Metabolic Adaptation to Nutritional Stress in Human Colorectal Cancer

Abstract

Tumor cells respond to their microenvironment, which can include hypoxia and malnutrition, and adapt their metabolism to survive and grow. Some oncogenes are associated with cancer metabolism via regulation of the related enzymes or transporters. However, the importance of metabolism and precise metabolic effects of oncogenes in colorectal cancer remain unclear. We found that colorectal cancer cells survived under the condition of glucose depletion, and their resistance to such conditions depended on genomic alterations rather than on KRAS mutation alone. Metabolomic analysis demonstrated that those cells maintained tricarboxylic acid cycle activity and ATP production under such conditions. Furthermore, we identified pivotal roles of GLUD1 and SLC25A13 in nutritional stress. GLUD1 and SLC25A13 were associated with tumor aggressiveness and poorer prognosis of colorectal cancer. In conclusion, GLUD1 and SLC25A13 may serve as new targets in treating refractory colorectal cancer which survive in malnutritional microenvironments.

Figure 1. Colorectal cancer cell lines survive under glucose depleted conditions.

(A) Cells were cultured in complete medium, which was replaced the following day with glucose- and glutamine-deficient media supplemented with 10% dialyzed fetal bovine serum and glucose (10 mM) or glutamine (2 mM), respectively. At the indicated time points, the cells were fixed in 80% methanol and stained with 0.1% crystal violet. OD determined the relative cell proliferation at 595 nm. Glc: glucose, Gln: glutamine. (B) RNA was extracted from DLD1 and HT29 cells, and the KRAS gene was PCR-amplified and sequenced. (C,D) Relative growth of DLD1 (C) and HT29 (D) under the indicated conditions. The difference in relative growth between Gln and Neither in DLD1 was significant. (E) The representative images are shown for day 3 (scale bar, 200 mm). (F) DLD1 cells survived for 2 weeks under the condition of glucose depletion. (G) The representative images are shown for day 14. (H) DLD1 and HT29 cells were cultured under the indicated conditions for 24 h. Apoptotic cells were analyzed by FACS using annexin V-FITC and propidium iodide. Representative results are shown. Data are presented as the mean ± SD of at least three independent experiments **P < 0.01).

Figure 2. Resistance to glucose-deprived conditions in colorectal cancer depended on whole genomic alterations rather than on KRAS mutation alone.

(A) The scheme shows the transfection methods of whole genomic DNA derived from a human colon cancer cell line (MA) harboring a KRAS mutation at codon 12 involving a nucleotide change from GGT to GAT. Transfection into NIH3T3 cells was performed using the calcium phosphate sedimentation method to establish Cle-H3 cells. (B,C) Relative growth of NIH3T3 (B) and Cle-H3 (C) under the indicated conditions. The difference in relative growth between Gln and Neither in Cle-H3 was significant. (D) Representative images are shown for day 3 (scale bar, 200 mm). (E) Western blot analysis of NIH3T3 cells transfected with control or KRAS overexpression (OE) vector. (F,G) Relative growth of control (F) and KRAS-overexpressing NIH3T3 cells (G) under the indicated conditions. Data are presented as the mean ± SD for at least three independent experiments (**P < 0.01).

Figure 3. Metabolomic analysis for identification of metabolic effects of oncogene KRAS.

(A,B) KRAS knockdown DLD1 cells and control cells were cultured at 3 × 10⁶ cells per 10-cm dish in 10 ml complete medium; the medium was replaced the following day with glucose(−) and glutamine(−) medium supplemented with 10% dialyzed fetal bovine serum. Glucose (10 mM) or glutamine (2 mM) was added, respectively. For hypoxia treatment, cells were incubated for 24 h with 1% O₂. Principal component analysis (A) and hierarchical cluster analysis (B) performed under the indicated conditions at the indicated time points. The red circles indicate the groups that have similar principal component scores. Data for two independent experiments are presented.

Figure 4. DLD1 cells retain TCA cycle activity under condition of glucose depletion.

DLD1 and HT29 cells were cultured at 3 × 10⁶ cells per 10-cm dish in 10 ml complete medium; this medium was replaced the following day with glucose(−) and glutamine(−) medium supplemented with 10% dialyzed fetal bovine serum. Glucose (10 mM) or glutamine (2 mM) was added, respectively. Cells incubated for 24 h were analyzed. Data are presented as mean ± SD for two independent experiments.

Figure 5. Increase in amino acids levels in colorectal cancer cells under condition of glucose depletion.

DLD1 and HT29 cells were plated at 3 × 10⁶ cells per 10-cm dish in 10 ml complete medium; the medium was replaced the following day with glucose(−) and glutamine(−) media supplemented with 10% dialyzed fetal bovine serum. Glucose (10 mM) or glutamine (2 mM) was added, respectively. Cells incubated for 24 h were analyzed. Data are presented as mean ± SD for two independent experiments.

Figure 6. Combined expression of GLUD1 and SLC25A13 was shown to be significantly associated with prognosis in colorectal cancer.

(A) Negative staining (−), positive staining (+), moderate staining (++), and strong staining (+++) for GLUD1 and SLC25A13 in colorectal cancer (scale bar, 200 mm). (B and C) Kaplan–Meier curves for recurrence-free survival (RFS) (B) and overall survival (OS) (C) according to GLUD1 expression. Differences between the two groups were evaluated by the log-rank test. Ordinate survival rate, abscissa years after surgery. (D,E) Kaplan–Meier curves for RFS (D) and OS (E) according to SLC25A13 expression. (F and G) Kaplan–Meier curves for RFS (F) and OS (G) according to combined expression of GLUD1 and SLC25A13. (H) Schematic overview of GLUD1-mediated Glu metabolism and SLC25A13-mediated ATP production. The citrin (SLC25A13) and aralar (SLC25A12; not shown) are components of aspartate-glutamate carrier (AGC), which functions collaboratively with oxoglutarate carrier (OGC) in the malate aspartate shuttle (MAS), a major intracellular pathway to transfer reducing equivalents. Glu: glutamic acid, Gln: glutamine, OAA: oxaloacetic acid, Asp: aspartic acid, α-KG: α-ketoglutaric acid, Mal: malic acid.