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The transcription factor EGR1 regulates metastatic potential of v-src transformed sarcoma cells

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Abstract

Metastatic spreading of cancer cells is a highly complex process directed primarily by the interplay between tumor microenvironment, cell surface receptors, and actin cytoskeleton dynamics. To advance our understanding of metastatic cancer dissemination, we have developed a model system that is based on two v-src transformed chicken sarcoma cell lines—the highly metastatic parental PR9692 and a non-metastasizing but fully tumorigenic clonal derivative PR9692-E9. Oligonucleotide microarray analysis of both cell lines revealed that the gene encoding the transcription factor EGR1 was downregulated in the non-metastatic PR9692-E9 cells. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from parental PR9692 cells. Microarray analysis of EGR1 reconstituted cells revealed the activation of genes that are crucial for actin cytoskeleton contractility (MYL9), filopodia formation (MYO10), the production of specific extracellular matrix components (HAS2, COL6A1-3) and other essential pro-metastatic abilities.

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Acknowledgments

We thank Dr. Alicia Corlett for the help with manuscript preparation, Dr. Robert Ivánek for the help with microarray analysis, and Dr. Michal Kolář for an expert advice on the statistical processing of qPCR data. This work was supported by grants AV0Z50520514 and KAN200520801 from GAAVCR and LC06061 from MEYS to M.D. and 204/07/1030 from GACR to J.P.

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Correspondence to Michal Dvořák.

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Supplementary Fig. S1.

Typical appearance of the cells cultured on uncoated and type I collagen-coated surface. The same cells as in Figure 1 shown in higher resolution. The culture of PR9692-E9 and PR9692-E9-mock cells on uncoated plastic surface is composed of floating clumps whereas the PR9692-E9-EGR1 cells adhere to the surface under the same conditions (JPEG 2474 kb)

Supplementary Fig. S2.

TRITC-falloidin staining of actin cytoskeleton in cells adhered to type I collagen-coated surface. Non-metastasizing PR9692-E9 and PR9692-E9-mock cells show absence of organized fibers and only short protruding spikes, whereas both metastasizing cell lines show polarized morphology, contain prominent parallel actin filaments across the whole cell volume and long filopodia can occasionally be observed (JPEG 1070 kb)

Supplementary Fig. S3.

qPCR analysis of differential gene expression. The relative differences in mRNA levels were assessed with ΔCt method. The data were further processed so that the value corresponding to mRNA level in PR9692 cells on uncoated plastic was set to 100% for each gene and the relative mRNA levels in the other samples were expressed as percents of this value. Each bar represents an average value of nine samples (three biological samples with three technical replicates each). Error bars, standard deviation, p values p1 (PR9692 vs. PR9692-E9) and p2 (PR9692-E9-EGR1 vs. PR9692-E9) were assessed with the Welch’s t test (JPEG 643 kb)

Supplementary Table S1.

Complete list of genes expressed differentially between PR9692 and PR9692-E9 cells. Log2 intensities, log2 change, change p values, accession numbers, gene ontology, and other data are provided (XLS 2553 kb)

Supplementary Table S2.

Complete list of genes expressed differentially between PR9692-E9 and PR9692-E9-EGR1 cells. Log2 intensities, log2 change, change p-values, accession numbers, gene ontology, and other data are provided (XLS 4385 kb)

Supplementary Table S3.

Detailed information about the genes presented in Figure 3 (results of clustering analysis). Probe set identities, gene accession numbers, gene ontology and other data are provided (XLS 55 kb)

Supplementary Table S4.

Primers used for PCR and qPCR verification of the differences in gene expression (XLS 22 kb)

Supplementary Movie SM1. Cells on a 6-well plate were cultivated in heated and humidity and CO2-conditioned incubator assembled on a microscope. Photographs were taken in 1 minute intervals in the course of approx. 24 hours. The video sequence is played 20 frames per second, so each second represents 20 minutes of cell culture. SM1 – PR9692. (MPG 78.4 MB)

Supplementary Movie SM2. Cells on a 6-well plate were cultivated in heated and humidity and CO2-conditioned incubator assembled on a microscope. Photographs were taken in 1 minute intervals in the course of approx. 24 hours. The video sequence is played 20 frames per second, so each second represents 20 minutes of cell culture. SM2 – PR9692-E9. (MPG 78.4 MB)

Supplementary Movie SM3. Cells on a 6-well plate were cultivated in heated and humidity and CO2-conditioned incubator assembled on a microscope. Photographs were taken in 1 minute intervals in the course of approx. 24 hours. The video sequence is played 20 frames per second, so each second represents 20 minutes of cell culture. SM3 – PR9692-E9-mock. (MPG 78.4 MB)

Supplementary Movie SM4. Cells on a 6-well plate were cultivated in heated and humidity and CO2-conditioned incubator assembled on a microscope. Photographs were taken in 1 minute intervals in the course of approx. 24 hours. The video sequence is played 20 frames per second, so each second represents 20 minutes of cell culture. SM4 – PR9692-E9-EGR1. (MPG 78.4 MB)

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Čermák, V., Kosla, J., Plachý, J. et al. The transcription factor EGR1 regulates metastatic potential of v-src transformed sarcoma cells. Cell. Mol. Life Sci. 67, 3557–3568 (2010). https://doi.org/10.1007/s00018-010-0395-6

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  • DOI: https://doi.org/10.1007/s00018-010-0395-6

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