Abstract LB-238: Posttranscriptional regulation of rictor by protein kinase D in human colon cancer cells

Abstract Background: mTORC2 phosphorylates Akt at Ser473 thus resulting in Akt activation; knockdown of rictor, a component of mTORC2, blocks Ser473 phosphorylation. Knockdown of rictor leads to growth inhibition and induces apoptosis in colorectal cancer (CRC) cells. However, the molecular mechanism(s) involved in the regulation of rictor expression remains unclear. Protein kinase D (PKD), a serine/threonine kinase family that includes PKD1, PKD2 and PKD3, has been implicated in the regulation of cell proliferation and apoptosis. Recently, PKD3 has been shown to increase Akt phosphorylation at Ser473. The purpose of this study was to investigate the regulation of rictor protein levels by protein kinase D (PKD) in CRC cells. Methods: HT29 and HCT116 CRC cells were treated with MG132, a proteasome inhibitor; cycloheximide (CHX), a protein synthesis inhibitor; Gö6976, a PKC and PKD inhibitor; Gö6983, a PKC inhibitor; or CID755673, a selective PKD inhibitor. Cells were transfected with plasmids encoding PKD1 or PKD2, or infected with an adenovirus construct expressing PKD3. Whole cell lysates were extracted and Western blot analysis performed for rictor protein expression. Total RNA was extracted and real-time RT-PCR was performed for rictor mRNA expression. To investigate whether PKD and rictor are co-localized, PKD and rictor expression was analyzed by immunohistochemistry in human CRCs, liver metastases and corresponding normal mucosa (AccuMax tissue array). Results: MG132 treatment increased rictor protein levels although raptor and mTOR expression was only slightly increased. Moreover, increased Akt Ser473 phosphorylation was observed. Treatment with CHX decreased rictor protein levels in a time-dependent fashion suggesting that rictor is a target of proteasomal degradation in CRC cells. Treatment of HT29 and HCT116 cells with Gö6976, but not Gö6983, decreased rictor protein expression. Consistently, treatment with the selective PKD inhibitor, CID755673, decreased rictor protein expression and inhibited CRC cell growth. Overexpression of PKD1, PKD2 or PKD3 increased rictor protein levels without affecting rictor mRNA expression. In addition, overexpression of PKD3 inhibited rictor degradation suggesting that PKDs are critical for rictor protein expression. Rictor as well as PKD1, PKD2 and PKD3 were up-regulated in primary CRCs and liver metastases compared with normal mucosa, implying a potential role of PKDs upstream of the rictor signaling pathway in the pathogenesis of CRC. Conclusions: Our results demonstrate that PKD post-transcriptionally regulates rictor protein levels in CRC cells. Importantly, these data suggest that PKD-mediated CRC growth is due, at least in part, to an mTORC2-dependent signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-238.