Mutational Damages in Malignant Lung Tumors

Background: Today, genomic changes are an important cause of the occurrence, growth and progression of cancer. Technological advances in cancer genomic analysis platforms have made it possible to identify genomic alterations that may influence response to lung cancer treatment. Methods: The study examined tumor growth-inhibiting oncogenes and genes responsible for cell growth and division to identify mutations characteristic of malignant lung tumors. The mutations were studied in 400 postoperative samples after amplifying p53 and HRAS fragments and p53, p21Waf1, MDM2 mRNA. p53 or p21Waf1 were expressed in 50% of squamous cell carcinomas and adenocarcinomas of the lung. Results: The study examined tumor growth-inhibiting oncogenes and genes responsible for cell growth and division to identify mutations characteristic of malignant lung tumors. The mutations were studied in 400 postoperative samples after amplifying p53 and HRAS fragments and p53, p21Waf1, MDM2 mRNA. p53 or p21Waf1 were expressed in 50% of squamous cell carcinomas and adenocarcinomas of the lung. HRAS mutations were present in most squamous cell carcinomas and adenocarcinomas of the lung. EcoR1- and Pst1- restriction enzymes destroyed the RT-PCR product of the p53 and p21Waf1 mRNA and increased the level of detected mutations in lung adenocarcinoma to 75% and 50 %, respectively. EGFR mutations were more frequent in lung adenocarcinoma than in lung squamous cell carcinoma. Mutations in EGFR exons 19 and 21 found in 65 of 263 lung tumor samples indicated the tumor sensitivity to EGFR tyrosine kinase inhibitors. EGFR deletions in exon 19 occurred mainly in adenocarcinoma, L858R mutations in EGFR exon 21 were quite common in lung adenocarcinoma. Conclusion: The mutations detected in most squamous cell carcinomas and adenocarcinomas of the lung could be used to diagnose and predict the disease severity and targeted therapy efficacy.     

Mice with alterations in both p53 and Ink4a/Arf display a striking increase in lung tumor multiplicity and progression: differential chemopreventive effect of budesonide in wild-type and mutant A/J mice

p53 transgenic mice carrying a dominant negative mutation were crossed with Ink4A/Arf heterozygous-deficient mice to investigate whether there is a synergy between these two germ-line mutations in promoting carcinogen-induced lung tumor progression in mice. Mice with a p53 dominant negative mutation and Ink4A/Arf heterozygous deficiency exhibited >20-fold increase in tumor volume compared with approximately 4-fold increase in Ink4A/Arf heterozygous-deficient mice and a 9-fold increase in mice with only the p53 dominant negative mutation. The effect of Ink4A/Arf heterozygous deficiency on lung tumor progression occurred late in the carcinogenesis process (>30 weeks after carcinogen treatment). In addition, most of the lung tumors (approximately 80%) from mice with a p53 mutation and deletion of Ink4A/Arf were lung adenocarcinomas. In contrast, lung adenocarcinomas were seen in <10% of the lung tumors from the wild-type mice and approximately 50% of the lung tumors from Ink4a/Arf heterozygous-deficient or p53 mutant mice. These results indicate a significant synergistic interaction between the presence of a mutant p53 transgene and the Ink4A/Arf deletion during lung tumor progression (P < 0.01). The usefulness of this new mouse model in lung cancer chemoprevention was examined. The chemopreventive efficacy of budesonide was examined in wild-type mice, mice with Ink4A/Arf heterozygous deficiency, mice with a mutation in the p53 gene, or mice with both a mutation in the p53 gene and deletion in the Ink4A/Arf locus. Mice treated with budesonide displayed an average of 90% inhibition of lung tumor progression in a standard 18-week chemoprevention assay, regardless of p53 and/or Ink4A/Arf status. However, the efficacy of budesonide against lung tumor progression decreased from 94 to 77% (P = 0.07) in mice with alterations in both p53 and Ink4A/Arf in a 40-week chemoprevention assay. Similarly, when mice bearing established lung adenomas were treated with budesonide, genotype-dependent differential effects of budesonide in wild-type and mutant mice were clearly revealed with a 82, 64, 45, and 33% decrease in tumor volume in wild-type mice, p53(+/+)Ink4a/Arf(+/-) mice, p53(+/-)Ink4a/Arf(+/+) mice, and p53(+/-)Ink4a/Arf(+/-), respectively. Thus, mutant mice with alterations in p53 and/or Ink4A/Arf exhibited a significant resistance to chemoprevention by budesonide. Because p53 and Ink4a/Arf mutations are the most prevalent mutations in human lung cancers, the effectiveness of chemopreventive agents on the mutant A/J mice containing alterations with p53 and Ink4a/Arf is the best preclinical estimate of their efficacy in humans. Thus, the mutant A/J mouse model should prove useful for chemoprevention studies.     

Prevention of lung cancer progression by bexarotene in mouse models

Bexarotene (Targretin), is a synthetic high-affinity RXR receptor agonist with limited affinity for RAR receptors. Bexarotene has shown efficacy in a phase I/II trial of non-small-cell lung cancers. However, the chemopreventive efficacy of bexarotene has not been determined in mouse lung cancer models. In this study, we have investigated the ability of bexarotene to inhibit lung tumor progression in the mutant A/J mouse models with genetic alterations in p53 or K-ras, two of the most commonly altered genes in human lung tumorigenesis. Mice were administered vinyl carbamate (VC), a carcinogen, by a single intraperitoneal injection (i.p.) at 6 weeks of age. Bexarotene was given by gavage starting at 16 weeks after VC and was continued for 12 weeks. Although all mice developed lung tumors, only 7% of lung tumors were adenocarcinomas in wild-type mice, whereas 22 and 26% of lung tumors were adenocarcinomas in p53 transgenic or K-ras heterozygous deficient mice. Bexarotene inhibited both tumor multiplicity and tumor volume in mice of all three genotypes. Furthermore, bexarotene reduced the progression of adenoma to adenocarcinoma by approximately 50% in both p53(wt/wt)K-ras(ko/wt) and p53(wt/wt)K-ras(wt/wt) mice. Thus, bexarotene appears to be an effective preventive agent against lung tumor growth and progression.     

Genetic Alterations in Thyroid Tumor Progression: Association with p53 Gene Mutations

To identify the genetic events that must be involved in thyroid tumor progression, we initially investigated p53 gene alterations in 10 papillary adenocarcinomas, 4 follicular adenocarcinomas, and 8 undifferentiated carcinomas. Base substitutional mutations in exons 5 to 8 and loss of heterozygosity (LOH) of the p53 gene were not detected in papillary or follicular adenocarcinomas. However, 7 of 8 undifferentiated carcinomas were carrying base substitutional mutations, and LOH was detected in 3 of 5 informative cases. Furthermore, to verify that the p53 gene alterations are truly involved in tumor progression, DNA from individual foci of the four undifferentiated carcinomas coexisting with a differentiated focus and from one follicular adenocarcinoma with an undifferentiated focus was analyzed by direct sequencing and polymerase-chain-reaction-restriction-fragment-length polymorphism (PCR-RFLP). Base substitutional mutations in the p53 gene from exons 5 to 8 were identified exclusively in the undifferentiated foci, but not in the differentiated foci. LOH was observed in 3 of 4 informative undifferentiated foci. In one of these positive cases, LOH was observed in both papillary adenocarcinoma and undifferentiated carcinoma. However, a p53 gene mutation at codon 248 was detected in the undifferentiated carcinoma but not in the papillary adenocarcinoma. The results imply that LOH occurs first in papillary adenocarcinoma followed by a p53 mutation during the transition from papillary adenocarcinoma to undifferentiated carcinoma. Maintenance of LOH during tumor progression excludes the possibility that these different histological foci are derived from different origins and represents molecular evidence that undifferentiated carcinoma is very likely derived from preexisting papillary adenocarcinoma. Furthermore, these results strongly suggest that the mutated p53 gene plays a crucial role in de-differentiation during the progression of thyroid tumors.     

Fate of UVB-induced p53 mutations in SKH-hr1 mouse skin after discontinuation of irradiation: relationship to skin cancer development

Chronic exposure to ultraviolet (UV) radiation causes skin cancer in humans and mice. We have previously shown that in hairless SKH-hr1 mice, UVB-induced p53 mutations arise very early, well before tumor development. In this study, we investigated whether discontinuation of UVB exposure before the onset of skin tumors results in the disappearance of p53 mutations in the skin of hairless SKH-hr1 mice. Irradiation of mice at a dose of 2.5 kJ/m2 three times a week for 8 weeks induced p53 mutations in the epidermal keratinocytes of 100% of the mice. UVB irradiation was discontinued after 8 weeks, but p53 mutations at most hotspot codons were still present even 22 weeks later. During that period, the percent of mice carrying p53(V154A/R155C), p53(H175H/H176Y), and p53R275C mutant alleles remained at or near 100%, whereas the percentage of mice with p53R270C mutation decreased by 45%. As expected, discontinuation of UVB after 8 weeks resulted in a delay in tumor development. A 100% of tumors carried p53(V154A/R155C) mutant alleles, 76% carried p53(H175H/H176Y) mutants, and 24 and 19% carried p53R270C and p53R275C mutants, respectively. These results suggest that different UVB-induced p53 mutants may provide different survival advantages to keratinocytes in the absence of further UVB exposure and that skin cancer development can be delayed but not prevented by avoidance of further exposure to UVB radiation.     

Association of p16 homozygous deletions with clinicopathologic characteristics and EGFR/KRAS/p53 mutations in lung adenocarcinoma

PURPOSE: The p16 gene is frequently inactivated in lung adenocarcinoma. In particular, homozygous deletions (HD) have been frequently detected in cell lines; however, their frequency and specificity is not well-established in primary tumors. The purpose of this study was to elucidate the prevalence and the timing for the occurrence of p16 HDs in lung adenocarcinoma progression in vivo. EXPERIMENTAL DESIGN: Multiple ligation-dependent probe amplification was used for the detection of p16 HDs in 28 primary small-sized lung adenocarcinomas and 22 metastatic lung adenocarcinomas to the brain. Cancer cells were isolated from primary adenocarcinoma specimens by laser capture microdissection. HDs were confirmed by quantitative real-time genomic PCR analysis. RESULTS: HDs were detected in 8 of 28 (29%) primary tumors, including 2 of 8 (25%) noninvasive bronchioloalveolar carcinomas, and 5 of 22 (26%) brain metastases, respectively. No significant associations were observed between p16 HDs and gender, age, smoking history, stage, and prognosis. HDs were detected with similar frequencies (17-29%) among adenocarcinomas with epidermal growth factor receptor (EGFR) mutations, with KRAS mutations, and without EGFR/KRAS mutations, and with similar frequencies (22-28%) between adenocarcinomas with and without p53 mutations. CONCLUSIONS: p16 HDs occur early in the development of lung adenocarcinomas and with similar frequencies among EGFR type, KRAS type, and non-EGFR/KRAS type lung adenocarcinomas. Tobacco carcinogens would not be a major factor inducing p16 HDs in lung adenocarcinoma progression.     

A mouse model for tumor progression of lung cancer in ras and p53 transgenic mice

Although ras and p53 are the most commonly found oncogene and tumor suppressor gene, respectively, in human cancers, their collective roles in tumor progression have yet to be defined in animal models. Here, we demonstrated the synergistic effect between ras and p53 in promoting tumor progression during lung tumorigenesis using bitransgenic mice. Mice with a heterozygous knockout of K-ras (K-ras(wt/ko)) were mated to p53 transgenic mice (p53(val135/wt)) in lung tumorigenesis (K-ras(wt/ko) x p53(val135/wt)). F(1) mice exhibited a significant increase in lung tumor load (tumor multiplicity x tumor volume) when compared to those seen in either K-ras(wt/ko) mice or p53(val135/wt) mice alone. Furthermore, over 50% of the lung tumors were lung adenocarcinomas in bitransgenic mice compared to only 3% in wild-type mice. Alterations of ras and p53 appear to promote the development of lung adenocarcinomas. These results provide the in vivo experimental evidence of synergistic interactions of ras and p53 in lung tumor progression.     

K-ras activation generates an inflammatory response in lung tumors

Activating mutations in K-ras are one of the most common genetic alterations in human lung cancer. To dissect the role of K-ras activation in bronchial epithelial cells during lung tumorigenesis, we created a model of lung adenocarcinoma by generating a conditional mutant mouse with both Clara cell secretory protein (CC10)-Cre recombinase and the Lox-Stop-Lox K-ras(G12D) alleles. The activation of K-ras mutant allele in CC10 positive cells resulted in a progressive phenotype characterized by cellular atypia, adenoma and ultimately adenocarcinoma. Surprisingly, K-ras activation in the bronchiolar epithelium is associated with a robust inflammatory response characterized by an abundant infiltration of alveolar macrophages and neutrophils. These mice displayed early mortality in the setting of this pulmonary inflammatory response with a median survival of 8 weeks. Bronchoalveolar lavage fluid from these mutant mice contained the MIP-2, KC, MCP-1 and LIX chemokines that increased significantly with age. Cell lines derived from these tumors directly produced MIP-2, LIX and KC. This model demonstrates that K-ras activation in the lung induces the elaboration of inflammatory chemokines and provides an excellent means to further study the complex interactions between inflammatory cells, chemokines and tumor progression.     

Mutant p53(R270H) gain of function phenotype in a mouse model for oncogene-induced mammary carcinogenesis

In human breast cancer, mutations in the p53 gene are associated with poor prognosis. However, analysis of patient data so far did not clarify, whether missense point mutations in the p53 gene, in addition to causing loss of wild-type p53 function, also confer a gain of function phenotype to the encoded mutant p53. As heterogeneity of patient material and data might obscure a clear answer, we studied the effects of a coexpressed mutant p53(R270H) in transgenic mice in which SV40 early proteins initiate the development of mammary adenocarcinoma (WAP-T mice). In such tumors the endogenous wild-type p53 is functionally compromised by complex formation with SV40 T-antigen, thereby constituting a loss of wild-type p53 function situation that allowed analysis of the postulated gain of function effects of mutant p53(R270H). We found that mutant p53(R270H) in bi-transgenic mice enhanced the transition from intraepithelial neoplasia to invasive carcinoma, resulting in a higher frequency of invasive carcinoma per gland and per mouse, a more severe tumor phenotype, and more frequent pulmonary metastasis. Surprisingly, mutant p53(R270H) in this system does not increase genomic instability. Therefore, other postulated gain of function activities of mutant p53 must be responsible for the effects described here.     

p53 mutations in human malignant gliomas: comparison of loss of heterozygosity with mutation frequency.

Mutations in the p53 gene were analyzed in 40 gliomas using the single strand conformation polymorphism assay together with restriction fragment length polymorphism analysis to assess loss of heterozygosity for 17p alleles in the same tumors. Mutations occurred in 40% of the gliomas and were found in exons 4-8 of the p53 gene. G:C to T:A transversions, which occur in high frequency in some lung (greater than 50%), liver (greater than 80%), breast (30%), and esophageal cancers (25%), were noted in greater than 25% of the gliomas studied here. These transversions were clustered in exon 5 from codons 156 to 168, a region of the p53 gene not previously associated with a high frequency of mutation, and may represent a new hot spot for mutations in certain cancers. The majority of gliomas (27 of 38) analyzed here retained both 17p alleles. The frequency of p53 mutations was 37% in this group of tumors and increased to 64% in tumors with one 17p allele. Allelic loss for chromosome 17p occurred in 4 of 11 gliomas independently of mutations in the p53 gene. Absence of p53 mutations in 36% of the tumors with one 17p allele suggests that a tumor suppressor gene other than p53 may be located on chromosome 17p and involved in progression to malignancy of some gliomas.     

P53 protein overexpression in lung adenocarcinomas in non-smokers

Greater than 55% of all lung cancers contain mutations in the p53 tumor suppressor gene, and the frequency of p53 mutations has been directly correlated with the use of tobacco and the smoking of cigarettes. To determine if environmental tobacco smoke (ETS) is associated with changes in p53 expression in human lung cancer in never-smokers, we determined the degree of p53 protein expression in ten lung tumors from never-smokers exposed to ETS. We show that 5 of 8 (62.5%) adenocarcinomas contained significant levels of p53 protein overexpression as detected by immunohistochemical staining. While these findings suggest that ETS is a possible inducer of altered p53 expression in lung adenocarcinoma in non-smokers, larger studies are needed to confirm this relationship.     

Aberrations of the p53 tumor suppressor gene in human non-small cell carcinomas of the lung.

Aberrations of the p53 gene in 115 surgical specimens of non-small cell carcinomas of the lung were examined by single-strand conformation polymorphism analysis of polymerase chain reaction products. Structural abnormalities of the p53 gene were observed in 60 tumors (52%), i.e., 8 of 14 large cell carcinomas, 24 of 58 adenocarcinomas, 25 of 37 squamous cell carcinomas, and 3 of 6 adenosquamous carcinomas. Direct sequencing of abnormal DNA fragments revealed 45 single-base substitutions, 9 deletions or insertion of a short nucleotide sequence, and 3 two-base substitutions in 57 tumors. In the other 3 tumors, loss of one of the p53 alleles was observed, with no mutation in the other allele. Allelic loss of the p53 gene was observed in 14 of 43 informative cases (33%), and in 11 of the 14 cases the remaining allele was mutated. The aberrations of the p53 gene were not limited to a particular histological type or clinical stage. Their high frequency suggests that they were involved in the genesis of non-small cell carcinomas of the lung. The mutation frequency (46%) of the p53 gene in tumors carrying mutated ras genes was essentially the same as the overall frequency in lung cancers, suggesting that accumulation of mutations in these two genes in a tumor is a random phenomenon.     

Evidence for nonautonomous effect of p53 tumor suppressor in carcinogenesis

Prostate, breast, and probably other epithelial tumors harbor inactivating mutations in the p53 tumor suppressor gene in the stromal cells, implying the nonautonomous action of p53 in carcinogenesis. We have tested this hypothesis by evaluating the tumorigenicity of MCF7 human breast cancer cells in severe combined immunodeficient mice that differ in their p53 status. Our results showed that, indeed, p53 ablation in the hosts reduced the latency for the development of MCF7 tumors. Furthermore, we show that heterozygous hosts frequently undergo loss of heterozygosity at the p53 locus in the tumor stroma tissue by mechanism that resembles the inactivation of p53 in primary tumors. To evaluate the impact of p53 ablation in the stromal fibroblasts, in tumorigenesis, tumors were reconstituted in mice bearing wild-type p53 alleles, by mixing MCF7 cells with fibroblasts isolated from mutant or wild-type p53 mice. Our results suggest that tumors containing p53-deficient fibroblasts developed faster and were more aggressive than their counterparts with wild-type fibroblasts, although their neoplastic component, namely MCF7 mammary carcinoma cells, was identical in both cases. These data strongly support the notion for the operation of a nonautonomous mechanism for p53 action in primary tumors and provide a mechanistic association between p53 mutations in the stromal component of epithelial tumors and carcinogenesis.     

A germ-line p53 mutation accelerates pulmonary tumorigenesis: p53-independent efficacy of chemopreventive agents green tea or dexamethasone/myo-inositol and chemotherapeutic agents taxol or adriamycin

Recent evidence indicates that individuals with a p53 germ-line mutation (Li-Fraumeni syndrome) have a 50% risk of developing lung cancer by age 60. In this study, p53 heterozygous knockout mice and p53 transgenic mice carrying a dominant negative mutant were crossed with the A/J mouse, which is highly susceptible to lung tumor induction, to investigate whether a p53 germ-line mutation is a predisposing gene for carcinogen-induced pulmonary adenomas in mice. The number of lung tumors was not significantly increased in (TSG-p53 x A/J)F1 p53 heterozygous knockout mice as compared with that in (TSG-p53 x A/J)F1 wt mice 16 weeks after exposure to N-nitrosomethylurea (MNU). In contrast, an average of 22 lung tumors were observed in (UL53-3 x A/J)F1 mice carrying a mutant p53 transgene (135Valp53) compared with an average of 7 lung tumors seen in (UL53-3 x A/J)F1 wt mice after treatment with N-nitrosomethylurea. Similar enhancement of lung tumor multiplicity (approximately 3-fold) was seen when mutant versus wt mice were treated with the tobacco-related carcinogens benzo[a]pyrene or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. These results suggest that the mutant p53 transgene may have a dominant negative effect on the wt p53. The potential usefulness of this new mouse model in lung cancer chemoprevention and chemotherapy was examined. The chemopreventive efficacy of the green tea or a combination of dietary dexamethasone and myoinositol and the chemotherapeutic efficacy of Taxol or Adriamycin was examined in wt mice or mice with a mutation in the p53 gene. Mice treated with dexamethasone/myo-inositol and green tea displayed an average of 70 and 50% inhibition of lung tumors, respectively, regardless of p53 status. Similarly, when mice bearing established lung adenomas were treated with Taxol or Adriamycin, a decrease in tumor volume of approximately 70% was observed independent of p53 mutation status. Thus, the (UL53-3 x A/J)F1 p53 transgenic mouse seems to be an excellent model for human carriers of p53 germ-line mutations (Li-Fraumeni syndrome). Furthermore, the lung adenomas generated in this model possess mutations in both the K-ras proto-oncogene and the p53 tumor suppressor gene. This model should prove directly useful for chemoprevention and chemotherapy studies.     

Targeted disruption of the 3p12 gene, Dutt1/Robo1, predisposes mice to lung adenocarcinomas and lymphomas with methylation of the gene promoter

The DUTT1 gene is located on human chromosome 3, band p12, within a region of nested homozygous deletions in breast and lung tumors. It is therefore a candidate tumor suppressor gene in humans and is the homologue (ROBO1) of the Drosophila axonal guidance receptor gene, Roundabout. We have shown previously that mice with a targeted homozygous deletion within the Dutt1/Robo1 gene generally die at birth due to incomplete lung development: survivors die within the first year of life with epithelial bronchial hyperplasia as a common feature. Because Dutt1/Robo1 heterozygous mice develop normally, we have determined their tumor susceptibility. Mice with a targeted deletion within one Dutt1/Robo1 allele spontaneously develop lymphomas and carcinomas in their second year of life with a 3-fold increase in incidence compared with controls: invasive lung adenocarcinomas are by far the predominant carcinoma. In addition to the mutant allele, loss of heterozygosity analysis indicates that these tumors retain the structurally normal allele but with substantial methylation of the gene's promoter. Substantial reduction of Dutt1/Robo1 protein expression in tumors is observed by Western blotting and immunohistochemistry. This suggests that Dutt1/Robo1 is a classic tumor suppressor gene requiring inactivation of both alleles to elicit tumorigenesis in these mice.     

The p53 codon 72 proline allele is associated with p53 gene mutations in non-small cell lung cancer.

The p53 gene plays a critical role in cell cycle control, the initiation of apoptosis, and in DNA repair. An Arg/Pro polymorphism at codon 72 of the p53 gene alters the ability of the p53 protein to induce apoptosis, influences the behavior of mutant p53, decreases DNA repair capacity, and may be linked with an increased risk of lung cancer. To further define the role of the p53 codon 72 polymorphism on DNA repair, lung cancer risk, and mutant p53 function, we examined the effect of this polymorphism on mutation of the p53 gene and patient survival in non-small cell lung cancer (NSCLC). Tumor and nonneoplastic (lung or lymphocyte) samples were collected from 182 patients with NSCLC. p53 mutations were detected by direct sequencing and/or the Gene Chip p53 assay in 93 of 182 (51%) tumors. p53 codon 72 polymorphisms were identified by PCR/RFLP analysis. p53 mutations were significantly (P = 0.01) associated with the number of codon 72 Pro alleles: Pro/Pro homozygotes, 17 of 26 (65%); Arg/Pro heterozygotes, 45 of 79 (57%); and Arg/Arg homozygotes, 31 of 77 (40%). The number of codon 72 Pro alleles was independently associated with p53 mutations (odds ratio, 1.97; 95% confidence interval, 1.14-3.40; P = 0.01) in a multiple logistic regression model. The codon 72 polymorphism did not influence patient survival in either the entire patient group or among patients with p53 mutant tumors. In summary, the p53 Pro allele is associated with an increased frequency of p53 mutations in NSCLC.