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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

Biochemical analysis of ErbB signaling.Immunoblot analysis performed on P90 kidneys from Cdh16Cre::Tfebfs mice (A) and P90 Cdh16CreErt2::Tfebfs animals induced with tamoxifen at P14 (B) and at P30 (C), respectively. Each replicate is a different biological sample. ErbB was analyzed by quantifying phosphoAKT (Ser473) to total AKT, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK; graphs are the densitometry quantifications of Western blot bands normalized to wild-type line and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test).DOI:http://dx.doi.org/10.7554/eLife.17047.011
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fig3s2: Biochemical analysis of ErbB signaling.Immunoblot analysis performed on P90 kidneys from Cdh16Cre::Tfebfs mice (A) and P90 Cdh16CreErt2::Tfebfs animals induced with tamoxifen at P14 (B) and at P30 (C), respectively. Each replicate is a different biological sample. ErbB was analyzed by quantifying phosphoAKT (Ser473) to total AKT, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK; graphs are the densitometry quantifications of Western blot bands normalized to wild-type line and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test).DOI:http://dx.doi.org/10.7554/eLife.17047.011

Mentions: Based on these results, we checked the activation of both ErbB and WNT signaling pathways. No evidence for an increase in the phosphorylation of AKT and ERK1/2 kinases (Arteaga and Engelman, 2014) was detected in P30 Cdh16Cre::Tfebfs kidneys or in primary kidney cells obtained from transgenic mice (Figure 3C and D), indicating that the ErbB pathway was not induced. Erk1/2 activation, as detected by pERK1/2, was observed only at late stages (Figure 3—figure supplement 2A). The same result was observed in P14 and P30 tam-treated Cdh16CreErt2::Tfebfs mice (Figure 3—figure supplement 2B and C). Conversely, we detected increased levels of total β-catenin and CCND1 in P30 renal tissues and primary kidney cells (Figure 3C and D) and increased levels of active β-catenin and of pLRP6 (Ser1490)/ LRP6 ratio in P30 and P90 renal tissues from Cdh16Cre::Tfebfsmice (Figure 4A and B) and in P14 and P30 tam-treated Cdh16CreErt2::Tfebfs mice (Figure 4—figure supplement 1). Moreover, β-catenin and active β-catenin staining of renal sections from Cdh16Cre::Tfebfs mice was significantly enhanced (Figure 4C). These results indicate the presence of a strong activation of the WNT signaling pathway in TFEB-overexpressing mice. Interestingly, the WNT pathway is known to play a role in renal cyst development (Vainio and Uusitalo, 2000; Rodova et al., 2002) and renal tumor formation, such as in VHL syndrome (Peruzzi and Bottaro, 2006) and Wilm’s tumor (Koesters et al., 1999; Zhu et al., 2000; Kim et al., 2000). To investigate the role of TFEB in WNT pathway activation, we performed luciferase assays using a TOP-FLASH Luciferase WNT-reporter on immortalized kidney cell lines (HEK293 and HK2) co-transfected with TFEB and with both β-catenin and TCF4 plasmids to stimulate WNT signaling. Luciferase activation was significantly higher in cells transfected with TFEB compared to controls without TFEB. No changes were observed when TFEB was transfected alone or only with β-catenin (Figure 5A and B). Together these data suggest that TFEB is able to enhance WNT pathway activation.10.7554/eLife.17047.014Figure 4.Molecular and histological analysis of WNT signaling.


Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling
Biochemical analysis of ErbB signaling.Immunoblot analysis performed on P90 kidneys from Cdh16Cre::Tfebfs mice (A) and P90 Cdh16CreErt2::Tfebfs animals induced with tamoxifen at P14 (B) and at P30 (C), respectively. Each replicate is a different biological sample. ErbB was analyzed by quantifying phosphoAKT (Ser473) to total AKT, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK; graphs are the densitometry quantifications of Western blot bands normalized to wild-type line and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test).DOI:http://dx.doi.org/10.7554/eLife.17047.011
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fig3s2: Biochemical analysis of ErbB signaling.Immunoblot analysis performed on P90 kidneys from Cdh16Cre::Tfebfs mice (A) and P90 Cdh16CreErt2::Tfebfs animals induced with tamoxifen at P14 (B) and at P30 (C), respectively. Each replicate is a different biological sample. ErbB was analyzed by quantifying phosphoAKT (Ser473) to total AKT, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK; graphs are the densitometry quantifications of Western blot bands normalized to wild-type line and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test).DOI:http://dx.doi.org/10.7554/eLife.17047.011
Mentions: Based on these results, we checked the activation of both ErbB and WNT signaling pathways. No evidence for an increase in the phosphorylation of AKT and ERK1/2 kinases (Arteaga and Engelman, 2014) was detected in P30 Cdh16Cre::Tfebfs kidneys or in primary kidney cells obtained from transgenic mice (Figure 3C and D), indicating that the ErbB pathway was not induced. Erk1/2 activation, as detected by pERK1/2, was observed only at late stages (Figure 3—figure supplement 2A). The same result was observed in P14 and P30 tam-treated Cdh16CreErt2::Tfebfs mice (Figure 3—figure supplement 2B and C). Conversely, we detected increased levels of total β-catenin and CCND1 in P30 renal tissues and primary kidney cells (Figure 3C and D) and increased levels of active β-catenin and of pLRP6 (Ser1490)/ LRP6 ratio in P30 and P90 renal tissues from Cdh16Cre::Tfebfsmice (Figure 4A and B) and in P14 and P30 tam-treated Cdh16CreErt2::Tfebfs mice (Figure 4—figure supplement 1). Moreover, β-catenin and active β-catenin staining of renal sections from Cdh16Cre::Tfebfs mice was significantly enhanced (Figure 4C). These results indicate the presence of a strong activation of the WNT signaling pathway in TFEB-overexpressing mice. Interestingly, the WNT pathway is known to play a role in renal cyst development (Vainio and Uusitalo, 2000; Rodova et al., 2002) and renal tumor formation, such as in VHL syndrome (Peruzzi and Bottaro, 2006) and Wilm’s tumor (Koesters et al., 1999; Zhu et al., 2000; Kim et al., 2000). To investigate the role of TFEB in WNT pathway activation, we performed luciferase assays using a TOP-FLASH Luciferase WNT-reporter on immortalized kidney cell lines (HEK293 and HK2) co-transfected with TFEB and with both β-catenin and TCF4 plasmids to stimulate WNT signaling. Luciferase activation was significantly higher in cells transfected with TFEB compared to controls without TFEB. No changes were observed when TFEB was transfected alone or only with β-catenin (Figure 5A and B). Together these data suggest that TFEB is able to enhance WNT pathway activation.10.7554/eLife.17047.014Figure 4.Molecular and histological analysis 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 &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