RESUMO
Immune exclusion predicts poor patient outcomes in multiple malignancies, including triple-negative breast cancer (TNBC)1. The extracellular matrix (ECM) contributes to immune exclusion2. However, strategies to reduce ECM abundance are largely ineffective or generate undesired outcomes3,4. Here we show that discoidin domain receptor 1 (DDR1), a collagen receptor with tyrosine kinase activity5, instigates immune exclusion by promoting collagen fibre alignment. Ablation of Ddr1 in tumours promotes the intratumoral penetration of T cells and obliterates tumour growth in mouse models of TNBC. Supporting this finding, in human TNBC the expression of DDR1 negatively correlates with the intratumoral abundance of anti-tumour T cells. The DDR1 extracellular domain (DDR1-ECD), but not its intracellular kinase domain, is required for immune exclusion. Membrane-untethered DDR1-ECD is sufficient to rescue the growth of Ddr1-knockout tumours in immunocompetent hosts. Mechanistically, the binding of DDR1-ECD to collagen enforces aligned collagen fibres and obstructs immune infiltration. ECD-neutralizing antibodies disrupt collagen fibre alignment, mitigate immune exclusion and inhibit tumour growth in immunocompetent hosts. Together, our findings identify a mechanism for immune exclusion and suggest an immunotherapeutic target for increasing immune accessibility through reconfiguration of the tumour ECM.
Assuntos
Colágeno/metabolismo , Receptor com Domínio Discoidina 1/metabolismo , Matriz Extracelular/metabolismo , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/metabolismo , Evasão Tumoral , Animais , Linhagem Celular Tumoral , Receptor com Domínio Discoidina 1/antagonistas & inibidores , Receptor com Domínio Discoidina 1/deficiência , Receptor com Domínio Discoidina 1/genética , Modelos Animais de Doenças , Matriz Extracelular/imunologia , Feminino , Deleção de Genes , Técnicas de Inativação de Genes , Humanos , Imunocompetência/imunologia , Imunoterapia , Camundongos , Linfócitos T/citologia , Linfócitos T/imunologia , Neoplasias de Mama Triplo Negativas/terapiaRESUMO
Medical castration that interferes with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, clinical progression is universal, and tumors with AR-independent resistance mechanisms appear to be increasing in frequency. Consequently, there is an urgent need to develop new treatments targeting molecular pathways enriched in lethal prostate cancer. Lysine-specific demethylase 1 (LSD1) is a histone demethylase and an important regulator of gene expression. Here, we show that LSD1 promotes the survival of prostate cancer cells, including those that are castration-resistant, independently of its demethylase function and of the AR. Importantly, this effect is explained in part by activation of a lethal prostate cancer gene network in collaboration with LSD1's binding protein, ZNF217. Finally, that a small-molecule LSD1 inhibitor-SP-2509-blocks important demethylase-independent functions and suppresses castration-resistant prostate cancer cell viability demonstrates the potential of LSD1 inhibition in this disease.
Assuntos
Redes Reguladoras de Genes , Histona Desmetilases/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de Próstata Resistentes à Castração/enzimologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/genética , Humanos , Hidrazinas/farmacologia , Masculino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Sulfonamidas/farmacologia , Transativadores/genética , Transativadores/metabolismoRESUMO
PURPOSE: Triple-negative breast cancer (TNBC)/basal-like breast cancer (BLBC) is a highly aggressive form of breast cancer. We previously reported that a small molecule agonist ligand for the orphan nuclear receptor estrogen-related receptor beta (ERRß or ESRRB) has growth inhibitory and anti-mitotic activity in TNBC cell lines. In this study, we evaluate the association of ESRRB mRNA, copy number levels, and protein expression with demographic, clinicopathological, and gene expression features in breast tumor clinical specimens. METHODS: ESRRB mRNA-level expression and clinical associations were analyzed using RNAseq data. Array-based comparative genomic hybridization determined ESRRB copy number in African-American and Caucasian women. Transcription factor activity was measured using promoter-reporter luciferase assays in TNBC cell lines. Semi-automatic quantification of immunohistochemistry measured ERRß protein expression on a 150-patient tissue microarray series. RESULTS: ESRRB mRNA expression is significantly lower in TNBC/BLBC versus other breast cancer subtypes. There is no evidence of ESRRB copy number loss. ESRRB mRNA expression is correlated with the expression of genes associated with neuroactive ligand-receptor interaction, metabolic pathways, and deafness. These genes contain G/C-rich transcription factor binding motifs. The ESRRB message is alternatively spliced into three isoforms, which we show have different transcription factor activity in basal-like versus other TNBC cell lines. We further show that the ERRß2 and ERRßsf isoforms are broadly expressed in breast tumors at the protein level. CONCLUSIONS: Decreased ESRRB mRNA expression and distinct patterns of ERRß isoform subcellular localization and transcription factor activity are key features in TNBC/BLBC.
Assuntos
Biomarcadores Tumorais , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Fatores Etários , Linhagem Celular Tumoral , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Lipid peroxidation of polyunsaturated fatty acids (PUFAs) is an endogenous source of α,ß-unsaturated aldehydes that react with DNA producing a variety of cyclic adducts. The mutagenic cyclic adducts, specifically those derived from oxidation of ω-6 PUFAs, may contribute to the cancer promoting activities associated with ω-6 PUFAs. ( E)-4-Hydroxy-2-nonenal (HNE) is a unique product of ω-6 PUFAs oxidation. HNE reacts with deoxyguanosine (dG) yielding mutagenic 1, N2-propanodeoxyguanosine adducts (HNE-dG). Earlier studies showed HNE can also be oxidized to its epoxide (EH), and EH can react with deoxyadenosine (dA) forming the well-studied εdA and the substituted etheno adducts. Using a liquid chromatography-based tandem mass spectroscopic (LC-MS/MS) method, we previously reported the detection of EH-derived 7-(1',2'-dihydroxyheptyl)-1, N6-ethenodeoxyadenosine (DHHεdA) as a novel endogenous background adduct in DNA from rodent and human tissues. The formation, repair, and mutagenicity of DHHεdA and its biological consequences in cells have not been investigated. To understand the roles of DHHεdA in carcinogenesis, it is important to develop an immuno-based assay to detect DHHεdA in cells and tissues. In this study we describe the development of monoclonal antibodies specifically against DHHεdA and its application to detect DHHεdA in human cells.
Assuntos
Anticorpos Monoclonais/imunologia , Adutos de DNA/química , Adutos de DNA/imunologia , Ácidos Graxos Ômega-6/química , Adenosina/análogos & derivados , Adenosina/química , Adenosina/imunologia , Aldeídos/química , Animais , Carcinógenos , Separação Celular , Cromatografia Líquida/métodos , DNA/química , Ensaio de Imunoadsorção Enzimática/métodos , Compostos de Epóxi/farmacologia , Citometria de Fluxo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Camundongos , Espectrometria de Massas em Tandem/métodosRESUMO
The unfolded protein response (UPR) monitors the folding environment within the endoplasmic reticulum (ER). Accumulation of misfolded proteins within the ER activates the UPR resulting in the execution of adaptive or non-adaptive signaling pathways. α-Synuclein (α-syn) whose accumulation and aggregation define the pathobiology of Parkinson's disease (PD) has been shown to inhibit ER-Golgi transit of COPII vesicles. ATF6, a protective branch of the UPR, is processed via COPII mediated ER-Golgi transit following its activation via ER stress. Using cellular PD models together with biochemical reconstitution assays, we showed that α-syn inhibited processing of ATF6 directly through physical interactions and indirectly through restricted incorporation into COPII vesicles. Impaired ATF6 signaling was accompanied by decreased ER-associated degradation (ERAD) function and increased pro-apoptotic signaling. The mechanism by which α-syn inhibits ATF6 signaling expands our understanding of the role ER stress and the UPR play in neurodegenerative diseases such as PD.
Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Doença de Parkinson/metabolismo , Resposta a Proteínas não Dobradas , alfa-Sinucleína/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Estresse do Retículo Endoplasmático , Degradação Associada com o Retículo Endoplasmático , Humanos , Neurônios/metabolismo , Transdução de Sinais , Substância Negra/metabolismoRESUMO
Mutations in the p53 gene are among the most frequent genetic events in human cancer and may be triggered by environmental and occupational exposures. We examined the association of clinical and pathological characteristics of breast tumors and breast cancer risk factors according to the prevalence and type of p53 mutations. Using tumor blocks from incident cases from a case-control study in western New York, we screened for p53 mutations in exons 2-11 using the Affymetrix p53 Gene Chip array and analyzed case-case comparisons using logistic regression. The p53 mutation frequency among cases was 28.1 %; 95 % were point mutations (13 % of which were silent) and the remainder were single base pair deletions. Sixty seven percent of all point mutations were transitions; 24 % of them are G:C>A:T at CpG sites. Positive p53 mutation status was associated with poorer differentiation (OR, 95 % CI 2.29, 1.21-4.32), higher nuclear grade (OR, 95 % CI 1.99, 1.22-3.25), and increased Ki-67 status (OR, 95 % CI 1.81, 1.10-2.98). Cases with P53 mutations were more likely to have a combined ER-positive and PR-negative status (OR, 95 % CI 1.65, 1.01-2.71), and a combined ER-negative and PR-negative status (OR, 95 % CI 2.18, 1.47-3.23). Body mass index >30 kg/m(2), waist circumference >79 cm, and waist-to-hip ratio >0.86 were also associated with p53 status; obese breast cancer cases are more likely to have p53 mutations (OR, 95 % CI 1.78, 1.19-2.68). We confirmed that p53 mutations are associated with less favorable tumor characteristics and identified an association of p53 mutation status and adiposity.
Assuntos
Adiposidade , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Genes p53 , Mutação , Adulto , Idoso , Alelos , Biomarcadores Tumorais , Neoplasias da Mama/epidemiologia , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , New York/epidemiologia , Vigilância da População , Fatores de Risco , Carga TumoralRESUMO
The mechanisms by which breast cancer (BrC) can successfully metastasize are complex and not yet fully understood. Our goal was to identify tumor-induced stromal changes that influence metastatic cell behavior, and may serve as better targets for therapy. To identify stromal changes in cancer-bearing tissue, dual-species gene expression analysis was performed for three different metastatic BrC xenograft models. Results were confirmed by immunohistochemistry, flow cytometry, and protein knockdown. These results were validated in human clinical samples at the mRNA and protein level by retrospective analysis of cohorts of human BrC specimens. In pre-clinical models of BrC, systemic recruitment of S100A8+ myeloid cells-including myeloid-derived suppressor cells (MDSCs)-was promoted by tumor-derived factors. Recruitment of S100A8+ myeloid cells was diminished by inhibition of tumor-derived factors or depletion of MDSCs, resulting in fewer metastases and smaller primary tumors. Importantly, these MDSCs retain their ability to suppress T cell proliferation upon co-culture. Secretion of macrophage inhibitory factor (MIF) activated the recruitment of S100A8+ myeloid cells systemically. Inhibition of MIF, or depletion of MDSCs resulted in delayed tumor growth and lower metastatic burden. In human BrC specimens, increased mRNA and protein levels of S100A8+ infiltrating cells are highly associated with poor overall survival and shorter metastasis free survival of BrC patients, respectively. Furthermore, analysis of nine different human gene expression datasets confirms the association of increased levels of S100A8 transcripts with an increased risk of death. Recruitment of S100A8+ myeloid cells to primary tumors and secondary sites in xenograft models of BrC enhances cancer progression independent of their suppressive activity on T cells. In clinical samples, infiltrating S100A8+ cells are associated with poor overall survival. Targeting these molecules or associated pathways in cells of the tumor microenvironment may translate into novel therapeutic interventions and benefit patient outcome.
Assuntos
Neoplasias da Mama/patologia , Carcinoma/patologia , Células Mieloides/patologia , Invasividade Neoplásica/patologia , Microambiente Tumoral , Animais , Calgranulina A/biossíntese , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Análise Serial de Tecidos , TranscriptomaRESUMO
BACKGROUND: Gene fusions between the ERG transcription factor and the androgen-regulated gene TMPRSS2 occur in a subset of prostate cancers and contribute to transformation of prostatic epithelial cells. Prior reports have used fluorescence in situ hybridization (FISH) or quantitative PCR (QPCR) to determine the presence of TMPRSS2-ERG fusions or ERG expression, respectively. Recently, several groups have reported on immunohistochemistry (IHC) to measure ERG expression, which is much more readily performed in clinical practice. However, the prior studies examining ERG expression by IHC had small sample sizes or they failed to clarify the association of ERG protein expression with important clinico-pathological features or prostate cancer-specific mortality. METHODS: To address these deficits, we evaluated ERG expression by IHC in 208 radical prostatectomy samples from the Kaiser Permanente Molecular Epidemiology of Fatal Prostate Cancer (MEFPC) study, a case-control study of prostate cancer-specific mortality. RESULTS: Nuclear ERG expression was seen in neoplastic prostate epithelia in 49 of the samples (23.7%). ERG expression in tumor cells was associated with higher tumor stage (OR = 2.0, 95% confidence interval 1.0-4.0, P value = 0.04). ERG immunoreactivity was positively associated with prostate cancer-specific mortality, although the confidence interval was wide (OR = 1.9, 95% confidence interval 0.88-4.0, P value = 0.10). CONCLUSIONS: Our results demonstrate that ERG protein expression is readily quantifiable with an existing commercial antibody. Evaluating ERG protein expression may improve our ability to identify the subset of more aggressive, invasive prostate cancers.
Assuntos
Proteínas de Fusão Oncogênica/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/mortalidade , Transativadores/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Próstata/patologia , Próstata/cirurgia , Prostatectomia , Neoplasias da Próstata/patologia , Regulador Transcricional ERGRESUMO
A prominent feature of late-onset neurodegenerative diseases is accumulation of misfolded protein in vulnerable neurons. When levels of misfolded protein overwhelm degradative pathways, the result is cellular toxicity and neurodegeneration. Cellular mechanisms for degrading misfolded protein include the ubiquitin-proteasome system (UPS), the main non-lysosomal degradative pathway for ubiquitinated proteins, and autophagy, a lysosome-mediated degradative pathway. The UPS and autophagy have long been viewed as complementary degradation systems with no point of intersection. This view has been challenged by two observations suggesting an apparent interaction: impairment of the UPS induces autophagy in vitro, and conditional knockout of autophagy in the mouse brain leads to neurodegeneration with ubiquitin-positive pathology. It is not known whether autophagy is strictly a parallel degradation system, or whether it is a compensatory degradation system when the UPS is impaired; furthermore, if there is a compensatory interaction between these systems, the molecular link is not known. Here we show that autophagy acts as a compensatory degradation system when the UPS is impaired in Drosophila melanogaster, and that histone deacetylase 6 (HDAC6), a microtubule-associated deacetylase that interacts with polyubiquitinated proteins, is an essential mechanistic link in this compensatory interaction. We found that compensatory autophagy was induced in response to mutations affecting the proteasome and in response to UPS impairment in a fly model of the neurodegenerative disease spinobulbar muscular atrophy. Autophagy compensated for impaired UPS function in an HDAC6-dependent manner. Furthermore, expression of HDAC6 was sufficient to rescue degeneration associated with UPS dysfunction in vivo in an autophagy-dependent manner. This study suggests that impairment of autophagy (for example, associated with ageing or genetic variation) might predispose to neurodegeneration. Morover, these findings suggest that it may be possible to intervene in neurodegeneration by augmenting HDAC6 to enhance autophagy.
Assuntos
Autofagia/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Histona Desacetilases/metabolismo , Doenças Neurodegenerativas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Autofagia/genética , Modelos Animais de Doenças , Drosophila melanogaster/genética , Desacetilase 6 de Histona , Humanos , Transtornos Musculares Atróficos/genética , Transtornos Musculares Atróficos/metabolismo , Doenças Neurodegenerativas/genética , Peptídeos/genética , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismoRESUMO
Bladder cancer is the fifth most common cancer in the United States. Most bladder cancers are early-stage lesions confined to the mucosa or submucosa and are therefore classified as non-muscle-invasive bladder cancer (NMIBC). A minority of tumors are diagnosed after they have invaded the underlying detrusor muscle and are classified as muscle-invasive bladder cancer (MIBC). Mutational inactivation of the STAG2 tumor suppressor gene is common in bladder cancer, and we and others have recently demonstrated that STAG2 mutation status can be used as an independent prognostic biomarker to predict whether NMIBC will recur and/or progress to MIBC. Here we describe an immunohistochemistry-based assay for identifying the STAG2 mutational status of bladder tumors.
Assuntos
Antígenos Nucleares , Neoplasias da Bexiga Urinária , Humanos , Imuno-Histoquímica , Antígenos Nucleares/genética , Proteínas de Ciclo Celular , Recidiva Local de Neoplasia/genética , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Invasividade NeoplásicaRESUMO
Background: Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer remains a significant clinical problem. Riluzole is FDA-approved for the treatment of amyotrophic lateral sclerosis. A benzothiazole-based glutamate release inhibitor with several context-dependent mechanism(s) of action, riluzole has shown antitumor activity in multiple malignancies, including melanoma, glioblastoma, and breast cancer. We previously reported that the acquisition of tamoxifen resistance in a cellular model of invasive lobular breast cancer is accompanied by the upregulation of GRM mRNA expression and growth inhibition by riluzole. Methods: We tested the ability of riluzole to reduce cell growth, alone and in combination with endocrine therapy, in a diverse set of ER+ invasive ductal and lobular breast cancer-derived cell lines, primary breast tumor explant cultures, and the estrogen-independent, ESR1-mutated invasive lobular breast cancer patient-derived xenograft model HCI-013EI. Results: Single-agent riluzole suppressed the growth of ER+ invasive ductal and lobular breast cancer cell lines in vitro, inducing a histologic subtype-associated cell cycle arrest (G0-G1 for ductal, G2-M for lobular). Riluzole induced apoptosis and ferroptosis and reduced phosphorylation of multiple prosurvival signaling molecules, including Akt/mTOR, CREB, and Fak/Src family kinases. Riluzole, in combination with either fulvestrant or 4-hydroxytamoxifen, additively suppressed ER+ breast cancer cell growth in vitro. Single-agent riluzole significantly inhibited HCI-013EI patient-derived xenograft growth in vivo, and the combination of riluzole plus fulvestrant significantly reduced proliferation in ex vivo primary breast tumor explant cultures. Conclusion: Riluzole may offer therapeutic benefits in diverse ER+ breast cancers, including lobular breast cancer.
RESUMO
UNLABELLED: Transforming growth factor beta (TGF-ß) is an important regulator of cell growth, and loss of TGF-ß signaling is a hallmark of carcinogenesis. The Smad3/4 adaptor protein ß2-spectrin (ß2SP) is emerging as a potent regulator of tumorigenesis through its ability to modulate the tumor suppressor function of TGF-ß. However, to date the role of the TGF-ß signaling pathway at specific stages of the development of hepatocellular carcinoma (HCC), particularly in relation to the activation of other oncogenic pathways, remains poorly delineated. Here we identify a mechanism by which ß2SP, a crucial Smad3 adaptor, modulates cyclin dependent kinase 4 (CDK4), cell cycle progression, and suppression of HCC. Increased expression of ß2SP inhibits phosphorylation of the retinoblastoma gene product (Rb) and markedly reduces CDK4 expression to a far greater extent than other CDKs and cyclins. Furthermore, suppression of CDK4 by ß2SP efficiently restores Rb hypophosphorylation and cell cycle arrest in G(1) . We further demonstrate that ß2SP interacts with CDK4 and Smad3 in a competitive and TGF-ß-dependent manner. In addition, haploinsufficiency of cdk4 in ß2sp(+/-) mice results in a dramatic decline in HCC formation compared to that observed in ß2sp(+/-) mice. CONCLUSION: ß2SP deficiency leads to CDK4 activation and contributes to dysregulation of the cell cycle, cellular proliferation, oncogene overexpression, and the formation of HCCs. Our data highlight CDK4 as an attractive target for the pharmacologic inhibition of HCC and demonstrate the importance of ß2sp(+/-) mice as a model of preclinical efficacy in the treatment of HCC.
Assuntos
Carcinoma Hepatocelular/patologia , Proliferação de Células , Quinase 4 Dependente de Ciclina/fisiologia , Neoplasias Hepáticas/patologia , Espectrina/fisiologia , Animais , Quinase 4 Dependente de Ciclina/genética , Haploinsuficiência , Camundongos , Proteína Smad3/fisiologia , Fator de Crescimento Transformador betaRESUMO
Acrolein (Acr) is a ubiquitous environmental pollutant found in cigarette smoke and automobile exhaust. It can also be produced endogenously by oxidation of polyunsaturated fatty acids. The Acr-derived 1,N(2)-propanodeoxyguanosine (Acr-dG) adducts in DNA are mutagenic lesions that are potentially involved in human cancers. In this study, monoclonal antibodies were raised against Acr-dG adducts and characterized using ELISA. They showed strong reactivity and specificity toward Acr-dG, weaker reactivity toward crotonaldehyde- and trans-4-hydroxy-2-nonenal-derived 1,N(2)-propanodeoxyguanosines, and weak or no reactivity toward 1,N(6)-ethenodeoxyadenosine and 8-oxo-deoxyguanosine. Using these antibodies, we developed assays to detect Acr-dG in vivo: first, a simple and quick FACS-based assay for detecting these adducts directly in cells; second, a highly sensitive direct ELISA assay for measuring Acr-dG in cells and tissues using only 1 µg of DNA without DNA digestion and sample enrichment; and third, a competitive ELISA for better quantitative measurement of Acr-dG levels in DNA samples. The assays were validated using Acr-treated HT29 cell DNA samples or calf thymus DNA, and the results were confirmed by LC-MS/MS-MRM. An immunohistochemical assay was also developed to detect and visualize Acr-dG in HT29 cells as well as in human oral cells. These antibody-based methods provide useful tools for the studies of Acr-dG as a cancer biomarker and of the molecular mechanisms by which cells respond to Acr-dG as a ubiquitous DNA lesion.
Assuntos
Acroleína/imunologia , Poluentes Atmosféricos/imunologia , Anticorpos Monoclonais/imunologia , Adutos de DNA/imunologia , Animais , Biomarcadores , Células Cultivadas , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Células HT29 , Humanos , Queratinócitos , Camundongos , Camundongos Endogâmicos BALB C , Boca/citologia , Espectrometria de Massas em TandemRESUMO
The role of dietary factors in inhibiting or delaying the development of non-melanoma skin cancer (NMSC) has been investigated for many years. Cardamom, which is a dietary phytoproduct, has been commonly used in cuisines for flavour and has numerous health benefits, such as improving digestion and stimulating metabolism and having antitumorigenic effects. We have investigated the efficacy of dietary cardamom against 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin papillomatogenesis in Swiss albino mice that closely resembles human NMSC. Mice were grouped into normal wild type (untreated), vehicle-treated (acetone), carcinogen-treated (DMBA), and DMBA and cardamom-treated (DMBA+CARD) to delineate the role of cardamom against DMBA-induced papillomatogenesis. Oral administration of cardamom to DMBA-treated mice up-regulated the phase II detoxification enzymes, such as glutathione-S-transferase and glutathione peroxidase, probably via activation of nuclear factor erythroid-2-related factor 2 transcription factor in 'DMBA+CARD' mice. Furthermore, reduced glutathione, glutathione reductase, superoxide dismutase and catalase were also up-regulated by cardamom in the same 'DMBA+CARD' group of mice compared with DMBA-treated mice. Cardamom ingestion in DMBA-treated mice blocked NF-κB activation and down-regulated cyclo-oxygenase-2 expression. As a consequence, both the size and the number of skin papillomas generated on the skin due to the DMBA treatment were reduced in the 'DMBA+CARD' group. Thus, the results from the present study suggest that cardamom has a potential to become a pivotal chemopreventive agent to prevent papillomagenesis on the skin.
Assuntos
Antioxidantes/uso terapêutico , Elettaria/química , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/prevenção & controle , Especiarias , 9,10-Dimetil-1,2-benzantraceno/antagonistas & inibidores , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Anticarcinógenos/uso terapêutico , Carcinógenos/antagonistas & inibidores , Carcinógenos/toxicidade , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Feminino , Desintoxicação Metabólica Fase II , Camundongos , NF-kappa B/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Papiloma/induzido quimicamente , Papiloma/metabolismo , Papiloma/patologia , Papiloma/prevenção & controle , Sementes/química , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Carga TumoralRESUMO
OBJECTIVE: Improvements to bladder cancer risk stratification guidelines are needed to better tailor post-operative surveillance and adjuvant therapy to individual patients. We previously identified STAG2 as a commonly mutated tumor suppressor gene in bladder cancer and an independent predictor of progression in NMIBC. Here we test the value of combining STAG2 immunostaining with other risk stratification biomarkers in NMIBC, and as an individual biomarker in MIBC. MATERIALS AND METHODS: STAG2 immunohistochemistry was performed on a progressor-enriched cohort of tumors from 297 patients with NMIBC, and on tumors from 406 patients with MIBC from Aarhus University Hospital in Denmark. Survival analysis was performed using Kaplan-Meier survival analysis, the log rank test, and Cox proportional hazards models. RESULTS: STAG2-negative low-grade NMIBC tumors were 2.5 times less likely to progress to muscle invasion than STAG2-positive low-grade NMIBC tumors (Log-rank test, Pâ¯=â¯0.008). In a composite group of patients with AUA intermediate and high-risk NMIBC tumors, STAG2-negative tumors were less likely to progress (Log-rank test, Pâ¯=â¯0.02). In contrast to NMIBC, we show that STAG2 is not useful as a prognostic biomarker in MIBC. CONCLUSIONS: STAG2 immunostaining can be used to subdivide low-grade NMIBC tumors into two groups with substantially different risks of disease progression. Furthermore, STAG2 immunostaining may be useful to enhance NMIBC risk stratification guidelines, though larger cohorts are needed to solidify this conclusion in individual risk groups. STAG2 is not useful as a biomarker in MIBC. Further study of the use of STAG2 immunostaining as a biomarker for predicting the clinical behavior in NMIBC is warranted.
Assuntos
Biomarcadores Tumorais/análise , Proteínas de Ciclo Celular/análise , Neoplasias da Bexiga Urinária/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Prognóstico , Estudos Retrospectivos , Medição de Risco , Análise de Sobrevida , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologiaRESUMO
BACKGROUND & AIMS: Obesity is a major cause of non-alcoholic fatty liver disease (NAFLD). NAFLD is an epidemic affecting nearly 34% of the adult population in the US. As a chronic inflammatory disease, NAFLD influences the immune system by dysregulating T-cell activity. Remedies for the adverse effects on the immune system are urgently needed. We studied Theaphenon E (TE), a standardized formulation of green tea extract, on the adverse effects of NAFLD in C57BL/6J mice fed a high fat diet (HFD). METHODS: Mice received HFD, low fat diet (LFD) or HFD+2% TE for 35 weeks. Hepatic lipid accumulation, cell proliferation, apoptosis and CD4+T lymphocytes were measured throughout the bioassay. The hepatic composition of fatty acids was determined. The effects of epigallocatechin gallate (EGCG) metabolites on lipid accumulation in mouse and primary human liver cells were studied. RESULTS: Unlike mice receiving HFD, mice on HFD+2% TE maintained normal liver to body weight ratios with low levels of alanine and aspartate aminotransferase (ALT and AST). Hepatic lipid accumulation was observed in HFD mice, accompanied by increased proliferation, reduced apoptosis and loss of CD4+ T lymphocytes. TE significantly inhibited lipid accumulation, decreased proliferation, induced apoptosis and increased CD4+ T cell survival in HFD mice. It was found that the EGCG metabolite EGC-M3 reduced lipid accumulation in mouse and human hepatocytes. Linoleic acid showed the largest increase (2.5-fold) in livers of mice on a HFD and this increase was significantly suppressed by TE. CONCLUSIONS: Livers of HFD-fed mice showed lipid accumulation, increased proliferation, reduced apoptosis, elevated linoleic acid and loss of CD4+ T cells. TE effectively ameliorated all of these adverse effects.
Assuntos
Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Catequina/análogos & derivados , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Obesidade/metabolismo , Animais , Catequina/metabolismo , Catequina/farmacologia , Proliferação de Células/efeitos dos fármacos , Dieta com Restrição de Gorduras , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Ácido Linoleico/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Obesidade/complicaçõesRESUMO
Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells. Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.
Assuntos
Técnicas de Cocultura/métodos , Linfócitos T Citotóxicos/citologia , Comunicação Celular , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Humanos , Invasividade Neoplásica , Esferoides Celulares/patologiaRESUMO
AIM: To analyze the clinicopathologic and prognostic significance of Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), a cancer stem cell marker expression in a cohort of colorectal cancer patients (CRC). PATIENTS & METHODS: A total of 76 formalin-fixed paraffin-embedded tissue blocks of primary or metastatic tumors from 49 CRC patients were collected for duration 2009-2015. LGR5 expression was assessed through immunohistochemical staining of a tissue microarray. RESULTS: LGR5 was significantly over expressed in CRC tissue samples and found to be a statistically significant independent prognostic marker for an improved overall survival. CONCLUSION: LGR5 expression was higher in colorectal cancer than in normal tissue. LGR5 was an independent prognostic marker for better clinical outcomes and might be used as a potential therapeutic target in CRCs.
RESUMO
BACKGROUND: We reported previously that phenethyl isothiocyanate (PEITC), a dietary compound, can reactivate p53R175H mutant in vitro and in SK-BR-3 (p53R175H) breast xenograft model resulting in tumor inhibition. Because of the diversity of human cancers with p53 mutations, these findings raise important questions whether this mechanism operates in different cancer types with same or different p53 mutations. In this study, we investigated whether PEITC recuses mutant p53 in prostate cancer cells harboring different types of p53 mutants, structural and contact, in vitro and in vivo. METHODS: Cell proliferation, cell apoptosis and cell cycle arrest assays were performed to examine the effects of PEITC on prostate cancer cell lines with p53 mutation(s), wild-type p53, p53 null or normal prostate cells in vitro. Western blot analysis was used to monitor the expression levels of p53 protein, activation of ATM and upregulation of canonical p53 targets. Immunoprecipitation, subcellular protein fraction and qRT-PCR was performed to determine change in conformation and restoration of transactivation functions/ inhibition of gain-of-function (GOF) activities to p53 mutant(s). Mice xenograft models were established to evaluate the antitumor efficacy of PEITC and PEITC-induced reactivation of p53 mutant(s) in vivo. Immunohistochemistry of xenograft tumor tissues was performed to determine effects of PEITC on expression of Ki67 and mutant p53 in vivo. RESULTS: We demonstrated that PEITC inhibits the growth of prostate cancer cells with different "hotspot" p53 mutations (structural and contact), however, preferentially towards structural mutants. PEITC inhibits proliferation and induces apoptosis by rescuing mutant p53 in p53R248W contact (VCaP) and p53R175H structural (LAPC-4) mutant cells with differential potency. We further showed that PEITC inhibits the growth of DU145 cells that co-express p53P223L (structural) and p53V274F (contact) mutants by targeting p53P223L mutant selectively, but not p53V274F. The mutant p53 restored by PEITC induces apoptosis in DU145 cells by activating canonical p53 targets, delaying cells in G1 phase and phosphorylating ATM. Importantly, PEITC reactivated p53R175H and p53P223L/V274F mutants in LAPC-4 and DU145 prostate xenograft models, respectively, resulting in significant tumor inhibition. CONCLUSION: Our studies provide the first evidence that PEITC's anti-cancer activity is cancer cell type-independent, but p53 mutant-type dependent.
Assuntos
Anticarcinógenos/administração & dosagem , Isotiocianatos/administração & dosagem , Neoplasias da Próstata/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Animais , Anticarcinógenos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Isotiocianatos/farmacologia , Masculino , Camundongos , Mutação , Fosforilação , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Oxidative stress and chronic inflammation can increase cellular levels of reactive oxygen species and lipid peroxidation (LPO) when associated with the pathogenesis of hepatocellular carcinoma (HCC), which can develop following the progression of steatosis, fibrosis and cirrhosis. Using a monoclonal antibody for cyclic γ-hydroxy-1, N2 -propanodeoxyguanosine (γ-OHPdG), a promutagenic DNA adduct formed endogenously by LPO, we examined its formation across liver disease stages to understand it's potential role in HCC development. METHODS: Formalin-fixed paraffin embedded (FFPE) liver tissue samples from 49 patients representing normal, steatosis, fibrosis, cirrhosis and HCC were stained for γ-OHPdG and 8-hydroxydeoxyguanosine (8-oxo-dG), an oxidative damage biomarker. Quantification of immunohistochemical (IHC) staining was performed using histological scoring of intensity and distribution. Using primary human hepatocytes (HH) and a stellate cell (SC) co-culture, immunocytochemical staining of γ-OHPdG and Nile Red was performed to determine if the formation of γ-OHPdG was consistent between the clinical sample disease stages and the in vitro steatotic and fibrotic conditions. RESULTS: γ-OHPdG levels varied significantly between the stages of normal and steatosis, steatosis and fibrosis, and steatosis and cirrhosis (P≤0.005). There was a trend, although not significant, of increased levels of γ-OHPdG in HCC compared to the other groups. A strong correlation was observed (Pearson's, R2 =0.85) between levels of γ-OHPdG and 8-oxo-dG across the disease spectrum. The increase of γ-OHPdG in steatosis and decrease in fibrosis was a pattern confirmed in an in vitro model using primary HH co-cultured with human SCs. CONCLUSIONS: γ-OHPdG was detected in FFPE liver tissues of patients with different stages of liver disease and in vitro studies, demonstrating that its formation is consistent with LPO in early stages of liver disease and suggesting that it may be a source of mutagenic DNA damage in liver disease progression.