Molecular analysis of metastasis in a polyomavirus middle T mouse model: the role of osteopontin

Breast Cancer Research

Jeffrey P Gregg³  Robert D Cardiff²  Alexander D Borowsky²  Lawrence JT Young²  Robert J Munn²  Andrea Rosner²  Erik T McGoldrick²  Juliana Karrim³  Clifford G Tepper¹  Stephenie Y Liu³  Katayoun Alavi Jessen³ 

[1] University of California, Davis, Cancer Center, Sacramento, California, USA;Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California, Davis, California, USA;Department of Pathology, University of California, Davis, School of Medicine, Sacramento, California, USA

关键词: osteopontin;    migration;    metastasis;    mammary gland;    breast cancer;   

 received in 2003-10-09, accepted in 2004-01-28,  发布年份 2004

【 摘 要 】

Introduction

In order to study metastatic disease, we employed the use of two related polyomavirus middle T transgenic mouse tumor transplant models of mammary carcinoma (termed Met and Db) that display significant differences in metastatic potential.

Methods

Through suppression subtractive hybridization coupled to the microarray, we found osteopontin (OPN) to be a highly expressed gene in the tumors of the metastatic mouse model, and a lowly expressed gene in the tumors of the lowly metastatic mouse model. We further analyzed the role of OPN in this model by examining sense and antisense constructs using in vitro and in vivo methods.

Results

With in vivo metastasis assays, the antisense Met cells showed no metastatic tumor formation to the lungs of recipient mice, while wild-type Met cells, with higher levels of OPN, showed significant amounts of metastasis. The Db cells showed a significantly reduced metastasis rate in the in vivo metastasis assay as compared with the Met cells. Db cells with enforced overexpression of OPN showed elevated levels of OPN but did not demonstrate an increase in the rate of metastasis compared with the wild-type Db cells.

Conclusions

We conclude that OPN is an essential regulator of the metastatic phenotype seen in polyomavirus middle T-induced mammary tumors. Yet OPN expression alone is not sufficient to cause metastasis. These data suggest a link between metastasis and phosphatidylinositol-3-kinase-mediated transcriptional upregulation of OPN, but additional phosphatidylinositol-3-kinase-regulated genes may be essential in precipitating the metastasis phenotype in the polyomavirus middle T model.

【 授权许可】

   
2004 Jessen et al., licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

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【 参考文献 】

• [1]Paigen K: A miracle enough: the power of mice. Nat Med 1995, 1:215-220.
• [2]Cardiff RD, Bern HA, Faulkin LJ, Daniel CW, Smith GH, Young LJ, Medina D, Gardner MB, Wellings SR, Shyamala G, Guzman RC, Rajkumar L, Yang J, Thordarson G, Nandi S, MacLeod CL, Oshima RG, Man AK, Sawai ET, Gregg JP, Cheung AT, Lau DH: Contributions of mouse biology to breast cancer research. Comp Med 2002, 52:12-31.
• [3]Hutchinson JN, Muller WJ: Transgenic mouse models of human breast cancer. Oncogene 2000, 19:6130-6137.
• [4]Muller WJ, Neville MC: Introduction: signaling in mammary development and tumorigenesis. J Mammary Gland Biol Neoplasia 2001, 6:1-5.
• [5]Guy CT, Cardiff RD, Muller WJ: Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol Cell Biol 1992, 12:954-961.
• [6]Siegel PM, Hardy WR, Muller WJ: Mammary gland neoplasia: insights from transgenic mouse models. Bioessays 2000, 22:554-563.
• [7]Hutchinson J, Jin J, Cardiff RD, Woodgett JR, Muller WJ: Activation of Akt (protein kinase B) in mammary epithelium provides a critical cell survival signal required for tumor progression. Mol Cell Biol 2001, 21:2203-2212.
• [8]Cardiff RD: Validity of mouse mammary tumour models for human breast cancer: comparative pathology. Microsc Res Tech 2001, 52:224-230.
• [9]Cardiff RD, Wagner U, Henninghausen L: Mammary cancer in humans and mice: a tutorial for comparative pathology. Vet Pathol 2001, 38:357-358.
• [10]Guy CT, Cardiff RD, Muller WJ: Activated neu induces rapid tumor progression. J Biol Chem 1996, 271:7673-7678.
• [11]Desai KV, Xiao N, Wang W, Gangi L, Greene J, Powell JI, Dickson R, Furth P, Hunter K, Kucherlapati R, Simon R, Liu ET, Green JE: Initiating oncogenic event determines gene-expression patterns of human breast cancer models. Proc Natl Acad Sci USA 2002, 99:6967-6972.
• [12]Muller WJ, Ho J, Siegel PM: Oncogenic activation of Neu/ErbB-2 in a transgenic mouse model for breast cancer. Biochem Soc Symp 1998, 63:149-157.
• [13]Barnes DM, Bartkova J, Camplejohn RS, Gullick WJ, Smith PJ, Millis RR: Overexpression of the c-erbB-2 oncoprotein: why does this occur more frequently in ductal carcinoma in situ than in invasive mammary carcinoma and is this of prognostic significance? Eur J Cancer 1992, 28:644-648.
• [14]Oostra BA, Harvey R, Ely BK, Markham AF, Smith AE: Transforming activity of polyoma virus middle-T antigen probed by site-directed mutagenesis. Nature 1983, 304:456-459.
• [15]Courtneidge SA, Smith AE: Polyoma virus transforming protein associates with the product of the c-src cellular gene. Nature 1983, 303:435-439.
• [16]Webster MA, Hutchinson JN, Rauh MJ, Muthuswamy SK, Anton M, Tortorice CG, Cardiff RD, Graham FL, Hassell JA, Muller WJ: Requirement for both Shc and phosphatidylinositol 3' kinase signaling pathways in polyomavirus middle T-mediated mammary tumorigenesis. Mol Cell Biol 1998, 18:2344-2359.
• [17]Whitman M, Kaplan DR, Schaffhausen B, Cantley L, Roberts TM: Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature 1985, 315:239-242.
• [18]Shaw LM, Rabinovitz I, Wang HH, Toker A, Mercurio AM: Activation of phosphoinositide 3-OH kinase by the alpha6beta4 integrin promotes carcinoma invasion. Cell 1997, 91:949-960.
• [19]Cheung ATW, Young LJT, Chen PCY, Chao CY, Ndoye A, Barry PA, Muller WJ, Cardiff RD: Microcirculation and metastasis in a new mouse mammary tumor model system. Int J Oncol 1997, 11:69-77.
• [20]Borowsky AD, Namba R, Niemand N, Tepper CG, McGoldrick ET, Cardiff RD, Gregg JP: Tumors from two related mouse models of mammary carcinoma show differential metastasis rate and regulation of Akt signal effectors. In In American Association for Cancer Research Annual Meeting: 2003; Toronto, Canada. Philadelphia, PA: AACR; 2003.
• [21]Welford SM, Gregg J, Chen E, Garrison D, Sorensen PH, Denny CT, Nelson SF: Detection of differentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray hybridization. Nucleic Acids Res 1998, 26:3059-3065.
• [22]Yang GP, Ross DT, Kuang WW, Brown PO, Weigel RJ: Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Nucleic Acids Res 1999, 27:1517-1523.
• [23]Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD: Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 1996, 93:6025-6030.
• [24]DeOme KB, Faulkin LJJ, Bern HA, Blair PB: Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Res 1959, 19:515-525.
• [25]Young LJT: The cleared mammary fat pad and the transplantation of mammary gland morphological structures and cells. In Methods in Mammary Gland Biology and Breast Cancer Research. Edited by Ip MMaABB. New York: Kluwer Academic/Plenum Publishers; 2000:67-74.
• [26]Bourguignon LY, Gunja-Smith Z, Iida N, Zhu HB, Young LJ, Muller WJ, Cardiff RD: CD44v(3,8-10) is involved in cytoskeleton-mediated tumor cell migration and matrix metalloproteinase (MMP-9) association in metastatic breast cancer cells. J Cell Physiol 1998, 176:206-215.
• [27]Young LJT, Cardiff RD, McGrath CM: Primary epithelial cell dome culture of mouse mammary tumors. In Tissue Culture Association Manual. Gaithersburg, MD: Tissue Culture Association; 1976:161-167.
• [28]Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem 1987, 162:156-159.
• [29]Schena M, Shalon D, Davis RW, Brown PO: Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995, 270:467-470.
• [30]Jessen KA, Satre MA: Induction of mouse retinol binding protein gene expression by cyclic AMP in Hepa 1-6 cells. Arch Biochem Biophys 1998, 357:126-130.
• [31]Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. New York: Wiley Press; 1991.
• [32]Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989.
• [33]Merzak A, Koocheckpour S, Pilkington GJ: CD44 mediates human glioma cell adhesion and invasion in vitro. Cancer Res 1994, 54:3988-3992.
• [34]Clark EA, Golub TR, Lander ES, Hynes RO: Genomic analysis of metastasis reveals an essential role for RhoC. Nature 2000, 406:532-535.
• [35]Soga N, Connolly JO, Chellaiah M, Kawamura J, Hruska KA: Rac regulates vascular endothelial growth factor stimulated motility. Cell Commun Adhes 2001, 8:1-13.
• [36]Rittling SR, Novick KE: Osteopontin expression in mammary gland development and tumorigenesis. Cell Growth Differ 1997, 8:1061-1069.
• [37]Nemir M, Bhattacharyya D, Li X, Singh K, Mukherjee AB, Mukherjee BB: Targeted inhibition of osteopontin expression in the mammary gland causes abnormal morphogenesis and lactation deficiency. J Biol Chem 2000, 275:969-976.
• [38]Baik MG, Lee MJ, Choi YJ: Gene expression during involution of mammary gland [review]. Int J Mol Med 1998, 2:39-44.
• [39]Singhal H, Bautista DS, Tonkin KS, O'Malley FP, Tuck AB, Chambers AF, Harris JF: Elevated plasma osteopontin in metastatic breast cancer associated with increased tumor burden and decreased survival. Clin Cancer Res 1997, 3:605-611.
• [40]Tuck AB, O'Malley FP, Singhal H, Harris JF, Tonkin KS, Kerkvliet N, Saad Z, Doig GS, Chambers AF: Osteopontin expression in a group of lymph node negative breast cancer patients. Int J Cancer 1998, 79:502-508.
• [41]Rudland PS, Platt-Higgins A, El-Tanani M, De Silva Rudland S, Barraclough R, Winstanley JH, Howitt R, West CR: Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer. Cancer Res 2002, 62:3417-3427.
• [42]Urquidi V, Sloan D, Kawai K, Agarwal D, Woodman AC, Tarin D, Goodison S: Contrasting expression of thrombospondin-1 and osteopontin correlates with absence or presence of metastatic phenotype in an isogenic model of spontaneous human breast cancer metastasis. Clin Cancer Res 2002, 8:61-74.
• [43]Sharp JA, Sung V, Slavin J, Thompson EW, Henderson MA: Tumor cells are the source of osteopontin and bone sialopro-tein expression in human breast cancer. Lab Invest 1999, 79:869-877.
• [44]Oates AJ, Barraclough R, Rudland PS: The identification of metastasis-related gene products in a rodent mammary tumour model. Biochem Soc Trans 1996, 24:353S.
• [45]Behrend EI, Craig AM, Wilson SM, Denhardt DT, Chambers AF: Reduced malignancy of ras-transformed NIH 3T3 cells expressing antisense osteopontin RNA. Cancer Res 1994, 54:832-837.
• [46]Gardner HA, Berse B, Senger DR: Specific reduction in osteopontin synthesis by antisense RNA inhibits the tumorigenicity of transformed Rat1 fibroblasts. Oncogene 1994, 9:2321-2326.
• [47]Sliva D, Rizzo MT, English D: Phosphatidylinositol 3-kinase and NF-kappaB regulate motility of invasive MDA-MB-231 human breast cancer cells by the secretion of urokinase-type plasminogen activator. J Biol Chem 2002, 277:3150-3157.
• [48]Tuck AB, Arsenault DM, O'Malley FP, Hota C, Ling MC, Wilson SM, Chambers AF: Osteopontin induces increased invasiveness and plasminogen activator expression of human mammary epithelial cells. Oncogene 1999, 18:4237-4246.
• [49]Baricos WH, Cortez SL, el-Dahr SS, Schnaper HW: ECM degradation by cultured human mesangial cells is mediated by a PA/plasmin/MMP-2 cascade. Kidney Int 1995, 47:1039-1047.
• [50]Mazzieri R, Masiero L, Zanetta L, Monea S, Onisto M, Garbisa S, Mignatti P: Control of type IV collagenase activity by components of the urokinase–plasmin system: a regulatory mechanism with cell-bound reactants. Embo J 1997, 16:2319-2332.
• [51]Zohar R, Suzuki N, Suzuki K, Arora P, Glogauer M, McCulloch CA, Sodek J: Intracellular osteopontin is an integral component of the CD44–ERM complex involved in cell migration. J Cell Physiol 2000, 184:118-130.
• [52]Maglione JE, Moghanaki D, Young LJ, Manner CK, Ellies LG, Joseph SO, Nicholson B, Cardiff RD, MacLeod CL: Transgenic Polyoma middle-T mice model premalignant mammary disease. Cancer Res 2001, 61:8298-8305.
• [53]Zhang G, He B, Weber GF: Growth factor signaling induces metastasis genes in transformed cells: molecular connection between Akt kinase and osteopontin in breast cancer. Mol Cell Biol 2003, 23:6507-6519.
• [54]Kansra V, Groves C, Gutierrez-Ramos JC, Polakiewicz RD: Phosphatidylinositol 3-kinase-dependent extracellular calcium influx is essential for CX(3)CR1-mediated activation of the mitogen-activated protein kinase cascade. J Biol Chem 2001, 276:31831-31838.
• [55]Sodhi CP, Batlle D, Sahai A: Osteopontin mediates hypoxia-induced proliferation of cultured mesangial cells: role of PKC and p38 MAPK. Kidney Int 2000, 58:691-700.
• [56]You J, Reilly GC, Zhen X, Yellowley CE, Chen Q, Donahue HJ, Jacobs CR: Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3-E1 osteoblasts. J Biol Chem 2001, 276:13365-13371.
• [57]Wang-Rodriguez J, Urquidi V, Rivard A, Goodison S: Elevated osteopontin and thrombospondin expression identifies malignant human breast carcinoma but is not indicative of metastatic status. Breast Cancer Res 2003, 5:R136-R143. BioMed Central Full Text