expression and tyrosine phosphorylation of ems1 in human breast cancer cell lines

8
, #$$& Publication of the InternationalUnion Against Cancer I.,\ Publication de I'Union lnternationale Contre le Cancer fnt. J. Cancer: 68,485-492 (1996) 0 1996 Wiley-Liss, Inc. EXPRESSION AND TYROSINE PHOSPHORYLATION OF EMSl IN HUMAN BREAST CANCER CELL LINES Douglas H. CAMPBELL', Anna DEFAZIO', Robert L. SUTHERLAND~ and Roger J. Day'.? lCancer Research Program, Garvan institute of Medical Research, St Vincent's Hospital, Darlinghurst, Sydney, Australia. The EMS1 gene encodes an 80185 kDa C-src substrate and localises with the CCNDl gene to chromosome I lq13. This locus is amplified in approximately 13% of human breast cancers. EMS I gene amplification and expression were charac- tensed in a panel of human breast cancer cell lines to determine at what levels expression is regulated. The degree of tyrosine phosphorylation of EMS1 protein was also determined and compared with the activity of src-family kinases. The EMS1 gene was amplified in 6 of 20 cell lines investigated MDA-MB- 134, - 157, - I75,453,ZR-75- I and MCF-7. In the MDA-MB- I57 and MCF-7 cell lines, EMS1 was amplified in the absence of CCND I gene amplification. EMS I protein levels were increased relative to normal breast epithelial cells in 6 cell lines (ZR-75- I, MDA-MB- 134, - 175. 453, MCF-7 and BT-474). Of these, BT- 474 is the only cell line that does not exhibit EMS I amplification or increased EMS1 mRNA levels. EMS1 tyrosine phosphoryla- tion was 3-fold higher in BT-474 and T-47D cells, which exhibited relatively high total src activity coupled with expres- sion of both c-fyn and c-yes, than in MDA-MB-453 cells, which expressed only c-yes. Our results therefore demonrtrate gene amplification to be the predominant mechanism underlying EMS1 over-expression in human breast cancer cell lines and identify tyrosine phosphotylation as a further level at which regulation of this protein may be perturbed. o 1996 Wiley-Liss, Inc. The EMSl gene encodes the human homologue of cortactin, an 80/85 kDa v-src substrate first identified in chick embryo cells transformed with the v-src oncogene (Wu et al., 1991; Schuuring et al., 1993). Both the chicken cortactin and human EMSl cDNAs encode proteins with 3 distinct domains. The N-terminal portion consists of 5 (p80) or 6 (p85) repeats of a 37 amino acid motif predicted to form a helix-turn-helix structure. Cortactin binds filamentous actin (F-actin) via this repeat region (Wu and Parsons, 1993) and localises with cortical actin in normal chick embryo cells. Furthermore, after v-src transfor- mation, cortactin relocaliscs with actin to the rosette/ podosome structures, abnormal focal adhesions rich in cytoskel- eta1 proteins. At the carboxy terminus of the protein is an src homology (SH)3 domain. SH3 domains are frequently found in signalling molecules (e.g., c-src, Grb2) as well as cytoskeletal proteins, such as a-spectrin, and bind to short proline-rich sequences within the target molecule (Pawson, 1995). There- fore, cortactin may play a role in linking signalling events to cytoskeletal reorganisation. The EMSl gene localises to the llq13 locus, a region frequently amplified in a number of human cancers, particu- larly those of the brcast and squamous-cell carcinomas of the head and neck and oesophagus (Schuuring et al., 1992a, b; Fantl et al., 1993; Williams el al., 1993; Karlseder et al., 1994; Peters et al., 1995). The llq13 locus is amplified in approxi- mately 13% of primary human breast cancers and has been consistently correlated with oestrogen receptor (ER) positivity (Fantl et al., 1993). Some studies have also suggested an association with increased lymph node involvement and poor patient prognosis (Schuuring et al., 199%). Two genes located in the llq13 region are fNT-2 and HST-1, which encode the FGF 3 and FGF 4 polypeptide growth factors, respectively. Howcvcr, neither gene is expressed in human breast cancer, despite clear evidence of gene amplification (Fantl et al., 1993). Two more likely candidate oncogenes located at 1lq13 are the genes encoding cyclin D1 (CCNDl) and EMS1. The formcr gene is often amplified and/or over-expressed in human breast cancer cell lines and primary brcast cancers (Buckley et al., 1993; Bartkova et al., 1995). Since ectopic over-expression of cyclin D l in T-47D breast cancer cells shortens the length of the GI phasc and in serum-starved cells is sufficient to promote entry into S phase (Musgrove et al., 1994), enhanced expres- sion of this gene may provide a proliferative advantage to cancer cells. However, the potential effects of EMSl over- expression remain speculative, though the properties and subcellular distribution of this protein suggest that it may play a role in tumour invasion and metastasis, in accordance with the reported increased invasive potential of 1 lql3-amplified breast cancers (Schuuring et al., 199%). In addition to being a substrate of v-src, EMSl is tyrosine phosphorylatcd by, and associates with, c-src during platelet activation (Wong et al., 1992). Also, EMSl is tyrosine- phosphorylated after activation of a number of growth factor receptors, and this is likely to be mediated by c-src (Maa et al., 1992; Zhan et al., 1994). Furthermore, in c-src (-/-), CSK (- / -) double knockout fibroblast cells, tyrosine phosphoryla- tion of cortactin was found to be dependent on c-src and not c-fyn (Thomas et al., 1995). Interestingly, high Levels of c-src activity have been detected in breast carcinomas relative to normal breast tissue, and amongst breast cancer patients, enhanced cytosolic tyrosine kinase activity, largely attributable to c-src, correlates with early systemic relapse (reviewed by Daly, 1995). Further evidence for the involvement of c-src in malignant breast disease is provided by transgenic mouse models (eg., Muthuswamy et al,, 1994). As EMSl is a known c-src substrate, it is possible that some of the effects of elevated c-src activity may be mediated by EMSl; however, the tyrosine phosphorylation of EMSl in normal and neoplastic breast tissue has not been investigated. In this study, amplification and expression of the EMSl gene was characterised in an extensive panel of breast cancer cell lines. The data are compared with our published data for CCNDl to determine if EMSl and CCNDl are always ampli- fied and over-expresscd in parallel. The degree of tyrosine phosphorylation of EMSl in these cell lines was also examined and compared to the activity of src family kinases. MATERIAL AND METHODS Cell culture The BT-20, -474, -483, -549, DU-4475, Hs-578T, MDA-MB- 134, -175, -361, -436, -453, -468, SK-BR-3 and ZR-75-1 human breast cell lines were obtained from the ATCC (Rockville, MD). The HBL-100, MDA-MB-157, -231, -330, MCF-7 and T-47D cell lines were obtained from the E. G. and G. Mason Research Institute (Worcester, MA). The normal rat kidney (NRK) cells transfected with the v-src oncogene were obtained from Dr. B. Mann (Ludwig Institute for Canccr Research, Melbourne, Australia). 2To whom corres ondence and reprint requests should be ad- dressed, at Cancer iesearch Pro ram, Garvan Institute of Medical Research. St Vincent's Hospital, barlinghurst, Sydney, N.S.W. 2010 Australia. Fax: 61-2-295-8321. Received: June 4, 1996 and in revised form July 30, 1996.

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Page 1: Expression and tyrosine phosphorylation of EMS1 in human breast cancer cell lines

,#$$& Publication of the International Union Against Cancer I.,\ Publication de I'Union lnternationale Contre le Cancer

fnt . J. Cancer: 68,485-492 (1996) 0 1996 Wiley-Liss, Inc.

EXPRESSION AND TYROSINE PHOSPHORYLATION OF EMSl IN HUMAN BREAST CANCER CELL LINES Douglas H. CAMPBELL', Anna DEFAZIO', Robert L. SUTHERLAND~ and Roger J. Day'.? lCancer Research Program, Garvan institute of Medical Research, St Vincent's Hospital, Darlinghurst, Sydney, Australia.

The EMS1 gene encodes an 80185 kDa C-src substrate and localises with the CCNDl gene to chromosome I lq13. Th is locus is amplified in approximately 13% of human breast cancers. E M S I gene amplification and expression were charac- tensed in a panel of human breast cancer cell lines to determine at what levels expression is regulated. The degree of tyrosine phosphorylation of EMS1 protein was also determined and compared with the activity of src-family kinases. The EMS1 gene was amplified in 6 of 20 cell lines investigated MDA-MB- 134, - 157, - I75,453,ZR-75- I and MCF-7. In the MDA-MB- I57 and MCF-7 cell lines, EMS1 was amplified in the absence of CCND I gene amplification. EMS I protein levels were increased relative to normal breast epithelial cells in 6 cell lines (ZR-75- I, MDA-MB- 134, - 175. 453, MCF-7 and BT-474). Of these, BT- 474 is the only cell line that does not exhibit E M S I amplification or increased EMS1 mRNA levels. EMS1 tyrosine phosphoryla- tion was 3-fold higher in BT-474 and T-47D cells, which exhibited relatively high total src activity coupled with expres- sion of both c-fyn and c-yes, than in MDA-MB-453 cells, which expressed only c-yes. Our results therefore demonrtrate gene amplification to be the predominant mechanism underlying EMS1 over-expression in human breast cancer cell lines and identify tyrosine phosphotylation as a further level at which regulation of this protein may be perturbed. o 1996 Wiley-Liss, Inc.

The EMSl gene encodes the human homologue of cortactin, an 80/85 kDa v-src substrate first identified in chick embryo cells transformed with the v-src oncogene (Wu et al., 1991; Schuuring et al., 1993). Both the chicken cortactin and human EMSl cDNAs encode proteins with 3 distinct domains. The N-terminal portion consists of 5 (p80) or 6 (p85) repeats of a 37 amino acid motif predicted to form a helix-turn-helix structure. Cortactin binds filamentous actin (F-actin) via this repeat region (Wu and Parsons, 1993) and localises with cortical actin in normal chick embryo cells. Furthermore, after v-src transfor- mation, cortactin relocaliscs with actin to the rosette/ podosome structures, abnormal focal adhesions rich in cytoskel- eta1 proteins. At the carboxy terminus of the protein is an src homology (SH)3 domain. SH3 domains are frequently found in signalling molecules (e.g., c-src, Grb2) as well as cytoskeletal proteins, such as a-spectrin, and bind to short proline-rich sequences within the target molecule (Pawson, 1995). There- fore, cortactin may play a role in linking signalling events to cytoskeletal reorganisation.

The EMSl gene localises to the llq13 locus, a region frequently amplified in a number of human cancers, particu- larly those of the brcast and squamous-cell carcinomas of the head and neck and oesophagus (Schuuring et al., 1992a, b; Fantl et al., 1993; Williams el al., 1993; Karlseder et al., 1994; Peters et al., 1995). The llq13 locus is amplified in approxi- mately 13% of primary human breast cancers and has been consistently correlated with oestrogen receptor (ER) positivity (Fantl et al., 1993). Some studies have also suggested an association with increased lymph node involvement and poor patient prognosis (Schuuring et al., 199%). Two genes located in the llq13 region are fNT-2 and HST-1, which encode the FGF 3 and FGF 4 polypeptide growth factors, respectively. Howcvcr, neither gene is expressed in human breast cancer, despite clear evidence of gene amplification (Fantl et al., 1993). Two more likely candidate oncogenes located at 1 lq13 are the genes encoding cyclin D1 (CCNDl) and EMS1. The formcr gene is often amplified and/or over-expressed in human breast

cancer cell lines and primary brcast cancers (Buckley et al., 1993; Bartkova et al., 1995). Since ectopic over-expression of cyclin Dl in T-47D breast cancer cells shortens the length of the GI phasc and in serum-starved cells is sufficient to promote entry into S phase (Musgrove et al., 1994), enhanced expres- sion of this gene may provide a proliferative advantage to cancer cells. However, the potential effects of EMSl over- expression remain speculative, though the properties and subcellular distribution of this protein suggest that it may play a role in tumour invasion and metastasis, in accordance with the reported increased invasive potential of 1 lql3-amplified breast cancers (Schuuring et al., 199%).

In addition to being a substrate of v-src, EMSl is tyrosine phosphorylatcd by, and associates with, c-src during platelet activation (Wong et al., 1992). Also, EMSl is tyrosine- phosphorylated after activation of a number of growth factor receptors, and this is likely to be mediated by c-src (Maa et al., 1992; Zhan et al., 1994). Furthermore, in c-src (-/-), CSK (- / -) double knockout fibroblast cells, tyrosine phosphoryla- tion of cortactin was found to be dependent on c-src and not c-fyn (Thomas et al., 1995). Interestingly, high Levels of c-src activity have been detected in breast carcinomas relative to normal breast tissue, and amongst breast cancer patients, enhanced cytosolic tyrosine kinase activity, largely attributable to c-src, correlates with early systemic relapse (reviewed by Daly, 1995). Further evidence for the involvement of c-src in malignant breast disease is provided by transgenic mouse models (eg., Muthuswamy et al,, 1994). As EMSl is a known c-src substrate, it is possible that some of the effects of elevated c-src activity may be mediated by EMSl; however, the tyrosine phosphorylation of EMSl in normal and neoplastic breast tissue has not been investigated.

In this study, amplification and expression of the EMSl gene was characterised in an extensive panel of breast cancer cell lines. The data are compared with our published data for CCNDl to determine if EMSl and CCNDl are always ampli- fied and over-expresscd in parallel. The degree of tyrosine phosphorylation of EMSl in these cell lines was also examined and compared to the activity of src family kinases.

MATERIAL AND METHODS Cell culture

The BT-20, -474, -483, -549, DU-4475, Hs-578T, MDA-MB- 134, -175, -361, -436, -453, -468, SK-BR-3 and ZR-75-1 human breast cell lines were obtained from the ATCC (Rockville, MD). The HBL-100, MDA-MB-157, -231, -330, MCF-7 and T-47D cell lines were obtained from the E. G. and G. Mason Research Institute (Worcester, MA). The normal rat kidney (NRK) cells transfected with the v-src oncogene were obtained from Dr. B. Mann (Ludwig Institute for Canccr Research, Melbourne, Australia).

2To whom corres ondence and reprint requests should be ad- dressed, at Cancer iesearch Pro ram, Garvan Institute of Medical Research. St Vincent's Hospital, barlinghurst, Sydney, N.S.W. 2010 Australia. Fax: 61-2-295-8321.

Received: June 4, 1996 and in revised form July 30, 1996.

Page 2: Expression and tyrosine phosphorylation of EMS1 in human breast cancer cell lines

486 CAMPBELL ETAI.

Cultures were maintained at 37T, 5% COz in RPMI 1640 medium, supplemented with 6 mM L-glutamine, 14 mM sodium bicarbonate, 20 mM HEPES [4-(2-hydroxyethyl)-l- piperazine-ethanesulphonic acid], 10 pg/ml human insulin and 10% FCS (CSL, Parkville, Australia). The NRK v-src cell line was grown in DMEM supplemented as for the RPMI 1640 medium. The normal breast epithelial cell strain HMEC-219-4 was obtained from Clonetics (San Diego, CA) and grown in medium supplied by the manufacturer. The HMEC-184, HMEC-184-B5 and HMEC-184-A1 cell lines were obtained from Dr. M. Stampfer (Berkeley, CA) and maintained as described for the HMEC-219-4 line.

cDNA probes A 1.8 kb EcoRI-BamHI restriction fragment harbouring the

full-length EMSl cDNA (Schuuringetal., 1993) was a gift from Dr. R. Michalides (Netherlands Cancer Institute, Amster- dam); 50 ng of this DNA fragment were labelled by random priming utilising [IX-'~P] dCTP (110 BTq/mmol; Amersham, Castle Hill, Australia) and hybridised at a final probe concen- tration of 1-2 ng/ml. A probe for the progesterone receptor (Misrahi et al., 1987) was used as a control for chromosome 11 copy number.

Isolation and analysis of DNA Genomic DNA was prepared from the CsCl gradient after

cells lysed in guanidinium isothiocyanate were centrifuged according to standard methods (Sambrook et al., 1989). DNA was digested with EcoRI, fractionated by gel electrophoresis in 1% agarose-TAE gels (1 X TAE: 40 mM Tris-acetate, 1 mM EDTA) and transferred to Zetaprobe membranes (BioRad, North Ryde, Australia) as previously described (Buckley et al., 1993). Hybridization was carried out for 16 hr at 65°C in 0.5 M sodium phosphate (pH 6.9), 7% (w/v) SDS, 0.5% (v/v) Blotto (stock solution: 10% [w/v] instant skim milk + 0.2% [w/v] sodium azide) and 1 mM EDTA. Filters were washed to a final stringency of 0.05 x SSC (20 x SSC: 3 M sodium chloride, 0.3 M sodium citrate [pH 7.01) + 0.1% (w/v) SDS for 30 min at 65°C. Autoradiography was performed at -70°C without intensifying screens. Densitometric analysis of the autoradio- graphs was performed by the IP Lab Gcl analysis program (Signal Analytics, Vienna, VA). Five restriction fragments were dctected (8.7,8.0,7.1,2.8 and 2 kb), and quantitation was based on the integrated signal obtained from all 5 fragments. Filters were stripped for reprobing by heating to 100°C for 5 min in a solution of 0.1 x SSC, 0.1% (w/v) SDS.

Isolation and analysis of RNA Total RNA was isolated from cells using the guanidinium

isothiocyanate-cesium chloride method (Sambrook et al., 1989). RNA was separated by formaldehyde/agarose gel electropho- resis, transferred to nylon membranes and hybridised with cDNA probes as previously described (Hall et al., 1990). Northcrn blots were normalised for loading by probing for the 18s ribosomal RNA (Buckley et al., 1993). Filters were stripped for reprobing by heating to 100°C for 5 min in a solution of 0.1 X SSC, 0.1% (w/v) SDS. Autoradiography was performed by exposing films overnight at -70°C (EMS1 Northcrn blot) or at room temperature for 15 rnin (18s Northern blot).

Antibodies A monoclonal antibody (MAb) against chicken p80/85

(cortactin) exhibiting cross-species reactivity (MAb 4Fll; UBI, Lake Placid, NY) (Wu et al., 1991) was used to immuno- precipitate EMSl protein from cell lysates. EMSl was de- tected on Western blots with the same antibody diluted 1:2,000. The c-sre protein was detectcd using MAb 327 (a gift from Dr. J. Brugge, Ariad Pharmaceuticals, Cambridge, MA) at a 1:1,000 dilution. The c-yes protein was detected with polyclonal anti-sera (Y35330; Transduction Laboratories, Lcx-

ington, KY) diluted 1:125. c-fyn was detected with a MAb (SC-434; Santa Cruz Bioteehnology, Santa Cruz, CA) diluted 1500. Anti-phosphotyrosine blots wcre performed with a recombinant antibody (RC20; ICN, Costa Mesa, CA) diluted 1:2,500 in buffer supplied by the manufacturer. All other antibody solutions for Western blotting were made in 5% (w/v) BSA (RIA grade; Sigma, Castle Hill, Australia)/TBS (10 mM Tris-CI, 150 mM NaCl [pH 7.41).

Immunoprecipitation and Western blotting Cell lysates were prepared using either lysis buffer, as

described previously (Janes el al., 1994), or RIPA buffer (20 mM Tris-HCI, 300 rnM NaCI, 2 mM EDTA, 1% [v/v] Triton X-100, 1% [w/v] sodium deoxycholate, 0.1% [w/v] SDS [pH 7.61). Protein concentrations were determined using the Bio- Rad Protein Assay Reagent, and samples were normalised by dilution with the appropriate lysis buffer. Immunoprecipita- tions were performed by incubation with 0.5-2 pg of antibody for a minimum of 2 hr at 4°C with constant mixing. Goat anti-mouse antibody-sepharose conjugate (20 pl) (Zymed, San Francisco, CA) or protein A-sepharose conjugate (Zymcd) (pre-washed twice in 20 mM HEPES [pH 7.51) was then added to thc lysate-antibody mixture for 1 hr. Immunoprecipitates wcre then washed 3 times in the lysis buffer and denatured in SDS-PAGE sample buffer by heating to 100°C for 3 min. Samples were analysed by SDS-PAGE (10% mini-gels), trans- ferrcd to 0.2 pm nitrocellulose (BioRad) and subjected to Western blot analysis. Equivalent loading of cell lysates was confirmed by Ponceau S staining of membranes after transfer. Detection of bound antibodies was by enhanced chemilumines- cence (ECL) (Amersham).

In vitro assay of c-src family kinase activity Acid-denatured enolase was prepared as follows. Rabbit

muscle enolase (Sigma) was obtained as an ammonium sul- phate precipitate; 20 pl of enolase suspension (200 pg) were centrifuged at 16,000g for 30 min at 4"C, then resuspended in 10 pl of buffer A (50 mM HEPES, 1 mM DTT, 10 mM MgClz [pH 7.01) on ice for 45 min; 10 p1 of glycerol were added and the sample stored at -70°C until required. Acid denaturation was accomplished by adding 1 pI of 50 mM acetic acid to 2 p1 of enolase solution and incubating for 10 min at 30°C. The enolase was stored on ice until required.

Immunoprecipitation of c-src or c-yes was performed from RIPA lysates as described above but with the following modifications. Following immunoprecipitation, immune com- plexes were washed 3 times with RIPA buffer and then 3 times with kinase buffer (100 mM HEPES, 5 mM MnClz [pH 7.01). Two-fifths of the irnmunoprecipitate was Western blotted to determine the amount of kinase prcscnt. The remainder was then resuspended in 10 p1 of kinase buffer, and 10 pg of acid-denatured enolase and 10 pCi of [y-32P] ATP were added. The reaction was incubated at 30°C for 10 min with constant agitation and terminated by addition of 3 x sample buffer. Proteins were resolved using SDS-PAGE (8% gels), visualised with Coomassie brilliant blue, dried, then either subjected to autoradiography for 30 min without intensifying screens or exposed to Phosphorlmager screens (Molecular Dynamics, Balwyn, Australia). Densitometric analysis of the autoradio- graphs was performed and the specific activity determined by correcting the enolasc phosphorylation for the amount of kinase present in the reaction.

RESULTS Amplification of the EMSl gene in human breast cancer cell lines

Amplification of the EMS1 gene was assessed in a panel of 20 breast cancer ccll lines and 4 lincs derived from normal breast epithelial cells by Southern blotting. A representative

Page 3: Expression and tyrosine phosphorylation of EMS1 in human breast cancer cell lines

487 EMSl IN BREAST CANCER CELL LINES

blot is shown in Figure la. Blots were normalised for chromo- some 11 copy number by hybridisation with a progesterone receptor probe. Peripheral blood leukocyte (PBL) DNA was used as a single-copy standard for both the EMSl and progesterone receptor genes. Six cell lines exhibited 2-fold or greater amplification of the EMSl gene (Fig. Ib): MDA-MB-

and MCF-7. There does not appear to be a correlation between amplification of the EMSl gene and ER status in these breast cancer cell lines, in concordance with data obtained for CCNDl amplification (Buckley et al., 1993), since 3 EMS1-amplified cell lines were ER-positive (MDA-MB-134, ZR-75-1 and MCF-7) and 3 ER-negative (MDA-MB-175, -453 and -157).

Expression of EMS1 mRNA in human breast cancer cell lines Gene amplification does not always result in ovcr-expression

of the corresponding mRNA (e.g., the INT-2 and HST-1 genes are amplified but not expressed in human breast cancers). Therefore, expression of EMSl mRNA was examined in a panel of 20 breast cancer cell lines and 1 control normal breast epithelial line. A representative blot (Fig. 2a) shows that all cell lines investigated expressed a single EMSl transcript of 3.8 kb. Four lines (MDA-MB-134, ZR-75-1, MDA-MB-175 and MDA-MB-453) over-expressed EMSl mRNA greater than 2-fold compared with the HMEC-219-4 control (Fig. B), whilst MCF-7 cells exhibited a small but reproducible over- expression. The only cell line amplified for EMSl which did not over-express EMSl mRNA was the MDA-MB-157 cell line.

Expression of EMSl protein in human breast cancer cell lines EMSl protein levels were determined in lysates from 10 cell

lines exhibiting a wide range of EMSl mRNA levels by Western blotting and compared with that of the HMEC-219-4 normal breast epithelial cell strain (Fig. 3a, b). EMSl protein was clearly over-expressed in the MDA-MB-134, -175, -453, ZR-75-1, MCF-7 and BT-474 cell lines. Of these, only the BT-474 line does not exhibit EMSl mRNA over-expression (Fig. 2b) and is not amplified for the EMS1 gene (Fig. 1). Another cross-reactive band of approximately 44 kDa was recognised by the EMSl antibody and has been observed by one other group (Durieu-Trautmann et a[., 1994). A similar profile of EMSl protein expression was obtained when the different cell lines were lysed in RIPA buffer (data not shown).

Tyrosine phosphorylation of EMS1 and activity of src family members in human breast cancer cell lines

Prior to investigation of EMSl tyrosine phosphorylation in breast cancer cell lines, we first determined the expression and specific activity of c-src. Western blotting of cell lysates revealed a hierarchy of c-src expression (a representative blot is shown in Fig. 4a). The T-47D, SK-BR-3 and ZR-75-1 cell lines expressed a similar level of c-src to that found in the HMEC-219-4 cell line (data not shown). However, MDA-MB- 134, MCF-7 and BT-474 cells expressed 2- to 3-, 5- and 10-fold, respectively, lower levels of c-src. Moreover, c-src was undetect- able in MDA-MB-453 cell lysates (Fig. 4a). To determine the specific kinase activity in the different cell lines, the ability of immunoprecipitated c-src to phosphorylatc the exogenous substrate enolase was determined using an in vitro kinase assay (Fig. 4b). The amount of c-src present in each reaction was also determined by Western blotting (Fig. 4c) and used to deter- mine the specific activity of the kinase (Fig. 4d). This varied over an approximately 10-fold range between the cell line with the highest specific activity (BT-474) and that with the lowest (SK-BR-3). Interestingly, the BT-474 cell line expressed the lowest levels of c-src protein. The MDA-MB-453 cells, which had no detectable c-src protein, failed to demonstrate kinase activity, confirming that the assay was specific for c-src.

134, ZR-75-1, MDA-MB-175, MDA-MB-453, MDA-MB-157

Tyrosine phosphorylation of EMSl was examined in 4 breast cancer cell lines exhibiting different levels of c-src activity: MCF-7, BT-474, MDA-MB-453 and T-47D. Following anti- phosphotyrosine blotting of EMSl immunoprecipitates, the filter was reblotted for EMSl to allow determination of relative tyrosine phosphorylation (Fig. 5a, c). NRK cells trans- formed with the v-src oncogcne were used as a positive control in this experiment (Fig. 5b). Tyrosine phosphorylation of EMSl was detected in all of the cell lines examined and, as expected, was most marked in the NRK v-src cells. In the breast cancer cell lines, the relative EMSl tyrosine phosphory- lation was significantly higher (approximately 3-fold) in BT- 474 and T-47D cells compared with MDA-MB-453 and MCF-7 cells. Similar results were obtained when cell lines were lysed in RIPA buffer (data not shown). The tyrosine phosphoryla- tion of EMSl in MDA-MB-453 cells, which do not express detectable levels of c-src, led us to suspect that a different src family member may act as an EMSl kinase in this cell line.

To identify a candidate kinase, the expression of 2 widely expressed src family kinase members, c-yes and c-fyn, was determined by Western blotting a panel of cell lysates (Fig. 6a). A band of the expected mobility for c-yes (62 kDa) was detected in all lines tested. An additional band of approxi- mately 56 kDa was notcd in a subset of lines. These 2 bands varied in their relative intensity between cell lines, and the origin of the 56-kDa species is unclear at present. Amongst the cell lines examined, MDA-MB-453 cells exhibited high expres- sion of the 62-kDa band only, whilst both species were expressed at high levels in ZR-75-1, MCF-7, BT-474 and SK-BR-3 cells. Active c-yes kinase could be detected in T-47D and MDA-MB-453 cells by an in vitro kinase assay (Fig. 6b), but we were unable to determine the amount of c-yes in the immunoprecipitate and, hence, thc specific kinase activity due to the high IgG signal obtained upon Western blot analysis. Expression of c-fyn was more limited than expression of either c-src or c-yes and restricted to the BT-474, SK-BR-3 and T-47D cell lines, in which it was expressed at approximately equal lcvcls (Fig. 6a). We were unable to develop an in vitro kinase assay for c-fyn using this antibody. These data strongly suggest that c-yes is responsible for tyrosine phosphorylation of EMSl in the MDA-MB-453 cell line, which does not express cither c-src or c-fyn, and that c-yes and c-fyn may contribute to EMSl tyrosine phosphorylation in other cell lines, e.g., BT- 474.

DISCUSSION

Human breast cancers amplified at the llq13 locus have been suggested to represent a subgroup with increased lymph node involvement and decreased relapse-free survival (Schuur- ing et al., 19926; Fantl et al., 1993). Of the potential oncogenes located at llq13, 2 are of particular interest with regard to breast cancer, those encoding cyclin D1 and EMS1. As a first part of a study of EMSl function in human breast cancer, we have investigated the expression and regulation of the EMSl gene in a well-defined series of human breast cancer cell lines.

Six cell lines (ZR-75-1, MDA-MB-134, -175, -453, MCF-7 and MDA-MB-157) were amplified 2-fold or greater for EMSl (Fig. l), and the first 5 of these cell lines over-expressed EMSl mRNA relative to normal breast epithelial cells at levels corresponding to that of the respective gene amplification (Fig. 2). EMSl protein levels were increased relative to normal breast epithelial cells in 6 cell lines (ZR-75-1, MDA-MB-134, -175, -453, MCF-7 and BT-474) (Fig. 3). Of these, the BT-474 cell line is the only one that does not exhibit EMSl amplifica- tion or increased EMSl mRNA levels. Therefore, amongst this wide panel of human breast cancer cell lines, EMSl amplifica- tion and protein over-expression are cssentially in accordance. In other studies of 1 lq13 amplification in human breast cancer, over-expression of the EMSl gene generally resulted from

Page 4: Expression and tyrosine phosphorylation of EMS1 in human breast cancer cell lines

488 CAMPBELL, ETAL

FIGURE 1 -Amplification of the EMSl gene in breast cancer cell lines. (a) Southern blot analysis of the EMSl gene in human breast cancer cell lines. Genomic DNA (15 pg) was digested with EcoRI, size-fractionated by agarosc gel electrophoresis and transferred to Zetaprobe membranes in 0.4 M NaOH. Southern blots were sequentially hybridized with EMS1 and progesterone receptor cDNA probes and autoradiographed for 16 hr without intensiQing screens. (b) Degree of amplification of EMSl in breast cancer cell lines. Densitometry of the autoradiographs shown above was performed and the EMS1 signal normalised for chromosome 11 copy number using the progesterone receptor control. The degree of amplification is expressed relative to the PBL control.

amplification events, but some examples of increased EMSl mRNA in the absence of amplification were observed (Schuur- ingetal., 1992~; Fantl et af., 1993). In squamous-cell carcinoma cell lines, EMS1 over-expression is tightly linked to gene

amplification (Schuuringet af., 1992a, 1993; Patel et al., 1996). However, increased expression of EMSl mRNA in the ab- sence of gene amplification occurs in m u r k plasmacytoma cells and in human bladder tumours (Miglarese et a/., 1994;

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EMSl IN BREAST CANCER CELL LINES 489

FIGURE 2 - Expression of EMSl mRNA in human breast cancer cell lines. (a) Northern blot analysis of EMSl mRNA expression in human breast cancer cell lines. Total RNA (20 Kg) was separated by formaldehyde/agarose electrophoresis, transferred to Zetaprobe membranes and hybridised with a cDNA probe for EMS1, followed by an oligonucleotide probe for the 18s ribosomal RNA. Autoradiography times were 16 hr for EMSl and 15 min for the 18s probe without intensifying screens. (b) Relative expression of EMS1 mRNA in breast cancer cell lines. Densitometric analysis of autoradiogra hs depicted in (a) was performed. Correction for RNA loading was made using the 18s signal, and the level of expression is shown reLtive to that in HMEC-219-4 normal breast epithelial cells.

Bringuier et al., 1996), demonstrating that the level of regula- tion of EMSl gene expression depends on cancer cell type.

The amplification and expression of the CCNDl gene has been well characterised in a large panel of breast cancer cell lines (Buckley et al., 1993). The EMS1 and CCNDl genes are co-amplified in 4 cell lines (MDA-MR-134, ZR-75-1, MDA- MB-175 and MDA-MB-453), whilst 4 lines show amplification of one gene alone: MDA-MB-157 and MCF-7 (EMSI) and MDA-MB-330 and -361 (CCND1). Karlseder et al. (1994) have

identified 4 separate amplicons in the 1 lq13 region important for breast cancer. Two of the amplicons encompassed either the EMSl and CCNDl genes, respectively, while the other 2 amplicons contained the as yet unassigned markers DllS97/ DllS146 and DllS833E. These results suggest that amplifica- tion and over-expression o f the CCNDl and EMSl genes confer separate and possibly complementary selective advan- tages to cancer cells. Our results appear to concur with this model, as independent amplification and over-expression of

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490

A

CAMPBELL ETAL

A

B C D

RGURE~ - Expression of the EMSl rotein in breast cancer cell lines. (a) Western blot analysis of EM& in selected cell lines. Cell lysates were prepared in lysis buffer as described in “Material and Methods”, and following normalisation of protein concentration subjected to SDS-PAGE. Proteins were then transferred to nitrocellulose membranes and Western blotted with the anti- EMSl 4Fl l MAb. Bound antibodies were detected using ECL. (b) Relative expression of EMSl protein in breast cancer cell lines. Densitometry was performed on the autoradiograph shown in (a) and the signals expressed relative to that obtained for HMEC- 219-4 normal breast epithelial cells.

either EMSl or CCNDl occur in a number of different cell lines.

In previous studies, tyrosine phosphorylation of EMSl was closely linked to activation of the c-src kinase. Therefore, prior to investigation of EMSl tyrosine phosphorylation in the breast cancer cell lines, we determined the protein levels and specific kinase activity of c-src. Both the levels and specific activity of c-src varied amongst the cell lines examined (Fig. 4). No c-src protein or kinase activity was detected in MDA-MB- 453 cells, whilst the BT-474 cell line displayed the highest specific activity and the lowest detectable levels of c-src. Down-regulation of c-src protein expression upon activation was also observed in a cell line derived from CSK gene knockout mice (Nada et al., 1994). A potential mechanism underlying c-src activation in the BT-474 cell line is via interaction of the c-src SH2 domain with the erbB2 receptor, which is over-expressed and highly tyrosine-phosphorylated in this cell line (Janes et al., 1994; Muthuswamy et al., 1994; Muthuswamy and Muller, 1995). However, the tyrosine phos- phorylation of EMSl in MDA-MB-453 cells, which d o not express detectable levels of src, suggested that other src family members may phosphorylate EMSl in human breast cancer cell lines. To address this, the expression of c-yes and c-fyn was invest igatcd.

Overall, the breast cancer cell lines exhibited qualitative and quantitative differences in their expression pattern of src family kinases (Figs. 4,6). For example, BT-474 cells expressed

FIGURE 4 - Expression and s ecific activity of c-src in breast cancer cell lines. (a) Western brot analysis of c-src expression in selected breast cancer cell lines. Cells were lysed in RIPA buffer, and equivalent amounts of protein were resolved b SDS-PAGE, transferred to nitrocellulose membranes and then bktted with the anti-src MAb 327. Bound antibodies were detected by ECL. (b) Kinase activity of c-src in selected breast cancer cell lines. The ability of immunoprecipitated c-src to phos horylate enolase was determined by an in vitro kinase assay, as d)escribed in “Material and Methods”. (c) Levels of c-src present in immunoprecipitates. The amount of c-src present in the immunopreci itates used for the kinase assays was determined by Western bEt analysis. (d) Specific kinase activity of c-src in breast cancer cell lines. Determi- nation of the amount of c-src present in the immunoprecipitate enabled calculation of the c-src-specific activity for each cell line, which is expressed relative to that in T-47D cells.

c-src, c-fyn and c-yes, whilst MDA-MB-453 cells expressed only detectable levels of c-yes. Interestingly, activation of c-yes occurs in parallel to that of c-src in murine mammary tumours induced in MMTV-erbBZ transgenic mice, and c-yes associates with erbB2 (Muthuswamy and Muller, 1995). Consequently, this kinase, in addition to c-src, might exhibit elevated activity in breast canccr cells over-expressing erbB2, eg., BT-474 cells. The tyrosine phosphorylation of EMSl is therefore likely to be determined by the expression and specific activity of the different src family members present. Our interpretation of the relative tyrosine phosphorylation of EMSl in the breast cancer cell lines examined (Fig. 5) is that in MDA-MB-453 cells the kinase responsible for EMSl phosphorylation is c-yes and that this kinase may also play a significant role in MCF-7 cells since these cells express only low amounts of c-src of low specific activity (Fig. 4). The increased tyrosine phosphorylation of

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EMSl IN BREAST CANCER CELL LINES 49 1

A B A

Blot:

C

B

FIGURE 5 - Tyrosine phosphorylation of EMSl in breast cancer cell lines. (a) EMSl was immunoprecipitated from equal amounts of cell lysates prepared in lysis buffer using the anti-EMS1 MAb 4Fll . Samples were resolved b SDS-PAGE, transferred to nitrocellulose membranes and then probed sequentially with anti-phosphotyrosine (RC-20) and anti-EMS1 (4Fl l ) antibodies. Detection of bound antibodies was by ECL. (b) Tyrosine phosphor- ylation of EMS I in v-src-transformed cells. EMSl was immunopre- cipitated from NRK v-src lysates prepared in RIPA buffer, resolved by SDS-PAGE, then probed sequentially with anti- phosphotyrosine or anti-EMS1 antibodies (as above). The same procedure was performed with T-47D cells as a comparison. (c) Relative tyrosine phosphorylation of EMSl in breast cancer cell lines. Densitometry was performed on the autoradiographs shown in Figure 5a to determine the relative tyrosine phosphorylation, which is expressed relative to the T-47D sample.

EMSl observed in BT-474 and T-47D cells is probably a combined effect of high total src activity (since the former line expresses low amounts of src of high specific activity and the latter, high amounts of src of a lower specific activity; Fig. 4) and the presence of both c-yes and c-fyn. Tyrosine phosphory- lation of EMSl by different src family members is not without precedent since it has been linked to c-fyn activation in differentiating keratinocytes (Calautti et al., 1995): is up- regulated in c-&-transfected fibroblasts (Sartor and Robbins, 1993) and occurs upon syk activation in megakaryocytes (Maruyama et al., 1996).

The transient tyrosine phosphorylation of EMSl in response to growth factor stimulation or platelet activation suggests a regulatory role, possibly linked to cytoskeletal reorganisation. This is supported by the redistribution of highly tyrosine- phosphorylated EMSl into podosome structures in v-src- transformed cells (Wu et al., 1991). Interestingly, cell lines which do not over-express EMSl protein (e.g., T-47D; Fig. 3) may contain similar total amounts of tyrosine-phosphorylated EMSl to a cell line which does (.g.. MDA-MB-453) due to the variation in activity of src family kinases. Moreover, both the expression of EMSl and the specific tyrosine phosphorylation

Flcum 6 - Expression and activity of c-src kinase family mem- bers in breast cancer cell lines. (a) Western blot analysis of c-yes and c-fyn expression in selected breast cancer cell lines. Cells were lysed in RIPA buffer, and e uivalent amounts of protein were resolved by SDS-PAGE, translerred to nitrocellulose membranes, and then blotted with either the anti-yes polyclonal antibody Y35330 or the anti-fyn MAb SC-434. Bound antibodies were detected by ECL. The position of the 58-kDa marker is shown. (b) Activity of c-yes in MDA-MB-453 and T-47D cell lines. The ability of immunoprecipitated c-yes to phosphorylate enolase in an in vitro kinase assay was determined as described in “Material and Methods”. No I”, control immunoprecipitation in which the anti-yes antibody was omitted.

may be increased, resulting in an additive effect (e.g., in BT-474 cells). Tyrosine phosphorylation, therefore, represents a further level a t which EMSl regulation is perturbed in human breast cancer cells and a mechanism of interaction between different proteins of potential oncogenic activity in human breast cancer.

ACKNOWLEDGEMENTS

We thank Ms. Y.-E. Chiew for the contribution of cell line DNA and RNA samples. This work was supported by research grants from the National Health and Medical Research Council of Australia and the New South Wales State Cancer Council.

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