Thao P. Dang

γ-Secretase Inhibitor Prevents Notch3 Activation and Reduces Proliferation in Human Lung Cancers

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.

Dominant-negative notch3 receptor inhibits mitogen-activated protein kinase pathway and the growth of human lung cancers

Notch3 is a member of an evolutionarily conserved family of cell surface receptors important in cell-fate determination in both vertebrates and invertebrates. Significant data support the role of Notch pathway in cancer development, although the conflicting role of Notch signaling pathways in tumorigenesis suggests that its action is highly context-dependent. Furthermore, although Notch receptors signal primarily through the regulation of hairy enhancer of split (HES) and HES-related (HRT) genes, they are known to crosstalk with other signaling pathways, including the epidermal growth factor (EGF) and the mitogen-activated protein kinase pathways. Whereas much is known about the role of Notch1 in human cancer, the role of Notch3 in epithelial tumors, such as lung carcinomas, has not been well established. In this study, we show that Notch3 is expressed in 80 of 207 (39%) resected human lung tumors and that its expression is positively correlated with EGF receptor expression. Inhibition of the Notch3 pathway using a dominant-negative receptor dramatically reduces growth in soft agar and increases growth factor dependence. We also find that Notch inhibition increases sensitivity to EGF receptor tyrosine kinase inhibition and decrease in phosphorylation of the mitogen-activated protein kinase. These observations support a role for Notch3 signaling in lung cancer, and one potential mechanism of maintaining the neoplastic phenotype is through the modulation of the EGF pathway.

Targeting cyclooxygenase-2 in recurrent non-small cell lung cancer : A phase II trial of celecoxib and docetaxel

Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in prostaglandin (PG) synthesis and is overexpressed in 70% to 90% of non–small cell lung cancers (NSCLC). Preclinical studies suggest inhibition of COX-2 can enhance the cytotoxic effect of docetaxel. To test this concept clinically, we administered celecoxib (400 mg p.o. twice daily) plus docetaxel (75 mg/m2 every 3 weeks) to a cohort of patients with recurrent, previously treated NSCLC. Patients first received single agent celecoxib for 5 to 10 days to ascertain the effectiveness of COX-2 inhibition, which was determined by measuring pre- and post-celecoxib levels of urinary 11α-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), the major metabolite of prostaglandin E2 (PGE2). We enrolled 56 patients (35 men, 21 women; median age, 61 years). All patients had received at least one prior chemotherapy regimen. The overall response rate was 11% and median survival was 6 months, similar to that observed with docetaxel alone. Pre-celecoxib urinary PGE-M decreased from a mean level of 27.2 to 12.2 ng/mg Cr after 5 to 10 days of celecoxib (P = 0.001). When grouped by quartile, patients with the greatest proportional decline in urinary PGE-M levels experienced a longer survival compared to those with no change or an increase in PGE-M (14.8 versus 6.3 versus 5.0 months). Our data suggest that combining celecoxib with docetaxel using the doses and schedule employed does not improve survival in unselected patients with recurrent, previously treated NSCLC. However, in light of the apparent survival prolongation in the subset with a marked decline in urinary PGE-M levels, further investigation of strategies designed to decrease PGE2 synthesis in NSCLC seems warranted.

Constitutive activation of Notch3 inhibits terminal epithelial differentiation in lungs of transgenic mice

Notch3 is a transmembrane receptor and a member of the Notch signaling pathway essential for cellular differentiation in a variety of developing tissues in both invertebrates and vertebrates. Emerging data support the role of the Notch signaling pathway in tumorigenesis. We have previously demonstrated the expression of Notch3 in a subset of lung adenocarcinomas. To further elucidate the role of Notch3 in development of lung cancer, we established a transgenic mouse model in which the intracellular domain of Notch3 is expressed using the surfactant protein C promoter/enhancer. Constitutive expression of Notch3 in the peripheral epithelium in the developing lung resulted in altered lung morphology and delayed development, leading to perinatal lethality in these transgenic mice. Cell-specific markers and electron microscopy examination showed that the majority of the epithelial cells are undifferentiated, with some maturation of type II pneumocytes. No type I alveolar cells were evident. Metaplasia of undifferentiated cells in the terminal airways was also observed. Although the mice did not live long enough to assess tumor development, these findings demonstrate that ectopic expression of Notch3 in airway epithelium potentially contributes to the multistep evolution of lung cancer through the inhibition of terminal differentiation.

Efficacy and Safety of Single-Agent Pertuzumab, a Human Epidermal Receptor Dimerization Inhibitor, in Patients with Non–Small Cell Lung Cancer

Purpose: Pertuzumab, a first-in-class human epidermal receptor 2 (HER2) dimerization inhibitor, is a humanized monoclonal anti-HER2 antibody that binds HER2s dimerization domain and inhibits HER2 signaling. Based on supporting preclinical studies, we undertook a Phase II trial of pertuzumab in patients with recurrent non–small cell lung cancer (NSCLC). Experimental Design: Patients with previously treated NSCLC accessible for core biopsy and naive to HER pathway inhibitors were treated with pertuzumab i.v. once every 3 weeks. Tumor assessments were done at 6 and 12 weeks and then every 3 months thereafter. The primary efficacy end point was overall response rate by Response Evaluation Criteria in Solid Tumors. Measurement of tumor glucose metabolism (SUVmax) by F-18-fluorodeoxyglucose positron emission tomography was used as an exploratory pharmacodynamic marker of drug activity. Results: Of 43 patients treated with pertuzumab, no responses were seen; 18 of 43 (41.9%) and 9 of 43 (20.9%) patients had stable disease at 6 and 12 weeks, respectively. The median and 3-month progression-free survival rates (PFS) were 6.1 weeks (95% confidence interval, 5.3-11.3 weeks) and 28.4% (95% confidence interval, 14.4-44.2%), respectively. Of 22 patients who underwent F-18-fluorodeoxyglucose positron emission tomography, six (27.3%) had a metabolic response to pertuzumab as evidenced by decreased SUVmax. These patients had prolonged PFS (HR = 0.11, log-rank P value = 0.018) compared with the 16 patients who had no metabolic response. Four patients (9.3%) experienced a grade 3/grade 4 adverse event judged related to pertuzumab; none exhibited grade 3/grade 4 cardiac toxicity. Conclusions: Pertuzumab is well tolerated as monotherapy. Pharmacodynamic activity correlated with prolonged PFS was detected in a moderate percentage of patients (27.3%). Further clinical development of pertuzumab should focus on rational combinations of pertuzumab with other drugs active in NSCLC.

Targeting Specific Regions of the Notch3 Ligand-Binding Domain Induces Apoptosis And Inhibits Tumor Growth In Lung Cancer

Like many signaling pathways in development, the Notch receptor pathway plays an important role in cancer pathobiology when it is dysregulated. Potential ligand-binding sites within the epidermal growth factor (EGF)-like repeats of Notch1 have been identified, but the ligand-binding domains in Notch3, which is implicated in lung cancer, are not known. In screening a library of 155 peptides representing all 34 EGF-like repeats in Notch3, we discovered two distinct ligand-binding regions involving the 7-10 and 21-22 repeats that are distinct from the putative ligand-binding domain of Notch1. In cell-based assays, peptides from these regions induced apoptosis and reduced expression of the Notch3-dependent gene Hey1. They also bound directly to the Notch ligand Jagged1, suggesting that their mechanism of action involves disrupting interactions between Notch3 and Jagged1. Recombinant Fc fusion peptides engineered for in vivo testing showed that the Notch3 peptides defined could trigger apoptosis and suppress tumor growth in tumor xenograft assays. These findings rationalize a mechanistic approach to lung cancer treatment based on Notch3 receptor-targeted therapeutic development.

Cloned fusion product from a rare t(15;19)(q13.2;p13.1) inhibit S phase in vitro

Background: Somatically acquired chromosomal translocation is a common mechanism of oncogene activation in many haematopoietic tumours and sarcomas. However, very few recurrent chromosomal translocations have been reported in more common epithelial tumours such as lung carcinomas. Methods: We established a cell line HCC2429 from an aggressive, metastatic lung cancer arising in a young, non-smoking woman, demonstrating a t(15;19)(q13.2;p13.1). The breakpoints on chromosomes 15 and 19 were cloned using long distance inverse PCR and we determined by RT-PCR that the translocation results in a novel fusion transcript in which the 3′ end Brd4 on chromosome 19p is fused to the 5′ end of NUT on chromosome 15q. Results: In total, 128 lung cancer tissues were screened using fluorescent in situ hybridisation, but none of the tumours screened demonstrated t(15;19), suggesting that this translocation is not commonly found in lung cancer. Consistent with previous literature, ectopic expression of wild type Brd4 was shown to inhibit G1 to S progression. However, we also found that the Brd4-NUT fusion augments the inhibition of progression to S phase compared with wild type Brd4. Conclusion: Alteration in cell cycle kinetics is important in tumorigenesis, although the exact role of Brd4-NUT fusion protein in the pathogenesis of lung cancers remains unclear and need to be further elucidated.

Classification by Mass Spectrometry Can Accurately and Reliably Predict Outcome in Patients with Non-small Cell Lung Cancer Treated with Erlotinib-Containing Regimen

Purpose: Although many lung cancers express the epidermal growth factor receptor and the vascular endothelial growth factor, only a small fraction of patients will respond to inhibitors of these pathways. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) has shown promise in biomarker discovery, potentially allowing the selection of patients who may benefit from such therapies. Here, we use a matrix-assisted laser desorption/ionization MS proteomic algorithm developed from a small dataset of erlotinib-bevacizumab treated patients to predict the clinical outcome of patients treated with erlotinib alone. Methods: Pretreatment serum collected from patients in a phase I/II study of erlotinib in combination with bevacizumab for recurrent or refractory non-small cell lung cancer was used to develop a proteomic classifier. This classifier was validated using an independent treatment cohort and a control population. Result: A proteomic profile based on 11 distinct m/z features was developed. This predictive algorithm was associated with outcome using the univariate Cox proportional hazard model in the training set (p = 0.0006 for overall survival; p = 0.0012 for progression-free survival). The signature also predicted overall survival and progression-free survival outcome when applied to a blinded test set of patients treated with erlotinib alone on Eastern Cooperative Oncology Group 3503 (n = 82, p < 0.0001 and p = 0.0018, respectively) but not when applied to a cohort of patients treated with chemotherapy alone (n = 61, p = 0.128). Conclusion: The independently derived classifier supports the hypothesis that MS can reliably predict the outcome of patients treated with epidermal growth factor receptor kinase inhibitors.