Decanoylcarnitine Inhibits Triple-Negative Breast Cancer Progression via Mmp9 in an Intermittent Fasting Obesity Mouse

Abstract

Purpose: Treatment of triple-negative breast cancer (TNBC) remains challenging. Intermittent fasting (IF) has emerged as a promising approach to improve metabolic health of various metabolic disorders. Clinical studies indicate IF is essential for TNBC progression. However, the molecular mechanisms underlying metabolic remodeling in regulating IF and TNBC progression are still unclear. Methods: In this study, we utilized a robust mouse model of TNBC and exposed subjects to a high-fat diet (HFD) with IF to explore its impact on the metabolic reprogramming linked to cancer progression. To identify crucial serum metabolites and signaling events, we utilized targeted metabolomics and RNA sequencing (RNA-seq). Furthermore, we conducted immunoblotting, real-time quantitative polymerase chain reaction (RT-qPCR), cell migration assays, lentivirus-mediated Mmp9 overexpression, and Mmp9 inhibitor experiments to elucidate the role of decanoylcarnitine/Mmp9 in TNBC cell migration. Results: Our observations indicate that IF exerts notable inhibitory effects on both the proliferation and cancer metastasis. Utilizing targeted metabolomics and RNA-seq, we initially identified pivotal serum metabolites and signaling events in the progression of TNBC. Among the 349 serum metabolites identified, decanoylcarnitine was picked out to inhibit TNBC cell proliferation and migration. RNA-seq analysis of TNBC cells treated with decanoylcarnitine revealed its suppressive effects on extracellular matrix-related protein components, with a notable reduction observed in Mmp9. Further investigations confirmed that decanoylcarnitine could inhibit Mmp9 expression in TNBC cells, primary tumors, lung, and liver metastasis tissues. Mmp9 overexpression abolished the inhibitory effect of decanoylcarnitine on cell migration. Conclusion: This study pioneers the exploration of IF intervention and the role of decanoylcarnitine/Mmp9 in the progression of TNBC in obese mice, enhancing our comprehension of the potential roles of various dietary patterns in the process of cancer treatment.

Keywords: cell migration; decanoylcarnitine; integrated multiomic analysis; intermittent fasting; triple-negative breast cancer.

Figure 1.

Impact of IF on obesity-related TNBC progression. (A) Schematic of the animal experiment procedure. Mice were fed a HFD for 8 weeks, then randomly divided into normal diet (ND, n = 6) and IF diet (InD, n = 6) groups. Two weeks later, all mice were inoculated with 1 × 10⁶ 4T1 cells in the fourth mammary pad for tumor progression analysis. (B) Graph showing body weight changes throughout the mouse modeling process. (C) Tumor volume comparison in different mice. (D) Weights of tissues from different mice. (E) Number of tumor metastases of different mice. (F) Hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) of Ki67 in various tissues. Data are mean ± SEM. *P < 0.05; **P < 0.01. Scale bars, 200 μm.

Figure 2.

Serum-targeted metabolomics in TNBC mice undergoing IF. (A) Workflow of serum-targeted metabolomics. (B) PCA plot summarizing biological sample data. (C) Bar chart classifying metabolites. Vertical axis: metabolite categories; Horizontal axis: metabolite count. (D) Bar chart of metabolic pathway classification. Vertical axis: pathway categories; horizontal axis: metabolite count. (E) Ring chart categorizing metabolites. Different colors represent various metabolite categories, with percentages indicating their proportion.

Figure 3.

Detailed analysis of differential metabolites. (A) Volcano plot showing differential metabolites. (B) Top 10 differential metabolites. (C) Heatmap illustrating correlations among differential metabolites. (D) Network diagram of metabolite correlations. (E, F) Concentrations of top upregulated metabolites (E) and downregulated metabolite (F), analyzed by HM700 metabolisome. Data were presented as mean ± SEM. *P < 0.05; **P < 0.01.

Figure 4.

Decanoylcarnitine's role in inhibiting TNBC cell migration and proliferation. (A–F) qPCR analysis of proliferative genes (Ki67, Pcna), mesenchymal genes (Cdh2, Vimentin), and epithelial genes (Cdh1, Zo-1) in 4T1 cells treated with top differential metabolites. (G) Transwell assay of cells treated with top differential metabolites. The concentration of each metabolite is 10 μM, and the vehicle used is an equal volume of DMSO. Data were presented as mean ± SEM. *P < 0.05; **P < 0.01. Scale bars, 50 μm.

Figure 5.

RNA-seq analysis of decanoylcarnitine-treated TNBC cells. (A) Schematic of RNA-seq in 10 μM decanoylcarnitine-treated 4T1 cells. (B) Differentially expressed genes in RNA-seq data. (C) Heatmap of 539 differentially expressed genes (DEGs). (D) Gene ontology (GO) enrichment for downregulated genes. (E) Transcription factor annotation for DEGs. (F) KEGG analysis of downregulated DEGs.

Figure 6.

Mmp9 is involved in inhibition of TNBC cell migration by decanoylcarnitine. (A) Volcano plot showing gene expression distribution. (B) Interaction network of the top DEGs. (C, D) qPCR and Immunoblotting analysis of Mmp9 expression in 10 μM Decanoylcarnitine-treated cells. (E) IHC analysis of Mmp9 in tissues from IF mice. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01. Scale bars, 200 μm.

Figure 7.

Mmp9-mediated inhibition of TNBC cell migration by decanoylcarnitine. (A–D) The CCK8 assay and qPCR analysis of cell proliferation, proliferative genes (Ki67, Pcna), mesenchymal genes (Cdh2, Vimentin, Fibronectin), and epithelial genes (Cdh1, Zo-1) in MDA-MB-231 (A, B) and 4T1 (C, D) cells treated with 10 nM Ilomastat for 24 hours. (E) Immunobloting assay to detect the expression of MMP9 in Mmp9 overexpressed 4T1 cells. (F) The Transwell assay to detect the effect of Mmp9 overexpression on decanoylcarnitine-inhibited cell migration. (G) The cell number per field of transwell assay. (H) Schematic depicting decanoylcarnitine's mechanism in TNBC inhibition. *P < 0.05; **P < 0.01. Scale bars, 50 μm.