Valine aminoacyl-tRNA synthetase promotes therapy resistance in melanoma.

Transfer RNA dynamics contribute to cancer development through regulation of codon-specific messenger RNA translation. Specific aminoacyl-tRNA synthetases can either promote or suppress tumourigenesis. Here we show that valine aminoacyl-tRNA synthetase (VARS) is a key player in the codon-biased translation reprogramming induced by resistance to targeted (MAPK) therapy in melanoma. The proteome rewiring in patient-derived MAPK therapy-resistant melanoma is biased towards the usage of valine and coincides with the upregulation of valine cognate tRNAs and of VARS expression and activity. Strikingly, VARS knockdown re-sensitizes MAPK-therapy-resistant patient-derived melanoma in vitro and in vivo. Mechanistically, VARS regulates the messenger RNA translation of valine-enriched transcripts, among which hydroxyacyl-CoA dehydrogenase mRNA encodes for a key enzyme in fatty acid oxidation. Resistant melanoma cultures rely on fatty acid oxidation and hydroxyacyl-CoA dehydrogenase for their survival upon MAPK treatment. Together, our data demonstrate that VARS may represent an attractive therapeutic target for the treatment of therapy-resistant melanoma.

Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization.

Macrophage polarization is a process whereby macrophages acquire distinct effector states (M1 or M2) to carry out multiple and sometimes opposite functions. We show here that translational reprogramming occurs during macrophage polarization and that this relies on the Elongator complex subunit Elp3, an enzyme that modifies the wobble uridine base U34 in cytosolic tRNAs. Elp3 expression is downregulated by classical M1-activating signals in myeloid cells, where it limits the production of pro-inflammatory cytokines via FoxO1 phosphorylation, and attenuates experimental colitis in mice. In contrast, alternative M2-activating signals upregulate Elp3 expression through a PI3K- and STAT6-dependent signaling pathway. The metabolic reprogramming linked to M2 macrophage polarization relies on Elp3 and the translation of multiple candidates, including the mitochondrial ribosome large subunit proteins Mrpl3, Mrpl13, and Mrpl47. By promoting translation of its activator Ric8b in a codon-dependent manner, Elp3 also regulates mTORC2 activation. Elp3 expression in myeloid cells further promotes Wnt-driven tumor initiation in the intestine by maintaining a pool of tumor-associated macrophages exhibiting M2 features. Collectively, our data establish a functional link between tRNA modifications, mTORC2 activation, and macrophage polarization.

The E3 ligase COP1 promotes ERα signaling and suppresses EMT in breast cancer.

ERα signaling drives proliferation, survival and cancer initiation in the mammary gland. Therefore, it is critical to elucidate mechanisms by which ERα expression is regulated. We show that the tumor suppressor E3 ligase COP1 promotes the degradative polyubiquitination of the microtubule-associated protein HPIP. As such, COP1 negatively regulates estrogen-dependent AKT activation in breast cancer cells. However, COP1 also induces ERα expression and ERα-dependent gene transcription, at least through c-Jun degradation. COP1 and ERα levels are positively correlated in clinical cases of breast cancer. COP1 also supports the metabolic reprogramming by estrogens, including glycolysis. On the other hand, COP1 suppresses EMT in breast cancer cells. COP1 deficiency also contributes to Tamoxifen resistance, at least through protective autophagy. Therefore, COP1 acts as an oncogenic E3 ligase by promoting ERα signaling but also acts as a tumor suppressor candidate by preventing EMT, which reflects a dual role of COP1 in breast cancer.

NF-κB Signaling in Ex-Vivo Mouse Intestinal Organoids.

We describe here a protocol to assess NF-κB activation in ex-vivo organoids generated from mouse intestinal crypts. These structures are maintained in culture as crypt-villus forming organoids. These ex-vivo organoids maintain both self-renewal and multilineage differentiation overtime. We also describe the generation of ex-vivo organoids from Apc-mutated mouse intestinal crypts. Both wild-type and Apc-mutated organoids respond very well to NF-κB-activating signals such as TNFα but not to LPS. The kinetic of NF-κB activation in response to these signals in ex-vivo intestinal organoids is very similar to what we see in 2D cell lines. This protocol provides investigators a powerful tool to assess NF-κB activation in both healthy and transformed intestinal epitheliums maintained in culture as 3D structures.

Wobble tRNA modification and hydrophilic amino acid patterns dictate protein fate.

Regulation of mRNA translation elongation impacts nascent protein synthesis and integrity and plays a critical role in disease establishment. Here, we investigate features linking regulation of codon-dependent translation elongation to protein expression and homeostasis. Using knockdown models of enzymes that catalyze the mcm5s2 wobble uridine tRNA modification (U34-enzymes), we show that gene codon content is necessary but not sufficient to predict protein fate. While translation defects upon perturbation of U34-enzymes are strictly dependent on codon content, the consequences on protein output are determined by other features. Specific hydrophilic motifs cause protein aggregation and degradation upon codon-dependent translation elongation defects. Accordingly, the combination of codon content and the presence of hydrophilic motifs define the proteome whose maintenance relies on U34-tRNA modification. Together, these results uncover the mechanism linking wobble tRNA modification to mRNA translation and aggregation to maintain proteome homeostasis.

Downregulation of the FTO m6A RNA demethylase promotes EMT-mediated progression of epithelial tumors and sensitivity to Wnt inhibitors.

Post-transcriptional modifications of RNA constitute an emerging regulatory layer of gene expression. The demethylase fat mass- and obesity-associated protein (FTO), an eraser of N6-methyladenosine (m6A), has been shown to play a role in cancer, but its contribution to tumor progression and the underlying mechanisms remain unclear. Here, we report widespread FTO downregulation in epithelial cancers associated with increased invasion, metastasis and worse clinical outcome. Both in vitro and in vivo, FTO silencing promotes cancer growth, cell motility and invasion. In human-derived tumor xenografts (PDXs), FTO pharmacological inhibition favors tumorigenesis. Mechanistically, we demonstrate that FTO depletion elicits an epithelial-to-mesenchymal transition (EMT) program through increased m6A and altered 3'-end processing of key mRNAs along the Wnt signaling cascade. Accordingly, FTO knockdown acts via EMT to sensitize mouse xenografts to Wnt inhibition. We thus identify FTO as a key regulator, across epithelial cancers, of Wnt-triggered EMT and tumor progression and reveal a therapeutically exploitable vulnerability of FTO-low tumors.

The X-linked trichothiodystrophy-causing gene RNF113A links the spliceosome to cell survival upon DNA damage.

Prolonged cell survival occurs through the expression of specific protein isoforms generated by alternate splicing of mRNA precursors in cancer cells. How alternate splicing regulates tumor development and resistance to targeted therapies in cancer remain poorly understood. Here we show that RNF113A, whose loss-of-function causes the X-linked trichothiodystrophy, is overexpressed in lung cancer and protects from Cisplatin-dependent cell death. RNF113A is a RNA-binding protein which regulates the splicing of multiple candidates involved in cell survival. RNF113A deficiency triggers cell death upon DNA damage through multiple mechanisms, including apoptosis via the destabilization of the prosurvival protein MCL-1, ferroptosis due to enhanced SAT1 expression, and increased production of ROS due to altered Noxa1 expression. RNF113A deficiency circumvents the resistance to Cisplatin and to BCL-2 inhibitors through the destabilization of MCL-1, which thus defines spliceosome inhibitors as a therapeutic approach to treat tumors showing acquired resistance to specific drugs due to MCL-1 stabilization.

Codon-specific translation reprogramming promotes resistance to targeted therapy.

Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation2,3. Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U34) tRNA (U34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U34 enzymes. Mechanistically, U34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U34 enzymes and HIF1α. Together, these results demonstrate that U34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.

The Endosomal Protein CEMIP Links WNT Signaling to MEK1-ERK1/2 Activation in Selumetinib-Resistant Intestinal Organoids.

MAPK signaling pathways are constitutively active in colon cancer and also promote acquired resistance to MEK1 inhibition. Here, we demonstrate that BRAFV600E -mutated colorectal cancers acquire resistance to MEK1 inhibition by inducing expression of the scaffold protein CEMIP through a ß-catenin- and FRA-1-dependent pathway. CEMIP was found in endosomes and bound MEK1 to sustain ERK1/2 activation in MEK1 inhibitor-resistant BRAFV600E-mutated colorectal cancers. The CEMIP-dependent pathway maintained c-Myc protein levels through ERK1/2 and provided metabolic advantage in resistant cells, potentially by sustaining amino acids synthesis. CEMIP silencing circumvented resistance to MEK1 inhibition, partly, through a decrease of both ERK1/2 signaling and c-Myc. Together, our data identify a cross-talk between Wnt and MAPK signaling cascades, which involves CEMIP. Activation of this pathway promotes survival by potentially regulating levels of specific amino acids via a Myc-associated cascade. Targeting this node may provide a promising avenue for treatment of colon cancers that have acquired resistance to targeted therapies.Significance: MEK1 inhibitor-resistant colorectal cancer relies on the scaffold and endosomal protein CEMIP to maintain ERK1/2 signaling and Myc-driven transcription. Cancer Res; 78(16); 4533-48. ©2018 AACR.

Elp3 links tRNA modification to IRES-dependent translation of LEF1 to sustain metastasis in breast cancer.

Quantitative and qualitative changes in mRNA translation occur in tumor cells and support cancer progression and metastasis. Posttranscriptional modifications of transfer RNAs (tRNAs) at the wobble uridine 34 (U34) base are highly conserved and contribute to translation fidelity. Here, we show that ELP3 and CTU1/2, partner enzymes in U34 mcm5s2-tRNA modification, are up-regulated in human breast cancers and sustain metastasis. Elp3 genetic ablation strongly impaired invasion and metastasis formation in the PyMT model of invasive breast cancer. Mechanistically, ELP3 and CTU1/2 support cellular invasion through the translation of the oncoprotein DEK. As a result, DEK promotes the IRES-dependent translation of the proinvasive transcription factor LEF1. Consistently, a DEK mutant, whose codon composition is independent of U34 mcm5s2-tRNA modification, escapes the ELP3- and CTU1-dependent regulation and restores the IRES-dependent LEF1 expression. Our results demonstrate that the key role of U34 tRNA modification is to support specific translation during breast cancer progression and highlight a functional link between tRNA modification- and IRES-dependent translation during tumor cell invasion and metastasis.

The Prosurvival IKK-Related Kinase IKKε Integrates LPS and IL17A Signaling Cascades to Promote Wnt-Dependent Tumor Development in the Intestine.

Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to establish a tumor microenvironment has not been extensively studied. Therefore, we assessed the role of the proinflammatory Ikk-related kinase Ikkε in Wnt-driven tumor development. We found that Ikkε was activated in intestinal tumors forming upon loss of the tumor suppressor Apc Genetic ablation of Ikkε in ß-catenin-driven models of intestinal cancer reduced tumor incidence and consequently extended survival. Mechanistically, we attributed the tumor-promoting effects of Ikkε to limited TNF-dependent apoptosis in transformed intestinal epithelial cells. In addition, Ikkε was also required for lipopolysaccharide (LPS) and IL17A-induced activation of Akt, Mek1/2, Erk1/2, and Msk1. Accordingly, genes encoding pro-inflammatory cytokines, chemokines, and anti-microbial peptides were downregulated in Ikkε-deficient tissues, subsequently affecting the recruitment of tumor-associated macrophages and IL17A synthesis. Further studies revealed that IL17A synergized with commensal bacteria to trigger Ikkε phosphorylation in transformed intestinal epithelial cells, establishing a positive feedback loop to support tumor development. Therefore, TNF, LPS, and IL17A-dependent signaling pathways converge on Ikkε to promote cell survival and to establish an inflammatory tumor microenvironment in the intestine upon constitutive Wnt activation. Cancer Res; 76(9); 2587-99. ©2016 AACR.

Elp3 drives Wnt-dependent tumor initiation and regeneration in the intestine.

Tumor initiation in the intestine can rapidly occur from Lgr5(+) crypt columnar stem cells. Dclk1 is a marker of differentiated Tuft cells and, when coexpressed with Lgr5, also marks intestinal cancer stem cells. Here, we show that Elp3, the catalytic subunit of the Elongator complex, is required for Wnt-driven intestinal tumor initiation and radiation-induced regeneration by maintaining a subpool of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Elp3 deficiency dramatically delayed tumor appearance in Apc-mutated intestinal epithelia and greatly prolonged mice survival without affecting the normal epithelium. Specific ablation of Elp3 in Lgr5(+) cells resulted in marked reduction of polyp formation upon Apc inactivation, in part due to a decreased number of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Mechanistically, Elp3 is induced by Wnt signaling and promotes Sox9 translation, which is needed to maintain the subpool of Lgr5(+)/Dclk1(+) cancer stem cells. Consequently, Elp3 or Sox9 depletion led to similar defects in Dclk1(+) cancer stem cells in ex vivo organoids. Finally, Elp3 deficiency strongly impaired radiation-induced intestinal regeneration, in part because of decreased Sox9 protein levels. Together, our data demonstrate the crucial role of Elp3 in maintaining a subpopulation of Lgr5-derived and Sox9-expressing cells needed to trigger Wnt-driven tumor initiation in the intestine.

EGFR and NF-κB: partners in cancer.

Oncogenic proteins cooperate to promote tumor development and progression by sustaining cell proliferation, survival and invasiveness. Constitutive epidermal growth factor receptor (EGFR) and nuclear factor κb (NF-κB) activities are seen in multiple solid tumors and combine to provide oncogenic signals to cancer cells. Understanding how these oncogenic pathways are connected is crucial, given their role in intrinsic or acquired resistance to targeted anticancer therapies. We review molecular mechanisms by which both EGFR- and NF-κB-dependent pathways establish positive loops to increase their oncogenic potential. We also describe how NF-κB promotes resistance to EGFR inhibitors.

A role for APPL1 in TLR3/4-dependent TBK1 and IKKε activation in macrophages.

Endosomes have important roles in intracellular signal transduction as a sorting platform. Signaling cascades from TLR engagement to IRF3-dependent gene transcription rely on endosomes, yet the proteins that specifically recruit IRF3-activating molecules to them are poorly defined. We show that adaptor protein containing a pleckstrin-homology domain, a phosphotyrosine-binding domain, and a leucine zipper motif (APPL)1, an early endosomal protein, is required for both TRIF- and retinoic acid-inducible gene 1-dependent signaling cascades to induce IRF3 activation. APPL1, but not early endosome Ag 1, deficiency impairs IRF3 target gene expression upon engagement of both TLR3 and TLR4 pathways, as well as in H1N1-infected macrophages. The IRF3-phosphorylating kinases TBK1 and IKKε are recruited to APPL1 endosomes in LPS-stimulated macrophages. Interestingly, APPL1 undergoes proteasome-mediated degradation through ERK1/2 to turn off signaling. APPL1 degradation is blocked when signaling through the endosome is inhibited by chloroquine or dynasore. Therefore, APPL1 endosomes are critical for IRF3-dependent gene expression in response to some viral and bacterial infections in macrophages. Those signaling pathways involve the signal-induced degradation of APPL1 to prevent aberrant IRF3-dependent gene expression linked to immune diseases.

NF-κB-induced KIAA1199 promotes survival through EGFR signalling.

Constitutive activation of EGFR- and NF-κB-dependent pathways is a hallmark of cancer, yet signalling proteins that connect both oncogenic cascades are poorly characterized. Here we define KIAA1199 as a BCL-3- and p65-dependent gene in transformed keratinocytes. KIAA1199 expression is enhanced on human papillomavirus (HPV) infection and is aberrantly expressed in clinical cases of cervical (pre)neoplastic lesions. Mechanistically, KIAA1199 binds Plexin A2 and protects from Semaphorin 3A-mediated cell death by promoting EGFR stability and signalling. Moreover, KIAA1199 is an EGFR-binding protein and KIAA1199 deficiency impairs EGF-dependent Src, MEK1 and ERK1/2 phosphorylations. Therefore, EGFR stability and signalling to downstream kinases requires KIAA1199. As such, KIAA1199 promotes EGF-mediated epithelial-mesenchymal transition (EMT). Taken together, our data define KIAA1199 as an oncogenic protein induced by HPV infection and constitutive NF-κB activity that transmits pro-survival and invasive signals through EGFR signalling.

NF-κB, stem cells and breast cancer: the links get stronger.

Self-renewing breast cancer stem cells are key actors in perpetuating tumour existence and in treatment resistance and relapse. The molecular pathways required for their maintenance are starting to be elucidated. Among them is the transcription factor NF-κB, which is known to play critical roles in cell survival, inflammation and immunity. Recent studies indicate that mammary epithelial NF-κB regulates the self-renewal of breast cancer stem cells in a model of Her2-dependent tumourigenesis. We will describe here the NF-κB-activating pathways that are involved in this process and in which progenitor cells this transcription factor is actually activated.

BCL-3 degradation involves its polyubiquitination through a FBW7-independent pathway and its binding to the proteasome subunit PSMB1.

The oncogenic protein BCL-3 activates or represses gene transcription through binding with the NF-kappaB proteins p50 and p52 and is degraded through a phospho- and GSK3-dependent pathway. However, the mechanisms underlying its degradation remain poorly understood. Yeast two-hybrid analysis led to the identification of the proteasome subunit PSMB1 as a BCL-3-associated protein. The binding of BCL-3 to PSMB1 is required for its degradation through the proteasome. Indeed, PSMB1-depleted cells are defective in degrading polyubiquitinated BCL-3. The N-terminal part of BCL-3 includes lysines 13 and 26 required for the Lys(48)-linked polyubiquitination of BCL-3. Moreover, the E3 ligase FBW7, known to polyubiquitinate a variety of substrates phosphorylated by GSK3, is dispensable for BCL-3 degradation. Thus, our data defined a unique motif of BCL-3 that is needed for its recruitment to the proteasome and identified PSMB1 as a key protein required for the proteasome-mediated degradation of a nuclear and oncogenic IkappaB protein.

The repressing function of the oncoprotein BCL-3 requires CtBP, while its polyubiquitination and degradation involve the E3 ligase TBLR1.

The nuclear and oncogenic BCL-3 protein activates or represses gene transcription when bound to NF-kappaB proteins p50 and p52, yet the molecules that specifically interact with BCL-3 and drive BCL-3-mediated effects on gene expression remain largely uncharacterized. Moreover, GSK3-mediated phosphorylation of BCL-3 triggers its degradation through the proteasome, but the proteins involved in this degradative pathway are poorly characterized. Biochemical purification of interacting partners of BCL-3 led to the identification of CtBP as a molecule required for the ability of BCL-3 to repress gene transcription. CtBP is also required for the oncogenic potential of BCL-3 and for its ability to inhibit UV-mediated cell apoptosis in keratinocytes. We also defined the E3 ligase TBLR1 as a protein involved in BCL-3 degradation through a GSK3-independent pathway. Thus, our data demonstrate that the LSD1/CtBP complex is required for the repressing abilities of an oncogenic I kappaB protein, and they establish a functional link between the E3 ligase TBLR1 and NF-kappaB.