RESUMEN
Disseminated tumor cells (dTCs) can frequently be detected in the bone marrow (BM) of colorectal cancer (CRC) patients, raising the possibility that the BM serves as a reservoir for metastatic tumor cells. Identification of dTCs in BM aspirates harbors the potential of assessing therapeutic outcome and directing therapy intensity with limited risk and effort. Still, the functional and prognostic relevance of dTCs is not fully established. We have previously shown that CRC cell clones can be traced to the BM of mice carrying patient-derived xenografts. However, cellular interactions, proliferative state and tumorigenicity of dTCs remain largely unknown. Here, we applied a coculture system modeling the microvascular niche and used immunofluorescence imaging of the murine BM to show that primary CRC cells migrate toward endothelial tubes. dTCs in the BM were rare, but detectable in mice with xenografts from most patient samples (8/10) predominantly at perivascular sites. Comparable to primary tumors, a substantial fraction of proliferating dTCs was detected in the BM. However, most dTCs were found as isolated cells, indicating that dividing dTCs rather separate than aggregate to metastatic clones-a phenomenon frequently observed in the microvascular niche model. Clonal tracking identified subsets of self-renewing tumor-initiating cells in the BM that formed tumors out of BM transplants, including one subset that did not drive primary tumor growth. Our results indicate an important role of the perivascular BM niche for CRC cell dissemination and show that dTCs can be a potential source for tumor relapse and tumor heterogeneity.
Asunto(s)
Médula Ósea/patología , Neoplasias Colorrectales/patología , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Mesenquimatosas/citología , Células Neoplásicas Circulantes/patología , Células Tumorales Cultivadas/citología , Animales , Médula Ósea/metabolismo , Rastreo Celular , Técnicas de Cocultivo , Neoplasias Colorrectales/metabolismo , Proteínas Fluorescentes Verdes/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Células Neoplásicas Circulantes/metabolismo , Imagen Óptica , Pronóstico , Nicho de Células Madre , Imagen de Lapso de Tiempo , Células Tumorales Cultivadas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Patient-derived cancer xenografts (PDX) are widely used to identify and evaluate novel therapeutic targets, and to test therapeutic approaches in preclinical mouse avatar trials. Despite their widespread use, potential caveats of PDX models remain considerably underappreciated. Here, we demonstrate that EBV-associated B-lymphoproliferations frequently develop following xenotransplantation of human colorectal and pancreatic carcinomas in highly immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice (18/47 and 4/37 mice, respectively), and in derived cell cultures in vitro. Strikingly, even PDX with carcinoma histology can host scarce EBV-infected B-lymphocytes that can fully overgrow carcinoma cells during serial passaging in vitro and in vivo. As serial xenografting is crucial to expand primary tumor tissue for biobanks and cohorts for preclinical mouse avatar trials, the emerging dominance of B-lymphoproliferations in serial PDX represents a serious confounding factor in these models. Consequently, repeated phenotypic assessments of serial PDX are mandatory at each expansion step to verify "bona fide" carcinoma xenografts.
Asunto(s)
Linfocitos B/trasplante , Carcinoma Ductal Pancreático/patología , Neoplasias Colorrectales/patología , Infecciones por Virus de Epstein-Barr/patología , Trastornos Linfoproliferativos/etiología , Neoplasias Pancreáticas/patología , Ensayo de Capsula Subrrenal , Animales , Antígenos de Neoplasias/análisis , Linfocitos B/patología , Linfocitos B/virología , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/virología , División Celular , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/virología , Medio de Cultivo Libre de Suero , Infecciones por Virus de Epstein-Barr/inmunología , Xenoinjertos/inmunología , Xenoinjertos/patología , Humanos , Huésped Inmunocomprometido , Antígenos Comunes de Leucocito/análisis , Trastornos Linfoproliferativos/patología , Trastornos Linfoproliferativos/virología , Ratones , Ratones Endogámicos NOD , Especificidad de Órganos , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/virología , Esferoides Celulares , Ensayo de Capsula Subrrenal/métodosRESUMEN
Novel treatment options for metastatic colorectal cancer (CRC) are urgently needed to improve patient outcome. Here, we screen a library of non-characterized small molecules against a heterogeneous collection of patient-derived CRC spheroids. By prioritizing compounds with inhibitory activity in a subset of-but not all-spheroid cultures, NCT02 is identified as a candidate with minimal risk of non-specific toxicity. Mechanistically, we show that NCT02 acts as molecular glue that induces ubiquitination of cyclin K (CCNK) and proteasomal degradation of CCNK and its complex partner CDK12. Knockout of CCNK or CDK12 decreases proliferation of CRC cells in vitro and tumor growth in vivo. Interestingly, sensitivity to pharmacological CCNK/CDK12 degradation is associated with TP53 deficiency and consensus molecular subtype 4 in vitro and in patient-derived xenografts. We thus demonstrate the efficacy of targeted CCNK/CDK12 degradation for a CRC subset, highlighting the potential of drug-induced proteolysis for difficult-to-treat types of cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteolisis , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Daño del ADN , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Proteómica , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
A hierarchically organized cell compartment drives colorectal cancer (CRC) progression. Genetic barcoding allows monitoring of the clonal output of tumorigenic cells without prospective isolation. In this study, we asked whether tumor clone-initiating cells (TcICs) were genetically heterogeneous and whether differences in self-renewal and activation reflected differential kinetics among individual subclones or functional hierarchies within subclones. Monitoring genomic subclone kinetics in three patient tumors and corresponding serial xenografts and spheroids by high-coverage whole-genome sequencing, clustering of genetic aberrations, subclone combinatorics, and mutational signature analysis revealed at least two to four genetic subclones per sample. Long-term growth in serial xenografts and spheroids was driven by multiple genomic subclones with profoundly differing growth dynamics and hence different quantitative contributions over time. Strikingly, genetic barcoding demonstrated stable functional heterogeneity of CRC TcICs during serial xenografting despite near-complete changes in genomic subclone contribution. This demonstrates that functional heterogeneity is, at least frequently, present within genomic subclones and independent of mutational subclone differences.
Asunto(s)
Neoplasias Colorrectales/genética , Variaciones en el Número de Copia de ADN , Células Madre Neoplásicas/metabolismo , Esferoides Celulares/metabolismo , Animales , Células Clonales/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Análisis Mutacional de ADN/métodos , Heterogeneidad Genética , Genómica/métodos , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Mutación , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Extensive prior research focused on somatic copy-number alterations (SCNAs) affecting cancer genes, yet the extent to which recurrent SCNAs exert their influence through rearrangement of cis-regulatory elements (CREs) remains unclear. Here we present a framework for inferring cancer-related gene overexpression resulting from CRE reorganization (e.g., enhancer hijacking) by integrating SCNAs, gene expression data and information on topologically associating domains (TADs). Analysis of 7,416 cancer genomes uncovered several pan-cancer candidate genes, including IRS4, SMARCA1 and TERT. We demonstrate that IRS4 overexpression in lung cancer is associated with recurrent deletions in cis, and we present evidence supporting a tumor-promoting role. We additionally pursued cancer-type-specific analyses and uncovered IGF2 as a target for enhancer hijacking in colorectal cancer. Recurrent tandem duplications intersecting with a TAD boundary mediate de novo formation of a 3D contact domain comprising IGF2 and a lineage-specific super-enhancer, resulting in high-level gene activation. Our framework enables systematic inference of CRE rearrangements mediating dysregulation in cancer.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Elementos de Facilitación Genéticos/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Sustrato del Receptor de Insulina/genética , Factor II del Crecimiento Similar a la Insulina/genética , Neoplasias/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Regiones Promotoras GenéticasRESUMEN
In recent years, numerous bacterial pathogens have been shown to inactivate the major tumour suppressor p53 during infection. This inactivation impedes the protective response of the host cell to the genotoxicity that often results from bacterial infection. Moreover, a new aspect of the antibacterial activity of p53 that has recently come to light - downregulation of host cell metabolism to interfere with intracellular bacterial replication - has further highlighted the crucial role of p53 in host-pathogen interactions, as host cell metabolism is relevant for all intracellular bacteria, as well as other pathogens that replicate inside host cells and use host metabolites. In this Progress article, we summarize recent work that has advanced our knowledge of the interaction between pathogenic bacteria and p53, and we discuss the known and expected outcomes of this interaction for pathogenesis.
Asunto(s)
Infecciones Bacterianas/inmunología , Interacciones Huésped-Patógeno , Proteína p53 Supresora de Tumor/metabolismo , Animales , Metabolismo Energético , Humanos , Estrés FisiológicoRESUMEN
CD46 and CD134 mediate attachment of Human Herpesvirus 6A (HHV-6A) and HHV-6B to host cell, respectively. But many cell types interfere with viral infection through rapid degradation of viral DNA. Hence, not all cells expressing these receptors are permissive to HHV-6 DNA replication and production of infective virions suggesting the involvement of additional factors that influence HHV-6 propagation. Here, we used a proteomics approach to identify other host cell proteins necessary for HHV-6 binding and entry. We found host cell chaperone protein GP96 to interact with HHV-6A and HHV-6B and to interfere with virus propagation within the host cell. In human peripheral blood mononuclear cells (PBMCs), GP96 is transported to the cell surface upon infection with HHV-6 and interacts with HHV-6A and -6B through its C-terminal end. Suppression of GP96 expression decreased initial viral binding but increased viral DNA replication. Transient expression of human GP96 allowed HHV-6 entry into CHO-K1 cells even in the absence of CD46. Thus, our results suggest an important role for GP96 during HHV-6 infection, which possibly supports the cellular degradation of the virus.
Asunto(s)
Herpesvirus Humano 6/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Virales/metabolismo , Internalización del Virus , Animales , Células CHO , Línea Celular Tumoral , Células Cultivadas , Cricetinae , Cricetulus , ADN Viral/metabolismo , Células HeLa , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/fisiología , Interacciones Huésped-Patógeno , Humanos , Immunoblotting , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Proteína Cofactora de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Microscopía Confocal , Modelos Biológicos , Unión Proteica , Proteolisis , Interferencia de ARN , Replicación ViralRESUMEN
Obligate intracellular bacteria depend entirely on nutrients from the host cell for their reproduction. Here, we show that obligate intracellular Chlamydia downregulate the central tumor suppressor p53 in human cells. This reduction of p53 levels is mediated by the PI3K-Akt signaling pathway, activation of HDM2, and subsequent proteasomal degradation of p53. The stabilization of p53 in human cells severely impaired chlamydial development and caused the loss of infectious particle formation. DNA-damage-induced p53 interfered with chlamydial development through downregulation of the pentose phosphate pathway (PPP). Increased expression of the PPP key enzyme glucose-6-phosphate dehydrogenase rescued the inhibition of chlamydial growth induced by DNA damage or stabilized p53. Thus, downregulation of p53 is a key event in the chlamydial life cycle that reprograms the host cell to create a metabolic environment supportive of chlamydial growth.
Asunto(s)
Infecciones por Chlamydia/metabolismo , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/fisiología , Interacciones Huésped-Patógeno , Proteína p53 Supresora de Tumor/metabolismo , Animales , Muerte Celular , Infecciones por Chlamydia/patología , Reparación del ADN , Regulación hacia Abajo , Fibroblastos/metabolismo , Fibroblastos/microbiología , Glucosafosfato Deshidrogenasa/metabolismo , Glucólisis , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/microbiología , Células Endoteliales de la Vena Umbilical Humana/ultraestructura , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismoRESUMEN
Human herpesvirus-6 (HHV-6) exists in latent form either as a nuclear episome or integrated into human chromosomes in more than 90% of healthy individuals without causing clinical symptoms. Immunosuppression and stress conditions can reactivate HHV-6 replication, associated with clinical complications and even death. We have previously shown that co-infection of Chlamydia trachomatis and HHV-6 promotes chlamydial persistence and increases viral uptake in an in vitro cell culture model. Here we investigated C. trachomatis-induced HHV-6 activation in cell lines and fresh blood samples from patients having Chromosomally integrated HHV-6 (CiHHV-6). We observed activation of latent HHV-6 DNA replication in CiHHV-6 cell lines and fresh blood cells without formation of viral particles. Interestingly, we detected HHV-6 DNA in blood as well as cervical swabs from C. trachomatis-infected women. Low virus titers correlated with high C. trachomatis load and vice versa, demonstrating a potentially significant interaction of these pathogens in blood cells and in the cervix of infected patients. Our data suggest a thus far underestimated interference of HHV-6 and C. trachomatis with a likely impact on the disease outcome as consequence of co-infection.
Asunto(s)
Infecciones por Chlamydia/microbiología , Infecciones por Chlamydia/virología , Chlamydia trachomatis/fisiología , Herpesvirus Humano 6/fisiología , Latencia del Virus/fisiología , Replicación Viral/fisiología , Carga Bacteriana/fisiología , Estudios de Casos y Controles , Línea Celular , Cuello del Útero/microbiología , Cuello del Útero/patología , Cuello del Útero/virología , Distribución de Chi-Cuadrado , Infecciones por Chlamydia/sangre , Infecciones por Chlamydia/patología , Cromosomas Humanos/genética , Replicación del ADN , ADN Bacteriano/sangre , ADN Bacteriano/genética , ADN Viral/sangre , ADN Viral/genética , Femenino , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones por Roseolovirus/microbiología , Infecciones por Roseolovirus/virología , Frotis Vaginal , Carga Viral/fisiología , Virión/ultraestructuraRESUMEN
Both human herpes viruses and Chlamydia are highly prevalent in the human population and are detected together in different human disorders. Here, we demonstrate that co-infection with human herpes virus 6 (HHV6) interferes with the developmental cycle of C. trachomatis and induces persistence. Induction of chlamydial persistence by HHV6 is independent of productive virus infection, but requires the interaction and uptake of the virus by the host cell. On the other hand, viral uptake is strongly promoted under co-infection conditions. Host cell glutathione reductase activity was suppressed by HHV6 causing NADPH accumulation, decreased formation of reduced glutathione and increased oxidative stress. Prevention of oxidative stress restored infectivity of Chlamydia after HHV6-induced persistence. We show that co-infection with Herpes simplex virus 1 or human Cytomegalovirus also induces chlamydial persistence by a similar mechanism suggesting that Chlamydia -human herpes virus co-infections are evolutionary shaped interactions with a thus far unrecognized broad significance.