3.1.3. Targeting Downstream Effectors of cAMP

Because of the various effects of cAMP in physiological responses, therapies targeting cAMP signaling result in side effects; thus, understanding downstream targets of cAMP signaling has been attempted in a number of studies [66,67]. The roles of Rap1 and Epac1 in *Prkar1a*-associated thyroid carcinogenesis have been studied [68]. *Rap1* is a small GTPase essential for effective signal transduction. There are two isoforms and each one is encoded by a separate gene, *Rap1a* and *Rap1b*, respectively. The activity of Rap1 has been shown to be regulated by both PKA and cAMP though signaling by TSH [69]. Increased Rap activity has been linked to various cancers, including thyroid cancer, while dysregulation of Rap1 has been postulated to contribute to the development of malignancy [70–74]. Epac (Exchange protein directly activated by cAMP) proteins are intracellular sensors for cAMP and mediate its effects to activate Rap1 [75,76]. The two isoforms include Epac1 which is ubiquitously expressed, with particularly high levels in the thyroid, among other tissues, and Epac2 which is not detected in the thyroid; however, it is expressed in a limited number of other tissues [76,77]. Epac regulates Rap activity in concert with and independently of PKA, and the effects—either stimulatory or inhibitory—seem to depend on the cellular context and the type of stimuli [69,75,78,79]. In addition, it has been shown that Epac1 plays a role in cell migration and invasion in other types of cancer [78,80]. Loss of *Rap*, specifically of the Rap1b isoform, in *Prkar1a* KO thyroids in the setting of overactivation of the PKA pathway, resulted in reduced risk of developing thyroid cancer by 65%; this occurred independently of Epac1 as its deletion did not have any effect in PKA-Rap1 associated thyroid tumorigenesis, underlying the essential role of PKA-Rap1 signaling in the development of FTC [68]. However, even though tumor suppression happened to a significant extent, the carcinogenic phenotype was not completely rescued, which led to the speculation that more complex signaling interactions may be involved [68].

These findings were further supported by other studies that showed that Rap proteins can be directly regulated by PKA using a specific phosphorylation site at serine 180 on Rap1a and serine 179 on Rap1b [81]. When PKA phosphorylates Rap, it regulates its subcellular localization, and its downstream effectors such as ERK and Rap-dependent regulation of cell migration [82,83]. These previous studies indicate that PKA can control Rap action and downstream cellular processes directly suggesting that PKA-Rap1 pathway is independent of Epac1 in thyroid cancer. On the other hand, previous studies have shown, that both PKA and Epac signal to Rap1 downstream of TSH [69,75], but it seems to be tissue-dependent [78,79].

In combination, these studies demonstrated that cAMP or PKA signaling or both play an important role in tumor development and that additional factors may contribute to *Prkar1a* haploinsufficiency in causing those tumors. *Trp53+/*− mice and other animal models for diseases like CNC, including Peutz–Jeghers and neurofibromatosis type 1, did not exhibit the same phenotype as in humans; it only occurred when one or more tumor suppressor genes were knocked out as well [84–86]. *Prkar1a* haploinsufficiency in addition to either *Trp53* or *Rb1* haploinsufficiency resulted in more tumors and decreased survival compared to *Trp53+/*− or *Rb1+/*− mice [87]. Specifically, *Rb1+/*− *Prkar1a+/*− mice developed more MTCs than *Rb1+/*− mice [87].
