RESUMEN
Galectin-8 is a small soluble lectin with two carbohydrate recognition domains (CRDs). N- and C-terminal CRDs of Gal-8 differ in their specificity for glycan ligands. Here, we wanted to find out whether oligomerization of individual CRDs of galectin-8 affects its biological activity. Using green fluorescent protein polygons (GFPp) as an oligomerization scaffold, we generated intrinsically fluorescent CRDs with altered valency. We show that oligomers of C-CRD are characterized by significant cell surface affinity. Furthermore, the multivalency of the resulting variants has an impact on cellular activities such as cell signaling, heparin binding and proliferation. Our data indicates that tunable valence is a useful tool for modifying the biological activity of CRDs of galectins.
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Galectinas , Galectinas/metabolismo , Galectinas/química , Humanos , Ligandos , Unión Proteica , Ingeniería de Proteínas/métodos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/química , Multimerización de Proteína , Proliferación Celular , Heparina/química , Heparina/metabolismoRESUMEN
FGF12 belongs to a subfamily of FGF proteins called FGF homologous factors (FHFs), which until recently were thought to be non-signaling intracellular proteins. Our recent studies have shown that although they lack a conventional signal peptide for secretion, they can reach the extracellular space, especially under stress conditions. Here, we unraveled that the long "a" isoform of FGF12 is secreted in a pathway involving the A1 subunit of Na(+)/K(+) ATPase (ATP1A1), Tec kinase and lipids such as phosphatidylinositol and phosphatidylserine. Further, we showed that the short "b" isoform of FGF12, which binds ATP1A1 and phosphatidylserine less efficiently, is not secreted from cells. We also indicated regions in the FGF12a protein sequence that are crucial for its secretion, including N-terminal fragment and specific residues, and proposed that liquid-liquid phase separation may be important in this process. Our results strongly suggest that the mechanism of this process is very similar for all unconventionally secreted FGF proteins.
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Factores de Crecimiento de Fibroblastos , Humanos , Factores de Crecimiento de Fibroblastos/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Fosfatidilserinas/metabolismo , Secuencia de AminoácidosRESUMEN
Fibroblast growth factors (FGFs) and their receptors (FGFRs) constitute plasma-membrane localized signaling hubs that transmit signals from the extracellular environment to the cell interior, governing pivotal cellular processes like motility, metabolism, differentiation, division and death. FGF/FGFR signaling is critical for human body development and homeostasis; dysregulation of FGF/FGFR units is observed in numerous developmental diseases and in about 10% of human cancers. Glycosylation is a highly abundant posttranslational modification that is critical for physiological and pathological functions of the cell. Glycosylation is also very common within FGF/FGFR signaling hubs. Vast majority of FGFs (15 out of 22 members) are N-glycosylated and few FGFs are O-glycosylated. Glycosylation is even more abundant within FGFRs; all FGFRs are heavily N-glycosylated in numerous positions within their extracellular domains. A growing number of studies points on the multiple roles of glycosylation in fine-tuning FGF/FGFR signaling. Glycosylation modifies secretion of FGFs, determines their stability and affects interaction with FGFRs and co-receptors. Glycosylation of FGFRs determines their intracellular sorting, constitutes autoinhibitory mechanism within FGFRs and adjusts FGF and co-receptor recognition. Sugar chains attached to FGFs and FGFRs constitute also a form of code that is differentially decrypted by extracellular lectins, galectins, which transform FGF/FGFR signaling at multiple levels. This review focuses on the identified functions of glycosylation within FGFs and FGFRs and discusses their relevance for the cell physiology in health and disease.
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Factores de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos , Transducción de Señal , Humanos , Glicosilación , Factores de Crecimiento de Fibroblastos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Procesamiento Proteico-PostraduccionalRESUMEN
Fibroblast growth factor receptor 1 (FGFR1) is a N-glycosylated cell surface receptor tyrosine kinase, which upon recognition of specific extracellular ligands, fibroblast growth factors (FGFs), initiates an intracellular signaling. FGFR1 signaling ensures homeostasis of cells by fine-tuning essential cellular processes, like differentiation, division, motility and death. FGFR1 activity is coordinated at multiple steps and unbalanced FGFR1 signaling contributes to developmental diseases and cancers. One of the crucial control mechanisms over FGFR1 signaling is receptor endocytosis, which allows for rapid targeting of FGF-activated FGFR1 to lysosomes for degradation and the signal termination. We have recently demonstrated that N-glycans of FGFR1 are recognized by a precise set of extracellular galectins, secreted and intracellular multivalent lectins implicated in a plethora of cellular processes and altered in immune responses and cancers. Specific galectins trigger FGFR1 clustering, resulting in activation of the receptor and in initiation of intracellular signaling cascades that shape the cell physiology. Although some of galectin family members emerged recently as key players in the clathrin-independent endocytosis of specific cargoes, their impact on endocytosis of FGFR1 was largely unknown.Here we assessed the contribution of extracellular galectins to the cellular uptake of FGFR1. We demonstrate that only galectin-1 induces internalization of FGFR1, whereas the majority of galectins predominantly inhibit endocytosis of the receptor. We focused on three representative galectins: galectin-1, -7 and -8 and we demonstrate that although all these galectins directly activate FGFR1 by the receptor crosslinking mechanism, they exert different effects on FGFR1 endocytosis. Galectin-1-mediated internalization of FGFR1 doesn't require galectin-1 multivalency and occurs via clathrin-mediated endocytosis, resembling in this way the uptake of FGF/FGFR1 complex. In contrast galectin-7 and -8 impede FGFR1 endocytosis, causing stabilization of the receptor on the cell surface and prolonged propagation of the signals. Furthermore, using protein engineering approaches we demonstrate that it is possible to modulate or even fully reverse the endocytic potential of galectins.
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Endocitosis , Galectina 1 , Galectinas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Animales , Humanos , Galectina 1/metabolismo , Galectina 1/genética , Galectinas/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de SeñalRESUMEN
Galectins constitute a class of lectins that specifically interact with ß-galactoside sugars in glycoconjugates and are implicated in diverse cellular processes, including transport, autophagy or signaling. Since most of the activity of galectins depends on their ability to bind sugar chains, galectins exert their functions mainly in the extracellular space or at the cell surface, which are microenvironments highly enriched in glycoconjugates. Galectins are also abundant inside cells, but their specific intracellular functions are largely unknown. Here we report that galectin-1, -3, -7 and -8 directly interact with the proteinaceous core of fibroblast growth factor 12 (FGF12) in the cytosol and in nucleus. We demonstrate that binding of galectin-1 to FGF12 in the cytosol blocks FGF12 secretion. Furthermore, we show that intracellular galectin-1 affects the assembly of FGF12-containing nuclear/nucleolar ribosome biogenesis complexes consisting of NOLC1 and TCOF1. Our data provide a new link between galectins and FGF proteins, revealing an unexpected glycosylation-independent intracellular interplay between these groups of proteins.
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Galectina 1 , Galectinas , Galectinas/metabolismo , Factores de Crecimiento de Fibroblastos , Glicoconjugados , Ribosomas/metabolismoRESUMEN
Breast cancer remains a significant global health challenge, necessitating the development of effective targeted therapies. This study aimed to create bispecific targeting molecules against HER2 and FGFR1, two receptors known to play crucial roles in breast cancer progression. By combining the high-affinity Affibody ZHER2:2891 and a modified, stable form of fibroblast growth factor 2 (FGF2), we successfully generated bispecific proteins capable of simultaneously recognizing HER2 and FGFR1. Two variants were designed: AfHER2-sFGF2 with a shorter linker and AfHER2-lFGF2 with a longer linker between the HER2 and FGFR1-recognizing proteins. Both proteins exhibited selective binding to HER2 and FGFR1, with AfHER2-lFGF2 demonstrating simultaneous binding to both receptors. AfHER2-lFGF2 exhibited superior internalization compared to FGF2 in FGFR-positive cells and significantly increased internalization compared to AfHER2 in HER2-positive cells. To enhance their therapeutic potential, highly potent cytotoxic agent MMAE was conjugated to the targeting proteins, resulting in protein-drug conjugates. The bispecific AfHER2-lFGF2-vcMMAE conjugate demonstrated exceptional cytotoxic activity against HER2-positive, FGFR-positive, and dual-positive cancer cell lines that was significantly higher compared to monospecific conjugates. These data indicate the beneficial effect of simultaneous targeting of HER2 and FGFR1 in precise anticancer medicine and contribute valuable insights into the design and potential of bispecific targeting molecules for breast cancer treatment.
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Antineoplásicos , Neoplasias de la Mama , Humanos , Femenino , Factor 2 de Crecimiento de Fibroblastos , Receptor ErbB-2/metabolismo , Línea Celular Tumoral , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológicoRESUMEN
Fibroblast growth factor 12 (FGF12) belongs to the fibroblast growth factor homologous factors (FHF) subfamily, which is also known as the FGF11 subfamily. The human FGF12 gene is located on chromosome 3 and consists of four introns and five coding exons. Their alternative splicing results in two FGF12 isoforms - the shorter 'b' isoform and the longer 'a' isoform. Structurally, the core domain of FGF12, is highly homologous to that of the other FGF proteins, providing the classical tertiary structure of ß-trefoil. FGF12 is expressed in various tissues, most abundantly in excitable cells such as neurons and cardiomyocytes. For many years, FGF12 was thought to be exclusively an intracellular protein, but recent studies have shown that it can be secreted despite the absence of a canonical signal for secretion. The best-studied function of FGF12 relates to its interaction with sodium channels. In addition, FGF12 forms complexes with signaling proteins, regulates the cytoskeletal system, binds to the FGF receptors activating signaling cascades to prevent apoptosis and interacts with the ribosome biogenesis complex. Importantly, FGF12 has been linked to nervous system disorders, cancers and cardiac diseases such as epileptic encephalopathy, pulmonary hypertension and cardiac arrhythmias, making it a potential target for gene therapy as well as a therapeutic agent.
RESUMEN
The unfolded protein response is a survival signaling pathway that is induced during various types of ER stress. Here, we determine IRE1's role in miRNA regulation during ER stress. During induction of ER stress in human bronchial epithelial cells, we utilized next generation sequencing to demonstrate that pre-miR-301a and pre-miR-106b were significantly increased in the presence of an IRE1 inhibitor. Conversely, using nuclear-cytosolic fractionation on ER stressed cells, we found that these pre-miRNAs were decreased in the nuclear fractions without the IRE1 inhibitor. We also found that miR-301a-3p targets the proapoptotic UPR factor growth arrest and DNA-damage-inducible alpha (GADD45A). Inhibiting miR-301a-3p levels or blocking its predicted miRNA binding site in GADD45A's 3' UTR with a target protector increased GADD45A mRNA expression. Furthermore, an elevation of XBP1s expression had no effect on GADD45A mRNA expression. We also demonstrate that the introduction of a target protector for the miR-301a-3p binding site in GADD45A mRNA during ER stress promoted cell death in the airway epithelial cells. In summary, these results indicate that IRE1's endonuclease activity is a two-edged sword that can splice XBP1 mRNA to stabilize survival or degrade pre-miR-301a to elevate GADD45A mRNA expression to lead to apoptosis. Video Abstract.
Asunto(s)
MicroARNs , Humanos , Apoptosis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , Regulación hacia ArribaRESUMEN
Fibroblast growth factor receptor 1 (FGFR1) is a heavily N-glycosylated cell surface receptor tyrosine kinase that transmits signals across the plasma membrane, in response to fibroblast growth factors (FGFs). Balanced FGF/FGFR1 signaling is crucial for the development and homeostasis of the human body, and aberrant FGFR1 is frequently observed in various cancers. In addition to its predominant localization to the plasma membrane, FGFR1 has also been detected inside cells, mainly in the nuclear lumen, where it modulates gene expression. However, the exact mechanism of FGFR1 nuclear transport is still unknown. In this study, we generated a glycosylation-free mutant of FGFR1, FGFR1.GF, and demonstrated that it is localized primarily to the nuclear envelope. We show that reintroducing N-glycans into the D3 domain cannot redirect FGFR1 to the plasma membrane or exclude the receptor from the nuclear envelope. Reestablishment of D2 domain N-glycans largely inhibits FGFR1 accumulation in the nuclear envelope, but the receptor continues to accumulate inside the cell, mainly in the ER. Only the simultaneous presence of N-glycans of the D2 and D3 domains of FGFR1 promotes efficient transport of FGFR1 to the plasma membrane. We demonstrate that while disturbed FGFR1 folding results in partial FGFR1 accumulation in the ER, impaired FGFR1 secretion drives FGFR1 trafficking to the nuclear envelope. Intracellular FGFR1.GF displays a high level of autoactivation, suggesting the presence of nuclear FGFR1 signaling, which is independent of FGF. Using mass spectrometry and proximity ligation assay, we identified novel binding partners of the nuclear envelope-localized FGFR1, providing insights into its cellular functions. Collectively, our data define N-glycosylation of FGFR1 as an important regulator of FGFR1 kinase activity and, most importantly, as a switchable signal for FGFR1 trafficking between the nuclear envelope and plasma membrane, which, due to spatial restrictions, shapes FGFR1 interactome and cellular function. Video Abstract.
Asunto(s)
Membrana Nuclear , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Humanos , Membrana Celular , Glicosilación , Factores de Crecimiento de FibroblastosRESUMEN
With almost 20 million new cases per year, cancer constitutes one of the most important challenges for public health systems. Unlike traditional chemotherapy, targeted anti-cancer strategies employ sophisticated therapeutics to precisely identify and attack cancer cells, limiting the impact of drugs on healthy cells and thereby minimizing the unwanted side effects of therapy. Protein drug conjugates (PDCs) are a rapidly growing group of targeted therapeutics, composed of a cancer-recognition factor covalently coupled to a cytotoxic drug. Several PDCs, mainly in the form of antibody-drug conjugates (ADCs) that employ monoclonal antibodies as cancer-recognition molecules, are used in the clinic and many PDCs are currently in clinical trials. Highly selective, strong and stable interaction of the PDC with the tumor marker, combined with efficient, rapid endocytosis of the receptor/PDC complex and its subsequent effective delivery to lysosomes, is critical for the efficacy of targeted cancer therapy with PDCs. However, the bivalent architecture of contemporary clinical PDCs is not optimal for tumor receptor recognition or PDCs internalization. In this review, we focus on multivalent PDCs, which represent a rapidly evolving and highly promising therapeutics that overcome most of the limitations of current bivalent PDCs, enhancing the precision and efficiency of drug delivery to cancer cells. We present an expanding set of protein scaffolds used to generate multivalent PDCs that, in addition to folding into well-defined multivalent molecular structures, enable site-specific conjugation of the cytotoxic drug to ensure PDC homogeneity. We provide an overview of the architectures of multivalent PDCs developed to date, emphasizing their efficacy in the targeted treatment of various cancers.
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Antineoplásicos , Inmunoconjugados , Neoplasias , Humanos , Preparaciones Farmacéuticas , Sistemas de Liberación de Medicamentos , Neoplasias/terapia , Anticuerpos Monoclonales/química , Antígenos/uso terapéuticoRESUMEN
FGF homologous factors (FHFs) are the least described group of fibroblast growth factors (FGFs). The FHF subfamily consists of four proteins: FGF11, FGF12, FGF13, and FGF14. Until recently, FHFs were thought to be intracellular, non-signaling molecules, despite sharing structural and sequence similarities with other members of FGF family that can be secreted and activate cell signaling by interacting with surface receptors. Here, we show that despite lacking a canonical signal peptide for secretion, FHFs are exported to the extracellular space. Furthermore, we propose that their secretion mechanism is similar to the unconventional secretion of FGF2. The secreted FHFs are biologically active and trigger signaling in cells expressing FGF receptors (FGFRs). Using recombinant proteins, we demonstrated their direct binding to FGFR1, resulting in the activation of downstream signaling and the internalization of the FHF-FGFR1 complex. The effect of receptor activation by FHF proteins is an anti-apoptotic response of the cell.
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Factores de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/fisiología , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
Fibroblast growth factors (FGFs) and their receptors (FGFRs) constitute complex signaling hubs that are crucial for the development and homeostasis of the human body. Most of FGFs are released by cells using the conventional secretory pathway and are N-glycosylated, yet the role of FGFs glycosylation is largely unknown. Here, we identify N-glycans of FGFs as binding sites for a specific set of extracellular lectins, galectins - 1, -3, -7 and - 8. We demonstrate that galectins attract N-glycosylated FGF4 to the cell surface, forming a reservoir of the growth factor in the extracellular matrix. Furthermore, we show that distinct galectins differentially modulate FGF4 signaling and FGF4-dependent cellular processes. Using engineered variants of galectins with altered valency we demonstrate that multivalency of galectins is critical for the adjustment of FGF4 activity. Summarizing, our data reveal a novel regulatory module within FGF signaling, in which the glyco-code in FGFs provides previously unanticipated information differentially deciphered by multivalent galectins, affecting signal transduction and cell physiology. Video Abstract.
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Factores de Crecimiento de Fibroblastos , Galectinas , Humanos , Galectinas/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , PolisacáridosRESUMEN
FGF/FGFR signaling is critical for the development and homeostasis of the human body and imbalanced FGF/FGFR contributes to the progression of severe diseases, including cancers. FGFRs are N-glycosylated, but the role of these modifications is largely unknown. Galectins are extracellular carbohydrate-binding proteins implicated in a plethora of processes in heathy and malignant cells. Here, we identified a precise set of galectins (galectin-1, -3, -7, and -8) that directly interact with N-glycans of FGFRs. We demonstrated that galectins bind N-glycan chains of the membrane-proximal D3 domain of FGFR1 and trigger differential clustering of FGFR1, resulting in activation of the receptor and initiation of downstream signaling cascades. Using engineered galectins with controlled valency, we provide evidence that N-glycosylation-dependent clustering of FGFR1 constitutes a mechanism for FGFR1 stimulation by galectins. We revealed that the consequences of galectin/FGFR signaling for cell physiology are markedly different from the effects induced by canonical FGF/FGFR units, with galectin/FGFR signaling affecting cell viability and metabolic activity. Furthermore, we showed that galectins are capable of activating an FGFR pool inaccessible for FGF1, enhancing the amplitude of transduced signals. Summarizing, our data identify a novel mechanism of FGFR activation, in which the information stored in the N-glycans of FGFRs provides previously unanticipated information about FGFRs' spatial distribution, which is differentially deciphered by distinct multivalent galectins, affecting signal transmission and cell fate.
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Galectinas , Transducción de Señal , Humanos , Galectinas/metabolismo , Transducción de Señal/fisiología , Fosforilación , Polisacáridos/metabolismo , GlicosilaciónRESUMEN
Among the FGF proteins, the least characterized superfamily is the group of fibroblast growth factor homologous factors (FHFs). To date, the main role of FHFs has been primarily seen in the modulation of voltage-gated ion channels, but a full picture of the function of FHFs inside the cell is far from complete. In the present study, we focused on identifying novel FGF12 binding partners to indicate its intracellular functions. Among the identified proteins, a significant number were nuclear proteins, especially RNA-binding proteins involved in translational processes, such as ribosomal processing and modification. We have demonstrated that FGF12 is localized to the nucleolus, where it interacts with NOLC1 and TCOF1, proteins involved in the assembly of functional ribosomes. Interactions with both NOLC1 and TCOF1 are unique to FGF12, as other FHF proteins only bind to TCOF1. The formation of nucleolar FGF12 complexes with NOLC1 and TCOF1 is phosphorylation-dependent and requires the C-terminal region of FGF12. Surprisingly, NOLC1 and TCOF1 are unable to interact with each other in the absence of FGF12. Taken together, our data link FHF proteins to nucleoli for the first time and suggest a novel and unexpected role for FGF12 in ribosome biogenesis. Video Abstract.
Asunto(s)
Factores de Crecimiento de Fibroblastos , Ribosomas , Proteínas Nucleares , FosforilaciónRESUMEN
Cancer drug resistance is a common, unpredictable phenomenon that develops in many types of tumors, resulting in the poor efficacy of current anticancer therapies. One of the most common, and yet the most complex causes of drug resistance is a mechanism related to dysregulation of tumor cell signaling. Abnormal signal transduction in a cancer cell is often stimulated by growth factors and their receptors, including fibroblast growth factors (FGFs) and FGF receptors (FGFRs). Here, we investigated the effect of FGF1 and FGFR1 activity on the action of drugs that disrupt tubulin polymerization (taltobulin, paclitaxel, vincristine) in FGFR1-positive cell lines, U2OS stably transfected with FGFR1 (U2OSR1) and DMS114 cells. We observed that U2OSR1 cells exhibited reduced sensitivity to the tubulin-targeting drugs, compared to U2OS cells expressing a negligible level of FGFRs. This effect was dependent on receptor activation, as inhibition of FGFR1 by a specific small-molecule inhibitor (PD173074) increased the cells' sensitivity to these drugs. Expression of functional FGFR1 in U2OS cells resulted in increased AKT phosphorylation, with no change in total AKT level. U2OSR1 cells also exhibited an elevated MDR1 and blocking MDR1 activity with cyclosporin A increased the toxicity of paclitaxel and vincristine, but not taltobulin. Analysis of tubulin polymerization pattern using fluorescence microscopy revealed that FGF1 in U2OSR1 cells partially reverses the drug-altered phenotype in paclitaxel- and vincristine-treated cells, but not in taltobulin-treated cells. Furthermore, we showed that FGF1, through activation of FGFR1, reduces caspase 3/7 activity and PARP cleavage, preventing apoptosis induced by tubulin-targeting drugs. Next, using specific kinase inhibitors, we investigated which signaling pathways are responsible for the FGF1-mediated reduction of taltobulin cytotoxicity. We found that AKT kinase is a key factor in FGF1-induced cell protection against taltobulin in U2OSR1 and DMS114 cells. Interestingly, only direct inhibition of AKT or dual-inhibition of PI3K and mTOR abolished this effect for cells treated with taltobulin. This suggests that both canonical (PI3K-dependent) and alternative (PI3K-independent) AKT-activating pathways may regulate FGF1/FGFR1-driven cancer cell survival. Our findings may contribute to the development of more effective therapies and may facilitate the prevention of drug resistance in FGFR1-positive cancer cells.
RESUMEN
Precise anticancer therapies employing cytotoxic conjugates constitute a side-effect-limited, highly attractive alternative to commonly used cancer treatment modalities, such as conventional chemotherapy, radiotherapy or surgical interventions. Receptor tyrosine kinases are a large family of N-glycoproteins intensively studied as molecular targets for cytotoxic conjugates in various cancers. At the cell surface, these receptors are embedded in a dense carbohydrate layer formed by numerous plasma membrane glycoproteins. The complexity of the cell surface architecture is further increased by galectins, secreted lectins capable of recognizing and clustering glycoconjugates, affecting their motility and activity. Cell surface N-glycosylation is intensively remodeled by cancer cells; however, the contribution of this phenomenon to the efficiency of treatment with cytotoxic conjugates is largely unknown. Here, we evaluated the significance of N-glycosylation for the internalization and toxicity of conjugates targeting two model receptor tyrosine kinases strongly implicated in cancer: HER2 and FGFR1. We employed three conjugates of distinct molecular architecture and specificity: AffibodyHER2-vcMMAE (targeting HER2), vcMMAE-KCK-FGF1.E and T-Fc-vcMMAE (recognizing different epitopes within FGFR1). We demonstrated that inhibition of N-glycosylation reduced the cellular uptake of all conjugates tested and provided evidence for a role of the galectin network in conjugate internalization. In vitro binding studies revealed that the reduced uptake of conjugates is not due to impaired HER2 and FGFR1 binding. Importantly, we demonstrated that alteration of N-glycosylation can affect the cytotoxic potential of conjugates. Our data implicate a key role for cell surface N-glycosylation in the delivery of cytotoxic conjugates into cancer cells.
Asunto(s)
Antineoplásicos , Neoplasias , Antineoplásicos/farmacología , Línea Celular Tumoral , Galectinas/metabolismo , Glicosilación , Humanos , Neoplasias/tratamiento farmacológico , Tirosina/metabolismoRESUMEN
Fibroblast growth factor 2 (FGF2) is a pleiotropic protein engaged in the regulation of key cellular processes in a wide spectrum of cells. FGF2 is an important object of basic research as well as a molecule used in regenerative medicine, in vitro cell culture maintenance, and as an anticancer drug carrier. However, the unsatisfactory stability and pleiotropic activities of the wild-type FGF2 largely limit its use as a medical product. To overcome these limitations, we have designed a set of FGF2-based macromolecules via sortase A-mediated cyclization and oligomerization. We obtained heparin-switchable FGF2 variants with enhanced stability and improved ability to stimulate cell proliferation and migration. We have shown that stimulation of glucose uptake by adipocytes is modulated by the architecture of FGF2 oligomers. Moreover, we used hyper-stable FGF2 variants for the construction of highly effective drug carriers for selective killing of FGFR1-overproducing cancer cells. The strategy for FGF2 engineering presented in this work provides novel insights into the design of growth factor variants for regenerative and anti-cancer precise medicine.
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Factor 2 de Crecimiento de Fibroblastos , Neoplasias , Proliferación Celular , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Heparina/farmacología , HumanosRESUMEN
Fibroblast growth factor receptor 1 (FGFR1) is an integral membrane protein that transmits prolife signals through the plasma membrane. Overexpression of FGFR1 has been reported in various tumor types, and therefore, this receptor constitutes an attractive molecular target for selective anticancer therapies. Here, we present a novel system for generation of intrinsically fluorescent, self-assembling, oligomeric cytotoxic conjugates with high affinity and efficient internalization targeting FGFR1. In our approach, we employed FGF1 as an FGFR1 recognizing molecule and genetically fused it to green fluorescent protein polygons (GFPp), a fluorescent oligomerization scaffold, resulting in a set of GFPp_FGF1 oligomers with largely improved receptor binding. To validate the applicability of using GFPp_FGF1 oligomers as cancer probes and drug carriers in targeted therapy of cancers with aberrant FGFR1, we selected a trimeric variant from generated GFPp_FGF1 oligomers and further engineered it by introducing FGF1-stabilizing mutations and by incorporating the cytotoxic drug monomethyl auristatin E (MMAE) in a site-specific manner. The resulting intrinsically fluorescent, trimeric cytotoxic conjugate 3xGFPp_FGF1E_LPET_MMAE exhibits nanomolar affinity for the receptor and very high stability. Notably, the intrinsic fluorescence of 3xGFPp_FGF1E_LPET_MMAE allows for tracking the cellular transport of the conjugate, demonstrating that 3xGFPp_FGF1E_LPET_MMAE is efficiently and selectively internalized into cells expressing FGFR1. Importantly, we show that 3xGFPp_FGF1E_LPET_MMAE displays very high cytotoxicity against a panel of different cancer cells overproducing FGFR1 while remaining neutral toward cells devoid of FGFR1 expression. Our data implicate that the engineered fluorescent conjugates can be used for imaging and targeted therapy of FGFR1-overproducing cancers.
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Antineoplásicos , Neoplasias , Antineoplásicos/farmacología , Línea Celular Tumoral , Unión Proteica , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismoRESUMEN
Increased expression of both FGF proteins and their receptors observed in many cancers is often associated with the development of chemoresistance, limiting the effectiveness of currently used anti-cancer therapies. Malfunctioning of the FGF/FGFR axis in cancer cells generates a number of molecular mechanisms that may affect the sensitivity of tumors to the applied drugs. Of key importance is the deregulation of cell signaling, which can lead to increased cell proliferation, survival, and motility, and ultimately to malignancy. Signaling pathways activated by FGFRs inhibit apoptosis, reducing the cytotoxic effect of some anti-cancer drugs. FGFRs-dependent signaling may also initiate angiogenesis and EMT, which facilitates metastasis and also correlates with drug resistance. Therefore, treatment strategies based on FGF/FGFR inhibition (using receptor inhibitors, ligand traps, monoclonal antibodies, or microRNAs) appear to be extremely promising. However, this approach may lead to further development of resistance through acquisition of specific mutations, metabolism switching, and molecular cross-talks. This review brings together information on the mechanisms underlying the involvement of the FGF/FGFR axis in the generation of drug resistance in cancer and highlights the need for further research to overcome this serious problem with novel therapeutic strategies.