Abstract LB-134: C-terminal PKCδ1 protein cleavage product acts as a tumor suppressor in human non-small cell lung carcinoma cells

Abstract

Abstract PKCδ1 is a member of family proteins of the lipid-activated serine/threonine kinases that have been implicated in a wide range of cellular functions, including apoptosis and cell proliferation. While the function of PKCδ1 in regulating apoptosis still remains controversial, it has been well documented that PKCδ1 can undergoe apoptosis-dependent protein cleavage. However, the function of its protein cleavage product has not been previously characterized. The PKCδ1 protein cleavage has also been frequently observed in our studies during apoptosis induced by various apoptosis stimuli, including proteasome inhibitors. To investigate the involvement of PKCδ1 protein cleavage product in apoptosis, in this study, we cloned 3′ portion of PKCδ1 cDNA, which encodes the predicted C-terminal PKCδ1 protein cleavage fragment starting from 318 asparagine residue to the stop codon of the protein, into pcDNA3.1Zeo(+) vector with Flag tag at its 5′end. The new vector was then named Flag-PKCδ1-Δ318. Subsequently, the Flag-PKCδ1-Δ318 demonstrated its ability to express a 35kd protein in COS7 cells following gene transfection. In human non-small cell lung carcinoma (NSCLC) NCI-H1703 and NCI-H358 cells, we observed that PKCδ1 underwent protein cleavage resulting in a 35kd cleavage product during proteasome inhibitor MG132-induced apoptosis. It was also observed that silencing of PKCδ1 expression by its siRNA mildly enhanced apoptosis induction in both NSCLC cell lines, suggesting that PKCδ1 can minimally inhibit MG132-induced apoptosis in NSCLC cells. However, unexpectedly, transfection with Flag-PKCδ1-Δ318, did not affect MG132-induced apoptosis. Instead, it caused a significant inhibition of cell proliferation as demonstrated by the slowed cell growth and reduced colony formation in both NSCLC cell lines. Moreover, it was found that expression of PKCδ1-Δ318 dramatically reduced in vitro migration and abolished in vivo tumor formation of NCI-H358 cells, strongly suggesting that PKCδ1-Δ318 acts as tumor suppressor. To further investigate the possible mechanisms underlying the tumor suppression function of PKCδ1-Δ318, we tested activity of MAPK, AKT and MLCK, the critical kinases associated with many oncogenic properties of tumor cells. It was then found that the activity of all these kinases were significantly reduced by PKCδ1-Δ318 in NCI-H358 cells, suggesting that the PKCδ1-Δ318 protein, different from its parental protein, exerts its tumor suppression function through inhibiting multiple oncogenic signaling pathways. Therefore, this study suggests that the apoptosis-dependent protein cleavage may represent a new mechanism in regulating tumorigenic activities and PKCδ1-Δ318 may be of great value of being a new cancer gene therapy agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-134. doi:10.1158/1538-7445.AM2011-LB-134