Cell proliferation and differentiation: genetics and epigenetics

Terminal differentiation is usually coupled to permanent exit from the cell cycle. The expression levels of many cell cycle regulators typically decline when cells exit the cell cycle and undergo differentiation. Moreover, induction of the expression of anti-proliferative molecules during cell differentiation prevents activation of major cell cycle engines such as cyclin–CDK complexes in terminally differentiated cells. This effect is mostly promoted by two types of molecules: a) enzymes involved in the proteasome-dependent degradation of cell cycle proteins; and b) protein phosphatases that counteract the function of cell cycle kinases. These enzymes dot not only modulate cell cycle progression but are critical players in the balance between cell proliferation and differentiation, a balance of major implications during development, tissue homeostasis or disease.

Dysregulation of cell cycle proteins is commonly found in several pathological conditions, including cancer, neurodegeneration and cardiac disease, but the relevance of non-canonical functions of cell cycle proteins in controlling processes other than cell proliferation — such as transcription, cell death, differentiation and metabolism — remains to be fully explored.

The Anaphase-promoting complex in differentiation

 

The Anaphase-Promoting Complex or Cyclosome (APC/C) is an E3 ubiquitin ligase whose activation requires the binding of a cofactor, either Cdc20 or Cdh1, which are critical for selecting the substrates that will be ubiquitinated for proteasome-dependent degradation. By selecting multiple cell cycle regulatory proteins, the APC/C plays major roles in cell cycle progression and exit. While APC/C-Cdc20 is a major player during the metaphase-to-anaphase transition and mitotic exit, APC/C-Cdh1 plays a central role in maintaining quiescence and controlling the onset of DNA replication. In addition, APC/C-Cdh1 is essential for endoreduplication, a process in which several rounds of DNA synthesis occur without mitosis. Recent data suggest that the APC/C is also involved in differentiation and metabolism, and plays important roles in postmitotic cells such as neurons.

In the last years, we also studied the relevance of the APC/C during tumor progression and its possible use as a target in cancer therapy. By participating in cell cycle exit and differentiation, Cdh1 may be considered as a tumor suppressor and its deletion contributes to unscheduled cell proliferation in several scenarios. On the other hand, Cdc20 may have specific uses as a cancer target owed to its essential role during mitotic progression.

Summary of multiple functions of the APC/C in a variety of cell types (Eguren et al. Semin. Cell Dev. Biol. 2011)

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.

It has been therefore proposed that blocking mitotic exit may have stronger effects than targeting the spindle or mitotic entry. In fact, we demonstrated 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. We also reported that prolonged mitotic arrest, such as the one induced by APC/C-Cdc20 inhibitors result in mitochondria exhaustion and strong dependence of glycolysis for survival. These data suggest that preventing mitotic slippage or modulating the metabolic pathways that support survival during mitotic arrest could enhance the efficacy of other antimitotic compounds.

Cell cycle phosphatases

 

Schematic representation of several kinases and phosphatases involved in cell cycle progression and several mouse models generated or to be generated in our lab for their analysis in vivo.

The mechanisms leading to cell cycle exit have been studied in detail in yeast. However, the information available in mammals is very limited, at least partially due to the use of in vitro models in which cultured cells are induced to exit the cell cycle in very special artifactual scenarios. In yeast, phosphatases of the Cdc14 family are critical effectors of mitotic exit by removing Cdk-dependent phosphates from cell cycle substrates. Two different Cdc14 phosphatases, Cdc14a and Cdc14b, exit in mammals although their specific role is still a mystery due to the lack of clear defects in cells depleted for one or the other member of the family.

We have recently generated mice deficient for Cdc14a, Cdc14b or both. Our data suggest certain level of compensation between these two family members in several cellular processes. Among them, cell cycle exit to quiescence or differentiation seem to be specially affected by the lack of these phosphatases. Our current efforts aim to characterize the relevance of these phosphatases in specific cell types in vivo.

A second family of phosphatases seem to be also involved in cell cycle and differentiation in mammals. PP2A is a major phosphatase whose activity is regulated by several families of regulatory subunits. Work in the last decade has established B55 proteins, B55alpha, beta, gamma and delta, as critical regulators of PP2A during the cell division cycle. We are currently generating mouse strains with loss-of-function models for these phosphatases to understand their relevance not only in cell cycle progression but also in tissue physiology and tumor development.

Cell Cycle regulators and the epigenetic code

During the chromosome cycle, the chromatin undergoes multiple changes required for the control of transcription, but also higher-level reorganizations required for the proper segregation of sister chromatids during cell division. The epigenetics code, based on a variety of post-translational modifications in histones, is critical for both of these processes. Mitotic kinases, such as Aurora B or Haspin are known to phosphorylate histones to promote some of the changes required for cell cycle progression.

We are currently investigating the regulatory crosslinks that may control chromatin structure during cell cycle progression, or differentiation/undifferentiation, including the changes required for reprogramming to pluripotent cells or the differentiation of these cells towards multiple tissue types.

 
Unconventional cell cycles

 

Variation in ploidy is commonly found in multiple organisms and polyploid cells are an essential part of the developmental program in many different species. In most cases, developmentally programmed polyploidy is an irreversible process linked to terminal cell differentiation and the acquisition of new functional capabilities. In addition, polyploidy has been proposed to buffer the genome against genetic damage. Somatic polyploidy can be achieved through multiple modifications of the basic cell division cycle. These special cycles include endocycles, originally described as a process in which rounds of DNA replication occur while mitosis is completely bypassed (successive DNA synthesis and gap phases), or endomitosis, an atypical cell cycle in which cells undergo an aberrant mitosis in the absence of segregation of the previously duplicated genomes. Other processes, such as re-replication, lead to non-uniform increased ploidy, as a consequence of alteration in the regulatory modules that impose a single round of DNA replication per cell cycle, and normally lead to cell death.

 

A summary of endocycles including endoreplication (placental trophoblast giant cells) or endomitosis (megakaryocytes)

Polyploidization is an essential part of the programmed developmental process required for the formation of placental trophoblast giant cells, which use endocycles to achieve chromatin-values (C-value, a multiple of the DNA content of the un-replicated haploid chromosome complement) of >1,000. Similarly, bone marrow megakaryocytes can reach up to 128C in humans or 64C in the mouse through endomitotic cell cycles. Whereas endocycling is characterized by low expression of mitotic genes, and low cyclin-dependent kinase (Cdk) activity, mitotic genes are normally expressed during endomitosis in megakaryocytes.

We recently performed a systematic genetic analysis of the relevance of specific endocycles using megakaryocytes as a model system. These studies allowed us to understand the relevance of the mitotic machinery during this very special process in megakaryocytes and the consequences of altering mitotic kinases in platelet levels. This study is relevant to the effect of many chemotherapy protocols resulting in thrombocytopenia as a consequence of impaired proliferation of megakaryocytes or their precursors. In addition, we recently studied the relevance of the cell cycle kinase MASTL in megakaryocytes. The corresponding human gene, MASTL, is actually mutated in specific thrombocytopenia patients and these studies allowed us to understand the function of MASTL in the re-organization of the actin cytoskeleton in postmitotic cells.

Further reading

Bueno, M.J., Pérez de Castro, I., Gómez de Cedrón, M., Santos, J., Calin, G.A., Cigudosa, J.C., Croce, C.M., Fernández-Piqueras, J. and Malumbres, M. (2008) Genetic and Epigenetic Silencing of microRNA-203 Enhances ABL1 and BCR-ABL1 Oncogene Expression. Cancer Cell 13, 496-506.

Eguren, M., Manchado, E. and Malumbres, M. (2011) Non-mitotic functions of the Anaphase-promoting complex. Semin. Cell Dev. Biol. 22, 572-578.

Eguren, M, Porlan, E., Manchado, E., García-Higuera, I., Cañamero, M., Fariñas, I. and Malumbres, M. (2013) The APC/C cofactor Cdh1 prevents replicative stress and p53-dependent cell death in neural progenitors. Nat. Commun. 4, 2880.

Fernández-Miranda, G., Trakala, M., Martín, J., Escobar, B., González, A., Ghyselinck, N.B., Ortega, S., Cañamero, M., Pérez de Castro, I. and Malumbres, M. (2011) Genetic disruption of Aurora B uncovers an essential role for Aurora C during early mammalian development. Development 138, 2661-2672.

García-Higuera, I., Manchado, E., Dubus, P., Cañamero, M., Mendez, J., Moreno, S. and Malumbres, M. (2008) Genomic Stability and Tumor Suppression by the APC/C Cofactor Cdh1. Nat. Cell Biol. 10, 802-811.

Guillamot, M., Manchado, E., Chiesa, M., Gómez-López, G., Pisano, D.G., Sacristán, M. and Malumbres, M. (2011) Cdc14b regulates mammalian RNA polymerase II and represses cell cycle transcription. Sci. Reports 1, 189.

Hurtado, B., Trakala, M., Ximénez-Embún, P., El Bakkali, A., Partida, D., Sanz-Castillo, B., Álvarez-Fernández, M., Maroto, M., Sánchez-Martínez, R., Martínez, L., Muñoz, J., García de Frutos, P., Malumbres, M. (2018) Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeletal dynamics in platelets. J. Clin. Invest. 128, 5351-5367.

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. (2013) miRNAs and cancer: An epigenetics view. Mol. Aspects Med. 34, 863–874.

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.

Trakala, M., Rodríguez-Acebes, S., Maroto, M., Symonds, C.E., Santamaría, D., Ortega, S., Barbacid, M., Méndez, J. and Malumbres, M. (2015) Functional reprogramming of polyploidization in megakaryocytes. Dev. Cell 32, 155-167.

Other Projects

Cell cycle targets & Cancer
Cell cycle & Metabolism
Pluripotency & Regenerative medicine