Cell Division and Cancer

 

Home

News

Research

Publications

Lab members

Lab. pictures

Opportunities

Links

Contact

 

Research

 

 

 

 


Collaborators
Databases
Protocols

The Cell Division and Cancer Group (CNIO) is interested in understanding the relevance of mitotic regulators not only during the cell cycle but also in physiological processes in different cell types or tissues. Mitosis is mostly regulated by posttranscriptional mechanisms such as protein phosphorylation or protein degradation. We are therefore interested in the study of mitotic kinases and phosphatases as well as regulatory complexes involved in ubiquitin-dependent degradation of proteins. The control of proper chromosome segregation by these regulators is essential to prevent genomic instability and may have critical implications in the generation of aneuploidy, an unbalance in the number of chromosomes commonly found in human tumor cells. In addition, we are interested in understanding how small, non-coding RNAs are involved in the control of the cell cycle and cell proliferation. Thus, we are using mouse models with genetic alterations in several mitotic regulators and microRNAs to gain insights into their relevance in vivo and their possible use in cancer therapy. Our interests also include the investigation of the regulatory mechanisms that control asymmetric cell division in progenitor/stem cells and their relevance in development, tissue homeostasis and cancer. General interests in our group include:

  • Understand the basic mechanisms of control of the mammalian cell cycle

  • Characterize the physiological and therapeutic consequences of cell cycle deregulation in animal models

  • Characterize the function of microRNAs in cell biology and tumour development

  • Understand the how progenitor cells and cancer stem cells control their self-renewal and proliferative properties



Control of the mammalian cell cycle

Mitosis is controlled by several families of protein kinases including cyclin-dependent kinases and the Aurora and Polo-like families (Malumbres & Barbacid, 2009; Malumbres, 2011). Many of these proteins control proper chromosome segregation between daughter cells during mitosis. Defects in this control may lead to aneuploidy or chromosomal instability, a condition commonly associated to cancer. The basic mechanisms leading to aneuploidy and the therapeutic possibilities arising from this condition are currently under intense research (see Malumbres, 2011 and Manchado and Malumbres, 2011).

We have generated several mouse models of gain- or loss-of-function of these kinases as well as other mitotic regulators with the aim of not only understanding their relevance during cell cycle progression but also in vivo.

 

Modelling the function of mitotic kinases and phosphatases

We have focused on the major cell cycle kinases that regulate progression through mitosis. All three Aurora kinases, A, B and C, contain a SUMOylation motif highly conserved through evolution. In collaboration with W.C. Earnshaw´s group (Edinburgh, UK) and the Macromolecular Crystallography Group (CNIO) we have observed that Aurora A and Aurora B are tagged by different SUMO peptides (Figure 1) and that interference with this post-translational modification results in defective Aurora function and genomic instability. Genetic ablation of Aurora B results in embryonic lethality after embryo implantation. Aurora C may complement for Aurora B loss during the first embryonic cell divisions or in rescue experiments in culture. We are currently analysing the cellular requirements for these proteins during the spindle assembly checkpoint using conditional knockout cells.

In collaboration with G. Manning at the Salk Institute (San Diego, USA) we have also identified a fifth member of the Polo-like kinase family in mammals. This protein, Plk5, also contains a Polo-box domain although it is not an efficient kinase in vitro. Plk5 is mostly expressed in brain cells and its overexpression interferes with entry into mitosis. In collaboration with the Hospital Nacional de Parapléjicos and the Hospital Virgen de la Salud, Toledo (Spain), we have demonstrated that Plk5 is necessary for neuronal function and the corresponding gene is epigenetically deregulated in brain tumours.

 

 

 

 

 

 

 

Figure 1: A model for the modification of Aurora B kinase by SUMO peptides (in collaboration with Guillermo Montoya from the CNIO´s Macromolecular Crystallography Group).

The anaphase-promoting complex and protein degradation

The Anaphase Promoting Complex (APC/C, or cyclosome) is an E3-ubiquitin ligase whose activity depends on two co-activators: Cdc20 and Cdh1/Fzr1. Along with Marta Cañamero´s Comparative Pathology Core Unit (CNIO), S. Moreno´s group (Instituto de Biología Molecular y Celular del Cáncer, Salamanca) and H. Yamano’s group (University College London, UK), we have demonstrated that genetic ablation of Cdc20 results in embryonic lethality at the two-cell stage of embryonic development. Cdc20-null cells arrest in metaphase in accordance with the function of this APC/C cofactor in the metaphase-to-anaphase transition. Cdc20 also appears to play an essential role in adult somatic cells since its acute genetic ablation results in mitotic arrest and proliferative defects in vivo. Genetic ablation of Cdc20 results in metaphase arrest and apoptosis in tumour cells. These Cdc20-null tumours regressed within a few days after loss of Cdc20. This strong effect contrasts with the partial therapeutic benefits of using current mitotic drugs such as Taxol or Plk1 inhibitors in similar assays. The molecular characterisation of Cdc20-null cells has allowed us to uncover a critical function for the PP2A-B55 phosphatase in mitotic exit. During a normal cell cycle, Cdc20 triggers the inactivation of the kinase Mastl, an inhibitor of PP2A, thus resulting in PP2A activation, removal of Cdkdependent mitotic phosphates and mitotic exit (Figure 2).

 




Figure 2: A working model for mitotic exit in mammalian cells.

 

 
Regulation of the mitotic checkpoint in progenitor cells

The mitotic checkpoint is the molecular mechanism that ensures proper segregation of chromosomes to daughter cells. Defects in this control may lead to abnormal chromosome segregation and aneuploidy. These problems may be tolerated in somatic cells but are specially dangerous in progenitor cells and actually aneuploidy is mostly lethal during embryonic development with only a few exceptions (Down syndrome). Although the mitotic checkpoint has been deeply studied in established cell lines or primary fibroblasts, its control in stem/progenitor cells is mostly unexplored. We plan to characterize the mitotic checkpoint in embryonic stem (ES) and induced pluripotent (iPS) cells and establish the putative differences between these two cell types. Given the promising uses of iPS in regenerative medicine, these studies will be crucial to evaluate the genomic stability of these cells or to improve possible instability.

 


 

 
 
The actin cytoskeleton and tumour development

With the Hereditary Endocrine Cancer Group (CNIO) we have recently characterised the importance of a protein, known as Brick1, in tumour development. The BRK1 gene encoding Brick1 is located close to the Von Hippel-Lindau (VHL) gene and both of these genes are frequently co-deleted in VHL patients. Interestingly, these patients are protected against renal cell carcinoma. We have recently demonstrated that Brick1 is required for cell transformation and tumour progression due to its critical role in actin dynamics. These data suggest the potential therapeutic uses of inhibiting the actin cytoskeleton in VHL patients that maintain a wild-type BRK1 gene or other tumour types (Figure 3).

 

 

 
  Identification of key mediators of mitotic cell death

Cell cycle is deregulated in most cancer cells, therefore a possible strategy to impair tumor cell proliferation is targeting the cell division cycle. Some approaches have been proposed in the last years;  ·        
Impairing cell cycle entry by inhibition of cyclin-dependent kinases (Cdks) results in minor effects given the compensation between multiple family members and are only usefull in certain cellular context. Besides inhibition of interphase Cdks induces cell cycle arrest but not apoptosis as a general feature. ·        
Arresting cells at the G1/S or G2/M transitions with DNA-damaging agents cannot proliferate but are viable. G0/G1 arrested cells can resume cell cycle proliferation upon activation of the appropriate stimuli.
Targeting the mitotic checkpoint (SAC) by inhibiting the mitotic kinases or with microtubule poison Checkpoint-mediated arrest can be transient once the damaging conditions have been eliminated or as a result of adaptation to the checkpoint. Moreover resistance microtubule poisons can be acquired by expression of particularly tubulin isoforms or microtubule-regulating proteins.

Whereas mitotic entry depends on the phosphorylation of a wide spectrum of substrates by Cdks and other kinases, mitotic exit requires Cdks inhibition and the activation of different phosphatases complexes such as PP2A/B5. Mitotic exit has been proposed as a relevant target given pro-apoptotic effect of Cdc20 depletion. We will use a Cdc20 conditional KO inducible by 4-OHT mouse model developed in our lab (Manchado et al., 2010). In the absence of Cdc20, cells are not able to exit mitosis and remain arrested in metaphase  during 6 to 36 hours. All Cdc20-deficient cells suffer mitotic arrest and are unable of exit mitosis, finally undergo apoptosis with an average latency of 20 ± 11 hours as detected by videomicroscopy, longer than taxanes and other microtulube drugs used in clinic. This death cascade, also known as mitotic cell death (MCD) or mitotic catastrophe, is not well-known at the molecular level. Cdc20 cKO model raises the possibility to analyze molecular pathways of mitotic exit and mitotic catastrophe with a genetic model in vitro as in vivo. MCD can be defined as a type of cell death resulting from aberrant mitosis, that may be different from apoptosis, necrosis or senescence. Is driven by a complex and poorly understood signalling cascade. MCD should be considered as an oncosuppressive mechanism avoiding the CIN by preventing aneuploidization. The disruption of MCD may lead to tumorigenesis and cancer progression and its induction constitutes a therapeutic endpoint. MCD seems to be a caspase- and mitochondria-dependent mechanism but it is not clear which are the apoptotic players involved. The objectives in this area include:

Identification of genes that regulate MCD in Cdc20-null MEFs and iPS, using siRNA libraries and videomicroscopy with apoptotic reporters in vivo.
 

Analyze the balance between kinases and phosphatases in mitotic exit and its relation to apoptosis.




 

Control of cell proliferation by microRNAs

We have studied the relevance of microRNAs (miRNAs) in the cell cycle at different levels. We have first analysed the expression of miRNAs in early cell cycle phases and have identified several clusters of miRNAs that are induced by E2F transcription factors during the early phases of the cell cycle. Several of these miRNAs modulate major proliferation pathways by controlling the expression of critical cell cycle regulators such as cyclins and cyclin-dependent kinases. The induction of these miRNAs prevents replicative stress upon cell cycle entry in the presence of strong mitogenic signals.

 

 

 

 

 

 

 

 

 

 
Cell Division and Cancer Group
 

Centro Nacional de Investigaciones Oncológicas (CNIO) Spanish National Cancer Research Centre

Melchor Fernández Almagro 3, E-28029 Madrid, Spain