but not by lomustine. Antagonism of irradiation-induced motility by TMZ is associated with the prevention of irradiation-induced alpha(v)beta(3)-integrin, matrix metalloproteinase-2 and MT1-matrix metalloproteinase-expression. Irradiation induces focal adhesion kinase (FAK) activation by phosphorylation, whereas TMZ promotes FAK cleavage. Inhibition of caspases prevents TMZ-induced FAK processing and restores the promigratory effect of irradiation, suggesting that the resistance of glioma cells to irradiation-induced caspase processing may determine the invasive responses of glioma cells to irradiation. In contrast, DAOY medulloblastoma cells, which respond with caspase activation to irradiation alone, do not show enhanced invasiveness when irradiated.     

Role of focal adhesion kinase and phosphatidylinositol 3'-kinase in integrin fibronectin receptor-mediated, matrix metalloproteinase-1-dependent invasion by metastatic prostate cancer cells

alpha(5)beta(1) Integrin interacts with the PHSRN sequence of plasma fibronectin, causing constitutive invasion by human prostate cancer cells. Inhibition of this process reduces tumorigenesis and prevents metastasis and recurrence. In this study, naturally serum-free basement membranes were used as in vitro invasion substrates. Immunoassays were employed to dissect the roles of focal adhesion kinase (FAK), phosphatidylinositol 3'-kinase (PI3K), and protein kinase Cdelta (PKC delta) in alpha(5)beta(1)-mediated, matrix metalloproteinase-1 (MMP-1)-dependent invasion by metastatic human DU 145 prostate cancer cells. We found that a peptide composed of the PHSRN sequence induced rapid FAK phosphorylation at Tyr(397) (Y397), a site whose phosphorylation is associated with kinase activation. The technique of RNA silencing [small interfering RNA (siRNA)] confirmed the role of FAK in PHSRN-induced invasion. PHSRN also induced the association of the p85-regulatory subunit of PI3K with FAK at a time corresponding to FAK phosphorylation and activation, and maximal PI3K activity occurred at this same time. The necessity of PI3K activity in both PHSRN-induced invasion and MMP-1 expression was confirmed by using specific PI3K inhibitors. By employing a specific inhibitor, Rottlerin, and by using siRNA, we also found that PKC delta, a PI3K substrate found in focal adhesions, functions in PHSRN-induced invasion. In addition, the induction of MMP-1 in PHSRN-treated DU 145 cells was shown by immunoblotting, and the role of MMP-1 in PHSRN-induced invasion was confirmed by the use of blocking anti-MMP-1 monoclonal antibody. Finally, a close temporal correspondence was observed between PHSRN-induced invasion and PHSRN-induced MMP-1 activity in DU 145 cells.     

Downregulation of PAK5 inhibits glioma cell migration and invasion potentially through the PAK5-Egr1-MMP2 signaling pathway

PAK5 (p21 activated kinase 5) is upregulated in human colorectal carcinoma cells and is a known tumor promoter in carcinogenesis of the colon. Little is known regarding the mechanisms underlying the downstream targets of PAK5, and information concerning its biological significance in glioma is lacking. In this study, we investigated the effects of PAK5 on proliferation, migration, invasion, and apoptosis in human U87 and U251 glioma cells and examined the underlying molecular mechanism. We performed cell growth assays and cell cycle analysis to observe the cell proliferation. Flow cytometry analysis was performed to evaluate apoptosis, and in vitro scratch assays, cell migration assays, and gelatin zymography were performed to examine cell migration. Western blot analysis was performed to examine signal transduction in the cells. We demonstrated that suppression of PAK5 in glioma cells significantly inhibited cell migration and invasion. We also observed that suppression of PAK5 in human glioma cell lines inhibited cell growth because of G1 phase arrest. Additionally, flow cytometry and Western blot analysis indicated that PAK5 could inhibit cell apoptosis. These results suggest that the PAK5–Egr1–MMP2 signaling pathway is involved in tumor progression and may have a potential role in cancer prevention and treatment.     

Emodin inhibits tumor cell adhesion through disruption of the membrane lipid Raft-associated integrin signaling pathway

Cell adhesion and spreading is a crucial step in the metastatic cascade of cancer cells, and interruption of this step is considered to be a logical strategy for prevention and treatment of tumor metastasis. Emodin is the major active component of the rhizome of Rheum palmatum L., with known anticancer activities. Here, we first found that emodin significantly inhibited cell adhesion of various human cancer cells. This inhibition was achieved through suppressing the recruitment of focal adhesion kinase (FAK) to integrin beta(1) as well as the phosphorylation of FAK followed by the decreased formation of focal adhesion complex (FAC). In understanding the underlying mechanisms, we found that emodin inhibited the lipid raft clustering and subsequent colocalization of integrin beta(1) and FAC proteins within lipid rafts. Lipid profile analysis revealed significant decrease of cholesterol and sphingolipids in raft fraction after emodin treatment. Cholesterol replenishment abolished the adverse effect of emodin on the translocation of integrin beta(1) and FAC proteins into the lipid raft fraction and cell adhesion. Therefore, data from this study provide novel evidence that emodin inhibits cell adhesion and spreading through disruption of the membrane lipid raft-associated integrin signaling pathway.     

CD155/PVR enhances glioma cell dispersal by regulating adhesion signaling and focal adhesion dynamics

We recently identified the immunoglobulin-CAM CD155/PVR (the poliovirus receptor) as a regulator of cancer invasiveness and glioma migration, but the mechanism through which CD155/PVR controls these processes is unknown. Here, we show that expression of CD155/PVR in rat glioma cells that normally lack this protein enhances their dispersal both in vitro and on primary brain tissue. CD155/PVR expression also reduced substrate adhesion, cell spreading, focal adhesion density, and the number of actin stress fibers in a substrate-dependent manner. Furthermore, we found that expression of CD155/PVR increased Src/focal adhesion kinase signaling in a substrate-dependent manner, enhancing the adhesion-induced activation of paxillin and p130Cas in cells adhering to vitronectin. Conversely, depletion of endogenous CD155/PVR from human glioma cells inhibited their migration, increased cell spreading, and down-regulated the same signaling pathway. These findings implicate CD155/PVR as a regulator of adhesion signaling and suggest a pathway through which glioma and other cancer cells may acquire a dispersive phenotype.     

Over-expression of cyclin D1 induces glioma invasion by increasing matrix metalloproteinase activity and cell motility.

In order to define the role of cyclin D1 in the progression of malignant glioma, cells over-expressing cyclin D1 were constructed (a-1 cells). They exhibited significantly increased invasiveness as compared with mock-transfected cells. Since cellular invasion is thought to depend on extracellular-matrix degradation, we determined whether cyclin-D1 expression modifies the activity of matrix metalloproteinases (MMPs). Increased gelatinolytic activity of latent type MMP-2 (proMMP-2) and active MMP-2 was observed in a-1 cells. Moreover, cyclin-D1 expression was associated with increased activation of proMMP-9 through MMP-3. Wound assays showed an increase of cell motility in a-1 cells. Cyclin-D1 expression was found to be associated with up-regulation of Rac1, which modulates the formation of ruffling membranes and cell motility. Our results show that cyclin D1 may modulate invasive ability by increasing MMP activity and cell motility, and suggests a novel function of cyclin D1 in the progression of malignant gliomas.     

Prostatic carcinoma cell migration via alpha(v)beta3 integrin is modulated by a focal adhesion kinase pathway

The highly invasive human prostate cancer PC3 cell line was found to express the alpha(v)beta3 integrin; in contrast, the noninvasive LNCaP prostate cancer cell line did not express alpha(v)beta3. PC3 cells adhered to and migrated on vitronectin (VN), an alpha(v)beta3 ligand expressed in mature bone where prostate cancer cells preferentially metastasize. In contrast, LNCaP cells did not adhere to or migrate on VN. Analysis of primary human prostate cancer cells isolated from 16 surgical specimens, showed that these cells expressed alpha(v)beta3, whereas normal prostate epithelial cells did not. In addition, only primary prostate cancer cells adhered to and migrated on VN. The role of alpha(v)beta3 in mediating prostate epithelial cell migration was confirmed using LNCaP cell transfectants expressing beta3 (beta3-LNCaP). Exogenous expression of alpha(v)beta3 induced LNCaP cells to adhere to and migrate on VN. In response to alpha(v)beta3 engagement, increased tyrosine phosphorylation of focal adhesion kinase (FAK), a signaling molecule activated by integrins and able to modulate cell migration, was detected. Transfection of FAK-related nonkinase, known to compete with FAK for its correct localization and phosphorylation, caused inhibition of beta3-LNCaP cell migration, specifically on VN. These data indicate that de novo expression of alpha(v)beta3 integrin in prostate cancer cells generates a migratory phenotype that is modulated by a FAK signaling pathway. This study points to alpha(v)beta3 as potential target in prostate cancer cell invasion and metastasis.     

Nicotinamide N-methyltransferase induces cellular invasion through activating matrix metalloproteinase-2 expression in clear cell renal cell carcinoma cells

Nicotinamide N-methyltransferase (NNMT) was recently identified as one clear cell renal cell carcinoma (ccRCC)-associated gene by analyzing full-length complementary DNA-enriched libraries of ccRCC tissues. The aim of this study is to investigate the potential role of NNMT in cellular invasion. A strong NNMT expression is accompanied with a high invasive activity in ccRCC cell lines, and small interfering RNA-mediated NNMT knockdown effectively suppressed the invasive capacity of ccRCC cells, whereas NNMT overexpression markedly enhanced that of human embryonic kidney 293 (HEK293) cells. A positive correlation between the expression of NNMT and matrix metallopeptidase (MMP)-2 was found in ccRCC cell lines and clinical tissues. The treatment of blocking antibody or inhibitor specific to MMP-2 significantly suppressed NNMT-dependent cellular invasion in HEK293 cells. Furthermore, SP-1-binding region of MMP-2 promoter was found to be essential in NNMT-induced MMP-2 expression. The specific inhibitors of PI3K/Akt signaling markedly decreased the binding of SP1 to MMP-2 promoter as shown by chromatin immunoprecipitation assay. We also demonstrated that PI3K/Akt pathway plays a role in NNMT-dependent cellular invasion and MMP-2 activation. Moreover, short hairpin RNA-mediated knockdown of NNMT expression efficiently inhibited the growth and metastasis of ccRCC cells in non-obese diabetic severe combined immunodeficiency mice. Taken together, the present study suggests that NNMT has a crucial role in cellular invasion via activating PI3K/Akt/SP1/MMP-2 pathway in ccRCC.     

Curcumin suppresses cell growth and invasion and induces apoptosis by down-regulation of Skp2 pathway in glioma cells

Studies have demonstrated that curcumin exerts its tumor suppressor function in a variety of human cancers including glioma. However, the exact underlying molecular mechanisms remain obscure. Emerging evidence has revealed that Skp2 (S-phase kinase associated protein 2) plays an oncogenic role in tumorigenesis. Therefore, we aim to determine whether curcumin suppresses the Skp2 expression, leading to the inhibition of cell growth, invasion, induction of apoptosis, and cell cycle arrest. To this end, we conducted multiple methods such as MTT assay, Flow cytometry, Wound healing assay, invasion assay, RT-PCR, Western blotting, and transfection to explore the functions and molecular insights of curcumin in glioma cells. We found that curcumin significantly inhibited cell growth, suppressed cell migration and invasion, induced apoptosis and cell cycle arrest in glioma cells. Furthermore, we observed that overexpression of Skp2 promoted cell growth, migration, and invasion, whereas depletion of Skp2 suppressed cell growth, migration, and invasion and triggered apoptosis in glioma cells. Mechanistically, we defined that curcumin markedly down-regulated Skp2 expression and subsequently up-regulated p57 expression. Moreover, our results demonstrated that curcumin exerts its antitumor activity through inhibition of Skp2 pathway. Collectively, our findings suggest that targeting Skp2 by curcumin could be a promising therapeutic approach for glioma prevention and therapy.     

Rictor regulates MMP-9 activity and invasion through Raf-1-MEK-ERK signaling pathway in glioma cells

Glioblastoma multiforme (GBM) is the most common and highly aggressive type of primary brain tumor. Tumor-associated macrophages (TAMs) secrete TNF-α that activates important survival pathways including Akt (PKB)/mTOR network. The mammalian target of rapamycin (mTOR) network functions downstream of PI3K/Akt pathway to regulate cell growth, proliferation and survival. mTOR exists in two distinct complexes-mTORC1 and mTORC2 that differ in their components and sensitivity to rapamycin. The rapamycin-insensitive complex (mTORC2) consists of mTOR, mLST8, Rictor, mSin1 and Protor and regulates the actin cytoskeleton in addition to activating Akt (protein kinase B). The present study aimed to investigate the role of Rictor-a core component of mTORC2 in regulating proliferation, survival, and invasion in gliomas. siRNA-mediated loss of Rictor function in human glioma cell lines, LN18 and LN229 and in primary GBM cells resulted in elevated expression and activity of MMP-9 and significant increase in the invasive potential of these cells. Mechanistic studies revealed that the activation of Raf-1-MEK-ERK pathway was essential for induction of MMP-9 activity and enhanced invasion. Interestingly, ablation of Rictor did not affect TNF-α-induced MMP-9 activity and invasiveness suggesting that TNF-α in the microenvironment of tumor might overrule the function of Rictor as a negative regulator of MMP-9 and invasion. Silencing Rictor had no effect on the survival or proliferation in the cell lines in the presence or absence of TNF-α. Our findings identify a role for Rictor in bridging two major pathways-Akt (PKB)/mTOR and Raf-1-MEK-ERK in regulating MMP-9 activity and invasion of glioma tumor cells.     

Down-regulation of integrin alpha2 surface expression by mutant epidermal growth factor receptor (EGFRvIII) induces aberrant cell spreading and focal adhesion formation

Elevated expression or activity of the epidermal growth factor receptor (EGFR) is common in ovarian cancer and is associated with poor patient prognosis. A naturally occurring EGFR mutation termed variant III (EGFRvIII) has been detected in many human tumors, including those of the ovary. This mutant receptor does not bind EGF; however, it is constitutively active as detected by receptor dimerization, autophosphorylation, and stimulation of signal transduction cascades. To identify the consequences of EGFRvIII expression in ovarian tumor cells, we introduced EGFRvIII into the epithelial ovarian cancer cell line OVCA 433. The EGFRvIII-transfected cells displayed a motile phenotype, defects in cell spreading, and decreased integrin alpha2 protein expression as detected by Western blot analysis and flow cytometry. Inhibition of EGFRvIII catalytic activity using the EGFR-selective tyrphostin AG1478 restored integrin alpha2 expression within 4 to 8 hours after treatment. The modulation of integrin alpha2 expression corresponded to marked changes in the actin cytoskeleton as detected by redistribution of filamentous-actin. Furthermore, focal adhesions were evident only when EGFRvIII activity was inhibited. Together, these findings suggest that expression of the constitutively active mutant EGFRvIII promotes changes in cell shape and focal adhesion formation, mediated in part through specific modulation of integrin alpha2 expression and function. We conclude that EGFR-activating mutations, such as EGFRvIII, in ovarian cancer may contribute to a more aggressive disease.     

Mda-9/syntenin promotes human brain glioma migration through focal adhesion kinase (FAK)-JNK and FAK-AKT signaling

Invasion is usually recognized as the main reason for the high recurrence and death rates of glioma and restricts the efficacy of surgery and other therapies. Therefore, we aimed to investigate the mechanism involved in promotion effects of mda-9/syntenin on human glioma cell migration. The wound healing method was used to test the migration ability of human glioma cells CHG-5 and CHG-hS, stably overexpressing mda-9/syntenin. Western blotting was performed to determine the expression and phosphorylation of focal adhesion kinase (FAK) and JNK in CHG-5 and CHG-hS cells. The migration ability of CHG-hS cells was significantly higher than that of CHG-5 cells in fibronectin (FN)-coated culture plates. Phosphorylation of FAK on tyrosine 397, 576, and 925 sites was increased with time elapsed in CHG-hS cells. However, phosphorylated FAK on the tyrosine 861 site was not changed. Phosphorylated Src, JNK and Akt levels in CHG-hS cells were also significantly upregulated. Phosphorylation of JNK and Akt were abolished by the specific inhibitors SP600125 and LY294002, respectively. and the migration ability of CHG-hS cells was decreased, indicating that the JNK and PI3K/Akt pathways play important roles in regulating mda-9/syntenin-induced human brain glioma migration. Our results indicate Mda- 9/syntenin overexpression could activate FAK-JNK and FAK-Akt signaling and then enhance the migration capacity of human brain glioma cells.