Human breast cancer growth inhibited in vivo by a dominant negative pleiotrophin mutant.

Nan Zhang,Rong Zhong,Thomas F Deuel,Zhao-Yi Wang

doi:10.1074/jbc.272.27.16733

Nan Zhang, Rong Zhong + Show 2 more

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https://doi.org/10.1074/jbc.272.27.16733

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Abstract

Pleiotrophin (PTN) is a recently described 18- kDa heparin binding growth/differentiation factor. It also is a proto-oncogene; cells transformed by the Ptn gene form highly angiogenic tumors when implanted into the nude mouse. PTN may be an important regulator of transformation in other tumors, because constitutively high levels of expression of the pleiotrophin (Ptn) gene are found in human breast cancer and other malignant cell lines, and its levels of expression are high in many human tumor specimens. To determine whether PTN is an important regulator of the malignant phenotype of human breast cancer cells, we constructed a mutant cDNA to encode a truncated PTN designed to heterodimerize with the product of the endogenous Ptn gene during processing. The mutant gene product blocked transformation of NIH 3T3 cells by the wild type (wt) Ptn gene product. The mutant Ptn cDNA was then introduced into human breast cancer MDA-MB-231 cells, and clonal lines that stably express the mutant Ptn cDNA were selected. The truncated PTN was shown to form heterodimers with the endogenous Ptn gene product in these cells. Furthermore, the MDA-MB-231 cells that express the mutant Ptn gene were no longer transformed; they failed to form plaques or colonies in soft agar and were unable to form tumors in the athymic nude mouse. The results establish an important role of PTN in the dysregulated growth of human breast cancer cells and suggest that constitutive expression of PTN may be essential to the malignant phenotype of human breast cancers in vivo.

Highlights

Identity and perfect conservation of 10 cysteine residues with the protein product of Mdk, a gene expressed in the early stages of retinoic acid-induced differentiation of mouse embryonal carcinoma cells [6], and with RI-HB, a protein isolated from chick basement membranes [7], linking Ptn with Mdk as a member of a new family of developmentally regulated genes [3]
It was shown subsequently that SW13 cells transformed by Ptn develop highly vascular tumors in the flanks of athymic nude mice [9], that high levels of the Ptn gene are expressed in specimens of many human tumors, including neuroblastoma, glioblastoma, prostate cancer, lung cancer, and Wilms’ tumor, and that many breast cancer cells and about one-fourth of over 40 human tumor cell lines of different origins express the Ptn gene in significant levels [9, 17, 18]
We demonstrate that expression of the truncated PTN reverts the transformed phenotype of clonally selected human breast cancer (MDA-MB-231) cells that constitutively express significant levels of the endogenous Ptn gene

Summary

Introduction

Identity and perfect conservation of 10 cysteine residues with the protein product of Mdk, a gene expressed in the early stages of retinoic acid-induced differentiation of mouse embryonal carcinoma cells [6], and with RI-HB, a protein isolated from chick basement membranes [7], linking Ptn with Mdk as a member of a new family of developmentally regulated genes [3]. It was shown subsequently that SW13 cells transformed by Ptn develop highly vascular tumors in the flanks of athymic nude mice [9], that high levels of the Ptn gene are expressed in specimens of many human tumors, including neuroblastoma, glioblastoma, prostate cancer, lung cancer, and Wilms’ tumor, and that many breast cancer cells and about one-fourth of over 40 human tumor cell lines of different origins express the Ptn gene in significant levels [9, 17, 18] These findings, strengthened by observations that the transformed phenotype of human melanoma cells (WM852) can be suppressed by Ptn-targeted ribozymes [19, 20], strongly support the view that PTN has a central regulatory role in neoplastic cell growth. The results establish that high levels of expression of PTN appear to be essential to maintain the transformed phenotype of MDA-MB231 cells and that the Ptn gene may be a central regulator of the dysregulated growth of many human tumors

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