Kojic Liliana D, Joshi Bharat, Lajoie Patrick, Le Phuong U, Cox Michael E, Turbin Dmitry A, Wiseman Sam M, Nabi Ivan R
Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada.
J Biol Chem. 2007 Oct 5;282(40):29305-13. doi: 10.1074/jbc.M704069200. Epub 2007 Aug 8.
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.
自分泌运动因子（AMF）通过受体介导、发动蛋白依赖性、胆固醇敏感的脂筏途径内化至滑面内质网，该途径受小窝蛋白-1负调控。AMF及其受体（AMFR）的表达与肿瘤进展和恶性程度相关；然而，AMF依赖脂筏的摄取在多大程度上具有肿瘤细胞特异性尚待研究。通过蛋白质印迹法和细胞表面荧光激活细胞分选仪（FACS）分析，相对于发育异常的MCF10A乳腺上皮细胞，致瘤性MCF7以及转移性MDA - 231和MDA - 435乳腺癌细胞系中AMFR表达增加。通过FACS测量蛋白酶不敏感的内化荧光素偶联AMF来确定AMF摄取，相对于MCF - 10A和表达小窝蛋白-1的MDA - 231细胞，MCF7和MDA - 435细胞中的AMF摄取增加。荧光素偶联AMF的摄取是发动蛋白依赖性的，对甲基-β-环糊精和染料木黄酮敏感，在MDA - 435细胞中过表达小窝蛋白-1时摄取减少，而在MDA - 231细胞中通过短发夹RNA降低小窝蛋白-1时摄取增加。对浸润性原发性人乳腺癌的组织芯片分析表明，AMFR表达对生存率没有影响，但与磷酸化Akt的表达显著相关。在摄取AMF的MCF7和MDA - 435乳腺癌细胞中磷酸化Akt表达增加。这些细胞中的AMF摄取通过磷脂酰肌醇3激酶抑制而减少，但不受巨胞饮作用调节剂如amiloride、佛波酯或细胞松弛素D对肌动蛋白细胞骨架的破坏的影响。因此，AMF依赖脂筏的内吞作用遵循一条独特的磷脂酰肌醇3激酶依赖性途径，该途径在侵袭性更强的肿瘤细胞中上调。
