Specific features of ß-catenin-mutated hepatocellular carcinomas.

CTNNB1, encoding the ß-catenin protein, is a key oncogene contributing to liver carcinogenesis. Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer in adult, representing the third leading cause of cancer-related death. Aberrant activation of the Wnt/ß-catenin pathway, mainly due to mutations of the CTNNB1 gene, is observed in a significant subset of HCC. In this review, we first resume the major recent advances in HCC classification with a focus on CTNNB1-mutated HCC subclass. We present the regulatory mechanisms involved in ß-catenin stabilisation, transcriptional activity and binding to partner proteins. We then describe specific phenotypic characteristics of CTNNB1-mutated HCC thanks to their unique gene expression patterns. CTNNB1-mutated HCC constitute a full-fledged subclass of HCC with distinct pathological features such as well-differentiated cells with low proliferation rate, association to cholestasis, metabolic alterations, immune exclusion and invasion. Finally, we discuss therapeutic approaches to target ß-catenin-mutated liver tumours and innovative perspectives for future drug developments.

@TOME 3.0: Interfacing Protein Structure Modeling and Ligand Docking.

Knowledge of protein-ligand complexes is essential for efficient drug design. Virtual docking can bring important information on putative complexes but it is still far from being simultaneously fast and accurate. Receptors are flexible and adapt to the incoming small molecules while docking is highly sensitive to small conformational deviations. Conformation ensemble is providing a mean to simulate protein flexibility. However, modeling multiple protein structures for many targets is seldom connected to ligand screening in an efficient and straightforward manner. @TOME-3 is an updated version of our former pipeline @TOME-2, in which protein structure modeling is now directly interfaced with flexible ligand docking. Sequence-sequence profile comparisons identify suitable PDB templates for structure modeling and ligands from these templates are used to deduce binding sites to be screened. In addition, bound ligand can be used as pharmacophoric restraint during the virtual docking. The latter is performed by PLANTS while the docking poses are analysed through multiple chemoinformatics functions. This unique combination of tools allows rapid and efficient ligand docking on multiple receptor conformations in parallel. @TOME-3 is freely available on the web at https://atome.cbs.cnrs.fr.

Emerging role of oncogenic ß-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma.

Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A, two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of ß-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response.

Loss of RND3/RHOE controls entosis through LAMP1 expression in hepatocellular carcinoma.

Entosis is a process that leads to the formation of cell-in-cell structures commonly found in cancers. Here, we identified entosis in hepatocellular carcinoma and the loss of Rnd3 (also known as RhoE) as an efficient inducer of this mechanism. We characterized the different stages and the molecular regulators of entosis induced after Rnd3 silencing. We demonstrated that this process depends on the RhoA/ROCK pathway, but not on E-cadherin. The proteomic profiling of entotic cells allowed us to identify LAMP1 as a protein upregulated by Rnd3 silencing and implicated not only in the degradation final stage of entosis, but also in the full mechanism. Moreover, we found a positive correlation between the presence of entotic cells and the metastatic potential of tumors in human patient samples. Altogether, these data suggest the involvement of entosis in liver tumor progression and highlight a new perspective for entosis analysis in medicine research as a novel therapeutic target.

Antagonism between wild-type and mutant ß-catenin controls hepatoblastoma differentiation via fascin-1.

Background & Aims: ß-catenin is a well-known effector of the Wnt pathway, and a key player in cadherin-mediated cell adhesion. Oncogenic mutations of ß-catenin are very frequent in paediatric liver primary tumours. Those mutations are mostly heterozygous, which allows the co-expression of wild-type (WT) and mutated ß-catenins in tumour cells. We investigated the interplay between WT and mutated ß-catenins in liver tumour cells, and searched for new actors of the ß-catenin pathway. Methods: Using an RNAi strategy in ß-catenin-mutated hepatoblastoma (HB) cells, we dissociated the structural and transcriptional activities of ß-catenin, which are carried mainly by WT and mutated proteins, respectively. Their impact was characterised using transcriptomic and functional analyses. We studied mice that develop liver tumours upon activation of ß-catenin in hepatocytes (APCKO and ß-cateninΔexon3 mice). We used transcriptomic data from mouse and human HB specimens, and used immunohistochemistry to analyse samples. Results: We highlighted an antagonistic role of WT and mutated ß-catenins with regard to hepatocyte differentiation, as attested by alterations in the expression of hepatocyte markers and the formation of bile canaliculi. We characterised fascin-1 as a transcriptional target of mutated ß-catenin involved in tumour cell differentiation. Using mouse models, we found that fascin-1 is highly expressed in undifferentiated tumours. Finally, we found that fascin-1 is a specific marker of primitive cells including embryonal and blastemal cells in human HBs. Conclusions: Fascin-1 expression is linked to a loss of differentiation and polarity of hepatocytes. We present fascin-1 as a previously unrecognised factor in the modulation of hepatocyte differentiation associated with ß-catenin pathway alteration in the liver, and as a new potential target in HB. Impact and implications: The FSCN1 gene, encoding fascin-1, was reported to be a metastasis-related gene in various cancers. Herein, we uncover its expression in poor-prognosis hepatoblastomas, a paediatric liver cancer. We show that fascin-1 expression is driven by the mutated beta-catenin in liver tumour cells. We provide new insights on the impact of fascin-1 expression on tumour cell differentiation. We highlight fascin-1 as a marker of immature cells in mouse and human hepatoblastomas.

Reuniting philosophy and science to advance cancer research.

Cancers rely on multiple, heterogeneous processes at different scales, pertaining to many biomedical fields. Therefore, understanding cancer is necessarily an interdisciplinary task that requires placing specialised experimental and clinical research into a broader conceptual, theoretical, and methodological framework. Without such a framework, oncology will collect piecemeal results, with scant dialogue between the different scientific communities studying cancer. We argue that one important way forward in service of a more successful dialogue is through greater integration of applied sciences (experimental and clinical) with conceptual and theoretical approaches, informed by philosophical methods. By way of illustration, we explore six central themes: (i) the role of mutations in cancer; (ii) the clonal evolution of cancer cells; (iii) the relationship between cancer and multicellularity; (iv) the tumour microenvironment; (v) the immune system; and (vi) stem cells. In each case, we examine open questions in the scientific literature through a philosophical methodology and show the benefit of such a synergy for the scientific and medical understanding of cancer.

Identification of an inhibitory domain in GTPase-activating protein p190RhoGAP responsible for masking its functional GAP domain.

The GTPase-activating protein (GAP) p190RhoGAP (p190A) is encoded by ARHGAP35 which is found mutated in cancers. p190A is a negative regulator of the GTPase RhoA in cells and must be targeted to RhoA-dependent actin-based structures to fulfill its roles. We previously identified a functional region of p190A called the PLS (protrusion localization sequence) required for localization of p190A to lamellipodia but also for regulating the GAP activity of p190A. Additional effects of the PLS region on p190A localization and activity need further characterization. Here, we demonstrated that the PLS is required to target p190A to invadosomes. Cellular expression of a p190A construct devoid of the PLS (p190AΔPLS) favored RhoA inactivation in a stronger manner than WT p190A, suggesting that the PLS is an autoinhibitory domain of p190A GAP activity. To decipher this mechanism, we searched for PLS-interacting proteins using a two-hybrid screen. We found that the PLS can interact with p190A itself. Coimmunoprecipitation experiments demonstrated that the PLS interacts with a region in close proximity to the GAP domain. Furthermore, we demonstrated that this interaction is abolished if the PLS harbors cancer-associated mutations: the S866F point mutation and the Δ865-870 deletion. Our results are in favor of defining PLS as an inhibitory domain responsible for masking the p190A functional GAP domain. Thus, p190A could exist in cells under two forms: an inactive closed conformation with a masked GAP domain and an open conformation allowing p190A GAP function. Altogether, our data unveil a new mechanism of p190A regulation.

Discoidin Domain Receptor 2 orchestrates melanoma resistance combining phenotype switching and proliferation.

Combined therapy with anti-BRAF plus anti-MEK is currently used as first-line treatment of patients with metastatic melanomas harboring the somatic BRAF V600E mutation. However, the main issue with targeted therapy is the acquisition of tumor cell resistance. In a majority of resistant melanoma cells, the resistant process consists in epithelial-to-mesenchymal transition (EMT). This process called phenotype switching makes melanoma cells more invasive. Its signature is characterized by MITF low, AXL high, and actin cytoskeleton reorganization through RhoA activation. In parallel of this phenotype switching phase, the resistant cells exhibit an anarchic cell proliferation due to hyper-activation of the MAP kinase pathway. We show that a majority of human melanoma overexpress discoidin domain receptor 2 (DDR2) after treatment. The same result was found in resistant cell lines presenting phenotype switching compared to the corresponding sensitive cell lines. We demonstrate that DDR2 inhibition induces a decrease in AXL expression and reduces stress fiber formation in resistant melanoma cell lines. In this phenotype switching context, we report that DDR2 control cell and tumor proliferation through the MAP kinase pathway in resistant cells in vitro and in vivo. Therefore, inhibition of DDR2 could be a new and promising strategy for countering this resistance mechanism.

Silencing of RND3/RHOE inhibits the growth of human hepatocellular carcinoma and is associated with reversible senescence.

Rnd3/RhoE is an atypical Rho GTPase family member, known to be deregulated in many types of cancer. Previously, we showed that RND3 expression is downregulated in hepatocellular carcinoma (HCC) cell lines and tissues. In cancer cells, Rnd3 is involved in the regulation of cell proliferation and cell invasion. The implication of Rnd3 in HCC invasion was importantly studied whereas its role in cell growth needs further investigation. Thus, in this work, we aimed to better understand the impact of Rnd3 on tumor hepatocyte proliferation. Our results indicate that the silencing of RND3 induces a cell growth arrest both in vitro in 2D and 3D culture conditions and in vivo in tumor xenografts. The growth alteration after RND3 silencing in HCC cells is not due to an increase of cell death but to the induction of senescence. This RND3 knockdown-mediated phenomenon is dependent on the decrease of hTERT expression. Interestingly, after re-expression of RND3, these cells are able to bypass senescence and regain the ability to proliferate, with a re-expression of hTERT. Given that a low expression of Rnd3 is linked to the presence of satellite nodules in HCC, the transient senescence state observed might play a role in cancer progression.

1D continuous gel electrophoresis composition for the separation of deamidated proteins.

Deamidation is a spontaneous modification of peptides and proteins that has potent repercussions on their activity and stability in vivo and in vitro. Being able to implement easy techniques to detect and quantify protein deamidation is a major goal in this field. Here we focus on electrophoretic methods that can be deployed to assess protein deamidation. We provide an update on the use of Taurine/Glycinate as trailing ions to assist the detection of several examples of deamidated proteins, namely the small GTPases RhoA, Rac1 and Cdc42, but also the oncogene Bcl-xL and calcium-binding Calmodulin. We also report on the use of imidazole as a counter ion to improve the focusing of deamidated bands. Finally, we provide examples of how these gels proved useful to compare on full-length proteins the effect of ions and pH on the catalytic rates of spontaneous deamidation. Taken together, the electrophoretic method introduced here proves useful to screen at once the effect of various conditions of pH, ionic strength and buffer ions on protein stability. Direct applications can be foreseen to tailor buffer formulations to control the stability of proteins drug products.

Reptin/RUVBL2 is required for hepatocyte proliferation in vivo, liver regeneration and homeostasis.

Previous studies have shown that Reptin is overexpressed in hepatocellular carcinoma and that it is necessary for in vitro proliferation and cell survival. However, its pathophysiological role in vivo remains unknown. We aimed to study the role of Reptin in hepatocyte proliferation after regeneration using a liver Reptin knock-out model (ReptinLKO ). Interestingly, hepatocyte proliferation is strongly impaired in ReptinLKO mice 36 h after partial hepatectomy, associated with a decrease of cyclin-A expression and mTORC1 and MAPK signalling, leading to an impaired liver regeneration. Moreover, in the ReptinLKO model, we have observed a progressive loss of Reptin invalidation associated with an atypical liver regeneration. Hypertrophic and proliferative hepatocytes gradually replace ReptinKO hypotrophic hepatocytes. To conclude, our results show that Reptin is required for hepatocyte proliferation in vivo and liver regeneration and that it plays a crucial role in hepatocyte survival and liver homeostasis.

Pathophysiological functions of Rnd proteins.

Rnd proteins constitute a subfamily of Rho GTPases represented in mammals by Rnd1, Rnd2 and Rnd3. Despite their GTPase structure, their specific feature is the inability to hydrolyse GTP-bound nucleotide. This aspect makes them atypical among Rho GTPases. Rnds are regulated for their expression at the transcriptional or post-transcriptional levels and they are activated through post-translational modifications and interactions with other proteins. Rnd proteins are mainly involved in the regulation of the actin cytoskeleton and cell proliferation. Whereas Rnd3 is ubiquitously expressed, Rnd1 and 2 are tissue-specific. Increasing data has described their important role during development and diseases. Herein, we describe their involvement in physiological and pathological conditions with a focus on the neuronal and vascular systems, and summarize their implications in tumorigenesis.

Rational programming of history-dependent logic in cellular populations.

Genetic programs operating in a history-dependent fashion are ubiquitous in nature and govern sophisticated processes such as development and differentiation. The ability to systematically and predictably encode such programs would advance the engineering of synthetic organisms and ecosystems with rich signal processing abilities. Here we implement robust, scalable history-dependent programs by distributing the computational labor across a cellular population. Our design is based on standardized recombinase-driven DNA scaffolds expressing different genes according to the order of occurrence of inputs. These multicellular computing systems are highly modular, do not require cell-cell communication channels, and any program can be built by differential composition of strains containing well-characterized logic scaffolds. We developed automated workflows that researchers can use to streamline program design and optimization. We anticipate that the history-dependent programs presented here will support many applications using cellular populations for material engineering, biomanufacturing and healthcare.

Improved Electrophoretic Separation to Assist the Monitoring of Bcl-xL Post-Translational Modifications.

Bcl-xL is an oncogene of which the survival functions are finely tuned by post-translational modifications (PTM). Within the Bcl-2 family of proteins, Bcl-xL shows unique eligibility to deamidation, a time-related spontaneous reaction. Deamidation is still a largely overlooked PTM due to a lack of easy techniques to monitor Asn→Asp/IsoAsp conversions or Glu→Gln conversions. Being able to detect PTMs is essential to achieve a comprehensive description of all the regulatory mechanisms and functions a protein can carry out. Here, we report a gel composition improving the electrophoretic separation of deamidated forms of Bcl-xL generated either by mutagenesis or by alkaline treatment. Importantly, this new gel formulation proved efficient to provide the long-sought evidence that even doubly-deamidated Bcl-xL remains eligible for regulation by phosphorylation.

Transient Activations of Rac1 at the Lamellipodium Tip Trigger Membrane Protrusion.

The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE regulatory complex (WRC), an effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Thus, activated Rac1 should operate at this location to activate WRC and trigger membrane protrusion. Yet correlation of Rho GTPase activation with cycles of membrane protrusion previously revealed complex spatiotemporal patterns of Rac1 and RhoA activation in the lamellipodium. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function mutants of Rho GTPases, we show that Rac1 immobilizations at the lamellipodium tip correlate with its activation, in contrast to RhoA. Using Rac1 effector loop mutants and wild-type versus mutant variants of WRC, we show that selective immobilizations of activated Rac1 at the lamellipodium tip depend on effector binding, including WRC. In contrast, wild-type Rac1 only displays slower diffusion at the lamellipodium tip, suggesting transient activations. Local optogenetic activation of Rac1, triggered by membrane recruitment of Tiam1, shows that Rac1 activation must occur close to the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. However, coupling tracking with optogenetic activation of Rac1 demonstrates that diffusive properties of wild-type Rac1 are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model whereby transient activations of Rac1 occurring close to the lamellipodium tip trigger WRC binding. This short-lived activation ensures a local and rapid control of Rac1 actions on its effectors to trigger actin-based protrusion.

PEPOP 2.0: new approaches to mimic non-continuous epitopes.

BACKGROUND: Bioinformatics methods are helpful to identify new molecules for diagnostic or therapeutic applications. For example, the use of peptides capable of mimicking binding sites has several benefits in replacing a protein which is difficult to produce, or toxic. Using peptides is less expensive. Peptides are easier to manipulate, and can be used as drugs. Continuous epitopes predicted by bioinformatics tools are commonly used and these sequential epitopes are used as is in further experiments. Numerous discontinuous epitope predictors have been developed but only two bioinformatics tools have been proposed so far to predict peptide sequences: Superficial and PEPOP 2.0. PEPOP 2.0 can generate series of peptide sequences that can replace continuous or discontinuous epitopes in their interaction with their cognate antibody. RESULTS: We have developed an improved version of PEPOP (PEPOP 2.0) dedicated to answer to experimentalists' need for a tool able to handle proteins and to turn them into peptides. The PEPOP 2.0 web site has been reorganized by peptide prediction category and is therefore better formulated to experimental designs. Since the first version of PEPOP, 32 new methods of peptide design were developed. In total, PEPOP 2.0 proposes 35 methods in which 34 deal specifically with discontinuous epitopes, the most represented epitope type in nature. CONCLUSION: Through the presentation of its user-friendly, well-structured new web site conceived in close proximity to experimentalists, we report original methods that show how PEPOP 2.0 can assist biologists in dealing with discontinuous epitopes.

p190RhoGAPs, the ARHGAP35- and ARHGAP5-Encoded Proteins, in Health and Disease.

Small guanosine triphosphatases (GTPases) gathered in the Rat sarcoma (Ras) superfamily represent a large family of proteins involved in several key cellular mechanisms. Within the Ras superfamily, the Ras homolog (Rho) family is specialized in the regulation of actin cytoskeleton-based mechanisms. These proteins switch between an active and an inactive state, resulting in subsequent inhibiting or activating downstream signals, leading finally to regulation of actin-based processes. The On/Off status of Rho GTPases implicates two subsets of regulators: GEFs (guanine nucleotide exchange factors), which favor the active GTP (guanosine triphosphate) status of the GTPase and GAPs (GTPase activating proteins), which inhibit the GTPase by enhancing the GTP hydrolysis. In humans, the 20 identified Rho GTPases are regulated by over 70 GAP proteins suggesting a complex, but well-defined, spatio-temporal implication of these GAPs. Among the quite large number of RhoGAPs, we focus on p190RhoGAP, which is known as the main negative regulator of RhoA, but not exclusively. Two isoforms, p190A and p190B, are encoded by ARHGAP35 and ARHGAP5 genes, respectively. We describe here the function of each of these isoforms in physiological processes and sum up findings on their role in pathological conditions such as neurological disorders and cancers.

Actin Depolymerization in Dedifferentiated Liver Sinusoidal Endothelial Cells Promotes Fenestrae Re-Formation.

Liver sinusoidal endothelial cells (LSECs) possess fenestrae, which are key for the exchange between blood and hepatocytes. Alterations in their number or diameter have important implications for hepatic function in liver diseases. They are lost early in the development of hepatic fibrosis through a process called capillarization. In this study, we aimed to demonstrate whether in vitro dedifferentiated LSECs that have lost fenestrae are able to re-form these structures. Using stimulated emission depletion super-resolution microscopy in combination with transmission electron microscopy, we analyzed fenestrae formation in a model mimicking the capillarization process in vitro. Actin is known to be involved in fenestrae regulation in differentiated LSECs. Using cytochalasin D, an actin-depolymerizing agent, we demonstrated that dedifferentiated LSECs remain capable of forming fenestrae. Conclusion: We provide a new insight into the complex role of actin in fenestrae formation and in the control of their size and show that LSEC fenestrae re-formation is possible, suggesting that this process could be used during fibrosis regression to try to restore exchanges and hepatocyte functions.

Regulation of Rho GTPase activity at the leading edge of migrating cells by p190RhoGAP.

Cell migration, a key feature of embryonic development, immunity, angiogenesis, and tumor metastasis, is based on the coordinated regulation of actin dynamics and integrin-mediated adhesion. Rho GTPases play a major role in this phenomenon by regulating the onset and maintenance of actin-based protruding structures at cell leading edges (i.e. lamellipodia and filopodia) and contractile structures (i.e., stress fibers) at their trailing edge. While spatio-temporal analysis demonstrated the tight regulation of Rho GTPases at the migration front during cell locomotion, little is known about how the main regulators of Rho GTPase activity, such as GAPs, GEFs and GDIs, play a role in this process. In this review, we focus on a major negative regulator of RhoA, p190RhoGAP-A and its close isoform p190RhoGAP-B, which are necessary for efficient cell migration. Recent studies, including our, demonstrated that p190RhoGAP-A localization and activity undergo a complex regulatory mechanism, accounting for the tight regulation of RhoA, but also other members of the Rho GTPase family, at the cell periphery.

Benchmarking the PEPOP methods for mimicking discontinuous epitopes.

BACKGROUND: Computational methods provide approaches to identify epitopes in protein Ags to help characterizing potential biomarkers identified by high-throughput genomic or proteomic experiments. PEPOP version 1.0 was developed as an antigenic or immunogenic peptide prediction tool. We have now improved this tool by implementing 32 new methods (PEPOP version 2.0) to guide the choice of peptides that mimic discontinuous epitopes and thus potentially able to replace the cognate protein Ag in its interaction with an Ab. In the present work, we describe these new methods and the benchmarking of their performances. RESULTS: Benchmarking was carried out by comparing the peptides predicted by the different methods and the corresponding epitopes determined by X-ray crystallography in a dataset of 75 Ag-Ab complexes. The Sensitivity (Se) and Positive Predictive Value (PPV) parameters were used to assess the performance of these methods. The results were compared to that of peptides obtained either by chance or by using the SUPERFICIAL tool, the only available comparable method. CONCLUSION: The PEPOP methods were more efficient than, or as much as chance, and 33 of the 34 PEPOP methods performed better than SUPERFICIAL. Overall, "optimized" methods (tools that use the traveling salesman problem approach to design peptides) can predict peptides that best match true epitopes in most cases.