Abstract 5249: Functional importance of IL-24 phosphorylation in regulating molecular signaling pathways associated with cancer cell survival and metastasis.

Abstract

Abstract Purpose of the Study: Interleukin (IL)-24 is a novel tumor suppressor/cytokine that has demonstrated potent anti-cancer activities against human tumors in both, preclinical and clinical studies. However, the importance of IL-24 phosphorylation for its anti-tumor effects remains in question. Therefore the present study was designed to identify the functional importance of IL-24 phosphorylation in mediating its anti-cancer activities. Experimental Procedure: Human H1299 lung tumor cell line was stably transfected with a tetracycline-inducible plasmid vector carrying the IL-24-wild-type (IL-24wt) or -IL-24 with global knock-down of all five phosphorylation sites (IL-24mt) cDNA. Upon addition of doxycycline (Dox; 1ug/ml), cells were induced to express wt or mt IL-24 protein. The functional importance of IL-24 phosphorylation was determined by assessing the Akt/mTOR signaling pathway and the consequence of its inhibition on cell viability, cell migration, and invasion. Results: Expression of IL-24wt but not IL-24mt resulted in inhibition of cell growth and reduced colony formation on soft-agar. Cell migration and invasion studies demonstrated that IL-24wt significantly suppressed the migration rate and invasion of tumor cells across matrigel compared to cells expressing IL-24mt. Confocal microscopy showed IL-24wt protein expression as punctate staining and present in the cytoplasm throughout the cell, whereas IL-24mt protein expression was mainly localized to the perinuclear space, endoplasmic reticulum (ER) and golgi. Reverse-phase protein array (RPPA) assay revealed differences in expression levels of Akt and its downstream targets between H1299 cells expressing IL-24wt and IL-24mt. Validation of RPPA data by Western blotting demonstrated expression of phosphorylated (p)-AKT and its downstream targets such as p-GSK-3β, p-mTOR, p-FOXO1, p-YAP1, and Cyclin D1 were markedly reduced with an increased expression of p27 and p-beta-catenin in H1299 cells expressing IL-24wt compared to H1299 cells expressing IL-24mt. Critical Akt/mTOR signaling pathways and their connected molecular transcriptional factors, which are important for cancer cell survival and metastasis were significantly suppressed in IL-24wt expressing H1299 cell lines thus reducing tumor survival and metastasis. Conclusion: Our results demonstrate for the first time, phosphorylation of IL-24, is functionally important and required for mediating its anticancer activities via inhibiting the Akt/mTOR pathway. Additional molecular studies are underway to identify the specific phosphorylation site (s) required for the IL-24-mediated anticancer activities. Citation Format: Janani Panneerselvam, Manish Shanker, Jiankang Jin, Cynthia Branch, Ranganayaki Muralidharan, Qi Wang, Anupama Munshi, Ramesh Rajagopal. Functional importance of IL-24 phosphorylation in regulating molecular signaling pathways associated with cancer cell survival and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5249. doi:10.1158/1538-7445.AM2013-5249