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1.
Cell ; 178(1): 160-175.e27, 2019 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-31155233

RESUMEN

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.


Asunto(s)
Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Microambiente Tumoral , Animales , Proliferación Celular , Técnicas de Cocultivo , Transición Epitelial-Mesenquimal , Femenino , Células HEK293 , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Quinasas Activadas por Mitógenos/metabolismo , RNA-Seq , Factor de Transcripción STAT3/metabolismo , Células del Estroma/metabolismo , Transfección
2.
Blood ; 139(18): 2770-2781, 2022 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-35226739

RESUMEN

Small ubiquitin-like modifier (SUMO) is a member of a ubiquitin-like protein superfamily. SUMOylation is a reversible posttranslational modification that has been implicated in the regulation of various cellular processes including inflammatory responses and expression of type 1 interferons (IFN1). In this report, we have explored the activity of the selective small molecule SUMOylation inhibitor subasumstat (TAK-981) in promoting antitumor innate immune responses. We demonstrate that treatment with TAK-981 results in IFN1-dependent macrophage and natural killer (NK) cell activation, promoting macrophage phagocytosis and NK cell cytotoxicity in ex vivo assays. Furthermore, pretreatment with TAK-981 enhanced macrophage phagocytosis or NK cell cytotoxicity against CD20+ target cells in combination with the anti-CD20 antibody rituximab. In vivo studies demonstrated enhanced antitumor activity of TAK-981 and rituximab in CD20+ lymphoma xenograft models. Combination of TAK-981 with anti-CD38 antibody daratumumab also resulted in enhanced antitumor activity. TAK-981 is currently being studied in phase 1 clinical trials (#NCT03648372, #NCT04074330, #NCT04776018, and #NCT04381650; www.clinicaltrials.gov) for the treatment of patients with lymphomas and solid tumors.


Asunto(s)
Linfoma , Sumoilación , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD20 , Línea Celular Tumoral , Humanos , Células Asesinas Naturales , Linfoma/tratamiento farmacológico , Linfoma/metabolismo , Macrófagos/metabolismo , Rituximab/metabolismo , Rituximab/farmacología , Rituximab/uso terapéutico
3.
Nat Chem Biol ; 13(11): 1164-1171, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28892090

RESUMEN

Small ubiquitin-like modifier (SUMO) family proteins regulate target-protein functions by post-translational modification. However, a potent and selective inhibitor targeting the SUMO pathway has been lacking. Here we describe ML-792, a mechanism-based SUMO-activating enzyme (SAE) inhibitor with nanomolar potency in cellular assays. ML-792 selectively blocks SAE enzyme activity and total SUMOylation, thus decreasing cancer cell proliferation. Moreover, we found that induction of the MYC oncogene increased the ML-792-mediated viability effect in cancer cells, thus indicating a potential application of SAE inhibitors in treating MYC-amplified tumors. Using ML-792, we further explored the critical roles of SUMOylation in mitotic progression and chromosome segregation. Furthermore, expression of an SAE catalytic-subunit (UBA2) S95N M97T mutant rescued SUMOylation loss and the mitotic defect induced by ML-792, thus confirming the selectivity of ML-792. As a potent and selective SAE inhibitor, ML-792 provides rapid loss of endogenously SUMOylated proteins, thereby facilitating novel insights into SUMO biology.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/antagonistas & inhibidores , Sumoilación , Proliferación Celular/efectos de los fármacos , Segregación Cromosómica/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes myc , Humanos , Mitosis/efectos de los fármacos , Neoplasias/genética , Neoplasias/patología , Procesamiento Proteico-Postraduccional , Células Tumorales Cultivadas
4.
Nature ; 487(7408): 510-3, 2012 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-22763454

RESUMEN

Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. Although these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single-molecule RNA sequencing, identifying Wnt2 as a candidate gene enriched in CTCs. Expression of WNT2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. This effect is correlated with fibronectin upregulation and suppressed by inhibition of MAP3K7 (also known as TAK1) kinase. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple WNT genes, and pancreatic CTCs revealed enrichment for WNT signalling in 5 out of 11 cases. Thus, molecular analysis of CTCs may identify candidate therapeutic targets to prevent the distal spread of cancer.


Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Metástasis de la Neoplasia/genética , Células Neoplásicas Circulantes/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/genética , Animales , Supervivencia Celular , Inhibición de Contacto , Modelos Animales de Enfermedad , Genes Relacionados con las Neoplasias/genética , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Ratones , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Análisis de Secuencia de ARN , Proteínas Wnt/genética , Proteína wnt2/genética , Proteína wnt2/metabolismo
5.
Proc Natl Acad Sci U S A ; 112(49): 15148-53, 2015 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-26575630

RESUMEN

Aberrant transcription of the pericentromeric human satellite II (HSATII) repeat is present in a wide variety of epithelial cancers. In deriving experimental systems to study its deregulation, we observed that HSATII expression is induced in colon cancer cells cultured as xenografts or under nonadherent conditions in vitro, but it is rapidly lost in standard 2D cultures. Unexpectedly, physiological induction of endogenous HSATII RNA, as well as introduction of synthetic HSATII transcripts, generated cDNA intermediates in the form of DNA/RNA hybrids. Single molecule sequencing of tumor xenografts showed that HSATII RNA-derived DNA (rdDNA) molecules are stably incorporated within pericentromeric loci. Suppression of RT activity using small molecule inhibitors reduced HSATII copy gain. Analysis of whole-genome sequencing data revealed that HSATII copy number gain is a common feature in primary human colon tumors and is associated with a lower overall survival. Together, our observations suggest that cancer-associated derepression of specific repetitive sequences can promote their RNA-driven genomic expansion, with potential implications on pericentromeric architecture.


Asunto(s)
Centrómero/genética , ADN Satélite/genética , Neoplasias/genética , Secuencias Repetitivas de Ácidos Nucleicos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Humanos , Hibridación de Ácido Nucleico , ARN/genética
6.
Biochemistry ; 52(34): 5920-7, 2013 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-23906287

RESUMEN

Inteins are intervening polypeptides that catalyze their own removal from flanking exteins, concomitant to the ligation of the exteins. The intein that interrupts the DP2 (large) subunit of DNA polymerase II from Methanoculleus marisnigri (Mma) can promote protein splicing. However, protein splicing can be prevented or reduced by overexpression under nonreducing conditions because of the formation of a disulfide bond between two internal intein Cys residues. This redox sensitivity leads to differential activity in different strains of E. coli as well as in different cell compartments. The redox-dependent control of in vivo protein splicing in an intein derived from an anaerobe that can occupy multiple environments hints at a possible physiological role for protein splicing.


Asunto(s)
Disulfuros/farmacología , Inteínas/genética , Empalme de Proteína/genética , Cisteína/química , ADN Polimerasa II/genética , Electroforesis en Gel de Poliacrilamida , Exteínas/genética , Oxidación-Reducción , Empalme de Proteína/efectos de los fármacos , Espectrometría de Masas en Tándem
7.
Sci Transl Med ; 13(611): eaba7791, 2021 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-34524860

RESUMEN

SUMOylation, the covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to protein substrates, has been reported to suppress type I interferon (IFN1) responses. TAK-981, a selective small-molecule inhibitor of SUMOylation, pharmacologically reactivates IFN1 signaling and immune responses against cancers. In vivo treatment of wild-type mice with TAK-981 up-regulated IFN1 gene expression in blood cells and splenocytes. Ex vivo treatment of mouse and human dendritic cells promoted their IFN1-dependent activation, and vaccination studies in mice demonstrated stimulation of antigen cross-presentation and T cell priming in vivo. TAK-981 also directly stimulated T cell activation, driving enhanced T cell sensitivity and response to antigen ex vivo. Consistent with these observations, TAK-981 inhibited growth of syngeneic A20 and MC38 tumors in mice, dependent upon IFN1 signaling and CD8+ T cells, and associated with increased intratumoral T and natural killer cell number and activation. Combination of TAK-981 with anti-PD1 or anti-CTLA4 antibodies improved the survival of mice bearing syngeneic CT26 and MC38 tumors. In conclusion, TAK-981 is a first-in-class SUMOylation inhibitor that promotes antitumor immune responses through activation of IFN1 signaling. TAK-981 is currently being studied in phase 1 clinical trials (NCT03648372, NCT04074330, NCT04776018, and NCT04381650) for the treatment of patients with solid tumors and lymphomas.


Asunto(s)
Inmunidad , Sumoilación
8.
Biomaterials ; 93: 71-82, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27082874

RESUMEN

Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating and painful cancers. It is often highly resistant to therapy owing to inherent chemoresistance and the desmoplastic response that creates a barrier of fibrous tissue preventing transport of chemotherapeutics into the tumor. The growth of the tumor in pancreatic cancer often leads to invasion of other organs and partial or complete biliary obstruction, inducing intense pain for patients and necessitating tumor resection or repeated stenting. Here, we have developed a delivery device to provide enhanced palliative therapy for pancreatic cancer patients by providing high concentrations of chemotherapeutic compounds locally at the tumor site. This treatment could reduce the need for repeated procedures in advanced PDAC patients to debulk the tumor mass or stent the obstructed bile duct. To facilitate clinical translation, we created the device out of currently approved materials and drugs. We engineered an implantable poly(lactic-co-glycolic)-based biodegradable device that is able to linearly release high doses of chemotherapeutic drugs for up to 60 days. We created five patient-derived PDAC cell lines and tested their sensitivity to approved chemotherapeutic compounds. These in vitro experiments showed that paclitaxel was the most effective single agent across all cell lines. We compared the efficacy of systemic and local paclitaxel therapy on the patient-derived cell lines in an orthotopic xenograft model in mice (PDX). In this model, we found up to a 12-fold increase in suppression of tumor growth by local therapy in comparison to systemic administration and reduce retention into off-target organs. Herein, we highlight the efficacy of a local therapeutic approach to overcome PDAC chemoresistance and reduce the need for repeated interventions and biliary obstruction by preventing local tumor growth. Our results underscore the urgent need for an implantable drug-eluting platform to deliver cytotoxic agents directly within the tumor mass as a novel therapeutic strategy for patients with pancreatic cancer.


Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Neoplasias Pancreáticas/tratamiento farmacológico , Adenocarcinoma/patología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Ratones , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Neoplasias Pancreáticas/patología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto
9.
Cell Rep ; 8(6): 1905-1918, 2014 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-25242334

RESUMEN

Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.


Asunto(s)
Matriz Extracelular/genética , Regulación Neoplásica de la Expresión Génica , Células Neoplásicas Circulantes/metabolismo , Neoplasias Pancreáticas/patología , Familia de Aldehído Deshidrogenasa 1 , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Movimiento Celular , Matriz Extracelular/metabolismo , Humanos , Ratones , Osteonectina/antagonistas & inhibidores , Osteonectina/genética , Osteonectina/metabolismo , Neoplasias Pancreáticas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Retinal-Deshidrogenasa/genética , Retinal-Deshidrogenasa/metabolismo , Análisis de Secuencia de ARN , Células Tumorales Cultivadas
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