Yiyu Zou

Reactive Oxygen Species Generation and Mitochondrial Dysfunction in the Apoptotic Response to Bortezomib, a Novel Proteasome Inhibitor, in Human H460 Non-small Cell Lung Cancer Cells

Bortezomib, a proteasome inhibitor, shows substantial anti-tumor activity in a variety of tumor cell lines, is in phase I, II, and III clinical trials and has recently been approved for the treatment of patients with multiple myeloma. The sequence of events leading to apoptosis following proteasome inhibition by bortezomib is unclear. Bortezomib effects on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration in the mitochondrial membrane potential (Δψm), and release of cytochrome c from mitochondria. With human H460 lung cancer cells, bortezomib exposure at 0.1 μm showed induction of apoptotic cell death starting at 24 h, with increasing effects after 48–72 h of treatment. After 3–6 h, an elevation in ROS generation, an increase in Δψm, and the release of cytochrome c into the cytosol, were observed in a time-dependent manner. Co-incubation with rotenone and antimycin A, inhibitors of mitochondrial electron transport chain complexes I and III, or with cyclosporine A, an inhibitor of mitochondrial permeability transition pore, resulted in inhibition of bortezomib-induced ROS generation, increase in Δψm, and cytochrome c release. Tiron, an antioxidant agent, blocked the bortezomib-induced ROS production, Δψm increase, and cytochrome c release. Tiron treatment also protected against the bortezomib-induced PARP protein cleavage and cell death. Benzyloxycarbonyl-VAD-fluoromethyl ketone, an inhibitor of pan-caspase, did not alter the bortezomib-induced ROS generation and increase in Δψm, although it prevented bortezomib-induced poly(ADP-ribose) polymerase cleavage and apoptotic death. In PC-3 prostate carcinoma cells (with overexpression of Bcl-2), a reduction of bortezomib-induced ROS generation, Δψm increase was correlated with cellular resistance to bortezomib and the attenuation of drug-induced apoptosis. The transient transfection of wild type p53 in p53 null H358 cells caused stimulation of the bortezomib-induced apoptosis but failed to enhance ROS generation and Δψm increase. Thus ROS generation plays a critical role in the initiation of the bortezomib-induced apoptotic cascade by mediation of the disruption of Δψm and the release of cytochrome c from mitochondria.

A Distinct Macrophage Population Mediates Metastatic Breast Cancer Cell Extravasation, Establishment and Growth

Background The stromal microenvironment and particularly the macrophage component of primary tumors influence their malignant potential. However, at the metastatic site the role of these cells and their mechanism of actions for establishment and growth of metastases remain largely unknown. Methodology/Principal Findings Using animal models of breast cancer metastasis, we show that a population of host macrophages displaying a distinct phenotype is recruited to extravasating pulmonary metastatic cells regardless of species of origin. Ablation of this macrophage population through three independent means (genetic and chemical) showed that these macrophages are required for efficient metastatic seeding and growth. Importantly, even after metastatic growth is established, ablation of this macrophage population inhibited subsequent growth. Furthermore, imaging of intact lungs revealed that macrophages are required for efficient tumor cell extravasation. Conclusion/Significance These data indicate a direct enhancement of metastatic growth by macrophages through their effects on tumor cell extravasation, survival and subsequent growth and identifies these cells as a new therapeutic target for treatment of metastatic disease.

The Autophagy Inhibitor Chloroquine Overcomes the Innate Resistance of Wild-Type EGFR Non-Small-Cell Lung Cancer Cells to Erlotinib

Introduction: The epidermal growth factor receptor (EGFR) inhibitor erlotinib is much less effective in non–small-cell lung cancer (NSCLC) tumors with wild-type EGFR, than in tumors with activating EGFR mutations. Autophagy is a tightly regulated lysosomal self-digestion process, which may alternatively promote cell survival or type II cell death. This study assessed the role of autophagy in erlotinib-mediated cytotoxicity. Methods: We used wild-type EGFR erlotinib-sensitive and erlotinib-resistant NSCLC cell lines to determine whether inhibiting autophagy by a therapeutic agent potentiated the antitumor activity of erlotinib in vitro and in vivo. Results: Erlotinib at a clinically relevant concentration (2 &mgr;M) induced autophagy in NSCLC cells with wild-type EGFR, and the degree of induction was greater in cells that were resistant than sensitive, suggesting that autophagy is cytoprotective. This was confirmed by knockdown of the autophagy-related gene Atg-5, and by using the autophagy inhibitor chloroquine (CQ), both of which increased the cytotoxicity of erlotinib. The synergistic activity of CQ was not because of the potentiation of erlotinib’s effects on autophagy, cell-cycle arrest, and inhibition of both EGFR or downstream signaling of EGFR. Rather, CQ markedly activated apoptosis in the cells. The ability of CQ to potentiate the antitumor activity of erlotinib was also seen in mice bearing NSCLC tumor xenografts. Conclusions: The ability to adapt to anti-EGFR therapy by triggering autophagy may be a key determinant for resistance to erlotinib in wild-type EGFR NSCLC. Inhibition of autophagy by CQ represents a novel strategy to broaden the spectrum of erlotinib efficacy in wild-type EGFR NSCLC tumors.

PM02734 (Elisidepsin) Induces Caspase-Independent Cell Death Associated with Features of Autophagy, Inhibition of the Akt/mTOR Signaling Pathway, and Activation of Death-Associated Protein Kinase

Purpose: PM02734 (elisidepsin) is a synthetic marine-derived cyclic peptide of the kahalalide family currently in phase II clinical development. The mechanisms of cell death induced by PM02734 remain unknown. Experimental Design: Human non–small-cell lung cancer (NSCLC) cell lines H322 and A549 were used to evaluate PM02734-induced cytotoxicity, apoptosis, and autophagy, as well as effects on cell death–related signaling pathways. Results: PM02734 at clinically achievable concentrations (0.5–1 μmol/L) was cytotoxic to H322 and A549 cells but did not cause nuclear fragmentation, PARP cleavage, or caspase activation, suggesting that classical apoptosis is not its main mechanism of cell death. In contrast, PM02734-induced cell death was associated with several characteristics of autophagy, including an increase in acidic vesicular organelle content, levels of GFP-LC3–positive puncta, elevation of the levels of Atg-5/12 and LC3-II, and an associated compromise of the autophagic flux resulting in increased number of autophagosomes and/or autolysosomes. Cotreatment with 3-methyladenine (3-MA) and downregulation of Atg-5 gene expression by siRNA partially inhibited PM02734-induced cell death. PM02734 caused inhibition of Akt/mTOR signaling pathways and cotreatment with the Akt inhibitor wortmannin or with the mTOR inhibitor rapamycin led to a significant increase in PM02734-induced cell death. Furthermore, PM02734 caused the activation of death-associated protein kinase (DAPK) by dephosphorylation at Ser308, and downregulation of DAPK expression with siRNA caused also a partial but significant reduction of PM02734-induced cell death. In vivo, PM02734 significantly inhibited subcutaneous A549 tumor growth in nude mice (P < 0.05) in association with induction of autophagy. Conclusions: Our data indicate that PM02734 causes cell death by a complex mechanism that involves increased autophagosome content, due for the most part to impairment of autophagic flux, inhibition of the Akt/mTOR pathway, and activation of DAPK. This unique mechanism of action justifies the continued development of this agent for the treatment of NSCLC. Clin Cancer Res; 17(16); 5353–66. ©2011 AACR.

p53 aerosol formulation with low toxicity and high efficiency for early lung cancer treatment

Purpose: To develop an optimal nonviral aerosol formulation for locoregional treatment of early lung cancer. Experimental Design: The formulation was made of polylysine/protamine combination (AND) as the carrier and p53 gene (p53sm) as therapeutic agent. To estimate the aerosol deposition, the aerodynamic size of the AND-p53sm was measured with extrusion-precipitation method. To accurately determine the dose, the aerosol efficiency in mice was measured with a fluorescent dye. The transfection efficiency and DNA protection function of the aerosolized formulation in cultured cells and mouse lungs were detected with reporter gene assays and/or reverse transcription-PCR. The preclinical safety and efficacy of AND-p53sm were studied in healthy mice and mice bearing orthotopic human non–small-cell lung cancer (NSCLC) xenograft. Results: After aerosolization, AND is 3- to 17-fold more effective than commonly used PEI or cationic lipid formulations in transfecting the NSCLC cells (relative light units, 1,494 versus 534 and 86; P < 0.003). Aerodynamic size of AND-p53sm ranged 0.2 to 3 μm is the optimal aerosol droplets for deposition in the entire human respiratory tract. Significant gene expression was detected in the lungs of mice given aerosolized AND-p53sm and AND-luciferase. Aerosolized AND-p53sm significantly prolonged the life of mice bearing orthotopic human NSCLC xenografts, and it was more effective than an optimal i.v. cisplatin chemotherapy (increased life span, 93% versus 25%; P = 0.014). Inhalation of AND produced low and reversible pulmonary toxicity and no systemic toxicity. Conclusions: This optimal formulation is suitable for delivering biological materials to human lung with aerosol administration. This therapeutic strategy is an option for patients with early lung cancer and bronchoalveolar carcinoma.

RICTOR amplification defines a novel subset of patients with lung cancer who may benefit from treatment with mTORC1/2 inhibitors

UNLABELLED We identified amplification of RICTOR, a key component of the mTOR complex 2 (mTORC2), as the sole actionable genomic alteration in an 18-year-old never-smoker with lung adenocarcinoma. Amplification of RICTOR occurs in 13% of lung cancers (1,016 cases) in The Cancer Genome Atlas and at a similar frequency in an independent cohort of 1,070 patients identified by genomic profiling. In the latter series, 11% of cases harbored RICTOR amplification as the only relevant genomic alteration. Its oncogenic roles were suggested by decreased lung cancer cell growth both in vitro and in vivo with RICTOR ablation, and the transforming capacity of RICTOR in a Ba/F3-cell system. The mTORC1/2 inhibitors were significantly more active against RICTOR-amplified lung cancer cells as compared with other agents targeting the PI3K-AKT-mTOR pathway. Moreover, an association between RICTOR amplification and sensitivities to mTORC1/2 inhibitors was observed. The index patient has been treated with mTORC1/2 inhibitors that led to tumor stabilization for more than 18 months. SIGNIFICANCE RICTOR amplification may define a novel and unique molecular subset of patients with lung cancer who may benefit from treatment with mTORC1/2 inhibitors.

Inhibition of FOXO3 tumor suppressor function by βTrCP1 through ubiquitin-mediated degradation in a tumor mouse model

Background The ubiquitin-proteasome system is the primary proteolysis machine for controlling protein stability of the majority of regulatory proteins including those that are critical for cancer development. The forkhead box transcription factor FOXO3 plays a key role in regulating tumor suppression; however, the control of FOXO3 protein stability remains to be established. It is crucial to elucidate the molecular mechanisms underlying the ubiquitin-mediated degradation of FOXO3 tumor suppressor. Methodology and Principal Findings Here we show that βTrCP1 oncogenic ubiquitin E3-ligase interacts with FOXO3 and induces its ubiquitin-dependent degradation in an IκB kinase-β phosphorylation dependent manner. Silencing βTrCP1 augments FOXO3 protein level, resulting in promoting cellular apoptosis in cancer cells. In animal models, increasing FOXO3 protein level by silencing βTrCP1 suppresses tumorigenesis, whereas decreasing FOXO3 by over-expressing βTrCP1 promotes tumorigenesis and tumor growth in vivo. Conclusions/Significance This is a unique demonstration that the βTrCP1-mediated FOXO3 degradation plays a crucial role in tumorigenesis. These findings significantly contribute to understanding of the control of FOXO3 stability in cancer cells and may provide opportunities for developing innovative anticancer therapeutic modalities.

Role of DNA methylation in renal cell carcinoma

Alterations in DNA methylation are seen in cancers and have also been examined in clear cell renal cell carcinoma (ccRCC). Numerous tumor suppressor genes have been reported to be partially or completely silenced due to hypermethylation of their promoters in single-locus studies, and the use of hypomethylating agents has been shown to restore the expression of many of these genes in vitro. In particular, members of the Wnt and TGF-beta pathways, pro-apoptotic genes such as APAF-1 and negative cell-cycle regulators such as KILLIN have been shown to be epigenetically silenced in numerous studies in ccRCC. Recently, TCGA analysis of a large cohort of ccRCC samples demonstrated that aberrant hypermethylation correlated with the stage and grade in kidney cancer. Our genome-wide studies also revealed aberrant widespread hypermethylation that affected regulatory regions of the kidney genome in ccRCC. We also observed that aberrant enhancer hypermethylation was predictive of adverse prognosis in ccRCC. Recent discovery of mutations affecting epigenetic regulators reinforces the importance of these changes in the pathophysiology of ccRCC and points to the potential of epigenetic modulators in the treatment of this malignancy.

Synergy of sequential administration of a deglycosylated ricin A chain-containing combined anti-CD19 and anti-CD22 immunotoxin (Combotox) and cytarabine in a murine model of advanced acute lymphoblastic leukemia.

Abstract The outcome for patients with refractory or relapsed acute lymphoblastic leukemia (ALL) treated with conventional therapy is poor. Immunoconjugates present a novel approach and have recently been shown to have efficacy in this setting. Combotox is a mixture of two ricin-conjugated monoclonal antibodies (RFB4 and HD37) directed against CD19 and CD22, respectively, and has shown activity in pediatric and adult ALL. We created a murine xenograft model of advanced ALL using the NALM/6 cell line to explore whether the combination of Combotox with the cytotoxic agent cytarabine (Ara-C) results in better outcomes. In our model the combination of both low- and high-dose Combotox and Ara-C resulted in significantly longer median survival. Sequential administration of Ara-C and Combotox, however, was shown to be superior to concurrent administration. These findings have led to a phase I clinical trial exploring this combination in adults with relapsed or refractory B-lineage ALL (ClinicalTrials.gov identifier NCT01408160).

Pexmetinib: A Novel Dual Inhibitor of Tie2 and p38 MAPK with Efficacy in Preclinical Models of Myelodysplastic Syndromes and Acute Myeloid Leukemia.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) suppress normal hematopoietic activity in part by enabling a pathogenic inflammatory milieu in the bone marrow. In this report, we show that elevation of angiopoietin-1 in myelodysplastic CD34(+) stem-like cells is associated with higher risk disease and reduced overall survival in MDS and AML patients. Increased angiopoietin-1 expression was associated with a transcriptomic signature similar to known MDS/AML stem-like cell profiles. In seeking a small-molecule inhibitor of this pathway, we discovered and validated pexmetinib (ARRY-614), an inhibitor of the angiopoietin-1 receptor Tie-2, which was also found to inhibit the proinflammatory kinase p38 MAPK (which is overactivated in MDS). Pexmetinib inhibited leukemic proliferation, prevented activation of downstream effector kinases, and abrogated the effects of TNFα on healthy hematopoietic stem cells. Notably, treatment of primary MDS specimens with this compound stimulated hematopoiesis. Our results provide preclinical proof of concept for pexmetinib as a Tie-2/p38 MAPK dual inhibitor applicable to the treatment of MDS/AML. Cancer Res; 76(16); 4841-9. ©2016 AACR.