RESUMEN
Protein kinase C (PKC)-θ is a serine/threonine kinase with both cytoplasmic and nuclear functions. Nuclear chromatin-associated PKC-θ (nPKC-θ) is increasingly recognized to be pathogenic in cancer, whereas its cytoplasmic signaling is restricted to normal T-cell function. Here we show that nPKC-θ is enriched in circulating tumor cells (CTCs) in patients with triple-negative breast cancer (TNBC) brain metastases and immunotherapy-resistant metastatic melanoma and is associated with poor survival in immunotherapy-resistant disease. To target nPKC-θ, we designed a novel PKC-θ peptide inhibitor (nPKC-θi2) that selectively inhibits nPKC-θ nuclear translocation but not PKC-θ signaling in healthy T cells. Targeting nPKC-θ reduced mesenchymal cancer stem cell signatures in immunotherapy-resistant CTCs and TNBC xenografts. PKC-θ was also enriched in the nuclei of CD8+ T cells isolated from stage IV immunotherapy-resistant metastatic cancer patients. We show for the first time that nPKC-θ complexes with ZEB1, a key repressive transcription factor in epithelial-to-mesenchymal transition (EMT), in immunotherapy-resistant dysfunctional PD1+/CD8+ T cells. nPKC-θi2 inhibited the ZEB1/PKC-θ repressive complex to induce cytokine production in CD8+ T cells isolated from patients with immunotherapy-resistant disease. These data establish for the first time that nPKC-θ mediates immunotherapy resistance via its activity in CTCs and dysfunctional CD8+ T cells. Disrupting nPKC-θ but retaining its cytoplasmic function may offer a means to target metastases in combination with chemotherapy or immunotherapy.
RESUMEN
Lysine specific demethylase 1 (LSD1) is a key epigenetic eraser enzyme implicated in cancer metastases and recurrence. Nuclear LSD1 phosphorylated at serine 111 (nLSD1p) has been shown to be critical for the development of breast cancer stem cells. Here we show that circulating tumor cells isolated from immunotherapy-resistant metastatic melanoma patients express higher levels of nLSD1p compared to responders, which is associated with co-expression of stem-like, mesenchymal genes. Targeting nLSD1p with selective nLSD1 inhibitors better inhibits the stem-like mesenchymal signature than traditional FAD-specific LSD1 catalytic inhibitors such as GSK2879552. We also demonstrate that nLSD1p is enriched in PD-1+CD8+ T cells from resistant melanoma patients and 4T1 immunotherapy-resistant mice. Targeting the LSD1p nuclear axis induces IFN-γ/TNF-α-expressing CD8+ T cell infiltration into the tumors of 4T1 immunotherapy-resistant mice, which is further augmented by combined immunotherapy. Underpinning these observations, nLSD1p is regulated by the key T cell exhaustion transcription factor EOMES in dysfunctional CD8+ T cells. EOMES co-exists with nLSD1p in PD-1+CD8+ T cells in resistant patients, and nLSD1p regulates EOMES nuclear dynamics via demethylation/acetylation switching of critical EOMES residues. Using novel antibodies to target these post-translational modifications, we show that EOMES demethylation/acetylation is reciprocally expressed in resistant and responder patients. Overall, we show for the first time that dual inhibition of metastatic cancer cells and re-invigoration of the immune system requires LSD1 inhibitors that target the nLSD1p axis.
Asunto(s)
Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/genética , Histona Demetilasas/genética , Neoplasias/etiología , Proteínas de Dominio T Box/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Animales , Biomarcadores , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Histona Demetilasas/metabolismo , Humanos , Inmunoterapia , Ratones , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/terapia , Proteínas de Dominio T Box/genética , Linfocitos T/inmunología , Resultado del TratamientoRESUMEN
DUSP6 is a dual-specificity phosphatase (DUSP) involved in breast cancer progression, recurrence, and metastasis. DUSP6 is predominantly cytoplasmic in HER2+ primary breast cancer cells, but the expression and subcellular localization of DUSPs, especially DUSP6, in HER2-positive circulating tumor cells (CTCs) is unknown. Here we used the DEPArray system to identify and isolate CTCs from metastatic triple negative breast cancer (TNBC) patients and performed single-cell NanoString analysis to quantify cancer pathway gene expression in HER2-positive and HER2-negative CTC populations. All TNBC patients contained HER2-positive CTCs. HER2-positive CTCs were associated with increased ERK1/ERK2 expression, which are direct DUSP6 targets. DUSP6 protein expression was predominantly nuclear in breast CTCs and the brain metastases but not pleura or lung metastases of TNBC patients. Therefore, nuclear DUSP6 may play a role in the association with cancer spreading in TNBC patients, including brain metastasis.
Asunto(s)
Biomarcadores de Tumor , Neoplasias Encefálicas/secundario , Fosfatasa 6 de Especificidad Dual/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Núcleo Celular/genética , Modelos Animales de Enfermedad , Fosfatasa 6 de Especificidad Dual/antagonistas & inhibidores , Fosfatasa 6 de Especificidad Dual/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Invasividad Neoplásica , Estadificación de Neoplasias , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Unión Proteica , Transporte de Proteínas , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Análisis de la Célula Individual , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/terapia , Ensayos Antitumor por Modelo de Xenoinjerto , Factores de Transcripción p300-CBP/metabolismoRESUMEN
BACKGROUND & AIMS: The early events by which inflammation promotes cancer are still not fully defined. The MCC gene is silenced by promoter methylation in colitis-associated and sporadic colon tumors, but its functional significance in precancerous lesions or polyps is not known. Here, we aimed to determine the impact of Mcc deletion on the cellular pathways and carcinogenesis associated with inflammation in the mouse proximal colon. METHODS: We generated knockout mice with deletion of Mcc in the colonic/intestinal epithelial cells (MccΔIEC) or in the whole body (MccΔ/Δ). Drug-induced lesions were analyzed by transcriptome profiling (at 10 weeks) and histopathology (at 20 weeks). Cell-cycle phases and DNA damage proteins were analyzed by flow cytometry and Western blot of hydrogen peroxide-treated mouse embryo fibroblasts. RESULTS: Transcriptome profiling of the lesions showed a strong response to colon barrier destruction, such as up-regulation of key inflammation and cancer-associated genes as well as 28 interferon γ-induced guanosine triphosphatase genes, including the homologs of Crohn's disease susceptibility gene IRGM. These features were shared by both Mcc-expressing and Mcc-deficient mice and many of the altered gene expression pathways were similar to the mesenchymal colorectal cancer subtype known as consensus molecular subtype 4 (CMS4). However, Mcc deletion was required for increased carcinogenesis in the lesions, with adenocarcinoma in 59% of MccΔIEC compared with 19% of Mcc-expressing mice (P = .002). This was not accompanied by hyperactivation of ß-catenin, but Mcc deletion caused down-regulation of DNA repair genes and a disruption of DNA damage signaling. CONCLUSIONS: Loss of Mcc may promote cancer through a failure to repair inflammation-induced DNA damage. We provide a comprehensive transcriptome data set of early colorectal lesions and evidence for the in vivo significance of MCC silencing in colorectal cancer.
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Neoplasias Colorrectales/genética , Eliminación de Gen , Genes MCC , Inflamación/genética , Animales , Cadherinas/metabolismo , Colon/efectos de los fármacos , Colon/patología , Neoplasias Colorrectales/patología , Reparación del ADN/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Femenino , GTP Fosfohidrolasas/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inflamación/patología , Interferón gamma/farmacología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , beta Catenina/metabolismoRESUMEN
Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates the adaptation of cells to hypoxic microenvironments, for example inside solid tumours. Stabilisation of HIF1α can also occur in normoxic conditions in inflamed tissue or as a result of inactivating mutations in negative regulators of HIF1α. Aberrant overexpression of HIF1α in many different cancers has led to intensive efforts to develop HIF1α-targeted therapies. However, the role of HIF1α is still poorly understood in chronic inflammation that predisposes the colon to carcinogenesis. We have previously reported that the transcription of HIF1α is upregulated and that the protein is stabilised in inflammatory lesions that are caused by the non-steroidal anti-inflammatory drug (NSAID) sulindac in the mouse proximal colon. Here, we exploited this side effect of long-term sulindac administration to analyse the role of HIF1α in colon inflammation using mice with a Villin-Cre-induced deletion of Hif1α exon 2 in the intestinal epithelium (Hif1α(ΔIEC)). We also analysed the effect of sulindac sulfide on the aryl hydrocarbon receptor (AHR) pathway in vitro in colon cancer cells. Most sulindac-treated mice developed visible lesions, resembling the appearance of flat adenomas in the human colon, surrounded by macroscopically normal mucosa. Hif1α(ΔIEC) mice still developed lesions but they were smaller than in the Hif1α-floxed siblings (Hif1α(F/F)). Microscopically, Hif1α(ΔIEC) mice had significantly less severe colon inflammation than Hif1α(F/F) mice. Molecular analysis showed reduced MIF expression and increased E-cadherin mRNA expression in the colon of sulindac-treated Hif1α(ΔIEC) mice. However, immunohistochemistry analysis revealed a defect of E-cadherin protein expression in sulindac-treated Hif1α(ΔIEC) mice. Sulindac sulfide treatment in vitro upregulated Hif1α, c-JUN and IL8 expression through the AHR pathway. Taken together, HIF1α expression augments inflammation in the proximal colon of sulindac-treated mice, and AHR activation by sulindac might lead to the reduction of E-cadherin protein levels through the mitogen-activated protein kinase (MAPK) pathway.
Asunto(s)
Neoplasias del Colon/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Inflamación , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cadherinas/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Exones , Femenino , Eliminación de Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunohistoquímica , Interleucina-8/metabolismo , Mucosa Intestinal/patología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Proteína Oncogénica p65(gag-jun)/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Sulindac/uso terapéutico , Regulación hacia ArribaRESUMEN
We describe a coculture model of a human intestinal epithelial cell line and human peripheral blood monocytes in which monocytes differentiate into cells with features of resident intestinal macrophages. Caco-2 cells are grown on the lower surface of a semipermeable filter with pore size of 3 µm (Transwells) until they differentiate into enterocytes. Peripheral-blood monocytes are added and the co-culture incubated for two days. Monocytes migrate through the pores of the membrane, come into direct contact with the basolateral surfaces of the epithelial cell monolayer, and develop characteristics of resident intestinal macrophages including downregulation of CD14 expression and reduced pro-inflammatory cytokine responses (IL-8, TNF and IL-1ß) to bacterial products. The apical application of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) resulted in an increased number of integrated monocytes, but abrogated the downregulation of CD14 expression and the diminished cytokine responses. MDP also reduced tight-junctional integrity, whilst LPS had no effect. These data indicate that LPS and MDP have significant pathophysiological effects on enterocyte-monocyte interactions, and confirm other studies that demonstrate that enterocytes and their products influence monocyte differentiation. This model may be useful in providing insights into the interaction between monocytes, epithelial cells and intestinal bacteria in health and disease.
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Acetilmuramil-Alanil-Isoglutamina/farmacología , Enterocitos/efectos de los fármacos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Bacterias , Células CACO-2 , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Técnicas de Cocultivo , Citocinas/metabolismo , Regulación hacia Abajo , Enterocitos/fisiología , Enterocitos/ultraestructura , Humanos , Receptores de Lipopolisacáridos/metabolismo , Macrófagos/fisiología , Macrófagos/ultraestructura , Monocitos/efectos de los fármacos , Monocitos/fisiología , Uniones EstrechasRESUMEN
Human fetal eyes 8-40 weeks gestation (WG) were examined using markers to hematopoietic stem cells (HSC), vascular precursor cells (VPC), monocytes/macrophages and endothelial cells (EC). Electron microscopy and bromo-deoxyuridene labeling were undertaken to confirm the existence of solid vascular cords and to demonstrate vasculogenesis and angiogenesis in developing choroidal tissue. Our results demonstrated that the earliest incipient choroid consisted of vimentin(+) mesenchymal precursor cells which downregulated vimentin expression with maturation. Our observations lead us to conclude that these vimentin(-)/CD34(+)/CD44(+)/CD133(+) HSCs then differentiated into three distinct lineages: single isolated CD34(-)/CD39(+) VPCs that formed solid vascular cords which lumenized and became lined with CD34(+) vascular ECs; CD34(--+)/CD14(+)/CD68(+) monocytes that differentiated into tissue macrophages; and CD133(+)/CD34(--+)/α-smooth muscle actin(+) mural precursor cells that matured into smooth muscle cells and pericytes. Blood vessel formation occurred throughout the whole choroid simultaneously, indicative of in situ differentiation. Vasculogenesis, as evidenced by lumenization of solid vascular cords, was responsible for the formation of the entire choroidal area with angiogenesis, in all three layers of the choroid, only adding to vascular density. These results suggest that formation of the human choroid involves three processes: HSC differentiation, vasculogenesis and angiogenesis. Since vasculogenesis takes place independently of VEGF(165), further insights regarding the molecular mechanisms of vasculogenesis are required to better inform future treatments of choroidal neovascularization.
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Diferenciación Celular/fisiología , Coroides/irrigación sanguínea , Coroides/embriología , Endotelio Vascular/citología , Células Madre Hematopoyéticas/citología , Neovascularización Fisiológica/fisiología , Actinas/metabolismo , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Capilares/citología , Capilares/metabolismo , Linaje de la Célula , Endotelio Vascular/metabolismo , Edad Gestacional , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Macrófagos/citología , Células Madre Mesenquimatosas/citología , Microscopía Confocal , Microscopía Electrónica , Vimentina/metabolismoRESUMEN
PURPOSE: To examine mural cell differentiation and pericyte ensheathment during human choroidal vascular formation and into adulthood. METHODS: Triple- and double-labeled immunohistochemistry (alpha-smooth muscle actin [αSMA], desmin, NG2, calponin, caldesmon, CD44, CD34, and CD39) were applied to human fetal (8-32 weeks' gestation) and adult choroidal and retinal wholemounts and histologic cross-sections. Transmission electron microscopy (TEM) was also undertaken. RESULTS: Early in development CD44+ stem cells also stained with αSMA and CD39, suggesting a common precursor. At 12 weeks' gestation, αSMA+ mural precursor cells, confirmed by TEM, were found scattered and isolated over the primordial vascular tree. During development, αSMA+ cells formed a continuous sheath around large arterioles; in veins there were gaps in αSMA expression. The choriocapillaris had an extensive vascular bed but limited coverage by αSMA+ and NG2+ mural cells. Calponin was expressed only on large vessels, and no caldesmon was detected. Pericyte ensheathment of adult capillaries was 11% for choroid versus 94% for retina. Remarkably, choroidal pericytes had no visible intermediate filaments (IFs) on TEM, though IFs were present in retinal pericytes. Neither retinal nor choroidal pericytes stained with desmin. CONCLUSIONS: CD44+ stem cells are involved in the formation of mural cells in the human choroidal vasculature. A marked reduction in pericyte ensheathment of human choroidal vessels suggests a permanently open "plasticity window" and a predisposition to vascular instability and poor autoregulatory ability.
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Coroides/irrigación sanguínea , Endotelio Vascular/embriología , Células Madre Hematopoyéticas/fisiología , Receptores de Hialuranos/metabolismo , Músculo Liso Vascular/embriología , Neovascularización Fisiológica/fisiología , Pericitos/citología , Actinas/metabolismo , Adulto , Antígenos/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Diferenciación Celular , Linaje de la Célula , Endotelio Vascular/metabolismo , Edad Gestacional , Humanos , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/ultraestructura , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Proteoglicanos/metabolismo , Vasos Retinianos/ultraestructura , Adulto Joven , CalponinasRESUMEN
BACKGROUND AND AIMS: The non-steroidal anti-inflammatory drug sulindac is an effective chemopreventive agent in sporadic colorectal cancer but its potential benefit in mismatch repair deficient cancers remains to be defined. We wanted to determine whether genetic defects that are relevant for colorectal cancer, such as Msh2 or p53 deficiency, would influence the efficiency of sulindac chemoprevention or increase the side effects. METHODS: Msh2 or p53 deficient and wild-type mice received feed containing 160-320 ppm sulindac for up to 25 weeks with or without a concurrent treatment with the carcinogen azoxymethane. Colon tissue was analysed by histopathology and molecular biology methods. RESULTS: We show that sulindac prevented azoxymethane-induced distal colon tumours in all mice. In the proximal colon, however, sulindac induced new inflammatory lesions on the mucosal folds, which further developed into adenocarcinoma in up to 18-25% of the p53 or Msh2 deficient mice but rarely in wild-type mice. This region in the proximal colon was characterised by a distinct profile of pro- and anti-inflammatory factors, which were modulated by the sulindac diet, including upregulation of hypoxia inducible factor 1α and macrophage inflammatory protein 2. CONCLUSIONS: These data show that the sulindac diet promotes carcinogenesis in the mouse proximal colon possibly through chronic inflammation. Sulindac has both beneficial and harmful effects in vivo, which are associated with different microenvironments within the colon of experimental mice. Deficiency for the Msh2 or p53 tumour suppressor genes increases the harmful side effects of long-term sulindac treatment in the mouse colon.
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Antiinflamatorios no Esteroideos/uso terapéutico , Anticarcinógenos/uso terapéutico , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/prevención & control , Sulindac/uso terapéutico , Adenocarcinoma/inducido químicamente , Adenocarcinoma/metabolismo , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/farmacocinética , Anticarcinógenos/efectos adversos , Anticarcinógenos/farmacocinética , Apoptosis/efectos de los fármacos , Azoximetano , Carcinógenos , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Colon/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Mediadores de Inflamación/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 2 Homóloga a MutS/deficiencia , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/patología , Sulindac/efectos adversos , Sulindac/farmacocinética , Proteína p53 Supresora de Tumor/deficienciaRESUMEN
AIM: To assess and compare immunohistochemical expression of epidermal growth factor receptor (EGFR) with gene amplification as demonstrated by chromogenic in situ hybridisation (CISH), in colorectal adenocarcinoma. METHODS: Sections from 100 consecutive colorectal cancer resection specimens were stained for EGFR using immunohistochemistry and CISH. Immunohistochemical assessment was independently performed at two laboratories, using the same antibody and protocols. RESULTS: With immunohistochemistry, strong circumferential membrane staining (3+ staining) was demonstrated in only 5% of cases, and this was only focal in three of five cases. At one laboratory, weak or incomplete staining (1+ or 2+) was observed in five further cases (5%), which had been negative at the other laboratory. CISH demonstrated high level gene amplification (>10 copies/nucleus) in the same five cases which had demonstrated 3+ staining with immunohistochemistry, and in those cases where the staining was focal, the amplification was demonstrated in the same foci of the tumour. Five further cases (5%) had low level amplification (5-10 copies per nucleus); these cases did not exhibit significant positive staining with immunohistochemistry. All the cases which demonstrated gene amplification (high or low level) arose in the distal colon. There was no correlation between gene amplification status and a variety of other variables, including stage at diagnosis, mucinous differentiation, neuroendocrine differentiation, or loss of expression of mismatch repair proteins. CONCLUSIONS: Immunohistochemical expression of EGFR is variable between laboratories, even using standardised protocols. 3+ staining is predictive of high level gene amplification, but correlates very poorly with low level amplification, which may still be clinically significant. In some cases gene amplification was only focal, offering a potential explanation for poor response to targeted therapy in patients with EGFR positive tumours.