Elsa M. Reyes-Reyes

Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways

Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinsons disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways.

LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells

Although several lines of evidence have established the central role of epithelial-to-mesenchymal-transition (EMT) in malignant progression of non-small cell lung cancers (NSCLCs), the molecular events connecting EMT to malignancy remain poorly understood. This study presents evidence that Long Interspersed Nuclear Element-1 (LINE-1) retrotransposon couples EMT programming with malignancy in human bronchial epithelial cells (BEAS-2B). This conclusion is supported by studies showing that: 1) activation of EMT programming by TGF-β1 increases LINE-1 mRNAs and protein; 2) the lung carcinogen benzo(a)pyrene coregulates TGF-β1 and LINE-1 mRNAs, with LINE-1 positioned downstream of TGF-β1 signaling; and, 3) forced expression of LINE-1 in BEAS-2B cells recapitulates EMT programming and induces malignant phenotypes and tumorigenesis in vivo. These findings identify a TGFβ1-LINE-1 axis as a critical effector pathway that can be targeted for the development of precision therapies during malignant progression of intractable NSCLCs.

Overview of Alterations in Cell Signaling

Cellular homeostasis involves the orchestration of complex biochemical events that ensure survival and preservation of differentiated functions. Toxic injury often alters cellular programming in ways that disrupt cellular signaling pathways and culminate in altered states that may or may not be consistent with cellular functions. For simplicity, this article has been organized to provide an overview of the major components of signal transduction: the specific signals, the sensors, and their corresponding signaling pathways. How toxic chemicals impinge on each of these components will be discussed in general terms with some references to the specifics covered in other articles provided when appropriate. The goal is to describe the signaling pathways that are often affected by carcinogenic compounds, specifically tumor promoters. The basic framework of the signal transduction cascade will be investigated. In subsequent articles, specific examples of xenobiotic alterations in signal transduction will be portrayed, as will the ultimate toxic responses of altered cell cycle control and apoptosis.

Plagiochiline A Inhibits Cytokinetic Abscission and Induces Cell Death

We previously reported on the isolation and biological activities of plagiochiline A (1), a 2,3-secoaromadendrane-type sesquiterpenoid from the Peruvian medicinal plant, Plagiochila disticha. This compound was found to have antiproliferative effects on a variety of solid tumor cell lines, as well as several leukemia cell lines. Other researchers have also noted the cytotoxicity of plagiochiline A (isolated from different plant species), but there are no prior reports regarding the mechanism for this bioactivity. Here, we have evaluated the effects of plagiochiline A on cell cycle progression in DU145 prostate cancer cells. A cell cycle analysis indicated that plagiochiline A caused a significant increase in the percentage of cells in the G2/M phase when compared with control cells. When cells were stained and observed by fluorescence microscopy to examine progress through the mitotic phase, we found a significant increase in the proportion of cells with features of late cytokinesis (cells connected by intercellular bridges) in the plagiochiline A-treated samples. These results suggest that plagiochiline A inhibits cell division by preventing completion of cytokinesis, particularly at the final abscission stage. We also determined that plagiochiline A reduces DU145 cell survival in clonogenic assays and that it induces substantial cell death in these cells.

mTOR/AMPK signaling in the brain: Cell metabolism, proteostasis and survival

The mechanistic (or mammalian) target of rapamycin (mTOR) and the adenosine monophosphate-activated protein kinase (AMPK) regulate cell survival and metabolism in response to diverse stimuli such as variations in amino acid content, changes in cellular bioenergetics, oxygen levels, neurotrophic factors and xenobiotics. This Opinion paper aims to discuss the current state of knowledge regarding how mTOR and AMPK regulate the metabolism and survival of brain cells and the close interrelationship between both signaling cascades. It is now clear that both mTOR and AMPK pathways regulate cellular homeostasis at multiple levels. Studies so far demonstrate that dysregulation in these two pathways is associated with neuronal injury, degeneration and neurotoxicity, but the mechanisms involved remain unclear. Most of the work so far has been focused on their antagonistic regulation of autophagy, but recent findings highlight that changes in protein synthesis, metabolism and mitochondrial function are likely to play a role in the regulatory effects of both mTOR and AMPK on neuronal health. Understanding the role and relationship between these two master regulators of cell metabolism is crucial for future therapeutic approaches to counteract alterations in cell metabolism and survival in brain injury and disease.

Abstract B26: Long interspersed nuclear element-1 regulates malignant transformation of lung bronchial epithelial cells through epithelial-to-mesenchymal transition

Lung cancer has the highest cancer-related mortality in the United States. Non-small cell lung cancer (NSCLC) is the predominant form of lung cancer. More than 65% of NSCLC patients show cancer progression presenting with locally advanced or metastatic disease. There is an imminent need to identify novel molecular targets that can help improve the precision of current therapies. Long interspersed nuclear element-1 (L1) is an abundant genetic element that mobilizes via retrotransposition using L1-encoded ORF1p and ORF2p proteins. L1 is reactivated by the carcinogen benzo(a)pyrene (BaP) which is a polycyclic aromatic hydrocarbon (PAH) present in tobacco smoke and environment pollutant. L1 reactivation is highly mutagenic by insertion into different locations in the genome. Recent studies have implicated L1 in the onset and progression of lung cancer, but the molecular bases of this response remain largely unknown. Moreover, the genome of lung cancer is one of the most frequently affected by L1 insertions, with reports showing that 50% of NSCLC have L1 ORF1p expression across a panel of different human lung neoplasms. L1 proteins expression correlates with downregulation of differentiation genes and appearance of undifferentiated phenotypes. Epithelial-to-mesenchymal transition (EMT) is a process that promotes undifferentiated phenotypes, loss of cell–cell adhesion and acquisition of cell motility, promoting invasiveness, metastasis and apoptotic resistance. To assess whether L1 induces EMT phenotypes to promote malignant transformation and cancer progression, non-malignant human bronchial epithelial BEAS-2B cells were stably transfected with vectors that constitutively expressed wildtype L1, a mutant counterpart that lacked reverse trascriptase activity and thus was unable to retrotranpose (mutant L1), or empty vector as control. Immunoblotting showed that cells expressing L1 and mutant L1 proteins exhibited increased expression of the mesenchymal markers, N-Cadherin and Snail, coupled with decreased expression of the epithelial marker ZO-1 compared to control cells. The expression of E-Cadherin or Vimentin was unaffected by ectopic expression of L1. Interestingly, Claudin-1 was selectively induced in cells expressing wildtype L1, but not cells expressing the mutant L1 or empty vector. The changes in ZO-1 and N-Cadherin expression, but not Claudin-1, were reversible upon genetic knockdown of L1. Importantly, ectopic expression of wildtype and mutant L1 in BEAS-2B cells enhanced tyrosine kinase inhibitor chemoresistance and induces a tumorigenicity in vivo. We conclude that L1 may contribute to lung carcinogenesis and progression by promoting cell transformation to induce tumorigenecity and changing cell plasticity to induce EMT-like phenotype via retrotransposition-dependent and -independent mechanisms in non-malignant bronchial epithelial. Citation Format: Ivan O. Aispuro, Elsa M. Reyes-Reyes, Minerva SantaCruz, Kenneth S. Ramos. Long interspersed nuclear element-1 regulates malignant transformation of lung bronchial epithelial cells through epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B26.

Overview of Receptor Systems

Receptors are defined as proteins that recognize a molecule or molecules (ligands) with some degree of specificity to initiate a biochemical signaling cascade that couples to secondary messenger systems. Receptor molecules transduce a signal from the exterior of the cell to the intracellular compartment, or reside within the cytosolic or nuclear compartment and convey signals that regulate gene expression. As such, receptor systems play critical roles in the regulation of development, cellular homeostasis, and disease onset and progression, to list a few. The ‘business end’ of virtually all receptor systems is often mediated by modulation of DNA functions and ultimately the regulation of protein/cellular functions. This article highlights major receptor systems that can be regulated by both endogenous and exogenous ligands, with a primary focus on xenobiotic receptor systems (XRSs) that modulate transcriptional events critical to the understanding the biology of the toxicological response. As appropriate, the molecular biology and human disease implications will be emphasized.