Tumor suppressor genes on chromosome 3p ... - Semantic Scholar

Oncogene (2002) 21, 6915 – 6935 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc

Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers Eugene R Zabarovsky*,1, Michael I Lerman*,2 and John D Minna*,3 1

Microbiology and Tumor Biology Center, Center for Genomics and Bioinformatics, Karolinska Institutet S-171 77, Stockholm, Sweden; 2Laboratory of Immunobiology, Center for Cancer Research, NCI-Frederick, Frederick, Maryland, MD 21702, USA; 3 Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas, TX 75390, USA

Loss of heterozygosity (LOH) involving several chromosome 3p regions accompanied by chromosome 3p deletions are detected in almost 100% of small (SCLCs) and more than 90% of non-small (NSCLCs) cell lung cancers. In addition, these changes appear early in the pathogenesis of lung cancer and are found as clonal lesions in the smoking damaged respiratory epithelium including histologically normal epithelium as well as in epithelium showing histologic changes of preneoplasia. These 3p genetic alterations lead to the conclusion that the short arm of human chromosome 3 contains several tumor suppressor gene(s) (TSG(s)). Although the first data suggesting that 3p alterations were involved in lung carcinogenesis were published more than 10 years ago, only recently has significant progress been achieved in identifying the candidate TSGs and beginning to demonstrate their functional role in tumor pathogenesis. Some of the striking results of these findings has been the discovery of multiple 3p TSGs and the importance of tumor acquired promoter DNA methylation as an epigenetic mechanism for inactivating the expression of these genes in lung cancer. This progress, combined with the well known role of smoking as an environmental causative risk factor in lung cancer pathogenesis, is leading to the development of new diagnostic and therapeutic strategies which can be translated into the clinic to combat and prevent the lung cancer epidemic. It is clear now that genetic and epigenetic abnormalities of several genes residing in chromosome region 3p are important for the development of lung cancers but it is still obscure how many of them exist and which of the numerous candidate TSGs are the key players in lung cancer pathogenesis. We review herein our current knowledge and describe the most credible candidate genes. Oncogene (2002) 21, 6915 – 6935. doi:10.1038/sj.onc. 1205835 Keywords: lung cancer; tumor supressor gene; human chromosome 3; deletion mapping; preneoplasia; gene therapy

*Correspondence: ER Zabarovsky, MTC and CGB, Box 280, Karolinska Institute, 171-77 Stockholm, Sweden; E-mail: [email protected] or MI Lerman; E-mail: [email protected] or JD Minna; E-mail: [email protected]

Allele loss (deletion) mapping of human chromosome 3 Allele loss and deletion mapping using microsatellite markers and the detection of homozygous deletions represented until now the most powerful method to localize potential TSGs. Recently new approaches based on the analysis of genomic microarrays have been developed. Comparative genome hybridization (CGH) microarrays allow the study of copy number changes but not LOH, while single nucleotide polymorphism (SNP) microarray approaches yield LOH but not copy number changes (Pinkel et al., 1998; Lindblad-Toh et al., 2000). Kok et al., 1997 summarized available human tumor LOH data and concluded that TSGs can be located anywhere in 3p12-p23. After 1997 a number of studies were done to perform fine mapping and localize TSG more precisely but these had low efficiency (Dahiya et al., 1997; Lounis et al., 1998; Sato et al., 1998; Uzawa et al., 1998; Fullwood et al., 1999; Caballero et al., 2001; Tschentscher et al., 2001; Fan et al., 2001; Acevedo et al., 2002, etc.). Some of the reasons for this low efficiency are discussed below. In addition, in most cases allele loss (loss of one of two polymorphic markers) was measured which could arise either by deletion or by other mechanisms such as mitotic recombination, while detection of a deletion would require other methods such as fluorescent in situ hybridization (FISH) to detect physical loss. We use the terms interchangably with the realization that a physical deletion may or may not have occurred to explain the allele loss. Reports differ, for example, on the extent of 3p losses in different lung tumors, with some papers reporting large terminal deletions, and others claiming interstitial deletions (see Kok et al., 1997; LindbladToh et al., 2000; Braga et al., 1999, 2002; Wistuba et al., 2000; Girard et al., 2000). Reports of the frequency of LOH can also differ for the same marker in the same type of tumor. For instance, D3S1284 (3p13p14.1) was deleted in only 8% of NSCLC biopsies studied by Pifarre et al., 1997, but in 63% of the NSCLCs studied by Wistuba et al. (2000). In contrast to this Tseng et al. (1999) reported deletion of marker D3S1234 (3p14.2-p14.3) in NSCLC biopsies (stage I) in 58%, but Wistuba et al. (2000) found this marker

Chromosome 3p TSGs ER Zabarovsky et al

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deleted in only 43% of NSCLC cell lines and in 52% of primary NSCLCs. Most of these studies did not use precise microdissection of tumor from other tissues. Thus, admixtures of stroma, blood vessels, lymphocytes and other normal cells in a macroscopically isolated tumor samples were unavoidable sources of error in LOH studies of solid tumors. In addition, it was demonstrated (Liu et al., 1999) that the high molecular weight allele (H-allele) is less sensitive to normal cell contamination than low molecular weight allele (Lallele). Random screening of 100 papers published between 1994 and 1999 revealed that the loss of L allele in tumors was detected at only 52% frequency of the loss of H allele (P50.00001) indicating a systematic artifactual bias. Rules have been developed to help account for this bias (Liu et al., 1999). Also recently it has been learned that amplification of one 3p allele can occur frequently leading to an erroneous conclusion that the other allele had suffered deletion (Zabarovsky et al., unpublished results) requiring reinterpretation of previous results. Another approach to solve this problem is to use microdissection. In this regard Wistuba et al. (2000) have performed the most detailed studies of such microdissected material using a high density of markers. They performed high resolution loss of heterozygosity (LOH) studies on 97 lung cancer and 54 preneoplastic/preinvasive microdissected respiratory epithelial samples using a panel of 28 3p markers. Allelic losses of 3p were detected in 96% of the lung cancers and in 78% of the preneoplastic/preinvasive lesions. The allele losses were often multiple and discontinuous, with areas of LOH interspersed with areas of retention of heterozygosity. Most SCLC (91%) and squamous cell carcinomas (95%) demonstrated larger 3p segments of allele loss, whereas most (71%) of the adenocarcinomas and preneoplastic/ preinvasive lesions had smaller chromosome areas of 3p allele loss. There was a progressive increase in the frequency and size of 3p allele loss regions with increasing severity of histopathological preneoplastic/ preinvasive changes. In analyses of the specific parental allele lost comparing preneoplastic/preinvasive foci with those lost in the lung cancer in the same patient, the same parental allele was lost in 88% of a large number of comparisons for 28 3p markers (a highly statistically significant finding). This indicates the occurrence of allele-specific loss in these foci similar to that seen in the tumor by a currently unknown mechanism. Analysis of all of the data indicated multiple regions of localized 3p allele loss including telomere-D3S1597, D3S1111-D3S2432, D3S2432-D3S1537, D3S1537, D3S1537-D3S1612, D3S4604/Luca19.1-D3S4622/Luca4.1, D3S4624/ Luca2.1, D3S4624/Luca2.1-D3S1582, D3S1766, D3S1234-D3S1300 (FHIT/FRA3B) region centered on D3S1300), D3S1284-D3S1577 (U2020/ROBO1-DUTT1 region centered on D3S1274), and D3S1511-centromere. A panel of six markers in the 600-kb 3p21.3 deletion region (discussed in detail below) showed loss Oncogene

in 77% of the lung cancers, 70% of normal or preneoplastic/preinvasive lesions associated with lung cancer, and 49% of 47 normal, mildly abnormal, or preneoplastic/preinvasive lesions found in smokers without lung cancer. By contrast, loss was never seen in 18 epithelial samples from seven life-time never smokers. The 600-kb 3p21.3 region and the 3p14.2 (FHIT/FRA3B) and 3p12 (ROBO1, also called DUTT1) regions (discussed below) were common, independent sites of breakpoints (retention of heterozygosity by some markers and LOH by other markers in the immediate region). They concluded that 3p allele loss is nearly universal in lung cancer pathogenesis; involves multiple, discrete, 3p LOH sites that often show a ‘discontinuous LOH’ pattern in individual tumors; occurs in preneoplastic/preinvasive lesions in smokers with and without lung cancer (multiple lesions often lose the same parental allele); frequently involves breakpoints in at least three very small defined genomic regions; and appears to have allele loss and breakpoints first occurring in the 600kb 3p21.3 region. These findings are consistent with previously reported LOH studies in a variety of tumors showing allele loss occurring by mitotic recombination and induced by oxidative damage. However, due to the scarcity of the available tumor material only a limited number of informative markers (rarely more than 30) could be used in the study of any one sample and this is not sufficient for scanning of a 100 Mb region. While combining studies is possible, most groups used different sets of markers and many of these are still not precisely positioned or correctly orient in the human genome draft sequence. For example, according to the present data at the http : / / www. ncbi.nlm.nih. gov/cgibin/Entrez/maps.cgi? org=hum&chr=3 orientation of the gene contig in the 3p21.3 homozygous deletion region (containing genes 3pK to RBM16/LUCA16) discussed below is reversed compared to that reported by precise study of the region in the literature (Wei et al., 1996). Homozygous deletions are excellent indicators of the locations of TSGs and several of these have been described at regions 3p12 (around the ROBO1/ DUTT1) gene), 3p14.2 (around the FHIT gene), 3p21.3 (several regions discussed below including around the RASSF1A gene, see also Kok et al., 1997; Todd et al., 1997). However, microsatellite deletion mapping is not very well suited for the detection of such deletions due to normal cell contamination. Balance between two alleles does not change in such cases and deletion will be undetected. In fact, this can lead to paradoxical results when LOH in homozygously deleted regions may be less than in surrounding regions. As will be seen, chromosome 3 appears to harbor many TSGs and these could be important in several different tumor types besides lung cancer or could be tumor type specific (Braga et al., 1999, 2002; Wistuba et al., 2000, 2001; Maitra et al., 2001; Angeloni and Lerman, 2001). For example, it is likely that within the same 3p region, TSGs for SCLC and NSCLC may


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differ. The role of a 3p TSG may also vary in different human populations or their role could be influenced by the genetic background or environmental factors besides cigarette smoking. Finally, technical differences between different investigations may initially confuse the candidacy of a specific gene. Aberrant tumor acquired DNA promoter region methylation constitutes an important mechanism in carcinogenesis and represents the main mechanism for inactivation of several TSGs. However, this mechanism of inactivation has only recently been studied during large-scale searches for chromosome 3 TSGs (see Wistuba et al., 2001; Esteller et al., 1999, 2001; Dammann et al., 2000; Burbee et al., 2001; Toyooka et al., 2001; Zo¨chbauerMuller et al., 2001a; Virmani et al., 2000, 2001, 2002; Li et al., 2002). Finally, most investigators thought there would be one or only a few TSGs residing on chromosome 3p. The current results suggest that there are many even in very small genomic areas. This number also has added to the complexity. Figure 1 and the rest of this article summarizes different studies searching for 3p TSGs including frequently affected by deletions and rearrangements regions (FARs) and candidate genes along with the arguments for their function as a TSG. Because of space limitations, some

other candidate 3p TSGs for which we do not currently have evidence of their direct involvement in lung cancer will be not discussed. Chromosome region 3p12-p13 (ROBO1/DUTT1 gene region) FAR and interstitial deletions were detected here by different researchers in lung and other cancers (Daly et al., 1991; Ganly et al., 1992; Pandis et al., 1993). Sanchez et al. (1994) demonstrated that 3p12-p14 region of chromosome 3 suppressed the tumor phenotype in RCC cell line. Rabbitts et al. (1990) reported a homozygous deletion at the locus D3S3 in the U2020 SCLC cell line. Later, Chen et al. (1994) detected a homozygous deletion in this region in breast cancer. Detailed mapping of the U2020 homozygous deletion indicated that it is about 8 Mb in size (Drabkin et al., 1992; Latif et al., 1992) has a complex origin (Heppell-Parton et al., 1999) and is actually located in 3p12 (Latif et al., 1992). Recently, two more homozygous overlapping lung and breast cancer deletions were reported in this low gene density region (Sundaresan et al., 1998a). A gene, DUTT1 (Deleted

Figure 1 Location of chromosome 3p regions most frequently affected in lung cancers (right panel). Candidate genes and information confirming their role in tumor suppression are also shown Oncogene


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in-U-Twenty-Twenty) that was disrupted by these deletions was identified and cloned (Sundaresan et al., 1998b). The same gene was also called ROBO1 gene (accession number AF040990). (Many discussed genes have different alternative forms and accession numbers, we will usually use only one for reference). ROBO1 was independently isolated as the human homolog of the Drosophila gene, Roundabout (Kidd et al., 1998). The gene, coding for a 1615 aa receptor with a domain structure of the neural-cell adhesion molecule (NCAM) family, is widely expressed (8 kb transcript) and has been implicated in the guidance and migration of axons, myoblasts, and leukocytes in vertebrates. A deleted form of the gene, which mimics a naturally occurring, tumor-associated human homozygous deletion of exon 2 of ROBO1, was introduced into the mouse germ line (Xian et al., 2001). Mice homozygous for this targeted mutation, which eliminates the first Ig domain of Robo1, frequently die at birth of respiratory failure because of delayed lung maturation. Lungs from these mice have reduced air spaces and increased mesenchyme, features that are present some days before birth. Survivors acquire extensive bronchial epithelial abnormalities including hyperplasia, providing evidence of a functional relationship between a 3p gene and the development of bronchial abnormalities associated with early lung cancer (Xian et al., 2001). Moreover, it was shown by karyotype analyses that deletions in this region are the only observable cytogenetic abnormalities in a short term culture isolated from apparently normal bronchial epithelial in a lung tumor-bearing patient (Sundaresan et al., 1995). These results are compatible with a role for ROBO1 in early stages of the multistep route to lung cancer. Furthermore, it was found that a fragment of human chromosome 3 overlapping with U2020 deletion mediates tumor suppression and rapid cell death of RCC cells in vivo (Lott et al., 1998; Lovell et al., 1999) and deletion of this region is associated with immortalization of human uroepithelial cells (Vieten et al., 1998) in vitro. On the other hand, it is not clear whether ROBO1 is the gene in this chromosome fragment responsible for these effects. Dallol et al. (2002) defined the genomic organization of the ROBO1 gene, performed mutation and expression analysis of ROBO1 in lung, breast and kidney cancers, and identified tumor specific promoter region methylation of ROBO1 in human cancers. The gene was found to contain 29 exons and spans at least 240 kb of genomic sequence. The 5’ region contains a CpG island, and the poly A tail has an atypical 5’GATAAA-3’ signal. ROBO1 was analysed for mutations in lung, breast and kidney cancers and no inactivating mutations were detected by PCR-SSCP. However, seven germline missense changes were found and characterized. ROBO1 expression was not detectable in 1/18 breast tumor lines analysed by RT – PCR. Bisulfite sequencing of the promoter region of ROBO1 gene in the HTB-19 breast tumor cell line (not expressing ROBO1) showed complete hypermethylation of CpG sites within the promoter region of the Oncogene

ROBO1 gene (7244 to+27 relative to the translation start site). The expression of the ROBO1 gene was reactivated in HTB-19 after treatment with the demethylating agent 5-aza-2’-deoxycytidine (AZA). The same region was also found to be hypermethylated in 19% of primary invasive breast carcinomas and 18% of primary clear cell renal cell carcinomas (CCRCC) and in 4% of primary NSCLC tumors. Furthermore 80% of breast and 75% of CC-RCC tumors showing DUTT1 methylation had allelic losses for 3p12 markers hence obeying Knudson’s two hit hypothesis. Our findings suggest that ROBO1/DUTT1 warrants further functional analysis as a candidate TSG at 3p12 in human cancers but its role in lung cancer appears to be limited. Chromosome region 3p14.2 (FHIT gene region) Deletions in this region are very frequent in lung and kidney carcinomas and were also observed in a variety of other common tumors, such as breast cancer, head and neck cancer, gastrointestinal cancer, esophageal cancer, and cervical cancer (see Croce et al., 1999; Huebner and Croce, 2001; Wistuba et al., 1997; Fong et al., 1997; Greenspan et al., 1997; Muller et al., 1998; Helland et al., 2000; Zo¨chbauer-Muller et al., 2001b). After careful mapping of this region, a gene at 3p14.2, designated FHIT (fragile histidine triad, NM_002012) gene was isolated. (Sozzi et al., 1996). This gene, covering more than 1 Mb, is composed of 10 exons, of which five are protein-coding (exons 5 – 9). FHIT encodes a small mRNA (1.1 kb) and a small protein (147 aa). Interestingly, the breakpoint at 3p14.2, involved in the t(3;8) chromosome translocation observed in the familial renal cell carcinomas, interrupts the third intron of the FHIT gene, inactivating one of the two FHIT alleles (see Huebner and Croce, 2001). The most common fragile site of the human genome, FRA3B, also maps within the FHIT gene. It had been speculated that fragile sites observed in the human genome correspond to chromosomal regions frequently involved in rearrangements in human cancer. It was suggested that the presence of the FRA3B fragile site within FHIT suggests that the fragility of this gene may make FHIT susceptible to rearrangements induced by a variety of environmental carcinogens. It was also suggested that the degree of chromosomal fragility at this site may contribute to the degree of cancer susceptibility. The observation that the degree of inducibility of the FRA3B fragile site with inhibitors of DNA synthesis, such as aphidicolin, differs in different individuals implies heterogeneity of the degree of fragility in the population, suggesting that it will be important to examine the correlation between degree of fragility and tumor susceptibility. It was demonstrated that this gene is frequently homozygously deleted in different cancers including lung cancer, aberrant FHIT transcripts are frequently found in lung cancer, and FHIT protein expression is frequently (*50%) lost in lung cancers (see Croce et


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al., 1999; Sozzi et al., 1997, 1998; Fong et al., 1997; Geradts et al., 2000 and other references). Multiple studies have shown that FHIT rarely suffers somatic amino acid missence mutations while aberrantly spliced mRNA giving whole exonic changes are frequent. By contrast epigenetic inactivation by tumor acquired promoter region DNA methylation of FHIT is common in several tumors and such 5’ CpG island methylation of the FHIT gene is correlated with loss of gene expression in esophageal, lung, and breast cancer (Tanaka et al., 1998; Zo¨chbauer-Muller et al., 2001b). Human lung and kidney cancer cell lines that overexpress exogenous FHIT grow more slowly in nude mice than their parental cancer-derived clones (Siprashvili et al., 1997). Infecting cells with a high-titre adenoviral vector that encodes FHIT inhibited cell growth in different FHIT-negative human cancer cell lines, but not in normal human bronchial epithelial cells (Ji et al., 1999; Dumon et al., 2001a; Ishii et al., 2001; Roz et al., 2002). FHIT overexpression caused the apoptotic cell population to increase markedly, and cells accumulated in S phase (Ji et al., 1999; Sard et al., 1999). Furthermore, adenoviral and adeno-associated viral vectors expressing FHIT markedly reduced tumorigenicity in vivo, indicating that FHIT functions as a tumor-suppressor gene both in vitro and in vivo (Ji et al., 1999; Dumon et al., 2001b). It was suggested that FHIT might, at least partly, exert its proapoptotic effect through a caspase-mediated pathway (Dumon et al., 2001a; Ishii et al., 2001; Roz et al., 2002). Fhit knockout mice (7/7) are, in general, healthy and fertile but have an increased susceptibility to spontaneous tumors and are exquisitely sensitive to carcinogens (Fong et al., 2000; Zanesi et al., 2001). However, the cancer sensitivity of Fhit knockout mice could be explained by some form of immune deficiency as 17 – 29% of Fhit (7/7) mice in contrast to Fhit (+/ +) mice showed evidence of infection (Zanesi et al., 2001). With a single NMBA (N-nitrosomethylbenzylamine) dose, around 80% of Fhit (+/7) and Fhit (7/ 7) mice have tumors of the forestomach, compared with fewer than 8% of wild-type mice. Susceptibility to spontaneous tumors was similar in Fhit+/7 and Fhit 7/7 mice. However, these mice did not develop any lung or kidney tumors. These results probably indicated that FHIT might be a haploinsufficient tumor suppressor in some tissues (Zanesi et al., 2001). Treatment of Fhit (+/7) mice, after NMBA exposure, with one oral dose of adeno-associated-FHIT virus (AAV-FHIT) caused a substantial reduction in size and number of tumors; 490% of control Fhit (+/7) mice showed forestomach tumors, whereas only 38% of AAV-FHIT treated mice developed these tumors (Dumon et al., 2001b; Huebner and Croce, 2001). Thus, a strong case can be made for FHIT as a TSG. However, other results are in conflict with this hypothesis (see Le Beau et al., 1998; Nelson et al., 1998; Tseng et al., 1999). Because of the enzymatic activity of FHIT (diadenosine triphosphate hydrolase activity) it was at first reasonable to consider that key

to the tumor suppressing function. However, ‘hydrolase dead’ FHIT mutant protein, with the central histidine of the triad mutated to asparagine (H96N), suppresses tumorigenicity just as well as wild-type FHIT (Siprashvili et al., 1997). Several investigators have transfected cancer cell lines with FHIT and have not seen suppression of cell growth in vitro or tumorigenicity in vivo (Otterson et al., 1998; Werner et al., 2000; Wu et al., 2000). FHIT coding exons can remain intact and expressed in cancer cells with deletions in FRA3B (Thiagalingam et al., 1996; Boldog et al., 1997; Wang et al., 1998; Muller et al., 1998). Boldog et al. (1997) analysed FRA3B region and found homozygous deletions in 90% of cervical and 50% of colorectal carcinoma cell lines and some of them did not involve FHIT exons. Moreover the same abnormal FHIT cDNA expression pattern detected by RT – PCR was observed in both normal and tumor cells (Boldog et al., 1997; Manning et al., 1999; van den Berg et al., 1997; Su et al., 2000). Familial t(3;8) and somatic t(3;12) chromosome translocations and FHIT The 3;8 chromosomal translocation, t(3;8)(p14.2;q24.1), was described in a family with classical features of hereditary renal cell carcinoma. Previous studies demonstrated that the 3p14.2 breakpoint interrupts FHIT in its 5’ noncoding region. It was shown that the 8q24.1 breakpoint region encodes a 664-aa multiple membrane spanning protein, TRC8, with similarity to the hereditary basal cell carcinoma/segment polarity gene, patched (Gemmill et al., 1998). This similarity involves two regions of patched, the putative sterolsensing domain and the second extracellular loop that participates in the binding of sonic hedgehog. In the 3;8 translocation, TRC8 is fused to FHIT and is disrupted within the sterol-sensing domain. In contrast, the FHIT coding region is maintained and expressed. In a series of sporadic renal carcinomas, an acquired TRC8 mutation was identified (Gemmill et al., 1998). The high mobility group protein gene, HMGIC, is implicated in a variety of salivary gland adenomas and other benign tumors. Thus, it is of interest that FHIT has also been involved in somatic rearrangement t(3;12) in a benign adenomas of the parotid gland (Geurts et al., 1997). Again this translocation produced a fusion HMGIC/FHIT transcript together with normal FHIT transcript. FOXP1 as a candidate TSG Recently isolation of a novel candidate TSG, FOXP1 (2.5 kb mRNA coding for 677aa, AF146696) from 3p14.1 (centromeric to FHIT) was reported (Banham et al., 2001). The FOXP1 protein sequence contains predicted domains characteristics of transcription factors, including a winged helix DNA-binding motif, a second potential DNA-binding motif, a C2H2 zinc finger, nuclear localization signals, coiled – coil regions, PEST sequences, and potential transactivation Oncogene


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domains. FOXP1 mRNA and protein are widely expressed in normal tissues and levels of FOXP1 mRNA were comparable in paired normal and tumor tissues from a variety of tissues and tumor types. FOXP1 was expressed in normal tissues while complete or reduced loss of expression seen in many tumors with 51% of samples showing a difference between tumor and normal tissues. In some cases increased expression was seen in tumors while in other cases there was cytoplasmic mislocalization of the predominantly nuclear FOXP1 protein. Analysis of the FOXP1 mRNA expression in normal tissues (not taken from cancer patients) indicated that loss of FOXP1 expression may occur in some histologically normal tissues adjacent to tumors. The region immediately telomeric to FHIT Several studies found that the region telomeric to FHIT gene is also a FAR (including homozygous deletions) for different epithelial cancers (Hu et al., 1996; Matsumoto et al., 1997; Orikasa et al., 1998). This region can also suppress tumorigenicity of a RCC cell line in nude mice and activate cellular senescence in vitro (Julicher et al., 1999). Thus, genes from this region will also need to be studied in lung cancer. Chromosome region 3p21.1-p21.2 (DRR1, BAP1, ARP) Various studies suggested that this region may contain a TSG as it was one of the FARs and homozygous deletions were detected here (see Kok et al., 1997; Buchhagen et al., 1994; Wistuba et al., 2000). Furthermore, it was also reported that this region suppressed the growth of tumor cells (Rimessi et al., 1994). Several candidate TSGs genes were isolated and suggested to be involved in the development of lung, kidney and other epithelial cancers. DRR1 as a candidate TSG DRR1 (downregulated in renal cell carcinoma) (also called TU3A) (NM_007177) from this region was identified (Yamato et al., 1999; Wang et al., 2000) which spans about 10 Kb of genomic DNA with a 3.5kb mature transcript expressed in many normal tissues including lung and kidney. The putative protein encoded by this gene is 144 amino acids and includes a nuclear localization signal and a coiled domain. Yamato et al. (1999) analysed 37 primary RCC and failed to find any mutations. However, two of five RCC cell lines totally lost DRR1 expression. Wang et al. (2000) found that DRR1 showed loss of expression in eight of eight RCC, one NSCLC and some other cancer cell lines. Southern blot analysis did not show any altered bands, indicating that gross structural changes or deletions did not cause the loss of expression. This gene was also found to have reduced expression in 23 of 34 paired primary renal cell carcinomas. Mutational analysis detected three polyOncogene

morphic sites within the gene, but no point mutations were identified in the 34 primary tumors. However, missense base substitutions were detected in five of 12 RCC and ovarian cell lines that had undetectable expression of the gene. Transfection of DRR1 into DRR1-negative RCC cell lines resulted in clear growth retardation. The authors suggested that loss of expression of the DRR1 may play an important role in the development of epithelial tumors and thus, this gene needs to be studied in lung cancer (Wang et al., 2000). BAP1 as a candidate TSG BRCA1 Associated Protein-1 (BAP1, NM_004656) binds to the RING finger domain of the Breast/ Ovarian Cancer Susceptibility Gene product, BRCA1 (Jensen et al., 1998). BAP1 (729 aa) is encoded by mRNAs of *4 and 4.8 kb that are expressed in variety of normal tissues. BAP1 is a nuclear-localized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING finger, but not to germline mutants of the BRCA1RING finger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially co-expressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. Intragenic homozygous rearrangements and deletions of BAP1 have been found in two (NCI-H226 and NCIH1466) of 77 lung carcinoma cell lines. Expression of BAP1 was lost only in the same two NSCLC cell lines. In gene replacement studies, BAP1 enhanced BRCA1mediated inhibition of breast cancer cell growth up to fourfold. It was suggested that BAP1 may be a new TSG which functions in the BRCA1 growth control pathway. ARP as a candidate TSG Arginine-rich protein (ARP, 1 kb transcript encodes 234 aa, NM_006010) is a highly conserved gene that maps to human chromosomal band 3p21.1 and is expressed in many normal and cancer tissues. This gene contains an imperfect trinucleotide repeat which encodes a string of arginines. A specific mutation (ATG50?AGG) within this region was detected in 10/ 21 RCC, 8/20 lung cancers (NSCLC and SCLC) and other solid tumors (Shridhar et al., 1996a,b, 1997). Rare mutations in other codons were also found. Other nucleotide changes were observed in a few PCR generated subclones, but their frequency was the same in both tumor and control samples, suggesting that many of these changes were PCR or subcloning artifacts rather than mutations in the tumor cells themselves. The wild-type allele was detected in many tumors (*50%). This is consistent with the notion that only a single copy of the gene is mutant in cancer cells. Data about aberrant transcription in cancer cells, growth inhibition or tumor suppression in vivo were not reported. The authors concluded that these results


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are compatible with the possibility that ARP could function either as a TSG or as an oncogene. Chromosome region 3p21.3C (centromeric) or lung cancer (LUCA) TSG region: a site of multiple potential TSGs This region appears to be the most FAR in lung and other epithelial cancers. It was found that fragment of human chromosome 3 containing sequences from this region has tumor suppressor activity in vivo (Killary et al., 1992; Daly et al., 1993; Cheng et al., 1998). Nested homozygous deletions in this region were detected in SCLC, NSCLC, breast, kidney and other epithelial tumors leading to delineation of a 630 kb region critical region with a working name of LUCA (lung cancer TSG region) (see Kok et al., 1997; Wei et al., 1996; Sekido et al., 1998; Zabarovsky et al., unpublished results). Loss of genetic material from this precise chromosome 3p21.3 by heterozygous or homozygous deletions is the earliest, most likely the first, genetic event observed in many types of lung pre-malignant lesions including histologically normal-looking, tobacco smoke-exposed, matching bronchial epithelium (Wistuba et al., 2000). Therefore, many researchers have formed a consortium to search for one or more TSGs in this defined region (see Lerman and Minna, 2000). Careful mapping of three homozygous deletions in SCLC cell lines, construction of a cosmid contig and complete genomic sequencing led to the detection, isolation, characterization, and annotation of a set of 25 genes that likely constitute the complete set of protein-coding genes residing in this smallest overlapping 630-kb sequence. A subset of 19 of these genes was found within the deleted overlap region of 370-kb. This region was further subdivided by a nesting 200-kb breast cancer homozygous deletion into two gene sets: eight genes lying in the proximal 120-kb segment and 11 genes lying in the distal 250-kb segment. These 19 genes were analysed extensively by computational methods and tested by experimental methods for loss of expression and mutations in lung cancers to identify candidate TSGs from within this group. All of the genes in the region have been shown to frequently undergo allele loss (LOH). However, none of the 19 genes tested for mutation showed a frequent (410%) mutation rate in lung cancer samples. Several of the genes showed loss-of-expression or reduced mRNA levels in NSCLC or SCLC cell lines. As it will be discussed further, several of the genes appear to be inactivated by tumor-acquired promoter hypermethylation. Alternatively, some may belong to the novel class of haploinsufficient genes that predispose to cancer in a hemizygous (+/7) state but do not show a second mutation in the remaining wild-type allele in the tumor. For several genes from this region additional functional data compatible with their role as TSGs has recently been obtained. The group of candidate tumor suppressor genes designated CACNA2D2 (AF042792, AF042793), PL6 (U09584), 101F6 (AF040704), NPRL2

(AF040707), BLU (U70880), RASSF1 (AF102770, AF040703), FUS1 (AF055479), HYAL2 (U09577), and HYAL1 (U03056) is defined by the nested lung and breast cancer homozygous deletions in the 120 kb region and along with the SEMA3B (U28369) gene that lies *50 kb telomeric of this region have received the first attention in promoter methylation and functional studies. These studies lead to the conclusion that multiple genes, often contiguous, have the properties of tumor suppressor genes for lung and other cancers. CACNA2D2 The calcium channel a2d2 subunit gene (CACNA2D2) encodes a functional calcium channel auxiliary regulatory subunit (Gao et al., 2000). The gene occupies *140-kb of genomic space, and is composed of at least 40 exons. It is expressed as a 5.5 – 5.7 kb mRNA (1146 aa). Three mRNA splice forms have been detected that code for two protein isoforms in several normal tissues. No mutations were detected in CACNA2D2 in detailed analysis of 100 lung cancers. In evaluating the potential of CACNA2D2 as a TSG its expression and ability to suppress the malignant phenotype of lung cancer cells was studied (Gao et al., submitted). CACNA2D2 mRNA is well expressed in normal human lung, while 64% of NSCLCs and 17% of SCLC lack CACNA2D2 mRNA and protein expression. The 2800 bp region 5’ and including the first exon of CACNA2D2 demonstrated promoter activity, and lack of expression of CACNA2D2 in lung cancer cells was associated with hypermethylation of CpG sites in this region. Furthermore, re-expression of CACNA2D2 was induced in lung tumor cell lines after 5’-aza-2’-deoxycytidine treatment. NSCLC cell line (NCI-H1299) stable transfectants expressing CACNA2D2 grew well in liquid culture but showed dramatically suppressed growth in soft agar and in nude mouse xenografts. From this information we conclude that CACNA2D2 is epigenetically inactivated in a large fraction of NSCLCs and in some SCLCs and can function to suppress the malignant phenotype of lung cancer cells in vitro and in vivo. Recently it has become clear that the mouse neurologic mutation ducky (du) represents a germline mutation of CACNA2D2. In addition, our group has created knockout mice for Cacna2D2 and these are being followed for the development of tumors (Ivanov et al., submitted). BLU This gene also residing in the 120 kb critical region is highly expressed in lung and testis and to a lesser extent in other tissues. The gene space of *4.5 kb contains 11 – 12 exons coding for a 2 kb mRNA (441 aa). It has two alternatively spliced isoforms (one being testis specific) both of which have a CpG island located in the 5’ end. It has sequence homology with MTG/ETO family of transcription factor proteins Oncogene


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and to suppressins which regulate entry into the cell cycle. Only three missense mutations in BLU were found in 61 lung cancer cell lines but many of these lung cancers showed loss of BLU mRNA expression. The BLU promoter region CpG islands were found to be methylated in *15% of 107 NSCLCs and in 5 – 20% of other human cancers such as kidney cancer and in a higher percentage of cancer cell lines including neuroblastomas (Agathanggelou et al., 2002) Promoter region DNA methylation was associated with down regulation of BLU expression which was reversed after 5-aza-2’ deoxycytidine treatment. Overexpression of BLU resulted in a decrease of colony formation in kidney cancer and neuroblastoma cell lines. Thus, BLU is involved in a small fraction of lung cancers and further functional studies of BLU are needed (Agathanggelou et al., 2002). RASSF1A The RASSF1 (initially called 123F2) gene can exist in different alternative splicing forms (at least six different isoforms). The gene space of 7.6 kb contains five exons coding for 2 kb alternatively spliced mRNAs (Wei et al., 1996; Lerman and Minna, 2000). Most common and probably the most important are RASSF1A and RASSF1C (Dammann et al., 2000; Burbee et al., 2001). They are driven from separate CpG-type promoters and share four terminal exons. The mRNA for both genes is well expressed in different normal tissues while the RASSF1C but not the RASSF1A mRNAs are well expressed in most lung cancers. Several studies have shown that this loss of RASSF1A expression occurs because of tumor acquired promoter DNA methylation. In addition, occasional missense mutations in RASSF1A have been reported. RASSF1A codes for 340 amino acids compared to 270 amino acids for the RASSF1C. The amino acid sequence of RASSF1A contains a predicted diacylglycerol (DAG) binding domain also found in the related gene NORE1 but not found in RASSF1C cDNA sequence. RASSF1A and RASSF1C are soluble cytoplasmic proteins that contain a Ras association domain (residues: 124 – 218). While not all Ras association domains bind RasGTP, the Ras association domain in the mouse paralog of RASSF1, NORE1 was found to bind RasGTP (Vavvas et al., 1998). Association of human RASSF1C and RASSF1A with RAS protein was also demonstrated (Vos et al., 2000; Ortiz-Vega et al., 2002). The NORE1 protein also contains the PKC-C1 and DAG/PE domains, that are found in the RASSF1A predicted protein but not in the RASSF1C protein. Recently, the Kastan group has identified a RASSF1 amino acid sequence (common to both the RASSF1C and 1A proteins) as a potential phosphorylation target for ATM (Kim et al., 1999). The presence of these functional domains suggests the RASSF1 proteins could play fundamental role(s) in signal transduction pathways from the cell surface to the nucleus. RASSF1A is silenced by promoter hypermethylation in over 90% of SCLCs and SCCs and in about 50% Oncogene

NSCLCs and is able to suppress growth of lung cancer cells in culture and tumor formation in mice (Dammann et al., 2000; Burbee et al., 2001). Furthermore, mutation in RASSF1A coding region significantly reduces its suppressor activity and this activity was demonstrated in a regulated manner, i.e. gene (and TSG activity) can be switched on and off using tetracycline-regulated system (Dreijerink et al., 2001; Protopopov et al., 2002; Kuzmin et al., 2002). Moreover, the gene is silenced in many other human cancers including kidney (Dreijerink et al., 2001; Morrissey et al., 2001), breast (Burbee et al., 2001; Dammann et al., 2001), nasopharyngeal (Lo et al., 2001), prostate (Maruyama et al., 2002; Kuzmin et al., 2002), bladder (Maruyama et al., 2001; Lee et al., 2001) and other cancers (Astuti et al., 2001; Agathanggelou et al., 2001). Shivakumar et al. (2002) found that RASSF1A can induce cell-cycle arrest by engaging the Rb-family cell cycle checkpoint. Re-expression in tumor cells of RASSF1A inhibited accumulation of native cyclin D1, and the RASSF1A-induced cell-cycle arrest was relieved by ectopic expression of cyclin D1 or of other downstream activators of the G1-S phase transition (cyclins A and E7). Regulation of cyclin D1 was responsive to native RASSF1A activity, as RNAi mediated downregulation of endogenous RASSF1A expression in human epithelial cells results in abnormal accumulation of cyclin D1 protein. Inhibition of cyclin D1 by RASSF1A occured post-transcriptionally and is likely at the level of translational control. Of importance, rare alleles of RASSF1A, isolated from tumor cell lines, encode proteins that fail to block cyclin D1 accumulation and cell cycle progression. These results strongly suggest that RASSF1A is an important human tumor suppressor protein acting at the level of G1 to S phase cell cycle progression. (Shivakumar et al., 2002). Overall these results suggest it is likely that RASSF1A is a TSG involved in the development or progression of a majority (probably about 75%) of human tumors. In lung cancer it is likely to play an early ‘gatekeeper’ role as one of the early events leading to pre-malignant lesions and then invasive cancer. FUS1 The FUS1 gene was isolated as the fusion (FUS=‘fusion’) junction of the ends part of a *30 kb homozygous deletion in SCLC NCI-H524 linking cosmid LUCA12 with cosmid LUCA13 sequences. (Despite its name, FUS1 does not ‘fuse’ two genes together.) The gene space of 3.3 kb contains three exons coding for a 1.8 kb mRNA that is well expressed in all analysed human tissues including lung, and in 20 lung cancer cell lines. Three mutations were discovered in 79 lung cancer cell lines DNAs leading to truncated products (Lerman and Minna, 2000). The FUS1 protein (110 amino acids) is probably a soluble cytoplasmic protein with no known domains or motifs. No evidence for FUS1 promoter region methylation in cancer cells was found. However, anti-Fus anti peptide


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antibody which readily detected exogenously expressed FUS1 failed to detect FUS1 protein in lung cancers (Kondo et al., 2001). Exogenously induced overexpression of FUS1 protein resulted in 60 – 80% inhibition of colony formation for NSCLC lines NCI-H1299 and NCI-H322 which did not express FUS1 gene. By contrast, a similar level of expression of a tumoracquired mutant form of FUS1 protein did not significantly suppress colony formation. Also, induced expression of Fus1 under the control of an Ecdysone regulated promoter decreased colony formation 75% and increased the doubling time twofold, and arrested H1299 cells in G1 (Kondo et al., 2001). FUS1 introduced by an adenovirus vector induced apoptosis in p53 wild-type A549 lung cancer cells and suppressed the xenograft growth of A549 and p53 null NCI-H1299 NSCLC cells both as a local tumor injection and following systemic administration to suppress the growth of metastatic disease in immunodeprived mice (Ji et al., 2002). Thus these data are consistent with the hypothesis that FUS1 functions as a 3p21.3 TSG probably in a haploinsufficient manner and FUS1 warrants further studies of its function in the pathogenesis of human cancers. HYAL2 The HYAL2 (working name LUCA2) gene also resides in the 120 kb critical 3p21.3 segment. The gene space of 2.8 kb contains three exons that encode a 2 kb mRNA (474 aa) well expressed in all analysed human tissues including lung, well expressed in lung cancer cell lines except SCLC line NCI-H524 due to a small (*30 kb) homozygous deletion/rearrangement. No mutations were detected in 40 lung cancer cell lines tested. The HYAL2 protein is a member of a large family of hyaluronidases and in fact the expressed recombinant protein was shown to have enzymatic activity (Lepperdinger et al., 1998). This gene has been identified as a cell-surface GPI-anchored receptor for jaagsiekte sheep retrovirus (JSRV) cell entry (see Angeloni and Lerman, 2001; Rai et al., 2001). JSRV is an ovine retrovirus. In sheep, it causes a contagious form of lung cancer that arises from epithelial cells in the lower airway including type II alveolar and bronchiolar epithelial cells (Palmarini et al., 1999). A recent study showed that antiserum directed against the JSRV capside protein cross-reacted with 30% of human pulmonary adenocarcinoma samples but not with normal lung tissue, or many adenocarcinoma samples from other tissues (De las Heras et al., 2000), supporting the proposition that related viruses may be involved in human lung cancer. While there is no known oncogene in JSRV, the Env of this virus could sequester HYAL2 and thus liberate an oncogenic factor negatively controlled by HYAL2 (Angeloni and Lerman, 2001). In this regard, it is important to remember that viral oncoproteins from both both SV40 and HPV viruses sequester TSG products, p53 and pRB inactivating the respective pathways. Additional experiments are needed to prove this hypothesis.

HYAL1 The HYAL1 gene, another hyaluronidase, is located in 250 kb segment of the 3p21.3 critical region. The gene space of *3.5 kb contains three exons coding for a 2.6 kb mRNA (436 aa) well expressed in all analysed human tissues including lung. However, it is not expressed in 18 out of 20 lung cancer cell lines. Two missense mutations were detected in 40 lung cancer cell lines. Triggs-Raine et al. (1999) identified two mutations in the HYAL1 alleles of a patient with newly described lysosomal disorder, mucopolysaccharidosis IX. So far, no increased incidence of cancer has been reported in these kindreds. Hyaluronan (HA) has been invoked as mechanisms for tumor invasion and metastatic spread. The levels of HA surrounding tumor cells often correlate with tumor aggressiveness and poor outcome (Zhang et al., 1995). Overproduction of HA enhances anchorage-independent tumor cell growth (Kosaki et al., 1999; Liu et al., 2001). Loss of hyaluronidase activity, permitting accumulation of HA, may be one of the several steps required by cells in the multi-step process of carcinogenesis (Csoka et al., 2001). Hyaluronidases would then qualify as candidate tumor suppressor gene products but no frequent inactivating mutations were identified so far. However, another inactivating mechanism was suggested and demonstrated in some oral cancers (Frost et al., 2000). The unusually large 5’ UTR, found by PCR, indicated the presence of a retained intron. Neither hyaluronidase enzyme activity nor Hyal-1 protein could be detected in a number of cancer cell lines, though the mRNA for HYAL1 was present. The retained intron prevented translation, possibly because of the large number of start and stop codons it contained. It is not known how widespread such a mode of gene silencing, epigenetic in nature is in carcinomas. In other malignancies such as breast cancer, hypermethylated CpG nucleotides in the 5’ HYAL1 promoter region have been identified by bisulfite sequencing, that correlates with loss of gene expression (Csoka et al., 2001). This loss of expression differed from previously examined oral malignancies, where transcripts with a retained intron predominated. HYAL2, a gene on 3p21.3 immediately centromeric to HYAL1, showed no similar loss of expression, ruling out a hypermethylation field effect. Treatment of the HYAL1 deficient breast carcinoma lines with the demethylating agent AZA and the histone deacetylase inhibitor, trichostatin-A (TSA) restored mRNA and protein expression. TSA proved more effective as a derepressing agent than AZA alone, and was independent of new protein synthesis as determined by cycloheximide pretreatment. Ectopic expression of HYAL1 in cell lines lacking a functional gene product resulted in cyclic arrest and/or apoptosis. Surviving clones were no longer tumorigenic in nude-mice (Angelborg et al., 2002). Loss of hyaluronidase might provide the cancer cell with the HA-rich environment that stimulates growth, movement, and metastatic spread. The absent expression in many cancer cell lines and occurrence of Oncogene


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mutations makes HYAL1 an attractive candidate for future promoter methylation and TSG functional studies in lung cancer. SEMA3B and SEMA3F Semaphorins/collapsins are a family of secreted and membrane-associated proteins involved in nerve growth cone migration. However, some are expressed widely in adult tissues suggesting additional functions. The semaphorin 3B (SEMA A/SEMA-V) gene is composed of 17 exons spread over 8 – 10 kb of genomic space coding for a 3.4 kb mRNA (750 aa) expressed in several normal tissues including lung and testis; and not expressed in many SCLC or NSCLCs lines (Sekido et al., 1996). Three missense mutations were found in 39 lung cancer cell lines; all mutations were in NSCLCs. The SEMA3B protein is an extracellular secreted protein. The second semaphorin gene (SEMA3F/SEMA IIIF/SEMA-IV) in the region was identified experimentally and cloned independently by several groups (Roche et al., 1996; Sekido et al., 1996; Xiang et al., 1996). The gene space of *28 kb (NM_004186) encodes a 3 and/or 4 kb mRNA (753 aa and 786 aa) composed of 18 exons. The gene is well expressed in several normal tissues including lung. Comparison of the cDNA sequences of Xiang et al. (1996) and Roche et al. (1996; U38276 and U33920) indicated that there are alternatively spliced forms of SEMA3F. SEMA3F was expressed in several but not all lung cancer cell lines (15/19, see Sekido et al., 1996). No mutations were found in 30 lung cancer lines by Sekido et al. (1996) testing 456 of the total 753 amino acids for mutations and in tests of the full open reading frame in 28 SCLC cell lines by Xiang et al. (1996). ProfileScan predicted an extracellular (secreted) protein containing several recognized domains. Interestingly, the PFAM: SEMA domain is also present in the extracellular part of the MET and RON oncoproteins belonging to the MET family of receptor tyrosine kinases (RTKs), as discovered by the PFAM (Bateman et al., 1999) program. Thus, it will be reasonable to test the hypothesis that interaction of SEMA3B and SEMA3F proteins with these oncogenes may disrupt the activation of MET and RON and therefore convey a negative growth signal or giving signal for invasive growth (Trusolino and Comoglio, 2002). On the other hand, SEMA3B and SEMA3F bind to Np-1 (neuropilin) and Np-2 receptors with high affinity (Fujisawa and Kitsukawa, 1998). Lung cancers (n=34) always express the neuropilin-1 receptor for secreted semaphorins, whereas 82% expressed the neuropilin-2 receptor (Tomizawa et al., 2001). Interestingly, the Np receptors serve as co-receptors for several isoforms of VEGFs (Fujisawa and Kitsukawa, 1998). VEGF is a very potent angiogenic as well as a mitogenic factor and has been found to be an essential initiator of tumor angiogenesis. Thus, SEMA3B/ SEMA3F action in tumorigenesis may also involve inhibition of tumor angiogenesis through interference Oncogene

with VEGF function (Keith et al., 2000; Tomizawa et al., 2001, Tse et al., 2002). To test the TSG candidacy of SEMA3B and SEMA3F, these genes were transfected into lung cancer NCI-H1299 cells, which do not express either gene (Tomizawa et al., 2001). Colony formation of H1299 cells was reduced 90% after transfection with wild-type SEMA3B compared with the control vector. By contrast, only 30 – 40% reduction in colony formation was seen after the transfection of SEMA3F or SEMA3B variants carrying lung cancer-associated single amino acid missense mutations. H1299 cells transfected with wild-type but not mutant SEMA3B underwent apoptosis. Conditioned medium from COS7 cells transfected with SEMA3B reduced the growth of several lung cancer lines 30 – 90%, whereas SEMA3B mutants or SEMA3F had little effect in the same assay. Sequencing of sodium bisulfite-treated DNA showed dense methylation of CpG sites in the SEMA3B 5’ region of lung cancers not expressing SEMA3B but no methylation in SEMA3B-expressing tumors. Similar results were obtained for SEMA3B by Tse et al., 2002 using HEY ovarian cancer cell line where they demonstrated that SEMA3B suppress tumor growth of human ovarian adenocarcinoma cell line HEY in nude mice. A P1 clone containing SEMA3F suppressed tumor growth of the mouse fibrosarcoma cell line A9 in nude mice (Todd et al., 1996). Brambilla et al. (2000) studied SEMA3F cellular localization using an anti-SEMA3F antibody. In normal lung, SEMA3F was found in all epithelial cells at the cytoplasmic membrane and, to a lesser extent, in the cytoplasm. In lung tumors, the localization was predominantly cytoplasmic, and the levels were comparatively reduced. In NSCLCs, low SEMA3F expression levels correlated with higher stage. In all tumors, an exclusive cytoplasmic localization of SEMA3F correlated with high levels of vascular endothelial growth factor and was related to the grade and aggressiveness. This suggests that vascular endothelial growth factor might compete with SEMA3F for binding to their common receptors, neuropilins-1 and -2 and might contribute to SEMA3F delocalization and deregulation in lung tumor. SEMA3F expression distribution was also examined in cell cultures by confocal microscopy. Marked staining was observed in pseudopods and in the leading edge or ruffling membranes of lamellipods or cellular protrusions in motile cells. SEMA3F was also observed at the interface of adjacent interacting cells suggesting a role in cell motility and cell adhesion. More recently these same authors have found that SEMA3F inhibited cell attachment and spreading in human mammary tumor cells indicating that the loss of SEMA3F expression in tumor cells may lead to increased metastatic properties (Roche et al., 2002). Exogenously expressed SEMA3F suppressed mouse fibrosarcoma line A9 xenograft growth, and also suprisingly blocked apoptosis induction in A9 cells, human ovarian adenocarcinoma cell line HEY but not human SCLC line GLC45 (Xiang et al., 2002.) These results are


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consistent with SEMA3B and potentially SEMA3F functioning as TSGs, and mean that these genes have to be considered in further functional and methylation analyses in lung cancer and other tumors. RBM5 and RBM6 Two genes (with the various names of G15/LUCA15/ RBM5 and G16/RBM6/DEF-3/NYLU12) with sequence homology to each other and which incode nuclear RNA binding proteins (RBM - RNA binding motif) were localized at the border of telomeric breakpoint in NCI-H740 SCLC cell line. The RBM5 gene (largest open reading frame, orf, is 815 aa protein, NM_005778) space of 18.5 kb encodes 18 exons expressed as a main 4 kb mRNA in several human tissues (a minor 7.5 kb and in some tissues 1.5 kb and 2 kb species are also expressed) and is well expressed in several lung cancer cell lines. No mutations were found in 18 lung cancer cell lines. The RBM6 gene (1123 aa, NM_005777) spans the distal breakpoint of the NCIH740 deletion (Wei et al., 1996), occupies more than 60 kb of genomic DNA, and encodes more than 16 exons expressed as a 4 kb mRNA in several human normal and lung cancer cell lines. Alternatively spliced forms have been described (Gure et al., 1998; Timmer et al., 1999). No mutations were found in 39 lung cancer cell lines. Timmer et al. (1999) using SSCP analysis of 16 lung cancer cell lines found a single presumably neutral mutation only in RBM5. However, by RT – PCR in the same study it was demonstrated the existence of two alternative splice variants of RBM6, one including and one excluding exon 5, in both normal lung tissue and lung cancer cell lines. Exclusion of exon 5 results in a frameshift which would cause a truncated protein of 520 amino acids instead of 1123 amino acids. In normal lung tissue, the relative amount of the shorter transcript was much greater than that in the lung tumor cell lines, which raised the question whether some tumor suppressor function may be attributed to the derived shorter protein. Serological analysis of a recombinant lung cancer cDNA expression library with the autologous patient serum led to an independent isolation of NYLU12 which turned out to be the RBM6 gene (Gure et al., 1998). Moreover these authors found that 2 of 21 lung cancer patients had anti-RBM6 antibodies. This fact raised the possibility that these antibodies can have antitumor effect (Drabkin et al., 1999). Interestingly, RNA recognition motifs (RRMs) of RBM5 and RBM6 show similarity to those of the Hu protein family. Paraneoplastic syndromes of neuropathy and encephalomyelitis are associated with high-titre antibodies directed against the RRMs of Hu-proteins (Dalmau et al., 1992). In the majority of cases, the associated tumor was lung cancer and most SCLC. However, RBM5 and RBM6 are immunologically distinct from the Hu proteins (Drabkin et al., 1999) and no paraneoplastic syndromes have yet been reported in patients with anti-RBM5 or anti-RBM6 antibodies.

Edamatsu et al. (2000) reported that in some of the human breast cancer tissues tested, mRNA levels of RBM5 were reduced. They suggested that changes of the expression level of the gene may be important for the tumor development. The authors observed that the mRNA level of RBM5 in the quiescent (serumstarved) Rat-1 cells is higher than that in the growing (not-starved) cells. This suggested that the expression of RBM5 may be down-regulated when the cells are growing, and that the expression may be up-regulated when cells are quiescent. The data in this work demonstrated that RBM5 is one of the downregulated genes in RAS-transformed cells, and suggested that the RBM5 may function as a negative regulator of cell proliferation by the alteration of its mRNA level. Moreover, ectopic overexpression of RBM5 in human fibrosarcoma HT1080 cells suppressed the cell growth in vitro up to 40%. RBM5 has different alternative splicing forms. Sutherland et al. (2000, 2001) reported that some of these splicing forms suppressed and other induced apoptosis in Jurkat T cells. These authors showed that RBM5 gene can be expressed in antisense orientation and this transcript inhibited apoptosis. These results suggested that RBM5 gene is involved in regulation of apoptosis and can have dual role as a TSG and oncogene. The lack of mutations and continued expression in most lung cancers means RBM5 and RBM6 are weak TSG candidates. Adenoviral transduction of 3p21.3 candidate TSGs (101F6, NPRL2, BLU, FUS1, HYAL2, HYAL1) The effects of six of these 3p21.3 genes (101F6, NPRL2, BLU, FUS1, HYAL2 and HYAL1) on tumor cell proliferation and apoptosis in human lung cancer cells was studied using recombinant adenovirusmediated gene transfer in vitro and in vivo (Ji et al., 2002). Forced expression of wild-type FUS1, 101F6, and NPRL2 genes significantly inhibited tumor cell growth by induction of apoptosis and alteration of cell cycle processes in 3p-deficient NCI-H1299 and A549 NSCLC cells, but not in the 3p-normal NCI-H358 NSCLC and normal human bronchial epithelial cells. Intratumoral injection of Ad-101F6, Ad-FUS1, AdNPRL2, and Ad-HYAL2 vectors or systemic administration of protamine-complexed vectors significantly suppressed growth of H1299 and A549 xenografts and inhibited A549 experimental lung metastases in nu/nu mice. Together, these results provide additional evidence that multiple genes in the 3p21.3 chromosomal region may exhibit tumor suppressor activity in vitro and in vivo. They also lead the way to clinical trials with these genes as systemically administered therapy for lung cancer. A pilot study evaluated whether 3p aberrations in the 3p21.3C region induced by benzo[a]pyrene diol epoxide (BPDE), the metabolic product of benzo[a]pyrene, a constituent of tobacco smoke, were more common in the peripheral blood lymphocytes of 40 lung cancer patients than they were in those of 54 Oncogene


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matched controls (Wu et al., 1998). The hypothesis was that 3p sensitivity to BPDE reflects the susceptibility of a specific locus to damage from carcinogens in tobacco smoke. BPDE- induced chromosome 3p21.3 aberrations were significantly more frequent in cases (34.1 per 1000) than they were in controls (22.1 per 1000; P50.0001). However, no such difference was observed for 6q27, a control locus. Using the median value in the controls (20 per 1000) as a cutoff point to classify BPDE-induced sensitivity at 3p21.3 and after adjustment by age, sex, ethnicity, and smoking status, 3p BPDE sensitivity was associated with an elevated risk of 14.1 (95% confidence interval: 3.5, 56.2) for lung cancer. There was also a dose- response relationship between the degree of BPDE sensitivity at 3p21.3 and increased risk for lung cancer. Therefore, this 3p21.3C region may be a molecular target for BPDE damage in lung cancer cases. Distal chromosome 3p21.3 regions LIMD1, LTF A region located distal to the 3p21.3 LUCA region between D3S32 and D3S2354, was reported to be frequently deleted in lung and other cancers (see Kok et al., 1997; Braga et al., 1999, 2002). By passaging mouse microcell hybrids (MCHs) containing human chromosome 3 (chr3) on A9 mouse fibrosarcoma background (mouse/human MCHs) through severe combined immunodeficient (SCID) mice it was found that this region was consistently eliminated from tumors growing as xenografts (elimination test) (Imreh et al., 1994; Kholodnyuk et al., 1997; Szeles et al., 1997). Fine mapping of the tumor xenograft deleted region resulted in identification of 1-Mb-long common eliminated region 1 (CER1) between D3S32 and D3S3582 at 3p21.3. A second eliminated region not always consistently deleted in tumor xenografts (ER2) at 3p21.1-p14 was also identified. Chromosome 3 was transferred by microcell fusion into the human nonpapillary renal cell carcinoma line KH39 that contained uniparentally disomic chromosome 3 to generate human/human MCHs. Compared with parental KH39 cells, these chromosome 3 microcell hybrids (MCHs) developed fewer and smaller tumors, which grew after longer latency periods in SCID mice. The tumors were analysed in comparison with corresponding MCHs. The results suggested that the human/human MCH-based elimination test identifies similar eliminated and retained regions on chromosome 3 as the human/murine MCH-based test (Yang et al., 2001a). From these data it was proposed that this region may contain genes antagonizing tumor growth. A detailed gene map for the region was constructed (Kiss et al., 2002) that included 19 active genes. Several genes can be considered as candidate TSGs on the basis of their structural and functional properties such as LIMD1 (LIM domain containing 1, AJ132408) and LTF (lactotransferrin, NM_002343). However, direct evidence of the involvement of any gene in tumor development, or Oncogene

tumor growth suppression activity in vitro or in vivo have not yet been reported. HD-PTP Close to this region two other genes were suggested as candidate TSGs. Slightly more centromeric HD-PTP (his-domain protein tyrosine phosphatase, AB025194) gene was localized. HD-PTP is highly similar to the rat PTP-TD14 gene which can suppress H-ras-mediated transformation. This gene encodes a polypeptide of at least 1636 aa although the true N-terminal methionine has not been definitively determined (Toyooka et al., 2000). A 6 kb mRNA was detected in every tissue examined indicating that the gene is ubiquitously expressed. In one of 12 SCLC cell lines with only one allele of HD-PTP a missence mutation was found. CTNNB1 Several megabases more distal, close to the centromeric border of the next FAR (see below), CTNNB1 (catenin beta 1-cadherin-associated protein) was mapped. The gene (781 aa) occupies more than 23 kb of genomic DNA, and is widely expressed in normal tissues. It is involved in the regulation of cell adhesion and in signal transduction through the wnt signaling pathway. CTNNB1 is engaged in a multiprotein complex that involves GSK, the TSG product APC and axin. It can heterodimerize with alphacatenin and also form a trimeric complex with cadherin. The beta-catenin gene (CTNNB1) has been shown to be genetically mutated to a dominant oncogenic form in various human malignancies, for instance colon, endometrium, thyroid, hepatoblastoma, uterine endometrioid carcinoma etc. (Blaker et al., 1999; Mirabelli-Primdahl et al., 1999; Ikeda et al., 2000; Schlosshauer et al., 2000; Garcia-Rostan et al., 2001). To determine whether the beta-catenin gene is responsible for oncogenesis in thoracic malignancies, 166 lung cancers (90 primary tumors and 76 cell lines) were searched for mutations (Shigemitsu et al., 2001). Four missense mutations in exon 3 were detected. One tested blastoma also had a somatic mutation from C to G, leading to a Ser to Cys substitution. One homozygous deletion in the NCIH28 cell line (involved all but one CTNNB1 exons) was found among 10 malignant mesotheliomas tested. Furthermore, Northern blot analysis of 26 lung cancer and eight mesothelioma cell line RNAs detected ubiquitous expression of the beta-catenin messages except NCI-H28, although Western blot analysis showed that relatively less amounts of protein products were expressed in some of lung cancer cell lines. The authors suggested that this gene is infrequently mutated in lung cancer and that the NCI-H28 homozygous deletion might indicate the possibility of a new tumor suppressor gene residing in this region at 3p21.3, where various types of human cancers show frequent allelic loss. In conclusion, CTNNB1 does not appear to be directly mutated in human lung cancer, but its expression may be deregulated by other changes in the wnt signaling pathway.


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Chromosome region 3p21.3T (telomeric) or AP20 region DLC1 Homozygous deletions in this region have been reported in five SCLC cell lines and three tumor biopsies (Murata et al., 1994; Roche et al., 1996). It was shown that this region is the most frequently hemyzygously and homozygously deleted region in CCRCC and other epithelial cancers (Alimov et al., 2000; Braga et al., 2002; Zabarovsky et al., unpublished results). Homozygous deletion in SCLC cell line ACCLC5 was carefully mapped and sequenced (Ishikawa et al., 1997). Four genes were found in this region but no evidence of their involvement in cancer development was reported. Further analysis led to the isolation of a new gene DLC1 or DLEC1 (deleted in lung cancer 1, Daigo et al., 1999a). This gene encodes 1755-amino acid polypeptide (NM_005106) and two main transcripts 6.0 and 8.0 kb were expressed in all examined normal tissues including lung and kidney. The gene consisted of at least 37 exons and numerous alternative splicing forms were present in different tissues. Mutational analysis of this gene by reverse transcription-PCR revealed the lack of functional transcripts and an increase of nonfunctional RNA transcripts in a significant proportion (33%) of cancer cell lines and primary cancers (four of 14 esophageal cancer cell lines, two of two renal cancer cell lines, 11 of 30 primary non-small cell lung cancers, and three of 10 primary squamous cell carcinomas of the esophagus). The abnormal transcription patterns of DLC1 in the tumors could be divided into two categories: (a) absence of normal transcript but presence of transcripts lacking one exon (exon 11 or 13) or containing an additional exon corresponding to part or all of intron 13, both mechanisms that disrupt the intact open reading frame; and (b) complete absence of DLC1 transcripts of any kind. In 20 primary cancer samples where abnormal expression was detected, 15 were classified as category (b) and five belonged to category (a), although no genetic alterations were detected in 5’ noncoding region, exons, or flanking introns, and no hypermethylation of the 5’ CpG island was detected in any of these cases. The mechanism that caused the loss of transcript(s) or aberrant splicing detected remains unclear. Introduction of the DLC1 cDNA significantly suppressed the growth of four of five different cancer cell lines, two of which produced no normal transcript on their own. No such effect occurred when DLC1 antisense cDNA, a cDNA corresponding to an aberrant transcript, or the vector DNA alone were transfected (Daigo et al., 1999a). Further studies of DLC1 tumor suppressing function in vivo are needed. Further sequencing of this region (Daigo et al., 1999b) resulted in identification and mapping of 14 genes but no evidence for their TSG activity was presented. In addition, we have located the border of a spontaneous deletion of human chromosome 3 in MCH939.2 human-mouse hybrid cell line that maps

exactly in this region (Kashuba et al., 1995). The physical map constructed with NotI jumping and linking clones revealed significant differences between our map and that of Daigo et al. (1999b) and the draft human genome sequence. Careful analysis of these differences resulted in identification of several new genes that are currently under analysis (Zabarovsky et al., unpublished results). Chromosome region 3p24-p26 VHL Several reports suggested that 3p25-p26 region may contain TSGs, although no candidate gene was reported. Frequent LOH and presence of homozygous deletions were reported here in lung, renal, breast and other solid tumors (Hu et al., 1996; Waber et al., 1996; Braga et al., 1999, 2002; Alimov et al., 2000; Hirano et al., 2001; Matsumoto et al., 2000). It was also reported that this region suppressed the growth of tumor cells (Rimessi et al., 1994) Moreover, for some cancers it was demonstrated that deletions here have prognostic value (Matsumoto et al., 2000; Hirano et al., 2001). The VHL gene located in this 3p25 region (213 aa; AF010238) at 3p25.3, associated with familial kidney cancer and multiple benign tumors of the retina, brain, and pancreas, was cloned in 1993 (Latif et al., 1993) and its function has been studied extensively. The mRNA is ubiquitously and abundantly expressed as a 4.8 – 5 kb species in adult and fetal tissues and cell lines. Two mRNAs are generated by alternative splicing of exon 2 and expressed in different normal tissues, including kidney. VHL is likely to have multiple and tissue specific functions. It is a recognition component of an E3 ubiquitin-protein ligase that targets HIF-1alpha for proteasomal degradation. VHL is also required for regulation of the HIF-1 transcriptional control and forms a complex with elongins B, C, and other proteins (Semenza, 2002). As expected for a ‘classic’ tumorsuppressor gene, both VHL alleles are inactivated through a combination of deletion, mutation, or promoter methylation mechanisms in the familial kidney cancers and in the majority of sporadic kidney cancers. However, mice with two inactivated Vhl alleles die early in embryogenesis; mice with one inactivated allele have no observable phenotype and therefore Vhl knockout mice are not a useful models for VHL disease in the human (Gnarra et al., 1997). It is believed that VHL is a cancer-specific gene (Waber et al., 1996). On the other hand it was found that patients with 3p-syndrome with deleted VHL gene never developed evidence of VHL disease (Drumheller et al., 1996). It is well known that HIF-1 activity is crucial for many cancers and thus the conclusion that VHL gene is important only for a limited number of cancers, may be, is premature (Ivanov et al., 2001). Nevertheless, detailed studies of lung cancer failed to find any mutation or expression Oncogene


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abnormalities (Sekido et al., 1994). Thus, it is unlikely that VHL is directly involved in abnormalities in lung cancer. RARb Several groups suggested that retinoic acid receptor beta (RAR-beta, NM_000965, 455 aa, 3p24) that functions as a receptor for retinoic acid (RA) may have TSG function and is involved in lung carcinogenesis. RARb is expressed in many normal tissues including lung and kidney. Retinoic acid (RA) possess profound effects in the control of many biological processes such as development, differentiation and cell proliferation (Lotan, 1980). Epidemiological studies demonstrated that RA has tumor suppressive effect (Graham, 1984). RARb has four alternative splicing forms and the beta-2 form appears to possess tumorantagonizing activity at least in some cancers. While RARb is not mutated it undergoes epigenetic inactivation by promoter methylation in tumors. Thus, it was reported that RARb underwent DNA promoter region methylation in 72% of SCLC and 41% of NSCLC (Virmani et al., 2000; Zo¨chbauer-Muller et al., 2001a) and in significant percent of several other tumors: breast (Yang et al., 2001b), cervical (Virmani et al., 2001), bladder (Maruyama et al., 2001), prostate (Maruyama et al., 2002). While Virmani et al. (2000) found reactivation of expression of RARb after treatment of lung cancers with AZA, Yang et al. (2001c) found no correlation between the hypermethylation and RAR-b2 loss of expression. Although no cancer associated mutations was found in lung cancers, most lung tumor lines in contrast to normal lung tissue were resistant to RA and did not show growth arrest after treatment with this ligand (Geradts et al., 1993). This contradiction can be now explained by loss of RARb expression by DNA promoter methylation. Moreover, it was demonstrated that RARb2 has in vitro growth suppression activity in lung cancer cell lines (Houle et al., 1993; Toulouse et al., 2000) and transgenic mice expressing antisense RARb2 transcripts develop lung tumors in contrast to nontransgenic control mice. Furthermore, a twofold higher incidence of lung tumors was seen in homozygous vs hemizygous antisense mice (Berard et al., 1996). Thus, RARb appears to have many of the characteristics of a TSG that is epigenetically inactivated in lung cancer. Conclusions Development of SCLC and NSCLC tumors is a complex process involving sometimes more than 20 genes from different chromosomes (Geradts et al., 1999; Girard et al., 2000; Zo¨chbauer-Muller et al., 2002). These genes belong to different classes of cancerassociated and cancer-causing genes: tumor suppressors, oncogenes, genes involved in apoptosis, angiogenesis, other processes. Despite significant progress achieved during the last few years we are still Oncogene

far away from a clear understanding of interaction between all genes involved in lung carcinogenesis. However, a large volume of information obtained during the past decade allows us to look with optimism toward the future. We know that human chromosome 3 contains several tumor suppressor genes some of which are cancer specific and others are common for different cancer types. It is clear that SCLC and NSCLC have different origins and various TSGs are involved in their development (Girard et al., 2000; Guo et al., 2000; Wistuba et al., 2001; Virmani et al., 2002; Braga et al., 1999, 2002; Zabarovsky et al., unpublished results). Identification and precise localization of these TSGs on human chromosome 3 was hampered by different obstacles. These problems were caused by inter-tumor heterogeneity and other technical problems. For instance, different techniques can be used to study promoter methylation and this can lead to slightly different results (Damman et al., 2000; Burbee et al., 2001; Dreijerink et al., 2001; Morrissey et al., 2001; Agathanggelou et al., 2001). Growth inhibition experiments using vectors with constitutive expression produce pharmacologic rather than physiologic levels of protein and therefore experiments with regulated expression are more valuable (Drejerink et al., 2001; Kondo et al., 2001; Kuzmin et al., 2002; Protopopov et al., 2002). LOH analysis detected allelic imbalance (AI) that can result both from amplification and from deletions. During the deletion mapping all such AI were considered as LOH that marked position of TSG. Human chromosome 3p probably contains many genes that can play a dominant oncogenic role. MST1 receptor (RON) and its ligand MST1 gene are located in 3p21 and their overproduction can result in autocrine stimulation and uncontrolled proliferation (Angeloni and Lerman, 2001). Therefore, amplification of some 3p21.3 regions can be induced by potential oncogenes contrary to the expected deletion of TSGs. It is important to mention that during the initial search for chromosome 3-specific TSGs one of the most important mechanisms for their inactivation, DNA promoter methylation was not tested because of lack of advances in this field. These and other new technological possibilities together with human genome draft sequence suggest that other major players involved in lung cancer development will be identified soon. Among chromosome 3-specific TSGs some probably will conform to the classical two-hit model requiring inactivation/or loss/or silencing of both alleles of the gene (homozygous, 7/7, phenotype), while others may function as haploinsufficient TSGs one allele of which is still expressed in the tumor (hemyzygous, 7/ +phenotype). Since the acquisition of other genetic modifications is still rate-limiting in causing cancer (Knudson, 1995), another mutation in a different gene would appear to be required in the haploinsufficient cancer model. It is tempting to assume that it could be a mutation in a second known TSG such as p16, p53, and RB, all of which are frequently mutated in many common cancers bearing 3p21.3 allele loss, including lung cancers. Consistently, allele loss at these genetic


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loci (at 9p21, 13q14 and 17p13) occurs after 3p allele loss at a later stage of carcinogenesis when histologically dysplastic or carcinoma in situ lesions become evident. The following model (Figure 2) of lung cancer pathogenesis based on these developing concepts and accumulated evidence was proposed (Zo¨chbauerMuller et al., 2002; Wistuba et al., 2001; Angeloni and Lerman, 2001). Consequent to smoking (or other environmental pollutants) damage, 3p21.3 allele loss occurs in thousands of different sites throughout the respiratory epithelium leaving the putative 3p21.3 TSGs haploinsufficient. These initiated (3p21.3 hemizygous) cells proliferate and spread throughout the lung epithelium. The next hit could occur either in the second allele of a classical TSG (e.g. RASSF1A) or, as required in the haploinsufficient model, in another cancer-causing gene (such as RB, p53, p16 or ‘gene X’), leading to the next stages of invasive cancer. In the classical homozygous model the 3p21.3 TSG first undergoes allele loss and then a second inactivating event (either as uncommon mutation or loss of expression through promoter hypermethylation) which is required to allow the clonal outgrowth of these initiated cells. Alternatively, the first event can be methylation inactivation of an initiating TSG following hemizygous deletion. Anyway, 3p21.3 allele loss in

premalignant lesions and the nearly universal loss of 3p21.3 DNA in SCLC and squamous cell carcinoma of the lung (SCC), and its occurrence in over 75% of adenocarcinomas of the lung, suggest that these deletions are obligatory, rate-limiting steps in the pathogenesis of many lung cancers, if not all. It should also be noted that allele loss that includes 3p21.3 and the immediately surrounding 3p21 regions would lead to a condition of hemyzygosity for many other predisposing genes residing in the area. These include MLH1, TGFRII, RARb, CTNNB1, WNT5, etc. which also could contribute to malignant transformation. Functional testing by gene transfer into lung cancer cells and gene disruption strategies in mice are necessary to test the theoretical model and discover all putative TSGs in 3p21.3, and also to produce mouse models of lung cancer. Such experiments are now in progress. It was also suggested (Figure 2) that in SCLC, in which no characteristic preneoplastic sequence of morphological changes has been described, tumors may arise directly either from normal or hyperplastic epithelia without passing through recognizable intermediate pathologic stages (parallel theory of lung cancer development). In contrast NSCLC would appear to develop after a sequence of morphologic intermediate steps (sequential theory) (Wistuba et al., 2001, Angeloni and Lerman, 2001).

Figure 2 Scheme explaining parallel and sequential hypotheses of different origin of SCLC and NSCLC. More intense colour reflects malignant progression, ‘3p’ – 3p deletions, ‘m’ designates methylation of chromosome 3-specific TSGs Oncogene


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Recent data suggest that significant progress in the early diagnosis of lung cancer can be achieved with methods based on detecting acquired abnormalities of gene-specific methylation (Wistuba et al., 2001; Adorjan et al., 2002; Zo¨chbauer-Muller et al., 2002; Li et al., 2002). Thus Palmisano et al. (2000) reported that aberrant methylation of the p16 and/or O6methyl-guanine-DNA methyltransferase (MGMT) promoters can be detected in DNA from sputum in 100% of patients with squamous cell lung carcinoma up to 3 years before clinical diagnosis. Such improved detection of people at greatest risk of lung cancer could greatly reduce mortality from this disease. In fact, the prognosis for patients with lung cancer strongly correlates with the stage of the disease at the time of presentation. Therefore, early identification and intervention strategies stemming from these developments are expected to improve the usually poor prognosis of this fatal disease even before efficient medical treatment of the disease will be developed. Because of the frequent occurrence of tumor acquired promoter methylation of 3p TSGs, reversal of this epigenetic effect with demethylating agents such as AZA with or without histone deacetylase inhibitors also offers a rationale treatment strategy for lung and other cancers. Finally, therapeutic trials in lung cancer patients with systemically administered 3p TSGs are being designed.

Abbreviations AI, allelic imbalance; aa, amino acid; BPDE, benzo[a]pyrene diol epoxide; CC-RCC, clear cell renal cell carcinomas; CER1, common eliminated region; FAR, frequently affected with rearrangements region; LOH, loss of heterozygosity; NSCLC, non-small cell lung cancer; RA, retinoic acid; SCLC, small cell lung cancer; TSA, trichostatin-A; TSG, tumor suppressor gene; AZA, 5-aza-2’-deoxycytidine; CGH,

comparative genome hybridization; SNP, single nucleotide polymorphism; FISH, fluorescent in situ hybridization

Acknowledgments We thank the many collaborators that made up the 3p lung cancer tumor suppressor gene consortium, particularly Yoshitaka Sekido and Farida Latif for all of their hard work in the initial identification of the 3p21.3 genes. The membership of the consortium includes: University of Texas Southwestern Medical Center at Dallas, and MD Anderson Cancer Center, Houston, USA Group: Yoshitaka Sekido, Scott Bader, David Burbee, Kwun Fong, Eva Forgacs, Boning Gao, Harold Garner, Adi F Gazdar, Luc Girard, Craig Kamibayashi, Masashi Kondo, Ashwini Pande, Vivek Ramalingam, Dwight Randle, Yoshio Tomizawa, Arvind Virmani, Ivan Wistuba and John D Minna (Dallas), and Lin Ji, Bingling Fang, Neely Atkinson, and Jack A Roth (Houston). University of Birmingham, UK Group: Donia Macartney, Sofia Honorio, Eamonn Maher and Farida Latif. The Karolinska Institute, Sweden, Group: Eleonora Braga, Vladimir Kashuba, Alexei Protopopov, Vera Senchenko, Jingfeng Li, Fuli Wang, Veronika Zabarovska, George Klein, and Eugene R Zabarovsky. NCI, USA Group: (1) Fuh-Mei Duh, Ming-Hui Wei, Laura Geil, Igor Kuzmin, Sergei Ivanov, Intramural Research Support Program, SAIC Frederick, and Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702; (2) Debora Angeloni, Alla Ivanova, Bert Zbar, Michael I Lerman, Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702; (3): Bruce E Johnson, Medicine Branch at the National Naval Medical Center, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. ER Zabarovsky was supported by research grants from the Swedish Cancer Society, the Swedish Research Council, STINT, Pharmacia Corporation and Karolinska Institute. JD Minna was supported by Lung Cancer SPORE P50 CA70907, and CA71618.

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