Heart Failure Post-Myocardial Infarction Promotes Mammary Tumor Growth Through the NGF-TRKA Pathway

Figures

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  Figure 1. MI Accelerates Mammary Tumor Growth in Mice (A) The experimental setup for the syngeneic mouse model mimicking tumor comorbidity with myocardial infarction (MI). Ligation to the left ascending coronary artery induced MI or sham surgery. Implantation of mammary tumor–derived 4T1 cells occurred 2 weeks after the surgeries in female BALB/c mice. (B) Tumor growth over a 3-week period following 4T1 cell implantation. (C) Quantification of tumor volume and tumor weight 3 weeks after 4T1 cell implantation (sham = 20, MI = 15). (D) Representative images of tumor tissues immunostained with an anti-Ki67 antibody. Scale bars: 200 μm. The ratio of Ki67-positive cells is presented in the graph (right) (sham = 7, MI = 6). (E) Volcano plot of RNA sequence depicting differentially expressed genes in tumor tissues in MI-operated mice. Red and green dots represent upregulated and downregulated genes, respectively. Volcano plots depict mean log2 fold change against the −log10 false discovery rate (FDR)–adjusted P values for all expressed genes. (F) Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of RNA sequencing from tumor tissues, with bubble plot demonstrating all KEGG enrichment…

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  Figure 2. Changes in the NGF-TRKA Axis After MI in Mice (A, B) Serum nerve growth factor (NGF) concentration at (A) 2 weeks and (B) 5 weeks after sham or myocardial infarction (MI) surgery (A: sham = 6, MI = 11; B: sham = 11, MI = 11). (C, D) Messenger RNA (mRNA) expressions of Ngf in the heart at the border area (MI -B) (n = 9 in each, C) and the remote area (MI -R) (n = 6 in each, D) 3 days after MI. (E, F) NGF protein expressions in the heart at (E) the border area (MI-B) and (F) the remote area (MI-R) 3 days after MI. The densitometric analyses are shown in the graphs (n = 6 in each). (G) Tropomyosin receptor kinase A (TRKA) and AKT phosphorylation in tumor tissues of the sham-operated and MI-operated mice. The ratios of phosphorylated TRKA (p-TRKA) to total TRKA and phosphorylated AKT (p-AKT) to total AKT are quantified, and the densitometric analyses are shown in the graphs (n = 5 in each). GAPDH was used as the loading control. Data are presented as mean ± SEM. ∗P < 0.05 and ∗∗∗P < 0.001, determined by 2-tailed unpaired Student’s t test.

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  Figure 3. Essential Role of NGF/TRKA Pathway in Cell Proliferation in 4T1 Cells (A) Effects of the NGF ligand on the phosphorylation of TRKA and AKT in cultured 4T1 cells. The cells were stimulated with NGF (100 ng/mL) for the indicated time, and cell lysates were subjected to Western blot analysis. The ratios of p-TRKA to total TRKA (n = 5 in each) and p-AKT to total AKT were quantified. Densitometric analyses results are shown in the graphs (n = 3 in each). (B) Impact of NGF on 4T1 cell proliferation. The cells were stimulated with NGF for 24 hours and stained with an anti-Ki67 antibody and DAPI. Scale bars: 50 μm. Quantitative analysis of Ki67-positive cells (n = 3 in each). (C) Cell numbers were determined by MTS cell proliferation assay and expressed as a relative ratio over the vehicle group (n = 6 in each). (D) 4T1 cells were transfected with TRKA-specific small interfering RNA (siTRKA) or nontargeting control small interfering RNA (scramble) with 120 nM for 72 hours. TRKA expression was assessed by immunoblotting, and densitometric analysis is shown in the graph (n = 4 in each). (E) Role of TRKA in NGF ligand–associated cell proliferation. Transfected cells were incubated with NGF…

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  Figure 4. Inhibition of TRKA Suppresses Tumor Progression in MI Mice (A) Experimental protocol. Two weeks after sham or myocardial infarction (MI) operation, 4T1 cells were implanted into female BALB/c mice. Intraperitoneal injections of either dimethyl sulfoxide (DMSO) or GW441756, a TRKA inhibitor (10 mg/kg), were administered twice a week for 3 weeks. (B) Tumor growth over a 3-week period following cell implantation (sham + DMSO = 9, sham + GW441756 = 15, MI + DMSO = 11, MI + GW441756 = 11). (C) Quantification of tumor volume at 3 weeks after 4T1 cell implantation. (D) Tumor tissues were stained with an anti-Ki67 antibody and DAPI. Representative images and quantification of Ki67-positive cells are shown (n = 9-15). Scale bars: 100 μm. (E) The phosphorylation of TRKA and AKT in tumor tissues in the GW441756-treated mice. The ratios of p-TRKA to total TRKA and p-AKT to total AKT are quantified, and the densitometric analyses are shown in the graphs (n = 4-5). Data are presented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, determined by (B) 2-way repeated-measures analysis of variance with Tukey’s multiple comparisons test or (C to E) 2-way analysis of variance with Tukey’s…

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  Central Illustration. Impact of NGF-TRKA Pathway in Tumor Progression After MI A syngeneic mouse model was established by implanting mammary tumor-derived 4T1 cells into a BALB/c mouse with myocardial infarction (MI). Tumor volume was increased in MI mice compared with sham-operated mice. Phosphorylation of tropomyosin receptor kinase A (TRKA), upstream of PI3K-AKT signaling, in tumor tissue was upregulated in MI mice. Circulating levels of nerve growth factor (NGF), a TRKA ligand, and NGF expressions in the myocardium were elevated after MI. Administration of a TRKA inhibitor, GW441756, suppressed tumor volume in the MI mice. Data are presented as mean ± SEM. ∗P < 0.05 and ∗∗∗P < 0.001, determined by a 1-way repeated-measures analysis of variance with Bonferroni’s multiple comparisons test or a 2-way repeated-measures analysis of variance with Tukey’s multiple comparisons test.