Colorectal polyps increase the glycolytic activity

Abstract

In colorectal cancer (CRC) energy metabolism research, the precancerous stage of polyp has remained rather unexplored. By now, it has been shown that CRC has not fully obtained the glycolytic phenotype proposed by O. Warburg and rather depends on mitochondrial respiration. However, the pattern of metabolic adaptations during tumorigenesis is still unknown. Understanding the interplay between genetic and metabolic changes that initiate tumor development could provide biomarkers for diagnosing cancer early and targets for new cancer therapeutics. We used human CRC and polyp tissue material and performed high-resolution respirometry and qRT-PCR to detect changes on molecular and functional level with the goal of generally describing metabolic reprogramming during CRC development. Colon polyps were found to have a more glycolytic bioenergetic phenotype than tumors and normal tissues. This was supported by a greater GLUT1, HK, LDHA, and MCT expression. Despite the increased glycolytic activity, cells in polyps were still able to maintain a highly functional OXPHOS system. The mechanisms of OXPHOS regulation and the preferred substrates are currently unclear and would require further investigation. During polyp formation, intracellular energy transfer pathways become rearranged mainly by increasing the expression of mitochondrial adenylate kinase (AK) and creatine kinase (CK) isoforms. Decreased glycolysis and maintenance of OXPHOS activity, together with the downregulation of the CK system and the most common AK isoforms (AK1 and AK2), seem to play a relevant role in CRC development.

Keywords: OXPHOS; Warburg effect; colonic adenoma; colorectal cancer; energy metabolism; metabolic phenotype.

Figure 1

Kinetic parameters of mitochondrial respiration in normal tissue, colorectal polyps, and colorectal cancer tissue (A) Comparative analysis of maximal ADP-stimulated respiratory rate (V_max ); and (B) apparent Michaelis-Menten constant values for ADP (K_{m (}ADP)) in control tissue (n=46), colorectal polyps (n=42), and colorectal cancer (CRC) tissue (n=57) ** p < 0,01, *** p < 0,001 (t-test).

Figure 2

Kinetic parameters of mitochondrial respiration in KRAS and BRAF mutated, and wild-type polyps (A) Comparative analysis of maximal ADP-activated respiratory rate (V_max ); and (B) apparent Michaelis-Menten constant values for ADP (K_m (ADP)) in KRAS mutated polyps (n=14), BRAF mutated polyps (n=6), and wild-type polyps (n=21) * p < 0,05, ** p < 0,01 (t-test).

Figure 3

The expression levels of genes coding for flux-controlling steps in glycolysis (A) GLUT 1 expression levels in healthy colon (n=16), colorectal polyps (n=8), and colorectal tumors (n=9) (B) Expression levels of hexokinase 1 (HK1) and hexokinase 2 (HK2) in healthy tissue (n=27), colorectal polyps (n=9), and colorectal cancer (CRC) tissue (n=27). * p < 0,05, ** p < 0,01, *** p < 0,001.

Figure 4

The expression levels of essential but non-controlling steps in glycolysis (A) The expression levels of lactate dehydrogenase A (LDHA) in healthy tissue (n=16), colorectal polyps (n=8), and colorectal cancer (CRC) tissue (n=9). (B) The xpression levels of monocarboxylate transporters MCT1 and (C) MCT2 in healthy tissue (n=16), colorectal polyps (n=8) and colorectal cancer (CRC) tissue (n=9); and expression levels of MCT4 in healthy tissue (n=24), colorectal polyps (n=8) and CRC tissue (n=22) * p < 0,05, ** p < 0,01, *** p < 0,001 (t-test).

Figure 5

The expression levels of (A) adenylate kinases AK1, AK2, AK4, and AK6; and creatine kinases (B) CKBB, CKMT1, and CKMT2 in the healthy colon (n=19), colorectal polyps (n=9), and colorectal cancer tissue (n=15) * p < 0,05, ** p < 0,01, *** p < 0,001 (t-test).

Figure 6

Changes of gene expression levels in colorectal polyps and tumors compared to normal colon mucosa. ATP production in normal colon mucosa is presented in the left. Glucose enters the cell through glucose transporters (GLUT1) and is then phosphorylated by hexokinase (HK). The glycolytic product pyruvate enters mitochondria through voltage dependent anion channel (VDAC) in mitochondrial outer membrane and is used to produce ATP through TCA cyle and oxidative phosphorylation (OXPHOS). Some of the pyruvate is converted into lactate by lactate dehydrogenase A (LDHA) and lactate is transported out of the cell using monocarboxylate transporter (MCT) 4. Additionally, colon cells uptake short fatty acids like butyrate using MCT1 and use them in fatty acid oxidation (FAO). To transport ATP and ADP between energy production and energy consumption sites, cells use creatine kinases (CKs) and adenylate kinases (AKs). In colorectal polyps, the energy metabolism is reprogrammed by increasing the glycolytic activity. This phenomenon could be facilitated by higher GLUT1, HK and LDHA expression boosting lactate production. The excessive amounts of lactate are transported out of the cell using MCT1 and MCT4 that were upregulated in polyps. Polyps also displayed higher levels of both CKs and AKs. However, colorectal cancer cells (right) seemed to depend more on OXPHOS suggested by the decreased expression levels of HKs and LDHA. Poor MCT levels indicate that the need for lactate transport is lower. Opposite to polyps, energy transport pathways in cancer cells showed signs of downregulation.