TOXICOLOGICAL HIGHLIGHT |
Focal Adhesion Kinase as a Potential Target in Arsenic Toxicity
Laboratory of Comparative Carcinogenesis, National Cancer Institute at NIEHS, 111 Alexander Drive, Research Triangle Park, North Carolina, 27709
1 To whom correspondence should be addressed. Fax: (919) 541-3970. E-mail: Liu6{at}niehs.nih.gov.
Received January 28, 2005; accepted February 3, 2005
Arsenic is one of the highest priority hazardous substances around the world, and is well known for its toxicity and carcinogenicity in humans (National Research Council, 1999
). Arsenic trioxide also finds use as a remarkably effective chemotherapeutic agent in the treatment of acute promyelocytic leukaemia (APL), and may find use in the treatment of certain solid tumors (Chen et al., 2002). This paradoxical nature of arsenic as a carcinogenic anti-cancer agent is dependent on the dose and duration of exposure, the status of various diseases, and the complex cellular interactions of this remarkable metalloid (National Research Council, 1999; Qian et al., 2003). In this issue of Toxicological Sciences, Yancy et al. (pp. 1–9) reported the effects of sodium arsenite on focal adhesion in H9C2 myoblasts. Sublethal concentrations of arsenic were shown to decrease cell migration, cell attachment, single cell-spreading area, and the distribution and the number of focal adhesions. The tyrosine phosphorylation of focal adhesion kinase (FAK) and the adhesion-related protein, paxillin, were also decreased, a mechanism critical for the formation of focal adhesions and important for signalling events of arsenic-induced toxicity.
FAK is a non-receptor tyrosine kinase residing at sites of cell attachment to the underlying extracellular matrix. The FAK protein has at least three major functional domains: (1) a focal adhesion targeting (FAT) domain, which is important for localization of FAK to focal adhesions and for binding integrin-associated proteins such as paxillin and talin; (2) a catalytic domain with tyrosine kinase activity; and (3) a N-terminal domain, important for the interaction with integrins and growth factor receptors (Parsons, 2003). When activated, the FAK catalytic domain facilitates autophosphorylation of Tyr-397, which is important for phosphorylation of focal adhesion-associated proteins and kinases. FAK has been shown to play an important role in signal transduction at integrin-linked cellular adhesions, as well as cell motility and invasion (Schlaepfer et al., 2004). FAK activation, demonstrated by an increase in phosphorylation of Tyr397 and subsequent phosphorylation of other proteins such as paxillin, mediates signalling to several downstream pathways (e.g., integrin, Src, Rho, Grb2, EGFR, ERK, cadherins) (Playford and Schaller, 2004; Schlaepfer et al., 2004; Yano et al., 2004). FAK-dependent activation of these pathways has been implicated in a diverse array of physiologic or toxic processes, including cell adhesion, cell migration, cell survival, cell cycle control, carcinogenesis, and tumor cell invasion (McLean et al., 2003; Parsons, 2003; Playford and Schaller, 2004; Schlaepfer et al., 2004). The study of Yancy et al. (2005) is the first to indicate interactions with FAK are a plausible mechanism for at least some aspects of arsenic toxicity.
The highlighted article lucidly describes a series of well-designed studies. The authors used an impressive battery of biochemical techniques to answer critical questions in the step by step manner. To begin with, the dose- and time-dependent cytotoxicity of arsenic in H9C2 cells is characterized using four different assays in order to clearly select the well-tolerated concentrations for the remaining studies. This is an important consideration, as in the in vitro studies with arsenic, all too often look at events occurring at near lethal or supra-lethal concentrations, while the low dose, long-term exposure is the major concern for humans, and data derived using high concentration may have little bearing on the mechanism of arsenic toxicity and carcinogenesis in human populations (Schoen et al., 2004). Yancy et al. (2005) clearly showed sublethal concentrations of sodium arsenite reduced cell migration rates. Cell migration is a complex and dynamic process that is dependent upon serial variety of events including the assembly and disassembly of focal adhesions. The authors found that arsenic reduced focal adhesions and altered their distribution pattern, as determined by immunofluorescence localization of the structural protein vinculin. This observation was further supported by cell attachment measures as arsenic treated cells adhered to the cell culture substrate at a slower rate, and the areas covered by a spread cell were decreased while the cell volume was unchanged. These results clearly demonstrate that sodium arsenite exposure at the sublethal concentrations reduced cell migration in the H9C2 myoblasts, possibly through alterations in focal adhesion function.
The highlighted article further examined the expression of several major focal adhesion proteins, including FAK, paxillin, talin, and vinculin by Western-blot analysis and the total F-actin content by phalloidin-binding assay. At sublethal concentrations, arsenic did not affect the amount of polymerized actin in cells, the rate of protein synthesis, or the amount of these adhesion proteins. However, the authors clearly demonstrated a decrease in tyrosine phosphorylation of FAK by immunoprecipitation with anti-FAK antibody, and further analysis with an anti-phosphotyrosine antibody. The FAK autophosphorylation at the site of tyrosine 397, a key indicator for FAK activation (Parsons, 2003), was also decreased by arsenic using a specific antibody against FAK Tyr397. The decreased Tyr397 phosphorylation of FAK subsequently led to a functional reduction of paxillin phosphorylation. These results demonstrate the importance of protein phosphorylation for signal transduction and in mediating cellular function. Overall, these results demonstrated that sublethal concentrations of arsenic can affect FAK phosphorylation, focal adhesion, and possibly signalling events in the cells.
The results of this study have multiple implications as FAK appears to be a "switch" for diverse signalling events in the cells (Parsons, 2003, Playford and Schaller, 2004; Schlaepfer et al., 2004). FAK has also been implicated in the development of cancer and is important for tumor metastasis (Gabarra-Niecko et al., 2003; Hecker and Gladson, 2003), and FAK is a proposed target for anticancer therapy (McLean et al., 2003). Thus, the effects of arsenic on FAK seen in this study form the basis for several additional studies. For example, FAK-null embryos showed cardiovascular defect (Schlaepfer et al., 2004) and arsenic is known to produce cardiovascular disorders in humans (National Research Council, 1999). Whether the arsenic suppression of FAK in the heart cells observed in this study could impair cardiovascular function in vivo warrants further investigation. In addition, because of the pivotal role of FAK in a number of processes critical to cancer cell progression, and since FAK suppression is known to reduce cell adhesion and to induce anoikis (apoptosis of cell after loss contact), the inhibition of FAK functioning may present therapeutic opportunities (McLean et al., 2003). Arsenic is very effective in the treatment of APL, and may also be effective for certain solid tumors (liver and gallbladder tumors) and tumor cell metastasis (Chen et al., 2002). Thus, whether arsenic could affect FAK in tumor cells as a part of its chemotherapeutic effects warrants further investigation. FAK also plays an important role in cadherin-based cell-cell adhesion (Playford and Schaller, 2004; Yano et al., 2004), and cadherin is known to be a potential target in metal toxicity and carcinogenesis (Prozialeck et al., 2002). The effect of arsenic on FAK and cell adhesion could vary depending on cell models; further investigations using other cell systems and in vivo models are desired. Furthermore, whether arsenic carcinogenesis is associated with alterations in FAK and FAK-related signalling molecules such as Src, Erb2, Ras, EGFR, c-Abl, and integrin, is an open question of future research.
In summary, the highlighted article demonstrated FAK as a potential target of arsenic. These novel findings could stimulate new investigations on arsenic effects on FAK in other cell models and in vivo. Further investigation on arsenic alterations in FAK and FAK-related signalling should provide further insight into the mechanism of arsenic toxicity and carcinogenesis.
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