Revistas
Revista:
JOURNAL FOR IMMUNOTHERAPY OF CANCER
ISSN:
2051-1426
Año:
2022
Vol.:
10
N°:
8
Págs.:
e004479
Background One of the main difficulties of adoptive cell therapies with chimeric antigen receptor (CAR)-T cells in solid tumors is the identification of specific target antigens. The tumor microenvironment can present suitable antigens for CAR design, even though they are not expressed by the tumor cells. We have generated a CAR specific for the splice variant extra domain A (EDA) of fibronectin, which is highly expressed in the tumor stroma of many types of tumors but not in healthy tissues. Methods EDA expression was explored in RNA-seq data from different human tumor types and by immunohistochemistry in paraffin-embedded tumor biopsies. Murine and human anti-EDA CAR-T cells were prepared using recombinant retro/lentiviruses, respectively. The functionality of EDA CAR-T cells was measured in vitro in response to antigen stimulation. The antitumor activity of EDA CAR-T cells was measured in vivo in C57BL/6 mice challenged with PM299L-EDA hepatocarcinoma cell line, in 129Sv mice-bearing F9 teratocarcinoma and in NSG mice injected with the human hepatocarcinoma cell line PLC. Results EDA CAR-T cells recognized and killed EDA-expressing tumor cell lines in vitro and rejected EDA-expressing tumors in immunocompetent mice. Notably, EDA CAR-T cells showed an antitumor effect in mice injected with EDA-negative tumor cells lines when the tumor stroma or the basement membrane of tumor endothelial cells express EDA. Thus, EDA CAR-T administration delayed tumor growth in immunocompetent 129Sv mice challenged with teratocarcinoma cell line F9. EDA CAR-T treatment exerted an antiangiogenic effect and significantly reduced gene signatures associated with epithelial-mesenchymal transition, collagen synthesis, extracellular matrix organization as well as IL-6-STAT5 and KRAS pathways. Importantly, the human version of EDA CAR, that includes the human 41BB and CD3 zeta endodomains, exerted strong antitumor activity in NSG mice challenged with the human hepatocarcinoma cell line PLC, which expresses EDA in the tumor stroma and the endothelial vasculature. EDA CAR-T cells exhibited a tropism for EDA-expressing tumor tissue and no toxicity was observed in tumor bearing or in healthy mice. Conclusions These results suggest that targeting the tumor-specific fibronectin splice variant EDA with CAR-T cells is feasible and offers a therapeutic option that is applicable to different types of cancer.
Revista:
JOURNAL FOR IMMUNOTHERAPY OF CANCER
ISSN:
2051-1426
Año:
2022
Vol.:
10
N°:
9
Págs.:
e004479corr1
Revista:
DISEASE MODELS & MECHANISMS
ISSN:
1754-8411
Año:
2022
Vol.:
15
N°:
1
Págs.:
dmm049137
There is a paucity of adequate mouse models and cell lines available to study lung squamous cell carcinoma (LUSC). We have generated and characterized two models of phenotypically different transplantable LUSC cell lines, i.e. UN-SCC679 and UN-SCC680, derived from A/J mice that had been chemically induced with N-nitroso-tris-chloroethylurea (NTCU). Furthermore, we genetically characterized and compared both LUSC cell lines by performing whole-exome and RNA sequencing. These experiments revealed similar genetic and transcriptomic patterns that may correspond to the classic LUSC human subtype. In addition, we compared the immune landscape generated by both tumor cells lines in vivo and assessed their response to immune checkpoint inhibition. The differences between the two cell lines are a good model for the remarkable heterogeneity of human squamous cell carcinoma. Study of the metastatic potential of these models revealed that both cell lines represent the organotropism of LUSC in humans, i.e. affinity to the brain, bones, liver and adrenal glands. In summary, we have generated valuable cell line tools for LUSC research, which recapitulates the complexity of the human disease.
Revista:
JOURNAL OF THORACIC ONCOLOGY
ISSN:
1556-0864
Año:
2022
Vol.:
17
N°:
12
Págs.:
1387 - 1403
Introduction: SCLC is an extremely aggressive subtype of lung cancer without approved targeted therapies. Here we identified YES1 as a novel targetable oncogene driving SCLC maintenance and metastasis.Methods: Association between YES1 levels and prognosis was evaluated in SCLC clinical samples. In vitro functional experiments for proliferation, apoptosis, cell cycle, and cytotoxicity were performed. Genetic and pharmacologic inhibition of YES1 was evaluated in vivo in cell-and patient -derived xenografts and metastasis. YES1 levels were eval-uated in mouse and patient plasma-derived exosomes.Results: Overexpression or gain/amplification of YES1 was identified in 31% and 26% of cases, respectively, across molecular subgroups, and was found as an independent predictor of poor prognosis. Genetic depletion of YES1 dramatically reduced cell proliferation, three-dimensional organoid formation, tumor growth, and distant metastasis, leading to extensive apoptosis and tumor regressions. Mechanistically, YES1-inhibited cells revealed alterations in the replisome and DNA repair processes, that conferred sensitivity to irradiation. Pharmacologic blockade with the novel YES1 inhibitor CH6953755 or dasatinib induced marked antitumor activity in organoid models and cell-and patient-derived xenografts. YES1 protein was detected in plasma exosomes from patients and mouse models, with levels matching those of tumors, suggesting that circulating YES1 could represent a biomarker for patient selection/ monitoring.Conclusions: Our results provide evidence that YES1 is a new druggable oncogenic target and biomarker to advance the clinical management of a subpopulation of patients with SCLC.
Autores:
Palanca-Ballester, C.; Rodríguez Casanova, A.; Torres, S.; et al.
Revista:
CANCERS
ISSN:
2072-6694
Año:
2021
Vol.:
13
N°:
12
Págs.:
3016
Simple Summary Apart from genetic changes, cancer is characterized by epigenetic alterations, which indicate modifications in the DNA (such as DNA methylation) and histones (such as methylation and acetylation), as well as gene expression regulation by non-coding (nc)RNAs. These changes can be used in biological fluids (liquid biopsies) for diagnosis, prognosis and prediction of cancer drug response. Although these alterations are not widely used as biomarkers in the clinical practice yet, increasing number of commercial kits and clinical trials are expected to prove that epigenetic changes are able to offer valuable information for cancer patients. Early alterations in cancer include the deregulation of epigenetic events such as changes in DNA methylation and abnormal levels of non-coding (nc)RNAs. Although these changes can be identified in tumors, alternative sources of samples may offer advantages over tissue biopsies. Because tumors shed DNA, RNA, and proteins, biological fluids containing these molecules can accurately reflect alterations found in cancer cells, not only coming from the primary tumor, but also from metastasis and from the tumor microenvironment (TME). Depending on the type of cancer, biological fluids encompass blood, urine, cerebrospinal fluid, and saliva, among others. Such samples are named with the general term "liquid biopsy" (LB).
Revista:
CANCERS
ISSN:
2072-6694
Año:
2021
Vol.:
13
N°:
11
Págs.:
2849
We have studied blood levels of cytokines/chemokines in patients with metastatic renal cell carcinoma treated with sunitinib or pazopanib, with the goal of identifying biomarkers that can predict efficacy and survival. We have found that high levels of CXCL10, CXCL11, HGF and IL-6 before treatment associate with poor prognosis in these patients. Moreover, these factors are correlated in patients with renal carcinoma, suggesting a coordinated expression and secretion. We have developed a prognostic signature including these factors that predicts very accurately prognosis. Our results may help defining better the group of renal cell carcinoma patients who may benefit from sunitinib/pazopanib.
Autores:
Anfray, C.; Mainini, F.; Digifico, E.; et al.
Revista:
JOURNAL FOR IMMUNOTHERAPY OF CANCER
ISSN:
2051-1426
Año:
2021
Vol.:
9
N°:
9
Págs.:
e002408
Background Tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the antitumoral efficacy of most treatments currently applied in the clinic. Previous studies have evaluated the antitumoral immune response triggered by (TLR) agonists, such as poly(I:C), imiquimod (R837) or resiquimod (R848) as monotherapies; however, their combination for the treatment of cancer has not been explored. This study investigates the antitumoral efficacy and the macrophage reprogramming triggered by poly(I:C) combined with R848 or with R837, versus single treatments. Methods TLR agonist treatments were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry using primary human and murine M-CSF-differentiated macrophages. Cytotoxic activity of TLR-treated macrophages toward cancer cells was evaluated with an in vitro functional assay by flow cytometry. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry, RT-qPCR, multispectral immunophenotyping, quantitative proteomic experiments, and protein-protein interaction analysis. Results Results demonstrated the higher efficacy of poly(I:C) combined with R848 versus single treatments or combined with R837 to polarize macrophages toward M1-like antitumor effectors in vitro.
Revista:
JOURNAL FOR IMMUNOTHERAPY OF CANCER
ISSN:
2051-1426
Año:
2021
Vol.:
9
N°:
3
Págs.:
e001496
Introduction The use of immune-checkpoint inhibitors has drastically improved the management of patients with non-small cell lung cancer (NSCLC), but innate and acquired resistances are hurdles needed to be solved. Immunomodulatory drugs that can reinvigorate the immune cytotoxic activity, in combination with antiprogrammed cell death 1 (PD-1) antibody, are a great promise to overcome resistance. We evaluated the impact of the SRC family kinases (SFKs) on NSCLC prognosis, and the immunomodulatory effect of the SFK inhibitor dasatinib, in combination with anti-PD-1, in clinically relevant mouse models of NSCLC. Methods A cohort of patients from University Clinic of Navarra (n=116) was used to study immune infiltrates by multiplex immunofluorescence (mIF) and YES1 protein expression in tumor samples. Publicly available resources (TCGA, Km Plotter, and CIBERSORT) were used to study patient's survival based on expression of SFKs and tumor infiltrates. Syngeneic NSCLC mouse models 393P and UNSCC680AJ were used for in vivo drug testing. Results Among the SFK members, YES1 expression showed the highest association with poor prognosis. Patients with high YES1 tumor levels also showed high infiltration of CD4+/FOXP3+ cells (regulatory T cells (Tregs)), suggesting an immunosuppressive phenotype. After testing for YES1 expression in a panel of murine cell lines, 393P and UNSCC680AJ were selected for in vivo studies. In the 393P model, dasatinib+anti-PD-1 treatment resulted in synergistic activity, with 87% tumor regressions and development of immunological memory that impeded tumor growth when mice were rechallenged. In vivo depletion experiments further showed that CD8+ and CD4+ cells are necessary for the therapeutic effect of the combination. The antitumor activity was accompanied by a very significant decrease in the number of Tregs, which was validated by mIF in tumor sections. In the UNSCC680AJ model, the antitumor effects of dasatinib+anti-PD-1 were milder but similar to the 393P model. In in vitro assays, we demonstrated that dasatinib blocks proliferation and transforming growth factor beta-driven conversion of effector CD4+ cells into Tregs through targeting of phospholymphocyte-specific protein tyrosine kinase and downstream effectors pSTAT5 and pSMAD3. Conclusions YES1 protein expression is associated with increased numbers of Tregs in patients with NSCLC. Dasatinib synergizes with anti-PD-1 to impair tumor growth in NSCLC experimental models. This study provides the preclinical rationale for the combined use of dasatinib and PD-1/programmed death-ligand 1 blockade to improve outcomes of patients with NSCLC.
Revista:
CANCERS
ISSN:
2072-6694
The development of predictive biomarkers of response to targeted therapies is an unmet clinical need for many antitumoral agents. Recent genome-wide loss-of-function screens, such as RNA interference (RNAi) and CRISPR-Cas9 libraries, are an unprecedented resource to identify novel drug targets, reposition drugs and associate predictive biomarkers in the context of precision oncology. In this work, we have developed and validated a large-scale bioinformatics tool named DrugSniper, which exploits loss-of-function experiments to model the sensitivity of 6237 inhibitors and predict their corresponding biomarkers of sensitivity in 30 tumor types. Applying DrugSniper to small cell lung cancer (SCLC), we identified genes extensively explored in SCLC, such as Aurora kinases or epigenetic agents. Interestingly, the analysis suggested a remarkable vulnerability to polo-like kinase 1 (PLK1) inhibition inCREBBP-mutant SCLC cells. We validated this association in vitro using four mutated and four wild-type SCLC cell lines and twoPLK1inhibitors (Volasertib and BI2536), confirming that the effect ofPLK1inhibitors depended on the mutational status ofCREBBP. Besides, DrugSniper was validated in-silico with several known clinically-used treatments, including the sensitivity of Tyrosine Kinase Inhibitors (TKIs) and Vemurafenib toFLT3andBRAFmutant cells, respectively. These findings show the potential of genome-wide loss-of-function screens to identify new personalized therapeutic hypotheses in SCLC and potentially in other tumors, which is a valuable starting point for further drug development and drug repositioning projects.
Revista:
MOLECULAR CELL
ISSN:
1097-2765
Año:
2020
Vol.:
80
N°:
6
Págs.:
1025 - 1038
The structural organization of chromosomes is a crucial feature that defines the functional state of genes and genomes. The extent of structural changes experienced by genomes of eukaryotic cells can be dramatic and spans several orders of magnitude. At the core of these changes lies a unique group of ATPases-the SMC proteins-that act as major effectors of chromosome behavior in cells. The Smc5/6 proteins play essential roles in the maintenance of genome stability, yet their mode of action is not fully understood. Here we show that the human Smc5/6 complex recognizes unusual DNA configurations and uses the energy of ATP hydrolysis to promote their compaction. Structural analyses reveal subunit interfaces responsible for the functionality of the Smc5/6 complex and how mutations in these regions may lead to chromosome breakage syndromes in humans. Collectively, our results suggest that the Smc5/6 complex promotes genome stability as a DNA micro-compaction machine.
Autores:
Zubeldia, Idoia G; Bleau, A. M; Redrado M; et al.
Revista:
EXPERIMENTAL CELL RESEARCH
ISSN:
0014-4827
Año:
2013
Vol.:
319
N°:
3
Págs.:
12-22
Colorectal cancer (CRC) frequently metastasizes to the liver, a phenomenon that involves the participation of transforming-growth-factor-beta(1) (TGF beta(1)). Blockade of the protumorigenic effects elicited by TGF beta(1) in advanced CRC could attenuate liver metastasis. We aimed in the present study to assess the antimetastatic effect of TGF beta(1)-blocking peptides P17 and P144, and to study mechanisms responsible for this activity in a mouse model. Colon adenocarcinoma cells expressing luciferase were pretreated with TGF beta(1) (Mc38-luc(TGF beta 1) cells), injected into the spleen of mice and monitored for tumor development. TGF beta(1) increased primary tumor growth and liver metastasis, whereas systemic treatment of mice with either P17 or P144 significantly reduced tumor burden (p < 0.01). In metastatic nodules, mitotic/apoptotic ratio, mesenchymal traits and angiogenesis (evaluated by CD-31, as well as circulating endothelial and progenitor cells) induced by TGF beta(1) were consistently reduced following injection of peptides. In vitro experiments revealed a direct effect of TGF beta(1) in Mc38 cells, which resulted in activation of Smad2, Smad3 and Smad1/5/8, and increased invasion and transendothelial migration, whereas blockade of TGF beta(1)-signaling reverted these features. Because TGF beta(1)-mediated epithelial -mesenchymal transition (EMT) has been suggested to induce a cancer stem cell (CSC) phenotype, we analyzed the ability of this cytokine to induce tumorsphere formation and the expression of CSC markers. In TGF beta(1)-treated cells, tumorspheres were enriched in CD44 and SOX2, which were diminished in the presence of P17. Our data provide a preclinical rationale to evaluate P17 and P144 as potential therapeutic options for the treatment of metastatic CRC. (c) 2012 Elsevier Inc. All rights reserved.
Revista:
INTEGRATIVE BIOLOGY
ISSN:
1093-4391
Año:
2013
Vol.:
5
N°:
2
Págs.:
402-413
Dyskerin is one of the three subunits of the telomerase ribonucleoprotein (RNP) complex. Very little is known about the role of dyskerin in the biology of the telomeres in cancer cells. In this study, we use a quantitative, multiscale 3D image-based in situ method and several molecular techniques to show that dyskerin is overexpressed in lung cancer cell lines. Furthermore, we show that dyskerin expression correlates with telomere length both at the cell population level--cells with higher dyskerin expression have short telomeres--and at the single cell level--the shortest telomeres of the cell are spatially associated with areas of concentration of dyskerin proteins. Using this in vitro model, we also show that exogenous increase in dyskerin expression confers resistance to telomere shortening caused by a telomerase inactivating drug. Finally, we show that resistance is achieved by the recovery of telomerase activity associated with dyskerin. In summary, using a novel multiscale image-based in situ method, we show that, in lung cancer cell lines, dyskerin responds to continuous telomere attrition by increasing the telomerase RNP activity, which in turn provides resistance to telomere shortening.
Revista:
MOLECULAR CANCER
ISSN:
1476-4598
Año:
2011
Vol.:
10
N°:
96
Págs.:
1 - 15
Background: Mortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs) are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC) cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312.
Results: The aldehyde dehydrogenase (ALDH) positive lung cancer cell fraction is enriched in markers of stemness and endowed with stem cell properties. ALDH+ CSCs display longer telomeres than the non-CSC population. Interestingly, MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in the whole tumor population. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (short-term effect) and through decrease in telomere length (long-term effect). Administration of this telomerase inhibitor (40 mg/kg) in the H460 xenograft model results in significant tumor shrinkage (70% reduction, compared to controls). Combination therapy consisting of irradiation (10Gy) plus administration of MST312 did not improve the therapeutic efficacy of the telomerase inhibitor alone. Treatment with MST312 reduces significantly the number of ALDH+ CSCs and their telomeric length in vivo.
Conclusions: We conclude that antitelomeric therapy using MST312 mainly targets lung CSCs and may represent a novel approach for effective treatment of lung cancer.
Revista:
Experimental Cell Research
ISSN:
0014-4827
Año:
2010
Vol.:
316
N°:
4
Págs.:
554 - 567
Vascular endothelial growth factor (VEGF) is overexpressed during the transition from prostate intraepithelial neoplasia (PIN) to invasive carcinoma. We have mimicked such a process in vitro using the PIN-like C3(1)/Tag-derived Pr-111 cell line, which expresses low levels of VEGF and exhibits very low tumorigenicity in vivo. Elevated expression of VEGF164 in Pr-111 cells led to a significant increase in tumorigenicity, invasiveness, proliferation rates and angiogenesis. Moreover, VEGF164 induced strong changes in cell morphology and cell transcriptome through an autocrine mechanism, with changes in TGF-beta1- and cytoskeleton-related pathways, among others. Further analysis of VEGF-overexpressing Pr-111 cells or following exogenous addition of recombinant VEGF shows acquisition of epithelial-mesenchymal transition (EMT) features, with an increased expression of mesenchymal markers, such as N-cadherin, Snail1, Snail2 (Slug) and vimentin, and a decrease in E-cadherin. Administration of VEGF led to changes in TGF-beta1 signaling, including reduction of Smad7 (TGF-beta inhibitory Smad), increase in TGF-betaR-II, and translocation of phospho-Smad3 to the nucleus. Our results suggest that increased expression of VEGF in malignant cells during the transition from PIN to invasive carcinoma leads to EMT through an autocrine loop, which would promote tumor cell invasion and motility. Therapeutic blockade of VEGF/TGF-beta1 in PIN lesions might impair not only tumor angiogenesis, but also the early dissemination of malignant cells outside the epithelial layer.
Nacionales y Regionales
Título:
GRANATE - GRUPO DE RADIOTERAPIA AVANZADA DE NAVARRA ¿ TERAPIA Y EFICACIA
Código de expediente:
0011-1411-2022-000066
Investigador principal:
Ana Patiño García
Financiador:
GOBIERNO DE NAVARRA
Convocatoria:
2022 GN PROYECTOS ESTRATEGICOS DE I+D 2022-2025
Fecha de inicio:
01/06/2022
Fecha fin:
31/12/2024
Importe concedido:
536.739,00€
Otros fondos:
-
Título:
Nuevas estrategias para el tratamiento del cáncer microcítico de pulmón basadas en la eliminación del microambiente inmunosupresor debido a factores intrínsecos de la célula tumora
Código de expediente:
PI22/01253
Investigador principal:
Alfonso Calvo González, Diego Serrano Tejero
Financiador:
INSTITUTO DE SALUD CARLOS III
Convocatoria:
2022 AES Proyectos de investigación
Fecha de inicio:
01/01/2023
Fecha fin:
31/12/2025
Importe concedido:
244.420,00€
Otros fondos:
-
Título:
2^2-INTRATARGET: nanomedicinas para la liberación de administrar anticuerpos
Código de expediente:
AC20/00117
Investigador principal:
Alfonso Calvo González
Financiador:
INSTITUTO DE SALUD CARLOS III
Convocatoria:
2020 AES Programación Conjunta Internacional
Fecha de inicio:
01/01/2021
Fecha fin:
31/12/2023
Importe concedido:
174.998,67€
Otros fondos:
-