Study of radiotherapy resistance associated to EGFR/MAPK signaling pathway and its application in the rational design of a treatment with simvastatin in combination with cetuximab and radiotherapy (in experimental models of carcinoma)

1. Generation and characterization of a radioresistant cell line In the first part of this project of Thesis, we subjected culture cells to fractionated radiation and clonal selection, to obtain a modified cohort of cells. The aim of this study was to develop an isogenic resistant cell line which could be used to identify molecular changes associated with acquired resistance to radiation and tumour aggressiveness in cancer. We developed the stable isogenic resistant A431-R cell line from parental A431 cells presented a diminution of the radiosensitivity (decreases in SF2 and α-component of LQ model). These changes were associated with an increased efficiency in DNA repair. We demonstrated that A431-R cells acquired higher cloning efficiency and faster growth and migration ability; these properties were observed to be associated with remarkable baseline levels of relevant oncoproteins and elevated angiogenic capabilities. 2. Development of a technique to irradiate mice bearing xenografts In the second part of this project of Thesis, we have reached four objectives during the design procedure described to administer fractionated radiotherapy to immunodeficient mice in the Radiotherapy Department of a Hospital. First, we have minimized irradiation of healthy tissues around the tumour. Second, we have fulfilled the standard irradiation principles for human treatments. Third, we reduced time and increase efficiency by irradiating several mice at a time. Finally, our procedure was easy to reproduce and repeat on a daily basis by only two people. We demonstrated the feasibility of fractionated irradiation using immunodeficient mice to evaluate the role of radiotherapy on experimental tumours simulating a clinical setting. 3. Simvastatin sensitizes to radiotherapy plus cetuximab In the third part of this project of Thesis, we pre-clinically explored whether a treatment regime involving the addition of simvastatin to XRT and C225 merits further research. We have shown that the addition of simvastatin significantly decreased proliferation and clonogenic survival of cells treated with XRT and C225. Moreover, we used an experimental model with tumor cells derived from squamous cell carcinoma of that suggests that simvastatin may increase the antitumor effect of XRT plus C225— at doses and fractions of XRT that mimic doses administered in the clinical setting. The addition of simvastatin was associated with an increase in apoptosis and a decrease in the levels of activated ERK1/2, AKT, and STAT3 oncoproteins, a set of observations that provide support to the higher anti-tumor effects produced by the triple treatment. An important consideration is that when we combined simvastatin with XRT + C225 in the treatment of the radioresistant generated A431-R, we observed that the initial resistance of these cells to the treatment is in part reverted. In fact, cell migration, cell proliferation and cell survival were decreased when simvastatin was combined to the treatment with XRT+C225. In addition, tumour growth was slowed down when simvastatin was added to XRT+C225 treatment. The model A431-WT/A431-R we have generated can be a useful tool to examine new treatments directed to revert radioresistance produced from a prior treatment with radiotherapy. Treatment effects can be studied in both cell sublines and comparison with the parental cells could give important informatio1. Generación de una línea celular con fenotipo radiorresitente Para el estudio de los posibles mecanismos implicados en la resistencia a radioterapia, se generó una línea celular resistente a radioterapia y se caracterizó: 1) Los tumores originados de las células resistentes mostraban un tamaño mayor al finalizar la radioterapia, mayor retraso del crecimiento y menor tasa de recrecimiento. 2) Aumento de la capacidad de migrar y reparar la herida. 3) Aumento de EGFR fosforilado basal y de AKT y ERK1/2. 4) Disminución de pERK1/2 tras irradiación (adquisición de una autosuficiencia de señales prosupervivencia). 5) Aumento de la secreción de VEGF en respuesta a radiación en las células radioresistentes (mayor angiogénesis asociada a tumor para resistir in vivo a la radioterapia. En conjunto, las cambios fenotípicos adquiridos son compatibles con la ganancia de un fenotipo agresivo asociados a la aparición de radiorresistencia. 2. Descripción de la metodología utilizada para irradiar ratones Debido a la falta de literatura existente sobre métodos para la irradiación de xenoimplantes tumorales, se diseñó un protocolo técnico adaptado a los recursos, sobre el que puede destacarse que: 1) Minimiza la irradiación de tejidos sanos. 2) Permite la irradiación según dosis y fraccionamientos estándar para el tratamiento humano. 3) Reduce el tiempo y aumenta la eficiencia de los experimentos irradiando varios ratones simultáneamente. 4) Es fácilmente reproducible. 5) Se puede exportar a otros centros para investigación en oncología radioterápica que dispongan de un acelerador lineal. 3. Estudio de la combinación de simvastatina con cetuximab y radioterapia En este estudio se ha explorado preclínicamente si un régimen de tratamiento que implique la adición de simvastatina al tratamiento de cetuximab y radioterapia merecería mayor atención. Los hallazgos experimentales sugieren que la simvastatina podría potenciar la respuesta antitumoral de la combinación concomitante de cetuximab y radioterapia. 1) Disminución de la proliferación. 2) Reducción de la tasa de migración celular. 3) Reduccuión de la supervivencia celular clonogénica. 4) Los tumores crecieron más despacio. La tasa de crecimiento de los tumores tratados con la estatina fue significativamente menor. 5) Incremento del número de células células apoptóticas in vitro e in vivo 6) Reducción de los niveles las proteínas ERK1/2, AKT y STAT3 en su forma activa fosforilada sin alterar los niveles totales

Estudos Artísticos

O 18º número da revista croma apresenta 15 artigos que prosseguem os objetivos editoriais desta revista. Trata-se de desafiar os autores ou criadores apresentar a obra de outros artistas preferencialmente originários dos países de expressão portuguesa ou espanhola.info:eu-repo/semantics/publishedVersio

An RNA polymerase III general transcription factor engages in cell type-specific chromatin looping

Transcription factors (TFs) bind DNA in a sequence-specific manner and are generally cell type-specific factors and/or developmental master regulators. In contrast, general TFs (GTFs) are part of very large protein complexes and serve for RNA polymerases' recruitment to promoter sequences, generally in a cell type-independent manner. Whereas, several TFs have been proven to serve as anchors for the 3D genome organization, the role of GTFs in genome architecture have not been carefully explored. Here, we used ChIP-seq and Hi-C data to depict the role of TFIIIC, one of the RNA polymerase III GTFs, in 3D genome organization. We find that TFIIIC genome occupancy mainly occurs at specific regions, which largely correspond to Alu elements; other characteristic classes of repetitive elements (REs) such as MIR, FLAM-C and ALR/alpha are also found depending on the cell's developmental origin. The analysis also shows that TFIIIC-enriched regions are involved in cell type-specific DNA looping, which does not depend on colocalization with the master architectural protein CTCF. This work extends previous knowledge on the role of TFIIIC as a bona fide genome organizer whose action participates in cell type-dependent 3D genome looping via binding to REs.This work was supported by Bando Galileo 2022 (G22-142) to R.F. and M.T. This work was also supported by the Spanish Ministry of Science and Innovation (PID2019-107185GB-I00) and Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya (2017SGR1163) to S.L. The CRG acknowledge the support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the support of the CERCA Programme/Generalitat de Cataluny

Unliganded progesterone receptor governs estrogen receptor gene expression by regulating DNA methylation in breast cancer cells

Breast cancer prognosis and response to endocrine therapy strongly depends on the expression of the estrogen and progesterone receptors (ER and PR, respectively). Although much is known about ERα gene (ESR1) regulation after hormonal stimulation, how it is regulated in hormone-free condition is not fully understood. We used ER-/PR-positive breast cancer cells to investigate the role of PR in ESR1 regulation in the absence of hormones. We show that PR binds to the low-methylated ESR1 promoter and maintains both gene expression and DNA methylation of the ESR1 locus in hormone-deprived breast cancer cells. Depletion of PR reduces ESR1 expression, with a concomitant increase in gene promoter methylation. The high amount of methylation in the ESR1 promoter of PR-depleted cells persists after the stable re-expression of PR and inhibits PR binding to this genomic region. As a consequence, the rescue of PR expression in PR-depleted cells is insufficient to restore ESR1 expression. Consistently, DNA methylation impedes PR binding to consensus progesterone responsive elements. These findings contribute to understanding the complex crosstalk between PR and ER and suggest that the analysis of ESR1 promoter methylation in breast cancer cells can help to design more appropriate targeted therapies for breast cancer patients.We received funding from the Spanish Ministry of Economy and Competitiveness, Plan Nacional Project SAF 2013-42497-P; Centro de Excelencia Severo Ochoa 2013–2017; the Centre de Recerca de Catalunya (CERCA) Programme/Generalitat de Catalunya; G.V. has received funding from the Spanish Ministry of Economy and Competitiveness, “Juan de la Cierva Incorporation” fellowship (Ref. IJCI-2014-20723), the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement Number 299429 and the European Molecular Biology Organization (EMBO long-term fellowship ALTF 1106-2011, cofunded with the European Commission EMBOCOFUND2010, GA-2010-267146)

Unliganded progesterone receptor governs estrogen receptor gene expression by regulating DNA methylation in breast cancer cells

Breast cancer prognosis and response to endocrine therapy strongly depends on the expression of the estrogen and progesterone receptors (ER and PR, respectively). Although much is known about ERα gene (ESR1) regulation after hormonal stimulation, how it is regulated in hormone-free condition is not fully understood. We used ER-/PR-positive breast cancer cells to investigate the role of PR in ESR1 regulation in the absence of hormones. We show that PR binds to the low-methylated ESR1 promoter and maintains both gene expression and DNA methylation of the ESR1 locus in hormone-deprived breast cancer cells. Depletion of PR reduces ESR1 expression, with a concomitant increase in gene promoter methylation. The high amount of methylation in the ESR1 promoter of PR-depleted cells persists after the stable re-expression of PR and inhibits PR binding to this genomic region. As a consequence, the rescue of PR expression in PR-depleted cells is insufficient to restore ESR1 expression. Consistently, DNA methylation impedes PR binding to consensus progesterone responsive elements. These findings contribute to understanding the complex crosstalk between PR and ER and suggest that the analysis of ESR1 promoter methylation in breast cancer cells can help to design more appropriate targeted therapies for breast cancer patients.We received funding from the Spanish Ministry of Economy and Competitiveness, Plan Nacional Project SAF 2013-42497-P; Centro de Excelencia Severo Ochoa 2013–2017; the Centre de Recerca de Catalunya (CERCA) Programme/Generalitat de Catalunya; G.V. has received funding from the Spanish Ministry of Economy and Competitiveness, “Juan de la Cierva Incorporation” fellowship (Ref. IJCI-2014-20723), the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement Number 299429 and the European Molecular Biology Organization (EMBO long-term fellowship ALTF 1106-2011, cofunded with the European Commission EMBOCOFUND2010, GA-2010-267146)

TFIIIC as a potential epigenetic modulator of histone acetylation in human stem cells

Regulation of histone acetylation dictates patterns of gene expression and hence cell identity. Due to their clinical relevance in cancer biology, understanding how human embryonic stem cells (hESCs) regulate their genomic patterns of histone acetylation is critical, but it remains largely to be investigated. Here, we provide evidence that acetylation of histone H3 lysine-18 (H3K18ac) and lysine-27 (H3K27ac) is only partially established by p300 in stem cells, while it represents the main histone acetyltransferase (HAT) for these marks in somatic cells. Our analysis reveals that whereas p300 marginally associated with H3K18ac and H3K27ac in hESCs, it largely overlapped with these histone marks upon differentiation. Interestingly, we show that H3K18ac is found at "stemness" genes enriched in RNA polymerase III transcription factor C (TFIIIC) in hESCs, whilst lacking p300. Moreover, TFIIIC was also found in the vicinity of genes involved in neuronal biology, although devoid of H3K18ac. Our data suggest a more complex pattern of HATs responsible for histone acetylations in hESCs than previously considered, suggesting a putative role for H3K18ac and TFIIIC in regulating "stemness" genes as well as genes associated with neuronal differentiation of hESCs. The results break ground for possible new paradigms for genome acetylation in hESCs that could lead to new avenues for therapeutic intervention in cancer and developmental diseases.This work was supported by Bando Galileo 2022 (G22-142) to R.F. and M.T. The research is also supported by the AIRC IG Grant 27712-A to R.F. This work was also supported by the Spanish Ministry of Science and Innovation (PID2019-107185GB-I00) to S.d.l.L. The CRG acknowledge the support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the support of the CERCA Programme/Generalitat de Catalunya. This work was also supported by the Ligue Contre le Cancer, committees des Landes et de la Dordogne to M.T

Stochastic particle unbinding modulates growth dynamics and size of transcription factor condensates in living cells

Liquid-liquid phase separation (LLPS) is emerging as a key physical principle for biological organization inside living cells, forming condensates that play important regulatory roles. Inside living nuclei, transcription factor (TF) condensates regulate transcriptional initiation and amplify the transcriptional output of expressed genes. However, the biophysical parameters controlling TF condensation are still poorly understood. Here we applied a battery of single-molecule imaging, theory, and simulations to investigate the physical properties of TF condensates of the progesterone receptor (PR) in living cells. Analysis of individual PR trajectories at different ligand concentrations showed marked signatures of a ligand-tunable LLPS process. Using a machine learning architecture, we found that receptor diffusion within condensates follows fractional Brownian motion resulting from viscoelastic interactions with chromatin. Interestingly, condensate growth dynamics at shorter times is dominated by Brownian motion coalescence (BMC), followed by a growth plateau at longer timescales that result in nanoscale condensate sizes. To rationalize these observations, we extended on the BMC model by including the stochastic unbinding of particles within condensates. Our model reproduced the BMC behavior together with finite condensate sizes at the steady state, fully recapitulating our experimental data. Overall, our results are consistent with condensate growth dynamics being regulated by the escaping probability of PR molecules from condensates. The interplay between condensation assembly and molecular escaping maintains an optimum physical condensate size. Such phenomena must have implications for the biophysical regulation of other nuclear condensates and could also operate in multiple biological scenarios.The research leading to these results has received funding from BIST-Ignite funding (PHASE-CHROM) (to C.R.-A. and J.A.T.-P.); the European Commission H2020 Program under grant agreement ERC Adv788546 (NANO-MEMEC) (to M.F.G.-P.), ERC AdG NOQIA and EU Horizon 2020 FET-OPEN OPTOlogic (Grant No 899794) (to M.L.), and ERC Synergy Grant 609989 (to M.B.); the Government of Spain (Severo Ochoa CEX2019-000910-S (to M.L. and to M.F.G.P.), JdC-IJCI-2017-33160 (to J.A.T.-P.), (PGC2018-097027-B-I00/10.13039/501100011033, CEX2019-000910-S/10.13039/501100011033 and QUSPIN RTC2019-007196-7) (to M.L.), the State Research Agency (FIDEUA PID2019-106901GB-I00/10.13039/501100011033) (to M.L.), PID2020-113068RB-I00/10.13039/501100011033 (to M.F.G.-P.) and (RYC-2017–22227 and PID2019-106232RB-I00/10.13039/501100011033) (to F.C.); QuantumCAT_U16-011424 (to M.L.), co-funded by the ERDF Operational Program of Catalonia 2014-2020; (QUANTERA MAQS (funded by the State Research Agency under PCI2019-111828-2/321 10.13039/501100011033) and QUANTERA DYNAMITE PCI2022-132919) (to M.L.); Barcelona Supercomputing Center MareNostrum (FI-2022-1-0042) (to M.L.); Obra Social La Caixa (LCF-ICFO) and the Austrian Science Fund (FWF) through SFB BeyondC F7102 (to G.M.-G.); National Science Centre, Poland (Symfonia Grant No. 2016/20/W/ST4/00314) (to M.L.); Fundació CELLEX (Barcelona); Fundació Mir-Puig; and the Generalitat de Catalunya through the CERCA program and AGAUR (grant no. 2017 SGR 1341 to M.L. and no. 2017SGR1000 to M.F.G.-P.)

A set of accessible enhancers enables the initial response of breast cancer cells to physiological progestin concentrations

Here, we report that in T47D breast cancer cells 50 pM progestin is sufficient to activate cell cycle entry and the progesterone gene expression program. At this concentration, equivalent to the progesterone blood levels found around the menopause, progesterone receptor (PR) binds only to 2800 genomic sites, which are accessible to ATAC cleavage prior to hormone exposure. These highly accessible sites (HAs) are surrounded by well-organized nucleosomes and exhibit breast enhancer features, including estrogen receptor alpha (ERα), higher FOXA1 and BRD4 (bromodomain containing 4) occupancy. Although HAs are enriched in RAD21 and CTCF, PR binding is the driving force for the most robust interactions with hormone-regulated genes. HAs show higher frequency of 3D contacts among themselves than with other PR binding sites, indicating colocalization in similar compartments. Gene regulation via HAs is independent of classical coregulators and ATP-activated remodelers, relying mainly on MAP kinase activation that enables PR nuclear engagement. HAs are also preferentially occupied by PR and ERα in breast cancer xenografts derived from MCF-7 cells as well as from patients, indicating their potential usefulness as targets for therapeutic intervention.Funding: Ministerio de Ciencia e Innovación [PID2019-105173RB-I00 and PID2019-110384GB-C21]; Spanish Ministry of Economy and Competitiveness [SAF2016-75006-P and G62426937]; Consejo Superior de Investigaciones Científicas [201820I131]; Centro de Excelencia Severo Ochoa [SEV-2012-2018]; European Research Council [609989]. Funding for open access charge: Ministerio de Ciencia e Innovació

TFIIIC binding to alu elements controls gene expression via chromatin looping and histone acetylation

How repetitive elements, epigenetic modifications, and architectural proteins interact ensuring proper genome expression remains poorly understood. Here, we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the activity-dependent neuroprotector homeobox Protein (ADNP) and located near cell-cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell-cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell-cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression.This work was supported by the Spanish Ministry of Economy and Competitiveness Centro de Excelencia Severo Ochoa 2013–2017 SEV-2012-0208 (to CRG) and BFU2016-76141-P (to S.d.l.L.); ACER (to CRG); Italian Association for Cancer Research (AIRC, grant IG16877 to G.D.); the Cancer Research UK Programme Foundation (CR-UK C47547/A21536 to A.V.); a Wellcome Trust Investigator Award (200818/Z/16/Z to A.V.); and the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP7/2007–2013/ERC Synergy grant agreement 609989-4DGenome to M.B.). We acknowledge the support of the Spanish Ministry of Science, Innovation, and Universities to the EMBL partnership, the CERCA Programme/Generalitat de Catalunya , and the Centro de Excelencia Severo Ochoa. The ERC provided funding for the open access charge. The proteomics analyses were performed at the CRG/UPF Proteomics Unit (part of the of Proteored, PRB3; supported by grant PT17/0019 [ISCIII and ERDF]). This work was also supported by the Ligue Contre le Cancer (to M.T.

Hormone-control regions mediate steroid receptor-dependent genome organization

In breast cancer cells, some topologically associating domains (TADs) behave as hormonal gene regulation units, within which gene transcription is coordinately regulated in response to steroid hormones. Here we further describe that responsive TADs contain 20- to 100-kb-long clusters of intermingled estrogen receptor (ESR1) and progesterone receptor (PGR) binding sites, hereafter called hormone-control regions (HCRs). In T47D cells, we identified more than 200 HCRs, which are frequently bound by unliganded ESR1 and PGR. These HCRs establish steady long-distance inter-TAD interactions between them and organize characteristic looping structures with promoters in their TADs even in the absence of hormones in ESR1+-PGR+ cells. This organization is dependent on the expression of the receptors and is further dynamically modulated in response to steroid hormones. HCRs function as platforms that integrate different signals, resulting in some cases in opposite transcriptional responses to estrogens or progestins. Altogether, these results suggest that steroid hormone receptors act not only as hormone-regulated sequence-specific transcription factors but also as local and global genome organizers.We received funding from the European Research Council under the European Union's Seventh Framework Program (FP7/2007–2013)/ERC Synergy grant agreement 609989 (4DGenome). The content of this manuscript reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein. We acknowledge support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013–2017’ and Plan Nacional (SAF2016-75006-P), as well as support of the CERCA Programme/Generalitat de Catalunya