AG Daniel

Clinical and Molecular Oncology

Forschungsschwerpunkt

Cell death and cell cycle deregulation in cancer and resistance to anticancer therapy

Virtually all medical anticancer therapies rely on the induction of cell cycle arrest or cell death in the malignant cells. Consequently, the analysis of such genetic events allows for the identification of patients at risk for an insufficient response to treatment with chemotherapeutic drugs, ionising irradiation or targeted cancer drugs.

Such analyses provide a rational basis for a molecular understanding of the response to anticancer therapies and the clinical use of cancer therapeutics. The aim of the group is, therefore, to define genetic defects in cancer that result in aggressive disease, poor prognosis, and resistance to clinical cancer therapy.

To this end, we have established an extensive genotyping program in solid tumors and leukemias. Recent pharmacogenomic data obtained from these screenings depict that defects in central regulatory genes, e.g. of the p53 pathway, do not result in global resistance to therapy but may be overcome by adequate therapeutic modalities.

Functional consequences of such cell death and cell cycle defects are analysed in vitro, often by the use of adenoviral gene transfer for complementation of disrupted genes. In addition, these systems are exploited to gain insights into novel aspects of cell cycle and cell death regulation and their intricate interactions.

Findings are translated into clinically applied innovative tumor genetic and molecular diagnostic applications in leukemias and lymphomas, myeloid neoplasias and selected solid tumors including gastrointestinal carcinomas, sof tissue sarcomas, renal and prostate cancer.

 

Understanding resistance to anticancer therapy

Anticancer therapies, i.e. chemotherapy and ionising irradiation, activate nuclear stress responses to induce cell cycle arrest and DNA repair. When repair fails, the same stress responses trigger cellular senescence or death and demise of the affected cell. The molecular basis of these events has been studied extensively during recent years and comprehensive models are now established for large parts of these signaling events. We have investigated the consequences of genetic defects in genes acting as effectors or inducers of p53 that trigger apoptosis and cell cycle arrest programs upon genotoxic stress. In this context, we recently described selective loss of multiple BH3-only proteins, pro-apoptotic homologs of the Bcl-2 family, including Nbk and Bim in renal carcinoma. This is a unifying feature of renal carcinoma and appears to be linked to the impressive clinical resistance of this tumor entity to anticancer therapy.

<b>Abbildung 1: Function of BH3-only proteins as death sensors </b>
A: BH3-only proteins act as functional interface between death signals and the mitochondrial apoptosis pathway. Anti-apoptotic Bcl-2 proteins put an at least dual layer of protection on activation of Bax/Bak that redistribute upon activation to form pores in the outer mitochondrial membrane for the release of pro-apoptotic factors such as cytochrome c. B: Binding of a BH3-domain to Bcl-xL: Bcl-2 Homology (BH) domains 1 (yellow), BH2 (red) and BH3 (green) of Bcl-xL form a cleft that binds a-helical BH3 domains (violet, BH3 domain of Bak). BH3-only proteins displace Bax/Bak from binding to e.g. Bcl-xL or Mcl-1. D: Conditional adenoviral expression of Nbk induces redistribution of Bax (EGFP, green) to mitochondria (TOM20, red) and a punctuate formation of Bax clusters due to oligomerization. Blue colour: DAPI stained nuclei. Mitochondria cluster around the nucleus and cells undergoing apoptosis shrink, detach and show a round shape

Regulation of cell death by pro-apoptotic Bcl-2 family members

Apoptosis is mediated through at least three major pathways that are regulated by (1) the death receptors, (2) the mitochondria, and (3) the endoplasmic reticulum (ER). In most cells, these pathways are controlled by the Bcl-2 family of proteins that can be divided into antiapoptotic and proapoptotic members. Although the overall amino acid sequence homology between the family members is relatively low, they contain highly conserved domains, referred to as Bcl-2 homology domains (BH1 to BH4) that are essential for homo- and heterocomplex formation as well as for their cell death inducing capacity. Structural and functional analyses revealed that the proapoptotic homologs can be subdivided into the Bax subfamily and the growing BH3-only subfamily. BH3-only proteins link upstream signals from different cellular or functional compartments to the mitochondrial apoptosis pathway (Figure 1). Puma, Noxa, Hrk, and Nbk (Bik) are induced by p53 and mediate cell death originating from the nucleus, e.g. upon DNA damage. Nbk localizes to the ER and activates Bax (but not Bak) indirectly, through a so far undefined ER-initiated death pathway.

The aim of our work is to gain structural and functional insights into how these subfamilies promote or inhibit cell death signals and how these properties may be utilized for development of apoptosis-promoting cancer therapies. Our studies therefore deal with questions such as how cell cycle stress responses including anticancer therapies and oncogene deregulation feed into the mitochondrial death pathway. We recently established that Nbk stabilizes the anti-apoptotic multidomain protein Mcl-1 that acts as an endogenous inhibitor of Bak. This fully explains the entirely Bax dependent induction of apoptosis by Nbk. Ongoing work addresses the transcriptional control of Nbk expression and its functional involvement in the regulation of cell death following ER stress responses.

 

Cell cycle and apoptosis

Using the apoptosis, cell cycle arrest and senescence inducing tumor suppressor gene p14ARF expression as a model system, we explore the intricate interconnections between cell cycle stress responses and apoptosis induction. P14ARF expression is induced upon cellular stress, especially following deregulation of oncogenes. While physical interaction of p14ARF with numerous regulatory proteins, induction of p53-dependent cell cycle phenomena and cellular senescence by p14ARF are well established, little is known how p14ARF induces cell death. Notably, we established that the induction of mitochondrial apoptosis by p14ARF is entirely independent from p53 and Bax in p53-deficient cells where Bak can complement for Bax function. In contrast to apoptosis induction, the triggering of a G1 cell cycle arrest (and presumably premature cellular senescence) by p14ARF is entirely dependent on p53 and p21CIP/WAF-1, indicating that the signalling pathways for p14ARF-induced G1 arrest and apoptosis induction dissociate upstream of p53. Noteworthy, loss of p21 strongly enhances apoptosis induction by p14ARF. In the same vein, loss of 14-3-3s or of both p21 and 14-3-3σ strongly augments p14ARF-induced apoptosis. Nonetheless, we recently demonstrated that, in the absence of functional p53 and/or p21, p14ARF triggers a G2 cell cycle arrest by downregulation of cdc2-kinase activity, protein expression, and cytoplasmic localization in these cells whereas p14ARF is localized to the nucleus (see figure 2), i.e. mediates cdc2 sequestration and induction of mitochondrial apoptosis through an indirect mechanism. Such p53-independent mechanisms of p14ARF induced apoptosis and arrest in the cell division cycle represent fail-safe mechanisms that allow for efficient grwoth suppression following induction of p14ARF-mediated stress responses in p53 pathway deficient cells.

<b>Abbildung 2: Network of p14ARF induced stress responses</b>
A-D: Subcellular localization of p14ARF to the nucleus but not to the mitochondria. A: Mitochondria are labeled red by stable expression of a DsRed fusion protein as an organelle marker. B: p14ARF expression is found in the nucleus and the nucleoli. C: Overlay for p14ARF and mitochondria. D: Overlay for red labelled mitochondria and DAPI. This experiment shows that p14ARF must triggers the mitochondrial apoptosis pathway via an indirect mechanism. This is unlike the case of p53 which had been described recently to localize not only to the nucleus but also to the mitochondria where p53 exerts transcription independent apoptotic functions.

Projekte

• EU
BMBF
DFG
• Deutsche Krebshilfe
• Berliner Krebsgesellschaft

Ausgewählte Publikationen

Hossbach J, Michalsky E, Henklein P, Jaeger M, Daniel PT, Preissner R.
Inhibiting the inhibitors: Retro-inverso Smac peptides..
Peptides [Epub ahead of print] 2009;
Füllbeck M, Dunkel M, Hossbach J, Daniel PT, Preissner R.
Cellular fingerprints: a novel approach using large-scale cancer cell line data for the identification of potential anticancer agents..
Chem Biol Drug Des 2009; 74(5):439-48.
le Coutre P, Reinke P, Neuhaus R, Trappe R, Ringel F, Lalancette M, Hemmati PG, Dörken B, Daniel PT
BCR-ABL positive cells and chronic myeloid leukaemia in immune suppressed organ transplant recipients..
Eur J Haematol [Epub ahead of print] 2009;
Hemmati PG, Normand G, Gillissen B, Wendt J, Dörken B, Daniel PT.
Cooperative effect of p21Cip1/WAF-1 and 14-3-3sigma on cell cycle arrest and apoptosis induction by p14ARF.
Oncogene. 2008; 27(53):6707-19.
Gryshchenko I, Hofbauer S, Stoecher M, Daniel PT, Steurer M, Gaiger A, Eigenberger K, Greil R, Tinhofer I.
MDM2 SNP309 is associated with poor outcome in B-cell chronic lymphocytic leukemia.
J Clin Oncol. 2008; 26(14):2252-7.
Grabowski P, Schrader J, Wagner J, Hörsch D, Arnold R, Arnold CN, Georgieva I, Stein H, Zeitz M, Daniel PT, Sturm I.
Loss of nuclear p27 expression and its prognostic role in relation to cyclin E and p53 mutation in gastroenteropancreatic neuroendocrine tumors..
Clin Cancer Res. 2008; 14(22):7378-84.
Gillissen B, Essmann F, Hemmati PG, Richter A, Richter A, Oztop I, Chinnadurai G, Dörken B, Daniel PT
Mcl-1 determines the Bax dependency of Nbk/Bik-induced apoptosis..
J Cell Biol. 2007; 179(4):701-15.
Sturm I, Stephan C, Gillissen B, Siebert R, Janz M, Radetzki S, Jung K, Loening S, Dörken B, Daniel PT.
Loss of the tissue-specific proapoptotic BH3-only protein Nbk/Bik is a unifying feature of renal cell carcinoma..
Cell Death Differ 2006; 13(4):619-27.
Daniel PT, Koert U, Schuppan J.
Apoptolidin: induction of apoptosis by a natural product..
Angew Chem Int Ed Engl. 2006; 45(6):872-93.
Sturm I, Rau B, Schlag PM, Wust P, Hildebrandt B, Riess H, Hauptmann S, Dörken B, Daniel PT.
Genetic dissection of apoptosis and cell cycle control in response of colorectal cancer treated with preoperative radiochemotherapy..
BMC Cancer 2006; 6:124.