The role of the long non coding RNA HAS2-AS1 in breast cancer cells

Extracellular matrix (ECM) is a network made by proteins and proteoglycans, whose structure is essential to maintain tissue architecture and to provide molecules diffusion and cellular communications. A deregulated synthesis of ECM components is often associated to a pathological status. Among various glycosaminoglycans, hyaluronan (HA) is a ubiquitous ECM component with a remarkable structural importance. It is able to modulate cell adhesion, motility, growth and inflammation after the binding with cellular receptors (CD44 and RHAMM) and the activation of different cellular pathways. In tumour microenvironment, the up-regulation of HAS2 and the overproduction of HA are often associated with tumour progression and metastasis. This also applies to breast cancer, where the accumulation of HA and the overexpression of hyaluronan synthases (HASes) in stromal and tumoral cells correlate with tumor malignancy and patients survival. The study of the regulation of HAS2, the main enzyme in the production of HA, is very important to understand the development and the progression of breast cancer. Recently, it has been discovered that the lncRNA HAS2-AS1 can modulate the expression of HAS2 and the production of hyaluronan in aortic smooth muscle cells via epigenetic modifications [1]. Although the role of HA and HAS2 in breast cancer is widely described, little is known about HAS2-AS1. Given this considerations, the aim of this project is to study the role of HAS2-AS1 in breast cancer. In particular, we compared the behaviour of MDA-MB-231 and MCF-7 cells after the modulation of HAS2-AS1 expression with functional assays evaluating cell proliferation, migration and invasion. In the same conditions, we analysed the expression of HA related genes and receptors in MDA-MB-231 cells. This analysis revealed that HAS2-AS1 knockdown stimulated the presence of a malignant phenotype, as its abrogation increased cell motility and invasion, as well as the expression of several HA related genes and the receptor CD44. These evidences suggested that HAS2-AS1 plays an important role breast tumor progression through alteration of HA metabolism. Further analysis were conducted to understand the molecular mechanisms at the basis of the changes observed. LncRNAs can orchestrate gene expression through a variety of mechanisms, regulating transcription and translation, chromatin remodelling and the interaction with other RNA species, i.e. miRNAs. HAS2-AS1 transcript contains a putative binding site for miRNA 186, a negative regulator of the pro-apoptotic receptor P2X7 [2]. In our results we demonstrated that the overexpression of HAS2-AS1 decreased the abundance of miR-186, while the transcript of P2X7 and other targets of miRNA 186 (involved in cell cycle and autophagy) raised. All together, these data suggest that the \u201csponge effect\u201d of HAS2-AS1 is able to antagonise the function of miRNA 186 on its downstream targets and could explain the presence of a malignant phenotype after HAS2-AS1 silencing in MDA-MB-231. 1. Vigetti D, Deleonibus S, Moretto P, Bowen T, Fischer JW, Grandoch M, et al. Natural antisense transcript for hyaluronan synthase 2 (HAS2-AS1) induces transcription of HAS2 via protein O-GlcNAcylation. J. Biol. Chem. 2014;289:28816\u201326. 2. Zhou L, Qi X, Potashkin JA, Abdul-Karim FW, Gorodeski GI. MicroRNAs miR-186 and miR-150 down-regulate expression of the pro-apoptotic purinergic P2X7 receptor by activation of instability sites at the 3\u2b9-untranslated region of the gene that decrease steady-state levels of the transcript. J. Biol. Chem. 2008;283:28274\u201386

The role of the long non coding RNA HAS2-AS1 in breast cancer cells

Extracellular matrix (ECM) is a network made by proteins and proteoglycans, whose structure is essential to maintain tissue architecture and to provide molecules diffusion and cellular communications. A deregulated synthesis of ECM components is often associated to a pathological status. Among various glycosaminoglycans, hyaluronan (HA) is a ubiquitous ECM component with a remarkable structural importance. It is able to modulate cell adhesion, motility, growth and inflammation after the binding with cellular receptors (CD44 and RHAMM) and the activation of different cellular pathways. In tumour microenvironment, the up-regulation of HAS2 and the overproduction of HA are often associated with tumour progression and metastasis. This also applies to breast cancer, where the accumulation of HA and the overexpression of hyaluronan synthases (HASes) in stromal and tumoral cells correlate with tumor malignancy and patients survival. The study of the regulation of HAS2, the main enzyme in the production of HA, is very important to understand the development and the progression of breast cancer. Recently, it has been discovered that the lncRNA HAS2-AS1 can modulate the expression of HAS2 and the production of hyaluronan in aortic smooth muscle cells via epigenetic modifications [1]. Although the role of HA and HAS2 in breast cancer is widely described, little is known about HAS2-AS1. Given this considerations, the aim of this project is to study the role of HAS2-AS1 in breast cancer. In particular, we compared the behaviour of MDA-MB-231 and MCF-7 cells after the modulation of HAS2-AS1 expression with functional assays evaluating cell proliferation, migration and invasion. In the same conditions, we analysed the expression of HA related genes and receptors in MDA-MB-231 cells. This analysis revealed that HAS2-AS1 knockdown stimulated the presence of a malignant phenotype, as its abrogation increased cell motility and invasion, as well as the expression of several HA related genes and the receptor CD44. These evidences suggested that HAS2-AS1 plays an important role breast tumor progression through alteration of HA metabolism. Further analysis were conducted to understand the molecular mechanisms at the basis of the changes observed. LncRNAs can orchestrate gene expression through a variety of mechanisms, regulating transcription and translation, chromatin remodelling and the interaction with other RNA species, i.e. miRNAs. HAS2-AS1 transcript contains a putative binding site for miRNA 186, a negative regulator of the pro-apoptotic receptor P2X7 [2]. In our results we demonstrated that the overexpression of HAS2-AS1 decreased the abundance of miR-186, while the transcript of P2X7 and other targets of miRNA 186 (involved in cell cycle and autophagy) raised. All together, these data suggest that the “sponge effect” of HAS2-AS1 is able to antagonise the function of miRNA 186 on its downstream targets and could explain the presence of a malignant phenotype after HAS2-AS1 silencing in MDA-MB-231. 1. Vigetti D, Deleonibus S, Moretto P, Bowen T, Fischer JW, Grandoch M, et al. Natural antisense transcript for hyaluronan synthase 2 (HAS2-AS1) induces transcription of HAS2 via protein O-GlcNAcylation. J. Biol. Chem. 2014;289:28816–26. 2. Zhou L, Qi X, Potashkin JA, Abdul-Karim FW, Gorodeski GI. MicroRNAs miR-186 and miR-150 down-regulate expression of the pro-apoptotic purinergic P2X7 receptor by activation of instability sites at the 3ʹ-untranslated region of the gene that decrease steady-state levels of the transcript. J. Biol. Chem. 2008;283:28274–86

Hyaluronan preconditioning of monocytes/macrophages affects their angiogenic behavior and regulation of TSG-6 expression in a tumor type-specific manner

Hyaluronan is a glycosaminoglycan normally present in the extracellular matrix in most tissues. Hyaluronan is a crucial player in many processes associated with cancer, such as angiogenesis, invasion and metastasis. However, little has been reported regarding the action of hyaluronan on monocytes/macrophages in tumor angiogenesis and its consequences on tumor development. In the present study, we investigated the effects of hyaluronan of different sizes on human monocytes/macrophages angiogenic behavior in colorectal and breast carcinoma. In vitro, treatment of monocytes/macrophages with lysates and conditioned media from a breast, but not from colorectal, carcinoma cell line plus high molecular weight hyaluronan induced: i) an increased expression of angiogenic factors VEGF, IL‐8, FGF‐2 and MMP‐2, ii) increased endothelial cell migration and iii) a differential expression of hyaluronan‐binding protein TSG‐6. Similar results were observed in monocytes/macrophages derived from breast cancer patients treated with tumor lysates. Besides, macrophages primed with high molecular weight hyaluronan and inoculated in human breast cancer xenograft tumor increased blood vessel formation and diminished TSG‐6 levels. In contrast, the effects triggered by high molecular weight hyaluronan on monocytes/macrophages in breast cancer context were not observed in the context of colorectal carcinoma. Taken together, these results indicate that the effect of high molecular weight hyaluronan as an inductor of the angiogenic behavior of macrophages in breast tumor context is in part consequence of the presence of TSG‐6.Fil: Spinelli, Fiorella Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; ArgentinaFil: Vitale, Daiana Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; ArgentinaFil: Icardi, Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; ArgentinaFil: Caon, Ilaria. Università degli Studi dell’Insubria; ItaliaFil: Brandone, Alejandra. Gobierno de la Provincia de Buenos Aires. Hospital Interzonal General de Agudos Doctor Abraham Felix Piñeyro.; ArgentinaFil: Giannoni, Ana Paula. No especifíca;Fil: Saturno, Maria Virginia. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Passi, Alberto. Università degli Studi dell’Insubria; ItaliaFil: García, Mariana. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Sevic, Ina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; ArgentinaFil: Alaniz, Laura Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentin

Regulation of Hyaluronan Synthesis in Vascular Diseases and Diabetes

Cell microenvironment has a critical role determining cell fate and modulating cell responses to injuries. Hyaluronan (HA) is a ubiquitous extracellular matrix glycosaminoglycan that can be considered a signaling molecule. In fact, interacting with several cell surface receptors can deeply shape cell behavior. In vascular biology, HA triggers smooth muscle cells (SMCs) dedifferentiation which contributes to vessel wall thickening. Furthermore, HA is able to modulate inflammation by altering the adhesive properties of endothelial cells. In hyperglycemic conditions, HA accumulates in vessels and can contribute to the diabetic complications at micro-and macrovasculature. Due to the pivotal role in favoring atherogenesis and neointima formation after injuries, HA could be a new target for cardiovascular pathologies. This review will focus on the recent findings regarding the regulation of HA synthesis in human vascular SMCs. In particular, the effects of the intracellular HA substrates availability, adenosine monophosphate-activated protein kinase (AMPK), and protein O-GlcNAcylation on the main HA synthetic enzyme (i.e., HAS2) will be discussed

The role of the long non coding RNA HAS2-AS1 in breast cancer cells

Extracellular matrix (ECM) is a network made by proteins and proteoglycans, whose structure is essential to maintain tissue architecture and to provide molecules diffusion and cellular communications. A deregulated synthesis of ECM components is often associated to a pathological status. Among various glycosaminoglycans, hyaluronan (HA) is a ubiquitous ECM component with a remarkable structural importance. It is able to modulate cell adhesion, motility, growth and inflammation after the binding with cellular receptors (CD44 and RHAMM) and the activation of different cellular pathways. In tumour microenvironment, the up-regulation of HAS2 and the overproduction of HA are often associated with tumour progression and metastasis. This also applies to breast cancer, where the accumulation of HA and the overexpression of hyaluronan synthases (HASes) in stromal and tumoral cells correlate with tumor malignancy and patients survival. The study of the regulation of HAS2, the main enzyme in the production of HA, is very important to understand the development and the progression of breast cancer. Recently, it has been discovered that the lncRNA HAS2-AS1 can modulate the expression of HAS2 and the production of hyaluronan in aortic smooth muscle cells via epigenetic modifications [1]. Although the role of HA and HAS2 in breast cancer is widely described, little is known about HAS2-AS1. Given this considerations, the aim of this project is to study the role of HAS2-AS1 in breast cancer. In particular, we compared the behaviour of MDA-MB-231 and MCF-7 cells after the modulation of HAS2-AS1 expression with functional assays evaluating cell proliferation, migration and invasion. In the same conditions, we analysed the expression of HA related genes and receptors in MDA-MB-231 cells. This analysis revealed that HAS2-AS1 knockdown stimulated the presence of a malignant phenotype, as its abrogation increased cell motility and invasion, as well as the expression of several HA related genes and the receptor CD44. These evidences suggested that HAS2-AS1 plays an important role breast tumor progression through alteration of HA metabolism. Further analysis were conducted to understand the molecular mechanisms at the basis of the changes observed. LncRNAs can orchestrate gene expression through a variety of mechanisms, regulating transcription and translation, chromatin remodelling and the interaction with other RNA species, i.e. miRNAs. HAS2-AS1 transcript contains a putative binding site for miRNA 186, a negative regulator of the pro-apoptotic receptor P2X7 [2]. In our results we demonstrated that the overexpression of HAS2-AS1 decreased the abundance of miR-186, while the transcript of P2X7 and other targets of miRNA 186 (involved in cell cycle and autophagy) raised. All together, these data suggest that the “sponge effect” of HAS2-AS1 is able to antagonise the function of miRNA 186 on its downstream targets and could explain the presence of a malignant phenotype after HAS2-AS1 silencing in MDA-MB-231. 1. Vigetti D, Deleonibus S, Moretto P, Bowen T, Fischer JW, Grandoch M, et al. Natural antisense transcript for hyaluronan synthase 2 (HAS2-AS1) induces transcription of HAS2 via protein O-GlcNAcylation. J. Biol. Chem. 2014;289:28816–26. 2. Zhou L, Qi X, Potashkin JA, Abdul-Karim FW, Gorodeski GI. MicroRNAs miR-186 and miR-150 down-regulate expression of the pro-apoptotic purinergic P2X7 receptor by activation of instability sites at the 3ʹ-untranslated region of the gene that decrease steady-state levels of the transcript. J. Biol. Chem. 2008;283:28274–86

HA and HS Changes in Endothelial Inflammatory Activation

Cardiovascular diseases are a group of disorders caused by the presence of a combination of risk factors, such as tobacco use, unhealthy diet and obesity, physical inactivity, etc., which cause the modification of the composition of the vessel's matrix and lead to the alteration of blood flow, matched with an inflammation condition. Nevertheless, it is not clear if the inflammation is a permissive condition or a consequent one. In order to investigate the effect of inflammation on the onset of vascular disease, we treated endothelial cells with the cytokine TNF-alpha that is increased in obese patients and is reported to induce cardiometabolic diseases. The inflammation induced a large change in the extracellular matrix, increasing the pericellular hyaluronan and altering the heparan sulfate Syndecans sets, which seems to be related to layer permeability but does not influence cell proliferation or migration nor induce blood cell recruitment or activation

Regulated Hyaluronan Synthesis by Vascular Cells

Cellular microenvironment plays a critical role in several pathologies including atherosclerosis. Hyaluronan (HA) content often reflects the progression of this disease in promoting vessel thickening and cell migration. HA synthesis is regulated by several factors, including the phosphorylation of HA synthase 2 (HAS2) and other covalent modifications including ubiquitination and O-GlcNAcylation. Substrate availability is important in HA synthesis control. Specific drugs reducing the UDP precursors are able to reduce HA synthesis whereas the hexosamine biosynthetic pathway (HBP) increases the concentration of HA precursor UDP-N-acetylglucosamine (UDP-GlcNAc) leading to an increase of HA synthesis. The flux through the HBP in the regulation of HA biosynthesis in human aortic vascular smooth muscle cells (VSMCs) was reported as a critical aspect. In fact, inhibiting O-GlcNAcylation reduced HA production whereas increased O-GlcNAcylation augmented HA secretion. Additionally, O-GlcNAcylation regulates HAS2 gene expression resulting in accumulation of its mRNA after induction of O-GlcNAcylation with glucosamine treatments. The oxidized LDLs, the most common molecules related to atherosclerosis outcome and progression, are also able to induce a strong HA synthesis when they are in contact with vascular cells. In this review, we present recent described mechanisms involved in HA synthesis regulation and their role in atherosclerosis outcome and development

Regulation of Hyaluronan Synthesis in Vascular Diseases and Diabetes

Cell microenvironment has a critical role determining cell fate and modulating cell responses to injuries. Hyaluronan (HA) is a ubiquitous extracellular matrix glycosaminoglycan that can be considered a signaling molecule. In fact, interacting with several cell surface receptors can deeply shape cell behavior. In vascular biology, HA triggers smooth muscle cells (SMCs) dedifferentiation which contributes to vessel wall thickening. Furthermore, HA is able to modulate inflammation by altering the adhesive properties of endothelial cells. In hyperglycemic conditions, HA accumulates in vessels and can contribute to the diabetic complications at micro- and macrovasculature. Due to the pivotal role in favoring atherogenesis and neointima formation after injuries, HA could be a new target for cardiovascular pathologies. This review will focus on the recent findings regarding the regulation of HA synthesis in human vascular SMCs. In particular, the effects of the intracellular HA substrates availability, adenosine monophosphate-activated protein kinase (AMPK), and protein O-GlcNAcylation on the main HA synthetic enzyme (i.e., HAS2) will be discussed

Biology and biotechnology of hyaluronan

The hyaluronan (HA) polymer is a critical component of extracellular matrix with a remarkable structure: is a linear and unbranched polymer without sulphate or phosphate groups. It is ubiquitous in mammals showing several biological functions, ranging from cell proliferation and migration to angiogenesis and inflammation. For its critical biological functions the amount of HA in tissues is carefully controlled by different mechanisms including covalent modification of the synthetic enzymes and epigenetic control of their gene expression. The concentration of HA is also critical in several pathologies including cancer, diabetes and inflammation. Beside these biological roles, the structural properties of HA allow it to take advantage of its capacity to form gels even at concentration of 1 % producing scaffolds with very promising applications in regenerative medicine as biocompatible material for advanced therapeutic uses. In this review we highlight the biological aspects of HA addressing the mechanisms controlling the HA content in tissues as well as its role in important human pathologies. In the second part of the review we highlight the different use of HA polymers in the modern biotechnology