Prostate cancer is one of the most prevalent cancers in men. It is critical to identify and chara... more 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...
The International Journal of Biochemistry & Cell Biology, 2015
MicroRNAs (miRNAs) are a family of short, non-coding RNA molecules (∼22nt) involved in post-trans... more MicroRNAs (miRNAs) are a family of short, non-coding RNA molecules (∼22nt) 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.
microRNAs (miRNAs) are a family of short, non-coding RNA molecules that drive a complex network o... more 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.
Elevated expression and activity of the epidermal growth factor receptor (EGFR)/protein kinase B ... more Elevated expression and activity of the epidermal growth factor receptor (EGFR)/protein kinase B (Akt) signaling pathway is associated with development, progression and treatment resistance of head and neck cancer (HNC). Several studies have demonstrated that microRNA-7 (miR-7) regulates EGFR expression and Akt activity in a range of cancer cell types via its specific interaction with the EGFR mRNA 3'-untranslated region (3'-UTR). In the present study, we found that miR-7 regulated EGFR expression and Akt activity in HNC cell lines, and that this was associated with reduced growth in vitro and in vivo of cells (HN5) that were sensitive to the EGFR tyrosine kinase inhibitor (TKI) erlotinib (Tarceva). miR-7 acted synergistically with erlotinib to inhibit growth of erlotinib-resistant FaDu cells, an effect associated with increased inhibition of Akt activity. Microarray analysis of HN5 and FaDu cell lines transfected with miR-7 identified a common set of downregulated miR-7 tar...
Aberrant expression of the epidermal growth factor receptor (EGFR) and/or human epidermal growth ... more Aberrant expression of the epidermal growth factor receptor (EGFR) and/or human epidermal growth factor receptor 2 (HER2) is a feature of many human tumors and is associated with disease progression, treatment resistance, and poor prognosis. Protein kinase B/Akt, an important downstream effector of these receptor tyrosine kinases, induces signaling pathways that control cancer cell proliferation, invasion, angiogenesis, and apoptosis resistance. MicroRNAs (miRNAs), small noncoding RNAs that bind to the 3'-untranslated region of target mRNAs, are now recognized to play key roles in the regulation of gene expression, particularly in tumor development and metastasis. We have shown that miRNA-7 (miR-7) and miRNA-331-3p (miR-331-3p) directly regulate expression of EGFR and HER2, respectively, in glioblastoma and prostate cancer cell lines. As a consequence, miR-7 and miR-331-3p reduce Akt activity and thus have the capacity to regulate a signaling pathway critical to the development and progression of glioblastoma and prostate cancer. This chapter provides a detailed approach outlining how to confirm that a putative target of a miRNA is a direct target, and subsequent assessment of downstream signaling mediators.
Proceedings of the National Academy of Sciences, 2013
The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including p... more The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.
Over-expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is regarded as an early ... more Over-expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is regarded as an early marker for several cancers. This protein is associated with proto-oncogenes and tumor suppressor genes and has itself been described as a proto-oncogene. Our earlier experiments drew a connection between hnRNP A2/B1 levels and cell proliferation and raised the possibility that this protein contributes to the uncontrolled cell division that characterizes cancer. Limited knowledge of the downstream targets of hnRNP A2/B1 has, however, precluded a clear understanding of their roles in cancer cell growth. To define the pathways in which this protein acts we have now carried out microarray experiments with total RNA from Colo16 epithelial cells transfected with an shRNA that markedly suppresses hnRNP A2/B1 expression. The microarray data identified 123 genes, among 22 283 human gene probe sets, with altered expression levels in hnRNP A2/B1-depleted cells. Ontological analysis showed that many of these downstream targets are involved in regulation of the cell cycle and cell proliferation and that this group of proteins is significantly over-represented amongst the affected proteins. The changes detected in the microarray experiments were confirmed by real-time PCR for a subset of proliferation-related genes. Immunoprecipitation-RT-PCR demonstrated that hnRNP A2/B1 formed complexes with the transcripts of many of the verified downstream genes, suggesting that hnRNP A2/B1 contributes to the regulation of these genes. These results reinforce the conclusion that hnRNP A2/B1 is associated with cellular processes that affect the cell cycle and proliferation.
Prostate cancer is one of the most prevalent cancers in men. It is critical to identify and chara... more 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...
The International Journal of Biochemistry & Cell Biology, 2015
MicroRNAs (miRNAs) are a family of short, non-coding RNA molecules (∼22nt) involved in post-trans... more MicroRNAs (miRNAs) are a family of short, non-coding RNA molecules (∼22nt) 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.
microRNAs (miRNAs) are a family of short, non-coding RNA molecules that drive a complex network o... more 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.
Elevated expression and activity of the epidermal growth factor receptor (EGFR)/protein kinase B ... more Elevated expression and activity of the epidermal growth factor receptor (EGFR)/protein kinase B (Akt) signaling pathway is associated with development, progression and treatment resistance of head and neck cancer (HNC). Several studies have demonstrated that microRNA-7 (miR-7) regulates EGFR expression and Akt activity in a range of cancer cell types via its specific interaction with the EGFR mRNA 3'-untranslated region (3'-UTR). In the present study, we found that miR-7 regulated EGFR expression and Akt activity in HNC cell lines, and that this was associated with reduced growth in vitro and in vivo of cells (HN5) that were sensitive to the EGFR tyrosine kinase inhibitor (TKI) erlotinib (Tarceva). miR-7 acted synergistically with erlotinib to inhibit growth of erlotinib-resistant FaDu cells, an effect associated with increased inhibition of Akt activity. Microarray analysis of HN5 and FaDu cell lines transfected with miR-7 identified a common set of downregulated miR-7 tar...
Aberrant expression of the epidermal growth factor receptor (EGFR) and/or human epidermal growth ... more Aberrant expression of the epidermal growth factor receptor (EGFR) and/or human epidermal growth factor receptor 2 (HER2) is a feature of many human tumors and is associated with disease progression, treatment resistance, and poor prognosis. Protein kinase B/Akt, an important downstream effector of these receptor tyrosine kinases, induces signaling pathways that control cancer cell proliferation, invasion, angiogenesis, and apoptosis resistance. MicroRNAs (miRNAs), small noncoding RNAs that bind to the 3'-untranslated region of target mRNAs, are now recognized to play key roles in the regulation of gene expression, particularly in tumor development and metastasis. We have shown that miRNA-7 (miR-7) and miRNA-331-3p (miR-331-3p) directly regulate expression of EGFR and HER2, respectively, in glioblastoma and prostate cancer cell lines. As a consequence, miR-7 and miR-331-3p reduce Akt activity and thus have the capacity to regulate a signaling pathway critical to the development and progression of glioblastoma and prostate cancer. This chapter provides a detailed approach outlining how to confirm that a putative target of a miRNA is a direct target, and subsequent assessment of downstream signaling mediators.
Proceedings of the National Academy of Sciences, 2013
The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including p... more The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.
Over-expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is regarded as an early ... more Over-expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is regarded as an early marker for several cancers. This protein is associated with proto-oncogenes and tumor suppressor genes and has itself been described as a proto-oncogene. Our earlier experiments drew a connection between hnRNP A2/B1 levels and cell proliferation and raised the possibility that this protein contributes to the uncontrolled cell division that characterizes cancer. Limited knowledge of the downstream targets of hnRNP A2/B1 has, however, precluded a clear understanding of their roles in cancer cell growth. To define the pathways in which this protein acts we have now carried out microarray experiments with total RNA from Colo16 epithelial cells transfected with an shRNA that markedly suppresses hnRNP A2/B1 expression. The microarray data identified 123 genes, among 22 283 human gene probe sets, with altered expression levels in hnRNP A2/B1-depleted cells. Ontological analysis showed that many of these downstream targets are involved in regulation of the cell cycle and cell proliferation and that this group of proteins is significantly over-represented amongst the affected proteins. The changes detected in the microarray experiments were confirmed by real-time PCR for a subset of proliferation-related genes. Immunoprecipitation-RT-PCR demonstrated that hnRNP A2/B1 formed complexes with the transcripts of many of the verified downstream genes, suggesting that hnRNP A2/B1 contributes to the regulation of these genes. These results reinforce the conclusion that hnRNP A2/B1 is associated with cellular processes that affect the cell cycle and proliferation.
PDF - 59KB, Supplementary Figure S3. Inhibition of Axl activity in HN5-ER cells and EGFR activity... more PDF - 59KB, Supplementary Figure S3. Inhibition of Axl activity in HN5-ER cells and EGFR activity in HN5 cells with the Axl inhibitor R428.
PDF - 313KB, Supplementary Table S2. List of genes downregulated in HN5-ER cells relative to HN5 ... more PDF - 313KB, Supplementary Table S2. List of genes downregulated in HN5-ER cells relative to HN5 cells.
PDF - 22KB, Supplementary Figure S1. Cross-resistance of HN5-ER cells to gefitinib and stability ... more PDF - 22KB, Supplementary Figure S1. Cross-resistance of HN5-ER cells to gefitinib and stability of erlotinib resistance in HN5-ER cells grown in the absence of erlotinib.
PDF - 13KB, Supplementary Figure S4. Sensitivity of HN5-ER cells to co-treatment with gefitinib a... more PDF - 13KB, Supplementary Figure S4. Sensitivity of HN5-ER cells to co-treatment with gefitinib and R428.
PDF - 59KB, Supplementary Figure S3. Inhibition of Axl activity in HN5-ER cells and EGFR activity... more PDF - 59KB, Supplementary Figure S3. Inhibition of Axl activity in HN5-ER cells and EGFR activity in HN5 cells with the Axl inhibitor R428.
PDF - 706KB, Supplementary Table S3. Functional pathways associated with acquired erlotinib resis... more PDF - 706KB, Supplementary Table S3. Functional pathways associated with acquired erlotinib resistance in HN5-ER cells.
PDF - 75KB, Supplementary Figure S6. Analysis of NF-kB activity between HN5 and HN5-ER cells and ... more PDF - 75KB, Supplementary Figure S6. Analysis of NF-kB activity between HN5 and HN5-ER cells and following transfection of HN5-ER cells with miR-34a or Axl siRNAs.
PDF - 47KB, Supplementary Figure S5. Sensitivity of HN5-ER cells to erlotinib following Axl knock... more PDF - 47KB, Supplementary Figure S5. Sensitivity of HN5-ER cells to erlotinib following Axl knockdown by RNAi.
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