Oncogenic KRAS modulates mitochondrial metabolism in human colon cancer cells by inducing HIF-1α and HIF-2α target genes - PubMed (original) (raw)

Oncogenic KRAS modulates mitochondrial metabolism in human colon cancer cells by inducing HIF-1α and HIF-2α target genes

Sang Y Chun et al. Mol Cancer. 2010.

Abstract

Background: Activating KRAS mutations are important for cancer initiation and progression; and have recently been shown to cause primary resistance to therapies targeting the epidermal growth factor receptor. Therefore, strategies are currently in development to overcome treatment resistance due to oncogenic KRAS. The hypoxia-inducible factors-1α and -2α (HIF-1α and HIF-2α) are activated in cancer due to dysregulated ras signaling.

Methods: To understand the individual and combined roles of HIF-1α and HIF-2α in cancer metabolism and oncogenic KRAS signaling, we used targeted homologous recombination to disrupt the oncogenic KRAS, HIF-1α, and HIF-2α gene loci in HCT116 colon cancer cells to generate isogenic HCT116WT KRAS, HCT116HIF-1α-/-, HCT116HIF-2α-/-, and HCT116HIF-1α-/-HIF-2α-/- cell lines.

Results: Global gene expression analyses of these cell lines reveal that HIF-1α and HIF-2α work together to modulate cancer metabolism and regulate genes signature overlapping with oncogenic KRAS. Cancer cells with disruption of both HIF-1α and HIF-2α or oncogenic KRAS showed decreased aerobic respiration and ATP production, with increased ROS generation.

Conclusion: Our findings suggest novel strategies for treating tumors with oncogenic KRAS mutations.

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Figures

Figure 1

Figure 1

Analyses of genes regulated by oncogenic KRAS, HIF-1α, HIF-2α, and both HIF-1α and HIF-2α. Expression of target genes in HCT116, HCT116HIF-1α-/-, HCT116HIF-2α-/-, HCT116HIF-1α-/-HIF-2α-/-, and HCT116WT KRAS cell lines. HK2, hexokinase 2; LDHA, lactate dehydrogenase A; MGLL, monoglyceride lipase; ANGPTL4, angiopoietin-like protein 4; and VEGFA, vascular endothelial growth factor A relative to β-actin were measured by real-time reverse transcription-PCR (n = 5). Bars, stdev. p < 0.05 by Student's t test comparing knockout cells with parental HCT116 cells. c, clone.

Figure 2

Figure 2

Analyses of metabolism gene set regulated by oncogenic KRAS and HIF. A, heatmap analysis comparing the following metabolism gene sets: HCT116 vs HCT116HIF-1α-/-, HCT116 vs HCT116HIF-2α-/-, HCT116 vs HCT116HIF-1α-/-HIF-2α-/-, and HCT116 vs HCT116WT KRAS cells. Expression values are averages of three samples. B, Venn diagrams analyzing the extent of overlap of the following metabolism gene sets: 1) HCT116 vs HCT116HIF-1α-/-, HCT116 vs HCT116HIF-2α-/-, and HCT116 vs HCT116HIF-1α-/-HIF-2α-/-; 2) HCT116 vs HCT116HIF-1α-/-HIF-2α-/- and HCT116 vs HCT116WT KRAS. Each circle represents a single gene set; and the number of genes common between the gene sets is denoted within the overlaps of the circles. C, Clonogenic survival assay of HCT116, HCT116HIF-1α-/-, HCT116HIF-2α-/-, HCT116HIF-1α-/-HIF-2α-/-, and HCT116WT KRAS cells. c, clone.

Figure 3

Figure 3

Analyses of target genes regulating phospholipids synthesis. A, overview of mitochondrial respiration and the contributory role of mitochondrial phospholipids synthesis. B, expression of genes regulating phospholipids synthesis in HCT116, HCT116HIF-1α-/-, HCT116HIF-2α-/-, HCT116HIF-1α-/-HIF-2α-/-, and HCT116WT KRAS cell lines. c, clone. ACSL5, acyl-CoA synthetase 5; AGPAT7, 1-acyl-sn-glycerol-3-phosphate acyltransferase 7; and PCK2, phosphoenolpyruvate carboxykinase 2 relative to β-actin were measured by real-time reverse transcription-PCR (n = 5). Bars, stdev. p < 0.05 by Student's t test comparing knockout cells with parental HCT116 cells. C, Expression of ACSL5, AGPAT7, and PCK2 in primary colon cancers. Gene expression, relative to β-ACTIN, in normal mucosa and primary tumor was determined by real-time RT-PCR. Log expression values were graphed as box and whiskers plots; showing median expression (horizontal line), surrounded by the first and third quartiles of those values (box), and the extreme values (whiskers). Hypothesis testing was performed using the Wilcoxon signed-rank test, with * = p < 0.05 considered statistically significant, comparing tumor to mucosa. D, western blot of ACSL5 in cell lines, with antibody to α-tubulin as loading control. c, clone. E, ACSL5 promoter activity in HCT116, HCT116HIF-1α-/-, HCT116HIF-2α-/-, and HCT116HIF-1α-/-HIF-2α-/- cells (n = 5). Diamond represents HRE site. The pGL3pro construct is a minimal promoter Firefly luciferase reporter. The ACSL5-pGL3pro construct contains an 1883 bp fragment of the 5' untranslated region of the ACSL5 gene with the HRE site. This construct is subjected to site-directed mutagenesis at the HRE site to generate the ACSL5(-HRE)-pGL3pro. Bars, stdev. p < 0.05 by Student's t test comparing transfection with pGL3pro construct versus ACSL5-pGL3pro or ACSL5(-HRE)-pGL3pro constructs.

Figure 4

Figure 4

Quantitation of total cellular (A) phosphatidyl choline (PC) and (B) cardiolipin (CL) levels in HCT116, HCT116HIF-1α-/-HIF-2α-/-, HCT116WT KRAS cells, HCT116 cells transduced with control scramble shRNA, and HCT116 cells transduced with ACSL5 shRNA (n = 3). Bars, stdev. p < 0.05 by Student's t test comparing knockout cells or shRNA transduced cells with parental HCT116 cells. Four lentiviral constructs carrying ACSL5 shRNA were tested for effectiveness in suppressing ACSL5 expression in comparison to the lentiviral construct carrying control scramble shRNA (Additional file 3, Fig. S3). Clone H45551 was chosen for subsequent experiments as it was the most effective (Additional file 3, Fig. S3).

Figure 5

Figure 5

Effects of genetic inactivation of oncogenic KRAS or both HIF-1α and HIF-2α on mitochondrial metabolism. A, change in cellular O2 consumption; B, change in MTT reduction; and C, change in intracellular ATP level in HCT116HIF-1α-/-HIF-2α-/- and HCT116WT KRAS relative to HCT116 cells (n = 5). Bars, stdev. p < 0.05 by Student's t test comparing knockout cells with parental HCT116 cells. D, cellular and mitochondrial ROS levels in HCT116, HCT116HIF-1α-/-HIF-2α-/-, and HCT116WT KRAS cells, as measured by the fluorescent dyes CM-H2DCFDA and MitoSOX.

Figure 6

Figure 6

Effects of ACSL5 gene knockdown on mitochondrial metabolism and tumorigenesis. A, change in cellular O2 consumption; and B, change in intracellular ATP level in HCT116 and LOVO cells transduced with ACSL5 shRNA relative to control scramble shRNA (n = 5). Bars, stdev. p < 0.05 by Student's t test comparing cells transduced with ACSL5 shRNA versus control scramble shRNA. C, cellular and mitochondrial ROS levels in HCT116 cells transduced with ACSL5 shRNA versus control scramble shRNA, as measured by the fluorescent dyes CM-H2DCFDA and MitoSOX.

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