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Coexpression of Transcripts Encoding EPHB Receptor Protein Tyrosine Kinases and Their Ephrin-B Ligands in Human Small Cell Lung Carcinoma¹

Division of Oncology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104 [X. X. T., G. M. B., N. I.], and Cancer Research Laboratories and Departments of Oncology and Pathology, Queen’s University, Kingston, Ontario, K7L 3N6 Canada [B. G. C.]

	ABSTRACT

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The EPH family is the largest subfamily of receptor protein tyrosine kinases, consisting of the EPHA and EPHB subgroups. Ephrin-B1, ephrin-B2, and ephrin-B3 are ligands of the EPHB subgroup and are encoded by the EFNB1, EFNB2, and EFNB3 genes, respectively. We have shown previously that EPHB2 transcripts are expressed in six small cell lung carcinoma (SCLC) cell lines. In this study, we examined the expression of EPHB1, EPHB2, EPHB3, EPHB4, and EPHB6 in 4 SCLC tumor specimens and 14 cell lines including 3 cell lines derived from these tumor specimens. To investigate whether potential autocrine loops of EPHB receptors and ephrin-B ligands exist in SCLC, the expression of EFNB1, EFNB2, and EFNB3 was also examined. Our data show that transcripts encoding multiple members of the EPHB subgroup and the ephrin-B subgroup are coexpressed in SCLC cell lines and tumors. These results suggest that the EPHB subgroup receptor kinases may modulate the biological behavior of SCLC through autocrine and/or juxtacrine activation by ephrin-B ligands that are expressed in the same or neighboring cells.

	INTRODUCTION

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EPH family receptor kinases and their ligands (ephrins) are involved in fundamental developmental processes in the nervous system, including axon guidance (1) , axon fasciculation (2) , neural crest cell migration (3) , acquisition of brain subregional identities (4) , and neuronal cell survival (5) . Evidence also suggests that some members of the EPH family and their ligands are involved in vascularization as well as oncogenesis (6, 7, 8, 9) . The EPH family is the largest subfamily of protein tyrosine kinases. To date, 14 members of the EPH gene family have been identified in various species (10) . Similarly, ephrin ligands constitute a large family, which includes eight members.

Based on the sequence relationships and structures, ephrin ligands are divided into two subgroups: (a) ephrin-A; and (b) ephrin-B (11) . The ephrin-A subgroup, which includes five members, is anchored to the cell membrane by the glycosylphosphatidylinositol link, whereas three members of the ephrin-B subgroup are transmembrane proteins. EFNA and EFNB genes encode for the ephrin-A and ephrin-B ligands, respectively. EPH family receptors can also be divided into two subgroups based on the relatedness of their extracellular domain sequences and on their ability to bind to the two subgroups of ephrins. The EPHA subgroup, which includes eight receptors, interacts preferentially with ephrin-A ligands, whereas the EPHB subgroup, which includes six receptors, interacts preferentially with ephrin-B ligands (11) .

It is known that growth/differentiation factor receptors and their cognate ligands can modulate the biological behavior of various types of tumors through autocrine and/or paracrine activation mechanisms. In SCLC,³ it was originally shown that bombesin-like peptides were involved in the growth stimulation of SCLC cells in culture and xenografts in nude mice through an autocrine activation of their receptors (12) . More recently, Krystal et al. (13) have shown that there is an autocrine stimulation of small cell lung cancer growth mediated through coexpression of c-kit and its ligand, the stem cell factor (13) . We previously reported that EPHB2 transcripts, which encode a member of the EPHB subgroup, are highly expressed in several SCLC cell lines (14) . In this study, we examined the expression of transcripts encoding five members of the EPHB subgroup as well as their ligands, ephrin-Bs, in SCLC tumor specimens and additional cell lines. We have found that transcripts encoding multiple members of the EPHB and ephrin-B subgroups are expressed together in SCLC tumors and cell lines, suggesting that EPHB receptors and their ligands may modulate the biological behavior of SCLC through an autocrine and/or juxtacrine activation mechanisms.

	MATERIALS AND METHODS

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SCLC Cell Lines.
NCIH and NCIN series of SCLC cell lines were obtained from Dr. John D. Minna (University of Texas, Dallas, TX) and from the American Type Culture Collection (Manassas, VA). These cell lines were maintained in RPMI 1640 supplemented with 5% fetal bovine serum.

SCLC Tumor Specimens.
All SCLC tumor specimens were effusion samples obtained from patients treated at the Kingston Regional Cancer Center as part of a study approved by the Research Ethics Board of Queen’s University. The samples were prepared as described previously (15) , and they all consisted of more than 90% tumor cells.

PT-S65 was a pleural effusion obtained from a patient with extensive stage SCLC with liver metastases. At the time that the pleural fluid was obtained, the patient had been treated with cyclophosphomide, Adriamycin, and vincristine, alternating with etoposide and cisplatin. After an initial partial response to this therapy, the tumor recurred, and the patient developed a large pleural effusion. A cell line was established from the pleural fluid and named WL-E.

PT-S169 was obtained from a pleural effusion of a patient who had presented with extensive stage SCLC with liver metastases. At the time that the pleural fluid was obtained, the patient had been treated with oral etoposide and had a minimal response, but the tumor then started to progress. The patient died shortly after the pleural fluid was obtained. A cell line was established from the pleural fluid and named LV-E.

PT-S228 was a pericardial effusion sample from a patient with extensive stage SCLC with liver metastases. The patient had initially been treated with cyclophosphamide, Adriamycin, and vincristine, alternating with etoposide and cisplatin. The patient had a good partial response to this treatment. After completion of treatment, the tumor recurred with a pleural and a pericardial effusion. The pericardial effusion was drained, and the PT-S228 tumor sample was isolated from this effusion. A cell line was established from the pleural fluid and named GL-E.

PT-S331 was a pleural fluid sample from a patient with extensive stage SCLC who had not received chemotherapy.

RNA Extraction and Northern Blot Analysis.
Total cellular RNA preparations and Northern blotting procedures were described in detail elsewhere (16) . Briefly, total cellular RNA was prepared by the method described by Auffray and Rougeon (17) or by the method of Chomczynski and Sacchi (18) . The expression of EPHB1, EPHB2, EFNB1, EFNB2, and EFNB3 was examined by Northern hybridization using DNA fragments of the corresponding cDNAs as probes (14 , 19) . The blots were washed in 0.1x SSC containing 1% SDS at 63°C and exposed to X-ray film overnight at -70°C.

RT.
Total RNA (1–2 µg) extracted from SCLC tumors or cell lines was mixed with 35 ng of random hexamers and 250 ng of oligodeoxythymidylic acid (15 mer) in a volume of 10 µl. The resultant mixture was heated at 70°C for 10 min and chilled on ice. Each RT reaction was carried out in a total volume of 30 µl containing the RNA-primer mixture, 10 mM dithiothreitol, 500 µM deoxynucleotide triphosphates, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1 unit of PRIME RNase inhibitor (3 PRIME ->5 PRIME, Inc.), and 200 units of Superscript II reverse transcriptase (Life Technologies, Inc.). The incubation was first carried out at 25°C for 10 min. The reaction temperature was then increased to 55°C at a rate of 1°C/20 s. The RT reaction was allowed to take place at 55°C for an additional 60 min. RNaseH (2 units; Life Technologies, Inc.) was then added to each RT reaction, followed by an incubation at 37°C for 20 min.

Semiquantitative RT-PCR Assay.
One-fifteenth of the RT reaction was subjected to PCR amplification. PCR primers were biotinylated at their 5' ends. GAPD primers were included in the PCR reaction as an internal control. PCR amplifications were performed in volumes of 20 µl and contained 0.4 µM primer, 200 µM deoxynucleotide triphosphates, one-fifteenth of the RT reaction, 50 mM KCl, 2 mM MgCl₂, 10 mM Tris-HCl (pH 8.3), and 1 unit of AmpliTaq Gold (PE Applied Biosystems). PCR conditions were as follows: (a) 95°C for 12 min; (b) 18 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 5 min; and (c) 72°C for 10 min. Under these conditions, PCR was at the exponential phase of amplification. Nucleotide sequences for the PCR primers are as follows: (a) EPHB1, 5'-GAGATGGACAGCTCCAGAGG-3' (sense primer) and 5'-CCAGCATGAGCTGGTGTAGA-3' (antisense primer); (b) EPHB2, 5'-AAAATTGAGCAGGTGATCGG-3' (sense primer) and 5'-TCACAGGTGTGCTCTTGGTC-3' (antisense primer); (c) EPHB3, 5'-AGCAACCTGGTCTGCAAAGT-3' (sense primer) and 5'-TCCATAGCTCATGACCTCCC-3' (antisense primer); (d) EPHB4, 5'-GTCTGACTTTGGCCTTTCCC-3' (sense primer) and 5'-TGACATCACCTCCCACATCA-3' (antisense primer); (e) EPHB6, 5'-TGCTGGTGAATAGCCACTTG-3' (sense primer) and 5'-CGGAACTCCTGCTCTATTGC-3' (antisense primer); (f) EFNB1, 5'-GGAGGCAGACAACACTGTCA-3' (sense primer) and 5'-GAACAATGCCACCTTGGAGT-3' (antisense primer); (g) EFNB2, 5'-GCAAGTT-CTGCTGGATCAAC-3' (sense primer) and 5'-AGGATG-TTGTTCCCCGAATG-3' (antisense primer); (h) EFNB3, 5'-CTGAAATGCCCATGGAAAGA-3' (sense primer) and 5'-ACGCCCAGCAAGAGCAGCGC-3' (antisense primer); (i) IGF1R, 5'-ACGCCAATAAGTTCGTCCAC-3' (sense primer) and 5'-TCCATCCTTGAGGGACTCAG-3' (antisense primer); (j) RET, 5'-ACCTCATCTCATTTGCCTGG-3' (sense primer) and 5'-CCTGGCTCCTCTTCACGTAG-3' (antisense primer); and (k) GAPD, 5'-GAAGGTGAAGGTCGGAGTCA-3' (sense primer) and 5'-TTGAGGTCAATGAAGGGGTC- 3' (antisense primer).

Chemiluminescent Detection of Biotinylated PCR Products.
PCR products (10 µl of a total of 20 µl of PCR products) were subjected to 6% PAGE. DNA bands were electrotransferred onto nylon membrane (Hybond N⁺; Amersham) and immobilized to the membrane by baking the filter for 30 min at 80°C, followed by 1 min of UV irradiation. The biotinylated PCR products were then detected using the Southern Light chemiluminescent detection procedure (Tropix, Inc.). Quantification of mRNA expression was performed by densitometric analysis on X-ray films. Relative levels of the expression of a given transcript were then determined by taking the ratio between the densitometric unit of the transcript and that of the internal control, GAPD.

	RESULTS

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Expression of Transcripts Encoding EPHB Receptor Tyrosine Kinases in SCLC Cell Lines.
We previously reported that EPHB2 transcripts were expressed at high levels in six SCLC cell lines (14) . This observation prompted us to examine the expression of transcripts encoding five members of the EPHB receptor kinase subgroup (EPHB1, EPHB2, EPHB3, EPHB4, and EPHB6) in additional SCLC cell lines and tumor specimens. The expression of EPHB5 transcripts was not examined in this study because the human EPHB5 gene has not been identified.

We first examined the expression of EPHB2 transcripts in 11 SCLC cell lines by Northern blot analysis (Fig. 1A) and by semiquantitative RT-PCR analysis (Fig. 1B) . Because transcripts encoding EPH family receptors as well as ephrin ligands are known to be highly expressed in the developing nervous system (14 , 16 , 19) , human fetal brain RNA was included in the RT-PCR analysis as a positive control. As shown in Fig. 1 , both methods showed compatible results. Four cell lines (NCIH69, NCIH82, NCIH345, and NCIH378) expressed high levels of EPHB2 (i.e., a level of expression similar to that found in the fetal brain). Five cell lines (NCIH187, NCIH209, NCIN417, NCIH510, and NCIH526) expressed EPHB2 at moderate to low levels. Two cell lines (NCIH889 and NCIH1688) expressed no detectable levels of EPHB2 transcripts.

View larger version (63K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Expression of EPHB2 in SCLC cell lines. The expression of EPHB2 transcripts was examined in 11 SCLC cell lines by (A) Northern blot analysis and by (B) semiquantitative RT-PCR analysis. EPHB2 is expressed into three transcripts of 4, 5, and 11 kb in size (arrows) that are generated by alternative splicing and by alternative use of polyadenylation signals (14 , 26) . Ethidium bromide staining of 18S rRNA shows the loading and integrity of RNA used in Northern analysis. The same RNA samples were used for the RT-PCR analysis, in which GAPD transcripts were used as an internal control.

We next examined the expression of EPHB1, EPHB3, EPHB4, and EPHB6 transcripts in the same set of 11 SCLC cell lines. As shown in Fig. 2 , the expression of EPHB1 transcripts was found in 8 of 11 SCLC cell lines at low levels. EPHB3 transcripts were expressed in nine cell lines at moderate to low levels, with the exception of NCIH526, which expressed EPHB3 at an even higher level than that in the fetal brain. EPHB4 transcripts were expressed in three cell lines at levels similar to that in the fetal brain, and seven cell lines expressed EPHB4 transcripts at very low levels. EPHB6 transcripts were expressed in NCIH345 at a high level, and three cell lines expressed EPHB6 transcripts at very low levels. Thus, these data showed that 10 of 11 SCLC cell lines examined expressed one or more EPHB subgroup transcripts at levels similar to that detected in the fetal brain. An exception to this was the NCIH889 cell line, which happened to express high levels of RET (see Fig. 5 ).

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Expression of EPHB1, EPHB3, EPHB4, and EPHB6 in SCLC cell lines. The expression of EPHB1, EPHB3, EPHB4, EPHB6 transcripts in SCLC cell lines was examined by RT-PCR analysis as described in the Fig. 1B legend.

View larger version (59K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Expression of IGF1R and RET in SCLC cell lines and tumors. The expression of IGF1R transcripts was examined as a control for a receptor protein tyrosine kinase gene expressed ubiquitously in various tumor cell lines. RET expression was examined as a control for a receptor tyrosine kinase gene expressed preferentially in the nervous system. RT-PCR analysis was carried out as described in the Fig. 1B legend.

Fig. 3 shows the expression pattern of EPHB transcripts in four SCLC tumor specimens and three corresponding cell lines. EPHB1 expression in these samples was either very low or absent, and none of the tumor specimens expressed EPHB1 transcripts. EPHB2 expression was detected in all of the SCLC cell lines and tumors examined at levels ranging from high (a similar level found in the fetal brain) to moderate. Three of four tumors expressed EPHB3 at moderate to low levels. Tumor PT-S169 did not express EPHB3, but its derivative cell line LV-E expressed detectable levels of EPHB3. All four tumors expressed EPHB4 at moderate levels, and it appeared that tumor specimens expressed higher levels of EPHB4 transcripts than the corresponding cell lines. It was noticed that although tumor PT-S65 expressed EPHB4, its derivative cell line WL-E showed no EPHB4 expression. Finally, EPHB6 expression was found in three of the four tumor specimens, but none of the corresponding cell lines expressed EPHB6 transcripts. Taken together, these data show that multiple EPHB subgroup transcripts are expressed in SCLC tumors.

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Expression of EPHB1, EPHB2, EPHB3, EPHB4, and EPHB6 in SCLC tumors and their corresponding cell lines. The expression of transcripts encoding the EPHB subgroup receptor kinases was examined in four SCLC tumor specimens and three cell lines derived from the tumors. RT-PCR analysis was carried out as described in the Fig. 1B legend.

The expression of EFNB transcripts was first examined in the same set of 11 SCLC cell lines shown in Figs. 1 and 2 . As shown in Fig. 4A , EFNB1 expression was detected in all 11 cell lines examined: (a) 6 cell lines expressed EFNB1 transcripts at moderate levels; and (b) others expressed low levels of EFNB1. Similarly, EFNB2 transcripts were detected in all 11 cell lines, ranging from high to low levels: (a) 2 cell lines expressed EFNB2 transcripts at high levels; (b) 3 cell lines expressed EFNB2 transcripts at moderate levels; and (c) 6 cell lines expressed EFNB2 at low levels. Finally, EFNB3 transcripts were detected in 10 of the 11 cell lines examined: (a) 5 cell lines expressed EFNB3 transcripts at moderate levels; and (b) the others expressed low levels of EFNB3. These data were consistent with those obtained by Northern blot analysis (data not shown).

View larger version (87K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Expression of EFNB1, EFNB2, and EFNB3 in SCLC cell lines and tumors. The expression of EFNB1, EFNB2, and EFNB3 transcripts was examined in the (A) SCLC cell lines and (B) tumor/cell line pairs shown in Figs. 1 2 3 . RT-PCR analysis was carried out as described in the Fig. 1B legend.

Expression of IGF1R and RET Transcripts in SCLC Cell Lines and Tumors.
In addition to EPHB and EFNB transcripts, we have examined the expression of IGF1R and RET transcripts in SCLC cell lines and tumor specimens as controls. IGF1R encodes a protein tyrosine kinase receptor for IGF-1 and IGF-2 and is ubiquitously expressed in various tumor cell lines. As shown in Fig. 5 , this was also the case for SCLC. IGF1R expression was found at relatively high levels across the tumors and cell lines examined, with the exception of one cell line (NCIH510) that expressed low levels of IGF1R transcripts.

RET was included in this study as a control for a receptor protein tyrosine kinase with preferential expression in the nervous system. As shown in Fig. 5 , RET expression was found in 5 of 14 SCLC cell lines. This observation is similar to the recent finding (21) in which RET expression was detected in 12 of 21 SCLC cell lines. In contrast, no detectable RET expression was found in SCLC tumor specimens. Because the number of SCLC tumor specimens examined is limited, it remains to be seen whether RET expression in SCLC is a cell culture-related phenomenon or whether it has certain contributions to the pathogenesis of SCLC in vivo.

	DISCUSSION

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Autocrine growth stimulation of SCLC has been recognized for some time. It was first reported that bombesin-like peptides such as gastrin-releasing peptide were involved in growth stimulation of SCLC cells in culture and xenografts in nude mice through an autocrine activation of their receptors (12) . Similarly, an autocrine loop mediated by insulin-like growth factor 1 and its receptor has been found in SCLC (20) . The frequent coexpression of the stem cell factor and its receptor, the gene product of the c-kit proto-oncogene, was then reported in SCLC cell lines and tumors (22) . Subsequently, an autocrine stimulation of SCLC growth mediated by stem cell factor and c-kit was demonstrated experimentally (13) . Furthermore, selective inhibition of protein tyrosine kinase activity in SCLC cells by genistein and a tyrphostin derivative was shown to stimulate apoptosis of the cells (23) . Taken together, these studies suggest that auto-activation of tyrosine kinases plays an important role in the growth and survival of SCLC cells.

In this study, we report that transcripts encoding EPHB receptor kinases and their ephrin-B ligands are expressed together in 13 of the 14 SCLC cell lines and all 4 of the tumors examined. These data suggest that there are multiple autocrine loops mediated by EPHB receptors and ephrin-B ligands in SCLC. These data also point to a possibility that additional autocrine loops mediated by EPHA receptor kinases and ephrin-A ligands may exist in SCLC. In fact, our preliminary data showed that EPHA2 transcripts were expressed in several SCLC cell lines, including NCIH1688, which expressed only low levels of EPHB3, EFNB1, and EFNB2. Additional analysis of the expression of eight EPHA receptor kinases and five ephrin-A ligands will be required to further address this question.

Accumulating evidence suggests that overexpression or coexpression of EPH family receptor tyrosine kinases and their ligands could promote tumor progression. For example, EPHA2 was found to be expressed at high levels in metastatic melanoma cells as compared to normal melanocytes (24) . Similarly, EFNB2 transcripts encoding the ephrin-B2 ligand were highly expressed in primary and metastatic melanomas compared to benign melanocytic nevi (25) . Furthermore, the expression of ephrin-A1 and the up-regulation of its receptor, EPHA2, were found during the course of melanoma progression (24) . In our study, all four SCLC tumor specimens, which coexpressed EPHB and EFNB transcripts, were collected from the patients with advanced-stage disease. Taken together, these observations suggest that EPH family receptor kinases and ephrin ligands may be involved in the progression of SCLC. Clearly, additional studies will be needed to address this possibility.

Finally, along with this study, we have also examined the expression of transcripts encoding EPHB receptors and ephrin-B ligands in neuroblastoma, a common childhood cancer of neural crest origin. We have found that EPHB and EFNB transcripts were also coexpressed in neuroblastoma cell lines and tumors, and that the expression of these transcripts may serve as a prognostic marker of neuroblastomas.⁴ Thus, it appears that the coexpression of EPH family receptors and their ephrin ligands is a common feature of neural crest-derived or neuroendocrine tumors, including melanoma, SCLC, and neuroblastoma. The expression of EPHB and EFNB may thus serve as a useful phenotypic and prognostic marker and as a therapeutic target for these tumors.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. Audrey E. Evans for support throughout this work.

	FOOTNOTES

 
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 F32 CA75748 (to X. X. T.), NS34514 (to G. M. B.), and CA70958 (to N. I.) and by the Children’s Cancer Research Foundation.

² To whom requests for reprints should be addressed, at Division of Oncology, The Children’s Hospital of Philadelphia, Abramson Research Center Room 902, 324 South 34th Street, Philadelphia, PA 19104-4318. Phone: (215) 590-5243; Fax: (215) 590-3770; E-mail: ikegaki{at}kermit.oncol.chop.edu

³ The abbreviations used are: SCLC, small cell lung carcinoma; RT, reverse transcription.

⁴ X. X. Tang, A. E. Evans, H. Zhao, A. Cnaan, W. London, S. L. Cohn, G. M. Brodeur, and N. Ikegaki. High level expression of EPHB6, EFNB2, and EFNB3 is associated with low tumor stage and high TrkA expression in human neuroblastoma, submitted for publication.

Received 8/18/98; revised 10/23/98; accepted 11/ 6/98.

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