Role of Akt2 and SHC1 proteins in 8-Cl-cAMP-induced cancer cell growth inhibition

Abstract

Cyclic AMP is a secondary messenger which plays a critical role(s) in various cellular physiological functions including regulation of cell growth and death. 8-chloro-cyclic AMP (8-Cl-cAMP) is one of the site-selective cAMP analogs that induce growth inhibition, apoptosis, differentiation and reverse transformation in a broad spectrum of cancer cell lines. Although 8-Cl-cAMP promotes tumor cell-specific growth inhibition and apoptosis, the exact signaling pathways for its action are still uncertain. Many research groups have made an effort to elucidate the signal pathways of 8-Cl-cAMP-induced cancer cell specific growth inhibition and apoptosis. Through several studies, we knew that 8-Cl-cAMP promotes the down-regulation of RI subunit of PKA, the activation of protein kinase C (PKC), Rap1, AMP-activated protein kinase (AMPK) as well as the p38 mitogen activated protein kinase (p38 MAPK) during the growth inhibition or apoptosis of cancer cells. Also, it was confirmed that the conversion of 8-Cl-cAMP to 8-Cl-adenosine is prerequisite for its cell growth inhibitory effects. In this thesis, I tried to show that Akt/protein kinase B (PKB) as well as Src homology 2 domain containing transforming protein 1 (SHC1) proteins were involved in 8-Cl-cAMP-induced cancer cell growth inhibition. Akt/PKB genes encode three isoforms, Akt1, Akt2 and Akt3, which belong to the serine/threonine-specific protein kinase family. Akt/PKB protein has been known for its ability to confer cells to enhance cell survival by inhibiting apoptosis. Accordingly, it is expected that the activation or phosphorylation of Akt/PKB would be decreased upon treatment with 8-Cl-cAMP. Contrary to the expectations, however, the phosphorylation of Akt/PKB was increased by treatment with 8-Cl-cAMP. The increased phosphorylation of Akt/PKB was repressed by treatments of ABT702 (an adenosine kinase inhibitor) and NBTI (an adenosine transporter inhibitor). Furthermore, the 8-Cl-cAMP-induced phosphorylation of Akt/PKB was not attenuated by the treatments of Compound C (an AMPK inhibitor), AMPK-DN (AMPK-dominant negative) mutant and SB203580 (a p38 MAPK inhibitor), whereas TCN (an Akt1/2/3 specific inhibitor) and an Akt2/PKB-targeted siRNA repressed the 8-Cl-cAMP- and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, an AMPK activator)-mediated phosphorylation of AMPK and p38 MAPK. TCN also restored the growth inhibitory effect mediated by 8-Cl-cAMP and AICAR, whereas, the Akt1/PKB-targeted siRNA did not reduce the 8-Cl-cAMP-induced phosphorylation of AMPK and p38 MAPK. The obtained results pointed to the direction that the treatments of 8-Cl-cAMP and AICAR increased the phosphorylation of Akt2/PKBβ, which in turn stimulated the activation of AMPK and p38 MAPK. SHC1 protein is expressed as three isoforms, 46, 52 and 66 kDa, which belong to the adaptor protein containing Src homology 2 (SH2) domains and each isoform has been shown to exert different functions in cellular response. In this thesis, it was shown that the treatment of 8-Cl-cAMP to cancer cells lower the phosphorylation level of SHC1, whereas the total amount of SHC1 protein was not altered. Furthermore, Compound C (an AMPK inhibitor) and SB203580 (a p38 MAPK inhibitor) did not block the 8-Cl-cAMP-induced decrease of SHC1 phosphorylation. However, when SHC1-targeted siRNAs were introduced in order to mimic the decrease of SHC1 phosphorylation, the decreased phosphorylaton and total amount of SHC1 protein seemed to stimulate the phosphorylation of AMPK and p38 MAPK similar to 8-Cl-cAMP treatment. These results suggest that reduced level of SHC1 phosphorylation acted upon the phosphorylation of AMPK and p38 MAPK as an upstream regulation factor during 8-Cl-cAMP-induced growth inhibition.