Advances in Brief Expression Profile of Tyrosine Kinases in Breast Cancer

The tyrosine kinase (TK) family includes many growth factor receptors, cell cycle regulators, and oncoproteins. Moreover, the receptor TKs HER2/neu and epidermal growth factor receptor are overexpressed in a subgroup of breast tumors and correlate with more aggressive behavior. Thus, TKs are being actively pursued as therapeutic targets. The purpose of this study was to determine the expression pattern of TKs in breast cancer. Reverse transcription-PCR was performed with degenerate primers based on conserved motifs of the catalytic domains of TKs, and the identities of the reverse transcription-PCR products were determined by digestion with a panel of restriction enzymes. Using a TK display assay, we studied the TK profiles of 13 breast cancer cell lines and two normal immortalized breast epithelial cell lines. The TK display assay reproducibly demonstrated known differences in HER-2/neu expression between cell 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, Rak, and YES were detected in breast cancer cells. Several kinases were differentially expressed among the cell lines. Similar TK profiles were found using RNA from human breast tumors. We conclude that there is significant variability in the TK expression pattern of breast cancers. This variability should be considered when selecting TK inhibitors to treat patients. Introduction TKs are regulatory proteins that play an important role in the cell growth and differentiation of normal cells. The TKs represent a major class of proto-oncogenes and may be involved in the progression and metastasis of cancer cells. Some TKs also affect the sensitivity of tumors to radiationand chemotherapyinduced apoptosis. As a result, TKs are being actively studied as targets for therapeutic intervention. The best studied are the 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). Several additional TKs have been found to be expressed or overexpressed in breast cancer (2) including BRK (3), c-Src (4), and FGFR (5). In contrast, loss of normal expression of other TKs such as Syk (6) and c-KIT (7) have been reported in breast cancer and been proposed to play a role in carcinogenesis. Trastuzumab, a monoclonal antibody against HER2/neu, is now in clinical use both as a single agent and in combination with chemotherapeutic agents. TK inhibitors targeting HER2/ neu and EGFR are also being tested in clinical trials. Understanding the TK expression patterns in breast cancer is essential for selecting particular TK inhibitors for clinical use and for identifying new therapeutic targets. We hypothesized that breast cancers have significant variability in their TK expression profiles, and that there may also be novel TKs that are differentially expressed in breast cancer and affect cancer biology. We used a differential display assay based on the conserved sequences in TKs to study their expression profiles in normal breast and breast cancer cell lines. Materials and Methods Cell Lines, Cell Cultures, and Breast Specimens. Breast cancer cell lines BT474, MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-453, MDA-MB-468, BT20, BT483, BT549, SBKBR3, T47D, ZR75-1, MDA-MB-330, and MDAMB-435, immortalized breast epithelial cell lines MCF10A and MCF12A, and human rhabdomyosarcoma cell line A204 were obtained from American Type Culture Collection (Manassas, VA) and maintained according to the manufacturer’s specifications. 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. TK Display Assay. Total RNA was isolated from cell lines and breast tissue with the TRIZOL reagent (Life Technologies, Inc., Carlsbad, CA), and the TKDA was carried out by a Received 9/19/01; revised 11/19/01; accepted 11/19/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked 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 Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 108, Houston, TX 77030. Phone: (713) 792-3668; Fax: (713) 794-0209; E-mail: [email protected]. 3 The abbreviations used are: TK, tyrosine kinase; TKDA, TK display assay; EGFR, epidermal growth factor receptor; FGFR fibroblast growth factor receptor; RT-PCR, reverse transcription-PCR; PDGFR, platelet-derived growth factor receptor; GAPDH, glyceraldehyde-3phosphate dehydrogenase; JAK, Janus kinase. 361 Vol. 8, 361–367, February 2002 Clinical Cancer Research Research. on July 28, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from modification of the method described by Robinson and colleagues (8, 9). Total RNA was reverse transcribed after annealing with the antisense degenerate primer Primer 3, 5 -CACAGGTTACCRHAIGMCCAIACRTC-3 , using the Superscript preamplification system (Life Technologies, Inc.) RT-PCR was performed using degenerate primers; the sense primers were Primer 1, 5 -CAGGTCACCAARRTIDCNGAYTTYGG-3 , and Primer 2, 5 -CCAGGTCACCAARRTTDCNGAYTTYGG3 , and the antisense primer was Primer 3. The mixed bases were defined as follows: N A C T G, D A T G, H A T C, R A G, Y C T, M A C, and I deoxyinosine. Alignment of the amino acid sequences corresponding to the primers and the TK consensus sequences are presented in Fig. 1A. RT-PCR was performed with DNA polymerase (FB-6000–60; Fisher Scientific, Pittsburgh, PA) in the presence of 25 mM MgCl2. Amplification was carried out at an annealing temperature of 44°C for 5 cycles and then at 55°C for 25 cycles. Amplified products were analyzed by gel electrophoresis on an 8% polyacrylamide gel (Fig. 1B). The primer sets yielded RT-PCR products that were consistently 154–170 bp because of the relatively constant spacing of the conserved motifs from which the primers were derived. A DNA was stained with 1 g/ml ethidium bromide. The 154–170-bp RTPCR products were excised from the polyacrylamide gel, eluted, precipitated, and dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). RT-PCR was repeated using a sense primer radiolabeled on the 5 end with [ -P]ATP (Amersham Life Science, Buckinghamshire, United Kingdom) and T4 polynucleotide kinase (New England Biolabs, Beverly, MA) before PCR. The 154–170-bp RT-PCR products were again excised from the gel and eluted. Equal amounts of radioactive DNA (10 cpm) were digested with a panel of restriction enzymes: AciI, AluI, CfoI, DdeI, HaeI1I, HinfI, MnlI, MspI, Sau3aI, Sau96I, ScrfI, Tru9I, and RsaI. Digestion products and uncut control DNA were resolved on a 6% acrylamide DNA sequencing gel and autoradiographed (Fig. 1B). A sequencing reaction was used as a size marker. The uncut kinases remained as tightly clustered 154–170-bp nucleotide bands (Fig. 2). Up to 10 kinases were cleaved with a given restriction enzyme, and different kinases were represented by bands of different sizes displayed in a DNA sequencing gel (Fig. 2). Cloning and Sequencing. PCR-amplified products were subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA). The recombinant plasmids were sequenced using the T7 Sequence version 2.0 DNA sequencing system (USB Corp., Cleveland, OH). The sequences obtained were compared with GenBank database sequences from the National Center for Biotechnology Information using the BLAST algorithm. RT-PCR for Specific Kinases. RT-PCR was performed at an annealing temperature of 55°C using primers 5 -GGTGGCTGTGAAGACGATGA-3 and 5 -CTCAGATACTCCATGCCACT-5 for Axl, 5 -TTGCTGCGCCAGAGCCGC-3 and 5 -GATGATGGCCTGCTCCAG-3 for Syk, and 5 -AGTCATCGTGGAGGATGATG-3 and 5 -AGGTTGACAGGTTCCAFig. 1 A, alignment of degenerate Primers 1 and 3 with the conserved catalytic domain of TK. B, TKDA. Primer 1 was radiolabeled with [ -P]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. 362 Tyrosine Kinases in Breast Cancer Research. on July 28, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from ACTG-3 for PDGFR . Amplification of GAPDH was performed as a control using primers 5 -AAGGTGAAGGTCGGAGTCAAC-3 and 5 -CATGAGTCCTTCCACGATACC-3 . PCR products were analyzed by 1.5% agarose gel electrophoresis. Western Blot Analysis. Whole-cell extracts were lysed using buffer A [20 mM Tris (pH 7.5), 10% glycerol, 1% Triton X-100, 0.15 mM NaCl, 1 mM -mercaptoethanol, 1 mM Na3VO4, 1 mM aprotinin, and 1 mM phenylmethylsulfonyl fluoride]. Samples were then centrifuged, and the supernatants were heated to 100°C for 5 min and separated by 8% SDS-PAGE. After transferring to nitrocellulose and blocking with 5% milk, the membranes were incubated with antibodies against Axl (Santa Cruz Biotechnology, Santa Cruz, CA). As a loading control, the membranes were stripped and incubated with an antibody against actin (Boehringer Mannheim, Indianapolis, IN). Immunocomplexes were detected with an ECL chemiluminescence system (Amersham Corp., Arlington Heights, IL). Northern Blot Analysis. Total RNA was extracted from cell lines using TRIZOL reagent as described previously. Ten g of total RNA from each cell line were resolved on a formaldehyde gel and transferred onto nylon membranes (GeneScreen; New England Nuclear Life Science Products, Boston, MA). A 303-bp Axl fragment was randomly labeled with [ -P]dCTP using the random primer DNA labeling system (Life Technologies, Inc.). The membranes were then stripped and rehybridized with a GAPDH probe as a loading control. Signals were detected with autoradiography.