MicroRNA-30c targets cytoskeleton genes involved in breast cancer cell invasion

Abstract

Metastasis remains a significant challenge in treating cancer. A better understanding of the molecular mechanisms underlying metastasis is needed to develop more effective treatments. Here, we show that human breast tumor biomarker miR-30c regulates invasion by targeting the cytoskeleton network genes encoding twinfilin 1 (TWF1) and vimentin (VIM). Both VIM and TWF1 have been shown to regulate epithelial-to-mesenchymal transition. Similar to TWF1, VIM also regulates F-actin formation, a key component of cellular transition to a more invasive mesenchymal phenotype. To further characterize the role of the TWF1 pathway in breast cancer, we found that IL-11 is an important target of TWF1 that regulates breast cancer cell invasion and STAT3 phosphorylation. The miR-30c-VIM/TWF1 signaling cascade is also associated with clinical outcome in breast cancer patients.

Fig. 1. miR-30c inhibits invasion of breast cancer cells

aqRT-PCR anlayses of miR-30c expression in triple-negative patient tumor-derived metastatic xenograft model (M1) and M1-derived less metastatic tumor model passaged in mice (M1-less met). U6 and RNU44 were used as internal controls. bInvasion of MDA-MB-231 and BT-20 cells transiently transfected with oligos of miR-30c or scrambled control (Scr), assessed by transwell invasion assays (n=6, *p<0.05, student’s t-test). cCell growth analyses on Day 1 and 5 of MDA-MB-231 cells, after transfections of mock, scramble (Scr) and miR-30c (100nM). dDown-regulated expression levels of VIM, TWF1 and IL-11 36hrs after transfection of miR-30c in MDA-MB-231 cells, measured by real-time PCR analyses. Scr, the scrambled control. ****p<0.00001, **p<0.001, ***p<0.0001 (n=3). eImmunoblots of VIM (top black band) and TWF1 (bottom green band) along with β-actin controls with protein lysates of MDA-MB-231 cells 36 hrs after transfection of mock control, scrambled (Scr), and miR-30c respectively (full blots see supplementary Fig S2) fLuciferase reporter activity assays of Hek293T cells co-transfected with scrambled (Scr) or miR-30c and the luciferase vector containing the 3’UTR of VIM or TWF1. **p<0.01 compared to Scr.

Fig. 2. Cytoskeleton genes important for miR-30c to regulate invasion

aIimages of F-actin staining of MDA-MB-231 cells inhibited by gene knockdowns mediated by siRNAs of VIM (1000x, green for F-actin staining and blue for DAPI staining). Scr, scrambled control. Cells were harvested 36 hrs after transfections. bOverexpression of TWF1 or VIM restored the invasion inhibited by miR-30c in MDA-MB-231 cells. **p<0.01 (n=4). cLeft panel: knockdown of TWF1 by siRNAs (siTWF1) suppressed the invasion of MDA-MB-231 cells and the inhibited invasion was reversed by overexpression of human IL-11 cDNA. Cell invasion were measured 24 hours after two transient transfections for four combinations: scrambled/vector control, scrambled/IL-11, siTWF1/vector, and siTWF1/IL-11. *p<0.05 (n=4). Right panel: Full image of the immunoblots of TWF1 (bottom green band) and beta-actin (top red band) with protein lysates of MDA-MB-231 cells 36hrs after transfections of scrambled (Scr) and siTWF1 respectively. dImmunoblots of phosphor-STAT3 (Y705) (green band), total STAT3 (black band) and β-actin control (red band) with protein lysates of MDA-MB-231 cells 36 hrs after transfections of mock control, scrambled (Scr), and miR-30c respectively (full blots see supplementary Fis. S3). eImmunoblots for pSTAT3 (Y705) (upper green band), total STAT3 (lower green band) and the loading control β-actin (red band) with lysates of MDA-MB-231 cells transfected with siTWF1, scrambled control (Scr) and siIL-11 (full blots see supplementary Fis. S3)

Fig. 3. miR-30c regulates metastasis of breast tumor in vivo

aLung metastasis analyses. Left panels: representative bioluminescence images of labeled breast tumors (top) and lung metastases (bottom). Right panel: a box plot of lung metastasis index between vector control group and miR-30c-overexpressing group (n=11, p<0.05, Wilcoxon rank sum test). Lung metastases were assessed by bioluminescence after lungs dissected from mice at the end point and were presented by normalized metastasis index, the total flux from the lungs divided by the total flux from the mammary tumors. bImages of immunohistochemistry staining against GFP for both L2G-vector-transduced and 30c-overexpressing primary breast tumor sections as well as respective mouse lung sections (40x). The invasive fronts of L2G tumors were shown with open arrows and the defined or contained boundary of 30c-expressing tumors were pointed by oval-arrows. GFP+ metastatic lesions were absent in the lungs of mice bearing 30c-overexpressing tumors. Scale bars=50 μm in all images. cReduced immunohistochemistry staining against human VIM in 30c-overexpressing primary breast tumor sections, compared to L2G-vector-transduced tumor sections (100x). Scale bars=20 μm in the images

Fig. 4. Clinical relevance of the miR-30c signaling pathway

aScatter plots between VIM single gene expression and the expression of miR-30c in GSE22220 and GSE22216 (n=210). Pearson correlation was estimated in each comparison and plots were drawn using the scatterplot function found in the Car package (http://cran.r-project.org). bBox plots of TWF1 expression compared between nodal metastasis positive (n=30) versus nodal-metastasis-negative (n=13) clinical breast tumors (UC set). A student’s t-test was used to calculate p values. cSignaling pathway scheme of miR30-TWF1(VIM)-IL-11-pSTAT3 regulation of breast cancer metastasis