MVP-mediated exosomal sorting of miR-193a promotes colon cancer progression

Abstract

Exosomes are emerging mediators of intercellular communication; whether the release of exosomes has an effect on the exosome donor cells in addition to the recipient cells has not been investigated to any extent. Here, we examine different exosomal miRNA expression profiles in primary mouse colon tumour, liver metastasis of colon cancer and naive colon tissues. In more advanced disease, higher levels of tumour suppressor miRNAs are encapsulated in the exosomes. miR-193a interacts with major vault protein (MVP). Knockout of MVP leads to miR-193a accumulation in the exosomal donor cells instead of exosomes, inhibiting tumour progression. Furthermore, miR-193a causes cell cycle G1 arrest and cell proliferation repression through targeting of Caprin1, which upregulates Ccnd2 and c-Myc. Human colon cancer patients with more advanced disease show higher levels of circulating exosomal miR-193a. In summary, our data demonstrate that MVP-mediated selective sorting of tumour suppressor miRNA into exosomes promotes tumour progression.

Figure 1. Identification of exosome miRNA profile that represents primary colon cancer and metastatic colon cancer in the liver.

(a) Schematic diagram for isolation of extracellular vesicles (EVs) from colon cancer CT26 cell line with multimodal imaging report. CT26 cells stably transduced with a lentiviral vector expressing membrane-bound Gaussia luciferase (GlucB) and biotin ligase (BirA). (b) Venn diagram summarizing unique and shared exosomal miRNAs detected in the tissues of naïve colon, primary colon cancer and metastatic mouse colon cancer in the liver using miRNA microarray data (n=5 mice per group). (c) Microarray data visualization by scatter plot comparing exosomal miRNAs detected in primary colon cancer (x axis) and metastatic colon cancer in the liver at day 3 (y axis) after a CT26 cell intrasplenic injection. (d) Heat map depicting changes in miRNAs with a statistically significant (P<0.05) change in the exosomal miRNAs from normal mouse colon, primary colon cancer tissue and metastatic colon cancer in the liver at days 3, 7 and 14 after injection of CT26 cells (n=3 mice per group). All tumour-derived exosomes were isolated with streptavidin magnetic beads. Microarray analysis results (e) and qPCR verification (f) of selected exosomal miRNAs from the source as described in d. (g) qPCR analysis of the plasma- (left panel) or faeces- (right panel) derived exosomes from the source are depicted in d. *P<0.05 versus naïve colon; ^#P<0.05 versus primary colon cancer (two-tailed t-test). Data are representative of three independent experiments (error bars, s.e.m.).

Figure 2. miRNA profile from exosomes is different from their donor cells.

(a) Comparative analysis of the miRNome in exosomes and exosome donor tissues using a microarray. miRNAs from exosomes and exosome donor tissues, including primary colon cancer (top panel) and liver metastasis (middle panel), were quantitatively analysed and expressed as a ratio of miRNAs from exosome donor tissues/exosomes. The similarity of each individual miRNA distribution in primary colon cancer and liver metastasis was analysed by overlaying each and are shown in yellow (bottom panel). Expression of miR-193a (b), miR-18a (c) and miR-21 (d) in the exosomes and exosome donor tissues, including primary colon cancer and liver metastasis of colon cancer, were assessed by qPCR. *P<0.05 (two-tailed t-test). Data are representative of three independent experiments (error bars, s.e.m.).

Figure 3. Microenvironment alters the composition of tumour exosome miRNA profiles.

Exosomal miRNAs isolated from the source listed in this figure were quantitatively analysed by qPCR. *P<0.05 (two-tailed t-test). Data are representative of three independent experiments (error bars, s.e.m.).

Figure 4. miR-193a suppresses the progression of CT26 colon cancer by directly targeting Caprin1.

(a) Schematic diagram of the putative binding sites of miR-193a in the wild-type (WT) Caprin1 3′ untranslated regions (UTR). The miR-193a seed matches in the Caprin1 3′UTR are mutated at the positions as indicated. CDS, coding sequence. (b) Potential miR-193a binding sites on Caprin1 (in grey) are broadly conserved among vertebrates. (c) Expression of miR-193a and candidate target gene Caprin1 as well as downstream genes (Ccnd2, c-myc) in CT26 cells assessed by qPCR following transfection of miR-193a mimic and control scramble miRNA. (d) Expression of candidate miR-193a and candidate target genes Caprin1 as well as downstream genes (Ccnd2, c-Myc, G3bp1) in CT26 cells assessed by western blot, following transfection of miR-193a mimic and control miRNA for 72 h. (e) Proliferation of CT26 cells with miR-193a and potential target Caprin1 knock down. Cell viability was detected from day 0 to 5 after transfection. (f) Luciferase activity assays of wild-type (WT) and mutated Caprin1 3′UTR luciferase reporters after co-transfection with miR-193a mimic, miRNA mimic control (scramble), anti-sense miR-193a or anti-sense negative control RNA in CT26 cells. The luciferase activity of each sample was normalized to the Renilla luciferase activity. The normalized luciferase activity of transfected control mimic miRNA was set as a relative luciferase activity of 1. (g) Survival of BALB/c mice after intrasplenic injection of CT26 cells with miR-193a overexpression (n=6 mice per group). (h) The cell cycle phase analysis of CT26 cells transfected with miR-193a mimic for 72 h using PI. The percentage of cells in the G1, S, and G2 phases are shown in the bar graph. (i) BrdU incorporation assay for cell cycle analysis of CT26 transfected with miR-193a mimic and control miRNA. Error bars represent s.e.m. *P<0.05 and **P<0.01 (two-tailed t-test). Each data point was measured in triplicate (error bars, s.e.m.).

Figure 5. Sorting of miR-193a from cell to exosomes through major vault protein (MVP).

(a) Biotin-miR-193a complex was pulled down from whole cell extracts using streptavidin beads and then analysed by electrophoresis followed by Coomassie blue staining (left panel). MALDI-TOF analysis of tryptic peptides (right panel) from the band indicated (left panel). (b) Western blot analysis expression of MVP proteins from before (top panel) and after streptavidin pulldown (bottom panel) of lysates of CT26 cells transfected with Bio-miR-193a or control miRNA. (c) MVP knockout (KO) CT26 cells were generated using the CRISPR/Cas9 system. qPCR-quantification of mature miR-193a, MVP, Caprin1, CyclinD and c-MYC expressed in CT26 cells (left panel) and CT26 exosomes (right panel) after the cells were treated as indicated. (d) Western blot analysis showing the level of MVP, Caprin1, CCND2 and c-MYC in cell lysates treated as indicated. (e) Proliferation of MVP KO CT26 cells treated as indicated. Cell viability was detected from day 0 to 5 after transfection. (f) Schematic representation (left panel) of treatment schedule as indicated. Representative livers (middle top panel) (metastatic nodules shown by arrows) and H&E-stained sections of livers (middle bottom panel, × 400 magnification, scale bar 200 μm) from tumour-bearing BALB/c mice (n=5 per group). Liver weight (right, top panel) and number of metastatic foci in liver (right bottom panel) were quantitatively analysed. (g) Mature miR-193a in tumour tissue (left panel) and tumour exosomes (middle panel) was quantified by qPCR. Survival analysis of BALB/c mice after intrasplenic injection of CT26 cells treated as indicated (right panel) (n=9 per group). (h) Representative images of xenografts in SW620 tumour-bearing nude mice (left panel) (n=5 mice per group). Changes of tumour volumes in an SW620 xenograft model (right panel). Liver tumour volume was used to evaluate tumour size using the following formula: nodule volume=(width)² × length/2. (i) qPCR-quantification of mature miR-193a in exosomes and tissues of tumour in SW620 xenograft mice. *P<0.05 (two-tailed t-test); NS represents non-significance. Each data point was measured in triplicate (error bars, s.e.m.).

Figure 6. Induction of exosome miR-193a in peripheral blood increases the risk of metastatic colon cancer in the liver of patients.

(a) qPCR-quantification of mature miR-193a, miR-126a, miR-148a and miR-196b in exosomes of plasma collected from colon cancer patients with (n=15) or without (n=25) liver metastasis. (b) qPCR analysis of miR-193a level in the exosomes from peripheral blood of colon cancer patients without metastasis and follow-up investigation carried out six months after diagnosis. (c) Representative H&E-stained sections of colon tumour tissue (× 200 magnification) from patients at various cancer stages. Scale bars, 200 μm (left panel). qPCR analysis of miR-193a, miR-126a and miR-148a expression in colon cancer tissue and adjacent non-tumour tissue from the same patients (right panel). (d) Representative MVP (red) expression in patient tumour sections with Alexa Fluor 594 dye labelling anti-MVP antibodies (red) visualized with confocal microscopy. (e) Western blot analysis showing the level of MVP, Caprin1, Cyclin D and c-MYC in colon cancer tissue (c) and adjacent non-tumour tissue (a) from the same patient at various stages (I, II and III) as indicated. GAPDH used as a loading control. Data are representative of three independent experiments. *P<0.05 and **P<0.01 (two-tailed t-test; error bars, s.e.m.); NS represents non-significance.

Figure 7. Proposed model for the mechanism of colon cancer metastasis to the liver involves exporting miR-193a via exosomes sorted by MVP.

Abbreviations: MVB, multivesicular bodies; RISC, RNA-induced silencing complex.