Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a rare genetic condition caused by mutations in fumarate hydratase (FH). HLRCC patients are at risk of developing type 2 papillary renal cell carcinoma (pRCC2) which typically has an early onset with high metastatic potential. Existing targeted treatments have very limited response rates in both primary and metastatic pRCC2 tumours. Developing more effective treatments relies on preclinical models such as the new FH-deficient cell line derived by Perrier-Trudova et al., (2015).

This cell line, NCCFH1, was derived from the pleural fluid of a pRRC2 lung metastasis in a 17 year old patient who, despite initially responding to sunitinib, died 19 months after diagnosis due to disease progression. The germline mutation in FH was a small deletion in exon 8 – c.1162delA – that results in a frameshift and protein truncation, with a loss of heterozygosity in the tumours and NCCFH1. Perrier-Trudova et al. confirmed a reduction in FH mRNA levels with no protein detected by western blot and no FH-enzymatic activity.

Loss-of-function mutations in the FH enzyme result in accumulation of fumarate and subsequent activation of oncogenetic pathways including upregulation of HIF-1α signalling. Previously two other FH-deficient cell lines have been derived from patient samples: UOK 262 from a renal metastasis (Yang et al., 2010) and UOK 268 from a primary renal tumour (Yang et al., 2012). Both of these cells lines display the Warburg effect with reduced oxidative phosphorylation, increased glucose-dependence and deregulated metabolism typical of cancer cells. UOK 262 and the healthy human kidney line HK-2 were used as controls during the characterisation of the NCCFH1 line.

To assess oxidative phosphorylation and glycolysis rates Perrier-Trudova et al. measured basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). HK-2 cells have an OCR of 130-140pMole/min (30,000 cells), significantly higher than in NCCFH1 (10pMole/min) and UOK 262 (<20pMole/min) identifying them as respiratory-deficient cell lines. Both FH-deficient cells lines had an OCR/ECAR ratio that was remarkably lower than HK-2 cells indicative of dependency on glycolysis. This dependency was confirmed using proliferation assay in media containing 0.5-4.5g/litre glucose. Whilst HK-2 cells survived and proliferated in as low as 0.5-1g/litre glucose, the FH-deficient cell lines required 2g/litre glucose for optimal proliferation. NCCFH1 showed lower proliferation compared to UOK 262 at 1-1.5g/litre glucose suggestive of a higher glucose dependency.

Using microarrays Perrier-Trudova et al. identified over 200 genes that are differentially regulated in the FH-deficient cell lines compared to HK-2. Functional annotation clustering analysis identified that the loss of FH alters expression of genes associated with AKT/ERK signalling, apoptosis, metabolism, ubiquitin-proteosome pathways, extracellular matrix activity and GTPase binding activity. Overexpression of one of the upregulated genes, AKR1B10, has previously been reported as a predominant feature of pRCC2 tumours (Ooi et al., 2011). Accordingly both NCCFH1 and UOK 262 showed significantly more AKR1B10 mRNA than HK-2 cells.

Based on survival assays NCCFH1 and UOK 262 cells show limited toxicity at 72 hours when incubated with 10μM of the commonly used sunitinib, everolimus and temsirolimus, as well as 12 other cytotoxic and cytostatic drugs. Although significant toxicity was seen when FH-deficient cells were incubated with mitoxantrone, epirubicin, topotecan and bortezomib, normal HK-2 cells showed similar levels of toxicity.

Similar to BHD, HLRCC is a rare condition and consequently there are relatively few pRCC2 patients available for clinical trials. It is therefore important to have reliable and representative in vitro cellular models that can be used to increase understanding of tumourigenesis and to develop new treatments. This new FH-deficient cell line provides another preclinical model, with a different FH mutation, for hereditary pRRC2.

• Ooi A, Wong JC, Petillo D, Roossien D, Perrier-Trudova V, Whitten D, Min BW, Tan MH, Zhang Z, Yang XJ, Zhou M, Gardie B, Molinié V, Richard S, Tan PH, Teh BT, Furge KA (2011). An antioxidant response phenotype shared between hereditary and sporadic type 2 papillary renal cell carcinoma. Cancer Cell. 20(4):511-23. PMID: 22014576.
• Perrier-Trudova V, Huimin BW, Kongpetch S, Huang D, Ong P, LE Formal A, Poon SL, Siew EY, Myint SS, Gad S, Gardie B, Couvé S, Foong YM, Choudhury Y, Poh J, Ong CK, Toh CK, Ooi A, Richard S, Tan MH, & Teh BT (2015). Fumarate Hydratase-deficient Cell Line NCCFH1 as a New In Vitro Model of Hereditary Papillary Renal Cell Carcinoma Type 2. Anticancer research, 35 (12), 6639-53 PMID: 26637880.
• Yang Y, Valera VA, Padilla-Nash HM, Sourbier C, Vocke CD, Vira MA, Abu-Asab MS, Bratslavsky G, Tsokos M, Merino MJ, Pinto PA, Srinivasan R, Ried T, Neckers L, Linehan WM (2010). UOK 262 cell line, fumarate hydratase deficient (FH-/FH-) hereditary leiomyomatosis renal cell carcinoma: in vitro and in vivo model of an aberrant energy metabolic pathway in human cancer. Cancer Genet Cytogenet. 196(1):45-55. PMID: 19963135.
• Yang Y, Valera V, Sourbier C, Vocke CD, Wei M, Pike L, Huang Y, Merino MA, Bratslavsky G, Wu M, Ricketts CJ, Linehan WM (2012). A novel fumarate hydratase-deficient HLRCC kidney cancer cell line, UOK268: a model of the Warburg effect in cancer. Cancer Genet. 205(7-8):377-90 PMID: 22867999.