Biology subjects

Huertas, P.

Publications and source records attributed to Huertas, P..

13 recordsLinked to original sources

bioRxiv2025

miRNA-mediated cell-to-cell communications boost DNA repair during theRadioadaptative Response

The Radioadaptive Response (RAR) is a phenomenon where a low, or priming, dose of ionizing radiation enhances cellular resistance to subsequent higher doses. We investigated whether RAR involves alterations in Homologous Recombination (HR), a high-fidelity DNA repair pathway. Using fibroblast models, we found that primed cells exhibit accelerated DNA end resection, an initial and essential HR step. This effect is mostly mediated by a bystander mechanism involving small extracellular vesicles (sEVs), as conditioned media fully replicated it. RNA profiling of sEVs identified miR-126-3p and miR-451a as key regulators of this response. Significantly, inhibiting miR-451a induced RAR in normally unresponsive cells. We further identified a miR-451a-p38-CCAR2 axis that enhances HR through suppression of CCAR2. These findings delineate a novel miRNA-mediated, sEV-driven mechanism that regulates HR during RAR, with potential therapeutic implications. Significance StatementWe uncover a previously unrecognized mechanism by which human fibroblasts enhance DNA double-strand break repair through homologous recombination following a priming dose of ionizing radiation-a phenomenon known as the radioadaptive response (RAR). We demonstrate that this enhanced repair capacity is driven by small extracellular vesicle (sEV)-mediated intercellular communication, through a transient reprograming of the DNA repair capacity of neighboring cells by modulating the levels of two key microRNAs. These provide new clues on how extracellular RNA signaling governs genome maintenance, with significant implications for genome stability in healthy and pathological context. The identification of actionable modulators further strengthens the translational potential of our work.

molecular biology↗

bioRxiv2025

Asparagine endopeptidase promotes radioresistance in breast cancer through ATR pathway modulation

BackgroundTumor resistance represents a major challenge in the current oncology landscape. Asparagine endopeptidase (AEP) overexpression correlates with worse prognosis and reduced overall survival in most human solid tumors. However, the underlying mechanisms of the connection between AEP and reduced overall survival in cancer patients remain unclear. MethodsHigh-throughput proteomics, cellular and molecular biology approaches and clinical data from breast cancer (BC) patients were used to identify novel, biologically relevant AEP targets. Immunoblotting and qPCR analyses were used to quantify mRNA and protein levels. Flow cytometry, confocal microscopy, chemical inhibitors, siRNA- and shRNA-silencing and DNA repair assays were used as functional assays. In-silico analyses using the TCGA BC dataset and immunofluorescence assays in an independent cohort of invasive ductal (ID) BC patients were used to validate the clinical relevance of our findings. ResultsHere we showed a dual role for AEP in genomic stability and radiotherapy resistance in BC patients by suppressing ATR and PPP1R10 levels. Reduced ATR and PPP1R10 levels were found in BC patients expressing high AEP levels and correlated with worst prognosis. Mechanistically, AEP suppresses ATR levels, reducing DNA damage-induced cell death, and PPP1R10 levels, promoting Chek1/P53 cell cycle checkpoint activation, allowing BC cells to efficiently repair DNA. Functional studies revealed AEP-deficiency results in genomic instability, increased DNA damage signaling, reduced Chek1/P53 activation, impaired DNA repair and cell death, with phosphatase inhibitors restoring the DNA damage response in AEP-deficient BC cells. Furthermore, AEP inhibition sensitized BC cells to the chemotherapeutic reagents cisplatin and etoposide. Immunofluorescence assays in an independent cohort of IDBC patients showed increased AEP levels in ductal cells. These analyses showed that higher AEP levels in radioresistant IDBC patients result in ATR nuclear eviction, revealing AEPhigh/ATRlow protein levels as an efficient predictive biomarker for the stratification of radioresistant patients. ConclusionThe newly identified AEP/ATR/PPP1R10 axis plays a dual role in genomic stability and radiotherapy resistance in BC. Our work provides new clues to the underlying mechanisms of tumor resistance and strong evidence validating the AEP/ATR axis as a novel predictive biomarker and therapeutic target for the stratification and treatment of radioresistant BC patients.

cancer biology↗

bioRxiv2024

Cx43 Enhances Response to BRAF/MEK Inhibitors by Reducing DNA Repair Capacity

BRAF and MEK inhibitors (BRAF/MEKi) have radically changed the treatment landscape of advanced BRAF mutation-positive tumours. However, limited efficacy and emergence of drug resistance are major handicaps for successful treatments. Here, by using relevant preclinical models, we found that Connexin43 (Cx43), a protein that plays a role in cell-to-cell communication, increases effectiveness of BRAF/MEKi by recruiting DNA repair complexes to lamin-associated domains and promoting persistent DNA damage and cellular senescence. The nuclear compartmentalization promoted by Cx43 contributes to genome instability and synthetic lethality caused by excessive DNA damage, which could lead to a novel therapeutic approach for these tumours to overcome drug resistance. Based on these findings, we designed an innovative drug combination using small extracellular vesicles (sEVs) to deliver the full-Cx43 in combination with the BRAF/MEKi. This study reveals Cx43 as a new player on DNA repair and BRAF/MEKi response, underlining the therapeutical potential that this approach could eventually have in the clinic to overcome the limitations of current therapies and improve treatment outcomes for patients with advanced BRAF mutant tumours.

cancer biology↗

bioRxiv2024

The signaling axis CDK12-BRCA1 mediates dinaciclib associated radiosensitivity through p53-mediated cellular senescence.

Pan-CDK inhibitors are a new class of targeted therapies that can act on multiple CDKs, with dinaciclib being one of the most promising compounds. Although used as monotherapy, an interesting approach could be to combine it with radiotherapy. Here we show that dinaciclib increases radiosensitivity in some experimental models of lung and colon cancer (A549 or HCT 116) but not in others (H1299 or HT-29). Dinaciclib did not alter responses such as ATM signalling, apoptosis or cell cycle profiling after ionising radiation exposure that have been described for other CDK inhibitors. Mechanistically, inhibition of CDK12 by dinaciclib abolishes BRCA1 expression, which blocks homologous recombination (HR), leaving only the non-homologous end joining repair process (NHEJ), which ultimately promotes the induction of ionising radiation-associated cellular senescence in a p53-dependent manner, explaining the lack of effect observed in some experimental models. In conclusion, our report explains the molecular mechanism involved in this novel therapeutic effect of dinaciclib and provides a rationale for more selective and personalised chemo/radiotherapy treatment according to the genetic background of the tumour. Highlights[tpltrtarr] Dinaciclib is a radiosensitising agent in lung and colon cancer cell lines. [tpltrtarr]Dinaciclib promotes radiosensitivity by inhibiting CDK12 leading to a decrease in BRCA1 expression. [tpltrtarr]Lack of BRCA1 blocks homologous recombination response to DNA damage, enhancing ionising radiation-associated senescence in a p53-dependent fashion. [tpltrtarr]The combination of dinaciclib plus radiotherapy could be a novel treatment for tumours holding functional BRCA1 and p53 signalling.

cancer biology↗

bioRxiv2024

EXO1-mediated ssDNA gap expansion is essential for ATR activation and to maintain viability in BRCA1-deficient cells.

DNA replication faces challenges from DNA lesions originated from endogenous or exogenous sources of stress, leading to the accumulation of single-stranded DNA (ssDNA) that triggers the activation of the ATR checkpoint response. To complete genome replication in the presence of damaged DNA, cells employ DNA damage tolerance mechanisms that operate not only at stalled replication forks but also at ssDNA gaps originated by repriming of DNA synthesis downstream of lesions. Here, we demonstrate that human cells accumulate post-replicative ssDNA gaps following replicative stress induction. These gaps, initiated by PrimPol repriming and expanded by the long-range resection factors EXO1 and DNA2, constitute the principal origin of the ssDNA signal responsible for ATR activation upon replication stress, in contrast to stalled forks. Furthermore, we show that EXO1-deficient cells exhibit marked sensitivity to translesion synthesis inhibition, a distinctive characteristic of mutations in proteins essential for repairing ssDNA gaps via template switching, such as BRCA1/2. Strikingly, EXO1 loss results in synthetic lethality when combined with BRCA1 deficiency, but not BRCA2. Indeed, BRCA1-deficient cells become addicted to the overexpression of EXO1 DNA2 or BLM. This dependence on long-range resection unveils a new vulnerability of BRCA1-mutant tumors, shedding light on potential therapeutic targets for these cancers.

molecular biology↗

bioRxiv2023

Human Cryptochrome1 dampens homologous recombination at nightfall

Genomic stability maintenance requires the repair of DNA breaks in the most accurate fashion. So, an exquisite regulatory network controls the choice between different repair mechanisms to maximize genome integrity. This relies mostly at the level of DNA end resection, the initial steps of the homologous recombination. On the other hand, numerous cellular activities follow a 24 h oscillation known as the circadian cycle. Thus, we explored the regulation of the choice between different DNA break repair pathways along the circadian cycle. Here we show that in human cells DNA resection shows a circadian oscillation, with a peak at early morning followed by a partial and progressive reduction until late afternoon. Such regulation depends on the circadian clock core component CRY1, which modulates the anti-resection activity of CCAR2 to limit CtIP at nightfall. Additionally, such regulation requires DNA-PK-mediated phosphorylation of CRY1. Finally, this circadian regulation impacts cancer progression and response to radiation therapy of specific tumours.

cell biology↗

bioRxiv2023

BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote replication fork stability and continuous DNA synthesis.

Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway, and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial and most efforts aimed to identify BRCA1/BARD1 ubiquitination substrates rely on indirect evidence. Here, we observed that the BRCA1/BARD1 ubiquitin E3 activity was not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identified substrates for BRCA1/BARD1. We found that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes replication fork stability epistatically to BRCA1 S114 phosphorylation, addressing the controversy about the function of BRCA1/BARD1 E3 activity in Homologous Recombination.

molecular biology↗

bioRxiv2022

Centriolar subdistal appendages promote double strand break repair through homologous recombination

The centrosome is a cytoplasmic organelle with roles in microtubule organization which has also been proposed to act as a hub for cellular signaling. Some centrosomal components are required for full activation of the DNA Damage Response. However, if the centrosome regulates specific DNA repair pathways is not known. Here, we show that centrosomes presence is required to fully activate recombination, specifically to completely license its initial step, the so-called DNA end resection. Furthermore, we identify a centriolar structure, the subdistal appendages, and a specific factor, CEP170, as the critical centrosomal component involved in the regulation of recombination and resection, albeit it does not control end-joining repair. Cells lacking centrosomes or depleted for CEP170 are, consequently, hyper-sensitive to DNA damaging agents. Moreover, low levels of CEP170 in multiple cancer types correlate with an increase of the mutation burden associated with specific mutational signatures and a better prognosis, suggesting that changes in CEP170 can act as a mutation driver but also could be targeted to improve current oncological treatments.

cell biology↗

bioRxiv2021

ADAR2-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution. One sentence summaryDNA recombination requires RNA editing of DNA:RNA hybrids to ease their melting and facilitate DNA end resection

genetics↗

bioRxiv2020

TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin stability

TRIM37 is an E3 ubiquitin ligase mutated in Mulibrey nanism, a disease characterized by impaired growth and increased tumorigenesis, whose cellular etiology is poorly understood. TRIM37 depletion from tissue culture cells results in supernumerary foci bearing the centriolar protein Centrin. Here, we characterized these centriolar protein assemblies (Cenpas) to uncover the mechanism of action of TRIM37. We established that an atypical de novo assembly pathway is notably involved in forming Cenpas, which can nevertheless trigger further centriole assembly and act as MTOCs. We found also that Cenpas are present and act similarly in Mulibrey patient cells. Through correlative light electron microscopy, we uncovered that Cenpas correspond to centriole related structures and elongated electron-dense structures with stripes. Importantly, we established that TRIM37 regulates the stability and solubility of the centriolar protein Centrobin. Our findings suggest that elongated Centrobin assemblies are a major constituent of the striped electron dense structures. Furthermore, we established that Cenpas formation upon TRIM37 depletion requires PLK4 activity, as well as two parallel pathways relying respectively on Centrobin and PLK1. Overall, our work uncovers how TRIM37 prevents the formation of Cenpas that would otherwise threaten genome integrity, including possibly in Mulibrey patients.

cell biology↗

bioRxiv2019

CtIP -mediated alternative mRNA splicing finetunes the DNA damage response

In order to survive to the exposure of DNA damaging agents, cells activate a complex response that coordinates the cellular metabolism, cell cycle progression and DNA repair. Among many other events, recent evidence has described global changes in mRNA splicing in cells treated with genotoxic agents. Here, we explore further this DNA damage-dependent alternative splicing. Indeed, we show that both the splicing factor SF3B2 and the repair protein CtIP contribute to the global pattern of splicing both in cells treated or not to DNA damaging agents. Additionally, we focus on a specific DNA damage- and CtIP-dependent alternative splicing event of the helicase PIF1 and explore its relevance for the survival of cells upon exposure to ionizing radiation. Indeed, we described how the nuclear, active form of PIF1 is substituted by a splicing variant, named vPIF1, in a fashion that requires both the presence of DNA damage and CtIP. Interestingly, timely expression of vPIF1 is required for optimal survival to exposure to DNA damaging agents, but early expression of this isoform delays early events of the DNA damage response. On the contrary, expression of the full length PIF1 facilitates those early events, but increases the sensitivity to DNA damaging agents if the expression is maintained long-term.

molecular biology↗

bioRxiv2019

PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks

Post-translational histone modifications and chromatin remodelling play a critical role in the mechanisms controlling the integrity of the genome. Here we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating the balance between DNA damage-induced focus formation by 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knock down leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knock down decreases CtIP and BRCA1 protein and mRNA levels and cells depleted of PHF2 display genome instability and are sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.

molecular biology↗

bioRxiv2018

RAD51 paralogs regulate double strand break repair pathway choice by limiting Ku complex retention

RAD51 paralogs are a group of conserved proteins in eukaryotes that are involved in the repair of DNA breaks at several levels. On one hand, they help the strand invasion step catalyzed by RAD51. Also, they play late roles in Holliday Junction metabolism. Here we uncover a new role of the RAD51 paralogs at an earlier event in the repair of broken chromosomes. All five RAD51 paralogs affect the balance between double strand break repair pathways. Specifically, they favor homology-mediated repair over non-homologous end-joining. Such role is independent of RAD51 or the checkpoint activity of these proteins. Moreover, it defines a novel control point of double strand break repair independent and subsequent to DNA-end resection initiation.\n\nMechanistically, RAD51 paralogs limit the retention of Ku80 at the sites of DNA breaks. Thus, our data extend the role of this family of proteins to the earliest event of double strand break repair.

molecular biology↗