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Supplementary Figure 5 from Oxygen Concentration Determines the Biological Effects of NOTCH-1 Signaling in Adenocarcinoma of the Lung

Supplementary Tables 1-2, Figures 1-8 from Opposite Effects of Notch-1 and Notch-2 on Mesothelioma Cell Survival under Hypoxia Are Exerted through the Akt Pathway

Supplementary Figures 1-5, Tables 1-2 from Inhibition of NOTCH Signaling by Gamma Secretase Inhibitor Engages the RB Pathway and Elicits Cell Cycle Exit in T-Cell Acute Lymphoblastic Leukemia Cells

NOTCH signaling is an evolutionarily conserved signaling pathway that regulates cell fate during development and postnatal life. It has been increasingly linked to carcinogenesis, although its role in cancer seems to be highly context and tissue specific. Although NOTCH signaling is required for lung development, little is known about its role in lung cancer. In this study, we show that NOTCH signaling, as measured by the γ-secretase cleavage product NIC-1, is active in both normal human and lung tumor samples; however, downstream NOTCH readouts (i.e., HES-1 and HES-5) are elevated in lung tumors. Levels of NOTCH signaling components in primary human lung cells reflect observations in tissue samples, yet lung tumor cell lines showed little NOTCH signaling. Because oxygen concentrations are important in normal lung physiology and lung tumors are hypoxic, the effect of low oxygen on these lung tumor cell lines was evaluated. We found that hypoxia dramatically elevates NOTCH signaling (especially NOTCH-1) in lung tumor cell lines and concomitantly sensitizes them to inhibition via small-molecule γ-secretase inhibitors or NOTCH-1 RNA interference. γ-Secretase inhibitor–induced apoptosis of lung tumor cells grown under hypoxic conditions could be rescued by reintroduction of active NOTCH-1. Our data strengthen the role of NOTCH in lung cancer and as a therapeutic target for the treatment of lung and other hypoxic tumor types. [Cancer Res 2007;67(17):7954–9]

Supplementary Legends 1-5 and Table 1 Legend from Oxygen Concentration Determines the Biological Effects of NOTCH-1 Signaling in Adenocarcinoma of the Lung

Supplementary Table 1 from Oxygen Concentration Determines the Biological Effects of NOTCH-1 Signaling in Adenocarcinoma of the Lung

Supplementary Figures 1-3, Tables 1-2 from Downregulation of Notch Pathway by a γ-Secretase Inhibitor Attenuates AKT/Mammalian Target of Rapamycin Signaling and Glucose Uptake in an ERBB2 Transgenic Breast Cancer Model

Supplementary Figure 3 from Oxygen Concentration Determines the Biological Effects of NOTCH-1 Signaling in Adenocarcinoma of the Lung

Supplementary Figure 2 from Oxygen Concentration Determines the Biological Effects of NOTCH-1 Signaling in Adenocarcinoma of the Lung

NOTCH signaling is deregulated in the majority of T-cell acute lymphoblastic leukemias (T-ALL) as a result of activating mutations in NOTCH1. Gamma secretase inhibitors (GSI) block proteolytic activation of NOTCH receptors and may provide a targeted therapy for T-ALL. We have investigated the mechanisms of GSI sensitivity across a panel of T-ALL cell lines, yielding an approach for patient stratification based on pathway activity and also providing a rational combination strategy for enhanced response to GSI. Whereas the NOTCH1 mutation status does not serve as a predictor of GSI sensitivity, a gene expression signature of NOTCH pathway activity does correlate with response, and may be useful in the selection of patients more likely to respond to GSI. Furthermore, inhibition of the NOTCH pathway activity signature correlates with the induction of the cyclin-dependent kinase inhibitors CDKN2D (p19INK4d) and CDKN1B (p27Kip1), leading to derepression of RB and subsequent exit from the cell cycle. Consistent with this evidence of cell cycle exit, short-term exposure of GSI resulted in sustained molecular and phenotypic effects after withdrawal of the compound. Combination treatment with GSI and a small molecule inhibitor of CDK4 produced synergistic growth inhibition, providing evidence that GSI engagement of the CDK4/RB pathway is an important mechanism of GSI action and supports further investigation of this combination for improved efficacy in treating T-ALL. [Cancer Res 2009;69(7):3060–8]

Supplementary Figure 4 from Oxygen Concentration Determines the Biological Effects of NOTCH-1 Signaling in Adenocarcinoma of the Lung

Supplementary Figure 1 from Oxygen Concentration Determines the Biological Effects of NOTCH-1 Signaling in Adenocarcinoma of the Lung

Proteins modified by oxidants are rapidly degraded by intracellular proteases. Oxidatively modified superoxide dismutase (Ox-SOD) was degraded 2-8 times faster at both acidic and alkaline pH than the native protein in bovine cardiac tissue extracts. At acidic pH, Ox-SOD hydrolysis was stimulated by ATP and by non-hydrolyzable ATP analogs by up to 50%, but degradation was not stimulated by ATP at alkaline pH. The aspartic protease inhibitor pepstatin completely inhibited the acid Ox-SOD hydrolyzing activity and its stimulation by ATP. This activity eluted from gel filtration with a molecular size of 34-48 kDa and contained the single chain and two mature forms of cathepsin D. Purified cathepsin D degraded Ox-SOD and ATP enhanced the affinity of cathepsin D for oxidatively modified proteins. Thus cardiac tissue proteins modified by oxidants may be substrates for the lysosomal protease cathepsin D.

It is well established that the functional properties of proteins can be compromised by oxidative damage and, in vivo, proteins modified by oxidants are rapidly degraded. It was hypothesized that oxidants may also affect the ability of proteases to hydrolyze peptides and proteins. We therefore examined the effect of oxidants on the endopeptidase activities of the 650 kDa 20S proteasome or multicatalytic endopeptidase (MCP), which is thought to play a central role in nonlysosomal protein breakdown. Treatment of the MCP with the oxidant system, FeSO4-EDTA-ascorbate, stimulated the peptidase activities of the MCP while H2O2 treatment showed little or no stimulation. However, treatment of the MCP with FeSO4-EDTA-ascorbate or H2O2 stimulated proteinase activity by 480% and 730%, respectively. An endogenous activator of the MCP, PA28, stimulated the acidic, basic, and hydrophobic peptidase activities of the MCP, but had no effect on proteolytic activity. Treatment of PA28 with oxidants in the presence of MCP or alone did not greatly affect PA28's ability to activate the peptidase activities of the MCP. Using nondenaturing polyacrylamide gel electrophoresis, structural alterations in the enzyme which may be responsible for the activation of peptidase and protease activities following exposure to oxidants were investigated. Treatment of the MCP with reagents that activate proteolysis, including H2O2, as well as the serine protease inhibitor 3,4-dichloroisocoumarin and the cysteine protease inhibitor p-(chloromercuri) benzenesulfonic acid, all caused dissociation of the 650 kDa MCP. However, exposure to FeSO4-EDTA-ascorbate resulted in little or no dissociation of the complex. The MCP complex dissociated by p-(chloromercuri) benzenesulfonic acid could be reassociated upon treatment with the reducing agent dithiothreitol, but dithiothreitol failed to completely reassociate 3,4-dichloroisocoumarin- or H2O2 treated MCP. Therefore, chemical modification of the MCP can cause activation with varying degrees of complex dissociation. These results suggest that metabolites, such as reactive oxygen species, in addition to endogenous proteins, such as PA28, are capable of modulating MCP activity.

Cancer stem cells (CSCs) are believed to be responsible for breast cancer formation and recurrence; therefore, therapeutic strategies targeting CSCs must be developed. One approach may be targeting signaling pathways, like Notch, that are involved in stem cell self-renewal and survival.Breast cancer stem-like cells derived from cell lines and patient samples were examined for Notch expression and activation. The effect of Notch inhibition on sphere formation, proliferation, and colony formation was determined.Breast cancer stem-like cells consistently expressed elevated Notch activation compared with bulk tumor cells. Blockade of Notch signaling using pharmacologic and genomic approaches prevented sphere formation, proliferation, and/or colony formation in soft agar. Interestingly, a gamma-secretase inhibitor, MRK003, induced apoptosis in these cells.Our findings support a crucial role for Notch signaling in maintenance of breast cancer stem-like cells, and suggest Notch inhibition may have clinical benefits in targeting CSCs.

Glioblastoma (GBM) is the most common malignant brain tumor that is characterized by high proliferative rate and invasiveness. Since dysregulation of Notch signaling is implicated in the pathogenesis of many human cancers, here we investigated the role of Notch signaling in GBM. We found that there is aberrant activation of Notch signaling in GBM cell lines and human GBM-derived neurospheres. Inhibition of Notch signaling via the expression of a dominant negative form of the Notch coactivator, mastermind-like 1 (DN-MAML1), or the treatment of a γ-secretase inhibitor, (GSI) MRK-003, resulted in a significant reduction in GBM cell growth in vitro and in vivo. Knockdown of individual Notch receptors revealed that Notch1 and Notch2 receptors differentially contributed to GBM cell growth, with Notch2 having a predominant role. Furthermore, blockade of Notch signaling inhibited the proliferation of human GBM-derived neurospheres in vitro and in vivo. Our overall data indicate that Notch signaling contributes significantly to optimal GBM growth, strongly supporting that the Notch pathway is a promising therapeutic target for GBM.

The oncoprotein hdm2 ubiquitinates p53, resulting in the rapid degradation of p53 through the ubiquitin (Ub)–proteasome pathway. Hdm2-mediated destabilization and inactivation of p53 are thought to play a critical role in a number of human cancers. We have used an in vitro enzyme assay, monitoring hdm2-catalyzed Ub transfer from preconjugated Ub-Ubc4 to p53, to identify small molecule inhibitors of this enzyme. Three chemically distinct types of inhibitors were identified this way, each with potency in the micromolar range. All three types of compounds display selective inhibition of hdm2 E3 ligase activity, with little or no effect on other Ub-using enzymes. Most strikingly, these compounds do not inhibit the autoubiquitination activity of hdm2. Steady-state analysis reveals that all three classes behave as simple reversible inhibitors of the enzyme and that they are noncompetitive with respect to both substrates, Ub-Ubc4 and p53. Studies of the effects of combinations of two inhibitory molecules on hdm2 activity indicate that the three types of compounds bind in a mutually exclusive fashion, suggesting a common binding site on hdm2 for all of these inhibitors. These compounds establish the feasibility of selectively blocking hdm2-mediated ubiquitination of p53 by small molecule inhibitors. Selective inhibitors of hdm2 E3 ligase activity could provide a novel mechanism for the development of new chemotherapeutics for the treatment of human cancers.

Cleavage of non-viral proteins is rarely observed with the HIV-1 protease (HIV pr). One such cleavage event occurs with bcl-2, an important cytoprotective protein. The loss of bcl-2 has biological consequences, leading to enhanced HIV replication and programmed death of the host cell. A strategy is proposed to suppress HIV with non-cleavable mutants of bcl-2.

1. 1. Two chromatographically distinct multicatalytic proteinases (MCP's) were isolated from the cytoplasm of chicken red blood cells and one MCP was purified from the nuclei. 2. 2. The nuclear and the majority (97–99%) of the cytoplasmic multicatalytic proteolytic activity were chromatographically similar and differed from the minor cytoplasmic activity in their elution from hydroxylapatite, number of subunits on 2D-SDS-PAGE, and in their sensitivity to proteinase inhibitors. 3. 3. Dichloroisocoumarin, a serine proteinase inhibitor, inhibited the hydrolysis of fluorogenic peptides but stimulated the degradation of casein by the multicatalytic proteinases suggesting that this enzyme has distinct active sites for protein and peptide hydrolysis.