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• W2023188630 abstract "Amphiregulin (AR) autocrine loops have been associated with several types of cancer. We demonstrate that SUM149 breast cancer cells have a self-sustaining AR autocrine loop. SUM149 cells are epidermal growth factor (EGF)-independent for growth, and they overexpress AR mRNA, AR membrane precursor protein, and secreted AR relative to the EGF-dependent human mammary epithelial cell line MCF10A. MCF10A cells made to overexpress AR (MCF10A AR) are also EGF-independent for growth. Treatment with the pan-ErbB inhibitor CI1033 and the anti-EGF receptor (EGFR) antibody C225 demonstrated that ligand-mediated activation of EGFR is required for SUM149 cell proliferation. AR-neutralizing antibody significantly reduced both SUM149 EGFR activity and cell proliferation, confirming that an AR autocrine loop is required for mitogenesis in SUM149 cells. EGFR tyrosine phosphorylation was dramatically decreased in both SUM149 and MCF10A AR cells after inhibition of AR cleavage with the broad spectrum metalloprotease inhibitor GM6001, indicating that an AR autocrine loop is strictly dependent on AR cleavage in culture. However, a juxtacrine assay where fixed SUM149 cells and MCF10A AR cells were overlaid on top of EGF-deprived MCF10A cells showed that the AR membrane precursor can activate EGFR. SUM149 cells, MCF10A AR cells, and MCF10A cells growing in exogenous AR were all considerably more invasive and motile than MCF10A cells grown in EGF. Moreover, AR up-regulates a number of genes involved in cell motility and invasion in MCF10A cells, suggesting that an AR autocrine loop contributes to the aggressive breast cancer phenotype. Amphiregulin (AR) autocrine loops have been associated with several types of cancer. We demonstrate that SUM149 breast cancer cells have a self-sustaining AR autocrine loop. SUM149 cells are epidermal growth factor (EGF)-independent for growth, and they overexpress AR mRNA, AR membrane precursor protein, and secreted AR relative to the EGF-dependent human mammary epithelial cell line MCF10A. MCF10A cells made to overexpress AR (MCF10A AR) are also EGF-independent for growth. Treatment with the pan-ErbB inhibitor CI1033 and the anti-EGF receptor (EGFR) antibody C225 demonstrated that ligand-mediated activation of EGFR is required for SUM149 cell proliferation. AR-neutralizing antibody significantly reduced both SUM149 EGFR activity and cell proliferation, confirming that an AR autocrine loop is required for mitogenesis in SUM149 cells. EGFR tyrosine phosphorylation was dramatically decreased in both SUM149 and MCF10A AR cells after inhibition of AR cleavage with the broad spectrum metalloprotease inhibitor GM6001, indicating that an AR autocrine loop is strictly dependent on AR cleavage in culture. However, a juxtacrine assay where fixed SUM149 cells and MCF10A AR cells were overlaid on top of EGF-deprived MCF10A cells showed that the AR membrane precursor can activate EGFR. SUM149 cells, MCF10A AR cells, and MCF10A cells growing in exogenous AR were all considerably more invasive and motile than MCF10A cells grown in EGF. Moreover, AR up-regulates a number of genes involved in cell motility and invasion in MCF10A cells, suggesting that an AR autocrine loop contributes to the aggressive breast cancer phenotype. The epidermal growth factor receptor (EGFR), 2The abbreviations used are: EGFR, epidermal growth factor receptor; AR, amphiregulin; EGF, epidermal growth factor; HB-EGF, heparin binding epidermal growth factor; TGF-α, transforming growth factor α; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; RT, reverse transcription. or ErbB1, is a transmembrane protein possessing intrinsic tyrosine kinase activity. There are several EGF family ligands that can bind and activate the EGFR including epidermal growth factor (EGF) (1Carpenter G. Cohen S. J. Biol. Chem. 1990; 265: 7709-7712Abstract Full Text PDF PubMed Google Scholar), amphiregulin (AR) (2Shoyab M. Plowman G.D. McDonald V.L. Bradley J.G. Todaro G.J. Science. 1989; 243: 1074-1076Crossref PubMed Scopus (488) Google Scholar), heparin-binding epidermal growth factor (HB-EGF) (3Higashiyama S. Abraham J.A. Miller J. Fiddes J.C. Klagsbrun M. Science. 1991; 251: 936-939Crossref PubMed Scopus (1045) Google Scholar), epiregulin (4Toyoda H. Komurasaki T. Uchida D. Takayama Y. Isobe T. Okuyama T. Hanada K. J. Biol. Chem. 1995; 270: 7495-7500Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), betacellulin (5Shing Y. Christofori G. Hanahan D. Ono Y. Sasada R. Igarashi K. Folkman J. Science. 1993; 259: 1604-1607Crossref PubMed Scopus (377) Google Scholar), transforming growth factor-α (TGF-α) (6Derynck R. Adv. Cancer Res. 1992; 58: 27-52Crossref PubMed Scopus (250) Google Scholar), and epigen (7Strachan L. Murison J.G. Prestidge R.L. Sleeman M.A. Watson J.D. Kumble K.D. J. Biol. Chem. 2001; 276: 18265-18271Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Ligand binding facilitates dimerization of the EGFR, which activates downstream pathways known to be involved in cell growth, proliferation, differentiation, and migration (8Wells A. Int. J. Biochem. Cell Biol. 1999; 31: 637-643Crossref PubMed Scopus (899) Google Scholar). Each EGF family ligand is expressed as a transmembrane precursor, which is proteolytically cleaved and released into the external milieu. There is no obvious homology in the predicted cleavage sites of the EGFR ligands, but it has been shown that metalloprotease activity is required for their release (9Hinkle C.L. Sunnarborg S.W. Loiselle D. Parker C.E. Stevenson M. Russell W.E. Lee D.C. J. Biol. Chem. 2004; 279: 24179-24188Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 12Arribas J. Coodly L. Vollmer P. Kishimoto T.K. Rose-John S. Massague J. J. Biol. Chem. 1996; 271: 11376-11382Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). The identities of the proteases involved in ligand cleavage are still obscure, but there is considerable evidence to suggest that tumor necrosis factor-α-converting enzyme/ADAM 17 is involved specifically in AR, HB-EGF, and TGF-α cleavage (9Hinkle C.L. Sunnarborg S.W. Loiselle D. Parker C.E. Stevenson M. Russell W.E. Lee D.C. J. Biol. Chem. 2004; 279: 24179-24188Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 10Sunnarborg S.W. Hinkle C.L. Stevenson M. Russell W.E. Raska C.S. Peschon J.J. Castner B.J. Gerhart M.J. Paxton R.J. Black R.A. Lee D.C. J. Biol. Chem. 2002; 277: 12838-12845Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). Although it is well known that soluble growth factors are biologically active, there is substantial debate in the current literature about the activity of EGF family precursor proteins. Certain EGF family members, such as HB-EGF, AR, TGF-α, and betacellulin, have been suggested to activate EGFR via juxtacrine interactions, whereas membrane-bound EGF has been shown to lack activity (13Anklesaria P. Teixido J. Laiho M. Pierce J.H. Greenberger J.S. Massague J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3289-3293Crossref PubMed Scopus (225) Google Scholar, 19Dong J. Opresko L.K. Chrisler W. Orr G. Quesenberry R.D. Lauffenburger D.A. Wiley H.S. Mol. Biol. Cell. 2005; 16: 2984-2998Crossref PubMed Scopus (29) Google Scholar). AR was originally purified from the conditioned media of MCF-7 breast cancer epithelial cells treated with the tumor promoter phorbol 12-myristate-13-acetate (20Shoyab M. McDonald V.L. Bradley J.G. Todaro G.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6528-6532Crossref PubMed Scopus (296) Google Scholar). It is synthesized as a 252-amino acid heparin-binding glycoprotein with an EGF-like domain and a basic NH2 terminus, which contains glycosylation sites and putative nuclear localization signals (21Plowman G.D. Green J.M. McDonald V.L. Neubauer M.G. Disteche C.M. Todaro G.J. Shoyab M. Mol. Cell Biol. 1990; 10: 1969-1981Crossref PubMed Scopus (327) Google Scholar). Due to differential processing and glycosylation, many different sizes of membrane-anchored AR (16-50 kDa) and secreted AR (9-60 kDa) have been found (2Shoyab M. Plowman G.D. McDonald V.L. Bradley J.G. Todaro G.J. Science. 1989; 243: 1074-1076Crossref PubMed Scopus (488) Google Scholar, 11Brown C.L. Meise K.S. Plowman G.D. Coffey R.J. Dempsey P.J. J. Biol. Chem. 1998; 273: 17258-17268Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 20Shoyab M. McDonald V.L. Bradley J.G. Todaro G.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6528-6532Crossref PubMed Scopus (296) Google Scholar, 24Martinez-Lacaci I. Johnson G.R. Salomon D.S. Dickson R.B. J. Cell Physiol. 1996; 169: 497-508Crossref PubMed Scopus (16) Google Scholar). In vivo, AR mRNA is expressed in many normal tissues, including placenta, testis, pancreas, spleen, kidney, lung, breast, ovary, and colon (21Plowman G.D. Green J.M. McDonald V.L. Neubauer M.G. Disteche C.M. Todaro G.J. Shoyab M. Mol. Cell Biol. 1990; 10: 1969-1981Crossref PubMed Scopus (327) Google Scholar). AR activation of EGFR appears to play a particularly relevant role in the developing breast, since AR is the critical EGFR ligand required for ductal morphogenesis in the mouse mammary gland, and it has been shown to act as an autocrine growth factor for some normal human mammary epithelial cells (25Sternlicht M.D. Sunnarborg S.W. Kouros-Mehr H. Yu Y. Lee D.C. Werb Z. Development. 2005; 132: 3923-3933Crossref PubMed Scopus (242) Google Scholar, 26Li S. Plowman G.D. Buckley S.D. Shipley G.D. J. Cell Physiol. 1992; 153: 103-111Crossref PubMed Scopus (69) Google Scholar). There is direct evidence that an EGFR/AR autocrine loop exists in pancreatic cancer, colon cancer, and hepatocellular carcinoma (27Castillo J. Erroba E. Perugorria M.J. Santamaria M. Lee D.C. Prieto J. Avila M.A. Berasain C. Cancer Res. 2006; 66: 6129-6138Crossref PubMed Scopus (123) Google Scholar, 29Funatomi H. Itakura J. Ishiwata T. Pastan I. Thompson S.A. Johnson G.R. Korc M. Int. J. Cancer. 1997; 72: 512-517Crossref PubMed Scopus (62) Google Scholar). AR expression was also found to be strongly correlated with inflammatory breast cancer, and a putative AR/EGFR autocrine loop is suggested to contribute to breast cancer progression (30Ma L. de Roquancourt A. Bertheau P. Chevret S. Millot G. Sastre-Garau X. Espie M. Marty M. Janin A. Calvo F. J. Pathol. 2001; 194: 413-419Crossref PubMed Scopus (37) Google Scholar). Indeed, AR may play a specific role in cancer progression, since it has been shown that AR activation of EGFR contributes to the synthesis, secretion, and activation of some proteins involved in invasion and metastasis, such as urokinase-type plasminogen activator, matrix metalloproteinase 9, and extracellular matrix metalloproteinase inducer (31Silvy M. Giusti C. Martin P.M. Berthois Y. Br. J. Cancer. 2001; 84: 936-945Crossref PubMed Scopus (25) Google Scholar, 33O-charoenrat P. Modjtahedi H. Rhys-Evans P. Court W.J. Box G.M. Eccles S.A. Cancer Res. 2000; 60: 1121-1128PubMed Google Scholar). We have developed the cell line SUM149 in our laboratory from an aggressive inflammatory breast cancer, and we used this cell line as a model to study the mechanism of AR action and its potential role as an autocrine growth factor in breast cancer (34Ignatoski K.M. Ethier S.P. Breast Cancer Res. Treat. 1999; 54: 173-182Crossref PubMed Scopus (42) Google Scholar). SUM149 cells are estrogen receptor-negative and overexpress EGFR but do not express any other active ErbB family members. In this report, we demonstrate that AR functions as an autocrine growth factor for a breast cancer cell line and show that this self-sustaining autocrine loop is dependent on EGFR activity. MCF10A cells are immortalized, nontransformed human mammary epithelial cells with an obligatory requirement for EGF for their growth and proliferation (35Soule H.D. Maloney T.M. Wolman S.R. Peterson Jr., W.D. Brenz R. McGrath C.M. Russo J. Pauley R.J. Jones R.F. Brooks S.C. Cancer Res. 1990; 50: 6075-6086PubMed Google Scholar). We show that overexpression of AR in MCF10A cells renders them EGF-independent for cell proliferation and increases both their cell motility and invasion capabilities. Similarly, SUM149 cells show increased cell motility and invasion relative to MCF10A cells. In addition, we provide evidence that membrane-anchored AR precursor can activate EGFR in a juxtacrine fashion, and therefore membrane-anchored AR may play an important role in the maintenance of an AR autocrine loop in breast cancer. Reagents and Antibodies—The antibodies used were mouse monoclonal anti-EGFR antibody Ab-5 (Oncogene) to immunoprecipitate EGFR, mouse monoclonal anti-EGFR antibody clone 31G7 (Zymed Laboratories, Inc.) to detect EGFR, anti-EGFR human (mouse) antibody clone 225 (C225) (Calbiochem) to block ligand binding to EGFR, goat polyclonal anti-AR antibody AF262 (R&D Systems) to detect AR and neutralize AR, mouse monoclonal antibody PY-20 (Calbiochem) to detect EGFR tyrosine phosphorylation, and mouse monoclonal anti-AR antibody MAB262 (R&D Systems) also used to neutralize AR. CI1033 was obtained from Pfizer, and GM6001 was obtained from Calbiochem. Recombinant AR was obtained from R&D Systems. Cell Culture—AR-overexpressing MCF10A cells (MCF10A AR) were made as previously described (36Berquin I.M. Dziubinski M.L. Nolan G.P. Ethier S.P. Oncogene. 2001; 20: 4019-4028Crossref PubMed Scopus (35) Google Scholar). SUM149 cells were maintained in Ham's F-12 medium with 5% fetal bovine serum, 5 μg/ml insulin, 2 μg/ml hydrocortisone, 5 μg/ml gentamicin, and 2.5 μg/ml fungizone. The serum-free base medium for MCF10A and MCF10A AR cells was SFIH (Ham's F-12 with 1 μg/ml hydrocortisone, 1 mg/ml bovine serum albumin, 10 mm Hepes, 5 mm ethanolamine, 5 μg/ml transferrin, 10 nm triiodothyronine, 50 nm sodium selenate, 5 μg/ml gentamicin, 2.5 μg/ml fungizone, and 5 μg/ml insulin), and MCF10A cells required 10 ng/ml EGF (SFIHE). MCF10A + AR cells were MCF10A cells grown in SFIH medium with 20 ng/ml exogenous AR (SFIHA), which was found previously to be the biologically equivalent concentration to EGF for these cells (36Berquin I.M. Dziubinski M.L. Nolan G.P. Ethier S.P. Oncogene. 2001; 20: 4019-4028Crossref PubMed Scopus (35) Google Scholar). All cells were maintained in a humidified incubator at 37 °C and 10% CO2. Quantitative RT-PCR—Total RNA was isolated from subconfluent cell culture plates using an RNeasy kit (Qiagen), and cDNA was reverse-transcribed from 4 μg of total RNA using the Superscript II kit (Invitrogen). Primers used were designed and synthesized by the Applied Biosystems Assays-by-Design service. AR primer sequences were 5′-GTTACTGCTTCCAGGTGCTCTA for the forward primer and 5′-GTTACTGCTTCCAGGTGCTCTA for the reverse primer. The probe sequence was 5′-ACGGAGAATGCAAATATA. A glyceraldehyde-3-phosphate dehydrogenase primer set was used as control. Real time quantitative RT-PCR was performed in 96-well plates using Taqman Universal PCR Master Mix (Applied Biosystems) at the University of Michigan Comprehensive Cancer Center Affymetrix and cDNA Microarray Core Facility. The reactions were done in replicates of 6. Calculation of the ΔΔCT values was performed as previously described (37Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (127155) Google Scholar). Briefly, for each cell line, the average number of cycles for glyceraldehyde-3-phosphate dehydrogenase control primer reactions to reach threshold fluorescence was calculated and subtracted from the average number of cycles for AR primer reactions to reach threshold fluorescence. These values for SUM149 and MCF10A AR cells were then subtracted from MCF10A values. These differences were then raised to the -2 power. Data are represented as -fold change relative to MCF10A control. Immunoprecipitation and Immunoblotting—The antibody anti-EGFR Ab-5 (Oncogene Research Products) was used for immunoprecipitation. Immunoprecipitation and Western blotting were performed as previously described (38Woods Ignatoski K.M. Livant D.L. Markwart S. Grewal N.K. Ethier S.P. Mol. Cancer Res. 2003; 1: 551-560PubMed Google Scholar). Protein from whole cell lysates was loaded onto 15% SDS-polyacrylamide gels for AR detection, and EGFR immunoprecipitates were loaded onto 7.5% SDS-polyacrylamide gels. After transferring proteins to polyvinylidene difluoride membranes, blots were probed with anti-EGFR antibody 31G7 (Zymed Laboratories), anti-phosphotyrosine antibody PY-20 (Calbiochem), or anti-AR antibody AF262 (R & D Systems) and visualized by enzymatic chemiluminescence (Pierce). Enzyme-linked Immunosorbent Assay (ELISA)—Conditioned medium was obtained from cells grown in 6-well plates. An AR DuoSet ELISA from R & D Systems was used to measure AR medium concentration. High binding ELISA plates were coated with 3 μg/ml MAB262 monoclonal AR antibody in sterile phosphate-buffered saline (PBS) overnight at room temperature. Absorbance was measured on a VERSAmax microplate reader (Molecular Devices Corp.). Cells were lysed, and nuclei were counted with a Z1 Coulter Counter (Beckman Coulter) for normalization. Samples were done in triplicate. Cell Proliferation Assays—Cells were seeded on day 0 in 6-well plates at ∼1.0×104 cells/well. Either 1 μg/ml AF262, 1 μg/ml MAB262, 1 μg/ml C225, or 1 μm CI1033 was added daily. After 5 or 7 days of treatment, plates were washed with PBS three times and agitated on a rocker table with 0.5 ml of a Hepes/MgCl2 buffer (0.01 m Hepes and 0.015 m MgCl2) for 5 min. Cells were then lysed for 10 min using a Bretol (ethyl hexadecyldimethylammonium) solution, and the nuclei were counted using a Z1 Coulter Counter (Beckman Coulter). Day 1 cells were counted for seeding counts. All experiments were done in triplicate. Juxtacrine Assays—MCF10A cells, SUM149 cells, and MCF10A AR cells were removed from confluent plates using 10 mm EDTA, washed three times with an acid wash (pH 4.0 Hanks' balanced salt solution), and fixed in formalin for 10 min. After fixation, the cells were washed three times in SFIH medium. The fixed cells were then resuspended in SFIH medium with either PBS or 1 μg/ml AF262 for 5 min and then overlaid on top of MCF10A cells that had been grown in EGF-free medium for 24 h. After 5 min, the plates were washed three times with PBS, followed by EGFR immunoprecipitation and Western analysis. Cell Motility Assay—The Cell Motility Bioapplication and Hitkit from Cellomics was used for these experiments. According to the manufacturer's instructions, collagen-I-coated 96-well plates were coated with prewashed blue fluorescent beads. ∼500 cells/well of MCF10A, SUM149, MCF10A AR, and MCF10A cells growing in AR were added to the wells of the plate. The plate was incubated for 24 h at 37 °C in 10% CO2. UV images were taken at ×20 using a Nikon inverted microscope. Cell Invasion Assay—Matrigel invasion chambers (BD Biosciences) were rehydrated with Dulbecco's modified Eagle's medium for 1 h in a 37°C incubator. ∼2.5 × 105 of MCF10A cells in SFIHE medium, SUM149 cells and MCF10A AR cells in SFIH medium, and MCF10A + AR cells in SFIHA medium were added to the upper chamber of both control and rehydrated Matrigel invasion chambers. 5% fetal bovine serum was added to media in the bottom chamber as a chemoattractant. After 24 h, membranes were fixed and stained using the Hema 3 Staining System (Fisher). Membranes were allowed to dry and then were placed onto slides for visualization. Cells on control and Matrigel membranes were counted from three microscopic fields after 24 h. Percentage of invasion was calculated by dividing the mean number of cells on the invasion membranes by the mean number of cells on the control membranes for each cell line. Experiments were repeated three times. Affymetrix Expression Array and Analysis—Total RNA was isolated from subconfluent cell culture plates using the RNeasy kit (Qiagen). The 28 and 18 S ribosomal RNA peak ratios were determined using microfluidics technology at the Wayne State University Applied Genomics Technology Center. The 28 S/18 S ratios of the RNA were determined to be higher than 1.3. 5-8 μg of RNA was then used for cDNA synthesis (Invitrogen). cRNA amplification was then performed using a kit from Enzo Diagnostics Inc. The cleaned cRNA was then hybridized to an Affymetrix human genome HGU133A array platform with 20,000 genes/chip. The expression array chip was scanned using an Agilent GeneArray scanner. The -fold change of fluorescence intensities was calculated relative to MCF10A. Analysis of array data was performed using Pathways-Express (39Khatri P. Sellamuthu S. Malhotra P. Amin K. Done A. Draghici S. Nucleic Acids Res. 2005; 33: W762-W765Crossref PubMed Scopus (94) Google Scholar) and the Database for Annotation, Visualization, and Integrated Discovery (40Dennis Jr., G. Sherman B.T. Hosack D.A. Yang J. Gao W. Lane H.C. Lempicki R.A. Genome Biol. 2003; 4: R60.1-R60.11Crossref Google Scholar). SUM149 Cells Require Ligand Activation of EGFR for Cell Proliferation—Constitutive EGFR signaling in cancer cells contributes to aberrant cell proliferation and tumor progression. Our laboratory has shown previously that EGF-independent SUM149 breast cancer cells overexpress constitutively active EGFR protein but do not express any other active ErbB family members (41Rao G.S. Murray S. Ethier S.P. Int. J. Radiat. Oncol. Biol. Phys. 2000; 48: 1519-1528Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Therefore, we utilized the pan-ErbB small molecule inhibitor CI1033 to determine whether SUM149 proliferation is dependent on EGFR activity. CI1033 prevents ATP binding in the kinase domain of the EGFR, thereby preventing activation of the receptor (42Smaill J.B. Rewcastle G.W. Loo J.A. Greis K.D. Chan O.H. Reyner E.L. Lipka E. Showalter H.D. Vincent P.W. Elliott W.L. Denny W.A. J. Med. Chem. 2000; 43: 1380-1397Crossref PubMed Scopus (293) Google Scholar). EGFR tyrosine phosphorylation was profoundly reduced in SUM149 cells treated with 1 μm of CI1033 for 1 h (Fig. 1A), which is in agreement with published data (41Rao G.S. Murray S. Ethier S.P. Int. J. Radiat. Oncol. Biol. Phys. 2000; 48: 1519-1528Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Moreover, treatment with 1 μm CI1033 every day for 5 days almost completely inhibited SUM149 cell proliferation (Fig. 1B). Therefore, SUM149 cells require EGFR activity for proliferation despite their EGF independence. Growth factor independence is a hallmark of malignancy, and one mechanism for this phenotype is the development of autocrine loops in cancer cells. Therefore, it is possible that SUM149 cells synthesize their own ligand in an autocrine fashion, which is necessary to maintain EGFR phosphorylation. To determine whether the constitutive EGFR activity in SUM149 cells can be attributed to ligand activation of the receptor, SUM149 cells were incubated for 15-min, 30-min, 1-h, or 2-h time periods with the clone 225 (C225) mouse monoclonal antibody to EGFR. The C225 antibody neutralizes EGFR activity by competing for the ligand binding domain of EGFR (43Thomas S.M. Grandis J.R. Cancer Treat. Rev. 2004; 30: 255-268Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). We observed that treatment with C225 antibody resulted in complete attenuation of EGFR tyrosine phosphorylation at 15 and 30 min. At 1 and 2 h after antibody, EGFR tyrosine phosphorylation had increased but not to the levels of control (Fig. 1C). These data are consistent with our hypothesis that the EGFR in SUM149 cells exhibits a dependence on an autocrine ligand for activity. To confirm that ligand activation of EGFR is required for SUM149 cell proliferation, a growth assay was performed in which the C225 antibody was added daily for 5 days and the cells were counted. The immortalized human mammary epithelial cell line MCF10A, which requires EGF for proliferation, was used as a positive control. The C225 antibody considerably reduced MCF10A cell proliferation, as expected. In addition, we found a significant reduction in SUM149 cell proliferation by growing cells in the presence of the antibody (Fig. 1D). The ability of C225 to decrease EGFR activity and cell proliferation suggests that endogenous EGFR ligands are regulating mitogenesis in SUM149 cells. AR Is Overexpressed at the Message and Protein Levels in SUM149 Cells—Previous expression cloning experiments performed in our laboratory identified candidate genes expressed by breast cancer cells that mediate growth factor autonomy. Using this expression cloning strategy with a cDNA library derived from SUM149 cells, we found that overexpression of the EGF family member AR could transduce the EGF-independent phenotype to MCF10A cells (36Berquin I.M. Dziubinski M.L. Nolan G.P. Ethier S.P. Oncogene. 2001; 20: 4019-4028Crossref PubMed Scopus (35) Google Scholar). The AR-overexpressing MCF10A cells derived in these experiments (MCF10A AR cells) are EGF-independent for growth and have ∼40-fold more AR mRNA than control MCF10A cells (Fig. 2A). AR was the only EGFR ligand isolated from the SUM149 cDNA library that enabled MCF10A cells to grow without EGF (36Berquin I.M. Dziubinski M.L. Nolan G.P. Ethier S.P. Oncogene. 2001; 20: 4019-4028Crossref PubMed Scopus (35) Google Scholar). Since our data show that SUM149 cells are dependent on ligand activation of EGFR for proliferation, we hypothesized that SUM149 cells overexpress AR, which enables their EGF independence. To begin to test this hypothesis, we measured AR mRNA expression in SUM149 cells relative to MCF10A cells using quantitative RT-PCR analysis and discovered that SUM149 cells overexpress AR mRNA at levels ∼2-fold higher than MCF10A cells (Fig. 2A). Although the level of AR message is not dramatically higher in SUM149 cells relative to MCF10A cells, this difference in message contributes to a more notable difference in AR protein between the two cell lines (Fig. 2B). Following whole cell lysis and Western blot analysis, we observed a 16- and 21-kDa AR form as well as a 26- and 28-kDa doublet in all three cell lines, which is in agreement with previously described AR membrane precursors in Madin-Darby canine kidney cells after wild type AR overexpression (11Brown C.L. Meise K.S. Plowman G.D. Coffey R.J. Dempsey P.J. J. Biol. Chem. 1998; 273: 17258-17268Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 44Brown C.L. Coffey R.J. Dempsey P.J. J. Biol. Chem. 2001; 276: 29538-29549Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). However, SUM149 cells and MCF10A AR cells overexpress the 21-, 26-, and 28-kDa forms of AR protein relative to MCF10A cells. It is notable that a 25-kDa form, which has been suggested to be a less glycosylated version of the 26-kDa AR, was the predominant AR form in both SUM149 and MCF10A AR cells (11Brown C.L. Meise K.S. Plowman G.D. Coffey R.J. Dempsey P.J. J. Biol. Chem. 1998; 273: 17258-17268Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In stark contrast, the 25-kDa form was not detectable in MCF10A cells, suggesting that this form in particular may play a significant role in EGF independence. An ELISA was performed to quantitate secreted AR protein present in the conditioned media of SUM149 and MCF10A AR cells. Our results show that SUM149 cells secrete ∼600 pg/ml AR into the medium/100,000 cells over a 24-h period, whereas MCF10A cells secrete ∼150 pg/ml AR/100,000 cells over 24 h (Fig. 3A). MCF10A AR cells secrete about 6-fold more AR than MCF10A cells alone (Fig. 3B). Taken together, these data show that AR is overexpressed at the message level as well as the membrane and secreted protein levels in EGF-independent SUM149 breast cancer cells and MCF10A AR cells and therefore may be permitting SUM149 EGF independence via an autocrine loop. Autocrine AR Is Required for EGFR Activity and SUM149 Cell Proliferation—AR was originally described as a bifunctional growth modulator, since it has both mitogenic and growth-inhibitory properties. Whether AR acts as a mitogen or a growth inhibitor is dependent upon several factors, including the concentration of AR and the nature of the target cells (19Dong J. Opresko L.K. Chrisler W. Orr G. Quesenberry R.D. Lauffenburger D.A. Wiley H.S. Mol. Biol. Cell. 2005; 16: 2984-2998Crossref PubMed Scopus (29) Google Scholar, 39Khatri P. Sellamuthu S. Malhotra P. Amin K. Done A. Draghici S. Nucleic Acids Res. 2005; 33: W762-W765Crossref PubMed Scopus (94) Google Scholar). Therefore, it was critical to confirm that AR is the autocrine ligand required to activate EGFR in SUM149 cells. To address this question, two neutralizing antibodies were used to sequester AR. Western analysis following an EGFR immunoprecipitation demonstrated that 15 min after the addition of the AF262 AR-neutralizing antibody (R & D Systems), EGFR tyrosine phosphorylation was reduced, and phosphorylation continued to decrease in a time-dependent fashion (Fig. 4A). SUM149 cell proliferation was also significantly reduced following the addition of both neutralizing antibodies daily for 7 days (Fig. 4B). Furthermore, MCF10A AR cells showed almost a 3-fold reduction in proliferation relative to control cells when the AR neutralizing antibody was added (Fig. 4C). Thus, SUM149 cells and MCF10A AR cells are dependent on AR for their EGF-independent proliferation. MCF10A cells, which express AR mRNA and detectable AR protein but still require exogenous EGF for growth, showed slightly reduced cell proliferation with AR-neutralizing antibody (Fig. 4C). This result suggests that secreted AR contributes in a small way to the growth of MCF10A cells that takes place in EGF-containing medium. To determine whether SUM149 EGFR activity contributes to the synthesis of AR, which would perpetuate the maintenance of an AR autocrine loop, CI1033 was added to SUM149 cells for 24 or" @default.
• W2023188630 created "2016-06-24" @default.
• W2023188630 creator A5057594429 @default.
• W2023188630 creator A5076801158 @default.
• W2023188630 date "2006-12-01" @default.
• W2023188630 modified "2023-10-15" @default.
• W2023188630 title "Autocrine and Juxtacrine Effects of Amphiregulin on the Proliferative, Invasive, and Migratory Properties of Normal and Neoplastic Human Mammary Epithelial Cells" @default.
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