Current Drug Targets - Volume 16, Issue 1, 2015

Saccharomyces cerevisiae is an optimal model to study stress responses for various reasons: i) budding yeast genome presents a high degree of homology with the human genome; ii) there are many proteins that show an elevated functional homology with specific human proteins; iii) it is a system whose genetic manipulation is reasonably easy and cheaper than other models; iv) the possibility of working with an haploid state facilitates the study of multiple processes; v) databases are the most complete of all the eukaryotic models. Due to the latest information derived from proteomic and genomic analyses, the genetic, biochemical and molecular information available relative to this biological system is extraordinarily big and complete. In this review, we present an overview of the mechanisms unravelling sensing and transducing oxidative stress. TOR, RAS/PKA, CWI, SNF1, and HOG are the main pathways involved both in the oxidative response and in the correct entry in stationary phase. In general, TOR and RAS/PKA dowregulation and SNF1 and CWI upregulation favour both a correct defence against oxidative damage and the entry in the quiescent state. All of these pathways have counterparts in humans. The actin cytoskeleton plays a dual function as sensor and target of oxidation, in tight connection with the former signalling cascades. In budding yeast, progression through stationary phase and quiescence constitute an accepted current model to study some of the mechanisms that determine life span. Aging is a process associated to oxidative stress and it is in tight relationship with bulk autophagy and mitophagy, both are mechanisms belonging to the oxidative defence and promoters of life extension when correctly regulated by, among other elements, the signalling cascades.

Elevated levels of both reactive oxygen species (ROS) and DNA methylation are characteristic of various types of cancer cells. However, the relation between these two is not well understood. Here we will discuss the cause-consequence relationship between ROS and DNA methylation. Cancer research reveals that disregulation of DNA methylation results in regional CpG island hypermethylation and generalized genomic hypomethylation. ROS-induced oxidative stress is associated with both aberrant hypermethylation of tumor suppressor gene (TSG) promoter regions and global hypomethylation. The DNA oxidation structure, 8-hydroxy-2'-deoxyguanosine (8-OHdG), can induce DNA hypomethylation by inhibiting DNA methylation at nearby cytosine bases, while another DNA oxidation structure, 5-hydroxymethylcytosine (5hmC), may achieve active DNA demethylation processes, thus, causing DNA hypomethylation. Recently, it has been found that ROS can function as catalysts of DNA methylation, further accounting for TSG promoter hypermethylation. Moreover, ROS may induce site-specific hypermethylation via either the up-regulation of expression of DNA methyltransferases (DNMTs) or the formation of a new DNMT containing complex. In addition, these ROS-induced DNA methylation pattern alterations have been implicated with not only malignant transformation, but also the progression of numerous tumors. In conclusion, ROS can influence both aspects of DNA methylation changes through different mechanisms, which play an important role of epigenetic regulation in cancer cells. Therefore, the comprehension of mechanisms leading to epigenetic modifications associated with ROS may help better understand the carcinogenesis and progression, as well as aid in the development of potential biomarkers for better cancer diagnostics and novel therapeutic strategies.

Neurotrophins are important neurotrophic factors involved in the survival, differentiation and function of a wide variety of neuron populations. A common feature for most neurotrophins is that they are synthesized as precursor proteins (pro-neurotrophins) that upon being processed by proteolysis render the mature active form responsible for most of their trophic functions. However, some of the pro-neurotrophin form of these proteins, such as the precursor form of NGF (pro-NGF), have been shown to induce opposite effects and trigger apoptosis on neurons through the p75NTR receptor. This suggests that the balance between the levels of proneurotrophin and neurotrophin must be tightly controlled. In this context, it has been shown that in conditions of oxidative stress due for instance to aging or the development of some neurodegenerative disease, neurotrophins are oxidatively modified at least by advanced glycation/lipoxidation end products (AGE/ALEs) which makes pro-NGF refractary to be processed. The lack of maturation and the imbalance in favor of the precursor form may change the pattern of active signaling pathways towards cell death, thus exacerbating the deleterious alterations, for instance during the development of neurodegenerative diseases. Besides that, AGE/ALEs also induce the processing of the pro-NGF receptor p75NTR by α- secretase which is followed by the processing by γ -secretase and the release of the intracellular domain of p75NTR (p75NTRICD). Once cleaved, p75NTRICD recruits two intracellular interactors, NRIF and TRAF6, which allows NRIF phosphorylation by JNK. The phosphorylated form of NRIF then translocates to the nucleus and induces the expression of pro-apoptotic proteins. In this chapter we will summarize the mechanisms by which ROS- induce protein modifications, which proteins are susceptible to be modified, how these modifications affect function and signaling and, finally, how they can be related to neurodegenerative diseases.

Aerobic metabolism of mammalian cells leads to the generation of reactive oxygen species (ROS). To cope with this toxicity, evolution provided cells with effective antioxidant systems like glutathione. Current anticancer therapies focus on the cancer dependence on oncogenes and non-oncogenes. Tumors trigger mechanisms to circumvent the oncogenic stress and to escape cell death. In this context we have studied 2-phenylethinesulfoxamine (PES), which disables the cell protective mechanisms to confront the proteotoxicity of damaged and unfolded proteins. Proteotoxic stress is increased in tumor cells, thus providing an explanation for the anticancer selectivity of PES. In addition, we have found that PES induces a severe oxidative stress and the activation of p53. The reduction of the cell content in glutathione by means of L-buthionine-sulfoximine (BSO) synergizes with PES. In conclusion, we have found that ROS constitutes a central element in a series of positive feed-back loops in the cell. ROS, p53, proteotoxicity, autophagy and mitochondrial dynamics are interconnected with the mechanisms leading to cell death, either apoptotic or necrotic. This network of interactions provides multiple targets for drug discovery and development in cancer.

NR4A1 (Nur77) belongs together with NR4A2 (Nurr1) and NR4A3 (NOR-1) to the nuclear orphan receptors of the NR4A-family. Their activation is generally short lived, the cellular outcome is a stimulus- and cell context-dependent differential activation of NR4A target genes that regulate cell cycle, apoptosis, inflammation, atherogenesis, metabolism, DNA repair and tumorigenesis. NR4A1 and NR4A3 were identified to function as tumor suppressors in acute myeloid leukemia (AML). Deletion of both nuclear receptors led to rapid development of AML in mice. Loss of NR4A1 and NR4A3 was a common feature in human AML patients. Additionally, NR4A1 and NR4A3 hypoallelic mice - mice with a reduced NR4A1 and NR4A3 expression - develop a chronic myeloid malignancy that recapitulates the pathological features of myelodysplastic/ myeloproliferative neoplasms with progression to AML in rare cases. Recently, a reduced NR4A1 and NR4A3 expression was described in aggressive lymphomas and low NR4A1 expression was associated with poor overall survival. Overexpression of NR4A1 in aggressive lymphoma cells led to induction of apoptosis and abrogated tumor growth in a xenograft mouse model. Recently, it was shown that NR4A inducing agents or NR4A agonist possess/induce apoptotic effects in AML and lymphoma cells. Due to this fact and the growing number of NR4A1 and NR4A3 inducing agents and NR4A agonists, both receptors represent new targets for anti tumor therapy.

Non-small cell lung cancer (NSCLC) is one of the most common causes of cancer-related death worldwide. Based on the patient’s stage of disease, treatment options include surgery, radiotherapy, and chemotherapy. Although chemotherapy remains the main therapeutic approach for advanced NSCLC, targeted therapy represents a good chance of treatment for this subgroup of patients. Currently this approach is based on previous evaluation of clinically relevant mutations and the Sanger sequencing is the main approach to assign mutational status and to guide the appropriate treatment; however this tool is characterized by a low sensitivity. Recently, the advent of next-generation sequencing (NGS) has dramatically revolutionized the molecular knowledge of cancer by increasing the feasibility and possibility to sequence DNA ranging from large scale studies to targeted regions. This review reports an overview of different applications of the NGS as novel approach to study NSCLC, thereby providing information about mutational spectrum of this cancer in order to identify novel targetable mutations and to predict the emergence of drug resistance. All studies demonstrated several advantages of this approach over the traditional tools. In particular the NGS was also able to reveal mutations in low percentage, and to screen the mutational status of different critical samples such as biopsies, cytological samples and circulating plasma DNA, offering innovative diagnostic opportunities. Despite several problems have to be overcome toward the personalized therapy, the NGS represents a highly attractive system to identify mutations improving the outcome of patients with this deadly disease.

Target discovery using the molecular approach, as opposed to the more traditional systems approach requires the study of the cellular or biological process underlying a condition or disease. The approaches that are employed by the “bench” scientist may be genetic, genomic or proteomic and each has its rightful place in the drug-target discovery process. Affinity-based proteomic techniques currently used in drug-discovery draw upon several disciplines, synthetic chemistry, cell-biology, biochemistry and mass spectrometry. An important component of such techniques is the probe that is specifically designed to pick out a protein or set of proteins from amongst the varied thousands in a cell lysate. A second component, that is just as important, is liquid-chromatography tandem massspectrometry (LC-MS/MS). LC-MS/MS and the supporting theoretical framework has come of age and is the tool of choice for protein identification and quantification. These proteomic tools are critical to maintaining the drug-candidate supply, in the larger context of drug discovery.

Cervical cancer is the third most common cancer among women worldwide and is responsible for 275.000 deaths each year. The development of cervical cancer has been linked to cell cycle disturbances caused by persistent expression of high- risk HPV oncoproteins (E5, E6 and E7), which modulate the expression of host genes and cellular microRNAs. An estimated 5 million women throughout the world are currently infected by HPV and several of them will develop invasive cervical cancer. Despite failures in conventional screening tests, approved therapies have no direct effect on HPV infection. In view of this, effective therapy for cervical cancer is still urgently needed, particularly in developing countries where more than 85% of fatal cases occur. In this paper we review the current molecular targeted therapies which are being explored and may have a significant impact on the treatment of HPV- related cervical dysplasia and carcinoma.