IL-24 AND ITS ROLE IN WOUND HEALING
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1 IL-24 AND ITS ROLE IN WOUND HEALING Nancy J. Poindexter, Ryan Williams, Garth Powis, Sunil Chada, and Elizabeth A. Grimm & Introgen Therapeutics, Inc., Houston, TX
2 IL-24/MDA 24/MDA-77 is a Tumor Suppressor mda-7 gene transfer inhibits growth of a broad spectrum of tumor cells while not affecting normal cells Ad-mda7 induces apoptosis in tumor cells and the mechanism of apoptosis is dependent on the cell type Ad-mda7 blocks tumor cell growth in vitro and in vivo through G2/M cell cycle blockade Ad-mda7 is currently being tested in Phase II clinical trial. Tumor suppressor functions have been duplicated in vitro and in mouse models with the pure protein.
3 Signaling pathways of IL-24/MDA 24/MDA-77 induced apoptosis LEGEND Ad-IL-24/mda7 IL-20R2 adhesion metastasis IL-22R1 IL-20R1 Secreted IL-24/MDA-7 Caspase 12 PKR UPR stress response PERK [Ca 2+ ] release Nucleus eif2α Bcl family STAT3 IL-20R Type I? NFκB? BAX inos IL-20R Type II Effector Caspases mitochondria APOPTOSIS NFκB? STAT3 G 1 arrest TUMOR CELLS OTHER? APOPTOSIS NO CELL DEATH NORMAL CELLS MELANOCYTES KERATINOCYTE IMMUNE CELLS
4 IL-24 Expression and Function in Normal Cells IL-24 gene maps to chromosome 1q32 in the IL-10 family cytokine cluster. It shares 19% amino acid identity with IL-10 as well as similar homology with other IL-10 family cytokines, IL-19, IL-20, IL-22, and IL-26. IL-24 is expressed and secreted by in cytokine activated monocytes. IL-24 is constituitively expressed by melanocytes in the skin. IL-24 protein stimulates the secretion of secondary cytokines from peripheral blood mononuclear cells. Regulation of expression in monocytes is at the posttranscriptional level with cytokine stimulation stabilizing IL-24 mrna
5 IL-24 in the Skin Is IL-24 involved in inflammatory responses in the skin? What cells express IL-24? What is the function of IL-24 in skin inflammation? How does this relate to melanoma development
6 Wound Healing hour: wound area filled with a blood clot with neutrophils 2-7 days: Blood vessel formation in the clot, lots of macrophages with keratinocytes proliferating at the wound edge 1-2 weeks: wound contraction and collagen deposition
7 Wounding Experiment n=4 Biopsy #1 Day 2 Biopsy #2 Day 6 Biopsy #3 Day 10
8 IL-24 Expression During Wound Repair Day 2 Day 6 Day 10
9 anti CD68 anti IL-24 anti Keratin HMB45
10 IL-24 Expression during Wound Healing Patient Day 2 Day 6 Day % cells staining positive % +++ >75%
11 IL-24 in the Skin Is IL-24 involved in inflammatory responses in the skin? What stimulates the expression of IL-24? What is the function of IL-24 in skin inflammation?
12 Signaling Pathways in Keratinocytes A. IL-1β activation of keratinocytes B. TNFα maintains activated state C. Growth factor ligation of EGFR results in proliferation and migration D. IFNγ contractions of newly formed extracellular matrix and stops proliferation of keratinocytes
13 Cytokine Induced Expression of IL-24 in Keratinocytes 4.00 IL-24 Expression Media IL-1beta TNF alpha TGFalpha GM-CSF IFN gamma TGF beta LPS
14 IL-24 in the Skin Is IL-24 involved in inflammatory responses in the skin? What cells express IL-24 and its receptors? What is the function of IL-24 in skin inflammation?
15 in vitro Wounding Assay Scratch Test Grow confluent monolayer of keratinocytes (NHEK) +/- Mitomycin C Wash well Scratch monolayer Incubate 18 + hr Measure relative growth = width of scratch at 0hr - width at 18 hr b a a-b=growth Koivisto L et al. HaCaT keratinocyte migration is dependent on EGFR signaling. Exp Cell Research 2006
16 NHEK in vitro Wounding Assay TGF alpha 100 ng/ml + TGF alpha 50 ng/ml TGF alpha 100 ng/ml + TGF alpha 50 ng/ml +IL ng/ml
17 Effect of IL-24 on TGFα stimulated Proliferation and Migration Media + IL-24 Growth and Migration * * * * 0 TGFalpha 0 TGFalpha + Mitomycin C *p<0.05 (media vs IL-24)
18 Effect of IL-24 on Keratinocytes Proliferation 3 Stimulation Index ** Media IL-24 TGF 5 ng/ml 25 ng/ml 100 ng/ml **p<0.05 IL-24 & TGFα 50 ng/ml
19 Effect of TGFα on NHEK expressing IL-24 Cell number x 10e NHEK NHEK-mock NHEK-IL TGF alpha All cells were > 90% 48 hr
20 Conclusions IL-24 is expressed during the later stages of wound healing in proliferating keratinocytes Cytokines involved in wound healing including TGFα, TGFβ and IFNγ, induce the expression of IL- 24 in keratinocytes. IL-24 protein inhibits growth factor stimulated proliferation and migration of keratinocytes. NHEK forced to express IL-24 do not proliferate in response to growth factors
21 Working Hypothesis We propose that the pro-inflammatory cytokine IL-24 functions in wound repair. Expression of this cytokine is upregulated by TGFα, TGFβ and IFNγ, factors involved in the later stages of wound healing. IL-24, produced by keratinocytes and immune cells, acts to inhibit proliferation and migration causing the contraction of the wound and return of keratinocytes to their normal differentiating processes. Loss of IL-24 expression will lead to uncontrolled keratinocyte proliferation abnormal healing.
22 Loss of IL-24 in the invasive front of a Primary melanoma superficial MDA-7 IHC of the Clark Level III and IV primary tumors shows significantly less staining in the deep portions of superficial layer n=84 tumors (p = 0.003) deep Ellerhorst et al. J Clin Oncol 20:1069,2002.
23 IL-24 and Melanoma Progression Cancer and wound healing are both characterized by cell proliferation, cell migration and invasion, and angiogenesis. We propose that IL-24 inhibits growth factorinduced proliferation and invasion in both wound repair and melanoma progression. Loss of IL-24 expression in invasive melanoma cells results in the loss of growth control and subsequent tumor progression.
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