The candidate tumor suppressor CST6 alters the gene expression profile of human breast carcinoma cells: Down-regulation of the potent mitogenic, motogenic, and angiogenic factor autotaxin

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Abstract

We recently coined CST6 as a novel candidate tumor suppressor gene for breast cancer. CST6 indeed is expressed in the normal human breast epithelium, but little or not at all in breast carcinomas and breast cancer cell lines. Moreover, ectopic expression of CST6 in human breast cancer cells suppressed cell proliferation, migration, invasion, and orthotopic tumor growth. To obtain insights into the molecular mechanism by which CST6 exhibits its pleiotropic effects on tumor cells, we compared global gene expression profiles in mock- and CST6-transfected human MDA-MB-435S cells. Out of 12,625 transcript species, 61 showed altered expression. These included genes for extracellular matrix components, cytokines, kinases, and phosphatases, as well as several key transcription factors. TaqMan PCR assays were used to confirm the microarray data for 7 out of 11 genes. One down-regulated gene product, secreted autotaxin/lyso-phospholipase D, was of particular interest because its down-regulation by CST6 could explain most of CST6’s effect on the breast cancer cells. This study thus provides the first evidence that CST6 plays a role in the modulation of genes, particularly, genes that are highly relevant to breast cancer progression.

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Materials and methods

Cell culture and transfection. Human breast carcinoma MDA-MB-435S cells (passage #400) were purchased from ATCC and, before transfection, cloned by limiting dilution. A single clone, representative of the parental cell ‘line’ with regard to morphology and cystatin expression (parental clone), was stably transfected with a pTracer-CMV2-based CST6 expression vector (CST6-clones) or the ‘empty’ control vector (mock-clones) as reported previously [6]. Cells were seeded into 100-mm Petri dishes and

CST6 does not alter expression of other cystatins or cathepsins

To determine the molecular mechanism(s) by which CST6 suppresses the malignant properties of tumor cells in vitro [6], we compared global gene expression profiles in two mock- and two CST6-transfected human MDA-MB-435S cell clones. For this, we used Affymetrix Hu95A v.2 oligonucleotide microarrays, which interrogate about 12,625 transcript species. First, we determined whether ectopic expression of CST6 in MDA-MB-435S cells was compensated by increased expression of a lysosomal cysteine

Discussion

Normal epithelial cells undergo multiple genetic and epigenetic changes to acquire the highly proliferative, invasive, vasculogenic/angiogenic, immunosuppressive, and metastatic properties that are characteristic of malignant tumors [28]. These genetic changes lead in turn to global changes in gene expression [29], and more specifically, in overexpression of tumor-promoting genes and loss of expression of tumor-suppressing genes. Close to 100 genes have so far been identified as tumor-promoting

Acknowledgments

This study was in part supported by a Research Grant CA91785 (DK) and by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research (TC). JS received a Takeda Scholar-in-Training Award from the American Association for Cancer Research to present part of this work at the Special Conference on Cancer Research: ‘New Directions in Angiogenesis Research,’ Chicago, October 15–19, 2003. RS was partly supported by Research Grant CA36481 (Bonnie F. Sloane, Wayne

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    Present address: Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 21231, USA.

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