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Phosphorylation of interleukin (IL)-24 is required for mediating its anti-cancer activity.

ABSTRACT: Interleukin (IL)-24 is a tumor suppressor/cytokine gene that undergoes post-translational modifications (PTMs). Glycosylation and ubiquitination are important for IL-24 protein stabilization and degradation respectively. Little is known about IL-24 protein phosphorylation and its role in IL-24-mediated anti-tumor activities. In this study we conducted molecular studies to determine whether IL-24 phosphorylation is important for IL-24-mediated anti-cancer activity.Human H1299 lung tumor cell line that was stably transfected with a doxycycline (DOX)-inducible (Tet-on) plasmid vector carrying the cDNA of IL-24-wild-type (IL-24wt) or IL-24 with all five phosphorylation sites replaced (IL-24mt) was used in the present study. Inhibition of tumor cell proliferation, cell migration and invasion, and induction of G2/M cell cycle arrest was observed in DOX-induced IL-24wt-expressing cells but not in IL-24mt-expressing cells. Secretion of IL-24mt protein was greatly reduced compared to IL-24wt protein. Further, IL-24wt and IL-24mt proteins markedly differed in their subcellular organelle localization. IL-24wt but not IL-24mt inhibited the AKT/mTOR signaling pathway. SiRNA-mediated AKT knockdown and overexpression of myristolyated AKT protein confirmed that IL-24wt but not IL-24mt mediated its anti-cancer activity by inhibiting the AKT signaling pathway.Our results demonstrate that IL-24 phosphorylation is required for inhibiting the AKT/mTOR signaling pathway and exerting its anti-cancer activities.

SUBMITTER: Panneerselvam J

PROVIDER: S-EPMC4599269 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Phosphorylation of interleukin (IL)-24 is required for mediating its anti-cancer activity.

Panneerselvam Janani J Shanker Manish M Jin Jiankang J Branch Cynthia D CD Muralidharan Ranganayaki R Zhao Yan D YD Chada Sunil S Munshi Anupama A Ramesh Rajagopal R

Oncotarget 20150601 18

Interleukin (IL)-24 is a tumor suppressor/cytokine gene that undergoes post-translational modifications (PTMs). Glycosylation and ubiquitination are important for IL-24 protein stabilization and degradation respectively. Little is known about IL-24 protein phosphorylation and its role in IL-24-mediated anti-tumor activities. In this study we conducted molecular studies to determine whether IL-24 phosphorylation is important for IL-24-mediated anti-cancer activity.Human H1299 lung tumor cell line ...[more]

PMID: 26009991

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Project description:BackgroundTransglutamiase-4 (TGase-4), also known as prostate transglutaminase, belongs to the TGase family and is uniquely expressed in the prostate gland. The functions of this interesting protein are not clearly defined. In the present study, we have investigated an unexpected link between TGase-4 and the melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24), a cytokine known to regulate the growth and apoptosis of certain cancer and immune cells.MethodsFrozen sections of normal and malignant human prostate tissues and human prostate cancer (PCa) cell lines PC-3 and CA-HPV-10, cell lines expressing low and high levels of TGase-4, and recombinant MDA-7/IL-24 (rhMDA-7/IL-24) were used. Expression construct for human TGase-4 was generated using a mammalian expression vector with full length human TGase-4 isolated from normal human prostate tissues. PC-3 cells were transfected with expression construct or control plasmid. Stably transfected cells for control transfection and TGase-4 over expression were created. Similarly, expression of TGase-4 in CA-HPV-10 cells were knocked down by way of ribozyme transgenes. Single and double immunofluorescence microscopy was used for localization and co-localization of TGase-4 and MDA-7/IL-24 in PCa tissues and cells with antibodies to TGase-4; MDA-7/IL-24; IL-20alpha; IL-20beta and IL-22R. Cell-matrix adhesion, attachment and migration were by electric cell substrate impedance sensing and growth by in vitro cell growth assay. A panel of small molecule inhibitors, including Akt, was used to determine signal pathways involving TGase-4 and MDA-7/IL-24.ResultsWe initially noted that MDA-7 resulted in inhibition of cell adhesion, growth and migration of human PCa PC-3 cells which did not express TGase-4. However, after the cells over-expressed TGase-4 by way of transfection, the TGase-4 expressing cells lost their adhesion, growth and migratory inhibitory response to MDA-7. On the other hand, CA-HPV-10 cells, a cell type naturally expressing high levels of TGase-4, had a contrasting response to MDA-7 when compared with PC-3 cells. Inhibitor to Akt reversed the inhibitory effect of MDA-7, only in PC-3 control cells, but not the TGase-4 expressing PC-3 cells. In human prostate tissues, TGase-4 was found to have a good degree of co-localization with one of the MDA-7 receptor complexes, IL-20Ra.ConclusionThe presence of TGase-4 has a biological impact on a prostate cancer cell's response to MDA-7. TGase-4, via mechanism(s) yet to be identified, blocked the action of MDA-7 in prostate cancer cells. This has an important implication when considering the use of MDA-7 as a potential anticancer cytokine in prostate cancer therapies.

Project description:Interleukin (IL)-24 plays a potential anti-tumor activity in colorectal cancer in a dose-dependent manner. However, the immunoregulatory role of IL-24 to peripheral and tumor-infiltrating T cell function in colorectal cancer was not fully elucidated. In this study, twenty-nine colorectal adenocarcinoma patients and fifteen healthy individuals were enrolled. IL-24 expression and IL-24 receptor (IL-20R1, IL-20R2, and IL-22R1) mRNA relative level was measured by ELISA and real-time PCR, respectively. CD4+ and CD8+ T cells were purified from peripheral bloods and cancer specimens, and were stimulated with low (10 ng/ml) and high (100 ng/ml) concentration of recombinant IL-24. CD4+ T cells activity was assessed by measurement of Th cell percentage, transcriptional factors, and cytokine production. CD8+ T cells activity was evaluated by investigation of cytotoxic molecules, target cell death, and interferon-? (IFN-?) secretion. IL-24 was decreasingly expressed in both peripheral bloods and cancer tissues in colorectal adenocarcinoma patients. However, IL-20R1 and IL-20R2 was comparable between healthy controls and colorectal adenocarcinoma patients. Low concentration of IL-24 suppressed CD4+ T cell proliferation. In contrast, high concentration of IL-24 not only promoted CD4+ T cell proliferation, but also enhanced CD4+ T cell activity, which mainly presented as up-regulation of Th1/Th17 frequency, T-bet/ROR?t mRNA, and IFN-?/IL-17 production but down-regulation of Treg percentage, FoxP3 mRNA, and IL-10/IL-35 secretion. Moreover, high concentration of IL-24 also increased perforin and granzyme B expression in CD8+ T cells, and elevated cytolytic and non-cytolytic activity of CD8+ T cells, which presented as induction of target cell death and elevation of IFN-? expression. However, low concentration of IL-24 did not affect bioactivity of CD8+ T cells. The current data indicated that IL-24 might regulate T cell function in a dose-dependent manner. High-concentration of IL-24 might promote anti-tumor immune responses in development novel therapeutic approaches to colorectal adenocarcinoma.

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Phosphorylation of interleukin (IL)-24 is required for mediating its anti-cancer activity.

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Phosphorylation of interleukin (IL)-24 is required for mediating its anti-cancer activity.

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