A Mesenchymal-Epithelial Transition Factor-Agonistic Antibody Accelerates Cirrhotic Liver Regeneration and Improves Mouse Survival Following Partial Hepatectomy.

Small-for-size syndrome (SFSS) is a common complication following partial liver transplantation and extended hepatectomy. SFSS is characterized by postoperative liver dysfunction caused by insufficient regenerative capacity and portal hyperperfusion and is more frequent in patients with preexisting liver disease. We explored the effect of the Mesenchymal-epithelial transition factor (MET)-agonistic antibody 71D6 on liver regeneration and functional recovery in a mouse model of SFSS. Male C57/BL6 mice were exposed to repeated carbon tetrachloride injections for 10 weeks and then randomized into 2 arms receiving 3 mg/kg 71D6 or a control immunoglobulin G (IgG). At 2 days after the randomization, the mice were subjected to 70% hepatectomy. Mouse survival was recorded up to 28 days after hepatectomy. Satellite animals were euthanized at different time points to analyze liver regeneration, fibrosis, and inflammation. Serum 71D6 administration significantly decreased mouse mortality consequent to insufficient regeneration of the cirrhotic liver. Analysis of liver specimens in satellite animals revealed that 71D6 promoted powerful activation of the extracellular signal-regulated kinase pathway and accelerated liver regeneration, characterized by increased liver-to-body weight, augmented mitotic index, and higher serum albumin levels. Moreover, 71D6 accelerated the resolution of hepatic fibrosis as measured by picrosirius red, desmin, and α-smooth muscle actin staining, and suppressed liver infiltration by macrophages as measured by CD68 and F4/80 staining. Analysis of gene expression by reverse-transcription polymerase chain reaction confirmed that 71D6 administration suppressed the expression of key profibrotic genes, including platelet-derived growth factor, tissue inhibitor of metalloproteinase 3, and transforming growth factor-ß1, and of key proinflammatory genes, including tumor necrosis factor-α, interleukin-1ß, chemokine (C-C motif) ligand 3, and chemokine (C-C motif) ligand 5. These results suggest that activating the MET pathway via an hepatocyte growth factor-mimetic antibody may be beneficial in patients with SFSS and possibly other types of acute and chronic liver disorders.

[Kidney transplantation in pediatric age.] / Trasplante renal pediátrico.

Kidney transplantation (KT) is the best treatment for children in end-stage renal disease. KT has less mortality than dialysis and provides a better quality of life. Thus, the inclusion criteria have been progressively broadened. Histocompatibility and the source of donation are the most relevant factors that influence graft survival. Graft and patient survival have improved dramatically in recent decades, coming close to the results of KT in adults. Some of the specific factors that differentiate it from the adult are: donor-recipient size mismatch,the impact on growth and therapeutic non-compliance. Overall graft survival at 5-years is 90% for living donor KT and 70% for cadaveric donor KT.The most frequent cause of graft loss is chronic rejection.Mortality in the first post-transplant years is less than 6.5%. Infections and cardiovascular complications are the main causes of transplant-related death.Despite the good results, it is imperative to continue investigating how to achieve immunological tolerance. In order to improve the long-term results of the kidney graftis necessary to reduce immunosuppressive treatment and its side effects, such as chronic rejection.

El Trasplante Renal (TR) es el tratamiento de elección para los niños que se encuentran en insuficiencia renal terminal. Los criterios de inclusión se han ido ampliando de manera progresiva al conocerse que su mortalidad es menor que la que ocurre en diálisis y proporciona una mejor calidad de vida. La histocompatibilidad y la fuente de donación son, de entre los numerosos factores que influyen en la supervivencia del injerto, los de mayor relevancia. La supervivencia del injerto y la del paciente han mejorado de forma espectacular en las últimas décadas, aproximándose a los resultados del TR en el adulto. La diferencia de tamaño entre donante y receptor, la afectación del crecimiento y la falta de cumplimiento terapéutico, son algunos de los factores específicos que lo diferencian del adulto.La supervivencia global del injerto a los 5 años es del 90% para el TR de donante vivo y del 70% para el TRde donante cadáver.La causa más frecuente de pérdida del injerto es el rechazo crónico. La mortalidad en los primeros años post-trasplante es inferior al 6,5%. La infección y las complicaciones cardiovasculares son las causas principales de muerte relacionada con el trasplante.Sin embargo, a pesar de estos buenos resultados, es preciso continuar investigando en cómo alcanzar la tolerancia inmunológica, que permita reducir el tratamiento inmunosupresor y sus efectos colaterales, entre los que se encuentra el rechazo crónico; y así poder mejorar los resultados a largo plazo del injerto renal.

Trasplante renal pediátrico / Pediatric kidney transplant

El Trasplante Renal (TR) es el tratamientode elección para los niños que se encuentran en insufi-ciencia renal terminal. Los criterios de inclusión se hanido ampliando de manera progresiva al conocerse quesu mortalidad es menor que la que ocurre en diálisis yproporciona una mejor calidad de vida. La histocompatibilidad y la fuente de donación son, de entre losnumerosos factores que influyen en la supervivencia delinjerto, los de mayor relevancia. La supervivencia delinjerto y la del paciente han mejorado de forma espectacular en las últimas décadas, aproximándose a losresultados del TR en el adulto. La diferencia de tamañoentre donante y receptor, la afectación del crecimientoy la falta de cumplimiento terapéutico, son algunos delos factores específicos que lo diferencian del adulto. La supervivencia global del injerto a los 5 años es del90% para el TR de donante vivo y del 70% para el TRde donante cadáver.La causa más frecuente de pérdida del injerto es elrechazo crónico. La mortalidad en los primeros añospost-trasplante es inferior al 6,5%. La infección y lascomplicaciones cardiovasculares son las causas principales de muerte relacionada con el trasplante.Sin embargo, a pesar de estos buenos resultados, espreciso continuar investigando en cómo alcanzar latolerancia inmunológica, que permita reducir el tratamiento inmunosupresor y sus efectos colaterales, entrelos que se encuentra el rechazo crónico; y así podermejorar los resultados a largo plazo del injerto renal.(AU)

Kidney transplantation (KT) is the besttreatment for children in end-stage renal disease. KT hasless mortality than dialysis and provides a better qualityof life. Thus, the inclusion criteria have been progressively broadened. Histocompatibility and the source ofdonation are the most relevant factors that influence graftsurvival. Graft and patient survival have improved dramatically in recent decades, coming close to the resultsof KT in adults. Some of the specific factors that differentiate it from the adult are: donor-recipient size mismatch,the impact on growth and therapeutic non-compliance.Overall graft survival at 5-years is 90% for living donorKT and 70% for cadaveric donor KT.The most frequent cause of graft loss is chronic rejection. Mortality in the first post-transplant years is less than 6.5%. Infections and cardiovascular complications arethe main causes of transplant-related death.Despite the good results, it is imperative to continue investigating how to achieve immunological tolerance. Inorder to improve the long-term results of the kidney graftis necessary to reduce immunosuppressive treatment andits side effects, such as chronic rejection.(AU)

Microscope-Based Automated Quantification of Liver Fibrosis in Mice Using a Deep Learning Algorithm.

In preclinical studies that involve animal models for hepatic fibrosis, accurate quantification of the fibrosis is of utmost importance. The use of digital image analysis based on deep learning artificial intelligence (AI) algorithms can facilitate accurate evaluation of liver fibrosis in these models. In the present study, we compared the quantitative evaluation of collagen proportionate area in the carbon tetrachloride model of liver fibrosis in the mouse by a newly developed AI algorithm to the semiquantitative assessment of liver fibrosis performed by a board-certified toxicologic pathologist. We found an excellent correlation between the 2 methods of assessment, most evident in the higher magnification (×40) as compared to the lower magnification (×10). These findings strengthen the confidence of using digital tools in the toxicologic pathology field as an adjunct to an expert toxicologic pathologist.

The conformational state of hERG1 channels determines integrin association, downstream signaling, and cancer progression.

Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell-extracellular matrix (ECM) transmembrane receptors called integrins. K+ flux through the human ether-à-go-go-related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in tumors, where it modulates integrin-mediated signaling. We found that hERG1 interacted with the ß1 integrin subunit at the plasma membrane of human cancer cells. This interaction was not detected in cardiomyocytes because of the presence of the hERG1 auxiliary subunit KCNE1 (potassium voltage-gated channel subfamily E regulatory subunit 1), which blocked the ß1 integrin-hERG1 interaction. Although open hERG1 channels did not interact as strongly with ß1 integrins as did closed channels, current flow through hERG1 channels was necessary to activate the integrin-dependent phosphorylation of Tyr397 in focal adhesion kinase (FAK) in both normal and cancer cells. In immunodeficient mice, proliferation was inhibited in breast cancer cells expressing forms of hERG1 with impaired K+ flow, whereas metastasis of breast cancer cells was reduced when the hERG1/ß1 integrin interaction was disrupted. We conclude that the interaction of ß1 integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression.

Stroma-derived HGF drives metabolic adaptation of colorectal cancer to angiogenesis inhibitors.

The role of paracrine Hepatocyte Growth Factor (HGF) in the resistance to angiogenesis inhibitors (AIs) is hidden in xenograft models because mouse HGF fails to fully activate human MET. To uncover it, we compared the efficacy of AIs in wild-type and human HGF knock-in SCID mice bearing orthotopic human colorectal tumors. Species-specific HGF/MET signaling dramatically impaired the response to anti-angiogenic agents and boosted metastatic dissemination. In cell-based assays mimicking the consequences of anti-angiogenic therapy, colorectal cancer cells were completely resistant to hypoxia but extremely sensitive to nutrient deprivation. Starvation-induced apoptosis could be prevented by HGF, which promoted GLUT1-mediated glucose uptake, sustained glycolysis and activated autophagy. Pharmacological inhibition of GLUT1 in the presence of glucose killed tumor cells as effectively as glucose deprivation, and this effect was antagonized by HGF. Concomitant targeting of GLUT1 and HGF potently suppressed growth and dissemination of AI-resistant human tumors in human HGF knock-in SCID mice without exacerbating tumor hypoxia. These data suggest that stroma-derived HGF protects CRC cells against glucose starvation-induced apoptosis, promoting resistance to both AIs and anti-glycolytic agents. Combined inhibition of glucose metabolism and HGF/MET signaling ('anti-METabolic therapy') may represent a more effective CRC treatment compared to utterly blocking tumor blood supply.

Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy.

Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo.

Depleting MET-Expressing Tumor Cells by ADCC Provides a Therapeutic Advantage over Inhibiting HGF/MET Signaling.

Hepatocyte growth factor (HGF) and its receptor MET represent validated targets for cancer therapy. However, HGF/MET inhibitors being explored as cancer therapeutics exhibit cytostatic activity rather than cytotoxic activity, which would be more desired. In this study, we engineered an antagonistic anti-MET antibody that, in addition to blocking HGF/MET signaling, also kills MET-overexpressing cancer cells by antibody-dependent cellular cytotoxicity (ADCC). As a control reagent, we engineered the same antibody in an ADCC-inactive form that is similarly capable of blocking HGF/MET activity, but in the absence of any effector function. In comparing these two antibodies in multiple mouse models of cancer, including HGF-dependent and -independent tumor xenografts, we determined that the ADCC-enhanced antibody was more efficacious than the ADCC-inactive antibody. In orthotopic mammary carcinoma models, ADCC enhancement was crucial to deplete circulating tumor cells and to suppress metastases. Prompted by these results, we optimized the ADCC-enhanced molecule for clinical development, generating an antibody (ARGX-111) with improved pharmacologic properties. ARGX-111 competed with HGF for MET binding, inhibiting ligand-dependent MET activity, downregulated cell surface expression of MET, curbing HGF-independent MET activity, and engaged natural killer cells to kill MET-expressing cancer cells, displaying MET-specific cytotoxic activity. ADCC assays confirmed the cytotoxic effects of ARGX-111 in multiple human cancer cell lines and patient-derived primary tumor specimens, including MET-expressing cancer stem-like cells. Together, our results show how ADCC provides a therapeutic advantage over conventional HGF/MET signaling blockade and generates proof-of-concept for ARGX-111 clinical testing in MET-positive oncologic malignancies.

Four individually druggable MET hotspots mediate HGF-driven tumor progression.

Activation of MET by HGF plays a key role in tumor progression. Using a recently developed llama platform that generates human-like immunoglobulins, we selected 68 different antibodies that compete with HGF for binding to MET. HGF-competing antibodies recognized 4 distinct hotspots localized in different MET domains. We identified 1 hotspot that coincides with the known HGF ß chain binding site on blades 2-3 of the SEMA domain ß-propeller. We determined that a second and a third hotspot lie within blade 5 of the SEMA domain and IPT domains 2-3, both of which are thought to bind to HGF α chain. Characterization of the fourth hotspot revealed a region across the PSI-IPT 1 domains not previously associated with HGF binding. Individual or combined targeting of these hotspots effectively interrupted HGF/MET signaling in multiple cell-based biochemical and biological assays. Selected antibodies directed against SEMA blades 2-3 and the PSI-IPT 1 region inhibited brain invasion and prolonged survival in a glioblastoma multiforme model, prevented metastatic disease following neoadjuvant therapy in a triple-negative mammary carcinoma model, and suppressed cancer cell dissemination to the liver in a KRAS-mutant metastatic colorectal cancer model. These results identify multiple regions of MET responsible for HGF-mediated tumor progression, unraveling the complexity of HGF-MET interaction, and provide selective molecular tools for targeting MET activity in cancer.

The mitochondrial chaperone TRAP1 promotes neoplastic growth by inhibiting succinate dehydrogenase.

We report that the mitochondrial chaperone TRAP1, which is induced in most tumor types, is required for neoplastic growth and confers transforming potential to noncancerous cells. TRAP1 binds to and inhibits succinate dehydrogenase (SDH), the complex II of the respiratory chain. The respiratory downregulation elicited by TRAP1 interaction with SDH promotes tumorigenesis by priming the succinate-dependent stabilization of the proneoplastic transcription factor HIF1α independently of hypoxic conditions. These findings provide a mechanistic clue to explain the switch to aerobic glycolysis of tumors and identify TRAP1 as a promising antineoplastic target.

Integrins and signal transduction.

Integrin signaling has a critical function in organizing cells in tissues during both embryonic development and tissue repair. Following their binding to the extracellular ligands, the intracellular signaling pathways triggered by integrins are directed to two major functions: organization of the actin cytoskeleton and regulation of cell behaviour including survival, differentiation and growth. Basic research conducted in the past twelve years has lead to remarkable breakthroughs in this field. Integrins are catalytically inactive and translate positional cues into biochemical signals by direct and/or functional association with intracellular adaptors, cytosolic tyrosine kinases or growth factor and cytokine receptors. The purpose of this chapter is to highlight recent experimental and conceptual advances in integrin signaling with particular emphasis on the ability of integrins to regulate Fak/Src family kinases (SFKs) activation and the cross-talk with soluble growth factors receptors and cytokines.

Convergence of integrins and EGF receptor signaling via PI3K/Akt/FoxO pathway in early gene Egr-1 expression.

The early gene early growth response (Egr-1), a broadly expressed member of the zing-finger family of transcription factors, is induced in many cell types by a variety of growth and differentiation stimuli, including epidermal growth factor (EGF). Here we demonstrate that Egr-1 expression is mainly regulated by integrin-mediated adhesion. Integrin-dependent adhesion plays a dual role in Egr-1 regulation, either being sufficient "per se" to induce Egr-1, or required for EGF-dependent expression of Egr-1, which occurs only in adherent cells and not in cells in suspension. To dissect the molecular basis of integrin-dependent Egr-1 regulation, we show by FLIM-based FRET that in living cells beta1-integrin associates with the EGF receptor (EGFR) and that EGF further increases the extent complex formation. Interestingly, Egr-1 induction depends on integrin-dependent PI3K/Akt activation, as indicated by the decrease in Egr-1 levels in presence of the pharmacological inhibitor LY294002, the kinase-defective Akt mutant and Akt1/2 shRNAs. Indeed, upon adhesion activated Akt translocates into the nucleus and phosphorylates FoxO1, a Forkhead transcription factors. Consistently, FoxO1silencing results in Egr-1-increased levels, indicating that FoxO1 behaves as a negative regulator of Egr-1 expression. These data demonstrate that integrin/EGFR cross-talk is required for expression of Egr-1 through a novel regulatory cascade involving the activation of the PI3K/Akt/Forkhead pathway.