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-β-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. 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-β-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. Autocrine motility factor (AMF) 4The abbreviations used are:AMFautocrine motility factorAMF-FITCfluorescein-conjugated AMFAMFRautocrine motility factor receptorCav1caveolin-1ERestrogen receptorFACSfluorescence-activated cell sorterHER2herceptin2Tf-FITCFITC-conjugated transferrinmβCDmethyl-β-cyclodextrinPI3Kphosphatidylinositol 3-kinasePRprogesterone receptorTMAtissue microarrayTPAphorbol ester12-O tetradecanoylphorbol 13-acetateshRNAshort hairpin RNAMFImean fluorescence intensityRTreverse transcriptionmAbmonoclonal antibody. is secreted by and stimulatory to tumor cells. AMF exhibits sequence identity to phosphoglucose isomerase, a glycolytic enzyme that also exhibits neurokine and lymphokine activities (1Watanabe H. Takehana K. Date M. Shinozaki T. Raz A. Cancer Res. 1996; 56: 2960-2963PubMed Google Scholar). AMFR expression is associated with poor survival and prognosis of patients with gastric, colorectal, bladder, and esophageal carcinomas, cutaneous malignant melanoma, and pulmonary adenocarcinoma (2Hirono Y. Fushida S. Yonemura Y. Yamamoto H. Watanabe H. Raz A. Br. J. Cancer. 1996; 74: 2003-2007Crossref PubMed Scopus (55) Google Scholar, 3Nakamori S. Watanabe H. Kameyama M. Imaoka S. Furukawa H. Ishikawa O. Sasaki Y. Kabuto T. Raz A. Cancer. 1994; 74: 1855-1862Crossref PubMed Scopus (75) Google Scholar, 4Otto T. Birchmeier W. Schmidt U. Hinke A. Schipper J. Rübben H. Raz A. Cancer Res. 1994; 54: 3120-3123PubMed Google Scholar, 5Maruyama K. Watanabe H. Hitoshi S. Takayama T. Gofuku J. Yano H. Inoue M. Tamura S. Raz A. Monden M. Int. J. Cancer. 1995; 64: 316-321Crossref PubMed Scopus (52) Google Scholar, 6Nagai Y. Ishikawa O. Miyachi Y. Watanabe H. Dermatology. 1996; 192: 8-11Crossref PubMed Scopus (20) Google Scholar, 7Takanami I. Takeuchi K. Naruke M. Kodaira S. Tanaka F. Watanabe H. Raz A. Tumor Biol. 1998; 19: 384-389Crossref PubMed Scopus (28) Google Scholar, 8Taniguchi K. Yonemura Y. Nojima N. Hirono Y. Fushida S. Fujimura T. Miwa K. Endo Y. Yamamoto H. Watanabe H. Cancer. 1998; 82: 2112-2122Crossref PubMed Scopus (101) Google Scholar). Importantly, in many of these studies, AMFR was absent or present at significantly reduced levels in adjacent normal tissue. Recently, a genome-wide analysis identified AMFR as one of 189 genes that present a high frequency of intragenic mutations in breast cancer (9Sjoblom T. Jones S. Wood L.D. Parsons D.W. Lin J. Barber T. Mandelker D. Leary R.J. Ptak J. Silliman N. Szabo S. Buckhaults P. Farrell C. Meeh P. Markowitz S.D. Willis J. Dawson D. Willson J.K.V. Gazdar A.F. Hartigan J. Wu L. Liu C. Parmigiani G. Park B.H. Bachman K.E. Papadopoulos N. Vogelstein B. Kinzler K.W. Velculescu V.E. Science. 2006; 314: 268-274Crossref PubMed Scopus (2870) Google Scholar). Altered expression of AMFR is therefore associated with breast tumor progression and metastasis. autocrine motility factor fluorescein-conjugated AMF autocrine motility factor receptor caveolin-1 estrogen receptor fluorescence-activated cell sorter herceptin2 FITC-conjugated transferrin methyl-β-cyclodextrin phosphatidylinositol 3-kinase progesterone receptor tissue microarray phorbol ester12-O tetradecanoylphorbol 13-acetate short hairpin RNA mean fluorescence intensity reverse transcription monoclonal antibody. AMFR localizes to cell surface caveolae and internalizes AMF to the smooth endoplasmic reticulum via a dynamin-dependent, raft-mediated endocytic pathway negatively regulated by caveolin-1 (Cav1) (10Benlimame N. Le P.U. Nabi I.R. Mol. Biol. Cell. 1998; 9: 1773-1786Crossref PubMed Scopus (104) Google Scholar, 11Le P.U. Nabi I.R. J. Cell Sci. 2003; 116: 1059-1071Crossref PubMed Scopus (171) Google Scholar, 12Le P.U. Guay G. Altschuler Y. Nabi I.R. J. Biol. Chem. 2002; 277: 3371-3379Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Multiple raft-dependent pathways have been described that vary in their dependence on expression of Cav1 and dynamin-2 (13Kirkham M. Parton R.G. Biochim. Biophys. Acta. 2005; 1745: 273-286Crossref PubMed Scopus (209) Google Scholar, 14Lajoie P. Nabi I.R. J. Cell Mol. Med. 2007; 11: 644-653Crossref PubMed Scopus (223) Google Scholar). The AMF pathway can be distinguished from the caveolae/raft-dependent endocytosis of SV40 virus to the endoplasmic reticulum (15Pelkmans L. Kartenbeck J. Helenius A. Nat. Cell Biol. 2001; 3: 473-483Crossref PubMed Scopus (1055) Google Scholar) and of cholera toxin b-subunit to the Golgi apparatus by the insensitivity of its uptake to brefeldin A, nocodazole, and a 20 °C temperature block (11Le P.U. Nabi I.R. J. Cell Sci. 2003; 116: 1059-1071Crossref PubMed Scopus (171) Google Scholar). Reduced expression of Cav1 upon transformation of NIH-3T3 cells with ras or abl oncogenes is associated with increased AMF uptake (12Le P.U. Guay G. Altschuler Y. Nabi I.R. J. Biol. Chem. 2002; 277: 3371-3379Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Conversely, AMF transfection of NIH-3T3 cells induces enhanced transformation and survival via PI3K/Akt signaling and Cav1 down-regulation (16Tsutsumi S. Hogan V. Nabi I.R. Raz A. Cancer Res. 2003; 63: 242-249PubMed Google Scholar). This suggests that AMF/AMFR expression and signaling are inversely related to Cav1 expression during tumor progression. However, AMF endocytosis in human tumor cells has yet to be studied, and factors that promote the raft-dependent endocytosis of AMF have not been identified. In this study, we show that AMF internalization is reduced in dysplastic MCF10A mammary cells relative to tumorigenic and metastatic tumor cell lines and that Cav1 is a critical regulator of the raft-dependent endocytosis of AMF in breast carcinoma cells. Tissue microarray analysis (TMA) of AMFR expression identified a highly significant association with p-Akt. AMF stimulates p-Akt expression, and PI3K inhibition prevents raft-dependent AMF uptake in breast carcinoma cells. PI3K is therefore identified as a positive regulator of the AMF raft pathway, indicative of a feedback loop linking AMFR signaling through PI3K to AMF uptake. Cells, Antibodies, and Reagents—MCF7, MDA-231, MDA-435, and MCF10A human breast cell lines (ATCC, Manassas, VA) were maintained in complete RPMI 1640 medium containing 10% FBS. Monoclonal rat IgM anti-AMFR (3F3A) was used as ascites fluid (17Nabi I.R. Watanabe H. Raz A. Cancer Res. 1990; 50: 409-414PubMed Google Scholar). Rabbit anti-Cav1/2 and mouse anti-Cav1 antibody were from Transduction Laboratories; rabbit anti-p-Akt and Akt were from Cell Signaling (Danvers, MA); Alexa488- and Alexa647-conjugated secondary antibodies, Alexa568-conjugated phalloidin, and rabbit anti-FITC antibodies were from Molecular Probes (Eugene, OR), and rhodamine red-X anti-rat IgM was from Jackson ImmunoResearch (West Grove, PA). Commercial rabbit phosphoglucose isomerase (referred to as AMF) was purchased from Sigma (P-9544) and conjugated to FITC using fluorescein-EX protein labeling kit (Molecular Probes). Monoclonal anti-β-actin, methyl-β-cyclodextrin (mβCD), genistein, FITC-conjugated transferrin (Tf-FITC), propidium iodide, goat serum, and Pronase were from Sigma, and LY294002 was from Calbiochem. Cav1 shRNA Lentiviral Constructs—Core sequences for Cav1 shRNA constructs (5′-CTGTTCCCATCCGGGAACAGGGCAACAT-3′) were cloned into pSHAG-1 plasmid (Dr. G. Hannon, Cold Spring Harbor, NY), and the shRNA cassette was transferred into pLenti6/BLOCK-iT™-DEST (Invitrogen) that carries a blasticidin selection marker. Lentiviral vector from conditioned media of HEK293T cells transfected with Cav1 shRNA-pLenti6/BLOCK-iT™-DEST (18Yu D. Jia W.W. Gleave M.E. Nelson C.C. Rennie P.S. Prostate. 2004; 59: 370-382Crossref PubMed Scopus (22) Google Scholar) was used to infect MDA-231 cells. A pooled population of blasticidin-selected MDA-231 cells exhibiting maximal Cav1 suppression was used for subsequent experiments. Western Blots and RT-PCR—Cell lysates were prepared as described (19Joshi B. Ordonez-Ercan D. Dasgupta P. Chellappan S. Oncogene. 2005; 24: 2204-2217Crossref PubMed Scopus (28) Google Scholar) and centrifuged for 15 min at 13,000 rpm at 4 °C. Supernatant and pellet, the latter passed 20 times through a 21.5-gauge needle, were separated on 10% SDS-PAGE, electroblotted onto nitrocellulose membranes, and revealed with the indicated primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and chemiluminescence. Band intensity was quantified by densitometry relative to β-actin. Total RNA was extracted by the TRIzol method, and RT-PCR was performed following standard protocols using SuperScript III reverse transcriptase (Invitrogen), oligo(dT), and primers for AMFR (5′-ATGCCGCTGCTCTTCCTCGAGCGC-3′;5′-TCAACCGAAAAACTCGGCAGCCAGCTC-3′) and β-actin (5′-GCCCTTTCTCACTGGTTCTC-3′;5′-CTTTACACCAGCCTCATGGC-3′). Immunofluorescence, FACS, and Electron Microscopy—Immunofluorescence, FACS, and electron microscopy were performed as described (10Benlimame N. Le P.U. Nabi I.R. Mol. Biol. Cell. 1998; 9: 1773-1786Crossref PubMed Scopus (104) Google Scholar, 12Le P.U. Guay G. Altschuler Y. Nabi I.R. J. Biol. Chem. 2002; 277: 3371-3379Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). For uptake analysis, adherent cells were incubated with 25 μg/ml AMF-FITC or 15 μg/ml Tf-FITC for 30 min at 37 °C. For FACS analysis, cell surface-bound conjugate was removed with Pronase (400 μg/ml) for 15 min. Flow cytometry measurements of at least 50,000 cells used Cellquest software on a FACSCalibur (BD Biosciences). Adenoviral constructs and infection were as described (12Le P.U. Guay G. Altschuler Y. Nabi I.R. J. Biol. Chem. 2002; 277: 3371-3379Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 20Altschuler Y. Liu S. Katz L. Tang K. Hardy S. Brodsky F. Apodaca G. Mostov K. J. Cell Biol. 1999; 147: 7-12Crossref PubMed Scopus (119) Google Scholar). Statistical Analysis—RT-PCR, Western blots, immunofluorescence, and FACS experiments were reproduced at least three times, and representative blots and images are presented. FACS experiments were performed in duplicate. Unless otherwise stated, all values are presented as mean ± S.E. of at least three independent experiments. Statistical significance was evaluated using the Student's t test for paired comparison; p < 0.05 was considered significant. Human Breast Tissue Microarrays— 438 sequential archival cases of invasive breast carcinoma, with available paraffin blocks, that had undergone treatment at Vancouver General Hospital between 1974 and 1995 were identified for TMA construction (21Parker R.L. D.W. M. J. 2002; PubMed Scopus Google Scholar) with tissue present for of The was by the of the of used and are in of TMA for and A. B. J. G. M. Cancer Res. 1990; 50: Google N. B. M. D. K. Cancer. 2005; PubMed Scopus Google and p-Akt J. Wu J. C. D. S. Nelson C. M. H. K. S. D. C. M. S.E. Oncogene. 2005; 24: PubMed Scopus Google Scholar) was as described AMFR was positive invasive carcinoma cell was For were using for and and a of for AMFR and to the for TMA were using a and to a are available on were evaluated and from images on a by to on all patients was from was 15 and all patients had invasive breast and tumor tumor tumor and were onto a TMA software W. Y. A. K. M. J. 2002; Full Text Full Text PDF PubMed Scopus Google and by software analysis used the were considered significant p < The was for survival analysis with test to significance in survival For the analysis, we used a AMFR Expression and in Human Cell and protein levels were significantly increased in MDA-231, and MDA-435 breast cancer cell lines relative to dysplastic epithelial MCF10A cells but not levels of AMFR were increased in MCF7 cells relative to metastatic MDA-231 and MDA-435 cells. By Western AMFR was in and and Cav1 in the Cav1 levels were in MDA-231 cells, in MDA-435 cells, and in MCF10A and MCF7 cells expression of Cav1 in MDA-231 cells was associated with a significantly increased of caveolae FACS analysis showed that dysplastic MCF10A cells were positive for surface AMFR expression relative to the three cancer cell metastatic MDA-435 and MDA-231 presented increased mean fluorescence intensity for surface AMFR expression relative to MCF7 and dysplastic MCF10A cell lines Cell surface of anti-AMFR was by of the cells with AMF at 4 °C as (17Nabi I.R. Watanabe H. Raz A. Cancer Res. 1990; 50: 409-414PubMed Google that the anti-AMFR antibody is for the AMF receptor incubated with cells for 30 min at 37 AMF-FITC showed increased association with MDA-435 cells relative to the breast cell lines by FACS of cell surface-bound AMF-FITC by MDA-435 cells showed significant uptake of AMF-FITC in of positive cell and AMF-FITC uptake was significantly increased in MCF7 relative to MCF10A and MDA-231 cells but reduced relative to MDA-435 cells. of AMFR Uptake by Cav1 in Breast Cancer of AMF-FITC to the smooth endoplasmic reticulum is by with and the FACS analysis that these of caveolae/raft-dependent endocytosis AMF-FITC internalization on uptake of Tf-FITC AMF uptake in these cell lines is as a of AMF for AMF-FITC uptake treatment on cell surface AMFR expression Adenoviral expression of Cav1 and but not or significantly AMF-FITC uptake in MDA-435 cells cell surface AMFR expression a expression of the and but not Cav1 or was to uptake of Tf-FITC Conversely, infection of MDA-231 cells with Cav1 Cav1 levels by in a in AMF uptake Cav1 expression therefore negatively the dynamin-dependent, raft-dependent uptake of AMF in invasive breast cancer cells. Tissue of Human Breast TMA analysis of cases of invasive breast cancer AMFR expression was not associated with survival were between AMFR expression and tumor tumor or AMFR had a with tumor and AMFR expression showed a highly significant with on the breast cancer TMA J. Wu J. C. D. S. Nelson C. M. H. K. S. D. C. M. S.E. Oncogene. 2005; 24: PubMed Scopus Google Scholar). of AMF high of of AMFR and p-Akt in the TMA analysis to p-Akt expression in the breast cancer cell in p-Akt levels are in MCF7 and MDA-435 relative to MCF10A and MDA-231 cells. in MDA-435 cells, AMF Akt with the of p-Akt in NIH-3T3 cells (16Tsutsumi S. Hogan V. Nabi I.R. Raz A. Cancer Res. 2003; 63: 242-249PubMed Google Scholar). with had no on in MDA-435 and MCF7 cells that of AMF uptake by is not to PI3K inhibition p-Akt expression to the increased AMF uptake in MDA-435 cells to a in MCF7 cells with the PI3K for min Akt to of significantly AMF uptake in MCF7 and MDA-435 cells PI3K inhibition AMFR surface expression uptake of Tf-FITC PI3K has been described as a regulator of macropinocytosis M. B. U. A. Mol. Biol. Cell. 11: PubMed Scopus Google Scholar, N. T. Furukawa A. J. Cell Sci. 2003; 116: PubMed Scopus Google Scholar, K. S. G. J. Cell Sci. 1998; PubMed Google Scholar). However, AMF uptake was not by of macropinocytosis such as or actin disruption with cytochalasin by the phorbol 13-acetate and AMFR expression was reduced in dysplastic MCF10A cells relative to breast cancer cell AMFR is at the cell surface and the smooth endoplasmic reticulum Nabi I.R. 2006; Scopus (52) Google Scholar). Reduced expression of AMFR by Western blot with its increased and cell surface expression by FACS in metastatic MDA-231 and MDA-435 cells relative to the MCF7 cells. AMFR is a critical in and can for by the S. M. C. S. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). The of the of AMFR in tumor cells However, that the cell surface expression of AMFR is more associated with its in metastasis. Uptake of AMF was in tumorigenic MCF7 and metastatic MDA-435 breast tumor cells J. 2001; Full Text Full Text PDF PubMed Scopus Google with increased AMFR internalization in NIH-3T3 and metastatic cells (12Le P.U. Guay G. Altschuler Y. Nabi I.R. J. Biol. Chem. 2002; 277: 3371-3379Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, H. Nabi I.R. Raz A. Cancer Res. Google Scholar). In uptake of AMF-FITC is dynamin-dependent, and and AMF uptake in these breast cancer cell lines therefore follows the caveolae/raft-dependent pathway to the smooth endoplasmic reticulum as in cell (10Benlimame N. Le P.U. Nabi I.R. Mol. Biol. Cell. 1998; 9: 1773-1786Crossref PubMed Scopus (104) Google Scholar, 12Le P.U. Guay G. Altschuler Y. Nabi I.R. J. Biol. Chem. 2002; 277: 3371-3379Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, I.R. Le P.U. J. Cell Biol. 2003; PubMed Scopus Google Scholar). Reduced uptake of AMF in metastatic MDA-231 cells was associated with increased expression of Cav1 overexpression and Cav1 shRNA in metastatic MDA-435 and MDA-231 cells, Cav1 as a regulator of AMF uptake in these invasive cancer cells. The high of of AMFR and p-Akt J. Wu J. C. D. S. Nelson C. M. H. K. S. D. C. M. S.E. Oncogene. 2005; 24: PubMed Scopus Google Scholar) on the breast tumor TMA to PI3K AMF uptake. In MDA-435 cells, AMF Akt and PI3K regulated AMF uptake. In p-Akt expression was in AMF MCF7 and MDA-435 cells but not in the Cav1 MDA-231 cells Multiple have been to SV40 uptake L. H. M. B. M. 2005; PubMed Scopus Google and the that AMF uptake but not Akt that are in the raft-dependent endocytosis of AMF of p-Akt in MDA-435 cells was the short of AMF to the smooth endoplasmic reticulum (11Le P.U. Nabi I.R. J. Cell Sci. 2003; 116: 1059-1071Crossref PubMed Scopus (171) Google Scholar). is therefore not PI3K its endocytosis or AMF uptake is for its of Multiple pathways of raft-dependent endocytosis have been dynamin-dependent, and pathways (13Kirkham M. Parton R.G. Biochim. Biophys. Acta. 2005; 1745: 273-286Crossref PubMed Scopus (209) Google Scholar, 14Lajoie P. Nabi I.R. J. Cell Mol. Med. 2007; 11: 644-653Crossref PubMed Scopus (223) Google Scholar). The latter raft-dependent but macropinocytosis (13Kirkham M. Parton R.G. Biochim. Biophys. Acta. 2005; 1745: 273-286Crossref PubMed Scopus (209) Google Scholar). However, inhibition of the uptake of AMF by the that the of AMF uptake at the cell surface and not at a subsequent AMF uptake was not by regulators of macropinocytosis the raft-dependent uptake of AMF as a distinct raft endocytic PI3K have been to uptake of by however, this endocytic to the AMF uptake pathway to be determined J. Biol. 1999; PubMed Scopus (55) Google Scholar). impact on macropinocytosis through of phosphatidylinositol of the actin cytoskeleton (13Kirkham M. Parton R.G. Biochim. Biophys. Acta. 2005; 1745: 273-286Crossref PubMed Scopus (209) Google Scholar, J. S. D. L. M. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). AMF uptake was not by disruption of the actin cytoskeleton with cytochalasin in MDA-435 cells or MCF7 cells not cytoskeleton disruption is associated with increased of Cav1 and uptake J. Cell Biol. 1995; PubMed Scopus Google Scholar, R.G. B. K. J. Cell Biol. 1994; PubMed Scopus Google Scholar, P. K. M. B. Mol. Biol. Cell. 2002; PubMed Scopus Google Scholar, T. J. Cell Sci. 2002; PubMed Scopus Google Scholar, L. D. Helenius A. Science. 2002; PubMed Scopus Google Scholar). The of AMF uptake on actin cytoskeleton this pathway from have that AMF uptake to the smooth endoplasmic reticulum is distinct from raft-dependent cholera toxin b-subunit uptake to the Golgi apparatus and SV40 uptake to the endoplasmic reticulum (11Le P.U. Nabi I.R. J. Cell Sci. 2003; 116: 1059-1071Crossref PubMed Scopus (171) Google Scholar). The PI3K dependence and actin of AMF internalization for the distinct of this dynamin-dependent, raft-dependent this pathway is to be tumor cell-specific and associated with enhanced AMFR expression in aggressive tumor cells. p-Akt in MDA-435 cells and AMF uptake is indicative of a feedback loop between these linking the of AMFR and p-Akt in the breast tumor transfection of NIH-3T3 with AMF, in autocrine of and to was to be associated with Akt and reduced Cav1 expression (16Tsutsumi S. Hogan V. Nabi I.R. Raz A. Cancer Res. 2003; 63: 242-249PubMed Google Scholar). Autocrine of AMFR and PI3K signaling in tumor cells to AMFR and Akt is associated with increased tumor progression and tumor cell Cell. 2002; PubMed Scopus Google Scholar) and is of aggressive tumor and reduced survival in breast cancer F. B. M. O. F. K.W. 2004; PubMed Scopus Google Scholar). AMFR in breast carcinomas did not correlate with as for p-Akt labeling of the TMA J. Wu J. C. D. S. Nelson C. M. H. K. S. D. C. M. S.E. Oncogene. 2005; 24: PubMed Scopus Google Scholar). This is with a of AMF and AMFR expression in breast Raz A. A. J. 2006; 54: PubMed Scopus Google Scholar). overexpression of AMFR and p-Akt a of human breast not the a significant of all invasive factors Cav1 and p-Akt AMF uptake, that internalization of cell surface AMFR be associated with of Akt and reduction of Cav1 levels in breast tumor cells. In invasive breast cancer AMFR tumor expression showed no significant with or expression, but did present a with with breast AMFR a of patients with breast cancer have from or The expression of AMFR by cancer cells in and in suggests that the and raft-mediated endocytosis of AMF a cancer cell-specific endocytic for

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.