DCLK1 and DNA Damage Response

Genome integrity is constantly monitored by sophisticated cellular networks, collectively termed as the DNA damage response (DDR). The DDR is a signaling network that includes cell cycle checkpoints and DNA repair and damage tolerance pathways. Failure of the DDR or associated events causes various diseases, including cancer. DDR is primarily mediated by phosphatidylinositol-3-kinase-like protein kinase (PIKKs) family members ataxia-telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR). However, one of the many unanswered questions regarding these signal-transduction pathways is: how does the cell turn the DDR signals on? There was no conclusive demonstration of the involvement of a specific sensory kinase in DDR signals until our recent research on the DCLK1 role in regulating ATM after genotoxic injury. Currently, various studies are demonstrating the importance of DCLK1 in DNA damage response. Here, we discuss the novel insights into the role of DCLK1 in DNA damage response

IL-24 Inhibits lung cancer cell migration and invasion by disrupting the SDF-1/CXCR4 signaling axis

© 2015 Panneerselvam et al. Background The stromal cell derived factor (SDF)-1/chemokine receptor (CXCR)-4 signaling pathway plays a key role in lung cancer metastasis and is molecular target for therapy. In the present study we investigated whether interleukin (IL)-24 can inhibit the SDF-1/CXCR4 axis and suppress lung cancer cell migration and invasion in vitro. Further, the efficacy of IL-24 in combination with CXCR4 antagonists was investigated. Methods Human H1299, A549, H460 and HCC827 lung cancer cell lines were used in the present study. The H1299 lung cancer cell line was stably transfected with doxycycline-inducible plasmid expression vector carrying the human IL-24 cDNA and used in the present study to determine the inhibitory effects of IL-24 on SDF-1/CXCR4 axis. H1299 and A549 cell lines w ere used in transient transfection studies. The inhibitory effects of IL-24 on SDF1/CXCR4 and its downstream targets were analyzed by quantitative RT-PCR, western blot, luciferase reporter assay, flow cytometry and immunocytochemistry. Functional studies included cell migration and invasion assays. Principal Findings Endogenous CXCR4 protein expression levels varied among the four human lung cancer cell lines. Doxycycline-induced IL-24 expression in the H1299-IL24 cell line resulted in reduced CXCR4 mRNA and protein expression. IL-24 post-transcriptionally regulated CXCR4 mRNA expression by decreasing the half-life of CXCR4 mRNA ( > 40%). Functional studies showed IL-24 inhibited tumor cell migration and invasion concomitant with reduction in CXCR4 and its downstream targets (pAKTS 473 , pmTORS 2448 , pPRAS40 T246 and HIF-1α). Additionally, IL-24 inhibited tumor cell migration both in the presence and absence of the CXCR4 agonist, SDF-1. Finally, IL-24 when combined with CXCR4 inhibitors (AMD3100, SJA5) or with CXCR4 siRNA demonstrated enhanced inhibitory activity on tumor cell migration. Conclusions IL-24 disrupts the SDF-1/CXCR4 signaling pathway and inhibits lung tumor cell migration and invasion. Additionally, IL-24, when combined with CXCR4 inhibitors exhibited enhanced anti-metastatic activity and is an attractive therapeutic strategy for lung metastasi

IL-24 Inhibits Lung Cancer Growth by Suppressing GLI1 and Inducing DNA Damage

Aberrant expression of GLI1 is responsible for aggressive tumor behavior and survival due to its effects on the DNA damage response (DDR). We investigated whether interleukin (IL)-24, a tumor suppressor, inhibits GLI1 and the associated DDR pathway in human NSCLCs. IL-24 treatment reduces mRNA and protein expression of GLI1 in lung tumor cells, but not in normal cells. GLI1 reporter assay and mRNA studies demonstrated that IL-24 regulates GLI1 at the post-transcriptional level by favoring mRNA degradation. Associated with GLI1 inhibition was marked suppression of the ATM-mediated DDR pathway resulting in increased DNA damage, as evidenced by γ-H2AX foci and Comet assay. Furthermore, attenuation of GLI1-associated DDR by IL-24 increased caspase-3 and PARP activity, resulting in cancer cell apoptosis. GLI1 inhibition and overexpression confirmed that IL-24-mediated anti-tumor effects involved the GLI-dependent pathway. Finally, we observed that IL-24-mediated alteration in GLI1 is independent of the canonical hedgehog-signaling pathway. Our study provides evidence that IL-24 treatment induces DNA damage, and reduces GLI1 expression and offers an opportunity for testing IL-24-based therapy for inhibiting GLI1 in lung cancer

Inflammatory Mediators and Gut Microbial Toxins Drive Colon Tumorigenesis by IL-23 Dependent Mechanism

Obesity-associated chronic inflammation predisposes colon cancer risk development. Interleukin-23 (IL-23) is a potential inflammatory mediator linking obesity to chronic colonic inflammation, altered gut microbiome, and colon carcinogenesis. We aimed to elucidate the role of pro-inflammatory eicosanoids and gut bacterial toxins in priming dendritic cells and macrophages for IL-23 secretion to promote colon tumor progression. To investigate the association of IL-23 with obesity and colon tumorigenesis, we utilized TCGA data set and colonic tumors from humans and preclinical models. To understand IL-23 production by inflammatory mediators and gut microbial toxins, we performed several in vitro mechanistic studies to mimic the tumor microenvironment. Colonic tumors were utilized to perform the ex vivo experiments. Our findings showed that IL-23 is elevated in obese individuals, colonic tumors and correlated with reduced disease-free survival. In vitro studies showed that IL-23 treatment increased the colon tumor cell self-renewal, migration, and invasion while disrupting epithelial barrier permeability. Co-culture experiments of educated dendritic cells/macrophages with colon cancer cells significantly increased the tumor aggression by increasing the secretory levels of IL-23, and these observations are further supported by ex vivo rat colonic tumor organotypic experiments. Our results demonstrate gut microbe toxins and eicosanoids facilitate IL-23 production, which plays an important role in obesity-associated colonic tumor progression. This newly identified nexus represents a potential target for the prevention and treatment of obesity-associated colon cancer

Targeting IL-23 for the interception of obesity-associated colorectal cancer

Inflammation and obesity are two major factors that promote Colorectal cancer (CRC). Our recent data suggests that interleukin (IL)-23, is significantly elevated in CRC tumors and correlates with patient obesity, tumor grade and survival. Thus, we hypothesize that obesity and CRC may be linked via inflammation and IL-23 may be a potential target for intervention in high-risk patients. TCGA dataset and patient sera were evaluated for IL-23A levels. IL-23A [IL-23 p19−/−] knockout (KO) mice were crossed to Apcmin/+ mice and progeny were fed low-fat or high-fat diets. At termination intestines were evaluated for tumorigenesis. Tumors, serum, and fecal contents were analyzed for protein biomarkers, cytokines, and microbiome profile respectively. IL-23A levels are elevated in the sera of patients with obesity and colon tumors. Genetic ablation of IL-23A significantly suppressed colonic tumor multiplicity (76–96 %) and incidence (72–95 %) in male and female mice. Similarly, small-intestinal tumor multiplicity and size were also significantly reduced in IL-23A KO mice. IL-23A knockdown in Apcmin/+ mice fed high-fat diet, also resulted in significant suppression of colonic (50–58 %) and SI (41–48 %) tumor multiplicity. Cytokine profiling showed reduction in several circulating pro-inflammatory cytokines including loss of IL-23A. Biomarker analysis suggested reduced tumor cell proliferation and immune modulation with an increase in tumor-infiltrating CD4+ and CD8+ T-lymphocytes in the IL-23A KO mice compared to controls. Fecal microbiome analysis revealed potentially beneficial changes in the bacterial population profile. In summary, our data indicates a tumor promoting role for IL-23 in CRC including diet-induced obesity. With several IL-23 targeted therapies in clinical trials, there is a great potential for targeting this cytokine for CRC prevention and therapy

siRNA mediated CXCR4 inhibition in combination with IL-24 produced greater inhibitory activity on tumor cell migration.

<p><b><i>A</i>,</b> siRNA-mediated CXCR4 knockdown when combined with IL-24 resulted in a significant suppression of SDF-1 induced tumor cell migration compared to cell migration in the control group (<i>P</i><0.05). Inhibitory effects on tumor cell migration mediated by IL-24 treatment alone and CXCR4 siRNA treatment alone were also significant when compared to control group. <b><i>B</i>,</b> Western blotting showed combination of CXCR4 siRNA and IL-24 produced greater reduction in the expression of CXCR4 and pAKT^S473 proteins when compared to all other groups. pPRAS40^T246 protein expression was reduced in all treatment groups when compared to control. However, its expression was slightly higher in the combination treatment group when compared to individual treatments. Beta actin was used as protein loading control. Differences in the expression of the proteins was determined by semi-quantitative analysis and represented in graphical format (<i>P</i><0.05). Bars denote standard deviation (SD).</p

IL-24 regulated CXCR4 at post-transcriptional level.

<p><b><i>A</i>,</b> RT-PCR analysis showed IL-24 reduced CXCR4 mRNA levels at 6 h and 24 h (<i>P</i><0.05). <b><i>B</i>,</b> CXCR4 promoter activity was determined using a luciferase reporter vector. Induction of IL-24 showed no significant reduction in luciferase activity indicating IL-24 did not affect the CXCR4 promoter. <b><i>C</i>,</b> mRNA stability studies showed IL-24 reduced the half-life of CXCR4 mRNA at approximately 4 h. Bars denote standard deviation (SD).</p

IL-24 suppresses SDF-1/CXCR4 signaling and tumor cell migration.

<p><b><i>A</i>,</b> IL-24 significantly inhibited tumor cell migration in the presence and absence of SDF-1. The inhibitory activity in the presence of SDF-1 however was less than that observed in the absence of SDF-1 (<i>P</i><0.05). Error bars denote standard deviation. <b><i>B</i>,</b> Induction of IL-24 protein expression in H1299-IL24 cells resulted in marked reduction in pAKT^S473 and pPRAS40^T246 protein expression at 24 h after doxycycline treatment. Beta actin was used as protein loading control. Differences in the expression of the proteins was determined by semi-quantitative analysis and represented in graphical format (<i>P</i><0.05). Bars denote standard deviation (SD).</p