Effects of Hyaluronan on Breast Cancer Aggressiveness.

The expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) in breast cancer cells is critical for determining tumor aggressiveness and targeting therapies. The presence of such receptors allows for the use of antagonists that effectively reduce breast cancer growth and dissemination. However, the absence of such receptors in triple-negative breast cancer (TNBC) reduces the possibility of targeted therapy, making these tumors very aggressive with a poor outcome. Cancers are not solely composed of tumor cells, but also include several types of infiltrating cells, such as fibroblasts, macrophages, and other immune cells that have critical functions in regulating cancer cell behaviors. In addition to these cells, the extracellular matrix (ECM) has become an important player in many aspects of breast cancer biology, including cell growth, motility, metabolism, and chemoresistance. Hyaluronan (HA) is a key ECM component that promotes cell proliferation and migration in several malignancies. Notably, HA accumulation in the tumor stroma is a negative prognostic factor in breast cancer. HA metabolism depends on the fine balance between HA synthesis by HA synthases and degradation yielded by hyaluronidases. All the different cell types present in the tumor can release HA in the ECM, and in this review, we will describe the role of HA and HA metabolism in different breast cancer subtypes.

Hyaluronan in the Cancer Cells Microenvironment.

The presence of the glycosaminoglycan hyaluronan in the extracellular matrix of tissues is the result of the cooperative synthesis of several resident cells, that is, macrophages and tumor and stromal cells. Any change in hyaluronan concentration or dimension leads to a modification in stiffness and cellular response through receptors on the plasma membrane. Hyaluronan has an effect on all cancer cell behaviors, such as evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and metastasis. It is noteworthy that hyaluronan metabolism can be dramatically altered by growth factors and matrikines during inflammation, as well as by the metabolic homeostasis of cells. The regulation of HA deposition and its dimensions are pivotal for tumor progression and cancer patient prognosis. Nevertheless, because of all the factors involved, modulating hyaluronan metabolism could be tough. Several commercial drugs have already been described as potential or effective modulators; however, deeper investigations are needed to study their possible side effects. Moreover, other matrix molecules could be identified and targeted as upstream regulators of synthetic or degrading enzymes. Finally, co-cultures of cancer, fibroblasts, and immune cells could reveal potential new targets among secreted factors.

Particle Exclusion Assay: A Tool for Measuring Hyaluronan Pericellular Matrix.

Hyaluronan (HA) is the most abundant glycosaminoglycan in the extracellular matrix, and its deposition is strictly related to changes in cellular behaviors, such as cell migration, proliferation, and adhesion. Pericellular HA is abundant in a variety of cell types, and its amount could reflect specific conditions, thus suggesting a particular cellular status.Particle exclusion assay is a useful tool to visualize pericellular matrices with a high HA content, simply employing microscope image analysis. This approach is quick and allows to visualize the presence of a clear pericellular region around single cells, where fixed red blood cells are excluded if the pericellular matrix has been deposited.

The hyaluronan-related genes HAS2, HYAL1-4, PH20 and HYALP1 are associated with prognosis, cell viability and spheroid formation capacity in ovarian cancer.

PURPOSE: Hyaluronan modulates tumour progression, including cell adhesion, cohesion, proliferation and invasion, and the cancer stem cell phenotype. In ovarian cancer, high levels of stromal hyaluronan are associated with poor prognosis. In this work, hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-4, PH-20, HYALP1) were examined with regard to different levels of gene expression and its influence on ovarian cancer patients' survival. The impact of a siRNA depletion of HAS2 was investigated in vitro. METHODS: Using the Kaplan-Meier Plotter tool, we investigated the influence of hyaluronic synthases and hyaluronidases on the survival of a collective of 1435 ovarian cancer patients. Differences in gene expression between normal (n = 46) and cancerous (n = 744) ovarian tissue were examined using the TNMplot database. Following an evaluation of hyaluronan-related gene expression in the ATCC ovarian cancer panel, we studied SKOV3 and SW 626 ovarian cancer cells subjected to HAS2 siRNA or control siRNA treatment in terms of HAS1-3, HYAL2 and HYAL3 mRNA expression. We investigated the ability to form spheroids using the Hanging Drop method and the response to chemotherapy at different concentrations using the MTT Assay. By STRING analysis, interactions within the enzymes of the hyaluronic acid system and with binding partners were visualized. RESULTS: HAS1, HYAL1 and HYAL4 mRNA expression is significantly upregulated, whereas HAS2, HYAL2 and HYAL3 mRNA expression is significantly downregulated in ovarian cancer tissue compared to controls. HAS2 improves cell viability, the capability to form tumour spheroids and has a negative prognostic value regarding overall survival. Lower HAS2 expression and high expression of HYAL2 and HYAL3 favours the survival of ovarian cancer patients. HAS2 knockdown cells and control cells showed a moderate response to combinatorial in vitro chemotherapy with taxol and cisplatin. CONCLUSION: In conclusion, our study shows that the hyaluronic acid system has a relevant influence on the survival of ovarian cancer patients and could therefore be considered as a possible prognostic factor.

Hyaluronan in pathophysiology of vascular diseases: specific roles in smooth muscle cells, endothelial cells, and macrophages.

One of the main components of the extracellular matrix (ECM) of blood vessels is hyaluronic acid or hyaluronan (HA). It is a ubiquitous polysaccharide belonging to the family of glycosaminoglycans, but, differently from other proteoglycan-associated glycosaminoglycans, it is synthesized on the plasma membrane by a family of three HA synthases (HAS). HA can be released as a free polymer in the extracellular space or remain associated with the plasma membrane in the pericellular space via HAS or HA-binding proteins. Several cell surface proteins can interact with HA working as HA receptors, like CD44, RHAMM, and LYVE-1. In physiological conditions, HA is localized in the glycocalyx and the adventitia where it is responsible for the loose and hydrated vascular structure favoring flexibility and allowing the stretching of vessels in response to mechanical forces. During atherogenesis, ECM undergoes dramatic alterations that have a crucial role in lipoprotein retention and in triggering multiple signaling cascades that induce the cells to exit from their quiescent status. HA becomes highly present in the media and neointima favoring smooth muscle cells dedifferentiation, migration, and proliferation that strongly contribute to vessel wall thickening. Furthermore, HA is able to modulate immune cell recruitment both within the vessel wall and on the endothelial cell layer. This review is focused on deeply analyzing the effects of HA on vascular cell behavior.

The natural antisense transcript HAS2-AS1 regulates breast cancer cells aggressiveness independently from hyaluronan metabolism.

Hyaluronan (HA) is a ubiquitous extracellular matrix component playing a crucial role in the regulation of cell behaviors, including cancer. Aggressive breast cancer cells tend to proliferate, migrate and metastatize. Notably, triple-negative breast cancer cells lacking the expression of estrogen receptor (ER) as well as progesterone receptor and HER2 are more aggressive than ER-positive ones. As currently no targeted therapy is available for triple-negative breast cancer, the identification of novel therapeutic targets has a high clinical priority. In ER-negative cells, tumoral behavior can be reduced by inhibiting HA synthesis or silencing the enzymes involved in its metabolism, such as HA synthase 2 (HAS2). HAS2-AS1 is a long non-coding RNA belonging to the natural antisense transcript family which is known to favor HAS2 gene expression and HA synthesis, thus bolstering malignant progression in brain, ovary, and lung tumors. As the role of HAS2-AS1 has not yet been investigated in breast cancer, in this work we report that ER-positive breast cancers had lower HAS2-AS1 expression compared to ER-negative tumors. Moreover, the survival of patients with ER-negative tumors was higher when the expression of HAS2-AS1 was elevated. Experiments with ER-negative cell lines as MDA-MB-231 and Hs 578T revealed that the overexpression of either the full-length HAS2-AS1 or its exon 2 long or short isoforms alone, strongly reduced cell viability, migration, and invasion, whereas HAS2-AS1 silencing increased cell aggressiveness. Unexpectedly, in these ER-negative cell lines, HAS2-AS1 is involved neither in the regulation of HAS2 nor in HA deposition. Finally, transcriptome analysis revealed that HAS2-AS1 modulation affected several pathways, including apoptosis, proliferation, motility, adhesion, epithelial to mesenchymal transition, and signaling, describing this long non-coding RNA as an important regulator of breast cancer cells aggressiveness.

Inflammation, Extracellular Matrix Remodeling, and Proteostasis in Tumor Microenvironment.

Cancer is a multifaceted and complex pathology characterized by uncontrolled cell proliferation and decreased apoptosis. Most cancers are recognized by an inflammatory environment rich in a myriad of factors produced by immune infiltrate cells that induce host cells to differentiate and to produce a matrix that is more favorable to tumor cells' survival and metastasis. As a result, the extracellular matrix (ECM) is changed in terms of macromolecules content, degrading enzymes, and proteins. Altered ECM components, derived from remodeling processes, interact with a variety of surface receptors triggering intracellular signaling that, in turn, cancer cells exploit to their own benefit. This review aims to present the role of different aspects of ECM components in the tumor microenvironment. Particularly, we highlight the effect of pro- and inflammatory factors on ECM degrading enzymes, such as metalloproteases, and in a more detailed manner on hyaluronan metabolism and the signaling pathways triggered by the binding of hyaluronan with its receptors. In addition, we sought to explore the role of extracellular chaperones, especially of clusterin which is one of the most prominent in the extracellular space, in proteostasis and signaling transduction in the tumor microenvironment. Although the described tumor microenvironment components have different biological roles, they may engage common signaling pathways that favor tumor growth and metastasis.

The role of the multifaceted long non-coding RNAs: A nuclear-cytosolic interplay to regulate hyaluronan metabolism.

In the extracellular matrix (ECM), the glycosaminoglycan (GAG) hyaluronan (HA) has different physiological roles favouring hydration, elasticity and cell survival. Three different isoforms of HA synthases (HAS1, 2, and 3) are responsible for the production of HA. In several pathologies the upregulation of HAS enzymes leads to an abnormal HA accumulation causing cell dedifferentiation, proliferation and migration thus favouring cancer progression, fibrosis and vascular wall thickening. An intriguing new player in HAS2 gene expression regulation and HA production is the long non-coding RNA (lncRNA) hyaluronan synthase 2 antisense 1 (HAS2-AS1). A significant part of mammalian genomes corresponds to genes that transcribe lncRNAs; they can regulate gene expression through several mechanisms, being involved not only in maintaining the normal homeostasis of cells and tissues, but also in the onset and progression of different diseases, as demonstrated by the increasing number of studies published through the last decades. HAS2-AS1 is no exception: it can be localized both in the nucleus and in the cytosol, regulating cancer cells as well as vascular smooth muscle cells behaviour.

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-α 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.

The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk.

Interaction between cancer cells and their microenvironment is central in defining the fate of cancer development. Tumour cells secrete signals (cytokines, chemokines, growth factors) that modify the surrounding area, while the niche supplies structures and activities necessary for tumour maintenance and growth. Hyaluronan (HA) is a glycosaminoglycan that constitute cancer cell niche and is known to influence tumour functions such as proliferation, migration and neoangiogenesis. The knowledge of the factors regulating HA synthesis and size is crucial in understanding the mechanisms sustaining tumour development. Here we show that a yet uncharacterized protein secreted by breast tumour cell lines, named c10orf118 (accession number NM_018017 in NCBI/BLAST, and Q7z3E2 according to the Uniprot identifier), with a predicted length of 898 amino acids, can induce the secretion of HA by stromal fibroblasts through the up-regulation of the hyaluronan synthase 2 gene (HAS2). Intracellularly, this protein is localized in the Golgi apparatus with a possible role in vesicle maturation and transport. The expression of c10orf118 was verified in breast cancer patient specimens and was found to be associated with the presence of estrogen receptor that characterizes a good patient survival. We suggest c10orf118 as a new player that influences the HA amount in breast cancer microenvironment and is associated with low aggressiveness of cancer.

Initial Identification of UDP-Glucose Dehydrogenase as a Prognostic Marker in Breast Cancer Patients, Which Facilitates Epirubicin Resistance and Regulates Hyaluronan Synthesis in MDA-MB-231 Cells.

UDP-glucose-dehydrogenase (UGDH) synthesizes UDP-glucuronic acid. It is involved in epirubicin detoxification and hyaluronan synthesis. This work aimed to evaluate the effect of UGDH knockdown on epirubicin response and hyaluronan metabolism in MDA-MB-231 breast cancer cells. Additionally, the aim was to determine UGDH as a possible prognosis marker in breast cancer. We studied UGDH expression in tumors and adjacent tissue from breast cancer patients. The prognostic value of UGDH was studied using a public Kaplan-Meier plotter. MDA-MB-231 cells were knocked-down for UGDH and treated with epirubicin. Epirubicin-accumulation and apoptosis were analyzed by flow cytometry. Hyaluronan-coated matrix and metabolism were determined. Autophagic-LC3-II was studied by Western blot and confocal microscopy. Epirubicin accumulation increased and apoptosis decreased during UGDH knockdown. Hyaluronan-coated matrix increased and a positive modulation of autophagy was detected. Higher levels of UGDH were correlated with worse prognosis in triple-negative breast cancer patients that received chemotherapy. High expression of UGDH was found in tumoral tissue from HER2--patients. However, UGDH knockdown contributes to epirubicin resistance, which might be associated with increases in the expression, deposition and catabolism of hyaluronan. The results obtained allowed us to propose UGDH as a new prognostic marker in breast cancer, positively associated with development of epirubicin resistance and modulation of extracellular matrix.

Human Primary Dermal Fibroblasts Interacting with 3-Dimensional Matrices for Surgical Application Show Specific Growth and Gene Expression Programs.

Several types of 3-dimensional (3D) biological matrices are employed for clinical and surgical applications, but few indications are available to guide surgeons in the choice among these materials. Here we compare the in vitro growth of human primary fibroblasts on different biological matrices commonly used for clinical and surgical applications and the activation of specific molecular pathways over 30 days of growth. Morphological analyses by Scanning Electron Microscopy and proliferation curves showed that fibroblasts have different ability to attach and proliferate on the different biological matrices. They activated similar gene expression programs, reducing the expression of collagen genes and myofibroblast differentiation markers compared to fibroblasts grown in 2D. However, differences among 3D matrices were observed in the expression of specific metalloproteinases and interleukin-6. Indeed, cell proliferation and expression of matrix degrading enzymes occur in the initial steps of interaction between fibroblast and the investigated meshes, whereas collagen and interleukin-6 expression appear to start later. The data reported here highlight features of fibroblasts grown on different 3D biological matrices and warrant further studies to understand how these findings may be used to help the clinicians choose the correct material for specific applications.

Cell Energy Metabolism and Hyaluronan Synthesis.

Hyaluronan (HA) is a linear glycosaminoglycan (GAG) of extracellular matrix (ECM) synthesized by three hyaluronan synthases (HASes) at the plasma membrane using uridine diphosphate (UDP)-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc) as substrates. The production of HA is mainly regulated by hyaluronan synthase 2 (HAS2), that can be controlled at different levels, from epigenetics to transcriptional and post-translational modifications. HA biosynthesis is an energy-consuming process and, along with HA catabolism, is strongly connected to the maintenance of metabolic homeostasis. The cytoplasmic pool of UDP-sugars is critical for HA synthesis. UDP-GlcNAc is an important nutrient sensor and serves as donor substrate for the O-GlcNAcylation of many cytosolic proteins, including HAS2. This post-translational modification stabilizes HAS2 in the membrane and increases HA production. Conversely, HAS2 can be phosphorylated by AMP activated protein kinase (AMPK), a master metabolic regulator activated by low ATP/AMP ratios, which inhibits HA secretion. Similarly, HAS2 expression and the deposition of HA within the pericellular coat are inhibited by sirtuin 1 (SIRT1), another important energetic sensor, confirming the tight connection between nutrients availability and HA metabolism.

Heparan Sulfate in the Tumor Microenvironment.

The biology of tumor cells strictly depends on their microenvironment architecture and composition, which controls the availability of growth factors and signaling molecules. Thus, the network of glycosaminoglycans, proteoglycans, and proteins known as extracellular matrix (ECM) that surrounds the cells plays a central role in the regulation of tumor fate. Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) are highly versatile ECM components that bind and regulate the activity of growth factors, cell membrane receptors, and other ECM molecules. These HS binding partners modulate cell adhesion, motility, and proliferation that are processes altered during tumor progression. Modification in the expression and activity of HS, HSPGs, and the respective metabolic enzymes results unavoidably in alteration of tumor cell microenvironment. In this light, the targeting of HS structure and metabolism is potentially a new tool in the treatment of different cancer types.

Sirtuin 1 reduces hyaluronan synthase 2 expression by inhibiting nuclear translocation of NF-κB and expression of the long-noncoding RNA HAS2-AS1.

Hyaluronan (HA) is one of the most prevalent glycosaminoglycans of the vascular extracellular matrix (ECM). Abnormal HA accumulation within blood vessel walls is associated with tissue inflammation and is prominent in most vascular pathological conditions such as atherosclerosis and restenosis. Hyaluronan synthase 2 (HAS2) is the main hyaluronan synthase enzyme involved in HA synthesis and uses cytosolic UDP-glucuronic acid and UDP-GlcNAc as substrates. The synthesis of UDP-glucuronic acid can alter the NAD+/NADH ratio via the enzyme UDP-glucose dehydrogenase, which oxidizes the alcohol group at C6 to the COO- group. Here, we show that HAS2 expression can be modulated by sirtuin 1 (SIRT1), the master metabolic sensor of the cell, belonging to the class of NAD+-dependent deacetylases. Our results revealed the following. 1) Treatments of human aortic smooth muscle cells (AoSMCs) with SIRT1 activators (SRT1720 and resveratrol) inhibit both HAS2 expression and accumulation of pericellular HA coats. 2) Tumor necrosis factor α (TNFα) induced HA-mediated monocyte adhesion and AoSMC migration, whereas SIRT1 activation prevented immune cell recruitment and cell motility by reducing the expression levels of the receptor for HA-mediated motility, RHAMM, and the HA-binding protein TNF-stimulated gene 6 protein (TSG6). 3) SIRT1 activation prevented nuclear translocation of NF-κB (p65), which, in turn, reduced the levels of HAS2-AS1, a long-noncoding RNA that epigenetically controls HAS2 mRNA expression. In conclusion, we demonstrate that both HAS2 expression and HA accumulation by AoSMCs are down-regulated by the metabolic sensor SIRT1.

Revisiting the hallmarks of cancer: The role of hyaluronan.

Extracellular matrix (ECM) is a complex network of macromolecules such as proteoglycans (PGs), glycosaminoglycans (GAGs) and fibrous proteins present within all tissues and organs. The main role of ECM is not only to provide an essential mechanical scaffold for the cells but also to mediate crucial biochemical cues that are required for tissue homeostasis. Dysregulations in ECM deposition alter cell microenvironment, triggering the onset or the rapid progression of several diseases, including cancer. Hyaluronan (HA) is a ubiquitous component of ECM considered as one of the main players of cancer initiation and progression. This review discusses how HA participate in and regulate several aspects of tumorigenesis, with particular attention to the hallmarks of cancer proposed by Hanahan and Weinberg such as sustaining of the proliferative signaling, evasion of apoptosis, angiogenesis, activation of invasion and metastases, reprogramming of energy metabolism and evasion of immune response.