Lori J. Kornberg

Focal adhesion kinase and its potential involvement in tumor invasion and metastasis

Integrins are cell surface receptors which, in part, mediate the adhesion of cells to the extracelluar matrix. In addition to providing a molecular “glue” essential for tissue organization and survival, integrins are dynamic signaling molecules. Integrins allow normal, nontransformed cells to sense that they are adhered to the extracellular matrix, thus providing a cell survival signal. This signal allows cells to proliferate in the presence of growth factors and in some instances prevents apoptosis. Integrins also mediate cell migration as it occurs in normal processes such as angiogenesis, wound healing, immune system function, and development. Aberrances in the expression and function of integrins contribute to many disease states including cancer.

Focal adhesion kinase expression in oral cancers

Evidence suggests that focal adhesion kinase (FAK) is involved in the pathogenesis of certain cancers. Focal adhesion kinase is overexpressed in invasive and metastatic cancers of the breast, colon, thyroid, and prostate. The objective of this study was to determine the presence and cellular distribution of FAK in oral cancer and determine whether there is a difference in FAK expression in preinvasive and invasive oral cancers.

Gene Expression Profiling in Squamous Cell Carcinoma of the Oral Cavity Shows Abnormalities in Several Signaling Pathways

Objectives/Hypothesis: To examine gene expression profiles in squamous cell carcinoma of the oral cavity (oral SCC) compared with histologically matched normal tissue.

Expression of focal adhesion kinase and phosphorylated focal adhesion kinase in squamous cell carcinoma of the larynx

Objectives: Focal adhesion kinase (FAK) is overexpressed in a variety of human cancers including those derived from the oral cavity. The purpose of this work is to determine the expression patterns of FAK and its activated form, FAK pY397, in squamous cell carcinoma of the larynx and to correlate FAK expression with tumor differentiation and clinical parameters. Study Design: A retrospective study using archival tissue. Methods: Thirty‐five paraffin embedded tissue specimens of laryngeal carcinoma were obtained from the Department of Pathology at the University of Florida College of Medicine. Immunohistochemical staining of the specimens for FAK and activated phospho‐FAK (FAK pY397) was performed. Intensity of staining, distribution of staining, and percentage of cells stained was determined by one pathologist. Results: There was a statistically significant correlation between FAK staining intensity and tumor differentiation. Poorly differentiated tumors stained more intensely than moderately differentiated tumors (P < .001). There was no correlation between FAK pY397 staining and differentiation (P = .163). However, FAK pY397 staining was unexpectedly found in the nuclei of many specimens. FAK was present in the basal layer of cells within nontransformed squamous mucosa derived from tonsillectomy specimens and in blood vessels. A small amount of FAK pY397 was also localized to blood vessels in nontransformed squamous mucosa. Conclusion: FAK and phospho‐FAK are overexpressed in squamous cell carcinoma of the larynx. FAK expression correlates with differentiation. Future investigations will examine the potential of FAK and FAK pY397 expression both as a prognostic indicator and a point of therapeutic inhibition.

Expression of GRP and Its Receptor in Well-differentiated Colon Cancer Cells Correlates with the Presence of Focal Adhesion Kinase Phosphorylated at Tyrosines 397 and 407

Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are not normally expressed by epithelial cells lining the colon but are aberrantly expressed in cancer, where they act as morphogens and regulate tumor cell differentiation. Studies of colon cancer formation in mice genetically incapable of synthesizing GRP-R suggested that this receptors morphogenic properties were mediated via focal adhesion kinase (FAK). We therefore set out to determine the presence of both total and phosphorylated forms of FAK in human colon cancer specimens as a function of tumor cell differentiation and GRP/GRP-R co-expression. Ten colon cancers containing 25 regions of distinct differentiation were randomly selected from our GI Cancer Tumor Bank. All specimens were immunohistochemically probed using antibodies recognizing GRP, GRP-R, total FAK, and FAK specifically phos-phorylated at tyrosine (Y) 397, 407, 576, 577, 861, and 925. Antibody-specific chromogen was determined by quantitative immunohistochemistry (IHC) for each region of defined differentiation. Here we confirm that GRP/GRP-R co-expression is a function of differentiation, with highest levels observed in well-differentiated tumor cells. We also show that the amount of total FAK and of FAK phosphorylated at Y397 and Y407 tightly correlates with differentiation and with the amount of GRP/GRP-R co-expression. These findings are consistent with GRP/GRP-R acting as a morphogen by activating FAK, and suggest that this occurs via phosphorylation of this enzyme at two specific tyrosine residues.

Phosphorylation of focal adhesion kinase tyrosine 397 critically mediates gastrin-releasing peptide's morphogenic properties.

We have proposed that gastrin‐releasing peptide (GRP) and its receptor (GRP‐R) are morphogens that when aberrantly re‐expressed in colon cancer promote tumor cell differentiation and retard metastasis. Because circumstantial evidence suggested that these properties were mediated via focal adhesion kinase (FAK), the purpose of this study was to elucidate the role of GRP‐induced activation of this enzyme on properties fundamental to metastasis including cell attachment, motility, and deformability. To do this, we studied 293 cells, a non‐malignant epithelial cell line that we show expresses GRP and GRPR. To dissect out the role of FAK, 293 cells were modified to inducibly express the dominant negative enzyme FAK‐related non‐kinase (FRNK) under control of a Tet‐On (i.e., doxycycline‐sensitive) promoter. Under serum‐free conditions, GRP acting in an autocrine manner caused FAK to be phosphorylated at Y397; and this could be completely inhibited either by incubating with the specific GRP‐R antagonist D‐Phe6(bombesin) methyl ester, or by upregulating FRNK using doxycycline. To measure cell attachment, we designed a cone‐plate viscometer that recorded the shear stress required to detach cells from their underlying matrix. To assess motility, confluent cells were wounded and behavior assessed by time‐lapse photography. To measure deformability, we recorded the ability of cells to be completely drawn into a micropipette <50% the size of the non‐deformed cell. Control 293 cells adhered more avidly to their underlying matrix, rapidly remodeled wounded tissues without any increase in overall proliferation, and were less distensible than cells treated with antagonist or doxycycline. Thus, these findings suggest that expression of GRP/GRPR in cancer inhibits metastasis by enhancing cell attachment to the matrix, regulating motility in the context of remodeling, and decreasing deformability. J. Cell. Physiol. 199: 77–88, 2004© 2003 Wiley‐Liss, Inc.

Expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases in laryngeal and pharyngeal squamous cell carcinoma: A quantitative analysis

Squamous cell carcinomas of the head and neck are known for their aggressive growth and propensity to metastasize. Invasion is facilitated by matrix metalloproteineases (MMPs). Tissue inhibitors of MMPs (TIMPs) negatively regulate MMP activity. MMP and TIMP expression in head and neck squamous cell carcinomas was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). qRT-PCR allows measurement of several mRNAs from as little as 4 μg of total cellular RNA. We measured MMP-1, MMP-2, MMP-9, and TIMP-1 expression in 8 specimens of primary tumors and adjacent normal tissue. MMP-1 was overexpressed in 6 of 8 tumors, and MMP-9 was over-expressed in 4 of 7 tumors. MMP-2 was expressed in 3 of 8 tumors and 3 of 8 normal samples. TIMP-1 was expressed in all specimens. This work demonstrates that qRT-PCR can be used to examine expression of specific mRNAs in clinical specimens. Therefore this method provides another tool for the molecular analysis of tumors.

Hepatocyte Growth Factor/Scatter Factor Stimulates Mitogenesis and Migration of a Head and Neck Squamous Cell Carcinoma Cell Line

OBJECTIVE: We sought to assess the effect of extracellular matrix and hepatocyte growth factor/scatter factor (HGF/SF) on the growth and motility of cultured squamous cell carcinoma of the head and neck (SCCHN) cells. METHODS: Cultured cells were incubated in the presence of HGF/SF. The effect of HFG/SF on cell growth, motility, and phosphorylation of the signaling proteins FAK and Erk was determined. RESULTS: HGF/SF is both mitogenic and motogenic to the human SCCHN cell line FaDu. Incubation of FaDu cells in the presence of HGF/SF led to a rapid increase in phosphorylation of both FAK and the growth-promoting kinase Erk. HGF/SF-induced phosphorylation of FAK and Erk was observed in both detached and attached SCCHN cells. However, phosphorylation was much greater in attached cells. CONCLUSIONS AND SIGNIFICANCE: The mitogenic and motogenic activities of HGF/SF may contribute to the pathogenesis of SCCHN in vivo.

Focal adhesion kinase in cancer

Focal adhesion kinase (FAK) has received much attention as a transducer of integrin-mediated signals. FAK becomes autophosphorylated on tyrosine 397 (Y397) in response to cell adhesion to the extracellular matrix (ECM) and in response to certain growth factors. Src-family kinases bind to FAK Y397 and mediate the phosphorylation of several additional tyrosine residues located within FAK. This leads to an enhancement of FAK activity and the recruitment of Grb2-SOS to the FAK signalling complex with subsequent activation of the Ras/ERK growth pathway in some cell types. Other signalling molecules such as Crk-associated substrate, Crk and paxillin can physically associate with FAK in response to adhesion to the ECM. FAK also seems to mediate cell migration and cell survival. Evidence obtained from several laboratories demonstrates that FAK is overexpressed in a variety of human cancers. FAK is not a classical oncogene and its overexpression appears to be a relatively late change in tumour progression. Because FAK can positively regulate the growth, migration and survival of cultured cells, it is likely that overexpression of FAK can contribute to the invasive/metastatic phenotype seen in late stage cancers. Therefore, FAK has received much attention as a point of therapeutic intervention in human cancers. The purpose of this article is to review the biology of FAK to date and to determine whether FAK inhibition might provide the much needed ‘magic bullet’ in cancer therapeutics.