A. Neuroendocrine differentiation and cellular lineages in the lung during injury repair and carcinogenesis. Exposure to naphthalene, a component of cigarette smoke, kills airway epithelial (Clara) cells in mice, but is rapidly followed by Clara cell reconstitution coincident with proliferation of pulmonary neuroendocrine cells. The reconstituting progenitor cells have been suggested to enter a transient neuroendocrine differentiation phase before differentiating to Clara cells. Furthermore, these progenitors were suggested to be the target population for transformation into the most common and virulent of all neuroendocrine tumors: small cell lung cancer (SCLC) whose precursor lesions remain unknown. It is currently thought that pulmonary neuroendocrine cells, like their counterparts in the gastrointestinal tract are derived from multipotent progenitors, not from neural crest and that all epithelial cells arise from a single cell. In many instances pulmonary neuroendocrine cells reveal a close relationship with non-neuroendocrine cells, which manifests as co-expression of neuroendocrine markers such as CGRP, PGP9.5 or synaptophysin and epithelial markers such as CC10 and surfactant proteins in the airway epithelium during embryonal development, injury repair and carcinogen exposure. We have previously shown that a neural transcription factor from Drosophila, achaete-schute homolog-1 (hASH1 in humans and Mash1 in mice) is expressed at high levels in neuroendocrine lung cancers and is essential for neuroendocrine differentiation in neoplastic and non-neoplastic lung. As the relative number of the pulmonary neuroendocrine cells peaks at the time of birth, it is plausible that prior to birth Mash1-defined progenitors may primarily contribute to the generation of neuroendocrine cells. On the other hand, we postulate that Mash1-expressing precursors will participate in epithelial expansion at times of significant growth and maturation of non-neuroendocrine epithelium which takes place immediately following birth and again at the time of injury repair. Specifically, in humans repeated injury and repair in smokers lungs is an integral part of carcinogenesis. We have now initiated in vivo lineage studies of Mash1-containing cells in the lungs using a dynamic approach utilizing novel genetically modified mouse models that have become available through collaboration. So far we have been able to demonstrate that, similarly to brain and spinal cord, Mash1- derived progenitors in the lung give rise to multiple cell types. We are currently in the process of determining the differentiation patterns of the cells utilizing neuroendocrine and epithelial (non-endocrine) markers. The significance of our research is that the results will provide a rational basis for early detection and novel molecular targets for therapeutic interventions for human lung small cell lung cancer by identifying specific markers and pathways as well as distinct models of multistep neuroendocrine carcinogenesis. B. Premalignant changes of peripheral lung adenocarcinoma We have previously established a mouse model for a potentially premalignant condition for peripheral lung adenocarcinomas composed of bronchialization of alveoli. This is a metaplastic lesion which we initially found in 12% of human lung cancer specimens as a proof for field cancerization of the alveolar compartment. Constitutive expression of the achaete-schute homolog-1 transcription factor under CC10 promoter in mice caused marked bronchialization of alveoli, which has provided us a continuous source of this abnormality for further studies. We recently discovered that matrilysin-1, also called matrix metalloproteinase-7, was expressed in both human and mouse lesions of bronchiolization of alveoli, but not in normal lungs. In vitro, forced expression of the matrilysin-1 gene in immortalized human airway cells promoted cellular migration, proliferation and resistance against apoptosis. Gene expression analysis uncovered several genes that were also related to cell growth, migration and death. In vivo, the bronchiolization of alveoli was reduced in mice that were deficient of matrilysin-1. We concluded that matrilysin-1 may play an important role in this candidate premalignant lesion by promoting proliferation, migration and attenuation of apoptosis and involving multiple genes in the MAP kinase pathway. We are currently assessing further the specific molecular mechanisms involved in cellular migration that are potentially regulated by hASH1 in cells derived both from airways and pulmonary carcinomas. We have also examined the neoplastic potential of the transgenic mice with bronchiolization of alveoli using the well-characterized tobacco-specific nitrosamine (NNK) induced lung tumorigenesis model. Interestingly, the transgenic mice revealed a delay in tumorigenesis. In order to examine the underlying mechanism, we focused on the interactions of achaete-scute homolog-1, matrilysin-1 and O-6-methylguanine-DNA methyltransferase which we had recently identified potentially being involved. Specifically, O-6-methylguanine-DNA methyltransferase was one of the genes strongly upregulated by matrilysin-1 in our previous microarray study. Our current results of the chromosome immunoprecipitation assay suggest that achaete-scute homolog-1 is able to directly upregulate both matrilysin-1 and O-6-methylguanine-DNA methyltransferase. The latter conveys a resistance to NNK-induced DNA damage in airway cells as indicated by the comet-assay thus delaying the tumorigenesis. However, exposure to NNK will eventually lead to silencing of O-6-methylguanine-DNA methyltransferase expression by promoter hypermethylation resulting in tumorigenesis. During field carcinogenesis that will lead to premalignant changes, the pulmonary epithelium is continuously exposed to cycles of injury and repair. In order to study the involvement of achaete-scute homolog-1 in regeneration of the conducting airway, we exposed the transgenic mice to naphthalene. Naphthalene, a component of cigarette smoke, kills airway epithelial (Clara) cells in mice, but is rapidly followed by Clara cell reconstitution coincident with proliferation of pulmonary neuroendocrine cells. We discovered enhanced epithelial cell proliferation with preservation of Clara cell markers CC10 and the principal naphthalene metabolizing enzyme cytochrome p4502F2 in bronchiolization of alveoli of the transgenic mice. In addition there was an overall attenuation of neuroendocrine maturation which was coupled with increased proliferation during the post-napthalene repair. These data suggest that achaete-scute homolog-1 may play an important role in maintaining a progenitor phenotype that promotes renewal of both epithelial and neuroendocrine cells. Moreover, it may propagate a stem cell microenvironment in bronchiolization of alveoli where epithelium becomes resistant to naphthalene toxicity. The significance of our findings is two-fold: First, we show that constitutive expression of hASH1 in lung epithelium promotes remodeling through the same pathways that are commonly activated during human lung carcinogenesis thus providing a novel model for the potential premalignant changes for lung adenocarcinomas. Second, our results suggest that the impact of achaete-scute homolog-1, a proneural basic helix-loop-helix factor, is not limited to neuroencocrine cells and differentiation, but is extended to multiple functions in epithelial (non-neuroendocrine) cells as well.
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