Deciphering Drug Resistance: Investigating the Emerging Role of Hyaluronan Metabolism and Signaling and Tumor Extracellular Matrix in Cancer Chemotherapy.

Hyaluronan (HA) has gained significant attention in cancer research for its role in modulating chemoresistance. This review aims to elucidate the mechanisms by which HA contributes to chemoresistance, focusing on its interactions within the tumor microenvironment. HA is abundantly present in the extracellular matrix (ECM) and binds to cell-surface receptors such as CD44 and RHAMM. These interactions activate various signaling pathways, including PI3K/Akt, MAPK, and NF-κB, which are implicated in cell survival, proliferation, and drug resistance. HA also influences the physical properties of the tumor stroma, enhancing its density and reducing drug penetration. Additionally, HA-mediated signaling contributes to the epithelial-mesenchymal transition (EMT), a process associated with increased metastatic potential and resistance to apoptosis. Emerging therapeutic strategies aim to counteract HA-induced chemoresistance by targeting HA synthesis, degradation, metabolism, or its binding to CD44. This review underscores the complexity of HA's role in chemoresistance and highlights the potential for HA-targeted therapies to improve the efficacy of conventional chemotherapeutics.

Proteoglycans: A Tool for Detecting Hyaluronan by ELISA-Like Methods.

Hyaluronan is a non-sulfated glycosaminoglycan synthesized on the plasma membrane of almost all mammalian cells, which can interact with different proteoglycans of the extracellular matrix. Aggrecan, versican, neurocan, and brevican are proteoglycans whose structures present a specific protein domain called "link module," which allows hyaluronan binding. Therefore, they can be helpful for assays that detect hyaluronan. For example, ELISA-like methods developed to measure hyaluronan amounts in solution are based on specific interactions between this molecule and the link module present in aggrecan or other hyaluronan-binding proteins (hyaladherins).

ß-Catenin is reduced in membranes of human prolactinoma cells and it is inhibited by temozolomide in prolactin secreting tumor models.

INTRODUCTION: Prolactinomas are the most frequent pituitary tumor subtype. Despite most of them respond to medical treatment, a proportion are resistant and become a challenge in clinical management. Wnt/ß-Catenin pathway has been implicated in several cancers including pituitary tumors and other sellar region malignancies. Interestingly, Wnt/ß-Catenin inhibition augments the cytotoxicity of the chemotherapeutic agent Temozolomide (TMZ) in different cancers. TMZ is now being implemented as rescue therapy for aggressive pituitary adenoma treatment. However, the molecular mechanisms associated with TMZ action in pituitary tumors remain unclear. OBJECTIVES: Our aims in the present study were to evaluate differential ß-Catenin expression in human resistant prolactinomas and Wnt/ß-Catenin signaling activation and involvement in Prolactin (PRL) secreting experimental models treated with TMZ. RESULTS: We first evaluated by immunohistochemistry ß-Catenin localization in human resistant prolactinomas in which we demonstrated reduced membrane ß-Catenin in prolactinoma cells compared to normal pituitaries, independently of the Ki-67 proliferation indexes. In turn, in vivo 15 mg/kg of orally administered TMZ markedly reduced PRL production and increased prolactinoma cell apoptosis in mice bearing xenografted prolactinomas. Intratumoral ß-Catenin strongly correlated with Prl and Cyclin D1, and importantly, TMZ downregulated both ß-Catenin and Cyclin D1, supporting their significance in prolactinoma growth and as candidates of therapeutic targets. When tested in vitro, TMZ directly reduced MMQ cell viability, increased apoptosis and produced G2/M cell cycle arrest. Remarkably, ß-Catenin activation and VEGF secretion were inhibited by TMZ in vitro. CONCLUSIONS: We concluded that dopamine resistant prolactinomas undergo a ß-Catenin relocalization in relation to normal pituitaries and that TMZ restrains experimental prolactinoma tumorigenicity by reducing PRL production and ß-Catenin activation. Together, our findings contribute to the understanding of Wnt/ß-Catenin implication in prolactinoma maintenance and TMZ therapy, opening the opportunity of new treatment strategies for aggressive and resistant pituitary tumors.

The effects of sulfated hyaluronan in breast, lung and colorectal carcinoma and monocytes/macrophages cells: Its role in angiogenesis and tumor progression.

Hyaluronan (HA) is a component of the extracellular matrix (ECM) it is the main non-sulfated glycosaminoglycan able to modulate cell behavior in the healthy and tumor context. Sulfated hyaluronan (sHA) is a biomaterial derived from chemical modifications of HA, since this molecule is not naturally sulfated. The HA sulfation modifies several properties of the native molecule, acquiring antitumor properties in different cancers. In this study, we evaluated the action of sHA of ~30-60 kDa with different degrees of sulfation (0.7 sHA1 and 2.5 sHA3) on tumor cells of a breast, lung, and colorectal cancer model and its action on other cells of the tumor microenvironment, such as endothelial and monocytes/macrophage cells. Our data showed that in breast and lung tumor cells, sHA3 is able to modulate cell viability, cytotoxicity, and proliferation, but no effects were observed on colorectal cancer cells. In 3D cultures of breast and lung cancer cells, sHA3 diminished the size of the tumorsphere and modulated total HA levels. In these tumor models, treatment of monocytes/macrophages with sHA3 showed a downregulation of the expression of angiogenic factors. We also observed a decrease in endothelial cell migration and modulation of the hyaluronan-binding protein TSG-6. In the breast in vivo xenograft model, monocytes/macrophages preincubated with sHA1 or sHA3 decreased tumor vasculature, TSG-6 and HA levels. Besides, in silico analysis showed an association of TSG-6, HAS2, and IL-8 with biological processes implicated in the progression of the tumor. Taken together, our data indicate that sHA in a breast and lung tumor context is able to induce an antiangiogenic action on tumor cells as well as in monocytes/macrophages (Mo/MØ) by modulation of endothelial migration, angiogenic factors, and vessel formation.

Antiproliferative and antiangiogenic effects of ammonium tetrathiomolybdate in a model of endometriosis.

AIMS: Copper (Cu) is involved in the endometriosis progression. Herein, an experimental endometriosis model was used to evaluate whether its chelation with ammonium tetrathiomolybdate (TM) affects the proliferation and angiogenesis in endometriotic-like lesions and the participation of oxidative stress in these processes. MAIN METHODS: Female C57BL/6 mice were divided into three groups: sham-operated mice, endometriosis-induced mice, and TM-treated endometriosis-induced mice. Each animal in the third group received 0.3 mg of TM/day in their drinking water from the postoperative 15th day. The samples were collected after one month of induced pathology. In peritoneal fluids, Cu and estradiol levels were determined by electrothermal atomic absorption spectrometry and electrochemiluminescence, respectively. Endometriotic-like lesions were processed for the analysis of cell proliferation by PCNA immunohistochemistry, the expression of angiogenic markers by RT-qPCR, the presence of endothelial cells by immunofluorescent staining, and oxidative stress applying spectrophotometric methods. KEY FINDINGS: TM treatment decreased Cu and estradiol levels, which were increased by this pathology. In lesions, TM induced: (a) a decrease in tissue weight and volume, (b) a decrease in PCNA-positive cells, (c) antiangiogenic effects by decreasing the number of blood vessels, the mRNA expression of fibroblast growth factor 2 (Fgf2) and platelet-derived growth factor subunit B (Pdgfb), and the presence of endothelial cells, (d) a decrease in antioxidant activity and an increase in lipid peroxidation. SIGNIFICANCE: TM is a highly effective antiproliferative and antiangiogenic agent, modulating oxidative imbalance in endometriosis. Its anti-endometriotic potential is an attractive feature of TM as a possible non-hormonal treatment.

Targeting the Tumor Extracellular Matrix by the Natural Molecule 4-Methylumbelliferone: A Complementary and Alternative Cancer Therapeutic Strategy.

In antineoplastic therapy, one of the challenges is to adjust the treatment to the needs of each patient and reduce the toxicity caused by conventional antitumor strategies. It has been demonstrated that natural products with antitumoral properties are less toxic than chemotherapy and radiotherapy. Also, using already developed drugs allows developing substantially less costly methods for the discovery of new treatments than traditional drug development. Candidate molecules proposed for drug repositioning include 4-methylumbelliferone (4-MU), an orally available dietetic product, derivative of coumarin and mainly found in the plant family Umbelliferae or Apiaceae. 4-MU specifically inhibits the synthesis of glycosaminoglycan hyaluronan (HA), which is its main mechanism of action. This agent reduces the availability of HA substrates and inhibits the activity of different HA synthases. However, an effect independent of HA synthesis has also been observed. 4-MU acts as an inhibitor of tumor growth in different types of cancer. Particularly, 4-MU acts on the proliferation, migration and invasion abilities of tumor cells and inhibits the progression of cancer stem cells and the development of drug resistance. In addition, the effect of 4-MU impacts not only on tumor cells, but also on other components of the tumor microenvironment. Specifically, 4-MU can potentially act on immune, fibroblast and endothelial cells, and pro-tumor processes such as angiogenesis. Most of these effects are consistent with the altered functions of HA during tumor progression and can be interrupted by the action of 4-MU. While the potential advantage of 4-MU as an adjunct in cancer therapy could improve therapeutic efficacy and reduce toxicities of other antitumoral agents, the greatest challenge is the lack of scientific evidence to support its approval. Therefore, crucial human clinical studies have yet to be done to respond to this need. Here, we discuss and review the possible applications of 4-MU as an adjunct in conventional antineoplastic therapies, to achieve greater therapeutic success. We also describe the main proposed mechanisms of action that promote an increase in the efficacy of conventional antineoplastic strategies in different types of cancer and prospects that promote 4-MU repositioning and application in cancer therapy.

Syndecan-1 Depletion Has a Differential Impact on Hyaluronic Acid Metabolism and Tumor Cell Behavior in Luminal and Triple-Negative Breast Cancer Cells.

Glycosaminoglycans (GAGs) and proteoglycans (PGs) are major components of the glycocalyx. The secreted GAG and CD44 ligand hyaluronic acid (HA), and the cell surface PG syndecan-1 (Sdc-1) modulate the expression and activity of cytokines, chemokines, growth factors, and adhesion molecules, acting as critical regulators of tumor cell behavior. Here, we studied the effect of Sdc-1 siRNA depletion and HA treatment on hallmark processes of cancer in breast cancer cell lines of different levels of aggressiveness. We analyzed HA synthesis, and parameters relevant to tumor progression, including the stem cell phenotype, Wnt signaling constituents, cell cycle progression and apoptosis, and angiogenic markers in luminal MCF-7 and triple-negative MDA-MB-231 cells. Sdc-1 knockdown enhanced HAS-2 synthesis and HA binding in MCF-7, but not in MDA-MB-231 cells. Sdc-1-depleted MDA-MB-231 cells showed a reduced CD24-/CD44+ population. Furthermore, Sdc-1 depletion was associated with survival signals in both cell lines, affecting cell cycle progression and apoptosis evasion. These changes were linked to the altered expression of KLF4, MSI2, and miR-10b and differential changes in Erk, Akt, and PTEN signaling. We conclude that Sdc-1 knockdown differentially affects HA metabolism in luminal and triple-negative breast cancer model cell lines and impacts the stem phenotype, cell survival, and angiogenic factors.

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.

Hyaluronan Metabolism is Associated with DNA Repair Genes in Breast and Colorectal Cancer. Screening of Potential Progression Markers Using qPCR.

In this work, we compared mRNA levels of Hyaluronan (HA) metabolism members and BRCA genes, known to be involved in the tumoral process, between tumor and non-tumor adjacent tissue and its correlation with previously proposed biomarkers (ER, PR, HER2 and KI67) in order to assess their value as a progression biomarkers. We show alteration in HA metabolism in colorectal but not breast cancer. However, we found a decrease in Hyaluronidase 1 HYAL1 levels in the breast but not colorectal cancer. We also show lower HA levels in tumor compared with normal tissue that could indicate a possible influence of tumor on its surrounding "normal" tissue. In both breast and colorectal cancer, CD44 and BRCA2 showed a strong positive correlation. Besides, our results show first indicators that qPCR of the analyzed genes could be used as an easy and low cost procedure for the evaluation of molecular markers we propose here.

Hyaluronan in the Tumor Microenvironment.

The extracellular matrix is part of the microenvironment and its functions are associated with the physical and chemical properties of the tissue. Among the extracellular components, the glycosaminoglycan hyaluronan is a key component, defining both the physical and biochemical characteristics of the healthy matrices. The hyaluronan metabolism is strictly regulated in physiological conditions, but in the tumoral tissues, its expression, size and binding proteins interaction are dysregulated. Hyaluronan from the tumor microenvironment promotes tumor cell proliferation, invasion, immune evasion, stemness alterations as well as drug resistance. This chapter describes data regarding novel concepts of hyaluronan functions in the tumor. Additionally, we discuss potential clinical applications of targeting HA metabolism in cancer therapy.

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.

Proteoglycans and glycosaminoglycans as regulators of cancer stem cell function and therapeutic resistance.

In contrast to the bulk of the tumor, a subset of cancer cells called cancer stem cells (CSC; or tumor-initiating cells) is characterized by self-renewal, unlimited proliferative potential, expression of multidrug resistance proteins, active DNA repair capacity, apoptosis resistance, and a considerable developmental plasticity. Due to these properties, CSCs display increased resistance to chemo- and radiotherapy. Recent findings indicate that aberrant functions of proteoglycans (PGs) and glycosaminoglycans (GAGs) contribute substantially to the CSC phenotype and therapeutic resistance. In this review, we summarize how the diverse functions of the glycoproteins and carbohydrates facilitate acquisition and maintenance of the CSC phenotype, and how this knowledge can be exploited to develop novel anticancer therapies. For example, the large transmembrane chondroitin sulfate PG NG2/CSPG4 marks stem cell (SC) populations in brain tumors. Cell surface heparan sulfate PGs of the syndecan and glypican families modulate the stemness-associated Wnt, hedgehog, and notch signaling pathways, whereas the interplay of hyaluronan in the SC niche with CSC CD44 determines the maintenance of stemness and promotes therapeutic resistance. A better understanding of the molecular mechanisms by which PGs and GAGs regulate CSC function will aid the development of targeted therapeutic approaches which could avoid relapse after an otherwise successful conventional therapy. Chimeric antigen receptor T cells, PG-primed dendritic cells, PG-targeted antibody-drug conjugates, and inhibitory peptides and glycans have already shown highly promising results in preclinical models.

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 (Mo/MØ) in tumor angiogenesis and its consequences on tumor development. In the present study, we investigated the effects of hyaluronan of different sizes on human Mo/MØ angiogenic behavior in colorectal and breast carcinoma. In vitro, the treatment of Mo/MØ with lysates and conditioned media from a breast but not from colorectal carcinoma cell line plus high-molecular weight hyaluronan induced: (a) an increased expression of angiogenic factors VEGF, IL-8, FGF-2, and MMP-2, (b) an increased endothelial cell migration, and (c) a differential expression of hyaluronan-binding protein TSG-6. Similar results were observed in Mo/MØ 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 Mo/MØ 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.

Determination of Cell-Surface Hyaluronan Through Flow Cytometry.

Hyaluronan is the major glycosaminoglycan present in the extracellular matrix of several cell types; its synthesis occurs on the cellular plasma membrane. Variations in its expression are related to alterations in cell proliferation, adhesion, and migration. It is able to interact with different binding proteins called hyaladherins, which can be conjugated to different fluorochromes and analyzed by flow cytometry.

Co-treatment of tumor cells with hyaluronan plus doxorubicin affects endothelial cell behavior independently of VEGF expression.

Hyaluronan, the main glycosaminoglycan of extracellular matrices, is concentrated in tissues with high cell proliferation and migration rates. In cancer, hyaluronan expression is altered and it becomes fragmented into low-molecular-weight forms, affecting mechanisms associated with cell proliferation, invasion, angiogenesis and multidrug resistance. Here, we analyzed the effect of low-molecular-weight hyaluronan on the response of T lymphoma, osteosarcoma, and mammary adenocarcinoma cell lines to the antineoplastic drug doxorubicin, and whether co-treatment with hyaluronan and doxorubicin modified the behavior of endothelial cells. Our aim was to associate the hyaluronan-doxorubicin response with angiogenic alterations in these tumors. After hyaluronan and doxorubicin co-treatment, hyaluronan altered drug accumulation and modulated the expression of ATP-binding cassette transporters in T-cell lymphoma cells. In contrast, no changes in drug accumulation were observed in cells from solid tumors, indicating that hyaluronan might not affect drug efflux. However, when we evaluated the effect on angiogenic mechanisms, the supernatant from tumor cells treated with doxorubicin exhibited a pro-angiogenic effect on endothelial cells. Hyaluronan-doxorubicin co-treatment increased migration and vessel formation in endothelial cells. This effect was independent of vascular endothelial growth factor but related to fibroblast growth factor-2 expression. Besides, we observed a pro-angiogenic effect on endothelial cells during hyaluronan and doxorubicin co-treatment in the in vivo murine model of T-cell lymphoma. Our results demonstrate for the first time that hyaluronan is a potential modulator of doxorubicin response by mechanisms that involve not only drug efflux but also angiogenic processes, providing an adverse tumor stroma during chemotherapy.

The immunological effect of hyaluronan in tumor angiogenesis.

The relationship between the immune system and angiogenesis has been described in several contexts, both in physiological and pathological conditions, as pregnancy and cancer. In fact, different types of immune cells, such as myeloid, macrophages and denditric cells, are able to modulate tumor neovascularization. On the other hand, tumor microenvironment also includes extracellular matrix components like hyaluronan, which has a deregulated synthesis in different tumors. Hyaluronan is a glycosaminoglycan, normally present in the extracellular matrix of tissues in continuous remodeling (embryogenesis or wound healing processes) and acts as an important modulator of cell behavior by different mechanisms, including angiogenesis. In this review, we discuss hyaluronan as a modulator of tumor angiogenesis, focusing in intracellular signaling mediated by its receptors expressed on different immune cells. Recent observations suggest that the immune system is an important component in tumoural angiogenesis. Therefore, immune modulation could have an impact in anti-angiogenic therapy as a new therapeutic strategy, which in turn might improve effectiveness of treatment in cancer patients.