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Department of Biomedical Engineering, Texas A&M University, 2121 W Holcombe St., Houston, TX, 77030, USADepartment of Biomedical Engineering, Texas A&M University, 101 Bizzell St., College Station, TX, 77843, USAArtie McFerrin Department of Chemical Engineering, Texas A&M University, 3122 TAMU, College Station, TX, 77843, USADepartment of Translational Medical Sciences, Texas A&M University, 2121 W Holcombe St., Houston, TX, 77030, USA
Although cancer is a genetic disease, physical changes such as stiffening of the extracellular matrix (ECM) also commonly occur in cancer. Cancer cells sense and respond to ECM stiffening through the process of mechanotransduction. Cancer cell mechanotransduction can enhance cancer-promoting cell behaviors such as survival signaling, proliferation, and migration. Glycans, carbohydrate-based polymers, have recently emerged as important mediators and/or modulators of cancer cell mechanotransduction. Stiffer tumors are characterized by increased glycan content on cancer cells and their associated extracellular matrix. Here we review the role of cancer- associated glycans in coupled mechanical and biochemical alterations during cancer progression. We discuss the recent evidence on how increased expression of different glycans, in the form of glycoproteins and proteoglycans, contributes to both mechanical changes in tumors and corresponding cancer cell responses. We conclude with a summary of emerging tools that can be used to modify glycans for future studies in cancer mechanobiology.
A tumor is a growing population of aberrant somatic cells. Over the course of tumorigenesis, the properties of the tumor microenvironment (TME) change dramatically. The progression of many solid cancers involves the recruitment of several different cell types to the TME, such as endothelial cells that form ‘leaky’ blood vessels (
), cancer-associated fibroblasts (CAFs) and mesenchymal stromal cells that secrete extracellular matrix (ECM) proteins such as collagen and glycans such as hyaluronan (
). These changes alter not only the biochemical composition of the TME, but also the mechanical stiffness of the ECM. Increased collagen secretion results in the assembly of higher concentration of collagen fibrils which result in a mechanically stiff ECM. In addition, non-enzymatic glycation of collagen and resulting accumulation of advanced glycation end products (AGEs) can increase stiffness of collagen independently of its concentration, by increasing the crosslinks between collagen fibrils (
). Such mechanical changes are common across different types of solid tumors, and are responsible for the presentation of solid tumors as stiff lumps within the tissue (
). Stiffening of the ECM, whether due to increased matrix deposition or due to increased matrix crosslinking, can enhance cancer cell growth, disrupt tissue structure and polarity and promote cell migration (
Cells can sense mechanical cues from their microenvironment, a process termed mechano-sensing, and adapt to these cues through changes in signaling pathways and gene expression, a process termed mechanotransduction (
). The simplest and robust canonical response of cells to ECM stiffening is increased cell spreading. Cell spreading is mediated by transmembrane integrin receptors, which preferentially cluster on stiff ECM compared to soft ECM, to form focal adhesions. Integrin clustering and focal adhesion formation is reciprocally coupled with the activation of signaling pathways including the Rho/Rho kinase (ROCK) pathway, PI3 kinase signaling, and YAP signaling (
) that in turn impact cell migration, traction force generation and cell proliferation.
Glycans have recently emerged as important mediators and/or modulators of mechano-transduction. The term ‘glycan’ refers to carbohydrate-based polymers that either exist as free molecules such as hyaluronan, or covalently bound to lipids or proteins. Protein-bound glycans fall into two classes, glycoproteins, and proteoglycans (
Glycocalyx regulates the strength and kinetics of cancer cell adhesion revealed by biophysical models based on high resolution label-free optical data.
). Glycoproteins are proteins that carry one or more branched glycan chains bound to either asparagine residues (via nitrogen linkages to form N-linked glycans) or serine/threonine residues (via oxygen linkages to form O-linked glycans) of the polypeptide (Fig. 1). The N- and O-linked glycans on glycoproteins can become further modified by terminal glycan motifs containing fucose (Lewis antigens) or sialic acids (
Differential expression of alpha-2,3-sialyltransferases and alpha-1,3/4-fucosyltransferases regulates the levels of sialyl Lewis a and sialyl Lewis x in gastrointestinal carcinoma cells.
) respectively. Proteoglycans consist of a core protein covalently bound to one or more glycan chains called glycosaminoglycans (such as chondroitin sulfate or heparan sulfate chains). Glycolipids consist of glycan chains bound to a lipid ceramide (glycosphingolipid), where the first sugar bound to the ceramide can be either glucose (glucosylceramide) or galactose (galactosylceramide) (Fig 1). Most of the glycoproteins/proteoglycans or glycolipids are either bound to the cell membrane or shed/secreted by the cells to become a part of the extracellular matrix (
Figure 1Schematic representation of different glycans involved in cancer progression. Glycans exist as either free forms or are attached to various proteins or lipids on cell surface. Glycosphingolipids consist of the lipid ceramide linked to a variable series of sugars that can be further modified with terminal sialic acids. Glycoproteins carry one or more branched glycan chains attached to either asparagine residues (via nitrogen linkages to form N-linked glycans) or serine/threonine residues (via oxygen linkages to form O-linked glycans) of the polypeptide. N-glycans share a common pentasaccharide core which is extended by high mannose, or both mannose and GlcNAc residues (hybrid) or complex types containing branched structures containing two or more antennae. O-Glycans are initiated by GalNAc with elongated GlcNAc containing glycans. Both N- and O-glycans are further modified by terminal sialic acid residues. Some glycoproteins called the glycosylphosphatidylinositol (GPI)-anchored proteins are anchored in the outer leaflet of the plasma membrane by a glycan linked to phosphatidylinositol. Glycosaminoglycans are linear polysaccharides consisting of repeating disaccharide units that exists as either free sugar chains (such as hyaluronan) or mostly found attached to proteoglycans (such as heparan sulfate or chondroitin sulfate chains). Sugars are represented by colored geometric symbols.
). This review focuses on the role of glycans in mediating cellular sensing of and response to changes in the mechanical stiffness of the tumor ECM, as well as in modulating the mechanical properties of the tumor ECM.
Mechanobiological pathways in cancer
Mechanotransduction pathways are complex, involving a range of molecular players and mechanisms of action. Cells adhere to the ECM through transmembrane integrin receptors whose extracellular domains bind to the ECM (
). The traction allows cells to maintain adhesion to the ECM as well as to move. Importantly, these integrin-mediated sites of adhesion are also key protein hubs that transmit externally applied mechanical force across the cell membrane (
For a cell that is adherent to the ECM, a mechanical change, such as a mechanical stretch of the ECM, causes transmission of force through the integrin receptors, through talin and other linker proteins, to the cytoskeleton. The force can unfold talin and other proteins in the adhesion (
), exposing cryptic binding sites for F-actin and structural proteins like vinculin, resulting in a higher local concentration of proteins localized to the adhesion including enzymes like focal adhesion kinase (FAK), and increased numbers of bonds with the actomyosin cytoskeleton. This remodeling response results in stronger adhesion, which is a classical adaptive response of cells to external mechanical force. The adaptive response, which can be triggered cell-wide (
). Force applied to integrin receptors activate the small GTPase Rho signaling pathway, further stimulating actomyosin force generation, by activating its effector mDia1 which promotes actin filament polymerization (
Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism.
The ECM-integrin-cytoskeleton linkage is key to sensing and transduction of changes in the mechanical stiffness of the ECM into intracellular responses (
), and a lack of translocation of Yes-associated protein(YAP)/ transcriptional co-activator with PDZ-binding motif (TAZ), transcriptional regulators of the hippo signaling pathway, to the nucleus (
). YAP/ TAZ get phosphorylated upon activation of the hippo pathway, and are retained in the cytoplasm, while inactivation of the hippo dephosphorylates YAP/TAZ and causes their nuclear translocation where they induce gene expression (
). Interestingly, the sensitivity of YAP/TAZ translocation to ECM stiffness, is independent of the hippo pathway. In fact, diverse mechanical stimuli impact YAP/TAZ translocation to the nucleus (
), highlighting its importance as a mechanotransduction pathway.
Altered glycan expression and cancer progression
The concentrations as well as the compositions of the different types of glycans are significantly altered in cancer (Fig. 1) and are specific to the type of cancer. Altered glycan composition and concentration impacts cancer cell adhesion, signaling, migration/invasion, tumor angiogenesis, drug resistance, epithelial to mesenchymal transition (EMT) and cancer metastasis (see reviews on this topic (
Correction: Perlecan/HSPG2 and matrilysin/MMP-7 as indices of tissue invasion: tissue localization and circulating perlecan fragments in a cohort of 288 radical prostatectomy patients.
Agrin promotes the proliferation, invasion and migration of rectal cancer cells via the WNT signaling pathway to contribute to rectal cancer progression.
The increased glycation of proteins and lipids in cancer is broadly due to increased expression of specific glycosyltransferases, which are enzymes responsible for the initiation and elongation of glycan chains. An additional mechanism that may contribute to increased glycation is synthesis of UDP-GlcNAc by the nutrient-sensing hexosamine biosynthetic pathway (HBP) (
), and the end product UDP-GlcNAc is the key substrate that is used for synthesis of glycans, including O-GlcNAcylation which is a single sugar conjugation (
). O-GlcNAcylation is a dynamic and reversible post-translational glycosylation that involves the addition of an N-acetylglucosamine (GlcNAc) from the precursor UDP-GlcNAc to the serine or threonine residues of a variety of cytoplasmic or nuclear proteins. A wide range of processes such as enzyme activity, protein stability, activation of survival signals and chemoresistance in cancer are regulated by O-GlcNAcylation (
). Below we discuss the role of different classes of glycans in modulating tumor cell response to mechanical changes in the ECM. We focus on the role of proteoglycans because most studies of the role of glycans in cancer mechanobiology have been performed in the context of proteoglycans.
Table 1Glycans regulated mechanical properties and cancer types
Glycan type
Mechanical properties
References
Cancer type
Sialylation
Increases integrin tension, enhance maturation of focal adhesions, and spreading and migration of cancer cells
Cytoplasmic domain binds to PDZ domain containing proteins such as syntenin-1 and recruits signaling and cytoskeletal proteins to the plasma membrane, promotes cytoskeletal rearrangements, cell-ECM interactions, migration, and metastasis.
Promotes formation of signaling complexes in cooperation with αVβ3, αVβ5, and α4β1 integrins, promotes ECM assembly and cancer cell spreading and migration, mediates flow stress induced changes in cell shape
Heparanase-induced shedding of syndecan-1/CD138 in myeloma and endothelial cells activates VEGFR2 and an invasive phenotype: prevention by novel synstatins.
Syndecan-1 and Syndecan-4 Capture Epidermal Growth Factor Receptor Family Members and the alpha3beta1 Integrin Via Binding Sites in Their Ectodomains: NOVEL SYNSTATINS PREVENT KINASE CAPTURE AND INHIBIT alpha6beta4-INTEGRIN-DEPENDENT EPITHELIAL CELL MOTILITY.
Promotes the formation of a denser collagen architecture, increase tissue stiffness, upregulates β1 integrin expression, promotes cancer cell invasion
). Agrin, which is expressed by several tissues, is known to cluster acetylcholine receptors in the neuromuscular junctions. It binds to lipoprotein-related receptor-4 (Lrp4) and mediates muscle-specific kinase (MuSK) signaling in neuromuscular junctions. A few studies have implicated a role for agrin in cancer. Agrin expression is elevated in oral squamous cell carcinoma and promotes tumor aggressiveness (
). This suggests that agrin may promote actomyosin force dependent assembly of cell-substrate adhesions, which is a crucial component of the cellular response mechanisms to mechanical cues from the ECM (
). Indeed, agrin levels are higher in cells cultured on stiff 2D collagen coated polyacrylamide hydrogels than in cells cultured on soft 2D polyacrylamide hydrogels (
). Knockdown of agrin prevents the nuclear translocation of YAP on stiff ECM, while addition of recombinant agrin causes the translocation of YAP to the nucleus even on soft ECM (
). Overall, the HSPG agrin is required for cancer cell response to mechanical stiffness of the ECM in hepatocellular carcinoma cells.
Syndecans
Syndecans are a four-member family of transmembrane proteoglycans that predominantly carry heparan sulfate GAG chains. Almost all cells, except for erythrocytes, express at least one member of the syndecan family (
). A marked alteration of syndecan expression occurs in cancer with syndecans acting either as a tumor suppressor or promoter depending upon the cancer types. Loss of syndecan-1 expression in most epithelial tumors such as cervical, lung, head and neck, squamous cell and esophageal cancers is associated with tumor progression and reduced patient survival (
), suggestive of a tumor suppressive role for syndecan-1. In contrast, increased syndecan-1 expression in breast, pancreatic, ovarian, thyroid, and endometrial tumors is associated with tumor progression and poor prognosis (
There is evidence that syndecan-4, a member of the four-member family of transmembrane HSPGs, can act as a transmitter of mechanical force in fibroblasts and pancreatic stellate cells (
). When pulsatile forces were applied to magnetic beads coated with a fragment of fibronectin that binds heparan on syndecan-4, or to beads coated with antibodies toward the core protein of syndecan-4, there was a reduction in bead displacement upon sustained force application, suggesting a mechanical stiffening response. This is similar to the stiffening response upon force application to integrins. Application of force to syndecan-4 altered the conformation of its cytoplasmic domain, promoting the binding of α-actinin, a scaffold protein that localizes to cell-ECM adhesions and also binds to F-actin (
). This linkage is likely responsible for the local mechanical stiffening response. Importantly, force application to syndecan-4 triggered the diffusion of PIP3 lipid second messengers, which in turn activated β1 integrins cell-wide by binding to kindlin-2 and promoting RhoA mediated actomyosin contractility (
). The promotion of adhesion assembly by mechanical activation of syndecan-4 is consistent with another report that syndecan-4 is required for assembly of focal adhesions and stress fibers in fibroblasts (
). Additionally, the extracellular core protein domains of syndecans 1 and 4 can bind to different integrin receptors - αvβ5, αvβ3, α3β1 and α4β1- forming diverse combinations (
Heparanase-induced shedding of syndecan-1/CD138 in myeloma and endothelial cells activates VEGFR2 and an invasive phenotype: prevention by novel synstatins.
Syndecan-1 and Syndecan-4 Capture Epidermal Growth Factor Receptor Family Members and the alpha3beta1 Integrin Via Binding Sites in Their Ectodomains: NOVEL SYNSTATINS PREVENT KINASE CAPTURE AND INHIBIT alpha6beta4-INTEGRIN-DEPENDENT EPITHELIAL CELL MOTILITY.
) that could facilitate mechanosensitive formation of focal adhesion complexes and downstream activation of signaling pathways. Thus, the syndecan-4 mediated mechanotransduction pathway is likely to be broadly important in cancer, as the expression of syndecan-4 is high in cancers such as glioblastoma (
). The GAG chain of serglycin can be either heparin or chondroitin sulfate depending on the cell type in which serglycin is expressed. Though serglycin is considered as an intracellular proteoglycan, recent studies have shown that serglycin can be secreted by cancer cells and bind to cell surface receptors (
). Initial studies reported increased levels of serglycin only in hematological malignancies like multiple myeloma and leukemia; however more recent findings suggest that serglycin is overexpressed in glioma and tumors of the breast, prostate, lung, and liver (
). Though the extent to which serglycin contributes to mechanotransduction remains understudied, a recent study reported that upregulation of serglycin expression in chemoresistant breast cancer cells activates focal adhesion kinase (FAK) signaling (
). Serglycin upregulates YAP expression in breast cancer cells by activating integrin α5/FAK/CREB signaling. YAP in turn upregulate HDAC2 expression via the transcription factor RUNX1 to maintain stemness and chemoresistance in these cells (
). Additionally, YAP positively regulate serglycin expression to form a feed-forward circuit in breast cancer cells. These findings highlight that the proteoglycan serglycin can mediate cancer cell adaptation to the changing mechanical environment through the FAK/YAP signaling axis; a possibility that deserves further exploration.
Glycoproteins
Mucins
Mucins are a family of highly glycosylated transmembrane glycoproteins produced by various epithelial cells and are categorized into membrane associated mucins, gel-forming mucins, and soluble mucins. The striking feature of cell surface mucins is their long, densely glycosylated ectodomain which can extend hundreds of nanometers from the plasma membrane. Mucins form a gel like mucus on the surface of the cells and impact integrin clustering, force sensing and signaling (
Localization of O-glycosylation sites on glycopeptide fragments from lactation-associated MUC1. All putative sites within the tandem repeat are glycosylation targets in vivo.
), and high amount of O-glycosylation on the central region of mucins makes them resistant to degradation during cancer progression. Cell surface MUC1 and MUC16 are consistently upregulated in epithelia cancers and are considered as biomarkers of the disease (
). Trimming mucins in glioblastoma cells, for example, represses integrin signaling, while increasing their size promotes tension-dependent integrin signaling (
). The dense glycan chains of mucins also promote cancer cell metastasis by enhancing integrin-FAK mechanosignaling, and cell cycle progression by the PI3K-Akt axis (
While glycans clearly impact mechanotransduction, the function of particular sugar modifications like sialylation of proteins remains understudied. Sialylation is a form of glycosylation that involves the addition of sialic acid to the terminal end of N- and O-linked glycan chains by sialyltransferase enzymes. Aberrant sialylation is a driver of malignant phenotype and regulates cell-cell and cell-matrix interactions, proliferation, invasion, angiogenesis, resistance to apoptosis and immune suppression (
). Using DNA-based tension probes and high resolution total internal reflection fluorescence (TIRF) microscopy, the study showed that high sialyltransferase activity increases forces on the ECM, promotes maturation of focal adhesions, and eventual spreading and migration of ovarian cancer cells (
). These outcomes were driven by membrane retention and activity of EGFR via sialylation and downstream activation of the ERK and PI3K-AKT signaling cascades. As sialylation is increased in many cancers (
), these findings highlight the need for further exploration of specific sugar modifications on proteins in the context of cancer cell mechanotransduction.
Glycans and tumor ECM structure and mechanics
There is a general over-expression of proteoglycans on tumor cells and in the ECM in cancer (
). Proteoglycans in the ECM can bind to a wide range of matrix proteins such as collagen, fibronectin, laminin, resulting in a more cross-linked matrix, and hence a stiffer matrix. For example, in breast cancer, even before tumor development there is an increased deposition of collagen I and proteoglycans in the ECM leading to increased breast density, which is a risk factor for breast cancer (
) and this in turn creates a tumor supporting matrix that contributes to the pathology of the tumor.
Accumulation of advanced glycation end products (AGEs) in tissues has recently gained attention because of their significant role in inflammation and tumor development (
). AGEs are formed when the carbonyl groups of endogenous reducing sugars (such as glucose-6-phosphate or ribose) non-enzymatically react with the free amino groups of proteins (
A quick, simple method for detecting circulating fluorescent advanced glycation end-products: Correlation with in vitro and in vivo non-enzymatic glycation.
). Stiffening of collagen gels by glycation, but not by increased density, enhances the angiogenic outgrowth and branching of endothelial cell spheroids (
); suggesting a potential role for collagen glycation in tumor angiogenesis. Furthermore a recent study reported that glucose can also promote glycation of collagen, AGE accumulation and stiffening of collagen without any change in collagen fiber density or architecture (
). Expectedly, glucose induced collagen stiffening promotes cancer cell contractility elongation and migration. The use of an AGE breaker alagebrium chloride (ALT711) reduces AGE mediated matrix stiffening and cancer cell migration, without altering collagen pore size, chemical composition, or architecture (
). These findings demonstrate that AGE formation may be one mechanism by which diabetes promotes cancer and disruptions of AGEs using AGE-breaker drugs can be therapeutically beneficial.
In addition, heparan sulfate PGs such as syndecan-1 can promote the assembly of parallel fibronectin and collagen-1 fibers facilitating the directional migration of cancer cells (
). The binding of the heparan sulfate chains of syndecan-1 to the heparin-II domain may facilitate the unfolding of dimeric fibronectin and expose fibronectin self-assembly sites promoting fibrillogenesis. Furthermore, small leucine rich proteoglycans (SLRPs) biglycan and decorin can also modulate collagen fibril structure, fiber realignment and matrix assembly (
Phenotypic effects of biglycan deficiency are linked to collagen fibril abnormalities, are synergized by decorin deficiency, and mimic Ehlers-Danlos-like changes in bone and other connective tissues.
) , although its role in cancer development is less clear.
The GAG hyaluronan (HA) is a non-sulfated GAG chain with no core protein and therefore is not considered a proteoglycan. HA is overexpressed in most cancers and accumulation of high levels of HA in the ECM triggers cancer progression and is associated with poor clinical outcome (
). Elevated ECM stiffness in patients with glioma is associated with a substantial increase in the levels of HA and the HA binding glycoprotein tenascin C, but not collagen (
). HA accumulation can contribute to growth induced solid mechanical stress and increase interstitial pressure through a retention of water within the tumor tissue due to the high negative charge on HA (
). Interestingly, soft HA substrates by themselves can elicit phenotypes from glioma cells like those when glioma cells are cultured on stiff ECM protein coated substrates (
), suggesting that HA may trigger mechanotransduction pathways independently of ECM stiffness. Hyaluronan can thus contribute in diverse ways to the mechanical changes in tumors.
In addition to their role in sensing extracellular mechanical cues and translating them to biochemical changes, cell surface glycans can induce membrane curvature and hence control plasma membrane architecture (
). Glycosaminoglycans and glycoproteins can generate crowding pressure strong enough to induce plasma membrane curvature, bending membranes into different shapes. This may contribute to the formation of functional cell surface structures such as microvilli, filopodia, lamellipodia, that are important for invasive migration, drug resistance, signaling, and secretion of extracellular vesicles (
Identification of PA2.26 antigen as a novel cell-surface mucin-type glycoprotein that induces plasma membrane extensions and increased motility in keratinocytes.
). More studies are needed to understand the extent to which glycan-induced membrane changes/instability contribute to cancer cell functions like invasive migration.
Tools for targeting glycans
Our understanding of the function of glycans in cancer progression, and particularly, in mechanobiology of cancer is steadily growing, but many more studies are needed if we are to ultimately develop effective therapeutic strategies directed against glycan-mediated cancer mechano-adaptation in cancer. Effective tools to inhibit glycosylation are still lacking with the major challenge being limited specificity. Here we discuss the variety of tools that have emerged to alter glycan chains for cell biology studies. Towards the end we discuss glycan targeting drugs used in clinical trials.
Tools to target glycans for in vitro applications
The small molecules that inhibit glycosylation prevent the formation of glycosylation precursors, inhibit the activity of glycan processing enzymes, or act as primers and decoys (
Esko, J. D., Bertozzi, C., and Schnaar, R. L. (2015) Chemical Tools for Inhibiting Glycosylation. in Essentials of Glycobiology (rd, Varki, A., Cummings, R. D., Esko, J. D., Stanley, P., Hart, G. W., Aebi, M., Darvill, A. G., Kinoshita, T., Packer, N. H., Prestegard, J. H., Schnaar, R. L., and Seeberger, P. H. eds.), Cold Spring Harbor (NY). pp 701-712
Esko, J. D., and Bertozzi, C. R. (2009) Chemical Tools for Inhibiting Glycosylation. in Essentials of Glycobiology (nd, Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., and Etzler, M. E. eds.), Cold Spring Harbor (NY). pp
Vocadlo, D. J., Lowary, T. L., Bertozzi, C. R., Schnaar, R. L., and Esko, J. D. (2022) Chemical Tools for Inhibiting Glycosylation. in Essentials of Glycobiology (th, Varki, A., Cummings, R. D., Esko, J. D., Stanley, P., Hart, G. W., Aebi, M., Mohnen, D., Kinoshita, T., Packer, N. H., Prestegard, J. H., Schnaar, R. L., and Seeberger, P. H. eds.), Cold Spring Harbor (NY). pp 739-752
). Small synthetic glycan mimetics can compete for glycan binding sites and inhibit their binding and signaling. A glutamine analog, 6-diazo-5-oxo-L-norleucine (DON) is an example of a small molecule inhibitor used to inhibit the formation of glucosamine. All of the major glycan families require N-acetylglucosamine or N-acetylgalactosamine for their synthesis, and inhibiting glucosamine synthesis alters glycan assembly. For example, tunicamycin inhibits glycosylation of glycoproteins entirely by inhibiting N-glycosylation. Tunicamycin blocks the transfer of GlcNAc-1-phosphoate to dolichol phosphate, by inhibiting GlcNAc phosphotransferase, during the first steps of N-glycan synthesis (
Vocadlo, D. J., Lowary, T. L., Bertozzi, C. R., Schnaar, R. L., and Esko, J. D. (2022) Chemical Tools for Inhibiting Glycosylation. in Essentials of Glycobiology (th, Varki, A., Cummings, R. D., Esko, J. D., Stanley, P., Hart, G. W., Aebi, M., Mohnen, D., Kinoshita, T., Packer, N. H., Prestegard, J. H., Schnaar, R. L., and Seeberger, P. H. eds.), Cold Spring Harbor (NY). pp 739-752
). The basic premise is that these sugar analogs can inhibit glycosyltransferase enzymes by serving as donor and substrate analogs. Sugar analogs that have been developed include analogs of sialic acid such as fluorinated sialic acid mimetic (
). Xylosides mimic the xylosylated serine residues on the core protein on PGs, where the priming of GAG chains occurs. Xylosides therefore divert the assembly of GAG chains from the core protein of PGs and thereby inhibit PG formation (
beta-D-xylosides and their analogues as artificial initiators of glycosaminoglycan chain synthesis. Aglycone-related variation in their effectiveness in vitro and in ovo.
). Strategies to terminate the elongation of glycan chains have also been employed. For example, compounds such as mannosamine prevent the elongation of glycan chains on the GPI anchor and thereby block the incorporation of GPI glycans to GPI anchored proteins (
). This method can be used to target a specific glycan by silencing specific glycotransferases or the entire glycan by silencing the chain initiating enzyme. For example, silencing specific enzymes such as sialyltransferase or fucosyltransferase inhibits the formation of sialofucosylated glycans on leukocytes and thereby blocks it binding to selectins (
). In addition to genome editing, tool kits for membrane incorporation of fully synthetic polymers that mimic key features of glycoproteins have been developed for precision editing of sugar chains (
). The approach is to engineer the structure and composition of the cellular glycans using genetically encoded glycoproteins and expression systems. This toolkit has been used to manipulate the shape and functions of the glycans and (
). Likewise, a tool kit based on CRISPR-Cas9 has been developed to prune specific glycan types, such as N-linked and O-linked glycans of glycoproteins and glycolipids (
Receptor for advanced glycation end products (RAGE) functions as receptor for specific sulfated glycosaminoglycans, and anti-RAGE antibody or sulfated glycosaminoglycans delivered in vivo inhibit pulmonary metastasis of tumor cells.
Glycan targeting drugs that progressed to clinical evaluation
Many highly sulfated and structurally defined heparan sulfate GAG analogs, that in part mimic the natural ligand, have been developed and tested in cancer (
Identification of sulfated oligosaccharide-based inhibitors of tumor growth and metastasis using novel in vitro assays for angiogenesis and heparanase activity.
). Heparin mimetics have high potential as anti-cancer agents due to their ability to inhibit heparanase activity, by competing with the HS GAGs for binding/signaling growth factors and chemokines. The heparin mimetic, PI-88, is in clinical trials for melanoma and liver cancer (
). Utilizing the technology underpinning PI-88, a set of heparan sulfate mimetics, called the PG500 series, such as PG545, have been developed to target both angiogenesis and heparanase activity (
). Viable approaches to target the GAG chains include the use of enzymes that can cleave specific GAGs. The use of a humanized mutant chondroitinase ABC enzyme (ChaseM) that depolymerizes chondroitin sulfate chains on a variety of CSPGs enhances glioma cell sensitivity to chemotherapeutic drugs (
). Depletion of HA by a PEGylated form of rHuPH20, PEGPH20, induced antitumor response and enhanced efficacy of anti-cancer drugs in pre-clinical models (
While a number of tools have now been developed as described above, tools are needed to specifically alter glycans. Such tools could greatly enhance understanding and targeting of glycans in cancer mechanobiology.
Conclusions
Stiffening of the ECM is common in cancer. Cancer cells sense and transduce mechanical stiffness of the ECM into intracellular responses by a process called mechanotransduction, which promotes aberrant cell functions and contributes to cancer progression. There is mounting evidence that a wide range of glycans, including proteoglycans like syndecans, agrin, serglycin, SLRPs and others, hyaluronan, sialylated proteins, and O-linked glycans, are involved in cancer cell mechanotransduction. Glycans modulate multiple steps in the mechanotransduction pathway, including integrin ligand binding and clustering, adhesion assembly, cytoskeletal dynamics, activation of the Rho/ROCK and YAP/TAZ pathway (Fig.2), and correlated cancer cell functions such as migration, proliferation, and drug resistance. Some glycans can directly stiffen the ECM by forming more crosslinks between ECM fibrils. We conclude that stiffening of tumor, as a normal part of cancer progression, is likely due directly to specific changes in glycan content/composition of the tumors. More comprehensive investigations of glycans in cancer cell mechanotransduction as well as development of new and more specific tools to modulate glycans both in vivo and in vitro will provide avenues for improved or novel cancer diagnostics and treatments in the future.
Figure 2Schematic illustration of mechanobiological pathways. Mechanotransduction in cancer cells is mediated by complex molecular pathways involving glycans, integrins, focal adhesion proteins and the actomyosin cytoskeleton. Cancer cells engage ECM proteins through the heterodimeric integrin receptors, enabling the assembly of cell-ECM adhesions. Integrin receptors function as mechanotransmitters, transmitting force through adhesion proteins to the actomyosin cytoskeleton. Glycans can act as mechanotransmitters themselves or modify mechanotransduction pathways including integrin clustering, adhesion formation, cytoskeletal remodeling, and YAP/TAZ translocation to the nucleus. Crowding of glycans can also induce morphological changes in the plasma membrane. Furthermore, binding of glycans to matrix proteins can promote matrix realignment and assembly which in turn can promote the stiffening of matrix. Accumulation of glycans such as hyaluronan in the ECM can increase interstitial pressure through retention of water within the confined tumor and thereby can contribute to mechanical stress. In addition, accumulation of advanced glycation end products can induce cross-linking and stiffening of collagen with minimal changes to the collagen fiber architecture. HAS, hyaluronan synthases; FAK, focal adhesion kinase; ECM, extracellular matrix.
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