RESUMO
BACKGROUND & AIMS: Patients with colon cancer with liver metastases may be cured with surgery, but the presence of additional lung metastases often precludes curative treatment. Little is known about the processes driving lung metastasis. This study aimed to elucidate the mechanisms governing lung vs liver metastasis formation. METHODS: Patient-derived organoid (PDO) cultures were established from colon tumors with distinct patterns of metastasis. Mouse models recapitulating metastatic organotropism were created by implanting PDOs into the cecum wall. Optical barcoding was applied to trace the origin and clonal composition of liver and lung metastases. RNA sequencing and immunohistochemistry were used to identify candidate determinants of metastatic organotropism. Genetic, pharmacologic, in vitro, and in vivo modeling strategies identified essential steps in lung metastasis formation. Validation was performed by analyzing patient-derived tissues. RESULTS: Cecum transplantation of 3 distinct PDOs yielded models with distinct metastatic organotropism: liver only, lung only, and liver and lung. Liver metastases were seeded by single cells derived from select clones. Lung metastases were seeded by polyclonal clusters of tumor cells entering the lymphatic vasculature with very limited clonal selection. Lung-specific metastasis was associated with high expression of desmosome markers, including plakoglobin. Plakoglobin deletion abrogated tumor cell cluster formation, lymphatic invasion, and lung metastasis formation. Pharmacologic inhibition of lymphangiogenesis attenuated lung metastasis formation. Primary human colon, rectum, esophagus, and stomach tumors with lung metastases had a higher N-stage and more plakoglobin-expressing intra-lymphatic tumor cell clusters than those without lung metastases. CONCLUSIONS: Lung and liver metastasis formation are fundamentally distinct processes with different evolutionary bottlenecks, seeding entities, and anatomic routing. Polyclonal lung metastases originate from plakoglobin-dependent tumor cell clusters entering the lymphatic vasculature at the primary tumor site.
Assuntos
Neoplasias do Colo , Neoplasias Hepáticas , Neoplasias Pulmonares , Camundongos , Animais , Humanos , gama Catenina/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias do Colo/genética , Neoplasias Hepáticas/patologiaRESUMO
Cancer evolution is predominantly studied by focusing on differences in the genetic characteristics of malignant cells within tumors. However, the spatiotemporal dynamics of clonal outgrowth that underlie evolutionary trajectories remain largely unresolved. Here, we sought to unravel the clonal dynamics of colorectal cancer (CRC) expansion in space and time by using a color-based clonal tracing method. This method involves lentiviral red-green-blue (RGB) marking of cell populations, which enabled us to track individual cells and their clonal outgrowth during tumor initiation and growth in a xenograft model. We found that clonal expansion largely depends on the location of a clone, as small clones reside in the center and large clones mostly drive tumor growth at the border. These dynamics are recapitulated in a computational model, which confirms that the clone position within a tumor rather than cell-intrinsic features, is crucial for clonal outgrowth. We also found that no significant clonal loss occurs during tumor growth and clonal dispersal is limited in most models. Our results imply that, in addition to molecular features of clones such as (epi-)genetic differences between cells, clone location and the geometry of tumor growth are crucial for clonal expansion. Our findings suggest that either microenvironmental signals on the tumor border or differences in physical properties within the tumor, are major contributors to explain heterogeneous clonal expansion. Thus, this study provides further insights into the dynamics of solid tumor growth and progression, as well as the origins of tumor cell heterogeneity in a relevant model system.
Assuntos
Neoplasias Colorretais/patologia , Animais , Linhagem da Célula , Células Clonais , Neoplasias Colorretais/genética , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Análise Espaço-TemporalRESUMO
The peritoneum is a common site of dissemination in patients with colorectal cancer. In order to identify high-risk patients and improve therapeutic strategies, a better understanding of the peritoneal dissemination process and the reasons behind the high heterogeneity that is observed between patients is required. We aimed to create a murine model to further elucidate the process of peritoneal dissemination and to provide an experimental platform for further studies. We developed an in vivo model to assess patterns of peritoneal dissemination of 15 colorectal cancer cell lines. Immune deficient mice were intraperitoneally injected with 10,000 human colorectal cancer cells. Ten weeks after injection, or earlier in case of severe discomfort, the mice were sacrificed followed by dissection including assessment of the outgrowth and localization of peritoneal metastases. Furthermore, using a color-based clonal tracing method, the clonal dynamics of peritoneal nodules were observed. The different cell lines showed great variation in the extent of peritoneal outgrowth, ranging from no outgrowth to localized or widespread outgrowth of cells. An association between KRAS pathway activation and the formation of peritoneal metastases was identified. Also, cell line specific tumor location preferences were observed, with similar patterns of outgrowth in anatomically related areas. Furthermore, different patterns regarding clonal dynamics were found, varying from monoclonal or polyclonal outgrowth to extensively dispersed polyclonal lesions. The established murine model recapitulates heterogeneity as observed in human peritoneal metastases, which makes it a suitable platform for future (intervention) studies.
Assuntos
Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Neoplasias Peritoneais/secundário , Peritônio/patologia , Animais , Feminino , Células HCT116 , Humanos , Camundongos Nus , Neoplasias ExperimentaisRESUMO
Colorectal cancer is the third most common cancer type worldwide. It is characterized by a high expression of aberrantly glycosylated ligands, such as the Tn antigen (GalNAcα1-Ser/Thr), which is a major ligand for the C-type lectin macrophage galactose-type lectin (MGL). We have previously determined that a high level of MGL ligands in colorectal tumors is associated with lower disease-free survival in patients with late stage disease, which we could attribute to the presence of oncogenic BRAFV600E mutations. Here we aimed to elucidate the downstream pathway of BRAFV600E governing high MGL ligand and Tn antigen expression. We focused on glycosylation-related enzymes involved in the synthesis or elongation of Tn antigen, N-acetylgalactosamine-transferases (GALNTs) and C1GalT1/COSMC, respectively. Both the activity and expression of C1GalT1 and COSMC were unrelated to the BRAF mutational status. In contrast, GALNT3, GALNT7 and GALNT12 were increased in colorectal cancer cells harboring the BRAFV600E mutation. Through CRISPR-Cas9 gene knockouts we could establish that GALNT3 increased MGL ligand synthesis in the HT29 cell line, while GALNT7 and GALNT12 appeared to have redundant roles. Together our results highlight a novel mechanistic pathway connecting BRAFV600E to aberrant glycosylation in colorectal cancer through GALNT3.
Assuntos
Neoplasias Colorretais/metabolismo , Lectinas Tipo C/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Colorretais/patologia , Células HT29 , Humanos , Lectinas Tipo C/genética , Ligantes , Proteínas Proto-Oncogênicas B-raf/genética , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
The C-type lectin macrophage galactose-type lectin (MGL) exerts an immunosuppressive role reflected by its interaction with terminal GalNAc moieties, such as the Tn antigen, on CD45 of effector T cells, thereby down-regulating T cell receptor signaling, cytokine responses, and induction of T cell death. Here, we provide evidence for the pathways that control the specific expression of GalNAc moieties on human CD4(+) T cells. GalNAc epitopes were readily detectable on the cell surface after T cell activation and required de novo protein synthesis. Expression of GalNAc-containing MGL ligands was completely dependent on PKC and did not involve NF-κB. Instead, activation of the downstream ERK MAPK pathway led to decreased mRNA levels and activity of the core 1 ß3GalT enzyme and its chaperone Cosmc, favoring the expression of Tn antigen. In conclusion, expression of GalNAc moieties mirrors the T cell activation status, and thus only highly stimulated T cells are prone to the suppressive action of MGL.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Linfócitos T CD4-Positivos/imunologia , Calcineurina/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Lectinas Tipo C/imunologia , Ativação Linfocitária/fisiologia , Sistema de Sinalização das MAP Quinases/imunologia , Antígenos Glicosídicos Associados a Tumores/genética , Antígenos Glicosídicos Associados a Tumores/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Galactosiltransferases/biossíntese , Galactosiltransferases/genética , Galactosiltransferases/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Glucosiltransferases/biossíntese , Glucosiltransferases/genética , Glucosiltransferases/imunologia , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/genética , Chaperonas Moleculares/imunologia , Proteína Quinase C/genética , Proteína Quinase C/imunologia , Proteína Quinase C/metabolismoRESUMO
The structure of cell-free DNA (cfDNA) is altered in the blood of patients with cancer. From whole-genome sequencing, we retrieve the cfDNA fragment-end composition using a new software (FrEIA [fragment end integrated analysis]), as well as the cfDNA size and tumor fraction in three independent cohorts (n = 925 cancer from >10 types and 321 control samples). At 95% specificity, we detect 72% cancer samples using at least one cfDNA measure, including 64% early-stage cancer (n = 220). cfDNA detection correlates with a shorter overall (p = 0.0086) and recurrence-free (p = 0.017) survival in patients with resectable esophageal adenocarcinoma. Integrating cfDNA measures with machine learning in an independent test set (n = 396 cancer, 90 controls) achieve a detection accuracy of 82% and area under the receiver operating characteristic curve of 0.96. In conclusion, harnessing the biological features of cfDNA can improve, at no extra cost, the diagnostic performance of liquid biopsies.
Assuntos
Ácidos Nucleicos Livres , Neoplasias , Humanos , Ácidos Nucleicos Livres/genética , Biomarcadores Tumorais/genética , Genômica , Biópsia Líquida , Curva ROCRESUMO
Background: The occurrence of peritoneal metastasis (PM) in patients with colorectal cancer (CRC) has a dismal prognosis. There is often limited response to systemic- and immunotherapy, even in microsatellite unstable (MSI) CRC. To overcome therapy resistance, it is critical to understand local immune environment in the peritoneal cavity, and to develop models to study anti-tumor immune responses. Here, we defined the peritoneal immune system (PerIS) in PM-CRC patients and evaluate the pre-clinical potential of a humanized immune system (HIS) mouse model for PM-CRC. Methods: We studied the human PerIS in PM-CRC patients (n=20; MSS 19/20; 95%) and in healthy controls (n=3). HIS mice (NODscid gamma background; n=18) were generated, followed by intraperitoneal injection of either saline (HIS control; n=3) or human MSS/MSI CRC cell lines HUTU80, MDST8 and HCT116 (HIS-PM, n=15). Immune cells in peritoneal fluid and peritoneal tumors were analyzed using cytometry by time of flight (CyTOF). Results: The human and HIS mouse homeostatic PerIS was equally populated by NK cells and CD4+- and CD8+ T cells, however differences were observed in macrophage and B cell abundance. In HIS mice, successful peritoneal engraftment of both MSI and MSS tumors was observed (15/15; 100%). Both in human PM-CRC and in the HIS mouse PM-CRC model, we observed that MSS PM-CRC triggered a CD4+ Treg response in the PerIS, while MSI PM-CRC drives CD8+ TEMs responses. Conclusion: In conclusion, T cell responses in PM-CRC in HIS mice mirror those in human PM-CRC, making this model suitable to study antitumor T cell responses in PM-CRC.
Assuntos
Neoplasias Colorretais , Modelos Animais de Doenças , Neoplasias Peritoneais , Animais , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/imunologia , Humanos , Camundongos , Masculino , Feminino , Linhagem Celular Tumoral , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Idoso , Microambiente Tumoral/imunologia , Células Matadoras Naturais/imunologiaRESUMO
Consensus Molecular Subtype (CMS) classification of colorectal cancer (CRC) tissues is complicated by RNA degradation upon formalin-fixed paraffin-embedded (FFPE) preservation. Here, we present an FFPE-curated CMS classifier. The CMSFFPE classifier was developed using genes with a high transcript integrity in FFPE-derived RNA. We evaluated the classification accuracy in two FFPE-RNA datasets with matched fresh-frozen (FF) RNA data, and an FF-derived RNA set. An FFPE-RNA application cohort of metastatic CRC patients was established, partly treated with anti-EGFR therapy. Key characteristics per CMS were assessed. Cross-referenced with matched benchmark FF CMS calls, the CMSFFPE classifier strongly improved classification accuracy in two FFPE datasets compared with the original CMSClassifier (63.6% versus 40.9% and 83.3% versus 66.7%, respectively). We recovered CMS-specific recurrence-free survival patterns (CMS4 versus CMS2: hazard ratio 1.75, 95% CI 1.24-2.46). Key molecular and clinical associations of the CMSs were confirmed. In particular, we demonstrated the predictive value of CMS2 and CMS3 for anti-EGFR therapy response (CMS2&3: odds ratio 5.48, 95% CI 1.10-27.27). The CMSFFPE classifier is an optimized FFPE-curated research tool for CMS classification of clinical CRC samples.
Assuntos
Neoplasias Colorretais , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/classificação , Neoplasias Colorretais/patologia , Inclusão em Parafina , Biomarcadores Tumorais/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Consenso , Fixação de Tecidos/métodos , Masculino , Perfilação da Expressão Gênica/métodos , Idoso , Pessoa de Meia-Idade , Prognóstico , Regulação Neoplásica da Expressão Gênica , FormaldeídoRESUMO
Peritoneal metastases (PMs) from colorectal cancer (CRC) respond poorly to treatment and are associated with unfavorable prognosis. For example, the addition of hyperthermic intraperitoneal chemotherapy (HIPEC) to cytoreductive surgery in resectable patients shows limited benefit, and novel treatments are urgently needed. The majority of CRC-PMs represent the CMS4 molecular subtype of CRC, and here we queried the vulnerabilities of this subtype in pharmacogenomic databases to identify novel therapies. This reveals the copper ionophore elesclomol (ES) as highly effective against CRC-PMs. ES exhibits rapid cytotoxicity against CMS4 cells by targeting mitochondria. We find that a markedly reduced mitochondrial content in CMS4 cells explains their vulnerability to ES. ES demonstrates efficacy in preclinical models of PMs, including CRC-PMs and ovarian cancer organoids, mouse models, and a HIPEC rat model of PMs. The above proposes ES as a promising candidate for the local treatment of CRC-PMs, with broader implications for other PM-prone cancers.
Assuntos
Neoplasias Colorretais , Mitocôndrias , Neoplasias Peritoneais , Neoplasias Colorretais/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/terapia , Animais , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Camundongos , Linhagem Celular Tumoral , Ratos , Feminino , Quimioterapia Intraperitoneal Hipertérmica/métodosRESUMO
Clonal growth and competition underlie processes of key relevance in etiology, progression and therapy response across all cancers. Here, we demonstrate a novel experimental approach, based on multi-color, fluorescent tagging of cell nuclei, in combination with picoliter droplet deposition, to study the clonal dynamics in two- and three-dimensional cell cultures. The method allows for the simultaneous visualization and analysis of multiple clones in individual multi-clonal colonies, providing a powerful tool for studying clonal dynamics and identifying clonal populations with distinct characteristics. Results of our experiments validate the utility of the method in studying clonal dynamics in vitro, and reveal differences in key aspects of clonal behavior of different cancer cell lines in monoculture conditions, as well as in co-cultures with stromal fibroblasts.
Assuntos
Técnicas de Cultura de Células , Neoplasias , Humanos , Células Clonais , Linhagem Celular , Técnicas de CoculturaRESUMO
BACKGROUND: Existing methods to detect tumor signal in liquid biopsy have focused on the analysis of nuclear cell-free DNA (cfDNA). However, non-nuclear cfDNA and in particular mitochondrial DNA (mtDNA) has been understudied. We hypothesize that an increase in mtDNA in plasma could reflect the presence of cancer, and that leveraging cell-free mtDNA could enhance cancer detection. RESULTS: We survey 203 healthy and 664 cancer plasma samples from three collection centers covering 12 cancer types with whole genome sequencing to catalogue the plasma mtDNA fraction. The mtDNA fraction is increased in individuals with cholangiocarcinoma, colorectal, liver, pancreatic, or prostate cancer, in comparison to that in healthy individuals. We detect almost no increase of mtDNA fraction in individuals with other cancer types. The mtDNA fraction in plasma correlates with the cfDNA tumor fraction as determined by somatic mutations and/or copy number aberrations. However, the mtDNA fraction is also elevated in a fraction of patients without an apparent increase in tumor-derived cfDNA. A predictive model integrating mtDNA and copy number analysis increases the area under the curve (AUC) from 0.73 when using copy number alterations alone to an AUC of 0.81. CONCLUSIONS: The mtDNA signal retrieved by whole genome sequencing has the potential to boost the detection of cancer when combined with other tumor-derived signals in liquid biopsies.
Assuntos
Ácidos Nucleicos Livres , Neoplasias da Próstata , Masculino , Humanos , Biópsia Líquida , Mitocôndrias/genética , DNA Mitocondrial/genética , Neoplasias da Próstata/genética , Biomarcadores Tumorais/genéticaRESUMO
Mutations in Bone Morphogenetic Protein (BMP) Receptor (BMPR)1A and SMAD4 are detected in 50% of juvenile polyposis syndrome (JPS) patients, who develop stroma-rich hamartomatous polyps. The established role of stromal cells in regulating BMP activity in the intestine implies a role for stromal cells in polyp development. We used conditional Cre-LoxP mice to investigate how specific loss of BMPR1A in endothelial cells, fibroblasts, or myofibroblasts/smooth muscle cells affects intestinal homeostasis. Selective loss of BMPR1A in fibroblasts causes severe histological changes in the intestines with a significant increase in stromal cell content and epithelial cell hyperproliferation, leading to numerous serrated polyps. This phenotype suggests that crucial changes occur in the fibroblast secretome that influences polyp development. Analyses of publicly available RNA expression databases identified CXCL12 as a potential candidate. RNAscope in situ hybridization showed an evident increase of Cxcl12-expressing fibroblasts. In vitro, stimulation of fibroblasts with BMPs resulted in downregulation of CXCL12, while inhibition of the BMP pathway resulted in gradual upregulation of CXCL12 over time. Moreover, neutralization of CXCL12 in vivo in the fibroblast-specific BMPR1A KO mice resulted in a significant decrease in polyp formation. Finally, in CRC patient specimens, mRNA-expression data showed that patients with high GREMLIN1 and CXCL12 expression had a significantly poorer overall survival. Significantly higher GREMLIN1, NOGGIN, and CXCL12 expression were detected in the Consensus Molecular Subtype 4 (CMS4) colorectal cancers, which are thought to arise from serrated polyps. Taken together, these data imply that fibroblast-specific BMP signaling-CXCL12 interaction could have a role in the etiology of serrated polyp formation.
Assuntos
Células Endoteliais , Pólipos , Camundongos , Animais , Transdução de Sinais , Fibroblastos , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismoRESUMO
Cell-free DNA (cfDNA) can be isolated and sequenced from blood and/or urine of cancer patients. Conventional short-read sequencing lacks deployability and speed and can be biased for short cfDNA fragments. Here, we demonstrate that with Oxford Nanopore Technologies (ONT) sequencing we can achieve delivery of genomic and fragmentomic data from liquid biopsies. Copy number aberrations and cfDNA fragmentation patterns can be determined in less than 24 h from sample collection. The tumor-derived cfDNA fraction calculated from plasma of lung cancer patients and urine of bladder cancer patients was highly correlated (R = 0.98) with the tumor fraction calculated from short-read sequencing of the same samples. cfDNA size profile, fragmentation patterns, fragment-end composition, and nucleosome profiling near transcription start sites in plasma and urine exhibited the typical cfDNA features. Additionally, a high proportion of long tumor-derived cfDNA fragments (> 300 bp) are recovered in plasma and urine using ONT sequencing. ONT sequencing is a cost-effective, fast, and deployable approach for obtaining genomic and fragmentomic results from liquid biopsies, allowing the analysis of previously understudied cfDNA populations.
Assuntos
Ácidos Nucleicos Livres , Neoplasias Pulmonares , Sequenciamento por Nanoporos , Humanos , Ácidos Nucleicos Livres/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Genômica/métodos , Análise de Sequência de DNA , DNA/genética , Biomarcadores Tumorais/genéticaRESUMO
BACKGROUND: Colorectal cancer (CRC) can be divided into four consensus molecular subtypes (CMS), each with distinct biological features. CMS4 is associated with epithelial-mesenchymal transition and stromal infiltration (Guinney et al., Nat Med 21:1350-6, 2015; Linnekamp et al., Cell Death Differ 25:616-33, 2018), whereas clinically it is characterized by lower responses to adjuvant therapy, higher incidence of metastatic spreading and hence dismal prognosis (Buikhuisen et al., Oncogenesis 9:66, 2020). METHODS: To understand the biology of the mesenchymal subtype and unveil specific vulnerabilities, a large CRISPR-Cas9 drop-out screen was performed on 14 subtyped CRC cell lines to uncover essential kinases in all CMSs. Dependency of CMS4 cells on p21-activated kinase 2 (PAK2) was validated in independent 2D and 3D in vitro cultures and in vivo models assessing primary and metastatic outgrowth in liver and peritoneum. TIRF microscopy was used to uncover actin cytoskeleton dynamics and focal adhesion localization upon PAK2 loss. Subsequent functional assays were performed to determine altered growth and invasion patterns. RESULTS: PAK2 was identified as a key kinase uniquely required for growth of the mesenchymal subtype CMS4, both in vitro and in vivo. PAK2 plays an important role in cellular attachment and cytoskeletal rearrangements (Coniglio et al., Mol Cell Biol 28:4162-72, 2008; Grebenova et al., Sci Rep 9:17171, 2019). In agreement, deletion or inhibition of PAK2 impaired actin cytoskeleton dynamics in CMS4 cells and, as a consequence, significantly reduced invasive capacity, while it was dispensable for CMS2 cells. Clinical relevance of these findings was supported by the observation that deletion of PAK2 from CMS4 cells prevented metastatic spreading in vivo. Moreover, growth in a model for peritoneal metastasis was hampered when CMS4 tumor cells were deficient for PAK2. CONCLUSION: Our data reveal a unique dependency of mesenchymal CRC and provide a rationale for PAK2 inhibition to target this aggressive subgroup of colorectal cancer.
Assuntos
Neoplasias Colorretais , Sarcoma , Humanos , Citoesqueleto de Actina , Carcinogênese , Linhagem CelularRESUMO
A significant proportion of colorectal cancer (CRC) patients develop peritoneal metastases (PM) in the course of their disease. PMs are associated with a poor quality of life, significant morbidity and dismal disease outcome. To improve care for this patient group, a better understanding of the molecular characteristics of CRC-PM is required. Here we present a comprehensive molecular characterization of a cohort of 52 patients. This reveals that CRC-PM represent a distinct CRC molecular subtype, CMS4, but can be further divided in three separate categories, each presenting with unique features. We uncover that the CMS4-associated structural protein Moesin plays a key role in peritoneal dissemination. Finally, we define specific evolutionary features of CRC-PM which indicate that polyclonal metastatic seeding underlies these lesions. Together our results suggest that CRC-PM should be perceived as a distinct disease entity.
Assuntos
Neoplasias Colorretais , Segunda Neoplasia Primária , Neoplasias Peritoneais , Neoplasias Colorretais/patologia , Humanos , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/secundário , Peritônio/metabolismo , Qualidade de VidaRESUMO
The p53 regulatory network is critically involved in preventing the initiation of cancer. In unstressed cells, p53 is maintained at low levels and is largely inactive, mainly through the action of its two essential negative regulators, HDM2 and HDMX. p53 abundance and activity are up-regulated in response to various stresses, including DNA damage and oncogene activation. Active p53 initiates transcriptional and transcription-independent programs that result in cell cycle arrest, cellular senescence, or apoptosis. p53 also activates transcription of HDM2, which initially leads to the degradation of HDMX, creating a positive feedback loop to obtain maximal activation of p53. Subsequently, when stress-induced post-translational modifications start to decline, HDM2 becomes effective in targeting p53 for degradation, thus attenuating the p53 response. To date, no clear function for HDMX in this critical attenuation phase has been demonstrated experimentally. Like HDM2, the HDMX gene contains a promoter (P2) in its first intron that is potentially inducible by p53. We show that p53 activation in response to a plethora of p53-activating agents induces the transcription of a novel HDMX mRNA transcript from the HDMX-P2 promoter. This mRNA is more efficiently translated than that expressed from the constitutive HDMX-P1 promoter, and it encodes a long form of HDMX protein, HDMX-L. Importantly, we demonstrate that HDMX-L cooperates with HDM2 to promote the ubiquitination of p53 and that p53-induced HDMX transcription from the P2 promoter can play a key role in the attenuation phase of the p53 response, to effectively diminish p53 abundance as cells recover from stress.
Assuntos
Íntrons/fisiologia , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Western Blotting , Proteínas de Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Doxiciclina/farmacologia , Etoposídeo/farmacologia , Evolução Molecular , Humanos , Imidazóis/farmacologia , Íntrons/genética , Camundongos , Proteínas Nucleares/metabolismo , Piperazinas/farmacologia , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Proteína Supressora de Tumor p53/genética , UbiquitinaçãoRESUMO
BACKGROUND: In around 50% of all human cancers the tumor suppressor p53 is mutated. It is generally assumed that in the remaining tumors the wild-type p53 protein is functionally impaired. The two main inhibitors of p53, hMDM2 (MDM2) and hMDMX (MDMX/MDM4) are frequently overexpressed in wild-type p53 tumors. Whereas the main activity of hMDM2 is to degrade p53 protein, its close homolog hMDMX does not degrade p53, but it represses its transcriptional activity. Here we study the role of hMDMX in the neoplastic transformation of human fibroblasts and embryonic retinoblasts, since a high number of retinoblastomas contain elevated hMDMX levels. METHODS: We made use of an in vitro transformation model using a retroviral system of RNA interference and gene overexpression in primary human fibroblasts and embryonic retinoblasts. Consecutive knockdown of RB and p53, overexpression of SV40-small t, oncogenic HRasV12 and HA-hMDMX resulted in a number of stable cell lines representing different stages of the transformation process, enabling a comparison between loss of p53 and hMDMX overexpression. The cell lines were tested in various assays to assess their oncogenic potential. RESULTS: Both p53-knockdown and hMDMX overexpression accelerated proliferation and prevented growth suppression induced by introduction of oncogenic Ras, which was required for anchorage-independent growth and the ability to form tumors in vivo. Furthermore, we found that hMDMX overexpression represses basal p53 activity to some extent. Transformed fibroblasts with very high levels of hMDMX became largely resistant to the p53 reactivating drug Nutlin-3. The Nutlin-3 response of hMDMX transformed retinoblasts was intact and resembled that of retinoblastoma cell lines. CONCLUSIONS: Our studies show that hMDMX has the essential properties of an oncogene. Its constitutive expression contributes to the oncogenic phenotype of transformed human cells. Its main function appears to be p53 inactivation. Therefore, developing new drugs targeting hMDMX is a valid approach to obtain new treatments for a subset of human tumors expressing wild-type p53.
Assuntos
Transformação Celular Neoplásica/patologia , Fibroblastos/patologia , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/metabolismo , Retina/patologia , Animais , Adesão Celular , Proteínas de Ciclo Celular , Proliferação de Células , Forma Celular , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/patologia , Fibroblastos/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imidazóis/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Transplante de Neoplasias , Oncogenes , Piperazinas/metabolismo , Cultura Primária de Células , Retina/embriologia , Retina/metabolismo , Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The MDM family proteins MDM2 and MDMX are two critical regulators of the p53 tumor suppressor protein. Expression of both proteins is necessary for allowing the embryonal development by keeping the activity of p53 in check. Upon stresses that need to activate p53 to perform its function as guardian of the genome, p53 has to be liberated from these two inhibitors. In this review, we will discuss the various mechanisms by which MDMX protein levels are downregulated upon various types of stress, including posttranslational modifications of the MDMX protein and the regulation of mdmx mRNA expression, including alternative splicing. In addition, the putative function(s) of the described MDMX splice variants, particularly in tumor development, will be discussed. Lastly, in contrast to common belief, we have recently shown the existence of a p53-MDMX feedback loop, which is important for dampening the p53-response at later phases after genotoxic stress.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Dano ao DNA , Humanos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/genética , Ribossomos/metabolismoRESUMO
Effective treatments for pancreatic ductal adenocarcinoma (PDAC) are lacking, and targeted agents have demonstrated limited efficacy. It has been speculated that a rare population of cancer stem cells (CSCs) drives growth, therapy resistance, and rapid metastatic progression in PDAC. These CSCs demonstrate high clonogenicity in vitro and tumorigenic potential in vivo. However, their relevance in established PDAC tissue has not been determined. Here, we use marker-independent stochastic clonal labeling, combined with quantitative modeling of tumor expansion, to uncover PDAC tissue growth dynamics. We find that in contrast to the CSC model, all PDAC cells display clonogenic potential in situ. Furthermore, the proximity to activated cancer-associated fibroblasts determines tumor cell clonogenicity. This means that the microenvironment is dominant in defining the clonogenic activity of PDAC cells. Indeed, manipulating the stroma by Hedgehog pathway inhibition alters the tumor growth mode, revealing that tumor-stroma crosstalk shapes tumor growth dynamics and clonal architecture.
Assuntos
Carcinoma Ductal Pancreático/patologia , Linhagem da Célula , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , Microambiente Tumoral , Anilidas/farmacologia , Animais , Antineoplásicos/farmacologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Comunicação Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Piridinas/farmacologia , Transdução de Sinais , Células Estromais/metabolismo , Células Estromais/patologia , Fatores de Tempo , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Survival rates of cancer patients vary widely within and between malignancies. While genetic aberrations are at the root of all cancers, individual genomic features cannot explain these distinct disease outcomes. In contrast, intra-tumour heterogeneity (ITH) has the potential to elucidate pan-cancer survival rates and the biology that drives cancer prognosis. Unfortunately, a comprehensive and effective framework to measure ITH across cancers is missing. Here, we introduce a scalable measure of chromosomal copy number heterogeneity (CNH) that predicts patient survival across cancers. We show that the level of ITH can be derived from a single-sample copy number profile. Using gene-expression data and live cell imaging we demonstrate that ongoing chromosomal instability underlies the observed heterogeneity. Analysing 11,534 primary cancer samples from 37 different malignancies, we find that copy number heterogeneity can be accurately deduced and predicts cancer survival across tissues of origin and stages of disease. Our results provide a unifying molecular explanation for the different survival rates observed between cancer types.