FERM control of FAK function: implications for cancer therapy

Abstract

Integrins are transmembrane receptors that bind to extracellular matrix proteins and convey anchorage-dependent signals regulating normal cell proliferation. Integrin signals within the tumor micro-environment also impact cancer cell survival and invasion during tumor progression. These integrin-associated signaling events are transduced in part through the activation of non-receptor protein-tyrosine kinases. Focal adhesion kinase (FAK) is activated by beta-subunit integrins in both normal and transformed cells. As genetic inactivation of beta1 integrin or FAK yield early embryonic lethal phenotypes associated with decreased cell proliferation, and dominant-negative inhibition of FAK can cause increased cell apoptosis, there is a concern that FAK inhibition may have cytotoxic effects on cell growth or survival. However, FAK-specific small molecule inhibitors do not directly impact cell growth in culture, but yet show potent anti-tumor growth effects in vivo. Additionally, recent studies have shed new insight into the FAK kinase-independent regulation of cell proliferation and survival mediated by the FAK N-terminal FERM (band 4.1, ezrin, radixin, moesin homology) domain. Herein, we review the role of the FAK FERM domain in both the intrinsic regulation of FAK kinase activity and how FERM-mediated nuclear localization of FAK promotes enhanced cell survival through the inhibition of tumor suppressor p53 activation during development and under conditions of cellular stress. As we find that FAK FERM-mediated regulation of p53 occurs in human carcinoma cells, elevated FAK expression in tumors may promote both kinase-dependent and -independent survival mechanisms. We discuss how the pharmacological inhibition of FAK kinase activity may impact tumor progression through combined effects of blocking both tumor- and stromal-associated signaling regulating neo-vascularization.

Figure 1

Signaling by integrins through FAK. (A) G-protein-linked, growth factor receptor, and cytokine stimuli activate intracellular signaling cascades and lead to diverse cell responses that require signals from integrin binding to extracellular matrix proteins such as fibronectin (FN). As integrins do not possess intrinsic catalytic activities, the association with and activation of protein-tyrosine kinases (PTKs) such as FAK can link integrins to modulation of cell responses. (B) FAK and Pyk2 contain a central kinase domain flanked by large N- and C-terminal domains with regions of sequence similarity surrounding phosphorylation sites (P) and proline-rich regions (Pro-1 to Pro-3) that are binding sites for Src-homology 3 (SH3) domain-containing proteins such as the adaptor protein, p130Cas. FAK contains 5 major sites of tyrosine phosphorylation (Y397, Y576, Y577, Y861, and Y925) where phosphorylation of either Y397 or Y925 facilitate the binding of Src-homology 2 (SH2) domain containing proteins such as Src kinase and the Grb2 adaptor protein, respectively. The corresponding and conserved phosphorylated tyrosines in Pyk2 are also indicated. The regional amino acid sequence identities between FAK and Pyk2 are indicated with the highest conservation (60%) in the kinase domains. Both proteins contain a FERM (band 4.1, ezrin, radixin, moesin homology) domain in the N-terminal region as well as a FAT (focal adhesion targeting) domain in the C-terminal region that indirectly links FAK to integrins. The FERM and FAT domains of FAK and Pyk2 may have conserved as well as divergent functions, .

Figure 2

FAK FERM regulation of p53 during development and in carcinoma cells. (A) Model of how loss of FAK affects mesenchymal cell survival during development. Depicted are FAK-null (FAK−/−, top) and normal (FAK+/+, bottom) mouse cells where knockout of FAK leads to early embryonic lethality and FAK−/− cells exhibit elevated tumor suppressor p53 activation, leading to enhanced p21(Cip/WAF1) expression, and a p21-dependent block in cell proliferation. Elevated p53 activity in FAK−/− cells inhibits proliferation, but does not directly promote apoptosis. In normal cells, FAK signaling occurs at sites of integrin clustering at the plasma membrane (focal adhesions) as well as FAK translocation to the nucleus upon conditions of reduced adhesion or cellular stress. FAK-mediated control of p53 and cell survival involves FAK FERM-mediated nuclear translocation, p53 binding, and FAK FERM-enhanced murine double minute-2 (Mdm2) ubiquitination leading to p53 proteosomal degradation. This regulatory connection between FAK and p53 is dependent on the FAK FERM domain but is independent of FAK kinase activity. (B) FAK expression was reduced by short-hairpin RNA (shRNA) interference in A549 lung or HCT116 colorectal carcinoma cells and compared to scrambled (Scr) shRNA-expressing controls. FAK knockdown was associated with increased p53 protein levels as determined by anti-FAK, p53, and actin immunoblotting. (C) Recombinant adenovirus was used to over-express the FAK FERM region in HCT116 carcinoma cells and this resulted in decreased p53 levels compared to Mock-infected cells as determined by anti-FAK, Myc-tag for FERM, and p53 immunoblotting.

Figure 3

Small molecule inhibitors of FAK and effects on basic fibroblast growth factor (bFGF) induced angiogenesis. (A) Compounds developed by Novartis (TAC-544 and TAE-226) or Pfizer (PF-228 and PF-562,271) inhibit FAK activity in vitro at low nanomolar concentrations and can reduce FAK Y397 phosphorylation in cells at sub-micromolar concentrations. TAC-544 and PF-228 show specificity toward FAK versus Pyk2 and the dual FAK-Pyk2 inhibitors (TAE-226 and PF-562,271) can act to reduce tumor growth in mouse models , , . (B) Angiogenesis in the chicken chorioallantoic membrane (CAM) assay is stimulated by bFGF addition and inhibited by administration of PF-562,271. Data represent average number of vessel branch points +/− SEM (n= 12). (C) Representative images of chicken CAMs after saline, bFGF, or bFGF plus PF-562,271 addition. Blood vessels are dark with backlit illumination of the CAM. Scale bar is 0.5 mm.