Rac1 promotes the reprogramming of glucose metabolism and the growth of colon cancer cells through upregulating SOX9

Abstract

Metabolic reprogramming is the survival rule of tumor cells, and tumor cells can meet their high metabolic requirements by changing the energy metabolism mode. Metabolic reprogramming of tumor cells is an important biochemical basis of tumor malignant phenotypes. Ras-related C3 botulinum toxin substrate 1 (Rac1) is abnormally expressed in a variety of tumors and plays an important role in the proliferation, invasion, and migration of tumor cells. However, the role of Rac1 in tumor metabolic reprogramming is still unclear. Herein, we revealed that Rac1 was highly expressed in colon cancer tissues and cell lines. Rac1 promotes the proliferation, migration, and invasion of colon cancer cells by upregulating SOX9, which as a transcription factor can directly bind to the promoters of HK2 and G6PD genes and regulate their transcriptional activity. Rac1 upregulates the expression of SOX9 through the PI3K/AKT signaling pathway. Moreover, Rac1 can promote glycolysis and the activation of the pentose phosphate pathway in colon cancer cells by mediating the axis of SOX9/HK2/G6PD. These findings reveal novel regulatory axes involving Rac1/SOX9/HK2/G6PD in the development and progression of colon cancer, providing novel promising therapeutic targets.

Keywords: Rac1; SOX9; glycolysis; metabolic reprogramming; pentose phosphate pathway.

FIGURE 1

Rac1 promotes the malignant biological behaviors of colon cancer. (A) Rac1 expression in colon cancer cell lines. The mRNA and protein expression of Rac1 in normal colonic epithelial cells (NCM460) and colon cancer cells with different invasive potential (HT‐29, HCT116, SW480, SW620, and DLD1) were detected by quantitative real‐time PCR (qRT‐PCR) and western blot analysis. (B) Rac1 overexpression and knockdown validation. Rac1 protein expression levels were measured by qRT‐PCR and western blot analysis. (C–H) The effect of altered Rac1 expression on colon cancer cell migration, invasion, and proliferation. Rac1 overexpression promoted migration, invasion, and proliferation of colon cancer cells HT‐29, and Rac1 interference inhibited migration, invasion, and proliferation of colon cancer cells SW620. (I, J) Growth of subcutaneous tumors after subcutaneous injection of colon cancer cells stably overexpressing Rac1 or stably interfering with Rac1. Data are representative images or expressed as the mean ± SD of each group of samples analyzed in triplicate from three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001. NC, negative control; NS, no statistical difference; OD, optical density.

FIGURE 2

Rac1 enhances glycolysis and the pentose phosphate pathway (PPP) in colon cancer cells. (A) Gene Set Enrichment Analysis of enriched glycolysis‐related genes in high Rac1 expressing colon cancer of the GSE78093 dataset. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of 879 DEGs. (C–H) Rac1 overexpression increased glucose consumption, lactate production, intracellular glucose‐6‐phosphate (G6P) level, and glucose‐6‐phosphate dehydrogenase (G6PD) activity and decreased the ratio of NADP⁺/NADPH in HT‐29 cells; however, after Rac1 silencing, the results were opposite. (I) After Rac1 overexpression or silencing, Western blot was used to detect the effects of glycolysis‐related enzymes hexokinase 2 (HK2), pyruvate kinase isoenzyme 2 (PKM2), and lactate dehydrogenase A (LDHA) and PPP‐related enzymes G6PD and 6‐phosphogluconate dehydrogenase (6PGD) protein expression levels in HT‐29 and SW620 colon cancer cells. (J) Immunohistochemistry was used to detect the expression of Rac1, HK2, and G6PD in colon cancer tissues and tumor sections (magnification 200×, scale bars 50 μm; magnification 400×, scale bars 20 μm). Data are representative images or expressed as the mean ± SD of each group of samples analyzed in triplicate from three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001. NS, no statistical difference; TK1, thymidine kinase 1.

FIGURE 3

Rac1 upregulates the expression of SOX9 in colon cancer cells. (A) Volcano plot and heatmap analysis of differentially expressed genes in GSE78093. Red indicates upregulation and green indicates downregulation, and the box in the volcano figure is identified as the SOX9 gene. (B) Western blot analysis was used to detect the effect of Rac1 overexpression or silencing on SOX9 protein expression in colon cancer cell lines HT‐29, HCT116, SW620, and DLD1. (C) Immunohistochemistry (IHC) was used to detect the expression of SOX9 in colon cancer tissues and xenografts (magnification 200×, scale bars 50 μm; magnification 400×, scale bars 20 μm). (D) IHC analysis of SOX9 expression in 184 colon cancer tissues. (E) Correlation between the relative levels of Rac1 and SOX9, determined by IHC in 184 colon cancer tissues. ***p < 0.001. NC, negative control.

FIGURE 4

SOX9 promotes colon cancer cell proliferation, invasion, and migration, and glycolysis and the pentose phosphate pathway (PPP). (A) SOX9 overexpression and knockdown validation. SOX9 mRNA and protein expression levels were measured by quantitative real‐time PCR and western blot analysis. (B–D) SOX9 overexpression significantly enhanced the colony formation and proliferation of colon cancer cells, while SOX9 silencing significantly inhibited colon cancer cell colony formation and proliferation. (E, F) SOX9 overexpression significantly enhanced the invasion and migration of colon cancer cells, and interference with SOX9 inhibited the invasion and migration of colon cancer cells (magnification 200×, scale bars 50 μm; magnification 400×, scale bars 20 μm). (G–K) SOX9 overexpression increased glucose consumption, lactate production, intracellular glucose‐6‐phosphate (G6P) level and glucose‐6‐phosphate dehydrogenase (G6PD) activity of colon cancer cells HT‐29 and decreased the NADP⁺/NADPH ratio in HT‐29 cells; however, silencing SOX9 yielded the opposite results (L) Western blot analysis was used to detect the effect of SOX9 overexpression or silencing on the protein expression levels of glycolysis‐related enzymes hexokinase 2 (HK2), pyruvate kinase isoenzyme 2 (PKM2), and lactate dehydrogenase A (LDHA) and PPP‐related enzymes G6PD and 6‐phosphogluconate dehydrogenase (6PGD) in HT‐29 and SW620 colon cancer cells. (M) Regulation of SOX9 overexpression on HK2 and G6PD promoter activities was detected in 293T, HT‐29, and SW620 cells. Data are representative images or expressed as the mean ± SD of each group of samples analyzed in triplicate from three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001. NC, negative control; NS, no statistical difference; OD, optical density.

FIGURE 5

Rac1 promotes colon cancer malignant biological behaviors through SOX9. (A–C) SOX9 overexpression partially reversed the inhibitory effect of Rac1 silencing on colon cancer cell colony formation and proliferation. (D–G) SOX9 overexpression partially reversed the inhibitory effect of Rac1 silencing on invasion and migration of colon cancer cells (magnification 200×, scale bars 50 μm; magnification 400×, scale bars 20 μm). (H–L) Overexpression of SOX9 significantly restored glucose consumption, lactate production, increased intracellular glucose‐6‐phosphate (G6P) level and glucose‐6‐phosphate dehydrogenase (G6PD) activity, and decreased NADP⁺/NADPH ratio in Rac1‐silenced colon cancer cells. (M) Western blot analysis showed that Rac1 overexpression enhanced the expression of glycolysis‐related enzymes hexokinase 2 (HK2), pyruvate kinase isoenzyme 2 (PKM2), and lactate dehydrogenase A (LDHA), and pentose phosphate pathway‐related enzymes G6PD and 6‐phosphogluconate dehydrogenase (6PGD) in HT‐29 cells, which were attenuated or abolished by SOX9 silencing. Data are representative images or expressed as the mean ± SD of each group of samples analyzed in triplicate from three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001. NC, negative control; NS, no statistical difference; OD, optical density.

FIGURE 6

Rac1 upregulated the expression of SOX9 through the PI3K/AKT signaling pathway. (A) Western blot analysis was used to detect the effect of Rac1 overexpression or silencing on PI3K, p‐AKT (Ser473), and SOX9 protein expression. (B) Rac1 overexpressing/silencing colon cancer cells were treated with LY294002 (10 μg/ml), an inhibitor of the PI3K/AKT signaling pathway, or 740Y‐P (20 μg/ml), an agonist, to further detect PI3K, AKT, p‐AKT, and SOX9 protein expression. (C–I) Overexpression of Rac1 reversed the inhibitory effects of PI3K inhibitor LY294002 on colon cancer cell proliferation, invasion, and migration, whereas silencing Rac1 weakened the promoting effects of PI3K agonist 740Y‐P on colon cancer cell proliferation, invasion, and migration (magnification 200×, scale bars 50 μm; magnification 400×, scale bars 20 μm). (J–N) Overexpression of Rac1 partially reversed the inhibition of glucose consumption, lactate production, intracellular glucose‐6‐phosphate (G6P) level, glucose‐6‐phosphate dehydrogenase (G6PD) activity and the promoting effect on the intracellular NADP⁺/NADPH ratio by the PI3K inhibitor LY294002. Data are representative images or expressed as the mean ± SD of each group of samples analyzed in triplicate from three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001. NC, negative control; NS, no statistical difference.

FIGURE 7

Increased levels of Rac1 activate the PI3K/AKT signaling pathway, thereby increasing SOX9 expression in colon cancer cells. Increased SOX9 expression activates the hexokinase (HK2) and glucose‐6‐phosphate dehydrogenase (G6PD) promoter and promotes the expression of HK2 and G6PD at the transcriptional level, then enhances glycolysis and the pentose phosphate pathway to enhance proliferation, invasion, and migration. We further revealed novel regulatory axes involving Rac1/SOX9/HK2/G6PD in the development and progression of colon cancer, providing novel promising therapeutic targets.