Metabolic remodeling in human colorectal cancer and surrounding tissues: alterations in regulation of mitochondrial respiration and metabolic fluxes

Abstract

The aim of the work was to evaluate whether or not there is glycolytic reprogramming in the neighboring cells of colorectal cancer (CRC). Using postoperative material we have compared the functional capacity of oxidative phosphorylation (OXPHOS) in CRC cells, their glycolytic activity and their inclination to aerobic glycolysis, with those of the surrounding and healthy colon tissue cells. Experiments showed that human CRC cannot be considered a hypoxic tumor, since the malignancy itself and cells surrounding it exhibited even higher rates of OXPHOS than healthy large intestine. The absence of acute hypoxia in colorectal carcinomas was also confirmed by their practically equal glucose-phosphorylating capacity as compared with surrounding non-tumorous tissue and by upregulation of VEGF family and their ligands. Studies indicated that human CRC cells in vivo exert a strong distant effect on the energy metabolism of neighboring cells, so that they acquire the bioenergetic parameters specific to the tumor itself. The growth of colorectal carcinomas was associated with potent downregulation of the creatine kinase system. As compared with healthy colon tissue, the tumor surrounding cells display upregulation of OXPHOS and have high values of basal and ADP activated respiration rates. Strong differences between the normal and CRC cells in the affinity of their mitochondria for ADP were revealed; the corresponding K_m values were measured as 93.6±7.7 µM for CRC cells and 84.9±9.9 µM for nearby tissue; both these apparent K_m (ADP) values were considerably (by almost 3 times) lower in comparison with healthy colon tissue cells (256±34 µM).

Keywords: AK, adenylate kinase; ANT, adenine nucleotide translocator; AP5A, diadenosine pentaphosphate; ATP-synthasome; BB-CK, – brain type creatine kinase; BSA, bovine serum albumin; CAT, carboxyatractyloside; CIMP, CpG island methylator phenotype; CK, creatine kinase; COX, cytochrome c oxidase; CRC, colorectal cancer; ETC, electron transport chain; Energy metabolism; FDG, 18-fluorodeoxyglucose; Glycolysis; HK, hexokinase; Human colorectal cancer; Km, Michaelis–Menten constant; MI, Mitochondrial Interactosome; MOM, mitochondrial outer membrane; Mitochondria; OXPHOS; OXPHOS, oxidative phosphorylation; PCr, phosphocreatine; PEP, phosphoenolpyruvate; PET, positron emission tomography; PYK, pyruvate kinase; Respiration; TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine; V0, basal respiration level; VDAC, voltage dependent anion channel; VEGF, vascular endothelial growth factor; Vm, maximal respiration rate; qPCR, real-time quantitative PCR; uMtCK, ubiquitous mitochondrial creatine kinase.

Fig. 1

Analysis of the mitochondrial respiratory chain function in permeabilized human colorectal cancer, junction area between cancer and normal mucosa (nearby) and healthy tissue samples. These studies were carried out in medium-B with 5 mM glutamate and 2 mM malate as respiratory substrates. TMPD is N,N,N′,N′-tetramethyl-phenylenediamine, and asc – ascorbate (bars are SEM, n=7, p<0.05). All respiratory substrates and inhibitors were added sequentially as indicated in the X-axis.

Fig. 2

(A) Western blot analysis of the level of voltage-dependent anion channel (VDAC) porin protein expression in human CRC, nearby and unaffected non-tumorous tissue. The VDAC bands in reference samples correspond to the molecular mass of 30 kDa. (B) Sample of Coomassie-stained PVDF membrane (cutoff between 35 and 63 kDa) after Western blot (loading control). (C) The level of VDAC expression in CRC and nearby tissues was normalized to that in unaffected (control) intestinal tissue. Mean values from 8 patients with clinically-diagnosed CRC; bars are SEM, * p<0.05.

Fig. 3

Q-PCR analysis of the levels of mRNA expression for hexokinase 1 and 2 in human CRC and surroundings tissues; bars are SEM; p=0.06 for HK 1 and p=0.01 for HK 2. Average results for 29 CRC patients.

Fig. 4

(A) Oxygraphic analysis of coupling of HK to OXPHOS in permeabilized CRC, nearby and control (healthy large intestine) tissue samples. The addition of 10 mM glucose to CRC fibers in the presence of 0.1 mM Mg-ATP caused a stimulatory effect on mitochondrial respiration in nearby and tumor tissue. (B) The comparison of glucose indexes of all permeabilized tissues samples; bars are SEM, n=5, * p<0.05.

Fig. 5

Western blot analysis of the level of various β-tubulin isotypes: relative expression compared to control. Bars are SEM, n=7, ^⁎p<0.05. Additional data on Supplementary Fig. 2.

Fig. 6

(A) Q-PCR analysis of the mRNA expression for some vascular endothelial growth factors and their receptors in human CRC and surrounding tissue samples. (B) VEGF(A-C) gene expressions normalized to the unaffected tissue. Bars are SEM, n=29, * p<0.05.

Fig. 7

Interrelationships between the level of VEGF-A expression in tissue samples of CRC patients and their respiratory parameters; rates of maximal ADP-activated respiration (V_m) and K_m values for ADP. (A) The relationship between the level of VEGF-A expression and V_m values for healthy (control) large intestine tissue samples. (B) Correlation between the level of VEGF-A and apparent K_m (ADP) value for nearby non-tumorous tissue fibers. On these figures each data point corresponds to the individual patient. Note: such interrelationships were not registered for colorectal cancer samples.

Fig. 8

Oxygraphic analysis of the functional coupling between adenylate kinase (AK) catalyzed processes and OXPHOS in permeabilized CRC, nearby and adjacent healthy intestine tissue samples. (A) Representative tracing of rates of O₂ consumption by CRC fibers; addition of 2 mM AMP in the presence of ATP led to activation of mitochondrial respiration due to formation of ADP in AK reactions. The involvement of AK(s) in stimulation of mitochondrial respiration was confirmed by subsequent addition of diadenosine pentaphosphate (AP5A)-an inhibitor of AK; as can be seen, the administration of AP5A resulted in a strong decrease in the rate of O₂ consumption by CRC samples. Similar experiments were performed with healthy colon and nearby tissue samples. Outer mitochondrial intactness was controlled by effect of exogenously-added cytochrome c (Cyt); usually, the stimulating effect of Cyt on mitochondrial respiration was <10%. CAT is carboxyatractyloside – a selective inhibitor of adenine nucleotide translocator. (B) Efficiency of the coupling was estimated by means of AK index. Bars are SEM, n=10; * p<0.05.

Fig. 9

Cutoff analysis of apparent K_m (ADP) for control-tumor (A), and control-nearby (B) permeabilized samples. V_m values cutoff analysis was also performed for these two datasets: (C) control-tumor and (D) control-nearby. For these calculations samples were taken from 46 patients.

Fig. 10

The dependence of the normalized values of respiration rates of CRC, nearby and unaffected colon tissue permeabilized samples: double reciprocal Lineweaver–Burk plots (n=9).