Contextual extracellular cues promote tumor cell EMT and metastasis by regulating miR-200 family expression

Abstract

Metastatic disease is a primary cause of cancer-related death, and factors governing tumor cell metastasis have not been fully elucidated. Here, we address this question by using tumor cell lines derived from mice that develop metastatic lung adenocarcinoma owing to expression of mutant K-ras and p53. Despite having widespread somatic genetic alterations, the metastasis-prone tumor cells retained a marked plasticity. They transited reversibly between epithelial and mesenchymal states, forming highly polarized epithelial spheres in three-dimensional culture that underwent epithelial-to-mesenchymal transition (EMT) following treatment with transforming growth factor-beta or injection into syngeneic mice. This transition was entirely dependent on the microRNA (miR)-200 family, which decreased during EMT. Forced expression of miR-200 abrogated the capacity of these tumor cells to undergo EMT, invade, and metastasize, and conferred transcriptional features of metastasis-incompetent tumor cells. We conclude that tumor cell metastasis is regulated by miR-200 expression, which changes in response to contextual extracellular cues.

Figure 1.

mRNA profiling of tumors reveals changes consistent with EMT. (A) Differentially expressed genes as identified by mRNA expression profiling of syngeneic tumors, with yellow representing increased expression and blue indicating decreased expression versus the 393P control: (393P) metastasis-incompetent; (393LN) intermediate; (344SQ) metastasis-prone. (B) Differences in subsets of genes related to polarity and EMT. (C) Q-PCR validation of EMT markers, polarity genes, and EMT-inducing transcription factors. (White) 393P; (gray) 393LN; (black) 344SQ.

Figure 2.

Metastatic 344SQ cells form polarized epithelial spheres in 3D Matrigel culture. (A) Morphology of day 12 spheres by contrast microscopy. (B) Sphere growth versus time from plating onto Matrigel (MG); diameter measured in microns. (C) Ki-67 (green) and Topro-3 (blue) staining of developing sphere, visualized by confocal microscopy. (D) Cleaved caspase 3 staining (green) and Topro-3 (blue) of developing sphere. (E) β-catenin (green) and Topro-3 (blue) staining of developing sphere. (F) E-cadherin (green) and Topro-3 (blue) staining of developing sphere. (G) Sphere stained for ZO-1 (green), α6-integrin (red), and Topro-3 (blue). (H) Sphere stained for ParD6B (green), α6-integrin (red), and Topro-3 (blue). (I) Sphere stained for GM130 (green) and Topro-3 (blue). Bar in confocal images represents 20 μm.

Figure 3.

Spheres are plastic in response to the in vitro and in vivo environment. (A) Spheres in Matrigel. (B) Sphere purified from Matrigel and plated on tissue culture plastic. Photo taken 20 h post-plating. (C) Sphere grown on tissue culture plastic for several days. (D) Second-generation spheres grown on Matrigel. Monolayer cells grown as in C were cultured in 3D Matrigel culture for 10 d. (E–H) Spheres purified from Matrigel were reimplanted by subcutaneous injection into syngeneic animals. Photos show H&E stain of tissue sections demonstrating metastases to the lung (E), heart (F), liver (G), and kidney (H). Bar, 100 μm. (I) Spheres in Matrigel prior to TGFβ treatment. (J) Spheres treated with TGFβ for 5 d. (K) Higher-power contrast image of spheres after 5 d of TGFβ treatment. (L) Confocal image of a plane through a TGFβ-treated structure, stained for ZO-1 (green), α6-integrin (red), and Topro-3 (blue). Bar, 20 μm. (M) Spheres were treated with the TGFβ inhibitor SB431542 (10 μM) for 3 h, followed by treatment with 5 ng/mL TGFβ in the presence of inhibitor for 5 d. (N) Quantitative RT–PCR of indicated markers, shown as fold change upon TGFβ treatment. Spheres were grown as in I and J, then harvested for analysis.

Figure 4.

miR profiling of tumors reveals miR-200 family differences. (A) Volcano plot showing differences in the miRs detected by microarray for the 344SQ and 393P tumor samples profiled in Figure 1A. The −log₁₀(P-value) is plotted against the fold change difference in expression between the samples for the indicated miR probes. The box indicates probes for the miR-200 family members. (B) Heat map showing differentially expressed miRs (P < 0.01, fold change >3, either 344SQ vs. 393LN or 393P vs. 393LN; average signal >100 U across tumors), from the three tumor types profiled in Figure 1A, with yellow representing increased expression and blue indicating decreased expression versus the 393P control. (C) TaqMan RT–PCR for the miR-200 family members as indicated, relative to a miR-16 control. (Gray) 393P; (black) 393LN.; (white) 344SQ. (D) RT–PCR assay of individual miR-200 members or miR-16 control, for 344SQ spheres in Matrigel (gray), or following TGFβ treatment (black). miR levels are normalized to snoRNA-135. Assay is of structures similar to those shown in Figure 3, I and J.

Figure 5.

miR-200 family and EMT marker levels in a panel of non-small-cell lung cancer cell lines. (A) Heat map of expression patterns for miRNAs in the miR-200 family and markers of EMT in a panel of 40 human lung cancer cell lines. Correlation of each gene or miR expression with that of miR-141 is indicated by Pearson's R- and P-values to the right. (B) Volcano plot showing the log-transformed P-values plotted against the log-transformed fold change in expression between cell lines derived from primary tumors relative to those derived from metastatic sites. miRs in red are differentially expressed more than twofold, with P < 0.05. (C) Table with x- and P-values corresponding to the highlighted miRs in B.

Figure 6.

Constitutive miR-200 expression converts 2D morphology, blocks TGFβ reponsiveness, and abrogates in vitro migration and invasion. (A) 344SQ cells grown on tissue culture plastic. (B) 344SQ cells treated for 10 d with TGFβ. (C) 344SQ_200b_1 transfectants on tissue culture plastic. (D) 344SQ_200b_1 transfectants treated for 10 d with TGFβ. (E) 344SQ_vector cells grown on Matrigel. (F) 344SQ_vector cells grown on Matrigel and stimulated with TGFβ for 5 d. (G) 344SQ_200b_1 cells grown on Matrigel. (H) 344SQ_200b_1 cells grown on Matrigel and stimulated with TGFβ for 5 d. (I,J) 344SQ_200b_1 cells grown on Matrigel were imaged by confocal microscopy after staining for ZO-1 (green), α6-integrin (red), and Topro-3 (blue). Bar, 50 μm. (K) In vitro Transwell migration (black) and invasion (gray) assays for the three cell lines. (L–Q) Q-PCR analysis of indicated EMT-related genes at baseline (black) or after TGFβ treatment (gray) for the 344SQ_vector cells or two different 344SQ_200b transfectants grown in monolayer culture as in A–D.

Figure 7.

Forced miR-200 expression reverts the cells to a nonmetastatic mRNA expression pattern and prevents metastasis. (A) Expression patterns for the genes significant between 344SQ and 393P (from Fig. 1A) or between the mir-200bc/429-overexpressing 344SQ cells (344SQ_200b_1) and the 344SQ_vector control. For 344SQ/393P data set, genes are centered on 393P group; for the 200bc/429 knockup data set, genes are centered on 344SQ_vector control group. Genes represented in both heat maps are the same and have the same ordering. Gray bars to the right highlight genes predicted to be miR-200 family targets, using Targetscan (version 5.0). (B) Table of gene probe sets intersecting between the 393P/393LN/344SQ gene signatures (rows, from Fig. 1A) and the genes induced or repressed by mir-200bc/429 expression. The last column indicates the number of genes predicted to be miR-200bc/429 targets using Targetscan (version 5.0). P-values by one-sided Fisher's exact test (using the population of ∼45,000 probe sets).