Background

1. Subcapsular Experimental Procedure Summary of results With more than 209,000 new cases and 102,000 deaths estimated worldwide annually, delayed detection of renal cell carcinoma (RCC) remains a key contributor to the high mortality associated with this disease As renal tumors are radiographically indistinct, typically insensitive to radiotherapy and chemotherapy, and are not associated with any known serum tumor markers, the standard of care remains to remove all suspicious enhancing renal masses. However, the majority of patients present with a mass less than 4cm and almost one-quarter of these will be benign The key genetic and epigenetic marker in RCC is mutation and inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene, but systemic treatments targeting the VHL pathway have yielded few durable responses and cures Prior work from our laboratory has demonstrated that the E3-ligase adaptor speckle-type POZ protein (SPOP) is involved in cell cycle regulation and over-expressed in the cytoplasm of 99% of clear cell RCC and absent in the cytoplasm of normal kidney tissue (Figure 1) Figure 2: Following (a) exteriorization of the right kidney, (b) 5x 104 cells are delivered underneath the renal capsule. Transabdominal micro-ultrasound (c) was performed after surgery and bi-weekly during the period of tumor growth (d) using 2-D and doppler measurements (e). Animals were sacrificed at 6 weeks (f). • Tumor formation occurred in 17/20 (85%) of SPOP implantation sites compared with 1/20 (5%) HEK293 and 0/20 (0%) pcDNA3 sites (Figure 3a; p<0.001) • Tumor volume increased exponentially in both the subcutaneous and subcapsular SPOP-NN implantation groups (Figure 3b) • Immunohistochemistry within SPOP-NN tumors demonstrated the following staining (Figure 4): • Daxx • DUSP6 • DUSP7 • PTEN • VEGF • HIF-ɑ a) b) c) Subcapsular wheal #145: SPECKLE-TYPE POZ PROTEIN CYTOPLASMIC MISLOCALIZATION AND OVEREXPRESSION PROMOTE TUMOR GROWTH IN AN ORTHOTOPIC MURINE RENAL CELL CANCER MODELSandip M. Prasad, MD, MPhil1, Jiang Liu, PhD2, Subhradip Karmaker,3, Yi Cai, PhD1, Donald VanderGriend, PhD1, Scott E. Eggener1, and Kevin P. White, PhD31. Section of Urology, University of Chicago, Chicago, IL; 2. Beijing Institute of Genomics, Beijing, China; 3. Institute for Genomics and System Biology, The University of Chicago and Argonne National Laboratory, Chicago, IL f) e) d) neovascularity tumor kidney necrosis necrosis Figure 1: SPOP expression by RCC subtype Results Discussion Background Figure 3: Tumor counts (a) and volume (b) for the SPOP-NN, SPOP, and pcDNA3 subcapsular (subcap) and subcutaneous (sq) groups • Cytoplasmic mislocalization of SPOP is a potent promoter of tumorigenesis in HEK293 cells • SPOP functions as an oncogene and upregulates the biological function of VEGF in vivo and is associated with the expression of proliferative and downregulation of apoptotic factors downstream within the VHL pathway (Figure 4) • These findings support further clinical development of SPOP as a therapeutic target for ccRCC a) b) Objective To investigate the sequalae of SPOP mislocalization in normal HEK293 embryonal kidney cells in an in vivo model Figure 4: Proposed schematic for SPOP hub activity Methods Figure 4: H&E (a) and selected Immunohistochemistry (b) of SPOP-NN associated renal tumors HEK293 cells were transfected with either an empty pcDNA3.1 vector (pcDNA3) or SPOP protein without the carboxy terminal nuclear location signal (SPOP-NN) Stable transfected HEK293 (SPOP), pcDNA3 or SPOP-NN cells were injected either subcutaneously (1x 106 cells or under the renal capsule (Figure 2) in 6 week old male BALB/c nude mice a) b) Daxx tumor High mitotic activity kidney control tumor - Cytoplasmic staining