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Vol. 27, No. 1, 2006
Issue release date: December 2005
Free Access
Tumor Biol 2006;27:17–26
(DOI:10.1159/000090152)

Silencing the hsp25 Gene Eliminates Migration Capability of the Highly Metastatic Murine 4T1 Breast Adenocarcinoma Cell

Bausero M.A.a, c · Bharti A.a · Page D.T.a · Perez K.D.a, b · Eng J.W.-L.a · Ordonez S.L.a · Asea E.E.a, b · Jantschitsch C.d · Kindas-Muegge I.e · Ciocca D.f · Asea A.a, b
aCenter for Molecular Stress Response, Boston University Medical Center and Boston University School of Medicine, Boston, Mass., bDivision of Investigative Pathology, Department of Pathology, Scott & White Clinic and Texas A&M University System Health Science Center College of Medicine, Temple, Tex., USA; cLaboratorio de Oncología Básica y Biología Molecular, Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; dDivision of Special and Environmental Dermatology, Department of Dermatology, University of Vienna, eInstitute of Cancer Research, Vienna, Austria; fInstitute of Experimental Medicine and Biology of Cuyo (IMBECU)-CONICET, Mendoza, Argentina
email Corresponding Author

Abstract

The 25-kDa heat shock protein (Hsp25) is associated with various malignancies and is expressed at high levels in biopsies as well as circulating in the serum of breast cancer patients. In this study, we used RNA interference technology to silence the hsp25 gene in 4T1 breast adenocarcinoma cells, known as a poorly immunogenic, highly metastatic cell line. We demonstrate that transfection of 4T1 cells with short interference RNA-Hsp25 dramatically inhibits proliferation as compared with control transfected cells. In addition, we show that 4T1 cells transfected with short interference RNA-Hsp25 abrogates tumor migration potential by a mechanism that is in part due to the repression of matrix metalloproteinase 9 expression and a concomitant upregulation of its antagonist, tissue inhibitor metalloproteinase 1. Taken together, these findings provide a model system for the study of metastatic potential of tumors and are suggestive of an earlier unrecognized role for Hsp25 in tumor migration.


 goto top of outline Key Words

  • Breast carcinoma
  • Chaperokine
  • Heat shock proteins
  • Metastasis
  • siRNA

 goto top of outline Abstract

The 25-kDa heat shock protein (Hsp25) is associated with various malignancies and is expressed at high levels in biopsies as well as circulating in the serum of breast cancer patients. In this study, we used RNA interference technology to silence the hsp25 gene in 4T1 breast adenocarcinoma cells, known as a poorly immunogenic, highly metastatic cell line. We demonstrate that transfection of 4T1 cells with short interference RNA-Hsp25 dramatically inhibits proliferation as compared with control transfected cells. In addition, we show that 4T1 cells transfected with short interference RNA-Hsp25 abrogates tumor migration potential by a mechanism that is in part due to the repression of matrix metalloproteinase 9 expression and a concomitant upregulation of its antagonist, tissue inhibitor metalloproteinase 1. Taken together, these findings provide a model system for the study of metastatic potential of tumors and are suggestive of an earlier unrecognized role for Hsp25 in tumor migration.

Copyright © 2006 S. Karger AG, Basel


 goto top of outline References
  1. Weir HK, Thun MJ, Hankey BF, Ries LA, Howe HL, Wingo PA, Jemal A, Ward E, Anderson RN, Edwards BK: Annual report to the nation on the status of cancer, 1975–2000, featuring the uses of surveillance data for cancer prevention and control. J Natl Cancer Inst 2003;95:1276–1299.
  2. Kim J, Yu W, Kovalski K, Ossowski L: Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell 1998;94:353–362.
  3. Egeblad M, Werb Z: New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2002;2:161–174.
  4. Neumark E, Sagi-Assif O, Shalmon B, Ben-Baruch A, Witz IP: Progression of mouse mammary tumors: MCP-1-TNFalpha cross-regulatory pathway and clonal expression of promalignancy and antimalignancy factors. Int J Cancer 2003;106:879–886.
  5. Lirdprapamongkol K, Sakurai H, Kawasaki N, Choo MK, Saitoh Y, Aozuka Y, Singhirunnusorn P, Ruchirawat S, Svasti J, Saiki I: Vanillin suppresses in vitro invasion and in vivo metastasis of mouse breast cancer cells. Eur J Pharm Sci 2005;25:57–65.
  6. Soldes OS, Kuick RD, Thompson IA 2nd, Hughes SJ, Orringer MB, Iannettoni MD, Hanash SM, Beer DG: Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas. Br J Cancer 1999;79:595–603.
  7. Jantschitsch C, Trautinger F: Heat shock and UV-B-induced DNA damage and mutagenesis in skin. Photochem Photobiol Sci 2003;2:899–903.
  8. Wieder R: Insurgent micrometastases: sleeper cells and harboring the enemy. J Surg Oncol 2005;89:207–210.
  9. Oesterreich S, Hilsenbeck SG, Ciocca DR, Allred DC, Clark GM, Chamness GC, Osborne CK, Fuqua SA: The small heat shock protein HSP27 is not an independent prognostic marker in axillary lymph node-negative breast cancer patients. Clin Cancer Res 1996;2:1199–1206.
  10. Ciocca DR, Vargas-Roig LM: Hsp27 as a prognostic and predictive factor in cancer. Prog Mol Subcell Biol 2002;28:205–218.
  11. Ciocca DR, Green S, Elledge RM, Clark GM, Pugh R, Ravdin P, Lew D, Martino S, Osborne CK: Heat shock proteins hsp27 and hsp70: lack of correlation with response to tamoxifen and clinical course of disease in estrogen receptor-positive metastatic breast cancer (a Southwest Oncology Group Study). Clin Cancer Res 1998;4:1263–1266.
  12. Ciocca DR, Rozados VR, Cuello Carrion FD, Gervasoni SI, Matar P, Scharovsky OG: Hsp25 and Hsp70 in rodent tumors treated with doxorubicin and lovastatin. Cell Stress Chaperones 2003;8:26–36.
  13. Cornford PA, Dodson AR, Parsons KF, Desmond AD, Woolfenden A, Fordham M, Neoptolemos JP, Ke Y, Foster CS: Heat shock protein expression independently predicts clinical outcome in prostate cancer. Cancer Res 2000;60:7099–7105.
  14. Bausero MA, Page DT, Osinaga E, Asea A: Surface expression of Hsp25 and Hsp72 differentially regulates tumor growth and metastasis. Tumor Biol 2004;25:243–251.
  15. Asea A: Exogenous Hsp70: principles and application of the chaperokine activity of Hsp70; in Henderson B, Pockley AG (eds): The Extracellular Biology of Molecular Chaperones. London, Cambridge University Press, 2005.
  16. Asea A, Ara G, Teicher BA, Stevenson MA, Calderwood SK: Effects of the flavonoid drug quercetin on the response of human prostate tumours to hyperthermia in vitro and in vivo. Int J Hyperthermia 2001;17:347–356.
  17. Jaattela M, Wissing D, Kokholm K, Kallunki T, Egeblad M: Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases. EMBO J 1998;17:6124–6134.
  18. Calderwood SK, Asea A: Targeting HSP70-induced thermotolerance for design of thermal sensitizers. Int J Hyperthermia 2002;18:597–608.
  19. Jaattela M: Programmed cell death: many ways for cells to die decently. Ann Med 2002;34:480–488.
  20. Farkas B, Hantschel M, Magyarlaki M, Becker B, Scherer K, Landthaler M, Pfister K, Gehrmann M, Gross C, Mackensen A, Multhoff G: Heat shock protein 70 membrane expression and melanoma-associated marker phenotype in primary and metastatic melanoma. Melanoma Res 2003;13:147–152.
  21. Asea A, Rehli M, Kabingu E, Boch JA, Bare O, Auron PE, Stevenson MA, Calderwood SK: Novel signal transduction pathway utilized by extracellular HSP70: role of toll-like receptor (TLR) 2 and TLR4. J Biol Chem 2002;277:15028–15034.
  22. Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC, Calderwood SK: HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med 2000;6:435–442.
  23. Asea A, Kabingu E, Stevenson MA, Calderwood SK: HSP70 peptide-bearing and peptide-negative preparations function as chaperokines. Cell Stress Chaperones 2000;5:425–431.
  24. Basu S, Suto R, Binder RJ, Srivastava PK: Heat shock proteins as novel mediators of cytokine secretion by macrophages. Cell Stress Chaperones 1998;3:11–16.
  25. Blackburn RV, Galoforo SS, Berns CM, Armour EP, McEachern D, Corry PM, Lee YJ: Comparison of tumor growth between hsp25- and hsp27-transfected murine L929 cells in nude mice. Int J Cancer 1997;72:871–877.
  26. Lemieux P, Oesterreich S, Lawrence JA, Steeg PS, Hilsenbeck SG, Harvey JM, Fuqua SA: The small heat shock protein hsp27 increases invasiveness but decreases motility of breast cancer cells. Invasion Metastasis 1997;17:113–123.
  27. Eskenazi AE, Powers J, Pinkas J, Oesterreich S, Fuqua SA, Frantz CN: Induction of heat shock protein 27 by hydroxyurea and its relationship to experimental metastasis. Clin Exp Metastasis 1998;16:283–290.
  28. Aldrian S, Trautinger F, Frohlich I, Berger W, Micksche M, Kindas-Mugge I: Overexpression of Hsp27 affects the metastatic phenotype of human melanoma cells in vitro. Cell Stress Chaperones 2002;7:177–185.
  29. Park SH, Lee YS, Osawa Y, Hachiya M, Akashi M: Hsp25 regulates the expression of p21(Waf1/Cip1/Sdi1) through multiple mechanisms. J Biochem (Tokyo) 2002;131:869–875.
  30. Lee YJ, Cho HN, Jeoung DI, Soh JW, Cho CK, Bae S, Chung HY, Lee SJ, Lee YS: HSP25 overexpression attenuates oxidative stress-induced apoptosis: roles of ERK1/2 signaling and manganese superoxide dismutase. Free Radic Biol Med 2004;36:429–444.
  31. Smith MC, Luker KE, Garbow JR, Prior JL, Jackson E, Piwnica-Worms D, Luker GD: CXCR4 regulates growth of both primary and metastatic breast cancer. Cancer Res 2004;64:8604–8612.
  32. Liang Z, Yoon Y, Votaw J, Goodman MM, Williams L, Shim H: Silencing of CXCR4 blocks breast cancer metastasis. Cancer Res 2005;65:967–971.
  33. Wang T, Yamashita K, Iwata K, Hayakawa T: Both tissue inhibitors of metalloproteinases-1 (TIMP-1) and TIMP-2 activate Ras but through different pathways. Biochem Biophys Res Commun 2002;296:201–205.
  34. Tanimura S, Asato K, Fujishiro SH, Kohno M: Specific blockade of the ERK pathway inhibits the invasiveness of tumor cells: down-regulation of matrix metalloproteinase-3/-9/-14 and CD44. Biochem Biophys Res Commun 2003;304:801–806.

 goto top of outline Author Contacts

Dr. Alexzander Asea, Division of Investigative Pathology
Scott & White Clinic and The Texas A&M University System Health Science Center College of Medicine, 2401 South 31st Street
Temple, TX 76508 (USA)
Tel. +1 254 743 0201, Fax +1 254 743 0247, E-Mail asea@medicine.tamhsc.edu


 goto top of outline Article Information

Received: April 6, 2005
Accepted: July 7, 2005
Published online: December 8, 2005
Number of Print Pages : 10
Number of Figures : 8, Number of Tables : 1, Number of References : 34


 goto top of outline Publication Details

Tumor Biology (Tumor Markers, Tumor Targeting and Translational Cancer Research)

Vol. 27, No. 1, Year 2006 (Cover Date: December 2005)

Journal Editor: Stigbrand, T. (Umeå)
ISSN: 1010–4283 (print), 1423–0380 (Online)

For additional information: http://www.karger.com/TBI


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

The 25-kDa heat shock protein (Hsp25) is associated with various malignancies and is expressed at high levels in biopsies as well as circulating in the serum of breast cancer patients. In this study, we used RNA interference technology to silence the hsp25 gene in 4T1 breast adenocarcinoma cells, known as a poorly immunogenic, highly metastatic cell line. We demonstrate that transfection of 4T1 cells with short interference RNA-Hsp25 dramatically inhibits proliferation as compared with control transfected cells. In addition, we show that 4T1 cells transfected with short interference RNA-Hsp25 abrogates tumor migration potential by a mechanism that is in part due to the repression of matrix metalloproteinase 9 expression and a concomitant upregulation of its antagonist, tissue inhibitor metalloproteinase 1. Taken together, these findings provide a model system for the study of metastatic potential of tumors and are suggestive of an earlier unrecognized role for Hsp25 in tumor migration.



 goto top of outline Author Contacts

Dr. Alexzander Asea, Division of Investigative Pathology
Scott & White Clinic and The Texas A&M University System Health Science Center College of Medicine, 2401 South 31st Street
Temple, TX 76508 (USA)
Tel. +1 254 743 0201, Fax +1 254 743 0247, E-Mail asea@medicine.tamhsc.edu


 goto top of outline Article Information

Received: April 6, 2005
Accepted: July 7, 2005
Published online: December 8, 2005
Number of Print Pages : 10
Number of Figures : 8, Number of Tables : 1, Number of References : 34


 goto top of outline Publication Details

Tumor Biology (Tumor Markers, Tumor Targeting and Translational Cancer Research)

Vol. 27, No. 1, Year 2006 (Cover Date: December 2005)

Journal Editor: Stigbrand, T. (Umeå)
ISSN: 1010–4283 (print), 1423–0380 (Online)

For additional information: http://www.karger.com/TBI


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

  1. Weir HK, Thun MJ, Hankey BF, Ries LA, Howe HL, Wingo PA, Jemal A, Ward E, Anderson RN, Edwards BK: Annual report to the nation on the status of cancer, 1975–2000, featuring the uses of surveillance data for cancer prevention and control. J Natl Cancer Inst 2003;95:1276–1299.
  2. Kim J, Yu W, Kovalski K, Ossowski L: Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell 1998;94:353–362.
  3. Egeblad M, Werb Z: New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2002;2:161–174.
  4. Neumark E, Sagi-Assif O, Shalmon B, Ben-Baruch A, Witz IP: Progression of mouse mammary tumors: MCP-1-TNFalpha cross-regulatory pathway and clonal expression of promalignancy and antimalignancy factors. Int J Cancer 2003;106:879–886.
  5. Lirdprapamongkol K, Sakurai H, Kawasaki N, Choo MK, Saitoh Y, Aozuka Y, Singhirunnusorn P, Ruchirawat S, Svasti J, Saiki I: Vanillin suppresses in vitro invasion and in vivo metastasis of mouse breast cancer cells. Eur J Pharm Sci 2005;25:57–65.
  6. Soldes OS, Kuick RD, Thompson IA 2nd, Hughes SJ, Orringer MB, Iannettoni MD, Hanash SM, Beer DG: Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas. Br J Cancer 1999;79:595–603.
  7. Jantschitsch C, Trautinger F: Heat shock and UV-B-induced DNA damage and mutagenesis in skin. Photochem Photobiol Sci 2003;2:899–903.
  8. Wieder R: Insurgent micrometastases: sleeper cells and harboring the enemy. J Surg Oncol 2005;89:207–210.
  9. Oesterreich S, Hilsenbeck SG, Ciocca DR, Allred DC, Clark GM, Chamness GC, Osborne CK, Fuqua SA: The small heat shock protein HSP27 is not an independent prognostic marker in axillary lymph node-negative breast cancer patients. Clin Cancer Res 1996;2:1199–1206.
  10. Ciocca DR, Vargas-Roig LM: Hsp27 as a prognostic and predictive factor in cancer. Prog Mol Subcell Biol 2002;28:205–218.
  11. Ciocca DR, Green S, Elledge RM, Clark GM, Pugh R, Ravdin P, Lew D, Martino S, Osborne CK: Heat shock proteins hsp27 and hsp70: lack of correlation with response to tamoxifen and clinical course of disease in estrogen receptor-positive metastatic breast cancer (a Southwest Oncology Group Study). Clin Cancer Res 1998;4:1263–1266.
  12. Ciocca DR, Rozados VR, Cuello Carrion FD, Gervasoni SI, Matar P, Scharovsky OG: Hsp25 and Hsp70 in rodent tumors treated with doxorubicin and lovastatin. Cell Stress Chaperones 2003;8:26–36.
  13. Cornford PA, Dodson AR, Parsons KF, Desmond AD, Woolfenden A, Fordham M, Neoptolemos JP, Ke Y, Foster CS: Heat shock protein expression independently predicts clinical outcome in prostate cancer. Cancer Res 2000;60:7099–7105.
  14. Bausero MA, Page DT, Osinaga E, Asea A: Surface expression of Hsp25 and Hsp72 differentially regulates tumor growth and metastasis. Tumor Biol 2004;25:243–251.
  15. Asea A: Exogenous Hsp70: principles and application of the chaperokine activity of Hsp70; in Henderson B, Pockley AG (eds): The Extracellular Biology of Molecular Chaperones. London, Cambridge University Press, 2005.
  16. Asea A, Ara G, Teicher BA, Stevenson MA, Calderwood SK: Effects of the flavonoid drug quercetin on the response of human prostate tumours to hyperthermia in vitro and in vivo. Int J Hyperthermia 2001;17:347–356.
  17. Jaattela M, Wissing D, Kokholm K, Kallunki T, Egeblad M: Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases. EMBO J 1998;17:6124–6134.
  18. Calderwood SK, Asea A: Targeting HSP70-induced thermotolerance for design of thermal sensitizers. Int J Hyperthermia 2002;18:597–608.
  19. Jaattela M: Programmed cell death: many ways for cells to die decently. Ann Med 2002;34:480–488.
  20. Farkas B, Hantschel M, Magyarlaki M, Becker B, Scherer K, Landthaler M, Pfister K, Gehrmann M, Gross C, Mackensen A, Multhoff G: Heat shock protein 70 membrane expression and melanoma-associated marker phenotype in primary and metastatic melanoma. Melanoma Res 2003;13:147–152.
  21. Asea A, Rehli M, Kabingu E, Boch JA, Bare O, Auron PE, Stevenson MA, Calderwood SK: Novel signal transduction pathway utilized by extracellular HSP70: role of toll-like receptor (TLR) 2 and TLR4. J Biol Chem 2002;277:15028–15034.
  22. Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC, Calderwood SK: HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med 2000;6:435–442.
  23. Asea A, Kabingu E, Stevenson MA, Calderwood SK: HSP70 peptide-bearing and peptide-negative preparations function as chaperokines. Cell Stress Chaperones 2000;5:425–431.
  24. Basu S, Suto R, Binder RJ, Srivastava PK: Heat shock proteins as novel mediators of cytokine secretion by macrophages. Cell Stress Chaperones 1998;3:11–16.
  25. Blackburn RV, Galoforo SS, Berns CM, Armour EP, McEachern D, Corry PM, Lee YJ: Comparison of tumor growth between hsp25- and hsp27-transfected murine L929 cells in nude mice. Int J Cancer 1997;72:871–877.
  26. Lemieux P, Oesterreich S, Lawrence JA, Steeg PS, Hilsenbeck SG, Harvey JM, Fuqua SA: The small heat shock protein hsp27 increases invasiveness but decreases motility of breast cancer cells. Invasion Metastasis 1997;17:113–123.
  27. Eskenazi AE, Powers J, Pinkas J, Oesterreich S, Fuqua SA, Frantz CN: Induction of heat shock protein 27 by hydroxyurea and its relationship to experimental metastasis. Clin Exp Metastasis 1998;16:283–290.
  28. Aldrian S, Trautinger F, Frohlich I, Berger W, Micksche M, Kindas-Mugge I: Overexpression of Hsp27 affects the metastatic phenotype of human melanoma cells in vitro. Cell Stress Chaperones 2002;7:177–185.
  29. Park SH, Lee YS, Osawa Y, Hachiya M, Akashi M: Hsp25 regulates the expression of p21(Waf1/Cip1/Sdi1) through multiple mechanisms. J Biochem (Tokyo) 2002;131:869–875.
  30. Lee YJ, Cho HN, Jeoung DI, Soh JW, Cho CK, Bae S, Chung HY, Lee SJ, Lee YS: HSP25 overexpression attenuates oxidative stress-induced apoptosis: roles of ERK1/2 signaling and manganese superoxide dismutase. Free Radic Biol Med 2004;36:429–444.
  31. Smith MC, Luker KE, Garbow JR, Prior JL, Jackson E, Piwnica-Worms D, Luker GD: CXCR4 regulates growth of both primary and metastatic breast cancer. Cancer Res 2004;64:8604–8612.
  32. Liang Z, Yoon Y, Votaw J, Goodman MM, Williams L, Shim H: Silencing of CXCR4 blocks breast cancer metastasis. Cancer Res 2005;65:967–971.
  33. Wang T, Yamashita K, Iwata K, Hayakawa T: Both tissue inhibitors of metalloproteinases-1 (TIMP-1) and TIMP-2 activate Ras but through different pathways. Biochem Biophys Res Commun 2002;296:201–205.
  34. Tanimura S, Asato K, Fujishiro SH, Kohno M: Specific blockade of the ERK pathway inhibits the invasiveness of tumor cells: down-regulation of matrix metalloproteinase-3/-9/-14 and CD44. Biochem Biophys Res Commun 2003;304:801–806.