JOURNAL OF VIROLOGY, Feb. 2003, p. 2056–2062 0022-538X/03/$08.00⫹0 DOI: 10.1128/JVI.77.3.2056–2062.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 3
Genome-Based Identification of Cancer Genes by Proviral Tagging in Mouse Retrovirus-Induced T-Cell Lymphomas Rachel Kim,1 Alla Trubetskoy,1 Takeshi Suzuki,2 Nancy A. Jenkins,2 Neal G. Copeland,2 and Jack Lenz1* Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York 10461,1 and Mouse Cancer Genetics Program, National Cancer Institute, Frederick, Maryland 217022 Received 16 August 2002/Accepted 19 October 2002
The identification of tumor-inducing genes is a driving force for elucidating the molecular mechanisms underlying cancer. Many retroviruses induce tumors by insertion of viral DNA adjacent to cellular oncogenes, resulting in altered expression and/or structure of the encoded proteins. The availability of the mouse genome sequence now allows analysis of retroviral common integration sites in murine tumors to be used as a genetic screen for identification of large numbers of candidate cancer genes. By positioning the sequences of inverse PCR-amplified, virus-host junction fragments within the mouse genome, 19 target genes were identified in T-cell lymphomas induced by the retrovirus SL3-3. The candidate cancer genes included transcription factors (Fos, Gfi1, Lef1, Myb, Myc, Runx3, and Sox3), all three D cyclins, Ras signaling pathway components (Rras2/ TC21 and Rasgrp1), and Cmkbr7/CCR7. The most frequent target was Rras2. Insertions as far as 57 kb away from the transcribed portion were associated with substantially increased transcription of Rras2, and no coding sequence mutations, including those typically involved in Ras activation, were detected. These studies demonstrate the power of genome-based analysis of retroviral insertion sites for cancer gene discovery, identify several new genes worth examining for a role in human cancer, and implicate the pathways in which those genes act in lymphomagenesis. They also provide strong genetic evidence that overexpression of unmutated Rras2 contributes to tumorigenesis, thus suggesting that it may also do so if it is inappropriately expressed in human tumors.
duces strictly T-cell lymphomas 2 to 4 months following inoculation of neonatal mice of susceptible strains such as NIH/ Swiss and AKR/J (15, 28). A genetic screen to identify CISs in SL3-induced T-cell lymphomas was undertaken.
The identification of genes that cause cancer is crucial for elucidation of the molecular mechanisms involved in tumorigenesis. Many retroviruses that cause tumors act by a mechanism of proviral insertional activation of cellular oncogenes or, less frequently, by inactivation of tumor suppressor genes (33). Retroviral DNA genomes integrate at essentially random sites within the host genome as part of the viral replication cycle (40). When a provirus, the integrated form of retroviral DNA, is situated adjacent to an appropriate cellular oncogene, resulting alterations in the level of expression and/or the structure of the gene contribute to clonal expansion of that infected cell as a tumor. Targeting of the same gene in multiple independent tumors provides compelling genetic evidence that the targeted gene plays a role in oncogenesis (20, 27, 29, 33). Identification of genes also implicates the molecular pathways in which they function in tumorigenesis. The availability of the mouse genome sequence offers a powerful means of rapid identification of novel oncogenes that flank common integration sites (CISs) of murine retroviruses. PCR-based methods can be used to isolate large numbers of provirus-host DNA junctions from tumors (13, 20). By determining the host sequences in these fragments, they now can be positioned within the mouse genome. Candidate oncogenes that are targeted in multiple independent tumors can thus be efficiently identified. The murine retrovirus SL3-3 (SL3) in-
MATERIALS AND METHODS Identification of CISs by I-PCR and cloning. Inverse PCR (I-PCR) was performed (20) by using 2 to 5 g of DNA from tumor tissue digested with 60 U of BamHI overnight in 40 l. After heat inactivation at 80°C for 20 min, the DNA was diluted to 200 l, circularized by ligation with 1,000 U of T4 DNA ligase at 16°C overnight, ethanol precipitated, and resuspended in 30 l of Tris-EDTA. PCR was performed in 25 l with 1 l of the DNA template, 0.2 mM deoxynucleoside triphosphates, 10 pmol of each primer, 1.3 U of Expand Long Template Polymerase, and Expand Buffer 1 (Roche). The primers used were I-1F (5⬘-CAATCAGTAAGTCTGAGTCCTGACCGAT-3⬘) and I-1R (5⬘-TCA TCTGGGGAACCTTGAGAC-3⬘). The cycling conditions were 94°C for 2 min, followed by 10 cycles of 94°C for 10 s, 65°C for 30 s, and 68°C for 6 min and 20 cycles of 94°C for 10 s, 65°C for 30 s, and 68°C for 6 min with a 20-s autoextension and a final extension at 68°C for 10 min. Amplified products were cloned into the TA cloning TOPO vector (Invitrogen). Parts of white colonies were placed in a PCR with the original primers, in a nested PCR, and in a 5-ml culture. The nested primers used were I-2F (5⬘-ACGTGGTTCTTTTAGGAGACGAGAGG TC-3⬘) and I-2R (5⬘-CTGAGAACATCAGCTCTGGT-3⬘). Clones were sequenced with primer I-2R. RT-PCR. Total RNA from tumor tissue extracted with TRIZOL (Invitrogen) was used in the SuperScript First-Strand Synthesis System for reverse transcriptase (RT) PCR (Invitrogen). Serial dilutions of 2, 1, 0.5, 0.25, and 0.125 g of RNA from each tumor sample were tested in parallel reaction mixtures. Two micrograms of RNA from each was also used as a negative control with no RT. RT products were then used in a PCR containing 10 pmol of Rras2 primers and 0.5 U of Taq polymerase (Amersham Pharmacia). Cycling conditions were 94°C for 2 min, followed by 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s for 20 cycles and a final extension at 72°C for 2 min. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used as an internal control under the same conditions. The Rras2 primers used were f2 (5⬘-GTGAAGCTAATGTAACGG
* Corresponding author. Mailing address: Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-3715. Fax: (718) 430-8778. E-mail:
[email protected]. 2056
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FIG. 1. Amplification of mouse genome sequences flanking proviruses by I-PCR. Viral DNA sequences are shown as black boxes. LTRs are thicker boxes. Small arrows show PCR and DNA sequencing primers. The dashed line indicates the I-PCR product. R, reverse; F, forward.
TC-3⬘) and r2 (5⬘-GAGTAACAGTAGGCAACATG-3⬘). The GAPDH primers used were GAPDH3 (5⬘-CACATTGGGGGTAGGAACAC-3⬘) and GAPDH5 (5⬘-ACCCAGAAGACTGTGGATGG-3⬘). Northern blot analysis. Eight to 12 g of RNA was suspended in 10 l of FORMAZOL (Molecular Research Center, Inc.) and incubated at 55°C for 10 min. Ten microliters of formaldehyde solution was added, and the mixture was incubated at 55°C for 15 min before being electrophoresed in a 1% denaturing formaldehyde gel in 1⫻ morpholinepropanesulfonic acid buffer. Samples were run overnight at 25 V in 1⫻ morpholinepropanesulfonic acid buffer and then transferred onto Sure Blot positively charged nylon membrane (Serologicals Corp.) in 20⫻ SSC (1⫻ SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Membranes were baked at 80°C in a vacuum oven for 2 h. Membranes were prehybridized in Hybrisol I buffer (Serologicals Corp.) with salmon sperm DNA and 15 to 30 g of mouse Cot-1 DNA (Invitrogen) at 45°C. Twenty-five nanograms of DNA was labeled with the RediPrime II random-labeling system (Amersham Pharmacia) and purified with QuantProbe G-50 columns (Amersham Pharmacia). Probes were heated to 100°C for 5 min with 100 g of salmon sperm DNA, 200 l of Hybrisol I buffer, and 30 to 40 g of mouse Cot-1 DNA; chilled on ice; added to the prehybridization buffer; and incubated overnight at 45°C. Serial washes were carried out at 65°C in 2⫻ to 0.1⫻ SSC–0.1% sodium dodecyl sulfate buffers, and membranes were autoradiographed at ⫺80°C. Sequencing of Rras2. RT-PCR was performed on tumor RNA by using primers r1 (5⬘-GGATGATGTAAACGGTAGCA-3⬘) and f3 (5⬘-TGCAGATCCGCGA AGAAGA ⫺3⬘) and the SuperScript First-Strand Synthesis System (Invitrogen) and the Advantage-GC 2 PCR kit (Clontech). Products were directly sequenced with r1 and f3. Exons 1 and 3 of genomic Rras2 were amplified from tumor genomic DNA by using primers f3 and r4 (5⬘-TGGATGAACTGGATGGTGA3⬘) and primers f4 (5⬘-TGATCGATGACCGAGCTGC-3⬘) and r10 (5⬘-CAGG AAGCCCTCGCCTGT-3⬘), respectively, and directly sequenced by using primer f3 or r10. Nucleotide sequence accession numbers. The sequences of all of the viral insertion sites in this study have been submitted to the mouse Retroviral Tagged Cancer Gene Database (http://genome2.ncifcrf.gov/RTCGD/) (37).
RESULTS AND DISCUSSION Analysis of integration sites by I-PCR and DNA sequencing. To isolate virus-host junction DNA fragments, I-PCR (20) using primers in the viral genome (Fig. 1) was applied to a panel of 48 SL3-induced T-cell lymphomas. The tumors involved the thymus, spleen, lymph nodes, and liver and sometimes other organs. Southern blot analysis of tumor DNAs indicated that there were usually about five proviruses per tumor, only one of which might be adjacent to a target oncogene. Host sequences in the I-PCR-amplified DNAs were determined by sequencing from primers in the long terminal repeat (LTR) of the viral genome. Inclusion of a small portion
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of the LTR in the sequence read verified that the amplified fragments were derived from bona fide proviral integration events that involved the deletion of 2 bp at the end of the viral LTR. It also confirmed that the fragments were from SL3 proviruses and not from any related, endogenous murine leukemia viruses (MuLVs) in the mouse germ line. One hundred twenty-eight junction fragments were analyzed, of which 95% were placed within the mouse genome by using the publicly available UCSC (University of California, Santa Cruz) (http: //genome.ucsc.edu/) and Ensembl (http://www.ensembl.org /Mus_musculus/) assemblies. This demonstrated the utility and depth of the public mouse genome databases. Genes tagged by SL3. A CIS was defined as two or more viral insertions anywhere within the transcribed portion of a gene or as two or more independent insertions in a 10-kb or smaller stretch of the mouse genome outside the transcribed portion of a gene. In a sample of 128 insertions (the number of viral insertions analyzed here), the possibility of two independent insertions occurring in a 10-kb segment by random chance is highly improbable (37). Candidate oncogenes were identified as the nearest gene. A total of 19 candidate cancer genes were tagged (Table 1). Ten CISs were identified as targets of multiple SL3 insertions. Six of these identified novel candidate oncogenes (Rrasgrp1, Rras2, Fos, 4933432H23Rik, Ccnd3, and Sox3), while four were previously known retroviral insertion targets (Runx3, Ahi1/ Epi1, Myc, and Pim1). In the case of Rasgrp1 and Rras2, additional insertions besides those in the ⱕ10-kb stretch were included in Table 1. A second set of candidate oncogenes was identified by single hits in this panel that matched previously identified retroviral CISs (Notch1, Gfi1, Ccnd2, Ccnd1, Myb, and Pvt1). A third set of genes (Lef1, 4921528H16Rik, and Cmkbr7) was identified by single hits in this panel and single viral insertions in a separate study (37). For Cmkbr7, the insertions were 16 bp apart; for 4921528H16Rik, they were 1.2 kb apart; while for Lef1, both insertions were within the transcribed portion of the gene. These observations emphasized the cumulative nature of this experimental approach. The genes identified here add substantially to the number of known retroviral targets in T-cell lymphomas. In all, nine new mouse genes targeted by retroviral insertions were identified. At least one candidate gene was identified in 31 (65%) of the 48 lymphomas in the panel. Each SL3 CIS identified was designated with the prefix Si (Table 1). These sites significantly increase the number of T-cell lymphoma CISs in the Retroviral Tagged Cancer Gene Database, which was established by analysis of MuLV insertion sites in tumors of BXH2 and AKXD recombinant inbred mice that were mostly myeloid leukemias or B-cell lymphomas. The genes targeted by SL3 are of types commonly involved in cancer. The genes identified as SL3 CISs encoded types of proteins frequently associated with cancer, including transcription activators-repressors, cell cycle regulators, or proteins involved in signal transduction. Several of these act in pathways previously associated with lymphomas, including the Notch and Ras signaling pathways. Notch1 and Notch4 are known retroviral insertion targets in murine lymphomas and mammary carcinomas, respectively (9, 10, 42). Notch1 (TAN1) is implicated in human T-lymphoid tumors (8). The oncogenic Hras, Kras, and Nras genes have been widely implicated in human
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KIM ET AL.
J. VIROL. TABLE 1. Genes targeted in SL3-induced T-cell lymphomas GenBank accession no.
Mouse chromosome
Mouse genome positionb
Rasgrp
NP_035376
2
118212807–118273056
5⬘ 5⬘ 5⬘ 5⬘ 5⬘
Runx3
AML2, Cbfa3
NP_062706
4
132732826–132759995
5⬘ 70.5 kb, inv 3⬘ 3.6 kb, same
Rras2
TC21
NP_080122
7
103667151–103680987
5⬘ 5⬘ 5⬘ 5⬘ 5⬘ 5⬘ 5⬘ 5⬘ 5⬘ 5⬘
3
ENSMUSGI00000043372
Ahi1, Epi1
10
20856481–20947204
3⬘ 15.8 kbg same Intron 10, inv Intron 10, same
6q23
Si4
2
Fos
c-fos
NP_034364
11
80175735–80178153
5⬘ 24.9 kb, inv 5⬘ 30.0 kb, same
14q24.3
Si10
4
Myc
c-myc
NP_034979
15
62430357–62435308
5⬘ 5⬘ 5⬘ 5⬘
8q24
Si22
4
4933432H23Rik
NP_080506
15
62662390–62664172
3⬘ 5.9 kb, same 3⬘ 4.0 kb, inv 3⬘ 2.1 kb, same
10q22
Si11
4
Pim1
Pim-1
NP_032868
17
28704562–28708120
3⬘ 3⬘ 3⬘ 3⬘
6p21.2
Si3
2
Ccnd3
Cyclin D3
NP_031658
17
46621807–46626765
5⬘ 66.5 kb, inv 5⬘ 67.0 kb, inv
6p21
Si15
2
Sox3
Sox-3
NP_033263
X
43983667–43984837
3⬘ 46.1 kb, same 3⬘ 46.0 kb, same
Xq26-q27
Si19
1
Notch1
Mis6, Tan 1
NP_032740
2
26744840–26789342
5⬘ 46.4 kb, same
9q34.3
Si17
1
Gfi1
Pal-1
NP_034408
5
105390226–105398083
3⬘ 14.9 kb, same
1p22
Si16
1
Ccnd2
Vin1, cyclin D2
NP_033959
6
128026656–128047570
5⬘ 3.4 kb, inv
12p13
Si18
1
Ccnd1
bcl-1, Cyclin D1
NP_031657
7
135581270–135389916
5⬘ 86.1 kb, same
11q15
Si13
1
Myb
c-myb
TVMSMY
10
21013821–21050000
Intron 5, same
6q22-q23
Si16
1
Pvt1
Mis-1, Mlvi-1
NP_035352
15
62623038–62629601
3⬘ 4.8 kb, same
8q24
5⬘ UTR, same
4q23-q25
Locusd
No. of tags
Si8
5
Rasgrp1
Si9
2
Si1
10
Si23
Si6
1
Candidate genea
Lef1
Si12
1
4921528H16Rik
Si7
1
Cmkbr7
a
Alias(es)
Lef-1
Ccr7
P27782
3
Provirus position orientation
f
131666779–131760166 f
9.8 kb, invc 50.2 kb, inv 57.4 kb, inv 57.6 kb, inv 78.4 kb, inv
16.0 17.8 17.8 18.0 47.0 47.4 47.8 48.4 57.0 63.0
239 455 684 722
kb, kb, kb, kb, kb, kb, kb, kb, kb, kb,
bp, bp, bp, bp,
UTR, UTR, UTR, UTR,
inve inve inve inve inve inve inve inve inve inve
inv inv inv inv
same same same same
Human chromosome position
15q15
1 p36 11p15.3
BAB29648
6
34198860–34306385
3⬘ 19.7 kb, same
7q36
NP_031745
11
99919090–99930067
5⬘ 12.8 kb, inv
17q12-q21.2
The gene nomenclature used is from http://www.informatics.jax.org/. Positions are from the UCSC Mouse February 2002 draft assembly (http://genome.ucsc.edu/), except where noted otherwise. inv indicates opposite transcriptional orientation relative to gene; UTR, untranslated region. d Common integration sites newly identified in this study are in bold type. e Exon 1 of mouse Rras2 was not positioned in the UCSC or Ensembl mouse genome assembly (June 20/02). Positions of Si1 and Si1 relative to Rras2 were determined relative to the position of exon 2 of the gene, estimating the size of exon 1 plus intron 1 (64 kb) to be similar to that of humans. f Positions are from the Ensembl version 3 draft sequence, frozen in February 2002 (http://www.ensembl.org/Mus musculus/). g Distance calculated from known cDNA sequences (GenBank accession numbers AI428154 and BB132089) that extend the gene further 3⬘ than ENSMUSG00000043372. b c
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and murine cancers, including lymphomas. The two most frequently targeted genes in this study were the Ras family member Rras2/TC21 and the Ras guanine nucleotide exchange factor Rasgrp1. Together, they accounted for a CIS in 30% of the tumors. This underscores the importance of Ras signaling in T-cell lymphomagenesis and also implicates these two relatively less studied Ras pathway components in tumorigenesis. Two of the targets identified in this study, Lef1 and Sox3, have been associated with the Wnt signaling pathway (43). Wnt signaling was previously implicated in lymphocyte development (21, 30). These observations raise the question of whether the Wnt pathway plays an unrecognized role in lymphomas, including those in humans. Cmkbr7 encodes chemokine receptor 7 (CCR7), which binds the chemokines CCL21/SLC and CCL19/ELC. Besides their roles in lymphoid development, maturation, and inflammation, certain CCRs, including CCR7, have been implicated in metastasis of human lymphomas and carcinomas (14, 24). Like other CCRs, CCR7 is a seven-transmembrane domain, G protein-coupled receptor. Several G protein-coupled receptors have been implicated in cell transformation and tumorigenesis (2, 35, 39). The identification of Cmkbr7 as a CIS suggests that CCR7 played a role in lymphomic transformation and/or metastasis of these tumors. Transcriptional regulatory molecules were the most common type of gene targeted by SL3. Sox3 and Lef1 were among the transcription factors identified as novel candidate lymphoma genes here. Sox3 is a high-mobility group (HMG) protein that binds -catenin and was demonstrated to have fibroblast-transforming activity (41, 43). The paralog Sox4 was the most frequently targeted gene in MuLV-induced tumors of BXH2 and AKXD recombinant inbred mice, although usually in myeloid leukemias or B-cell lymphomas and only uncommonly in T-cell lymphomas (37). Lef1 is a member of the T-cell factor (TCF) family of HMG proteins that interact with -catenin. Thus, HMG proteins appear to be important in hematopoietic tumors. Fos was initially characterized as the transforming gene of the osteosarcoma-inducing FBJ and FBR viruses (22) and has been widely studied as a component of the AP-1 transcription factor. Identification here of Fos as a CIS indicates a role for this gene in lymphomagenesis. Runx3 (also called AML2 and Cbfa3) was the only member of the Runx family targeted here. It was recently identified as a CIS in Moloney leukemia virusinduced rat thymic lymphomas (36). One of its paralogs, Runx1/AML1, is important in human leukemias and was also targeted in MuLV-induced myeloid leukemias (20). Another paralog, Runx2/AML3, strongly cooperates with Myc in MuLVinduced T-cell lymphomas (5). The basis for the apparent selectivity for specific Runx paralogs in different tumors is an interesting question. Among the transcription factors targeted were the previously known retroviral insertion targets Myb, Myc, and Gfi1. Tagging of Myb emphasizes its importance for oncogenesis in the T-cell lineage (1). Both Myc and Gfi1 are frequent targets during T-cell lymphomagenesis by Moloney MuLV (34, 38). While Myc was tagged four times, Gfi1 was hit only once in this panel of SL3-induced lymphomas, suggesting that there may be subtle differences in how the two MuLVs cause T lymphomas. In addition to four viral insertions within 1 kb of the first exon
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TABLE 2. Multiple CISs per tumor CISs
No. of tumors
Rasgrp1, Runx3...................................................................................... 2 Rasgrp1, Fos, Pim1, Pim1 .................................................................... 1 Ccnd2, Ccnd3 ........................................................................................ 1 Pim1, Myc .............................................................................................. 1 Rras2, Myc ............................................................................................. 1 Rras2, Pvt1............................................................................................. 1 Rras2, 4921528H16Rik ......................................................................... 1 Rras2, ENSMUSG00000043372 .......................................................... 1 Rras2, Ccnd1, 4933432H23Rik ............................................................ 1
of Myc, there was one near the Pvt1 locus that is situated 188 kb 3⬘ to Myc (Table 1). Viral insertions or chromosomal translocations involving immunoglobulin loci at Pvt1 activate Myc expression (19). The cell cycle regulatory molecules targeted by SL3 included all three cyclin D genes. No other cyclins were tagged. Along with previous studies implicating cyclins D1 and D2 in lymphomagenesis (12, 18), this indicates the importance of the Rb-cyclin D-cdk4,6 G1 checkpoint in regulation of T cells and is consistent with the idea that the products of the three cyclin D genes might function similarly in lymphomagenesis. Among the genes targeted by SL3 were a few of unknown function. Ahi1/Epi1 is a previously identified CIS located 30 to 40 kb 3⬘ to Myb (4, 17). Retroviral insertions at this locus did not affect Myb expression (4, 17), suggesting that a different gene was involved in lymphomagenesis. One SL3 insertion was situated in Ahi1/Epi1. The mouse genome assemblies located Ahi1/Epi1 just 5⬘ to a gene predicted on the basis of many known transcripts. Two additional SL3 insertions were identified (Table 1) in a central intron of the presumptive gene that encodes a protein predicted to have an SH3 domain and four G protein  subunit-like WD40 repeats. Another gene of unknown function targeted by multiple SL3 insertions was 4933432H23Rik (Table 1). It encodes a macro-H2A histonelike protein (Table 1) and is located about 227 kb 3⬘ to Myc. A third locus identified in this study lay between a predicted UDP galactose transporter and a gene of unknown function (4921528H16Rik) predicted to encode a protein with both a leucine repeat domain characteristic of protein phosphatase regulatory subunits and a guanylate kinase domain. Although considerable further experimentation is required to analyze the expression and functions of these genes in tumorigenesis, these observations highlight the power of this genomics-based approach to identify candidate cancer genes. Several of the loci identified in this study are known to be altered by chromosomal rearrangements in human cancer, including Myc, Ccnd3, Notch1, Ccnd1, and Pvt1 (http://cgap .nci.nih.gov/Chromosomes/Mitelman). Coding sequence mutations have been identified in the Rras2 gene in a few human tumors (6, 16). Expression of several additional genes tagged here has been correlated with certain types of human cancer. Thus, genes identified by retroviral tagging in mice are frequently implicated in human cancer as well. The possible role of several candidate genes identified here, including Rasgrp1, Sox3, 4933432H23Rik, and 4921528H16Rik, in human cancer
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KIM ET AL.
J. VIROL.
FIG. 2. Analysis of the transcription and coding sequence of Rras2/TC21. (A) The upper line shows the structure of the Rras2 gene. Exons are shown as boxes and are not on the same scale as the introns. Translational start and stop sites are indicated. Triangles show the clusters of SL3 insertions and point in the viral 5⬘-to-3⬘ orientation. The lower line shows Rras2 mRNA. Amino acids that are frequently mutated in oncogenic H, N, and K Ras proteins are shown in single-letter code above the Rras2 mRNA, along with their positions in the protein. PCR primers are shown as small arrows below the mRNA. The PCR-amplified probe used for Northern blotting is also indicated. (B) Northern blot analysis of Rras2 RNA in lymphomas. Numbers indicate specific tumors. Tumors with insertions at cluster A, cluster B, or ⫺57 kb are noted. The same blots were also hybridized to a -actin probe. (C) Semiquantitative RT-PCR analysis of Rras2 RNA in lymphomas. Twofold serial dilutions of RNA isolated from tumors were subjected to RT-PCR with primers r2 and f2. Numbers indicate specific tumors.
has not been reported. These genes warrant further investigation for potential roles in human cancer. Most viral insertions were upstream or downstream of the transcribed parts of the candidate oncogenes (Table 1). This is
consistent with transcriptional enhancement by elements such as those in the viral LTR being the most common mechanism of proviral insertional activation. Most viral insertions were situated tens of kilobases away from the transcribed portions of
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genes (Table 1). This suggests that retroviruses are capable of activating many genes from such distances, as was previously demonstrated for distances much greater than 100 kb for Myc and Ccnd1 (18, 19). In some cases, SL3 insertions were clustered over relatively small stretches of the genome situated a relatively long distance from the transcribed portion of the gene, as best illustrated by Rras2/TC21 (Table 1). It is possible that such clusters define key regulatory elements of the genes. Thus, the analysis of retroviral CISs in tumors not only identifies candidate cancer genes but may also identify regulatory elements within them. Multiple CISs in tumors. Ten tumors (21%) contained two or more CISs (Table 2). Some cases were presumably due to two independent lymphoma clones in the same tumor mass, such as the one with two independent insertions in Pim1 and the one with insertions in the Ccnd2 and Ccnd3 genes. However, it is also possible that viral insertions in two or more candidate cancer genes occurred within a single lymphoma clone. Precedent for cooperativity between provirus-targeted genes is amply documented (3, 26, 38). The lymphomas with insertions in Rras2 plus Myc, Rras2 plus Pvt1, or Myc plus Pim1 (Table 2) are good candidates to be such cases. Thus, the retroviral tagging approach allows the identification of candidate cooperating oncogenes. Long-distance activation of the transcription of Rras2/ TC21without coding sequence mutations. The gene most commonly targeted by SL3 was Rras2/TC21 (Table 1). Viral insertions were detected in two clusters, designated A and B (Fig. 2A), about 16 and 47 kb upstream of the transcribed part of the gene, respectively. Additional insertions were detected about 57 and 63 kb 5⬘ to the gene (Table 1). Proviral insertions at these sites were correlated with high levels of Rras2 mRNA (Fig. 2). Both Northern blotting (Fig. 2B) and semiquantitative RT-PCR (Fig. 2C) showed that insertions at cluster A, cluster B, or the 57-kb position were associated with high relative levels of Rras2 mRNA. Two Rras2/TC21 transcripts of about 1.7 and 2.5 kb were detected, similar to the sizes reported for human Rras2 previously (6). Both were elevated when a flanking provirus was present. Rras2 transcripts were much lower in normal spleen or normal thymus. They were also low in most SL3-induced lymphomas that lacked I-PCR-detected proviral insertions near Rras2 (Fig. 2B and C). However, in 3 of 12 such lymphomas (tumors 29, 47, and 50 in Fig. 2B), relatively high levels of Rras2 transcripts were detected. It is unknown whether these lymphomas had proviral insertions near Rras2 that were missed in the I-PCR screen or whether a different molecular mechanism is responsible for increased Rras2 transcription in them. We conclude that proviral insertions as far as 57 kb 5⬘ to the first exon of Rras2 were associated with greatly elevated levels of Rras2 transcription. Tumorigenic activation of the oncogenic ras genes Hras1, Kras2, and Nras in human and murine tumors is widely due to mutations that cause single amino acid substitutions in one of four key positions (Fig. 2A). Substitutions at the comparable positions also greatly increased the transforming activity of Rras2 in rodent fibroblasts (6, 11, 23). Therefore, we tested whether the Rras2 genes adjacent to SL3 proviruses in lymphomas had acquired such mutations. First, we performed RT-PCR to isolate transcripts from tumors, hypothesizing that the elevated transcripts in lymphomas were derived principally
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from the provirus-containing alleles. The RT-PCR products were directly sequenced without subcloning. No mutations were detected in any of the tumors. The four key amino acid positions (23, 24, 70, and 72 in Rras2) are encoded in exons 1 and 3 (Fig. 2A). Each of these exons was PCR amplified from genomic DNA of the tumors with proviruses at cluster A or B (Fig. 2A). The products were directly sequenced to test for heterozygosity, but again, no mutations were found. Therefore, we conclude that oncogenic activity in these tumors was due to expression of inappropriately high levels of normal Rras2. Overexpression of Rras2 without amino acid substitutions was observed frequently in human breast cancer cell lines (7). In addition, Rras2 without coding sequence changes was shown to have measurable transforming activity in cultured rodent cells, although less than that of Ras proteins with typical activating mutations (7, 23). Studies of the pathways activated by Rras2 widely implicate activation of the phosphatidylinositol 3-kinase/Akt pathway, although it is less agreed how widely the Raf/mitogen-activated protein kinase, jun kinase/p38, and RalGDS pathways contribute to Rras2 activity (23, 25, 31, 32) The genetic evidence presented here that unmutated Rras2 is a frequent target for tumorigenic activation by retroviruses substantially strengthens the hypothesis that normal Rras2 protein may contribute to human cancer in instances where its expression is elevated. ACKNOWLEDGMENTS This work was supported by National Institutes of Health grant CA44822 (J.L.) and National Institutes of Health training grant CA09060 (R.K.). We thank Bisram Deocharan, David Garbe, Keiko Akagi, Geoff Turner, Anders Lund, Maarten van Lohuizen, Laura Levy, Channing Der, and Celia Brosnan for help. REFERENCES 1. Badiani, P. A., D. Kioussis, D. M. Swirsky, I. A. Lampert, and K. Weston. 1996. T-cell lymphomas in v-Myb transgenic mice. Oncogene 13:2205–2212. 2. Bais, C., B. Santomasso, O. Coso, L. Arvanitakis, E. G. Raaka, J. S. Gutkind, A. S. Asch, E. Cesarman, M. C. Gershengorn, E. A. Mesri, and M. C. Gerhengorn. 1998. G-protein-coupled receptor of Kaposi’s sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator. Nature 391:86–89. 3. Bear, S. E., A. Bellacosa, P. A. Lazo, N. A. Jenkins, N. G. Copeland, C. Hanson, G. Levan, and P. N. Tsichlis. 1989. Provirus insertion in Tpl-1, an Ets-1-related oncogene, is associated with tumor progression in Moloney murine leukemia virus-induced rat thymic lymphomas. Proc. Natl. Acad. Sci. USA 86:7495–7499. 4. Blaydes, S. M., S. C. Kogan, B.-T. Truong, D. J. Gilbert, N. A. Jenkins, N. G. Copeland, D. A. Largaespada, and C. I. Brannan. 2001. Retroviral integration at the Epi1 locus cooperates with Nf1 gene loss in the progression to acute myeloid leukemia. J. Virol. 75:9427–9434. 5. Blyth, K., A. Terry, N. Mackay, F. Vaillant, M. Bell, E. R. Cameron, J. C. Neil, and M. Stewart. 2001. Runx2: a novel oncogenic effector revealed by in vivo complementation and retroviral tagging. Oncogene 20:295–302. 6. Chan, A. M., T. Miki, K. A. Meyers, and S. A. Aaronson. 1994. A human oncogene of the RAS superfamily unmasked by expression cDNA cloning. Proc. Natl. Acad. Sci. USA 91:7558–7562. 7. Clark, G. J., M. S. Kinch, T. M. Gilmer, K. Burridge, and C. J. Der. 1996. Overexpression of the Ras-related TC21/R-Ras2 protein may contribute to the development of human breast cancers. Oncogene 12:169–176. 8. Ellisen, L. W., J. Bird, D. C. West, A. L. Soreng, T. C. Reynolds, S. D. Smith, and J. Sklar. 1991. TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 66:649–661. 9. Gallahan, D., and R. Callahan. 1997. The mouse mammary tumor associated gene INT3 is a unique member of the NOTCH gene family (NOTCH4). Oncogene 14:1883–1890. 10. Girard, L., Z. Hanna, N. Beaulieu, C. D. Hoemann, C. Simard, C. A. Kozak, and P. Jolicoeur. 1996. Frequent provirus insertional mutagenesis of Notch1 in thymomas of MMTVD/myc transgenic mice suggests a collaboration of c-myc and Notch1 for oncogenesis. Genes Dev. 10:1930–1944.
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