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. 2015 Oct 20;6(32):32761-73.
doi: 10.18632/oncotarget.5018.

Flcn-deficient renal cells are tumorigenic and sensitive to mTOR suppression

Mingsong Wu  1 , Shuhui Si  2 , Yan Li  3 , Susan Schoen  2 , Guang-Qian Xiao  4 , Xueying Li  1 , Bin Tean Teh  5 , Guan Wu  1   4 , Jindong Chen  1   2
Affiliations

Flcn-deficient renal cells are tumorigenic and sensitive to mTOR suppression

Mingsong Wu et al. Oncotarget. .

Abstract

Deficiency of tumor suppressor FLCN leads to the activation of the mTOR signaling pathway in human BHD-associated renal cell carcinomas (RCC). We have previously developed a renal distal tubule-collecting duct-Henle's loop-specific Flcn knockout (KO) mouse model (Flcnflox/flox/Ksp-Cre). This mouse model can only survive for three weeks after birth due to the development of polycystic kidney and uremia. Whether these cystic solid hyperplasia changes seen in those KO mice are tumorigenic or malignant is unknown. In this study, we demonstrated that genetic disruption of Flcn in mouse kidney distal tubule cells could lead to tumorigenic transformation of these cells to develop allograft tumors with an aggressive histologic phenotype. Consistent with previous reports, we showed that the mTOR pathway plays an important role in the growth of these Flcn-deficient allograft and human UOK 257-1 xenograft tumors. We further demonstrated that the mTOR inhibitor, sirolimus, suppresses the tumor's growth, suggesting that mTOR inhibitors might be effective in control of FLCN-deficient RCC, especially in BHD renal tumorigenesis.

Keywords: BHD; RCC; kidney cancer; mTOR; sirolimus.

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Conflict of interest statement

CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. Generation of tumorigenic Flcn-deficient renal distal tubule cell lines
A. phenotype of the KO (Flcnflox/flox/Ksp-Cre knockout) mice. KO kidneys were enlarged due to polycystic changes compared to WT ones. B. H&E staining of the polycystic kidneys of Flcnflox/flox/Ksp-Cre mice at age of 10 days. C. hyperplasia/micro-tumors identified in a Flcnflox/flox/Ksp-Cre mouse kidney (indicated by arrows). D. No Flcn expression observed in the hyperplasia/micro-tumors (indicated by arrows). Note that the hyperplasia/micro-tumors were Flcn negative compared to the proximal tubules stained positively (indicated by arrow heads). E, F. representative cells lines isolated from two Flcnflox/flox/Ksp-Cre polycystic/micro-tumor kidneys and cultured in DMEM medium. G. PCR genotyping demonstrated that cell lines derived from four KO kidneys (C1-C4) displayed KO band (152 bp), indicated that Flcn had been disrupted. Wild-type kidney (Flcnflox/flox) showed only a wild-type band (1001 bp). Cystic kidney (Cys) had both wild-type band and KO band due to the mixture of Flcn disrupted and undisrupted renal cells. H. Western blot analysis demonstrated that the cells (C1–C4) have no Flcn expression. Cystic kidney tissue showed weak Flcn expression. WT, wild type; KO, knockout. Cys, cystic kidney. Bar scale, 50 μm.
Figure 2
Figure 2. Flcn-deficient renal distal tubule cells are tumorigenic
A. inoculation of Flcn-deficient renal distal tubule cells developed into allograft tumors in nude mice. B. allograft tumors obtained in the nude mice. C, D. H&E staining of these tumor tissue sections revealed that all tumors were high-grade renal cell carcinomas (sarcomatoid RCC). E. IHC analysis with FLCN antibody showed no Flcn expression in the allograft tumors. F, G. and H. IHC analysis by renal cell carcinoma makers (AQP1, PAX2, and CK7) revealed that the allograft tumors were positive for these markers, indicating that the allograft tumors were renal cell carcinomas. Bar scale, 50 μm.
Figure 3
Figure 3. The mTOR pathway is activated in allograft tumors developed from Flcn-deficient renal distal tubule cell lines
A, B. H&E staining of tumors. C, D. Flcn was not expressed in allograft tumor. E, F. mTOR was activated by phosphorylation in Flcn-deficient tumor. G, H. phosphorylated activation of S6 was also observed in tumors. Bar scale, 50 μm.
Figure 4
Figure 4. Sirolimus inhibits mouse allograft Flcn-deficient tumor growth
A. representative tumors photographed from both sirolimus-treated mice (upper panel) and vehicle-treated control mice (lower panel). Sirolimus (7.5 mg/kg) was given every other day when allograft tumor size reached 200 mm3. B. growth curve of sirolimus-treated mice and vehicle-treated control mice. C. tumor growth was inhibited significantly by sirolimus compared to vehicle-treated controls (p < 0.01, t-test, showed in C). At the end of the treatment, tumors continued to grow slowly. D. representative tumors photographed from both sirolimus-treated mice (upper panel) and vehicle-treated control mice (lower panel). Sirolimus at a high dose (15 mg/kg) was given to treat the allograft tumors when the tumor size reached 450 mm3. E. growth curves of sirolimus-treated mice with high dosage of sirolimus treatment (15 mg/kg) and vehicle-treated control mice. F. tumor growth was inhibited significantly by sirolimus compared to vehicle-treated controls (p < 0.01, showed in F). Note that tumor growth had essentially stopped when treated with a higher dose of sirolimus.
Figure 5
Figure 5. Sirolimus inhibits tumor growth
A. representative tumors photographed from both sirolimus-treated mice (upper panel) and vehicle-treated control mice (lower panel). B, C. sirolimus (7.5 mg/kg) was given to the treatment group when allograft tumor size reached 150 mm3. Tumor growth was inhibited significantly by sirolimus compared to vehicle-treated controls (C). At the end of the treatment, allograft tumors shrank slightly.
Figure 6
Figure 6. High dosages of sirolimus and early treatment lead to better treatment effects on tumor
A. high dosage of sirolimus (15 mg/kg) exerted stronger inhibition on allograft tumor growth compared with low dose (7.5 mg/kg) (p < 0.05). B. sirolimus exerted more efficient suppression on smaller tumors at an early-stage (tumor size, <150 mm3).
Figure 7
Figure 7. Sirolimus inhibits human UOK 257-1 xenograft tumor growth
A. representative tumors photographed from both sirolimus-treated mice (upper panel) and vehicle-treated control mice (lower panel) at the end of the experiments (day 22). Sirolimus (15 mg/kg) was given every other day for 21 days when allograft tumor size reached 200 mm3. B. growth curves of sirolimus-treated mice and vehicle-treated control mice. C. at the end of treatment, tumors were significantly smaller in sirolimus-treated group compared to vehicle-treated controls (p < 0.05).
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