ABSTRACT
BACKGROUND: Epigenetic silencing of tumor suppressor genes (TSGs) is a key feature of oncogenesis in hepatocellular carcinoma (HCC). Liver-targeted delivery of CRISPR-activation (CRISPRa) systems makes it possible to exploit chromatin plasticity, by reprogramming transcriptional dysregulation. RESULTS: Using The Cancer Genome Atlas HCC data, we identify 12 putative TSGs with negative associations between promoter DNA methylation and transcript abundance, with limited genetic alterations. All HCC samples harbor at least one silenced TSG, suggesting that combining a specific panel of genomic targets could maximize efficacy, and potentially improve outcomes as a personalized treatment strategy for HCC patients. Unlike epigenetic modifying drugs lacking locus selectivity, CRISPRa systems enable potent and precise reactivation of at least 4 TSGs tailored to representative HCC lines. Concerted reactivation of HHIP, MT1M, PZP, and TTC36 in Hep3B cells inhibits multiple facets of HCC pathogenesis, such as cell viability, proliferation, and migration. CONCLUSIONS: By combining multiple effector domains, we demonstrate the utility of a CRISPRa toolbox of epigenetic effectors and gRNAs for patient-specific treatment of aggressive HCC.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Clustered Regularly Interspaced Short Palindromic Repeats , DNA Methylation , Epigenesis, Genetic , Genes, Tumor Suppressor , Cell Line, Tumor , Gene Expression Regulation, NeoplasticABSTRACT
The MYC proto-oncogene (MYC) is one of the most frequently overexpressed genes in breast cancer that drives cancer stem cell-like traits, resulting in aggressive disease progression and poor prognosis. In this study, we identified zinc finger transcription factor 148 (ZNF148, also called Zfp148 and ZBP-89) as a direct target of MYC. ZNF148 suppressed cell proliferation and migration and was transcriptionally repressed by MYC in breast cancer. Depletion of ZNF148 by short hairpin RNA (shRNA) and CRISPR/Cas9 increased triple-negative breast cancer (TNBC) cell proliferation and migration. Global transcriptome and chromatin occupancy analyses of ZNF148 revealed a central role in inhibiting cancer cell de-differentiation and migration. Mechanistically, we identified the Inhibitor of DNA binding 1 and 3 (ID1, ID3), drivers of cancer stemness and plasticity, as previously uncharacterized targets of transcriptional repression by ZNF148. Silencing of ZNF148 increased the stemness and tumorigenicity in TNBC cells. These findings uncover a previously unknown tumor suppressor role for ZNF148, and a transcriptional regulatory circuitry encompassing MYC, ZNF148, and ID1/3 in driving cancer stem cell traits in aggressive breast cancer.
ABSTRACT
Improving tumor access for drug delivery is challenging, particularly in poorly perfused tumors. The availability of functional tumor blood vessels for systemic access is vital to allow drugs or imaging agents to accumulate in the tumor parenchyma. We subjected mice engineered to develop hepatocellular carcinoma (HCC), to treatment with tumor necrosis factor alpha (TNFα) conjugated to a CSG peptide (CSGRRSSKC). CSG binds to the laminin-nidogen-1 complex of the extracellular matrix (ECM) in HCC. When produced as a recombinant fusion protein, the TNFα-CSG functions as an ECM depletion agent via an immune-mediated mechanism to improve tumor perfusion. Tumor perfusion in HCC was dramatically improved after daily intravenous (i.v.) injection of 5 µg TNFα-CSG for five consecutive days. Following treatment, we assessed the tumor accessibility to accumulate an imaging agent, superparamagnetic iron-oxide nanoparticles (IO-NP). Here, we compared the passive delivery of an i.v. dose of IO-NP in HCC following ECM depletion after TNFα-CSG treatment, to the intratumoral accumulation of a comparable dose of CSG-targeted IO-NP in HCC with intact ECM. Magnetic resonance imaging (MRI) T2-weighted scans and T2 relaxation times indicate that when the tumor ECM is intact, HCC was resistant to the intratumoral uptake of IO-NP, even when the particles were tagged with CSG peptide. In contrast, pre-treatment with TNFα-CSG resulted in the highest IO-NP accumulation in tumors. These findings suggest poorly perfused HCC may be resistant to molecular-targeted imaging agents including CSG-IO-NP. We demonstrate that specific ECM depletion using TNFα-CSG improves nanoparticle delivery into poorly perfused tumors such as HCC.
ABSTRACT
RNA-based therapeutics are emerging as innovative options for cancer treatment, with microRNAs being attractive targets for therapy development. We previously implicated microRNA-642a-5p (miR-642a-5p) as a tumor suppressor in prostate cancer (PCa), and here we characterize its mode of action, using 22Rv1 PCa cells. In an in vivo xenograft tumor model, miR-642a-5p induced a significant decrease in tumor growth, compared to negative control. Using RNA-Sequencing, we identified gene targets of miR-642a-5p which were enriched for gene sets controlling cell cycle; downregulated genes included Wilms Tumor 1 gene (WT1), NUAK1, RASSF3 and SKP2; and upregulated genes included IGFBP3 and GPS2. Analysis of PCa patient datasets showed a higher expression of WT1, NUAK1, RASSF3 and SKP2; and a lower expression of GPS2 and IGFBP3 in PCa tissue compared to non-malignant prostate tissue. We confirmed the prostatic oncogene WT1, as a direct target of miR-642a-5p, and treatment of 22Rv1 and LNCaP PCa cells with WT1 siRNA or a small molecule inhibitor of WT1 reduced cell proliferation. Taken together, these data provide insight into the molecular mechanisms by which miR-642a-5p acts as a tumor suppressor in PCa, an effect partially mediated by regulating genes involved in cell cycle control; and restoration of miR-642-5p in PCa could represent a novel therapeutic approach.
Subject(s)
Cell Cycle/genetics , MicroRNAs/genetics , Prostate/metabolism , Prostatic Neoplasms/genetics , WT1 Proteins/genetics , 3' Untranslated Regions , Animals , Base Pairing , Base Sequence , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Insulin-Like Growth Factor Binding Protein 3/genetics , Insulin-Like Growth Factor Binding Protein 3/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mice, SCID , MicroRNAs/metabolism , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/metabolism , Prostate/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/mortality , Prostatic Neoplasms/pathology , Protein Kinases/genetics , Protein Kinases/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolism , Signal Transduction , Survival Analysis , Tumor Burden , WT1 Proteins/antagonists & inhibitors , WT1 Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
The aim of this preclinical study was to directly compare [18F]PSMA-1007 with both [68Ga]Ga-PSMA-11 and [18F]AlF-PSMA-11 in mice bearing PSMA-positive tumor xenografts. Uptake was assessed by PET/CT at 1, 2 and 4 h post-injection, and by ex vivo measurement after 4 h. [18F]PSMA-1007 demonstrated the highest tumor uptake of the three tracers. The high uptake in bone for mice injected with [18F]AlF-PSMA-11 suggested rapid in vivo decomposition. This was confirmed by an in vitro plasma stability study.
Subject(s)
Fluorine Radioisotopes/pharmacokinetics , Gallium Radioisotopes/pharmacokinetics , Niacinamide/analogs & derivatives , Oligopeptides/pharmacokinetics , Prostatic Neoplasms/metabolism , Animals , Blood Proteins/metabolism , Cell Line, Tumor , Heterografts , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Niacinamide/genetics , Niacinamide/pharmacokinetics , Oligopeptides/genetics , Positron Emission Tomography Computed Tomography/methods , Prostatic Neoplasms/diagnostic imaging , Tissue DistributionABSTRACT
Iron is an essential nutrient that plays a complex role in cancer biology. Iron metabolism must be tightly controlled within cells. Whilst fundamental to many cellular processes and required for cell survival, excess labile iron is toxic to cells. Increased iron metabolism is associated with malignant transformation, cancer progression, drug resistance and immune evasion. Depleting intracellular iron stores, either with the use of iron chelating agents or mimicking endogenous regulation mechanisms, such as microRNAs, present attractive therapeutic opportunities, some of which are currently under clinical investigation. Alternatively, iron overload can result in a form of regulated cell death, ferroptosis, which can be activated in cancer cells presenting an alternative anti-cancer strategy. This review focuses on alterations in iron metabolism that enable cancer cells to meet metabolic demands required during different stages of tumorigenesis in relation to metastasis and immune response. The strength of current evidence is considered, gaps in knowledge are highlighted and controversies relating to the role of iron and therapeutic targeting potential are discussed. The key question we address within this review is whether iron modulation represents a useful approach for treating metastatic disease and whether it could be employed in combination with existing targeted drugs and immune-based therapies to enhance their efficacy.
ABSTRACT
Erythroid maturation requires the concerted action of a core set of transcription factors. We previously identified the Krüppel-type zinc finger transcription factor Zfp148 (also called ZBP-89) as an interacting partner of the master erythroid transcription factor GATA1. Here we report the conditional knockout of Zfp148 in mice. Global loss of Zfp148 results in perinatal lethality from nonhematologic causes. Selective Zfp148 loss within the hematopoietic system results in a mild microcytic and hypochromic anemia, mildly impaired erythroid maturation, and delayed recovery from phenylhydrazine-induced hemolysis. Based on the mild erythroid phenotype of these mice compared with GATA1-deficient mice, we hypothesized that additional factor(s) may complement Zfp148 function during erythropoiesis. We show that Zfp281 (also called ZBP-99), another member of the Zfp148 transcription factor family, is highly expressed in murine and human erythroid cells. Zfp281 knockdown by itself results in partial erythroid defects. However, combined deficiency of Zfp148 and Zfp281 causes a marked erythroid maturation block. Zfp281 physically associates with GATA1, occupies many common chromatin sites with GATA1 and Zfp148, and regulates a common set of genes required for erythroid cell differentiation. These findings uncover a previously unknown role for Zfp281 in erythroid development and suggest that it functionally overlaps with that of Zfp148 during erythropoiesis.
Subject(s)
Cell Differentiation/genetics , DNA-Binding Proteins/genetics , Erythroid Cells/cytology , Erythroid Cells/metabolism , Erythropoiesis/genetics , Transcription Factors/genetics , Animals , Cell Line, Tumor , DNA-Binding Proteins/metabolism , GATA1 Transcription Factor/metabolism , Gene Expression Regulation, Developmental , Genotype , Humans , Mice , Mice, Knockout , Protein Binding , Transcription Factors/metabolism , gamma-Globins/genetics , gamma-Globins/metabolismABSTRACT
RNA-binding proteins (RBPs) and microRNAs (miRNAs) are the most important regulators of mRNA stability and translation in eukaryotic cells; however, the complex interplay between these systems is only now coming to light. RBPs and miRNAs regulate a unique set of targets in either a positive or negative manner and their regulation is mainly opposed to each other on overlapping targets. In some cases, the levels of RBPs or miRNAs regulate the cellular levels of one another and decreased levels of either results in changes in translation of their targets. There is growing evidence that these regulatory circuits are crucial in the development and progression of cancer; however, the rules underlying synergism and antagonism between miRNAs and RNA-binding proteins remain unclear. Synthetic biology seeks to develop artificial systems to better understand their natural counterparts and to develop new, useful technologies for manipulation of gene expression at the RNA level. The recent development of artificial RNA-binding proteins promises to enable a much greater understanding of the importance of the functional interactions between RNA-binding proteins and miRNAs, as well as enabling their manipulation for therapeutic purposes.
Subject(s)
MicroRNAs/metabolism , Neoplasms/genetics , RNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Genetic Diseases, Inborn/therapy , Humans , Neoplasms/metabolism , Neoplasms/therapy , Protein Engineering , RNA Processing, Post-Transcriptional , RNA-Binding Proteins/geneticsABSTRACT
Expression of the transcriptional regulator, E26 transformation-specific 1 (ETS1), is elevated in human prostate cancers, and this is associated with more aggressive tumor behavior and a rapid progression to castrate-resistant disease. Multiple ETS1 isoforms with distinct biological activities have been characterized and in 44 matched nonmalignant and malignant human prostate specimens, messenger RNAs for two ETS1 isoforms, ETS1p51 and ETS1p42, were detected, with ETS1p51 levels significantly lower in prostate tumor compared to matched nonmalignant prostate tissues. In contrast, ETS1p51 protein, the only ETS1 isoform detected, was expressed at significantly higher levels in malignant prostate. Analysis of epithelial-to-mesenchymal transition (EMT)-associated genes regulated following overexpression of ETS1p51 in the LNCaP prostate cancer cell line predicted promotion of transforming growth factor ß (TGFß) signaling and of EMT. ETS1p51 overexpression upregulated cellular levels of the EMT transcriptional regulators, ZEB1 and SNAIL1, resulted in reduced expression of the mesenchymal marker vimentin with concomitantly elevated levels of claudin 1, an epithelial tight junction protein, and increased prostate cancer cell migration and invasion. ETS1p51-induced activation of the pro-EMT TGFß signaling pathway that was predicted in polymerase chain reaction arrays was verified by demonstration of elevated SMAD2 phosphorylation following ETS1p51 overexpression. Attenuation of ETS1p51 effects on prostate cancer cell migration and invasion by inhibition of TGFß pathway signaling indicated that ETS1p51 effects were in part mediated by induction of TGFß signaling. Thus, overexpression of ETS1p51, the predominant ETS1 isoform expressed in prostate tumors, promotes an EMT program in prostate cancer cells in part via activation of TGFß signaling, potentially accounting for the poor prognosis of ETS1-overexpressing prostate tumors.
Subject(s)
Epithelial-Mesenchymal Transition/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Proto-Oncogene Protein c-ets-1/genetics , Proto-Oncogene Protein c-ets-1/metabolism , Transforming Growth Factor beta1/metabolism , Benzamides/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Dioxoles/pharmacology , Gene Expression Regulation, Neoplastic , Humans , Male , Neoplasm Invasiveness , Phosphorylation , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/genetics , Receptor, Transforming Growth Factor-beta Type I/antagonists & inhibitors , Smad2 Protein/metabolism , Snail Family Transcription Factors/metabolism , Transfection , Transforming Growth Factor beta1/pharmacology , Zinc Finger E-box-Binding Homeobox 1/metabolismABSTRACT
OBJECTIVE: Guidelines for thyroid function evaluation recommend testing TSH first, then assessing fT4 only if TSH is out of the reference range (two-step), but many clinicians initially request both TSH and fT4 (one-step). Given limitations of previous studies, we aimed to compare the two-step with the one-step approach in an unselected community-dwelling study population, and develop a prediction score based on clinical parameters that could identify at-risk patients for thyroid dysfunction. DESIGN: Cross-sectional analysis of the population-based Busselton Health Study. METHODS: We compared the two-step with the one-step approach, focusing on cases that would be missed by the two-step approach, i.e. those with normal TSH, but out-of-range fT4. We used likelihood ratio tests to identify demographic and clinical parameters associated with thyroid dysfunction and developed a clinical prediction score by using a beta-coefficient based scoring method. RESULTS: Following the two-step approach, 93.0% of all 4471 participants had normal TSH and would not need further testing. The two-step approach would have missed 3.8% of all participants (169 of 4471) with a normal TSH, but a fT4 outside the reference range. In 85% (144 of 169) of these cases, fT4 fell within 2 pmol/l of fT4 reference range limits, consistent with healthy outliers. The clinical prediction score that performed best excluded only 22.5% of participants from TSH testing. CONCLUSION: The two-step approach may avoid measuring fT4 in as many as 93% of individuals with a very small risk of missing thyroid dysfunction. Our findings do not support the simultaneous initial measurement of both TSH and fT4.
Subject(s)
Thyroid Diseases/blood , Thyroid Diseases/diagnosis , Thyrotropin/blood , Thyroxine/blood , Adolescent , Adult , Aged , Aged, 80 and over , Cross-Sectional Studies , Female , Humans , Hyperthyroidism/blood , Hypothyroidism/blood , Likelihood Functions , Male , Middle Aged , Reference Values , Retrospective Studies , Sensitivity and Specificity , Western Australia , Young AdultABSTRACT
BACKGROUND: microRNAs (miRNAs) are short non-coding RNAs that fine-tune gene expression. The aberrant expression of miRNAs is associated with many diseases and they have both therapeutic and biomarker potential. However, our understanding of their usefulness is dependent on the tools we have to study them. Previous studies have identified the need to optimise and standardise RNA extraction methods in order to avoid biased results. Herein, we extracted RNA from murine lung, liver and brain tissues using five commercially available total RNA extraction methods. These included either: phenol: chloroform extraction followed by alcohol precipitation (TRIzol), phenol:chloroform followed by solid-phase extraction (column-based; miRVana and miRNeasy) and solid-phase separation with/without affinity resin (Norgen total and Isolate II). We then evaluated each extraction method for the quality and quantity of RNA recovered, and the expression of miRNAs and target genes. RESULTS: We identified differences between each of the RNA extraction methods in the quantity and quality of RNA samples, and in the analysis of miRNA and target gene expression. For the purposes of consistency in quantity, quality and high recovery of miRNAs from tissues, we identified that Phenol:chloroform phase separation combined with silica column-based solid extraction method was preferable (miRVana microRNA isolation). We also identified a method that is not appropriate for miRNA analysis from tissue samples (Bioline Isolate II). For target gene expression any of the kits could be used to analyse mRNA, but if interested in analysing mRNA and miRNA from the same RNA samples some methods should be avoided. CONCLUSIONS: Different methods used to isolate miRNAs will yield different results and therefore a robust RNA isolation method is required for reproducibility. Researchers should optimise these methods for their specific application and keep in mind that "total RNA" extraction methods do not isolate all types of RNA equally.
Subject(s)
Biochemistry/methods , Gene Expression Profiling/methods , MicroRNAs/genetics , RNA/isolation & purification , Animals , Brain Chemistry , Chloroform/chemistry , ErbB Receptors/genetics , Liver/chemistry , Lung/chemistry , Male , Mice, Inbred C57BL , Phenol/chemistry , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Solid Phase Extraction , Workflow , Axl Receptor Tyrosine KinaseABSTRACT
Sorafenib remains the only approved drug for treating patients with advanced hepatocellular carcinoma (HCC). However, the therapeutic effect of sorafenib is transient, and patients invariably develop sorafenib resistance (SR). Recently, TYRO3, a member of the TYRO3-AXL-MER family of receptor tyrosine kinases, was identified as being aberrantly expressed in a significant proportion of HCC; however, its role in SR is unknown. In this study, we generated two functionally distinct sorafenib-resistant human Huh-7 HCC cell lines in order to identify new mechanisms to abrogate acquired SR as well as new potential therapeutic targets in HCC. Initially, we investigated the effects of a microRNA (miR), miR-7-5p (miR-7), in both in vitro and in vivo preclinical models of human HCC and identified miR-7 as a potent tumor suppressor of human HCC. We identified TYRO3 as a new functional target of miR-7, which regulates proliferation, migration, and invasion of Huh-7 cells through the phosphoinositide 3-kinase/protein kinase B pathway and is markedly elevated with acquisition of SR. Furthermore, miR-7 effectively silenced TYRO3 expression in both sorafenib-sensitive and sorafenib-resistant Huh-7 cells, inhibiting TYRO3/growth arrest specific 6-mediated cancer cell migration and invasion. CONCLUSION: We identified a mechanism for acquiring SR in HCC that is through the aberrant expression of the TYRO3/phosphoinositide 3-kinase/protein kinase B signal transduction pathway, and that can be overcome by miR-7 overexpression. Taken together, these data suggest a potential role for miR-7 as an RNA-based therapeutic to treat refractory and drug-resistant HCC. (Hepatology 2018;67:216-231).
Subject(s)
Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/drug effects , MicroRNAs/metabolism , Niacinamide/analogs & derivatives , Phenylurea Compounds/pharmacology , Receptor Protein-Tyrosine Kinases/genetics , Analysis of Variance , Blotting, Western , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor/drug effects , Cell Line, Tumor/pathology , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/genetics , Drug Resistance, Neoplasm/drug effects , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , MicroRNAs/drug effects , Molecular Targeted Therapy/methods , Niacinamide/pharmacology , RNA, Small Interfering/drug effects , RNA, Small Interfering/metabolism , Real-Time Polymerase Chain Reaction , Signal Transduction/drug effects , SorafenibABSTRACT
MicroRNAs (miRNAs) are a family of short noncoding RNA molecules that fine-tune expression of mRNAs. Often their altered expression is associated with a number of diseases, including cancer. Given that miRNAs target multiple genes and "difficult to drug" oncogenes, they present attractive candidates to manipulate as an anti-cancer strategy. MicroRNA-7 (miR-7) is a tumor suppressor miRNA that has been shown to target oncogenes overexpressed in cancers, such as the epidermal growth factor receptor (EGFR) and the nuclear factor-κ B subunit, RelA. Here, we describe methods for evaluating systemic delivery of miR-7 using a lipid nanoparticle formulation in an animal model. The microRNA is delivered three times, over 1 week and tissues collected 24 h after the last injection. RNA and protein are extracted from snap frozen tissues and processed to detect miRNA distribution and subsequent assessment of downstream targets and signaling mediators, respectively. Importantly, variability in efficiency of miRNA delivery will be observed between organs of the same animal and also between animals. Additionally, delivering the microRNA to organs other than the liver, particularly the brain, remains challenging. Furthermore, large variation in miRNA targets is seen both within tissues and across tissues depending on the lysis buffer used for protein extraction. Therefore, analyzing protein expression is dependent upon the method used for isolation and requires optimization for each individual application. Together, these methods will provide a foundation for those planning on assessing the efficacy of delivery of a miRNA in vivo.
Subject(s)
Drug Delivery Systems/methods , MicroRNAs/administration & dosage , MicroRNAs/pharmacokinetics , Nanoparticles/administration & dosage , Animals , ErbB Receptors/genetics , ErbB Receptors/metabolism , Injections, Intravenous , Lipids/chemistry , Mice , Mice, Inbred C57BL , MicroRNAs/chemistry , Nanoparticles/chemistry , Proteins/isolation & purification , RNA/isolation & purification , Tissue Distribution , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolismABSTRACT
RNA-based therapeutics could represent a new avenue of cancer treatment. miRNA 331-3p (miR-331-3p) is implicated in prostate cancer (PCa) as a putative tumor suppressor, but its functional activity and synergy with other anti-tumor agents is largely unknown. We found miR-331-3p expression in PCa tumors was significantly decreased compared to non-malignant matched tissue. Analysis of publicly available PCa gene expression data sets showed miR-331-3p expression negatively correlated with Gleason Score, tumor stage, lymph node involvement and PSA value, and was significantly down regulated in tumor tissue relative to normal prostate tissue. Overexpression of miR-331-3p reduced PCa cell growth, migration and colony formation, as well as xenograft tumor initiation, proliferation and survival of mice. Microarray analysis identified seven novel targets of miR-331-3p in PCa. The 3'-untranslated regions of PLCγ1 and RALA were confirmed as targets of miR-331-3p, with mutation analyses confirming RALA as a direct target. Expression of miR-331-3p or RALA siRNA in PCa cells reduced RALA expression, proliferation, migration and colony formation in vitro. RALA expression positively correlated with Gleason grade in two separate studies, as well as in a PCa tissue microarray. Co-treatment using siRALA with an Aurora Kinase inhibitor (AKi-II) decreased colony formation of PCa cells while the combination of AKi-II with miR-331-3p resulted in significant reduction of PCa cell proliferation in vitro and PCa xenograft growth in vivo. Thus, miR-331-3p directly targets the RALA pathway and the addition of the AKi-II has a synergistic effect on tumor growth inhibition, suggesting a potential role as combination therapy in PCa.
ABSTRACT
Prostate cancer is one of the most prevalent cancers in men. It is critical to identify and characterize oncogenes that drive the pathogenesis of human prostate cancer. The current study builds upon previous research showing that a disintegrin and metallproteinase (ADAM)28 is involved in the pathogenesis of numerous cancers. Our novel study used overexpression, pharmacological, and molecular approaches to investigate the biological function of ADAM28 in human prostate cancer cells, with a focus on cell proliferation and migration. The results of this study provide important insights into the role of metalloproteinases in human prostate cancer.The expression of ADAM28 protein levels was assessed within human prostate tumors and normal adjacent tissue by immunohistochemistry. Immunocytochemistry and western blotting were used to assess ADAM28 protein expression in human prostate cancer cell lines. Functional assays were conducted to assess proliferation and migration in human prostate cancer cells in which ADAM28 protein expression or activity had been altered by overexpression, pharmacological inhibition, or by siRNA gene knockdown.The membrane bound ADAM28 was increased in human tumor biopsies and prostate cancer cell lines. Pharmacological inhibition of ADAM28 activity and/or knockdown of ADAM28 significantly reduced proliferation and migration of human prostate cancer cells, while overexpression of ADAM28 significantly increased proliferation and migration.ADAM28 is overexpressed in primary human prostate tumor biopsies, and it promotes human prostate cancer cell proliferation and migration. This study supports the notion that inhibition of ADAM28 may be a potential novel therapeutic strategy for human prostate cancer.
Subject(s)
ADAM Proteins/genetics , DNA, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques/methods , Prostatic Neoplasms/genetics , ADAM Proteins/biosynthesis , Cell Proliferation , Enzyme-Linked Immunosorbent Assay , Humans , Immunohistochemistry , Male , Prostatic Neoplasms/pathology , Real-Time Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
BACKGROUND: The TSH-T4 relationship was thought to be inverse log-linear, but recent cross-sectional studies of selected populations report a complex, nonlinear relationship. The TSH-T4 relationship has not been evaluated in an unselected, community-based cohort, and there are limited data regarding clinical factors which affect it. OBJECTIVE: To analyse the TSH-free T4 relationship in a community-based cohort. DESIGN, PARTICIPANTS AND METHODS: In a cross-sectional, retrospective study, we analysed serum TSH and free T4 concentrations from 4427 participants (55% female) in the 1994 Busselton Health Study who were not taking thyroxine. Simple linear, segmented-linear and nonlinear regression models of log10 TSH on free T4 were compared for goodness of fit. RESULTS: All 5 log TSH-free T4 models tested (separate lines, segmented conterminal line, quartic, error function, double-sigmoid curve) fitted significantly better than a simple linear model (each P < 0·01 by Vuong test). Ranking by Akaike information criterion indicated that the segmented conterminal line and double-sigmoid models provided best fit, followed by the error function, quartic and separate lines models. From multiple regression analysis, age tertile, current smoking and TPOAb status each significantly influenced the TSH-free T4 relationship, whereas BMI category and diabetes did not. A sex difference in the TSH-free T4 relationship was apparent only in the lower part of the free T4 reference range. CONCLUSION: In a community-based setting, the relationship between log TSH and free T4 is complex, nonlinear and influenced by age, smoking and TPOAb status.
Subject(s)
Thyrotropin/blood , Thyroxine/blood , Adolescent , Adult , Age Factors , Aged , Aged, 80 and over , Autoantibodies , Cohort Studies , Cross-Sectional Studies , Female , Humans , Male , Middle Aged , Regression Analysis , Retrospective Studies , Smoking , Young AdultABSTRACT
microRNA-7-5p (miR-7-5p) is a tumor suppressor in multiple cancer types and inhibits growth and invasion by suppressing expression and activity of the epidermal growth factor receptor (EGFR) signaling pathway. While melanoma is not typically EGFR-driven, expression of miR-7-5p is reduced in metastatic tumors compared to primary melanoma. Here, we investigated the biological and clinical significance of miR-7-5p in melanoma. We found that augmenting miR-7-5p expression in vitro markedly reduced tumor cell viability, colony formation and induced cell cycle arrest. Furthermore, ectopic expression of miR-7-5p reduced migration and invasion of melanoma cells in vitro and reduced metastasis in vivo. We used cDNA microarray analysis to identify a subset of putative miR-7-5p target genes associated with melanoma and metastasis. Of these, we confirmed nuclear factor kappa B (NF-κB) subunit RelA, as a novel direct target of miR-7-5p in melanoma cells, such that miR-7-5p suppresses NF-κB activity to decrease expression of canonical NF-κB target genes, including IL-1ß, IL-6 and IL-8. Importantly, the effects of miR-7-5p on melanoma cell growth, cell cycle, migration and invasion were recapitulated by RelA knockdown. Finally, analysis of gene array datasets from multiple melanoma patient cohorts revealed an association between elevated RelA expression and poor survival, further emphasizing the clinical significance of RelA and its downstream signaling effectors. Taken together, our data show that miR-7-5p is a potent inhibitor of melanoma growth and metastasis, in part through its inactivation of RelA/NF-κB signaling. Furthermore, miR-7-5p replacement therapy could have a role in the treatment of this disease.
Subject(s)
Cell Movement , Cell Proliferation , Melanoma/metabolism , MicroRNAs/metabolism , Skin Neoplasms/metabolism , Transcription Factor RelA/metabolism , 3' Untranslated Regions , Animals , Binding Sites , Cell Cycle Checkpoints , Cell Line, Tumor , Down-Regulation , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Interleukins/genetics , Interleukins/metabolism , Kaplan-Meier Estimate , Male , Melanoma/genetics , Melanoma/mortality , Melanoma/secondary , Mice, Inbred NOD , MicroRNAs/genetics , Neoplasm Invasiveness , Prognosis , RNA Interference , Signal Transduction , Skin Neoplasms/genetics , Skin Neoplasms/mortality , Skin Neoplasms/pathology , Time Factors , Transcription Factor RelA/genetics , Transcriptome , TransfectionABSTRACT
MicroRNAs (miRNAs) are a family of short, non-coding RNA molecules (â¼22nt) involved in post-transcriptional control of gene expression. They act via base-pairing with mRNA transcripts that harbour target sequences, resulting in accelerated mRNA decay and/or translational attenuation. Given miRNAs mediate the expression of molecules involved in many aspects of normal cell development and functioning, it is not surprising that aberrant miRNA expression is closely associated with many human diseases. Their pivotal role in driving a range of normal cellular physiology as well as pathological processes has established miRNAs as potential therapeutics, as well as potential diagnostic and prognostic tools in human health. MicroRNA-7 (miR-7) is a highly conserved miRNA which displays restricted spatiotemporal expression during development and in maturity. In humans and mice, mature miR-7 is generated from three different genes, illustrating unexpected redundancy and also the importance of this miRNA in regulating key cellular processes. In this review we examine the expanding role of miR-7 in the context of health, with emphasis on organ differentiation and development, as well as in various mammalian diseases, particularly of the brain, heart, endocrine pancreas and skin, as well as in cancer. The more we learn about miR-7, the more we realise the complexity of its regulation and potential functional application both from a biomarker and therapeutic perspective.
Subject(s)
MicroRNAs/physiology , Animals , Base Sequence , Brain/growth & development , Conserved Sequence , Diabetes Mellitus/metabolism , Gene Expression , Gene Expression Regulation, Developmental , Heart/growth & development , Humans , Neoplasms/genetics , Neoplasms/metabolism , Pancreas/growth & development , RNA Interference , Skin Diseases/metabolismABSTRACT
microRNAs (miRNAs) are a family of short, non-coding RNA molecules that drive a complex network of post-transcriptional gene regulation by enhancing target mRNA decay and/or inhibiting protein synthesis from mRNA transcripts. They regulate genes involved in key aspects of normal cell growth, development and the maintenance of body homeostasis and have been closely linked to the development and progression of human disease, in particular cancer. Over recent years there has been much interest regarding their potential as biomarkers and as therapeutic agents or targets. microRNA-7 (miR-7) is a 23 nucleotide (nt) miRNA known primarily to act as a tumour suppressor. miR-7 directly inhibits a number of oncogenic targets and impedes various aspects of cancer progression in vitro and in vivo, however, some studies have also implicated miR-7 in oncogenic roles. This review summarises the role of miR-7 in cancer, its potential in miRNA-based replacement therapy and its capacity as both a diagnostic and prognostic biomarker.
ABSTRACT
Cumulative exposure to estrogen (E) and progesterone (P) over the menstrual cycle significantly influences the risk of developing breast cancer. Despite the dogma that PR in the breast merely serves as a marker of an active estrogen receptor (ER), and as an inhibitor of the proliferative actions of E, it is now clear that in the breast P increases proliferation independently of E action. We show here that the progesterone receptor (PR) and ER are expressed in different epithelial populations, and target non-overlapping pathways in the normal human breast. In breast cancer, PR becomes highly correlated with ER, and this convergence is associated with signaling pathways predictive of disease metastasis. These data challenge the established paradigm that ER and PR function co-operatively in normal breast, and have significant implications not only for our understanding of normal breast biology, but also for diagnosis, prognosis and/or treatment options in breast cancer patients.