Flcn-deficient renal cyst cells can be tumourigenic

Kidney-specific Flcn knockout in mice results in the development of large polycystic kidneys and uremia causing renal failure and death within three weeks (Chen et al., 2008). As this short time frame is insufficient for substantial solid tumour growth Wu et al., (2015) extracted renal cyst cells to assess their tumourigenic potential and response to mTOR inhibitors.

Microtumour and cystic hyperplasia cells from 20-day-old Flcn-knockout mouse kidneys were cultured in vitro for at least 35 passages to establish new cell lines; a process that might preferentially select pre-malignant and malignant cells. Genotyping and western blots confirmed that these lines were Flcn-null. Within one month of inoculation into nude mice the cells developed into tumours – sarcomatoid renal cell carcinomas (SRCC) based on histology. SRCCs are highly malignant neoplasms that can evolve from all RCC subtypes but more commonly from chromophobe RCC (Parada et al., 2006) – the most common form in BHD patients.

Disrupted mTOR signaling has been identified as a cause of renal tumourigenesis and has seen in both BHD cell and animal models associated with the loss of FLCN (Baba et al., 2006, Baba et al., 2008, Hasumi et al., 2009Hartman et al., 2009). Wu et al. reported increased mTOR activity, indicated by phosphorylation of S6, in the allografted Flcn-negative tumours. Sirolimus, an mTOR inhibitor, inhibits tumour cell growth in vitro and in vivo by reducing cell proliferation and angiogenesis. The allografted mice were treated with sirolimus to assess its efficacy on the Flcn-null tumours.

Mice with tumours approximately 200mm3 were treated with 7.5mg/kg sirolimus for 21 days and their tumours grew significantly less than those in untreated controls (average 700mm3 vs. 2200mm3). Tumours treated later but with a higher dose of sirolimus -15mg/kg when 450mm3 – also showed slowed growth. To assess the timing effects on sirolimus efficacy mice with tumours only 150mm3 were treated with the lower dosage; the tumours shrank to average of only 117mm3. Treatment groups each contained 15 treated and 15 control mice.

Although these results are encouraging Wu et al. do not provide detailed information on the age of the individual mice when treatment was started. Without this it is difficult to assess the broad efficacy of sirolimus in these tumours as it is possible that variable tumorigenicity could bias responses; for example the smaller tumours, which had the greatest response, could have been inherently slower growing and potentially more susceptible to inhibition. Wu et al. also propose that, based on the dosage response seen, early treatment with a higher dosage would be more effective at reducing and reversing tumour growth but provide no data to support this. As all mice were euthanised at the end of treatment the long term effects of sirolimus treatment on tumour growth could also not be assessed.

Wu et al. also xenografted human FLCN-null UOK-257 cells into nude mice. The resulting tumours showed slowed growth when mice were treated with the higher sirolimus dose. However, the efficacy was lower than in the allograft animals: the treated tumours began to grow again, albeit slower than control tumours, after an average of 12 days. This suggests that they had escaped sirolimus-associated growth inhibition.

The UOK-257 cell line was derived from a human RCC sample (Yang et al., 2008), and like most cell lines is likely to carry uncharacterised mutations in proliferation pathways. Mutations in alternative tumourigenic pathways would enable the cells to escape inhibition of mTOR-associated growth. As such it is debatable whether such cell lines, which have been biasedly selected and accumulate mutations during in vitro culture, can be accurate representations of patient tumours and provide valid data on tumour development and drug responses.

The advanced RCC guidelines list mTOR inhibitors as a first line option for poor risk patients or as a second line option after TKI treatment (Escudier et al., 2014). However, this work and others provide evidence that mTOR inhibitors – potentially in combination with other inhibitors to limit the possibility of escape via alternative pathways – could be a valid treatment for BHD-associated renal tumours. Therefore, given the different genetic background and histology of BHD tumours, it would be advisable to assessment potential treatments in BHD patients to create specific guidelines.

  • Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, Zbar B (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proc Natl Acad Sci U S A. Oct 17;103(42):15552-7. PMID: 17028174.
  • Baba M, Furihata M, Hong SB, Tessarollo L, Haines DC, Southon E, Patel V, Igarashi P, Alvord WG, Leighty R, Yao M, Bernardo M, Ileva L, Choyke P, Warren MB, Zbar B, Linehan WM, Schmidt LS. Kidney-targeted Birt-Hogg-Dube gene inactivation in a mouse model: Erk1/2 and Akt-mTOR activation, cell hyperproliferation, and polycystic kidneys. J Natl Cancer Inst. 2008 Jan 16;100(2):140-54. PubMed PMID: 18182616.
  • Chen J, Futami K, Petillo D, Peng J, Wang P, Knol J, Li Y, Khoo SK, Huang D, Qian CN, Zhao P, Dykema K, Zhang R, Cao B, Yang XJ, Furge K, Williams BO, Teh BT. Deficiency of FLCN in mouse kidney led to development of polycystic kidneys and renal neoplasia. PLoS One. 2008;3(11) PubMed PMID: 18974783.
  • Escudier B, Eisen T, Porta C, Patard JJ, Khoo V, Algaba F, Mulders P, Kataja V; ESMO Guidelines Working Group (2012). Renal cell carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. Oct;23 Suppl 7:vii65-71. PMID: 22997456.
  • Hartman TR, Nicolas E, Klein-Szanto A, Al-Saleem T, Cash TP, Simon MC, Henske EP (2009). The role of the Birt-Hogg-Dubé protein in mTOR activation and renal tumorigenesis. Oncogene. Apr 2;28(13):1594-604. PubMed PMID: 19234517.
  • Hasumi Y, Baba M, Ajima R, Hasumi H, Valera VA, Klein ME, Haines DC, Merino MJ, Hong SB, Yamaguchi TP, Schmidt LS, Linehan WM (2009). Homozygous loss of BHD causes early embryonic lethality and kidney tumor development with activation of mTORC1 and mTORC2. Proc Natl Acad Sci U S A. Nov 3;106(44):18722-7. PubMed PMID: 19850877.
  • Parada D, Peña K, Moreira O (2006). Sarcomatoid chromophobe renal cell carcinoma. A case report and review of the literature. Arch Esp Urol. Mar;59(2):209-14. . PubMed : 16649532.
  • Wu M, Si S, Li Y, Schoen S, Xiao GQ, Li X, Teh BT, Wu G, & Chen J (2015). Flcn-deficient renal cells are tumorigenic and sensitive to mTOR suppression. Oncotarget PMID: 26418749.
  • Yang Y, Padilla-Nash HM, Vira MA, Abu-Asab MS, Val D, Worrell R, Tsokos M, Merino MJ, Pavlovich CP, Ried T, Linehan WM, Vocke CD (2008). The UOK 257 cell line: a novel model for studies of the human Birt-Hogg-Dubé gene pathway. Cancer Genet Cytogenet. Jan 15;180(2):100-9. PMID: 18206534.

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