Characterization of the coiled-coil domain-containing protein 124 (Ccdc124) as a novel centrosome and midbody component involved in cytokinesis

Ankara : The Department of Molecular Biology and Genetics and the Graduate School of Engineering and Science of Bilkent Univ., 2013.Thesis (Ph. D.) -- Bilkent University, 2013.Includes bibliographical references leaves 114-124.Cytokinetic abscission is the cellular process leading to physical separation of two postmitotic sister cells by severing the intercellular bridge. During cell division several functional complexes accumulate at the bridge connecting the two sister cells. The most noticeable structural component of the intercellular bridge is a transient organelle termed as midbody. This novel organelle is localized at a central region, which marks the site of cytokinetic abscission. Despite its major role in completion of cell divison, our understanding of spatiotemporal regulation of midbody assembly is incomplete. In this thesis work, we first characterizated the coiled-coil domain-containing protein-124 (Ccdc124), a eukaryotic protein conserved from fungi-to-man, at the molecular level. We identified that at the sub-cellular level Ccdc124 is localized at centrosomes and the midbody depending on stages of the cell cycle. In interphase cells, as well as in mitosis, the protein is localized to centrosomes. However at later stages of cytokinesis (lateanaphase/ telophase) Ccdc124 translocates to the midbody. Knockdown of Ccdc124 in human HeLa cells leads to accumulation of enlarged and multinucleated cells; however, centrosome maturation was not affected. Similarly, in preliminary in vivo assays involving down-regulation of Ccdc124-homologue in zebra fish early embryos, we observed multinuclear embryonic cells. Furthermore, we have validated a previously observed in vitro interaction in our laboratory between Ccdc124 and the Ras guanine nucleotide exchange factor 1B (RasGEF1B) by co-immunoprecipitation assays. As RasGEF1B is strictly a Rap2 GTP-binding protein specific nucleotide exchange factor, this result has suggested a possible involvement of Rap2 in cytokinesis related events. Thus, subsequently, we assessed the sub-cellular localization of Rap2 in synchronized cells during cytokinesis. We found that even though it does not play a role in cell division, Rap2 is localized to the midbody. This result establishes a functional link between cytokinesis and activation of localized Rap2 signaling at the midbody. Data presented in this thesis work indicate that Ccdc124 is a novel factor operating both for proper progression of late cytokinetic stages in eukaryotes, and for establishment of Rap2 signaling dependent cellular functions proximal to the abscission site.Akıllılar, Pelin TelkoparanPh.D

TURKISH JOURNAL of ONCOLOGY Role of Circulating Tumor Cells and Cell-Free Tumor Deoxyribonucleic Acid Fragments as Liquid Biopsy Materials in Breast Oncology

SUMMARY Breast cancer is the most commonly diagnosed type of cancer among females worldwide. It is also the leading cause of female cancer-related death in developing countries. Though novel biomarkers and new strategies for diagnosis, monitoring course of disease, and treatment of breast cancer have been found, these methods are invasive, costly, labor-intensive, and inadequate to fully gauge treatment response and disease recurrence. Therefore, novel biomarkers with greater sensitivity and specificity, and which are easy to perform are needed in breast cancer oncology. There is growing interest in the potential use of circulating tumor cells and circulating tumor deoxyribonucleic acid fragments in liquid biopsy as non-invasive biopsy materials for early detection of breast cancer, monitoring disease progression, and understanding reasons for treatment resistance. This review is a discussion of current status of utilization of these liquid biopsy materials in breast oncology

The NRF2/KEAP1 axis in the regulation of tumor metabolism: mechanisms and therapeutic perspectives

The NRF2/KEAP1 pathway is a fundamental signaling cascade that controls multiple cytoprotective responses through the induction of a complex transcriptional program that ultimately renders cancer cells resistant to oxidative, metabolic and therapeutic stress. Interestingly, accumulating evidence in recent years has indicated that metabolic reprogramming is closely interrelated with the regulation of redox homeostasis, suggesting that the disruption of NRF2 signaling might represent a valid therapeutic strategy against a variety of solid and hematologic cancers. These aspects will be the focus of the present review

Therapeutic targeting of the NRF2 signaling pathway in cancer

Cancer is one of the most fatal diseases with an increasing incidence and mortality all over the world. Thus, there is an urgent need for novel therapies targeting major cancer-related pathways. Nuclear factor-erythroid 2-related factor 2 (NRF2) and its major negative modulator Kelch-like ECH-associated protein 1 (KEAP1) are main players of the cellular defense mechanisms against internal and external cell stressors. However, NRF2/KEAP1 signaling pathway is dysregulated in various cancers, thus promoting tumor cell survival and metastasis. In the present review, we discuss the mechanisms of normal and deregulated NRF2 signaling pathway focusing on its cancer-related functions. We further explore activators and inhibitors of this pathway as cancer targeting drug candidates in order to provide an extensive background on the subject

Recoding of nonsense mutation as a pharmacological strategy

Approximately 11% of genetic human diseases are caused by nonsense mutations that introduce a premature termination codon (PTC) into the coding sequence. The PTC results in the production of a potentially harmful shortened polypeptide and activation of a nonsense-mediated decay (NMD) pathway. The NMD pathway reduces the burden of unproductive protein synthesis by lowering the level of PTC mRNA. There is an endogenous rescue mechanism that produces a full-length protein from a PTC mRNA. Nonsense suppression therapies aim to increase readthrough, suppress NMD, or are a combination of both strategies. Therefore, treatment with translational readthrough-inducing drugs (TRIDs) and NMD inhibitors may increase the effectiveness of PTC suppression. Here we discuss the mechanism of PTC readthrough and the development of novel approaches to PTC suppression. We also discuss the toxicity and bioavailability of therapeutics used to stimulate PTC readthrough

Potential applications of NRF2 modulators in cancer therapy

The nuclear factor erythroid 2-related factor 2 (NRF2)–Kelch-like ECH-associated protein 1 (KEAP1) regulatory pathway plays an essential role in protecting cells and tissues from oxidative, electrophilic, and xenobiotic stress. By controlling the transactivation of over 500 cytoprotective genes, the NRF2 transcription factor has been implicated in the physiopathology of several human diseases, including cancer. In this respect, accumulating evidence indicates that NRF2 can act as a double-edged sword, being able to mediate tumor suppressive or pro-oncogenic functions, depending on the specific biological context of its activation. Thus, a better understanding of the mechanisms that control NRF2 functions and the most appropriate context of its activation is a prerequisite for the development of effective therapeutic strategies based on NRF2 modulation. In line of principle, the controlled activation of NRF2 might reduce the risk of cancer initiation and development in normal cells by scavenging reactive-oxygen species (ROS) and by preventing genomic instability through decreased DNA damage. In contrast however, already transformed cells with constitutive or prolonged activation of NRF2 signaling might represent a major clinical hurdle and exhibit an aggressive phenotype characterized by therapy resistance and unfavorable prognosis, requiring the use of NRF2 inhibitors. In this review, we will focus on the dual roles of the NRF2-KEAP1 pathway in cancer promotion and inhibition, describing the mechanisms of its activation and potential therapeutic strategies based on the use of context-specific modulation of NRF2

Intronic elements in the Na+/I- symporter gene (NIS) interact with retinoic acid receptors and mediate initiation of transcription

Activity of the sodium/iodide symporter (NIS) in lactating breast is essential for iodide (I–) accumulation in milk. Significant NIS upregulation was also reported in breast cancer, indicating a potential use of radioiodide treatment. All-trans-retinoic acid (tRA) is a potent ligand that enhances NIS expression in a subset of breast cancer cell lines and in experimental breast cancer models. Indirect tRA stimulation of NIS in breast cancer cells is very well documented; however, direct upregulation by tRA-activated nuclear receptors has not been identified yet. Aiming to uncover cis-acting elements directly regulating NIS expression, we screened evolutionary-conserved non-coding genomic sequences for responsiveness to tRA in MCF-7. Here, we report that a potent enhancer in the first intron of NIS mediates direct regulation by tRA-stimulated nuclear receptors. In vitro as well as in vivo DNA–protein interaction assays revealed direct association between retinoic acid receptor-α (RARα) and retinoid-X-receptor (RXR) with this enhancer. Moreover, using chromatin immunoprecipitation (ChIP) we uncovered early events of NIS transcription in response to tRA, which require the interaction of several novel intronic tRA responsive elements. These findings indicate a complex interplay between nuclear receptors, RNA Pol-II and multiple intronic RAREs in NIS gene, and they establish a novel mechanistic model for tRA-induced gene transcription

The Ability to Generate Senescent Progeny as a Mechanism Underlying Breast Cancer Cell Heterogeneity

Background Breast cancer is a remarkably heterogeneous disease. Luminal, basal-like, "normal-like", and ERBB2+ subgroups were identified and were shown to have different prognoses. The mechanisms underlying this heterogeneity are poorly understood. In our study, we explored the role of cellular differentiation and senescence as a potential cause of heterogeneity. Methodology/Principal Findings A panel of breast cancer cell lines, isogenic clones, and breast tumors were used. Based on their ability to generate senescent progeny under low-density clonogenic conditions, we classified breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All SCP cell lines expressed estrogen receptor (ER). Loss of ER expression combined with the accumulation of p21Cip1 correlated with senescence in these cell lines. p21Cip1 knockdown, estrogen-mediated ER activation or ectopic ER overexpression protected cells against senescence. In contrast, tamoxifen triggered a robust senescence response. As ER expression has been linked to luminal differentiation, we compared the differentiation status of SCP and ICP cell lines using stem/progenitor, luminal, and myoepithelial markers. The SCP cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some ICP cell lines generated only CD44+/CD24-/ER-/ASMA- progenitor/stem-like cells, and others also produced CD24+/ER- luminal-like, but not ASMA+ myoepithelial-like cells. Furthermore, gene expression profiles clustered SCP cell lines with luminal A and "normal-like" tumors, and ICP cell lines with luminal B and basal-like tumors. The ICP cells displayed higher tumorigenicity in immunodeficient mice. Conclusions/Significance Luminal A and "normal-like" breast cancer cell lines were able to generate luminal-like and myoepithelial-like progeny undergoing senescence arrest. In contrast, luminal B/basal-like cell lines acted as stem/progenitor cells with defective differentiation capacities. Our findings suggest that the malignancy of breast tumors is directly correlated with stem/progenitor phenotypes and poor differentiation potential. © 2010 Mumcuoglu et al

UNDERSTANDING THE CROSSTALK BETWEEN ENDOPLASMIC RETICULUM STRESS AND OXIDATIVE STRESS IN DISEASES

The endoplasmic reticulum (ER) is a vital organel with many critical cell functions. Various intracellular and environmental factors may disrupt its function and lead to the accumulation of unfolded or misfolded proteins that cause ER stress and activate the unfolded protein response (UPR). UPR leads to disruption of intracellular calcium and redox homeostasis and mitochondrial dysfunction. When the redox is unbalanced, the antioxidant mechanism is disrupted and leads to oxidative stress. ER stress mediates oxidative stress and increases reactive oxygen species (ROS) by affecting mitochondrial function, thus causing apoptosis in various metabolic diseases including cancer. In this talk, I will discuss how ER stress mediates oxidative stress in various metabolic diseases, including cancer, and the potential development of drugs that target these integrated signaling pathways

Pharmacological Applications of Nrf2 Inhibitors as Potential Antineoplastic Drugs

Oxidative stress (OS) is associated with many diseases ranging from cancer to neurodegenerative disorders. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is one of the most effective cytoprotective controller against OS. Modulation of Nrf2 pathway constitutes a remarkable strategy in the antineoplastic treatments. A big number of Nrf2-antioxidant response element activators have been screened for use as chemo-preventive drugs in OS associated diseases like cancer even though activation of Nrf2 happens in a variety of cancers. Research proved that hyperactivation of the Nrf2 pathway produces a situation that helps the survival of normal as well as malignant cells, protecting them against OS, anticancer drugs, and radiotherapy. In this review, the modulation of the Nrf2 pathway, anticancer activity and challenges associated with the development of an Nrf2-based anti-cancer treatment approaches are discussed