Open Access

Maspin is a tumour suppressor that inhibits breast cancer tumour metastasis in vivo

  • Charles H Streuli1
Breast Cancer Research20024:137

DOI: 10.1186/bcr437

Accepted: 01 May 2002

Published: 1 August 2002

Abstract

Maspin is a member of the serpin family of serine proteases and functions as a tumour suppressor. A study using a new syngeneic mouse model for breast cancer suggests that maspin can inhibit metastasis in vivo.

Keywords

breast cancer maspin metastasis protease syngeneic model tumour suppressor

Introduction

Proteases that remodel the extracellular matrix (ECM) play an important role in the progression of neoplasia [1]. Excessive protease activity can lead to major changes within the microenvironment of tumour tissue to promote cell migration, and it thereby contributes to metastasis. Moreover, subtle changes in the levels and activities of proteases can expose cryptic sites in ECM molecules that alter integrin usage, and release matrix-bound growth factors, which both potentiate proliferation and survival of tumour cells, and induce angiogenesis [24]. Thus, several of the hallmarks of tumour progression occur as a result of alteration in protease activity within the extracellular environment of a nascent tumour [5]. Direct evidence that inappropriate expression of both matrix metalloproteinases (MMPs) and serine proteases, the two main classes of ECM-degrading proteases, is involved in tumour progression comes from misexpression studies in genetically altered mice [68].

It is little wonder, then, that nature has devised means of keeping ECM-degrading proteases under tight control. Tissue inhibitors of MMPs suppress the activity of MMPs, whereas serpins are a class of serine protease inhibitor. The expression of these protease inhibitors is closely regulated in developmental morphogenetic processes. For example, tissue inhibitors of MMPs control ECM remodelling during mammary gland development, suppressing excess MMP activity and therefore preventing matrix remodelling from occurring prematurely in postlactational involution [9, 10]. However, there can be disastrous consequences if the expression of matrix proteinase suppressing enzymes is misregulated. Just as over-expression of MMPs and serine proteases can contribute to carcinogenesis, so can down-regulation of their inhibitors. Levels of one such inhibitor of serine proteases, namely maspin, are frequently reduced or even absent in invasive cancer [11].

Maspin: a serine protease inhibitor

Maspin was identified by subtractive hybridization of cDNAs from normal versus tumourigenic breast cells [11]. This 42-kDa protein has significant homology to serpin and contains a carboxyl terminal reactive serpin loop domain, which is essential for its antiprotease activity. Several features of its expression and function, which are discussed below, indicate that maspin is a tumour suppressor.

First, maspin is strongly down-regulated in some cancers. Its levels inversely correlate with the stage of malignancy during breast cancer progression [12, 13]. Moreover, maspin levels are also reduced in prostate and oral squamous carcinoma, and in mouse models of mammary tumourigenesis [1416]. Two mechanisms for its altered expression in cancer have thus far been identified. The gene that encodes maspin is silenced in some tumours through hypermethylation at CpG islands [17]. Moreover, maspin is under the control of p53 and may therefore not be expressed in tumour cells with abnormal p53 function [18].

Second, recombinant maspin blocks the invasion of several tumour cell lines in Matrigel culture assays [19], indicating that it has a migration suppression function. One possible mechanism for this is by reducing the cell surface proteolytic activities required for breaking and making cell-matrix adhesions during migration [20]. Tissue plasminogen activator, localized to the cell surface, is implicated in migration of tumour cells [21], whereas membrane type 1 MMP stimulates migration of MCF7 cells [22]. An alternative possibility is that maspin, through an unknown mechanism, prevents invasion by increasing the strength of integrin mediated adhesion to the ECM. Recombinant maspin elevates the cell surface levels of α5β1 integrin in MDA-MB-435 cells, thereby reducing their motility on fibronectin [23]. As with many cell regulatory factors, maspin may be presented to the cell surface by matrix molecules themselves, because it binds collagen types I and III [24].

The migration suppression function of maspin for cancer cells is potent, with a median effective dose of 0.2–0.3 mol/l, and requires the reactive serpin loop [25]. Importantly, maspin also has an equally effective but quite different function, because it blocks endothelial cell migration in culture and neovascularization in vivo, independently of the reactive serpin loop [25]. Also implicated in tumour angiogenesis are MMPs [26], and analogous antiangiogenic activity is provided by a novel membrane anchored MMP inhibitor, RECK [27]. Thus, a third tumour suppressor activity of maspin is to inhibit angiogenesis, a role that now appears to be extended to other classes of protease inhibitor [28].

Maspin and tumour progression

Given all of these intriguing properties, it would be useful to know whether maspin really can affect the progression of tumours toward malignancy in vivo. Not many good orthotopic models for human metastatic breast cancer are available to study this. MCF7 breast cancer cells can grow and metastasize from an orthotopic site [29]. In a further model, MDA-MB-435 cells that stably express hepatocyte growth factor rapidly metastasize to the lung [30]. However, MDA-MB-435 has now been shown to be derived from a melanoma rather than a breast cancer, thus reducing the number of available models for studying metastasis from human breast tumours [31]. By contrast, several mammary orthotopic metastatic syngeneic models have been described for both mice and rats [3237]. A recent study [38] described an additional syngeneic tumour implantation model to investigate the role of maspin in tumour progression in vivo.

The new model involves transplantation of TM40D cells derived from the Balb/c mammary epithelial strain FSK-4 [39] into the orthotopic site of syngeneic hosts [38]. Primary tumours develop 3–4 weeks after inoculation with 5 × 105 cells and become large 2 weeks later. The tumours are aggressive, showing little encapsulation, and metastasize to the intestine and lung in approximately 75% of cases. The model appears to be valuable, and hopefully further studies that involve more mice and more detailed pathology of the tumours and metastases will eventually be forthcoming. For the purpose of the present commentary, however, the important result concerns the effect of maspin expression in the tumour model.

Two approaches were used in the study conducted by Shi et al. [38]. In the first approach, maspin was transfected into TM40D cells under the control of the strongly expressed elongation factor promoter and stable clones were isolated. In the second, cells infected with a retrovirus expressing maspin and green fluorescent protein (GFP) were selected by flow cytometry on the basis of GFP expression. Maspin significantly reduced the percentage of mice that developed tumours, increased the time taken for tumours to become established, and reduced their growth rate. The effect was more marked using the cells selected after viral infection. These results are impressive but not as dramatic as those from the subsequent metastasis study, in which metastasis was completely abrogated by maspin expression [38]. In each case the primary tumour was encapsulated with a fibrous sheath, suggesting a possible mechanism for inhibition of secondary tumour formation.

These are certainly interesting data and point to a potentially important role for maspin as a tumour suppressor for breast cancer in vivo. However, a significant number of questions remain that will hopefully be resolved by future studies. First, the data sets are very small (12 mice with TM40D implants and only three with maspin transduced cells in the metastasis study), and so statistical analysis is not really possible at this stage. Second, control experiments using cells transduced with catalytically altered maspin have not yet been done. Finally, the mechanism responsible for maspin mediated suppression of tumour growth has not been elucidated; is it due to maspin's function in blocking epithelial cell migration or angiogenesis, or both?

Intriguingly, maspin is normally produced by myoepithelial cells [40, 41]. In the early stages of breast cancer progression, tumours are ensheathed by a layer of myoepithelial cells that have tumour suppressor activity [42], and are themselves subtended by a laminin-rich basement membrane [43, 44]. The transition from ductal carcinoma in situ to malignant lesions results in loss of this myoepithelial cell layer and consequent disappearance of the basement membrane [42]. This myoepithelial cell and basement membrane loss is likely to have several implications for tumour progression. For example, cells that would normally depend on basement membrane for preventing apoptosis [45] are selected for their ability to survive in an inappropriate ECM environment by over-expression of integrin signalling enzymes, such as focal adhesion kinase [46], which is frequently up-regulated in breast cancer [47]. A further consequence of myoepithelial cell disappearance, based on the study using the new syngeneic breast cancer model [38], is that the reduction in maspin's function in vivo results in an increase in serine protease activity and thereby contributes to metastasis and/or the associated neovascularization to feed the growing tumour.

Conclusion

We are left with a tantalizing image of maspin as a potent tumour suppressor, both in culture and now in vivo. Hopefully, future studies on the structure of maspin and the molecular details of how it interacts with substrates may ultimately yield novel therapeutics that mimic its activities.

Abbreviations

ECM: 

= extracellular matrix

MMP: 

= matrix metalloproteinase.

Declarations

Acknowledgements

Thanks to Emma Marshman for commenting on the text and to Teresa Klinowska for insight into orthotopic models of breast cancer.

Authors’ Affiliations

(1)
School of Biological Sciences, University of Manchester

References

  1. Werb Z: ECM and cell surface proteolysis: regulating cellular ecology. Cell. 1997, 91: 439-442. 10.1016/S0092-8674(00)80429-8.View ArticlePubMedGoogle Scholar
  2. Streuli CH: Extracellular matrix remodelling and cellular differentiation. Curr Opin Cell Biol. 1999, 11: 634-640. 10.1016/S0955-0674(99)00026-5.View ArticlePubMedGoogle Scholar
  3. Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z: MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell. 1998, 93: 411-422. 10.1016/S0092-8674(00)81169-1.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Stetler-Stevenson WG: Matrix metalloproteinases in angiogenesis: a moving target for therapeutic intervention. J Clin Invest. 1999, 103: 1237-1241.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Hanahan D, Weinberg RA: The hallmarks of cancer. Cell. 2000, 100: 57-70. 10.1016/S0092-8674(00)81683-9.View ArticlePubMedGoogle Scholar
  6. Wilson CL, Heppner KJ, Labosky PA, Hogan BLM, Matrisian LM: Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin. Proc Natl Acad Sci USA. 1997, 94: 1402-1407. 10.1073/pnas.94.4.1402.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Johnsen M, Lund LR, Romer J, Almholt K, Dano K: Cancer invasion and tissue remodeling: common themes in proteolytic matrix degradation. Curr Opin Cell Biol. 1998, 10: 667-671. 10.1016/S0955-0674(98)80044-6.View ArticlePubMedGoogle Scholar
  8. Sternlicht MD, Lochter A, Sympson CJ, Huey B, Rougier JP, Gray JW, Pinkel D, Bissell MJ, Werb Z: The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis. Cell. 1999, 98: 137-146.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Talhouk RS, Bissell MJ, Werb Z: Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution. J Cell Biol. 1992, 118: 1271-1282.View ArticlePubMedGoogle Scholar
  10. Fata JE, Leco KJ, Voura EB, Yu HY, Waterhouse P, Murphy G, Moorehead RA, Khokha R: Accelerated apoptosis in the Timp-3-deficient mammary gland. J Clin Invest. 2001, 108: 831-841. 10.1172/JCI200113171.View ArticlePubMedPubMed CentralGoogle Scholar
  11. Zou Z, Anisowicz A, Hendrix MJ, Thor A, Neveu M, Sheng S, Rafidi K, Seftor E, Sager R: Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Science. 1994, 263: 526-529.View ArticlePubMedGoogle Scholar
  12. Maass N, Teffner M, Rosel F, Pawaresch R, Jonat W, Nagasaki K, Rudolph P: Decline in the expression of the serine proteinase inhibitor maspin is associated with tumour progression in ductal carcinomas of the breast. J Pathol. 2001, 195: 321-326. 10.1002/path.948.View ArticlePubMedGoogle Scholar
  13. Maass N, Hojo T, Rosel F, Ikeda T, Jonat W, Nagasaki K: Down regulation of the tumor suppressor gene maspin in breast carcinoma is associated with a higher risk of distant metastasis. Clin Biochem. 2001, 34: 303-307. 10.1016/S0009-9120(01)00220-X.View ArticlePubMedGoogle Scholar
  14. Machtens S, Serth J, Bokemeyer C, Bathke W, Minssen A, Kollmannsberger C, Hartmann J, Knuchel R, Kondo M, Jonas U, Kuczyk M: Expression of the p53 and Maspin protein in primary prostate cancer: correlation with clinical features. Int J Cancer. 2001, 95: 337-342. 10.1002/1097-0215(20010920)95:5<337::AID-IJC1059>3.3.CO;2-T.View ArticlePubMedGoogle Scholar
  15. Reddy KB, McGowen R, Schuger L, Visscher D, Sheng S: Maspin expression inversely correlates with breast tumor progression in MMTV/TGF-alpha transgenic mouse model. Oncogene. 2001, 20: 6538-6543. 10.1038/sj.onc.1204796.View ArticlePubMedGoogle Scholar
  16. Yasumatsu R, Nakashima T, Hirakawa N, Kumamoto Y, Kuratomi Y, Tomita K, Komiyama S: Maspin expression in stage I and II oral tongue squamous cell carcinoma. Head Neck. 2001, 23: 962-966. 10.1002/hed.1139.View ArticlePubMedGoogle Scholar
  17. Domann FE, Rice JC, Hendrix MJ, Futscher BW: Epigenetic silencing of maspin gene expression in human breast cancers. Int J Cancer. 2000, 85: 805-810. 10.1002/(SICI)1097-0215(20000315)85:6<805::AID-IJC12>3.0.CO;2-5.View ArticlePubMedGoogle Scholar
  18. Zou Z, Gao C, Nagaich AK, Connell T, Saito S, Moul JW, Seth P, Appella E, Srivastava S: p53 regulates the expression of the tumor suppressor gene maspin. J Biol Chem. 2000, 275: 6051-6054. 10.1074/jbc.275.9.6051.View ArticlePubMedGoogle Scholar
  19. Sheng S, Pemberton PA, Sager R: Production, purification, and characterization of recombinant maspin proteins. J Biol Chem. 1994, 269: 30988-30993.PubMedGoogle Scholar
  20. Sheng S, Carey J, Seftor EA, Dias L, Hendrix MJ, Sager R: Maspin acts at the cell membrane to inhibit invasion and motility of mammary and prostatic cancer cells. Proc Natl Acad Sci USA. 1996, 93: 11669-11674. 10.1073/pnas.93.21.11669.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Rabbani SA, Mazar AP: The role of the plasminogen activation system in angiogenesis and metastasis. Surg Oncol Clin N Am. 2001, 10: 393-415.PubMedGoogle Scholar
  22. Deryugina EI, Ratnikov BI, Postnova TI, Rozanov DV, Strongin AY: Processing of integrin alpha v subunit by membrane type 1 matrix metalloproteinase stimulates migration of breast carcinoma cells on vitronectin and enhances tyrosine phosphorylation of focal adhesion kinase. J Biol Chem. 2002, 277: 9749-9756. 10.1074/jbc.M110269200.View ArticlePubMedGoogle Scholar
  23. Seftor RE, Seftor EA, Sheng S, Pemberton PA, Sager R, Hendrix MJ: Maspin suppresses the invasive phenotype of human breast carcinoma. Cancer Res. 1998, 58: 5681-5685.PubMedGoogle Scholar
  24. Blacque OE, Worrall DM: Evidence for a direct interaction between the tumour suppressor serpin maspin, and types I and III collagen. J Biol Chem. 2002, 277: 10783-10788. 10.1074/jbc.M110992200.View ArticlePubMedGoogle Scholar
  25. Zhang M, Volpert O, Shi YH, Bouck N: Maspin is an angiogenesis inhibitor. Nat Med. 2000, 6: 196-199. 10.1038/72303.View ArticlePubMedGoogle Scholar
  26. Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, Tanzawa K, Thorpe P, Itohara S, Werb Z, Hanahan D: Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol. 2000, 2: 737-744. 10.1038/35036374.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Oh J, Takahashi R, Kondo S, Mizoguchi A, Adachi E, Sasahara RM, Nishimura S, Imamura Y, Kitayama H, Alexander DB, Ide C, Horan TP, Arakawa T, Yoshida H, Nishikawa S, Itoh Y, Seiki M, Itohara S, Takahashi C, Noda M: The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis. Cell. 2001, 107: 789-800. 10.1016/S0092-8674(01)00597-9.View ArticlePubMedGoogle Scholar
  28. Welm B, Mott J, Werb Z: Developmental biology: vasculogenesis is a wreck without RECK. Curr Biol. 2002, 12: R209-R211. 10.1016/S0960-9822(02)00752-2.View ArticlePubMedGoogle Scholar
  29. Thompson EW, Brunner N, Torri J, Johnson MD, Boulay V, Wright A, Lippman ME, Steeg PS, Clarke R: The invasive and metastatic properties of hormone-independent but hormone-responsive variants of MCF-7 human breast cancer cells. Clin Exp Metastasis. 1993, 11: 15-26.View ArticlePubMedGoogle Scholar
  30. Meiners S, Brinkmann V, Naundorf H, Birchmeier W: Role of morphogenetic factors in metastasis of mammary carcinoma cells. Oncogene. 1998, 16: 9-20. 10.1038/sj/onc/1201486.View ArticlePubMedGoogle Scholar
  31. Ellison G, Klinowska T, Westwood R, Docter E, French T, Fox JC: Further evidence to support the melanocytic origin of MDA-MB-435. Mol Pathol. 2002.Google Scholar
  32. Hossain A, Sarkar NH: Colonization characteristics of a murine mammary tumor cell line that metastasizes frequently to the heart. Clin Exp Metastasis. 1991, 9: 351-361.View ArticlePubMedGoogle Scholar
  33. Murthy MS, Scanlon EF, Jelachich ML, Klipstein S, Goldschmidt RA: Growth and metastasis of human breast cancers in athymic nude mice. Clin Exp Metastasis. 1995, 13: 3-15.View ArticlePubMedGoogle Scholar
  34. Hall DG, Stoica G: Characterization of brain and bone-metastasizing clones selected from an ethylnitrosourea-induced rat mammary carcinoma. Clin Exp Metastasis. 1994, 12: 283-295.View ArticlePubMedGoogle Scholar
  35. Neri A, Ruoslahti E, Nicolson GL: Distribution of fibronectin on clonal cell lines of a rat mammary adenocarcinoma growing in vitro and in vivo at primary and metastatic sites. Cancer Res. 1981, 41: 5082-5095.PubMedGoogle Scholar
  36. Xing RH, Rabbani SA: Overexpression of urokinase receptor in breast cancer cells results in increased tumor invasion, growth and metastasis. Int J Cancer. 1996, 67: 423-429. 10.1002/(SICI)1097-0215(19960729)67:3<423::AID-IJC18>3.0.CO;2-8.View ArticlePubMedGoogle Scholar
  37. Lelekakis M, Moseley JM, Martin TJ, Hards D, Williams E, Ho P, Lowen D, Javni J, Miller FR, Slavin J, Anderson RL: A novel orthotopic model of breast cancer metastasis to bone. Clin Exp Metastasis. 1999, 17: 163-170. 10.1023/A:1006689719505.View ArticlePubMedGoogle Scholar
  38. Shi HY, Zhang W, Liang R, Abraham S, Kittrell FS, Medina D, Zhang M: Blocking tumor growth, invasion, and metastasis by maspin in a syngeneic breast cancer model. Cancer Res. 2001, 61: 6945-6951.PubMedGoogle Scholar
  39. Kittrell FS, Oborn CJ, Medina D: Development of mammary preneoplasias invivo from mouse mammary epithelial-cell lines invitro. Cancer Res. 1992, 52: 1924-1932.PubMedGoogle Scholar
  40. Sternlicht MD, Kedeshian P, Shao ZM, Safarians S, Barsky SH: The human myoepithelial cell is a natural tumor suppressor. Clin Cancer Res. 1997, 3: 1949-1958.PubMedGoogle Scholar
  41. Reis-Filho JS, Milanezi F, Silva P, Schmitt FC: Maspin expression in myoepithelial tumors of the breast. Pathol Res Pract. 2001, 197: 817-821.View ArticlePubMedGoogle Scholar
  42. Gudjonsson T, Ronnov-Jessen L, Villadsen R, Rank F, Bissell MJ, Petersen OW: Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition. J Cell Sci. 2002, 115: 39-50.PubMedPubMed CentralGoogle Scholar
  43. Lakhani SR, O'Hare MJ: The mammary myoepithelial cell: Cinderella or ugly sister?. Breast Cancer Res. 2001, 3: 1-4. 10.1186/bcr260.View ArticlePubMedGoogle Scholar
  44. Prince JM, Klinowska TC, Marshman E, Lowe ET, Mayer U, Miner J, Aberdam D, Vestweber D, Gusterson B, Streuli CH: Cell-matrix interactions during development and apoptosis of the mouse mammary gland in vivo. Dev Dyn. 2002, 223: 497-516. 10.1002/dvdy.10070.View ArticlePubMedGoogle Scholar
  45. Pullan S, Wilson J, Metcalfe A, Edwards GM, Goberdhan N, Tilly J, Hickman JA, Dive C, Streuli CH: Requirement of basement membrane for the suppression of programmed cell death in mammary epithelium. J Cell Sci. 1996, 109: 631-642.PubMedGoogle Scholar
  46. Gilmore AP, Metcalfe AM, Romer LH, Streuli CH: Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization. J Cell Biol. 2000, 149: 431-445. 10.1083/jcb.149.2.431.View ArticlePubMedPubMed CentralGoogle Scholar
  47. Cance WG, Harris JE, Iacocca MV, Roche E, Yang X, Chang J, Simkins S, Xu L: Immunohistochemical analyses of focal adhesion kinase expression in benign and malignant human breast and colon tissues: correlation with preinvasive and invasive phenotypes. Clin Cancer Res. 2000, 6: 2417-2423.PubMedGoogle Scholar

Copyright

© BioMed Central Ltd 2002