A. Lineage studies revealed multi-directional differentiation of Ascl1-defined progenitors in lung development and injury repair We have previously shown that achaete-scute homolog 1, a proneural basic helix-loop-helix transcription factor, is critical for pulmonary neuroendocrine cell development. We postulated that achaete-scute homolog 1-defined cells might be progenitors, and traced their fate during development and injury repair in genetically engineered mice. R26R-stop-lacZ (Rosa) reporter mice were crossed with achaete-scute homolog 1-Cre or inducible Cre mice, in which the Ascl1 promoter drives the expression of Cre or inducible Cre recombinase, respectively. As a result, achaete-scute homolog 1-defined cells and their descendants will be permanently labeled. The labeled cells were characterized by immunohistochemistry using highly specific differentiation markers. The analyses revealed a population that proliferates prior to the pseudoglandular stage and widely contributes to epithelial and different other compartments. Notably, their distribution extended beyond the highly specialized neuroendocrrine cells. When achaete-scute homolog 1-defined cells were labeled earlier, at the embryonic day 9.5 they gave rise to both airway and alveolar cells, but when labeled later, at embryonic day 11.5 they only gave rise to the airway cells. In conclusion, achaete-scute homolog 1-defined cells in the lung represent a novel multipotent lineage indicating a close relationship of neuroendocrine cells with other cell types. In a well characterized lung injury and repair model, 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 whose precursor lesions remain unknown. In humans repeated injury and repair in smokers lungs is an integral part of carcinogenesis. Our lineage studies showed that achaete-scute homolog 1-defined precursors participate in epithelial expansion at times of significant growth and maturation of non-neuroendocrine epithelium which takes place immediately following birth. When we examined the same genetically modified mouse models following naphthalene injury, we were able to demonstrate that achaete-scute homolog 1-defined progenitors in the repairing adult lung also give rise to not only to neuroendocrine cells but multiple other cell types such as Clare cells. We are currently in the process of further defining the differentiation patterns of the cells utilizing neuroendocrine and epithelial (non-endocrine) markers. The significance of our research is that the this the first study to provide experimental evidence for the fate of neuroendocrine cells which 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. Establishing the role of neuroendocrine and achaete-scute homolog 1-defined cells in lung carcinogenesis We have previously established that constitutive expression of the achaete-schute homolog-1 transcription factor in the airway lining of mice caused marked bronchialization of alveoli, which has provided us a continuous source of this abnormality for further studies. Such metaplastic lesions occur in 12% of human lung cancer specimens and serve as a proof for field cancerization of the alveolar compartment. During field carcinogenesis the pulmonary epithelium is continuously exposed to cycles of injury and repair that will lead to premalignant changes. Gene expression analysis uncovered several genes that were related to cell growth, migration and death. We are currently assessing further the specific molecular mechanisms involved in cellular migration that are potentially regulated by achaete-scute homolog-1 in cells derived both from airways and pulmonary carcinomas. We and others have shown that achaete-scute homolog-1 is important for neuroendocrine differentiation and growth of small cell lung cancers which prompted us to examine whether deletion of pulmonary neuroendocrine cells prevents small cell lung cancer. Since achaete-scute homolog-1 mutant mice die shortly after birth due central nervous system abnormalities we obtained through collaboration a novel Cre-inducible achaete-scute homolog-1 knockout mouse. We have been able to show that this construct was able to delete neuroendocrine cells in the lung. We and others have previously shown that partial deletion of pulmonary neuroendocrine cells in adults will be compatible with a near normal life span. We are currently comparing methods to most efficiently delete the cells in the recently established genetically engineered mouse model of small cell lung cancer.

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Naizhen, Xu; Linnoila, R Ilona; Kimura, Shioko (2016) Co-expression of Achaete-Scute Homologue-1 and Calcitonin Gene-Related Peptide during NNK-Induced Pulmonary Neuroendocrine Hyperplasia and Carcinogenesis in Hamsters. J Cancer 7:2124-2131
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