The tumor suppressor gene Zac1 is essential for the antiproliferative action of somatostatin

Somatostatin is a neuropeptide which inhibits multiple functions, such as, hormone secretion, angiogenesis, cell proliferation and tumor progression. Octreotide is one of the most commonly used somatostatin analogues in the treatment of neuroendocrine tumors. Acting mainly through the somatostatin receptor 2, which belongs to G-protein coupled receptor superfamily, it limits cell growth by inhibiting the proliferative activity of growth factor receptors. Although the events taking place after somatostatin binding to SSTR have been subject of extensive studies, little is known about which transcription factors mediate the antiproliferative action of somatostatin and its analogues. In this study, we used the rat pituitary tumor cell line GH3 to study the effect of octreotide on Zac1 gene expression. ZAC/Zac1 is a tumor suppressor gene which is highly expressed in normal pituitary, mammary, and ovarian glands, but downregulated in pituitary, breast, and ovarian tumors. Octreotide induced Zac1 gene expression, in a concentration dependent manner, 6 hours after stimulation and the effect remained for 24 hours thereafter. Pre-treatment with pertussis toxin or with the tyrosine phosphatase inhibitor orthovanadate completely abolished this effect, indicating involvement of the Giα subunit and a phosphotyrosine phosphatase, respectively. Both phosphotyrosine phosphatases that are involved in somatostatin's signaling, i.e. SHP-1 and SHP-2, are expressed in GH3 cells. Transfection with dominant negative SHP-1 and 2, abolished the effect of octreotide on Zac1 gene expression in cells transfected with dnSHP-1, but not with dnSHP-2. This indicates the involvement of SHP-1. To check the significance of Zac1 in the antiproliferative action of octreotide we knocked-down Zac1 gene expression by RNA interference. Knocking-down Zac1 abolished the effect of octreotide on cell growth, but not on cAMP downregulation, indicating that the loss of the antiproliferative action of octreotide was not due to loss of function of the receptor. We have observed that Zac1 levels increase after treatment with the PI3K inhibitors wortmannin and LY-249002 suggesting that (a) Zac1 lies downstream to PI3K/Akt and (b) octreotide may upregulate Zac1 gene expression by inhibiting this pathway. Indeed, 3 hours treatment with octreotide resulted in decreased PDK1 and Akt phosphorylation levels without affecting basal Akt. Furthermore it dephosphorylated, and therefore activated, GSK3 beta, which regulates cell cycle by activating p53 and inhibiting Cyclin D1. In conclusion, this study highlights the role of Zac1 as an essential mediator of octreotide's antiproliferative signaling downstream to PI3K/Akt.