Showing papers by "Eileen White published in 2012"

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

Abstract: In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

Deconvoluting the context-dependent role for autophagy in cancer

Abstract: Autophagy (also known as macroautophagy) captures intracellular components in autophagosomes and delivers them to lysosomes, where they are degraded and recycled. Autophagy can have two functions in cancer. It can be tumour suppressive through the elimination of oncogenic protein substrates, toxic unfolded proteins and damaged organelles. Alternatively, it can be tumour promoting in established cancers through autophagy-mediated intracellular recycling that provides substrates for metabolism and that maintains the functional pool of mitochondria. Therefore, defining the context-specific role for autophagy in cancer and the mechanisms involved will be important to guide autophagy-based therapeutic intervention.

Autophagy suppresses RIP kinase-dependent necrosis enabling survival to mTOR inhibition.

Abstract: mTOR inhibitors are used clinically to treat renal cancer but are not curative. Here we show that autophagy is a resistance mechanism of human renal cell carcinoma (RCC) cell lines to mTOR inhibitors. RCC cell lines have high basal autophagy that is required for survival to mTOR inhibition. In RCC4 cells, inhibition of mTOR with CCI-779 stimulates autophagy and eliminates RIP kinases (RIPKs) and this is blocked by autophagy inhibition, which induces RIPK- and ROS-dependent necroptosis in vitro and suppresses xenograft growth. Autophagy of mitochondria is required for cell survival since mTOR inhibition turns off Nrf2 antioxidant defense. Thus, coordinate mTOR and autophagy inhibition leads to an imbalance between ROS production and defense, causing necroptosis that may enhance cancer treatment efficacy.

Immunohistochemical detection of cytoplasmic LC3 puncta in human cancer specimens

Abstract: Autophagy is an evolutionarily conserved catabolic process that involves the entrapment of cytoplasmic components within characteristic vesicles for their delivery to and degradation within lysosomes. Alterations in autophagic signaling are found in several human diseases including cancer. Here, we describe a validated immunohistochemical protocol for the detection of LC3 puncta in human formalin-fixed, paraffin-embedded cancer specimens that can also be applied to mouse tissues. In response to systemic chemotherapy, autophagy-competent mouse tumors exhibited LC3 puncta, which did not appear in mouse cancers that had been rendered autophagy-deficient by the knockdown of Atg5 or Atg7. As compared with normal tissues, LC3 staining was moderately to highly elevated in the large majority of human cancers studied, albeit tumors of the same histological type tended to be highly heterogeneous in the number and intensity of LC3 puncta per cell. Moreover, tumor-infiltrating immune cells often were highly positive for LC3. Altogether, this protocol for LC3 staining appears suitable for the specific detection of LC3 puncta in human specimens, including tissue microarrays. We surmise that this technique can be employed for retrospective or prospective studies involving large series of human tumor samples.

Effect of dual inhibition of apoptosis and autophagy in prostate cancer.

Abstract: PURPOSE Targeting multiple anti-apoptotic proteins is now possible with the small molecule BH3 domain mimetics such as ABT-737. Given recent studies demonstrating that autophagy is a resistance mechanism to multiple therapeutic agents in the setting of apoptotic inhibition, we hypothesized that hydroxychloroquine (HCQ), an anti-malarial drug that inhibits autophagy, will increase cytotoxicity of ABT-737. EXPERIMENTAL DESIGN Cytotoxicity of ABT-737 and HCQ was assessed in vitro in PC-3 and LNCaP cells, and in vivo in a xenograft mouse model. The role of autophagy as a resistance mechanism was assessed by siRNA knockdown of the essential autophagy gene beclin1. ROS was measured by flow cytometry, and mitophagy assessed by the mCherry-Parkin reporter. RESULTS Induction of autophagy by ABT-737 was a mechanism of resistance in prostate cancer cell lines. Therapeutic inhibition of autophagy with HCQ increased cytotoxicity of ABT-737 both in vitro and in vivo. ABT-737 induced LC-3 and decreased p62 expression by immunoblot in cell lines and by immunohistochemistry in tumors in vivo. Assessment of ROS and mitochondria demonstrated that ROS production by ABT-737 and HCQ was a mechanism of cytotoxicity. CONCLUSIONS We demonstrated that autophagy inhibition with HCQ enhances ABT-737 cytotoxicity in vitro and in vivo, that LC-3 and p62 represent assessable markers in human tissue for future clinical trials, and that ROS induction is a mechanism of cytotoxicity. These results support a new paradigm of dual targeting of apoptosis and autophagy in future clinical studies. Prostate 72:1374–1381, 2012. © 2012 Wiley Periodicals, Inc.

PEA-15 potentiates H-Ras-mediated epithelial cell transformation through phospholipase D

Abstract: The small GTPase H-Ras is a proto-oncogene that activates a variety of different pathways including the extracellular-signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway. H-Ras is mutated in many human malignancies, and these mutations cause the protein to be constitutively active. Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) blocks ERK-dependent gene transcription and inhibits proliferation by sequestering ERK in the cytoplasm. We therefore investigated whether PEA-15 influences H-Ras-mediated transformation. We found that PEA-15 does not block H-Ras-activated proliferation when H-Ras is constitutively active. We show instead that in H-Ras-transformed mouse kidney epithelial cells, co-expression of PEA-15 resulted in enhanced soft agar colony growth and increased tumor growth in vivo. Overexpression of both H-Ras and PEA-15 resulted in accelerated G1/S cell cycle transition and increased activation of the ERK signaling pathway. PEA-15 mediated these effects through activation of its binding partner phospholipase D1 (PLD1). Inhibition of PLD1 or interference with PEA-15/PLD1 binding blocked PEA-15's ability to increase ERK activation. Our findings reveal a novel mechanism by which PEA-15 positively regulates Ras/ERK signaling and increases the proliferation of H-Ras-transformed epithelial cells through enhanced PLD1 expression and activation. Thus, our work provides a surprising mechanism by which PEA-15 augments H-Ras-driven transformation. These data reveal that PEA-15 not only suppresses ERK signaling and tumorigenesis but also alternatively enhances tumorigenesis in the context of active Ras.

Ammonificins C and D, Hydroxyethylamine Chromene Derivatives from a Cultured Marine Hydrothermal Vent Bacterium, Thermovibrio ammonificans

Abstract: Chemical and biological investigation of the cultured marine hydrothermal vent bacterium, Thermovibrio ammonifican led to the isolation of two hydroxyethylamine chromene derivatives, ammonificins C and D. Their structures were elucidated using combination of NMR and mass spectrometry. Absolute stereochemistry was ascertained by comparison of experimental and calculated CD spectra. Biological evaluation and assessment were determined using the patented ApopScreen cell-based screen for apoptosis-induction. Ammonificins C and D induce apoptosis in micromolar concentrations. To our knowledge, this finding is the first report of chemical compounds that induce apoptosis from the cultured deep-sea marine organism, hydrothermal vent bacterium, Thermovibrio ammonificans.

Ras-driven cancer cells can scavenge exogenous lipids to support their proliferation

Abstract: Background Lipids are an important structural component of the cell, making up the cell’s membranes. Cancer cells need lipids in large quantities to enable their rapid proliferation. Here we aim to quantitatively evaluate the routes by which cancer cells acquire lipids (de novo synthesis, uptake from environment) under various conditions (specific oncogenic activations, nutrient availability). We developed a mass spectrometry-based method to quantitate the relative contributions of the various fatty acid acquisition routes and applied this and other methods to study fatty acid metabolism in cell lines, both in vitro and in vivo.

Q&A: Eileen White on understanding autophagy.

Abstract: They’re in development. Many companies are interested in autophagy inhibitors, possibly to augment the activity of their targeted therapies. A lot of people are using hydroxychloroquine as a shortcut. It interferes with lysosome function. Lysosomes are the garbage cans of the cell, and they degrade the cargo that’s delivered to them. The process of autophagy captures and delivers cargo—cellular trash, unfolded proteins, bad organelles—to the lysosome to be degraded. If you block lysosome function, you block the degradation of cargo in the autophagy pathway. A bunch of clinical trials are using hydroxychloroquine in different cancers and in combination with different agents. Overall, there is a lot of interest in targeting the metabolism of cancer cells, because metabolism is different in a cancer cell than it is in a normal cell. When you inhibit a metabolic pathway, one common response is the upregulation of autophagy as a compensatory mechanism. I think another use of autophagy inhibition will be to augment interference in metabolism.

Abstract 4104: Moderate calorie restriction activates autophagy during tumor growth suppression

Abstract: Multiple mouse models of pancreatic, colon, prostate, skin and breast cancer have shown that calorie restriction (CR) prevents obesity and suppresses tumor development and growth. In normal muscle, studies have shown that mild CR attenuates the age-related impairment of autophagy, cellular damage, and cell death. Autophagy is a survival response in solid tumors that co-localizes with hypoxic regions, allowing tolerance to metabolic stress. Autophagy also protects cells from genome damage and limits both inflammation and cell death as possible means to tumor suppression. Thus, we hypothesized that autophagy plays a major role in CR-mediated tumor inhibition. To test this hypothesis, we utilized immortalized baby mouse kidney epithelium (iBMK) tumor cell lines. iBMK cells were first immortalized by E1A and dominant negative p53 (p54DD) transfection, and rendered apoptosis-deficient by Bcl-2 overexpression. They were then transfected with oncogenic Ras and EGFP-LC3 plasmids. Cell lines were either autophagy deficient through knockout of the essential autophagy gene Atg5 (Atg5 -/-) or were autophagy competent (Atg5 +/+). Female nude mice were fed either control AIN-76A diet ad-libitum or a CR regimen (isonutrient; 30% reduction in calories relative to control group) for 8 weeks prior to subcutaneous flank injection of cell lines (n=15 per group). CR mice were significantly lighter, had decreased body fat percentage, and had lower fasting glucose levels (p CR; p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4104. doi:1538-7445.AM2012-4104

Identification of Modulators of Autophagy

Abstract: Methods for identifying compounds that inhibit or stimulate the autophagy pathway are described. Devices for detecting the expression of autophagy-related genes and kits for assaying the expression of autophagy-related genes are also described. Also described are methods for identifying individuals susceptible to or afflicted with a disease state associated with an autophagy pathway defect.