Gabriel Leprivier

The AMPK stress response pathway mediates anoikis resistance through inhibition of mTOR and suppression of protein synthesis.

Suppression of anoikis after detachment of cancer cells from the extracellular matrix is a key step during metastasis. Here we show that, after detachment, mouse embryonic fibroblasts (MEFs) transformed by K-Ras(V12) or ETV6-NTRK3 (EN) activate a transcriptional response overrepresented by genes related to bioenergetic stress and the AMP-activated protein kinase (AMPK) energy-sensing pathway. Accordingly, AMPK is activated in both transformed and non-transformed cells after detachment, and AMPK deficiency restores anoikis to transformed MEFs. However, AMPK activation represses the mTOR complex-1 (mTORC1) pathway only in transformed cells, suggesting a key role for AMPK-mediated mTORC1 inhibition in the suppression of anoikis. Consistent with this, AMPK−/− MEFs transformed by EN or K-Ras show sustained mTORC1 activation after detachment and fail to suppress anoikis. Transformed TSC1−/− MEFs, which are incapable of suppressing mTORC1, also undergo anoikis after detachment, which is reversed by mTORC1 inhibitors. Furthermore, transformed AMPK−/− and TSC1−/− MEFs both have higher total protein synthesis rates than wild-type controls, and translation inhibition using cycloheximide partially restores their anoikis resistance, indicating a mechanism whereby mTORC1 inhibition suppresses anoikis. Finally, breast carcinoma cell lines show similar detachment-induced AMPK/mTORC1 activation and restoration of anoikis by AMPK inhibition. Our data implicate AMPK-mediated mTORC1 inhibition and suppression of protein synthesis as a means for bioenergetic conservation during detachment, thus promoting anoikis resistance.

Translational Activation of HIF1α by YB-1 Promotes Sarcoma Metastasis

Metastatic dissemination is the leading cause of death in cancer patients, which is particularly evident for high-risk sarcomas such as Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. Previous research identified a crucial role for YB-1 in the epithelial-to-mesenchymal transition (EMT) and metastasis of epithelial malignancies. Based on clinical data and two distinct animal models, we now report that YB-1 is also a major metastatic driver in high-risk sarcomas. Our data establish YB-1 as a critical regulator of hypoxia-inducible factor 1α (HIF1α) expression in sarcoma cells. YB-1 enhances HIF1α protein expression by directly binding to and activating translation of HIF1A messages. This leads to HIF1α-mediated sarcoma cell invasion and enhanced metastatic capacity in vivo, highlighting a translationally regulated YB-1-HIF1α axis in sarcoma metastasis.

YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1

YB-1, which is upregulated in human sarcomas, controls the availability of the stress granule nucleator G3BP1 and thereby controls stress granule assembly.

Stress-mediated translational control in cancer cells

Tumor cells are continually subjected to diverse stress conditions of the tumor microenvironment, including hypoxia, nutrient deprivation, and oxidative or genotoxic stress. Tumor cells must evolve adaptive mechanisms to survive these conditions to ultimately drive tumor progression. Tight control of mRNA translation is critical for this response and the adaptation of tumor cells to such stress forms. This proceeds though a translational reprogramming process which restrains overall translation activity to preserve energy and nutrients, but which also stimulates the selective synthesis of major stress adaptor proteins. Here we present the different regulatory signaling pathways which coordinate mRNA translation in the response to different stress forms, including those regulating eIF2α, mTORC1 and eEF2K, and we explain how tumor cells hijack these pathways for survival under stress. Finally, mechanisms for selective mRNA translation under stress, including the utilization of upstream open reading frames (uORFs) and internal ribosome entry sites (IRESes) are discussed in the context of cell stress. This article is part of a Special Issue entitled: Translation and Cancer.

HACE1 reduces oxidative stress and mutant Huntingtin toxicity by promoting the NRF2 response.

Significance Oxidative stress is an important contributor to aging-associated diseases including cancer and neurodegeneration, and antioxidant stress responses are critical to limit manifestations of these diseases. We report that the tumor suppressor Homologous to the E6-AP Carboxyl Terminus domain and Ankyrin repeat containing E3 ubiquitin–protein ligase 1 (HACE1) promotes activity of the transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the antioxidative stress response. In Huntington disease patients, HACE1 is lost in the brain region most affected by the disease, namely the striatum, and restoring HACE1 functions in striatal cells expressing mutant Huntingtin protein provides protection against oxidative stress. Therefore, the tumor suppressor HACE1 is a new regulator of NRF2 and an emerging player in neurodegeneration. Oxidative stress plays a key role in late onset diseases including cancer and neurodegenerative diseases such as Huntington disease. Therefore, uncovering regulators of the antioxidant stress responses is important for understanding the course of these diseases. Indeed, the nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the cellular antioxidative stress response, is deregulated in both cancer and neurodegeneration. Similar to NRF2, the tumor suppressor Homologous to the E6-AP Carboxyl Terminus (HECT) domain and Ankyrin repeat containing E3 ubiquitin–protein ligase 1 (HACE1) plays a protective role against stress-induced tumorigenesis in mice, but its roles in the antioxidative stress response or its involvement in neurodegeneration have not been investigated. To this end we examined Hace1 WT and KO mice and found that Hace1 KO animals exhibited increased oxidative stress in brain and that the antioxidative stress response was impaired. Moreover, HACE1 was found to be essential for optimal NRF2 activation in cells challenged with oxidative stress, as HACE1 depletion resulted in reduced NRF2 activity, stability, and protein synthesis, leading to lower tolerance against oxidative stress triggers. Strikingly, we found a reduction of HACE1 levels in the striatum of Huntington disease patients, implicating HACE1 in the pathology of Huntington disease. Moreover, ectopic expression of HACE1 in striatal neuronal progenitor cells provided protection against mutant Huntingtin-induced redox imbalance and hypersensitivity to oxidative stress, by augmenting NRF2 functions. These findings reveal that the tumor suppressor HACE1 plays a role in the NRF2 antioxidative stress response pathway and in neurodegeneration.

Identification and quantification of newly synthesized proteins translationally regulated by YB-1 using a novel Click–SILAC approach

Messenger RNA-binding translational regulatory proteins determine in large part the spectrum of transcripts that are translated under specific cellular contexts. Y-box binding protein-1 (YB-1) is a conserved eukaryotic translational regulator that is implicated in cancer progression. To identify specific proteins that are translationally regulated by YB-1, we established a pulse-labelling approach combining Click chemistry and stable isotope labelling by amino acids in cell culture (SILAC). The proteome of TC32 human Ewing sarcoma cells, which robustly express YB-1, was compared with or without YB-1 siRNA knockdown. Cells labelled with light or heavy isotopologs of Arg and Lys were then cotranslationally pulsed with the methionine derivative, azidohomoalanine (AHA). Cells were lysed and newly synthesized proteins were selectively derivatized via a Click (3+2 cycloaddition) reaction to add an alkyne biotin tag. They were then affinity purified and subjected to liquid chromatography-tandem mass spectrometry. This combined Click-SILAC approach enabled us to catalog and quantify newly synthesized proteins regulated by YB-1 after only 45 min of labelling. Bioinformatic analysis revealed that YB-1 regulated proteins are involved in diverse biological pathways. We anticipate that this Click-SILAC strategy will be useful for studying short-term protein synthesis in different cell culture systems and under diverse biological contexts.

Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1): a master regulator of mRNA translation involved in tumorigenesis.

Protein synthesis activity is abnormally enhanced in cancer cells to support their uncontrolled growth. However, this process needs to be tightly restricted under metabolic stress-a condition often found within the tumor microenvironment-to preserve cell viability. mTORC1 is critical to link protein synthesis activity to nutrient and oxygen levels, in part by controlling the 4E-BP1-eIF4E axis. Whereas mTORC1 and eIF4E are known pro-tumorigenic factors, whose expression or activity is increased in numerous cancers, the role of 4E-BP1 in cancer is not yet definitive. On the one hand, 4E-BP1 has tumor suppressor activity by inhibiting eIF4E and, thus, blocking mRNA translation and proliferation. This is corroborated by elevated levels of phosphorylated and hence inactive 4E-BP1, which are detected in various cancers. On the other hand, 4E-BP1 has pro-tumorigenic functions as it promotes tumor adaptation to metabolic and genotoxic stress by selectively enhancing or preventing the translation of specific transcripts. Here we describe the molecular and cellular functions of 4E-BP1 and highlight the distinct roles of 4E-BP1 in cancer depending on the microenvironmental context of the tumor.

A possible role for long non-coding RNA in modulating signaling pathways.

Signaling proteins often engage in multiple protein-protein interactions that are dependent upon cellular context. Little is known about how signaling proteins select their interacting targets. The Ras GTPase is an example of a protein that can activate a large number of distinct and interconnected downstream signaling pathways. Hyperactive forms of Ras are commonly found in a variety of different cancers, often due to somatic mutations within the RAS gene. Despite extensive studies to identify Ras-regulated pathways, it is still not known exactly which pathways might be activated by hyperactive Ras in a given cellular and disease context. Long non-coding RNAs (lncRNAs) are RNA transcripts longer than 200 bp exhibiting spatially and temporally-regulated expression patterns. LncRNAs have been shown to harbor biological activities but the functions of the great majority of lncRNAs are not known. We hypothesize that long non-coding RNAs serve as signaling modulators linking Ras and potentially other signaling proteins to their specific downstream targets and may therefore play a key role in how signals are propagated in a specific cellular environment. In support of our hypothesis we argue that lncRNAs have been shown to bind and regulate protein complexes targeting their enzymatic activity towards specific substrates. It has also been demonstrated that specific lncRNAs are expressed in particular types of cancers where they may influence tumor progression. Studies suggest that lncRNAs have evolved to help regulate complex biological processes that require the ability to stringently discriminate between a large number of potential effectors. If our hypothesis is correct, we envision that it will be possible to predict the target pathway of a mutant protein based on the lncRNA profile in a specific cancer. More generally, this will expand our understanding of how signal transduction networks are wired within a given biological context.

Mutation of the Salt Bridge-forming Residues in the ETV6-SAM Domain Interface Blocks ETV6-NTRK3-induced Cellular Transformation *

Background: SAM domain-mediated polymerization is essential for ETV6-NTRK3 (EN)-induced cellular transformation. Results: Mutation of a salt bridge at the SAM polymer interface weakens SAM polymerization and abrogates EN transformation. Conclusion: Intermolecular electrostatic interactions are important for SAM domain polymerization and EN transformation. Significance: These studies provide further insights into the mechanisms by which ETV6-SAM domain mediates EN transformation. The ETV6-NTRK3 (EN) chimeric oncogene is expressed in diverse tumor types. EN is generated by a t(12;15) translocation, which fuses the N-terminal SAM (sterile α-motif) domain of the ETV6 (or TEL) transcription factor to the C-terminal PTK (protein-tyrosine kinase) domain of the neurotrophin-3 receptor NTRK3. SAM domain-mediated polymerization of EN leads to constitutive activation of the PTK domain and constitutive signaling of the Ras-MAPK and PI3K-Akt pathways, which are essential for EN oncogenesis. Here we show through complementary biophysical and cellular biological techniques that mutation of Lys-99, which participates in a salt bridge at the SAM polymer interface, reduces self-association of the isolated SAM domain as well as high molecular mass complex formation of EN and abrogates the transformation activity of EN. We also show that mutation of Asp-101, the intermolecular salt bridge partner of Lys-99, similarly blocks transformation of NIH3T3 cells by EN, reduces EN tyrosine phosphorylation, inhibits Akt and Mek1/2 signaling downstream of EN, and abolishes tumor formation in nude mice. In contrast, mutations of Glu-100 and Arg-103, residues in the vicinity of the interdomain Lys-99–Asp-101 salt bridge, have little or no effect on these oncogenic characteristics of EN. Our results underscore the importance of specific electrostatic interactions for SAM polymerization and EN transformation.

MYCN amplified neuroblastoma requires the mRNA translation regulator eEF2 kinase to adapt to nutrient deprivation

MYC family proteins are implicated in many human cancers, but their therapeutic targeting has proven challenging. MYCN amplification in childhood neuroblastoma (NB) is associated with aggressive disease and high mortality. Novel and effective therapeutic strategies are therefore urgently needed for these tumors. MYC-driven oncogenic transformation impairs cell survival under nutrient deprivation (ND), a characteristic stress condition within the tumor microenvironment. We recently identified eukaryotic Elongation Factor 2 Kinase (eEF2K) as a pivotal mediator of the adaptive response of tumor cells to ND. We therefore hypothesized that eEF2K facilitates the adaptation of MYCN amplified NB to ND, and that inhibiting this pathway can impair MYCN-driven NB progression. To test our hypothesis, we first analyzed publicly available genomic databases and tissue microarrays for eEF2K expression in NB, and for links between eEF2K, MYCN, and clinical outcome in NB. Effects of eEF2K inhibition were evaluated on survival of MYCN amplified versus non-amplified NB cell lines under ND. Finally, NB xenograft mouse models were used to confirm in vitro observations. Our results indicate that high eEF2K expression and activity are strongly predictive of poor outcome in NB, and correlates significantly with MYCN amplification. Inhibition of eEF2K markedly decreases survival of MYCN amplified NB cell lines in vitro under ND. Growth of MYCN amplified NB xenografts is markedly impaired by eEF2K knockdown, particularly under caloric restriction. In summary, eEF2K protects MYCN overexpressing NB cells from ND in vitro and in vivo, highlighting this kinase as a critical mediator of the adaptive response of MYCN amplified NB cells to metabolic stress.