ASN's Mission

ASN leads the fight to prevent, treat, and cure kidney diseases throughout the world by educating health professionals and scientists, advancing research and innovation, communicating new knowledge, and advocating for the highest quality care for patients.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on Twitter

Kidney Week

Abstract: FR-PO1034

Stromal Signaling Promotes ccRCC Tumor Growth Through FOXD1 Overexpression

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Authors

  • Bond, Kyle H., Maine Medical Center Research Institute, Scarborough, Maine, United States
  • Gupta, Ashwani Kumar, Maine Medical Center Research Institute, Scarborough, Maine, United States
  • Oxburgh, Leif, Maine Medical Center Research Institute, Scarborough, Maine, United States

Group or Team Name

  • Oxburgh Lab
Background

Clear cell renal cell carcinoma (ccRCC) is the 8th most common cancer in the U.S.. Although tyrosine kinase inhibitors (TKIs) have improved survival in patients, many patients are not responsive to TKI are in need of more options. The tumor microenvironment (TME) in ccRCC is not well understood and may be a potential target. The transcription factor FOXD1 has been shown to be upregulated in lung, breast, and kidney cancer. Functionally, FOXD1 regulates epithelial-stromal signaling in the developing kidney. We hypothesize that FOXD1 overexpression in ccRCC would promote tumor growth through stromal signaling.

Methods

The relevancy FOXD1 was first determined through RNA-seq and staining analyses. ccRCC tumor microarrays (TMAs) were stained for FOXD1, PECAM, aSMA, PDGFRb, and NG2. A Kaplain-Meier survival analysis for FOXD1 expression was performed using data from The Cancer Genome Atlas (TCGA). A potential FOXD1 binding site analyses using the TRANSFAC FOXD1 binding site matrix was performed. Binding site were compared to previously reported FOXD1-/- RNA-microarray data to determine direct binding targets. Results were confirmed by qPCR in renal proximal tubule cells (RPTECs) after transfection with a FOXD1 adenovirus. SLIT2 was found to be a functionally relevant target in kidney cancer and was assessed for its role in fibroblast signaling through scratch assays, 3D migration assays, and multiplex proximity ligation assays. Knockdown of FOXD1 in the 786-O cell line followed by qPCR, western blot, and migration analyses.

Results

65% of TMA samples stained positively for FOXD1. FOXD1 expression within the cancer cells correlated with stromal PDGFRβ expression (p<4.5x10-6). FOXD1-high ccRCC patients had a worse survival outcome (FOXD1 low= 2830 days; FOXD1 high= 1913 days). FOXD1 binding site analysis, RNA-microarray, and qPCR found FGF1, SLIT2, and Decorin to be potential secreted signaling molecules repressed by FOXD1. Scratch assays on fibroblasts showed that SLIT2 reduced PDGFBB-induced cell migration (p<0.27). Additionally, 3D migration co-culture with 786-O cancer cells determined a repulsive effect of SLIT2 in 3D culture as well (5µm p=1.34x10-4; 50µm p=1.64x10-2).

Conclusion

FOXD1 is an important prognostic marker for aggressiveness in ccRCC. The importance of fibroblast recruitment by cancer cells in ccRCC is still not known.

Funding

  • NIDDK Support