KRAS Lung Research Articles

Abstract LB-353: From worms to humans: understanding the role of microRNAs in cancer progression

Abstract MicroRNAs (miRNAs) regulate important developmental events and are often misexpressed in human cancers, but little is known regarding how these small RNAs contribute to tumor formation. Our laboratory uses a combination of nematode and mammalian model systems to functionally characterize miRNAs and determine the role these factors play in controlling cancer progression pathways. We have successfully used the nematode to analyze how members of the lin-4 and let-7 miRNA families functionally overlap to direct common developmental events associated with cell cycle progression and differentiation. Specifically, animals that carry a triple deletion for the lin-4 homologue, mir-237, and the let-7 homologues, mir-48 and mir-84, exhibit morphological defects and fertility problems related to abnormal mitotic and meiotic abnormalities in the germline that were not found in the single or double deletion combinations. We have also begun to determine how the let-7 miRNA family functions as tumor suppressor genes in mammals. Our experiments showing that exogenous let-7 miRNA administration in the lung using a conditionally activated KRAS lung cancer mouse model result in reduced lung tumor load in these animals highlight the therapeutic potential of miRNAs. We are also studying the role of let-7 together with other miRNA families in directing cancer progression pathways in human tissues of urothelial origin. For instance, we find that let-7 is dysregulated in human prostate cancer cell lines differing in their metastatic status and indicates a novel role for this miRNA in prostate cancer progression. Due to our access to a vast repository of human prostate specimens from the Virginia Prostate Center at Eastern Virginia Medical School, we have begun to analyze the miRNA expression profiles in FFPE specimens taken from the primary prostate tumor, normal adjacent tissue and the lymph node metastasis of individual prostate cancer patients as well as in tumors from patients presenting insignificant and organ confined cancer. We are also determining the “miRNA signatures” in blood, urine, and expressed prostatic secretions (EPS) that were obtained non-invasively from both normal and cancer patients. Our work investigating the role of miRNAs during cancer progression promises to reveal novel therapeutic approaches and diagnostic and prognostic biomarkers for human cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-353.

Abstract A255: Activity of VEGFR inhibitor tivozanib as a single agent or in combination with EGFR inhibitor erlotinib in engineered lung adenocarcinoma models

Abstract Tivozanib (AV-951 or KRN951) is an ATP competitive small molecule VEGFR antagonist that exhibits picomolar inhibitory activity against all three VEGF receptors. It has demonstrated robust clinical activity in renal cell carcinoma and is currently being evaluated in multiple clinical studies including a monotherapy phase 1b/2a trial in NSCLC. NSCLC is a highly heterogeneous disease, in which tumor histology and genetics have defined sensitivities and settings to targeted therapeutics. To model and explore potential clinical settings, we created genetically engineered lung adenocarcinoma models driven by EGFRL858R, EGFRL858R/T790M or KRAS G12V, propagated these models in vivo both subcutaneously and orthotopically and evaluated tumor inhibitory activity of tivozanib as a single agent or in combination with EGFR inhibitor erlotinib. To further explore activity of metastatic lesions to the lung, we also evaluated the activity of tivozanib using an engineered breast tumor metastatic model. Treatment of subcutaneous lung KRAS tumors or lung EGFRL858R/T790M tumors with tivozanib alone resulted in complete tumor growth inhibition. Histological analysis revealed substantial central necrosis with proliferating (Ki67+) tumor cells present only at the tumor periphery, typical of an anti-angiogenesis mechanism. When tivozanib was given to treat lung EGFRL858R+T790M tumors in an orthotopic setting in the lungs after tumors were well established, significant antiangiogenic and antitumor effects, as demonstrated by histology and extended survival period, were observed. However, the necrotic lung lesions showed no sign of resorption or elimination after 6 weeks of treatment. Similar results were also observed in a HER2 driven breast tumor lung metastatic model. This prompted us to further explore the activity of tivozanib in an orthotopic lung tumor minimal disease model. Tivozanib treatment in this setting did not alter tumor lesion numbers but effectively blocked the progression of micro pre-angiogenic lesions. Finally, we explored the combined activity of tivozanib and erlotinib in a model constructed to reflect the emergence of erlotinib resistance by implanting tumor material comprising both EGFRL858R and EGFRL858R/T790M tumor cells. In this setting, the combination of tivozanib and erlotinib resulted in significantly greater control of tumor progression than erlotinib alone. Our results suggest that tivozanib may have activity as a single agent in lung tumors with KRAS or EGFR L858R/T790M mutations and that combinations with other targeted lung cancer drugs such as erlotinib may provide meaningful clinical benefit. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A255.

A Gene Expression Signature Associated with “K-Ras Addiction” Reveals Regulators of EMT and Tumor Cell Survival

K-ras mutations occur frequently in epithelial cancers. Using short hairpin RNAs to deplete K-Ras in lung and pancreatic cancer cell lines harboring K-ras mutations, two classes were identified-lines that do or do not require K-Ras to maintain viability. Comparing these two classes of cancer cells revealed a gene expression signature in K-Ras-dependent cells, associated with a well-differentiated epithelial phenotype, which was also seen in primary tumors. Several of these genes encode pharmacologically tractable proteins, such as Syk and Ron kinases and integrin beta6, depletion of which induces epithelial-mesenchymal transformation (EMT) and apoptosis specifically in K-Ras-dependent cells. These findings indicate that epithelial differentiation and tumor cell viability are associated, and that EMT regulators in "K-Ras-addicted" cancers represent candidate therapeutic targets.

High dietary inorganic phosphate increases lung tumorigenesis and alters Akt signaling.

Phosphate (Pi) is an essential nutrient to living organisms. Recent surveys indicate that the intake of Pi has increased steadily. Our previous studies have indicated that elevated Pi activates the Akt signaling pathway. An increased knowledge of the response of lung cancer tissue to high dietary Pi may provide an important link between diet and lung tumorigenesis. The current study was performed to elucidate the potential effects of high dietary Pi on lung cancer development. Experiments were performed on 5-week-old male K-ras(LA1) lung cancer model mice and 6-week-old male urethane-induced lung cancer model mice. Mice were fed a diet containing 0.5% Pi (normal Pi) and 1.0% Pi (high Pi) for 4 weeks. At the end of the experiment, all mice were killed. Lung cancer development was evaluated by diverse methods. A diet high in Pi increased lung tumor progression and growth compared with normal diet. High dietary Pi increased the sodium-dependent inorganic phosphate transporter-2b protein levels in the lungs. High dietary consumption of Pi stimulated pulmonary Akt activity while suppressing the protein levels of tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 as well as Akt binding partner carboxyl-terminal modulator protein, resulting in facilitated cap-dependent protein translation. In addition, high dietary Pi significantly stimulated cell proliferation in the lungs of K-ras(LA1) mice. Our results showed that high dietary Pi promoted tumorigenesis and altered Akt signaling, thus suggesting that careful regulation of dietary Pi may be critical for lung cancer prevention as well as treatment.

Deficiency of a potential 3p21.3 tumor suppressor gene UBE1L (UBA7) does not accelerate lung cancer development in K-rasLA2 mice

Genetic lesions in chromosomal region 3p21.3 marks one of the earliest events in human lung cancer development. It is hypothesized that one or more tumor suppressor genes reside in this region. Identification and characterization of these genes are important for the understanding of lung cancer initiation. UBE1L (UBA7) is a long-suspected 3p21.3 residing tumor suppressor gene. It encodes the key enzyme that activates ISGylation, a novel, ubiquitination-like, post-translational protein modification system that is inducible by interferon. It has been implicated that ISGylation plays a variety of biological roles ranging from viral defense to tumor surveillance. Here we tested the possible function of ISGylation during lung cancer development by using the Ube1l-deficient mice and the K-ras(LA2) lung cancer mice. Protein ISGylation levels were largely unchanged during lung cancer progression. Ube1l deficiency neither altered the lung cancer progression nor affected the overall survival of K-ras(LA2) lung cancer mice. Our study suggests that Ube1l is not a tumor suppressor gene in K-ras(LA2) lung cancer mouse model. However, as described in the discussion, additional studies with other lung cancer mouse models will be necessary to elucidate the potential tumor suppressor function of UBE1L in K-RAS mutation independent human lung cancers.

Urocanic acid-modified chitosan-mediated PTEN delivery via aerosol suppressed lung tumorigenesis in K-rasLA1 mice

The low efficiency of conventional therapies in achieving long-term survival of lung cancer patients calls for development of novel options. Revisiting of aerosol gene delivery may provide an alternative for safe and effective treatment for lung cancer. In this study, imidazole ring-containing urocanic acid-modified chitosan (UAC) designed in the previous study was used as a gene carrier. The potential effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) on Akt-related signals and cell cycle regulation were evaluated. Aerosols of UAC-PTEN were delivered into K-ras(LA1) lung cancer model mice through the nose-only inhalation system twice a week for total 4 weeks. Delivered PTEN suppressed lung tumor development significantly through nuclear complex formation between PTEN and p53, suppressing Akt-related signals as well as cell cycle regulation. Together, our results suggest that aerosol delivery of UAC-PTEN may be compatible with noninvasive in vivo gene therapy.

Mutually Exclusive Inactivation of DMP1 and ARF/p53 in Lung Cancer

Dmp1 (Dmtf1) is activated by oncogenic Ras-Raf signaling and induces cell-cycle arrest in an Arf, p53-dependent fashion. The survival of K-ras(LA) mice was shortened by approximately 15 weeks in both Dmp1(+/-) and Dmp1(-/-) backgrounds, the lung tumors of which showed significantly decreased frequency of p53 mutations compared to Dmp1(+/+). Approximately 40% of K-ras(LA) lung tumors from Dmp1(+/+) mice lost one allele of the Dmp1 gene, suggesting the primary involvement of Dmp1 in K-ras-induced tumorigenesis. Loss of heterozygosity (LOH) of the hDMP1 gene was detectable in approximately 35% of human lung carcinomas, which was found in mutually exclusive fashion with LOH of INK4a/ARF or that of P53. Thus, DMP1 is a pivotal tumor suppressor for both human and murine lung cancers.

Genetic pathways and mutation profiles of human cancers: site- and exposure-specific patterns.

Cancer is a complex disease that involves the accumulation of both genetic and epigenetic alterations of numerous genes. Data in the Genetic Alterations in Cancer database for gene mutations and allelic loss [loss of heterozygosity (LOH)] in human tumors (e.g. lung, oral, esophagus, stomach and colon/rectum) were reviewed. Results for the genes and pathways implicated in tumor development at these sites are presented. Mutation incidence, spectra and codon specificity are described for lung, larynx and oral tumors. LOH occurred more frequently than gene mutations in tumors from all sites examined. The cell cycle gene, TP53 (all sites), and cell signaling gene, APC (colorectal and gastric cancers), were the only genes with similar incidences of LOH and mutation. Alterations of one or more cell cycle and cell signaling genes were reported for tumors from each site. Site-specific activation was apparent in the cell signaling mitogen-activated protein kinase oncogenes (KRAS in lung, HRAS in oral cancers and BRAF in esophageal and colorectal cancers). Analysis of genetic changes in lung tumors showed that the incidence of mutations in the TP53 and KRAS genes and the incidence of LOH in the FHIT gene were significantly greater in smokers versus non-smokers (P < 0.01). In lung and oral cancers, the TP53 GC --> TA transversion frequency increased with tobacco smoke exposure (P < 0.05). Furthermore, the TP53 mutational hot spots for lung and laryngeal cancers from smokers included codons 157, 245 and 273, whereas for oral tumors included codons 280 and 281.

Elevated K-ras activity with cholestyramine and lovastatin, but not konjac mannan or niacin in lung—–Importance of mouse strain

Our previous work established that hypocholesterolemic agents altered K-ras intracellular localization in lung. Here, we examined K-ras activity to define further its potential importance in lung carcinogenesis. K-ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For 3 weeks, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% konjac mannan (KM), or were injected with lovastatin 25 mg/kg three or five times weekly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After 3 weeks, serum cholesterol was assayed with a commercial kit. Activated K-ras protein from lung was affinity precipitated with a Raf-1 ras binding domain-glutathione- S-transferase fusion protein bound to glutathione-agarose beads, followed by Western blotting, K-ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in two of three mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-ras activity increased 1.8-fold versus control ( p = 0.009). In normal lung with wild-type K-ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-ras activity increased 2.1-fold ( p = 0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-ras activity. These data are consistent with the concept that K-ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type.

1129. Ad.Interferon Beta Is an Effective Treatment in an Orthotopic Model of Broncho-Alveolar Non-Small Cell Lung Cancer Via NK Cell and CD 8+ T Cell Mechanisms

Top of pageAbstract Introduction: Broncho-Alveolar Carcinoma (BAC) is a diffuse (but non-metastasizing) highly lethal lung tumor. To date, it has been difficult to model in mice. However, an orthotopic mouse model for BAC involving a conditionally expressed mutated K ras protein has recently been developed (Jackson et al. Genes & Dev. 15: 3243, 2001). After removal of a lox-flanked stop codon by intra-pulmonary administration of an adenovirus expressing Cre-recombinase (Ad.Cre), localized mutant Kras transcription occurs leading to the development of lung cancer. The tumors progress in a controlled fashion from small diffuse adenomas to widespread sheets of lung cancer. These mice thus offer the ideal model to study new therapeutic approaches for BAC. We have previously shown in mesothelioma models that Ad.interferon |[beta]| (Ad.IFN |[beta]|) can eradicate tumors. We hypothesized that Ad.IFN |[beta]| could be similarly effective in the Kras lung cancer mouse model. Methods: K ras mutant mice were given Ad.Cre intra-nasally. At 3 and 4 weeks after Ad.Cre instillation, groups of mice (n=8) were either untreated or treated intra-nasally with 109 plaque forming units (PFU) of Ad.IFN |[beta]| or a control virus, Ad.LacZ. The mice were studied for survival. In addition, cells derived from K ras mutated lung tumors were grown in the flanks of mice to study some of the mechanisms behind these responses. Results: The untreated tumor-bearing mice all died by day 57 (median survival of 49 days); the mice treated with Ad.LacZ were all dead by day 71 (median survival of 65 days); all mice treated with Ad.IFN |[beta]| were long term survivors (> 105 days) (p<0.001). This experiment was independently repeated with virtually identical results. Using tumor-derived cells grown in vitro and in the flanks of syngeneic mice, we found that Ad. IFN |[beta]| worked via both direct cell killing, and by stimulation of natural killer (NK) cells and CD+8 T cells; depleting NK or CD+8 T cells, but not CD+4 T cells with antibodies attenuated the effect of Ad. IFN |[beta]|. Conclusions: Two doses of Ad.IFN |[beta]| delivered intranasally into mice pre-sensitized with adenovirus lead to remarkably effective anti-tumor activity in an orthotopic model of BAC. Effects were dependent on activation of both CD8+ T cells and NK cells. These studies provide strong preclinical support for a trial of Ad.IFN |[beta]| to treat human BAC.

Levels and membrane localization of the c-K-ras p21 protein in lungs of mice of different genetic strains and effects of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) and Aroclor 1254

Mutational activation of the K-ras oncogene often occurs in human and mouse lung adenocarcinomas. Since K-ras p21 functions in trans-membrane signaling, we have investigated whether the amount of this protein in lung cell membranes is a variable that could influence lung tumorigenesis, either due to genetic differences or in response to tumor promoters. The six mouse strains assessed showed little difference in the total lung K-ras p21 after immunoprecipitation and immunoblotting. However, amount of ras p21 in the membrane fraction showed significant differences, with C57BL/6 and BALB/c having 3-5-fold more than NIH Swiss, AKR and DBA mice. Interestingly, a congenic AKR strain having the Ahr(b-1) Ah receptor allele from C57BL/6 mice (designated AKR.B6Ah) had high lung membrane K-ras p21 similar to that of C57BL/6. To test for possible changes related to lung tumor promotion, mice were treated with a promotional dose of TCDD (5 nmol/kg). After 48 h C57BL/6 lungs showed an increase in p21 in both total and membrane fractions. BALB/c, DBA and Swiss mice showed an increase only in membranes. There was no change in the AKR and AKR.B6Ah. Aroclor 1254 (250 mg/kg) caused an increase in membrane/cytosol ratio in Swiss mice. Thus the membrane:cytosol K-ras p21 ratio may be influenced by the Ahr phenotype, and TCDD and PCBs can induce p21 or increase its membrane level in certain strains, but these properties are not fully dependent on Ahr receptor type. In confirmation of the relevance of these findings for the tumor target cell type, the immortalized alveolar type 2 E10 cell line presented K-ras p21 in membrane, and this was increased 4-fold by treatment with 10 nM TCDD.