Cell Cycle targets and Cancer
Cyclin-dependent kinases (CDKs) are serine/threonine kinases whose activity depends on a regulatory subunit -a cyclin. Based on the sequence of the kinase domain, CDKs belong to the CMGC group of kinases. To aid analysis of CDKs, we proposed in 2009 a consensus nomenclature for proteins belonging to this family as Cdk1 through to Cdk20.
CDKs were first discovered by genetic and biochemical studies in model organisms such as yeasts and frogs. This work established the importance of CDKs in promoting transitions through the cell cycle. In addition, these studies showed that the catalytic subunit, the CDK, must associate with a regulatory subunit, the cyclin, whose protein levels are subject to regulation during the cell cycle (this oscillation lent these regulators their cyclin name). Since these pioneer studies conducted in the 1980s, the importance of CDKs acting as a major eukaryotic protein kinase family involved in the integration of extracellular and intracellular signals to modulate gene transcription and cell division has been clearly established. CDKs probably originated as a system to modulate cell-cycle-promoting activity in response to various cellular scenarios. Over the course of evolution, both CDK and cyclin gene families have independently undergone a significant number of functional specializations.
Schematic representation of the function of Cyclin-dependent kinases in the cell (Malumbres, Genome Biol. 2014)
As a consequence of their importance in multiple processes, CDKs are frequently mutated or deregulated in disease. A classic example is the almost universal deregulation of the CDK-cyclin-Rb pathway in cell-cycle entry during malignant transformation. Underlining the significance of CDKs, inhibitors of Cdk4 and Cdk6 have been recently approved for treatment of patients with breast cancer and multiple clinical trials have been design to test the effect of these inhibitors in many other tumor types. Other members of the CDK family can also be considered as interesting targets for therapeutics in cancer or other diseases. Cdk5 displays multiple roles in neurodegenerative diseases and in other tissues with relevance to diabetes, cardiovascular disease or cancer. Cdk8 exhibits copy-number gains in colon cancers, and recently it has been characterized as a coactivator of the beta-catenin pathway in colon cancer cell proliferation. Cdk10 is a major determinant of resistance to endocrine therapy for breast cancer, and inhibition of Cdk12 confers sensitivity to inhibitors of poly (ADP-ribose) polymerases PARP1 and PARP2. Cdk14 confers motility advantages and metastatic potential in hepatocellular carcinoma motility and metastasis. Finally, as indicated above, cyclin Y kinases regulate the Wnt pathway, providing new therapeutic opportunities that are yet to be explored. Hence, it seems very likely that new targets within the CDK family will be explored in the near future for therapy of cancer or other diseases.
Our laboratory is firmly interested in understanding the differential role of CDK-family members in normal and tumor cells. Our ultimate goal is contribute to the validation of Cdk proteins as cancer targets, define their contribution to specific tumor types, and help in the identification of patients that could benefit from the use of specific inhibitors against various members of the CDK family.
Mitotic kinases of the Polo and Aurora families
Among the different stages of the cell cycle, mitosis has attracted significant attention, not only because many mitotic regulators have been linked to tumorigenesis but also because the success of microtubule poisons in cancer therapy. Multiple enzymatic activities are required for mitosis. These include centrosomal and mitotic kinases of the Polo and Aurora families, required for the maintenance of the mitotic state, generation of the mitotic spindle, and proper attachment of chromosomes to microtubules for segregation.
Polo-like kinases were initially identified for their role in centrosome function and mitotic progression. Five Polo-like kinases (PLK1-5) exist in mammals whereas a single family member is present in yeast (CDC5). These kinases are divided in three subfamilies: PLK1, PLK4, and PLK2,3,5. Only PLK1 and PLK4 play diverse roles during centrosome maturation and mitosis whereas PLK2, PLK3, and PLK5 are mostly involved in stress responses during interphase or in neuron biology.
By phosphorylating different substrates, Plk1 controls a number of processes throughout the cell cycle including centrosome maturation, mitotic entry, chromosome segregation, and cytokinesis. The requirement for Plk1 during mitotic entry and progression led to the development of small-molecule inhibitors of this kinase, some of which are now in clinical trials. However, Plk1 is an essential protein in mammals and the therapeutic windows of Plk1 inhibitors may be limited by toxicity. In addition, we have recently reported that Plk1 overexpression, commonly found in tumors, may act as a tumor suppressor mechanism, rather than an oncogene, due to the induction of chromosomal instability. In 2017, we also reported an essential role of Plk1 in blood pressure by controlling contraction of smooth muscle cells in arteria walls, adding a note of caution to the use of Plk1 inhibitors in the clinic.
Aurora family kinases are of special relevance during mitosis because they play essential roles in centrosome maturation and chromosome segregation. The founding member of the Aurora family, Ipl1 (Increase in ploidy 1), was described in 1993 in a screen for Saccharomyces cerevisiae mutants that failed to undergo normal chromosome segregation. Two Ipl1-like proteins (Aurora kinases A and B) have been found in Drosophila melanogaster, Caenorhabditis elegans and Xenopus laevis. Three members of this family of serine-threonine kinases, Aurora A, B and C are encoded in humans by the genes AURKA, AURKB and AURKC. Although they are highly conserved at the protein level, Aurora kinases have very distinct expression and localization patterns as well as functions.
Aurora A is amplified and/or overexpressed in primary tumors of the breast, ovary, colon, pancreas, prostate, as well as in neuroblastoma among others, and high levels of Aurora A are associated with poor prognosis. Interfering with Aurora A expression or activity by siRNA expression, immunodepletion or specific inhibitors induces mitotic alterations that impair cell cycle progression, suggesting the potential use of Aurora A inibitors in cancer therapy. We actually reported that genetic ablation of Aurora A results in a significant increase in mitotic abnormalities and DNA damage markers and with the presence of aneuploid cells, eventually resulting in impaired proliferation and senescence. Conditional deletion of Aurora A in adult tissues induces premature aging and prevents tumor growth in vivo.
A significant number of Aurora kinase inhibitors have been analyzed in preclinical and clinical studies. Most pre-clinical studies showed the potent antiproliferative effect of these compounds. However, their inclusion in routine antitumor therapies is still far to be achieved, and a number of issues need to be addressed to improve the efficacy of AURKA inhibition for the treatment of cancer diseases. These include the definition of the proper therapeutic window to avoid toxicities due to effects in non-tumoral cells; and the analysis of possible biomarkers that may guide in the selection of target patients. Finally, although some clinical trials are already testing the cooperative effect of different antitumoral drugs, additional preclinical studies are also necessary to establish the best combinations.
Targeting mitosis for killing cancer cells (Doménech & Malumbres, Curr. Opin. Pharmacol. 2013)
Mitotic exit and The Anaphase-promoting complex (APC/C)
One of the mechanisms that explain resistance to mitotic drugs is mitotic slippage. In the presence of antimitotic drugs such as microtubule poisons or Plk1 inhibitors, the activity of the SAC prevents the degradation of cyclin B1, inactivation of Cdk1 and mitotic exit. However, this arrest is transient as a consequence of the slow but maintained degradation of cyclin B1 even in the presence of an active checkpoint. This arrest is likely insufficient for an efficient therapeutic response and cells that exit mitosis can remain viable. Cell fate during or after mitotic arrest is dictated by multiple networks but preventing mitotic exit invariably results in cell death.
It has been therefore proposed that blocking mitotic exit may have stronger effects than targeting the spindle or mitotic entry. In fact, we demonstrated in 2010 that elimination of Cdc20, the APC/C cofactor required for cyclin B1 degradation and mitotic exit, is highly efficient in killing cells in mitosis and preventing tumor growth in vivo. Small-molecule inhibitors of the APC/C are now available for preclinical studies.
Available data suggest that preventing mitotic slippage or modulating the cell death pathways that trigger cell death in mitosis should enhance the efficacy of other antimitotic compounds.
iLUNG: New Targets in Lung Cancer
As an example of research projects aimed to identify new therapeutic options, we are coordinating the iLUNG-CM consortium of the Comunidad de Madrid, focused on Tailored Therapies and Nanotechnology in Lung Cancer.
This initiative aims to combine the efforts of Physics and Cell Biology, along the preclinical and clinical advances, to improve therapies for patients with lung cancer , one of the most frequent and worse prognosis tumors.
The iLUNG Program aims to develop and apply for the first time novel techniques of nanotechnology from a perspective of molecular and cellular biology in which clinical samples can be diagnosed and stratified molecularly based on their genomic alterations. On the other hand, preclinical models will be used in genetically modified mice to evaluate possible therapies directed against such genomic alterations, especially in the oncogenic pathway that drives the cellular signals from the membrane receptors, to the Ras oncogenes and to cell cycle regulators, as well as its implications in immunotherapy. To this end, the objectives of the iLUNG Program will focus on:
- a) development of new molecular diagnostic methods based on nanotechnology.
- b) stratification of molecular alterations based on massive genomic studies in patients.
- c) in vivo evaluation of new therapeutic strategies directed to groups of patients with specific alterations.
This project has been funded by the Comunidad de Madrid (B2017-BMD-3884) with support from FEDER and FSE.
Marcos Malumbres, Centro Nacional de Investigaciones Oncológicas (CNIO)
Mariano Barbacid, Centro Nacional de Investigaciones Oncológicas (CNIO)
Javier Tamayo, Instituto de Micro y Nanotecnología-CSIC
Luis Paz-Ares, Univ. Complutense de Madrid y Hospital 12 de Octubre
Fernando López-Ríos, Hospital Universitario Sanchinarro
Álvarez-Fernández, M. & Malumbres, M. (2014) Preparing a cell for nuclear envelope breakdown: Spatio-temporal control of phosphorylation during mitotic entry. Bioessays 36, 757-765.
Álvarez-Fernández, M., Sánchez-Martínez, R., Sanz-Castillo, B., Gan, P.P., Sanz-Flores, M., Trakala, M., Ruiz-Torres, M., Lorca, T., Castro., A. and Malumbres, M. (2013) Greatwall is essential to prevent mitotic collapse after nuclear envelop breakdown in mammals. Proc. Natl. Acad. Sci. USA. 110, 17374-17379.
Álvarez-Fernández, M., Sanz-Flores, M., Sanz-Castillo, B., Salazar-Roa, M., Partida, D., Zapatero-Solana, E., Ali, H.R., Manchado, E., Lowe, S., VanArsdale, T., Shields, D., Caldas, C., Quintela-Fandino, M. and Malumbres, M. (2018) Therapeutic relevance of the PP2A-B55 inhibitory kinase MASTL/Greatwall in breast cancer. Cell Death Differ, 25, 828-840.
Burgess, A., Vuong, J., Rogers, S., Malumbres, M. and O’Donoghue, S.I. (2017) SnapShot: Phosphoregulation of mitosis. Cell 169, 1358-1358e1.
de Cárcer, G. and Malumbres, M. (2014) A centrosomal route for cancer genome instability. Nat. Cell Biol. 16, 504-506.
de Cárcer, G., Wachowicz, P., Martínez-Martínez, S., Oller, J., Méndez-Barbero, N., Escobar, B., González-Loyola, A., Takaki, T., El Bakkali, A., Cámara, J.A., Jiménez-Borreguero, L.J., Bustelo, X., Cañamero, M., Mulero, F., Sevilla, M.d.l.A., Montero, M.J., Redondo, J.M. and Malumbres, M. (2017) Plk1 regulates contraction of postmitotic smooth muscle cells and is required for vascular homeostasis. Nat. Med. 23, 964-974.
Eguren, M., Álvarez-Fernández, M., García, F., López-Contreras, A.J., Fujimitsu, K., Yaguchi, H., Luque-García, J.L., Fernández-Capetillo, O., Muñoz, J., Yamano, H. and Malumbres, M. (2014). A synthetic lethal interaction between APC/C and topoisomerase poisons uncovered by proteomic screens. Cell Reports 6, 670-683.
Hydbring, P., Malumbres, M. and Sicinski, P. (2016) Non-canonical functions of cell cycle cyclins and cyclin-dependent kinases. Nat. Rev. Mol. Cell. Biol. 17, 280-292.
Malumbres, M. (2012) Cell cycle-based therapies move forward. Cancer Cell 22, 419-420.
Malumbres, M. (2014). Cyclin-dependent kinases. Genome Biol. 15, 122.
Malumbres, M. (2014) Control of the cell cycle. In: Abeloff’s Clinical Oncology, 5th edition. (Niederhuber, J.E., Armitage, J.O., Doroshow, J.H., Kastan, M.B., and Tepper, J.E., eds.) Elsevier, pp. 52-68.
Malumbres, M. (2016) CDK4/6 inhibitors resTORe therapeutic sensitivity in HER2+ breast cancer. Cancer Cell 29, 243-244.
Malumbres, M. and Barbacid, M. (2009) Cell cycle, CDKs and cancer: a changing paradigm. Nat. Rev Cancer 9, 153-166.
Malumbres, M., Edward Harlow, Tim Hunt, Tony Hunter, Jill M. Lahti, Gerard Manning, David O. Morgan, Li-Huei Tsai, and Debra J. Wolgemuth (2009) Cyclin-dependent kinases: a family portrait. Nat. Cell Biol. 11, 1275-1276.
Manchado, E. and Malumbres, M. (2011) Targeting aneuploidy for cancer therapy. Cell 144, 465-466.
Manchado, E., Guillamot, M., de Cárcer, G., Eguren, M., Trickey, M., García-Higuera, I., Moreno, S., Yamano, H., Cañamero, M. and Malumbres, M. (2010) Targeting mitotic exit leads to tumor regression in vivo: modulation by Cdk1, Mastl, and the PP2A/B55alpha, delta phosphatase. Cancer Cell 18, 641-654.
Pérez de Castro, I., Aguirre-Portolés, C., Fernández-Miranda, G., Cañamero, M., Cowley, D.O., van Dyke, T. and Malumbres, M. (2013) Requirements for Aurora-A in tissue regeneration and tumor development in adult mammals. Cancer Res. 73, 6804-6815.
Trakala, M. and Malumbres, M. (2014). Cyclin C surprises in tumor suppression. Nat. Cell Biol. 16, 1031-1033.