Session I:

Mechanisms of metastasis

 

TUMOR ANGIOGENESIS NORMALIZATION IN THE PREVENTION OF METASTASIS

 

Claudine Kieda

 

Centre for Molecular Biophysics, UPR 4301 CNRS, 45071 Orleans, France; email: claudine.kieda@cnrs-orleans.fr

 

In tumors, the contribution of angiogenesis to metastasic spreading is documented in several distinct aspects which contribute to efficient  tumor cell distribution.

Among these parameters, of particular importance, is the oxygen partial pressure (pO₂), a key component of any organ physiology. It results from the balance between oxygen delivery and consumption. Red blood cell-borne O₂ delivery depends upon metabolic requirements and functional status of a tissue, characterized by its own “tissue normoxia” called: “physioxia”. This is severely disturbed in pathologic conditions as cancer, diabetes, coronary heart disease, stroke etc. that are associated with decreased pO₂ : “hypoxia”. Monitoring tissue oxygen grade has both prognostic and therapeutic values.

O₂ delivery is impaired in tumors, by chaos and leakiness of vessels contributing to tumor cells spreading. Tumor angiogenesis, through its characteristics, both cellular and chemical, is the key to the portal that allow metastasis. Counteracting tumor hypoxia is highly promising and appears as a challenging issue with important consequences to control selection of cancer stem-like cells. Those are aggressively metastatic, resistant to low pH, immune cell cytotoxicity and drugs.

Long considered as “The” powerful therapeutic approach, antiangiogenesis strategies are being revisited. Indeed, the actual challenge in angiogenesis-related therapy favors blood vessels normalization/maturation vs. destruction. New strategies aiming to tumor targeted expression of the soluble VEGF receptor-2 (sVEGF-R2) in a manner restricted to tumor hypoxia should normalize the tumor vasculature and restore an efficient blood flow thus reducing tumor hypoxia. Furthermore, tumor targeting is performed by the use of endothelial precursor cells that incorporate the newly developing blood vessels thus contributing to tumor angiogenesis. These cells will be used as a carrier for the above described vector to express the sVEGF-R2 inside the tumor. Another, approach is to make them express new regulators of angiogenesis as miRNAs. These attempts  switch the endothelial precursors from pro- into angiogenesis controls.

Tumor hypoxia reduction is a challenge for efficient radiotherapy and/or chemotherapy. The hypoxia compensation strategy is a new approach using the allosteric effector of hemoglobin to revert pathologic angiogenesis. This strategy is strongly active against metastasis development. The mechanism was shown mainly due to the vessels normalization /maturation. It has also potent applications in repair mechanisms of ischemia mediated diseases.

BRAIN METASTASIS PROTEINS AND THEIR INTERACTIONS: A RATIONAL PREDICTIVE-BIOMARKERS RESEARCH   

 

Antonio Martínez, Rebeca Sanz-Pamplona, Baldo Oliva, Miguel Gil, Susana Boluda,
Víctor Moreno, Angels Sierra

 

Bellvitge Biomedical Research Institute-IDIBELL, Barcelona

 

Predicting the risk of metastasis remains a critical point in daily clinical practice. Of the various markers (or tools) that are currently available, none gives information about the risk of developing organ-specific metastasis. This knowledge can help clinicians to plan more carefully the treatment of patients, help them to decide which aggressive treatments to use, avoid unnecessary treatment and adjust therapy according to the organ affected by metastasis.

Indeed, in breast cancer only hormone receptors and HER-2 expression are predictors used to decide hormonetherapy or Trastuzumab therapy (respectively). However, around 10% of patients with a diagnosis of early breast cancer will develop distant relapse, 10-15% of them will develop brain metastasis [1, 2]. The increase in this rate could be linked to greater survival in patients receiving chemotherapy and the fact that it is difficult to overcome the blood brain barrier (BBB) with current systemic treatments [3]. Thus, there is a need of predictive biomarkers useful to assess the probability that a patient will benefit from a particular treatment at the first diagnosis to refining patient care.

To obtain an accurate classification of brain metastasis proteins, we mapped organ-specific brain metastasis gene expression signatures onto an experimental protein-protein interaction network based on brain metastatic cells [4]. We identified 37 proteins that were differently expressed between brain metastases non-brain metastases [5]. Analysis of metastatic tissues, the use of bioinformatic approaches and the characterization of protein expression in tumors with or without metastasis identified candidate markers. We performed a multivariate analysis based on stepwise logistic regression which revealed GRP94, FN14 and Inhibin as the best combination to discriminate between brain and non-brain metastases (aROC 0.85, 95% CI 0.73 – 0.96, for the combination of the 3 proteins). These markers substantially improve the discrimination of brain metastasis compared to ErbB2 alone (aROC 0.76, 95% CI 0.60 – 0.93). Furthermore GRP94 was a better negative marker (LR 0.16) than ErbB2 (LR 0.42). These predictive biomarkers can help in the selection of treatment strategies, since the current ambitious aim is to identify treatment strategies that will cure patients with ErbB2-positive and negative disease ensuring minimal toxicity for each individual patient. This could also lead to preventive therapy for brain metastases at initial diagnosis.

 

References:

[1] Bendell J.C., Domchek S.M., Burstein H.J., Harris L., Younger J., Kuter I., Bunnell C.,
Rue M., Gelman R., Winer E.: Central nervous system metastases in women who receive trastuzumab-based therapy for metastatic breast carcinoma, Cancer, 97, 2972-2977, 2003.

[2] Palmieri D., Bronder J.L., Herring J.M., Yoneda T., Weil R.J., Stark A.M., Kurek R., Vega-Valle E., Feigenbaum L., Halverson D., Vortmeyer A.O., Steinberg S.M., Aldape K., Steeg, P.S: Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain, Cancer Res, 67, 4190-4198, 2007

[3] Carey L.A., Ewend M.G., Metzger R., Sawyer L., Dees E.C., Sartor C.I., Moore D.T., Graham M.L.: Central nervous system metastases in women after multimodality therapy for high risk breast cancer. Breast Cancer Res Treat, 88, 273-280, 2004.

[4] Martin B., Aragues R., Sanz R., Oliva B., Boluda S., Martinez A., Sierra A Biological pathways contributing to organ-specific phenotype of brain metastatic cells, J Proteome Res, 7, 908-920, 2008.

[5] Sanz-Pamplona R., Aragüés R., Driouch K., Martín B., Oliva B., Gil M., Boluda S., Fernández P.L., Martínez A., Moreno V., Acebes, J.J., Lidereau R., Reyal F., Van de Vijver M. Sierra A.: Expression of endoplasmic reticulum stress proteins is a candidate marker of brain metastasis in both ErbB2-positive and -negative primary breast tumors, Am J Pathol, 179, 564-579, 2011.

Incidence and prognostic role of circulating tumor cells in squamous cell carcinoma of the head and neck region (SCCHN)

 

Inge Tinhofer¹, Tsvetana Hristozova¹, Carmen Stromberger¹, Robert Konschak¹,
Volker Budach¹, Ulrich Keilholz²

 

¹Laboratory for Translational Radiobiology and Radiooncology, Department
of Radiooncology, Charité Campus Mitte; ²Department of Hematology and Oncology, Campus Benjamin Franklin Universitätsmedizin-Berlin, Germany

 

Introduction: The presence of circulating tumor cells (CTCs) in peripheral blood (PB) of patients with solid tumors has previously been associated with a more aggressive disease, increased risk of local or distant metastasis and reduced overall survival. In a former study, we detected CTCs in 43% of locally advanced, inoperable SCCHN cases. More recently, we developed the protocol for CTC analysis by flow cytometry further in order to study their basal expression and activation of the epidermal growth factor receptor (EGFR) signalling pathway. Given the important role of EGFR signalling in metastasis and treatment efficacy in SCCHN, we then assessed the influence of treatment on the detection rate of CTCs and their expression of EGFR/phospho-EGFR.

Methods: SCCHN patients with locally advanced, unresectable tumors who participated in a clinical study comparing induction chemotherapy followed by radiotherapy plus cetuximab with standard concurrent radiochemotherapy were included in this study. Blood samples were collected and analysed for the presence of CTCs and their EGFR/phospho-EGFR expression at predefined time points – prior treatment, after completion of 3 cycles induction chemotherapy, at the end of treatment and at the first and the second follow-up. The absolute numbers of CTCs defined as EpCAM+ cytokeratin+ CD45− in 3.75 ml blood and their EGFR/phosphor-EGFR expression were determined by flow cytometry.

Results: Overall, prior treatment CTCs were detected in the blood of 7 of 24 patients (29.2%). Administration of induction chemotherapy seemed not to have a major influence in the frequency of CTCs, since 4 of 13 patients (30.1%) were positive for CTCs after its completion. Interestingly, the frequency of CTC+ cases significantly increased after radiotherapy combined with either cetuximab or cisplatin/5-FU and at the end of treatment CTCs could be detected in peripheral blood samples of 10 of 18 patients (56%). During the second follow up visit, 2 of 10 patients (20%) still had detectable CTCs in their blood which is slightly above the detection limit of the method. All detected CTCs expressed EGFR on their membrane before therapy which was not influenced by the treatment. However, the frequency of CTC+ cases expressing phospho-EGFR was reduced after radiotherapy/cetuximab treatment and remained unchanged after concurrent radiochemotherapy

Conclusions: Detection of CTCs might represent a novel non-invasive prognostic and predictive tool in SCCHN. Their potential in monitoring tumor response and predicting treatment outcome needs further evaluation.

 

THE BIOLOGICAL ROLE OF ADAPTOR/SCAFFOLD PROTEIN RUK/CIN85 IN BREAST CARCINOGENESIS

 

L.B. Drobot

 

Palladin Institute of Biochemisry, National Academy of Sciences of Ukraine,Kyiv, 01601, Ukraine; drobot@biochem.kiev.ua

 

Adaptors are proteins of multi-modular structure without enzymatic activity. Their capacity to organize large, temporary protein complexes by linking proteins together in a regulated and selective fashion makes them of outstanding importance in the establishment and maintenance of specificity and efficiency in all known signal transduction pathways. Given the important role of adaptor proteins in propagating cellular signals, it is quite likely that their dysfunction may be involved in carcinogenesis. The prooncogenic or tumor-suppressor activities of a set of adaptor proteins have been already demonstrated. The adaptor/scaffold protein Ruk/CIN85, containing multiple SH3 domains, was implicated in carcinogenesis by influencing a number of processes such as cell adhesion, motility and apoptosis. Although Ruk/CIN85 appears to modulate tyrosine kinase receptors and PI3 kinase signalling, the exact molecular mechanisms by which Ruk/CIN85 affects carcinogenesis are largely unknown.

Using Western-blot analysis, the statistically significant increase in expression level of Ruk/CIN85 full-length form was detected in human invasive ductal breast adenocarcinoma samples in comparison with surrounding conditionally normal tissues. Therefore, we decided to investigate the oncogenic potential of Ruk/CIN85 by stably overexpressing the full-length isoform in weakly invasive MCF-7 breast adenocarcinoma cells. The Ruk_l/CIN85 overexpressing cells showed a slower growth rate, decreased cell adhesion, and an enhanced anchorage-independent growth in soft agar. Further, overexpression of Ruk_l/CIN85 also affected EGF-dependent signalling: activation of Src, Akt and ERK1/2 was faster than in the control cells and all kinases remained in their active state for up to 30 min after EGF treatment. Transwell migration and wound healing assays revealed that Ruk_l/CIN85 overexpressing cells possessed increased motility. The EGF-induced motility was attenuated in Ruk_l/CIN85-overexpressing cells but could be restored upon knock-down of Ruk_l/CIN85 with specific shRNA. It was found also that Ruk/CIN85 induced PAI-1 mRNA and protein expression both under normoxia and hypoxia. The induction of PAI-1 expression by Ruk/CIN85 occurred at the transcriptional level since the half-life of PAI-1 mRNA was not affected in cells overexpressing Ruk/CIN85 and reporter gene assays using wild-type and mutant human PAI-1 promoter luciferase constructs showed that the hypoxia responsive element was responsible for Ruk/CIN85 effects. Further, knocking down HIF-1α abolished not only the hypoxia-dependent but also the Ruk/CIN85-dependent PAI-1 induction. In addition, transient or stable overexpression of Ruk/CIN85 also induced HIF-1α protein levels and HIF-1 activity and knocking down Ruk/CIN85 reversed these effects. Thereby, Ruk/CIN85 interfered with the proline hydroxylation-dependent HIF-1α protein destabilisation.

Together, these findings suggest that high levels of Ruk_l/CIN85 can modulate EGF-and hypoxia-dependent signalling and contribute to the conversion of breast adenocarcinoma cells into a more malignant phenotype.

 

Attempts to target cancer stem cells – Salinomycin as an example

 

Jaganmohan R. Jangamreddy and Marek Los

 

Department of Clinical and Experimental Medicine (IKE), Integrative Regenerative Medicine Center (IGEN), Linköping Univ., Linköping, Sweden

 

Malignant tumors are composed of cells with varying potential for renewal and proliferation. Accumulating evidence suggests that a subset of cells within tumor, called cancer stem cells (CSCs) are responsible for tumor growth, metastasis development, and recurrence upon therapy. Cancer stem cells are also sufficient to initiate full re-grow of tumors in tumor-transfer experiments in animal models, and they are more resistant to conventional forms of therapy. Thus, several labs conduct search for molecules that show preferential- or selective toxicity towards CSCs. Recent reports show that human breast cancer stem-like cells are more sensitive to the treatment with an antibiotic salinomycin. However, the mechanism of salinomycin-induced cell death is not well-defined. Salinomycin is a polyether antibiotic and acts in different biological membranes as an ionophore with a preference for potassium. To decipher molecular mechanism(s) of salinomycin toxicity, we have used serum-free mammosphere culture to enrich for cancer stem cells in four breast cancer cell lines: MCF7, SKBR3, BT474 and MDA-MB468. Furthermore, we have utilized model cell lines that either lack- or over-express regulators or effectors of cell death, including: the p53-, Bax/Bak-, and Apaf1-deficient cell lines, as well as Bcl-2, Bcl-xL and caspase-3 over-expressed cell lines. We have found that, salinomycin-induced cell death was p53- and caspase-independent, however, Bax/Bak, Bcl-2, Bcl-xL and Apaf1 were involved. Our data indicate that multiple cell death pathways were engaged upon salinomycin treatment, including apoptosis, necrosis and autophagy. In particular, detailed electron-microscopy studies revealed that the induction of autophagy (autophagosome formation) was very dramatic, vastly exceeding changes induced by i.e. Rapamycin, used as a “positive control” for autophagy induction. The involvement of mitoptosis, the irreversible changes of mitochondrial structure, and detrimental impairment of crucial mitochondrial functions have also readily been documented. Thus, better understanding of molecular mechanisms of Salinomycin’s anticancer-stem cell activity may assist the discovery of new generation of anticancer drugs that target CSC.

 

THE ROLE OF CERAMIDE GALACTOSYLTRANSFERASE (UGT8), A NEW MOLECULAR MARKER OF BREAST CANCER MALIGNANCY, IN CANCER PROGRESSION

 

Maciej Ugorski^1,2, Tomasz Owczarek^1,2

 

¹Laboratory of Glycobiology and Cell Interactions, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wroclaw, Poland; ²Department
of Biochemistry, Pharmacology and Toxicology, Wroclaw University of Environmental
and Life Sciences, 50-375 Wroclaw, Norwida 31, Poland

 

The UDP-galactose:ceramide galactosyltransferase (UGT8) is an enzyme responsible for the synthesis of galactosylceramide (GalCer) which is known mostly as the constituent of myelin. Recently, we have shown that increased level of UGT8 in breast cancerous tissue is associated with progression to a more malignant phenotype; however very little is known about the possible functions of UGT8 and GalCer in tumor cells. On the basis of the existing data, it was proposed that accumulation of GalCer in tumor cells inhibits apoptosis, which facilitated metastatic cells to survive in the hostile microenvironment of the target organ. Therefore, to verify this hypothesis and study the role of this glycosphingolipid in breast cancer progression, we have used MCF7 cells overexpressing UGT8 and GalCer after transfection with UGT8 mRNA and MDA-MB-231 cells with highly decreased expression of UGT8 and GalCer after stable expression of shRNA directed against UGT8 mRNA. It was found that accumulation of GalCer in MCF7 cells increased their resistance to apoptosis induced by N-(4-hydroxyphenyl)retinamide (4-HPR). In the case of MDA-MB-231 cells, the down-regulation of GalCer resulted in increased sensitivity of breast cancer cells to doxorubicin-induced apoptosis.

To further reveal the role of UGT8 and GalCer in breast cancer progression, the tumorigenicity and metastatic potential of parental MDA-MB-231 cells and GalCer-negative MDA-MB-231 cells was studied in vivo in athymic nu/nu mice. It was found that subcutaneous transplantation of tumor cells with no expression of GalCer, resulted in the formation of tumors with highly reduced volumes in comparison with mice inoculated with parental MDA-MB-231 cells. GalCer-negative breast cancer MDA-MD-231 cells were also characterized by markedly decreased ability to form metastases. When these cells were transplanted intracardiacally, the average time of metastasis occurrence was markedly longer (14 weeks) in comparison with appearance of metastases after implantation of the parental MDA-MB-231 cells (6 weeks).

In conclusion, our data support the thesis on the importance of UGT8 and GalCer in the drug-induced apoptosis and the development of lung metastases by breast cancer cells.

 

Session II:

Regulation of gene expression
at the transcript level

Understanding RNA silencing pathways through the Argonaute proteins

 

Martin Simard

 

Laval University, Quebec

 

Since their discoveries twenty years ago, 20-30 nucleotides long small RNAs species have rapidly emerged as essential contributors in the maintenance of cell homeostasis in nearly all eukaryotes. Despite their impressive abundance, we poorly understand how these small RNAs, particularly microRNAs, tightly control gene expression in animals. To elucidate how these small molecules can regulate gene expression, we initiated characterization of key players of the RNA silencing pathways: the Argonaute gene family. During this talk, I will discuss our recent experimental progress towards the identification of new cellular components important for the microRNA pathway as well as our characterization of molecular features of the Argonaute proteins. 

Analysis of Argonaute interactions in mammalian cells

 

Gunter Meister

 

University of Regensburg, 93053 Regensburg

 

Argonaute (Ago) proteins interact with small regulatory RNAs such as microRNAs (miRNAs) and facilitate gene-silencing processes. miRNAs guide Ago proteins to specific mRNAs leading to translational silencing or mRNA decay. In order to understand the mechanistic details of miRNA-guided gene silencing in mammals, it is important to characterize Ago protein interactors. Although several proteomic studies have been performed, it is not clear how the Ago interactome changes upon miRNA or mRNA binding. Here, we report the quantitative analysis of Ago protein interactions in miRNA-containing and miRNA-depleted cells. Using Stable Isotope Labeling in Cell Culture (SILAC) in conjunction with Dicer knock out mouse embryonic fibroblasts (MEFs), we identify proteins that interact with Ago2 in the presence or the absence of miRNAs. In contrast to our current view on miRNAs as guides for Ago proteins, we find that Ago proteins can interact with mRNAs in the absence of miRNAs as well. Our proteomics approach provides a detailed basis for further functional studies on the cellular roles of Ago proteins.

Unique regulation of small RNAs during oocyte-to-embryo transition in mammals

 

Matyas Flemr, Ma Jun, Radek Malik, Radislav Sedlacek, Richard M. Schultz, Petr Svoboda

 

Institute of Molecular Genetics, ASCR, Prague

 

In mouse, a major portion of time during the oocyte-to-zygote transition occurs in the absence of transcription, and thus depends on post-transcriptional control of the maternal mRNA pool synthesized during oocyte growth. Many maternal mRNAs are stored in the cytoplasm and are translationally inactive. In somatic cells, the translationally repressed mRNAs are targeted to different RNA granules, such as stress granules or Processing bodies (P-bodies). P-bodies are cytoplasmic foci enriched in mRNA-destabilizing proteins, translational repressors and other RNA binding proteins, and microRNAs (miRNAs). To understand the regulation of maternal mRNA storage, recruitment, and degradation, we investigated the distribution of messenger-ribonucleoprotein (mRNP) complexes in mouse oocytes. We found that P-bodies disassemble early during oocyte growth and several P-body components, including RNA helicase DDX6 and polyadenylation regulator CPEB, form a novel type of mRNA storage granules in the cortex of fully-grown oocytes. Because P-bodies form as a consequence of miRNA pathway activity, we analyzed activity of maternal miRNA. We found that P-body disappearance correlates with reduced ability of let-7 and miR-30c miRNAs to repress translation although they are present and loaded on AGO2 in the oocyte. In addition, zygotic expression of let-7 is also suppressed by maternally provided LIN28. Furthermore, transcriptome analysis of oocytes lacking miRNA processing enzyme Dicer did not reveal any miRNA specific footprint in the set of differentially expressed transcripts. These data suggest that miRNA function is suppressed in mouse oocytes, perhaps in order to support mRNA-stabilizing environment of the oocyte cytoplasm and reprogramming of differentiated oocytes into pluripotent cells of the early embryo.

 

MODULATION OF MICRO-RNA BIOGENESIS BY USING SHORT OLIGO-RNA MOLECULES

 

A. Kurzyńska-Kokorniak¹, N. Koralewska¹, A. Tyczewska¹, T. Twardowski¹,
M. Figlerowicz^1,2

 

¹Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12/14,
61-704 Poznań, Poland; ²Institute of Computing Science, Poznań University of Technology, Piotrowo 3A, 60-965 Poznań, Poland

 

During the last decade several types of small regulatory RNA (srRNA) have been discovered. Their significant role in the regulation of eukaryotic gene expression has been well documented. In addition, numerous proteins involved in the biogenesis of srRNA have been identified. One of them is human ribonuclease Dicer, which excises srRNA from perfectly or partially double-stranded RNA precursors.

           Although Dicer substrates and products have already been quite well characterized, our knowledge about cellular factors regulating the activities of this enzyme is still limited. To learn more about this problem, we attempted to determine whether RNA can function not only as a Dicer substrate but also as its regulator. To this end, we applied an in vitro selection method. We identified 120 RNA oligonucleotides binding human Dicer. Sixteen of them were subjected to more detailed in vitro studies. The influence of the chosen RNAs on Dicer’s nuclease activity was tested in the reactions involving one of two human miRNA precursors, either pre-miR-33a or pre-miR-210. We found that all oligomers affected Dicer ability to digest pre-miRNAs, although most of them were cleaved by this enzyme. For six the most active oligomers the putative mechanism of Dicer inhibition was determined. Three oligomers were classified as typical competitive inhibitors and one as an allosteric inhibitor. Interestingly, the remaining two oligomers acted as selective inhibitors. They affected the production of one miRNA, whereas the formation of other miRNAs was hardly influenced. In general, the data obtained suggest that one can modulate the production of specific miRNAs by using RNA oligomers. The performed bioinformatic analysis also suggests that riboregulators similar to the selected oligomers can be encoded in the human genome.

The role of microRNA sequence variation in thyroid cancer

 

Krystian Jazdzewski

 

Comprehensive Cancer Center, Columbus

 

MicroRNAs (miRs) are small (19-25 nucleotides) non-coding RNA molecules that typically function as negative regulators of the expression of protein-encoding genes (Bartel 2009). It is speculated that miRs altogether regulate around 30% of the human genome, which highlights their potential importance as global regulators of gene expression (Santarpia, Nicoloso et al. 2010). MiRs regulate such major processes as development, apoptosis, cell proliferation, immune response, and hematopoiesis (Croce and Calin 2005); they also may act as tumor suppressor genes and oncogenes (Esquela-Kerscher and Slack 2006; Zhang, Pan et al. 2007). Mature miRs target and inhibit translation or promote mRNA degradation by annealing to complementary sequences in mRNA 3’untranslated regions (UTRs). Watson-Crick complementarity between the target and the “seed” region comprising 2-8 nucleotides of the mature miR is both necessary and sufficient for targeting and regulation of mRNAs by miRs. The sequence of this “seed” region is the basis of most genome-wide predictions of miR binding sites within miR-regulated genes (Nielsen, Shomron et al. 2007). A single nucleotide polymorphism (SNP) located in the crucial “seed” sequence affects its complementarity to potential target genes determining the functionality of a miR (Jazdzewski, Liyanarachchi et al. 2009). Individual miRNAs typically target dozens of mRNAs, often encoding proteins with related functions (Santarpia, Nicoloso et al. 2010). Therefore, although their inhibitory effects on individual mRNAs are generally modest, their combined effects on multiple mRNAs can induce strong biological responses (Lim, Lau et al. 2005; Negrini, Nicoloso et al. 2009; Volinia, Galasso et al. 2010). As they were discovered only relatively recently, the study of miRs is a young and rapidly changing field in which many genes remain to be discovered and the sequence modifications are not well understood.

For further clarification of roles of miRs in tumorigenesis it is crucial to generate more complete lists of miRs variations and expression changes of miRs isoforms in cancer tissues. In thyroid cancer we surveyed the whole human transcriptome of small RNAs, including miRs, using next-generation deep-sequencing technology. Application of this approach to RNA from paired normal-tumor thyroid tissue led to revealing the sequences and expression levels of all known miRs and discovering previously unidentified miR genes. This method allows identifying all germline and somatic variability in pre- and mature miRs sequences. We believe that the findings will create an opportunity for new approaches to the diagnosis and treatment not only thyroid carcinoma but also other types of cancers.

 

Hypothalamic miRNAs suppress obesity in mice

 

Witold Konopka

 

Molecular Biology of the Cell I, German Cancer Research Center (DKFZ) Heidelberg, Germany

 

Nowadays, obesity problem (BMI ≥ 30 kg/m²) affects more than 10% of people all over the world with a special increase in the number of cases in developed countries. Control of energy homeostasis is performed by both peripheral tissues and the brain. In the latter, especially important regions for metabolism are located in the hypothalamus. With the advantage of a inducible Cre/loxP system we have deleted Dicer1 gene from forebrain neurons (including hypothalamic nuclei) in the adult mice. This resulted in the disruption of processing of a mature form of miRNAs in the brain of Dicer1^CaMKCreERT2 mice, which revealed unexpected phenotype. Mutant mice start to gain weight three weeks after induction of the mutation with tamoxifen administration. Within the next 3 weeks mutant mice achieve 70-100% increase of initial body weight, the phenomenon observed for both genders. This was followed by an increase in food and water consumption during a period of weight gain. In Dicer1^{CaMKII-CreERT2} mice we have observed increased level of pS6 protein in many nuclei of the hypothalamus, including POMC neurons, already 3 weeks after induction of the mutation. This indicates that over-activation of insulin-mTOR signaling, due to loss of miRNAs in those neurons, may be responsible for the observed phenotype. Additionally,  rapamycin (inhibitor of mTOR protein) treatment of mutant mice attenuated the obesity phenotype in terms of both weight gain and increased food intake. Moreover, the preliminary analysis of another mutant mouse line Pten ^{CaMKII-CreERT2} (that has up-regulaed insulin pathway in the same neurons) showed a phenotype very similar to the Dicer mutants. All the above data raise an intriguing possibility that miRNAs may be specifically involved in the central nervous system regulation of body weight in humans, which may have implications for treatment of the obesity syndrome.

ROQUIN PROMOTES RAPID TNFa mRNA DEGENERATION via a STEM-LOOP RECOGNITION ELEMENT

 

Kathrin Leppek, Georg Stöcklin

 

Junior Research Group Posttranscriptional Control of Gene Expression, German Cancer Research Center (DKFZ) and the Center for Molecular Biology of Heidelberg University (ZMBH), 69120 Heidelberg, Im Neuenheimer Feld 280 and Im Neuenheimer Feld 282, Germany

 

Tumor necrosis factor alpha (TNFa) is the most potent pro-inflammatory cytokine of the mammalian organism. Numerous posttranscriptional mechanisms participate in controlling the expression of this potentially harmful cytokine, including a constitutive decay element (CDE). The CDE is located in the 3’UTR of the TNFa transcript and mediates rapid mRNA degradation independently of the well characterized AU-rich element. We now mapped the CDE to a 15 nt long sequence and provide evidence that it does not serve as a microRNA binding site. Rather, we found that in its active conformation, the CDE folds into a stem-loop. Inline probing was used to solve the secondary structure of the CDE, and compensatory mutations allowed confirmation of our structure model.

In order to identify CDE-binding effector proteins, we optimized a protocol for RNP affinity purification by developing S1m, an improved streptavidin-binding RNA aptamer. Cellular extracts were applied to CDE-S1m and control-S1m RNA samples followed by RNP purification. Mass spectrometry was then used to identify proteins associated with the CDE. Thereby, we found the CCCH-type zinc and RING finger protein Roquin (Rc3h1) to be a CDE-binding protein. Roquin is known to destabilize the inducible costimulator (ICOS) mRNA via a 3’UTR interaction. Using RNA-IP, we provide evidence that Roquin specifically binds to CDE-containing mRNAs. Importantly, overexpression of Roquin enhances the deadenylation of a CDE-reporter mRNA and promotes its degradation, whereas Roquin-knockdown abolishes CDE-mRNA decay. In conclusion, we solved the secondary structure of the CDE and identified Roquin as a CDE-binding protein that accelerates TNFa mRNA decay.

 

MICRO-RNA AND OXIDATIVE DAMAGE

 

Joanna Rzeszowska, Roman Jaksik, Artur Cieślar-Pobuda, Magdalena Skonieczna,
Anna Lalik, Sebastian Student

 

Biosystems Group, Faculty of Automatic Control, Electronics and Informatics, Silesian Univerisity of Technology, Akademicka 16, 44-100 Gliwice

 

We examined transcript profiles changes in a few human cell lines at different time points after exposure of cells to ionizing radiation. A short time after treatment, both increase and decrease of transcript levels resulted from changes in stability of some mRNAs. Because binding of miRNAs and proteins to mRNAs are important factors in regulating mRNA stability, we explored if this up- or down-regulation is correlated with the presence of sequence motifs which bind miRNAs and proteins. In all cell lines, more transcripts were up- than down-regulated one hour after irradiation. The up-regulated transcripts contained significantly more (p<10^-10) target motifs for miRNAs and also, in three cell lines, for protein-binding AU-rich motifs in their 3' untranslated region compared with those down-regulated or unchanged.  At the same time, an increase in 8-oxo-7,8-dihydro-guanosine (8-oxoG)  in RNA  and changes of reactive oxygen species (ROS) level in irradiated cells were observed, suggesting that oxidative damage introduced into RNA by direct irradiation or by signals released by irradiated cells can modulate the specific interactions between RNA and miRNA or mRNA- binding proteins and cause a change in mRNA stability.

 

 

 

Session III:

Response to radiation:
 low doses, bystander effects

Emerging issues in radiobiology – the impact of non-targeted effects

 

Carmel Mothersill

 

McMaster University, Hamilton, Ontario, Canada

Email: mothers@mcmaster.ca

 

Introduction: Since the acceptance that non-targeted effects  (NTE) can be measured in unirradiated cells or distant progeny of irradiated cells, the discussion has developed about the relevance of these effects for radiobiology and radiation protection since they increase the complexity of the radiation response and allow for outcomes which are not as predictable as they were under the “old rules”.

Methods: Specific examples will be presented and analysed which challenge accepted paradigms.

Results: Data show that bystander mechanisms are either on or off in cells and that the “on” threshold appears to be at a very low dose (mGy range). Data suggest that adaptive responses are induced not only in neighbouring cells but in organisms which receive bystander signals. Data show that chronic exposures to alpha or gamma irradiation lead to complex responses in organisms which can be adaptive and protective. Evidence suggests that mixed contaminant exposures which include radiation can have sub-additive or synergistic effects.

Conclusions: A key consequence of findings in NTE biology is that at any given level of organization, from gene to ecosystem – communication of stress signals and heritability of stress adaptations provide the bridges linking one hierarchical level to the next and enable the rapid propagation of change triggered by stress at one level, resulting in change at a higher (or lower?) level. Evolution could thus be regulated through communicated signals between cells, individuals, and populations which control and optimize responses coordinating the emergence of exquisitely tuned systems which can adapt rapidly to micro or macro environmental change.

Antineoplastic effects of low-level exposures to ionizing radiation

 

Marek K. Janiak

 

Military Institute of Hygiene & Epidemiology, Warsaw

 

According to the linear, no threshold (LNT) hypothesis any radiation dose, no matter how small, is presumed to cause cancer in at least one person in a large population exposed to ionizing radiation (IR). In other words, there is no threshold dose below which radiation exposure can be considered innocuous and harm is the focus of the prevailing paradigm of the cancer risk assessment. This notion is consistent with the general view of IR as a universal, even though a week carcinogen, in which no radiation benefit is usually considered.

However, results of numerous epidemiological and experimental studies from the last twenty or so years have repeatedly demonstrated that exposures to a low-dose or low-dose rate IR are not only associated with increased risk of cancer, but the reverse is often true, that is low-level exposures protect against the development of malignancy. Evidence form such studies, which invalidate the LNT model and support the hormetic response of people and laboratory animals to low-level IR, will be presented and discussed taking into account the general limitations of the epidemiological estimations of cancer risk at low-level exposures. Also, possible mechanisms of the beneficiary effects of such irradiations will be dealt with.

 

Spatial and stochastic effects in models of cell interaction

 

Marek Kimmel

 

Department of Statistics, Rice University, Houston, TX, USA

 

 

 

One of the more spectacular mathematical inventions of Alan Turing was that of diffusion driven instabilities (DDI). In brief, suppose that a stable system of ordinary differential equations (the reaction kinetics equations) is parametrized by spatial location and at least one of the unknown functions is allowed to diffuse in space (the reaction-diffusion equations). It may happen that as a result, the system loses stability around the space-homogeneous solutions and displays spatially distributed patterns, stable or not. Many examples of pattern-forming realistic systems have been found, and some of them serve as explanations of natural and man-made phenomena, such as segmentation in organisms, scroll waves in chemical reactions, cloud arrangements, and other. 

However, many systems in physics and particularly in biology are, at a smaller scale, not continuous, but composed of a large number of discrete units, some of which execute random walks in space. Summing up the number of particles present in each of the grid locations at any given time, we obtain a discrete stochastic process. Will the behaviour of such system be approximated by the continuous and deterministic reaction-diffusion system? The answer is not straightforward, but we will try to find some clues. If the qualitative behavior of the deterministic-continuous description of the spatial systems is different than that of the stochastic-discrete one, this could have an impact on the interpretation of the already existing reaction-diffusion models and on the future development of modelling theory and practice.

We will present three biological models, in which spatial aspects play a major role: (1) Early carcinogenesis, with growth factors affecting creation of primary malignant foci. (2) Spread of viral infection and interferon defences. (3) A model of paracrine regulation, being a metaphore of the bystander effect in irradiated cells. Mathematical and computational analysis of these systems indicates that interactions between spatial and stochastic effects may invalidate some naïve spatial models.

 Bystander effect modeling using evolutionary games

 

Andrzej Świerniak

 

 Institute of Automatic Control, Silesian University of Technology, Akademicka 16 Street,
44-100 Gliwice, Poland

 

We present an application of evolutionary game theory to modelling carcinogenesis processes. The studied phenomenon is a radiation-induced bystander effect; the game theoretic model which we have proposed may be viewed as a follower of the angiogenic model considered by Bach et al.  We consider three different strategies/phenotypes of cells: escape to apoptosis, production of growth and mutation factors, and neutrality.

The proposed payoff table of fitness, related to environment adaptation and genetical cell behaviour, contains costs/profits of bystander effect, choice of apoptotic pathway, producing growth factors and resistance against bystander effect.

We consider also a game theory model including spatial cells allocation (the game is played on lattice). We discuss different polymorphic equilibrium points dependent on model

parameters, types of spatial games and players distribution.

 

Key words: Bystander effect, biomathematical modelling, evolutionary games, spatial evolutionary games, cellular automata, replicator dymamics, irradiation

INVESTIGATING THE GENEALOGICAL RELATIONSHIP AMONG  sLAVS, BALTS AND FINNS USING DEMOGRAPHIC NETWORK MODEL

 

Tomasz Wojdyła¹, Marek Kimmel^1,2

 

¹Silesian University of Technology, Poland;  ² Rice University, USA

 

           Demographic network is defined as a set of populations evolving from a single ancestral population with a beginning at the time 0. The structure of the network is described by two types of events: split of a single population into two populations and merger of two populations. Additionally, we incorporate migration between populations coexisting in the model. Our forward-time and time-continuous model allows to calculate the exact values of the entries of the matrices R_ij(t) being the joint distributions of pairs of alleles sampled at the time t from populations i (first allele from a pair) and j (second allele). We assume that individuals in each population in the network are described by the same allelic space model and we introduce mutation to the model using intensity matrices Q_i of the Markov chain of the mutation process in population i. Mutation model is assumed unchanged between two adjacent demographic events. Population size growth can be specified for each population. Evolution of the joint distributions between network events is described by the Lyapunov differential equations.

           In our work we apply the model to three Eastern European populations: Slavic, Baltic and Finnish. We investigate the values of several parameters (such as the time of split of the Finns from other Indo-European tribes or the Balt-Slav migration rate) and their impact on the genetic distance between populations. We compare our results to the estimates of the Slatkin's R_ST distance obtained from the genetic data. We consider nine Y-STR loci and the genetic data from 1216 unrelated male individuals (919 of them come from six geographical regions of Poland and 297 are Balts' descendants from Riga and Vilnius).

 

PROTECTIVE BYSTANDER EFFECT

 

Maria Wideł

 

Institute of Automatic Control, Silesian University of Technology, Akademicka 16 Street,
44-100 Gliwice, Poland

 

The radiation-induced bystander effect has been well documented in a variety of biological systems in vitro and in vivo. Available experimental data show that irradiated cells are capable of providing signals to the neighboring non-irradiated cells through intercellular gap junctions or via culture medium resulting in some change to neighboring cells, often similar to changes in directly targeted cells. Bystander responses include damage-inducible stress responses such as sister chromatid exchanges, decrease of clonogenic survival, increase of micronuclei and apoptosis, double strand breaks, changes in gene expression, and others.

Although the most radiation-induced bystander effects have been shown to be associated with cellular damage, there is evidence for protective cell signaling involved in the bystander effects. These protective effects disclose as: significant increase in proliferation of bystander cells after treatment with medium harvested from irradiated cells or after co-incubation with radiation exposed cells, an adaptive response, where bystander cells that are subsequently irradiated become more radioresistant than cells not exposed to bystander signals and as induction of terminal differentiation with loss of proliferative potential.

Recently, new data including our own, have indicated that not only radiation targeted cells influence the status of bystander cells but inversely, bystander cells can also modify response of targeted cells to radiation, indicating that mutual signaling between both types of cells exists. We observed in co-culture system of irradiated cancer cells with non irradiated fibroblasts that damage induced in cancer cells expressed as micronuclei and apoptosis was significantly diminished due to co-incubation with fibroblasts. This observation was true for murine carcinoma (LLC) cells co-cultured with murine NIH3T3 fibroblasts and also for human malignant melanoma Me45 irradiated cells co-incubated with normal human dermal fibroblasts (NHDF). Such a radioprotection was not observed in the case of irradiated Me45 cells co-cultured with cells of the same melanoma line. Looking for the explanation of the mechanism(s) underlying this phenomenon, we measured intracellular level of ROS induced in irradiated and bystander cells and we observed that diminution of micronuclei and apoptosis in radiation-targeted melanoma cells is mediated by, up today undefined, “rescue signals”, which lessen intracellular level of ROS. Similar effect is likely to occur during cancer radiotherapy, and cause some protective effect to cancer cells owing to fibroblasts present in tumor tissue. In contrast, non irradiated melanoma cells co-incubated with irradiated NHDF, did not ameliorate damage in radiation-hit fibroblasts.

In conclusion, our data indicate that in addition to targeted effects which induce damage to directly irradiated cells, some interplay between adjacent irradiated and non-irradiated cell populations may modulate the direct radiation effect and may contribute to overall outcome after radiation exposures.

 

Supported by the grant No N N518 497 639 from Polish Ministry of Science and Higher Education.

Radiation-related changes in serum proteome profiles detected by mass spectrometry in blood of patients treated with radiotherapy due to larynx cancer

 

P. Widłak¹,  M. Pietrowska¹, K. Wojtkiewicz², T. Rutkowski¹, A. Wygoda¹, K. Składowski¹,
 Ł. Marczak³, M. Marczyk², J. Polańska², A. Polański²

 

¹Maria Sklodowska - Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland; ²Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland; ³Institute of Bioorganic Chemistry, Polish Academy of Science, Poznań, Poland

 

The study was aimed to detect features of human serum proteome that were associated with exposure to ionizing radiation. The analyzed group consisted of 46 patients treated with radical radiotherapy because of larynx cancer; patients were irradiated with total doses lying in 51-72 Gy range. Three consecutive blood samples were collected from each patient: before the start, 2 weeks after the start, and 1-2 months after the end of radiotherapy. The low-molecular-weight fraction of the serum proteome (2000-13000 Da) was analyzed by the MALDI-ToF mass spectrometry. Proteome profiles of serum samples collected before the start of radiotherapy and during early stage of the treatment were similar. In marked contrast, mass profiles of serum samples collected several weeks after the end of the treatment revealed clear changes. We found that 41 out of 312 registered peptide ions changed their abundance significantly when serum samples collected after the final irradiation were compared with samples collected at two earlier time points. We also found that abundances of certain serum peptides were associated with total doses of radiation received by patients. The results of this pilot study indicate that features of serum proteome analyzed by mass spectrometry have potential applicability as a retrospective marker of exposure to ionizing radiation.

 

The Ego, The Id, and Radiotherapy

 

Colin Seymour

 

McMaster University, Canada

 

Tumours can be successfully treated by radiation in animals, but results in humans may not be as consistent. This may be because of psycho-social complexity.

Humans are largely thought to be distinct within animal species because of their ability to think and reason. In Freud’s topography, this reasoning part of the brain struggles to control the unconscious (the Id), which is full of rage and drives, including the Death Instinct.

Many bodily functions are controlled by the autonomic nervous system, but are not exclusively controlled, and there is growing evidence that the immune system (for example) can be modulated by lifestyle.

One of the components of lifestyle is group support, as are lifestyle choices such as diet and exercise.

Cancer is a chronic disease often treated by radiotherapy, and the standard treatment regime has the patient meeting his or her treatment team many times a week. In effect, a treatment group is set up, with the patient as a member. It is proposed that if the patient feels part of this group that treatment results can be modified. Whether the patient feels part of the group will depend in part on the interactions of personalities within the groups, and it will be argued that “caring” and “empathy” are more important in those groups who regularly interact with the patients. Talking and listening to the patient then become as important as the physical treatment, which might conflict with the optimum machine utilization often demanded in modern radiotherapy departments.

A corollary is that animal models for treatment will never give the complete picture, as they lack the psycho-social complexity that allows for human specific adaptive responses to the disease treatment.