Expression Profile of Tyrosine Kinases in Breast Cancer1

Vol. 8, 361–367, February 2002 Clinical Cancer Research 361

Advances in Brief Expression Profile of Tyrosine Kinases in Breast Cancer1

Funda Meric, Wei-Ping Lee, Aysegul Sahin, Introduction Haixia Zhang, Hsing-Jien Kung, and TKs3 are regulatory proteins that play an important role in Mien-Chie Hung2 the cell growth and differentiation of normal cells. The TKs represent a major class of proto-oncogenes and may be involved Departments of Surgical Oncology [F. M., H. Z., M-C. H.], Molecular in the progression and metastasis of cancer cells. Some TKs also and Cellular Oncology [W-P. L., M-C. H.], and Pathology [A. S.], The University of Texas M. D. Anderson Cancer Center, Houston, affect the sensitivity of tumors to radiation- and chemotherapy- Texas 77030, and Department of Biological Chemistry, University of induced apoptosis. As a result, TKs are being actively studied as California at Davis Cancer Center, Sacramento, California 95817 targets for therapeutic intervention. The best studied are the [H-J. K.] receptor TKs HER2/neu and EGFR. Overexpression of HER2/ neu occurs in 20% of invasive breast cancers and has been found to predict a poorer prognosis than does normal expression (1). Abstract Several additional TKs have been found to be expressed or The tyrosine kinase (TK) family includes many growth overexpressed in breast cancer (2) including BRK (3), c-Src (4), factor receptors, cell cycle regulators, and oncoproteins. and FGFR (5). In contrast, loss of normal expression of other Moreover, the receptor TKs HER2/neu and epidermal TKs such as Syk (6) and c-KIT (7) have been reported in breast growth factor receptor are overexpressed in a subgroup of cancer and been proposed to play a role in carcinogenesis. breast tumors and correlate with more aggressive behavior. Trastuzumab, a monoclonal antibody against HER2/neu, is Thus, TKs are being actively pursued as therapeutic targets. now in clinical use both as a single agent and in combination The purpose of this study was to determine the expression with chemotherapeutic agents. TK inhibitors targeting HER2/ pattern of TKs in breast cancer. Reverse transcription-PCR neu and EGFR are also being tested in clinical trials. Under- was performed with degenerate primers based on conserved standing the TK expression patterns in breast cancer is essential motifs of the catalytic domains of TKs, and the identities of for selecting particular TK inhibitors for clinical use and for the reverse transcription-PCR products were determined by identifying new therapeutic targets. digestion with a panel of restriction enzymes. Using a TK We hypothesized that breast cancers have significant var- display assay, we studied the TK profiles of 13 breast cancer iability in their TK expression profiles, and that there may also cell lines and two normal immortalized breast epithelial cell be novel TKs that are differentially expressed in breast cancer and affect cancer biology. We used a differential display assay lines. The TK display assay reproducibly demonstrated based on the conserved sequences in TKs to study their expres- known differences in HER-2/neu expression between cell sion profiles in normal breast and breast cancer cell lines. lines. Several TKs, including receptor TKs Axl, Cak, fibroblast growth factor receptor 4, HEK8, HER2/neu, c-MET, RET, and nonreceptor TKs ARG, BRK, Janus kinase 1, Materials and Methods Rak, and YES were detected in breast cancer cells. Several Cell Lines, Cell Cultures, and Breast Specimens. Breast cancer cell lines BT474, MCF7, MDA-MB-231, kinases were differentially expressed among the cell lines. MDA-MB-361, MDA-MB-453, MDA-MB-468, BT20, BT483, Similar TK profiles were found using RNA from human BT549, SBKBR3, T47D, ZR75-1, MDA-MB-330, and MDA- breast tumors. We conclude that there is significant varia- MB-435, immortalized breast epithelial cell lines MCF10A and bility in the TK expression pattern of breast cancers. This MCF12A, and human rhabdomyosarcoma cell line A204 were variability should be considered when selecting TK inhibi- obtained from American Type Culture Collection (Manassas, tors to treat patients. VA) and maintained according to the manufacturer’s specifica- tions. Cells were cultured in a humidified atmosphere of 5%

CO2 and at 37°C. Cells were harvested for RNA and protein analysis at 70–80% confluence. The breast specimens were obtained from the University of Texas M. D. Anderson Tumor Bank. Received 9/19/01; revised 11/19/01; accepted 11/19/01. TK Display Assay. Total RNA was isolated from cell The costs of publication of this article were defrayed in part by the lines and breast tissue with the TRIZOL reagent (Life Technol- payment of page charges. This article must therefore be hereby marked ogies, Inc., Carlsbad, CA), and the TKDA was carried out by a advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grants 5T32CA09599-11 and 1K08-CA91895-01 (to F. M.) and NIH Grants R0-1 CA58880 and P50 CA83639 (to M-C. H.). 2 To whom requests for reprints should be addressed, at Department of 3 The abbreviations used are: TK, tyrosine kinase; TKDA, TK display Molecular and Cellular Oncology, The University of Texas M. D. assay; EGFR, epidermal growth factor receptor; FGFR fibroblast Anderson Cancer Center, 1515 Holcombe Boulevard, Box 108, Hous- growth factor receptor; RT-PCR, reverse transcription-PCR; PDGFR, ton, TX 77030. Phone: (713) 792-3668; Fax: (713) 794-0209; E-mail: platelet-derived growth factor receptor; GAPDH, glyceraldehyde-3- [email protected]. phosphate dehydrogenase; JAK, Janus kinase. Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2002 American Association for Cancer Research. 362 Tyrosine Kinases in Breast Cancer

Fig. 1 A, alignment of degenerate Primers 1 and 3 with the conserved catalytic domain of TK. B, TKDA. Primer 1 was radiolabeled with [␥-32P]ATP before RT-PCR. RT-PCR products were separated by 8% PAGE. The 154–170-bp products were excised and eluted. DNAs of equal radioactivity were digested with a panel of restriction enzymes.

modification of the method described by Robinson and col- Science, Buckinghamshire, United Kingdom) and T4 polynu- leagues (8, 9). Total RNA was reverse transcribed after anneal- cleotide kinase (New England Biolabs, Beverly, MA) before ing with the antisense degenerate primer Primer 3, 5Ј-CACAG- PCR. The 154–170-bp RT-PCR products were again excised GTTACCRHAIGMCCAIACRTC-3Ј, using the Superscript from the gel and eluted. Equal amounts of radioactive DNA (105 preamplification system (Life Technologies, Inc.) RT-PCR was cpm) were digested with a panel of restriction enzymes: AciI, performed using degenerate primers; the sense primers were AluI, CfoI, DdeI, HaeI1I, HinfI, MnlI, MspI, Sau3aI, Sau96I, Primer 1, 5Ј-CAGGTCACCAARRTIDCNGAYTTYGG-3Ј, ScrfI, Tru9I, and RsaI. Digestion products and uncut control and Primer 2, 5Ј-CCAGGTCACCAARRTTDCNGAYTTYGG- DNA were resolved on a 6% acrylamide DNA sequencing gel 3Ј, and the antisense primer was Primer 3. The mixed bases and autoradiographed (Fig. 1B). A sequencing reaction was used were defined as follows: N ϭ A ϩ C ϩ T ϩ G, D ϭ A ϩ T ϩ as a size marker. The uncut kinases remained as tightly clustered G, H ϭ A ϩ T ϩ C, R ϭ A ϩ G, Y ϭ C ϩ T, M ϭ A ϩ C, 154–170-bp nucleotide bands (Fig. 2). Up to 10 kinases were and I ϭ deoxyinosine. Alignment of the amino acid sequences cleaved with a given restriction enzyme, and different kinases corresponding to the primers and the TK consensus sequences were represented by bands of different sizes displayed in a DNA are presented in Fig. 1A. RT-PCR was performed with DNA sequencing gel (Fig. 2). polymerase (FB-6000–60; Fisher Scientific, Pittsburgh, PA) in Cloning and Sequencing. PCR-amplified products were

the presence of 25 mM MgCl2. Amplification was carried out at subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA). an annealing temperature of 44°C for 5 cycles and then at 55°C The recombinant plasmids were sequenced using the T7 Se- for 25 cycles. Amplified products were analyzed by gel elec- quence version 2.0 DNA sequencing system (USB Corp., Cleve- trophoresis on an 8% polyacrylamide gel (Fig. 1B). The primer land, OH). The sequences obtained were compared with Gen- sets yielded RT-PCR products that were consistently 154–170 Bank database sequences from the National Center for bp because of the relatively constant spacing of the conserved Biotechnology Information using the BLAST algorithm. motifs from which the primers were derived. A DNA was RT-PCR for Specific Kinases. RT-PCR was performed stained with 1 ␮g/ml ethidium bromide. The 154–170-bp RT- at an annealing temperature of 55°C using primers 5Ј-GGTG- PCR products were excised from the polyacrylamide gel, eluted, GCTGTGAAGACGATGA-3Ј and 5Ј-CTCAGATACTCCAT- precipitated, and dissolved in TE buffer (10 mM Tris, 1 mM GCCACT-5Ј for Axl, 5Ј-TTGCTGCGCCAGAGCCGC-3Ј and EDTA, pH 8.0). RT-PCR was repeated using a sense primer 5Ј-GATGATGGCCTGCTCCAG-3Ј for Syk, and 5Ј-AGTCAT- radiolabeled on the 5Ј end with [␥-32P]ATP (Amersham Life CGTGGAGGATGATG-3Ј and 5Ј-AGGTTGACAGGTTCCA-

Fig. 2 A, TKDA of normal breast MCF10A, breast cancer MCF7, and MDA-MB-361 cell lines using two sets of primers (Lanes 1–3, Primers 1 and 3; Lanes 4–6, Primers 2 and 3). PCR products of equal radioactivity were digested with HinfI. Arrow, digestion product corresponding to the HER2/neu TK. B, TKDA of normal breast cell lines MCF10A and MCF12A and a panel of breast cancer cell lines. PCR products of equal radioactivity were digested with CfoI and separated by 6% PAGE. Identities of digestion products were determined by comparing their sizes with those of a sequencing reaction marker (left lane) and comparing their patterns with the restriction digest patterns of 11 other restriction enzymes (not shown). Kinases digested by CfoI included GCK, Rak, BRK/HEK8, and HER2/neu (arrows). BRK and HEK8 gave the same-sized products. Arrows, two PCR digestion products that did not correspond to known kinases.

ACTG-3Ј for PDGFR␣. Amplification of GAPDH was performed derived from the conserved amino acid motifs DFG and DVW as a control using primers 5Ј-AAGGTGAAGGTCGGAG- of the subdomains VII and IX in the activation loop of TKs (Fig. TCAAC-3Ј and 5Ј-CATGAGTCCTTCCACGATACC-3Ј. PCR 1A). TK profiles of normal breast cell lines MCF10A and products were analyzed by 1.5% agarose gel electrophoresis. MCF12A and breast cancer cell lines BT474, MCF7, MDA- Western Blot Analysis. Whole-cell extracts were lysed MB-231, MDA-MB-361, MDA-MB-453, MDA-MB-468, using buffer A [20 mM Tris (pH 7.5), 10% glycerol, 1% Triton BT20, BT483, BT549, SBKBR3, T47D, ZR75-1, MDA-MB- ␤ X-100, 0.15 mM NaCl, 1 mM -mercaptoethanol, 1 mM Na3VO4, 330, and MDA-MB-435 were performed. TKDA was performed 1mM aprotinin, and 1 mM phenylmethylsulfonyl fluoride]. Sam- by loading equal amounts of radioactive DNA (i.e., equal ples were then centrifuged, and the supernatants were heated to amounts of RT-PCR products) for each cell line onto the se- 100°C for 5 min and separated by 8% SDS-PAGE. After trans- quencing gel. Thus, the assays were standardized using the total ferring to nitrocellulose and blocking with 5% milk, the mem- TK content rather than the total RNA content as a reference. branes were incubated with antibodies against Axl (Santa Cruz Although results of this PCR-based assay were not quantitative, Biotechnology, Santa Cruz, CA). As a loading control, the we found that the HER2/neu expression detected by TKDA in membranes were stripped and incubated with an antibody MCF10A cells, in low HER2/neu-expressing MCF7 cells, and against actin (Boehringer Mannheim, Indianapolis, IN). Immu- in high HER2/neu-expressing MDA-MB-361 cells was consist- nocomplexes were detected with an ECL chemiluminescence ent with the expected expression profile (Fig. 2A). Furthermore, system (Amersham Corp., Arlington Heights, IL). when RT-PCR was performed with two primer sets (Primers 1 Northern Blot Analysis. Total RNA was extracted from and 3 versus Primers 2 and 3), similar results were obtained, cell lines using TRIZOL reagent as described previously. Ten demonstrating that the results of TKDA were reproducible. Fig. ␮ g of total RNA from each cell line were resolved on a form- 2B shows one TKDA after restriction enzyme digestion with aldehyde gel and transferred onto nylon membranes (Gene- CfoI. HER2/neu was detected with the expected expression Screen; New England Nuclear Life Science Products, Boston, pattern. The expression of several other kinases, including ger- MA). A 303-bp Axl fragment was randomly labeled with minal center kinase (a serine-threonine kinase), were detected ␣ 32 [ - P]dCTP using the random primer DNA labeling system simultaneously. (Life Technologies, Inc.). The membranes were then stripped TKDA products corresponding to receptor TKs Axl, cell and rehybridized with a GAPDH probe as a loading control. adhesion kinase (Cak or trkE), FGFR4, MET, and RET and Signals were detected with autoradiography. nonreceptor TKs Arg, BRK, JAK1, Rak, and YES were identified (Fig. 3A). Their identities were confirmed by at least two Results separate restriction enzyme digestions. Furthermore, the expres- TKDA. We used TKDA to profile TKs expressed in sion patterns of Arg, Axl, BRK, Cak, HEK8, JAK1, MET, Rak, breast cancer cell lines. The catalytic domains of TKs have and YES were confirmed by subcloning and sequencing of conserved motifs that have been commonly used for amplifica- RT-PCR products. tion of TKs using degenerate primers (2). We used primer sets Seven RT-PCR products, the digestion patterns of which did

Fig. 3 TK profiles of commonly used breast cancer cell lines. A, TKDA of normal MCF10A and MCF12A breast cell lines and breast cancer cell lines simultaneously detected several receptor (upper panel) and nonreceptor (lower panel) TK. The right panel and left panel represent separate experiments, each using MCF10A and MCF12A cells as controls. The TK profiles were confirmed with at least one other TKDA. The identity of each TK was confirmed by digestion with at least one other restriction enzyme. B, three of the RT- PCR digest products that do not correspond to any known kinases. Upper and middle panels, frag- ments 65 and 90 bp in size upon digestion with DdeI. Lower panel, a fragment 85 bp in size upon digest with RsaI.

not correspond to known kinases, were also identified; we hypoth- sense degenerate primer, possibly because that method was the esized that these represented either novel kinases or RT-PCR least vulnerable to RNA degradation. products of nonspecific annealing of the primers. Fig. 3B demon- We additionally studied the TK profiles of five normal strates the expression patterns of three of the most abundant po- breast specimens and seven invasive carcinomas (six ductal and tential novel kinases. Two nonkinase products, 18S rRNA and one lobular) with TKDA. Overall, the TK profile of the breast mitochondrial cytochrome c oxidase subunit II, were also ampli- tissues was remarkably similar to that of the breast cell lines. All fied, most likely because of primer sequence homology to them. TKs detected in breast cell lines were detected in breast tissue. The TK profiles were compared using cDNAs generated An additional TK, Tie, was detected only in the tissue; this from RT-PCR of cell lines using antisense degenerate Primer 1, kinase may be endothelial in origin. an oligo dT primer, or random hexamers. The TK profiles were RT-PCR Analysis of Specific Kinases. TKDA revealed reproducible regardless of the method used to produce the differential expression of Axl in breast cancer cell lines; there- cDNA. However, optimal results were obtained using the anti- fore, we further evaluated Axl expression at the RNA and

Fig. 5 A, Syk expression in breast cancer cell lines. RT-PCR was performed using Syk primers and cDNA from normal breast cell lines MCF10A and MCF12A breast cancer cell lines MCF7, BT20, BT549, MDA-MB-231, MDA-MB-435, and MDA-MB-453 with a 327-bp product (top panel). RT-PCR was performed with primers for GAPDH as a control (lower panel). B, PDGFR␣ expression in breast cancer. RT-PCR was performed with primers for PDGFR␣ and cDNA from normal breast and breast cancer cell lines (upper panel). Rhabdomyosarcoma cell line A204 was used as a positive control for PDGFR␣ expression and yielded a 577-bp product (right lane). RT-PCR was performed with Fig. 4 Expression of the receptor TK Axl. A, detection of Axl mRNA primers for GAPDH as a control (lower panel). by sequence-specific RT-PCR. RT-PCR was performed using Axl primers and cDNA from normal breast cell lines MCF10A and MCF12A and breast cancer cell lines MCF7, BT20, BT549, MDA-MB-231, MDA- MB-435, and MDA-MB-453, yielding a 303-bp product (top panel). breast cells. Syk mRNA was also not detected in four of six RT-PCR was performed with primers for GAPDH as a control (lower breast cancer cell lines tested (Fig. 5A). PDGFR␣ mRNA was panel). B, expression of Axl protein. Cell extracts were obtained from normal breast cell line MCF10A and breast cancer cell lines MCF7, not detected in normal breast or breast cancer cell lines, but it BT20, BT549, MDA-MB-231, MDA-MB-435, and MDA-MB-453. was detected in rhabdomyosarcoma cell line A204 (Fig. 5B). Western blot analysis was performed using anti-Axl antibodies (top panel) and anti-actin antibodies as a loading control (bottom panel). C, detection of Axl mRNA by Northern blot analysis. Ten ␮g of total RNA Discussion from MCF10A, MCF12A, BT549, and MDA231 cell lines were ana- Given the tremendous current interest in the use of TK lyzed. Hybridization was carried out with an Axl probe (top panel)ora inhibitors for breast cancer therapy, we felt it was important to GAPDH probe (bottom panel). determine the profile of TKs expressed in breast cancer. We performed TKDA on breast cancer cell lines and detected several TKs including receptor TKs Axl, Cak, FGFR4, HEK8, protein level. RT-PCR using Axl-specific primers detected Axl HER2/neu, c-MET, RET, and nonreceptor TKs ARG, BRK, mRNA in normal MCF10A and MCF12A breast cell lines and JAK1, Rak, and YES (Fig. 3A). To our knowledge, this is the in breast cancer cell lines BT549 and MDA-MB-231 (Fig. 4A). first time the expression of Arg, HEK8, and YES has been However, Northern blot analysis demonstrated that the mean reported in breast cancer. In addition to identifying known expression level of Axl was higher in BT549 cells and 12-fold kinases, we identified some RT-PCR products that may repre- higher in MDA-MB-231 cells than in normal breast cells sent novel kinases. Overall, breast cancer cell lines had a similar MCF10A and MCF12A (Fig. 4B). Overexpression of Axl in TK profile to that of breast tissue specimens, with one excep- BT549 and MDA-MB-231 cells was confirmed by Western blot tion: the TK Tie was expressed in breast tissue but not in cell analysis with polyclonal anti-Axl antibodies (Fig. 4C). lines (suggesting that it has a vascular endothelial origin). The two sets of degenerate primers we used in this study Several TKs are known to be expressed in human malig- did not allow us to detect all TKs. For example, our primers did nancies, and some are thought to be involved in the development not detect EGFR. Sequence-specific primers were used to de- of specific malignancies, including Bcr-Abl in chronic myelog- termine the expression of Syk, reported recently to be down- enous leukemia and the RET receptor TK in medullary thyroid regulated in breast cancer (6), and PDGR␣, one of the thera- carcinoma. Overall, TK activity in invasive breast cancer has peutic targets of signal transduction inhibitor 571. Syk been shown to be greater than that in normal breast tissue (10, expression was detected in normal MCF12A but not MCF10A 11). Moreover, several TKs that have been reported to be

expressed in breast cancer are proposed to play a role in breast thus, attention must be paid to ensure that all digestions actually cancer biology. For example, EGFR and HER2/neu are overex- work, and consideration must be given to performing confirm- pressed in 35–50% and 20–30% of invasive breast cancers, atory double digests if needed. Another limitation of the assay is respectively, and predict a poor prognosis (1, 12, 13). BRK, a that it is not quantitative: it is more useful for determining the TK cloned from a metastatic breast tumor (3), has been reported presence or absence of TK expression than for determining to be overexpressed in two thirds of breast tumors (14), the relative expression levels. TKDA is fairly labor intensive and c-Met receptor has been found to be expressed at high levels in requires large amounts of RNA to produce reproducible results. poorly differentiated breast cancer cells (5), and c-Src has also Therefore, it is unlikely to be used routinely in clinical practice; been reported to be overexpressed in breast cancer (4). In however, it is a valuable research tool. Overall, the kinase addition, McLeskey et al. (5) showed that several breast cancer profiles found were fairly reproducible in cell lines. However, cell lines express high levels of FGFR; 10% of primary breast the expression of four protein kinases (RET, Axl, GCK, and one tumor samples have been found to have amplification of the potential novel kinase) were found to be more variable. The FGFR4 gene (15), and 9% amplification of FGFR1 (16). In expression of these kinases may be sensitive to culture condi- contrast, c-kit has been found to be expressed significantly less tions, such as cell confluence, pH, and relative serum starvation, in breast cancer than in normal breast tissue (7, 17). Further- or may represent a variation in assay conditions. more, Syk has been reported to be lost in invasive breast cancer In summary, we used a TKDA to profile the TK expression and is proposed to be a potential tumor suppressor in human pattern in breast cancer. Several TKs were found to be differ- breast carcinomas (6). Thus, several TKs are expressed in breast entially expressed in commonly used breast cancer cell lines. cancer, potentially exerting some opposing effects on cell This heterogeneity should be taken into consideration when growth and proliferation. studying breast cancer biology. Tailoring TK inhibitors to the In the present study, we found that the receptor TK Axl was expression profile of each tumor may enhance their therapeutic overexpressed at the mRNA and protein level in some breast efficacy. cancer cell lines. Axl was initially identified as a protein en- coded by a transforming gene from primary human myeloid Acknowledgments leukemia cells (18). Growth arrest-specific gene 6, which is the We thank Kerry Wright of the Department of Scientific Publica- ligand for Axl (19), mediates mitogenic activity in Axl-overex- tions, The University of Texas M. D. Anderson Cancer Center in Hous- pressing cells (20). Overexpression of Axl has been reported in ton, TX for editorial assistance, and Dan Robinson from the University thyroid cancer (21) and hepatocellular cancer (22). The expres- of California at Davis Cancer Center in Sacramento, CA for providing sion of Axl has also been found to be higher in a metastatic the tyrosine kinase restriction enzyme site database. prostate cell line than in a less aggressive prostate carcinoma cell line or in normal prostate cells (23). Axl has also been References reported to be expressed 10-fold more in colon cancer metasta- 1. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A., ses than in other normal and malignant tissues (24). Our study and McGuire, W. L. Human breast cancer: correlation of relapse and suggests that Axl is overexpressed in a subgroup of breast survival with amplification of the HER-2/neu oncogene. Science (Wash. cancers. We have reported recently that adenovirus type 5 E1A DC), 235: 177–182, 1987. down-regulates the expression of Axl at a transcriptional level, 2. Cance, W. G., Craven, R. J., Bergman, M., Xu, L., Alitalo, K., and and that down-regulation of Axl is involved in E1A-mediated Liu, E. T. Rak, a novel nuclear tyrosine kinase expressed in epithelial cells. Cell Growth Differ., 5: 1347–1355, 1994. growth suppression and E1A-induced apoptosis (25). Further 3. Mitchell, P. J., Barker, K. T., Martindale, J. E., Kamalati, T., Lowe, study is needed to determine whether Axl will be useful as a P. N., Page, M. J., Gusterson, B. A., and Crompton, M. R. Cloning and therapeutic target in a subgroup of breast tumors. characterisation of cDNAs encoding a novel nonreceptor tyrosine ki- Craven et al. (24) and Cance et al. (26) have previously nase, brk, expressed in human breast tumours. Oncogene, 9: 2383–2390, attempted to systematically identify TKs expressed in breast 1994. cancer cell line 600PEI and in a primary tumor library by 4. Verbeek, B. S., Vroom, T. M., Adriaansen-Slot, S. S., Ottenhoff- Kalff, A. E., Geertzema, J. G., Hennipman, A., and Rijksen, G. c-Src performing RT-PCR with a different set of degenerate primers protein expression is increased in human breast cancer. An immunohis- and constructing and sequencing these kinase-specific cDNA tochemical and biochemical analysis. J. Pathol., 180: 383–388, 1996. libraries. The advantages of TKDA are that it allows for simul- 5. McLeskey, S. W., Ding, I. Y., Lippman, M. E., and Kern, F. G. taneous profiling of up to 40–50 TKs (8), as well as provide a MDA-MB-134 breast carcinoma cells overexpress fibroblast growth potential for novel gene discovery. It may be less sensitive to factor (FGF) receptors and are growth-inhibited by FGF ligands. Cancer RNA degradation than are other RNA-based assays, because the Res., 54: 523–530, 1994. primers chosen for TKDA are close to the 3Ј end of the RNA 6. Coopman, P. J., Do, M. T., Barth, M., Bowden, E. T., Hayes, A. J., Basyuk, E., Blancato, J. K., Vezza, P. R., McLeskey, S. W., Mangeat, and flank a short RNA sequence (120 bp without primers). The P. H., and Mueller, S. C. The Syk tyrosine kinase suppresses malignant disadvantages of TKDA are that it, similar to other PCR-based growth of human breast cancer cells. Nature (Lond.), 406: 742–747, assays, has a contamination potential, and there is also a poten- 2000. tial for nonspecific priming with degenerate primers, as we 7. Chui, X., Egami, H., Yamashita, J., Kurizaki, T., Ohmachi, H., observed when they amplified with 18S rRNA and mitochon- Yamamoto, S., and Ogawa, M. Immunohistochemical expression of the Ͼ c-kit proto-oncogene product in human malignant and non-malignant drial cytochrome c oxidase subunit II; however, 90% of clones breast tissues. Br. J. Cancer, 73: 1233–1236, 1996. obtained with degenerate primers are protein kinases (8). There 8. Robinson, D., He, F., Pretlow, T., and Kung, H. J. A tyrosine kinase is also a potential for misidentification of TKs with the assay, profile of prostate carcinoma. Proc. Natl. Acad. Sci. USA, 93: 5958– because identification is dependent on restriction digest pattern; 5962, 1996.

9. Kung, H. J., Chen, H. C., and Robinson, D. Molecular profiling of leukemia cells, encodes a novel receptor tyrosine kinase. Mol. Cell. tyrosine kinases in normal and cancer cells. J. Biomed. Sci., 5: 74–78, Biol., 11: 5016–5031, 1991. 1998. 19. Mark, M. R., Chen, J., Hammonds, R. G., Sadick, M., and 10. Kopreski, M. S., Witters, L., Brennan, W. A., Jr., Buckwalter, E. A., Godowsk, P. J. Characterization of Gas6, a member of the superfamily Chinchilli, V. M., Demers, L. M., and Lipton, A. Protein tyrosine kinase of G domain-containing proteins, as a ligand for Rse and Axl. J. Biol. activity in breast cancer and its relation to prognostic indicators. Anti- Chem., 271: 9785–9789, 1996. cancer Res., 16: 3037–3041, 1996. 20. Tanaka, K., Nagayama, Y., Nakano, T., Takamura, N., Namba, H., 11. Hennipman, A., van Oirschot, B. A., Smits, J., Rijksen, G., and Fukada, S., Kuma, K., Yamashita, S., and Niwa, M. Expression profile Staal, G. E. Tyrosine kinase activity in breast cancer, benign breast of receptor-type protein tyrosine kinase genes in the human thyroid. disease, and normal breast tissue. Cancer Res., 49: 516–521, 1989. Endocrinology, 139: 852–858, 1998. 12. Sainsbury, J. R., Farndon, J. R., Needham, G. K., Malcolm, A. J., and Harris, A. L. Epidermal-growth-factor receptor status as predictor of 21. Ito, T., Ito, M., Naito, S., Ohtsuru, A., Nagayama, Y., Kanematsu, early recurrence of and death from breast cancer. Lancet, 1: 1398–1402, T., Yamashita, S., and Sekine, I. Expression of the Axl receptor tyrosine 1987. kinase in human thyroid carcinoma. Thyroid, 9: 563–567, 1999. 13. Gusterson, B. A., Gelber, R. D., Goldhirsch, A., Price, K. N., 22. Tsou, A. P., Wu, K. M., Tsen, T. Y., Chi, C. W., Chiu, J. H., Lui, Save-Soderborgh, J., Anbazhagan, R., Styles, J., Rudenstam, C. M., W. Y., Hu, C. P., Chang, C., Chou, C. K., and Tsai, S. F. Parallel Golouh, R., Reed, R., et al. Prognostic importance of c-erbB-2 expres- hybridization analysis of multiple protein kinase genes: identification of sion in breast cancer. International (Ludwig) Breast Cancer Study gene expression patterns characteristic of human hepatocellular carci- Group. J. Clin. Oncol., 10: 1049–1056, 1992. noma. Genomics, 50: 331–340, 1998. 14. Barker, K. T., Jackson, L. E., and Crompton, M. R. BRK tyrosine 23. Jacob, A. N., Kalapurakal, J., Davidson, W. R., Kandpal, G., kinase expression in a high proportion of human breast carcinomas. Dunson, N., Prashar, Y., and Kandpal, R. P. A receptor tyrosine kinase, Oncogene, 15: 799–805, 1997. UFO/Axl, and other genes isolated by a modified differential display 15. Jaakkola, S., Salmikangas, P., Nylund, S., Partanen, J., Armstrong, PCR are overexpressed in metastatic prostatic carcinoma cell line E., Pyrhonen, S., Lehtovirta, P., and Nevanlinna, H. Amplification of DU145. Cancer Detect. Prev., 23: 325–332, 1999. fgfr4 gene in human breast and gynecological cancers. Int. J. Cancer, 54: 24. Craven, R. J., Xu, L. H., Weiner, T. M., Fridell, Y. W., Dent, G. A., 378–382, 1993. Srivastava, S., Varnum, B., Liu, E. T., and Cance, W. G. Receptor 16. Jacquemier, J., Adelaide, J., Parc, P., Penault-Llorca, F., Planche, J., tyrosine kinases expressed in metastatic colon cancer. Int. J. Cancer, 60: de Lapeyriere, O., and Birnbaum, D. Expression of the FGFR1 gene in 791–797, 1995. human breast-carcinoma cells. Int. J. Cancer, 59: 373–378, 1994. 25. Lee, W. P., Liao, Y., Robinson, D., Kung, H. J., Liu, E. T., and 17. Natali, P. G., Nicotra, M. R., Sures, I., Mottolese, M., Botti, C., and Hung, M. C. Axl-gas6 interaction counteracts E1A-mediated cell Ullrich, A. Breast cancer is associated with loss of the c-kit oncogene growth suppression and proapoptotic activity. Mol. Cell. Biol., 19: product. Int. J. Cancer, 52: 713–717, 1992. 8075–8082, 1999. 18. O’Bryan, J. P., Frye, R. A., Cogswell, P. C., Neubauer, A., Kitch, 26. Cance, W. G., Craven, R. J., Weiner, T. M., and Liu, E. T. Novel B., Prokop, C., Espinosa, R., III, Le Beau, M. M., Earp, H. S., and Liu, protein kinases expressed in human breast cancer. Int. J. Cancer, 54: E. T. axl, a transforming gene isolated from primary human myeloid 571–577, 1993.

Funda Meric, Wei-Ping Lee, Aysegul Sahin, et al.

Clin Cancer Res 2002;8:361-367.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/8/2/361

Cited articles This article cites 26 articles, 9 of which you can access for free at: http://clincancerres.aacrjournals.org/content/8/2/361.full#ref-list-1

Citing articles This article has been cited by 26 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/8/2/361.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/8/2/361. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.