Raft-dependent endocytosis of autocrine motility factor is phosphatidylinositol 3-kinase-dependent in breast carcinoma cells.
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-beta-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.
Kojic
Liliana D
LD
Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada.
Joshi
Bharat
B
Lajoie
Patrick
P
Le
Phuong U
PU
Cox
Michael E
ME
Turbin
Dmitry A
DA
Wiseman
Sam M
SM
Nabi
Ivan R
IR
D001940:Breast / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D002277:Carcinoma / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002469:Cell Separation; D015638:Cytochalasin D / Q000494:pharmacology; D053938:DNA Fragmentation; D004705:Endocytosis; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D005956:Glucose-6-Phosphate Isomerase / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*
D015638:Cytochalasin D; D019869:Phosphatidylinositol 3-Kinases; D005956:Glucose-6-Phosphate Isomerase