Studies on Proteins Influencing Cancer Progression and Regulating Endocytic Lipid Trafficking

Endocytosis is a form of active cellular transport by which cells internalize molecules from the extracellular environment. This thesis studies proteins involved in endocytosis that influence the progression of cancers in humans. Two of the three articles summarized here employ cell biological experiments to elucidate the role of the proteins N-muc downstream-regulated gene 1 (NDRG1) and StAR-related lipid transfer protein 3 (StARD3) in the regulation of endocytic trafficking. The third article describes a novel data analysis method and its application to a large data set of breast cancer patients. We studied the effects of NDRG1 protein depletion on cellular cholesterol content and distribution, low-density lipoprotein receptor (LDLR) localization and turn-over, and the morphology of endosomal organelles. We found that depletion of the NDRG1 protein leads to reduced abundance of LDLR on the plasma membrane (PM) and as a result, reduced LDL uptake. When NDRG1 is depleted, LDLR accumulates in multi-vesicular bodies (MVBs), and while LDLR ubiquitination is increased, its degradation is decreased. The PM levels of LDLR and LDL uptake are rescued upon co-depletion of the inducible degrader of the LDL-receptor (IDOL). Our findings identify NDRG1 as regulator of MVB formation and trafficking. We also found that elevated levels of StARD3 protein alter the cholesterol balance and adhesiveness of breast cancer cells. We studied the effects of StARD3 overexpression in cells, as well as the association of StARD3 protein levels with cancer progression markers in Finnish breast cancer patients. We found that in MCF-7 cells, stable StARD3 overexpression altered the morphological features and the cholesterol balance of the cells. In the breast cancer tumor samples from patients, high StARD3 protein levels associated with ERBB2 (receptor tyrosine-protein kinase erbB-2) amplification. High protein levels also associated with proto-oncogene tyrosine-protein kinase Src (Src) activation and increased 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) transcript levels. Our findings suggest that elevated StARD3 levels contribute to breast cancer aggressiveness. Finally, a systematic approach to data analysis was developed and demonstrated by applying it to an existing data set of breast cancer patients. High-throughput molecular level data and clinical data were integrated in the study. This revealed a coamplification of NDRG1 and the gene lysosomal-associated transmembrane protein 4B (LAPTM4B), encoding an endosomal protein that regulates ceramide trafficking, and discovered new genes potentially coregulated with LAPTM4B.Solut ottavat sisäänsä ja kierrättävät ravinteita ja signaalimolekyylejä endosytoosin avulla. Vesikkeleitä muodostuu solun pinnassa kun solukalvon sisäänpainaumat kuroutuvat irti solukalvosta ja siirtyvät solun sisälle. Näissä endosomeissa (rakkuloissa) on kuljetuskoneisto. Endosomien kalvoilla on proteiineja, mukaan lukien solukalvon reseptoreja. Resoptorit voidaan hajoittaa lysosomissa tai ne voidaan kierrättää takaisin solun pintaan, joka toimii myös säätelymekanismina. Syöpäsolut pystyvät välttämään elimistön kasvun, erilaistumisen ja solukierron tiukan säätelyn. Tämä väitöskirjatutkimus osoittaa, miten endosytoosin epätyypillinen säätely voi vaikuttaa syövän etenemiseen. Kokeellisen työn keskiössä oli kaksi geeniä: StARD3 ja NDRG1. Tulokset osoittavat StARD3-geenin mahdollisen kytköksen rintasyövän aggressiivisuuteen. NDRG1-geeni näytti säätelevän erityisesti LDL-reseptorin kuljetusta, mutta sillä voi olla yleisempi tehtävä solun pintareseptorin hajoamisen säätelyssä. Ymmärtääksemme laajemmin endosytoosin säätelyn roolia syövässä, analysoimme kokogenomista DNA- ja mRNA-dataa, sekä kliinistä potilasdataa tietokannasta (The Cancer Genome Atlas). Tunnistimme geeniryppään joita säädellään yhdessä LAPTM4B:n kanssa, joka on myöhäisessä endosomissa tapahtuvaan lipidien kuljetukseen liittyvä proteiini. Lopuksi, näytimme miten data-analyysejä voi järjestää ja esittää käytännöllisesti toistettavissa olevassa muodossa

Identification of genes that are essential to restrict genome duplication to once per cell division.

Nuclear genome duplication is normally restricted to once per cell division, but aberrant events that allow excess DNA replication (EDR) promote genomic instability and aneuploidy, both of which are characteristics of cancer development. Here we provide the first comprehensive identification of genes that are essential to restrict genome duplication to once per cell division. An siRNA library of 21,584 human genes was screened for those that prevent EDR in cancer cells with undetectable chromosomal instability. Candidates were validated by testing multiple siRNAs and chemical inhibitors on both TP53+ and TP53- cells to reveal the relevance of this ubiquitous tumor suppressor to preventing EDR, and in the presence of an apoptosis inhibitor to reveal the full extent of EDR. The results revealed 42 genes that prevented either DNA re-replication or unscheduled endoreplication. All of them participate in one or more of eight cell cycle events. Seventeen of them have not been identified previously in this capacity. Remarkably, 14 of the 42 genes have been shown to prevent aneuploidy in mice. Moreover, suppressing a gene that prevents EDR increased the ability of the chemotherapeutic drug Paclitaxel to induce EDR, suggesting new opportunities for synthetic lethalities in the treatment of human cancers

MultiDecision-2: A Multicriteria Decision Support System

The paper presents a multicriteria decision support system, called MultiDecision-2, which consists of two independent parts - MKA-2 subsystem and MKO-2 subsystem. MultiDecision-2 software system supports the decision makers (DMs) in the solving process of different problems of multicriteria analysis and linear (continues and integer) problems of multicriteria optimization. The two subsystems MKA-2 and MKO-2 of of MultiDecision-2 are briefly described in the paper in the terms of the class of the problems being solved, the system structure, the operation with the interface modules for input data entry and the information about DM’s local preferences, as well as the operation with the interface modules for visualization of the current and final solutions

Language-Agnostic Reproducible Data Analysis Using Literate Programming

A modern biomedical research project can easily contain hundreds of analysis steps and lack of reproducibility of the analyses has been recognized as a severe issue. While thorough documentation enables reproducibility, the number of analysis programs used can be so large that in reality reproducibility cannot be easily achieved. Literate programming is an approach to present computer programs to human readers. The code is rearranged to follow the logic of the program, and to explain that logic in a natural language. The code executed by the computer is extracted from the literate source code. As such, literate programming is an ideal formalism for systematizing analysis steps in biomedical research. We have developed the reproducible computing tool Lir (literate, reproducible computing) that allows a tool-agnostic approach to biomedical data analysis. We demonstrate the utility of Lir by applying it to a case study. Our aim was to investigate the role of endosomal trafficking regulators to the progression of breast cancer. In this analysis, a variety of tools were combined to interpret the available data: a relational database, standard command-line tools, and a statistical computing environment. The analysis revealed that the lipid transport related genes LAPTM4B and NDRG1 are coamplified in breast cancer patients, and identified genes potentially cooperating with LAPTM4B in breast cancer progression. Our case study demonstrates that with Lir, an array of tools can be combined in the same data analysis to improve efficiency, reproducibility, and ease of understanding. Lir is an open-source software available at github. com/borisvassilev/lir.Peer reviewe

Fuzzy Tandem Repeats Containing p53 Response Elements May Define Species-Specific p53 Target Genes

Evolutionary forces that shape regulatory networks remain poorly understood. In mammals, the Rb pathway is a classic example of species-specific gene regulation, as a germline mutation in one Rb allele promotes retinoblastoma in humans, but not in mice. Here we show that p53 transactivates the Retinoblastoma-like 2 (Rbl2) gene to produce p130 in murine, but not human, cells. We found intronic fuzzy tandem repeats containing perfect p53 response elements to be important for this regulation. We next identified two other murine genes regulated by p53 via fuzzy tandem repeats: Ncoa1 and Klhl26. The repeats are poorly conserved in evolution, and the p53-dependent regulation of the murine genes is lost in humans. Our results indicate a role for the rapid evolution of tandem repeats in shaping differences in p53 regulatory networks between mammalian species

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

The European Eel Anguilla anguilla (Pisces, Anguillidae). Native or Alien in the Black Sea?

Abstract. In 2006 the Scientific, Technical and Economic Committee for Fisheries (STEFC) of EU suggested the Black Sea region should be excluded from the list of natural areas, where the European eel (Anguilla anguilla) is distributed. The basic conception of this idea was that the eels caught in the Black Sea region represent escaped specimens from fish farming in some Danubian countries. This article illustrates an effort to be given an indirect answer on the question if Black Sea is the end of natural distribution of European eel. The species is present but never been abundant in the region and do not represent an object of commercial fishing