miR-155, identified as anti-metastatic by global miRNA profiling of a metastasis model, inhibits cancer cell extravasation and colonization in vivo and causes significant signaling alterations

Abstract

To gain insight into miRNA regulation in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using global miRNA profiling, 28 miRNAs were found to exhibit significantly altered expression between isogenic metastasizing and non-metastasizing cancer cells, with miR-155 being the most differentially expressed. Highly metastatic mesenchymal-like CL16 cancer cells showed very low miR-155 expression, and miR-155 overexpression in these cells lead to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. Our experiments addressing the underlying mechanism of the altered tumor burden revealed that miR-155-overexpressing CL16 cells were less invasive than CL16 control cells in vitro, while miR-155 overexpression had no effect on cancer cell proliferation or apoptosis in established lung tumors. To identify proteins regulated by miR-155 and thus delineate its function in our cell model, we compared the proteome of xenograft tumors derived from miR-155-overexpressing CL16 cells and CL16 control cells using mass spectrometry-based proteomics. >4,000 proteins were identified, of which 92 were consistently differentially expressed. Network analysis revealed that the altered proteins were associated with cellular functions such as movement, growth and survival as well as cell-to-cell signaling and interaction. Downregulation of the three metastasis-associated proteins ALDH1A1, PIR and PDCD4 in miR-155-overexpressing tumors was validated by immunohistochemistry. Our results demonstrate that miR-155 inhibits the ability of cancer cells to extravasate and/or colonize at distant organs and brings additional insight into the complexity of miR-155 regulation in metastatic seeding.

Keywords: LNA miRNA microarray; cancer; colonization; in vivo metastasis cell line model; miR-155.

Figure 1. Heat map depicting unsupervised hierarchical clustering of metastatic and non-metastatic cell lines based on the differentially expressed miRNA measured by microarray and validation of miRNA alteration by qRT-PCR

A. Top: Blue: Non-metastatic cell line NM-2C5, Green: Low metastatic cell line M-4A4, Red: Intermediate metastatic cell line LM3. Red squares in the hierarchical clustering represent higher expression and green squares lower expression of a given miRNA. B. Relative fold change of nine miRNAs in the non-metastatic and metastatic cell line groups measured by microarray and qRT-PCR. Values above one correspond to higher expression in the non-metastatic vs. metastatic cell line groups. Standard deviation is measured between the two biological replicates, each performed in triplicates.

Figure 2. miR-155 decreases the tumor burden in lungs after i.v. injection

CL16-miR-155 or CL16-Ctrl cells (7.8 × 10⁵) were injected into the tail vein of groups of female CB-17 SCID mice and tumor burden in lungs was monitored by bioluminescence imaging using an IVIS Spectrum instrument. A. IVIS images of the animals (photon radiance per area from the lung region) from the initial study (n = 6 in each group) at week 3, and B. comparison of the two groups using the Mann-Whitney statistical test (p = 0.0087). C. Increased tumor burden in lungs over time in the initial experiment measured by bioluminescence imaging starting one week after tumor cell injection. D, E. For the second animal study (n = 6 in each group), the IVIS scans (photon radiance per area from the lung region) at week 3 were also compared using the Mann-Whitney test (p = 0.041). F. Tumor growth in lungs in the second experiment measured by bioluminescence imaging over time starting one week after tumor cell injection. G. The difference in lung tumor burdens between the two groups of animals in the initial experiment was also visualized by staining the excised and FFPE lungs using an anti-human vimentin antibody. H. Quantitative evaluation of the differences in pulmonary foci/nodules (n = 6) in the initial experiment. Only tumors larger than 100 μm were included. (*p < 0.05).

Figure 3. miR-155 overexpression inhibits invasion of metastatic CL16 cancer cells in vitro, but has no effect on proliferation or apoptosis

A. Immunohistochemical staining of lung tumors derived from i.v. injected CL16-miR-155 or CL16-Ctrl cells for the apoptotic marker cleaved Caspase-3 showed very few apoptotic cells and no difference between the two groups. B. Staining of the same lung tumors for the proliferation marker Ki-67 showed similar frequency of Ki-67-positive tumors cells in the two groups. C. CL16-miR-155 cells showed significantly decreased invasion capability compared to CL16-Ctrl (student t-test, p = 0.028) when evaluated in an in vitro invasion assay. A representative experiment out of 3 is shown. D. In the same experiment, no difference in proliferation in vitro was observed between the CL16-miR-155 and CL16-Ctrl cells.

Figure 4. No difference in morphology, E-cadherin or vimentin expression was observed between CL16-miR-155 and CL16-Ctrl cells

A. Phase-contrast images showing that both CL16-miR-155 and CL16-Ctrl cells exhibit mesenchymal-like morphology in vitro. Objective 10x, numerical aperture NA 0.3. Scale bar 100 μm. B. Staining for E-cadherin and vimentin in metastasis from i.v. injection model and cells grown in vitro as determined by immunohistochemistry. Objective 100x, numerical aperture NA 1.25 Scale bar 20 μm.

Figure 5. IPA network based on proteins exhibiting altered expression between CL16-miR-155- and CL16-Ctrl-derived xenograft tumors

A. Network 1 with the highest score was related to Lipid Metabolism, Small Molecule Biochemistry, and Cellular Movement and B. Network 2 with the second highest score was related to Cell-To-Cell Signaling and Interaction, Cellular Growth and Proliferation, Cell Death and Survival. Red symbols indicate increased expression and green Red symbols indicate lower expression in CL16-miR-155- vs CL16-Ctrl-derived xenograft tumors.

Figure 6. Lower expression of putative miR-155 targets in xenograft tumors with high miR-155 expression

Immunohistochemical staining of ALDH1A1 A. PIR B. and PDCD4 C. in tumors derived from CL16 cells transduced with either miR-155 or Ctrl vector. Expression of the three targets in non-metastatic NM-2C5 (high miR-155) and metastatic CL16 cells (low miR-155) grown in vitro was also evaluated. Objective 40x, Scale bar 50 μm.