Advertisement

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

  • 394 Accesses

  • 20 Citations

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.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2

References

  1. 1.

    Deryugina EI, Quigley JP (2006) Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev 25:9–34

  2. 2.

    Olson MF, Sahai E (2009) The actin cytoskeleton in cancer cell motility. Clin Exp Metastasis 26:273–287

  3. 3.

    Brooks SA, Lomax-Browne HJ, Carter TM, Kinch CE, Hall DM (2010) Molecular interactions in cancer cell metastasis. Acta Histochem 112:3–25

  4. 4.

    Yokota J (2000) Tumor progression and metastasis. Carcinogenesis 21:497–503

  5. 5.

    Yilmaz M, Christofori G (2009) EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev 28:15–33

  6. 6.

    Scheel C, Onder T, Karnoub A, Weinberg RA (2007) Adaptation versus selection: the origins of metastatic behavior. Cancer Res 67:11476–11479 (discussion 11479-80)

  7. 7.

    Gadd MA, Casper ES, Woodruff JM, McCormack PM, Brennan MF (1993) Development and treatment of pulmonary metastases in adult patients with extremity soft tissue sarcoma. Ann Surg 218:705–712

  8. 8.

    Songur N, Dinc M, Ozdilekcan C, Eke S, Ok U, Oz M (2003) Analysis of lung metastases in patients with primary extremity sarcoma. Sarcoma 7:63–67

  9. 9.

    Tournay O, Benezra R (1996) Transcription of the dominant-negative helix-loop-helix protein Id1 is regulated by a protein complex containing the immediate-early response gene Egr-1. Mol Cell Biol 16:2418–2430

  10. 10.

    Zhu X, Lin Y, Bacanamwo M, Chang L, Chai R, Massud I, Zhang J, Garcia-Barrio MT, Thompson WE, Chen YE (2007) Interleukin-1 beta-induced Id2 gene expression is mediated by Egr-1 in vascular smooth muscle cells. Cardiovasc Res 76:141–148

  11. 11.

    Shingu T, Bornstein P (1994) Overlapping Egr-1 and Sp1 sites function in the regulation of transcription of the mouse thrombospondin 1 gene. J Biol Chem 269:32551–32557

  12. 12.

    Copertino DW, Edelman GM, Jones FS (1997) Multiple promoter elements differentially regulate the expression of the mouse tenascin gene. Proc Natl Acad Sci USA 94:1846–1851

  13. 13.

    Chen SJ, Ning H, Ishida W, Sodin-Semrl S, Takagawa S, Mori Y, Varga J (2006) The early-immediate gene EGR-1 is induced by transforming growth factor-beta and mediates stimulation of collagen gene expression. J Biol Chem 281:21183–21197

  14. 14.

    Silverman ES, Khachigian LM, Lindner V, Williams AJ, Collins T (1997) Inducible PDGF A-chain transcription in smooth muscle cells is mediated by Egr-1 displacement of Sp1 and Sp3. Am J Physiol 273:H1415–H1426

  15. 15.

    Bhattacharyya S, Ishida W, Wu M, Wilkes M, Mori Y, Hinchcliff M, Leof E, Varga J (2009) A non-Smad mechanism of fibroblast activation by transforming growth factor-beta via c-Abl and Egr-1: selective modulation by imatinib mesylate. Oncogene 28:1285–1297

  16. 16.

    Padua D, Massague J (2009) Roles of TGFbeta in metastasis. Cell Res 19:89–102

  17. 17.

    Svoboda J, Dvorak M, Guntaka R, Geryk J (1986) Transmission of (LTR, v-src, LTR) without recombination with a helper virus. Virology 153:314–317

  18. 18.

    Fuerstenberg SM, Vennstrom B (1993) Versatile avian retrovirus vectors. Anal Biochem 209:375–376

  19. 19.

    Plachy J, Vilhelmova M (1984) Syngeneic lines of chickens. VII. The lines derived from the recombinants at the B complex (MHC) of Rous sarcoma regressor and progressor inbred lines of chickens. Folia Biol (Praha) 30:189–201

  20. 20.

    Svoboda J, Plachy J, Hejnar J, Karakoz I, Guntaka RV, Geryk J (1992) Tumor induction by the LTR, v-src, LTR DNA in four B (MHC) congenic lines of chickens. Immunogenetics 35:309–315

  21. 21.

    Cosset FL, Legras C, Chebloune Y, Savatier P, Thoraval P, Thomas JL, Samarut J, Nigon VM, Verdier G (1990) A new avian leukosis virus-based packaging cell line that uses two separate transcomplementing helper genomes. J Virol 64:1070–1078

  22. 22.

    Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP (2003) Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res 31:e15

  23. 23.

    Smyth GK (2004) Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3, Article 3

  24. 24.

    Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5:R80

  25. 25.

    Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B 57:289–300

  26. 26.

    Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Trush V, Quackenbush J (2003) TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34:374–378

  27. 27.

    Heyer LJ, Kruglyak S, Yooseph S (1999) Exploring expression data: identification and analysis of coexpressed genes. Genome Res 9:1106–1115

  28. 28.

    Kobayashi D, Yamada M, Kamagata C, Kaneko R, Tsuji N, Nakamura M, Yagihashi A, Watanabe N (2002) Overexpression of early growth response-1 as a metastasis-regulatory factor in gastric cancer. Anticancer Res 22:3963–3970

  29. 29.

    Suzuki T, Inoue A, Miki Y, Moriya T, Akahira J, Ishida T, Hirakawa H, Yamaguchi Y, Hayashi S, Sasano H (2007) Early growth responsive gene 3 in human breast carcinoma: a regulator of estrogen-meditated invasion and a potent prognostic factor. Endocr Relat Cancer 14:279–292

  30. 30.

    Totsukawa G, Yamakita Y, Yamashiro S, Hartshorne DJ, Sasaki Y, Matsumura F (2000) Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts. J Cell Biol 150:797–806

  31. 31.

    Gutjahr MC, Rossy J, Niggli V (2005) Role of Rho, Rac, and Rho-kinase in phosphorylation of myosin light chain, development of polarity, and spontaneous migration of Walker 256 carcinosarcoma cells. Exp Cell Res 308:422–438

  32. 32.

    Vicente-Manzanares M, Koach MA, Whitmore L, Lamers ML, Horwitz AF (2008) Segregation and activation of myosin IIB creates a rear in migrating cells. J Cell Biol 183:543–554

  33. 33.

    Wolf K, Mazo I, Leung H, Engelke K, von Andrian UH, Deryugina EI, Strongin AY, Bröcker EB, Friedl P (2003) Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J Cell Biol 160:267–277

  34. 34.

    Sahai E, Marshall CJ (2003) Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol 5:711–719

  35. 35.

    Rosel D, Brábek J, Tolde O, Mierke CT, Zitterbart DP, Raupach C, Bicanová K, Kollmannsberger P, Panková D, Vesely P, Folk P, Fabrym B (2008) Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces. Mol Cancer Res 6:1410–1420

  36. 36.

    Lafuente EM, van Puijenbroek AA, Krause M, Carman CV, Freeman GJ, Berezovskaya A, Constantine E, Springer TA, Gertler FB, Boussiotis VA (2004) RIAM, an Ena/VASP and profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion. Dev Cell 7:585–595

  37. 37.

    Lee HS, Lim CJ, Puzon-McLaughlin W, Shattil SJ, Ginsberg MH (2009) RIAM activates integrins by linking talin to ras GTPase membrane-targeting sequences. J Biol Chem 284:5119–5127

  38. 38.

    Berg JS, Cheney RE (2002) Myosin-X is an unconventional myosin that undergoes intrafilopodial motility. Nat Cell Biol 4:246–250

  39. 39.

    Tokuo H, Ikebe M (2004) Myosin X transports Mena/VASP to the tip of filopodia. Biochem Biophys Res Commun 319:214–220

  40. 40.

    Bohil AB, Robertson BW, Cheney RE (2006) Myosin-X is a molecular motor that functions in filopodia formation. Proc Natl Acad Sci USA 103:12411–12416

  41. 41.

    Mattila PK, Lappalainen P (2008) Filopodia: molecular architecture and cellular functions. Nat Rev Mol Cell Biol 9:446–454

  42. 42.

    Bassi DE, Mahloogi H, Klein-Szanto AJ (2000) The proprotein convertases furin and PACE4 play a significant role in tumor progression. Mol Carcinog 28:63–69

  43. 43.

    Hubbard FC, Goodrow TL, Liu SC, Brilliant MH, Basset P, Mains RE, Klein-Szanto AJ (1997) Expression of PACE4 in chemically induced carcinomas is associated with spindle cell tumor conversion and increased invasive ability. Cancer Res 57:5226–5231

  44. 44.

    Zocchi MR, Vidal M, Poggi A (1993) Involvement of CD56/N-CAM molecule in the adhesion of human solid tumor cell lines to endothelial cells. Exp Cell Res 204:130–135

  45. 45.

    Lehembre F, Yilmaz M, Wicki A, Schomber T, Strittmatter K, Ziegler D, Kren A, Went P, Derksen PW, Berns A, Jonkers J, Christofori G (2008) NCAM-induced focal adhesion assembly: a functional switch upon loss of E-cadherin. EMBO J 27:2603–2615

  46. 46.

    Simpson MA, Wilson CM, Furcht LT, Spicer AP, Oegema TR Jr, McCarthy JB (2002) Manipulation of hyaluronan synthase expression in prostate adenocarcinoma cells alters pericellular matrix retention and adhesion to bone marrow endothelial cells. J Biol Chem 277:10050–10057

  47. 47.

    Udabage L, Brownlee GR, Waltham M, Blick T, Walker EC, Heldin P, Nilsson SK, Thompson EW, Brown TJ (2005) Antisense-mediated suppression of hyaluronan synthase 2 inhibits the tumorigenesis and progression of breast cancer. Cancer Res 65:6139–6150

  48. 48.

    Cook AC, Chambers AF, Turley EA, Tuck AB (2006) Osteopontin induction of hyaluronan synthase 2 expression promotes breast cancer malignancy. J Biol Chem 281:24381–24389

  49. 49.

    Daniels KJ, Boldt HC, Martin JA, Gardner LM, Meyer M, Folberg R (1996) Expression of type VI collagen in uveal melanoma: its role in pattern formation and tumor progression. Lab Invest 75:55–66

  50. 50.

    Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, Savagner P, Gitelman I, Richardson A, Weinberg RA (2004) Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117:927–939

  51. 51.

    Peinado H, Olmeda D, Cano A (2007) Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 7:415–428

  52. 52.

    Medjkane S, Perez-Sanchez C, Gaggioli C, Sahai E, Treisman R (2009) Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis. Nat Cell Biol 11:257–268

  53. 53.

    Miano JM, Long X, Fujiwara K (2007) Serum response factor: master regulator of the actin cytoskeleton and contractile apparatus. Am J Physiol Cell Physiol 292:C70–C81

Download references

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.

Author information

Correspondence to Michal Dvořák.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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)

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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) doi:10.1007/s00018-010-0395-6

Download citation

Keywords

  • EGR1
  • Sarcoma
  • Metastasis
  • Regulation of expression
  • Transcription
  • Microarrays