Tyrolean Cancer Research Institute

Innsbruck, Austria

Tyrolean Cancer Research Institute

Innsbruck, Austria
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Obexer P.,Innsbruck Medical University | Obexer P.,Tyrolean Cancer Research Institute | Ausserlechner M.,Tyrolean Cancer Research Institute | Ausserlechner M.,Innsbruck Medical University
Frontiers in Oncology | Year: 2014

Defects in apoptosis regulation are one main cause of cancer development and may result from overexpression of anti-apoptotic proteins such as Inhibitor of Apoptosis Proteins (IAPs). IAPs are cell death regulators that, among other functions, bind caspases and interfere with apoptotic signaling via death receptors or intrinsic cell death pathways. All IAPs share one to three common structures, the so called baculovirus-IAP-repeat (BIR)-domains that allow them to bind caspases and other proteins. X-linked inhibitor of apoptosis protein (XIAP) is the most potent and best-defined anti-apoptotic IAP-family member that directly neutralizes caspase-9 via its BIR3 domain and the effector caspases-3 and -7 via its BIR2 domain. A natural inhibitor of XIAP is SMAC/Diablo which is released from mitochondria in apoptotic cells and displaces bound caspases from the BIR2/BIR3 domains of XIAP thereby reactivating cell death execution. The central apoptosis-inhibitory function of XIAP and its overexpression in many different types of advanced cancers have led to significant efforts to identify therapeutics that neutralize its anti-apoptotic effect. Most of these drugs are chemical derivatives of the N-terminal part of SMAC/Diablo. These "SMAC mimetics" either specifically induce apoptosis in cancer cells or act as drug-sensitizers. Several "SMAC mimetics" are currently tested by the pharmaceutical industry in Phase I and Phase II trials. In this review we will discuss recent advances in understanding the function of IAPs in normal and malignant cells and focus on approaches to specifically neutralize XIAP in cancer cells © 2014 Obexer and Ausserlechner.

Mikolcevic P.,Innsbruck Medical University | Mikolcevic P.,Barcelona Institute for Research in Biomedicine | Rainer J.,Innsbruck Medical University | Rainer J.,Tyrolean Cancer Research Institute | Geley S.,Innsbruck Medical University
Cell Cycle | Year: 2012

PCTAIRE kinases (PCTK) are a highly conserved, but poorly characterized, subgroup of cyclin-dependent kinases (CDK). They are characterized by a conserved catalytic domain flanked by N- and C-terminal extensions that are involved in cyclin binding. Vertebrate genomes contain three highly similar PCTAIRE kinases (PCTK1,2,3, a.k.a., CDK16,17,18), which are most abundant in post-mitotic cells in brain and testis. Consistent with this restricted expression pattern, PCTK1 (CDK16) has recently been shown to be essential for spermatogenesis. PCTAIRE s are activated by cyclin Y (CCNY), a highly conserved single cyclin fold protein. By binding to N-myristoylated CCNY, CDK16 is targeted to the plasma membrane. Unlike conventional cyclin-CDK interactions, binding of CCNY to CDK16 not only requires the catalytic domain, but also domains within the N-terminal extension. Interestingly, phosphorylation within this domain blocks CCNY binding, providing a novel means of cyclin-CDK regulation. By using these functional characteristics, we analyzed "PCTAIRE" sequence containing protein kinase genes in genomes of various organisms and found that CCNY and CCNY-dependent kinases are restricted to eumetazoa and possibly evolved along with development of a central nervous system. Here, we focus on the structure and regulation of PCTAIRE s and discuss their established functions. ©2012 Landes Bioscience.

Hagenbuchner J.,Tyrolean Cancer Research Institute | Kuznetsov A.,Cardiac Research Laboratory | Hausott B.,Innsbruck Medical University | Obexer P.,Tyrolean Cancer Research Institute | Ausserlechner M.J.,Tyrolean Cancer Research Institute
Journal of Cell Science | Year: 2012

FOXO transcription factors induce apoptosis and regulate cellular production of reactive oxygen species (ROS). To identify the sequence of molecular events underlying FOXO3 (FKHRL1)-induced apoptosis, we studied the regulation and function of FOXO3 by expressing an ECFP-tagged FOXO3 or a 4OH-tamoxifen (4OHT)-inducible FOXO3-ERtm fusion protein in SH-EP and STA-NB15 neuronal cells. After knockdown of FOXO3 or expression of a dominant-negative FOXO3 mutant we observed that etoposide- and doxorubicin-induced elevation of cellular ROS depends on FOXO3 activation and induction of its transcriptional target BCL2L11 (Bim). Activation of FOXO3 on its own induced two sequential ROS waves as measured by reduced MitoTrackerRed in live cell microscopy. Induction of Bim by FOXO3 is essential for this phenomenon because Bim knockdown or ectopic expression of BCL2L1 (BclxL) prevented FOXO3-mediated overproduction of ROS and apoptosis. Tetracycline-controlled expression of Bim impaired mitochondrial respiration and caused ROS production, suggesting that FOXO3 induces uncoupling of mitochondrial respiration through Bim. FOXO3 also activated a ROS rescue pathway by inducing the peroxiredoxin SESN3 (Sestrin3), which is responsible for the biphasic ROS accumulation. Knockdown of SESN3 caused an increase of FOXO3-induced ROS and accelerated apoptosis. The combined data clearly demonstrate that FOXO3 activates overproduction of ROS as a consequence of Bim-dependent impairment of mitochondrial respiration in neuronal cells, which leads to apoptosis. © 2012.

Martowicz A.,Tyrolean Cancer Research Institute | Spizzo G.,Tyrolean Cancer Research Institute | Spizzo G.,Day Hospital of Haematology and Oncology | Gastl G.,Tyrolean Cancer Research Institute | And 2 more authors.
BMC Cancer | Year: 2012

Background: The epithelial cell adhesion molecule (EpCAM) has been shown to be overexpressed in breast cancer and stem cells and has emerged as an attractive target for immunotherapy of breast cancer patients. This study analyzes the effects of EpCAM on breast cancer cell lines with epithelial or mesenchymal phenotype.Methods: For this purpose, shRNA-mediated knockdown of EpCAM gene expression was performed in EpCAMhigh breast cancer cell lines with epithelial phenotype (MCF-7, T47D and SkBR3). Moreover, EpCAMlow breast carcinoma cell lines with mesenchymal phenotype (MDA-MB-231, Hs578t) and inducible overexpression of EpCAM were used to study effects on proliferation, migration and in vivo growth.Results: In comparison to non-specific silencing controls (n/s-crtl) knockdown of EpCAM (E#2) in EpCAMhigh cell lines resulted in reduced cell proliferation under serum-reduced culture conditions. Moreover, DNA synthesis under 3D culture conditions in collagen was significantly reduced. Xenografts of MCF-7 and T47D cells with knockdown of EpCAM formed smaller tumors that were less invasive. EpCAMlow cell lines with tetracycline-inducible overexpression of EpCAM showed no increased cell proliferation or migration under serum-reduced growth conditions. MDA-MB-231 xenografts with EpCAM overexpression showed reduced invasion into host tissue and more infiltrates of chicken granulocytes.Conclusions: The role of EpCAM in breast cancer strongly depends on the epithelial or mesenchymal phenotype of tumor cells. Cancer cells with epithelial phenotype need EpCAM as a growth- and invasion-promoting factor, whereas tumor cells with a mesenchymal phenotype are independent of EpCAM in invasion processes and tumor progression. These findings might have clinical implications for EpCAM-based targeting strategies in patients with invasive breast cancer. © 2012 Martowicz et al.; licensee BioMed Central Ltd.

Tuzlak S.,Innsbruck Medical University | Kaufmann T.,University of Bern | Villunger A.,Innsbruck Medical University | Villunger A.,Tyrolean Cancer Research Institute
Genes and Development | Year: 2016

“Programmed cell death or ‘apoptosis’ is critical for organogenesis during embryonic development and tissue homeostasis in the adult. Its deregulation can contribute to a broad range of human pathologies, including neurodegeneration, cancer, or autoimmunity…” These or similar phrases have become generic opening statements in many reviews and textbooks describing the physiological relevance of apoptotic cell death. However, while the role in disease has been documented beyond doubt, facilitating innovative drug discovery, we wonder whether the former is really true. What goes wrong in vertebrate development or in adult tissue when the main route to apoptotic cell death, controlled by the BCL2 family, is impaired? Such scenarios have been mimicked by deletion of one or more prodeath genes within the BCL2 family, and gene targeting studies in mice exploring the consequences have been manifold. Many of these studies were geared toward understanding the role of BCL2 family proteins and mitochondrial apoptosis in disease, whereas fewer focused in detail on their role during normal development or tissue homeostasis, perhaps also due to an irritating lack of phenotype. Looking at these studies, the relevance of classical programmed cell death by apoptosis for development appears rather limited. Together, these many studies suggest either highly selective and context-dependent contributions of mitochondrial apoptosis or significant redundancy with alternative cell death mechanisms, as summarized and discussed here. © 2016 Tuzlak et al.

Martowicz A.,Tyrolean Cancer Research Institute | Rainer J.,Innsbruck Medical University | Lelong J.,Innsbruck Medical University | Spizzo G.,Tyrolean Cancer Research Institute | And 3 more authors.
Molecular Cancer | Year: 2013

Introduction: The Epithelial Cell Adhesion Molecule (EpCAM) has been shown to be strongly expressed in human breast cancer and cancer stem cells and its overexpression has been supposed to support tumor progression and metastasis. However, effects of EpCAM overexpression on normal breast epithelial cells have never been studied before. Therefore, we analyzed effects of transient adenoviral overexpression of EpCAM on proliferation, migration and differentiation of primary human mammary epithelial cells (HMECs).Methods: HMECs were transfected by an adenoviral system for transient overexpression of EpCAM. Thereafter, changes in cell proliferation and migration were studied using a real time measurement system. Target gene expression was evaluated by transcriptome analysis in proliferating and polarized HMEC cultures. A Chicken Chorioallantoic Membrane (CAM) xenograft model was used to study effects on in vivo growth of HMECs.Results: EpCAM overexpression in HMECs did not significantly alter gene expression profile of proliferating or growth arrested cells. Proliferating HMECs displayed predominantly glycosylated EpCAM isoforms and were inhibited in cell proliferation and migration by upregulation of p27KIP1 and p53. HMECs with overexpression of EpCAM showed a down regulation of E-cadherin. Moreover, cells were more resistant to TGF-β1 induced growth arrest and maintained longer capacities to proliferate in vitro. EpCAM overexpressing HMECs xenografts in chicken embryos showed hyperplastic growth, lack of lumen formation and increased infiltrates of the chicken leukocytes.Conclusions: EpCAM revealed oncogenic features in normal human breast cells by inducing resistance to TGF-β1-mediated growth arrest and supporting a cell phenotype with longer proliferative capacities in vitro. EpCAM overexpression resulted in hyperplastic growth in vivo. Thus, we suggest that EpCAM acts as a prosurvival factor counteracting terminal differentiation processes in normal mammary glands. © 2013 Martowicz et al.; licensee BioMed Central Ltd.

Neumann B.,Leibniz Institute for Primate Research | Klippert A.,Leibniz Institute for Primate Research | Raue K.,Leibniz Institute for Primate Research | Raue K.,University of Veterinary Medicine Hannover | And 4 more authors.
Journal of Leukocyte Biology | Year: 2015

B cells, as an important part of the humoral immune response, are generated in the BM, migrate to secondary lymphoid organs, and upon activation, differentiate into antibody-producing memory B cells or plasma cells. Despite the pivotal roles that they play in different diseases, a comprehensive characterization in healthy rhesus macaques, which serve as valuable models for a variety of human diseases, is still missing. With the use of multiparameter flow cytometry, we analyzed B cells in BM collected from two locations, i.e., the iliac crest (BMca) and the femur (BMfem), PB, as well as second-ary lymphoid organs of healthy rhesus macaques. We assessed the frequencies of immature and mature B cells, as well as CD19+ CD20- CD38+/++ CD138+/++ Plasmablasts/plasma cells. Furthermore, we found site-specific differences in the expression of markers for B cell activation and proliferation, chemokine receptors and Igs, as well as the distribution of memory B cell subpopulations. As secondary lymphoid organs harbor the highest frequencies of naive B cells, expression of CD80, CD95, and Ki67 was lower compared with B cells in the periphery and BM, whereas expression of IgD, CXCR4 (CD184), and CCR7 (CD197) was higher. Interestingly, BMca differed from BMfem regarding frequencies of b cells, their expression of CD80 and CXCR4, T cells, and plasma cells. In summary, these data identify baseline values for the above-mentioned parameters and provide the foundation for future studies on B arid plasma cells in different diseases. © Society for Leukocyte Biology.

Hagenbuchner J.,Innsbruck Medical University | Hagenbuchner J.,Tyrolean Cancer Research Institute | Kuznetsov A.V.,Innsbruck Medical University | Obexer P.,Innsbruck Medical University | And 3 more authors.
Oncogene | Year: 2013

Gain of chromosome 17q correlates with high-stage disease, an adverse clinical outcome and leads to the overexpression of the antiapoptotic protein BIRC5/Survivin in neuroblastoma (NB). We have shown before that Survivin defines a threshold for the sensitivity of NB cells to DNA-damaging chemotherapeutic agents that require FOXO3 activation for apoptosis induction. To investigate the molecular basis of apoptosis inhibition we analyzed the function of Survivin at mitochondria and uncovered that Survivin induces mitochondrial fragmentation, reduces mitochondrial respiration and represses BCL2L11/Bim. Mitochondrial fission depends on Survivin-induced recruitment of the fission regulator DNM1L/Drp1 to mitochondria. In parallel, Survivin expression inhibits the respiratory complex-I, thereby preventing reactive oxygen species accumulation and, as a consequence, FOXO3-induced apoptosis. The loss of energy production via oxidative phosphorylation is compensated by increased glycolysis in Survivin-overexpressing NB tumor cells. Glycolysis inhibitors neutralize the antiapoptotic effect of Survivin and sensitize high-stage NB to DNA-damaging drugs. This suggests that glycolysis inhibitors target an 'archilles heel' of Survivin-overexpressing NB and may be highly useful as chemosensitizers in the treatment of high-stage NB. © 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13.

Hagenbuchner J.,Innsbruck Medical University | Hagenbuchner J.,Tyrolean Cancer Research Institute | Ausserlechner M.J.,Tyrolean Cancer Research Institute | Ausserlechner M.J.,Innsbruck Medical University
Frontiers in Physiology | Year: 2013

Forkhead box O (FOXO) transcription factors are regulators of cell-type specific apoptosis and cell cycle arrest but also control longevity and reactive oxygen species (ROS). ROS-control by FOXO ismediated by transcriptional activation of detoxifying enzymes such as Superoxide dismutase 2 (SOD2), Catalase or Sestrins or by the repression of mitochondrial respiratory chain proteins resulting in reduced mitochondrial activity. FOXO3 also regulates the adaptation to hypoxia by reducing mitochondrial mass and oxygen consumption during HIF-1a activation. In neuronal tumor cells, FOXO3 triggers ROS-accumulation as a consequence of transient mitochondrial outer membrane permeabilization, which is essential for FOXO3-induced apoptosis in these cells. Cellular ROS levels are affected by the FOXO-targets Bim, BclxL, and Survivin. All three proteins localize to mitochondria and affect mitochondrial membrane potential, respiration and cellular ROS levels. Bim-activation by FOXO3 causes mitochondrial depolarization resulting in a transitory decrease of respiration and ROS production. Survivin, on the other hand, actively changes mitochondrial architecture, respiration-efficacy and energy metabolism. This ability distinguishes Survivin from other anti-apoptotic proteins such as BclxL, which inhibits ROS by inactivating Bim but does not alter mitochondrial function. Importantly, FOXO3 simultaneously also activates ROS-detoxification via induction of SESN3. In this paper we discuss the hypothesis thatthe delicate balance between ROS-accumulation by Bim-triggered mitochondrial damage, mitochondrial architecture and ROS-detoxifying proteins determines cell fate. We provide evidence for a FOXO self-reactivating loop and for novel functions of FOXO3 in controlling mitochondrial respiration of neuronal cells, which further supports the current view that FOXO transcription factors are information-integrating sentinels of cellular stress and critical modulators of cell homeostasis. © 2013 Hagenbuchner and Ausserlechner.

Saez A.J.G.,University of Tübingen | Villunger A.,Innsbruck Medical University | Villunger A.,Tyrolean Cancer Research Institute
Genes and Development | Year: 2016

The minimum requirement for mitochondrial apoptosis has been controversial ever since the discovery of BCL-2 as a cell death regulator. In this issue of Genes & Development, O’Neill and colleagues (pp. 973–988) end a long-standing debate by creating a cellular system free of BCL-2 family proteins, thereby identifying the outer mitochondrial membrane rather than BH3-only proteins as the only requirement for BAX/BAK activation and mitochondrial outer membrane permeabilization (MOMP). © 2016 Sáez and Villunger.

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