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M10-01: Molecular pathogenesis of lung cancer with translation to the clinic

2007; Elsevier BV; Volume: 2; Issue: 8 Linguagem: Inglês

10.1097/01.jto.0000282957.22684.90

1556-1380

John D. Minna, Luc Girard, Mitsuo Sato, Michael Peyton, Woochang Lee, David S. Shames, Sofia Honorio, Yang Xie, Xian-Jin Xie, David Lam, Will Lockwood, Wan L. Lam, Yuzhuo Wang, Stephen Lam, Edward Kim, Jonathan R. Pollack, Rachel M. Greer, Robin E. Frink, James P. Sullivan, Boning Gao, Monica Spinola, Ignacio I. Wistuba, Kevin R. Coombes, John V. Heymach, Meera Nanjundan, Li Mao, Christopher I. Amos, Bingliang Fang, Jack A. Roth, Alex Pertsemlidis, Chaitanya S. Nirodi, Michael D. Story, Harold R. Garner, Michael A. White, Jef K. De Brabander, Patrick G. Harran, Xiaodong Wang, Yangsik Jeong, David J. Mangelsdorf, J. Michael DiMaio, Joan H. Schiller, Jerry W. Shay, Adi F. Gazdar,

MicroRNA in disease regulation

The pathogenesis of clinically evident lung cancer involves the acquisition of multiple genetic and epigenetic changes (on the order of 10–20 genes) in lung epithelial cells. This usually occurs after smoking exposure. Genome wide approaches as well as study of multiple individual genes have identified several key genes which fall into different components of the “Hallmarks of Cancer”. Genome wide approaches include genome wide mRNA expression profiling using microarrays, genome wide array based CGH analysis (aCGH), genome wide epigenetic approaches, genome wide microRNA (miRNA) expression profiling, large scale gene sequencing for mutation detection, genome wide siRNA and shRNA knockdowns (e.g. knockdown of all kinases), and large scale proteomics analysis such as with reverse phase protein arrays (RPPAs). These have led to the identification of a large number of new genes, miRNAs, and activated proteins involved in lung cancer pathogenesis. Some examples include discovery of new mutated and amplified genes likely serving as oncogenes such as TITF1, MET, and PI3K, changes in miRNA expression profiles, and multiple new genes that are methylated in lung cancer pathogenesis and thus are likely to function as tumor suppressor genes. In addition, there are changes in gene expression of key genes likely to be involved in the pathogenesis of different types of lung cancer such as differences in patterns of expression of nAChRs subunit genes in adenocarcinomas arising in smokers vs. never smokers. The challenge is to apply these findings to improve diagnosis, prognosis, therapy and prevention of lung cancer. One way this translation to the clinic occurs early is testing the expression of the genes via immunohistochemistry or through FISH in tissue microarrays (TMAs) of lung cancer involving large numbers of clinically annotated (1,000) specimens including preneoplastic lesions. Biomarkers for Early Diagnosis For early diagnosis studies of preneoplastic lesions and even histologically normal epithelium have identified some of the changes as occurring at very early stages including loss of heterozygosity at chromosome regions 3p (multiple sites) and 9p (p16 site). The development of molecular markers that could be detected in sputum or bronchial brushes of persons at highest risk of developing lung cancer would be advantageous. Methylated DNA sequences associated with RASSF1A and p16 are such candidates. Several groups have demonstrated detection of methylated DNA sequences in the sputum of patients going onto develop lung cancer. Recently, proteomic analysis of lung cancers and serum from lung cancer patients have identified markers and proteomic patterns that should aid in predicting which persons are at highest risk of developing lung cancer. As part of this detecting cytokine angiogenic factors (CAFs) that are produced by tumor cells and can be detected in the blood of patients are particularly attractive markers. All of these need to be integrated with ongoing studies of CT screening for lung cancer. Inherited Susceptibility to Lung Cancer The Genetic Epidemiology of Lung Cancer Consortium (GELCC) has collected families with a strong family history of lung cancer and from these performed linkage studies which have identified a locus at 6q23 which in affected individuals leads to a very high risk of developing lung cancer. The region has been narrowed down to ~100 genes which are now being sequenced. The identification of this locus has provided for tests of the role of smoking in obligate carriers vs. non-carriers and led to the finding that carriers who are non-smoking have an increased risk of developing lung cancer, but more importantly that any degree of smoking (even light smoking) greatly increases this risk. Such genetic differences between individuals are being incorporated with epidemiology data to derive new risk assessment models of predicting who is most likely to develop lung cancer. Targeted Therapy for Lung Cancer Mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) have been discovered in ~5-10% of lung cancer patients, particularly in patients with adenocarcinoma of the lung arising in never smokers, females, and East Asians. These mutations appear to make the EGFR oncogenic but also make the tumor cells exquisitely sensitive to TK inhibitors (TKIs) such as gefitinib and erlotinib (oral agents with little toxicity). These therapies provide substantial clinical remissions for such patients. In addition, other mutations (such as EGFR T790M, and MET amplification) can give resistance to EGFR TKIs and drugs which can bypass this resistance are being sought. However, even more important are developing drugs that would targeted other oncogenes that are mutated or amplified in lung cancer and to which lung cancer cells are “addicted” such as MET, TITF1, PI3K, BRAF, and oncogenic KRAS. Lung Cancer Expression Profiles of Clinical Benefit Genome wide oligonucleotide mRNA and protein (proteomic) expression profiles of lung cancer have shown that different lung cancer types have different profiles, that lung cancers with different prognoses have different profiles, and more recently that lung tumors that have different sensitivities to chemotherapy have distinct gene and protein expression signatures. This offers the possibility of testing a patient's tumor before treatment to help with prognosis and to select individualized therapy. The key to this will be to obtain clinically annotated specimens that link a patient's response to therapy with a tumor tissue sample. As part of this is developing genetic biomarkers predicting response to radiation of tumor cells. One new example of this, is the recently discovered (an unexpected) sensitivity of lung cancers with EGFR TK domain mutations to radiation. Preclinical Model Lung Cancer System for Drug and Expression Profile Signature Development An important component of developing such predictive biomarker signatures is the validation of preclinical model systems for testing drugs and combinations of therapy including lung cancer cell lines, xenografts, including orthotopic (lung) xenografts and xenografts made directly from patient samples without intervening cell culture. For this reason we have developed a very large (>200) panel of lung cancer cell lines of all histologic types and including all of the various genetic changes found in lung cancer. Coupled with this we have developed xenograft models (including orthotopic models) of lung cancer in >50 lung cancers including xenografts made from lung cancer lines and from primary patient materials. We can determine expression profiles in these preclinical models as a step toward developing biomarker signatures that can be tested in the clinic. The use of bioluminescence imaging (BLI) to help follow the growth and response of xenografts to therapy is an important new tool. From all of these we need to know how good or bad such preclinical model systems are in both identifying drug response phenotypes and in developing predictive signatures that will work in patient specimens. For example the whole EGFR TKI story coupled with EGFR TK domain mutations as well as EGFR TKI resistance was present in the large panel of lung cancer cell lines and xenografts we have developed. Because of this we have been testing several new drugs in development alone and in combination such as a SMAC mimetic and Peloruside A. We have found not only dramatic differences between lung cancer cells in response to the drugs as single agents, but complex patterns of synergy (which are often dramatic) when they are combined with standard drugs. Genome Wide Approaches to Discovery of New Therapeutic Targets Including Synthetic Lethal Screens Large scale DNA sequencing and DNA amplification screens are going on to find oncogenic changes that represent “druggable” targets. However, another approach that is gaining attention are genome wise functional screens for such targets such as genome wide screening of siRNA and shRNA libraries for genes that when knocked down sensitize tumor cells to low doses of chemotherapy agents. This approach has led to the discovery of 87 genes, which when knocked down by siRNAs dramatically sensitize lung cancer cells to paclitaxel. This information could provide “signatures” for predicting paclitaxel response, but also targets that could be addressed in combination with paclitaxel. Some of these (such as a panel of cancer testis antigen genes) are selectively expressed in tumor cells. Of course these siRNA/shRNA screens can be coupled with screening of small molecule libraries. Related to this is miRNA profiling which identified two miRNAs whose expression was inversely correlated with paclitaxel resistance. By re expressing these two miRNAs, paclitaxel resistant lung cancers were converted to a paclitaxel sensitive phenotype. Thus, such miRNAs can be considered for therapeutic development. Lung Cancer Stem Cells There is considerable evidence mounting that a small subset of lung cancer cells (~1% or less of the total tumor cell population) has the properties consistent to give immortal and metastatic disease and are referred to as “cancer stem cells” or “stem-like” cells. We have developed ways to isolate and study these cells and they have clear differences in gene expression programs for a variety of stem cell proliferation genes as well as increased ability to clone in soft agar, form tumors in xenografts, and give metastatic disease in xenografts. Key issues will be to develop good, easy to use markers (such as monoclonal antibodies) to identify these cells, to see how their response to therapy compares with the bulk of the tumor (since it is likely they may be more resistant to therapy), and whether their presence or amount is of prognostic significance. Of course their identification in preneoplastic stages will be important as new molecular markers for early lung cancer detection. New Model Systems for Studying Lung Cancer Pathogenesis In order to determine which steps are most important we have developed a new system for immortalizing human bronchial epithelial cells (HBECs) in the absence of viral oncoproteins using hTERT (human telomerase providing part of the immortalization pathway) and cdk4 (bypassing the p16/Rb checkpoint) expression vectors. We have established over 30 such strains from persons with and without lung cancer. These expressed stem cell markers, can differentiate in 3 dimensional cultures into ciliated epithelium and are do not expression malignant properties. We have added oncogenic KRAS (found in 30% of non-small cell lung cancers, NSCLC) and knocked out p53 (found in 50% of all lung cancers) alone or together. These (together with hTERT and cdk4) only partially progress the HBECs toward malignancy. We have also made changes in multiple other genes such as CMYC, BCL2, PTEN, and EGFR and find that again these only partially progress HBECs towards malignancy. However, combining KRASV12, p53 knockdown, and CMYC coupled with biologic selection in SCID mice can identify a subset of fully malignant tumor cells. IN addition, the addition of these oncogenic changes leads to changes in cytokine secretion patterns that provide new information for early lung cancer detection through screening for these cytokines in blood. These HBECs are providing a new model for systematically testing the importance of additional genetic changes in lung cancer pathogenesis. In developments by several other groups, mouse models of lung cancer have been developed based on genetic abnormalities found in human lung cancer with development of mouse systems for specific development of these abnormalities in lung as a target tissue. These have been developed for non-small cell lung cancer (NSCLC) (based on oncogenic KRAS) and for small cell lung cancer (SCLC) (based on p53 and Rb abnormalities). These models are proving extremely valuable in the preclinical testing of early detection, chemoprevention, and new treatment strategies.