Ciclo Celular y Estabilidad Genómica Versión en español

We work under the hypothesis that DNA replication is a valid target for cancer therapy. When replication templates are suboptimal, normal cells do not enter S phase; instead, cancer cells are more likely to attempt DNA replication regardless of such limited templates' quality. Also, the molecular pathways in charge of sensing damaged DNA are impaired in cancer cells. Such a strive to undergo constant DNA replication creates a window of opportunity for treatment: if chemotherapy spoils the quality of DNA templates strongly, DNA duplication is irreversibly halted, triggering cell death. However, decades of chemotherapy usage on human subjects have revealed that such a treatment is not as successful as initially expected. Such a limitation results from the activation of a molecular network, the DDR (DNA damage response). Such a collection of molecular pathways enables the duplication of DNA damaged by metabolic and environmental stress in healthy cells. Unfortunately, cancer cells use the DDR to complete DNA replication, survive and adapt to chemotherapy. 
Our laboratory's primary goal is to identify the specific contribution to cancer cell survival and genomic instability of different factors in this network, looking for druggable targets that selectively sensitize cancer cells to cell death and/or regulate genomic instability.


Molecular biology and cell biology tools are instrumental for our research. We use cancer cell lines as a model to recapitulate the response of tumor cells to treatment. While simplified and artificial when attempting to recapitulate cancer tissues, cancer cells in culture are easy to manipulate when attempting to alter the expression of specific genes. By doing so, we can establish causal relationships between a given gene and a phenotypic output, revealing aspects of the DDR network that are not known yet. To monitor such a network, we study DNA replication at different levels, including the evaluation of single molecules of newly synthesized DNA (Figure 1), different direct or indirect markers of DNA damage (Figure 2), and parameters of chromosome instability (Figure 3) and cell death. By analyzing such parameters, we assess the impact of the inhibition of a given cellular factor on cell death or cancer cells' ability to adapt to anti-cancer treatments.

Figure 1: DNA Fiber spreading assay reveals single nascent DNA molecules. A) Nascent DNA is labeled by the incubation of cells with two sequential pulses of thymidine analogs. B) Cells are trypsinized, seeded on the end of a coverslip, and lysed under denaturing conditions while tilting the coverslip to make the drop fall slowly down the surface, extending the DNA fibers. C) Lastly, the nascent DNA is revealed by immunostaining. D) Representative panel of a fiber spread field (Speroni et al., PNAS, 2012). E) Representative panels of single fibers showing the different labeling that can be found in the samples, indicating how they should be interpreted (panels A-C y E are extracted from the Ph.D. Thesis from Nicolás Calzetta).

Figure 2: Examples of direct and indirect markers of DNA damage. A) Focal and pan nuclear staining of the phosphorylation of histone variant H2AX (γH2AX) that reveal sites of DNA damage and cells committed to cell death respectively. B) Focal accumulation of γH2AX on condensed chromosomes of mitotic cells. C) Formation of comet tails on intact nuclei subjected to electrophoretic fields. Such a field does not alter the nuclei with undamaged DNA, but nuclei containing lighter DNA, resulting from the formation of DSB, form a tail of DNA that resembles a comet tail. D) Chromosomes spread (labeled in red) combined with a label for DNA síntesis in M phase (EdU incorporation during M phase- labeled in green). Zoom on the left corresponds to the indicated chromosome and depicts semiconservative DNA synthesis (EdU labeling in gree). The photographs in B and C are extracted from Calzetta et al., 2020, Sci Adv.

Figure 3: Examples of genomic instability outputs. A) Anaphase aberrations including lagging chromosomes and bridges. B) ultrafine DNA bridges that reveal underreplicated DNA regions at the end of metaphase. C) FormEjemplos de parámetros de inestabilidad cromosómica. A) Defectos de segregación de cromosomas revelados al analizar la organización de metafases. B) puentes ultrafinos que revelan la acumulación de regiones de ADN no duplicadas al final de la metafase. C) representative image of micronuclated cells. Such structures are formed with entire or broken chromosomes that failed to align on metaphase plates during mitosis. D) mitotic spread showing the acumulation of aberrant chromosomes. Images A-B are extracted from Calzetta et al., 2020, Sci Adv and C-D from Federico et al., PloS Genetics.


Cancer cells survive when challenged by the DNA damage caused by chemotherapy because of the availability of pathways such as translesion DNA synthesis (TLS), which prevents the persistent halting of DNA synthesis at damaged DNA sites. During a TLS event, stalled replicative polymerases are replaced by TLS polymerases capable of using damaged DNA as templates. We have demonstrated that the cyclin kinase inhibitor p21 is a negative regulator of TLS. Specifically, the C-terminal domain of p21 binds to the auxiliary platform for DNA polymerase, PCNA (proliferating cell nuclear antigen), displacing TLS polymerases from the replisome (Soria et al., 2006, JCS). In undamaged cells, p21 prevents the dysregulated participation of TLS polymerases in the duplication of undamaged DNA (Mansilla et al., eLIFE, 2016) (Figure 4). In contrast, when DNA damage increases, TLS is needed, and p21 must be degraded (Soria et al., 2006 JCS; Mansilla et al., Nucleic Acid Research 2013). The stable expression of p21 causes cell death in cells dealing with high DNA damage levels. Hence, the overexpression of p21 or the downregulation of TLS are candidates that could be targeted to improve the therapeutic outcomes of treatments involving DNA damaging agents. 

Figure 4: The primary function of p21 is not inhibiting cyclin-dependent kinases (CDKs). P21 inhibits CDKs only when p21 levels augment abruptly in non-proliferating cells. In contrast, the low levels of p21 expressed in cyclin cells promote, rather than inhibit, proliferation by preventing the dysregulated loading to replisomes of TLS polymerases that are less processive and more mutagenic than replicative polymerases. Such low levels of p21 must be removed when TLS activation is needed. The figure is extracted from Mansilla et al., Genes, 2020.


We have also participated in the validation node of a public-private consortium in collaboration with GlaxoSmithKline, that has conducted a screening evaluating the capacity of different kinase inhibitors to selectively kill cells deficient in the BRCA1and 2 tumor suppressors (the mutation of the BRCA tumor suppressors is frequently associated with triple-negative based cancers). The screening node identified kinase inhibitors that cause selective cell death either in BRCA1-deficient backgrounds (PLK1 inhibition) (Carbajosa et al., 2019) and BRCA2-deficient backgrounds (unpublished results). Later on, our laboratory explored the mechanism of action-MOA of such kinase inhibitors. We discovered that such an MOA is strikingly different from the one caused by inhibitors of PARPs (PARP-Poly ADP ribose polymerase-inhibition), currently available to treat BRCA-deficient cancers. Remarkably, while PARP inhibitors cause the accumulation of many markers of chromosome instability, PLK1 inhibition does not (Figure 5). We will further investigate if such a lack of CIN represents an opportunity to improve the treatment of cancers carrying BRCA-deficiency.


Figure 5: the cell death of BRCA1 deficient cells is not always dependent on the accumulation of replication stress and chromosome instability. While the inhibitors of PARPs cause a rise in the markers of replication stress (e.g. γH2AX y 53BP1) and genomic instability (e.g., micronuclei and aberrant chromosomes), the inhibition of PLK1 triggers a type of cell death that is not preceded by those alterations. 


We also used the Chk1 inhibitor to explore cell death mechanisms and genomic instability caused by DDR inhibition. We chose Chk1 inhibition because cancer cells rely heavily on this enzyme in terms of transducing the replication status to CDK activity levels. We found that Chk1 inhibition not only inhibits the kinase activity of Chk1 but also causes the formation of replication barriers mediated by the increased recruitment to DNA of CDC45, a component of the origin firing complex. Also, such replication barriers are triggers for cell death (González Besteiro et al., EMBO, 2019). The cell death trigger by Chk1 depletion is preceded by double-strand break formation in S phase, but is unrelated to the mitotic chromosome instability observed after Chk1 depletion. We also show that aberrant DNA synthesis in M phase triggers chromosome instability, which can be reverted by adding extra nucleosides. The implications are that by controlling mitosis-triggered chromosome instability, Chk1 inhibition may still efficiently reduce the cancer cell load, preventing, however, genetic changes and adaptation to treatment in the surviving remnants (Calzetta et al., Sci Adv., 2020). 

Figure 6: The association between genomic instability and cell death during cancer treatment. A) it is broadly accepted that cell death and genomic instability are interlinked variables triggered by the same molecular pathway. It is also accepted that genomic instability preceded cell death and that tumor adaptation to treatment results from a moderate genomic instability that is insufficient to cause cell death. B) Instead, we have shown that the triggers for genomic instability and cell death after Chk1 downregulation are different. Hence, cell death and genomic instability could be, at least after treatment with Chk1 inhibitors, selectively inhibited.