Role of NADPH oxidases in the redox biology of liver fibrosis

Liver fibrosis is the pathological consequence of chronic liver diseases, where an excessive deposition of extracellular matrix (ECM) proteins occurs, concomitantly with the processes of repair and regeneration. It is characterized by increased production of matrix proteins, in particular collagens, and decreased matrix remodelling. The principal source of ECM accumulation is myofibroblasts (MFB). Most fibrogenic MFB are endogenous to the liver, coming from hepatic stellate cells (HSC) and portal fibroblasts. Dysregulated inflammatory responses have been associated with most (if not all) hepatotoxic insults and chronic oxidative stress play a role during the initial liver inflammatory phase and its progression to fibrosis. Redox-regulated processes are responsible for activation of HSC to MFB, as well as maintenance of the MFB function. Increased oxidative stress also induces hepatocyte apoptosis, which contributes to increase the liver injury and to transdifferentiate HSC to MFB, favouring the fibrogenic process. Mitochondria and other redox-active enzymes can generate superoxide and hydrogen peroxide as a by-product in liver cells. Moreover, accumulating evidence indicates that NADPH oxidases (NOXs), which play a critical role in the inflammatory response, may contribute to reactive oxygen species (ROS) production during liver fibrosis, being important players in HSC activation and hepatocyte apoptosis. Based on the knowledge of the pathogenic role of ROS, different strategies to prevent or reverse the oxidative damage have been developed to be used as therapeutic tools in liver fibrosis. This review will update all these concepts, highlighting the relevance of redox biology in chronic fibrogenic liver pathologies

A preclinical pipeline to evaluate migrastatics as therapeutic agents in metastatic melanoma

© The Author(s) 2021.[Background]: Metastasis is a hallmark of cancer and responsible for most cancer deaths. Migrastatics were defined as drugs interfering with all modes of cancer cell invasion and thus cancers’ ability to metastasise. First anti-metastatic treatments have recently been approved. [Methods]: We used bioinformatic analyses of publicly available melanoma databases. Experimentally, we performed in vitro target validation (including 2.5D cell morphology analysis and mass spectrometric analysis of RhoA binding partners), developed a new traceable spontaneously metastasising murine melanoma model for in vivo validation, and employed histology (haematoxylin/eosin and phospho-myosin II staining) to confirm drug action in harvested tumour tissues. [Results]: Unbiased and targeted bioinformatic analyses identified the Rho kinase (ROCK)-myosin II pathway and its various components as potentially relevant targets in melanoma. In vitro validation demonstrated redundancy of several RhoGEFs upstream of RhoA and confirmed ROCK as a druggable target downstream of RhoA. The anti-metastatic effects of two ROCK inhibitors were demonstrated through in vivo melanoma metastasis tracking and inhibitor effects also confirmed ex vivo by digital pathology. [Conclusions]: We proposed a migrastatic drug development pipeline. As part of the pipeline, we provide a new traceable spontaneous melanoma metastasis model for in vivo quantification of metastasis and anti-metastatic effects by non-invasive imaging.GOF’s lab was supported by Cancer Research UK [C48390/A21153], Worldwide Cancer Research [16-1153], and King’s Health Partners [King’s Medical Research Trust Joint Research Committee studentship to A.V.]. B.F. was supported by a King’s Health Partners studentship to V.S.M. and G.O.F. V.S.M.’s lab was supported by Cancer Research UK [C33043/A12065] and [C33043/A24478] (V.S.M., E.C.M., J.L.O., L.B. and GC), the Royal Society [RG110591] (V.S.M.), The Harry J. Lloyd Charitable Trust (J.L.O. and V.S.M.), the Barts Charity (V.S.M., J.L.O., O.M., I.R.H. and E.C.M.), the Fundacion Alfonso Martin Escudero and Marie Sklodowska-Curie Action [H2020-MSCA-IF-2014-EF-ST] (I.R.H.), and Fundacion Ramon Areces (E.C.M.). F.M. was supported by an MRC Career Development Award (MR/P009417/1). This work was further supported by the Department of Health (DoH) via the National Institute for Health Research (NIHR) Comprehensive Biomedical Research Centre award to King’s Health Partners, and the Wellcome/EPSRC Centre for Medical Engineering [WT203148/Z/16/Z]. Views expressed are those of the authors and not necessarily those of the NHS, NIHR or DoH

Downregulation of epidermal growth factor receptor in hepatocellular carcinoma facilitates transforming growth factor-β-induced epithelial to amoeboid transition

The Epidermal Growth Factor Receptor (EGFR) and the Transforming Growth Factor-beta (TGF-β) are key regulators of hepatocarcinogenesis. Targeting EGFR was proposed as a promising therapy; however, poor success was obtained in human hepatocellular carcinoma (HCC) clinical trials. Here, we describe how EGFR is frequently downregulated in HCC patients while TGF-β is upregulated. Using 2D/3D cellular models, we show that after EGFR loss, TGF-β is more efficient in its pro-migratory and invasive effects, inducing epithelial to amoeboid transition. EGFR knock-down promotes loss of cell-cell and cell-to-matrix adhesion, favouring TGF-β-induced actomyosin contractility and acquisition of an amoeboid migratory phenotype. Moreover, TGF-β upregulates RHOC and CDC42 after EGFR silencing, promoting Myosin II in amoeboid cells. Importantly, low EGFR combined with high TGFB1 or RHOC/CDC42 levels confer poor patient prognosis. In conclusion, this work reveals a new tumour suppressor function for EGFR counteracting TGF-β-mediated epithelial to amoeboid transitions in HCC, supporting a rational for targeting the TGF-β pathway in patients with low EGFR expression. Our work also highlights the relevance of epithelial to amoeboid transition in human tumours and the need to better target this process in the clinic

Non-muscle Myosin II reactivation and cytoskeletal remodelling as a new vulnerability in therapy-resistant melanoma

Trabajo presentado en el 3rd ASEICA Educational Symposium, celebrado en modalidad virtual del 23 al 25 de noviembre de 2021.MAPK-targeted therapies (MAPKi) and immune checkpoint blockers (ICB) improve survival of subsets of melanoma patients. However, therapy resistance is a persistent problem. Cross-resistance to MAPKi and ICB may be driven by common transcriptomic alterations in pathways controlling invasion and metastasis. Using phosphoproteomic and transcriptomic analyses, we find that adaptation to treatment and acquisition of resistance to MAPKi involve cytoskeletal remodelling and changes in levels in the ROCK-non-muscle Myosin II (NMII) pathway, which is essential for cancer invasion and metastasis. NMII activity is decreased shortly after MAPK is blocked. However, persister cells promptly restore NMII activity to increase survival, and this becomes a vulnerability, since survival of MAPKi- and ICB-resistant cells is highly dependent on ROCK-NMII. Efficacy of MAPKi and ICB can be improved by combination with ROCK inhibitors, which have a dual action by impairing melanoma cell survival (through induction of lethal reactive oxygen species and unresolved DNA damage) and reducing myeloid- and lymphoid-driven immunosuppression, ultimately overcoming cross-resistance in vivo. In human tumours, high ROCK-NMII levels identify MAPKi-, ICB-resistant melanomas, and treatment-naïve melanomas with worse prognosis. Therefore, a subset of MAPKi- and ICB-resistant melanomas is more susceptible to ROCK-NMII blockade, suggesting clinical opportunities for combination therapies

The Myosin II cytoskeleton as a new vulnerability in therapy-resistant melanoma

Trabajo presentado en VIB Conference: Tumor Heterogeneity, Plasticity and Therapy, celebrado en modalidad virtual del 05 al 06 de mayo de 2021.MAPK-targeted therapies (MAPKi) and immune checkpoint blockers (ICB) improve survival of subsets of melanoma patients. However, therapy resistance is a persistent problem. Cross-resistance to MAPKi and ICB has been suggested to be driven, in part, by common transcriptomic alterations in pathways controlling invasion and metastasis. We find that adaptation to treatment and acquisition of resistance to MAPKi involve cytoskeletal remodelling and changes in expression levels in the ROCK-Myosin II pathway, which plays a key role in cancer invasion and metastasis. Myosin II activity is decreased shortly after MAPK is blocked. However, resistant cells promptly restore Myosin II activity to increase survival, and this becomes a vulnerability, since survival of MAPKi- and ICB-resistant cells is highly dependent on ROCK-Myosin II. Efficacy of MAPKi and ICB can be improved by combination with ROCK inhibitors, which have a dual action by impairing melanoma cell survival (through induction of lethal reactive oxygen species and unresolved DNA damage) and myeloid- and lymphoiddriven immunosuppression, overcoming cross-resistance. In human tumours, high ROCK-Myosin II activity and their associated transcriptome identify MAPKi-, ICBresistant melanomas, and treatment-naïve melanomas with worse prognosis. Therefore, a subset of MAPKi- and ICB-resistant melanomas is intrinsically more susceptible to ROCK-Myosin II inhibition, suggesting clinical opportunities for combination therapies

The Myosin II cytoskeleton as a new vulnerability in therapy-resistant melanoma

Trabajo presentado en la EACR-AstraZeneca Virtual Conference ‘Drug Tolerant Persister Cells’, celebrada del 07 al 08 de diciembre de 2021.MAPK-targeted therapies (MAPKi) and immune checkpoint blockers (ICB) improve survival of subsets of melanoma patients. However, therapy resistance is a persistent problem. Cross-resistance to MAPKi and ICB may be driven by common transcriptomic alterations in pathways controlling invasion and metastasis. We find that adaptation to treatment and acquisition of resistance to MAPKi involve cytoskeletal remodelling and changes in expression levels in the ROCK-non-muscle Myosin II (NMII) pathway, which is essential for cancer invasion and metastasis. NMII activity is decreased shortly after MAPK is blocked. However, persister cells promptly restore NMII activity to increase survival, and this becomes a vulnerability, since survival of MAPKi- and ICB-resistant cells is highly dependent on ROCK-NMII. Efficacy of MAPKi and ICB can be improved by combination with ROCK inhibitors, which have a dual action by impairing melanoma cell survival (through induction of lethal reactive oxygen species and unresolved DNA damage) and reducing myeloid- and lymphoid-driven immunosuppression, ultimately overcoming cross-resistance. In human tumours, high ROCK-NMII levels identify MAPKi-, ICB-resistant melanomas, and treatment-naïve melanomas with worse prognosis. Therefore, a subset of MAPKi- and ICB-resistant melanomas is more susceptible to ROCK-NMII blockade, suggesting clinical opportunities for combination therapies

The Myosin II cytoskeleton as a new vulnerability in therapy-resistant melanoma

Trabajo presentado en el XIX Congreso de la Sociedad Española de Biología Celular, celebrado en Boadilla del Monte (España) del 26 al 29 de octubre de 2021.MAPK-targeted therapies (MAPKi) and immune checkpoint blockers (ICB) improve survival of subsets of melanoma patients. However, therapy resistance is a persistent problem. Cross-resistance to MAPKi and ICB has been suggested to be driven, in part, by common transcriptomic alterations in pathways controlling invasion and metastasis. We find that adaptation to treatment and acquisition of resistance to MAPKi involve cytoskeletal remodelling and changes in expression levels in the ROCK-Myosin II pathway, which plays a key role in cancer invasion and metastasis. Myosin II activity is decreased shortly after MAPK is blocked. However, resistant cells promptly restore Myosin II activity to increase survival, and this becomes a vulnerability, since survival of MAPKi- and ICB-resistant cells is highly dependent on ROCK-Myosin II. Efficacy of MAPKi and ICB can be improved by combination with ROCK inhibitors, which have a dual action by impairing melanoma cell survival (through induction of lethal reactive oxygen species, unresolved DNA damage and cell cycle arrest) and myeloid- and lymphoid-driven immunosuppression, ultimately overcoming cross-resistance. In human tumours, high ROCK-Myosin II activity and their associated transcriptome identify MAPKi-, ICB-resistant melanomas, and treatment-naïve melanomas with worse prognosis. Therefore, a subset of MAPKi- and ICB-resistant melanomas is intrinsically more susceptible to ROCK-Myosin II inhibition, suggesting clinical opportunities for combination therapies

AMPK is a mechano-metabolic sensor linking cell adhesion and mitochondrial dynamics to Myosin-dependent cell migration

Cell migration is crucial for cancer dissemination. We find that AMP-activated protein kinase (AMPK) controls cell migration by acting as an adhesion sensing molecular hub. In 3-dimensional matrices, fast-migrating amoeboid cancer cells exert low adhesion/low traction linked to low ATP/AMP, leading to AMPK activation. In turn, AMPK plays a dual role controlling mitochondrial dynamics and cytoskeletal remodelling. High AMPK activity in low adhering migratory cells, induces mitochondrial fission, resulting in lower oxidative phosphorylation and lower mitochondrial ATP. Concurrently, AMPK inactivates Myosin Phosphatase, increasing Myosin II-dependent amoeboid migration. Reducing adhesion or mitochondrial fusion or activating AMPK induces efficient rounded-amoeboid migration. AMPK inhibition suppresses metastatic potential of amoeboid cancer cells in vivo, while a mitochondrial/AMPK-driven switch is observed in regions of human tumours where amoeboid cells are disseminating. We unveil how mitochondrial dynamics control cell migration and suggest that AMPK is a mechano-metabolic sensor linking energetics and the cytoskeleton. Cell metabolism must adapt to the energy needs of migrating cells. This study finds that fast amoeboid migrating cells harbor high AMPK activity, which controls both mitochondrial dynamics and cytoskeletal remodeling, enabling reduced energy needs

Role of the NADPH Oxidase NOX4 in Liver Fibrosis and Hepatocarcinogenesis

[spa] La familia de las NADPH oxidasas (NOXes) se considera una de las principales fuentes de radicales libres de oxígeno (ROS) involucrados en señalización celular. Se ha descrito que en el hígado se expresan las isoformas NOX1, NOX2 y NOX4, y trabajos previos del grupo han demostrado que estas pueden jugar papeles diferentes e incluso antagónicos. En este trabajo analizamos el papel de NOX4 en diferentes procesos fisiopatológicos del hígado. Describimos la relevancia de NOX4 en la inducción de la fibrosis hepática, mediando los efectos del TGF-ß, tanto a nivel de transactivación de las células hepáticas estrelladas a miofibroblastos, como a nivel de inducción de apoptosis en hepatocitos. También analizamos la relevancia de la vía del TGF-ß en los procesos de hepatocarcinogenesis. Niveles altos de TGF-ß autocrino provocan la pérdida de características epiteliales, la ganancia de características mesenquimales y la resistencia a los efectos del TGF-ß como supresor tumoral. Además mostramos una correlación inversa entre los niveles de expresión de TGF-ß y NOX4. Por otro lado demostramos que NOX4 juega un papel como inhibidor del crecimiento tanto en células de hepatocarcinoma humano como en hepatocitos no transformados. Además, mostramos una caída en los niveles de NOX4 durante la regeneración hepática así como durante la hepatocarcinogenesis inducida por Dietilnitrosamina (DEN) en modelos murinos. También mostramos una mayor capacidad tumorigénica de las células cuando la expresión de NOX4 esta atenuada. Además, los niveles de NOX4 se encuentran disminuidos en muestras de tejidos humanos de hepatocarcinoma. Finalmente, analizamos el papel de NOX4 en la capacidad metastásica de células hepatocarcinoma. Describimos que la disminución en los niveles de NOX4 incrementa la capacidad de crecimiento invasivo de las células en una matriz de colágeno. Además, niveles bajos de NOX4 correlacionan con una mayor contractilidad de la actomiosina y una destrucción de las estructuras parenquimáticas de las células, disminuyendo las adhesiones célula-célula y célula-matriz extracelular, incrementando la capacidad invasiva de las mismas. En resumen, en este trabajo describimos que en el hígado, NOX4 tiene un papel pro­fibrótico mediando la activación de las HSC a miofibroblastos y la muerte de los hepatocitos inducidas por TGF-ß, pero también tiene un papel como supresor tumoral durante la carcinogénesis, por ser un regulador negativo del crecimiento tumoral y de la invasión.[eng] Reactive oxygen species (ROS) are short-lived, highly electrophilic molecules generated by the partial reduction of oxygen to form superoxide, hydrogen peroxide and hydroxyl radical as well as other secondary metabolites. For years, ROS have been considered as damaging molecules; however, in the last 20 years it has been proved that low levels of ROS can provoke transient and reversible modifications, leading to the regulation of signalling pathways. Within the cell there are numerous potential sources of ROS. They can be produced as by-products of enzymatic processes, which is considered the case of mitochondrial ROS, but they can also be produced as primary species, which is the case of ROS from the NADPH oxidase (NOX) family of enzymes that has emerged in the last years as an important source of ROS in signal transduction. NOX-derived ROS have been implicated in regulation of cytoskeletal remodelling, gene expression, proliferation, differentiation, migration and cell death. The NOX family is composed of seven members, NOX1-5 and two dual oxidases (DUOX1-2), which share analogies in structure and catalytic function, since all of them are transmembrane flavoproteins that mediate the reduction of oxygen using NADPH as an electron donor. However, important differences in regulation and cellular functions are observed among them. It has been reported that NOX1, NOX2 and NOX4 are expressed in the liver. Indeed, previous works from our group demonstrated that different NOX isoforms play different functions, sometimes even opposite roles, in hepatocytes. In this work we analyse the role of the NADPH oxidase NOX4 in different physiological and pathological situations of the liver such as liver regeneration, liver fibrosis and hepatocarcinogenesis. First, we describe the role of NOX4 during liver fibrosis. We show that Nox4 expression is up-regulated during liver fibrosis in two different mice models, and it is required for TGF-ß­induced transdifferentiation of hepatic stellate cells (HSC) to myofibroblasts, as well as for hepatocyte apoptosis. Secondly, we analyse the relevance of the autocrine activation of the Transforming Growth Factor-beta (TGF-ß) pathway during hepatocarcinogenesis and its relation with NOX4. We show that high levels of autocrine TGF-ß provoke loss of epithelial characteristics, gain of mesenchymal features and resistance to TGF-ß suppressor effects, in different human hepatocellular carcinoma (HCC) cells. Surprisingly, there is an inverse correlation between autocrine TGF-ß and NOX4 expression in HCC cell lines. Another important point in this work is the study of the role of NOX4 in the proliferative capacity of hepatocytes, as well as in their tumorigenic capacity. Results show that NOX4 is a negative regulator of proliferation in both untransformed hepatocytes and liver tumour cells. Moreover, its expression is down-regulated during liver regeneration after partial hepatectomy in mice. Furthermore, Nox4 expression is also down-regulated during Diethylnitrosamine (DEN)­induced liver tumorigenesis in mice, and targeting knock-down of NOX4 in HCC cells confers them advantage for tumour progression in xenograft models in mice. Low levels of NOX4 are found in human HCC cell lines and in a relevant percentage of human liver tumour tissues. Finally, we analyse the role of NOX4 in the metastatic capacity of HCC cell lines. We describe that down-regulation of NOX4 increases the invasive growth capacity of HCC cells when embedded in a pliable matrix, increasing both proliferation and invasion. Interestingly, low levels of NOX4 correlate with high actomyosin contractility, which provokes loss of epithelial parenchymal characteristics, decreasing both cell-to-cell contacts and cell-to-matrix adhesion, and increasing invasive capacity. In summary, in the liver, NOX4 plays pro-fibrotic roles, mediating TGF-ß-induced HSC activation and hepatocyte death, but plays a tumour suppressor role during carcinogenesis, being a negative regulator of liver tumour growth and invasion

Role of NADPH oxidases in the redox biology of liver fibrosis

AbstractLiver fibrosis is the pathological consequence of chronic liver diseases, where an excessive deposition of extracellular matrix (ECM) proteins occurs, concomitantly with the processes of repair and regeneration. It is characterized by increased production of matrix proteins, in particular collagens, and decreased matrix remodelling. The principal source of ECM accumulation is myofibroblasts (MFB). Most fibrogenic MFB are endogenous to the liver, coming from hepatic stellate cells (HSC) and portal fibroblasts. Dysregulated inflammatory responses have been associated with most (if not all) hepatotoxic insults and chronic oxidative stress play a role during the initial liver inflammatory phase and its progression to fibrosis. Redox-regulated processes are responsible for activation of HSC to MFB, as well as maintenance of the MFB function. Increased oxidative stress also induces hepatocyte apoptosis, which contributes to increase the liver injury and to transdifferentiate HSC to MFB, favouring the fibrogenic process. Mitochondria and other redox-active enzymes can generate superoxide and hydrogen peroxide as a by-product in liver cells. Moreover, accumulating evidence indicates that NADPH oxidases (NOXs), which play a critical role in the inflammatory response, may contribute to reactive oxygen species (ROS) production during liver fibrosis, being important players in HSC activation and hepatocyte apoptosis. Based on the knowledge of the pathogenic role of ROS, different strategies to prevent or reverse the oxidative damage have been developed to be used as therapeutic tools in liver fibrosis. This review will update all these concepts, highlighting the relevance of redox biology in chronic fibrogenic liver pathologies