Serotonin is hypothesized to play a significant role in the development of carcinoid syndrome, and many treatments for the symptoms inhibit the release of serotonin and other hormones from carcinoid tumors. Our results demonstrate an inverse relationship between PI3K/Akt signaling and serotonin synthesis and secretion from the BON carcinoid cell line. Our in vivo studies further suggest that the secretion of serotonin is decreased in more aggressive carcinoid tumors.
PTEN activity directly correlates with serotonin secretion and synthesis. One of the central hypotheses of cancer biology is that cellular dedifferentiation is concomitant with tumor progression (21
), and deletion of Pten
has been demonstrated to induce the dedifferentiation of cells into a “cancer stem cell” phenotype (22
). By inhibiting the tumorigenic activity of PI3K/Akt signaling, PTEN may also contribute to the maintenance of the differentiated, secretory state of BON cells. One of the markers of the differentiated carcinoid phenotype TPH1, the rate-limiting enzyme in the synthesis of serotonin, is expressed in the enterochromaffin cells of the gastrointestinal tract (18
), from which carcinoid tumors arise. Overactivation of the Notch signaling pathway, known to inhibit endocrine differentiation, represses TPH1 expression in BON cells (23
). Furthermore, TPH expression is restricted to cells whose distinct function is serotonin synthesis, and in serotonergic neurons, its expression is regulated by several transcription factors (24
). Gata3, a transcription factor that regulates TPH expression, is downregulated, while PI3K/Akt signaling is enhanced, in metastatic breast cancers (26
). Interestingly, we found that inhibition of Akt1 had no effect on serotonin secretion, while inhibition of Akt2 increased serotonin secretion from BON cells. While the role of Akt2 in cancer progression and metastasis is well-established, its role in secretion is less well understood, and further investigation is warranted. Similar to our results of Akt2-mediated inhibition of BON cell serotonin secretion, Akt2 was recently shown to be a suppressor of gastric acid secretion (27
We demonstrated that molecular inhibition of PTEN concomitantly reduced TPH1 expression, while pharmacological inhibition of PTEN decreased serotonin secretion but not its synthesis. A possible explanation is that pharmacological inhibition of PTEN only transiently increases PI3K/Akt signaling, due to degradation of the inhibitor, thereby affecting secretion but not gene transcription. Consistent with our in vitro
studies, mice with tumors derived from BON shPTEN cells secreted less serotonin and excreted less 5-HIAA than mice with tumors derived from BON shControl cells. Interestingly, the expression of TPH1 in primary tumors derived from BON shControl and BON shPTEN cells was similar. However, we observed that metastatic tumors had decreased expression of TPH1 compared to primary tumors, suggesting that as carcinoid cells adopt a more metastatic phenotype there is a concurrent loss in the secretory phenotype. This can be explained by the complexity of the regulation of serotonin synthesis and secretion in cancer cells. It has been demonstrated in breast cancers that serotonin has tumor suppressive and tumor promoting actions and that the expression of TPH1 has a nonlinear association with tumor stage (29
). As breast tumors grow, they lose expression of TPH1, followed by a regain in TPH1 expression as the tumors become invasive (29
). The hypothesis for this phenomenon is that early on in breast tumor development, TPH1 expression is reduced so that tumors can evade the tumor-suppressive functions of serotonin, such as growth inhibition and apoptosis, and maintain a proliferative state. Since serotonin has also been demonstrated to promote epithelial-to-mesenchymal transition (EMT) (30
), the increase in TPH1 expression as tumors expand is proposed to be a marker of increased metastatic capability (29
). We noted previously that carcinoid tumors with reduced PTEN expression have an increased metastatic potential (16
). Thus, in the more aggressive BON shPTEN primary tumors, it is possible that during the first weeks of tumor growth, the expression of TPH1, and, hence, secretion of serotonin, was much lower than at the time of sacrifice and more comparable to our in vitro
results, shown in . At the time of sacrifice, cells from the BON shPTEN tumors are becoming more invasive and may have increased the expression of TPH1. After tumor cells migrate to a distant site, they undergo mesenchymal-to-epithelial transition (MET) to form a micrometastasis resembling the primary tumor (32
). Thus, the decreased expression of TPH1 in the carcinoid liver metastases may be associated with the resurgence of a proliferative, non-invasive phenotype.
While carcinoid tumors generally maintain a differentiated phenotype, the carcinoid syndrome does not present until liver metastases have developed and the hormone products secreted by the cancer cells are able to bypass degradation in the liver. Paradoxically, we observed that metastatic tumors in the liver had decreased expression of TPH1. A possibility for this paradox is that the secretory products responsible for carcinoid syndrome are actually secreted from the primary tumor. Carcinoid tumors are highly vascular (33
) and upon angiogenesis may receive blood from the systemic, in addition to the portal circulation, enabling the secreted products to circulate throughout the body without being degraded in the liver.
In summary, we have identified a unique relationship between PI3K/Akt/PTEN signaling and the secretion and synthesis of serotonin in the BON carcinoid cell line. Furthermore, the expression of TPH1 and secretion of serotonin from carcinoid cells is directly related to the activity of PTEN. With the evolving complexity of PI3K/Akt/PTEN signaling, we have identified a novel function for this cell signaling pathway, which may be utilized in the development of new treatments for carcinoid disease and the carcinoid syndrome. Our findings suggest the need for a multi-targeted approach in treating carcinoid tumors, as targeting the PI3K/Akt pathway may inhibit the growth of the tumor but also results in enhanced secretion of serotonin and other peptide products which may have deleterious clinical effects.