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Notch signaling regulates cell-fate decisions during development and postnatal life. Little is known, however, about the role of Delta-like-4 (Dll4)-Notch signaling between cancer cells, or how this signaling affects cancer metastasis. We, therefore, assessed the role of Dll4-Notch signaling in cancer metastasis. We generated a soluble Dll4 fused to the IgG1 constant region (Dll4-Fc) that acts as a blocker of Dll4-Notch signaling and introduced it into human small cell lung cancer (SCLC) cell lines expressing either high levels (SBC-3 and H1048) or low levels (SBC-5) of Dll4. The effects of Dll4-Fc on metastasis of SCLC were evaluated using a mouse model. Although Dll4-Fc had no effect on the liver metastasis of SBC-5, the number of liver metastasis inoculated with SBC-3 and H1048 cells expressing Dll4-Fc was significantly lower than that injected with control cells. To study the molecular mechanisms of the effects of Dll4-Fc on liver metastasis, a PCR array analysis was conducted. Because the expression of NF-κB target genes was affected by Dll4-Fc, we conducted an electrophoretic mobility shift assay and observed that NF-κB activities, both with and without stimulation by TNF-α, were downregulated in Dll4-Fc–overexpressing SBC-3 and H1048 cells compared with control cells. Moreover, Dll4-Fc attenuates, at least in part, the classical and alternative NF-κB activation pathway by reducing Notch1 signaling. These results suggest that Dll4-Notch signaling in cancer cells plays a critical role in liver metastasis of SCLC by regulating NF-κB signaling. Mol Cancer Ther; 11(12); 2578–87. ©2012 AACR.

PDF file - 330K, Dll4-Fc reduced NF-κB/DNA binding in SBC-3 and H1048 cells

Supplementary Table 1, Figures 1-5 from E7080 Suppresses Hematogenous Multiple Organ Metastases of Lung Cancer Cells with Nonmutated Epidermal Growth Factor Receptor

While epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors improve the prognosis of patients with EGFR mutant lung cancer, the prognosis of patients with nonmutant EGFR lung cancer, especially those with metastases, is still extremely poor. We have assessed the therapeutic efficacy of E7080, an orally available inhibitor of multiple tyrosine kinases including VEGF receptor 2 (VEGFR-2) and VEGFR-3, in experimental multiple organ metastasis of lung cancer cell lines without EGFR mutations. E7080 markedly inhibited the in vitro proliferation of VEGF-stimulated microvascular endothelial cells. Intravenous inoculation into natural killer cell–depleted severe combined immunodeficient mice of the small cell lung cancer cell lines H1048 (producing low amounts of VEGF) and SBC-5 (producing intermediate amounts of VEGF) resulted in hematogenous metastases into multiple organs, including the liver, lungs, kidneys, and bones, whereas intravenous inoculation of PC14PE6, a non–small cell lung cancer cell line producing high amounts of VEGF, resulted in lung metastases followed by massive pleural effusion. Daily treatment with E7080 started after the establishment of micrometastases significantly reduced the number of large (>2 mm) metastatic nodules and the amount of pleural effusion, and prolonged mouse survival. Histologically, E7080 treatment reduced the numbers of endothelial and lymph endothelial cells and proliferating tumor cells and increased the number of apoptotic cells in metastatic nodules. These results suggest that E7080 has antiangiogenic and antilymphangiogenic activity and may be of potential therapeutic value in patients with nonmutant EGFR lung cancer and multiple organ metastases. Mol Cancer Ther; 10(7); 1218–28. ©2011 AACR.

PDF file - 245K, Dll4-Fc promoted excessive angiogenesis of liver metastasis without affecting VEGFA expression and HUVECs proliferation

PDF file - 50K, The effect of recombinant Dll4-Fc on the NF-κB activity of SBC-3-vector cells

PDF file - 116K, Extracellular domain of murine Dll4-fused to Fc (Dll4-Fc) expressed in H1048 and SBC-5 cells

PDF file - 83K

PDF file - 174K, Dll4-Fc suppressed TNF-α induced NF-κB activity without attenuation of the cytoplasmic sequestration of NF-κB subunit

PDF file - 69K, Dll4-Fc suppresses genes-associated with metastasis in SCLC cells

PDF file - 47K, Taqman gene expression assays

Supplementary Data from Hepatocyte Growth Factor Reduces Susceptibility to an Irreversible Epidermal Growth Factor Receptor Inhibitor in EGFR-T790M Mutant Lung Cancer

Purpose: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib and erlotinib, show favorable response to EGFR mutant lung cancer. However, the responders acquire resistance almost without exception. We recently reported that hepatocyte growth factor (HGF) induces EGFR-TKI resistance by activating MET that restores downstream mitogen activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK)1/2 and phosphoinositide 3-kinase (PI3K)/Akt signaling. The purpose of this study was to determine whether inhibition of PI3K, a downstream molecule of both EGFR and MET, could overcome HGF-mediated EGFR-TKI resistance in EGFR mutant lung cancer cells PC-9 and HCC827.Experimental Design: We explored therapeutic effect of a class I PI3K inhibitor PI-103 on HGF-induced EGFR-TKI resistance in vitro and in vivo.Results: Unlike gefitinib or erlotinib, continuous exposure with PI-103 inhibited proliferation of PC-9 and HCC827 cells, even in the presence of HGF. On the other hand, in gefitinib-resistant xenograft model by using PC-9 cells mixed with HGF high producing fibroblasts, PI-103 monotherapy did not inhibit tumor growth. However, PI-103 combined with gefitinib successfully regressed gefitinib-resistant tumor. In vitro experiments by considering short half-life of PI-103 reveal that transient exposure of PI-103 combined with gefitinib caused sustained inhibition of Akt phosphorylation, but not ERK1/2 phosphorylation, resulting in induction of tumor cell apoptosis even in the presence of HGF.Conclusions: These results indicate that transient blockade of PI3K/Akt pathway by PI-103 and gefitinib could overcome HGF-mediated resistance to EGFR-TKIs by inducing apoptosis in EGFR mutant lung cancer. Clin Cancer Res; 17(8); 2260–9. ©2011 AACR.

Supplementary Figure S1 from Reveromycin A Inhibits Osteolytic Bone Metastasis of Small-Cell Lung Cancer Cells, SBC-5, through an Antiosteoclastic Activity

Purpose: Hepatocyte growth factor (HGF) induces resistance to reversible and irreversible epidermal growth factor receptor–tyrosine kinase inhibitors (EGFR-TKI) in EGFR mutant lung cancer cells by activating Met and the downstream phosphoinositide 3-kinase (PI3K)/Akt pathway. Moreover, continuous exposure to HGF accelerates the emergence of EGFR-TKI–resistant clones. We assayed whether a new Met kinase inhibitor, E7050, which is currently being evaluated in clinical trials, could overcome these three mechanisms of resistance to EGFR-TKIs.Experimental Design: The effects of E7050 on HGF-induced resistance to reversible (gefitinib), irreversible (BIBW2992), and mutant-selective (WZ4002) EGFR-TKIs were determined using the EGFR mutant human lung cancer cell lines PC-9 and HCC827 with an exon 19 deletion and H1975 with an T790M secondary mutation. PC-9 cells were mixed with HGF-producing fibroblasts, MRC-5 cells, and subcutaneously inoculated into severe combined immunodeficient mice, and the therapeutic effects of E7050 plus gefitinib were assayed.Results: E7050 circumvented resistance to all of the reversible, irreversible, and mutant-selective EGFR-TKIs induced by exogenous and/or endogenous HGF in EGFR mutant lung cancer cell lines, by blocking the Met/Gab1/PI3K/Akt pathway in vitro. E7050 also prevented the emergence of gefitinib-resistant HCC827 cells induced by continuous exposure to HGF. In the in vivo model, E7050 plus gefitinib resulted in marked regression of tumor growth associated with inhibition of Akt phosphorylation in cancer cells.Conclusions: A new Met kinase inhibitor, E7050, reverses the three HGF-induced mechanisms of gefitinib resistance, suggesting that E7050 may overcome HGF-induced resistance to gefitinib and next-generation EGFR-TKIs. Clin Cancer Res; 18(6); 1663–71. ©2012 AACR.

Supplementary Data from Crosstalk to Stromal Fibroblasts Induces Resistance of Lung Cancer to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors

Supplementary Figures S1-S4.

Purpose: Lung cancers with epidermal growth factor receptor (EGFR)–activating mutations show good clinical response to gefitinib and erlotinib, selective tyrosine kinase inhibitors (TKI) to EGFR, but these tumors invariably develop drug resistance. Host stromal cells have been found to have a considerable effect on the behavior of cancer cells. Little is known, however, about the role of host cells on the sensitivity of cancer cells to receptor TKIs. We have therefore assessed the effect of crosstalk between stromal cells and lung cancer cells harboring EGFR mutations on susceptibility to EGFR-TKIs.Experimental Design: We evaluated the gefitinib sensitivity of lung cancer cells with EGFR-activating mutations, PC-9 and HCC827, when cocultured with fibroblasts and coinjected into severe combined immunodeficient mice. We also examined the effect of lung cancer cells to fibroblast recruitment.Results: Both human fibroblast cell lines and primary cultured fibroblasts produced various levels of hepatocyte growth factor (HGF). Lung cancer cells markedly recruited fibroblasts. The lung cancer cells became resistant to EGFR-TKIs when cocultured in vitro with HGF-producing fibroblasts and coinjected into severe combined immunodeficient mice. Importantly, combined use of gefitinib plus anti-HGF antibody or the HGF antagonist, NK4, successfully overcame the fibroblast-induced EGFR-TKI resistance both in vitro and in vivo. Colocalization of fibroblasts and HGF was detected in both xenograft tumors in mouse model and lung cancer patient specimens.Conclusions: These findings indicate that crosstalk to stromal fibroblasts plays a critical role in lung cancer resistance to EGFR-TKIs and may be an ideal therapeutic target in lung cancer with EGFR-activating mutations. (Clin Cancer Res 2009;15(21):6630–8)

Purpose: The purpose of this study was to determine therapeutic effect of a novel antibiotic, reveromycin A, against osteolytic bone metastasis of human small cell lung cancer (SBC-5) cells.Results: Reveromycin A induced apoptosis specifically in osteoclasts in vitro. Although reveromycin A did not inhibit SBC-5 cell proliferation, it suppressed the expression of parathyroid hormone–related peptide. Intravenous inoculation of SBC-5 cells in natural killer cell–depleted severe combined immunodeficient mice produced experimental metastases in multiple organs, including the bone. Daily administration of reveromycin A inhibited the bone metastasis, but not visceral metastasis, in a dose-dependent manner. Histologic analyses revealed that although treatment with reveromycin A did not affect the number of proliferating tumor cells, it decreased the number of osteoclasts and increased apoptotic cells in bone lesions.Conclusions: These findings suggest that reveromycin A may inhibit osteolytic bone metastasis through suppression of osteoclast activity by directly inducing apoptosis and indirectly inhibiting tumor cell–derived parathyroid hormone–related peptide production. Therefore, reveromycin A may be a novel, potent therapeutic agent against osteolytic bone metastasis of lung cancer in humans.

Supplementary Figure 3 from Hepatocyte Growth Factor Induces Gefitinib Resistance of Lung Adenocarcinoma with Epidermal Growth Factor Receptor–Activating Mutations

Supplementary Figure 2 from Hepatocyte Growth Factor Induces Gefitinib Resistance of Lung Adenocarcinoma with Epidermal Growth Factor Receptor–Activating Mutations

Supplementary Figure S3 from The Neuromedin U-Growth Hormone Secretagogue Receptor 1b/Neurotensin Receptor 1 Oncogenic Signaling Pathway as a Therapeutic Target for Lung Cancer

Supplementary Figure S4 from The Neuromedin U-Growth Hormone Secretagogue Receptor 1b/Neurotensin Receptor 1 Oncogenic Signaling Pathway as a Therapeutic Target for Lung Cancer

Supplementary Figure Legends 1-5 from Intrinsic Cooperation between p16INK4a and p21Waf1/Cip1 in the Onset of Cellular Senescence and Tumor Suppression In vivo

Supplementary Figure 4 from Hepatocyte Growth Factor Induces Gefitinib Resistance of Lung Adenocarcinoma with Epidermal Growth Factor Receptor–Activating Mutations

Supplementary Figure Legends 1-5 from Hepatocyte Growth Factor Induces Gefitinib Resistance of Lung Adenocarcinoma with Epidermal Growth Factor Receptor–Activating Mutations

Supplementary Figure 5 from Hepatocyte Growth Factor Induces Gefitinib Resistance of Lung Adenocarcinoma with Epidermal Growth Factor Receptor–Activating Mutations

Supplementary Figure S2A-D from The Neuromedin U-Growth Hormone Secretagogue Receptor 1b/Neurotensin Receptor 1 Oncogenic Signaling Pathway as a Therapeutic Target for Lung Cancer

Supplementary Figure S6 from The Neuromedin U-Growth Hormone Secretagogue Receptor 1b/Neurotensin Receptor 1 Oncogenic Signaling Pathway as a Therapeutic Target for Lung Cancer

Supplementary Figure S5 from The Neuromedin U-Growth Hormone Secretagogue Receptor 1b/Neurotensin Receptor 1 Oncogenic Signaling Pathway as a Therapeutic Target for Lung Cancer

Supplementary Figures 1-5 from Intrinsic Cooperation between p16INK4a and p21Waf1/Cip1 in the Onset of Cellular Senescence and Tumor Suppression In vivo

Using a genome-wide cDNA microarray to search for genes that were specifically up-regulated in non–small cell lung cancers (NSCLC), we identified an abundant expression of neuromedin U (NMU) in the great majority of lung cancers. Immunohistochemical analysis showed a significant association of NMU expression with poorer prognosis of patients with NSCLC. Treatment of NSCLC cells with short interfering RNA against NMU suppressed its expression and inhibited the growth of the cells; on the other hand, the induction of exogenous expression of NMU conferred growth-promoting activity and enhanced cell mobility in vitro. We found that two G protein–coupled receptors, growth hormone secretagogue receptor 1b and neurotensin receptor 1, were also overexpressed in NSCLC cells, and that a heterodimer complex of these receptors functioned as an NMU receptor. The NMU-receptor interaction subsequently induced the generation of a second messenger, cyclic AMP, to activate its downstream genes including transcription factors and cell cycle regulators. Treatment of NSCLC cells with short interfering RNAs for growth hormone secretagogue receptor or neurotensin receptor 1 suppressed the expression of those genes and the growth of NSCLC cells. These data strongly implied that targeting the NMU signaling pathway would be a promising therapeutic strategy for the treatment of lung cancers. (Cancer Res 2006; 66(19): 9408-19)

Supplementary Figure 1 from Hepatocyte Growth Factor Induces Gefitinib Resistance of Lung Adenocarcinoma with Epidermal Growth Factor Receptor–Activating Mutations

Supplementary Figure S1A-C from The Neuromedin U-Growth Hormone Secretagogue Receptor 1b/Neurotensin Receptor 1 Oncogenic Signaling Pathway as a Therapeutic Target for Lung Cancer