Abstract 200: FOXD1 promotes stromal investment in clear cell renal cell carcinoma

Abstract

Abstract Forkhead transcription factors (FOX) play a role in the development of many cancers, including clear cell renal cell carcinoma (ccRCC). FOXD1, essential for angiogenesis in the developing kidney, has not previously been implicated in ccRCC. Tumor microarrays (TMAs) containing 41 patient-derived ccRCC tumors and 26 healthy cortex samples were stained for FOXD1, with 65% of tumor samples staining positively. Due to the effect of FOXD1 on angiogenesis, staining for stromal markers PECAM, αSMA, PDGFRβ and NG2 was also performed. A direct correlation was found only between FOXD1 and PDGFRβ (p<4.5x10-6), suggesting interstitial fibroblast recruitment by FOXD1+ cancer cells. Increased expression of FOXD1 was found to correlate with increased stage (p=9.1x10-5) and reduced survival (FOXD1 low= 2830 days; FOXD1 high= 1913 days) from a Kaplan-Meier analysis of RNA-seq data from The Cancer Genome Atlas (TCGA). Potential FOXD1 target binding sites were determined using the TRANSFAC FOXD1 binding site matrix. SLIT2, a factor known to inhibit migration of pericytes, was uncovered from the analysis and was further found to be downregulated in response to FOXD1 overexpression in renal proximal tubule cells (RPTECs). To test the influence of SLIT2 on interstitialfibroblast migration, scratch assays on NRK-49F (rat kidney fibroblast) and Gli-1 (cardiac fibroblast) cells were performed and showed that SLIT2 reduced PDGFBB-induced cell migration (p<0.027). A multiplex proximity ligation assay for multiple signaling pathways showed that SLIT2 treatment reduced the STAT signaling response to PGDFBB. To model the influence of SLIT2 on ccRCC stromal invasion in vitro, we devised a novel 3D invasion assay. In summary, 786-O ccRCC cancer cells were seeded into a 200um thick silk scaffold and cultured for 3 days to allow deposition of ECM. Scaffolds were then placed on top of a monolayer of interstitial fibroblasts and invasion into the cancer cell-filled scaffold was measured using confocal microscopy. The results showed a significant reduction of cell invasion into the cancer-filled matrix with the addition of recombinant SLIT2 to media (5µm p=1.34x10-4; 50µm p=1.64x10-2). In conclusion, we show that FOXD1 expression has prognostic relevance to patient survival in ccRCC, and that this may be due to modulation of SLIT2 expression. Citation Format: Kyle H. Bond, Jennifer Fetting, Ashwani Gupta, Christine W. Duarte, Michele Karolak, Clare B. Congdon, Ivette Emery, Eoghainín Ó hAinmhire, Benjamin D. Humphreys, Leif Oxburgh. FOXD1 promotes stromal investment in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 200.