Yian Wang

Wildtype Kras2 can inhibit lung carcinogenesis in mice

Although the ras genes have long been established as proto-oncogenes, the dominant role of activated ras in cell transformation has been questioned. Previous studies have shown frequent loss of the wildtype Kras2 allele in both mouse and human lung adenocarcinomas. To address the possible tumor suppressor role of wildtype Kras2 in lung tumorigenesis, we have carried out a lung tumor bioassay in heterozygous Kras2-deficient mice. Mice with a heterozygous Kras2 deficiency were highly susceptible to the chemical induction of lung tumors when compared to wildtype mice. Activating Kras2 mutations were detected in all chemically induced lung tumors obtained from both wildtype and heterozygous Kras2-deficient mice. Furthermore, wildtype Kras2 inhibited colony formation and tumor development by transformed NIH/3T3 cells and a mouse lung tumor cell line containing an activated Kras2 allele. Allelic loss of wildtype Kras2 was found in 67% to 100% of chemically induced mouse lung adenocarcinomas that harbor a mutant Kras2 allele. Finally, an inverse correlation between the level of wildtype Kras2 expression and extracellular signal–regulated kinase (ERK) activity was observed in these cells. These data strongly suggest that wildtype Kras2 has tumor suppressor activity and is frequently lost during lung tumor progression.

LOH of chromosome 12p correlates with Kras2 mutation in non-small cell lung cancer

Previous observation has shown that the wild-type Kras2 allele is a suppressor of lung cancer in mice. Here we report that loss of heterozygosity (LOH) of chromosome 12p was detected in ∼50% of human lung adenocarcinomas and large cell carcinomas, and Kras2 mutations were detected at codon 12 in ∼40% of adenocarcinomas and large cell carcinomas. Interestingly, all of the lung adenocarcinomas and large cell carcinomas containing a Kras2 mutation exhibited allelic loss of the wild-type Kras2 allele when a correlation between LOH of the region on chromosome 12p and Kras2 mutation was made. These results from human lung cancer tissues provide a strong evidence in support of our previous observation in mouse models that the wild-type Kras2 is a tumor suppressor of lung cancer.

Differentially expressed genes associated with mouse lung tumor progression.

To detect altered gene expression associated with mouse lung tumor progression, we compared the gene expression profile of lung adenocarcinomas with that of lung adenomas and normal lungs. Autoradiographic analysis showed that among the 588 genes surveyed, 152 genes were detected and the remaining 436 genes did not give any signals. A gene-specific semiquantitative reverse transcription polymerase chain reaction method was used to confirm the expression profile. A total of 29 genes was found to be differentially expressed in mouse lung tumors when compared to normal lungs. The pattern of expression, either underexpression or overexpression, was the same for 10 genes between adenocarcinomas and adenomas. Among them, seven genes were overexpressed, two genes were underexpressed and one gene was lost. Interestingly, 19 genes showed differential expression or increased incidence or difference in level of change between lung adenomas and adenocarcinomas, including Stat1, ADAP, IGFBP-6, PDGF-A, TGF-β2, Int-3, VEGFR2, BAX, BAG-1, c-Jun, FasL, TRAIL, YB-1, CD31, Cdc42, B-raf, Rab-2, Abi-1, and ACE. These genes can be designated as candidate ‘lung tumor progression’ (LTP) genes because their expression changes may specifically affect lung tumor progression in mice. Further analyses of these candidate LTP genes may provide new leads for elucidation of lung tumor progression in mice.

Chemopreventive effect of perillyl alcohol on 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone induced tumorigenesis in (C3H/HeJ X A/J)F1 mouse lung

This study was designed to test the chemopreventive potential of perillyl alcohol, an inhibitor of farnesyltransferase, in a mouse lung tumor bioassay. Perillyl alcohol is a naturally occurring monoterpene found in lavender, cherries, and mint. We have shown previously that the majority of lung tumors in this bioassay have an activating mutation in the K‐ras gene, which occurs early in the development of mouse lung carcinogenesis. The Ras protein undergoes a series of post‐translational modifications, the first of which is farnesylation at the cysteine of the C‐terminal CAAX motif. These modifications lead to the anchoring of Ras p21 to the plasma membrane in its biologically active state. Activated Ras p21 couples growth regulatory signals from receptor tyrosine kinases to cytoplasmic second messengers. In a preliminary study, we determined the maximum tolerated dose of perillyl alcohol to be 75 mg/kg body weight. For the bioassay, 5‐week‐old male (C3H/HeJ X A/J) F1 hybrid mice were randomized into trial groups, and treated with perillyl alcohol three times per week i.p., starting 1 week prior to initiation with the carcinogen NNK, and continuing for 22 weeks after initiation. Our results show a 22% reduction in tumor incidence, and a 58% reduction in tumor multiplicity. Our study demonstrates that perillyl alcohol is an effective chemopreventive compound in the mouse lung tumor bioassay. J. Cell. Biochem. Suppl. 27:20–25.

Prevention of lung cancer progression by bexarotene in mouse models

Bexarotene (Targretin®, Ligand Pharmaceuticals Inc.) 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 ∼50% in both p53wt/wtK-rasko/wt and p53wt/wtK-raswt/wt mice. Thus, bexarotene appears to be an effective preventive agent against lung tumor growth and progression.

Characterization of epidermal growth factor receptor mutations in non-small-cell lung cancer patients of African-American ancestry

Epidermal growth factor receptor (EGFR) mutations are predictive markers for response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). The most common mutations, exon 19 short deletions and exon 20 point mutation (L858R), activate the tyrosine kinase and confer sensitivity to EGFR-TKIs. However, the function and sensitivity of rare mutations to EGFR-TKIs are unknown. In this study, we found five EGFR mutations out of 16 patients with NSCLC of African-American descent. The frequency of such mutations in this patient population appears to be significantly higher than previously reported. Two of them (N771GY and A767-V769dup) are rare insertion mutations located in exon 20. Using YFP-tagged EGFR mutants, we demonstrated that the mutations confer increased kinase activity, but no sensitivity to erlotinib at clinically available concentrations. In addition, we examined efficacy of PF00299804, an irreversible EGFR-TKI. Although the drug failed to show efficacy to T790M and S768N mutations, the exon 20 insertion mutations were sensitive to PF00299804. These data suggest that rare mutations in exon 20 are resistant to erlotinib but may be sensitive to irreversible inhibitors.

CHEMOPREVENTIVE EFFICACY OF PROMISING FARNESYLTRANSFERASE INHIBITORS

The studies presented were designed to test the efficacy of farnesyltransferase inhibitors (FTIs) as potential chemopreventive compounds in the mouse lung tumor model, and in tumor cell lines. The compounds included manumycin, gliotoxin, dihydroepiandrosterone (DHEA), perillyl alcohol (POH), and FTI-276. Each of these compounds had the potential, based on in vitro and limited in vivo evidence, to inhibit mouse lung tumorigenesis. In vitro studies were conducted with both K-ras-transformed NIH-3T3 cells and mouse lung tumor epithelial cell lines. Weutilized 2 primary mouse lung tumor models that reliably produce lung tumors with an oncogenic K-ras mutation when induced by 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). Manumycin, gliotoxin, DHEA, and POH were administered 3 times per week peritoneally (IP), starting 1 week prior to carcinogen treatment, and throughout the test period (4.5 months). FTI-276 was delivered daily for 4 months by a time-release pellet method. Both the manumycin and gliotoxin treatment groups demonstrated 100% incidence and an increase in tumor multiplicity over control, of 66% and 58% increase respectively (P<.05). Although DHEA showed no significant chemopreventive effect, POH treatment demonstrated a 22% reduction in tumor incidence (P<.05) and a 58% reduction in tumor multiplicity (P<.05). Finally, FTI-276 reduced both the tumor multiplicity by 41.7% (P <.005), and the total tumor volume/burden per mouse by 79.4% (P<.0001). The apoptotic index in FTI-276-treated tumors showed an increase of 77% over control tumors (P<.05). In vitro, all compounds demonstrated growth inhibition at a dose-response manner; however, manumycin, gliotoxin, and DHEA demonstrated an initial increase in growth rate at lower doses. In summary, we have shown that POH and FTI-276 are chemopreventive in a primary mouse lung tumor model. In contrast, DHEA was not significantly chemopreventive at the dosage utilized, and treatment of an immunocompetent host with manumycin or gliotoxin demonstrated a significant increase in tumorigenicity over carcinogen control.

A strong candidate gene for the Papg1 locus on mouse chromosome 4 affecting lung tumor progression

Lung cancer is the leading cause of cancer death among both men and women, accounting for more than 28% of all cancer deaths. In fact, more people die of lung cancer than of colon, breast, and prostate cancers combined. Although lung cancer is largely induced by smoking, there is strong evidence for genetic susceptibility and gene-environment interactions in the development of lung cancer. Inbred mouse models offer an effective means of identifying candidate lung cancer susceptibility loci since genetic heterogeneity and enormous variation in exposure levels to environmental agents make it difficult to identify lung cancer susceptibility loci in humans. Papg-1 (pulmonary adenoma progression 1) was previously mapped to a region on mouse chromosome 4. This locus contains a candidate gene, Cdkn2a also referred to as Ink4a/Arf, which dually encodes two established tumor suppressors p16INK4a and ARF. Cdkn2a became a primary candidate for Papg-1 for two reasons: (1) two haplotypes of mouse Cdkn2a were found to segregate with differential genetic susceptibility to lung tumor progression in mice; and (2) in vitro studies showed that the p16INK4a allele from the BALB/cJ mouse had a significantly decreased ability to bind and inhibit CDK6 and to suppress cell growth when compared with the p16INK4a allele from the A/J mouse. Here, we report that mice with a heterozygous deficiency for the A/J Cdkn2a allele were significantly more susceptible to lung tumor progression than mice with a heterozygous deficiency for a BALB/cJ Cdkn2a allele, when compared to their respective wild type mice. These results offer strong evidence that naturally occurring variation of p16INK4a influences susceptibility to enhance lung tumor progression making it a strong candidate for the lung tumor progression locus, Papg-1.

Altered gene expression profile in chemically induced rat mammary adenocarcinomas and its modulation by an aromatase inhibitor.

In the present study, competitive cDNA library screening (CCLS) and cDNA microarray analyses were employed to identify differentially expressed genes in methylnitrosourea-induced rat mammary adenocarcinomas. The preliminary screening of 100u2009000 plaques by CCLS identified 1217 clones with differential expression. Dot–blot analysis of the isolated clones verified differential expression in 471 distinct genes. Confirmation of these 471 genes was conducted by performing reverse transcription-polymerase chain reactions, and a total of 160 genes were confirmed after comparing six rat mammary adenocarcinomas and three normal rat mammary glands. Fifty-nine of these showed lower expression in the adenocarcinomas while the remaining 101 were overexpressed in the tumors. Employing a cDNA microarray containing 588 known genes revealed an additional 33 differentially expressed genes in these tumors. Importantly, most of the identified genes demonstrated relatively reproducible overexpression or underexpression in individual tumors. Many of the altered genes determined by cDNA microarray analysis were oncogenes, tumor suppressor genes, or genes involved in cell cycle control and apoptosis. CCLS identified many others not previously associated with mammary carcinogenesis, including a novel gene named RMT-7. Preliminary studies to determine the applicability of this gene expression approach for detecting potential biomarkers for cancer chemoprevention was evaluated in rat mammary tumors obtained from animals treated with vorozole, a potent aromatase inhibitor. When genes exhibiting differential expression as determined by CCLS or cDNA microarray analysis were examined in control and vorozole-treated tumors, expression of 19 genes was found to be modulated significantly in tumors treated with vorozole. Further investigations into these identified genes should contribute significantly to our understanding of the molecular mechanisms of rat mammary tumorigenesis. In addition, the identified genes may become useful targets for drug development and potential biomarkers for monitoring treatment and prevention of breast cancer in humans.

Differential Gene Expression in Chemically Induced Mouse Lung Adenomas

Because of similarities in histopathology and tumor progression stages between mouse and human lung adenocarcinomas, the mouse lung tumor model with lung adenomas as the endpoint has been used extensively to evaluate the efficacy of putative lung cancer chemopreventive agents. In this study, a competitive cDNA library screening (CCLS) was employed to determine changes in the expression of mRNA in chemically induced lung adenomas compared with paired normal lung tissues. A total of 2555 clones having altered expression in tumors were observed following competitive hybridization between normal lung and lung adenomas after primary screening of over 160,000 clones from a mouse lung cDNA library. Among the 755 clones confirmed by dot blot hybridization, 240 clones were underexpressed, whereas 515 clones were overexpressed in tumors. Sixty-five clones with the most frequently altered expression in six individual tumors were confirmed by semiquantitative RT-PCR. When examining the 58 known genes, 39 clones had increased expression and 19 had decreased expression, whereas the 7 novel genes showed overexpression. A high percentage (>60%) of overexpressed or underexpressed genes was observed in at least two or three of the lesions. Reproducibly overexpressed genes included ERK-1, JAK-1, surfactant proteins A, B, and C, NFAT1, alpha-1 protease inhibitor, helix-loop-helix ubiquitous kinase (CHUK), alpha-adaptin, alpha-1 PI2, thioether S-methyltransferase, and CYP2C40. Reproducibly underexpressed genes included paroxanase, ALDH II, CC10, von Ebner salivary gland protein, and alpha- and beta-globin. In addition, CCLS identified several novel genes or genes not previously associated with lung carcinogenesis, including a hypothetical protein (FLJ11240) and a guanine nucleotide exchange factor homologue. This study shows the efficacy of this methodology for identifying genes with altered expression. These genes may prove to be helpful in our understanding of the genetic basis of lung carcinogenesis and in developing biomarkers for lung cancer chemoprevention studies in mice.