Cellular and genetic dissection of pulmonary neuroendocrine stem cells and small cell lung cancer initiation

Pulmonary neuroendocrine (NE) cells are specialized neurosensory cells sparsely distributed throughout the bronchial epithelium of all terrestrial vertebrates. NE cells coalesce during embryonic development into clusters of ~20-30 cells termed neuroepithelial bodies (NEBs) mainly at airway branchpoints, where they are proposed to sense a variety of physiological stimuli and signal their presence both locally to other lung-resident cells and through synapse-mediated activation of pulmonary sensory neurons leading to breathing control centers in the brainstem. Consistent with their morphological and molecular specialization, NE cells normally divide rarely if ever under homeostatic conditions. However, following severe lung injury, NE cells can proliferate and generate other bronchial cell types to promote epithelial repair. Here I probe NE stem cell function at single-cell resolution in the adult mouse lung. I show that only a minor subpopulation (~17%) of differentiated NE cells, typically 2-4 cells per NEB, has stem cell function, which I term "NEstem." I chart the behavior of these differentiated stem cells after activation by airway injury, revealing an elaborate but modular and temporally phased sequence of cellular events rivaling those of classical stem cells. These include self-renewal within the NEB niche, dispersal (cell migration) away from the niche, and the deprogramming of a single daughter cell and its amplification to generate a large clonal patch of regenerated epithelium, complete with appropriately restored cell types, surrounding the parental NEB. In addition to their physiological and stem cell functions, NE cells can also serve as the cell of origin for small cell lung cancer (SCLC), the deadliest lung cancer subtype. SCLC patient tumors universally harbor loss-of-function mutations in the tumor suppressors Rb and p53, and commonly mutations in at least one of the four Notch receptors and in the insulin-like growth factor signaling pathway protein, IRS2. I provide evidence that all four of these tumor suppressors or oncogenes control the NEstem injury-dependent activation program and define the steps they regulate, leading to identification of genes with either pleiotropic (e.g., p53) or exquisitely specific (e.g., Notch) effects. The results suggest that NEstem are prominent cells of origin in SCLC, and that transformation results from constitutive activation of stem cell renewal and dispersal and concomitant inhibition of tumor suppressive deprogramming. Findings herein provide direct insight into basic questions concerning lung stem cell biology and lung cancer, and identify generalizable principles relevant to stem cells and cancer types across adult mammalian organs. I show that a differentiated stem cell can exhibit many or all of the salient features classically ascribed to "professional" stem cells, and though single cells can perform the full sequence, the activation program is modular and controlled by distinct but interacting signals. To generate lung cancer, the stem cell program itself is corrupted through module-specific genetic activation or inactivation, in order to promote uncontrolled growth and invasion while preventing unwanted shunt pathways. I propose that findings in stem cell and cancer biology should each be leveraged to better understand the other, and to hasten the arrival of discoveries that will ultimately improve the human condition both present and future.

Advisor: Krasnow Mark
Beachy Philip Arden 
Desai Tushar
Sage Julien