Direct targeting of Sec23a by miR-200s influences cancer cell secretome and promotes metastatic colonization

Abstract

Although the role of miR-200s in regulating E-cadherin expression and epithelial-to-mesenchymal transition is well established, their influence on metastatic colonization remains controversial. Here we have used clinical and experimental models of breast cancer metastasis to discover a pro-metastatic role of miR-200s that goes beyond their regulation of E-cadherin and epithelial phenotype. Overexpression of miR-200s is associated with increased risk of metastasis in breast cancer and promotes metastatic colonization in mouse models, phenotypes that cannot be recapitulated by E-cadherin expression alone. Genomic and proteomic analyses revealed global shifts in gene expression upon miR-200 overexpression toward that of highly metastatic cells. miR-200s promote metastatic colonization partly through direct targeting of Sec23a, which mediates secretion of metastasis-suppressive proteins, including Igfbp4 and Tinagl1, as validated by functional and clinical correlation studies. Overall, these findings suggest a pleiotropic role of miR-200s in promoting metastatic colonization by influencing E-cadherin-dependent epithelial traits and Sec23a-mediated tumor cell secretome.

Figure 1

MiR-200s are associated with poor prognosis in breast cancer. (a) Kaplan-Meier curves showing the distant relapse-free survival of 210 patients with high or low expression of the entire miR-200 family (top panel), miR-429 (middle panel) and miR-200a (bottom panel) in breast tumors. P values were computed by a likelihood ratio test. (b) Box plots showing miR-200 expression levels in ten human primary and metastasis samples as assessed by qRT-PCR analysis. Data is normalized to U6 and P values were computed by Student’s t-test. (c) Heat map showing miRNA expression levels in 4T1 series. 168: 168FARN. (d) Phase contrast images (left panel) and immunofluorescence (IF) images of 4TO7 and 4T1 cells stained for E-cadherin (right panel). (e) Phase contrast images (left panel) and IF images for E-cadherin (right panel) of MCFCA1h and MCFCA1a cells. Inserts highlight the membrane localization of E-cadherin. (f) Kaplan-Meier curves showing the distant-relapse free survival of 210 patients with high or low CDH1 expression. P values were computed by a likelihood ratio test.

Figure 2

Ectopic miR-200 expression enhances spontaneous metastasis and colonization of distant organs. (a) Western blot showing expression of indicated proteins in various genetically modified 4TO7 cell lines. (b) Phase contrast and IF images of cell lines stained for E-cadherin and N-cadherin. Yellow outline emphasizes cell morphology. (c) Various cell lines were used to generate orthotopic mammary gland tumors. After one month, lungs were excised, dissociated and plated in selective media for colony formation. Average numbers of colonies were listed below representative plate images. Data represent mean ± s.e.m. from a single representative experiment out of three independent experiments. (n = 9–10). (d) Dot plot showing relative expression of puromycin resistance gene, an indicator of circulating tumor cells, by qRT-PCR analysis of genomic DNA from whole blood samples. Red lines represent median values. P = 0.02 (Student’s t-test). (e) Representative gross lung and H&E stained lung sections from animals intravenously injected with various 4TO7 cell lines. Red arrowheads and dotted lines highlight metastatic nodules. (f) Immunohistochemical staining for E-cadherin of lung nodules established from indicated cells. (g) Bar graph showing fold increase in number of pulmonary metastasis nodules for each group. Data represent mean fold increase ± s.e.m. from a single representative experiment out of three independent experiments. (n = 9–10). (h) Left: RT-PCR showing expression of Cdh1 in C1C2 cells with or without stable Cdh1knockdown. Right: Bar graph showing number of pulmonary lesions following intravenous inoculation of tumor cells. * P < 0.05, ** P < 0.01 (Student’s t-test).

Figure 3

Ectopic miR-200 expression promotes global changes in gene expression. (a) Unsupervised clustering highlighting genome-wide changes in gene expression upon miR-200 expression in 4TO7 cells. Experiment was performed twice in duplicates. (b) GSEA showing influence of miR-200 overexpression on the overall gene expression profile of 4TO7 cells. Gene sets used are the top 100 (left panel) and bottom 100 (right panel) differentially expressed genes in the test (C1, C2, C1+C2 and CDH1) vs control lines. Gene list used include all mouse genes ranked by their differential expression between 4T1 and 4TO7. Enrichment of top and bottom 100 genes from 4T1 vs. 4TO7 ranked list is shown as an example of maximum possible enrichment. ES: enrichment score. NES: normalized enrichment score. Red and blue arrows denote the relative number of core genes for each analysis. (c) Venn diagram showing significant overlap of core genes from top 100 gene sets for C1 (red circle), C2 (blue circle) and C1+C2 (green circle) lines from (b). Common core genes between C1, C2 and C1+C2 lines are presented. Cdh1 is highlighted in red font to emphasize the positive influence of miR-200s on E-cadherin expression.

Figure 4

Identification of putative miR-200 targets using mass spectrometry. (a) Scatter plot shows the comparison between protein abundance and mRNA abundance for 1562 proteins in C1+C2 versus control cells. Red dots represent genes with miR-200 target sites. (b) Comparison between protein abundance and mRNA abundance for genes containing miR-200 target sites (n =130). Genes highlighted by red dots represent those that are significantly reduced in expression at both mRNA and protein level (n = 9). Dots confined within orange dashed circle represent genes harboring miR-200 target sites showing little or no change in gene expression. (c) qRT-PCR validation of reduced expression of the nine candidate genes highlighted by red dots in panel (b) in C1+C2 line compared to the control. Data represent mean ± s.e.m. * P < 0.05 (Student’s t-test). (d) Heat map showing expression of nine candidate genes in MDA-MB-231 (left) and TSU-PR1 (right) cells upon transient transfection of miR-200s. ZEB1 and ZEB2 were included as positive controls. (e) Heat map showing negative correlation between average expression of nine-candidate gene signature and miRs-200b and -200c in NCI-60 panel of cell lines. (f) Direct targeting of eight of nine candidate genes was tested by luciferase assays in HeLa cells. Data represents percent change in normalized luciferase activity after co-transfection of miR-200s relative to the negative control pre-miR ± s.e.m. * P < 0.05, ** P < 0.01 (Student’s t-test).

Figure 5

Sec23a knockdown phenocopies miR-200s in inhibiting migration and promoting metastatic colonization. (a) Transwell migration assays were performed in triplicate and is presented as the mean migration of experimental lines as a ratio relative to parental 4TO7 cells ± s.e.m. * P < 0.05 (Student’s t-test). (b) Bar graph showing fold change in number of pulmonary nodules relative to 4TO7 parental line. Data presented as mean ± s.e.m. * P < 0.05 (Student’s t-test). (c) Representative gross lung images and H&E stained lung sections (lower panel) from mice intravenously injected with various cell lines as indicated. Red arrows were used to highlight metastatic nodules, except in Sec23a-KD samples which contained large numbers of nodules. (d) Box plot showing relative expression of SEC23A in ten human primary tumors versus ten metastases. GAPDH was used to normalize expression. P value computed by Student’s t-test. (e) Bar graph showing relative expression of SEC23A in matched primary and metastasis samples collected from six individuals. GAPDH was used to normalize expression. * P < 0.05 (Student’s t-test).

Figure 6

Sec23a knockdown disrupts secretion of proteins that are correlated with suppression of clinical metastasis. (a) Scatter plot showing correlation of secretome profiles between two different Sec23a KD lines and between Sec23a KD and C1+C2 lines. Proteins in common between different lines were used to generate the plots. Orange dots represent proteins reduced in abundance in both conditions; green dots represent proteins increased in abundance in both conditions whereas gray dots represent those proteins that show discordant expression patterns. (b) Kaplan-Meier curves showing relapse-free survival of patients with high and low median expression level of 35 genes reduced in secretion in Sec23a KD lines. (c) Bar graph showing fold increase in number of pulmonary metastases in 4TO7-derived lines stably knocking down Axl, Tinagl1 or Igfbp4 relative to vector control. (d) Representative gross lung images from animals injected via lateral tail vein with various knockdown lines from (c) along with vector control. ** P < 0.01 (Student’s t-test). (e) Kaplan-Meier plots of distant metastasis-free survival of patients in the EMC286 dataset stratified by expression of TINAGL1 (top) or IGFBP4 (bottom). P values were computed by log-rank test. (f) Schematic model of miR-200 function during metastasis. MiR-200s simultaneously target several genes including Zeb1, ZEB2 and Sec23a to inhibit local invasion but promote metastatic colonization. Targeting of Zeb1/2 influences cell-intrinsic epithelial traits whereas targeting of Sec23a modulates tumor-derived secretion of factors such as Igfbp4 and Tinagl1, which influence metastatic colonization by altering tumor-stromal interactions.