The ubiquitin E3/E4 ligase, UBE4A, fine-tunes protein ubiquitylation and accumulation at sites of DNA damage facilitating double-strand break repair

Double-strand breaks (DSBs) are critical DNA lesions that robustly activate the elaborate DNA damage response (DDR) network. We identified a critical player in DDR fine-tuning - the E3/E4 ubiquitin ligase, UBE4A. UBE4A’s recruitment to sites of DNA damage is dependent on primary E3 ligases in the DDR and promotes enhancement and sustainment of K48- and K63-linked ubiquitin chains at these sites. This step is required for timely recruitment of the RAP80 and BRCA1 proteins and proper organization of RAP80- and BRCA1-associated protein complexes at DSB sites. This pathway is essential for optimal end-resection at DSBs, and its abrogation leads to up-regulation of the highly mutagenic alternative end-joining repair at the expense of error-free homologous recombination repair. Our data uncover a critical regulatory level in the DSB response and underscore the importance of fine-tuning of the complex DDR network for accurate and balanced execution of DSB repai

Prognostic value of CtIP/RBBP8 expression in breast cancer

CtIP/RBBP8 is a multifunctional protein involved in transcription, DNA replication, DNA repair by homologous recombination and the G1 and G2 checkpoints. Its multiple roles are controlled by its interaction with several specific factors, including the tumor suppressor proteins BRCA1 and retinoblastoma. Both its functions and interactors point to a putative oncogenic potential of CtIP/RBBP8 loss. However, CtIP/RBBP8 relevance in breast tumor appearance, development, and prognosis has yet to be established. We performed a retrospective analysis of CtIP/RBBP8 and RB1 levels by immunohistochemistry using 384 paraffin-embedded breast cancer biopsies obtained during tumor removal surgery. We have observed that low or no expression of CtIP/RBBP8 correlates with high-grade breast cancer and with nodal metastasis. Reduction on CtIP/RBBP8 is most common in hormone receptor (HR)-negative, HER2-positive, and basal-like tumors. We observed lower levels of RB1 on those tumors with reduced CtIP/RBBP8 levels. On luminal tumors, decreased but not absence of CtIP/RBBP8 levels correlate with increased disease-free survival when treated with a combination of hormone, radio, and chemo therapies.España Ministerio de Economía y Competitividad SAF2010-14877Junta de Andalucía, Consejería de Salud AI2013-A-000

DNA end resection requires constitutive sumoylation of CtIP by CBX4

DNA breaks are complex DNA lesions that can be repaired by two alternative mechanisms: non-homologous end-joining and homologous recombination. The decision between them depends on the activation of the DNA resection machinery, which blocks non-homologous end-joining and stimulates recombination. On the other hand, post-translational modifications play a critical role in DNA repair. We have found that the SUMO E3 ligase CBX4 controls resection through the key factor CtIP. Indeed, CBX4 depletion impairs CtIP constitutive sumoylation and DNA end processing. Importantly, mutating lysine 896 in CtIP recapitulates the CBX4-depletion phenotype, blocks homologous recombination and increases genomic instability. Artificial fusion of CtIP and SUMO suppresses the effects of both the non-sumoylatable CtIP mutant and CBX4 depletion. Mechanistically, CtIP sumoylation is essential for its recruitment to damaged DNA. In summary, sumoylation of CtIP at lysine 896 defines a subpopulation of the protein that is involved in DNA resection and recombination

The Helicase PIF1 Facilitates Resection over Sequences Prone to Forming G4 Structures

DNA breaks are complex lesions that can be repaired either by non-homologous end joining (NHEJ) or by homologous recombination (HR). The decision between these two routes of DNA repair is a key point of the DNA damage response (DDR) that is controlled by DNA resection. The core machinery catalyzing the resection process is well established. However, little is known about the additional requirements of DNA resection over DNA structures with high complexity. Here, we found evidence that the human helicase PIF1 has a role in DNA resection, specifically for defined DNA regions, such as those prone to form G-quadruplexes. Indeed, PIF1 is recruited to the site of DNA damage and physically interacts with proteins involved in DNA resection, and its depletion causes DNA damage sensitivity and a reduction of HR efficiency. Moreover, G4 stabilization by itself hampers DNA resection, a phenomenon suppressed by PIF1 overexpressio

New insights into CtIP protein: expression in breast cancer and regulation by sumoylation

Las roturas de doble cadena representan el tipo más nocivo entre todas las posibles lesiones que pueden ocurrir en el ADN, ya que ponen en peligro la viabilidad celular y la estabilidad genómica. Las células eucariotas han desarrollado dos vías principales de reparación para minimizar el riesgo de las roturas de doble cadena: la unión de extremos no homólogos y la recombinación homóloga. Un correcto balance entre ambas rutas es de vital importancia para asegurar la correcta reparación de los cortes y la estabilidad genómica. Por lo tanto, la desregulación en la elección entre ambas vías de reparación puede causar distintas enfermedades humanas, entre las que destaca el cáncer. La resección de los extremos de ADN es un mecanismo que promueve la reparación de los cortes de doble cadena a través de recombinación homóloga e impide la actuación de la maquinaria de la unión de extremos no homólogos. Por lo tanto, la resección supone un importante punto de control y de elección entre ambas rutas, que a su vez es controlada por la proteína nuclear CtIP. La actividad de CtIP en células humanas está sujeta a una fuerte regulación a nivel de diversas modificaciones postraduccionales e interacciones con otras proteínas. En esta Tesis investigamos el papel de CtIP en el mantenimiento de la estabilidad genómica desde dos perspectivas distintas. En primer lugar, analizamos los niveles de CtIP a nivel de proteína en tejidos de cáncer de mama, y encontramos que la expresión de CtIP es nula en una alta proporción de tumores, especialmente en los tumores clasificados como triple negativos. En segundo lugar, demostramos que CtIP es un sustrato de sumoilación en células humanas. Esta modificación postraduccional de CtIP, mediada por la ligasa de SUMO CBX4, es necesaria para el proceso de resección y de recombinación homóloga de las roturas de doble cadena. Por lo tanto, en esta Tesis identificamos una nueva modificación reguladora de CtIP que participa en el mantenimiento de la estabilidad genómica, y demostramos la existencia de una fuerte conexión entre la pérdida de CtIP y el desarrollo de tumores de mama de carácter agresivo.DNA double strand breaks (DSBs) are highly cytotoxic DNA lesions that threaten cellular viability and genomic stability. To avoid the deleterious outcomes of persistent DSBs, eukaryotic cells have developed two main repair pathways: Non homologous end joining (NHEJ) and Homologous recombination (HR). The choice between these two pathways is a key event in the accurate repair of the breaks and the maintenance of genome stability. Consequently, a deregulation in the repair pathway choice can result in the development of several human diseases, including cancer. The best-known decision point resides in the DNA end resection process, which channel DSB repair through HR, and depends on the nuclear protein CtIP. The activity of CtIP is extensively modulated in human cells by post-translational modifications and interaction with many different proteins. In this Thesis, we investigated the role of CtIP in the maintenance of genomic stability at two different levels. First, we studied CtIP protein levels in breast cancer, and we showed that CtIP is frequently lost in breast tumors, especially in the triple negative subtype. Moreover, we demonstrated that CtIP is sumoylated in human cells by the SUMO ligase CBX4, and that this post-translational modification is required for DNA end resection and homologous recombination. Thus, here we identify a new regulatory modification of CtIP involved in the maintenance of genomic stability and provide a strong connection of CtIP loss to aggressive phenotypes in breast cancer

Prognostic value of CtIP/RBBP8 expression in breast cancer

CtIP/RBBP8 is a multifunctional protein involved in transcription, DNA replication, DNA repair by homologous recombination and the G1 and G2 checkpoints. Its multiple roles are controlled by its interaction with several specific factors, including the tumor suppressor proteins BRCA1 and retinoblastoma. Both its functions and interactors point to a putative oncogenic potential of CtIP/RBBP8 loss. However, CtIP/RBBP8 relevance in breast tumor appearance, development, and prognosis has yet to be established. We performed a retrospective analysis of CtIP/RBBP8 and RB1 levels by immunohistochemistry using 384 paraffin-embedded breast cancer biopsies obtained during tumor removal surgery. We have observed that low or no expression of CtIP/RBBP8 correlates with high-grade breast cancer and with nodal metastasis. Reduction on CtIP/RBBP8 is most common in hormone receptor (HR)-negative, HER2-positive, and basal-like tumors. We observed lower levels of RB1 on those tumors with reduced CtIP/RBBP8 levels. On luminal tumors, decreased but not absence of CtIP/RBBP8 levels correlate with increased disease-free survival when treated with a combination of hormone, radio, and chemo therapies. © 2013 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.This work has been funded by a R+D+I grant from the Spanish Ministry of Economy and Competitivity (SAF2010-14877) and a European Research Council Starting Grant (DSBRECA). C. S. is supported by a contract of Nicolas Monardes program, and M. A. J. by grant AI2013-A-0006, Consejerıa de Salud, Junta de AndalucíaPeer Reviewe

The Frontline of Courland Pocket and Its Impact on Landscape: a Case Study of Dobele – Lestene Area

Ainava ir vēstures notikumu telpiskās materializēšanās glabātāja un līdzīgi kā vēsture rakstīta grāmatās, tāpat tā mūsu priekšā guļ ierakstīta ainavas izskatā. Bakalaura darba pētījumu centrā ir ainavas izmaiņas posmā starp Dobeli un Lesteni Otrā Pasaules kara cīņu Kurzemes „cietoksnī” iespaidā. Kā vienas no ainavas izmaiņu indikatoriem izvēlētas viensētas, kuras kara laikā bija spiesti pamest to saimnieki un daudzas no kurām cīņās tika nopostītas. Lai novērtētu karadarbības ietekmi uz viensētu izvietojuma izmaiņām, tika analizēta situācija 30. gados, frontes līniju izplatība Otrajā Pasaules karā un viensētu novietojums un skaits 60. gadu sākumā. Karadarbības pēdas šodienas ainavā tika noteiktas apsekojot pētījumu teritoriju un izmantojot ainavas lasīšanas pieeju. Atslēgas vārdi: ainava, ainavas izmaiņas, karadarbības ietekme, frontes līnija, Kurzemes „cietoksnis”, Otrais Pasaules karšA landscape is merely a three dimensional holder of materialization of historical events and just as history that is written and published in books, it lies before us written in the form of a landscape. The bachelor thesis research focuses on how landscape has been changed in various locations between Dobele and Lestene during the World War II, more specifically, during the battles in Courland Pocket. Cottages that were abandoned by their inhabitants and many of which were destroyed, have been selected as the factors indicating the change in the landscape. In order to investigate the change of the placement of the cottages, the situation in the 1930s, the expansion of the frontlines during WWII and the number and placement of cottages in the beginning of the 1960s were closely researched. The aftermath of the war in present day's scenery was studied by exploring the territory of the research and by using the method of landscape reading. Key words: landscape, landscape change, warfare’s impact, frontline, Courland Pocket, World War

CtIP as a novel tumor suppressor and its relevance for initiation, prognosis and treatment of cancer

Póster presentado al 22nd IUBMB & 37th FEBS Congress: From Single Molecules to Systems Biology, celebrado en Sevilla (España) del 4 al 9 de septiembre de 2012DNA damage and repair are closely linked to cancer, not only during tumor initiation and progression, but also as a potent antitumoral therapeutical opportunity. From the many types of damaged DNA, double strand breaks (DSBs) are specially relevant in oncology. DSBs are repaired by two major mechanisms that compete for the same substrate. Both ends of the DSB can be simple re-joined with little or no processing, a mechanism known as non-homologous end-joining. On the other hand, DSBs can be processed and engaged in a more complex repair pathway called homologous recombination. This pathway uses the information present in a homologue sequence. The balance between these two pathways is exquisitely controlled. Alterations in the DSBs repair pathways facilitate tumor progression and are selected early on during cancer development. On the other hand, DSBs are the molecular base of radiotherapies and several chemotherapies. Little is known about how cells chose between these two repair pathways, or the relevance of such choice in cancer initiation, progression or treatment. A main player for this election is the protein known as CtIP, which has been loosely implicated with cancer. We are studying the role of CtIP as a tumor suppressor and its importance in tumor initiation, progression, prognosis and treatment using cancer samples and cancer cell models.Peer Reviewe

Alkylphospholipids deregulate cholesterol metabolism and induce cell-cycle arrest and autophagy in U-87 MG glioblastoma cells

Glioblastoma is the most common malignant primary brain tumour in adults and one of the most lethal of all cancers. Growing evidence suggests that human tumours undergo abnormal lipid metabolism, characterised by an alteration in the mechanisms that regulate cholesterol homeostasis. We have investigated the effect that different antitumoural alkylphospholipids (APLs) exert upon cholesterol metabolism in the U-87 MG glioblastoma cell line. APLs altered cholesterol homeostasis by interfering with its transport from the plasma membrane to the endoplasmic reticulum(ER), thus hindering its esterification. At the same time they stimulated the synthesis of cholesterol from radiolabelled acetate and its internalisation from low-density lipoproteins (LDLs), inducing both 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and LDL receptor (LDLR) genes. Fluorescent microscopy revealed that these effects promoted the accumulation of intracellular cholesterol. Filipin staining demonstrated that this accumulation was not confined to the late endosome/lysosome (LE/LY) compartment since it did not colocalise with LAMP2 lysosomal marker. Furthermore, APLs inhibited cell growth, producing arrest at the G2/M phase. We also used transmission electron microscopy (TEM) to investigate ultrastructural alterations induced by APLs and found an abundant presence of autophagic vesicles and autolysosomes in treated cells, indicating the induction of autophagy. Thus our findings clearly demonstrate that antitumoural APLs interfere with the proliferation of the glioblastoma cell line via a complex mechanism involving cholesterol metabolism, cell-cycle arrest or autophagy. Knowledge of the interrelationship between these processes is fundamental to our understanding of tumoural response and may facilitate the development of novel therapeutics to improve treatment of glioblastoma and other types of cancer. © 2013 Elsevier B.V.This research was aided by the Andalusian regional government (CTS-236). The authors thank Xiomara Gálvez for her technical support and Dr. Francisco D. Martín for his help in our fluorescence microscopy experiments. Pablo Ríos-Marco holds a fellowship from the Spanish Ministry of EducationPeer Reviewe

The ubiquitin E3/E4 ligase UBE4A adjusts protein ubiquitylation and accumulation at sites of DNA damage, facilitating double-strand break repair

Double-strand breaks (DSBs) are critical DNA lesions that robustly activate the elaborate DNA damage response (DDR) network. We identified a critical player in DDR fine-tuning: the E3/E4 ubiquitin ligase UBE4A. UBE4A’s recruitment to sites of DNA damage is dependent on primary E3 ligases in the DDR and promotes enhancement and sustainment of K48- and K63-linked ubiquitin chains at these sites. This step is required for timely recruitment of the RAP80 and BRCA1 proteins and proper organization of RAP80- and BRCA1-associated protein complexes at DSB sites. This pathway is essential for optimal end resection at DSBs, and its abrogation leads to upregulation of the highly mutagenic alternative end-joining repair at the expense of error-free homologous recombination repair. Our data uncover a critical regulatory level in the DSB response and underscore the importance of fine-tuning the complex DDR network for accurate and balanced execution of DSB repair.Work in the Y.S. laboratory is funded by research grants from the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the A-T Children’s Project, the Israel Science Foundation Joint ISF-NSFC Research Program (jointly funded by the Israel Science Foundation and the National Natural Science Foundation of China - Grant No. 998/14), and the Israel Cancer Research Fund (Professorship). T.M. and T.C.V. were supported by funding from the Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute, and the Center for Cancer Research. Work in the K.R. laboratory is supported by the Swiss National Science Foundation (31003A_141197) and the Medical Research Council, UK (MC_PC_12001/1). Work in the G.D. laboratory is supported by a discovery grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN 05616). Work in the P.H. lab was supported by an R+D+I grant from the Spanish Ministry of Economy and Competitivity (SAF2013-43255-P) and an ERC starting grant (DSBRECA). Work in the E.R. laboratory is supported by NIH grants GM108119 and CA187612 and American Cancer Society grant ACS130304-RSG-16-241-01-DMC. I.S.-B. is the recipient of a Ph.D. fellowship from the University of Sevilla. D.C. was supported by a Nova Scotia graduate scholarship. K.B.B. is a Jack and Florence Berlin fellow. Y.S. is a Research Professor of the Israel Cancer Research Fund.Peer reviewe