While the frequency of Ras mutations in human tumors is only 20% overall, constitutive signaling through Ras is a conserved feature of a much higher proportion of human tumors. Mutations giving rise to increased production of growth factors or sustained activation of growth factor receptors are frequent events in human tumors. Over the past year, mutations in a Ras effector, B-Raf, have been identified in several types of human cancer (Davies et al., 2002; Rajagopalan et al., 2002). Together, mutations that give rise to constitutive signaling through Ras-mediated pathways comprise a significant proportion of human tumors. The fundamental role of Ras in tumori-genesis is particularly evident in the thyroid gland where mutations in Ras, B-Raf and RET/PTC have now been identified. Expression of activated Ras in thyroid cells in vitro elicits morphological transformation, sustained proliferation, apoptosis and genomic instability, hallmarks of human tumor cells (Figure 3). Despite the significant advances that have been made in identifying regulators and targets of Ras, these recent advances highlight how little we know regarding the signaling pathways activated by Ras, as well as the consequences of sustained Ras activity in the thyroid cell.
d«differentiation genomic instability proliferation . apoptosis morphologic transformation
Figure 3. Ras elicits multiple effects in thyroid cells.
Overexpression studies have relied extensively on H- and K- rather than N-Ras, which appears to be a more frequent target for mutation in thyroid cancer. With accumulating evidence that individual Ras proteins localize to different cellular microdomains (Prior et al., 2001; Chiu et al., 2002; Matallanas et al., 2003) and signal in distinct ways (Maher et al., 1995; Villalonga et al., 2001; Walsh et al., 2001), it will be important to assess the specific consequences of N-ras activity in thyroid cells. It is interesting that N-Ras provides a survival signal in fibroblasts (Wolfman et al., 2002).
The recent discovery of B-Raf mutations in thyroid tumors paves the way for studies of the contribution of this specific Raf isoform to tumorigenesis. Although cAMP impairs Ras signaling to Raf-1, its effects on B-Raf activity in thyroid cells are largely unknown. Moreover, cAMP itself stimulates several signaling pathways with the potential to regulate B-Raf activity. Thyroid cells are a rich source of Epac, a Rap1-specific GEF (DeRooij et al., 1998; Kawasaki et al., 1998). TSH and cAMP activate endogenous Rap1 in rat (Tsygankova et al., 2001) and canine (Dremier et al., 1997) thyroid cells, effects that are PKA-independent. In other cells, active Rap1 binds to Raf-1 and B-Raf, but with differing consequences. Association between Rap1 and B-Raf stimulates B-Raf activity, whereas Rap-1:Raf-1 complexes are inactive. B-Raf is a substrate for protein kinase A, the other arm of the cAMP signaling pathway. This raises interesting avenues for regulation of B-Raf by cAMP and PKA. The signaling pathways activated by B-Raf in thyroid cells are unknown. While B-Raf binds and activates MEK1/2, it remains to be determined whether this pathway is active in TSH-treated cells.
Rap1 has been linked to TSH effects on differentiated gene expression (Tsygankova et al., 2001) and proliferation (Ribeiro-Neto et al., 2002), although the mechanism through which it elicits these effects is not clear. A limited mutational analysis failed to reveal mutations in Rap 1 or Epac in follicular adenomas (Vanvooren et al., 2001). Nonetheless, the contribution of Rap 1 to thyroid cell biology is important to pursue based on its ability to signal through B-Raf and to affect Ras-mediated signaling. Rap1 was initially isolated as K-rev1, an inhibitor of K-Ras transformation (Kitayama et al., 1989). Although Rap 1 clearly functions in Ras-independent pathways, crosstalk between Ras and Rap 1 has been shown to modulate the ability of Ras to activate discrete effector pathways. Competition between Rap1 and Ras for downstream signaling molecules may provide a mechanism for balancing the activities of these two signaling molecules and for channeling their effects to discrete effector pathways.
The notion that Ras stimulates genomic instability, predisposing thyroid cells to the acquisition of additional mutations, promises to provide further insight into the molecular mechanisms through which Ras contributes to thyroid cell transformation. Thyroid cancer cell lines and tumors have been shown to exhibit mitotic checkpoint dysfunction, however the genetic and/or epigenetic changes responsible for this have not been identified. A recent analysis failed to reveal mutations in the candidate checkpoint genes, BUB1 or BUBR1 (Ouyang et al., 2002). The relationship between tumors harboring mutations in Ras or B-Raf to DNA damage and effects on p53 deserves further attention given genetic evidence for the acquisition of p53 mutations secondary to mutations in N-Ras in thyroid tumors (Asakawa et al., 2002). In experimental models, stable expression of activated Ras has been shown to induce p53 mutations (Chen et al., 1998). The effects of Ras on cell cycle regulatory proteins needs to be examined in more detail given the unusual effects of Ras on these molecules in human and rat thyroid cells, together with the identification of mutations in cyclins and cyclin-dependent kinase inhibitors in thyroid tumors. It is noteworthy that overexpression of cyclins D1 (Lung et al., 2002) and E (Spruck et al., 1999) has also been linked to genomic instability.
The TSH-dependent nature and relative ease with which rat thyroid cells can be manipulated in vitro affords an important cell model for future studies. Transgenic and knock out animal models for discrete Ras and Raf isoforms hold enormous promise for understanding the contributions of these signaling molecules to thyroid tumorigenesis. Whatever is learned from the rodent model systems must be validated in human thyroid cells. With increasing evidence that Ras signals through discrete pathways in rodent versus human cells (Hamad et al., 2002), studies ofthe signal transduction mechanisms and consequences of Ras and B-Rafactivity in human thyroid cells are essential. Finally, while numerous studies have examined the cellular consequences that arise following transient or stable expression of activated Ras, few studies have attempted to model both the primary and secondary adaptive changes that occur in response to sustained Ras activity in the same cells.
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