RESUMO
Metastatic castration-resistant prostate cancer (mCRPC) is an immunologically cold disease with dismal outcomes. Cryoablation destroys cancer tissue, releases tumor-associated antigens and creates a pro-inflammatory microenvironment, while dendritic cells (DCs) activate immune responses through processing of antigens. Immunotherapy combinations could enhance the anti-tumor efficacy. This open-label, single-arm, single-center phase I trial determined the safety and tolerability of combining cryoablation and autologous immature DC, without and with checkpoint inhibitors. Immune responses and clinical outcomes were evaluated. Patients with mCRPC, confirmed metastases and intact prostate gland were included. The first participants underwent prostate cryoablation with intratumoral injection of autologous DCs in a 3 + 3 design. In the second part, patients received cryoablation, the highest acceptable DC dose, and checkpoint inhibition with either ipilimumab or pembrolizumab. Sequentially collected information on adverse events, quality of life, blood values and images were analyzed by standard descriptive statistics. Neither dose-limiting toxicities nor adverse events > grade 3 were observed in the 18 participants. Results indicate antitumor activity through altered T cell receptor repertoires, and 33% durable (> 46 weeks) clinical benefit with median 40.7 months overall survival. Post-treatment pain and fatigue were associated with circulating tumor cell (CTC) presence at inclusion, while CTC responses correlated with clinical outcomes. This trial demonstrates that cryoimmunotherapy in mCRPC is safe and well tolerated, also for the highest DC dose (2.0 × 108) combined with checkpoint inhibitors. Further studies focusing on the biologic indications of antitumor activity and immune system activation could be considered through a phase II trial focusing on treatment responses and immunologic biomarkers.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Humanos , Masculino , Células Dendríticas , Ipilimumab/uso terapêutico , Estudos Prospectivos , Neoplasias de Próstata Resistentes à Castração/terapia , Qualidade de Vida , Microambiente TumoralRESUMO
Wnt (wingless)/ß-catenin signaling is critical for tumor progression and is frequently activated in colorectal cancer as a result of the mutation of adenomatous polyposis coli (APC); however, therapeutic agents targeting this pathway for clinical use are lacking. Here we report that nitazoxanide (NTZ), a clinically approved antiparasitic drug, efficiently inhibits Wnt signaling independent of APC. Using chemoproteomic approaches, we have identified peptidyl arginine deiminase 2 (PAD2) as the functional target of NTZ in Wnt inhibition. By targeting PAD2, NTZ increased the deamination (citrullination) and turnover of ß-catenin in colon cancer cells. Replacement of arginine residues disrupted the transcriptional activity, and NTZ induced degradation of ß-catenin. In Wnt-activated colon cancer cells, knockout of either PAD2 or ß-catenin substantially increased resistance to NTZ treatment. Our data highlight the potential of NTZ as a modulator of ß-catenin citrullination for the treatment of cancer patients with Wnt pathway mutations.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Tiazóis/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Citrulinação , Neoplasias do Colo/patologia , Técnicas de Inativação de Genes , Humanos , Nitrocompostos , Proteína-Arginina Desiminase do Tipo 2 , Desiminases de Arginina em Proteínas/genética , Via de Sinalização Wnt/genética , beta Catenina/genéticaRESUMO
Oncogenic mutations of the Wnt (wingless)/ß-catenin pathway are frequently observed in major cancer types. Thus far, however, no therapeutic agent targeting Wnt/ß-catenin signaling is available for clinical use. Here we demonstrate that axitinib, a clinically approved drug, strikingly blocks Wnt/ß-catenin signaling in cancer cells, zebrafish, and Apc(min/+) mice. Notably, axitinib dramatically induces Wnt asymmetry and nonrandom DNA segregation in cancer cells by promoting nuclear ß-catenin degradation independent of the GSK3ß (glycogen synthase kinase3ß)/APC (adenomatous polyposis coli) complex. Using a DARTS (drug affinity-responsive target stability) assay coupled to 2D-DIGE (2D difference in gel electrophoresis) and mass spectrometry, we have identified the E3 ubiquitin ligase SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase) as the direct target of axitinib in blocking Wnt/ß-catenin signaling. Treatment with axitinib stabilizes SHPRH and thereby increases the ubiquitination and degradation of ß-catenin. Our findings suggest a previously unreported mechanism of nuclear ß-catenin regulation and indicate that axitinib, a clinically approved drug, would provide therapeutic benefits for cancer patients with aberrant nuclear ß-catenin activation.
Assuntos
Divisão Celular/efeitos dos fármacos , Imidazóis/farmacologia , Indazóis/farmacologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/fisiologia , Animais , Axitinibe , DNA Helicases/fisiologia , Glicogênio Sintase Quinase 3 beta/fisiologia , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regeneração/efeitos dos fármacos , Ubiquitina-Proteína Ligases/fisiologia , Peixe-ZebraRESUMO
BACKGROUND: Despite successful implementation of drugs targeting the human epidermal growth factor receptor 2 (HER2) receptor in breast and gastric cancers, the potential of HER2 as a therapeutic target in other cancers has been less studied, including endometrial cancer. We investigated expression levels of HER2 (ERBB2) in a large cohort of endometrial cancer lesions, also including complex atypical hyperplasia and metastatic lesions. METHODS: 67 precursor lesions, 790 primary endometrial cancers and 383 metastatic lesions were investigated for HER2 expression in relation to clinicopathologic features and outcome. Protein levels were assessed by immunohistochemistry (using the HercepTest and staining index (SI) criteria), mRNA levels by microarrays and amplification status by chromogenic in situ hybridisation. RESULTS: High HER2 protein levels were significantly associated with features of aggressive disease and increased mRNA ERBB2 levels. HER2 expression defined by the SI proved to be a better predictor of survival compared with the HercepTest. A discordant HER2 expression pattern between paired primary and metastatic lesions was detected, revealing substantial reduction in HER2 expression from primary to metastatic disease. CONCLUSIONS: Loss of HER2 expression is common in metastatic endometrial cancer lesions and assessment of HER2 levels in the metastatic lesions may be important to define the potential benefit of anti-HER2 treatments in endometrial cancer patients.
Assuntos
Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Lesões Pré-Cancerosas/metabolismo , RNA Mensageiro/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Idoso , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Metástase Neoplásica , Lesões Pré-Cancerosas/genética , Taxa de SobrevidaRESUMO
To investigate if viruses are involved in the pathogenesis of vestibular schwannomas (VS), we have screened biopsies from VS patients using different molecular techniques. Screening for the presence of known viruses using a pan-viral microarray assay (ViroChip) indicated the presence of several viruses including human endogenous retrovirus K (HERV-K) and human herpes virus 2 (HHV2). But with the exception of HERV-K, none of the findings could be verified by other methods. Whole transcriptome sequencing showed only the presence of HERV-K transcripts and whole genome sequencing showed only the presence of Epstein-Barr virus, most likely originating from infiltration of lymphocytes. We therefore conclude that it is less likely that viruses are involved in the pathogenesis of vestibular schwannomas.
Assuntos
DNA Viral/análise , Neuroma Acústico/virologia , RNA Viral/análise , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Little is known about the role of glial host cells in brain tumours. However, supporting stromal cells have been shown to foster tumour growth in other cancers. METHODS: We isolated stromal cells from patient-derived glioblastoma (GBM) xenografts established in GFP-NOD/scid mice. With simultaneous removal of CD11b+ immune and CD31+ endothelial cells by fluorescence activated cell sorting (FACS), we obtained a population of tumour-associated glial cells, TAGs, expressing markers of terminally differentiaed glial cell types or glial progenitors. This cell population was subsequently characterised using gene expression analyses and immunocytochemistry. Furthermore, sphere formation was assessed in vitro and their glioma growth-promoting ability was examined in vivo. Finally, the expression of TAG related markers was validated in human GBMs. RESULTS: TAGs were highly enriched for the expression of glial cell proteins including GFAP and myelin basic protein (MBP), and immature markers such as Nestin and O4. A fraction of TAGs displayed sphere formation in stem cell medium. Moreover, TAGs promoted brain tumour growth in vivo when co-implanted with glioma cells, compared to implanting only glioma cells, or glioma cells and unconditioned glial cells from mice without tumours. Genome-wide microarray analysis of TAGs showed an expression profile distinct from glial cells from healthy mice brains. Notably, TAGs upregulated genes associated with immature cell types and self-renewal, including Pou3f2 and Sox2. In addition, TAGs from highly angiogenic tumours showed upregulation of angiogenic factors, including Vegf and Angiopoietin 2. Immunohistochemistry of three GBMs, two patient biopsies and one GBM xenograft, confirmed that the expression of these genes was mainly confined to TAGs in the tumour bed. Furthermore, their expression profiles displayed a significant overlap with gene clusters defining prognostic subclasses of human GBMs. CONCLUSIONS: Our data demonstrate that glial host cells in brain tumours are functionally distinct from glial cells of healthy mice brains. Furthermore, TAGs display a gene expression profile with enrichment for genes related to stem cells, immature cell types and developmental processes. Future studies are needed to delineate the biological mechanisms regulating the brain tumour-host interplay.
Assuntos
Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Glioblastoma/metabolismo , Transcriptoma , Animais , Biomarcadores Tumorais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise em Microsséries , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The purpose of this study was to evaluate invasive and metastatic potential of urothelial cancer by investigating differential expression of various clock genes/proteins participating in the 24 h circadian rhythms and to compare these gene expressions with transcription of other cancer-associated genes. METHODS: Twenty seven paired samples of tumour and benign tissue collected from patients who underwent cystectomy were analysed and compared to 15 samples of normal bladder tissue taken from patients who underwent cystoscopy for benign prostate hyperplasia (unrelated donors). Immunohistochemical analyses were made for clock and clock-related proteins. In addition, the gene-expression levels of 22 genes (clock genes, casein kinases, oncogenes, tumour suppressor genes and cytokeratins) were analysed by real-time quantitative PCR (qPCR). RESULTS: Considerable up- or down-regulation and altered cellular distribution of different clock proteins, a reduction of casein kinase1A1 (CSNK1A1) and increase of casein kinase alpha 1 E (CSNK1E) were found. The pattern was significantly correlated with simultaneous up-regulation of stimulatory tumour markers, and a down-regulation of several suppressor genes. The pattern was mainly seen in aneuploid high-grade cancers. Considerable alterations were also found in the neighbouring bladder mucosa. CONCLUSIONS: The close correlation between altered expression of various clock genes and common tumour markers in urothelial cancer indicates that disturbed function in the cellular clock work may be an important additional mechanism contributing to cancer progression and malignant behaviour.
Assuntos
Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Urotélio/patologia , Aneuploidia , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Fatores de Transcrição/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Urotélio/metabolismoRESUMO
BACKGROUND: Expression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells, thus androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. A clearer understanding of the context dependent activation of the AR and its target genes is therefore desirable. METHODS: Immortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts. RESULTS: A variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen- and AR-dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial cells, but did not exert a positive feedback on endogenous AR expression. Treatment of basal prostate epithelial cells with inhibitors of epigenetic gene silencing was not efficient in inducing androgen-dependent transcription of AR target genes, suggesting the importance of missing cofactor(s). CONCLUSIONS: Regulatory mechanisms of AR and androgen-dependent AR target gene transcription are insufficiently understood and may be critical for prostate cancer initiation, progression and escape from standard therapy. The present model is useful for the study of context dependent activation of the AR and its transcriptome.
Assuntos
Redes Reguladoras de Genes , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Neoplasias da Próstata/genética , Ligação Proteica , Análise de Sequência de RNA/métodos , Análise de Célula ÚnicaRESUMO
The mechanisms underlying tumor dormancy have been elusive and not well characterized. We recently published an experimental model for the study of human tumor dormancy and the role of angiogenesis, and reported that the angiogenic switch was preceded by a local increase in VEGF-A and basic fibroblast growth factor. In this breast cancer xenograft model (MDA-MB-436 cells), analysis of differentially expressed genes revealed that heat shock protein 27 (HSP27) was significantly up-regulated in angiogenic cells compared with nonangiogenic cells. The effect of HSP27 down-regulation was further evaluated in cell lines, mouse models, and clinical datasets of human patients with breast cancer and melanoma. Stable down-regulation of HSP27 in angiogenic tumor cells was followed by long-term tumor dormancy in vivo. Strikingly, only 4 of 30 HSP27 knockdown xenograft tumors initiated rapid growth after day 70, in correlation with a regain of HSP27 protein expression. Significantly, no tumors escaped from dormancy without HSP27 expression. Down-regulation of HSP27 was associated with reduced endothelial cell proliferation and decreased secretion of VEGF-A, VEGF-C, and basic fibroblast growth factor. Conversely, overexpression of HSP27 in nonangiogenic cells resulted in expansive tumor growth in vivo. By clinical validation, strong HSP27 protein expression was associated with markers of aggressive tumors and decreased survival in patients with breast cancer and melanoma. An HSP27-associated gene expression signature was related to molecular subgroups and survival in breast cancer. Our findings suggest a role for HSP27 in the balance between tumor dormancy and tumor progression, mediated by tumor-vascular interactions. Targeting HSP27 might offer a useful strategy in cancer treatment.
Assuntos
Neoplasias da Mama/metabolismo , Regulação para Baixo , Proteínas de Choque Térmico HSP27/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Animais , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP27/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos SCID , Neovascularização Patológica/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
MicroRNAs play critical roles in tumorigenesis and metastasis. Here, we report the dual functions of miR-182 and miR-203 in our previously described prostate cell model. MiR-182 and miR-203 were completely repressed during epithelial to mesenchymal transition (EMT) from prostate epithelial EP156T cells to the progeny mesenchymal nontransformed EPT1 cells. Re-expression of miR-182 or miR-203 in EPT1 cells and prostate cancer PC3 cells induced mesenchymal to epithelial transition (MET) features. Simultaneously, miR-182 and miR-203 provided EPT1 cells with the ability to self-sufficiency of growth signals, a well-recognized oncogenic feature. Gene expression profiling showed high overlap of the genes affected by miR-182 and miR-203. SNAI2 was identified as a common target of miR-182 and miR-203. Knock-down of SNAI2 in EPT1 cells phenocopied re-expression of either miR-182 or miR-203 regarding both MET and self-sufficiency of growth signals. Strikingly, considerable overlaps of changed genes were found between the re-expression of miR-182/203 and knock-down of SNAI2. Finally, P-cadherin was identified as a direct target of SNAI2. We conclude that miR-182 and miR-203 induce MET features and growth factor independent growth via repressing SNAI2 in prostate cells. Our findings shed new light on the roles of miR-182/203 in cancer related processes.
Assuntos
MicroRNAs/metabolismo , Próstata/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Animais , Caderinas/metabolismo , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Lentivirus/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , Próstata/citologia , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução GenéticaRESUMO
The presence of tumor cells entering vascular channels is a prognostic marker for many cancers, including endometrial carcinoma. Vascular invasion is considered to be an early step in the metastatic process and important for the progress of malignant tumors. Here, we investigated the gene expression patterns related to vascular involvement in 57 primary endometrial cancers, using DNA microarray and quantitative PCR techniques. A vascular invasion signature of 18 genes was significantly associated with patient survival and clinicopathological phenotype. Vascular involvement was also related to gene sets for epithelial-mesenchymal transition, wound response, endothelial cells, and vascular endothelial growth factor (VEGF) activity. With immunohistochemical validation, both collagen 8 and matrix metalloproteinase 3 (MMP3) were associated with vascular invasion, whereas ANGPTL4 and IL-8 were associated with patient survival. Our findings indicate that vascular involvement within primary tumors is associated with gene expression profiles related to angiogenesis and epithelial-mesenchymal transition. These data could contribute to an improved understanding of potential targets for metastatic spread and may provide clinically important information for better management of endometrial cancer.
Assuntos
Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neovascularização Patológica/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Análise por Conglomerados , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Fenótipo , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Análise de SobrevidaRESUMO
BACKGROUND: Increased expression of lipocalin 2 (LCN2) has been observed in several cancers. The aim of the present study was to investigate LCN2 in endometrial cancer in relation to clinico-pathologic phenotype, angiogenesis, markers of epithelial-mesenchymal transition (EMT), and patient survival. METHODS: Immunohistochemical staining was performed using a human LCN2 antibody on a population-based series of endometrial cancer patients collected in Hordaland County (Norway) during 1981-1990 (n = 256). Patients were followed from the time of primary surgery until death or last follow-up in 2007. The median follow-up time for survivors was 17 years. Gene expression data from a prospectively collected endometrial cancer series (n = 76) and a publicly available endometrial cancer series (n = 111) was used for gene correlation studies. RESULTS: Expression of LCN2 protein, found in 49% of the cases, was associated with non-endometrioid histologic type (p = 0.001), nuclear grade 3 (p = 0.001), >50% solid tumor growth (p = 0.001), ER and PR negativity (p = 0.028 and 0.006), and positive EZH2 expression (p < 0.001). LCN2 expression was significantly associated with expression of VEGF-A (p = 0.021), although not with other angiogenesis markers examined (vascular proliferation index, glomeruloid microvascular proliferation, VEGF-C, VEGF-D or bFGF2 expression). Further, LCN2 was not associated with several EMT-related markers (E-cadherin, N-cadherin, P-cadherin, ß-catenin), nor with vascular invasion (tumor cells invading lymphatic or blood vessels). Notably, LCN2 was significantly associated with distant tumor recurrences, as well as with the S100A family of metastasis related genes. Patients with tumors showing no LCN2 expression had the best outcome with 81% 5-year survival, compared to 73% for intermediate and 38% for the small subgroup with strong LCN2 staining (p = 0.007). In multivariate analysis, LCN2 expression was an independent prognostic factor in addition to histologic grade and FIGO stage. CONCLUSION: Increased LCN2 expression is associated with aggressive features and poor prognosis in endometrial cancer.
Assuntos
Proteínas de Fase Aguda/biossíntese , Biomarcadores Tumorais/biossíntese , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Lipocalinas/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas de Fase Aguda/genética , Biomarcadores Tumorais/genética , Neoplasias do Endométrio/irrigação sanguínea , Neoplasias do Endométrio/genética , Transição Epitelial-Mesenquimal , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Lipocalina-2 , Lipocalinas/genética , Análise Multivariada , Invasividade Neoplásica , Metástase Neoplásica , Prognóstico , Proteínas Proto-Oncogênicas/genéticaRESUMO
BACKGROUND: The tumor microenvironment is pivotal in tumor progression. Thus, we aimed to develop a mammary tumor model to elucidate molecular characteristics in the stroma versus the tumor cell compartment by global gene expression. Secondly, since tumor hypoxia influences several aspects of tumor pathophysiology, we hypothesized that hyperoxia might have an inhibitory effect on tumor growth per se. Finally, we aimed to identify differences in gene expression and key molecular mechanisms, both in the native state and following treatment. METHODS: 4T1 dsRed breast cancer cells were injected into eGFP expressing NOD/SCID mice. Group 1 was exposed to 3 intermittent HBO treatments (Day 1, 4 and 7), Group 2 to 7 daily HBO treatments (both 2.5 bar, 100% O2, à 90 min), whereas the controls were exposed to a normal atmosphere. Tumor growth, histology, vascularisation, cell proliferation, cell death and metastasis were assessed. Fluorescence-activated cell sorting was used to separate tumor cells from stromal cells prior to gene expression analysis. RESULTS: The purity of sorted cells was verified by fluorescence microscopy. Gene expression profiling demonstrated that highly expressed genes in the untreated tumor stroma included constituents of the extracellular matrix and matrix metalloproteinases. Tumor growth was significantly inhibited by HBO, and the MAPK pathway was found to be significantly reduced. Immunohistochemistry indicated a significantly reduced microvessel density after intermittent HBO, whereas daily HBO did not show a similar effect. The anti-angiogenic response was reflected in the expression trends of angiogenic factors. CONCLUSIONS: The present in vivo mammary tumor model enabled us to separate tumor and stromal cells, and demonstrated that the two compartments are characterized by distinct gene expressions, both in the native state and following HBO treatments. Furthermore, hyperoxia induced a significant tumor growth-inhibitory effect, with significant down-regulation of the MAPK pathway. An anti-angiogenic effect after intermittent HBO was observed, and reflected in the gene expression profile.
Assuntos
Hipóxia Celular/fisiologia , Perfilação da Expressão Gênica , Neoplasias Mamárias Experimentais/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Matriz Extracelular/genética , Feminino , Proteínas de Fluorescência Verde/metabolismo , Oxigenoterapia Hiperbárica , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Metaloproteinases da Matriz/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.
Assuntos
Núcleo Celular/química , Cromatina/química , DNA/análise , Transcrição Gênica , Animais , Células COS , Chlorocebus aethiops , Hibridização In Situ/métodos , RNA/biossíntese , Sondas RNARESUMO
Epithelial to mesenchymal transition (EMT) is pivotal in tumor metastasis. Our previous work reported an EMT model based on primary prostate epithelial cells (EP156T) which gave rise to cells with mesenchymal phenotype (EPT1) without malignant transformation. To promote prostate cell transformation, cells were maintained in saturation density cultures to select for cells overriding quiescence. Foci formed repeatedly following around 8 weeks in confluent EPT1 monolayers. Only later passage EPT1, but not EP156T cells of any passage, could form foci. Cells isolated from the foci were named EPT2 and formed robust colonies in soft agar, a malignant feature present neither in EP156T nor in EPT1 cells. EPT2 cells showed additional malignant traits in vitro, including higher ability to proliferate following confluence, higher resistance to apoptosis and lower dependence on exogenous growth factors than EP156T and EPT1 cells. Microarray profiling identified gene sets, many of which belong to cell junction modules, that changed expression from EP156T to EPT1 cells and continued to change from EPT1 to EPT2 cells. Our findings provide a novel stepwise cell culture model in which EMT emerges independently of transformation and is associated with subsequent accumulation of malignant features in prostate cells. Reprogramming of cell junction modules is involved in both steps.
Assuntos
Desdiferenciação Celular , Transformação Celular Neoplásica/patologia , Células Epiteliais/citologia , Transição Epitelial-Mesenquimal , Junções Intercelulares/patologia , Próstata/citologia , Apoptose , Técnicas de Cultura de Células , Linhagem Celular , Movimento Celular , Proliferação de Células , Células Clonais , Células Epiteliais/patologia , Perfilação da Expressão Gênica , Humanos , Cariotipagem , Masculino , Repetições de Microssatélites , Próstata/metabolismo , Próstata/patologiaRESUMO
GATA2 has been shown to be an important transcription factor together with androgen receptor (AR) in prostate cancer cells. Less is known about GATA2 in benign prostate epithelial cells. We have investigated if GATA2 exogenous expression in prostate epithelial basal-like cells could induce AR transcription or luminal differentiation. Prostate epithelial basal-like (transit amplifying) cells were transduced with lentiviral vector expressing GATA2. Luminal differentiation markers were assessed by RT-qPCR, Western blot and global gene expression microarrays. We utilized our previously established AR and androgen-dependent fluorescence reporter assay to investigate AR activity at the single-cell level. Exogenous GATA2 protein was rapidly and proteasome-dependently degraded. GATA2 protein expression was rescued by the proteasome inhibitor MG132 and partly by mutating the target site of the E3 ligase FBXW7. Moreover, MG132-mediated proteasome inhibition induced AR mRNA and additional luminal marker gene transcription in the prostate transit amplifying cells. Different types of intrinsic mechanisms restricted GATA2 expression in the transit amplifying cells. The appearance of AR mRNA and additional luminal marker gene expression changes following proteasome inhibition suggests control of essential cofactor(s) of AR mRNA expression and luminal differentiation at this proteolytic level.
RESUMO
Conventional type 1 dendritic cells (cDC1) and conventional type 2 dendritic cells (cDC2) have attracted increasing attention as alternatives to monocyte-derived dendritic cells (moDCs) in cancer immunotherapy. Use of cDCs for therapy has been hindered by their low numbers in peripheral blood. In the present study, we found that extensive spontaneous apoptosis and cDC death in culture within 24hrs represent an additional challenge. Different media conditions that maintain cDC viability and function were investigated. CD141+ cDC1 and CD1c+ cDC2 were isolated from healthy blood donor buffy coats. Low viabilities were found with CellGenix DC, RPMI-1640, and X-VIVO 15 standard culture media and with several supplements at 24hrs and 48hrs. Among multiple factors it was found that GM-CSF improved both cDC1 and cDC2 viability, whereas Flt3-L and IL-4 only increased viability of cDC1 and cDC2, respectively. Combinations of these three cytokines improved viability of both cDCs further, both at 24hrs and 48hrs time points. Although these cytokines have been extensively investigated for their role in myeloid cell differentiation, and are also used clinically, their effects on mature cDCs remain incompletely known, in particular effects on pro-inflammatory or tolerogenic cDC features. HLA-DR, CD80, CD83, CD86, PD-L1 and PD-L2 cDC membrane expressions were relatively little affected by GM-CSF, IL-4 and Flt3-L cytokine supplements compared to the strong induction following Toll-like receptor (TLR) stimulation for 24hrs. With minor exceptions the three cytokines appeared to be permissive to the TLR-induced marker expression. Allogeneic mixed leukocyte reaction showed that the cytokines promoted T-cell proliferation and revealed a potential to boost both Th1 and Th2 polarizing cytokines. GM-CSF and Flt3-L and their combination improved the capability of cDC1 for dextran uptake, while in cDC2, dextran capture was improved by GM-CSF. The data suggest that GM-CSF, IL-4 and Flt3-L and combinations might be beneficial for DC viability and function in vitro. Limited viability of cDCs could be a confounding variable experimentally and in immunotherapy.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos , Interleucina-4 , Citocinas/metabolismo , Células Dendríticas/metabolismo , Dextranos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-4/farmacologia , Interleucina-4/metabolismo , HumanosRESUMO
Our drug discovery model has identified two novel STAT3 SH2 domain inhibitors 323-1 and 323-2 (delavatine A stereoisomers) in a series of experiments. In silico computational modeling, drug affinity responsive target stability (DARTS), and fluorescence polarization (FP) assays altogether determined that 323-1 and 323-2 directly target the STAT3 SH2 domain and inhibited both phosphorylated and non-phosphorylated STAT3 dimerization. Computational docking predicted that compound 323s bind to three subpockets of the STAT3 SH2 domain. The 323s inhibition of STAT3 dimerization was more potent than the commercial STAT3 SH2 domain inhibitor S3I-201 in the co-immunoprecipitation assay, correlating with computational docking data. The fluorescence polarization assay further confirmed that the compound 323s target the STAT3 SH2 domain by competitively abrogating the interaction between STAT3 and the SH2-binding peptide GpYLPQTV. Compared with S3I-201, the 323 compounds exhibited stronger inhibition of STAT3 and reduced the level of IL-6-stimulated phosphorylation of STAT3 (Tyr705) in LNCaP cells over the phosphorylation of STAT1 (Tyr701) induced by IFN-É£ in PC3 cells or the phosphorylation of STAT1 (Ser727) in DU145 cells. Both compounds downregulated STAT3 target genes MCL1 and cyclin D1. Thus, the two compounds are promising lead compounds for the treatment of cancers with hyper-activated STAT3.
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Modulation of ß-catenin signaling has attractive therapeutic potential in cancer immunotherapy. Several studies have found that ß-catenin can mediate immune evasion in cancer and promote anti-inflammatory features of antigen-presenting dendritic cells. Many small molecular compounds that inhibit Wnt/ß-catenin signaling are currently in clinical development, but none have entered routine clinical use. New inhibitors of ß-catenin signaling are consequently desirable. Here, we have tested, in monocyte-derived dendritic cells, the effects of two small molecular compounds, axitinib and nitazoxanide, that previously have been discovered to inhibit ß-catenin signaling in colon cancer cells. Immature and lipopolysaccharide-matured dendritic cells prepared from healthy blood donor buffy coats were stimulated with 6-bromoindirubin-3'-oxime (6-BIO) to boost basal ß-catenin activity, and the effects of axitinib and nitazoxanide were compared with the commercial ß-catenin inhibitor ICG-001. Assays, including genome-wide RNA-sequencing, indicated that neither axitinib nor nitazoxanide demonstrated considerable ß-catenin inhibition. Both compounds were found to be less toxic to monocyte-derived dendritic cells than either 6-BIO or ICG-001. Axitinib stimulated several aspects of dendritic cell function, such as IL12-p70 secretion, and counteracted IL-10 secretion, according to the present study. However, neither axitinib nor nitazoxanide were found to be efficient ß-catenin inhibitors in monocyte-derived dendritic cells.
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Liquid biopsies have emerged as a potential new diagnostic tool, providing detailed information relevant for characterization and treatment of solid cancers. We here present an overview of current evidence supporting the clinical relevance of liquid biopsy assessments. We also discuss the implementation of liquid biopsies in clinical studies and their current and future clinical role, with a special reference to the Nordic healthcare systems. Our considerations are restricted to the most established liquid biopsy specimens: circulating tumor DNA (ctDNA) and circulating tumor cells (CTC). Both ctDNA and CTCs have been used for prognostic stratification, treatment choices, and treatment monitoring in solid cancers. Several recent publications also support the role of ctDNA in early cancer detection. ctDNA seems to provide more robust clinically relevant information in general, whereas CTCs have the potential to answer more basic questions related to cancer biology and metastasis. Epidermal growth factor receptor-directed treatment of non-small-cell lung cancer represents a clinical setting where ctDNA already has entered the clinic. The role of liquid biopsies in treatment decisions, standardization of methods, diagnostic performance and the need for further research, as well as cost and regulatory issues were identified as factors that influence further integration in the clinic. In conclusion, substantial evidence supports the clinical utility of liquid biopsies in cancer diagnostics, but further research is still required for a more general application in clinical practice.