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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

Generation of transgenic mouse lines with kidney-specific Tfeb overexpression.(A) Map of the transgene: Tfeb-3xFLAG was inserted after the chicken actin promoter (CAG) and the chloramphenicol acetyltransferase (CAT)-SV40pA flanked by loxP sites. The latter can be removed by CRE recombinase, resulting in the overexpression of the Tfeb gene under the control of the strong CAG promoter. Two different CRE lines were used: (1) a constitutive kidney-specific Cdh16Cre(Cadherin 16) and (2) a tamoxifen-inducible Cdh16CreErt2. (B, C) Representative genotypes of littermates. (B) Lanes 1 and 4 indicate double heterozygous Cdh16Cre::Tfebfs mice as they carry both the Cdh16Cre (420 bp CRE band, 200 bp wt band) and the Tfebfs transgenes (700 bp) (M, marker; B, Blank). (C) Lane 1 indicates a double heterozygous Cdh16CreErt2::Tfebfs mouse as it carries both the Cdh16CreErt2 (507 bp Cre band, 388 bp wt band) and the Tfebfs transgenes (700 bp). (D) Real-time PCR analysis of Tfeb-3xFLAG mRNA levels performed on Cdh16Cre and Cdh16Cre::Tfebfs mice at different stages (P0, P12, P30) and on Cdh16CreErt2and Cdh16CreErt2::Tfebfs mice induced with tamoxifen at P12 and sacrificed at P90. Values are shown as the average (± SEM) of at least three animals per time point and genotype (*p<0. 05, **p<0.01, two-sided, Student’s t test). (E) Immunoblot analysis using an anti-Flag antibody to determine the expression of Tfeb-3xFLAG protein in Cdh16Cre::Tfebfs and tam-treated Cdh16CreErt2::Tfebfs mice. Each replicate is a different biological sample.DOI:http://dx.doi.org/10.7554/eLife.17047.003
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fig1s1: Generation of transgenic mouse lines with kidney-specific Tfeb overexpression.(A) Map of the transgene: Tfeb-3xFLAG was inserted after the chicken actin promoter (CAG) and the chloramphenicol acetyltransferase (CAT)-SV40pA flanked by loxP sites. The latter can be removed by CRE recombinase, resulting in the overexpression of the Tfeb gene under the control of the strong CAG promoter. Two different CRE lines were used: (1) a constitutive kidney-specific Cdh16Cre(Cadherin 16) and (2) a tamoxifen-inducible Cdh16CreErt2. (B, C) Representative genotypes of littermates. (B) Lanes 1 and 4 indicate double heterozygous Cdh16Cre::Tfebfs mice as they carry both the Cdh16Cre (420 bp CRE band, 200 bp wt band) and the Tfebfs transgenes (700 bp) (M, marker; B, Blank). (C) Lane 1 indicates a double heterozygous Cdh16CreErt2::Tfebfs mouse as it carries both the Cdh16CreErt2 (507 bp Cre band, 388 bp wt band) and the Tfebfs transgenes (700 bp). (D) Real-time PCR analysis of Tfeb-3xFLAG mRNA levels performed on Cdh16Cre and Cdh16Cre::Tfebfs mice at different stages (P0, P12, P30) and on Cdh16CreErt2and Cdh16CreErt2::Tfebfs mice induced with tamoxifen at P12 and sacrificed at P90. Values are shown as the average (± SEM) of at least three animals per time point and genotype (*p<0. 05, **p<0.01, two-sided, Student’s t test). (E) Immunoblot analysis using an anti-Flag antibody to determine the expression of Tfeb-3xFLAG protein in Cdh16Cre::Tfebfs and tam-treated Cdh16CreErt2::Tfebfs mice. Each replicate is a different biological sample.DOI:http://dx.doi.org/10.7554/eLife.17047.003

Mentions: In addition, to assess the effects of Tfeb overexpression during kidney development, we generated a second transgenic line by crossing the Tfebfs/fs mice with a mouse line that carries a tamoxifen-inducible CreErt2 element under the control of a Cdh16 promoter (Cdh16CreErt2promoter) (Lantinga-van Leeuwen et al., 2006) (Figure 1—figure supplement 1A). Cdh16Cre::Tfebfs and Cdh16CreErt2::Tfebfs double heterozygous mice were generated from these crossings (Figure 1—figure supplement 1B and C). We checked both the constitutive and inducible lines for renal Tfeb overexpression and confirmed that Tfeb mRNA levels were highly increased, and further increasing with time (Figure 1—figure supplement 1D). Consistently, immunoblot experiments revealed increased levels of Tfeb-3xFLAG protein in kidneys from Cdh16Cre::Tfebfs and Cdh16CreErt2::Tfebfs mice (Figure 1—figure supplement 1E).


Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling
Generation of transgenic mouse lines with kidney-specific Tfeb overexpression.(A) Map of the transgene: Tfeb-3xFLAG was inserted after the chicken actin promoter (CAG) and the chloramphenicol acetyltransferase (CAT)-SV40pA flanked by loxP sites. The latter can be removed by CRE recombinase, resulting in the overexpression of the Tfeb gene under the control of the strong CAG promoter. Two different CRE lines were used: (1) a constitutive kidney-specific Cdh16Cre(Cadherin 16) and (2) a tamoxifen-inducible Cdh16CreErt2. (B, C) Representative genotypes of littermates. (B) Lanes 1 and 4 indicate double heterozygous Cdh16Cre::Tfebfs mice as they carry both the Cdh16Cre (420 bp CRE band, 200 bp wt band) and the Tfebfs transgenes (700 bp) (M, marker; B, Blank). (C) Lane 1 indicates a double heterozygous Cdh16CreErt2::Tfebfs mouse as it carries both the Cdh16CreErt2 (507 bp Cre band, 388 bp wt band) and the Tfebfs transgenes (700 bp). (D) Real-time PCR analysis of Tfeb-3xFLAG mRNA levels performed on Cdh16Cre and Cdh16Cre::Tfebfs mice at different stages (P0, P12, P30) and on Cdh16CreErt2and Cdh16CreErt2::Tfebfs mice induced with tamoxifen at P12 and sacrificed at P90. Values are shown as the average (± SEM) of at least three animals per time point and genotype (*p<0. 05, **p<0.01, two-sided, Student’s t test). (E) Immunoblot analysis using an anti-Flag antibody to determine the expression of Tfeb-3xFLAG protein in Cdh16Cre::Tfebfs and tam-treated Cdh16CreErt2::Tfebfs mice. Each replicate is a different biological sample.DOI:http://dx.doi.org/10.7554/eLife.17047.003
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fig1s1: Generation of transgenic mouse lines with kidney-specific Tfeb overexpression.(A) Map of the transgene: Tfeb-3xFLAG was inserted after the chicken actin promoter (CAG) and the chloramphenicol acetyltransferase (CAT)-SV40pA flanked by loxP sites. The latter can be removed by CRE recombinase, resulting in the overexpression of the Tfeb gene under the control of the strong CAG promoter. Two different CRE lines were used: (1) a constitutive kidney-specific Cdh16Cre(Cadherin 16) and (2) a tamoxifen-inducible Cdh16CreErt2. (B, C) Representative genotypes of littermates. (B) Lanes 1 and 4 indicate double heterozygous Cdh16Cre::Tfebfs mice as they carry both the Cdh16Cre (420 bp CRE band, 200 bp wt band) and the Tfebfs transgenes (700 bp) (M, marker; B, Blank). (C) Lane 1 indicates a double heterozygous Cdh16CreErt2::Tfebfs mouse as it carries both the Cdh16CreErt2 (507 bp Cre band, 388 bp wt band) and the Tfebfs transgenes (700 bp). (D) Real-time PCR analysis of Tfeb-3xFLAG mRNA levels performed on Cdh16Cre and Cdh16Cre::Tfebfs mice at different stages (P0, P12, P30) and on Cdh16CreErt2and Cdh16CreErt2::Tfebfs mice induced with tamoxifen at P12 and sacrificed at P90. Values are shown as the average (± SEM) of at least three animals per time point and genotype (*p<0. 05, **p<0.01, two-sided, Student’s t test). (E) Immunoblot analysis using an anti-Flag antibody to determine the expression of Tfeb-3xFLAG protein in Cdh16Cre::Tfebfs and tam-treated Cdh16CreErt2::Tfebfs mice. Each replicate is a different biological sample.DOI:http://dx.doi.org/10.7554/eLife.17047.003
Mentions: In addition, to assess the effects of Tfeb overexpression during kidney development, we generated a second transgenic line by crossing the Tfebfs/fs mice with a mouse line that carries a tamoxifen-inducible CreErt2 element under the control of a Cdh16 promoter (Cdh16CreErt2promoter) (Lantinga-van Leeuwen et al., 2006) (Figure 1—figure supplement 1A). Cdh16Cre::Tfebfs and Cdh16CreErt2::Tfebfs double heterozygous mice were generated from these crossings (Figure 1—figure supplement 1B and C). We checked both the constitutive and inducible lines for renal Tfeb overexpression and confirmed that Tfeb mRNA levels were highly increased, and further increasing with time (Figure 1—figure supplement 1D). Consistently, immunoblot experiments revealed increased levels of Tfeb-3xFLAG protein in kidneys from Cdh16Cre::Tfebfs and Cdh16CreErt2::Tfebfs mice (Figure 1—figure supplement 1E).

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