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

Abstract

Energy deregulation and abnormalities of tumor cell metabolism are critical issues in understanding cancer. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is an aggressive form of RCC characterized by germline mutation of the Krebs cycle enzyme fumarate hydratase (FH), and one known to be highly metastatic and unusually lethal. There is considerable utility in establishing preclinical cell and xenograft models for study of disorders of energy metabolism, as well as in development of new therapeutic approaches targeting of tricarboxylic acid (TCA) cycle enzyme-deficient human cancers. Here we describe a new immortalized cell line, UOK 262, derived from a patient having aggressive HLRCC-associated recurring kidney cancer. We investigated gene expression, chromosome profiles, efflux bioenergetic analysis, mitochondrial ultrastructure, FH catabolic activity, invasiveness, and optimal glucose requirements for in vitro growth. UOK 262 cells have an isochromosome 1q recurring chromosome abnormality, i(1)(q10), and exhibit compromised oxidative phosphorylation and in vitro dependence on anaerobic glycolysis consistent with the clinical manifestation of HLRCC. The cells also display glucose-dependent growth, an elevated rate of lactate efflux, and overexpression of the glucose transporter GLUT1 and of lactate dehydrogenase A (LDHA). Mutant FH protein was present primarily in edematous mitochondria, but with catalytic activity nearly undetectable. UOK 262 xenografts retain the characteristics of HLRCC histopathology. Our findings indicate that the severe compromise of oxidative phosphorylation and rapid glycolytic flux in UOK 262 are an essential feature of this TCA cycle enzyme-deficient form of kidney cancer. This tumor model is the embodiment of the Warburg effect. UOK 262 provides a unique in vitro and in vivo preclinical model for studying the bioenergetics of the Warburg effect in human cancer.

Fig.1

(A) Abdominal CT scan reveals a recurrent, right retroperitoneal tumor mass. (B) FDG PET imaging of HLRCC patient reveals strong glucose uptake in the lymph node tumor metastasis. (C) The in vitro growth of UOK 262 in 0.5g/L, 2.5g/L, 5g/L, and 10g/L D-glucose. The cells' proliferation and survival were glucose dependent. (D) The morphology of UOK 262 cells in culture, showing active branching and the unique HLRCC pathological features: large nuclei with predominant nucleoli and atypical perinucleolar clearing (white arrows; 40× magnification). (E) Real-time assay of the invasive potential of UOK 262 cells.

Fig. 2

(A) Hematoxylin-eosin (H&E) staining of tumor tissue from xenograft derived from UOK 262 cells (3×10⁶); (B) H&E staining of tissue from metastatic HLRCC kidney cancer removed surgically from the patients retroperitoneum; (C) Gene-specific FISH and (D) SKY analysis of UOK 262 cells: FH gene genomic probe RP11-527D7 (157 Kb) is present in both normal copies of chromosome 1 and in an i(1)(q10), band 1q42.3. Karyotyping by SKY revealed clonal and also multiple numerical and structural aberrations. Composite karyotype: 47, X, -X, +1, i(1)(q10), +5, der(21)t(15;21)(q15;p11.2), +22

Fig. 3

(A) DNA chromatograms of fumarate hydratase (FH) gene sequence surrounding nucleotide 1187. DNA was prepared from Wild type control sequence (top panel), early passage 6 UOK 262 (middle panel), and late passages 49 (lower panel) UOK 262. (B) Transmission electron microscopy images of mitochondria from UOK262 cells (top panels) and from normal human renal cortical epithelial (HRCE) cells (bottom panels). Mitochondria of UOK 262 tumor cells are edematous and show disruption of internal membrane cristae. Magnification indicated as a scale bar on each image.

Fig. 4

(A) Double immunofluorescence labeling for protein expression in situ. Mutant FH is primarily localized to mitochondria of UOK 262 cells. Alexa-Fluor^®- 488 labeling with mouse monoclonal antibody to human FH (top panel); Alexa Fluor^®- 594 labeling with rabbit antibody to human superoxide dismutase 2 (SOD2) which is a marker of the mitochondria matrix (middle panel); overlap of signals obtained with both FH and SOD2 antibodies showing co-localization FH and SOD2 (lower panel). (B) RT-PCR (top panel) and Western blot (middle panel) showing that both mutant FH mRNA and protein are present in UOK 262 cells. FH enzymatic activity in UOK 262 cells (bottom panel) is undetectable.

Fig. 5

Immunohistochemistry, real time PCR analysis, and real time measurement of mitochondrial respiration rate and lactate efflux (glycolysis rate) in UOK 262 cells: (A) GLUT-1 protein is strongly expressed at branching tumor cell membrane; (B) LDH 5 is strongly expressed in a murine xenograft of UOK 262 cells; (C) Comparison of gene expression in UOK262 cells (green columns) with HRCE normal renal epithelial cells (black columns). (D) Basal cellular respiration rate (Oxygen consumption rate, OCR) and (E) Basal extracellular acidification rate (ECAR) of UOK 262 and control cells. Results from (D) and (E) were expressed as means ±SD; n=5. Both OCR (pMoles/min) and ECAR (mpH/min) were normalized against cell number and expressed as rate per 5×10⁴ cells.