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Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling

View Article: PubMed Central - PubMed

ABSTRACT

TFE-fusion renal cell carcinomas (TFE-fusion RCCs) are caused by chromosomal translocations that lead to overexpression of the TFEB and TFE3 genes (Kauffman et al., 2014). The mechanisms leading to kidney tumor development remain uncharacterized and effective therapies are yet to be identified. Hence, the need to model these diseases in an experimental animal system (Kauffman et al., 2014). Here, we show that kidney-specific TFEB overexpression in transgenic mice, resulted in renal clear cells, multi-layered basement membranes, severe cystic pathology, and ultimately papillary carcinomas with hepatic metastases. These features closely recapitulate those observed in both TFEB- and TFE3-mediated human kidney tumors. Analysis of kidney samples revealed transcriptional induction and enhanced signaling of the WNT β-catenin pathway. WNT signaling inhibitors normalized the proliferation rate of primary kidney cells and significantly rescued the disease phenotype in vivo. These data shed new light on the mechanisms underlying TFE-fusion RCCs and suggest a possible therapeutic strategy based on the inhibition of the WNT pathway.

Doi:: http://dx.doi.org/10.7554/eLife.17047.001

No MeSH data available.


Related in: MedlinePlus

Inhibition of autophagy in Tfeb overexpressing mice (Atg7flox/flox::Cdh16Cre::Tfebfs) does not affect the cystic phenotype.(A) Real-time PCR validation of well-known TFEB direct gene targets whose function is related to the lysosomal and autophagic pathways performed on P30 Cdh16Cre::Tfebfs mice. Values are shown as the average (± SEM) of at least three Cdh16Cre::Tfebfs mice and are normalized to wild-type mice (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test). (B) P14 and P30 kidney lysates from Cdh16Cre::Tfebfs animals were evaluated by LC3I/II immunoblot. LC3 active / LC3 total ratios were quantified via densitometry of the Western blot bands (graph). Each replicate is a different biological sample. Values are shown as the average (± SEM) of at least three Cdh16Cre::Tfebfs mice and are normalized to wild-type lines. (*p<0.05, **p<0.01, ***p<0.001, two-sided, Student’s t test). (C) Renal images of (Atg7flox/flox::Cdh16Cre::Tfebfs) double transgenic mice and controls at P30. (D) Kidney-to-body weight ratio (KW/BW) from the different genotypes obtained. Values are normalized to the Atg7flox/flox::Cdh16Cre line and are shown as the average (± SEM) of at least three mice per genotype. One-way Anova was applied (factors: genotype) (*p<0.05, **p<0.01, ***p<0.001). (E) PAS and ATG7 staining of kidneys from the different mouse lines.DOI:http://dx.doi.org/10.7554/eLife.17047.021
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fig6s3: Inhibition of autophagy in Tfeb overexpressing mice (Atg7flox/flox::Cdh16Cre::Tfebfs) does not affect the cystic phenotype.(A) Real-time PCR validation of well-known TFEB direct gene targets whose function is related to the lysosomal and autophagic pathways performed on P30 Cdh16Cre::Tfebfs mice. Values are shown as the average (± SEM) of at least three Cdh16Cre::Tfebfs mice and are normalized to wild-type mice (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test). (B) P14 and P30 kidney lysates from Cdh16Cre::Tfebfs animals were evaluated by LC3I/II immunoblot. LC3 active / LC3 total ratios were quantified via densitometry of the Western blot bands (graph). Each replicate is a different biological sample. Values are shown as the average (± SEM) of at least three Cdh16Cre::Tfebfs mice and are normalized to wild-type lines. (*p<0.05, **p<0.01, ***p<0.001, two-sided, Student’s t test). (C) Renal images of (Atg7flox/flox::Cdh16Cre::Tfebfs) double transgenic mice and controls at P30. (D) Kidney-to-body weight ratio (KW/BW) from the different genotypes obtained. Values are normalized to the Atg7flox/flox::Cdh16Cre line and are shown as the average (± SEM) of at least three mice per genotype. One-way Anova was applied (factors: genotype) (*p<0.05, **p<0.01, ***p<0.001). (E) PAS and ATG7 staining of kidneys from the different mouse lines.DOI:http://dx.doi.org/10.7554/eLife.17047.021

Mentions: Considering the known role of TFEB as a master regulator of the lysosomal-autophagy pathway (Argani et al., 2001, 2005; Camparo et al., 2008; Davis et al., 2003), and the recent evidence indicating that activation of autophagy driven by MiT/TFE genes plays an important role in pancreatic cancer (Perera et al., 2015), we tested whether autophagy plays a role in TFE-tRCC development. We analyzed the expression levels of a well-characterized panel of TFEB target genes known to be involved in lysosomal biogenesis and autophagy in Cdh16Cre::Tfebfs mice. Surprisingly, no significant changes in the expression levels of these genes were detected in Cdh16Cre::Tfebfs compared to wild type mice, with a few exceptions (Figure 6—figure supplement 3A). Consistently, immunoblot analysis of the autophagy marker LC3 in kidneys from transgenic mice did not reveal any significant changes compared to control littermates (Figure 6—figure supplement 3B). Furthermore, to test the role of autophagy in the pathogenesis of TFE-tRCC we crossed Cdh16Cre::Tfebfs mice with autophagy-deficient Atg7flox/flox mice. No changes in kidney size or in the cystic phenotype were observed in TFEB overexpressing/autophagy-deficient double transgenic mice (Atg7flox/flox::Cdh16Cre::Tfebfs), herein referred to Atg7flox/flox::Cdh16Cre::Tfebfs, compared to Cdh16Cre::Tfebfs mice (Figure 6—figure supplement 3C–E). Interestingly, most of the double transgenic animals died at approximately 1 month of age, suggesting that the combination of TFEB overexpression with autophagy inhibition in the kidney is toxic. This may be due to the previously described increase in sensitivity to oxidative stress of kidney-specific autophagy-deficient mice (Liu et al., 2012). These results suggest that autophagy does not play a critical role in the development of TFE-tRCC phenotype.


Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling
Inhibition of autophagy in Tfeb overexpressing mice (Atg7flox/flox::Cdh16Cre::Tfebfs) does not affect the cystic phenotype.(A) Real-time PCR validation of well-known TFEB direct gene targets whose function is related to the lysosomal and autophagic pathways performed on P30 Cdh16Cre::Tfebfs mice. Values are shown as the average (± SEM) of at least three Cdh16Cre::Tfebfs mice and are normalized to wild-type mice (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test). (B) P14 and P30 kidney lysates from Cdh16Cre::Tfebfs animals were evaluated by LC3I/II immunoblot. LC3 active / LC3 total ratios were quantified via densitometry of the Western blot bands (graph). Each replicate is a different biological sample. Values are shown as the average (± SEM) of at least three Cdh16Cre::Tfebfs mice and are normalized to wild-type lines. (*p<0.05, **p<0.01, ***p<0.001, two-sided, Student’s t test). (C) Renal images of (Atg7flox/flox::Cdh16Cre::Tfebfs) double transgenic mice and controls at P30. (D) Kidney-to-body weight ratio (KW/BW) from the different genotypes obtained. Values are normalized to the Atg7flox/flox::Cdh16Cre line and are shown as the average (± SEM) of at least three mice per genotype. One-way Anova was applied (factors: genotype) (*p<0.05, **p<0.01, ***p<0.001). (E) PAS and ATG7 staining of kidneys from the different mouse lines.DOI:http://dx.doi.org/10.7554/eLife.17047.021
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fig6s3: Inhibition of autophagy in Tfeb overexpressing mice (Atg7flox/flox::Cdh16Cre::Tfebfs) does not affect the cystic phenotype.(A) Real-time PCR validation of well-known TFEB direct gene targets whose function is related to the lysosomal and autophagic pathways performed on P30 Cdh16Cre::Tfebfs mice. Values are shown as the average (± SEM) of at least three Cdh16Cre::Tfebfs mice and are normalized to wild-type mice (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test). (B) P14 and P30 kidney lysates from Cdh16Cre::Tfebfs animals were evaluated by LC3I/II immunoblot. LC3 active / LC3 total ratios were quantified via densitometry of the Western blot bands (graph). Each replicate is a different biological sample. Values are shown as the average (± SEM) of at least three Cdh16Cre::Tfebfs mice and are normalized to wild-type lines. (*p<0.05, **p<0.01, ***p<0.001, two-sided, Student’s t test). (C) Renal images of (Atg7flox/flox::Cdh16Cre::Tfebfs) double transgenic mice and controls at P30. (D) Kidney-to-body weight ratio (KW/BW) from the different genotypes obtained. Values are normalized to the Atg7flox/flox::Cdh16Cre line and are shown as the average (± SEM) of at least three mice per genotype. One-way Anova was applied (factors: genotype) (*p<0.05, **p<0.01, ***p<0.001). (E) PAS and ATG7 staining of kidneys from the different mouse lines.DOI:http://dx.doi.org/10.7554/eLife.17047.021
Mentions: Considering the known role of TFEB as a master regulator of the lysosomal-autophagy pathway (Argani et al., 2001, 2005; Camparo et al., 2008; Davis et al., 2003), and the recent evidence indicating that activation of autophagy driven by MiT/TFE genes plays an important role in pancreatic cancer (Perera et al., 2015), we tested whether autophagy plays a role in TFE-tRCC development. We analyzed the expression levels of a well-characterized panel of TFEB target genes known to be involved in lysosomal biogenesis and autophagy in Cdh16Cre::Tfebfs mice. Surprisingly, no significant changes in the expression levels of these genes were detected in Cdh16Cre::Tfebfs compared to wild type mice, with a few exceptions (Figure 6—figure supplement 3A). Consistently, immunoblot analysis of the autophagy marker LC3 in kidneys from transgenic mice did not reveal any significant changes compared to control littermates (Figure 6—figure supplement 3B). Furthermore, to test the role of autophagy in the pathogenesis of TFE-tRCC we crossed Cdh16Cre::Tfebfs mice with autophagy-deficient Atg7flox/flox mice. No changes in kidney size or in the cystic phenotype were observed in TFEB overexpressing/autophagy-deficient double transgenic mice (Atg7flox/flox::Cdh16Cre::Tfebfs), herein referred to Atg7flox/flox::Cdh16Cre::Tfebfs, compared to Cdh16Cre::Tfebfs mice (Figure 6—figure supplement 3C–E). Interestingly, most of the double transgenic animals died at approximately 1 month of age, suggesting that the combination of TFEB overexpression with autophagy inhibition in the kidney is toxic. This may be due to the previously described increase in sensitivity to oxidative stress of kidney-specific autophagy-deficient mice (Liu et al., 2012). These results suggest that autophagy does not play a critical role in the development of TFE-tRCC phenotype.

View Article: PubMed Central - PubMed

ABSTRACT

TFE-fusion renal cell carcinomas (TFE-fusion RCCs) are caused by chromosomal translocations that lead to overexpression of the TFEB and TFE3 genes (Kauffman et al., 2014). The mechanisms leading to kidney tumor development remain uncharacterized and effective therapies are yet to be identified. Hence, the need to model these diseases in an experimental animal system (Kauffman et al., 2014). Here, we show that kidney-specific TFEB overexpression in transgenic mice, resulted in renal clear cells, multi-layered basement membranes, severe cystic pathology, and ultimately papillary carcinomas with hepatic metastases. These features closely recapitulate those observed in both TFEB- and TFE3-mediated human kidney tumors. Analysis of kidney samples revealed transcriptional induction and enhanced signaling of the WNT &beta;-catenin pathway. WNT signaling inhibitors normalized the proliferation rate of primary kidney cells and significantly rescued the disease phenotype in vivo. These data shed new light on the mechanisms underlying TFE-fusion RCCs and suggest a possible therapeutic strategy based on the inhibition of the WNT pathway.

Doi:: http://dx.doi.org/10.7554/eLife.17047.001

No MeSH data available.


Related in: MedlinePlus