Tumor stroma, including macrophages and other cells, actively influence cancer through soluble factors that alter signaling in tumor cells. Specifically, this document discusses: 1) Colon cancer cells stimulate macrophages to release IL-1β which mediates crosstalk between tumor cells and macrophages. 2) IL-1β inhibits TRAIL-induced apoptosis in tumor cells through NFkB and Wnt signaling pathways. 3) Vitamin D3 can inhibit IL-1β release from macrophages and restore TRAIL-induced apoptosis in a VDR-dependent manner.

1. Tumor stroma: an active player in cancer

2. Soluble factors produced by stromal cells alter signaling pathways in tumor cells HCT116- NFkB-LUC HCT116- Hes1/5-LUC Macrophages 0.4µm HCT116- TOP-LUC HCT116- STAT1-LUC HCT116- STAT3-LUC HCT116- AP1-LUC Tumor cells

3. Colon cancer cells stimulate macrophages to release IL1b; the role of pSTAT1 ( siRNA) STAT1 NSP pSTAT1 STAT3 -actin (IL-1b) 0 8h 24h 48h pGSK3 c-myc tGSK3b

4. Vitamin D3 inhibits the protumorigenic activity of macrophages THP1/IL1 -/- THP1/D3 CTRL THP1 pGSK3 c-myc tGSK3b

5. Vitamin D3: an inhibitor of IL1b release from macrophages 24h 48h 1, 25(OH)2D3 (nM) 50 50 0 0 25 25 pSTAT1 STAT1 -actin CTRL 1, 25(OH)2D3 (nM)

6. IL1b mediates the crosstalk between tumor cells and macrophages ?? Wnt targets b-catenin TCF STAT1 pSTAT1 GSK3b NFkB AKT PDK1 IL-1b Proliferation

7. Macrophages interfere with TRAIL-induced apoptosis Hke3 HCT116 Trail-Mo Trail –IL1 Trail -Mo Trail-Mo CTRL Trail CTRL Trail Cl caspase 8 (41,43 kDa) Cl caspase 8 (18 kDa) Cl caspase 9 bactin

8. IL1 is sufficient/required to inhibit TRAIL-induced apoptosis

9. Vitamin D3 restores TRAIL induced apoptosis in VDR dependent manner A C Trail-THP1-VDR-/--D3 Trail-THP1-D3-IL1 Trail-THP1-VDR-/- Trail-THP1-D3 Trail-THP1 CTRL Trail Cl caspase 8 (41,43 kDa) Cl caspase 8 (18 kDa) B Cleaved caspase 9 PARP (FL) PARP (Cleaved) Total -catenin (LE) Total -catenin (HE) actin

10. CTRL TRAIL Trail/THP1 Trail/IL1 neo dnIkB dnAKT dnTCF4 Macrophage/IL1 inhibit TRAIL-induced apoptosis through NFkB/Wnt signaling

11. b catenin, an important target of TRAIL signaling Neo dnIB dnAKT dnTCF4 Trail-THP1 Trail–THP1 Trail-THP1 Trail-THP1 Trail–IL1 Trail-IL1 Trail-IL1 Trail-IL1 CTRL CTRL CTRL CTRL Trail Trail Trail Trail Cleaved caspase 8 Cleaved caspase-9 -catenin PARP (FL) PARP (Cleaved) bactin

12. Neo dnAKT dnIkB dnTCF4 CTRL CTRL THP1 THP1 CTRL CTRL TNF THP1 THP1 TNF TNF IL1 IL1 TNF IL1 IL1 Snail c-Jun bactin Macrophages stabilize Snail in tumor cells THP1-STAT1 siRNA THP1-IL1 siRNA THP1-D3 CTRL THP1 THP1 CTRL Snail Snail bactin bactin

13. IL1b mediates the crosstalk between tumor cells and macrophages ?? Snail Wnt targets b-catenin TCF STAT1 pSTAT1 GSK3b NFkB AKT IL-1b Proliferation Survival

14. Versican Normal (71) Breast, colon, esophageal, hepatocellular, lung (235) Thyroid (35) Yu et al, PloS Genet 2008 Rectal cancer Colon cancer

15. VCAN stimulates macrophages to release IL1b A siRNA NSP VERSICAN B Top-Luc

16. Snail regulates the expression of VCAN NSP Snail VER (siRNA) VCAN bcatenin (siRNA)

17. Co-expression of VCAN and Snail in colon cancer patients Lin, Clinical Cancer research, 2007 (55 colon cancer patients, Affymetrics)

18. IL1b: a messenger that perpetuates the crosstalk between colon cancer cells and macrophages VCAN EMT VCAN Vit D3 Growth c-myc ?? Snail Wnt targets b-catenin TCF pSTAT1 GSK3b TRAIL NFkB AKT PDK1 IL-1b

19. Thank you: Pawan Kaler, PhD Malika Godasi, MD Vincent Galea Leonard Augenlicht, PhD