Genetic Alterations of TRAF Proteins in Human Cancers

The tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family of cytoplasmic adaptor proteins regulate the signal transduction pathways of a variety of receptors, including the TNF-R superfamily, Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and cytokine receptors. TRAF-dependent signaling pathways participate in a diverse array of important cellular processes, including the survival, proliferation, differentiation, and activation of different cell types. Many of these TRAF-dependent signaling pathways have been implicated in cancer pathogenesis. Here we analyze the current evidence of genetic alterations of TRAF molecules available from The Cancer Genome Atlas (TCGA) and the Catalog of Somatic Mutations in Cancer (COSMIC) as well as the published literature, including copy number variations and mutation landscape of TRAFs in various human cancers. Such analyses reveal that both gain- and loss-of-function genetic alterations of different TRAF proteins are commonly present in a number of human cancers. These include pancreatic cancer, meningioma, breast cancer, prostate cancer, lung cancer, liver cancer, head and neck cancer, stomach cancer, colon cancer, bladder cancer, uterine cancer, melanoma, sarcoma, and B cell malignancies, among others. Furthermore, we summarize the key in vivo and in vitro evidence that demonstrates the causal roles of genetic alterations of TRAF proteins in tumorigenesis within different cell types and organs. Taken together, the information presented in this review provides a rationale for the development of therapeutic strategies to manipulate TRAF proteins or TRAF-dependent signaling pathways in different human cancers by precision medicine

Identification of novel modulators of protein synthesis and nucleolar biology using high throughput phenotypic screens

Protein synthesis and ribosome biogenesis are fundamental steps in gene expression and constitute the most energy demanding processes in living cells. Dysregulation of these processes is associated to a variety of human disorders including cancer, metabolic diseases, immunodeficiency, neurological and developmental disorders, and physiological aging. Therapeutic strategies modulating protein synthesis and ribosome biogenesis or nucleolar biology, have proven to be efficient for several of these disorders, and some of them are already used in the clinic, predominantly in the context of cancer. However, the success of these drugs has been limited due to activation of mechanisms of resistance or lack of general effects among different cancer types. Additionally, the application of modulators of protein and ribosome production in other disease contexts is just starting to be explored. This is particularly important for disorders where altered translation control is a hallmark, such as in the case of some neurodegenerative diseases. Moreover, different disorders may require different therapeutic approaches, hence, research in less known disease areas opens possibilities of finding new ways of regulating protein synthesis and ribosome biogenesis, and perhaps new biology. In this thesis we have used high throughput phenotypic screens to discover new modulators of protein synthesis and nucleolar biology. Phenotypic screening allows for the systematic identification of regulators of an organismal feature (phenotype) without having any prior knowledge. In paper I we benefited from novel technologies allowing visualization of changes in protein synthesis to evaluate the effects of medically approved and well-characterized drugs in mRNA translation. Our screen failed to identify small molecules stimulating translation in cancer cells growing in complete media. Yet, it seems that translation can only be boosted when the translation machinery of cells is challenged, such as when cells are grown under starvation conditions. Nevertheless, our screen identified known down-regulators of translation, supporting the validity of our approach, and a new translation inhibitor, SKI-II. SKI-II was developed as a sphingosine kinase inhibitor (SPHK), and this group of compounds has been explored extensively as anticancer drugs. However, in our hands, SKI-II inhibited translation by inducing the integrated stress response (ISR), causing physical damage to the endoplasmic reticulum (ER), which resulted in cell death. The toxicity of SKI-II and its clinically relevant analog ABC294640 was not abrogated when knocking out sphingosine kinases, while it was partially rescued upon inhibition of the ISR. Our work is the first to systematically examine the effect of known drugs in translation in cells and to report cytotoxic properties of SPHK inhibitors that are independent of SPHKs. In paper II we conducted a chemical screen to identify compounds limiting the toxicity of amyotrophic lateral sclerosis (ALS)-related dipeptide repeats (DPRs). ALS is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurons, leading to muscular paralysis and death, within 3 to 5 years after diagnosis. The expansion of G4C2 repeats within the first intron of the C9ORF72 gene constitutes the most common cause of ALS and frontotemporal dementia (FTD). Through repeat-associated non-ATG (RAN) translation, these expansions are translated into DPRs, some of which, poly-proline arginine (PR) and poly-glycine arginine (GR), bind to the nucleoli and lead to cell death. Here we conducted a screen to identify compounds reducing toxicity of twenty-repeats poly-PR peptides (PR20) added exogenously to cells in culture. Our screen identified two BET bromodomain inhibitors (Bromosporine-1 and PFI-1) and sodium phenylbutyrate (Na-Phen), currently in clinical trials, as modifiers of PR20 toxicity in different cell lines and in developing zebrafish embryos. Our work shows that BET Bromodomain inhibitors rescue the nucleolar stress induced by PR20 and the known nucleolar stressor Actinomycin D (ActD). To our knowledge, this is the first time that compounds able to protect nucleolar integrity are reported in the literature, and therefore, they might have beneficial effects in diseases associated to nucleolar stress, such as ALS/FTD. Inspired by our results, we conducted four additional screens that are collected in the section preliminary results. Following paper I, we applied the same screening pipeline to identify novel modulators of translation among natural compounds (preliminary results I). Related to paper II, the literature points to two main issues with current modulators of ribosome biogenesis, promiscuity, even among the so-called selective modulators, and heterogeneity in the efficacy of compounds across different cancer types. Regarding the first, the discovery of regulators of ribosome biogenesis has advanced in parallel with the technology allowing their study. Current methods allow better characterization of the activities of these drugs and development of strategies to find more selective modulators, which we reviewed in annex I. Nevertheless, there is a growing need for novel modulators of nucleolar activity, and we benefited from publicly available image datasets to explore the effects of known drugs in the nucleolus (preliminary results II). Also, we conducted a genome-wide CRISPR/Cas9 screen to identify vulnerabilities to nucleolar stressors and systematically interrogate in which genetic backgrounds these drugs are suitable anticancer therapies (preliminary results III). Lastly, triggered by the discovery of “nucleolar protectors” in paper II, we conducted a chemical screen to explore novel nucleolar functions of known drugs using the Drug Repurposing Hub library 1 from the Broad Institute (preliminary results IV). Altogether, here we have used high throughput phenotypic screens to discover new modulators of protein synthesis and nucleolar biology relevant for disease contexts, and to uncover new biology linked to these processes

Features of Dormancy in Metastatic Ovarian Cancer Cells

The most prevalent subtype of ovarian cancer – high-grade serous (HGS) carcinoma – is also the most lethal, since the majority of cases are characterized by advanced-stage (metastatic) presentation. Metastasis of this cancer proceeds by an intra-peritoneal route, involving detachment of cells from the primary tumour and dissemination throughout the peritoneal cavity as multicellular aggregates, or spheroids. Herein, we demonstrate that HGS patient-derived tumour cells cultured to form in vitro spheroids exhibit features of cancer dormancy, a cellular state known to promote therapeutic resistance and disease recurrence. We discovered that upon spheroid formation, cells became non-proliferative, exhibiting a cell cycle profile and protein expression pattern (elevated p27Kip1 and RBL2/p130) that was consistent with quiescence. This was accompanied by decreased AKT kinase activity, which may be important in mediating cell cycle exit via the SCF ubiquitin-ligase complex member p45/SKP2. Moreover, when spheroids were re-attached to an adherent substratum, quiescence was rapidly reversed in an AKT-dependent manner. Aside from quiescence, we also discovered that the cellular self-digestion mechanism autophagy was upregulated during spheroid formation. Induction of this process was also observed in adherent cells (and augmented in spheroids) by pharmacologic AKT inhibition. To determine autophagy’s effect on cell viability, we attempted to block it using siRNAs targeting critical autophagy-related (ATG) genes. Interestingly, depletion of Beclin1/ATG6 had no effect, despite its role as a canonical inducer of the process. Conversely, depletion of ATG5 and ATG7 led to efficient autophagy blockade, as did treatment with the classical autophagy inhibitor Chloroquine and the novel agent Spautin-1. These approaches caused a loss of viability in both adherent and spheroid cultures. Moreover, combining autophagy blockade with AKT inhibition synergistically reduced viability, thus implying that autophagy upregulation functions as a survival mechanism. Taken together, these data reveal that two cellular processes, quiescence and autophagy, are readily induced by metastatic ovarian cancer cells as features of a dormant phenotype. We therefore propose that therapeutically targeting these dormant cells will prove highly effective in combating metastasis, resistance, and recurrence in patients with metastatic HGS ovarian cancer

Lysosome Biogenesis and Autophagy

Lysosomes degrade biological components acquired by endocytosis, the major cellular pathway for internalization of extracellular material, and macroautophagy. This chapter presents an overview of these two major degradative intracellular pathways, and highlights the emerging cross talks between them, in healthy and diseased conditions. The pathways to lysosomes include the biosynthetic transport routes, endocytic pathways, and the autophagy pathways. The central actors of the autophagy process are the ATG genes. Based on their organization in complexes and interactions, the ATG genes have been divided into many functional clusters that compose the core autophagy machinery. Cross talk between the endocytic and autophagic pathways occurs at many levels: transcriptional regulation, protein sharing, and compartmental connections. The chapter focuses on the fusion and fission events between compartments of the endolysosomal system and autophagic membranes, respectively. Lysosome-related disorders are caused by mutations in genes encoding for proteins that directly affect lysosomal functioning, including lysosomal hydrolases and lysosomal membrane proteins

Regulation of the MDM4 oncogene by the ribosomal protein L22 through alternative mRNA splicing

p53 is the most frequently mutated tumor suppressor in human cancer. By acting as a transcription factor for its target genes, it causes downstream effects such as cell cycle arrest or apoptosis. To avoid excessive p53 activity under unstressed conditions, negative regulation of p53 by its antagonists MDM2 and MDM4 is essential. Regulation of MDM2 and MDM4 in turn allows for p53 activation upon different stresses, including nucleolar stress resulting from an impairment of ribosome biogenesis. An important mechanism of how MDM4 is regulated consists in alternative splicing of the MDM4 pre-mRNA and particularly the differential inclusion of its exon 6. MDM4 exon 6 inclusion results in the formation of the MDM4-FL variant encoding full-length, functional MDM4 protein. In contrast, upon exon 6 skipping, the truncated, nonfunctional MDM4-S variant is formed. Correlation analyses of differential RNA splicing and gene mutations in large panels of cancer cell lines revealed a correlation of mutations of the ribosomal protein L22 (RPL22; eL22) and enhanced MDM4 exon 6 inclusion. Based on this, we asked whether L22 regulates MDM4 splicing, promoting exon 6 skipping. We found that L22 indeed regulates MDM4 splicing, promoting exon 6 skipping. This effect was particularly pronounced when impaired ribosome biogenesis caused nucleolar stress and redistribution of L22 to the nucleoplasm. L22 depletion not only led to the presence of functional MDM4 despite nucleolar stress, but it also reduced p53 activity. Previous work revealed that some RNAs bind L22, and that this is mediated by a specific L22-binding consensus motif within RNA. We identified three L22-binding consensus sequences in MDM4 intron 6, and these directly associated with L22. Deletion of these sequences abolished the regulation of MDM4 splicing by L22, reduced p53 activity, and caused resistance of cells to nucleolar stress-induced growth arrest. Overlapping sequences were required for the MDM4 splicing regulation by ZMAT3, but this involved a mechanism that was at least partially independent of L22. Finally, L22 regulated the splicing of additional genes besides MDM4, i.e. L22L1 (RPL22L1) and UBAP2L. In summary, we identified a new mechanism of how p53 is activated and cell proliferation is reduced upon disrupted ribosome biogenesis and nucleolar stress. In this context, L22 redistributes to the nucleoplasm of cells. This allows L22 to bind MDM4 pre-mRNA at its consensus sequence and promote the skipping of exon 6, thereby lowering functional MDM4 levels. This increases p53 activity and suppresses cell proliferation. Through this mechanism, L22 interconnects different layers of gene expression, i.e. ribosome biogenesis, RNA splicing, and transcription.2024-10-0

The hypoxic tumor microenvironment regulates the LPA signaling axis to promote cancer cell invasion and metastasis

Le développement des métastases est la cause principale de mortalité des patients atteints de cancer, mais demeure un obstacle majeur aux traitements. L'hypoxie, une caractéristique commune des tumeurs solides, est fortement impliquée dans l'invasion cellulaire et le développement des métastases, mais les mécanismes sous-jacents demeurent méconnus. La signalisation du LPA joue un rôle important au cours de la tumorigenèse et du développement des métastases, les membres de cette voie étant souvent régulés à la hausse dans les cellules tumorales. La signalisation du LPA a également été impliquée dans la production de structures de dégradation, les invadopodes, qui sont nécessaires à la formation de métastases. Des études récentes indiquent que la formation d'invadopodes est également induite par l'hypoxie. Par conséquent, nous avons voulu élucider l'influence du microenvironnement hypoxique sur l'axe de signalisation du LPA et si celui-ci joue un rôle dans la production d'invadopodes et la formation de métastases. Nous avons découvert que le LPA1 est un récepteur utilisé de façon commune et majoritaire pour la production d'invadopodes induite par l'hypoxie, et ce, dans diverses lignées cellulaires cancéreuses. Nous avons démontré que l'hypoxie favorise la formation d'invadopodes en utilisant une voie de signalisation distincte qui implique une communication croisée entre le récepteur LPA1 et l'EGFR, médiée par la kinase Src, Dans ce contexte, l'inhibition combinée du LPA1 et de l'EGFR agit en synergie afin d’empêcher la formation de métastases spontanées. Étant donné que la toxicité et la résistance aux inhibiteurs de l'EGFR représentent un défi important pour les patients atteints de cancer, ce travail permet l’identification d’une cible potentielle, le LPA1, qui pourrait être inhibée conjointement avec l'EGFR dans le but d’améliorer la survie de ces patients. D'autres études sur la régulation hypoxique de l'axe de signalisation du LPA ont démontré que l'hypoxie peut contrôler les niveaux d'expression des enzymes impliqués dans la production (ATX) et la dégradation (LPP1 / LPP3) du LPA, des évènements qui conduisent à une production accrue d'invadopodes. L'hypoxie permet également de modifier la localisation de ces protéines, ce qui pourrait constituer un mécanisme additionnel de régulation de l’axe de signalisation du LPA en hypoxie. Notre travail suggère que l'hypoxie est un régulateur important de l'axe de signalisation du LPA menant à l’invasion et à la formation de métastases. Par conséquent, les thérapies ciblant cet axe pourraient être bénéfiques pour contrer les effets néfastes de l'hypoxie tumorale sur la survie des patients atteints de cancer. De plus, un traitement combiné, ciblant le LPA1 et l’EGFR, pourrait être utile afin de réduire les effets secondaires et la résistance aux inhibiteurs de l'EGFR. Des études supplémentaires seront nécessaires afin de valider le potentiel thérapeutique de ce type de traitement.Abstract : Metastasis is the leading cause of cancer patient mortality yet remains a major hurdle for treatment. Hypoxia, a common feature of solid tumors, has been critically involved in cell invasion and metastasis but the underlying mechanisms remain poorly understood. The LPA signaling axis plays an important role during tumorigenesis and metastasis, with members of this pathway often being upregulated in tumor cells. LPA signaling has also been implicated in production of the degradative structures invadopodia, which are known to be required for metastasis. Interestingly, formation of invadopodia can also be induced by hypoxia. Therefore, we endeavoured to elucidate the influence of the hypoxic tumor microenvironment on the LPA signaling axis and whether this could play a role in invadopodia production and metastasis. We uncovered LPA1 as a common and major receptor used for hypoxia-induced invadopodia production in various cancer cell lines. We demonstrated that hypoxia promotes invadopodia formation through a distinct signaling pathway that involves Src-mediated cross-communication between LPA1 and EGFR, and that combined inhibition of LPA1 and EGFR acts synergistically to impede spontaneous metastasis. Since EGFR inhibitor toxicity and resistance represents a current challenge for cancer patients, this work identifies a potential target, LPA1 that could be inhibited in conjunction with EGFR to improve patient outcomes. Further studies into hypoxic regulation of the LPA signaling axis demonstrated that hypoxia can control the expression levels of LPA producing (ATX) and degrading (LPP1/LPP3) enzymes, events that lead to increased invadopodia production. Hypoxia was also found to alter the localization of these proteins, uncovering an additional mechanism of hypoxic regulation. Our work suggests that hypoxia is a master regulator of the LPA signaling axis that leads to metastasis, therefore therapies targeting this axis could be beneficial to counteract the detrimental effects of tumor hypoxia on cancer patient survival. Furthermore, LPA1-EGFR combination therapy could be a useful strategy to reduce EGFR inhibitor side effects and resistance and therefore warrants further studies to evaluate the potential of combination therapies in cancer patients

Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

A dramatic increase in knowledge regarding the molecular biology of brain tumors has been established over the past few years. In particular recent new avenues regarding the role of stem cells and microRNAs along with further understanding of the importance of angiogenesis, immunotherapy and explanations for the resistance of the tumors to chemotherapeutic agents and radiation therapy has been developed. It is hopeful that this new information will lead to efficacious treatment strategies for these tumors which remain a challenge. In this book a review of the latest information on these topics along with a variety of new therapeutic treatment strategies with an emphasis on molecular targeted therapies is provided