RESUMO
WNT2 acts as a pro-angiogenic factor in placental vascularization and increases angiogenesis in liver sinusoidal endothelial cells (ECs) and other ECs. Increased WNT2 expression is detectable in many carcinomas and participates in tumor progression. In human colorectal cancer (CRC), WNT2 is selectively elevated in cancer-associated fibroblasts (CAFs), leading to increased invasion and metastasis. However, if there is a role for WNT2 in colon cancer, angiogenesis was not addressed so far. We demonstrate that WNT2 enhances EC migration/invasion, while it induces canonical WNT signaling in a small subset of cells. Knockdown of WNT2 in CAFs significantly reduced angiogenesis in a physiologically relevant assay, which allows precise assessment of key angiogenic properties. In line with these results, expression of WNT2 in otherwise WNT2-devoid skin fibroblasts led to increased angiogenesis. In CRC xenografts, WNT2 overexpression resulted in enhanced vessel density and tumor volume. Moreover, WNT2 expression correlates with vessel markers in human CRC. Secretome profiling of CAFs by mass spectrometry and cytokine arrays revealed that proteins associated with pro-angiogenic functions are elevated by WNT2. These included extracellular matrix molecules, ANG-2, IL-6, G-CSF, and PGF. The latter three increased angiogenesis. Thus, stromal-derived WNT2 elevates angiogenesis in CRC by shifting the balance towards pro-angiogenic signals.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/patologia , Neovascularização Patológica/induzido quimicamente , Proteína Wnt2/metabolismo , Proteína Wnt2/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Microambiente Tumoral/fisiologiaRESUMO
A key question in precision medicine is how functional heterogeneity in solid tumours informs therapeutic sensitivity. We demonstrate that spatial characteristics of oncogenic signalling and therapy response can be modelled in precision-cut slices from Kras-driven non-small-cell lung cancer with varying histopathologies. Unexpectedly, profiling of in situ tumours demonstrated that signalling stratifies mostly according to histopathology, showing enhanced AKT and SRC activity in adenosquamous carcinoma, and mitogen-activated protein kinase (MAPK) activity in adenocarcinoma. In addition, high intertumour and intratumour variability was detected, particularly of MAPK and mammalian target of rapamycin (mTOR) complex 1 activity. Using short-term treatment of slice explants, we showed that cytotoxic responses to combination MAPK and phosphoinositide 3-kinase-mTOR inhibition correlate with the spatially defined activities of both pathways. Thus, whereas genetic drivers determine histopathology spectra, histopathology-associated and spatially variable signalling activities determine drug sensitivity. Our study is in support of spatial aspects of signalling heterogeneity being considered in clinical diagnostic settings, particularly to guide the selection of drug combinations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Carcinogênese/genética , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologiaRESUMO
The neuropeptide CGRP, acting through the G-protein coupled receptor CALCRL and its coreceptor RAMP1, plays a key role in migraines, which has led to the clinical development of several inhibitory compounds. Recently, high CALCRL expression has been shown to be associated with a poor prognosis in acute myeloid leukemia (AML). We investigate, therefore, the functional role of the CGRP-CALCRL axis in AML. To this end, in silico analyses, human AML cell lines, primary patient samples, and a C57BL/6-based mouse model of AML are used. We find that CALCRL is up-regulated at relapse of AML, in leukemic stem cells (LSCs) versus bulk leukemic cells, and in LSCs versus normal hematopoietic stem cells. CGRP protects receptor-positive AML cell lines and primary AML samples from apoptosis induced by cytostatic drugs used in AML therapy, and this effect is inhibited by specific antagonists. Furthermore, the CGRP antagonist olcegepant increases differentiation and reduces the leukemic burden as well as key stem cell properties in a mouse model of AML. These data provide a basis for further investigations into a possible role of CGRP-CALCRL inhibition in the therapy of AML.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteína Semelhante a Receptor de Calcitonina/antagonistas & inibidores , Linhagem Celular Tumoral , Daunorrubicina/farmacologia , Daunorrubicina/uso terapêutico , Dipeptídeos/farmacologia , Dipeptídeos/uso terapêutico , Feminino , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Piperazinas , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Transdução de SinaisRESUMO
BACKGROUND: Several studies in animal models demonstrated that obligate and facultative anaerobic bacteria of the genera Bifidobacterium, Salmonella, or Clostridium specifically colonize solid tumors. Consequently, these and other bacteria are discussed as live vectors to deliver therapeutic genes to inhibit tumor growth. Therapeutic approaches for cancer treatment using anaerobic bacteria have been investigated in different mouse models. In the present study, solid three-dimensional (3D) multicellular tumor spheroids (MCTS) of the colorectal adenocarcinoma cell line HT-29 were generated and tested for their potential to study prodrug-converting enzyme therapies using bacterial vectors in vitro. RESULTS: HT-29 MCTS resembled solid tumors displaying all relevant features with an outer zone of proliferating cells and hypoxic and apoptotic regions in the core. Upon incubation with HT-29 MCTS, Bifidobacterium bifidum S17 and Salmonella typhimurium YB1 selectively localized, survived and replicated in hypoxic areas inside MCTS. Furthermore, spores of the obligate anaerobe Clostridium sporogenes germinated in these hypoxic areas. To further evaluate the potential of MCTS to investigate therapeutic approaches using bacteria as gene delivery vectors, recombinant bifidobacteria expressing prodrug-converting enzymes were used. Expression of a secreted cytosine deaminase in combination with 5-fluorocytosine had no effect on growth of MCTS due to an intrinsic resistance of HT-29 cells to 5-fluorouracil, i.e. the converted drug. However, a combination of the prodrug CB1954 and a strain expressing a secreted chromate reductase effectively inhibited MCTS growth. CONCLUSIONS: Collectively, the presented results indicate that MCTS are a suitable and reliable model to investigate live bacteria as gene delivery vectors for cancer therapy in vitro.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/genética , Técnicas In Vitro/métodos , Esferoides Celulares/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , CamundongosRESUMO
BACKGROUND: Clinical management of prostate cancer (PC) is still highly demanding on the identification of robust biomarkers which will allow a more precise prediction of disease progression. METHODS: We profiled both mRNA expression and DNA copy number alterations (CNAs) from laser capture microdissected cells from 31 PC patients and 17 patients with benign prostatic hyperplasia using Affymetrix GeneChip® technology. PC patients were subdivided into an aggressive (Gleason Score 8 or higher, and/or T3/T4 and/or N+/M+) and non-aggressive (all others) form of PC. Furthermore, we correlated the two datasets, as genes whose varied expression is due to a chromosomal alteration, may suggest a causal implication of these genes in the disease. All statistical analyses were performed in R version 2.15.0 and Bioconductor version 1.8.1., respectively. RESULTS: We confirmed several common altered chromosomal regions as well as recently discovered loci such as deletions on chromosomes 3p14.1-3p13 and 13q13.3-13q14.11 supporting a possible role for RYBP, RGC32, and ELF1 in tumor suppression. Integrative analysis of expression and CN data combined with data retrieved from online databases propose PTP4A3 and ELF1 as possible factors for tumor progression. CONCLUSIONS: Copy number data analysis revealed some significant differences between aggressive and non-aggressive tumors, while gene expression data alone could not define an aggressive group of patients. The assessment of CNA may have diagnostic and prognostic value in PC.
Assuntos
Perfilação da Expressão Gênica , Próstata/patologia , Neoplasias da Próstata/patologia , Adulto , Idoso , Variações do Número de Cópias de DNA , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Microdissecção e Captura a Laser , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA MensageiroRESUMO
The growing number of recently identified negative feedback regulators of receptor tyrosine kinases (RTKs) highlights the importance of signal attenuation and modulation for correct signaling outcome. Mitogen-inducible gene 6 (Mig6 also known as RALT or Gene 33) is a multiadaptor protein thought to be involved in the regulation of RTK and stress signaling. Here, we show that deletion of the mouse gene encoding Mig6 (designated Errfi1, which stands for ERBB receptor feedback inhibitor 1) causes hyperactivation of endogenous epidermal growth factor receptor (EGFR) and sustained signaling through the mitogen-activated protein kinase (MAPK) pathway, resulting in overproliferation and impaired differentiation of epidermal keratinocytes. Furthermore, Errfi1-/- mice develop spontaneous tumors in various organs and are highly susceptible to chemically induced formation of skin tumors. A tumor-suppressive role for Mig6 is supported by our finding that MIG6 is downregulated in various human cancers. Inhibition of endogenous Egfr signaling with the Egfr inhibitor gefitinib (Iressa) or replacement of wild-type Egfr with the kinase-deficient protein encoded by the hypomorphic Egfr(wa2) allele completely rescued skin defects in Erffi1-/- mice. Carcinogen-induced tumors displayed by Errfi1-/- mice were highly sensitive to gefitinib. These results indicate that Mig6 is a specific negative regulator of Egfr signaling in skin morphogenesis and is a novel tumor suppressor of Egfr-dependent carcinogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptores ErbB/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Morfogênese , Neoplasias/metabolismo , Pele/crescimento & desenvolvimento , Pele/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proliferação de Células , Gefitinibe , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Quinazolinas/metabolismo , Pele/citologia , Pele/metabolismoRESUMO
Treatment with anaplastic lymphoma kinase (ALK) inhibitors significantly improves outcome for non-small-cell lung cancer (NSCLC) patients with ALK-rearranged tumors. However, clinical resistance typically develops over time and, in the majority of cases, resistance mechanisms are ALK-independent. We generated tumor cell cultures from multiple regions of an ALK-rearranged clinical tumor specimen and deployed functional drug screens to identify modulators of ALK-inhibitor response. This identified a role for PI3Kß and EGFR inhibition in sensitizing the response regulating resistance to ALK inhibition. Inhibition of ALK elicited activation of EGFR, and subsequent MAPK and PI3K-AKT pathway reactivation. Sensitivity to ALK targeting was enhanced by inhibition or knockdown of PI3Kß. In ALK-rearranged primary cultures, the combined inhibition of ALK and PI3Kß prevented the EGFR-mediated ALK-inhibitor resistance, and selectively targeted the cancer cells. The combinatorial effect was seen also in the background of TP53 mutations and in epithelial-to-mesenchymal transformed cells. In conclusion, combinatorial ALK- and PI3Kß-inhibitor treatment carries promise as a treatment for ALK-rearranged NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Fosfatidilinositol 3-Quinases , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico/genética , Inibidores de Proteínas Quinases/efeitos adversos , Receptores ErbB/genéticaRESUMO
The bromodomain and PHD-finger containing transcription factor (BPTF) is part of the nucleosome remodeling factor (NURF) complex and has been implicated in multiple cancer types. Here, we report the discovery of a potent and selective chemical probe targeting the bromodomain of BPTF with an attractive pharmacokinetic profile enabling cellular and inâ vivo experiments in mice. Microarray-based transcriptomics in presence of the probe in two lung cancer cell lines revealed only minor effects on the transcriptome. Profiling against a panel of cancer cell lines revealed that the antiproliferative effect does not correlate with BPTF dependency score in depletion screens. Both observations and the multi-domain architecture of BPTF suggest that depleting the protein by proteolysis targeting chimeras (PROTACs) could be a promising strategy to target cancer cell proliferation. We envision that the presented chemical probe and the related negative control will enable the research community to further explore scientific hypotheses with respect to BPTF bromodomain inhibition.
Assuntos
Neoplasias Pulmonares , Fatores de Transcrição , Animais , Camundongos , Proliferação de Células , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
During the past 15 years, a plethora of innovative 3D in vitro systems has been developed. They offer the possibility of identifying crucial cellular and molecular contributors to the disease by permitting manipulation of each in isolation. However, improvements are needed particularly with respect to the predictivity and validity of those models. The major challenge now is to identify which assay and readout combination(s) best suits the current scientific question(s). A deep understanding of the different platforms along with their pros and cons is a prerequisite to make this decision. This review aims to give an overview of the most prominent systems with a focus on applications, translational relevance and adoption drivers from an industry perspective.
Assuntos
Neoplasias , HumanosRESUMO
Antibody-mediated cancer immunotherapy targets inhibitory surface molecules, such as PD1, PD-L1, and CTLA-4, aiming to re-invigorate dysfunctional T cells. We purified and characterized tumor-infiltrating lymphocytes (TILs) and their patient-matched non-tumor counterparts from treatment-naïve NSCLC patient biopsies to evaluate the effect of PD1 expression on the functional and molecular profiles of tumor-resident T cells. We show that PD1+ CD8+ TILs have elevated expression of the transcriptional regulator ID3 and that the cytotoxic potential of CD8 T cells can be improved by knocking down ID3, defining it as a potential regulator of T cell effector function. PD1+ CD4+ memory TILs display transcriptional patterns consistent with both helper and regulator function, but can robustly facilitate B cell activation and expansion. Furthermore, we show that expanding ex vivo-prepared TILs in vitro broadly preserves their functionality with respect to tumor cell killing, B cell help, and TCR repertoire. Although purified PD1+ CD8+ TILs generally maintain an exhausted phenotype upon expansion in vitro, transcriptional analysis reveals a downregulation of markers of T-cell dysfunction, including the co-inhibitory molecules PD1 and CTLA-4 and transcription factors ID3, TOX and TOX2, while genes involved in cell cycle and DNA repair are upregulated. We find reduced expression of WNT signaling components to be a hallmark of PD1+ CD8+ exhausted T cells in vivo and in vitro and demonstrate that restoring WNT signaling, by pharmacological blockade of GSK3ß, can improve effector function. These data unveil novel targets for tumor immunotherapy and have promising implications for the development of a personalized TIL-based cell therapy for lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Antígeno CTLA-4 , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Receptor de Morte Celular Programada 1/genéticaRESUMO
BACKGROUND: Although T cell abundance in solid tumours is associated with better outcomes, it also correlates with a stroma-mediated source of immune suppression driven by TGFß1 and poor overall survival. Whether this also is observed in non-small cell lung cancer (NSCLC) is unknown. METHODS: We utilized molecular analysis of The Cancer Genome Atlas (TCGA) NSLCC cohort to correlate immune activation (IA) gene expression and extracellular matrix/stromal (ECM/stromal) gene expression with patient survival. In an independent cohort of NSCLC samples, we used flow cytometry to identify mesenchymal subsets and ex vivo functional studies to characterize their immune regulatory function. FINDINGS: We observed a high enrichment in a core set of genes defining an IA gene expression signature in NSCLC across TCGA Pan-cancer cohort. High IA signature score correlates with enrichment of ECM/stromal gene signature across TCGA NSCLC datasets. Importantly, a higher ratio of ECM/stromal to IA gene signature score was associated with shorter overall survival. In tumours resected from a separate cohort of NSCLC patients, we identified CD90+CD73+ peritumoral cells that were enriched in the ECM/stromal gene signature, which was amplified by TGFß1. IFNγ and TNFα-primed peritumoral CD90+CD73+ cells upregulate immune checkpoint molecules PD-L1 and IDO1 and secrete an array of cytokines/chemokines including TGFß1. Finally, immune primed peritumoral CD90+CD73+ cells suppress T cell function, which was relieved following combined blockade of PD-L1 and TGFß1 with IDO1 inhibition but not PD-L1 or anti-CD73 alone. INTERPRETATION: Our findings suggest that targeting PD-L1 together with independent biological features of the stroma may enhance host antitumor immunity in NSCLC. FUNDING: LW and HY are supported by a 4-year China Scholarship Council award. This work was funded, in part, by a grant from the Cancer League of Bern, Switzerland to SRRH. Laser scanning microscopy imaging was funded by the R'Equip grant from the Swiss National Science Foundation Nr. 316030_145003.
Assuntos
5'-Nucleotidase/metabolismo , Carcinoma Pulmonar de Células não Pequenas/etiologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Imunomodulação , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Antígenos Thy-1/metabolismo , Microambiente Tumoral , Biomarcadores , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/patologia , Biologia Computacional , Bases de Dados Genéticas , Matriz Extracelular , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Imunomodulação/genética , Imunofenotipagem , Neoplasias Pulmonares/patologia , Modelos Biológicos , Células Estromais/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Transcriptoma , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Colorectal cancer (CRC) accounts for about 10% of cancer deaths worldwide. Colon carcinogenesis is critically influenced by the tumor microenvironment. Cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs) represent the major components of the tumor microenvironment. TAMs promote tumor progression, angiogenesis and tissue remodeling. However, the impact of the molecular crosstalk of tumor cells (TCs) with CAFs and macrophages on monocyte recruitment and their phenotypic conversion is not known in detail so far. In a 3D human organotypic CRC model, we show that CAFs and normal colonic fibroblasts are critically involved in monocyte recruitment and for the establishment of a macrophage phenotype, characterized by high CD163 expression. This is in line with the steady recruitment and differentiation of monocytes to immunosuppressive macrophages in the normal colon. Cytokine profiling revealed that CAFs produce M-CSF, and IL6, IL8, HGF and CCL2 secretion was specifically induced by CAFs in co-cultures with macrophages. Moreover, macrophage/CAF/TCs co-cultures increased TC invasion. We demonstrate that CAFs and macrophages are the major producers of CCL2 and, upon co-culture, increase their CCL2 production twofold and 40-fold, respectively. CAFs and macrophages expressing high CCL2 were also found in vivo in CRC, strongly supporting our findings. CCL2, CCR2, CSF1R and CD163 expression in macrophages was dependent on active MCSFR signaling as shown by M-CSFR inhibition. These results indicate that colon fibroblasts and not TCs are the major cellular component, recruiting and dictating the fate of infiltrated monocytes towards a specific macrophage population, characterized by high CD163 expression and CCL2 production.
Assuntos
Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Quimiocina CCL2/genética , Colo/metabolismo , Neoplasias Colorretais/genética , Receptores de Superfície Celular/genética , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Diferenciação Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , Colo/patologia , Neoplasias Colorretais/sangue , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Humanos , Fator Estimulador de Colônias de Macrófagos/genética , Masculino , Células Mieloides/metabolismo , Células Mieloides/patologia , Transdução de Sinais/genética , Microambiente Tumoral/genéticaRESUMO
Organometallic metal(arene) anticancer agents were believed to confer low selectivity for potential cellular targets. However, the ruthenium(arene) pyridinecarbothioamide (plecstatin-1) showed target selectivity for plectin, a scaffold protein and cytolinker. We employed a three-dimensional cancer spheroid model and showed that plecstatin-1 limited spheroid growth, induced changes in the morphology and in the architecture of tumour spheroids by disrupting the cytoskeletal organization. Additionally, we demonstrated that plecstatin-1 induced oxidative stress, followed by the induction of an immunogenic cell death signature through phosphorylation of eIF2α, exposure of calreticulin, HSP90 and HSP70 on the cell membrane and secretion of ATP followed by release of high mobility group box-1.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Morte Celular Imunogênica/efeitos dos fármacos , Rutênio/farmacologia , Antineoplásicos/química , Neoplasias Colorretais/patologia , Células HCT116 , Células HT29 , Humanos , Rutênio/química , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Tioamidas/química , Tioamidas/farmacologia , Células Tumorais CultivadasRESUMO
SYK has been reported to possess both tumour promotor and repressor activities and deletion has been linked to a pro-proliferative / pro-invasive phenotype in breast tumours. It is unclear whether this is a consequence of protein deletion or loss of kinase activity. The SYK inhibitor, BI 1002494, caused no increase in proliferation in breast cancer cells or primary mammary epithelial cells in 2D or 3D cultures, nor changes in proliferation (CD1/2, CDK4, PCNA, Ki67) or invadopodia markers (MMP14, PARP, phospho-vimentin Ser56). BI 1002494 did not alter SYK protein expression. There was no change in phenotype observed in 3D cultures after addition of BI 1002494. Thirteen weeks of treatment with BI 1002494 resulted in no ductal branching or cellular proliferation in the mammary glands of mice. An in silico genetic analysis in breast tumour samples revealed no evidence that SYK has a typical tumour suppressor gene profile such as focal deletion, inactivating mutations or lower expression levels. Furthermore, SYK mutations were not associated with reduction in survival and disease-free period in breast cancer patients. In conclusion, small molecule inhibition of the kinase function of SYK does not contribute to a typical tumour suppressor profile.
RESUMO
The oncogenic KRAS mutation has a critical role in the initiation of human pancreatic ductal adenocarcinoma (PDAC) since it rewires glutamine metabolism to increase reduced nicotinamide adenine dinucleotide phosphate (NADPH) production, balancing cellular redox homeostasis with macromolecular synthesis1,2. Mitochondrial glutamine-derived aspartate must be transported into the cytosol to generate metabolic precursors for NADPH production2. The mitochondrial transporter responsible for this aspartate efflux has remained elusive. Here, we show that mitochondrial uncoupling protein 2 (UCP2) catalyses this transport and promotes tumour growth. UCP2-silenced KRASmut cell lines display decreased glutaminolysis, lower NADPH/NADP+ and glutathione/glutathione disulfide ratios and higher reactive oxygen species levels compared to wild-type counterparts. UCP2 silencing reduces glutaminolysis also in KRASWT PDAC cells but does not affect their redox homeostasis or proliferation rates. In vitro and in vivo, UCP2 silencing strongly suppresses KRASmut PDAC cell growth. Collectively, these results demonstrate that UCP2 plays a vital role in PDAC, since its aspartate transport activity connects the mitochondrial and cytosolic reactions necessary for KRASmut rewired glutamine metabolism2, and thus it should be considered a key metabolic target for the treatment of this refractory tumour.
Assuntos
Ácido Aspártico/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Glutamina/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína Desacopladora 2/metabolismo , Animais , Transporte Biológico Ativo , Linhagem Celular Tumoral , Citosol/metabolismo , Feminino , Humanos , Camundongos , Camundongos SCID , Mitocôndrias/metabolismo , NADP/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Translation initiation of eukaryotic mRNAs generally occurs by cap-dependent ribosome scanning. However, certain mRNAs contain internal ribosome entry sites (IRES) allowing cap-independent translation. Several of these IRES-competent transcripts and their corresponding proteins are involved in tumourigenesis. This study focused on IRES-driven translation control during the epithelial to mesenchymal transition (EMT) of hepatocytes that reflects crucial aspects of carcinoma progression. Expression profiling of EMT revealed Laminin B1 (LamB1) to be translationally upregulated. The 5'-untranslated region (UTR) of LamB1 was potent to direct IRES-dependent mRNA utilization of a bicistronic reporter construct. Stringent assays for cryptic promoter and splice sites showed no aberrantly expressed transcripts, suggesting that the reporter activity provided by the leader region of LamB1 mRNA exclusively depends on IRES. In accordance, LamB1 expression increased upon negative interference with cap-dependent translation by expression of human rhinovirus 2A protease or heat shock of cells. Finally, the enhanced expression of LamB1 during EMT correlated with an elevated IRES activity. Together, these data provide first evidence that the 5'-UTR of LamB1 contains a bona fide IRES that directs translational upregulation of LamB1 during stress conditions and neoplastic progression of hepatocytes.
Assuntos
Regiões 5' não Traduzidas/química , Hepatócitos/metabolismo , Laminina/genética , Iniciação Traducional da Cadeia Peptídica , Animais , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Epitélio/metabolismo , Genes Reporter , Resposta ao Choque Térmico , Hepatócitos/citologia , Humanos , Laminina/biossíntese , Mesoderma/metabolismo , Camundongos , Regiões Promotoras Genéticas , Capuzes de RNA/metabolismo , Sítios de Splice de RNA , Regulação para Cima , Proteínas Virais/metabolismoRESUMO
The cross talk between tumor cells and other cells present in the tumor microenvironment such as stromal and immune cells highly influences the behavior and progression of disease. Understanding the underlying mechanisms of interaction is a prerequisite to develop new treatment strategies and to prevent or at least reduce therapy failure in the future. Specific reactivation of the patient's immune system is one of the major goals today. However, standard two-dimensional (2D) cell culture techniques lack the necessary complexity to address related questions. Novel three-dimensional (3D) in vitro models-embedded in a matrix or encapsulated in alginate-recapitulate the in vivo situation much better. Cross talk between different cell types can be studied starting from co-cultures. As cancer immune modulation is becoming a major research topic, 3D in vitro models represent an important tool to address immune regulatory/modulatory questions for T, NK, and other cells of the immune system. The 3D systems consisting of tumor cells, fibroblasts, and immune cells (3D-3) already proved as a reliable tool for us. For instance, we made use of those models to study the molecular mechanisms of the cross talk of non-small cell lung cancer (NSCLC) and fibroblasts, to unveil macrophage plasticity in the tumor microenvironment and to mirror drug responses in vivo. Generation of those 3D models and how to use them to study immune cell infiltration and activation will be described in the present book chapter.
Assuntos
Técnicas de Cocultura/métodos , Descoberta de Drogas/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Antineoplásicos/farmacologia , Reatores Biológicos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Células Imobilizadas/efeitos dos fármacos , Células Imobilizadas/imunologia , Células Imobilizadas/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/patologia , Humanos , Imunidade Celular/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/imunologia , Esferoides Celulares/patologia , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacosRESUMO
In up to 30% of non-small cell lung cancer (NSCLC) patients, the oncogenic driver of tumor growth is a constitutively activated epidermal growth factor receptor (EGFR). Although these patients gain great benefit from treatment with EGFR tyrosine kinase inhibitors, the development of resistance is inevitable. To model the emergence of drug resistance, an EGFR-driven, patient-derived xenograft (PDX) NSCLC model was treated continuously with Gefitinib in vivo. Over a period of more than three months, three separate clones developed and were subsequently analyzed: Whole exome sequencing and reverse phase protein arrays (RPPAs) were performed to identify the mechanism of resistance. In total, 13 genes were identified, which were mutated in all three resistant lines. Amongst them the mutations in NOMO2, ARHGEF5 and SMTNL2 were predicted as deleterious. The 53 mutated genes specific for at least two of the resistant lines were mainly involved in cell cycle activities or the Fanconi anemia pathway. On a protein level, total EGFR, total Axl, phospho-NFκB, and phospho-Stat1 were upregulated. Stat1, Stat3, MEK1/2, and NFκB displayed enhanced activation in the resistant clones determined by the phosphorylated vs. total protein ratio. In summary, we developed an NSCLC PDX line modelling possible escape mechanism under EGFR treatment. We identified three genes that have not been described before to be involved in an acquired EGFR resistance. Further functional studies are needed to decipher the underlying pathway regulation.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos , Gefitinibe/farmacologia , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Feminino , Gefitinibe/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Mutação , NF-kappa B/genética , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Regulação para CimaRESUMO
PREDECT, a European IMI consortium, has assumed the task to generate robust 2D and 3D culture platforms. Protocols established for 2D and 3D monoculture and stromal coculture models of increasing complexity (spheroid, stirred-tank bioreactor, Matrigel- and collagen-embedded cultures) have been established between six laboratories within academia, biotech, and pharma. These models were tested using three tumor cell lines (MCF7, LNCaP, and NCI-H1437), covering three pathologies (breast, prostate, and lung), but should be readily transferable to other model systems. Fluorescent protein tagged cell lines were used for all platforms, allowing for online measurement of growth curves and drug responses to treatments. All methods, from culture setup to phenotypic characterization and gene expression profiling are described in this chapter.The adaptable methodologies and detailed protocols described here should help to include these models more readily to the drug discovery pipeline.
Assuntos
Técnicas de Cultura de Células , Reatores Biológicos , Linhagem Celular Tumoral , Citometria de Fluxo , Imunofluorescência , Expressão Gênica , Ordem dos Genes , Genes Reporter , Vetores Genéticos/genética , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Software , Esferoides Celulares , Transdução Genética , Células Tumorais CultivadasRESUMO
The ruthenium complex sodium trans-[tetrachloridobis(1H-indazole)ruthenate(iii)] (KP1339/IT-139) showed preclinical activity in a variety of in vivo tumor models including a highly predictive colon cancer model. The compound has entered clinical trials, where patients experienced disease stabilization accompanied by mild side effects. KP1339, a GRP78 inhibitor, disrupts endoplasmic reticulum (ER) homeostasis leading to cell death. The PERK/eIF2α-branch of the ER plays an essential role in the cascade of events triggering immunogenic cell death (ICD). ICD makes dying cancer cells 'visible' to the immune system, initiating a prolonged immune response against the tumor. As some metal-based chemotherapeutics such as oxaliplatin are able to induce ICD, we investigate whether KP1339 could also trigger induction of the ICD signature. For this, we employ a three-dimensional colon cancer spheroid model and show for the first time that the treatment with KP1339, a ruthenium-based complex, triggers an ICD signature hallmarked by phosphorylation of PERK and eIF2α, exposure of calreticulin on the cell membrane, release of high mobility group box 1 and secretion of ATP.