Animal models can be useful for understanding disease pathology and as preclinical models for drug testing. As BHD patients develop renal cell carcinomas (RCCs) of varied histologies, associated with a loss of FLCN, BHD animal models could be used to study of a wide range of renal cancer subtypes. Current BHD mouse models include kidney-specific Flcn-knockouts (Chen et al., 2008, Baba et al., 2008) and ubiquitous knockouts (Hasumi et al., 2009, Hartman et al., 2009, Hudon et al., 2010). The former develop polycystic kidneys and die within three weeks, the latter can only be studied as heterozygotes with tumourigenesis dependent on a “second hit” resulting in variable penetrance and making them less suitable for drug studies.
A new mouse model from Chen et al., (2015) uses the proximal tubule-specific promoter, Sglt2, with Cre-Lox recombination (Sauer & Henderson, 1988) to knockout Flcn. Western blot confirmed the loss of Flcn in homozygous knockout (Flcnflox/flox/Sglt2-Cre) mouse proximal tubules with sustained expression in other renal tissues. These mice show bilateral renal cyst and tumour formation within the first year. Survival is impaired compared to the heterozygous knockout (Flcnflxo/+/Sglt2-Cre) or wildtype mice (<24 months vs >24 months) but is significantly longer than other kidney-specific Flcn knockout mice (<21 days; Chen et al., 2008, Baba et al., 2008).
Renal cysts developed in all homozygous knockout mice (n=100) – in over 50% within the first month – but were rarely seen in heterozygous knockout (n=5/33) or wildtype (n=1/38) mice. Immunohistochemistry confirmed that the cysts had developed from proximal tubule cells and were Flcn-deficient. Where complete Flcn knockout was not achieved, mainly in younger mice, there was no evidence of cyst formation (Chen et al., 2015).
The majority of homozygous knockout mice developed RCC (n=41/54). Lower grade chromophobe RCC, oncocytomas and hybrid RCCs (most often seen in BHD patients) were more frequently identified in younger mice with increased prevalence of higher grade papillary RCC and clear cell RCC in aging mice. Whether high grade tumours are the result of lower grade tumour transformation over time is an area that requires further investigation. Only two heterozygous knockout mice developed renal tumours (at 27 and 29 months), a lower tumourigenic frequency than the ubiquitous heterozygous Flcn knockout models (Hasumi et al., 2009).
Tumours from homozygous knockout mice were negative for Flcn and showed increased levels of AKT, mTOR, MMP and TGFβ signalling compared to healthy tissue. Activation of the mTOR pathway has previously been reported as a feature of BHD-mouse RCC development (Chen et al., 2008, Baba et al., 2008). However, a loss of Flcn has previously been linked to reduced TGFβ signalling in human UOK-257 cells (Hong et al., 2010) and mouse embryonic stem cells (Cash et al., 2011); this discrepancy could represent a species-specific role for TGFβ in mouse renal tumourigenesis or be the result of tissue-specific functions.
Chen et al., confirmed the applicability of their mice to drug testing by treating mice with rapamycin for ten months (started at two months old). Comparison of drug and sham treated mice showed a vast reduction in cyst and tumour development; the kidneys of treated mice did contain cysts but the authors suggest that these developed before treatment began. Based on this they claim “rapamycin effectively inhibited the development of new cysts and tumours”, but was unable to reverse pre-existing damage. However, as the number of cysts was only assessed at one time point it is possible that cysts continued to develop but treatment with rapamycin resulted in markedly slower growth and restricted transformation into tumours.
This new Flcnflox/flox/Sglt2-Cre knockout BHD-RCC model clearly represents the developmental stages of human renal tumour progression: cyst development (months 1-4), hyperplasia (month 5), microtumours (from 6 months) and large tumours (from 12 months).The high and constant penetrance afforded by highly-tissue specific homozygous knockout coupled with early onset and longer lifespan compared to other kidney specific Flcn-knockout mice make them a powerful tool for understanding tumourigenesis and for preclinical testing.
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