Methylmalonic acid promotes colorectal cancer progression via activation of Wnt/β-catenin pathway mediated epithelial-mesenchymal transition

Abstract

Background: It has been manifested in several studies that age-related metabolic reprogramming is associated with tumor progression, in particular, colorectal cancer (CRC). Here we investigated the role of upregulated metabolites of the aged serum, including methylmalonic acid (MMA), phosphoenolpyruvate (PEP), and quinolinate (QA), in CRC.

Methods: Functional assays including CCK-8, EdU, colony formation and transwell experiments were used to ascertain which upregulated metabolite of elderly serum was related to tumor progression. RNA-seq analysis was conducted to explore the potential mechanisms of MMA-induced CRC progression. Subcutaneous tumorigenesis and metastatic tumor models were constructed to verify the function of MMA in vivo.

Results: Among three consistently increased metabolites of the aged sera, MMA was responsible for tumorigenesis and metastasis in CRC, according to functional assays. The promotion of Epithelial-mesenchymal transition (EMT) was observed in CRC cells treated with MMA, on the basis of protein expression of EMT markers. Moreover, combined with transcriptome sequencing, Wnt/β-catenin signaling pathway was activated in CRC cells treated with MMA, which was verified by western blot and qPCR experiments. Furthermore, animal assays demonstrated the pro-proliferation and promotion of metastasis role of MMA in vivo.

Conclusion: We have identified that age-dependent upregulation of MMA in serum promoted the progression of CRC via Wnt/β-catenin signaling pathway mediated EMT. These collective findings provide valuable insights into the vital role of age-related metabolic reprogramming in CRC progression and propose a potential therapeutic target for elderly CRC.

Keywords: Age; Colorectal cancer; Epithelial–mesenchymal transition; Methylmalonic acid; Wnt; β-catenin.

Fig. 1

MMA promotes cell proliferation in CRC cells. A, B CCK-8 assay showed that MMA was the most significant metabolite promoting the proliferation of both HCT116 (A) and SW480 (B) cells. C–F EdU assay indicated that MMA was significant for the increasing proliferation in both HCT116 (C, E) and SW480 (D, F) cells. Magnification: × 200. G–I Colony formation assay indicated that cell colony formation was increased in both HCT116 (G, H) and SW480 (G, I) cells. (**P < 0.01, ***P < 0.001, ****P < 0.0001)

Fig. 2

MMA promotes cell migration, invasion and regulates EMT in CRC cells. A–F Transwell assays indicated that MMA promoted the migration and invasion of both HCT116 (A–C) and SW480 (D–F) cells most remarkably. (G–J) Western blot analysis of alterations in protein levels of EMT -related markers showed that epithelial markers were downregulated and mesenchymal markers were upregulated in both HCT116 (G, I) and SW480 (H, J) cells treated with MMA. (**P < 0.01, ***P < 0.001, ****P < 0.0001)

Fig. 3

Transcriptomic analysis in HCT116 cells treated with MMA. A Gene Ontology (GO) analysis showed that MMA positively regulated cell population proliferation in HCT116 cells treated with MMA. B KEGG pathway enrichment analysis revealed that Wnt signaling pathway may be involved in MMA-induced HCT116 cells. C The volcano plot visualized the genes that were significantly changed ≥ 1.5 fold in HCT116 cells treated with MMA

Fig. 4

MMA drives EMT in CRC cells through Wnt/β-catenin signaling. A, B Wnt signaling pathway related genes, including FZD3, DKK1, PRKCA and FZD4, were upregulated in both HCT116 (A) and SW480 (B) cells treated with MMA, as observed with gene set enrichment analysis (GSEA) and RT-PCR. C–F Western blot analysis of the protein levels of Wnt/β-catenin signaling -related proteins showed that the expression of β-catenin increased while p-GSK-3β (Ser9) decreased in both HCT116 (C, D) and SW480 (E, F) cells treated with MMA. (**P < 0.01, ***P < 0.001, ****P < 0.0001)

Fig. 5

MMA promotes tumor growth in vivo. A The images of primary tumor samples obtained from mice subcutaneously injected with HCT116 cells treated with MMA and control cell groups. B, C The relative tumor volumes (B) and weights (C) at the endpoint were analyzed (n = 5). D, E Western blot analysis of the tumor samples of EMT-related proteins showed that epithelial markers were downregulated and mesenchymal markers were upregulated in the MMA-induced group. F, G Western blot analysis of the tumor samples of Wnt/β-catenin signaling-related proteins showed that the expression of β-catenin increased while p-GSK-3β (Ser9) decreased in the MMA-induced group. (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 6

MMA promotes tumor metastasis in vivo. A–D Representative IHC images showing expression of Ki67 (B), E-cadherin (C) and N-cadherin (D) in subcutaneous tumor tissues. E, F Bioluminescence intensity of the metastases in mice that were injected through the tail vein with mCherry- luciferase HCT116 cells (treated with MMA or ddH₂O for 10 days). G, H Representative images of metastatic lung tumors (G) and the number of lung tumors were quantitatively analyzed (H). (*P < 0.05, ***P < 0.001)