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ICA~CER RESEARCH $7, 2~21-2123..lure Advances in Brief Association between Cigarette Smoking and FHIT Gene Alterations in Lung Cancer1 Gabriella Sozzi,2 Laura Sard, Laura De Gregorio, Antonio Marchetti, Katia Musso, Fiamma Buttitta, Silvana Tornielli, Siivia Pellegrini, Maria Luisa Veronese, Giacomo Manenti, Matteo Incarbone, Antonio Chella, Carlo A. Angelettl, Ugo Pastorino, Kay Huebner, Generoso Bevilaqua, Silvana Pilotti, Carlo M. Croee, and Marco A. Pierotti Divisions of Experimental Oncology A [G. S.. !.. S., I.. D. G., K. M., S. T., G. M., M. A. P.] Anatomical Pathology [S. Pi.], and Chest Surgery. [M. L ] lstimto Nazionale Tumorl, Via Venezian 1, 20133 Milan, and Departmems of Oncology [A. M., F. B., S. Pc.] and Surgery [A. C., C. A. A., G. B.], Universit,~ di Pisa, Pisa, Italy; Royal Bromptan Hospital, London. United Kingdom [U. P.]: and Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Penn~.'lvania 19107 [M. L. V., K. H., C. M. C.] Abstract Patients and Methods Epidemiologtc data have strongly indicated that cigarette smoking is linked to the development of lung cancer. However, little is known of the molecular targets of carcinogens contained in tobacco smoke. To identify genetic lesions characteristic of tobacco damage, we undertook a molec- ular analysis of microsateliite alterations within the FHIT gene and FRA3B, as well as at an independent locus on chromosome 10, D10S197, in lung tumors from heavy smokers and in tumors from never smokers. Loss of heterozygosity affecting at least one locus of the FHIT gene was observed in 41 of 51 tumors in the smokers group (80%) but in only 9 of' 40 tumors in nonsmokers (22%). The comparison between the frequency of losses in FHIT in smokers and nonsmokers was statistically significant (P = 0.0001), whereas no difference in Io~ of heterozygosity rate was observed at D10S197 locus. These findings suggest that FHIT is a candi- date molecular target of carcinogens contained in tobacco smoke. Introduction Smoking is recognized as a major cause of cancer-related death worldwide (1). Lung cancer, which represents the most common tumor type in men, is directly associated with tobacco smoking (2), and lung tumors in never smokers account for only 5-10% of all lung cancer. Carcinogens in cigarette smoke may leave "fingerprints" in the bronchial tissue in the form of specific mutations that initiate cancer development. The recently cloned FHIT gene at 3p14.2 con- tains the most common fragile site of the human genome, FRA3B (3). FHIT, a 5',5"-Pl,P3-triphosphate hydrolase (4), is a putative tumor suppressor gene. Given the concordance between the occurrence of LOH3 affecting microsatellite markers within the FHIT gehe and abnormal FHIT transcripts in tobacco-related cancers such as lung (5) and head and neck tumors (6, 7), loss of one FHIT allele is likely to be a crucial step leading to loss of function of the gene. Abnormalities of the FHIT gene in carcinogen-related tumors provided the fir~.t molecular evidence linking the instability of fragile sites to cancer. Here, we undertook a molecular study of FHIT and RA3B micro- satellite alterations in lung tumors from heavy smokers and in tumors developed in never smokers to seek genetic damage attributable to tobacco smoking. Received 3/26/97; accepted ~7. The costs of publication of ~ article were defrayed in part by the payment of page charges. This article must therefm~ be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely Io indicate this fact. ' This work was supported by grants from the Italian Association for Cancer Research and Special Project Applicazioni Cliniche delh Ricerca Oncologiea of the Italian National Research Council. L. S. is an Italian Association for Cancer Research Fellow. 2 To whom requests for reprin~ should be addressed. Phone: 39-2-2390232; lax: 39-2-2390764: E-mail: sozzi@istitutotumod.mi.it. 3 The abbreviations used are: LOH, loss of betemzygusity; Bfa)P, benzo~a)pyrene. Patient and Sample Collection. Tumor specimens were obtained from surgically resected lung cancer patients at Istituto Nazionale Tumori (Milan, Italy) and Universit~ di Pisa (Pisa, Italy). The minors were classified according • to the WHO Histological Typing of Long Tumors (8) and staged according to the TNM classification of malignant tumors defined by the International Union against Cancer. Among tumors in smokers, 31 were in stage I, 9 were in stage II, and 11 were in stage III, whereas in nonsmokers, 16 tumors were in stage I, 9 were in stage I1, and 15 were in stage HI. The mean ages of patients at presentation were 62 in smokers and 58 in nonsmokers. Matched normal lung parenchyma tissue samples were taken at a most distant site of the tumor or in a different segment or lobe as a source for the normal DNA. LOH Analysis. DNAs were extracted from frozen tumor and normal tissues using standard methods (9). Analysis of allelic losses was performed using a PCR-based approach (5). Primers that amplify polymorphic microsat- ellite markers were used for the following loci: D3S4103, D3S1300, and D3S1234. all internal to the FHIT gene, and Di0S197 on the short arm of chromosome 10. The sequences of all nucleotide primers are available through. the C-enome Data Base. We carried out 22 cycles of amplification at 57-60"C annealing temperature, as appropriate for each primer. Products were separated in 6% urca-polyacrylamide gels, and autoradiography was then performed. For informative cases, allelie loss was scored if the autoradiographic signal of one allele was approximatelff 50% reduced in the tumor DNA, compared with the corresponding normal allele. The loci displaying microsatellite instability were not scored for allelie loss. Results and Discussion LOH at D3S1300 and D3S4103 microsatellite markers, located in the epicenter of the fragile region encompassing exon 5 and intron 5 of the FHIT gene (Fig. 1), and at D3S1234, in the mor[ distal 3' end of the gene, was analyzed in tumor tissues. To test the effect of smoking at a genomic region other then 3p14.2, we scored LOH at the D10S197 locus on the short arm of chromosome 10. We found LOH affecting at least one locus of the FHIT gene in 41 of 51 (80%) tumors in the smokers group (80%), whereas only 9 of 40 nonsmokers (22%) showed FHIT allelic losses in tumor DNA (Fig. 2). The comparison between the frequency of losses in smokers and nonsmokers was statistically significant (80 versus 22%; P = 0.0001). The results did not change following adjustment for histological type (73 versus 22% of losses in adenocarcinoma from smokers and nonsmokers, respectively; P = 0.0001). All the tumors with loss of one FHIT marker had lost all the informative 0actcrozygous) markers (Fig. 2), suggesting that the tumor cells had lost an entire FH/T allele. No difference in the LOH rate was found at locus D10S197 be- tween smokers (7 of 33 informative cases; 21%) and nonsmokers (5 of 27 informative cases, 19%). These observations indicate that the 2121 C)

FH/T ALTERATIONS IN TUMORS OF SMOKERS AND NONSMOKERS Telomere. I EIOE9 E8 E7 E6 <~,~,o ~ Centrome.re ~ ............ L_.J. 5' E5 E4 E3 E2E1 Fig. ]. FElT gene organization showing the position of the internal microsat¢llite markers and a FRA3B site represented by the hybrid clone 3 (cI3) break. I, FHIT protein-coding exons; r-i, untranslated exons. preferential involvement of the FHIT gene in smokers is a specific event and not a result of a more general genotoxic effect of tobacco smoke. These data indicate that FRA3B is a preferential target of tobacco smoke damage at a molecular level, although we can not exclude involvement of other 3p loci because we have not delineated the extent of the deletion on the short arm of chromosome 3. It is well known that LOH on 3p in lung cancer generally involves most of 3p, complicating identification of specific target regions. The level of LOH at the FHITgene in lung cancer from heavy smokers, among the highest observed for other markers on 3p and for other tumor sup- pressor genes, and the extent of allelic losses, involving not only markers in the epicenter of the fragile region (10), but also the more distal D3S1234 marker (Fig. 1), strongly implicate FHITas a target of carcinogens contained in tobacco smoke. Notably, an accurate history of smoking exposure in six of eight nonsmoker patients with FHIT abnormalities revealed a significant exposure to passive smoke, either at home or at work. The lower incidence of FHIT genetic alterations in lung tumors from riever smokers indicates" that different somatic or inherited genetic mechanisms could underlie cancer development in these pa- tients. On the other hand, the frequency of mutations in the p53 gene among lung tumors in smokers was similar to that reported among nonsmokers (11-13). However, a significant relationship between p53 mutation and cigarette ~moke is indicated by the type of mutations detected in smokers' tumors, G:C to T:A transversions, whereas C:T to A:T transitions are more frequent in tumors from nonsmokers (14), Tumors from smokers Tumors from non smokers CASES pter D3S1234 D3S4103 D3S1300 can llADC 2/SCLC 3/ADC 4/ADC 51SQC 6ISCLC 71ADC 81SCLC 91SQC 10/ADC 11/SQC 121SQC 131ADC 14/SCLC 15/SQC 16/SQC 171SQC 18/SQC 19/A D C 20/SQC 21/LC 22/ADC 231ADC 24/ADC 2~ILC 261ADC 27/ADC 28/ADC 291SQC 301SQC 31/SQC 32/ADC 33/LC 341SQC 35/ADC 361ADC 37/ADC 38/ADC 39/ADC 40/ADC 41/ADC 421ADC 43/ADC 44/ADC 451SQC 46/5QC 471ADC 48/ADC 49/SQC 50/ADC 511SQC CASES pter D3St234 D3S4t03 D3S1300 ten 1lINT 3/INT 4lINT 5lINT 6lINT 7lINT 9lINT 10lINT 11lINT I~/INT 13lINT 16lINT 18lINT 2qlINT 14/INT 19lINT 4941P 4471P 402/P 2811P 961P 811P 50glP 484/P 4421P 390/P 2331P 1321P 891P 611P 500/P 460/P 4041P 488/P 2221P 1201P 88/P 39/P 22/INT 8/RBH Fig. 2. Results of micmsatellite analysis of the 51 lung tumors in smokers and of the 40 lung tumors in nonsmokers with three polymorphic markers internal to the FHIT gcnc. Min, mierosatellite instability, tie, not evaluable, nd, not done; O, hetemzygous; O, LOH; @, not informative. 2122

El'liT ALTERATIONS IN TUMORS suggesting that mutational mechanisms involving DNA polymerase infidelity, deamination of 5-methylcytosine, and spontaneous depuri- nation could play a role in lung tumor development in nonsmokers. Tobacco smoke contains a mixture of highly mutagenic compounds such as polycyclic aromatic hydrocarbons; in particular, B(a)P, a major constituent of tobacco smoke, has been reported to be one of the most potent carcinogenic compounds in vivo and in vitro (15). Ben- zo(a)pyrene diol-epoxide, the ultimate carcinogen of B(a)P and the most reactive with DNA, specifically binds guanine-rich sequences of active genes and induces fragile sites (16). In vitro evidence for B(a)P-induced formation of DNA adducts at the major mutational hot spots of the p53 gene in human lung cancer has recently been provided (17). Taken together, these results provide a direct link between specific genetic alterations and exposure to tobacco carcin- ogens. In lung cancer, aberrant FHIT transcripts, lacking key coding exons, and LOH, afecfing raicrosatellite markers within the FHIT gene, have been detected in >70% of all types of lung cancer (5). Moreover, the occurrence of LOH at 3p14.2 (18, 19) and FHIT gene alterations in precancerous lesions and nonneoplastic bronchial mu- cosa4 argue in favor of FHIT deledon as an early molecular event in lung carcinogenesis, The ability to perform routine microsatelfite analyses of cytological specimens will allow use of these genetic changes as early molecular indicators of damage related to tobacco smoke in screening high-risk individuals, such as those belonging to the heavy smokers category. Acknowledgments We thank Mario A~zini for the art work and Anna Grassi for secretarial assistance. References 1. IARC. Tobacco Smoking. Monographs on the Evaluntion of the Carcinogenic Risk of Chemicals to Humans. IARC $ci. Publ., 38: 35-394. 1986. 2. Shopland" D. R.. Eyre, H. I., and Puchacek, T. F.. Smoking-aun'butable cancer mortality in 1991: is lung cancer now the leading cause of death among ~moke~ in the United States.'? J. Nail. Cancer Inst.. (Bethesd~), 83:1142-114.8, 1991. 4 G. Sozzi. unpublished ~esults. OF SMOKER~ AND NONSMOKERS 3. Ohi~ M., Indue, H., Cotticelli. M. G., Kastury. K., Barfs, R.. Pal~.zo, .I.. Siprashvili, Z,, Mod, M., McCue, P., D~uck. T., Croce, C. M.. and Huebner, K. The FHIT gene, sl~nning the chromosome 3p14.2 fragile site and renal carcinoma-t~ocia~ed b~t' is abnormal in digestive tract cancer. Cell. 84: 587-597, 1996, 4. Bame~ L D.. Garrison. P. N.0 Siprashvill, Z.. Guranowski. A.. Robinson, A. Ingrem. S. W.. Croce. C. M., Otha. M.. and Hnebner. K. FHIT. a putative tumor supp~e~z~" in humans, is a dinuclcoside 5',5"-Pt.PLttiphosphate hydmlase. Bio- chemistry. 35: 11529-11535. 1996. 5. So~i. G., Vcronese. M. L., Ncgrini. M.. Baffa, R., Cotticelli, M. G., Inouc. Toraielli, S., Pilotti, S., De Gregodo, L.. Pastotinn, U., PicroRi, M. A., Othm M., Hnebner, K., and Croce, C. M. The FHIT gene at 3p14.2 is abnormni in lung cancer. Cell, 85: 17-26, 1996. 6. Virgilio, L, Shuster. M., Gollin, S. M.. Veronese, M. L., Ohia, M., Huebnex, IC, and Crocc, C. M. FHIT gene alterations in head and neck squamons cell carcinomas. Proc. Nail. Acad. Sci. USA, 93: 9770--9775, 1996. 7. Mao, L, Fan, Y-H., Lotan, R., and Hong, W. IC Frequent abnormalities of FHIT, a ~mdidate tumor suppressor gene, in head and neck cancer cell lines. Cancer Res., 56: 5128-5131, 1996. 8. WHO. Histological Typing of Lung Tumors. Geneva: WHO, 1981. 9. Sambrook, J., Frhsch, E. F., and Maniafis, T. (eds.). Molecular Cloning:. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Ha~eor Laboratory, 1989. I0. Zimonji¢, D. B., Druck, T., Ohta, M., Kastury, K.. Croce, C. M., popesun, N. C., and Huebner. K, Positions of chromosome 3pl4.2 fragile sites (FRA3B) wiilfm the FHIT gene. Cancer Res., 57: 1166-1170, 1997. I 1. Suzuki, H., Takabashi, T., Kuroishi, T., Suyama, M., Ariynshi, Y., and Ueda, R. 1)53 mutafiuns in nun-smO! cell lung cancer in Japan: association between mutations and smoking. Cancer Rf~,:~" 734-736, 1992. 12. Takabashi, T., Stm/Id,~H~, Hida, T., Selddo, Y., AriyoshL Y, and Ueda, R. The p53 geae is very frequently mutated in small-cell lung cancer with a distinct nucleotide suhsiltution pattern. Oncogene, 6: 1775-1778, 1991. 13. Takeshima, Y., Seyam.a, T., Bennett, W. P., Akiyama, M., Tokuoka, S., Inni, K., Mabuchi, K., Land, C. E., and Harris, C. C. p53 mutations in lung cancers from non-smoking atomic-bomb survivors (Published en'atum in Lancet, 343: 1302, 1994). Lancet, 342: 1520-1521, 1993. 14. Hollstein, M., Sidransky, H., Vogelstein, B., and Hanis, C. C. p53 mutations in human cancen. Science (Washington DC), 253: 49-53, 1991. 15. Hecht. S. S., Carmelia, S. G., Murphy, S. E., Folles, P. G., and Chung, F. L Carcinogen biomerkers related to smoking and upper aerodigestive tract cancer. J. Cell. Biochem. Suppl. 17F: 27-35, 1993. 16. Ytmis, J. J., Soreag. A. L., and Bowe. A. E. Fragile sites are t~gets of diverse mutagens and carcinogens. Oncogene, l: 59-69, 1987. 17. Danissanko, M. F., Pan, A., Tang, M., and Pfeifef, G. P. Preferential formation of beazo[o~]pyrene adducts at lung cancer mutational hctspots in P53. Science (Wash- ington DC), 274: 430-432, 1996. .18. Hung. J., Kishimoto, Y., Sugio, K., Virmaai, A., Mcinthe, D., I~finna, J. D., and Gazdar, A. F. Allele-specific chromosome 3p deletions occur at an early stage in the pathogenesis of lung carcinoma. J. Am. Med. Assoc., 273: 1928, 1995. 19. Sundaresan, V., Ganly, P.. Haselton, P., Rudd" R., Sinha, G., Bieahen, N. M., and Rabbits, P. p53 and obmmosome 3 abnormalities, characteristic of malignant lung tumoers, are detectable in preinvasive lesions of the b~onchus. O~cogene, 7: 1989- 1997, 1992. 2123