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J. Biol. Chem., Vol. 282, Issue 40, 29305-29313, October 5, 2007

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Raft-dependent Endocytosis of Autocrine Motility Factor Is Phosphatidylinositol 3-Kinase-dependent in Breast Carcinoma Cells^*

Liliana D. Kojic, Bharat Joshi, Patrick Lajoie¹, Phuong U. Le, Michael E. Cox^¶, Dmitry A. Turbin^||, Sam M. Wiseman^**¶||2, and Ivan R. Nabi, Canadian Institutes of Health Research Investigator³

From the Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, National Research Council, Biotechnology Research Institute, Montreal, Quebec H4P 2R2, ^¶Prostate Research Centre, British Columbia Cancer Agency and University of British Columbia, Vancouver, British Columbia V6H 3Z6, ^||Genetic Pathology Evaluation Centre, Department of Pathology, Vancouver General Hospital, British Columbia Cancer Agency, and University of British Columbia, Vancouver, British Columbia V6H 3Z6, and the ^**Department of Surgery, St. Paul's Hospital/University of British Columbia, Vancouver, British Columbia V6T NZ, Canada

Autocrine motility factor (AMF) is internalized via a receptor-mediated, dynamin-dependent, cholesterol-sensitive raft pathway to the smooth endoplasmic reticulum that is negatively regulated by caveolin-1. Expression of AMF and its receptor (AMFR) is associated with tumor progression and malignancy; however, the extent to which the raft-dependent uptake of AMF is tumor cell-specific has yet to be addressed. By Western blot and cell surface fluorescence-activated cell sorter (FACS) analysis, AMFR expression is increased in tumorigenic MCF7 and metastatic MDA-231 and MDA-435 breast cancer cell lines relative to dysplastic MCF10A mammary epithelial cells. AMF uptake, determined by FACS measurement of protease-insensitive internalized fluorescein-conjugated AMF, was increased in MCF7 and MDA-435 cells relative to MCF-10A and caveolin-1-expressing MDA-231 cells. Uptake of fluorescein-conjugated AMF was dynamin-dependent, methyl--cyclodextrin- and genistein-sensitive, reduced upon overexpression of caveolin-1 in MDA-435 cells, and increased upon short hairpin RNA reduction of caveolin-1 in MDA-231 cells. Tissue microarray analysis of invasive primary human breast carcinomas showed that AMFR expression had no impact on survival but did correlate significantly with expression of phospho-Akt. Phospho-Akt expression was increased in AMF-internalizing MCF7 and MDA-435 breast carcinoma cells. AMF uptake in these cells was reduced by phosphatidylinositol 3-kinase inhibition but not by regulators of macropinocytosis such as amiloride, phorbol ester, or actin cytoskeleton disruption by cytochalasin D. The raft-dependent endocytosis of AMF therefore follows a distinct phosphatidylinositol 3-kinase-dependent pathway that is up-regulated in more aggressive tumor cells.

Received for publication, May 16, 2007 , and in revised form, July 25, 2007.

^* This work was supported in part by a grant from the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Research student of the Terry Fox Foundation through an award from the National Cancer Institute of Canada.

² A Michael Smith Scholar.

³ To whom correspondence should be addressed: Dept. of Cellular and Physiological Sciences, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. E-mail: irnabi{at}interchange.ubc.ca.

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