Myristoylation of Src kinase mediates Src-induced and high-fat diet-accelerated prostate tumor progression in mice

Abstract

Exogenous fatty acids provide substrates for energy production and biogenesis of the cytoplasmic membrane, but they also enhance cellular signaling during cancer cell proliferation. However, it remains controversial whether dietary fatty acids are correlated with tumor progression. In this study, we demonstrate that increased Src kinase activity is associated with high-fat diet-accelerated progression of prostate tumors and that Src kinases mediate this pathological process. Moreover, in the in vivo prostate regeneration assay, host SCID mice carrying Src(Y529F)-transduced regeneration tissues were fed a low-fat diet or a high-fat diet and treated with vehicle or dasatinib. The high-fat diet not only accelerated Src-induced prostate tumorigenesis in mice but also compromised the inhibitory effect of the anticancer drug dasatinib on Src kinase oncogenic potential in vivo We further show that myristoylation of Src kinase is essential to facilitate Src-induced and high-fat diet-accelerated tumor progression. Mechanistically, metabolism of exogenous myristic acid increased the biosynthesis of myristoyl CoA and myristoylated Src and promoted Src kinase-mediated oncogenic signaling in human cells. Of the fatty acids tested, only exogenous myristic acid contributed to increased intracellular myristoyl CoA levels. Our results suggest that targeting Src kinase myristoylation, which is required for Src kinase association at the cellular membrane, blocks dietary fat-accelerated tumorigenesis in vivo Our findings uncover the molecular basis of how the metabolism of myristic acid stimulates high-fat diet-mediated prostate tumor progression.

Keywords: Src; diet; fatty acid; prostate cancer; protein myristoylation.

Figure 1.

Elevated Src kinase activity is associated with high-fat diet–accelerated prostate tumor growth. PC-3 human prostate cancer cells (3 × 10⁵ cells/xenograft) were subcutaneously injected into both flanks of SCID mice (n = 9). The SCID mice were fed a 10%, 45%, or 60% fat diet for 8 weeks. A and B, representative images from H&E staining of white adipose and liver tissue from host SCID mice on a 10%, 45%, or 60% fat diet. The accumulation of ectopic fat in hepatocytes and the size of adipocytes increased in mice on the 45% and 60% fat diets compared with 10% fat diet. Scale bars = 200 μm. C, images of representative tumor-bearing mice and isolated tumors. D, the size and weight of tumors were measured. E, expression levels of total Src, pSrc(Tyr-416), Erk, pErk, and GAPDH in three representative PC-3 xenografts from 10%, 45%, and 60% fat diets. The expression levels of pSrc/total Src and pErk/total Erk were quantified. #, p < 0.05; ##, p < 0.01; ###, p < 0.005.

Figure 2.

Src kinase mediates accelerated prostate tumor growth by dietary fat. A, PC-3 cells were transduced with shRNA-Control or shRNA-Src and subcutaneously injected into both flanks of SCID mice (n = 6/group). The mice were placed on a 10% or 45% fat diet for 8 weeks. Representative images of mice and of excised xenografts are shown (scale bar = 8 mm). B, the size and weight of xenografts are represented as mean ± S.E. (n = 6/group). Statistics were performed using two-way analysis of variance by the SAS system. C–F, RFP fluorescence and IHC staining of total Src kinase, pSrc(Tyr-416), and pErk1/2 of PC-3 xenografts from A (scale bars = 200 μm). G and H, the expression levels of pSrc and pErk in E and F were analyzed by image analysis software. #, p < 0.05; ##, p < 0.01; ###, p < 0.005; N.S., not significant.

Figure 3.

Dietary fat accelerates Src-induced prostate tumor progression. A, schematic of the in vivo prostate regeneration assay. Host SCID mice carrying Src(Y529F)-transduced regeneration tissues were placed on a 10% or 60% fat diet for 8 weeks and treated with vehicle or dasatinib from week 5 to week 8 (n = 4/group). UGSM, urogenital sinus mesenchyme. B, total calorie intake was not significantly different between the diet groups. C, representative images of regenerated prostate tumors. The dashed lines show the regenerated prostate grafts on the kidney (scale bar = 4 mm). D, prostate tumor weight from A represented as mean ± S.E. (n = 4/group). Asterisks indicate unpaired, two-tailed t test. E, representative H&E (panoramic view, scale bars = 400 μm) and IHC staining (selected tumorigenic region, scale bars = 100 μm) of total Src, pSrc(Tyr-416), and pErk1/2 and co-staining of CK5, a basal epithelial cell marker, CK8, a luminal epithelial cell marker, and DAPI of tumors from C. F, the expression levels of pSrc and pErk were analyzed in IHC samples based on image analysis. #, p < 0.05; ##, p < 0.01; N.S., not significant.

Figure 4.

Loss of myristoylation inhibits Src-mediated HFD-accelerated tumor progression. A, the in vivo prostate regeneration assay was performed with Src(Y529F) and Src(Y529F/G2A) under 10%, 45%, and 60% fat diets with vehicle or dasatinib treatment (75 mg/kg/day weeks 5 to 8), a similar experimental setting as described in Fig. 1. The dashed lines represent the regenerated prostate tumors grown on the kidney (scale bars = 4 mm). B–F, H&E panoramic view (B, scale bars = 300 μm) and selected region (C, scale bars = 100 μm) and IHC staining of total Src kinase, pSrc(Tyr-416), and pErk1/2 of regenerated prostate tissue (D–F, scale bars = 100 μm) in A. See also supplemental Fig. S5.

Figure 5.

Exogenous MA or an HFD increases intracellular myristoyl-CoA in cells or xenograft tumors. A, 293T+TRE/Src(Y529F) cells were treated with DMSO, DA (C10:0), LA (C12:0), MA (C14:0), or PA (C16:0) for 2 or 24 h (three repeats in each group). Con, control. B, PC-3 or DU145 cells were treated similarly for 24 h. The levels of myristoyl-CoA were analyzed by LC/MS-MS, and the levels in the control were set as 1 (three repeats in each group). C, the levels of myristoyl-CoA in PC-3 xenograft tumors from Fig. 1 were analyzed by LC/MS-MS. The amount of myristoyl-CoA was standardized to the amount of total protein in the tumors. Sixteen tumors per group were analyzed. #, p < 0.05; ##, p < 0.01. See also supplemental Fig. S6.

Figure 6.

Exogenous MA elevates myristoylated Src kinase and promotes Src oncogenic signaling. A, schematic of click chemistry to detect the myristoylation of Src kinase. Cells were grown with myristic acid-azide overnight. NMT1/2 catalyzes the cellular acylation of Src kinase. As a result, myristic acid-azide was processed intracellularly and incorporated into de novo synthesized Src kinase (Step 1). Myristic acid-azide–modified Src kinase in the cell lysate was reacted with a biotin-conjugated alkyne in vitro in a click reaction (Step 2). The biotin-myristoylated Src was immunoprecipitated and visualized by immunoblotting using streptavidin-HRP (Step 3). Total Src was visualized by immunoblotting. B, SYF1 (Src^−/−Yes^−/−Fyn^−/−) cells expressing Src(WT), Src(G2A), or control were grown in medium containing 100 μm myristic acid-azide. Myristoylated proteins were detected by a click chemistry reaction. C, identification of myristoylated Src(WT) from click chemistry reactions by immunoprecipitation (IP, using a Src antibody) and immunoblotting with streptavidin-HRP. D, SYF1 cells expressing Src(WT) were cultured with 0, 20, and 100 μm of myristic acid-azide. Myristoylated proteins and the expression of Src were detected by click chemistry reactions (left) and immunoblotting (right). WB, Western blot. E, PC-3 cells expressing control or shRNA-Src were cultured with 0, 20, or 60 μm myristic acid-azide. The arrowhead indicates the band of Src kinase. F and G, 293T cells expressing doxycycline-inducible Src(Y529F) (293T+TRE/Src(Y529F)) were grown in DMEM with 2% BSA and treated with MA (F) with/without Dox (1 μg/ml) or dasatinib (10 nm) for 24 h. PC-3 cells were grown in F-12K medium with 2% BSA and treated with 0, 100, 300, 600, or 1200 μm MA overnight (G). The levels of total Src, pSrc(Tyr-416), total FAK, pFAK (Tyr-925), total Erk, and pErk1/2 were determined by immunoblotting. The data represent three independent experiments.

Figure 7.

Exogenous myristate elevates the expression levels of Src kinase at the membrane fraction. A, SYF1 cells expressing Src(WT) were grown in medium containing 400 μm myristic acid. The expression levels of Src kinase in the cytosol (Cyt) and membrane (Mem) fraction and total lysate were analyzed by immunoblotting. The amount of Src expression in the cytosol fraction treated with DMSO was set as 1. B, SYF1 cells expressing Src(WT) were transduced with shRNA-NMT1 by lentiviral infection with increased amounts of lentivirus. SYF1 and SYF1 cells expressing Src(WT) and different expression levels of NMT1 were grown in medium containing 60 μm myristic acid-azide. Myristoylated proteins were detected by a click chemistry reaction. Additionally, the levels of total Src, NMT1, and GAPDH were determined by immunoblotting. The amount of myristoyl-Src expression in cells without shNMT1 was set as 1. The data represent three independent experiments.

Figure 8.

Mechanism of Src-mediated prostate tumor progression by exogenous myristic acid. Exogenous myristic acid is converted into the corresponding myristoyl-CoA. Myristoyl-CoA will be incorporated into the de novo synthesized Src kinase, which is essential for its kinase activity, by N-myristoyltransferases. The elevation of Src myristoylation enhances Src oncogenic signaling, including the phosphorylation of MAPK and FAK, thereby accelerating Src-mediated tumor progression.