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

Human CRC cells HCT116 and SW480 were purchased from Cell Bank of Chinese Academy of Sciences in Shanghai. HCT116 cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Yeasen Biotechnology, Shanghai, China) and 1% penicillin–streptomycin (Gibco). SW480 cells were cultured in L15 medium (Fuheng, Shanghai, China) supplemented with 10% FBS and 1% penicillin–streptomycin. All cell lines were maintained in a thermostatic incubator with 5% CO₂ at 37 ℃.

To explore the effects of age on the progression of CRC, HCT116 and SW480 cells were plated in normal culture media and the next day treated with the top 3 upregulated metabolites in the aged serum, including 5 mM QA (Sigma-Aldrich), 5 mM PEP (Sigma-Aldrich), 5 mM MMA (Sigma-Aldrich), or vehicle (0.1% DMSO for QA; double distilled water for MMA and PEP). For all acidic treatments, 25 mM HEPES (Sigma-Aldrich) was added to the treatment media to buffer potential changes in pH, and the media were replaced every day during the treatments.

Total RNA was extracted with TRIzol reagent (Life, USA) and reverse transcribed to cDNA using a specific cDNA synthesis kit (Yeasen). SYBR green master mix (Yeasen) was used for quantitative PCR assay (Roche). The determination conditions are as follows: pre-denaturation at 95℃ for 5 min, followed by 35 cycles of denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, and extension at 72℃ for 30 s. The gene expression results were normalized to GAPDH and analyzed using GraphPad Prism 6 software. The specific primers were shown in Additional file 1: Table S1.

Total proteins were extracted from cultured cells or tumor tissue using NP40 lysis buffer containing 1% 100 mM phenylmethanesulfonyl fluoride (Beyotime, Nantong, China). Samples were separated by 10% SDS-PAGE and transferred onto nitrocellulose (NC) filter membrane (Millipore, USA). Membranes were blocked with Tris-Buffered Saline Tween-20 buffer (TBST) containing 8% defatted milk for 1–2 h and incubated with the corresponding primary antibody (listed in Additional file 1: Table S2) at 4 ℃ overnight. The membranes were washed with TBST for three times, and then incubated with the appropriate secondary antibodies for 1 h at room temperature. After washing with TBST for three times, the membranes were exposed to enhanced chemiluminescence substrate detection solution (NCM Biotech, Suzhou, China). Image J software was used to quantify the gray value of the bands.

To assess the proliferation of HCT116 and SW480 cells, cell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), and colony formation assay were performed. CCK-8 assay was executed according to the manufacturer’s recommendations. About 5 × 10³ cells/well of HCT116 and SW480 cells were seeded in 96-well plates and incubated for 0, 24, 48, and 72 h, followed by the addition of 10 ul CCK8 reagent (Yeasen) to each well and incubated for 2 h in a constant temperature incubator. After that, the optical cell density was detected in 450 nm microplate reader (Thermo Fisher, USA).

For EDU assay, cells were treated with 50 μM EDU (1:1000, RiboBo, Guangzhou, China) medium at 37 ℃ for 2 h, then fixed with 4% paraformaldehyde for 30 min. After permeabilized with 0.5% Triton-X, the cells were successively reacted with 1 × Apollo dyeing reaction solution and Hoechst 33,342 (RiboBo) for 30 min. Finally, images were visualized using fluorescence microscope and analyzed by Image J and GraphPad Prism 6 software.

For the colony formation assay, 3 × 10³ cells were seeded in each 6-well plate cultured with normal medium for 14 days in a constant temperature incubator. The cells were fixed with 4% paraformaldehyde for 30 min and then stained with 0.25% crystal violet for 30 min.

Transwell chambers (Corning, USA) with 8 μm micropore were used. 2 × 10⁵ cells/ well for the cell migration experiments and 4 × 10⁵ cells/ well with a layer of matrigel (Becton, Dickinson) covering for the cell invasion experiments, were separately seeded into upper wells without FBS, whereas the medium in the bottom section was supplemented with 30% FBS. After incubation for 48 h, the cells that migrated or invaded to the lower chamber were fixed with 4% paraformaldehyde for 30 min and stained with 0.25% crystal violet for 30 min.

Total RNA was obtained from HCT116 cells treated with 5 mM MMA for 10 days and control groups with TRIzol^® (Takara Bio, Inc.). Total RNA was sent to Hangzhou Lianchuan Biotechnology Co., Ltd for further processing and RNA-seq analysis. Briefly, Illumina Paired End Sample Prep kits (Illumina, Inc.) were used to prepare libraries. Each cDNA library was sequenced using an Illumina Hiseq 4000 (cat. no. PE150; Illumina, Inc.). Differential expression levels of mRNA transcripts between the MMA-treated and control groups were measured and subsequent GO and KEGG analysis were performed.

Male athymic nude mice (4–6 weeks of age) were bred in laminar flow hoods using plastic cages with filter caps. The mice were maintained in compliance with Nanjing Medical University Institutional Animal Care and Use Committee (IACUC) protocols. The tumor size limit on the protocol was 15 mm on the largest dimension or 2.5 cm³ tumor volume or 10% of body weight, whichever was reached first. To achieve increased circulatory MMA concentrations and ascertain the effect of MMA on tumor growth in vivo, mice were treated with MMA ((200 μg MMA/g/day) through the drinking water.

For the in vivo xenograft model, a total of 1 × 10⁶ HCT116 cells (treated with MMA or ddH₂O for 10 days) were suspended in 100 μl PBS and subcutaneously injected into the axillary region of the mice. Four weeks after cell injection, the mice were anesthetized by inhaling carbon dioxide and sacrificed by cervical dislocation, and the volume and weight of each excised subcutaneous tumor were measured. The tumor volumes were calculated by the following formula: tumor volume (mm³) = 0.5 × length × width². Besides, tumor tissues were fixed and sectioned for immunohistochemistry of Ki67, E-cadherin and N-cadherin.

For secondary organ colonization assay in mice, 1 × 10⁵ mCherry- luciferase HCT116 cells (treated with MMA or ddH₂O for 10 days) were suspended in 100 μl PBS and injected into the tail vein. Metastases were monitored using IVIS Spectrum CT Pre-Clinical In Vivo Imaging System (Perkin-Elmer), and luminescence was measured and quantified to determine secondary organ colonization after 7 weeks.

In order to ascertain the effect of serum metabolic changes of the elderly on CRC, we treated HCT116 and SW480 cells with three metabolites respectively, including MMA, PEP and QA, which were significantly elevated in the old serum. The results of the CCK-8 assay showed that MMA was the most significant metabolite promoting the capacity of both HCT116 and SW480 cells to proliferate (Fig. 1A, B). To further investigate the promoting influence of age-induced circulatory factors on cell proliferation, the EdU assay was performed in two CRC cell lines. The results indicated that only MMA was significant for the increasing capacity of proliferation in CRC cells (Fig. 1C–F). Furthermore, the colony formation assays revealed that MMA was the most significant serum metabolite promoting cell colony formation (Fig. 1G–I). Taken together, these results demonstrated that among the top three significant up-regulated metabolites of the old sera, only MMA was responsible for promoting cell proliferation in CRC cells.

To further investigate the pro-aggressive effects of age-induced serum metabolic changes on migration and invasion of CRC cells, transwell assays were performed. It showed that MMA was the most remarkable serum metabolite to facilitate cell migration and invasion in both HCT116 and SW480 cells (Fig. 2A–F). EMT is a key process in cancer cell metastasis. According to the analysis of cellular phenotypes, EMT promotion may be involved in the pro-aggressive effects of MMA on CRC cells. Western blot was used to analyze EMT markers induced by the three metabolites in HCT116 and SW480 cells. It showed that epithelial markers, including E-cadherin and ZO-1, were remarkably downregulated only in MMA-induced cells both in HCT116 and SW480 cell lines. Meanwhile, the mesenchymal markers, including fibronectin and vimentin, was upregulated by MMA in the two cell lines. Consistent with these findings, the expression of aggressive markers,including MMP2 and Serpine1, increased significantly in MMA-induced cells compared to its expression in control cells (Fig. 2G–J). These results suggested that MMA promoted EMT-mediated migration and invasion in CRC cells.

To demonstrate how MMA promoted EMT-mediated migration and invasion in CRC cells, we conducted a global transcriptomic analysis in HCT116 cells treated with MMA for 10 days. Gene Ontology (GO) analysis showed that MMA positively regulated cell population proliferation (Fig. 3A), which was consistent with the cellular phenotypes above. KEGG pathway enrichment analysis revealed that the Wnt signaling pathway might be involved in MMA-induced cells (Fig. 3B). A volcano plot visualizing the genes that were significantly changed ≥ 1.5 fold was plotted (Fig. 3C). Furthermore, four genes related to Wnt pathway were significantly upregulated according to the gene set enrichment analysis (GSEA), including FZD3, DKK1, PRKCA and FZD4, which was validated by qPCR assay (Additional file 2: Table S3, Fig. 4A, B). It is well-known that Wnt signaling is a common upstream signal of EMT [19]. To further clarify whether MMA regulated Wnt signaling pathway, we verified the expression of the key point β-catenin and downstream protein p-GSK-3β by western blot assay, and the results showed that β-catenin increased while p-GSK-3β (Ser9) decreased only in MMA induced CRC cells (Fig. 4C–F). These results prompted that MMA facilitated the aggressivity of CRC cells by inducing EMT through Wnt/β-catenin signaling pathway.

Subcutaneous tumorigenesis model was used to assess the effects of MMA on the capacity of tumourigenicity in CRC in vivo. HCT116 cells were treated with MMA for 10 days prior to injection into the nude mice, and the volume and weight of each excised subcutaneous tumor were measured after four weeks. The results showed that MMA accelerated tumor xenograft growth in vivo (Fig. 5A–C). Western blot assay of excised tumor tissue further confirmed that MMA induced a decrease in ZO-1, E-cadherin and p-GSK-3β (Ser9) expression and an increase in ZEB-1, N-cadherin, Vimentin, Serpine1 and β-catenin expression, which demonstrated that MMA induced EMT through Wnt/β-catenin signaling pathway in CRC in vivo (Fig. 5D–G). In addition, Ki67 expression levels were significantly higher in MMA-induced tumors according to the immunohistochemistry results. Furthermore, decreased E-cadherin and increased N-cadherin expression levels were shown by immunohistochemistry in situ in the MMA-induced tumors of nude mice (Fig. 6A–D). In order to assess the effect of MMA on metastatic ability in CRC in vivo, mCherry-luciferase HCT116 cells treated with MMA or ddH₂O for 10 days were injected through the tail vein. The bioluminescence intensity of metastases in the MMA-treated mice was significantly higher than that in the control group (Fig. 6E, F). The number of lung metastases in the MMA-treated mice was significantly higher than that in the control group (Fig. 6G, H). Overall, these data supported that MMA promoted tumorigenicity and tumor metastasis in vivo.