Galen B. Schneider

Biological significance of focal adhesion kinase in ovarian cancer: Role in migration and invasion

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is activated by integrin clustering. There are limited data regarding the functional role of FAK in ovarian cancer migration and invasion. In the current study, FAK expression was evaluated in ovarian cell lines (nontransformed and cancer), 12 benign ovarian samples, and in 79 invasive epithelial ovarian cancers. All three ovarian cancer cell lines overexpressed FAK compared to the nontransformed cells. The dominant-negative construct called FAK-related nonkinase (FRNK) was introduced into two ovarian cancer cell lines (SKOV3 and 222). FRNK promoted FAK dephosphorylation without changing total FAK levels in these cell lines. Furthermore, FRNK decreased the in vitro invasive ability of ovarian cancer cells by 56 to 85% and decreased migration by 52 to 68%. FRNK-transfected cells also displayed poor cell spreading. Immunohistochemical analysis revealed that the surface epithelium from all benign ovarian samples had weak FAK expression. In contrast, 68% of invasive ovarian cancers overexpressed FAK. FAK overexpression was significantly associated with high tumor stage, high tumor grade, positive lymph nodes and presence of distant metastasis (all P values <0.05). FAK overexpression was also associated with shorter overall survival (P = 0.008). Multivariate analysis revealed that FAK overexpression and residual disease >1 cm were independent predictors of poor survival. These data indicate that FAK is overexpressed in most invasive ovarian cancers and plays a functionally significant role in ovarian cancer migration and invasion. Thus, FAK may be an important therapeutic target in ovarian carcinoma.

Direct Association of Protein-tyrosine Phosphatase PTP-PEST with Paxillin

Tyrosine phosphorylation of focal adhesion-associated proteins may be involved in the regulation of the cytoskeleton and in the control of signals for growth and survival. The focal adhesion kinase (FAK) functions in regulating tyrosine phosphorylation of several of these proteins, including paxillin, tensin, and p130 cas . Protein- tyrosine phosphatases, the counterparts of protein-tyrosine kinases, also presumably regulate phosphorylation of these proteins. We have tested the hypothesis that FAK intimately associates with a protein-tyrosine phosphatase. Protein-tyrosine phosphatase activity associated with the recombinant C-terminal domain of FAK in vitro and could be coimmunoprecipitated with both FAK and paxillin from lysates of chicken embryo cells. However, the interaction with FAK appeared to be indirect and mediated via paxillin. The protein-tyrosine phosphatase was subsequently identified as protein-tyrosine phosphatase-PEST, a nonreceptor protein-tyrosine phosphatase. The C-terminal noncatalytic domain of protein-tyrosine phosphatase-PEST directly bound to paxillinin vitro. The association of both a protein-tyrosine kinase and a protein-tyrosine phosphatase with paxillin suggests that paxillin may play a critical role in the regulation of the phosphotyrosine content of proteins in focal adhesions.

Implant Surface Roughness Affects Osteoblast Gene Expression

The transcription factor Cbfa1 regulates osteoblast differentiation and expression of genes necessary for the development of a mineralized phenotype. The purpose of this study was to determine if Cbfa1 and BSPII gene expression are influenced by implant surface microtopography. Osteoblasts were cultured on 600-grit (grooved) or sandblasted (roughened) cpTi implant discs. Mineralization was evaluated by Alizarin-Red-S staining. Real Time PCR was used for quantitative analysis of Cbfa1 and BSPII gene expression. Enhanced mineralization was seen in osteoblasts grown on roughened implant surfaces relative to tissue culture plastic. Real Time PCR showed significant (P < 0.05) increases in Cbfa1 gene expression in cells grown on roughened, as compared with grooved, implant surfaces. BSPII gene expression was also increased on rough surfaces in the UMR cells, but was reduced in the rat calvarial osteoblast cultures. These results suggest that osteoblast gene expression and mineralization are affected by roughened implant surface microtopographies during osseointegration of dental implants.

Osteoblast Integrin Adhesion and Signaling Regulate Mineralization

Integrin adhesion and signaling events may contribute to the progressive differentiation of the osteoblast and to the initiation of a mineralized matrix. The purpose of our study was to begin to analyze the role of integrin receptors, in particular α2β1, α5β1, and αVβ3, regarding mediation of the initiation of a mineralized matrix. Integrin-perturbation assays were conducted in microdot cultures of UMR-106-01 Bone Sialoprotein (BSP) osteoblast cells. For phenotypic analysis, we performed bright-field microscopy and Aliziran Red S staining to analyze effects on mineralization initiation. Mineralization was reduced significantly (P < 0.001) following the addition of a5- or β1-integrin subunit antibody by approximately 20% and 45%, respectively—αVβ3 integrin by nearly 65%, and α2β1 integrin by nearly 95%. This effect was reversible following the removal of the anti-integrin antibody. These results suggest that integrin adhesion and signaling events may contribute to the ability of this cell line to mediate the initiation of the mineralization phenotype biologically.

Focal Adhesion Kinase Promotes the Aggressive Melanoma Phenotype

Malignant melanoma continues to remain a significant health threat, with death often occurring as a result of metastasis. The metastatic phenotype typically is characterized by augmented tumor cell invasion and migration in addition to tumor cell plasticity as shown by vasculogenic mimicry. Therefore, understanding the molecular mechanisms that promote an aggressive phenotype is essential to predicting the likelihood of metastasis at a stage when intervention may be possible. This study focuses on the role of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase important for many cellular processes, including cell survival, invasion, and migration. We found FAK to be phosphorylated on its key tyrosine residues, Tyr397 and Tyr576, in only aggressive uveal and cutaneous melanoma cells, which correlates with their increased invasion, migration, and vasculogenic mimicry plasticity. Additionally, we confirmed the presence of FAK phosphorylated on Tyr397 and Tyr576 in both cutaneous and uveal melanoma tumors in situ. Examination of a functional role for FAK in aggressive melanoma revealed that disruption of FAK-mediated signal transduction pathways, through the expression of FAK-related nonkinase (FRNK), results in a decrease in melanoma cell invasion, migration, and inhibition of vasculogenic mimicry. Moreover, we found that FRNK expression resulted in a down-regulation of Erk1/2 phosphorylation resulting in a decrease in urokinase activity. Collectively, these data suggest a new mechanism involved in promoting the aggressive melanoma phenotype through FAK-mediated signal transduction pathways, thus providing new insights into possible therapeutic intervention strategies.

In vivo evaluation of hsp27 as an inhibitor of actin polymerization: Hsp27 limits actin stress fiber and focal adhesion formation after heat shock

The role of hsp27 as an inhibitor of actin polymerization was considered in the context of the actin cytoskeleton and its relationship with focal adhesion formation. The aim of this study was to evaluate the potential effects of hsp27 on focal adhesion formation as a relevant biological consequence of actin stress fiber formation. When hsp27 was overexpressed in stably transfected cells, cell attachment was delayed and recovery of disrupted stress fibers and focal adhesions was limited. In ROS 17/2.8 cells, heat shock caused the reversible disruption of stress fibers and focal adhesions. The loss of stress fibers and focal adhesions was associated with reduced phosphotyrosine on the focal adhesion kinase (FAK). Microinjection of recombinant 6‐His hsp27 and phosphorylated 6‐His hsp27 was used to demonstrate that nonphosphorylated hsp27 prevented the recovery of stress fibers and focal adhesions. These results provide in vivo evidence that hsp27 acts as an inhibitor of actin polymerization that can alter cellular interactions with extracellular environments by perturbation of stress fibers, and subsequently focal adhesions. J Cell Physiol 177:575–584, 1998.

Elevated focal adhesion kinase expression facilitates oral tumor cell invasion

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.

Apatite/Amelogenin Coating on Titanium Promotes Osteogenic Gene Expression

Osteoblast differentiation and extracellular matrix production are pivotal processes for implant osseointegration or bone tissue engineering. We hypothesized that a biomimetic coating on titanium surfaces, consisting of apatite and amelogenin, would promote such processes. Human Embryonic Palatal Mesenchymal pre-osteoblasts were used as a model for the evaluation of cell adhesion and spreading patterns, as well as mRNA expression of certain osteoblastic gene products. Real-time PCR showed significant (p < 0.05) increase in expression of type I collagen, alkaline phosphatase, and osteocalcin from cells grown on titanium with an apatite/amelogenin composite, as compared with that from cells grown on a pure titanium or apatite coating only. Osteocalcin expression was specifically stimulated by amelogenin added to the culture media. Enhanced attachment and cell spreading were also observed. The biomimetic coating promoting cell adhesion and osteoblast differentiation may have great potential for future dental and biomedical applications.

Early cell adhesion events differ between osteoporotic and non-osteoporotic osteoblasts.

In osteoporosis, the regenerative capacity of bone is compromised, which may involve altered osteoblast (OB) activity. This could be attributed to an inappropriate synthesis and assembly of an extracellular matrix (ECM), altered cell adhesion to the ECM, or be due to inappropriate downstream activation of adhesion‐mediated signaling cascades through proteins such as focal adhesion kinase (FAK). The purpose of our study was to compare early adhesion‐mediated events using previously described and characterized clinically derived OBs obtained from human patients undergoing major joint arthroplasty for osteoporosis or osteoarthritis. The presence or absence of osteoporosis was established with a radiographic index. Using light microscopy and crystal violet staining, we show that OB cells derived from sites of osteoporosis do not attach and spread as well as non‐osteoporotic (OP) OB cells. OP cells initially have a more rounded morphology, and show significantly less (P < 0.001) attachment to serum‐coated tissue culture plastic over a 24 h time period. Immunofluorescent labeling after 24 h of attachment showed that OP OB focal adhesions (FAs) and stress fibers were less defined, and that the OP cells were smaller and had a more motile phenotype. When normalized protein lysates were Western blotted for phosphotyrosine (PY) a band corresponding to pp125FAK was identified. FAK tyrosine phosphorylation was evident at 6 h in both the OP and non‐OP OBs, but decreased or was absent through 24 h in OP OBs. These results suggest early adhesion‐mediated events, such as cell adhesion, attachment, and FAK signaling via PY may be altered in OP OBs.

Rotary Culture Enhances Pre-osteoblast Aggregation and Mineralization

Three-dimensional environments have been shown to enhance cell aggregation and osteoblast differentiation. Thus, we hypothesized that three-dimensional (3D) growth environments would enhance the mineralization rate of human embryonic palatal mesenchymal (HEPM) pre-osteoblasts. The objective of this study was to investigate the potential use of rotary cell culture systems (RCCS) as a means to enhance the osteogenic potential of pre-osteoblast cells. HEPM cells were cultured in a RCCS to create 3D enviroments. Tissue culture plastic (2D) cultures served as our control. 3D environments promoted three-dimensional aggregate formations. Increased calcium and phosphorus deposition was significantly enhanced three- to 18-fold (P < 0.001) in 3D cultures as compared with 2D environments. 3D cultures mineralized in 1 wk as compared with the 2D cultures, which took 4 wks, a decrease in time of nearly 75%. In conclusion, our studies demonstrated that 3D environments enhanced osteoblast cell aggregation and mineralization.