Ganoderiol A-Enriched Extract Suppresses Migration and Adhesion of MDA-MB-231 Cells by Inhibiting FAK-SRC-Paxillin Cascade Pathway

Cell adhesion, migration and invasion are critical steps for carcinogenesis and cancer metastasis. Ganoderma lucidum, also called Lingzhi in China, is a traditional Chinese medicine, which exhibits anti-proliferation, anti-inflammation and anti-metastasis properties. Herein, GAEE, G. lucidum extract mainly contains ganoderiol A (GA), dihydrogenated GA and GA isomer, was shown to inhibit the abilities of adhesion and migration, while have a slight influence on that of invasion in highly metastatic breast cancer MDA-MB-231 cells at non-toxic doses. Further investigation revealed that GAEE decreased the active forms of focal adhesion kinase (FAK) and disrupted the interaction between FAK and SRC, which lead to deactivating of paxillin. Moreover, GAEE treatment downregulated the expressions of RhoA, Rac1, and Cdc42, and decreased the interaction between neural Wiskott-Aldrich Syndrome protein (N-WASP) and Cdc42, which impair cell migration and actin assembly. To our knowledge, this is the first report to show that G.lucidum triterpenoids could suppress cell migration and adhesion through FAK-SRC-paxillin signaling pathway. Our study also suggests that GAEE may be a potential agent for treatment of breast cancer.     

17β-Estradiol enhances breast cancer cell motility and invasion via extra-nuclear activation of actin-binding protein ezrin

Estrogen promotes breast cancer metastasis. However, the detailed mechanism remains largely unknown. The actin binding protein ezrin is a key component in tumor metastasis and its over-expression is positively correlated to the poor outcome of breast cancer. In this study, we investigate the effects of 17β-estradiol (E2) on the activation of ezrin and its role in estrogen-dependent breast cancer cell movement. In T47-D breast cancer cells, E2 rapidly enhances ezrin phosphorylation at Thr(567) in a time- and concentration-dependent manner. The signalling cascade implicated in this action involves estrogen receptor (ER) interaction with the non-receptor tyrosine kinase c-Src, which activates the phosphatidylinositol-3 kinase/Akt pathway and the small GTPase RhoA/Rho-associated kinase (ROCK-2) complex. E2 enhances the horizontal cell migration and invasion of T47-D breast cancer cells in three-dimensional matrices, which is reversed by transfection of cells with specific ezrin siRNAs. In conclusion, E2 promotes breast cancer cell movement and invasion by the activation of ezrin. These results provide novel insights into the effects of estrogen on breast cancer progression and highlight potential targets to treat endocrine-sensitive breast cancers.     

Differential regulation of components of the focal adhesion complex by heregulin: role of phosphatase SHP-2.

Heregulin (HRG) has been implicated in the progression of breast cancer cells to a malignant phenotype, a process that involves changes in cell motility and adhesion. Here we demonstrate that HRG differentially regulates the site-specific phosphorylation of the focal adhesion components focal adhesion kinase (FAK) and paxilin in a dose-dependent manner. HRG at suboptimal doses (0.01 and 0.1 nM) increased adhesion of cells to the substratum, induced phosphorylation of FAK at Tyr-577, -925, and induced formation of well-defined focal points in breast cancer cell line MCF-7. HRG at a dose of 1 nM, increased migratory potential of breast cancer cells, selectively dephosphorylated FAK at Tyr-577, -925, and paxillin at Tyr-31. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by HRG stimulation. FAK associated with HER2 only in response to 0.01 nM HRG. In contrast, 1 nM HRG induced activation and increased association of tyrosine phosphatase SHP-2 with HER2 but decreased association of HER2 with FAK. Expression of dominant-negative SHP-2 blocked HRG-mediated dephosphorylation of FAK and paxillin, leading to persistent accumulation of mature focal points. Our results suggest that HRG differentially regulates signaling from focal adhesion complexes through selective phosphorylation and dephosphorylation and that tyrosine phosphatase SHP-2 has a role in the HRG signaling.     

Regulation of Focal Adhesion Kinase Activation,Breast Cancer Cell Motility,and Amoeboid Invasion by the RhoA Guanine Nucleotide Exchange Factor Net1

Net1 is a RhoA guanine nucleotide exchange factor (GEF) that is overexpressed in a subset of human cancers and contributes to cancer cell motility and invasion in vitro. However, the molecular mechanism accounting for its role in cell motility and invasion has not been described. In the present work, we show that expression of both Net1 isoforms in breast cancer cells is required for efficient cell motility. Although loss of Net1 isoform expression only partially blocks RhoA activation, it inhibits lysophosphatidic acid (LPA)-stimulated migration as efficiently as knockdown of RhoA itself. However, we demonstrate that the Net1A isoform predominantly controls myosin light-chain phosphorylation and is required for trailing edge retraction during migration. Net1A interacts with focal adhesion kinase (FAK), localizes to focal adhesions, and is necessary for FAK activation and focal adhesion maturation during cell spreading. Net1A expression is also required for efficient invasion through a Matrigel matrix. Analysis of invading cells demonstrates that Net1A is required for amoeboid invasion, and loss of Net1A expression causes cells to shift to a mesenchymal phenotype characterized by high β₁-integrin activity and membrane type 1 matrix metalloproteinase (MT1-MMP) expression. These results demonstrate a previously unrecognized role for the Net1A isoform in controlling FAK activation during planar cell movement and amoeboid motility during extracellular matrix (ECM) invasion.     

Heregulin and HER2 signaling selectively activates c-Src phosphorylation at tyrosine 215

To elucidate the molecular mechanisms by which human epidermal growth factor receptor/heregulin (HER2/HRG) influence the migratory potential of breast cancer cells, we have used phospho-specific antibodies against c-Src kinase and focal adhesion kinase (FAK). This study establishes that HER2/HRG signaling selectively upregulates Tyr phosphorylation of c-Src at Tyr-215 located within the SH2 domain, increases c-Src kinase activity and selectively upregulates Tyr phosphorylation of FAK at Tyr-861. HER2-overexpressing tumors showed increased levels of c-Src phosphorylation at Tyr-215. These findings suggest that HER2/HRG influence metastasis of breast cancer cells through a novel signaling pathway involving phosphorylation of FAK tyrosine 861 via activation of c-Src tyrosine 215.     

Contortrostatin,a Homodimeric Disintegrin,Actively Disrupts Focal Adhesion and Cytoskeletal Structure and Inhibits Cell Motility Through a Novel Mechanism

Irttegrins play a major role in the regulation of cell motility. They physically link the extracellular environment to the cytoskeleton and participate in large protein complexes known as focal adhesions. In this report, it is demonstrated that treatment of tumor cells with the homodimeric disintegrin contortrostatin induces integrin-mediatcd tyrosine phosphorylation events and causes severe disruptions in the actin cytoskeleton and disassembly of focal adhesion structures without affecting cellular adhesion to a reconstituted basement membrane. Included in this disruption is the tyrosine phosphorylation and altered subcellular localization of FAK. Through use of transfected 293 cells with specific integrin expression profiles and anti-αvβ3 mAbs, we demonstrate that these events are mediated exclusively by the αvβ3 integrin and are likely the result of contortrostatin-mediated crosslinking of this receptor at the cell surface, since monovalent disintegrins, flavoridin or echistatin do not induce such effects. Further, it is shown that contortrostatin potently inhibits motility in cells expressing the αvβ3 integrin. The results of this study describe a novel integrin-mediated mechanism by which cell motility can be inhibited and suggest an alternative approach to therapeutic intervention for cancer invasion and metastasis.     

FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion

The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression. We demonstrate that focal adhesion kinase (FAK) is essential for oncogenic transformation and cell invasion that is induced by ErbB-2 and -3 receptor signaling. ErbB-2/3 overexpression in FAK-deficient cells fails to promote cell transformation and rescue chemotaxis deficiency. Restoration of FAK rescues both oncogenic transformation and invasion that is induced by ErbB-2/3 in vitro and in vivo. In contrast, the inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis formation. The activation of ErbB-2/3 regulates FAK phosphorylation at Tyr-397, -861, and -925. ErbB-induced oncogenic transformation correlates with the ability of FAK to restore ErbB-2/3-induced mitogen-activated protein kinase (MAPK) activation; the inhibition of MAPK prevented oncogenic transformation. In contrast, the inhibition of Src but not MAPK prevented ErbB-FAK-induced chemotaxis. In migratory cells, activated ErbB-2/3 receptors colocalize with activated FAK at cell protrusions. This colocalization requires intact FAK. In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.     

Differential regulation of cell motility and invasion by FAK

Cell migration and invasion are fundamental components of tumor cell metastasis. Increased focal adhesion kinase (FAK) expression and tyrosine phosphorylation are connected with elevated tumorigenesis. Null mutation of FAK results in embryonic lethality, and FAK-/- fibroblasts exhibit cell migration defects in culture. Here we show that viral Src (v-Src) transformation of FAK-/- cells promotes integrin-stimulated motility equal to stable FAK reexpression. However, FAK-/- v-Src cells were not invasive, and FAK reexpression, Tyr-397 phosphorylation, and FAK kinase activity were required for the generation of an invasive cell phenotype. Cell invasion was linked to transient FAK accumulation at lamellipodia, formation of a FAK-Src-p130Cas-Dock180 signaling complex, elevated Rac and c-Jun NH2-terminal kinase activation, and increased matrix metalloproteinase expression and activity. Our studies support a dual role for FAK in promoting cell motility and invasion through the activation of distinct signaling pathways.     

黏着斑激酶与肿瘤侵袭转移

黏着斑激酶（focal adhesion kinase,FAK）是一种非受体型蛋白酪氨酸激酶,在肿瘤细胞的侵袭和转移中起着重要的作用。FAK是整合素介导的或生长因子受体诱导的调节细胞迁移的信号通路的关键组分。FAK通过与相关分子作用可以调节细胞骨架重构、胞外基质降解、细胞黏附更新以及质膜突出,进而参与肿瘤细胞的运动等多个过程,所以FAK与肿瘤发展的关系已经越来越受到重视。     

Oleic acid promotes migration on MDA-MB-231 breast cancer cells through an arachidonic acid-dependent pathway

An association between dietary fatty, obesity and an increased risk of developing breast cancer has been suggested. In breast cancer cells, free fatty acids (FFAs) mediate biological effects including cell proliferation and ERK1/2 activation. However, the contribution of FFAs to tumor progression and metastasis through the regulation of cell migration has not been studied. We demonstrated here that stimulation on MDA-MB-231 breast cancer cells with oleic acid (OA) promotes an increase in focal adhesion kinase (FAK) phosphorylation, as revealed by site-specific antibodies that recognize the phosphorylation state of FAK at tyrosine-397 (Tyr-397), Tyr-577 and in vitro kinase assays. OA also promotes the migration of MDA-MB-231 cells. Treatment with Gi/Go proteins, phospholipase C (PLC), lipoxygenases (LOXs) and Src inhibitor prevents FAK phosphorylation and cell migration. In summary, our findings delineate a new signal transduction pathway, where OA mediates the production of arachidonic acid (AA), and then AA metabolites mediate FAK phosphorylation and cell migration in MDA-MB-231 breast cancer cells.     

Akt directly regulates focal adhesion kinase through association and serine phosphorylation: implication for pressure-induced colon cancer metastasis

Although focal adhesion kinase (FAK) is typically considered upstream of Akt, extracellular pressure stimulates cancer cell adhesion via Akt-dependent FAK activation. How Akt regulates FAK is unknown. We studied Akt-FAK interaction in colon cancer cells under 15 mmHg increased extracellular pressure. Pressure enhanced Akt-FAK association, blocked by inhibiting FAK or silencing Akt1 but not Akt2, and stimulated FAK serine phosphorylation in Caco-2 and human colon cancer cells from surgical specimens Akt1-dependently. FAK includes three serine (S517/601/695) and one threonine (T600)-containing consensus sequences for Akt phosphorylation. Studying S->A nonphosphorylatable point mutants suggests that these sites coordinately upregulate FAK Y397 tyrosine phosphorylation, which conventionally initiates FAK activation, and mediate pressure-induced cancer cell adhesion. FAK(T600A) mutation did not prevent pressure-induced FAK(Y397) phosphorylation or adhesion. Akt1 appeared to directly bind FAK, and this binding did not depend on the FAK autophosphorylation site (Y397). In addition, our results demonstrated that Akt phosphorylated FAK at three novel serine phosphorylation sites, which were also not required for FAK-Akt binding. This novel interaction suggests that FAK and Akt may be dual kinase targets to prevent cancer cell adhesion and metastasis.     

Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233–620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer.     

Ezrin regulates focal adhesion and invadopodia dynamics by altering calpain activity to promote breast cancer cell invasion

Up-regulation of the cytoskeleton linker protein ezrin frequently occurs in aggressive cancer types and is closely linked with metastatic progression. However, the underlying molecular mechanisms detailing how ezrin is involved in the invasive and metastatic phenotype remain unclear. Here we report a novel function of ezrin in regulating focal adhesion (FA) and invadopodia dynamics, two key processes required for efficient invasion to occur. We show that depletion of ezrin expression in invasive breast cancer cells impairs both FA and invadopodia turnover. We also demonstrate that ezrin-depleted cells display reduced calpain-mediated cleavage of the FA and invadopodia-associated proteins talin, focal adhesion kinase (FAK), and cortactin and reduced calpain-1–specific membrane localization, suggesting a requirement for ezrin in maintaining proper localization and activity of calpain-1. Furthermore, we show that ezrin is required for cell directionality, early lung seeding, and distant organ colonization but not primary tumor growth. Collectively our results unveil a novel mechanism by which ezrin regulates breast cancer cell invasion and metastasis.     

Progesterone enhances vascular endothelial cell migration via activation of focal adhesion kinase

The mechanisms of progesterone on endothelial cell motility are poorly investigated. Previously we showed that progesterone stimulated endothelial cell migration via the activation of actin-binding protein moesin, leading to actin cytoskeleton remodelling and the formation of cell membrane structures required for cell movement. In this study, we investigated the effects of progesterone on the formation of focal adhesion complexes, which provide anchoring sites for cell movement. In cultured human umbilical endothelial cells, progesterone enhanced focal adhesion kinase (FAK) phosphorylation at Tyr(397) in a dose- and time-dependent manner. Several signalling inhibitors interfered with progesterone-induced FAK activation, including progesterone receptor (PR) antagonist ORG 31710, specific c-Src kinase inhibitor PP2, phosphatidylinosital-3 kinase (PI3K) inhibitor wortmannin as well as ρ-associated kinase (ROCK-2) inhibitor Y27632. It suggested that PR, c-Src, PI3K and ROCK-2 are implicated in this action. In line with this, we found that progesterone rapidly promoted c-Src/PI3K/Akt activity, which activated the small GTPase RhoA/ρ-associated kinase (ROCK-2) complex, resulting in FAK phosphorylation. In the presence of progesterone, endothelial cells displayed enhanced horizontal migration, which was reversed by small interfering RNAs abrogating FAK expression. In conclusion, progesterone promotes endothelial cell movement via the rapid regulation of FAK. These findings provide new information on the biological actions of progesterone on human endothelial cells that are relevant for vascular function.     

Fibronectin promotes cervical cancer tumorigenesis through activating FAK signaling pathway

Cervical cancer is a cancer arising from the cervix, and it is the fourth most common cause of death in women. Overexpression of fibronectin 1 (FN1) was observed in many tumors and associated with the survival and metastasis of cancer cells. However, the mechanism by which FN1 promotes cervical cancer cell viability, migration, adhesion, and invasion, and inhibits cell apoptosis through focal adhesion kinase (FAK) signaling pathway remains to be investigated. Our results demonstrated that FN1 was upregulated in patients with cervical cancer and higher FN1 expression correlated with a poor prognosis for patients with cervical cancer. FN1 knockdown by small interfering RNA (siRNA) inhibited SiHa cell viability, migration, invasion, and adhesion, and promoted cell apoptosis. FN1 overexpression in CaSki cell promoted cell viability, migration, invasion, and adhesion, and inhibited cell apoptosis. Further, phosphorylation of FAK, a main downstream signaling molecule of FN1, and the protein expression of Bcl-2/Bax, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), and N-cadherin was upregulated in CaSki cells with FN1 overexpression, but caspase-3 protein expression was downregulated. The FAK phosphorylation inhibitor PF573228 inhibited FN1 overexpression-induced expression of those proteins in CaSki cells with FN1 overexpression. In vivo experiment demonstrated that FN1 knockdown significantly inhibited FN1 expression, phosphorylation of FAK, and tumor growth in xenograft from the nude mice. These results suggest that FN1 regulates the viability, apoptosis, migration, invasion, and adhesion of cervical cancer cells through the FAK signaling pathway and is a potential therapeutic target in the treatment of cervical cancer.     

Tetraspanin CD9 in cell migration

Tetraspanin CD9 is associated with integrin adhesion receptors and it was reported that CD9 regulates integrin-dependent cell migration and invasion. Pro- and anti-migratory effects of CD9 have been linked to adhesion-dependent signalling pathways, including phosphorylation of FAK (focal adhesion kinase) and activation of phosphoinositide 3-kinase, p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase). In the present paper, we describe a novel mechanism whereby CD9 specifically controls localization of talin1, one of the critical regulators of integrin activation, to focal adhesions: CD9-deficiency leads to impaired localization of talin1 to focal adhesions and correlates with increased motility of breast cancer cells.     

A novel role of ERK5 in integrin‐mediated cell adhesion and motility in cancer cells via Fak signaling

In metastatic cancer, high expression levels of vitronectin (VN) receptors (integrins), FAK, and ERK5 are reported. We hypothesized that integrin‐mediated ERK5 activation via FAK may play a pivotal role in cell adhesion, motility, and metastasis. ERK5 and FAK phosphorylation when metastatic MDA‐MB‐231 and PC‐3 cells were plated on VN was enhanced. Further experiments showed co‐immunoprecipitation of integrins β1, αVβ3, or αVβ5 with ERK5 and FAK. To gain better insight into the mechanism of ERK5, FAK, and VN receptors in cell adhesion and motility, we performed loss‐of‐function experiments using integrin blocking antibodies, and specific mutants of FAK and ERK5. Ectopic expression of dominant negative ERK5/AEF decreased ERK5 and FAK (Y397) phosphorylation, cell adhesion, and haptotactic motility (micromotion) on VN. Additionally, DN FAK expression attenuated ERK5 phosphorylation, cell adhesion, and motility. This study documents the novel finding that in breast and prostate cancer cells, ERK5 is a critical target of FAK in cell adhesion signaling. Using different cancer cells, our experiments unveil a novel mechanism by which VN receptors and FAK could promote cancer metastasis via ERK5 activation. J. Cell. Physiol. 219: 152–161, 2009. © 2008 Wiley‐Liss, Inc.     

FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion

Focal adhesion kinase (FAK) is important for breast cancer progression and invasion and is necessary for the dynamic turnover of focal adhesions. However, it has not been determined whether FAK also regulates the dynamics of invasive adhesions formed in cancer cells known as invadopodia. In this study, we report that endogenous FAK functions upstream of cellular Src (c-Src) as a negative regulator of invadopodia formation and dynamics in breast cancer cells. We show that depletion of FAK induces the formation of active invadopodia but impairs invasive cell migration. FAK-deficient MTLn3 breast cancer cells display enhanced assembly and dynamics of invadopodia that are rescued by expression of wild-type FAK but not by FAK that cannot be phosphorylated at tyrosine 397. Moreover, our findings demonstrate that FAK depletion switches phosphotyrosine-containing proteins from focal adhesions to invadopodia through the temporal and spatial regulation of c-Src activity. Collectively, our findings provide novel insight into the interplay between FAK and Src to promote invasion.     

PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway.

The tumor suppressor PTEN is a phosphatase with sequence homology to tensin. PTEN dephosphorylates phosphatidylinositol 3,4, 5-trisphosphate (PIP3) and focal adhesion kinase (FAK), and it can inhibit cell growth, invasion, migration, and focal adhesions. We investigated molecular interactions of PTEN and FAK in glioblastoma and breast cancer cells lacking PTEN. The PTEN trapping mutant D92A bound wild-type FAK, requiring FAK autophosphorylation site Tyr397. In PTEN-mutated cancer cells, FAK phosphorylation was retained even in suspension after detachment from extracellular matrix, accompanied by enhanced PI 3-K association with FAK and sustained PI 3-K activity, PIP3 levels, and Akt phosphorylation; expression of exogenous PTEN suppressed all five properties. PTEN-mutated cells were resistant to apoptosis in suspension, but most of the cells entered apoptosis after expression of exogenous PTEN or wortmannin treatment. Moreover, overexpression of FAK in PTEN-transfected cells reversed the decreased FAK phosphorylation and PI 3-K activity, and it partially rescued PIP3 levels, Akt phosphorylation, and PTEN-induced apoptosis. Our results show that FAK Tyr397 is important in PTEN interactions with FAK, that PTEN regulates FAK phosphorylation and molecular associations after detachment from matrix, and that PTEN negatively regulates the extracellular matrix-dependent PI 3-K/Akt cell survival pathway in a process that can include FAK.