The intracellular interplay between galectin-1 and FGF12 in the assembly of ribosome biogenesis complex.

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.

FGF12: biology and function.

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.

Intracellular FGF1 protects cells from apoptosis through direct interaction with p53.

Fibroblast growth factor 1 (FGF1) acts by activating specific tyrosine kinase receptors on the cell surface. In addition to this classical mode of action, FGF1 also exhibits intracellular activity. Recently, we found that FGF1 translocated into the cell interior exhibits anti-apoptotic activity independent of receptor activation and downstream signaling. Here, we show that expression of FGF1 increases the survival of cells treated with various apoptosis inducers, but only when wild-type p53 is present. The p53-negative cells were not protected by either ectopically expressed or translocated FGF1. We also confirmed the requirement of p53 for the anti-apoptotic intracellular activity of FGF1 by silencing p53, resulting in loss of the protective effect of FGF1. In contrast, in p53-negative cells, intracellular FGF1 regained its anti-apoptotic properties after transfection with wild-type p53. We also found that FGF1 directly interacts with p53 in cells and that the binding region is located in the DBD domain of p53. We therefore postulate that intracellular FGF1 protects cells from apoptosis by directly interacting with p53.

N-glycosylation acts as a switch for FGFR1 trafficking between the plasma membrane and nuclear envelope.

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.

FGF1 Protects MCF-7 Cells against Taltobulin through Both the MEKs/ERKs and PI3K/AKT Signaling Pathway.

Breast cancer is a widespread and complex disease characterized by abnormal signaling pathways that promote tumor growth and progression. Despite significant medical advances and the development of increasingly effective therapies for breast cancer, drug resistance and reduced sensitivity to prior therapies remain persistent challenges. Dysregulation of growth factors such as FGFs and EGF and their receptors is a contributing factor to reduced response to treatment, promoting cell survival and proliferation, metastasis, EMT or increased expression of ABC transporters. Our study demonstrates a protective role for FGF1 in MCF-7 breast cancer cells against taltobulin-induced cytotoxicity, mediated by activation of its receptors and compares its activity to EGF, another growth factor involved in breast cancer development and progression. The mechanisms of action of these two proteins are different: FGF1 exerts its effects through the activation of both ERKs and AKT, whereas EGF acts only through ERKs. FGF1 action in the presence of the drug promotes cell viability, reduces apoptosis and increases cell migration. Although EGF and its receptors have received more attention in breast cancer research to date, our findings highlight the key role played by FGFs and their receptors in promoting drug resistance to tubulin polymerization inhibitors in FGFR-positive tumors.

Multivalent protein-drug conjugates - An emerging strategy for the upgraded precision and efficiency of drug delivery to cancer cells.

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.

FGF homologous factors are secreted from cells to induce FGFR-mediated anti-apoptotic response.

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.

Short report galectins use N-glycans of FGFs to capture growth factors at the cell surface and fine-tune their signaling.

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.

Receptor clustering by a precise set of extracellular galectins initiates FGFR signaling.

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.

FGF12 is a novel component of the nucleolar NOLC1/TCOF1 ribosome biogenesis complex.

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.

FGF1 protects FGFR1-overexpressing cancer cells against drugs targeting tubulin polymerization by activating AKT via two independent mechanisms.

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.

The Significance of Cell Surface N-Glycosylation for Internalization and Potency of Cytotoxic Conjugates Targeting Receptor Tyrosine Kinases.

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.

Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells.

Fibroblast growth factor 1 (FGF1) is considered primarily as a ligand for FGF surface receptors (FGFRs) through which it activates a number of cellular responses. In addition to its canonical mode of action, FGF1 can act intracellularly, before secretion or after internalization and translocation from the cell exterior. The role of FGF1 inside the cell is to provide additional protection against apoptosis and promote cell survival. The FGF1 protein contains a specific N-terminal nuclear localization sequence (NLS) that is essential for its efficient transport to the nucleus. Here, we investigated the role of this sequence in the anti-apoptotic response of FGF1. To this end, we produced recombinant FGF1 variants with mutated or deleted NLS and added them to apoptosis-induced cells in which FGFR1 was inactive, either as a result of chemical inhibition or kinase-dead mutation. After internalization, all FGF1 variants were able to protect the differentiated cells from serum starvation-induced apoptosis. To verify the results obtained for NLS mutants, we knocked down LRRC59, a protein that mediates the nuclear transport of FGF1. Upon LRRC59 silencing, we still observed a decrease in caspase 3/7 activity in cells treated exogenously with wild-type FGF1. In the next step, FGF1 variants with mutated or deleted NLS were expressed in U2OS cells, in which apoptosis was then induced by various factors (e.g., starvation, etoposide, staurosporine, anisomycin and actinomycin D). Experiments were performed in the presence of specific FGFR inhibitors to eliminate FGFR-induced signaling, potentially activated by FGF1 proteins released from damaged cells. Again, we found that the presence of NLS in FGF1 is not required for its anti-apoptotic activity. All NLS variants tested were able to act as wild type FGF1, increasing the cell viability and mitochondrial membrane potential and reducing the caspase 3/7 activity and PARP cleavage in cells undergoing apoptosis, both transiently and stably transfected. Our results indicate that the nuclear localization of FGF1 is not required for its intracellular anti-apoptotic activity in differentiated cells and suggest that the mechanism of the stress response differs according to the level of cell differentiation.

Intrinsically Fluorescent Oligomeric Cytotoxic Conjugates Toxic for FGFR1-Overproducing Cancers.

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.

FGF/FGFR-Dependent Molecular Mechanisms Underlying Anti-Cancer Drug Resistance.

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.

Modular self-assembly system for development of oligomeric, highly internalizing and potent cytotoxic conjugates targeting fibroblast growth factor receptors.

BACKGROUND: Overexpression of FGFR1 is observed in numerous tumors and therefore this receptor constitutes an attractive molecular target for selective cancer treatment with cytotoxic conjugates. The success of cancer therapy with cytotoxic conjugates largely relies on the precise recognition of a cancer-specific marker by a targeting molecule within the conjugate and its subsequent cellular internalization by receptor mediated endocytosis. We have recently demonstrated that efficiency and mechanism of FGFR1 internalization are governed by spatial distribution of the receptor in the plasma membrane, where clustering of FGFR1 into larger oligomers stimulated fast and highly efficient uptake of the receptor by simultaneous engagement of multiple endocytic routes. Based on these findings we aimed to develop a modular, self-assembly system for generation of oligomeric cytotoxic conjugates, capable of FGFR1 clustering, for targeting FGFR1-overproducing cancer cells. METHODS: Engineered FGF1 was used as FGFR1-recognition molecule and tailored for enhanced stability and site-specific attachment of the cytotoxic drug. Modified streptavidin, allowing for controlled oligomerization of FGF1 variant was used for self-assembly of well-defined FGF1 oligomers of different valency and oligomeric cytotoxic conjugate. Protein biochemistry methods were applied to obtain highly pure FGF1 oligomers and the oligomeric cytotoxic conjugate. Diverse biophysical, biochemical and cell biology tests were used to evaluate FGFR1 binding, internalization and the cytotoxicity of obtained oligomers. RESULTS: Developed multivalent FGF1 complexes are characterized by well-defined architecture, enhanced FGFR1 binding and improved cellular uptake. This successful strategy was applied to construct tetrameric cytotoxic conjugate targeting FGFR1-producing cancer cells. We have shown that enhanced affinity for the receptor and improved internalization result in a superior cytotoxicity of the tetrameric conjugate compared to the monomeric one. CONCLUSIONS: Our data implicate that oligomerization of the targeting molecules constitutes an attractive strategy for improvement of the cytotoxicity of conjugates recognizing cancer-specific biomarkers. Importantly, the presented approach can be easily adapted for other tumor markers.

Fibroblast Growth Factor 2 Conjugated with Monomethyl Auristatin E Inhibits Tumor Growth in a Mouse Model.

Worldwide, cancer is the second leading cause of death. Regardless of the continuous progress in medicine, we still do not have a fully effective anti-cancer therapy. Therefore, the search for new targeted anti-cancer drugs is still an unmet need. Here, we present novel protein-drug conjugates that inhibit tumor growth in a mouse model of human breast cancer. We developed conjugates based on fibroblast growth factor (FGF2) with improved biophysical and biological properties for the efficient killing of cancer cells overproducing fibroblast growth factor receptor 1 (FGFR1). We used hydrophilic and biocompatible PEG4 or PEG27 molecules as a spacer between FGF2 and the toxic agent monomethyl auristatin E. All conjugates exhibited a cytotoxic effect on FGFR1-positive cancer cell lines. The conjugate with the highest hydrodynamic size (42 kDa) and cytotoxicity was found to efficiently inhibit tumor growth in a mouse model of human breast cancer.

Roles of the FGF-FGFR Signaling System in Cancer Development and Inflammation.

For multi-cellular organisms to organize tissues, their cells must communicate with each other [...].

FGF1 Fusions with the Fc Fragment of IgG1 for the Assembly of GFPpolygons-Mediated Multivalent Complexes Recognizing FGFRs.

FGFRs are cell surface receptors that, when activated by specific FGFs ligands, transmit signals through the plasma membrane, regulating key cellular processes such as differentiation, division, motility, metabolism and death. We have recently shown that the modulation of the spatial distribution of FGFR1 at the cell surface constitutes an additional mechanism for fine-tuning cellular signaling. Depending on the multivalent, engineered ligand used, the clustering of FGFR1 into diverse supramolecular complexes enhances the efficiency and modifies the mechanism of receptor endocytosis, alters FGFR1 lifetime and modifies receptor signaling, ultimately determining cell fate. Here, we present a novel approach to generate multivalent FGFR1 ligands. We functionalized FGF1 for controlled oligomerization by developing N- and C-terminal fusions of FGF1 with the Fc fragment of human IgG1 (FGF1-Fc and Fc-FGF1). As oligomerization scaffolds, we employed GFPpolygons, engineered GFP variants capable of well-ordered multivalent display, fused to protein G to ensure binding of Fc fragment. The presented strategy allows efficient assembly of oligomeric FGFR1 ligands with up to twelve receptor binding sites. We show that multivalent FGFR1 ligands are biologically active and trigger receptor clustering on the cell surface. Importantly, the approach described in this study can be easily adapted to oligomerize alternative growth factors to control the activity of other cell surface receptors.