Folliculin contributes to VHL tumor suppressing activity in renal cancer through regulation of autophagy

Abstract

Von Hippel-Lindau tumor suppressor (VHL) is lost in the majority of clear cell renal cell carcinomas (ccRCC). Folliculin (FLCN) is a tumor suppressor whose function is lost in Birt-Hogg-Dubé syndrome (BHD), a disorder characterized by renal cancer of multiple histological types including clear cell carcinoma, cutaneous fibrofolliculoma, and pneumothorax. Here we explored whether there is connection between VHL and FLCN in clear cell renal carcinoma cell lines and tumors. We demonstrate that VHL regulates expression of FLCN at the mRNA and protein levels in RCC cell lines, and that FLCN protein expression is decreased in human ccRCC tumors with VHL loss, as compared with matched normal kidney tissue. Knockdown of FLCN results in increased formation of tumors by RCC cells with wild-type VHL in orthotopic xenografts in nude mice, an indication that FLCN plays a role in the tumor-suppressing activity of VHL. Interestingly, FLCN, similarly to VHL, is necessary for the activity of LC3C-mediated autophagic program that we have previously characterized as contributing to the tumor suppressing activity of VHL. The results show the existence of functional crosstalk between two major tumor suppressors in renal cancer, VHL and FLCN, converging on regulation of autophagy.

Figure 1. VHL induces expression of FLCN mRNA and protein.

Reconstitution of VHL in 786-O and A498 cells led to induction of FLCN and p53 mRNAs (A) and proteins (B). Knockdown of VHL in Caki-1 cells resulted in decreased FLCN mRNA (C) and protein (D) levels; *** P<0.001, **P<0.01. Scr- scramble control virus.

Figure 2. FLCN protein levels are reduced in human ccRCC with lost VHL.

Analysis of FLCN protein (A–C) and mRNA (D) in samples of human ccRCC with known VHL status, as compared with adjacent kidney tissue. A. A representative western blot analysis of FLCN protein in pairs of kidney (K) and tumor with lost VHL (VHL-DEL) separated on 10% PAGE. The lower band may represent a degradation or posttranslational modification of FLCN. B and C. Quantification of FLCN protein expression in tumors that had lost VHL (VHL-DEL) and tumors with wild-type VHL (VHL-WT). The boxes represent lower and upper quartiles separated by the median (thick horizontal line) and the whiskers extend up to the minimum and maximum values, excluding points that are outside the 1.5 interquartile range from the box (marked as circles). Means ± SD of each distribution are indicated by closed dots and crosses on the whiskers, respectively. D. Quantitative RT-PCR measurement of FLCN mRNA levels in kidneys and ccRCC VHL-DEL and VHL-WT tumors.

Figure 3. FLCN is necessary for the VHL-mediated tumor suppression.

ShRNA-induced knockdown of FLCN promotes tumor formation by 786-O cells with reconstituted VHL. A. Western blot demonstrating a decrease in FLCN protein expression in cells stably expressing three different FLCN shRNAs as compared to cells transfected with scramble (Scr) control. B. Quantification of incidence and weight of tumors formed by the indicated cell lines in orthotopic xenografts. NK- average weight of normal kidneys. C. H&E staining of representative sections for tumors grown by VHL(+)/scramble control cells and VHL(+) cells with FLCN knockdowns. Scale bars = 50 µm.

Figure 4. Knockdown of FLCN inhibits the mTOR pathway.

A. Western blot analysis of mTOR downstream targets in 786-O VHL(+) cells stably expressing either scramble or two different FLCN shRNAs. The GAPDH loading control is shown for each group of immunoblots processed from the same gel. B. Representative sections of indicated tumors stained for S6 and S6P. Scale bar = 50 µm.

Figure 5. FLCN regulates autophagy.

Knockdown of FLCN (shRNA 3) in 786-O (A) or RCC4 (B) VHL(+) cells reduces accumulation of tumor suppressive LC3C-II but induces expression of oncogenic LC3B-II. Western blots were probed with indicated antibodies. C. Quantification of 3 independent experiments as shown in panel A and B. LC3 protein levels measured by optical density were first normalized to GAPDH. Then the ratio of LC3 was calculated between LC3 levels measured in chloroquine treated samples in cells with FLCN knocked down and control cells (compare lane 6 to lane 3 in panels A and B). D. Model of the proposed regulation.