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Amplification and rearrangement of L-myc in human small-cell lung cancer
Mutation Research, 276 (1992) 307-315
307
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1110/92/$05.00
MUT 00324
Amplification and rearrangement of L-myc in human small-cell lung cancer T o m i P. Miikelii, K a l l e S a k s e l a * a n d K a r i A l i t a l o Laboratory of Cancer Biology, Departments of Virology and Pathology, University of Helsinki, Helsinki (Finland) (Accepted 15 November 1991)
Keywords: Small-cell lung cancer; L-myc proto-oncogene; Rearrangement of L-myc
Summary DNA amplification of cellular proto-oncogenes is a well-established and common mechanism of oncogene activation in several types of human tumors, including the rapidly fatal small-cell lung cancer (SCLC). Approximately one fourth of primary SCLC tumors contain amplified copies of one of the three myc proto-oncogenes. Occasionally DNA amplification of the myc genes is associated with DNA rearrangements. Specifically, a novel locus named rlf is often involved in intrachromosomal L-myc rearrangements in SCLC. The structurally similar rearrangements are probably due to a highly repetitive region upstream of the L-rnyc gene, and result in the formation of a chimeric rlf-L-myc fusion protein. The consistent finding of the rlf-L-myc rearrangement in SCLC suggests that it may provide a selective advantage to the cells harboring it.
Amplification of myc genes in human tumors Cellular proto-oncogenes can be activated into oncogenes by several molecular mechanisms resulting in either qualitative or quantitative changes in the corresponding oncoprotein products. The discovery of amplification of drug-resis-
* Present address: Rockefeller University, 1230 York Avenue, New York, NY 10021-6399 (USA). Correspondence: Dr. K. Alitalo, Laboratory of Cancer Biology, Departments of Virology and Pathology, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki (Finland). Tel.: 358-0-434 6434; Fax: 358-0-434 6448.
tance genes in cell culture (reviewed in Schimke, 1984; Stark and Wahl, 1984) raised the possibility that an increase in the copy number of a protooncogene could provide one means of increasing the amount of the proto-oncogene product. Amplified proto-oncogenes were then identified from cell lines with karyotypic changes (double minutes and homologously staining regions; Alitalo et al., 1983; Little et al., 1983; Schwab et al., 1983) suggesting the presence of amplified genes, but also from lines that did not show cytogenetic abnormalities associated with gene amplification (e.g., HL-60: Dalla-Favera et al., 1982; COLO205: Alitalo et al., 1984). The consistent finding of amplified oncogenes in a variety of human tumors has thereafter provided abundant evidence
308 indicating that the amplified proto-oncogenes are implicated in tumorigenesis (reviewed in Alitalo and Schwab, 1986). When first discovered, the amplified oncogenes were identified as cellular counterparts of oncogenes isolated from transducing retroviruses (e.g., myc, ras, myb). Later examples were identified as amplified genes homologous to known proto-oncogenes. In this way the N-myc and Lmyc genes became known as c-myc-related amplified sequences from neuroblastoma (Schwab et al., 1983) and small-cell lung cancer (SCLC; Nau et al., 1985), respectively. Further studies have indicated that both of these genes are commonly found amplified and overexpressed also in vivo in the corresponding tumors. Moreover, amplification of at least N-myc correlates with a poor prognosis of the patient (Brodeur et al., 1984), suggesting that overexpression of N-myc augments the malignancy of neuroblastomas during tumor progression. c-myc has been found oncogenically activated in several fashions including retroviral transduction (v-myc; reviewed in Bishop, 1985), deregulation due to chromosomal translocations in lymphomas of the B-cell lineage and analogous translocations involving the T-cell lineage (reviewed in Cory, 1986) as well as more or less sporadic gene amplifications. Reported oncogenic insults also include retroviral and hepadnaviral damage of N-myc in mouse T-cell lymphomas (van Lohuizen et al., 1989) and woodchuck hepatomas (Fourel et al., 1990), respectively. However, the human N-myc and L-myc genes have only been found activated through gene amplification (reviewed in Alitalo et al., 1987; Saksela, 1990). Studies on the structure of the amplified DNA including the myc genes indicate that DNA rearrangements commonly accompany amplifications (Amler and Schwab, 1989; Kinzler et al., 1986; Shiloh et al., 1985; Trent et al., 1986; Zehnbauer et al., 1988). Most of the rearrangements involve only a portion of the amplified DNA, which is therefore heterogenous in structure. These rearrangements have been suggested to result from heterogenous integration of amplified DNA sequences into chromosomal DNA. However, in a few cases rearrangements appear homogenous
(Hiyama et al., 1991; Shtivelman and Bishop, 1989; Takahashi et al., 1989), suggesting that they have occurred prior to gene amplification. A number of lung cancer cell lines have been found to contain amplified DNA from one of the myc genes (reviewed in Alitalo et al., 1987; Saksela, 1990). Because initial reports suggested that the frequency of myc gene amplification is higher in cell lines than in tumors (Gazdar et al., 1985; Little et al., 1983; Wong et al., 1986), several studies have approached the important question of myc gene amplification in primary lung tumors (reviewed in M~ikel~i et al., 1991d). A compilation of the data from these reports indicates that amplifications of N-myc and L-myc are found in primary SCLC tumors at prevalences of 7% and 15%, respectively (Brennan et al., 1991; Gazzeri et al., 1991; Johnson et al., 1988; Nau et al., 1985; Noguchi et al., 1990; Shiraisi et al., 1989; Takahashi et al., 1989; Yokota et al., 1988), while the frequency of c-myc amplifications in these tumors is low, and comparable to c-myc activations in other types of lung cancer (Gazzeri et al., 1991; Shiraisi et al., 1989; Taya et al., 1984). This may reflect the restricted expression pattern of N-myc and L-myc in contrast to the more generalized expression of c-myc (Hirvonen et al., 1990; Zimmerman et aI., 1986). A fusion protein by L.myc and a novel gene in SCLC
We have been interested in small-cell lung cancer cell lines with amplified myc genes, because they provide a useful system to analyze the myc proteins (reviewed in Alitalo et al., 1991), and may also prove fruitful in in vivo studies of myc binding proteins, such as the recently identified Max protein (Blackwood and Eisenman, 1991). During our studies of such cells (M~ikel~i et al., 1989; Saksela et al., 1989), we observed an abnormally large L-myc protein in the GLC28 SCLC cell line (a kind gift from Dr. Charles Buys, University of Groningen, The Netherlands). Comparison of this protein to a previously identified abnormal L-myc protein (Gerard Evan, personal communication) identified from the CORL47 SCLC cell line (Baillie-Johnson et al., 1985) revealed these proteins to be virtually iden-
309 r/fexon I
L-myc
~-:
i
i
,A ,A
3.7 kb 3.2 kb
...........
446 aa
rlf-L-myc chimeric mRNAs
rlf-L-myc Transactivation, Rb binding
DNA-binding dimerization
chimeric protein
Fig. 1. The rlf-L-myc fusion protein is formed by joining rlf exon I sequences to L-myc. Schematic structures of the rlf-L-
myc gene fusion (top), the resulting mRNAs (middle) and the fusion protein (bottom). The 3.7- and 3.2-kb mRNAs are formed by alternative splicing of L-myc exon II as shown. Exon I is excluded from the mRNAs because it does not contain a splice acceptor. The rlf sequences are shown in gray. The intervening three amino acids from the normally untranslated 5' end of L-myc exon II are marked black, and the L-rnyc polypeptide part is white. The regions homologous to c-myc domains for DNA-binding/dimerization or transactivation and retinoblastoma binding are indicated with stippled bars.
the switch from the L-myc transcriptional control elements or those of rlf, resulting in deregulation of the normally strictly controlled expression of L-rnyc. Deregulation by a similar p r o m o t e r switch has been suggested to be the activating mechanism of c-myc in a woodchuck liver tumor, where the hcr locus fuses upstream of c-myc (Etiemble et al., 1989). Alternatively, the alteration of the amino terminus of L-myc could affect the activity or substrate specificity of the L-myc protein. Concerning this it is interesting to note that the N-terminal region of the c-myc (Kato et al., 1990) and L-myc (K. Saksela, unpublished) proteins are involved in transcription activation in a heterologous system. In addition, N-terminal domains of both c-myc and N-rnyc have recently been shown to associate with the retinoblastoma protein (Rustgi et al., 1991). Therefore it will be interesting to explore the possibility that the abnormal N-terminal sequences in the rlf-L-myc fusion protein interfere with this association. Structure of the
tical (M~ikel~i et al., 1991a). Characterization of L-myc c D N A clones from the GLC28 cell line indicated that the abnormal protein resulted from a gene fusion between L-myc and a previously uncharacterized gene, which we have n a m e d rlf (for rearranged L-myc fusion: M~ikel~i et al., 1991a). As shown schematically in Fig. 1, the rlf-L-myc fusion gene directs the synthesis of a fusion m R N A with the first exon of rlf spliced either to the second (3.7-kb m R N A ) or to the third exon of L-myc (3.2-kb mRNA). The first exon of L-myc is not included in either m R N A , because it does not contain a splice acceptor. The 3.7-kb m R N A produces the observed N-terminally extended L-myc protein of 446 amino acids instead of the normal L-myc protein of 364 amino acids. In a collaborative study with Dr. Takahashi's group we afterwards identified three additional SCLC cell lines that produce similar chimeric transcripts (Sekido et al., 1991). The finding of rlf-L-myc rearrangements in five SCLC cell lines and importantly also in a primary SCLC tumor (M~ikel~i et al., 1991c) suggests the presence of selective pressure for this rearrangement during tumor growth. This could be attributed to
rlf-L-myc
gene fusions
In all cases identified to date the rlf-L-myc rearrangement has been associated with gene amplification. Due to the relatively long distance between exon I of rearranged rlf and L-myc, initial studies on the structure of the fusion genes were conducted using pulsed-field gel electrophoresis (PFGE; M~ikel~i et al., 1991b). Closer analysis of the breakpoint regions on both the rlf and L-myc sides was possible only after mapping of these regions in normal DNA. For this purpose, normal genomic clones were obtained from both regions. Restriction analysis of these breakpoint regions enabled their characterization in more detail, and indicated that the breakpoints differ considerably on the L-myc and rlf sides (M~ikel~i et al., 1991b,c). The structures of all three characterized rlf-L-myc fusion genes are shown in Fig. 2. The breakpoint in GLC28 is in a 100-bp region 3.5 kb downstream from rlf exon I and 16 kb upstream of L-myc. Although the breakpoint is also close to the first exon of rlf in CORL47, it is more than 40 kb upstream of the L-myc gene. The opposite situation can be seen in the LuC194 tumor DNA; there the breakpoint
310
rlf GLC28
I
CORLA7
I
LuC194
I
L-myc ::
~_~
"
::
:: ~
1
in the upstream region of L-myc, and they clearly indicate that the mechanism of rearrangement is not sequence-specific. However, in all cases the rearrangements occur upstream of the L-myc gene, and in the first intron of the rlf gene, and they would thus be expected to encode similar fusion proteins.
~.
=
0
kb
Fig. 2. Similar structure but distinct rearrangements characterize the rlf-L-myc gene fusions. The three different characterized rlf-L-myc fusion genes are shown. The introns between rlf exon I (white box) and L-myc (three exons shown as black boxes) are 21, 2 4 a n d > 31 k b long in G L C 2 8 , L u C 1 9 4 , and CORL47, respectively. The thin line represents sequences from the rlf locus, and the thick line sequences from L-myc. The white thick line represents the region where the rearrangements have occurred. Slashed lines in CORL47 indicate that the exact length of the first intron of rlf-L-myc is unknown.
The rlf-L-myc fusions are caused by intrachromosomal rearrangements A previous cytogenetic study indicated that one of the cell lines with the rearrangement (CORL47) did not contain abnormalities of chromosome 1 (Waters et al., 1988). Because L-myc is located at lp32 (Nau et al., 1985), this suggested that at least in this case the rearrangement is not due to a chromosomal translocation. Accordingly, we could map the rlf locus to chromosome 1 using human-rodent somatic cell hybrids. Therefore we also decided to explore the possible physical linkage of rlf and L-myc. Using PFGE analysis we found that the rlf and L-myc genes are
lies furthest downstrean from rlf exon I, but relatively close to L-myc. These results delineate the rearrangement region in rlf intron I to span at least 10 kb, and correspondingly at least 35 kb
TABLE DNA
1
REARRANGEMENTS
Class
I
II
III
IV
T,
T/C
INVOLVING
rlf AND L-rnyc I N
CANCER
Tumor/
Rearrangement
cell line
rlf
C
CORL47
+
+
+
C
GLC28
+
+
+
T
LuC194
C
ACC-LC-49
C C
Amplification
L-myc
+
+
+
+
+
+
ACC-LC-48
+
+
+
SK-LC-17
+
+
+
C
CORL88
-
-
+
T
RO
-
-
+
T
AS
-
-
+
T
PA
-
-
+
C
ACC-LC-177
-
-
+
C
ACC-LC-178
-
-
+
C
ACC-LC-52
-
-
+
-
+
+
-
+
+
C
U1690
C
ACC-LC-49
*
*
T
LuC49
+
-
-
T
LuC26
-
+
-
primary tumor; C, tumor cell line. line has other rearrangements involving
* The ACC-LC-49 al. (1991).
LUNG
L-myc
besides the
rlf-L-myc
rearrangement as described in Sekido et
311 r/f ~t
~l
IIII
L-myc Nt (~t)
II
M
Nt
I
I
100 kb r/fexon I
breakpoints
Fig. 3. The rlf and L-myc genes are located within 480 kb of each other in germ-line DNA. A long-range restriction map encompassing the rlf and L-rnyc loci. In this scale the three exons of L-myc are shown as a single black box, whereas the putative exons of rlf are only shown schematically.Their exact number is not known, but the gene is known to span over 60 kb of DNA, and the length of the first intron of rlf is over 15 kb (Miikelii et al., 1991b). This is also the region where the breakpoints occur, as shown. M = MluI, N = NotI. Sites in parentheses are partially digested in PFGE analysis.
both present in a 780-kb MluI D N A fragment (Miikel~i et al., 1991b). A refined long-range restriction map surrounding these two loci shows that the first exon of rlf is approximately 480 kb upstream of L-myc (Fig. 3). This physical linkage was later confirmed by the finding of coamplification of unrearranged rlf and L-myc in several SCLCs (Miikel~i et al., 1991c; Sekido et al., 1991; Table 1).
Relationship of rearrangement and gene amplification All intrachromosomal rearrangements leading to the rlf-L-myc fusions have also been involved in D N A amplification. However, in all cases with the rearrangement, only a single amplified band is present with several different digestions, suggesting that the rearrangement has occurred before D N A amplification. In addition to the rlf-Lmyc rearrangements, the L-myc gene has been found involved in two other amplification-associated rearrangements. The ACC-LC-49 SCLC cell line contains complex rearrangements, which apparently include both the rlf-L-myc rearrangement and a rearrangement between the upstream region of L-myc and a novel locus EX (Sekido et al., 1991; Takahashi et al., 1989). The U1690 SCLC cell line (Bergh et al., 1985) also contains a rearrangement in the upstream region of L-myc which is fused to unknown sequences (unpublished data). However, both of these L-myc rearrangements, which do not involve rlf, still produce normal L-myc mRNAs (Miikel~i et al., 1991a; Sekido et al., 1991), and have only been found from a single cell line.
Homogenous rearranged amplified D N A has been described for the N-myc gene in a primary neuroblastoma (Hiyama et al., 1991), and for the c-myc gene in the COLO320 DM cell line (the pvt-myc fusion; Shtivelman and Bishop, 1989), although in both cases amplified copies of the unrearranged gene can also be found (Alitalo et al., 1983; Hiyama et al., 1991; Schwab et al., 1986). In all rlf-L-myc rearrangements, in the pvt-myc rearrangement, and in several neuroblastomas with amplifications and rearrangements (Shiloh et al., 1985) the rearranged D N A has been located in the vicinity of the corresponding myc gene, suggesting that specific local rearrangements occur as initial amplification steps. Such a model is inherent in the currently held models for gene amplification (Kinzler et al., 1986; Stark et al., 1989; Trask and Hamlin, 1989). Accordingly, intrachromosomal single-copy rearrangements involving c-myc described in a giant cell carcinoma of the lung (Iizuka et al., 1990) may represent an early step of this process as suggested by the finding of a similar rearrangement amplified in a giant cell carcinoma cell line (Yoshimoto et al., 1986).
A reeombinogenic DNA region upstream of L.myc Because the breakpoints in the different rlf-Lmyc fusions differ considerably on the rlf and the L-myc side, the rearrangement mechanism probably is not sequence-specific. Sequence analysis of one of the breakpoint regions on the L-myc side indicated that it contains an Alu repeat sequence. Further mapping and sequencing of the L-myc upstream region revealed a large D N A region
312
produce the chimeric rlf-L-myc mRNAs. Class II contains SCLCs with coamplification of rlf and L-myc with no rearrangements detected in conventional Southern analysis. They also appear to produce normal L-myc transcripts. Class III represents cases where the L-myc gene is rearranged with another locus. The ACC-LC-49 cell line appears to have two distinct rearrangements (see above), and is therefore included in both class I and class II. Class IV SCLCs contain rearrangements of either rlf or L-myc without DNA amplification. According to the material analyzed (14 cases) it would appear that the rlf gene is commonly involved in coamplification with L-myc. This might be expected due to the short distance between the two genes. Interestingly, rearrangements fusing intron I of rlf to the upstream region of L-myc and leading to the production of chimeric rlf-L-myc mRNAs are found in six out of 14 analyzed cases. The consistent finding of this rearrangement suggests that it may provide a selective growth advantage to the SCLC cells.
clustered with repeated sequences as shown in Fig. 4. In addition to associated KpnI (DiGiovanni et al., 1983) and Alu sequences (Deininger et al., 1981), at least one conserved member of the family of SINE-R endogenous retrovirusrelated retroposons (Ono et al., 1987) was identified (unpublished data). The region mapped so far contains the breakpoints of at least three L-myc amplified cell lines as shown in Fig. 4. The breakpoint in the GLC28 cell line involves the rlf-L-myc rearrangement. The characterized breakpoint in the ACC-LC-49 cell line does not involve rlf, but another novel locus (EX). However, the ACC-LC-49 cell line also contains a less well characterized rlf-L-myc rearrangement not shown in Fig. 4. The U1690 breakpoint also involves a new locus and not rlf (see above). All three breakpoint regions are located in the cluster of repeats which thus represents a recombinatorial hotspot of repetitive sequence, and the rearrangements may have occurred as homologous recombinations between repetitive sequences as described previously (Lehrman et al., 1987). Interestingly, a recombinogenic A + T-rich region containing Alu repeats has also been found to be frequently involved in early amplificationassociated rearrangements of the Chinese hamster adenylate deaminase gene (Hyrien et al., 1987). Table 1 contains a summary of the different configurations of the L-myc a n d / o r rlf genes in SCLC cell lines and tumors. Several classes can be distinguished according to the amplification and rearrangement status of L-myc and rlf. Class I SCLCs contain a rlf-L-myc rearrangement associated with D N A amplification. All analyzed cases (excluding the LUC194 tumor, from which material was not available) have also been found to
?? repeats I breakpoints
Acknowledgements We gratefully acknowledge our collaborators Gerard Evan, Juha Kere, Robert Winqvist, Takao Sekiya, Masahiko Shiraishi, Maria G. Borrello, Yoshitada Sekido, Takashi Takahashi, and Toshitada Takahashi. We express our gratitude to Fred Alt, Charles Buys, Pamela Rabbitts, Jonas Bergh, and Naohiko Ikegaki for cell lines and reagents, and to Kirsi Pylkkanen, Elina Roimaa, Tapio Tainola, Merja Virta, Raili Taavela, and Ritva Tolvanen for technical assistance. The work from our laboratory reviewed here was supported by the Finnish Cancer Organizations, Finnish Cultural Foundation, Emil Aaltonen Foundation, Ida
?? ?
S1NE-R r,~l I I I l l GLC28 U1690
I
L-myc
,.?
I [] ACC-LC-49
? I-'-I 1 kb
Fig. 4. A highly repetitive region of D N A u p s t r e a m of the L-myc gene. Sequence analysis of normal genomic clones immediately u p s t r e a m of the L-myc gene has revealed several clusters of highly repetitive D N A (shown schematically as white boxes) including one m e m b e r of the S I N E - R e n d o g e n o u s retrovirus-derived r e t r o t r a n s p o s o n ( O n o et al., 1987). All characterized rlf-L-myc breakpoint regions (shown as gray boxes) coincide with these repetitive regions. Balls m a r k EcoRl sites and black boxes L-myc exons.
313
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Sekido, Y., T. Takahashi, T.P. M~ikelii, Y. Obata, R. Ueda, T. Hida, K. Hibi, K. Shimokata, K. Alitalo and T. Takahashi (1991) Complex rearrangements and amplification of the L-myc locus involving rlf and jal, Submitted. Shiloh, Y., J. Shipley, G.M. Brodeur, G. Bruns, B. Korf, T. Donlon, R.R. Schreck, R. Seeger, K. Sakai and S.A. Latt (1985) Differential amplification, assembly, and relocation of multiple DNA sequences in human neuroblastomas and neuroblastoma cell lines, Proc. Natl. Acad. Sci. (U.S.A.), 82, 3761-3765. Shiraisi, M., M. Noguchi, Y. Shimosato and T. Sekiya (1989) Amplification of proto-oncogenes in surgical specimens of human lung carcinomas, Cancer Res., 49, 6474-6479. Shtivelman, E., and J.M. Bishop (1989) The PVT gene frequently amplifies with myc in tumor cells, Mol. Cell. Biol., 9, 1148-1154. Stark, G.R., and G.M. Wahl (1984) Gene amplification, Annu. Rev. Biochem., 53, 447-491. Stark, G.R., M. Debatisse, E. Giulotto and G.M. Wahl (1989) Recent progress in understanding mechanisms of mammalian DNA amplification, Cell, 57, 901-908. Takahashi, T., Y. Obata, Y. Sekido, T. Hida, R. Ueda, H. Watanabe, Y. Ariyoshi, T. Sugiura and T. Takahashi (1989) Expression and amplification of myc gene family in small cell lung cancer and its relation to biological characteristics, Cancer Res., 49, 2683-2688. Taya, Y., K. Hosogai, S. Hirohashi, Y. Shimosato, Tsuchi, T. Sekiya and S. Nishimura (1984) A novel combination of K-ras and myc amplification accompanied by point mutational activation of K-ras in a human lung cancer, EMBO J., 3, 2943-2946. Trask, B.J., and J.L. Hamlin (1989) Early dihydrofolate reductase gene amplification events in CHO cells usually occur on the same chromosome arm as the original locus, Genes Dev., 3, 1913-1925. Trent, J., P. Meltzer, M. Rosenblum, G. Harsh, K. Kinzler, R. Mashal, A. Feinberg and B. Vogelstein (1986) Evidence for rearrangement, amplification, and expression of c-myc in a human glioblastoma, Proc Natl. Acad. Sci. (U.S.A.), 83, 470-473. van Lohuizen, M., M. Breuer and A. Berns (1989) N-myc is frequently activated by proviral insertion in MuLV-induced T cell lymphomas, EMBO J., 8, 133-136. Waters, J.J., J.M. Ibson, P.R. Twentyman, N.M. Bleehen and P.H. Rabbitts (1988) Cytogenetic abnormalities in human small cell lung carcinoma: cell lines characterized for myc gene amplification, Cancer Genet. Cytogenet., 30, 213-223. Wong, A.J., J.M. Ruppert, J. Eggleston, S.R. Hamilton, S.B. Baylin and B. Vogelstein (1986) Gene amplification of c-myc and N-myc in small cell carcinoma of the lung, Science, 233, 461-464. Yokota, J., M. Wada, T. Yoshida, M. Noguchi, T. Terasaki, Y. Shimosato, T. Sugimura and M. Terada (1988) Heterogeneity of lung cancer cells with respect to the amplification and rearrangement of myc family oncogenes, Oncogene, 2, 607-611.
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Zimmerman, K.A., G.D. Yancopoulos, R.G. Collum, R.K. Smith, N.E. Kohl, K.A. Denis, M.M. Nau, O.N. Witte, D. Toran-Allerand, C.E. Gee, J.D. Minna and F.W. Alt (1986) Differential expression of the myc family genes during murine development, Nature (London), 319, 780-783.
307
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1110/92/$05.00
MUT 00324
Amplification and rearrangement of L-myc in human small-cell lung cancer T o m i P. Miikelii, K a l l e S a k s e l a * a n d K a r i A l i t a l o Laboratory of Cancer Biology, Departments of Virology and Pathology, University of Helsinki, Helsinki (Finland) (Accepted 15 November 1991)
Keywords: Small-cell lung cancer; L-myc proto-oncogene; Rearrangement of L-myc
Summary DNA amplification of cellular proto-oncogenes is a well-established and common mechanism of oncogene activation in several types of human tumors, including the rapidly fatal small-cell lung cancer (SCLC). Approximately one fourth of primary SCLC tumors contain amplified copies of one of the three myc proto-oncogenes. Occasionally DNA amplification of the myc genes is associated with DNA rearrangements. Specifically, a novel locus named rlf is often involved in intrachromosomal L-myc rearrangements in SCLC. The structurally similar rearrangements are probably due to a highly repetitive region upstream of the L-rnyc gene, and result in the formation of a chimeric rlf-L-myc fusion protein. The consistent finding of the rlf-L-myc rearrangement in SCLC suggests that it may provide a selective advantage to the cells harboring it.
Amplification of myc genes in human tumors Cellular proto-oncogenes can be activated into oncogenes by several molecular mechanisms resulting in either qualitative or quantitative changes in the corresponding oncoprotein products. The discovery of amplification of drug-resis-
* Present address: Rockefeller University, 1230 York Avenue, New York, NY 10021-6399 (USA). Correspondence: Dr. K. Alitalo, Laboratory of Cancer Biology, Departments of Virology and Pathology, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki (Finland). Tel.: 358-0-434 6434; Fax: 358-0-434 6448.
tance genes in cell culture (reviewed in Schimke, 1984; Stark and Wahl, 1984) raised the possibility that an increase in the copy number of a protooncogene could provide one means of increasing the amount of the proto-oncogene product. Amplified proto-oncogenes were then identified from cell lines with karyotypic changes (double minutes and homologously staining regions; Alitalo et al., 1983; Little et al., 1983; Schwab et al., 1983) suggesting the presence of amplified genes, but also from lines that did not show cytogenetic abnormalities associated with gene amplification (e.g., HL-60: Dalla-Favera et al., 1982; COLO205: Alitalo et al., 1984). The consistent finding of amplified oncogenes in a variety of human tumors has thereafter provided abundant evidence
308 indicating that the amplified proto-oncogenes are implicated in tumorigenesis (reviewed in Alitalo and Schwab, 1986). When first discovered, the amplified oncogenes were identified as cellular counterparts of oncogenes isolated from transducing retroviruses (e.g., myc, ras, myb). Later examples were identified as amplified genes homologous to known proto-oncogenes. In this way the N-myc and Lmyc genes became known as c-myc-related amplified sequences from neuroblastoma (Schwab et al., 1983) and small-cell lung cancer (SCLC; Nau et al., 1985), respectively. Further studies have indicated that both of these genes are commonly found amplified and overexpressed also in vivo in the corresponding tumors. Moreover, amplification of at least N-myc correlates with a poor prognosis of the patient (Brodeur et al., 1984), suggesting that overexpression of N-myc augments the malignancy of neuroblastomas during tumor progression. c-myc has been found oncogenically activated in several fashions including retroviral transduction (v-myc; reviewed in Bishop, 1985), deregulation due to chromosomal translocations in lymphomas of the B-cell lineage and analogous translocations involving the T-cell lineage (reviewed in Cory, 1986) as well as more or less sporadic gene amplifications. Reported oncogenic insults also include retroviral and hepadnaviral damage of N-myc in mouse T-cell lymphomas (van Lohuizen et al., 1989) and woodchuck hepatomas (Fourel et al., 1990), respectively. However, the human N-myc and L-myc genes have only been found activated through gene amplification (reviewed in Alitalo et al., 1987; Saksela, 1990). Studies on the structure of the amplified DNA including the myc genes indicate that DNA rearrangements commonly accompany amplifications (Amler and Schwab, 1989; Kinzler et al., 1986; Shiloh et al., 1985; Trent et al., 1986; Zehnbauer et al., 1988). Most of the rearrangements involve only a portion of the amplified DNA, which is therefore heterogenous in structure. These rearrangements have been suggested to result from heterogenous integration of amplified DNA sequences into chromosomal DNA. However, in a few cases rearrangements appear homogenous
(Hiyama et al., 1991; Shtivelman and Bishop, 1989; Takahashi et al., 1989), suggesting that they have occurred prior to gene amplification. A number of lung cancer cell lines have been found to contain amplified DNA from one of the myc genes (reviewed in Alitalo et al., 1987; Saksela, 1990). Because initial reports suggested that the frequency of myc gene amplification is higher in cell lines than in tumors (Gazdar et al., 1985; Little et al., 1983; Wong et al., 1986), several studies have approached the important question of myc gene amplification in primary lung tumors (reviewed in M~ikel~i et al., 1991d). A compilation of the data from these reports indicates that amplifications of N-myc and L-myc are found in primary SCLC tumors at prevalences of 7% and 15%, respectively (Brennan et al., 1991; Gazzeri et al., 1991; Johnson et al., 1988; Nau et al., 1985; Noguchi et al., 1990; Shiraisi et al., 1989; Takahashi et al., 1989; Yokota et al., 1988), while the frequency of c-myc amplifications in these tumors is low, and comparable to c-myc activations in other types of lung cancer (Gazzeri et al., 1991; Shiraisi et al., 1989; Taya et al., 1984). This may reflect the restricted expression pattern of N-myc and L-myc in contrast to the more generalized expression of c-myc (Hirvonen et al., 1990; Zimmerman et aI., 1986). A fusion protein by L.myc and a novel gene in SCLC
We have been interested in small-cell lung cancer cell lines with amplified myc genes, because they provide a useful system to analyze the myc proteins (reviewed in Alitalo et al., 1991), and may also prove fruitful in in vivo studies of myc binding proteins, such as the recently identified Max protein (Blackwood and Eisenman, 1991). During our studies of such cells (M~ikel~i et al., 1989; Saksela et al., 1989), we observed an abnormally large L-myc protein in the GLC28 SCLC cell line (a kind gift from Dr. Charles Buys, University of Groningen, The Netherlands). Comparison of this protein to a previously identified abnormal L-myc protein (Gerard Evan, personal communication) identified from the CORL47 SCLC cell line (Baillie-Johnson et al., 1985) revealed these proteins to be virtually iden-
309 r/fexon I
L-myc
~-:
i
i
,A ,A
3.7 kb 3.2 kb
...........
446 aa
rlf-L-myc chimeric mRNAs
rlf-L-myc Transactivation, Rb binding
DNA-binding dimerization
chimeric protein
Fig. 1. The rlf-L-myc fusion protein is formed by joining rlf exon I sequences to L-myc. Schematic structures of the rlf-L-
myc gene fusion (top), the resulting mRNAs (middle) and the fusion protein (bottom). The 3.7- and 3.2-kb mRNAs are formed by alternative splicing of L-myc exon II as shown. Exon I is excluded from the mRNAs because it does not contain a splice acceptor. The rlf sequences are shown in gray. The intervening three amino acids from the normally untranslated 5' end of L-myc exon II are marked black, and the L-rnyc polypeptide part is white. The regions homologous to c-myc domains for DNA-binding/dimerization or transactivation and retinoblastoma binding are indicated with stippled bars.
the switch from the L-myc transcriptional control elements or those of rlf, resulting in deregulation of the normally strictly controlled expression of L-rnyc. Deregulation by a similar p r o m o t e r switch has been suggested to be the activating mechanism of c-myc in a woodchuck liver tumor, where the hcr locus fuses upstream of c-myc (Etiemble et al., 1989). Alternatively, the alteration of the amino terminus of L-myc could affect the activity or substrate specificity of the L-myc protein. Concerning this it is interesting to note that the N-terminal region of the c-myc (Kato et al., 1990) and L-myc (K. Saksela, unpublished) proteins are involved in transcription activation in a heterologous system. In addition, N-terminal domains of both c-myc and N-rnyc have recently been shown to associate with the retinoblastoma protein (Rustgi et al., 1991). Therefore it will be interesting to explore the possibility that the abnormal N-terminal sequences in the rlf-L-myc fusion protein interfere with this association. Structure of the
tical (M~ikel~i et al., 1991a). Characterization of L-myc c D N A clones from the GLC28 cell line indicated that the abnormal protein resulted from a gene fusion between L-myc and a previously uncharacterized gene, which we have n a m e d rlf (for rearranged L-myc fusion: M~ikel~i et al., 1991a). As shown schematically in Fig. 1, the rlf-L-myc fusion gene directs the synthesis of a fusion m R N A with the first exon of rlf spliced either to the second (3.7-kb m R N A ) or to the third exon of L-myc (3.2-kb mRNA). The first exon of L-myc is not included in either m R N A , because it does not contain a splice acceptor. The 3.7-kb m R N A produces the observed N-terminally extended L-myc protein of 446 amino acids instead of the normal L-myc protein of 364 amino acids. In a collaborative study with Dr. Takahashi's group we afterwards identified three additional SCLC cell lines that produce similar chimeric transcripts (Sekido et al., 1991). The finding of rlf-L-myc rearrangements in five SCLC cell lines and importantly also in a primary SCLC tumor (M~ikel~i et al., 1991c) suggests the presence of selective pressure for this rearrangement during tumor growth. This could be attributed to
rlf-L-myc
gene fusions
In all cases identified to date the rlf-L-myc rearrangement has been associated with gene amplification. Due to the relatively long distance between exon I of rearranged rlf and L-myc, initial studies on the structure of the fusion genes were conducted using pulsed-field gel electrophoresis (PFGE; M~ikel~i et al., 1991b). Closer analysis of the breakpoint regions on both the rlf and L-myc sides was possible only after mapping of these regions in normal DNA. For this purpose, normal genomic clones were obtained from both regions. Restriction analysis of these breakpoint regions enabled their characterization in more detail, and indicated that the breakpoints differ considerably on the L-myc and rlf sides (M~ikel~i et al., 1991b,c). The structures of all three characterized rlf-L-myc fusion genes are shown in Fig. 2. The breakpoint in GLC28 is in a 100-bp region 3.5 kb downstream from rlf exon I and 16 kb upstream of L-myc. Although the breakpoint is also close to the first exon of rlf in CORL47, it is more than 40 kb upstream of the L-myc gene. The opposite situation can be seen in the LuC194 tumor DNA; there the breakpoint
310
rlf GLC28
I
CORLA7
I
LuC194
I
L-myc ::
~_~
"
::
:: ~
1
in the upstream region of L-myc, and they clearly indicate that the mechanism of rearrangement is not sequence-specific. However, in all cases the rearrangements occur upstream of the L-myc gene, and in the first intron of the rlf gene, and they would thus be expected to encode similar fusion proteins.
~.
=
0
kb
Fig. 2. Similar structure but distinct rearrangements characterize the rlf-L-myc gene fusions. The three different characterized rlf-L-myc fusion genes are shown. The introns between rlf exon I (white box) and L-myc (three exons shown as black boxes) are 21, 2 4 a n d > 31 k b long in G L C 2 8 , L u C 1 9 4 , and CORL47, respectively. The thin line represents sequences from the rlf locus, and the thick line sequences from L-myc. The white thick line represents the region where the rearrangements have occurred. Slashed lines in CORL47 indicate that the exact length of the first intron of rlf-L-myc is unknown.
The rlf-L-myc fusions are caused by intrachromosomal rearrangements A previous cytogenetic study indicated that one of the cell lines with the rearrangement (CORL47) did not contain abnormalities of chromosome 1 (Waters et al., 1988). Because L-myc is located at lp32 (Nau et al., 1985), this suggested that at least in this case the rearrangement is not due to a chromosomal translocation. Accordingly, we could map the rlf locus to chromosome 1 using human-rodent somatic cell hybrids. Therefore we also decided to explore the possible physical linkage of rlf and L-myc. Using PFGE analysis we found that the rlf and L-myc genes are
lies furthest downstrean from rlf exon I, but relatively close to L-myc. These results delineate the rearrangement region in rlf intron I to span at least 10 kb, and correspondingly at least 35 kb
TABLE DNA
1
REARRANGEMENTS
Class
I
II
III
IV
T,
T/C
INVOLVING
rlf AND L-rnyc I N
CANCER
Tumor/
Rearrangement
cell line
rlf
C
CORL47
+
+
+
C
GLC28
+
+
+
T
LuC194
C
ACC-LC-49
C C
Amplification
L-myc
+
+
+
+
+
+
ACC-LC-48
+
+
+
SK-LC-17
+
+
+
C
CORL88
-
-
+
T
RO
-
-
+
T
AS
-
-
+
T
PA
-
-
+
C
ACC-LC-177
-
-
+
C
ACC-LC-178
-
-
+
C
ACC-LC-52
-
-
+
-
+
+
-
+
+
C
U1690
C
ACC-LC-49
*
*
T
LuC49
+
-
-
T
LuC26
-
+
-
primary tumor; C, tumor cell line. line has other rearrangements involving
* The ACC-LC-49 al. (1991).
LUNG
L-myc
besides the
rlf-L-myc
rearrangement as described in Sekido et
311 r/f ~t
~l
IIII
L-myc Nt (~t)
II
M
Nt
I
I
100 kb r/fexon I
breakpoints
Fig. 3. The rlf and L-myc genes are located within 480 kb of each other in germ-line DNA. A long-range restriction map encompassing the rlf and L-rnyc loci. In this scale the three exons of L-myc are shown as a single black box, whereas the putative exons of rlf are only shown schematically.Their exact number is not known, but the gene is known to span over 60 kb of DNA, and the length of the first intron of rlf is over 15 kb (Miikelii et al., 1991b). This is also the region where the breakpoints occur, as shown. M = MluI, N = NotI. Sites in parentheses are partially digested in PFGE analysis.
both present in a 780-kb MluI D N A fragment (Miikel~i et al., 1991b). A refined long-range restriction map surrounding these two loci shows that the first exon of rlf is approximately 480 kb upstream of L-myc (Fig. 3). This physical linkage was later confirmed by the finding of coamplification of unrearranged rlf and L-myc in several SCLCs (Miikel~i et al., 1991c; Sekido et al., 1991; Table 1).
Relationship of rearrangement and gene amplification All intrachromosomal rearrangements leading to the rlf-L-myc fusions have also been involved in D N A amplification. However, in all cases with the rearrangement, only a single amplified band is present with several different digestions, suggesting that the rearrangement has occurred before D N A amplification. In addition to the rlf-Lmyc rearrangements, the L-myc gene has been found involved in two other amplification-associated rearrangements. The ACC-LC-49 SCLC cell line contains complex rearrangements, which apparently include both the rlf-L-myc rearrangement and a rearrangement between the upstream region of L-myc and a novel locus EX (Sekido et al., 1991; Takahashi et al., 1989). The U1690 SCLC cell line (Bergh et al., 1985) also contains a rearrangement in the upstream region of L-myc which is fused to unknown sequences (unpublished data). However, both of these L-myc rearrangements, which do not involve rlf, still produce normal L-myc mRNAs (Miikel~i et al., 1991a; Sekido et al., 1991), and have only been found from a single cell line.
Homogenous rearranged amplified D N A has been described for the N-myc gene in a primary neuroblastoma (Hiyama et al., 1991), and for the c-myc gene in the COLO320 DM cell line (the pvt-myc fusion; Shtivelman and Bishop, 1989), although in both cases amplified copies of the unrearranged gene can also be found (Alitalo et al., 1983; Hiyama et al., 1991; Schwab et al., 1986). In all rlf-L-myc rearrangements, in the pvt-myc rearrangement, and in several neuroblastomas with amplifications and rearrangements (Shiloh et al., 1985) the rearranged D N A has been located in the vicinity of the corresponding myc gene, suggesting that specific local rearrangements occur as initial amplification steps. Such a model is inherent in the currently held models for gene amplification (Kinzler et al., 1986; Stark et al., 1989; Trask and Hamlin, 1989). Accordingly, intrachromosomal single-copy rearrangements involving c-myc described in a giant cell carcinoma of the lung (Iizuka et al., 1990) may represent an early step of this process as suggested by the finding of a similar rearrangement amplified in a giant cell carcinoma cell line (Yoshimoto et al., 1986).
A reeombinogenic DNA region upstream of L.myc Because the breakpoints in the different rlf-Lmyc fusions differ considerably on the rlf and the L-myc side, the rearrangement mechanism probably is not sequence-specific. Sequence analysis of one of the breakpoint regions on the L-myc side indicated that it contains an Alu repeat sequence. Further mapping and sequencing of the L-myc upstream region revealed a large D N A region
312
produce the chimeric rlf-L-myc mRNAs. Class II contains SCLCs with coamplification of rlf and L-myc with no rearrangements detected in conventional Southern analysis. They also appear to produce normal L-myc transcripts. Class III represents cases where the L-myc gene is rearranged with another locus. The ACC-LC-49 cell line appears to have two distinct rearrangements (see above), and is therefore included in both class I and class II. Class IV SCLCs contain rearrangements of either rlf or L-myc without DNA amplification. According to the material analyzed (14 cases) it would appear that the rlf gene is commonly involved in coamplification with L-myc. This might be expected due to the short distance between the two genes. Interestingly, rearrangements fusing intron I of rlf to the upstream region of L-myc and leading to the production of chimeric rlf-L-myc mRNAs are found in six out of 14 analyzed cases. The consistent finding of this rearrangement suggests that it may provide a selective growth advantage to the SCLC cells.
clustered with repeated sequences as shown in Fig. 4. In addition to associated KpnI (DiGiovanni et al., 1983) and Alu sequences (Deininger et al., 1981), at least one conserved member of the family of SINE-R endogenous retrovirusrelated retroposons (Ono et al., 1987) was identified (unpublished data). The region mapped so far contains the breakpoints of at least three L-myc amplified cell lines as shown in Fig. 4. The breakpoint in the GLC28 cell line involves the rlf-L-myc rearrangement. The characterized breakpoint in the ACC-LC-49 cell line does not involve rlf, but another novel locus (EX). However, the ACC-LC-49 cell line also contains a less well characterized rlf-L-myc rearrangement not shown in Fig. 4. The U1690 breakpoint also involves a new locus and not rlf (see above). All three breakpoint regions are located in the cluster of repeats which thus represents a recombinatorial hotspot of repetitive sequence, and the rearrangements may have occurred as homologous recombinations between repetitive sequences as described previously (Lehrman et al., 1987). Interestingly, a recombinogenic A + T-rich region containing Alu repeats has also been found to be frequently involved in early amplificationassociated rearrangements of the Chinese hamster adenylate deaminase gene (Hyrien et al., 1987). Table 1 contains a summary of the different configurations of the L-myc a n d / o r rlf genes in SCLC cell lines and tumors. Several classes can be distinguished according to the amplification and rearrangement status of L-myc and rlf. Class I SCLCs contain a rlf-L-myc rearrangement associated with D N A amplification. All analyzed cases (excluding the LUC194 tumor, from which material was not available) have also been found to
?? repeats I breakpoints
Acknowledgements We gratefully acknowledge our collaborators Gerard Evan, Juha Kere, Robert Winqvist, Takao Sekiya, Masahiko Shiraishi, Maria G. Borrello, Yoshitada Sekido, Takashi Takahashi, and Toshitada Takahashi. We express our gratitude to Fred Alt, Charles Buys, Pamela Rabbitts, Jonas Bergh, and Naohiko Ikegaki for cell lines and reagents, and to Kirsi Pylkkanen, Elina Roimaa, Tapio Tainola, Merja Virta, Raili Taavela, and Ritva Tolvanen for technical assistance. The work from our laboratory reviewed here was supported by the Finnish Cancer Organizations, Finnish Cultural Foundation, Emil Aaltonen Foundation, Ida
?? ?
S1NE-R r,~l I I I l l GLC28 U1690
I
L-myc
,.?
I [] ACC-LC-49
? I-'-I 1 kb
Fig. 4. A highly repetitive region of D N A u p s t r e a m of the L-myc gene. Sequence analysis of normal genomic clones immediately u p s t r e a m of the L-myc gene has revealed several clusters of highly repetitive D N A (shown schematically as white boxes) including one m e m b e r of the S I N E - R e n d o g e n o u s retrovirus-derived r e t r o t r a n s p o s o n ( O n o et al., 1987). All characterized rlf-L-myc breakpoint regions (shown as gray boxes) coincide with these repetitive regions. Balls m a r k EcoRl sites and black boxes L-myc exons.
313
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