Rolf J. Craven

Expression of focal adhesion kinase gene and invasive cancer

The focal adhesion kinase (FAK) gene produces a tyrosine kinase that localises to contact points between cells and extracellular matrix. It is believed to be an important signal molecule in cell adhesion. We have isolated a human homologue of the FAK gene from primary sarcomas and looked for FAK mRNA in 49 human tissue samples, including paired normal and neoplastic samples. We found increased levels of FAK in 1 of 8 adenomatous tissues, in 17 of 20 invasive tumours, and in all 15 metastatic tumours. There was no detectable FAK mRNA in 6 normal tissue samples. These observations suggest that FAK overexpression may accompany changes in signal pathways involved in tumour cell invasion.

Focal adhesion kinase as a marker of invasive potential in differentiated human thyroid cancer.

AbstractBackground: The FAK gene encodes a 125-kDa tyrosine kinase (p125FAK) involved in signal transduction pathways used in cell adhesion, motility, and anchorage-independent growth. Because thyroid carcinomas have a wide variability in their propensity for invasion and metastasis, we studied the expression of FAK in a variety of thyroid tissues. Methods: We synthesized a recombinant N-terminal fragment of the human FAK protein and developed a specific polyclonal antisera. Using Western blot analysis, we assessed the levels of p125FAK expression in 30 human thyroid tissue samples from 27 patients that included paired normal and malignant specimens. Levels of FAK protein in individual tumors were quantitated by densitometric scanning of the immunoblots, and the results were correlated with tumor histology and biologic behavior. Results: The levels of FAK expression were directly correlated with thyroid carcinomas demonstrating the most aggressive phenotypes. The highest levels of p125FAK were seen in follicular carcinomas and tumors associated with distant metastatic foci. In contrast, neoplastic thyroid tissues with limited invasive potential, such as papillary carcinomas, follicular adenomas, and other nonmalignant thyroid lesions, showed minimal p125FAK expression. Conclusions: Overexpression of FAK may be part of a mechanism for invasion and metastasis of thyroid cancer. Furthermore, the levels of p125FAK may serve as a marker of biologic behavior in this disease.

The Focal Adhesion Kinase Suppresses Transformation-associated, Anchorage-independent Apoptosis in Human Breast Cancer Cells INVOLVEMENT OF DEATH RECEPTOR-RELATED SIGNALING PATHWAYS

The focal adhesion kinase (FAK) is a mediator of cell-extracellular matrix signaling events and is overexpressed in tumor cells. In order to rapidly down-regulate FAK function in normal and transformed mammary cells, we have used adenoviral gene transduction of the carboxyl-terminal domain of FAK (FAK-CD). Transduction of adenovirus containing FAK-CD in breast cancer cells caused loss of adhesion, degradation of p125FAK, and induced apoptosis. Furthermore, breast tumor cells that were viable without matrix attachment also underwent apoptosis upon interruption of FAK function, demonstrating that FAK is a survival signal in breast tumor cells even in the absence of matrix signaling. In addition, both anchorage-dependent and anchorage-independent apoptotic signaling required Fas-associated death domain and caspase-8, suggesting that a death receptor-mediated apoptotic pathway is involved. Finally, FAK-CD had no effect on adhesion or viability in normal mammary cells, despite the loss of tyrosine phosphorylation of p125FAK. These results indicate that FAK-mediated signaling is required for both cell adhesion and anchorage-independent survival and the disruption of FAK function involves the Fas-associated death domain and caspase-8 apoptotic pathway.

Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor-interacting protein.

ABSTRACT Tumor cells resist the apoptotic stimuli associated with invasion and metastasis by activating survival signals that suppress apoptosis. Focal adhesion kinase (FAK), a tyrosine kinase that is overexpressed in a variety of human tumors, mediates one of these survival signals. Attenuation of FAK expression in tumor cells results in apoptosis that is mediated by caspase 8- and FADD-dependent pathways, suggesting that death receptor pathways are involved in the process. Here, we report a functional link between FAK and death receptors. We have demonstrated that FAK binds to the death domain kinase receptor-interacting protein (RIP). RIP is a major component of the death receptor complex and has been shown to interact with Fas and tumor necrosis factor receptor 1 through its binding to adapter proteins. We have shown that RIP provides proapoptotic signals that are suppressed by its binding to FAK. We thus propose that FAK overexpression in human tumors provides a survival signal function by binding to RIP and inhibiting its interaction with the death receptor complex.

Rak Functions as a Tumor Suppressor by Regulating PTEN Protein Stability and Function

Expression of the PTEN tumor suppressor is frequently lost in breast cancer in the absence of mutation or promoter methylation through as yet undetermined mechanisms. In this study, we demonstrate that the Rak tyrosine kinase physically interacts with PTEN and phosphorylates PTEN on Tyr336. Knockdown of Rak enhanced the binding of PTEN to its E3 ligase NEDD4-1 and promoted PTEN polyubiquitination, leading to PTEN protein degradation. Notably, ectopic expression of Rak effectively suppressed breast cancer cell proliferation, invasion, and colony formation in vitro and tumor growth in vivo. Furthermore, Rak knockdown was sufficient to transform normal mammary epithelial cells. Therefore, Rak acts as a bona fide tumor suppressor gene through the mechanism of regulating PTEN protein stability and function.

PGRMC1 (progesterone receptor membrane component 1): A targetable protein with multiple functions in steroid signaling, P450 activation and drug binding

Hormone signaling is important in a number of disease states, and hormone receptors are effective therapeutic targets. PGRMC1 (progesterone receptor membrane component 1) is a member of a multi-protein complex that binds to progesterone and other steroids, as well as pharmaceutical compounds. In spite of its name, PGRMC1 shares homology with cytochrome b5-related proteins rather than hormone receptors, and heme binding is the sole biochemical activity of PGRMC1. PGRMC1 and its homologues regulate cholesterol synthesis by activating the P450 protein Cyp51/lanosterol demethylase, and the cholesterol synthetic pathway is an important target in cardiovascular disease and in treating infections. PGRMC1 binding partners include multiple P450 proteins, PAIR-BP1, Insig, and an uncharacterized hormone/drug-binding protein. PGRMC1 is induced in a spectrum of cancers, where it promotes cell survival and damage resistance, and PGRMC1 is also expressed in the nervous system and tissues involved in drug metabolism, cholesterol synthesis and hormone synthesis and turnover. One of the appealing features of PGRMC1 and its associated protein complex is its affinity for steroids and drugs. Together with its biological role in promoting tumor survival, PGRMC1 is an attractive target for therapeutic intervention in cancer and related malignancies.

High focal adhesion kinase expression in invasive breast carcinomas is associated with an aggressive phenotype

Focal adhesion kinase (FAK) is a protein tyrosine kinase expressed in invasive breast cancer that regulates antiapoptotic signaling. We have examined FAK expression by immunohistochemistry using anti-FAK 4.47 in breast tumor samples from a large population-based, case–control study of women participating in the University of North Carolina Breast Specialized Programs of Research Excellence (SPORE), Carolina Breast Cancer Study. In this population, 629 formalin-fixed, paraffin-embedded tissue sections were stained for FAK and scored as high (3+ or 4+ intensity and ≥90% positive cells) or otherwise. High FAK expression was associated with poor prognostic indicators including high mitotic index (>10 mitoses per 10 consecutive high-power fields), nuclear grade 3, architectural grade 3, estrogen and progesterone receptor negative, and HER-2/neu overexpressed using CB11 antibody. The association of high FAK expression with HER-2/neu overexpression lends further support that HER-2/neu and FAK collaborate to promote tumorigenesis. The presence of strong FAK expression in many high grade, estrogen- and progesterone-negative breast carcinomas indicates that FAK may be an attractive target for therapeutic intervention.

Expression of growth factor receptors, the focal adhesion kinase, and other tyrosine kinases in human soft tissue tumors

AbstractBackground: The tyrosine kinases are a family of genes that includes many growth factor receptors and protooncogenes. They appear to have a role in many cancers, but have not been systematically studied in the pathogenesis and progression of human sarcomas. Methods: To characterize the protein tyrosine kinases that are expressed in human sarcomas, we used a polymerase chain reaction (PCR)-based method to construct kinase-specific cDNA libraries from low-grade and high-grade primary tumors. Thereafter, individual tyrosine kinase gene expression was assessed in a panel of sarcoma cell lines and primary tumors using Northern blotting and PCR. Results: We identified 19 species of tyrosine kinase genes, including many growth factor receptors, the human homolog of the focal adhesion kinase (FAK) gene, and a noveltrk-related kinase designated HGK2. Messenger RNA expression analyses showed relative overexpression of the two forms of the platelet-derived growth factor receptors (PDGFRs) with expression of the α form restricted to a subgroup of high-grade and metastatic sarcomas. We were unable to demonstrate coexpression of the PDGF isoforms in primary tumors that overexpressed the receptors, suggesting that a PDGF/PDGFR autocrine pathway may not be a central mechanism in the malignant transformation of sarcomas in vivo. FAK expression was observed in a variety of sarcomas, with increased levels in several high-grade and metastatic leiomyosarcomas. Conclusions: When grouped together by histologic cell type and grade, the expression data of the 19 kinases in primary tumors described a greater degree of heterogeneity than is generally appreciated by clinicopathologic classification schemes. This diversity suggests that sarcomas, even those that appear to be clinically similar, arise through a variety of molecular pathways involving tyrosine kinases.

Progesterone Receptor Membrane Component 1 (Pgrmc1): A Heme-1 Domain Protein That Promotes Tumorigenesis and Is Inhibited by a Small Molecule

Tumorigenesis requires the concerted action of multiple pathways, including pathways that stimulate proliferation and increase metabolism. Progesterone receptor membrane component 1 (Pgrmc1) is related to cytochrome b5, binds to heme, and is associated with DNA damage resistance and apoptotic suppression. Pgrmc1 is induced by carcinogens, including dioxin, and is up-regulated in multiple types of cancer. In the present study, we found that Pgrmc1 increased in vivo tumor growth, anchorage-independent growth, and migration. Pgrmc1 also promoted proliferation in the absence of serum in A549 non-small cell lung cancer cells but enhanced proliferation regardless of serum concentration in MDA-MB-468 breast cancer cells. Pgrmc1 promotes cholesterol synthesis and binds to Insig (insulin-induced gene), Scap (sterol regulatory element binding protein cleavage activating protein), and P450 proteins, but Pgrmc1 did not affect cholesterol synthesis in lung cancer cells. Pgrmc1 is also associated with progesterone signaling and plasminogen activator inhibitor (PAI1) RNA binding protein, but neither progesterone activity nor PAI1 transcript levels were altered in Pgrmc1-knockdown lung cancer cells. Pgrmc1 homologues bind to aryl ligands identified in an in silico screen, and we have found that a Pgrmc1 ligand induced cell death in a Pgrmc1-specific manner in multiple breast and lung tumor cell lines. Our data support a role for Pgrmc1 in promoting cancer-associated phenotypes and provide a therapeutic approach for targeting Pgrmc1 with a small molecule in lung and breast cancer.

Upregulation of focal adhesion kinase (FAK) expression in ductal carcinoma in situ (DCIS) is an early event in breast tumorigenesis.

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is overexpressed in a subset of invasive breast cancers. FAK transmits signals that mediate several functions including tumor cell proliferation, migration, adhesion and survival. We used immunohistochemical techniques to assess FAK expression in patients with fibrocystic disease (FCD), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC). Formalin-fixed, paraffin-embedded (FFPE) tissue sections were obtained from 119 patients (12 FCD, 38 ADH, 51 DCIS and 18 IDC). The anti-FAK 4.47 monoclonal antibody was used to detect FAK expression. FAK expression was scored as high (3 or 4 intensity and ≥90 positive cells) or low. The DCIS tissue sections demonstrated high FAK expression in 34/51 (66) of the sections. High FAK expression was demonstrated in 6/18 (33) of the IDC tissue sections and 8/38 (21)of the ADH tissue sections. None (0/12) of the FCD tissues sections stained high for FAK. The pattern of FAK expression in DCIS was significantly higher than ADH (p < 0.0001) and IDC (p =0.02). We conclude that FAK overexpression in preinvasive, DCIS tumors precedes tumor cell invasion or metastasis, suggesting that FAK may function as a survival signal and be an early event in breast tumorigenesis.