Non-canonical mRNA translation initiation in cell stress and cancer.

The now well described canonical mRNA translation initiation mechanism of m7G 'cap' recognition by cap-binding protein eIF4E and assembly of the canonical pre-initiation complex consisting of scaffolding protein eIF4G and RNA helicase eIF4A has historically been thought to describe all cellular mRNA translation. However, the past decade has seen the discovery of alternative mechanisms to canonical eIF4E mediated mRNA translation initiation. Studies have shown that non-canonical alternate mechanisms of cellular mRNA translation initiation, whether cap-dependent or independent, serve to provide selective translation of mRNAs under cell physiological and pathological stress conditions. These conditions typically involve the global downregulation of canonical eIF4E1/cap-mediated mRNA translation, and selective translational reprogramming of the cell proteome, as occurs in tumor development and malignant progression. Cancer cells must be able to maintain physiological plasticity to acquire a migratory phenotype, invade tissues, metastasize, survive and adapt to severe microenvironmental stress conditions that involve inhibition of canonical mRNA translation initiation. In this review we describe the emerging, important role of non-canonical, alternate mechanisms of mRNA translation initiation in cancer, particularly in adaptation to stresses and the phenotypic cell fate changes involved in malignant progression and metastasis. These alternate translation initiation mechanisms provide new targets for oncology therapeutics development.

Coiled-Coil Protein Hydrogels Engineered with Minimized Fiber Diameters for Sustained Release of Doxorubicin in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) lacks expressed protein targets, making therapy development challenging. Hydrogels offer a promising new route in this regard by improving the chemotherapeutic efficacy through increased solubility and sustained release. Moreover, subcutaneous hydrogel administration reduces patient burden by requiring less therapy and shorter treatment times. We recently established the design principles for the supramolecular assembly of single-domain coiled-coils into hydrogels. Using a modified computational design algorithm, we designed Q8, a hydrogel with rapid assembly for faster therapeutic hydrogel preparation. Q8 encapsulates and releases doxorubicin (Dox), enabling localized sustained release via subcutaneous injection. Remarkably, a single subcutaneous injection of Dox-laden Q8 (Q8•Dox) significantly suppresses tumors within just 1 week. This work showcases the bottom-up engineering of a fully protein-based drug delivery vehicle for improved TBNC treatment via noninvasive localized therapy.

Engineered coiled-coil HIF1α protein domain mimic.

The development of targeted anti-cancer therapeutics offers the potential for increased efficacy of drugs and diagnostics. Utilizing modalities agnostic to tumor type, such as the hypoxic tumor microenvironment (TME), may assist in the development of universal tumor targeting agents. The hypoxia-inducible factor (HIF), in particular HIF1, plays a key role in tumor adaptation to hypoxia, and inhibiting its interaction with p300 has been shown to provide therapeutic potential. Using a multivalent assembled protein (MAP) approach based on the self-assembly of the cartilage oligomeric matrix protein coiled-coil (COMPcc) domain fused to the critical residues of the C-terminal transactivation domain (C-TAD) of the α subunit of HIF1 (HIF1α), we generate HIF1α-MAP (H-MAP). The resulting H-MAP demonstrates picomolar binding affinity to p300, the ability to downregulate hypoxia-inducible genes, and in vivo tumor targeting capability.

Breast cancer cell mesenchymal transition and metastasis directed by DAP5/eIF3d-mediated selective mRNA translation.

Cancer cell plasticity enables cell survival in harsh physiological environments and fate transitions such as the epithelial-to-mesenchymal transition (EMT) that underlies invasion and metastasis. Using genome-wide transcriptomic and translatomic studies, an alternate mechanism of cap-dependent mRNA translation by the DAP5/eIF3d complex is shown to be essential for metastasis, EMT, and tumor directed angiogenesis. DAP5/eIF3d carries out selective translation of mRNAs encoding EMT transcription factors and regulators, cell migration integrins, metalloproteinases, and cell survival and angiogenesis factors. DAP5 is overexpressed in metastatic human breast cancers associated with poor metastasis-free survival. In human and murine breast cancer animal models, DAP5 is not required for primary tumor growth but is essential for EMT, cell migration, invasion, metastasis, angiogenesis, and resistance to anoikis. Thus, cancer cell mRNA translation involves two cap-dependent mRNA translation mechanisms, eIF4E/mTORC1 and DAP5/eIF3d. These findings highlight a surprising level of plasticity in mRNA translation during cancer progression and metastasis.

eIF2Bδ blocks the integrated stress response and maintains eIF2B activity and cancer metastasis by overexpression in breast cancer stem cells.

Breast cancer (BC) metastasis involves cancer stem cells (CSCs) and their regulation by micro-RNAs (miRs), but miR targeting of the translation machinery in CSCs is poorly explored. We therefore screened miR expression levels in a range of BC cell lines, comparing non-CSCs to CSCs, and focused on miRs that target translation and protein synthesis factors. We describe a unique translation regulatory axis enacted by reduced expression of miR-183 in breast CSCs, which we show targets the eIF2Bδ subunit of guanine nucleotide exchange factor eIF2B, a regulator of protein synthesis and the integrated stress response (ISR) pathway. We report that reduced expression of miR-183 greatly increases eIF2Bδ protein levels, preventing strong induction of the ISR and eIF2α phosphorylation, by preferential interaction with P-eIF2α. eIF2Bδ overexpression is essential for BC cell invasion, metastasis, maintenance of metastases, and breast CSC expansion in animal models. Increased expression of eIF2Bδ, a site of action of the drug ISRIB that also prevents ISR signaling, is essential for breast CSC maintenance and metastatic capacity.

DDX60 selectively reduces translation off viral type II internal ribosome entry sites.

Co-opting host cell protein synthesis is a hallmark of many virus infections. In response, certain host defense proteins limit mRNA translation globally, albeit at the cost of the host cell's own protein synthesis. Here, we describe an interferon-stimulated helicase, DDX60, that decreases translation from viral internal ribosome entry sites (IRESs). DDX60 acts selectively on type II IRESs of encephalomyocarditis virus (EMCV) and foot and mouth disease virus (FMDV), but not by other IRES types or by 5' cap. Correspondingly, DDX60 reduces EMCV and FMDV (type II IRES) replication, but not that of poliovirus or bovine enterovirus 1 (BEV-1; type I IRES). Furthermore, replacing the IRES of poliovirus with a type II IRES is sufficient for DDX60 to inhibit viral replication. Finally, DDX60 selectively modulates the amount of translating ribosomes on viral and in vitro transcribed type II IRES mRNAs, but not 5' capped mRNA. Our study identifies a novel facet in the repertoire of interferon-stimulated effector genes, the selective downregulation of translation from viral type II IRES elements.

Ribosome profiling reveals novel regulation of C9ORF72 GGGGCC repeat-containing RNA translation.

GGGGCC (G4C2) repeat expansion in the first intron of C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-containing RNA is translated into dipeptide repeat (DPR) proteins, some of which are neurotoxic. Using dynamic ribosome profiling, we identified three translation initiation sites in the intron upstream of (G4C2) repeats; these sites are detected irrespective of the presence or absence of the repeats. During translocation, ribosomes appear to be stalled on the repeats. An AUG in the preceding C9ORF72 exon initiates a uORF that inhibits downstream translation. Polysome isolation indicates that unspliced (G4C2) repeat-containing RNA is a substrate for DPR protein synthesis. (G4C2) repeat-containing RNA translation is 5' cap-independent but inhibited by the initiation factor DAP5, suggesting an interplay with uORF function. These results define novel translational mechanisms of expanded (G4C2) repeat-containing RNA in disease.

A DAP5/eIF3d alternate mRNA translation mechanism promotes differentiation and immune suppression by human regulatory T cells.

Regulatory T cells (Treg cells) inhibit effector T cells and maintain immune system homeostasis. Treg cell maturation in peripheral sites requires inhibition of protein kinase mTORC1 and TGF-beta-1 (TGF-beta). While Treg cell maturation requires protein synthesis, mTORC1 inhibition downregulates it, leaving unanswered how Treg cells achieve essential mRNA translation for development and immune suppression activity. Using human CD4+ T cells differentiated in culture and genome-wide transcription and translation profiling, here we report that TGF-beta transcriptionally reprograms naive T cells to express Treg cell differentiation and immune suppression mRNAs, while mTORC1 inhibition impairs translation of T cell mRNAs but not those induced by TGF-beta. Rather than canonical mTORC1/eIF4E/eIF4G translation, Treg cell mRNAs utilize the eIF4G homolog DAP5 and initiation factor eIF3d in a non-canonical translation mechanism that requires cap-dependent binding by eIF3d directed by Treg cell mRNA 5' noncoding regions. Silencing DAP5 in isolated human naive CD4+ T cells impairs their differentiation into Treg cells. Treg cell differentiation is mediated by mTORC1 downregulation and TGF-beta transcriptional reprogramming that establishes a DAP5/eIF3d-selective mechanism of mRNA translation.

AUF1 gene transfer increases exercise performance and improves skeletal muscle deficit in adult mice.

Muscle function and mass begin declining in adults long before evidence of sarcopenia and include reduced mitochondrial function, although much remains to be characterized. We found that mRNA decay factor AU-rich mRNA binding factor 1 (AUF1), which stimulates myogenesis, is strongly reduced in skeletal muscle of adult and older mice in the absence of evidence of sarcopenia. Muscle-specific adeno-associated virus (AAV)8-AUF1 gene therapy increased expression of AUF1, muscle function, and mass. AAV8 AUF1 muscle gene transfer in 12-month-old mice increased the levels of activated muscle stem (satellite) cells, increased muscle mass, reduced markers of muscle atrophy, increased markers of mitochondrial content and muscle fiber oxidative capacity, and enhanced exercise performance to levels of 3-month-old mice. With wild-type and AUF1 knockout mice and cultured myoblasts, AUF1 supplementation of muscle fibers was found to increase expression of Peroxisome Proliferator-activated Receptor Gamma Co-activator 1-alpha (PGC1α), a major effector of skeletal muscle mitochondrial oxidative metabolism. AUF1 stabilized and increased translation of the pgc1α mRNA, which is strongly reduced in adult muscle in the absence of AUF1 supplementation. Skeletal muscle-specific gene transfer of AUF1 therefore restores muscle mass, increases exercise endurance, and may provide a therapeutic strategy for age-related muscle loss.

m7G tRNA modification reveals new secrets in the translational regulation of cancer development.

Orellana et al. (2021) and Dai et al. (2021) demonstrate that increased m7G modification of a subset of tRNAs by the METTL1/WDR4 complex stabilizes these mRNAs against decay, increases translation efficiency, reduces ribosome pausing, is associated with poor survival in human cancers, and is directly transforming.

Translation Regulation by eIF2α Phosphorylation and mTORC1 Signaling Pathways in Non-Communicable Diseases (NCDs).

Non-communicable diseases (NCDs) are medical conditions that, by definition, are non-infectious and non-transmissible among people. Much of current NCDs are generally due to genetic, behavioral, and metabolic risk factors that often include excessive alcohol consumption, smoking, obesity, and untreated elevated blood pressure, and share many common signal transduction pathways. Alterations in cell and physiological signaling and transcriptional control pathways have been well studied in several human NCDs, but these same pathways also regulate expression and function of the protein synthetic machinery and mRNA translation which have been less well investigated. Alterations in expression of specific translation factors, and disruption of canonical mRNA translational regulation, both contribute to the pathology of many NCDs. The two most common pathological alterations that contribute to NCDs discussed in this review will be the regulation of eukaryotic initiation factor 2 (eIF2) by the integrated stress response (ISR) and the mammalian target of rapamycin complex 1 (mTORC1) pathways. Both pathways integrally connect mRNA translation activity to external and internal physiological stimuli. Here, we review the role of ISR control of eIF2 activity and mTORC1 control of cap-mediated mRNA translation in some common NCDs, including Alzheimer's disease, Parkinson's disease, stroke, diabetes mellitus, liver cirrhosis, chronic obstructive pulmonary disease (COPD), and cardiac diseases. Our goal is to provide insights that further the understanding as to the important role of translational regulation in the pathogenesis of these diseases.

mTOR-mediated inactivation of 4E-BP1, an inhibitor of translation, precedes cartilage degeneration in rat osteoarthritic knees.

Proper control of protein synthesis is vital for tissue homeostasis and its deregulation is characteristic of many disorders including osteoarthritis (OA). The objectives of this work were to analyze and correlate changes in activity of the translation apparatus associated with cartilage degeneration in an animal model of OA. Osteoarthritis was induced surgically in rats by anterior cruciate ligament transection (ACLT). Using a modified Mankin scoring system and analysis of protein expression we demonstrated, that mechanistic target of rapamycin complex 1 (mTORC1)-mediated 4E-BP1 phosphorylation was detected significantly earlier than other mTORC1-mediated modifications, such as p70S6K and ULK1 phosphorylation. 4E-BP1 is an inhibitor of cap-dependent translation those functions are inhibited by mTORC1 mediated phosphorylation. This signaling event not only preceded prominent signs of cartilage degeneration but also the increase in global protein synthesis rate. These results suggest that abnormal mTORC1 activity is one of the primary dysregulations observed in OA cartilage. Importantly, it is distributed disproportionately between targets, with 4E-BP1 being phosphorylated earlier than other downstream targets. Thus, our work provides new insights into the sequence of molecular events leading to cartilage destruction in OA and identifies translational control as an important regulatory hub involved in initiating OA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2728-2735, 2018.

Phosphoproteomics of Fibroblast Growth Factor 1 (FGF1) Signaling in Chondrocytes: Identifying the Signature of Inhibitory Response.

Fibroblast growth factor (FGF) signaling is vital for many biological processes, beginning with development. The importance of FGF signaling for skeleton formation was first discovered by the analysis of genetic FGFR mutations which cause several bone morphogenetic disorders, including achondroplasia, the most common form of human dwarfism. The formation of the long bones is mediated through proliferation and differentiation of highly specialized cells - chondrocytes.Chondrocytes respond to FGF with growth inhibition, a unique response which differs from the proliferative response of the majority of cell types; however, its molecular determinants are still unclear. Quantitative phosphoproteomic analysis was utilized to catalogue the proteins whose phosphorylation status is changed upon FGF1 treatment. The generated dataset consists of 756 proteins. We could localize the divergence between proliferative (canonical) and inhibitory (chondrocyte specific) FGF transduction pathways immediately upstream of AKT kinase. Gene Ontology (GO) analysis of the FGF1 regulated peptides revealed that many of the identified phosphorylated proteins are assigned to negative regulation clusters, in accordance with the observed inhibitory growth response. This is the first time a comprehensive subset of proteins involved in FGF inhibitory response is defined. We were able to identify a number of targets and specifically discover glycogen synthase kinase3ß (GSK3ß) as a novel key mediator of FGF inhibitory response in chondrocytes.

Increased Activity of the Chondrocyte Translational Apparatus Accompanies Osteoarthritic Changes in Human and Rodent Knee Cartilage.

OBJECTIVE: Degeneration of articular cartilage is central to the pathology of osteoarthritis (OA). However, the molecular mechanisms leading to these irreversible changes are still poorly understood. This study was undertaken to investigate how changes in the chondrocyte translational apparatus may contribute to the development and progression of knee OA. METHODS: Articular cartilage from the knees of normal healthy subjects and patients with OA was used to analyze the activity of different components of the translational machinery. Chondrocytes isolated from lesional and nonlesional areas of the human OA cartilage were used to estimate the relative rate of protein synthesis by metabolic labeling. Experimental OA was induced by transection of the anterior cruciate ligament of rats to investigate changes in the translational apparatus associated with OA. The role of interleukin-1ß (IL-1ß) signaling was assessed in vitro using rat articular chondrocytes. In human or rodent knee cartilage, messenger RNA expression was analyzed by quantitative polymerase chain reaction, and protein levels were determined by immunohistochemistry and Western blotting. RESULTS: Several novel traits of OA chondrocytes were identified, including up-regulation of the serine/threonine kinases Akt-2 and Akt-3 at the posttranscriptional level and an increased rate of total protein synthesis, likely attributable to inactivation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1), a known repressor of cap-dependent translation. Inactivation of 4E-BP1 was dependent on the activity of mechanistic target of rapamycin and was crucial for the up-regulation of protein synthesis in general and expression of matrix metalloproteinase 13 and ADAMTS-5 in particular. In addition, treatment of articular chondrocytes with IL-1ß led to inactivation of 4E-BP1 and up-regulation of protein synthesis. CONCLUSION: Precise control of protein synthesis is vital for cartilage homeostasis, and its dysregulation contributes to the molecular pathology of OA. The results of this study therefore identify a novel set of potential therapeutic targets to ameliorate the effects of knee OA.

Cell type-specific control of protein synthesis and proliferation by FGF-dependent signaling to the translation repressor 4E-BP.

Regulation of protein synthesis plays a vital role in posttranscriptional modulation of gene expression. Translational control most commonly targets the initiation of protein synthesis: loading 40S ribosome complexes onto mRNA and AUG start codon recognition. This step is initiated by eukaryotic initiation factor 4E (eIF4E) (the m7GTP cap-binding protein), whose binding to eIF4G (a scaffolding subunit) and eIF4A (an ATP-dependent RNA helicase) leads to assembly of active eIF4F complex. The ability of eIF4E to recognize the cap is prevented by its binding to eIF4E binding protein (4E-BP), which thereby inhibits cap-dependent translation by sequestering eIF4E. The 4E-BP activity is, in turn, inhibited by mTORC1 [mTOR (the mechanistic target of rapamycin) complex 1] mediated phosphorylation. Here, we define a previously unidentified mechanism of mTOR-independent 4E-BP1 regulation that is used by chondrocytes upon FGF signaling. Chondrocytes are responsible for the formation of the skeleton long bones. Unlike the majority of cell types where FGF signaling triggers proliferation, chondrocytes respond to FGF with inhibition. We establish that FGF specifically suppresses protein synthesis in chondrocytes, but not in any other cells of mesenchymal origin. Furthermore, 4E-BP1 repressor activity is necessary not only for suppression of protein synthesis, but also for FGF-induced cell-cycle arrest. Importantly, FGF-induced changes in the 4E-BP1 activity observed in cell culture are likewise detected in vivo and reflect the action of FGF signaling on downstream targets during bone development. Thus, our findings demonstrate that FGF signaling differentially impacts protein synthesis through either stimulation or repression, in a cell-type-dependent manner, with 4E-BP1 being a key player.

IGF2BP1 expression in human mesenchymal stem cells significantly affects their proliferation and is under the epigenetic control of TET1/2 demethylases.

Mesenchymal stem cells (MSCs) are a population of cells harboring in many tissues with the ability to differentiate toward many different lineages. Unraveling the molecular profile of MSCs is of great importance due to the fact that these cells are very often used in preclinical and clinical studies. We have previously reported the expression of insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) an oncofetal mRNA-binding protein-in different stem cell types such as bone marrow (BM)-MSC and umbilical cord blood (UCB)-hematopoietic stem cells. Here, we demonstrate that MSCs of adipose tissue, BM, and UC origin have a differential pattern of IGF2BP1 and ten-eleven-translocate 1/2 (TET1/2) expression that could correlate with their proliferation potential. Upon IGF2BP1 interference, a significant reduction of cell proliferation is observed, accompanied by reduced expression of c-MYC and GLI1 and increased p21. We also present, for the first time, evidence that IGF2BP1 is epigenetically regulated by TET1 and TET2 demethylases. Specifically, we show that TET1 directly binds to the promoter of IGF2BP1 gene and affects the hydroxymethylation status of its promoter. These results indicate that IGF2BP1 and TET1/2 contribute to the stemness of MSCs, at least regarding their proliferative potential.

Effects of donor age, gender, and in vitro cellular aging on the phenotypic, functional, and molecular characteristics of mouse bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are a very important adult stem cell population with a multitude of potential applications in regenerative medicine. The thorough characterization of the bone marrow MSC (BM-MSC) population derived from the BALB/c species was essential, considering the significance of the murine model amongst animal models. In the present study, we examined the effect of gender, age, and in vitro culture on the basic properties (proliferation, differentiation, and immunosuppressive potential) of BM-MSCs. We found a decline in the progenitor frequencies from the BM of adult mice, lower MSC frequencies in all female donors, and an increase in the BM-MSC proliferation rate upon in vitro propagation. We also examined BM-MSCs for the expression of the 3 major embryonic stem cell transcription factors, Oct3/4, Sox-2, and Nanog, as well as 2 mRNA binding proteins, coding region determinant binding protein/insulin-like growth factor 2 mRNA binding protein 1 (Crd-bp/Imp1) and Deleted in azoospermia-like (Dazl), which are expressed in primitive stem cells, umbilical cord blood-hematopoietic stem cells and amniotic fluid stem cells, respectively. Further, it has been reported that these 2 genes are critical for embryonic development. In this study, therefore, we report, for the first time, the expression of Crd-bp/Imp1 and Dazl in BM-MSCs. Dazl, Oct3/4, and Sox2 were detected in relatively low levels in contrast to Crd-bp/Imp1, its major target c-Myc, as well as Nanog, which were expressed redundantly, irrespective of sex, donor age, or in vitro passaging. These findings could further support the extrinsic theory of aging of the MSC population and the potential implication of embryonic genes in adult stem cell physiology.