ANTICANCER RESEARCH 26: 4969-4974 (2006)

Diagnostic Relevance of Overexpressed NOK mRNA in Breast Cancer

RYOSUKE MORIAI*, DAISUKE KOBAYASHI*, TOMOKO AMACHIKA, NAOKI TSUJI and NAOKI WATANABE

Department of Clinical Laboratory Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan

Abstract. A novel oncogene with a kinase domain (NOK), a acted (2, 3). Since many RPTK are overexpressed or receptor protein , has been reported to cause mutated in various cancers (1), RPTKs can serve as markers proliferation of normal cells, suggesting its possible use as a for the diagnosis of cancer that conventional diagnostic diagnostic marker in human cancer. To determine the methods cannot detect or confirm. significance of NOK expression in cancer cells, the effect of The expression profile of an epidermal growth factor NOK inhibition was first examined on cell proliferation in receptor family member, c-erbB2/HER2/neu, has been well- vitro. The degree of expression in 52 clinical breast cancer investigated, confirming clinical significance in breast samples was then correlated with clinical features. The cancers. Amplification or overexpression of c-erbB2 in breast transduction of NOK small inhibitory (si) RNA in T47D breast cancer was associated with a poor prognosis (4, 5). From this cancer cells decreased NOK mRNA expression, thereby evidence, antibody therapy targeting c-erbB2/HER2/neu was inhibiting growth. When the mean expression in non-cancerous carried out in breast cancer patients, not only with tissues from the same breast resection specimens ±2SD was metastasis, but also at early stages. Expression of the c-erbB2 used as a cut-off value, 67.3% of breast cancers were positive or protein was examined in tumors by fluorescence in for NOK expression – a higher positivity rate than that found situ hybridization or immunohistochemistry to determine for c-erbB2 (28.8%). NOK mRNA expression did not correlate whether such therapy is indicated (6, 7). However, with c-erbB2 expression, indicating the independence of NOK amplification or overexpression of c-erbB2 and other RPTK as a diagnostic marker. Furthermore, NOK mRNA was highly was detected in only 20% to 30% of breast cancers, and expressed even at early clinical stages. NOK mRNA might be many patients still have poor outcomes in the absence of an ideal target to support the diagnosis of breast cancer overexpression. especially in tiny tumors in which the malignancy cannot be The novel oncogene with a kinase domain (NOK), an confirmed by other means. RPTK-like protein recently identified by Liu et al., shares 20% to 30% homology with members of the fibroblast Receptor protein tyrosine kinases (RPTK) are key (FGFR)/platelet-derived growth molecules in cell proliferation, affecting cell cycle factor receptor (PDGFR) family (8). NOK has a single progression, as well as resistance to apoptotic stimuli in putative transmembrane domain and an intracellular cancer cells (1). After RPTK are activated by self- tyrosine kinase domain, but lacks an extracellular domain. phosphorylation, with or without ligand binding, they Overexpression of NOK gene resulted in growth factor- initiate the growth signals before most other molecules have independent cell proliferation in BaF3 cells, and also surface adhesion-independent growth and colony formation in both NIH3T3 and BaF3 cell (8). Furthermore, the overexpression of NOK in BaF3 cells induced tumorigenesis *Both authors contributed equally to this study. and metastasis in nude mice. These observations indicate that NOK is a critical molecule in promoting cell Correspondence to: Naoki Watanabe, MD, Ph.D., Department of proliferation and suggest that NOK expression could be up- Clinical Laboratory Medicine, Sapporo Medical University School regulated in cancer cells. NOK was hypothesized to serve as of Medicine, South-1, West-16, Chuo-Ku, Sapporo 060-8543, a novel marker for the genetic diagnosis of breast cancer. Japan. Tel: +81 11 611 2111 (ext. 3640), Fax: +81 11 622 7502, e- To date, however, the degree of importance of NOK mail: [email protected] expression in cancer cells and the differences in NOK Key Words: NOK, c-erbB2, mRNA expression, breast cancer, expression between human cancer and non-cancerous diagnosis. tissues remain unclear. The effect of small inhibitory (si)

0250-7005/2006 $2.00+.40 4969 ANTICANCER RESEARCH 26: 4969-4974 (2006)

RNA against NOK (NOK-siRNA) on the proliferation of at 37ÆC. As a transduction control, non-silencing control RNA breast cancer cells was examined. The degree of NOK (QIAGEN,) was used. The siRNA duplexes used in this study were mRNA expression in breast cancer tissues was then as follows: NOK; 5’-GCA GGA CAU GGA GGA AAU G TT’, 3’- TT CGU CCU GUA CCU CCU UUA C-5’. The transduction of correlated with clinical features and the prevalence of siRNA was performed using HiPerFect Transfection Reagent positivity in breast cancers for NOK and c-erbB2 mRNA (QIAGEN) according to the manufacturer's protocol. Briefly, expression was compared. 1x105 of T47D cells were cultured in 6-well culture plates (Costar, Tokyo, Japan) in 2.3 ml of RPMI supplemented with 10% FBS. Materials and Methods After 24 h, 100 Ìl of HiPerFect Transfection Reagent with the addition of 20 nM of either RNA were added and the cells were incubated for an additional 72 h. After 72 h, the cells were Patients and frozen tissue samples. Samples of breast cancer and harvested, silenced mRNA expression was estimated and cell paired samples of non-cancerous tissue from the same resected number was also counted on hemocytometer chambers. breast were obtained from patients undergoing surgery at Sapporo Medical University Hospital and Sapporo Breast Surgical Clinic (Sapporo, Japan). Before the acquisition of these tissues, informed Results consent was obtained explaining the investigational nature of the study. Tissues were immediately frozen and stored in liquid Effect of transducing NOK-siRNA on cell proliferation. To nitrogen. Tissues were also stained with hematoxylin/eosin and determine the importance of NOK expression in cancer cell were reviewed by well-experienced pathologists. The clinico- growth, NOK-siRNA was transduced into breast cancer cells. pathological factors and clinical stages were evaluated according to The extent of NOK mRNA expression was determined in four the criteria of the Japanese Society of Breast Cancer. breast cancer cell lines to select cells for siRNA transduction. Cell culture. The human breast cancer cell lines, T47D and MCF7 The highest expression was observed in the T47D cells (Figure were obtained from the American Type Culture Collection (ATCC; 1A). Transduction of NOK siRNA into T47D cells decreased Manassas, VA, USA). HMC-1 and SKBR-3 were kindly provided the expression of NOK mRNA compared to the cells from 1st Department of Pathology, Sapporo Medical University transduced with non-silencing control RNA (NSC; Figure 1B). School of Medicine (9). All breast cancer cell lines were cultured in Accordingly, after 3 days of transduction, growth was inhibited RPMI-1640 (BioWhittaker, Walkersville, MD, USA) supplemented by approximately 50% in NOK siRNA-transduced cells with 10% heat-inactivated FBS (Invitrogen, Carlsbad, CA, USA) compared with NSC-transduced cells (Figure 1C). and grown at 37ÆC in a humidified atmosphere of 5% CO2. C-erbB2 and NOK mRNA expression in breast cancer tissues. To Quantification of NOK and c-erbB2 mRNA expression. The expression compare NOK expression, the mRNA expression for of NOK and c-erbB2 mRNA was determined by a quantitative RT- PCR using an ABI PRISM 7700 sequence detector system (Applied c-erbB2 was first measured in the breast tissues. The Biosystems, Foster City, CA, USA). Isolation of total RNA were expression of c-erbB2 mRNA was significantly higher in breast performed using ISOGEN reagent (Nippon Gene, Toyama, Japan) cancer tissues (mean±SD, 0.511±0.971) than in adjacent non- according to the manufacturer’s protocol. Gene-specific primers and cancerous breast tissues (0.059±0.077, p<0.002; Figure 2). fluorescent hybridization probes used in quantitative PCR were as When a cut-off value was set as the mean±2SD of expression follows: NOK forward primer, 5’-CAT CTT TCG AGC CAA TAT in non-cancerous breast tissues, c-erbB2 mRNA expression GAA CAC-3’; reverse primer, 5’-TGG AAT TGG ATT CGC CCT was positive in 28.8% of cancers (15 out of 52; Figure 2). AA’; and probe, 5’-(FAM) CCA GCT GGG CTC CAT GAG GTA CAA GAT (TAMRA)-3’, c-erbB2 forward primer, 5’-AGG ATG The degree of NOK mRNA expression compared to TGC GGC TCG TAC AC-3’; reverse primer, 5’-TAA TTT TGA c-erbB2 was subsequently examined. As shown in Figure 3, CAT GGT TGG GAC TCT T-3’; and probe, 5’-(FAM) CTT GGC NOK mRNA expression in breast cancer tissues CGC TCG GAA CGT GC (TAMRA)-3’. Quantitative RT-PCR was (0.088±0.085) was six times higher than that in non- performed using TaqMan One Step RT-PCR Master Mix Reagents cancerous tissues (0.015±0.013, p<0.0001). With positivity (Applied Biosystems). To compare amounts of mRNA encoding determined using a definition for cut-off value analogous NOK and c-erbB2 in different samples, the quantity of specific that for c-erbB2 mRNA expression, the positivity rate for mRNA was normalized as a ratio to the amount of 18S ribosomal RNA (18S rRNA), which was determined using TaqMan Ribosomal NOK mRNA in cancer tissues was 67.3% (35 out of 52), RNA Control Reagents (Applied Biosystems) according to much higher than that for c-erbB2 mRNA expression. manufacturer's protocol. The relationships between clinicopathological factors and NOK mRNA expression in breast cancer tissues were Transduction of small inhibitory RNA (siRNA) for NOK. A siRNA further analyzed. No correlations were found between age, was designed to target the coding region of the NOK gene menopausal state, or the expression of estrogen (GenBank accession no. NM-018423, which was originally progesterone receptors or NOK mRNA. As for expression expressed as STYK1, nucleotides 224 to 244, relative to the start codon), and prepared by QIAGEN (Tokyo, Japan). Single-strand at clinical stages determined by tumor size, invasion, and RNAs were annealed by incubating each strand in siRNA metastasis to lymph nodes and distant organs, NOK mRNA suspension buffer for 1 min at 90ÆC, followed by 1 h of incubation expression was high, even at an early stage (Figure 4).

4970 Moriai et al: NOK mRNA in Breast Cancers

Figure 1. In vitro experiments. A) Expression of NOK mRNA in human breast cancer cell lines including T47D. NOK mRNA expression as measured by quantitative RT-PCR is reported relative to expression by MCF7 cells. Data are the means of triplicate measurements. B) NOK mRNA expression in T47D cells transduced with siRNA against NOK (NOK-siRNA). C) Proliferation of these cells. Cells were transduced with non-silencing control RNA (NSC) or NOK-siRNA. Total RNA was extracted at 72 h after transduction to measure mRNA expression. NOK mRNA expression in siRNA transfectants is reported relative to expression in NSC transfectants. Cells were counted in hemocytometer chambers at 72 h after transfection.

Correlation between NOK and c-erbB2 mRNA expression in of breast cancer. No previous studies have compared breast cancer tissues. The correlation between NOK and amounts of NOK expression in resected human cancers with c-erbB2 mRNA expression was examined in breast cancer adjacent non-cancerous tissues. Our comparison tissues. As shown in Figure 5, no correlation (r=0.017) was demonstrated NOK mRNA to be overexpressed in breast found between NOK and c-erbB2 mRNA expression, cancer tissues. The expression of mRNA encoding c-erbB2, indicating that each mRNA was an independent marker. a well known receptor protein tyrosine kinase (RPTK) Further, the positivity rate of both molecules was analyzed targeted therapeutically in HER2 antibody therapy, was also in four groups defined by the cut-off values for these two found to be overexpressed in cancer tissues – but far less molecules. The plot shows that most NOK-positive tumors frequently than NOK mRNA. Previous studies using various were c-erbB2-negative, reflecting the difference in positivity cut-off values have indicated nearly the same positivity rate rate between these molecules. Only 5 tumors (9.6%) were for c-erbB2 mRNA, 17% to 20% by quantitative RT-PCR, positive for c-erbB2 alone, while positivity for NOK alone as we found (10, 11). The positivity for NOK mRNA among showed a rate of 48.1% (25 out of 52). Both c-erbB2 and in breast cancers was also quite high compared to mRNA NOK were positive in 19.2% of tumors (10 out of 52). expression of other RPTK, such as epidermal growth factor Accordingly, combining NOK with c-erbB2 resulted in a receptor, FGFR and PDGFR(12-14). higher positivity rate (76.9%; 40 out of 52). As previously reported, RPTK generally require ligand binding for tyrosine phosphorylation and growth signal Discussion transmission, with the exception of variant molecules, such as FGFR4 and hepatocyte growth factor receptor (Met) with This study was performed to investigate whether NOK alterations in the extracellular domain (15, 16). In contrast to could be a useful gene expression marker for the diagnosis these RPTK, NOK has been reported to lack an extracellular

4971 ANTICANCER RESEARCH 26: 4969-4974 (2006)

Figure 3. Expression of NOK mRNA in breast cancer and adjacent Figure 2. Expression of c-erbB2 mRNA in breast cancers and adjacent noncancerous tissues. The expression of NOK mRNA was measured using noncancerous tissues. The expression of c-erbB2 mRNA was measured quantitative RT-PCR and is reported relative to the amount of 18S rRNA. using quantitative RT-PCR and is reported relative to the amount of 18S The dotted line shows the mean±2SD of NOK mRNA in noncancerous rRNA. The dotted line shows the mean±2SD of c-erbB2 mRNA in tissues. NC, noncancerous tissues; C, cancer tissues. Statistical analysis noncancerous tissues. NC, noncancerous tissues; C, cancer tissues. was performed using a Mann-Whitney rank-sum test. Statistical analysis was performed using a Mann-Whitney rank-sum test. domain. Instead, growth signals are thought to be transmitted by auto-activation of NOK (8, 17). Thus, the presence of NOK even without a specific ligand may lead to abnormal cell proliferation. Li et al. have reported that not only tumorigenesis but also metastasis were observed in mice transplanted with normal cells overexpressing NOK by gene transduction. However, the extent to which NOK expression by cancer cells participates in cell proliferation has been unclear. We, therefore, examined the significance of NOK expression in cancer cells, demonstrating that introduction of an siRNA against NOK leads to growth inhibition in breast cancer cells. Furthermore, the extent of growth inhibition was exceeded by the percentage decrease in mRNA expression. Taken together with the observation by Liu et al. that NOK gene overexpression led to progressive cell growth via the Figure 4. NOK mRNA expression at each clinical stage of breast cancer. signaling pathway of phosphatidylinositol 3-kinase and NOK mRNA expression was measured using quantitative RT-PCR and mitogen activated (8), NOK expression itself was expressed relative to the amount of 18S rRNA. could be critical for the proliferation of breast cancer cells. In addition to our observations in vitro, NOK mRNA was also found to be overexpressed even at early clinical stages of supporting the diagnosis of breast cancer especially when only breast cancer. Amounts of NOK mRNA were elevated in small numbers of cells can be obtained in tiny tumors in which 92.2% of tumor samples (47/51). Accordingly, NOK may play a the malignancy cannot be confirmed by other means. significant role in the initial carcinogenesis of breast cancer. The It was found that mRNA expression of NOK did not quantitative measurement of NOK mRNA might be useful in correlate with that of c-erbB2, indicating that NOK could

4972 Moriai et al: NOK mRNA in Breast Cancers

Santiago A, Park JM, Riva A and Slamon DJ: Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials. Clin Cancer Res 11: 6598- 6607, 2005. 7 Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, Slamon DJ, Murphy M, Novotny WF, Burchmore M, Shak S, Stewart SJ and Press M: Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 20: 719-726, 2002. 8 Liu L, Yu XZ, Li TS, Song LX, Chen PL, Suo TL, Li YH, Wang SD, Chen Y, Ren YM, Zhang SP, Chang ZJ and Fu XY: A novel protein tyrosine kinase NOK that shares homology with platelet- derived growth factor/fibroblast growth factor receptors induces tumorigenesis and metastasis in nude mice. Cancer Res 64: 3491- 3499, 2004. 9 Asanuma H, Torigoe T, Kamiguchi K, Hirohashi Y, Ohmura T, Hirata K, Sato M and Sato N: Survivin expression is regulated by coexpression of human epidermal growth factor receptor 2 and epidermal growth factor receptor via phosphatidylinositol 3- kinase/AKT signaling pathway in breast cancer cells. Cancer Res 65: 11018-11025, 2005. 10 Izawa A, Kobayashi D, Nasu S, Saito K, Moriai R, Asanuma K, Nakamura M, Yagihashi A and Watanabe N: Relevance of c- Figure 5. Correlation between NOK mRNA and c-erbB2 mRNA erbB2, PLU-1 and survivin mRNA expression to diagnostic expression in breast cancer tissues. The dotted line shows the mean±2SD assessment of breast cancer. Anticancer Res 22: 2965-2969, 2002. of NOK or c-erbB2 mRNA expression in noncancerous tissues. Pearson’s 11 Bieche I, Onody P, Laurendeau I, Olivi M, Vidaud D, Lidereau R correlation was used to calculate the coefficients. and Vidaud M: Real-time reverse transcription-PCR assay for future management of ERBB2-based clinical applications. Clin Chem 45: 1148-1156, 1999. 12 Tsutsui S, Kataoka A, Ohno S, Murakami S, Kinoshita J and be an independent marker for the diagnosis of breast Hachitanda Y: Prognostic and predictive value of epidermal cancer. A discrepancy in expression profiles between the growth factor receptor in recurrent breast cancer. Clin Cancer molecules may reflect the structured difference that NOK Res 8: 3454-3460, 2002. lacks extracellular domain and does not require specific 13 Coltrera MD, Wang J, Porter PL and Gown AM: Expression of ligand binding for growth signal transmission. Tumors platelet-derived growth factor B-chain and the platelet-derived showing high c-erbB2 expression can be treated by growth factor receptor beta subunit in human breast tissue and ® breast carcinoma. Cancer Res 55: 2703-2708, 1995. Trastuzumab (Herceptin ), but some c-erbB2-negative 14 Penault-Llorca F, Bertucci F, Adelaide J, Parc P, Coulier F, cases also show poor outcomes. Since most of our c-erbB2- Jacquemier J, Birnbaum D and deLapeyriere O: Expression of negative cases were NOK-positive, these patients might be FGF and FGF receptor genes in human breast cancer. Int J treated in the future by inhibition of NOK expression. Cancer 61: 170-176, 1995. 15 Ezzat S, Zheng L, Zhu XF, Wu GE and Asa SL: Targeted References expression of a human pituitary tumor-derived isoform of FGF receptor-4 recapitulates pituitary tumorigenesis. J Clin Invest 109: 69-78, 2002. 1 Blume-Jensen P and Hunter T: Oncogenic kinase signalling. 16 Wallenius V, Hisaoka M, Helou K, Levan G, Mandahl N, Meis- Nature 411: 355-365, 2001. Kindblom JM, Kindblom LG and Jansson JO: Overexpression of 2 Hubbard SR, Mohammadi M and Schlessinger J: the hepatocyte growth factor (HGF) receptor (Met) and presence Autoregulatory mechanisms in protein-tyrosine kinases. J Biol of a truncated and activated intracellular HGF receptor fragment Chem 273: 11987-11990, 1998. in locally aggressive/malignant human musculoskeletal tumors. 3 Schlessinger J: Cell signaling by receptor tyrosine kinases. Cell Am J Pathol 156: 821-829, 2002. 103: 211-225, 2000. 17 Chen Y, Li YH, Chen XP, Gong LM, Zhang SP, Chang ZJ, 4 Gusterson BA, Gelber RD, Goldhirsch A, Price KN, Save- Zhang XF, Fu XY and Liu L: Point mutation at single tyrosine Soderborgh J, Anbazhagan R et al: Prognostic importance of c- residue of novel oncogene NOK abrogates tumorigenesis in nude erbB-2 expression in breast cancer. International (Ludwig) mice. Cancer Res 65: 10838-10846, 2005. Breast Cancer Study Group. J Clin Oncol 10: 1049-1056, 1992. 5 Duffy MJ: Predictive markers in breast and other cancers: a review. Clin Chem 51: 494-503, 2005. 6 Press MF, Sauter G, Bernstein L, Villalobos IE, Mirlacher M, Received September 19, 2006 Zhou JY, Wardeh R, Li YT, Guzman R, Ma Y, Sullivan-Halley J, Accepted October 10, 2006

4973