Decreased Focal Adhesion Kinase Phosphorylation Research Articles

Tumor removal limits prostate cancer cell dissemination in bone and osteoblasts induce cancer cell dormancy through focal adhesion kinase

BackgroundDisseminated tumor cells (DTCs) can enter a dormant state and cause no symptoms in cancer patients. On the other hand, the dormant DTCs can reactivate and cause metastases progression and lethal relapses. In prostate cancer (PCa), relapse can happen after curative treatments such as primary tumor removal. The impact of surgical removal on PCa dissemination and dormancy remains elusive. Furthermore, as dormant DTCs are asymptomatic, dormancy-induction can be an operational cure for preventing metastases and relapse of PCa patients.MethodsWe used a PCa subcutaneous xenograft model and species-specific PCR to survey the DTCs in various organs at different time points of tumor growth and in response to tumor removal. We developed in vitro 2D and 3D co-culture models to recapitulate the dormant DTCs in the bone microenvironment. Proliferation assays, fluorescent cell cycle reporter, qRT-PCR, and Western Blot were used to characterize the dormancy phenotype. We performed RNA sequencing to determine the dormancy signature of PCa. A drug repurposing algorithm was applied to predict dormancy-inducing drugs and a top candidate was validated for the efficacy and the mechanism of dormancy induction.ResultsWe found DTCs in almost all mouse organs examined, including bones, at week 2 post-tumor cell injections. Surgical removal of the primary tumor reduced the overall DTC abundance, but the DTCs were enriched only in the bones. We found that osteoblasts, but not other cells of the bones, induced PCa cell dormancy. RNA-Seq revealed the suppression of mitochondrial-related biological processes in osteoblast-induced dormant PCa cells. Importantly, the mitochondrial-related biological processes were found up-regulated in both circulating tumor cells and bone metastases from PCa patients’ data. We predicted and validated the dormancy-mimicking effect of PF-562,271 (PF-271), an inhibitor of focal adhesion kinase (FAK) in vitro. Decreased FAK phosphorylation and increased nuclear translocation were found in both co-cultured and PF-271-treated C4-2B cells, suggesting that FAK plays a key role in osteoblast-induced PCa dormancy.ConclusionsOur study provides the first insights into how primary tumor removal enriches PCa cell dissemination in the bones, defines a unique osteoblast-induced PCa dormancy signature, and identifies FAK as a PCa cell dormancy gatekeeper.

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling.

BackgroundS100A9, which is expressed in prostate cancer, has been reported in association with prostate cancer progression. However, the role of S100A9 in prostate cancer metastasis is largely unknown. The aim of this study was to investigate the effect of S100A9 on prostate cancer cell invasion and the involved mechanisms.Materials and methodsIntegrin β1 expression in PC-3 and DU-145 cells was determined by quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) and Western blot. Cellular invasion was measured by transwell invasion assay. Western blot was used to determine protein expression. Concentrations of S100A9 and fibronectin were analyzed by enzyme-linked immunosorbent assay. The protein interaction was detected by immunoprecipitation. The NF-κB activity was measured by luciferase reporter assay. The DU-145 cells metastasis in vivo was determined in mice xenograft models after S100A9 overexpression.ResultsS100A9 promoted prostate cancer cells invasion, integrin β1 expression and fibronectin secretion. Further investigation evidenced that S100A9 interacted with Toll-like receptor 4 (TLR4) and activated NF-κB, which was responsible for tumor cell invasion, integrin β1 up-regulation and focal adhesion kinase (FAK) phosphorylation. Furthermore, integrin β1 inhibition led to decreased FAK phosphorylation and reduced tumor cell invasion. Overexpression of S100A9 increased xenograft tumor micro-metastases, integrin β1 expression and induced NF-κB and FAK activation in vivo.ConclusionOur study demonstrated that S100A9 promotes prostate cancer cell invasion, and one of the underlying molecular mechanisms is that S100A9 activates integrin β1/FAK through TLR4/NF-κB signaling leading to metastasis of prostate cancer cell.

P1.07-028 Dual Role of the Focal Adhesion Kinase in Small-Cell Lung Cancer

Small cell-lung cancer (SCLC) is a devastating illness with five-year overall survival as low as 5%. The molecular steps leading to SCLC development and progression are still poorly understood and this has translated into the absence of targeted therapies. Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase which regulates integrin and growth factor signaling pathways involved in cell proliferation, survival, migration, and invasion. FAK is overexpressed and/or activated in many cancers, including SCLC. We hypothesized that FAK overexpression/activation in SCLC contributes to its aggressive behavior and that FAK may represent a therapeutic target in SCLC. Two SCLC cell lines growing in suspension (NCI-H82, NCI-H146), and adherent SCLC cell lines (NCI-H196, NCI-H446) were treated with PF-228. NCI-H446 and H82 cells were stably transfected with FAK shRNA and/or FRNK using lentivirus vector. Cell proliferation was evaluated by WST-1 assay; cell cycle by flow cytometry with propidium iodide and bromodeoxyuridine; apoptosis by caspase 3 staining in flow cytometry and by cleaved PARPp85 Western blotting (WB); motility by wound healing assay; and invasion by Boyden chambers. FAK expression/activity was evaluated by WB. While PF-228 did not modify total FAK expression, it decreased FAK phosphorylation (Y397). Inhibition of FAK activity by PF-228 decreased cell proliferation, DNA synthesis, induced cell cycle arrest in G2/M phases, and increased apoptosis in dose-dependently. PF-228 also decreased motility in the adherent H196-H446 cells. To confirm the specificity of the antitumoral effects of PF-228, we stably transfected SCLC cells with FAK shRNA and FRNK and then analyzed the phenotypic changes induced by these approaches. Knockdown of total FAK protein by transfection of FAK shRNA inhibited FAK activity, but did not have any effect on cell proliferation, DNA synthesis, and cell cycle. However, reintroduction of FRNK in cells stably transfected with FAK shRNA inhibited cell proliferation and DNA synthesis. Expression of FRNK decreased cell proliferation and DNA synthesis in SCLC cells. Inhibition of FAK activity by PF-228 in SCLC cell lines demonstrates that FAK activity is required for cell proliferation, cycle progression, survival, and motility, suggesting that FAK inhibition may represent a suitable therapeutic target for SCLC. Inhibition of FAK by a genomic approach suggests that FAK has a dual role in SCLC biology, namely (i) a pro-tumoral effect related to the kinase domain, which induces downstream signals (ii) an anti-tumoral effect mediated by the non-kinase C-terminal domain (FRNK domain), which keeps inactive other pro-tumoral effectors.

Novel anticancer agent, SQAP, binds to focal adhesion kinase and modulates its activity.

SQAP is a novel and promising anticancer agent that was obtained by structural modifications from a natural compound. SQAP inhibits angiogenesis in vivo resulting in increased hypoxia and reduced tumor volume. In this study, the mechanism by which SQAP modifies the tumor microenvironment was revealed through the application of a T7 phage display screening. This approach identified five SQAP-binding proteins including sterol carrier protein 2, multifunctional enzyme type 2, proteasomal ubiquitin receptor, UV excision repair protein and focal adhesion kinase (FAK). All the interactions were confirmed by surface plasmon resonance analysis. Since FAK plays an important role in cell turnover and angiogenesis, the influence of SQAP on FAK was the principal goal of this study. SQAP decreased FAK phosphorylation and cell migration in human umbilical vein endothelial cells and A549 cancer cells. These findings suggest that inhibition of FAK phosphorylation works as the mechanism for the anti-angiogenesis activity of SQAP.

Cellular fibronectin 1 promotes VEGF-C expression, lymphangiogenesis and lymph node metastasis associated with human oral squamous cell carcinoma.

Lymph node metastasis (LNM) is associated with poor survival in patients with oral squamous cell carcinoma (OSCC). Vascular endothelial growth factor-C (VEGF-C) is thought to be responsible for increased lymphangiogenesis and LNM. Understanding of the mechanism by which VEGF-C expression is regulated in OSCC is thus important to design logic therapeutic interventions. We showed that inoculation of the SAS human OSCC cells expressing the venus GFP (V-SAS cells) into the tongue in nude mice developed LNM. V-SAS cells in LNM were isolated by FACS and re-inoculated into the tongue. This procedure was repeated eight times, establishing V-SAS-LM8 cells. Differential metastasis PCR array between the parental V-SAS and V-SAS-LM8 was performed to identify a molecule responsible for lymphangiogenesis and LNM. Fibronectin 1 (FN1) expression was elevated in V-SAS-LM8 cells compared to V-SAS-cells. V-SAS-LM8 tongue tumor showed increased expression of FN1 and VEGF-C, and promoted lymphangiogenesis and LNM compared with V-SAS tumor. Further, phosphorylation of focal adhesion kinase (FAK), a main downstream signaling molecule of FN1, was up-regulated, and epithelial-mesenchymal transition (EMT) was promoted in V-SAS-LM8 cells. Silencing of FN1 by shRNA in V-SAS-LM8 cells decreased FAK phosphorylation, VEGF-C expression and inhibited lymphangiogenesis and LNM. EMT was also reversed. The FAK phosphorylation inhibitor PF573228 also decreased VEGF-C expression and reversed EMT in V-SAS-LM8 cells. Finally, we detected intense FN1 expression in some clinical specimens obtained from OSCC patients with LNM. These results demonstrate that elevated expression of cellular FN1 and following activation of FAK lead to increased VEGF-C expression, lymphangiogenesis and LNM and promoted EMT in SAS human OSCC cells and suggest that FN1-phosphorylated FAK signaling cascade is a potential therapeutic target in the treatment of LNM in OSCC.

Novel anticancer agent, SQAP, binds to focal adhesion kinase and modulate its activity

Sulfoquynovosyl acylpropanediol (SQAP) is a novel and promising anticancer agent that was obtained by structural modifications from a natural compound. SQAP has the ability to trigger antiangiogenesis in vivo with very few side effects. In this study, the mechanism by which SQAP modifies the tumor microenvironment was revealed through the application of a T7 phage display screening. We could identify five SQAP-binding proteins including among them the FAT domain of focal adhesion kinase (FAK). Two independent experiments were used to verify the interactions. SQAP decreased FAK phosphorylation and cell migration in human umbilical vein endothelial cells and A549 cancer cells. The obtained data suggest that SQAP antiangiogenic activity may be due the modulation of FAK phosphorylation. Additionally, we hypothesized that SQAP radiosensitizing activity might be at least partly due to ROS generation. Indeed, we observed that SQAP romotes ROS formation on HeLa cancer cells.

JNK Pathway-associated Phosphatase Dephosphorylates Focal Adhesion Kinase and Suppresses Cell Migration

JNK pathway-associated phosphatase (JKAP, also named DUSP22) is expressed in various tissues, indicating that JKAP may have an important biological function. We showed that JKAP localized in the actin filament-enriched region. Expression of JKAP reduced cell migration, whereas a JKAP mutant lacking catalytic activity promoted cell motility. JKAP efficiently removed tyrosine phosphorylation of several proteins. We have identified focal adhesion kinase (FAK) as a substrate of JKAP. Overexpression of JKAP, but not JKAP mutant lacking catalytic activity, decreased FAK phosphorylation at tyrosines 397, 576, and 577 in H1299 cells. Consistent with these results, decreasing JKAP expression by RNA interference promoted cell migration and Src-induced FAK phosphorylation. Taken together, this study identified a new role for JKAP in the modulation of FAK phosphorylation and cell motility.

Activation of focal adhesion kinase and JNK contributes to the extracellular matrix and cAMP-GEF mediated survival from bile acid induced apoptosis in rat hepatocytes

Adherence to an extracellular matrix (ECM) rescues hepatocytes from apoptosis, but how hepatocytes adhered to different ECM and respond to apoptotic and cytoprotective stimuli is unknown. Rat hepatocytes were plated on type 1 collagen (CI), laminin (LM) or polylysine (PL) and the amount of apoptosis induced by glycochenodeoxycholate (GCDC), deoxycholate (DCA), Fas ligand or serum withdrawal was determined by Hoechst staining. The response to cytoprotection by cAMP-guanine exchange factor (cAMP-GEF) activation was determined. Kinase activation was determined by immunoblotting with phosphospecific antibodies. Hepatocytes on LM and PL had more apoptosis in response to all apoptotic stimuli. GCDC increased c-jun-N-terminal kinase (JNK) phosphorylation 2-fold in hepatocytes on CI, but 15- and 30-fold in hepatocytes on PL or LM. SP-600125, a JNK inhibitor, prevented LM and PL potentiation of bile acid apoptosis. GCDC induced dephosphorylation of focal adhesion kinase (FAK) was prevented by cAMP-GEF activation. Cytochalasin B which decreased FAK phosphorylation prevented cAMP-GEF cytoprotection. JNK activation augments apoptosis in hepatocytes plated on PL and LM. Decreased FAK phosphorylation as seen in cells treated with bile acids or attached to PL and LM promotes hepatocyte apoptosis.

NG2, a novel proapoptotic receptor, opposes integrin α4 to mediate anoikis through PKCα-dependent suppression of FAK phosphorylation

Disruption of cell-matrix interactions can lead to anoikis - apoptosis due to loss of matrix contacts. Altered fibronectin (FN) induces anoikis of primary human fibroblasts by a novel signaling pathway characterized by reduced phosphorylation of focal adhesion kinase (FAK). However, the receptors involved are unknown. FAK phosphorylation is regulated by nerve/glial antigen 2 (NG2) receptor signaling through PKCalpha a point at which signals from integrins and proteoglycans may converge. We found that an altered FN matrix induced anoikis in fibroblasts by upregulating NG2 and downregulating integrin alpha4. Suppressing NG2 expression or overexpressing alpha4 rescued cells from anoikis. NG2 overexpression alone induced apoptosis and, by reducing FAK phosphorylation, increased anoikis induced by an altered matrix. NG2 overexpression or an altered matrix also suppressed PKCalpha expression, but overexpressing integrin alpha4 enhanced FAK phosphorylation independently of PKCalpha. Cotransfection with NG2 cDNA and integrin alpha4 siRNA did not lower PKCalpha and pFAK levels more than transfection with either alone. PKCalpha was upstream of FAK phosphorylation, as silencing PKCalpha decreased FAK phosphorylation. PKCalpha overexpression reversed this behavior and rescued cells from anoikis. Thus, NG2 is a novel proapoptotic receptor, and NG2 and integrin alpha4 oppositely regulate anoikis in fibroblasts. NG2 and integrin alpha4 regulate FAK phosphorylation by PKCalpha-dependent and -independent pathways, respectively.

504 THE EFFECT OF FAK-RELATED NON-KINASE PLASMID TRANSFECTION ON FIBRONECTIN-STIMULATED HEPATIC STELLATE CELLS BEHAVIOURS

BACKGROUND AND AIMS The main cells responsible for liver fibrosis are hepatic stellate cells (HSC), which undergo activation, proliferation and increased production of extracellular matrix following liver injury. Focal adhesion kinase (FAK) plays an important role in integrinmediated signal transduction pathways and its C-terminal noncatalytic domain FAK-related non-kinase (FRNK), which is autonomously expressed, acts as an inhibitor of FAK phosphorylation. In the present study, we therefore investigated the effects of FRNK on proliferation, apoptosis and cell cycle in HSC stimulated by fibronectin (FN). The signal transduction molecules-extracellular signal-regulated kinase 1/2(ERK1/2) was also investigated. METHODS We have used rat immortalized hepatic stellate cell lines CFSC, and cells were divided into 5 groups as below: blank control group; FN-treated group; liposome group; empty plasmid group and FRNK plasmid group. FRNK plasmids were transfected into HSCs mediated by lipofectamine 2000 reagent. Improved MTT assay was used to determine cell proliferation, and flow cytometry (FCM)was used to detect cell cycle phase. Apoptosis was examined by Annexin-V/PI double-labelled FCM, gel electrophoresis and transmission electron microscope. The expression of FRNK, FAK, P-FAK (Tyr397), ERK1/2 and cell cycle related protein (Cyclin D1 and CDK4) were detected by Western blot and RT-PCR. RESULTS Transfection of FRNK decreased FAK phosphorylation on Tyr397 without changing total FAK level, coincident with a large increase in FRNK expression. After FRNK transfection, the proliferation inhibition rates in HSC were 20.07%, 26.16% and 29.77% at 12 h, 24 h, and 48 h, respectively. Meanwhile, the apoptotic rate in HSC exposed to FRNK plasmid for 48 h was higher than that in the empty plasmid group (25.37%±1.92% vs 9.28%±1.05%), P<0.01. The morphology changes of HSC observed by transmission electron microscopy also revealed that the apoptosis of HSC was increased by FRNK. Consistent with this, apoptotic DNA Ladder was detected in HSC exposed to FRNK. The percentage of cells at G0/G1 phase was significantly higher in FRNK plasmid group than that in empty plasmid group (71.4±2.81 vs 48.9±1.66), P<0.01, accompanied by down-regulation of Cyclin D1 and CDK4. Additionally, we found that FRNK attenuated ERK1/2 phosphorylation and resulted in a down-regulation of ERK1/2 mRNA. CONCLUSION FRNK may inhibit the proliferation and induce the apoptosis of HSC and arrested HSC at G0/G1 phase via decreasing phosphorylation levels at FAK and ERK1/2. The inhibition of FAK/ERK1/2 pathway may be a potential therapeutic strategy for preventing liver fibrogenesis.

Focal Adhesion Kinase is Activated in Invading Fibrosarcoma Cells and Regulates Metastasis

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is overexpressed in several human cancers, and induces survival, proliferation and motility of cells in culture. Phosphorylation of FAK has been studied extensively in vitro, but little is known about its regulation during tumor invasion in vivo. In the current study, green fluorescent protein (GFP) was expressed stably in an invasive murine fibrosarcoma cell line for the purpose of discrimination between tumor and normal cells. Under fluorescence microscopy, the tumor was highly fluorescent, and the margin between the tumor and normal tissue was clearly demarcated. Using this invasion model, we showed localization of pY397-FAK expression in the infiltrative edge of tumors. We reproduced local invasion in vivo using a tumor tissue culture method in a three dimensional collagen gel. Phosphorylation of FAK is also upregulated in invading fibrosarcoma cells under in vitro conditions. Expression of the FAK C-terminal domain termed FRNK (FAK-related non-kinase) in 2,472 cells decreased FAK phosphorylation without changing total FAK levels. FRNK inhibited the motility of 2,472 cells, and reduced invasion in vitro. Although FRNK did not affect cell growth, it inhibited experimental metastases in syngenic mice. These results demonstrate that the phosphorylation of FAK might be specifically upregulated in invading fibrosarcoma cells and regulate their invasion and metastasis.

Focal adhesion kinase and Src phosphorylations in HGF-induced proliferation and invasion of human cholangiocarcinoma cell line, HuCCA-1

To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression. Previously isolated HuCCA-1 cells were re-characterized by immunofluorescent staining and reverse transcriptase-polymerase chain reaction assay for the expression of cytokeratin 19, HGF and c-Met mRNA. Cultured HuCCA-1 cells were treated with HGF and determined for cell proliferation and invasion effects by MTT and invasion assays. Western blotting, immunoprecipitation, and co-immunoprecipitation were also performed to study the phosphorylation and interaction of FAK and Src. A novel Src inhibitor (AZM555130) was applied in cultures to investigate the effects on FAK phosphorylation inhibition and on cell proliferation and invasion. HGF enhanced HuCCA-1 cell proliferation and invasion by mediating FAK and Src phosphorylations. FAK-Src interaction occurred in a time-dependent manner that Src was proved to be an upstream signaling molecule to FAK. The inhibitor to Src decreased FAK phosphorylation level in correlation with the reduction of cell proliferation and invasion. FAK plays a significant role in signaling pathway of HGF-responsive cell line derived from cholangiocarcinoma. Autophosphorylated Src, induced by HGF, mediates Src kinase activation, which subsequently phosphorylates its substrate, FAK, and signals to cell proliferation and invasion.

Squamous Cell Carcinoma Cell Aggregates Escape Suspension-induced, p53-mediated Anoikis: FIBRONECTIN AND INTEGRIN αv MEDIATE SURVIVAL SIGNALS THROUGH FOCAL ADHESION KINASE

Resistance to anoikis, or apoptosis triggered by detachment from the extracellular matrix (ECM), lengthens the survival of malignant cells, facilitating reattachment and colonization of secondary sites. To examine the molecular mechanisms underlying resistance to anoikis in human oral squamous cell carcinoma (SCC) cells, we cultured human squamous carcinoma (HSC-3) cells in suspension on plates coated with poly-2-hydroxyethyl methacrylate, which blocks access to the ECM. Cells in suspension that formed multicellular aggregates had significantly lower levels of apoptosis than single cells. Aggregates, but not single cells, had high levels of fibronectin. Preincubation with a cyclic arginine-glycine-aspartic acid peptide or fibronectin-blocking antibody significantly increased anoikis. Single cells had markedly lower expression of the integrin alpha(v) receptor than aggregates. Blocking alpha(v) function with a blocking antibody or by transfection with an antisense oligonucleotide increased apoptosis and inhibited aggregation. In single cells but not aggregates, phosphorylation of the integrin-associated focal adhesion kinase (FAK) at tyrosine 397 was reduced, and p53 levels were increased. Apoptosis was increased by blocking FAK with an antisense oligonucleotide and reduced by blocking p53. These findings show that SCC cells escape suspension-induced anoikis by forming multicellular aggregates that avail themselves of fibronectin survival signals mediated by integrin alpha(v). Single cells in suspension that do not form aggregates undergo anoikis because of decreased FAK phosphorylation and increased p53 levels. Thus, SCC cells appear to use neighboring cells and the ECM molecule FN to promote the metastatic phenotype.

This month in Gastroenterology

This month in Gastroenterology

The C-terminal domain of focal adhesion kinase reduces the tumor cell invasiveness in chondrosarcoma cell lines

Human chondrosarcoma is a malignancy that has no effective systemic therapy, making the interruption of the metastatic cascade critical to enhance patient survival. The processes of local invasion and metastases share similar mechanisms at a cellular level. Focal adhesion kinase (FAK) has been implicated in local invasion of malignant tumor cells. In the current manuscript we examine the effect of FAK inhibition on cell attachment to extracellular matrix (ECM) and in vitro invasion. Bovine articular chondrocytes and two human chondrosarcoma cell lines were utilized to examine FAK activity in tumor cell invasiveness. Endogenous FAK activity was inhibited by adenoviral transfection with the C-terminal domain of FAK. This inhibition resulted from decreased FAK phosphorylation, while FAK expression remained unchanged. Inhibition of FAK phosphorylation and hence its activity lead to decreased cell adhesion to Type II collagen and decreased cell invasiveness. These effects were not due to changes in integrin expression, indicating that the inhibition was the result of disruption of outside: in signaling. There are three important aspects to these results. The first is that interruption of transmembrane signaling can affect cell attachment. The second is that in chondrosarcoma, cell differentiation correlates with FAK expression and metastatic potential. Thirdly, that cell invasiveness correlates with FAK activity and implies a mechanistic role for this molecular complex in local invasion and metastasis.

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.