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BACKGROUND: After resection, colorectal cancer liver metastases (CRLM) surrounded by a desmoplastic rim carry a better prognosis than the metastases replacing the adjacent liver. However, these histopathological growth patterns (HGPs) are insufficient to guide clinical decision-making. We explored whether the adaptive immune features of HGPs could refine prognostication. METHODS: From 276 metastases resected in 176 patients classified by HGPs, tissue microarrays were used to assess intratumoral T cells (CD3), antigen presentation capacity (MHC class I) and CD73 expression producing immunosuppressive adenosine. We tested correlations between these variables and patient outcomes. RESULTS: The 101 (57.4%) patients with dominant desmoplastic HGP had a median recurrence-free survival (RFS) of 17.1 months compared to 13.3 months in the 75 patients (42.6%) with dominant replacement HGP (p = 0.037). In desmoplastic CRLM, high vs. low CD73 was the only prognostically informative immune parameter and was associated with a median RFS of 12.3 months compared to 26.3, respectively (p = 0.010). Only in dominant replacement CRLM, we found a subgroup (n = 23) with high intratumoral MHC-I expression but poor CD3+ T cell infiltration, a phenotype associated with a short median RFS of 7.9 months. CONCLUSIONS: Combining the assessments of HGP and adaptive immune features in resected CRLM could help identify patients at risk of early recurrence.
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Neoplasias Colorrectales , Neoplasias Hepáticas , Neoplasias Colorrectales/patología , Hepatectomía , Humanos , Neoplasias Hepáticas/patología , PronósticoRESUMEN
[This corrects the article DOI: 10.3389/fimmu.2024.1355945.].
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The anticancer efficacy of Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate internalized through a sortilin-mediated process, was assessed in a triple-negative breast cancer-derived MDA-MB-231 immunocompromised xenograft tumor model where complete tumor regression was observed for more than 40 days after the last treatment. Surprisingly, immunohistochemistry analysis revealed high staining of STING, a master regulator in the cancer-immunity cycle. A weekly administration of TH1902 as a single agent in a murine B16-F10 melanoma syngeneic tumor model demonstrated superior tumor growth inhibition than did docetaxel. A net increase in CD45 leukocyte infiltration within TH1902-treated tumors, especially for tumor-infiltrating lymphocytes and tumor-associated macrophages was observed. Increased staining of perforin, granzyme B, and caspase-3 was suggestive of elevated cytotoxic T and natural killer cell activities. Combined TH1902/anti-PD-L1 treatment led to increases in tumor growth inhibition and median animal survival. TH1902 inhibited cell proliferation and triggered apoptosis and senescence in B16-F10 cells in vitro, while inducing several downstream effectors of the cGAS/STING pathway and the expression of MHC-I and PD-L1. This is the first evidence that TH1902 exerts its antitumor activity, in part, through modulation of the immune tumor microenvironment and that the combination of TH1902 with checkpoint inhibitors (anti-PD-L1) could lead to improved clinical outcomes.
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Antígeno B7-H1 , Nucleotidiltransferasas , Humanos , Ratones , Animales , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Muerte CelularRESUMEN
CCN4 (also known as WNT1-Inducible Signaling Pathway Protein 1 or WISP1) is a 367 amino acid, 40 kDa protein located on chromosome 8q24.1-8q24.3. Prior studies have provided support for a pro-inflammatory role for CCN4. We have shown recently that CCN4 expression is associated with advanced disease, epithelial-mesenchymal transition, and an inflamed tumor microenvironment in multiple solid tumors. We detail here the CCN4 tissue microarray immunofluorescence protocol related to these findings.
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Proteínas CCN de Señalización Intercelular , Neoplasias , Humanos , Proteínas CCN de Señalización Intercelular/genética , Proteínas CCN de Señalización Intercelular/metabolismo , Microambiente Tumoral , Transición Epitelial-Mesenquimal , Técnica del Anticuerpo FluorescenteRESUMEN
CD73 is an ectonucleotidase overexpressed on tumor cells that suppresses anti-tumor immunity. Accordingly, several CD73 inhibitors are currently being evaluated in the clinic, including in large randomized clinical trials. Yet, the tumor cell-intrinsic impact of CD73 remain largely uncharacterized. Using metabolomics, we discovered that CD73 significantly enhances tumor cell mitochondrial respiration and aspartate biosynthesis. Importantly, rescuing aspartate biosynthesis was sufficient to restore proliferation of CD73-deficient tumors in immune deficient mice. Seahorse analysis of a large panel of mouse and human tumor cells demonstrated that CD73 enhanced oxidative phosphorylation (OXPHOS) and glycolytic reserve. Targeting CD73 decreased tumor cell metabolic fitness, increased genomic instability and suppressed poly ADP ribose polymerase (PARP) activity. Our study thus uncovered an important immune-independent function for CD73 in promoting tumor cell metabolism, and provides the rationale for previously unforeseen combination therapies incorporating CD73 inhibition.
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Ácido Aspártico , Neoplasias , Humanos , Línea Celular Tumoral , Neoplasias/patología , Animales , RatonesRESUMEN
Inhibition of adenosine A2A receptor (A2AR) is a promising approach for cancer immunotherapy currently evaluated in several clinical trials. We here report that anti-obesogenic and anti-inflammatory functions of A2AR, however, significantly restrain hepatocellular carcinoma (HCC) development. Adora2a deletion in mice triggers obesity, non-alcoholic steatohepatitis (NASH), and systemic inflammation, leading to spontaneous HCC and promoting dimethylbenzyl-anthracene (DMBA)- or diethylnitrosamine (DEN)-induced HCC. Conditional Adora2a deletion reveals critical roles of myeloid and hepatocyte-derived A2AR signaling in restraining HCC by limiting hepatic inflammation and steatosis. Remarkably, the impact of A2AR pharmacological blockade on HCC development is dependent on pre-existing NASH. In support of our animal studies, low ADORA2A gene expression in human HCC is associated with cirrhosis, hepatic inflammation, and poor survival. Together, our study uncovers a previously unappreciated tumor-suppressive function for A2AR in the liver and suggests caution in the use of A2AR antagonists in patients with NASH and NASH-associated HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Enfermedad del Hígado Graso no Alcohólico , Humanos , Animales , Ratones , Carcinoma Hepatocelular/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Receptor de Adenosina A2A/genética , Neoplasias Hepáticas/genética , InflamaciónRESUMEN
The ectonucleotidases CD39 and CD73 catalyze extracellular ATP to immunosuppressive adenosine, and as such, represent potential cancer targets. We investigated biological impacts of CD39 and CD73 in pancreatic ductal adenocarcinoma (PDAC) by studying clinical samples and experimental mouse tumors. Stromal CD39 and tumoral CD73 expression significantly associated with worse survival in human PDAC samples and abolished the favorable prognostic impact associated with the presence of tumor-infiltrating CD8+ T cells. In mouse transplanted KPC tumors, both CD39 and CD73 on myeloid cells, as well as CD73 on tumor cells, promoted polarization of infiltrating myeloid cells towards an M2-like phenotype, which enhanced tumor growth. CD39 on tumor-specific CD8+ T cells and pancreatic stellate cells also suppressed IFNγ production by T cells. Although therapeutic inhibition of CD39 or CD73 alone significantly delayed tumor growth in vivo, targeting of both ectonucleotidases exhibited markedly superior antitumor activity. CD73 expression on human and mouse PDAC tumor cells also protected against DNA damage induced by gemcitabine and irradiation. Accordingly, large-scale pharmacogenomic analyses of human PDAC cell lines revealed significant associations between CD73 expression and gemcitabine chemoresistance. Strikingly, increased DNA damage in CD73-deficient tumor cells associated with activation of the cGAS-STING pathway. Moreover, cGAS expression in mouse KPC tumor cells was required for antitumor activity of the CD73 inhibitor AB680 in vivo. Our study, thus, illuminates molecular mechanisms whereby CD73 and CD39 seemingly cooperate to promote PDAC progression.
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Adenosina , Neoplasias Pancreáticas , Animales , Humanos , Ratones , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Adenosina/metabolismo , Apirasa , Linfocitos T CD8-positivos/metabolismo , Línea Celular , Neoplasias PancreáticasRESUMEN
Although inflammatory mechanisms driving hepatocellular carcinoma (HCC) have been proposed, the regulators of anticancer immunity in HCC remain poorly understood. We found that IL27 receptor (IL27R) signaling promotes HCC development in vivo. High IL27EBI3 cytokine or IL27RA expression correlated with poor prognosis for patients with HCC. Loss of IL27R suppressed HCC in vivo in two different models of hepatocarcinogenesis. Mechanistically, IL27R sig-naling within the tumor microenvironment restrains the cytotoxicity of innate cytotoxic lymphocytes. IL27R ablation enhanced their accumulation and activation, whereas depletion or functional impairment of innate cytotoxic cells abrogated the effect of IL27R disruption. Pharmacologic neutralization of IL27 signaling increased infiltration of innate cytotoxic lymphocytes with upregulated cytotoxic molecules and reduced HCC development. Our data reveal an unexpected role of IL27R signaling as an immunologic checkpoint regulating innate cytotoxic lymphocytes and promoting HCC of different etiologies, thus indicating a therapeutic potential for IL27 pathway blockade in HCC. SIGNIFICANCE: HCC, the most common form of liver cancer, is characterized by a poor survival rate and limited treatment options. The discovery of a novel IL27-dependent mechanism controlling anticancer cytotoxic immune response will pave the road for new treatment options for this devastating disease. This article is highlighted in the In This Issue feature, p. 1825.
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Antineoplásicos , Carcinoma Hepatocelular , Interleucina-27 , Neoplasias Hepáticas , Linfocitos T Citotóxicos , Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/inmunología , Humanos , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Interleucina-27/inmunología , Interleucinas/inmunología , Neoplasias Hepáticas/inmunología , Pronóstico , Receptores de Interleucina/inmunología , Transducción de Señal , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
EMSY links the BRCA2 pathway to sporadic breast/ovarian cancer. It encodes a nuclear protein that binds to the BRCA2 N-terminal domain implicated in chromatin/transcription regulation, but when sporadically amplified/overexpressed, increased EMSY level represses BRCA2 transactivation potential and induces chromosomal instability, mimicking the activity of BRCA2 mutations in the development of hereditary breast/ovarian cancer. In addition to chromatin/transcription regulation, EMSY may also play a role in the DNA-damage response, suggested by its ability to localize at chromatin sites of DNA damage/repair. This implies that EMSY overexpression may also repress BRCA2 in DNA-damage replication/checkpoint and recombination/repair, coordinated processes that also require its interacting proteins: PALB2, the partner and localizer of BRCA2; RPA, replication/checkpoint protein A; and RAD51, the inseparable recombination/repair enzyme. Here, using a well-characterized recombination/repair assay system, we demonstrate that a slight increase in EMSY level can indeed repress these two processes independently of transcriptional interference/repression. Since EMSY, RPA and PALB2 all bind to the same BRCA2 region, these findings further support a scenario wherein: (a) EMSY amplification may mimic BRCA2 deficiency, at least by overriding RPA and PALB2, crippling the BRCA2/RAD51 complex at DNA-damage and replication/transcription sites; and (b) BRCA2/RAD51 may coordinate these processes by employing at least EMSY, PALB2 and RPA. We extensively discuss the molecular details of how this can happen to ascertain its implications for a novel recombination mechanism apparently conceived as checkpoint rather than a DNA repair system for cell division, survival, death, and human diseases, including the tissue specificity of cancer predisposition, which may renew our thinking about targeted therapy and prevention.
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Proteína BRCA2/metabolismo , Inestabilidad Cromosómica/genética , Daño del ADN , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Recombinasa Rad51/metabolismo , Recombinación Genética , Proteínas Represoras/metabolismo , Transducción de Señal , Proteína BRCA2/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Replicación del ADN/genética , Humanos , Secuencias Invertidas Repetidas/genética , Modelos Biológicos , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Recombinasa Rad51/genética , Proteínas Represoras/genética , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Hydrolysis of extracellular ATP to adenosine (eADO) is an important immune checkpoint in cancer immunology. We here investigated the impact of the eADO pathway in high-grade serous ovarian cancer (HGSC) using multiparametric platforms. METHODS: We performed a transcriptomic meta-analysis of eADO-producing CD39 and CD73, an eADO signaling gene signature, immune gene signatures and clinical outcomes in approximately 1200 patients with HGSC. Protein expression, localization and prognostic impact of CD39, CD73 and CD8 were then performed on approximately 1000 cases on tissue microarray, and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry and single-cell RNA sequencing on a subset of patients. RESULTS: Concomitant CD39 and CD73 gene expression, as well as high levels of an eADO gene signature, were associated with worse prognosis in patients with HGSC, notably in the immunoregulatory molecular subtype, characterized by an immune-active microenvironment. CD39 was further associated with primary chemorefractory and chemoresistant human HGSC and platinum-based chemotherapy of murine HGSC was significantly more effective in CD39-deficient mice. At protein level, CD39 and CD73 were predominantly expressed by cancer-associated fibroblasts, and CD39 was expressed on severely exhausted, clonally expanded and putative tissue-resident memory TILs. CONCLUSIONS: Our study revealed the clinical, immunological, subtype-specific impacts of eADO signaling in HGSC, unveiled the chemoprotective effect of CD39 and supports the evaluation of eADO-targeting agents in patients with ovarian cancer.
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5'-Nucleotidasa/genética , Adenosina/metabolismo , Antígenos CD/metabolismo , Apirasa/genética , Apirasa/metabolismo , Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , Neoplasias Quísticas, Mucinosas y Serosas/genética , Neoplasias Ováricas/genética , Transcriptoma , 5'-Nucleotidasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antígenos CD/genética , Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Bases de Datos Genéticas , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Hidrólisis , Ratones Noqueados , Persona de Mediana Edad , Clasificación del Tumor , Neoplasias Quísticas, Mucinosas y Serosas/tratamiento farmacológico , Neoplasias Quísticas, Mucinosas y Serosas/inmunología , Neoplasias Quísticas, Mucinosas y Serosas/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/metabolismo , RNA-Seq , Transducción de Señal , Análisis de la Célula Individual , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Immune checkpoint blockade has not yet been effective in patients with mismatch repair proficient metastatic colorectal cancer. Targeting immunosuppressive metabolic pathways is being explored as a new immunotherapeutic approach. We assessed whether CD73, the rate limiting enzyme that catalyzes the degradation of extracellular AMP into immunosuppressive adenosine, could be an immunological determinant of colorectal liver metastases (CRLMs). By immunofluorescence on tissue microarrays, intratumoral CD73 expression (tCD73) was analyzed in 391 CRLMs resected in 215 patients, and soluble CD73 (sCD73) was measured by ELISA in the pre-operative serum of 193 patients. High tCD73 was associated with worse pathological features, such as multiple and larger CRLMs, and poorer pathologic response to pre-operative chemotherapy. The median time to recurrence and disease-specific survival after CRLM resection was significantly shorter in patients with high tCD73 (11.0 and 46.4 months, respectively) compared with low tCD73 (19.0 and 61.5 months, respectively). tCD73 was strongly associated with patient outcomes independently of clinicopathological variables. sCD73 did not correlate with tCD73. Patients with high levels of sCD73 also had shorter disease-specific survival. Our results suggested that CD73 in CRLMs may be prognostically informative and may help select patients more likely to respond to adenosine pathway blocking agents.
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Neoplasias Hepáticas , Neoplasias del Recto , Humanos , Neoplasias Hepáticas/cirugía , Recurrencia Local de Neoplasia , PronósticoRESUMEN
CD73 is a membrane-anchored ectoenzyme that degrades extracellular AMP into adenosine, a potent immunosuppressive factor. In physiological conditions, induction of the CD73-adenosine pathway acts as natural feedback mechanism to prevent excessive immune reactions and subsequent tissue damage. In the past few years, the CD73-adenosine pathway has emerged as a major immunosuppressive mechanism by which multiple types of cancer evade anti-tumor immunity. Research from our group and others have established that blocking the CD73-adenosine pathway represents a promising approach to improve cancer immunotherapy. In this context, an increasing number of research laboratories are becoming interested in CD73 biology and in the development/characterization of CD73 inhibitors. Implementation of simple, rapid and HTS-compatible assays to evaluate CD73 enzymatic active is a critical step for any laboratory willing to study the CD73-adenosine pathway. Over the years, we developed, optimized or adapted various methodologies to assess CD73 enzymatic activity using in vitro assays. In this chapter, we describe two different in vitro assays adapted to the measurement of CD73 enzymatic activity. Both assays are simple, robust, HTS-compatible and can be used in a cell-based fashion.
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5'-Nucleotidasa/análisis , Pruebas de Enzimas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Neoplasias/tratamiento farmacológico , 5'-Nucleotidasa/antagonistas & inhibidores , 5'-Nucleotidasa/metabolismo , Adenosina/inmunología , Adenosina/metabolismo , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Línea Celular Tumoral , Colorimetría/instrumentación , Colorimetría/métodos , Colorantes/química , Pruebas de Enzimas/instrumentación , Estudios de Factibilidad , Proteínas Ligadas a GPI/análisis , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Luciferasas/química , Luciferasas/metabolismo , Luminiscencia , Neoplasias/inmunología , Neoplasias/patología , Receptores Purinérgicos P1/inmunología , Receptores Purinérgicos P1/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/metabolismo , Colorantes de Rosanilina/química , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
The formation of new lymphatic vessels, or lymphangiogenesis, is a critical step of the tissue repair program. In pathological conditions involving chronic inflammation or tumorigenesis, this process is often dysregulated and can contribute to disease progression. Yet, lymphangiogenesis is still incompletely understood. In this study, we identified A2a adenosinergic signaling as an important regulator of inflammatory and tumor-associated lymphangiogenesis. Using Adora2a (A2a)-deficient mice, we demonstrated that A2a signaling was involved in the formation of new lymphatic vessels in the context of peritoneal inflammation. We also demonstrated that tumor-associated and sentinel lymph node lymphangiogenesis were impaired in A2a-deficient mice, protecting them from lymph node metastasis. Notably, A2a signaling in both hematopoietic and non-hematopoietic cells contributed to sentinel lymph node metastasis. In A2a-deficient tumor-draining lymph nodes, impaired lymphangiogenesis was associated with a reduced accumulation of B cells and decreased VEGF-C levels. Supporting a role for non-hematopoietic A2a signaling, we observed that primary murine lymphatic endothelial cells (LEC) predominantly expressed A2a receptor and that A2a signaling blockade altered LEC capillary tube formation in vitro. Finally, we observed that Adora2a, Nt5e and Entpd1 gene expression positively correlated with Lyve1, Pdpn and Vegfc in several human cancers, thereby supporting the notion that adenosine production and A2a receptor activation might promote lymphangiogenesis in human tumors. In conclusion, our study highlights a novel pathway regulating lymphangiogenesis and further supports the use of A2a or adenosine blocking agents to inhibit pathological lymphangiogenesis in cancers and block the dissemination of tumor cells through the lymphatic system.
RESUMEN
The breast/ovarian cancer susceptibility proteins BRCA1 and BRCA2 maintain genome stability, at least in part, through a functional role in DNA damage repair. They both colocalize with RAD51 at sites of DNA damage/replication and activate RAD51-mediated homologous recombination repair of DNA double-strand breaks (DSB). Whereas BRCA2 interacts directly with and regulates RAD51, the role of BRCA1 in this process is unclear. However, BRCA1 may regulate RAD51 in response to DNA damage or through its ability to interact with and regulate MRE11/RAD50/NBS1 (MRN) during the processing of DSBs into single-strand DNA (ssDNA) ends, prerequisite substrates for RAD51, or both. To test these hypotheses, we measured the effect of BRCA1 on the competition between RAD51-mediated homologous recombination (gene conversion and crossover) versus RAD51-independent homologous recombination [single-strand annealing (SSA)] for ssDNA at a site-specific chromosomal DSB within a DNA repeat, a substrate for both homologous recombination pathways. Expression of wild-type BRCA1 in BRCA1-deficient human recombination reporter cell lines promoted both gene conversion and SSA but greatly enhanced gene conversion. In addition, BRCA1 also suppressed both spontaneous gene conversion and deletion events, which can arise from either crossover or sister chromatid replication slippage (SCRS), a RAD51-independent process. BRCA1 does not seem to block crossover. From these results, we conclude that (a) BRCA1 regulates RAD51 function in response to the type of DNA damage and (b) BRCA1 suppresses SCRS, suggesting a role for this protein in sister chromatid cohesion/alignment. Loss of such control in response to estrogen-induced DNA damage after BRCA1 inactivation may be a key initial event that triggers genome instability and carcinogenesis.
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Proteína BRCA1/farmacología , Neoplasias de la Mama/genética , Daño del ADN/genética , Inestabilidad Genómica , Recombinasa Rad51/metabolismo , Recombinación Genética , Intercambio de Cromátides Hermanas , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Intercambio Genético , ADN/genética , ADN/metabolismo , Reparación del ADN/genética , Replicación del ADN , Femenino , Conversión Génica , Humanos , Recombinasa Rad51/genéticaRESUMEN
BRCA2 has been implicated in the maintenance of genome stability and RAD51-mediated homologous recombination repair of chromosomal double-strand breaks (DSBs), but its role in these processes is unclear. To gain more insight into its role in homologous recombination, we expressed wild-type BRCA2 in the well-characterized BRCA2-deficient human cell line CAPAN-1 containing, as homologous recombination substrates, either direct or inverted repeats of two inactive marker genes. Whereas direct repeats monitor a mixture of RAD51-dependent and RAD51-independent homologous recombination events, inverted repeats distinguish between these events by reporting RAD51-dependent homologous recombination, gene conversion, and crossover events only. At either repeats, BRCA2 decreases the rate and frequency of spontaneous homologous recombination, but following chromosomal DSBs, BRCA2 increases the frequency of homologous recombination. At direct repeats, BRCA2 suppresses both spontaneous gene conversion and deletions, which can arise either from crossover or RAD51-independent sister chromatid replication slippage (SCRS), but following chromosomal DSBs, BRCA2 highly promotes gene conversion with little effect on deletions. At inverted repeats, spontaneous or DSB-induced crossover events were scarce and BRCA2 does not suppress their formation. From these results, we conclude that (i) BRCA2 regulates RAD51 recombination in response to the type of DNA damage and (ii) BRCA2 suppresses SCRS, suggesting a role for BRCA2 in sister chromatids cohesion and/or alignment. Loss of such control in response to estrogen-induced DNA damage after BRCA2 inactivation may be a key initial event triggering genome instability and carcinogenesis.
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Adenocarcinoma/genética , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Daño del ADN/genética , Neoplasias Pancreáticas/genética , Recombinación Genética/genética , Adenocarcinoma/metabolismo , Proteína BRCA2/biosíntesis , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Rotura Cromosómica , Reparación del ADN/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Genes BRCA2 , Genoma Humano , Humanos , Neoplasias Pancreáticas/metabolismo , Recombinasa Rad51RESUMEN
Mutations in the mismatch repair (MMR) genes hMSH2 and hMLH1 have been associated with hereditary nonpolyposis colorectal cancer. Tumor cell lines that are deficient in MMR exhibit a high mutation rate, a defect in the response to certain types of DNA damage and in transcription-coupled repair, as well as an increase in the rate of gene amplification. We show here that hMSH2-deficient tumor cell lines lost most of their ability to accurately repair plasmid DNA double-strand breaks (DSBs) by homologous recombination, compared with MMR-proficient or hMLH1-deficient tumor cell lines. In all of these cell lines, DSB repair occurred almost exclusively by nonreciprocal homologous recombination: gene conversion (GC). However, there were two types of GC products: precise and rearranged. The rearranged products contained deletions or insertions of sequences and represented GC intermediates trapped at various stages and shunted to nonhomologous end joining. In MMR-proficient or MLH1-deficient cells, >50% of GC products were of the precise type, whereas in two MSH2-deficient backgrounds, this proportion decreased to 8%, whereas that of rearranged GC products increased by 2-fold. These results seem to predict a novel way by which MSH2-deficiency could promote mutation: deletion or insertion mutations associated with DSB repair, which may also contribute to cancer predisposition.
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Disparidad de Par Base/genética , Reparación del ADN/genética , Proteínas de Unión al ADN , Conversión Génica , Mutagénesis , Proteínas Proto-Oncogénicas/deficiencia , Recombinación Genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Replicación del ADN , ADN Bacteriano/metabolismo , Predisposición Genética a la Enfermedad , Humanos , Proteína 2 Homóloga a MutS , Mutagénesis Insercional , Plásmidos/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Eliminación de Secuencia , Células Tumorales Cultivadas/metabolismoRESUMEN
The cell surface nucleotidase CD73 is an immunosuppressive enzyme involved in tumor progression and metastasis. Although preclinical studies suggest that CD73 can be targeted for cancer treatment, the clinical impact of CD73 in ovarian cancer remains unclear. In this study, we investigated the prognostic value of CD73 in high-grade serous (HGS) ovarian cancer using gene and protein expression analyses. Our results demonstrate that high levels of CD73 are significantly associated with shorter disease-free survival and overall survival in patients with HGS ovarian cancer. Furthermore, high levels of CD73 expression in ovarian tumor cells abolished the good prognosis associated with intraepithelial CD8(+) cells. Notably, CD73 gene expression was highest in the C1/stromal molecular subtype of HGS ovarian cancer and positively correlated with an epithelial-to-mesenchymal transition gene signature. Moreover, in vitro studies revealed that CD73 and extracellular adenosine enhance ovarian tumor cell growth as well as expression of antiapoptotic BCL-2 family members. Finally, in vivo coinjection of ID8 mouse ovarian tumor cells with mouse embryonic fibroblasts showed that CD73 expression in fibroblasts promotes tumor immune escape and thereby tumor growth. In conclusion, our study highlights a role for CD73 as a prognostic marker of patient survival and also as a candidate therapeutic target in HGS ovarian cancers.
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5'-Nucleotidasa/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/mortalidad , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/mortalidad , 5'-Nucleotidasa/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos CD/metabolismo , Apirasa/metabolismo , Biomarcadores de Tumor/genética , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia sin Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Fibroblastos/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Escape del Tumor/inmunologíaRESUMEN
Humans heterozygous for BRCA1 mutations have a high risk of losing the remaining wild-type BRCA1 allele and developing breast/ovarian cancer, but a molecular basis for this has not yet been determined. It is thought that heterozygosity status-reduced wild-type BRCA1 protein dosage (haploinsufficiency) and/or the presence of a mutant BRCA1 protein-may affect BRCA1 functions and heighten the risk of cancer promoting mutations. BRCA1 maintains genome stability, at least in part, by regulating homologous recombination according to the type of DNA damage. To investigate whether this BRCA1 function is affected by heterozygosity status, we employed, as recombination reporters, human breast cancer MCF-7 cells known to have a single wild-type BRCA1 allele and reduced BRCA1 protein dosage. These cells revealed: (1) a spontaneous hyper-recombination phenotype; (2) reduced efficiency in homologous recombination repair of DNA double-strand breaks (DSBs); and (3) sensitivity to the DSB-inducing chemotherapeutic agent mitomycin C. Correction of BRCA1 protein dosage to the wild-type level reversed all these phenotypes, whereas physiological expression of the cancer-eliciting BRCA1 5382insC mutant allele had no effect on either phenotype. These findings implicate BRCA1 C-terminal domain in recombination control, and indicate that BRCA1 haploinsufficiency alone, which is also a feature of sporadic breast/ovarian cancer, is sufficient to compromise genome stability by triggering spontaneous recombination events that are likely to account for the loss of the remaining wild-type BRCA1 allele and increased cancer risk. Our observations may also have implications for the medical management of cancer patients and cancer prevention.
Asunto(s)
Proteína BRCA1/genética , Codón sin Sentido/fisiología , Recombinación Genética/genética , Proteína BRCA1/química , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Humanos , Pérdida de Heterocigocidad , Modelos Genéticos , Estructura Terciaria de ProteínaRESUMEN
In addition to its well established role in the maintenance of genome integrity by regulating transcription of genes involved in cell cycle arrest and programmed cell death, the tumour suppressor p53 has also been shown to inhibit spontaneous chromosomal homologous recombination (HR) between adjacent transcription units, raising the possibility that p53 may prevent chromosomal rearrangements by suppressing HR between repetitive DNA elements (ectopic HR). Consistent with its role in the maintenance of genome integrity is that p53 does not suppress HR between homologous chromosomes (allelic HR) or identical sister chromatids, raising the question of how p53 discriminates between ectopic and allelic HR events. Here, we report that disruption of human p53 by the viral oncoprotein HPV16-E6 does not result in increased rates of chromosomal HR between adjacent DNA repeats in a transcriptional interference-free assay system in which a HR reporter gene can escape transcription repression. These results argue against a direct role for p53 in the regulation of HR mechanisms, imply that HR assay systems may be important determinants of the outcome, and suggest that p53 may suppress ectopic HR through its known ability to repress transcription and alter chromatin structure.