mrk-003 and Lung-Neoplasms

The cancer stem cell theory postulates that tumors contain a subset of cells with stem cell properties of self-renewal, differentiation, and tumor initiation. The purpose of this study is to determine the role of Notch activity in identifying lung cancer stem cells.. We investigated the role of Notch activity in lung adenocarcinoma using a Notch GFP reporter construct and a γ-secretase inhibitor (GSI), which inhibits Notch pathway activity.. Transduction of lung cancer cells with Notch GFP reporter construct identified a subset of cells with high Notch activity (GFP-bright). GFP-bright cells had the ability to form more tumor spheres in serum-free media and were able to generate both GFP-bright and GFP-dim (lower Notch activity) cell populations. GFP-bright cells were resistant to chemotherapy and were tumorigenic in serial xenotransplantation assays. Tumor xenografts of mice treated with GSI had decreased expression of downstream effectors of Notch pathway and failed to regenerate tumors upon reimplantation in NOD/SCID mice. Using multivariate analysis, we detected a statistically significant correlation between poor clinical outcome and Notch activity (reflected in increased Notch ligand expression or decreased expression of the negative modulators), in a group of 443 patients with lung adenocarcinoma. This correlation was further confirmed in an independent group of 89 patients with adenocarcinoma in which Hes-1 overexpression correlated with poor overall survival.. Notch activity can identify lung cancer stem cell-like population and its inhibition may be an appropriate target for treating lung adenocarcinoma.

Topics: Adenocarcinoma; Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Cell Cycle; Cell Line, Tumor; Cyclic S-Oxides; Flow Cytometry; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Homeodomain Proteins; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Multivariate Analysis; Neoplastic Stem Cells; Protein Isoforms; Receptors, Notch; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Thiadiazoles; Transcription Factor HES-1; Xenograft Model Antitumor Assays

The Notch signaling pathway is a critical embryonic developmental regulatory pathway that has been implicated in oncogenesis. In non-small cell lung cancer (NSCLC), recent evidence suggests that Notch signaling may contribute to maintenance of a cancer stem or progenitor cell compartment required for tumorigenesis. We explored whether intact Notch signaling is required for NSCLC clonogenic and tumorigenic potential in vitro and in vivo using a series of genetically modified model systems. In keeping with previous observations, we find that Notch3 in particular is upregulated in human lung cancer lines and that downregulation of Notch signaling using a selective γ-secretase inhibitor (MRK-003) is associated with decreased proliferation and clonogenic capacity in vitro. We show that this phenotype is rescued with the expression of NICD3, a constitutively active cleaved form of Notch3 not affected by γ-secretase inhibition. Using an inducible LSL-KRAS(G12D) model of lung cancer in vivo, we show a transient upregulation of Notch pathway activity in early tumor precursor lesions. However, a more rigorous test of the requirement for Notch signaling in lung oncogenesis, crossing the LSL-KRAS(G12D) mouse model with a transgenic with a similarly inducible global dominant-negative suppressor of Notch activity, LSL-DNMAML (dominant-negative mastermind-like), reveals no evidence of Notch pathway requirement for lung tumor initiation or growth in vivo. Distinct Notch family members may have different and potentially opposing activities in oncogenesis, and targeted inhibition of individual Notch family members may be a more effective anticancer strategy than global pathway suppression.

Topics: Amyloid Precursor Protein Secretases; Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Colony-Forming Units Assay; Cyclic S-Oxides; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms, Experimental; Receptor, Notch3; Receptors, Notch; Signal Transduction; Thiadiazoles

Notch receptors are key regulators of development by controlling cell-fate determination in many multicellular organisms. Genes that are important for normal differentiation play a role in cancer when their normal functions became dysregulated. Notch signaling has been shown to promote and maintain survival of many types of cancers, and we previously have shown that Notch3 plays an important role in lung cancer. In this study, we showed that a high percentage of lung cancer lines expressed Jagged1, Notch receptors, and their transcriptional target genes (HES1, Hey1), suggesting that the Notch pathway plays an important role in lung cancer biology. Thus, inhibition of Notch receptor activation represents a compelling treatment strategy. Notch activation requires proteolytic cleavage of the receptor by gamma-secretase protein complex. In this study, we determined the ability of MRK-003, a gamma-secretase inhibitor, to inhibit Notch3 signaling, growth, and apoptosis of lung cancer cell lines in vitro and in vivo using mouse xenograft models. We also found that MRK-003 inhibited Notch3 signaling, reduced tumor cell proliferation, inhibited serum independence, and induced apoptosis. This drug had no effect when Notch3 expression was knocked down using small interfering RNA (siRNA), suggesting that the observed effects were mediated by specific action on this receptor. In conclusion, these results support the hypothesis that inhibition of Notch activation using a gamma-secretase inhibitor represents a potential new approach for the targeted therapy of lung cancer.

Topics: Amyloid Precursor Protein Secretases; Animals; Antineoplastic Agents; Apoptosis Regulatory Proteins; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cyclic S-Oxides; Disease Progression; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mice, Nude; Receptor, Notch3; Receptors, Notch; Signal Transduction; Thiadiazoles; Xenograft Model Antitumor Assays