Abstract 5096: Pre- and early postnatal loss of tsc1/2 induces kidney hybrid oncocytic/chromophobe tumors (HOCT) via c-JUN
Authors: Du Heng , Liu Heng-jia , Khabibullin Damir , Zarei Mahsa , Dreier John , Wu Chin-Lee , Henske Elizabeth , Kwiatkowski David 
Published: August 2018

Abstract
Renal cell carcinoma (RCC) occurs in up to 4% of tuberous sclerosis complex (TSC) patients, often affecting children and young adults. The mechanisms of TSC-associated RCC are not well understood. We explored the pathogenesis of TSC-associated RCC using three mouse models. First, loss of Tsc2 was induced by i.p. tamoxifen (TM) injection (10mg/kg) at embryonic day 17.5 (E17.5) in Tsc2flox/floxROSA26-CreER+ mice (n=24), examined at age 6 m. Second, loss of Tsc1 was induced by injecting Cre-encoding adenovirus directly into the left kidney of Tsc1flox/flox mice at P1 (n=19), examined at age 1 year. Third, Tsc2 was knock out through 5 consecutive times of i.p. tamoxifen injection (1mg/mouse/day) when the Tsc2flox/floxROSA26-CreER+ mice were 6 weeks old (n=7), examined at age 6 month. We found the following: In the E17.5 TM injection mouse model, each kidney had more than 200 tumor lesions, which almost occupied the whole kidney cortex and medulla. Extensive TSC-associated kidney papillary RCC (PRCC) and hybrid oncocytic/chromophobe tumors (HOCT) were seen in every kidney. The same types of tumor but fewer tumor lesions (~40/kidney) were found in the Cre-encoding adenovirus group. All the females in the P6 week group had the same phenotype as E17.5 TM injection group, but only single cyst was found in the male kidneys. So, the earlier those mice lost TSC1/2, the more severe their tumor burdens. IHC was performed to further validate the diagnosis. All the PRCC showed strong and diffuse expression of phospho-S6-S240/244 (pS6), CK7, and carbonic anhydrase-IX, with negative vimentin staining, while all the HOCT were positive for pS6, S-100 (a diagnostic marker used for human HOCT) and vimentin. The single cysts in the P6 week TM injection male kidneys only showed pS6 and CK7 staining. To further explore the mechanism of tumorigenesis, a phospho-RTK screen was done using lysates from kidney tumors (PRCC and HOCT) and normal kidneys. The pAXL level was 2.8-fold higher in comparison to normal kidney. Immunoblotting revealed high levels of p-AXL and total AXL in kidney tumor lysates compared to normal kidney. AXL mRNA, measured by real-time RT-PCR, was increased 2-fold (p=0.011). c-Jun, a known driver of AXL expression, was also highly expressed in those tumors but negative in normal kidneys as shown by immunoblot. Immunoblot analysis of a cell line established from the kidney tumors showed TSC-dependent c-Jun and AXL expression. These novel mouse models of TSC-related RCC closely resemble those found in TSC patients and elucidate distinct mechanisms of disease development and potential therapeutic targets.
Citation Format: Heng Du, Heng-jia Liu, Damir Khabibullin, Mahsa Zarei, John Dreier, Chin-Lee Wu, Elizabeth Henske, David Kwiatkowski. Pre- and early postnatal loss of tsc1/2 induces kidney hybrid oncocytic/chromophobe tumors (HOCT) via c-JUN [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 5096.