CRC-derived exosomes containing the RNA binding protein HuR promote lung cell proliferation by stabilizing c-Myc mRNA

Abstract

HuR overexpression is related to poor survival in patients with colon cancer. HuR overexpression leads to stabilization of tumor-promoting mRNAs by binding to 3'UTR-resident AREs. Exosomes, nanosized lipid bilayer vesicles, mediate many steps in cancer progression. The potential role of exosomal HuR in colon cancer lung metastasis is unclear. HuR expression was assessed immunohistochemically in tumor tissue samples from 20 patients with metastatic or nonmetastatic colon cancer and colon cancer lung metastasis and benign lung disease samples from ten patients. Exosomes were isolated from HCT116 WT and HuR KO colon cancer cells, and uptake of PKH67- and PKH26-labeled exosomes by BEAS-2B cells was evaluated using fluorescence and confocal microscopy. C-Myc and p21protein and mRNA levels were measured by western blotting and RT-qPCR, respectively. In clinical patients, HuR overexpression was significantly enhanced in colon tissues of patients with lung metastasis. HuR expression was higher in lung tissue with metastasis of colonic origin than with benign lung disease. The effect of HuR-containing CRC exosomes compared to HuR-deficient exosomes on wound closure was observed as enhanced proliferation. BEAS-2B cell migration and invasion were enhanced after HuR-containing exosomes treatment. BEAS-2B cells showed similar uptake of PKH67 (HCT116 WT)- and PKH26 (HCT116 HuR KO)-labeled exosomes. Exosomal HuR stabilized c-Myc mRNA and downregulated p21 expression, leading to G1/S transition, in human bronchial epithelial cells. HuR overexpression is associated with lung metastasis in colon cancer patients. Exosomal HuR derived from colon cancer cells alter the biological effect on normal lung epithelial cells.

Keywords: colon cancer; exosomes; extracellular vesicle; human antigen R; proliferation.

Figure 1.

HuR expression in CRC patients with metastasis was detected using IHC. (a) CRC patients without metastasis (magnification, 200×) and (b) CRC patients with metastasis (magnification, 200×). (c) HuR IRS in CRC patients (stage II–III and stage IV). (d) HuR expression in benign lung disease and (e) HuR expression in lung metastases from the colon. (E) HuR IRS in lung metastases from the colon and benign lung disease. The student’s t-test was used to analyze differences between the two groups. The data are presented as the mean ± SEM value. **p < .01. BLD: benign lung disease, LMC: lung metastasis from the colon.

Figure 2.

Identification and characterization of HCT116 exosomes. (a) Western blot analysis of HCT116 WT cells and HCT116 HuR KO cells showed the absence of the HuR protein in HCT116 HuR KO cells. (b) Western blot analysis of exosomes isolated from HCT116 WT and HuR KO cell supernatants showing the presence of the traditional exosomal markers CD81, HSP90 and flotillin-1, but absence of calnexin, in both cells lines, with absence of the HuR protein specifically in HuR KO cells. (c and d) Electron micrographs of the exosomes revealed rounded structures with a size of approximately 50–150 nm. The scale bar indicates 100 nm.

Figure 3.

Exosomal HuR promotes the migration and invasion of BEAS-2B cells. Migration assay of BEAS-2B cells (5 × 10³ cells/well) incubated with HCT116 WT and HuR KO exosomes (20 µg/ml) or TGF-β1 (5 ng/ml) compared with LHC-9 medium as a control for 72 hours. Cells that migrated into the lower chamber (falcon cell culture insert) were photographed (a) and quantified (c). Matrigel invasion assay of BEAS-2B cells (5 × 10³ cells/well) incubated with HCT116 WT and HuR KO exosomes (20 µg/ml) or TGF-β1 (5 ng/ml) compared with LHC-9 medium as a control for 72 hours. Cells that invaded through the Matrigel were photographed (b) and quantified (d).TGF-β1 as a positive control. For each condition, cells were counted from 5 different fields. The student’s t-test was used to analyze differences between the two groups. *p < .05, **p < .01, ***p < .001. The data are presented as the mean ± SEM values. All experiments were repeated at least three times.

Figure 4.

Exosomal HuR promotes the migration and proliferation of BEAS-2B cells. Alterations in migration induced by HCT116 WT and HuR KO exosomes (20 µg/ml) or TGF-β1 (5 ng/ml) were evaluated by a wound healing assay (a and b). (c) An MTT assay was performed to evaluate the proliferation of BEAS-2B cells stimulated with HCT116 WT or HuR KO exosomes (20 µg/ml) or TGF-β1 (5 ng/ml). Scale bar, 400 µm. The student’s t-test was used to analyze differences between the two groups. One-way ANOVA followed by Dunnett’s test was used to analyze differences among three groups. *p < .05, **p < .01, ***p < .001. The data are presented as the mean ± SEM values. All experiments were repeated at least three times.

Figure 5.

Uptake of HCT116 WT and KO exosomes by BEAS-2B cells. Ten micrograms of PKH67-labeled HCT116 WT exosomes, PKH26-labeled HCT116 HuR KO exosomes or a PKH67/26-PBS control were added per 5 × 10⁴ BEAS-2B cells and incubated at 37°C for 24 hours. Uptake of the fluorescently labeled exosomes by BEAS-2B cells was detected with fluorescence microscopy and confocal microscopy (at 24 hours). (a) Uptake of PKH67/26-PBS control, PKH67-labeled HCT116 WT exosomes (green) and PKH26-labeled HCT116 HuR KO exosomes (red) in BEAS-2B cells at 37°C was evaluated with fluorescence microscopy. Nuclei were stained with DAPI (blue) (scale bar, 100 μm). (b) Uptake of PKH67-labeled HCT116 WT exosomes and PKH26-labeled HCT116 HuR KO exosomes was evaluated with spinning disc confocal microscopy (scale bar, 50 μm). Nuclei were stained with DAPI (blue). (c) Exosomes uptake was quantified by determining the fluorescence intensity, and intensity values are shown as the means ± SEMs (N = 3). The student’s t-test was used to analyze differences between two groups.n.s., no significant difference; u.d., undetected.

Figure 6.

Exosomal HuR promotes cell cycle progression in BEAS-2B cells by inhibiting p21 expression. (a and b) Cell cycle histograms of unexposed BEAS-2B cells and BEAS-2B cells incubated with HCT116 WT exosomes or HuR KO exosomes (20 µg/ml). (c) BEAS-2B cells were incubated with HCT116 WT or HuR KO exosomes (20 µg/ml). p21, Cyclin A and Cdk2 expression was assessed by Western blotting, and to normalize protein loading, samples were also probed for β-Actin. (d) Relative p21 mRNA expression in BEAS-2B cells treated with HCT116 WT or HuR KO exosomes (20 µg/ml) for 72 hours. (e) c-Myc and p53 expression were assessed by Western blotting, and to normalize protein loading, samples were also probed for β-Actin. (f) Relative c-Myc mRNA expression in BEAS-2B cells treated with HCT116 WT or HuR KO exosomes (20 µg/ml) for 72 hours. (g) The decay rates of c-Myc mRNA and GAPDH in BEAS-2B cells, as indicated, were assessed by RT-qPCR after treatment with HCT116 WT or KO exosomes for 72 hours and inhibition of transcription with actinomycin D. The data are presented as the mean ± SEM values. The student’s t-test was used to analyze differences between the two groups.*p < .05, **p < .01; n = 3.

Figure 7.

Schematic diagram showing that colon cancer cells can release exosomes that can be taken up by BEAS-2B cells. In BEAS-2B cells, these exosomes can stabilize c-Myc mRNA and influence the expression of p21. p21 expression, therefore, decreases in the cells and accelerates cell cycle progression. Eventually, exosomal HuR from colon cancer cells causes proliferation, migration and invasion of lung cells.