Tohoku
J. Exp.
The
Med., 1992,
168, 239-245
Biology
Molecular
of Lung
Cancer
ADI F. GAZDAR Simmons Cancer Center, and Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA
GAZDAR,A.F. The Molecular Biology of Lung Cancer. Tohoku J. Exp. Med., 1992, 168 (2), 239-245 Lung cancer arises atter a series of morphological changes, which take several years to progress from normal epithelium to invasive cancer. The morphological changes progress from hyperplasia, to metaplasia, to dysplasia, to carcinoma in situ, to invasive cancer and finally to metastatic cancer. Multiple molecular changes have been documanted in lung cancers, both small cell (SCLC) and non-small cell (NSCLC) types. The number of changes has been estimated to be in double digits. These changes include activation of dominant oncogenes myc family, (K-ras and neu genes), as well as loss of recessive growth regulatory genes or anti-oncogenes (p53, and RB as well as unidentified gene or genes on chromosome 3). However, cytogenetic and molecular genetic studies indicate that multiple other specific sites of actual or potential DNA loss may be present in lung cancers. Other changes may include development of drug resistance, and production of growth factors and their receptors. It is tempting to associate specific molecular changes with specific morphological changes, as has been attempted in the colon. However, because of the difficulties in serially sampling the respiratory tract, such studies have not been performed to date. Documentation of molecular changes in premalignant lesions and prospective studies of their prognostic effects will be necessary for the design of rational chemoprevention trials. anti-oncogenes ; K-ras ; lung cancer ; myc family ; neu
It is generally accepted that most adult epithelial cancers do not arise de novo, and that carcinogenesis is a multistep process. For centrally arising lung cancers, the morphological steps include hyperplasia, metaplasia, dysplasia, carcinoma in situ (CIS), invasive carcinoma and metastatic carcinoma (Saccomanno et al. 1974). Lung cancers, both SCLC and NSCLC, are characterized by multiple genetic changes, including activation or over expression of dominantly acting oncogenes, as well as loss or mutation of growth regulatory genes (anti-oncogenes). Specifically, these changes include loss or inactivation of genes on chromosomes 3p (unknown gene or genes), 13q (retinoblastoma gene), Pip (p53 gene) and multiple other sites. Activation or over expression of myc, ras and neu genes have also been associated with subsets of SOLO and/or NSCLC. It is tempting to speculate that specific genetic changes are associated with specific morphological alterations. Address
for reprints
; 5323
Harry
Hines
Blvd., 239
Dallas,
Texas
75235-8590,
USA.
240
A.F. Gazdar
Attempts to demonstrate such a sequence in colon cancer have resulted in the identification of early and late appearing changes (Fearon and Vogelstein 1990). There is little or no knowledge about the sequence of events in lung cancer, because identification and sequential sampling of premalignant lesions is difficult. In this report we will discuss some of the dominant (oncogene) and recessive (anti-oncogene) changes associated with lung cancer. ras gene family The ras gene family consists of three genes, K-, H- and N-ras which encode similar 2lkD membrane bound proteins. These proteins bind guanine nucleotide, have GTPase activity and are involved in signal transduction pathways (Per 1989 ; Barbacid 1990). The ras genes can be activated by point mutations at codon 12, 13, or 61, which result in an enhanced ability to retain its GTP-bound form and oncogenic potential. A large number of human tumors have been screened for mutated ras genes and the incidence and type of mutations varies considerably between different tumor types. More than 90% of pancreatic cancers and 50% of colon cancers have an activated K-ras gene, whereas about 20% of seminomas have an activated N-ras gene. In primary resected NSCLC tumors, the incidence of mutations is about 20%, mostly in adenocarcinomas, which have an incidence of about 30% (Rodenhuis and Slebos 1990; Suzuki et al. 1990). We screened a panel of 103 human lung cancer cell lines for the presence of point mutations at codons 12,13 or 61 of the human K-, H- and N-ras genes, using restriction fragment length polymorphisms (RFLP), created through mismatched primers during polymerase chain reaction (PCR) of genomic DNA (Mitsudomi et al. 1991b). We found ras mutations in 22/61 (36%) non-small-cell lung cancer (NSCLC) cell lines, predominantly in K-ras colon 12. Identical mutations were present in uncultured tumor materials corresponding to 11 cell lines containing mutated ras genes. ras mutations were found not only in adenocarcinoma cell lines (9/32, 28%), but also in cell lines derived from other types of NSCLC (13/ 29, 45%). In contrast, none of 37 small-cell lung cancer (SCLC) cell lines and five extrapulmonary small-cell cancer cell lines had ras mutations. ras mutations were not correlated with sex of the patients, tumor extent, prior therapy status or in vitro culture time. G to T or A to T transversions were the most common base substitutions, occurring in codons 12 and 61, respectively. We conclude that ras mutations play a role in the pathogenesis of a subset of NSCLC but are not involved in SOLO. Slebos et al. reported that ras mutations in early stage NSCLC tumors is an important negative prognostic factor. We have confirmed and extended these findings (Mitsudomi et al. 1991a). By univariate analysis of 66 NSCLC lines, the presence of ras mutations was associated with a shortened survival for patients who received curative intent (p2=0.002) as well as for patients who received palliative therapy (p2 =0.01). The Cox proportional hazards model also predict-
The
ed a higher
risk for patients
Molecular
Biology
with any type
of Lung
Cancer
of ras mutations.
present in a subset of NSCLC cancers are independently survival, irrespective of treatment intent.
241
Thus, associated
ras mutations, with
shortened
myc genes and lung cancer In contrast to ras genes, which are activated by point mutations in the open reading frame, deregulated or over expression of an apparently normal myc protein appears to be the usual mechanism associated with tumors. While dysregulation may occur by several methods, the usual mechanism in SOLO is amplification of one of the myc family genes. Amplification is associated with prior chemotherapy. c-myc amplification is associated with the variant form of SOLO, which is characterized by altered morphology, rapid in vitro growth, radioresistance and shortened survival (Little et al. 1983; Gazdar et al. 1985; Brennan et al. 1991). However, over expression may occur with or without amplification in both SOLO and NSCLC (Takahashi et al. 1989b ; Saksela 1990). The clinical and biological effects (if any) of over expression without amplification are not known. The neu gene and lung cancer The neu protooncogene is a recently described transforming gene originally isolated from a rat neuroblastoma. It is frequently amplified in breast carcinomas, and occasionally in other epithelial tumors. Amplification of c-erbB-2 may contribute to the pathogenesis of some forms of node-negative breast cancer and thus may serve as a useful genetic marker to identify a subset of high-risk patients (Paterson et al. 1991). The neu protein product (pl85neu) was present in eight of 22 non-small carcinoma cell lines derived from human lung tumors (Weiner et al. 1990). Expression of pl85neu was found in all histological subtypes of non-small cell carcinomas including large cell carcinomas, squamous cell carcinomas, and adenocarcinomas. The protein is expressed in normal ciliated bronchial epithelium of the peripheral airways expressed pl85neu at low levels. Neoplastic cells in four of 12 adenocarcinomas and three of five squamous cell carcinomas also expressed pl85neu at levels higher than the normal ciliated bronchial epithelium. Together these studies indicate that pl85neu expression is a common feature of human lung tumors. Overexpression of neu protein in lung adenocarcinomas (but not in squamous cell tumors) may be a negative prognostic factor (Kern et al. 1990).
The retinoblastoma (RB) gene and lung cancer Abnormalities of the retinoblastoma gene or protein have been found in the majority of SOLOtumors and cell lines, and in a subset of NSCLC (Yokota et al. 1988; Hensel et al. 1990). As with p53, the RB gene (located at 13814) encodes a nuclear phosphoprotein with DNA and viral oncoprotein binding activities.
242
A.F. Gazdar
RFLP analyses indicate that nearly all SCLC tumors demonstrate loss of heterozygosity at 13q, but are present in about 30% of NSCLC (Yokota et al. 1987). While most SCLC lines demonstrate no RB protein expression, NSCLC lines show more variable patterns, including relatively normal expression, absent expression or predominant or sole expression of the nonphosphorylated band (M. Nau, J. Minna and author's unpublished data). Many mutant forms are underphosphorylated and fail to bind to SV 40 T antigen. Phosphorylation is cell cycle dependent (Mihara et al. 1989), and T antigen binds only to the unphosphorylated form. The p53 gene and lung cancer Mutations of the p53 gene are the most frequent genetic abnormality found in human cancers to date (Harris 1990). The gene lies on chromosome band 17p13 and has been examined in a wide variety of primary tumors and cell lines derived from tumors. Of these, colon carcinomas have been studies in greater detail : 7580% showed a "loss" of both p53 alleles, one through deletion and the other through a point mutation. The point mutations are usually missense, giving rise to an altered protein. Similarly, the use of polymorphic DNA probes for chromosome 17p markers demonstrate loss of heterozygosity in approximately 60% of NSCLC and almost 100% of SCLC (Takahashi et al. 1989a; Chiba et al. 1990). Several of the mutant p53 genes have been cloned and the resulting cDNA and genomic clones tested for their biological properties. The wild-type allele can suppress the growth of transformed cells in culture and the formation of tumors in animals. Low to modest amounts of wild-type (WT) p53 protein are expressed in the nucleus of all normal dividing cells. WT protein has other important properties, including the ability to bind to several viral oncoproteins. Many mutant proteins have conformational changes resulting in long half lives, and consequently their levels are increased many fold. Some mutant proteins fail to enter the nucleus, and localize to the perinuclear areas. Mutants vary in their efficiency in cooperating with mutant ras genes in transforming primary rat cells in culture. Mutant p53 may gain a new function that overcomes the negative regulation by small quantities of wild-type p53 thus acting to stimulate cell growth and tumor formation. Alternatively, mutant protein could form an oligomeric complex with wide-type p53 resulting in loss of normal function. The ratio of mutant to wild type p53 in a cell could be critical in regulating cell division. Mutant proteins lose the ability to bind oncoproteins, but acquire the ability to bind to heat shock protein 70. Concluding The
remarks molecular
changes
complex
and
multiple.
recessive
anti-oncogenes.
that
These Other
precede changes changes
the
development
involve may
both include
of lung
dominant development
cancer
are
oncogenes
and
of drug
resis-
The
Molecular
Biology
of Lung
Cancer
243
tance, and production of growth factors and their receptors. The estimated number of mutations required for the development of invasive cancer is in double digits (Minna et al. 1990). How can so many lesions develop in a single cell ? One possible explanation is a so-called "mutator phenotype", with some (?early) mutations predisposing to the development of other changes. Another possible explanation is the "field cancerization" theory (Slaughter et al. 1954). The latter developed from observations that the surface changes in head and neck cancers were extensive, and that the incidence of synchronous, metachronous or second malignancies was very high, suggesting that much or all of the epithelium has undergone one or more genetic alterations (presumably as the result of repeated exposure to carcinogens such as tobacco), and is at increased risk for developing multiple, independent morphological alterations and malignancies. The concept of field cancerization is equally applicable to the respiratory tract. Lung cancers may be associated with extensive surface changes (Carter 1978), and secondary primaries are relatively frequent (Boice and Fraumeni 1985; Thomas et al. 1990). The annual incidence of second malignancies after resections for stage I NSCLC is 2-5% (Thomas et al. 1990). It is tempting to associate specific molecular changes with specific morphological changes, as has been attempted in the colon. However, because of the difficulties in serially sampling the respiratory tract, such studies have not been performed to date. Documentation of molecular changes in premalignant lesions and prospective clinical studies of their prognostic effects will be necessary for the design of rational chemoprevention trials. References 1) Barbacid, M. (1990) ras oncogenes : Their role in neoplasia. Eur. J. Clin. Invest., 20, 225-235. 2) Boice, JD. & Fraumeni, J.F. (1985) Second cancer following cancer of the respiratory system in Connecticut, 1935-1982. Natl. Cancer Inst. Monogr., 68, 83-98. 3) Brennan, J., O'Connor, T., Makuchi, R.W., Simmons, A.M., Russell, E., Linnoila, R.I., Phelps, R.M., Gazdar, A.F., Ihde, D.C. & Johnson, BE. (1991) myc family DNA amplification in 107 tumors and tumor cell lines from patients with small cell lung cancer treated with different combination chemotherapy regimens. Cancer Res., 51, 1708-1712. 4) Carter, D. (1978) Pathology of the early squamous cell carcinoma of the lung. Pathol. Annu., 13, 131-147. i) Chiba, I., Takahashi, T., Nau, M.M., D'Amico, D., Curiel, D., Mitsudomi, T., Buchhagen, D., Carbone, D., Koga, H., Reissmann, P., Slamon, D., Homes, E. & Minna, J. (1990) Mutations in the p53 gene are frequent in primary, resected non-small cell lung cancer. Oncogene,5, 1603-1610. 6) Der, C.J. (1989) The ras family of oncogenes. Cancer Treat. Res., 47, 73-119. 7) Fearon, ER. & Vogelstein, B. (1990) A genetic model for colorectal tumorigenesis. Cell, 61, 759-767. 8) Gazdar, A.F., Carney, D.N., Nau, M.M. & Minna, J.D. (1985) Characterization of variant subclasses of cell lines derived from small cell lung cancer having distinctive biochemical, morphological and growth properties. Cancer Res., 45, 2924-2930. 9) Harris, A.L. (1990) Mutant p53-the commonest genetic abnormality in human can-
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cer ? [editorial] . J. Pathol., 162, 5-6. 10) Hensel, C.H., Hsieh, C.L., Gazdar, A.F., Johnson, B.E., Sakaguchi, AY., Naylor, S.L., Lee, W.-H. & Lee, E.Y.-H.P. (1990) Altered structure and expression of the human retinoblastoma susceptibility gene in small cell lung cancer. Cancer Res., 50, 30673072. 11) Kern, J.A., Schwarts, D.A., Nordberg, J.E., Weiner, D.B., Greene, MI., Torney, L. & Robinson, R.A. (1990) pl85neu expression in human lung adenocarcinomas predicts shortened survival. Cancer Res., 50, 5184-5187. 12) Little, C.D., Nau, MM., Carney, D.N., Gazdar, A.F. & Minna, J.D. (1983) Amplification and expression of the c-myc oncogene in human lung cancer cell lines. Nature, 306, 194-196. 13) Mihara, K., Cao, X.R., Yen, A., Chandler, S., Driscoll, B., Murphree, AL., T'Ang, A. & Fung, Y.K. (1989) Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science,246, 1300-1303. 14) Minna, J.D., Nau, MM., Takahashi, T., Shutte, J., Chiba, I., Viallet, J., Kaye, F., Whang-Peng, J., Oie, H., Russell, E. & Gazdar, A.F. (1990) Molecular pathogenesis of lung cancer. In : Molecular Mechanisms and Their Clinical Applications in Malignancies, edited by D.E. Bergsagel & T.W. Mak, Academic Press, New York, pp. 63-83. 15) Mitsudomi, T., Steinberg, S.M., Oie, HK., Mulshine, J.L., Phelps, R., Viallet, J., Pass, H., Minna, J.D. & Gazdar, A.F. (1991a) ras gene mutations in non-small cell lung cancers are associated with shortened survival irrespective of treatment intent. Cancer Res., 51, 4999-5002. 16) Mitsudomi, T., Viallet, J., Mulshine, J.L., Linnoila, R.I., Minna, J.D. & Gazdar, A.F. (1991b) Mutations of ras genes distinguish a subset of non-small-cell lung cancer lines from small cell lung cancer cell lines. Oncogene,6, 1353-1362. 17) Paterson, M.C., Dietrich, K.D., Danyluk, J., Paterson, A.H.G., Lees, A.W., Jamil, N., Hanson, J., Jenkins, H., Krause, B.E., McBlain, W.A., Slamon, D.J. & Fourney, R.M. (1991) Correlation between c-erbB-2 amplication and risk of recurrent disease in node-negative breast cancer. Cancer Res., 51, 556-567. 18) Rodenhuis, S. & Slebos, R.J. (1990) The ras oncogenes in human lung cancer. Am. Rev. Respir. Dis., 142, S27-S30. 19) Saccomanno, G., Archer, V.E., Auerbach, 0., Saunders, R.P. & Brenan, L.M. (1974) Development of carcinoma of the lung as reflected in exfoliated cells. Cancer, 33, 256-270. 20) Saksela, K. (1990) myc genes and their deregulation in lung cancer. J. Cell. Biochem., 42, 153-180. 21) Slaughter, D.P., Southwick, H.W. & Smejkal, W. (1954) "Field cancerization" in oral stratified squamous epithelium : Clinical implications of multicentric origin. Cancer, 6, 963-968. 22) Suzuki, Y., Orita, M., Shiraishi, M., Hayashi, K. & Sekiya, T. (1990) Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene,5, 1037-1043. 23) Takahashi, T., Nau, MM., Chiba, I., Birrer, M., Rosenberg, R., Vinocour, M., Levitt, M., Pass, H., Gazdar, A.F. & Minna, J. (1989a) p53: A frequent target for genetic abnormalities in lung cancer. Science,246, 491-494. 24) Takahashi, T., Obata, Y., Sekido, Y., Hida, T., Ueda, R., Watanabe, H., Ariyoshi, Y., Sugiura, T. & Takahashi, T. (1989b) Expression and amplification of myc gene family in small cell lung cancer and its relation to biological characteristics. Cancer Res., 49, 2683-2688. 25) Thomas, P., Rubinstein, L. & Lung Cancer Study Group (1990) Cancer recurrence after resection : T1N0 non-small cell lung cancer. Ann. Thorac. Surg., 49, 242-246. 26) Weiner, D.B., Nordberg, J., Robinson, R., Nowell, P.C., Gazdar, A.F., Greene, MI.,
The Molecular Biology of Lung Cancer
27)
28)
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Williams, W.V., Cohen, J.A. & Kern, J.A. (1990) Expression of the neu geneencoded protein (pl85neu) in human non-small cell carcinomas of the lung. Cancer Res., 50, 421-425. Yokota, J., Wada, M., Shimosato, Y., Terada, M. & Sugimura, T. (1987) Loss of heterozygosity on chromosomes 3, 13, and 17 in small-cell carcinoma and on chromosome 3 in adenocarcinoma of the lung. Proc. Natl. Acad. Sci. USA, 84, 9252-9256. Yokota, J., Akiyama, T., Fung, Y.-K., Benedict, W.F., Namba, Y., Hanaoka, M., Wada, M., Terasaki, T., Shimosato, Y., Sugimura, T. & Terada, M. (1988) Altered expression of the retinoblastoma (RB) gene in small cell carcinoma of the lung. Oncogene,3, 471-475.
J. Exp.
The
Med., 1992,
168, 239-245
Biology
Molecular
of Lung
Cancer
ADI F. GAZDAR Simmons Cancer Center, and Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA
GAZDAR,A.F. The Molecular Biology of Lung Cancer. Tohoku J. Exp. Med., 1992, 168 (2), 239-245 Lung cancer arises atter a series of morphological changes, which take several years to progress from normal epithelium to invasive cancer. The morphological changes progress from hyperplasia, to metaplasia, to dysplasia, to carcinoma in situ, to invasive cancer and finally to metastatic cancer. Multiple molecular changes have been documanted in lung cancers, both small cell (SCLC) and non-small cell (NSCLC) types. The number of changes has been estimated to be in double digits. These changes include activation of dominant oncogenes myc family, (K-ras and neu genes), as well as loss of recessive growth regulatory genes or anti-oncogenes (p53, and RB as well as unidentified gene or genes on chromosome 3). However, cytogenetic and molecular genetic studies indicate that multiple other specific sites of actual or potential DNA loss may be present in lung cancers. Other changes may include development of drug resistance, and production of growth factors and their receptors. It is tempting to associate specific molecular changes with specific morphological changes, as has been attempted in the colon. However, because of the difficulties in serially sampling the respiratory tract, such studies have not been performed to date. Documentation of molecular changes in premalignant lesions and prospective studies of their prognostic effects will be necessary for the design of rational chemoprevention trials. anti-oncogenes ; K-ras ; lung cancer ; myc family ; neu
It is generally accepted that most adult epithelial cancers do not arise de novo, and that carcinogenesis is a multistep process. For centrally arising lung cancers, the morphological steps include hyperplasia, metaplasia, dysplasia, carcinoma in situ (CIS), invasive carcinoma and metastatic carcinoma (Saccomanno et al. 1974). Lung cancers, both SCLC and NSCLC, are characterized by multiple genetic changes, including activation or over expression of dominantly acting oncogenes, as well as loss or mutation of growth regulatory genes (anti-oncogenes). Specifically, these changes include loss or inactivation of genes on chromosomes 3p (unknown gene or genes), 13q (retinoblastoma gene), Pip (p53 gene) and multiple other sites. Activation or over expression of myc, ras and neu genes have also been associated with subsets of SOLO and/or NSCLC. It is tempting to speculate that specific genetic changes are associated with specific morphological alterations. Address
for reprints
; 5323
Harry
Hines
Blvd., 239
Dallas,
Texas
75235-8590,
USA.
240
A.F. Gazdar
Attempts to demonstrate such a sequence in colon cancer have resulted in the identification of early and late appearing changes (Fearon and Vogelstein 1990). There is little or no knowledge about the sequence of events in lung cancer, because identification and sequential sampling of premalignant lesions is difficult. In this report we will discuss some of the dominant (oncogene) and recessive (anti-oncogene) changes associated with lung cancer. ras gene family The ras gene family consists of three genes, K-, H- and N-ras which encode similar 2lkD membrane bound proteins. These proteins bind guanine nucleotide, have GTPase activity and are involved in signal transduction pathways (Per 1989 ; Barbacid 1990). The ras genes can be activated by point mutations at codon 12, 13, or 61, which result in an enhanced ability to retain its GTP-bound form and oncogenic potential. A large number of human tumors have been screened for mutated ras genes and the incidence and type of mutations varies considerably between different tumor types. More than 90% of pancreatic cancers and 50% of colon cancers have an activated K-ras gene, whereas about 20% of seminomas have an activated N-ras gene. In primary resected NSCLC tumors, the incidence of mutations is about 20%, mostly in adenocarcinomas, which have an incidence of about 30% (Rodenhuis and Slebos 1990; Suzuki et al. 1990). We screened a panel of 103 human lung cancer cell lines for the presence of point mutations at codons 12,13 or 61 of the human K-, H- and N-ras genes, using restriction fragment length polymorphisms (RFLP), created through mismatched primers during polymerase chain reaction (PCR) of genomic DNA (Mitsudomi et al. 1991b). We found ras mutations in 22/61 (36%) non-small-cell lung cancer (NSCLC) cell lines, predominantly in K-ras colon 12. Identical mutations were present in uncultured tumor materials corresponding to 11 cell lines containing mutated ras genes. ras mutations were found not only in adenocarcinoma cell lines (9/32, 28%), but also in cell lines derived from other types of NSCLC (13/ 29, 45%). In contrast, none of 37 small-cell lung cancer (SCLC) cell lines and five extrapulmonary small-cell cancer cell lines had ras mutations. ras mutations were not correlated with sex of the patients, tumor extent, prior therapy status or in vitro culture time. G to T or A to T transversions were the most common base substitutions, occurring in codons 12 and 61, respectively. We conclude that ras mutations play a role in the pathogenesis of a subset of NSCLC but are not involved in SOLO. Slebos et al. reported that ras mutations in early stage NSCLC tumors is an important negative prognostic factor. We have confirmed and extended these findings (Mitsudomi et al. 1991a). By univariate analysis of 66 NSCLC lines, the presence of ras mutations was associated with a shortened survival for patients who received curative intent (p2=0.002) as well as for patients who received palliative therapy (p2 =0.01). The Cox proportional hazards model also predict-
The
ed a higher
risk for patients
Molecular
Biology
with any type
of Lung
Cancer
of ras mutations.
present in a subset of NSCLC cancers are independently survival, irrespective of treatment intent.
241
Thus, associated
ras mutations, with
shortened
myc genes and lung cancer In contrast to ras genes, which are activated by point mutations in the open reading frame, deregulated or over expression of an apparently normal myc protein appears to be the usual mechanism associated with tumors. While dysregulation may occur by several methods, the usual mechanism in SOLO is amplification of one of the myc family genes. Amplification is associated with prior chemotherapy. c-myc amplification is associated with the variant form of SOLO, which is characterized by altered morphology, rapid in vitro growth, radioresistance and shortened survival (Little et al. 1983; Gazdar et al. 1985; Brennan et al. 1991). However, over expression may occur with or without amplification in both SOLO and NSCLC (Takahashi et al. 1989b ; Saksela 1990). The clinical and biological effects (if any) of over expression without amplification are not known. The neu gene and lung cancer The neu protooncogene is a recently described transforming gene originally isolated from a rat neuroblastoma. It is frequently amplified in breast carcinomas, and occasionally in other epithelial tumors. Amplification of c-erbB-2 may contribute to the pathogenesis of some forms of node-negative breast cancer and thus may serve as a useful genetic marker to identify a subset of high-risk patients (Paterson et al. 1991). The neu protein product (pl85neu) was present in eight of 22 non-small carcinoma cell lines derived from human lung tumors (Weiner et al. 1990). Expression of pl85neu was found in all histological subtypes of non-small cell carcinomas including large cell carcinomas, squamous cell carcinomas, and adenocarcinomas. The protein is expressed in normal ciliated bronchial epithelium of the peripheral airways expressed pl85neu at low levels. Neoplastic cells in four of 12 adenocarcinomas and three of five squamous cell carcinomas also expressed pl85neu at levels higher than the normal ciliated bronchial epithelium. Together these studies indicate that pl85neu expression is a common feature of human lung tumors. Overexpression of neu protein in lung adenocarcinomas (but not in squamous cell tumors) may be a negative prognostic factor (Kern et al. 1990).
The retinoblastoma (RB) gene and lung cancer Abnormalities of the retinoblastoma gene or protein have been found in the majority of SOLOtumors and cell lines, and in a subset of NSCLC (Yokota et al. 1988; Hensel et al. 1990). As with p53, the RB gene (located at 13814) encodes a nuclear phosphoprotein with DNA and viral oncoprotein binding activities.
242
A.F. Gazdar
RFLP analyses indicate that nearly all SCLC tumors demonstrate loss of heterozygosity at 13q, but are present in about 30% of NSCLC (Yokota et al. 1987). While most SCLC lines demonstrate no RB protein expression, NSCLC lines show more variable patterns, including relatively normal expression, absent expression or predominant or sole expression of the nonphosphorylated band (M. Nau, J. Minna and author's unpublished data). Many mutant forms are underphosphorylated and fail to bind to SV 40 T antigen. Phosphorylation is cell cycle dependent (Mihara et al. 1989), and T antigen binds only to the unphosphorylated form. The p53 gene and lung cancer Mutations of the p53 gene are the most frequent genetic abnormality found in human cancers to date (Harris 1990). The gene lies on chromosome band 17p13 and has been examined in a wide variety of primary tumors and cell lines derived from tumors. Of these, colon carcinomas have been studies in greater detail : 7580% showed a "loss" of both p53 alleles, one through deletion and the other through a point mutation. The point mutations are usually missense, giving rise to an altered protein. Similarly, the use of polymorphic DNA probes for chromosome 17p markers demonstrate loss of heterozygosity in approximately 60% of NSCLC and almost 100% of SCLC (Takahashi et al. 1989a; Chiba et al. 1990). Several of the mutant p53 genes have been cloned and the resulting cDNA and genomic clones tested for their biological properties. The wild-type allele can suppress the growth of transformed cells in culture and the formation of tumors in animals. Low to modest amounts of wild-type (WT) p53 protein are expressed in the nucleus of all normal dividing cells. WT protein has other important properties, including the ability to bind to several viral oncoproteins. Many mutant proteins have conformational changes resulting in long half lives, and consequently their levels are increased many fold. Some mutant proteins fail to enter the nucleus, and localize to the perinuclear areas. Mutants vary in their efficiency in cooperating with mutant ras genes in transforming primary rat cells in culture. Mutant p53 may gain a new function that overcomes the negative regulation by small quantities of wild-type p53 thus acting to stimulate cell growth and tumor formation. Alternatively, mutant protein could form an oligomeric complex with wide-type p53 resulting in loss of normal function. The ratio of mutant to wild type p53 in a cell could be critical in regulating cell division. Mutant proteins lose the ability to bind oncoproteins, but acquire the ability to bind to heat shock protein 70. Concluding The
remarks molecular
changes
complex
and
multiple.
recessive
anti-oncogenes.
that
These Other
precede changes changes
the
development
involve may
both include
of lung
dominant development
cancer
are
oncogenes
and
of drug
resis-
The
Molecular
Biology
of Lung
Cancer
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tance, and production of growth factors and their receptors. The estimated number of mutations required for the development of invasive cancer is in double digits (Minna et al. 1990). How can so many lesions develop in a single cell ? One possible explanation is a so-called "mutator phenotype", with some (?early) mutations predisposing to the development of other changes. Another possible explanation is the "field cancerization" theory (Slaughter et al. 1954). The latter developed from observations that the surface changes in head and neck cancers were extensive, and that the incidence of synchronous, metachronous or second malignancies was very high, suggesting that much or all of the epithelium has undergone one or more genetic alterations (presumably as the result of repeated exposure to carcinogens such as tobacco), and is at increased risk for developing multiple, independent morphological alterations and malignancies. The concept of field cancerization is equally applicable to the respiratory tract. Lung cancers may be associated with extensive surface changes (Carter 1978), and secondary primaries are relatively frequent (Boice and Fraumeni 1985; Thomas et al. 1990). The annual incidence of second malignancies after resections for stage I NSCLC is 2-5% (Thomas et al. 1990). It is tempting to associate specific molecular changes with specific morphological changes, as has been attempted in the colon. However, because of the difficulties in serially sampling the respiratory tract, such studies have not been performed to date. Documentation of molecular changes in premalignant lesions and prospective clinical studies of their prognostic effects will be necessary for the design of rational chemoprevention trials. References 1) Barbacid, M. (1990) ras oncogenes : Their role in neoplasia. Eur. J. Clin. Invest., 20, 225-235. 2) Boice, JD. & Fraumeni, J.F. (1985) Second cancer following cancer of the respiratory system in Connecticut, 1935-1982. Natl. Cancer Inst. Monogr., 68, 83-98. 3) Brennan, J., O'Connor, T., Makuchi, R.W., Simmons, A.M., Russell, E., Linnoila, R.I., Phelps, R.M., Gazdar, A.F., Ihde, D.C. & Johnson, BE. (1991) myc family DNA amplification in 107 tumors and tumor cell lines from patients with small cell lung cancer treated with different combination chemotherapy regimens. Cancer Res., 51, 1708-1712. 4) Carter, D. (1978) Pathology of the early squamous cell carcinoma of the lung. Pathol. Annu., 13, 131-147. i) Chiba, I., Takahashi, T., Nau, M.M., D'Amico, D., Curiel, D., Mitsudomi, T., Buchhagen, D., Carbone, D., Koga, H., Reissmann, P., Slamon, D., Homes, E. & Minna, J. (1990) Mutations in the p53 gene are frequent in primary, resected non-small cell lung cancer. Oncogene,5, 1603-1610. 6) Der, C.J. (1989) The ras family of oncogenes. Cancer Treat. Res., 47, 73-119. 7) Fearon, ER. & Vogelstein, B. (1990) A genetic model for colorectal tumorigenesis. Cell, 61, 759-767. 8) Gazdar, A.F., Carney, D.N., Nau, M.M. & Minna, J.D. (1985) Characterization of variant subclasses of cell lines derived from small cell lung cancer having distinctive biochemical, morphological and growth properties. Cancer Res., 45, 2924-2930. 9) Harris, A.L. (1990) Mutant p53-the commonest genetic abnormality in human can-
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