Background: The cancer stem cell hypothesis postulates that tumors contain a subset of cells with stem cell properties that can initiate and recapitulate original tumor heterogeneity in serial xenotransplantation assays. A number of developmental signaling pathways, including Notch, have been implicated in regulating stem cell properties of self-renewal and differentiation. In this study, we investigated the role of Notch activity in lung cancer stem cells utilizing a Notch GFP-reporter construct and a gamma secretase inhibitor (MRK003), which inhibits the activation of Notch through preventing the cleavage of its intracellular domain (NICD).

Results: Transduction of lung cancer cell line NCI-H1299 with a Notch GFP-reporter construct identified a subset of cells with high Notch activity (GFP-positive). We compared the tumor initiating capacity of GFP-positive and -negative cells and only cells with high Notch activity, as evidenced by GFP expression, were tumorigenic in serial transplantation assays in NOD/SCID mice. Furthermore, GFP-positive cells were capable of generating both GFP-positive and GFP-negative cell populations. In contrast, GFP-negative cells generated primary xenograft tumors only and failed to generate the GFP-positive population or secondary tumors in serial xenotransplantation. Furthermore, a single GFP-positive cell but not a GFP-negative cell was able to generate a xenograft in NOD/SCID mice. In vitro, the gamma secretase inhibitor MRK003 induced cell cycle inhibition and apoptosis in lung cancer cell lines NCI-H1299, NCI-H460, NCI-H358, and NCI-H82. To determine the specificity of MRK003, we introduced NICD expressing vectors that rescued the effects of MRK003, thus demonstrating a direct role in Notch inhibition. MRK003 treatment of mice with lung tumor xenografts (100mg/kg, 3 days per week) decreased the expression of downstream effectors of Notch. Furthermore, cells derived from MRK003 treated tumors failed to induce tumors upon secondary reimplantation in NOD/SCID mice, consistent with effects of this treatment on the cancer stem cell population. Utilizing multivariate analysis, we assessed the association of Notch pathway components with clinical outcome of 441 lung adenocarcinoma patients. We found a statistically significant correlation between poor overall survival and increased Notch ligands expression (p=0.042) or decreased expression of negative modulators (p=0.00016).

Conclusion: These studies suggest that the Notch pathway is an important regulator of lung cancer stem cells and that its inhibition may specifically target the cancer stem cell population. The correlation between Notch pathway activation and patient survival, suggests a potential role for gamma secretase inhibitors in the treatment of lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 496. doi:10.1158/1538-7445.AM2011-496