Differential cellular responses to adhesive interactions with galectin-8- and fibronectin-coated substrates.

The mechanisms underlying the cellular response to extracellular matrices (ECMs) that consist of multiple adhesive ligands are still poorly understood. Here, we address this topic by monitoring specific cellular responses to two different extracellular adhesion molecules - the main integrin ligand fibronectin and galectin-8, a lectin that binds ß-galactoside residues  - as well as to mixtures of the two proteins. Compared with cell spreading on fibronectin, cell spreading on galectin-8-coated substrates resulted in increased projected cell area, more-pronounced extension of filopodia and, yet, the inability to form focal adhesions and stress fibers. These differences can be partially reversed by experimental manipulations of small G-proteins of the Rho family and their downstream targets, such as formins, the Arp2/3 complex and Rho kinase. We also show that the physical adhesion of cells to galectin-8 was stronger than adhesion to fibronectin. Notably, galectin-8 and fibronectin differently regulate cell spreading and focal adhesion formation, yet act synergistically to upregulate the number and length of filopodia. The physiological significance of the coherent cellular response to a molecularly complex matrix is discussed. This article has an associated First Person interview with the first author of the paper.

Amyloid Precursor Protein and Tau Peptides Linked Together Ameliorate Loss of Cognition in an Alzheimer's Disease Animal Model.

The major proteins involved in Alzheimer's disease (AD) are amyloid precursor protein (APP) and Tau. We demonstrate that APP1 (390-412) and Tau1 (19-34), linked together with either a flexible or a rigid peptide bridge, are able to inhibit, in vitro, the interaction between APP and Tau proteins. Furthermore, nasal administration of biotin-labelled Flex peptide for two weeks indicated the localization of the peptide around and close to plaques in the hippocampus area. In vivo studies in 5xFAD transgenic (Tg) mice, which exhibit plaque load and mild cognitive decline at four months of age, show that nasal administration of the flexible linked peptide reduced amyloid plaque burden. Additionally, nasal treatment with either flexible or rigid linked peptides prevented cognitive function deterioration. A significant treatment effect was achieved when either treatment was initiated at the age of three months, before severe cognitive deficiency is evident, or at five months, when such deficiency is already observed. The nasally treated mice demonstrated a cognitive ability not significantly different from the non-Tg littermate controls. Testing the effect of the flexible peptide by gavage feeding on the cognitive function of 5xFAD Tg mice demonstrated that feeding as well as nasal treatment significantly improves the cognitive ability of Tg mice compared to control PBS-treated mice.

Ablation of the mammalian lectin galectin-8 induces bone defects in mice.

Mice overexpressing galectin-8 [gal-8 transgenic (Tg)], a secreted mammalian lectin, exhibit enhanced bone turnover and reduced bone mass, similar to cases of postmenopausal osteoporosis. Here, we show that gal-8 knockout (KO) mice have increased bone mass accrual at a young age but exhibit accelerated bone loss during adulthood. These phenotypes can be attributed to a gal-8-mediated increase in receptor activator of NF-κB ligand (RANKL) expression that promotes osteoclastogenesis, combined with direct inhibition of osteoblast differentiation, evident by reduced bone morphogenetic protein (BMP) signaling, reduced phosphorylation of receptor regulated mothers against decapentaplegic homolog (R-SMAD) and reduced expression of osteoblast differentiation markers osterix, osteocalcin, runt-related transcription factor 2 (RUNX2), dentin matrix acidic phosphoprotein-1 (DMP1), and alkaline phosphatase. At the same time, gal-8 promotes expression of estrogen receptor α (ESR1). Accordingly, the rate of bone loss is accelerated in ovariectomized, estrogen-deficient gal-8 Tg mice, whereas gal-8 KO mice, having low levels of ESR1, are refractory to ovariectomy. Finally, gal-8 mRNA positively correlates with the mRNA levels of osteoclastogenic markers RANKL, tartrate-resistant acid phosphatase, and cathepsin K in human femurs. Collectively, these findings identify gal-8 as a new physiologic player in the regulation of bone mass.-Vinik, Y., Shatz-Azoulay, H., Hiram-Bab, S., Kandel, L., Gabet, Y., Rivkin, G., Zick, Y. Ablation of the mammalian lectin galectin-8 induces bone defects in mice.

Nedd4 family interacting protein 1 (Ndfip1) promotes death of pancreatic beta cells.

High-throughput siRNA screening was employed to identify novel genes that regulate cytokine-induced death of pancreatic ß-cells. One of the 'hits' was Nedd4 family interacting protein 1 (Ndfip1), an adaptor and activator of Nedd4-family ubiquitin ligases. Silencing of Ndfip1 inhibited cytokine-induced apoptosis of mouse and human pancreatic islets and promoted glucose-stimulated insulin secretion. These effects were associated with an increase in the cellular content of JunB, a potent inhibitor of ER stress and apoptosis. Silencing of Ndfip1 also increased the expression of ATF4, IRE-1α, and the spliced form of XBP that govern the unfolded protein response (UPR) and relieve cytokine-induced ER stress, while overexpression of Ndfip1 exerted opposite effects. These findings implicate Ndfip1 in the degradation of JunB; inhibition of the UPR and insulin secretion; and promotion of cytokine-induced death of pancreatic ß-cells.

Computational pipeline predicting cell death suppressors as targets for cancer therapy.

Identification of promising targets for cancer therapy is a global effort in precision medicine. Here, we describe a computational pipeline integrating transcriptomic and vulnerability responses to cell-death inducing drugs, to predict cell-death suppressors as candidate targets for cancer therapy. The prediction is based on two modules; the transcriptomic similarity module to identify genes whose targeting results in similar transcriptomic responses of the death-inducing drugs, and the correlation module to identify candidate genes whose expression correlates to the vulnerability of cancer cells to the same death-inducers. The combined predictors of these two modules were integrated into a single metric. As a proof-of-concept, we selected ferroptosis inducers as death-inducing drugs in triple negative breast cancer. The pipeline reliably predicted candidate genes as ferroptosis suppressors, as validated by computational methods and cellular assays. The described pipeline might be used to identify repressors of various cell-death pathways as potential therapeutic targets for different cancer types.

Programming a Ferroptosis-to-Apoptosis Transition Landscape Revealed Ferroptosis Biomarkers and Repressors for Cancer Therapy.

Ferroptosis and apoptosis are key cell-death pathways implicated in several human diseases including cancer. Ferroptosis is driven by iron-dependent lipid peroxidation and currently has no characteristic biomarkers or gene signatures. Here a continuous phenotypic gradient between ferroptosis and apoptosis coupled to transcriptomic and metabolomic landscapes is established. The gradual ferroptosis-to-apoptosis transcriptomic landscape is used to generate a unique, unbiased transcriptomic predictor, the Gradient Gene Set (GGS), which classified ferroptosis and apoptosis with high accuracy. Further GGS optimization using multiple ferroptotic and apoptotic datasets revealed highly specific ferroptosis biomarkers, which are robustly validated in vitro and in vivo. A subset of the GGS is associated with poor prognosis in breast cancer patients and PDXs and contains different ferroptosis repressors. Depletion of one representative, PDGFA-assaociated protein 1(PDAP1), is found to suppress basal-like breast tumor growth in a mouse model. Omics and mechanistic studies revealed that ferroptosis is associated with enhanced lysosomal function, glutaminolysis, and the tricarboxylic acid (TCA) cycle, while its transition into apoptosis is attributed to enhanced endoplasmic reticulum(ER)-stress and phosphatidylethanolamine (PE)-to-phosphatidylcholine (PC) metabolic shift. Collectively, this study highlights molecular mechanisms underlying ferroptosis execution, identified a highly predictive ferroptosis gene signature with prognostic value, ferroptosis versus apoptosis biomarkers, and ferroptosis repressors for breast cancer therapy.

TM7SF3 controls TEAD1 splicing to prevent MASH-induced liver fibrosis.

The mechanisms of hepatic stellate cell (HSC) activation and the development of liver fibrosis are not fully understood. Here, we show that deletion of a nuclear seven transmembrane protein, TM7SF3, accelerates HSC activation in liver organoids, primary human HSCs, and in vivo in metabolic-dysfunction-associated steatohepatitis (MASH) mice, leading to activation of the fibrogenic program and HSC proliferation. Thus, TM7SF3 knockdown promotes alternative splicing of the Hippo pathway transcription factor, TEAD1, by inhibiting the splicing factor heterogeneous nuclear ribonucleoprotein U (hnRNPU). This results in the exclusion of the inhibitory exon 5, generating a more active form of TEAD1 and triggering HSC activation. Furthermore, inhibiting TEAD1 alternative splicing with a specific antisense oligomer (ASO) deactivates HSCs in vitro and reduces MASH diet-induced liver fibrosis. In conclusion, by inhibiting TEAD1 alternative splicing, TM7SF3 plays a pivotal role in mitigating HSC activation and the progression of MASH-related fibrosis.

Mouse Modeling Dissecting Macrophage-Breast Cancer Communication Uncovered Roles of PYK2 in Macrophage Recruitment and Breast Tumorigenesis.

Macrophage infiltration in mammary tumors is associated with enhanced tumor progression, metastasis, and poor clinical outcome, and considered as target for therapeutic intervention. By using different genetic mouse models, the authors show that ablation of the tyrosine kinase PYK2, either in breast cancer cells, only in the tumor microenvironment, or in both, markedly reduces the number of infiltrating tumor macrophages and concomitantly inhibits tumor angiogenesis and tumor growth. Strikingly, PYK2 ablation only in macrophages is sufficient to induce similar effects. These phenotypic changes are associated with reduced monocyte recruitment and a substantial decrease in tumor-associated macrophages (TAMs). Mechanistically, the authors show that PYK2 mediates mutual communication between breast cancer cells and macrophages through critical effects on key receptor signaling. Specifically, PYK2 ablation inhibits Notch1 signaling and consequently reduces CCL2 secretion by breast cancer cells, and concurrently reduces the levels of CCR2, CXCR4, IL-4Rα, and Stat6 activation in macrophages. These bidirectional effects modulate monocyte recruitment, macrophage polarization, and tumor angiogenesis. The expression of PYK2 is correlated with infiltrated macrophages in breast cancer patients, and its effects on macrophage infiltration and pro-tumorigenic phenotype suggest that PYK2 targeting can be utilized as an effective strategy to modulate TAMs and possibly sensitize breast cancer to immunotherapy.

A seven-transmembrane protein-TM7SF3, resides in nuclear speckles and regulates alternative splicing.

The seven-transmembrane superfamily member 3 protein (TM7SF3) is a p53-regulated homeostatic factor that attenuates cellular stress and the unfolded protein response. Here we show that TM7SF3 localizes to nuclear speckles; eukaryotic nuclear bodies enriched in splicing factors. This unexpected location for a trans -membranal protein enables formation of stable complexes between TM7SF3 and pre-mRNA splicing factors including DHX15, LARP7, HNRNPU, RBM14, and HNRNPK. Indeed, TM7SF3 regulates alternative splicing of >330 genes, mainly at the 3'end of introns by directly modulating the activity of splicing factors such as HNRNPK. These effects are observed both in cell lines and primary human pancreatic islets. Accordingly, silencing of TM7SF3 results in differential expression of 1465 genes (about 7% of the human genome); with 844 and 621 genes being up- or down-regulated, respectively. Our findings implicate TM7SF3, as a resident protein of nuclear speckles and suggest a role for seven-transmembrane proteins as regulators of alternative splicing.

The AXL-PYK2-PKCα axis as a nexus of stemness circuits in TNBC.

Cancer stem cells (CSCs) are implicated in tumor initiation, metastasis and drug resistance, and considered as attractive targets for cancer therapy. Here we identified a clinically relevant signaling nexus mediated by AXL receptor, PYK2 and PKCα and show its impact on stemness in TNBC. AXL, PYK2, and PKCα expression correlates with stemness signature in basal-like breast cancer patients, and their depletion in multiple mesenchymal TNBC cell lines markedly reduced the number of mammosphere-forming cells and cells harboring CSCs characteristic markers. Knockdown of PYK2 reduced the levels of AXL, PKCα, FRA1, and PYK2 proteins, and similar trend was obtained upon PKCα depletion. PYK2 depletion decreased AXL transcription through feedback loops mediated by FRA1 and TAZ, whereas PKCα inhibition induced redistribution of AXL to endosomal/lysosomal compartment and enhanced its degradation. PYK2 and PKCα cooperate at a convergence point of multiple stemness-inducing pathways to regulate AXL levels and concomitantly the levels/activation of STAT3, TAZ, FRA1, and SMAD3 as well as the pluripotent transcription factors Nanog and Oct4. Induction of stemness in TNBC sensitized cells to PYK2 and PKCα inhibition suggesting that targeting the AXL-PYK2-PKCα circuit could be an efficient strategy to eliminate CSCs in TNBC.

BCL-XL blockage in TNBC models confers vulnerability to inhibition of specific cell cycle regulators.

Cell cycle regulators are frequently altered in Triple-Negative Breast Cancer (TNBC). Emerging agents targeting these signals offer the possibility to design new combinatorial therapies. However, preclinical models that recapitulate TNBC primary resistance and heterogeneity are essential to evaluate the potency of these combined treatments. Methods: Bioinformatic processing of human breast cancer datasets was used to analyse correlations between expression levels of cell cycle regulators and patient survival outcome. The MMTV-R26Met mouse model of TNBC resistance and heterogeneity was employed to analyse expression and targeting vulnerability of cell cycle regulators in the presence of BCL-XL blockage. Robustness of outcomes and selectivity was further explored using a panel of human breast cancer cells. Orthotopic studies in nude mice were applied for preclinical evaluation of efficacy and toxicity. Alterations of protein expression, phosphorylation, and/or cellular localisation were analysed by western blots, reverse phase protein array, and immunocytochemistry. Bioinformatics was performed to highlight drug's mechanisms of action. Results: We report that high expression levels of the BCL2L1 gene encoding BCL-XL and of specific cell cycle regulators correlate with poor survival outcomes of TNBC patients. Blockage of BCL-XL confers vulnerability to drugs targeting CDK1/2/4, but not FOXM1, CDK4/6, Aurora A and Aurora B, to all MMTV-R26Met and human TNBC cell lines tested. Combined blockage of BCL-XL and CDK1/2/4 interfered with tumour growth in vivo. Mechanistically, we show that, co-targeting of BCL-XL and CDK1/2/4 synergistically inhibited cell viability by combinatorial depletion of survival and RTK/AKT signals, and concomitantly restoring FOXO3a tumour suppression actions. This was accompanied by an accumulation of DNA damage and consequently apoptosis. Conclusions: Our studies illustrate the possibility to exploit the vulnerability of TNBC cells to CDK1/2/4 inhibition by targeting BCL-XL. Moreover, they underline that specificity matters in targeting cell cycle regulators for combinatorial anticancer therapies.

Modeling Heterogeneity of Triple-Negative Breast Cancer Uncovers a Novel Combinatorial Treatment Overcoming Primary Drug Resistance.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype characterized by a remarkable molecular heterogeneity. Currently, there are no effective druggable targets and advanced preclinical models of the human disease. Here, a unique mouse model (MMTV-R26Met mice) of mammary tumors driven by a subtle increase in the expression of the wild-type MET receptor is generated. MMTV-R26Met mice develop spontaneous, exclusive TNBC tumors, recapitulating primary resistance to treatment of patients. Proteomic profiling of MMTV-R26Met tumors and machine learning approach show that the model faithfully recapitulates intertumoral heterogeneity of human TNBC. Further signaling network analysis highlights potential druggable targets, of which cotargeting of WEE1 and BCL-XL synergistically kills TNBC cells and efficiently induces tumor regression. Mechanistically, BCL-XL inhibition exacerbates the dependency of TNBC cells on WEE1 function, leading to Histone H3 and phosphoS33RPA32 upregulation, RRM2 downregulation, cell cycle perturbation, mitotic catastrophe, and apoptosis. This study introduces a unique, powerful mouse model for studying TNBC formation and evolution, its heterogeneity, and for identifying efficient therapeutic targets.

The Animal Lectin Galectin-8 Promotes Cytokine Expression and Metastatic Tumor Growth in Mice.

Secreted animal lectins of the galectin family are key players in cancer growth and metastasis. Here we show that galectin-8 (gal-8) induces the expression and secretion of cytokines and chemokines such as SDF-1 and MCP-1 in a number of cell types. This involves gal-8 binding to a uPAR/LRP1/integrin complex that activates JNK and the NFkB pathway. Cytokine and chemokine secretion, induced by gal-8, promotes migration of cancer cells toward cells treated with this lectin. Indeed, immune-competent gal-8 knockout (KO) mice express systemic lower levels of cytokines and chemokines while the opposite is true for gal-8 transgenic animals. Accordingly, gal-8 KO mice experience reduced tumor size and smaller and fewer metastatic lesions when injected with cancer cells. These results suggest the existence of a 'vicious cycle' whereby gal-8 secreted by the tumor microenvironment, promotes secretion of chemoattractants at the metastatic niche that promote further recruitment of tumor cells to that site. This study further implicate gal-8 in control of cancer progression and metastasis through its effects on the production of immunoregulatory cytokines.

Rational Design and Synthesis of Methyl-ß-d-galactomalonyl Phenyl Esters as Potent Galectin-8N Antagonists.

Galectin-8 is a ß-galactoside-recognizing protein having an important role in the regulation of bone remodeling and cancer progression and metastasis. Methyl ß-d-galactopyranoside malonyl aromatic esters have been designed to target and engage with particular amino acid residues of the galectin-8N extended carbohydrate-binding site. The chemically synthesized compounds had in vitro binding affinity toward galectin-8N in the range of 5-33 µM, as evaluated by isothermal titration calorimetry. This affinity directly correlated with the compounds' ability to inhibit galectin-8-induced expression of chemokines and proinflammatory cytokines in the SUM159 breast cancer cell line. X-ray crystallographic structure determination revealed that these monosaccharide-based compounds bind galectin-8N by engaging its unique arginine (Arg59) and simultaneously cross-linking to another arginine (Arg45) located across the carbohydrate-binding site. This structure-based drug design approach has led to the discovery of novel monosaccharide galactose-based antagonists, with the strongest-binding compound (Kd 5.72 µM) holding 7-fold tighter than the disaccharide lactose.

Proteomic analysis of circulating extracellular vesicles identifies potential markers of breast cancer progression, recurrence, and response.

Proteomic profiling of circulating small extracellular vesicles (sEVs) represents a promising, noninvasive approach for early detection and therapeutic monitoring of breast cancer (BC). We describe a relatively low-cost, fast, and reliable method to isolate sEVs from plasma of BC patients and analyze their protein content by semiquantitative proteomics. sEV-enriched fractions were isolated from plasma of healthy controls and BC patients at different disease stages before and after surgery. Proteomic analysis of sEV-enriched fractions using reverse phase protein array revealed a signature of seven proteins that differentiated BC patients from healthy individuals, of which FAK and fibronectin displayed high diagnostic accuracy. The size of sEVs was significantly reduced in advanced disease stage, concomitant with a stage-specific protein signature. Furthermore, we observed protein-based distinct clusters of healthy controls, chemotherapy-treated and untreated postsurgery samples, as well as a predictor of high risk of cancer relapse, suggesting that the applied methods warrant development for advanced diagnostics.

Synthetic lethal combination targeting BET uncovered intrinsic susceptibility of TNBC to ferroptosis.

Identification of targeted therapies for TNBC is an urgent medical need. Using a drug combination screen reliant on synthetic lethal interactions, we identified clinically relevant combination therapies for different TNBC subtypes. Two drug combinations targeting the BET family were further explored. The first, targeting BET and CXCR2, is specific for mesenchymal TNBC and induces apoptosis, whereas the second, targeting BET and the proteasome, is effective for major TNBC subtypes and triggers ferroptosis. Ferroptosis was induced at low drug doses and was associated with increased cellular iron and decreased glutathione levels, concomitant with reduced levels of GPX4 and key glutathione biosynthesis genes. Further functional studies, analysis of clinical datasets and breast cancer specimens revealed a unique vulnerability of TNBC to ferroptosis inducers, enrichment of ferroptosis gene signature, and differential expression of key proteins that increase labile iron and decrease glutathione levels. This study identified potent combination therapies for TNBC and unveiled ferroptosis as a promising therapeutic strategy.

Structure-Based Design of a Monosaccharide Ligand Targeting Galectin-8.

Galectin-8 is a ß-galactoside-recognising protein that has a role in the regulation of bone remodelling and is an emerging new target for tackling diseases with associated bone loss. We have designed and synthesised methyl 3-O-[1-carboxyethyl]-ß-d-galactopyranoside (compound 6) as a ligand to target the N-terminal domain of galectin-8 (galectin-8N). Our design involved molecular dynamics (MD) simulations that predicted 6 to mimic the interactions made by the galactose ring as well as the carboxylic acid group of 3'-O-sialylated lactose (3'-SiaLac), with galectin-8N. Isothermal titration calorimetry (ITC) determined that the binding affinity of galectin-8N for 6 was 32.8 µm, whereas no significant affinity was detected for the C-terminal domain of galectin-8 (galectin-8C). The crystal structure of the galectin-8N-6 complex validated the predicted binding conformation and revealed the exact protein-ligand interactions that involve evolutionarily conserved amino acids of galectin and also those unique to galectin-8N for recognition. Overall, we have initiated and demonstrated a rational ligand design campaign to develop a monosaccharide-based scaffold as a binder of galectin-8.

Prolonged Elimination of Negative Feedback Control Mechanisms Along the Insulin Signaling Pathway Impairs ß-Cell Function In Vivo.

Cellular stress and proinflammatory cytokines induce phosphorylation of insulin receptor substrate (IRS) proteins at Ser sites that inhibit insulin and IGF-I signaling. We therefore examined the effects of mutation of five "inhibitory" Ser phosphorylation sites on IRS2 function in transgenic mice that overexpress, selectively in pancreatic ß-cells, either wild-type (WT) or a mutated IRS2 protein (IRS25A). Islets size, number, and mRNA levels of catalase and superoxide dismutase were increased, whereas those of nitric oxide synthase were decreased, in 7- to 10-week-old IRS25A-ß mice compared with IRS2WT-ß mice. However, glucose homeostasis and insulin secretion in IRS25A-ß mice were impaired when compared with IRS2WT-ß mice or to nontransgenic mice. This was associated with reduced mRNA levels of Glut2 and islet ß-cell transcription factors such as Nkx6.1 and MafA Similarly, components mediating the unfolded protein response were decreased in islets of IRS25A-ß mice in accordance with their decreased insulin secretion. The beneficial effects of IRS25A on ß-cell proliferation and ß-cell transcription factors were evident only in 5- to 8-day-old mice. These findings suggest that elimination of inhibitory Ser phosphorylation sites of IRS2 exerts short-term beneficial effects in vivo; however, their sustained elimination leads to impaired ß-cell function.

The mammalian lectin galectin-8 induces RANKL expression, osteoclastogenesis, and bone mass reduction in mice.

Skeletal integrity is maintained by the co-ordinated activity of osteoblasts, the bone-forming cells, and osteoclasts, the bone-resorbing cells. In this study, we show that mice overexpressing galectin-8, a secreted mammalian lectin of the galectins family, exhibit accelerated osteoclasts activity and bone turnover, which culminates in reduced bone mass, similar to cases of postmenopausal osteoporosis and cancerous osteolysis. This phenotype can be attributed to a direct action of galectin-8 on primary cultures of osteoblasts that secrete the osteoclastogenic factor RANKL upon binding of galectin-8. This results in enhanced differentiation into osteoclasts of the bone marrow cells co-cultured with galectin-8-treated osteoblasts. Secretion of RANKL by galectin-8-treated osteoblasts can be attributed to binding of galectin-8 to receptor complexes that positively (uPAR and MRC2) and negatively (LRP1) regulate galectin-8 function. Our findings identify galectins as new players in osteoclastogenesis and bone remodeling, and highlight a potential regulation of bone mass by animal lectins.