In 2010 over 160,000 Americans died of lung cancer, more than from prostate, breast, and colon cancers combined, making this by far the leading cause of cancer deaths (1
). Approximately 85% of all lung cancers are of non-small cell histology. Although current therapies have improved survival and quality of life for patients with advanced non-small cell lung cancer (NSCLC), this remains a nearly universally fatal disease. New therapeutic approaches are critically needed.
Progenitor subpopulations of cancer cells with high clonogenic and tumorigenic potential, termed cancer stem cells or tumor progenitor cells, have recently been described within many solid tumors including cancers of the breast, prostate, and brain (2
). Differentiated tissues of the body are all derived from pluripotent precursors, the most primitive of which are termed stem cells. Stem cells maintain a unique ability for self-renewal, and for production of progeny capable of differentiation along multiple developmental lineages. Similarly, recent data from a large number of laboratories using a variety of approaches suggest that the differentiated and heterogeneous cells that comprise human tumors are derived from relatively small populations of precursors with selective self-renewal and differentiative potential (3
). These self-renewing precursors appear to have unique tumorigenic capacity in in vivo
models. Taken together these several lines of evidence support what has become known as the cancer stem cell hypothesis: that a small but variable percentage of cells in a tumor are capable of extensive self-renewal and tumor propagation, and that tumors are comprised of at least two functionally and phenotypically distinct populations: 1) a small population of cells with stem cell-like characteristics and extensive proliferative capability, and 2) a more differentiated population with limited proliferative (and essentially no long-term tumorigenic) potential.
The isolation and initial phenotypic characterization of stem-like cells within cancers with unique clonogenic/tumorigenic potential was first demonstrated in the context of Acute Myeloid Leukemia and more recently in breast, brain, prostate and other malignancies (4
). Definitive isolation and characterization of precursor populations for NSCLC has not been reported, although data from a murine model of lung adenocarcinoma suggests that tumors arise from a small compartment of specialized cells in the terminal bronchoalveolar junctions (9
). The authors of this report suggest that similar cells may give rise to human adenocarcinoma.
Emerging data from multiple groups suggests that survival, proliferation, and differentiation of cancer stem cells are regulated by differential activity of key embryonic signaling pathways (10
). Survival, proliferation and differentiation of normal stem cells and somatic precursors are tightly regulated by key developmental signal pathways. Many of these pathways, including the Notch, Wnt, and Hedgehog pathways, appear to be active, and aberrantly regulated, in cancers and in defined cancer precursor populations (2
). Elevated expression of Notch family members, as well as expression of Hes1
(hairy and enhancer of split 1), a downstream target of the Notch pathway, has been reported in NSCLC, consistent with functional pathway activity (11
The Notch signaling pathway is a highly evolutionarily conserved developmental regulatory pathway (14
). In mammalian species including mice and humans, there are four Notch receptors: Notch1, Notch2, Notch3 and Notch4 (17
). Notch receptors are single-pass transmembrane proteins with a large extracellular portion and relatively small intracellular domain. Notch signaling is initiated upon Notch ligand (Jagged or Delta-like) binding to the extracellular domain. Because Notch ligands are also transmembrane proteins, the Notch cascade is normally triggered by direct cell-to-cell contact. Upon ligand/receptor interaction, two sequential proteolytic cleavage events of the engaged Notch receptor occur, involving α-secretase and γ-secretase, releasing the Notch intracellular domain (NICD). The NICD can then translocate to the nucleus were it converts the complex CSL (comprised of CBF1 and RB) from a transcriptional repressor into a transcriptional activator (18
). The NICD-CBF1 complex is bound by Mastermind-like (MAML) proteins, which serve as a scaffold to recruit co-activators (i.e. p300), driving the expression of downstream targets such as Hes1
, encoding a basic helix loop helix transcriptional repressor, and other genes that promote cell growth and proliferation.
In addition to its multiple roles in controlling cell fate and differentiation decisions in embryogenesis, dysregulation of the Notch pathway has been implicated in many cancers, first and most definitively T-cell acute lymphoblastic leukemia (T-ALL) (20
). Approximately 60% of T-ALL harbor activating mutations in Notch1
, which functions as a critical driver mutation for this type of cancer. The roles of aberrant Notch signaling in other malignancies are more complex, with both Notch pathway activating mutations, and inactivating mutations being defined (21
Dang and colleagues first linked the dysregulation of Notch3 to human lung cancer when they observed an upstream chromosome translocation in tumor from a nonsmoker and later observed overexpression of Notch3
in 40% of NSCLC (24
). Oncogenic mutations in Notch1
in lung cancer have also been described (11
). Among other suggested effects, it has been reported that Notch activity promotes NSCLC survival through inhibition of pro-apoptotic Bim, and through induction of anti-apoptotic Survivin (26
). A putative tumor progenitor cell subset in NSCLC lines, defined by aldehyde dehydrogenase upregulation, was recently found to be specifically dependent on Notch activity for maintenance of clonogenic potential (12
). Taken together, these observations suggest that the inhibition of Notch signaling represents a potential therapeutic strategy in NSCLC.
In summary several lines of indirect evidence suggest that Notch signaling may regulate proliferation, survival, and differentiation of a subpopulation of clonogenic precursors or cancer stem cells in NSCLC. We sought to explore the roles of Notch signaling in lung cancer development, and in particular to more definitively test the hypothesis that Notch signaling was critical to initial stages of lung carcinogenesis in early precursor lesions using in vivo models of NSCLC tumorigenesis.