c-Myc Translation Research Articles

LINC01764 promotes colorectal cancer cells proliferation, metastasis, and 5-fluorouracil resistance by regulating glucose and glutamine metabolism via promoting c-MYC translation.

Few biomarkers are available for predicting chemotherapeutic response and prognosis in colorectal cancer (CRC). Long-noncoding RNAs (lncRNAs) are essential for CRC development and growth. Therefore, studying lncRNAs may reveal potential predictors of chemotherapy response and prognosis in CRC. LINC01764 was analyzed using datasets from Fudan University Shanghai Cancer Center's advanced CRC patients' RNA-seq and The Cancer Genome Atlas datasets. Gene set enrichment analysis was employed to detect related pathways. Cotransfection experiments, RNA pulldown assays, RNA-binding protein immunoprecipitation, protein synthesis activity, and dual-luciferase reporter assays were performed to determine interactions among LINC01764, hnRNPK, and c-MYC. High LINC01764 expression correlates with metastasis, a poor response to FOLFOX/XELOX chemotherapy, and a poor prognosis in CRC. LINC01764 enhance glycolysis and glutamine metabolism to promote CRC cells proliferation, metastasis, and 5-fluorouracil (5-FU) resistance. LINC01764 specifically binds to hnRNPK, facilitating its interaction with c-MYC mRNA and promoting internal ribosome entry site (IRES)-dependent translation of c-MYC, thereby exerting oncogenic effects. LINC01764 induced 5-FU chemoresistance by upregulating the c-MYC, glucose, and glutamine metabolism pathways, which downregulated UPP1, crucial for activating 5-FU. Conclusively, LINC01764 promotes CRC progression and 5-FU resistance through hnRNPK-mediated-c-MYC IRES-dependent translational regulation, which suggests its potential as a predictor of CRC chemotherapy response and prognosis.

Circular RNA hsa_circ_0000467 promotes colorectal cancer progression by promoting eIF4A3-mediated c-Myc translation

BackgroundColorectal cancer (CRC) is the second most common malignant tumor worldwide, and its incidence rate increases annually. Early diagnosis and treatment are crucial for improving the prognosis of patients with colorectal cancer. Circular RNAs are noncoding RNAs with a closed-loop structure that play a significant role in tumor development. However, the role of circular RNAs in CRC is poorly understood.MethodsThe circular RNA hsa_circ_0000467 was screened in CRC circRNA microarrays using a bioinformatics analysis, and the expression of hsa_circ_0000467 in CRC tissues was determined by in situ hybridization. The associations between the expression level of hsa_circ_0000467 and the clinical characteristics of CRC patients were evaluated. Then, the role of hsa_circ_0000467 in CRC growth and metastasis was assessed by CCK8 assay, EdU assay, plate colony formation assay, wound healing assay, and Transwell assay in vitro and in a mouse model of CRC in vivo. Proteomic analysis and western blotting were performed to investigate the effect of hsa_circ_0000467 on c-Myc signaling. Polysome profiling, RT‒qPCR and dual-luciferase reporter assays were performed to determine the effect of hsa_circ_0000467 on c-Myc translation. RNA pull-down, RNA immunoprecipitation (RIP) and immunofluorescence staining were performed to assess the effect of hsa_circ_0000467 on eIF4A3 distribution.ResultsIn this study, we found that the circular RNA hsa_circ_0000467 is highly expressed in colorectal cancer and is significantly correlated with poor prognosis in CRC patients. In vitro and in vivo experiments revealed that hsa_circ_0000467 promotes the growth and metastasis of colorectal cancer cells. Mechanistically, hsa_circ_0000467 binds eIF4A3 to suppress its nuclear translocation. In addition, it can also act as a scaffold molecule that binds eIF4A3 and c-Myc mRNA to form complexes in the cytoplasm, thereby promoting the translation of c-Myc. In turn, c-Myc upregulates its downstream targets, including the cell cycle-related factors cyclin D2 and CDK4 and the tight junction-related factor ZEB1, and downregulates E-cadherin, which ultimately promotes the growth and metastasis of CRC.ConclusionsOur findings revealed that hsa_circRNA_0000467 plays a role in the progression of CRC by promoting eIF4A3-mediated c-Myc translation. This study provides a theoretical basis and molecular target for the diagnosis and treatment of CRC.

Linc00941 fuels ribogenesis and protein synthesis by supporting robust cMYC translation in malignant pleural mesothelioma

Malignant pleural mesothelioma is a rare and lethal cancer caused by exposure to asbestos. The highly inflammatory environment caused by fibers accumulation forces cells to undergo profound adaptation to gain survival advantages. Prioritizing the synthesis of essential transcripts is an efficient mechanism coordinated by multiple molecules, including long non-coding RNAs. Enhancing the knowledge about these mechanisms is an essential weapon in combating mesothelioma. Linc00941 correlates to bad prognosis in various cancers, but it is reported to partake in distinct and apparently irreconcilable processes. In this work, we report that linc00941 supports the survival and aggressiveness of mesothelioma cells by influencing protein synthesis and ribosome biogenesis. Linc00941 binds to the translation initiation factor eIF4G, promoting the selective protein synthesis of cMYC, which, in turn, enhances the expression of key genes involved in translation. We analyzed a retrospective cohort of 97 mesothelioma patients' samples from our institution, revealing that linc00941 expression strongly correlates with reduced survival probability. This discovery clarifies linc00941's role in mesothelioma and proposes a unified mechanism of action for this lncRNA involving the selective translation of essential oncogenes, reconciling the discrepancies about its function.

LncRNA SNHG26 promotes gastric cancer progression and metastasis by inducing c-Myc protein translation and an energy metabolism positive feedback loop

Metastasis is a bottleneck in cancer treatment. Studies have shown the pivotal roles of long noncoding RNAs (lncRNAs) in regulating cancer metastasis; however, our understanding of lncRNAs in gastric cancer (GC) remains limited. RNA-seq was performed on metastasis-inclined GC tissues to uncover metastasis-associated lncRNAs, revealing upregulated small nucleolar RNA host gene 26 (SNHG26) expression, which predicted poor GC patient prognosis. Functional experiments revealed that SNHG26 promoted cellular epithelial–mesenchymal transition and proliferation in vitro and in vivo. Mechanistically, SNHG26 was found to interact with nucleolin (NCL), thereby modulating c-Myc expression by increasing its translation, and in turn promoting energy metabolism via hexokinase 2 (HK2), which facilitates GC malignancy. The increase in energy metabolism supplies sufficient energy to promote c-Myc translation and expression, forming a positive feedback loop. In addition, metabolic and translation inhibitors can block this loop, thus inhibiting cell proliferation and mobility, indicating potential therapeutic prospects in GC.

Translational enhancement of target endogenous mRNA in mammalian cells using programmable RNA-binding pentatricopeptide repeat proteins

Programmable protein scaffolds are invaluable in the development of genome engineering tools. The pentatricopeptide repeat (PPR) protein is an attractive platform for RNA manipulation because of its programmable RNA-binding selectivity, which is determined by the combination of amino acid species at three specific sites in the PPR motif. Translation is a key RNA regulatory step that determines the final gene expression level and is involved in various human diseases. In this study, designer PPR protein was used to develop a translational enhancement technique by fusion with the translation initiation factor eIF4G. The results showed that the PPR-eIF4G fusion protein could activate the translation of endogenous c-Myc and p53 mRNAs and control cell fate, indicating that PPR-based translational enhancement is a versatile technique applicable to various endogenous mRNAs in mammalian cells. In addition, the translational enhancement was dependent on both the target position and presence of eIF4G, suggesting the presence of an unknown translation activation mechanism.

USP47 inhibits m6A-dependent c-Myc translation to maintain regulatory T cell metabolic and functional homeostasis.

The functional integrity of Tregs is interwoven with cellular metabolism; however, the mechanisms governing Treg metabolic programs remain elusive. Here, we identified that the deubiquitinase USP47 inhibited c-Myc translation mediated by the RNA N6-methyladenosine (m6A) reader YTHDF1 to maintain Treg metabolic and functional homeostasis. USP47 positively correlated with the tumor-infiltrating Treg signature in samples from patients with colorectal cancer and gastric cancer. USP47 ablation compromised Treg homeostasis and function in vivo, resulting in the development of inflammatory disorders, and boosted antitumor immune responses. USP47 deficiency in Tregs triggered the accumulation of the c-Myc protein and in turn exacerbated hyperglycolysis. Mechanistically, USP47 prevented YTHDF1 ubiquitination to attenuate the association of YTHDF1 with translation initiation machinery, thereby decreasing m6A-based c-Myc translation efficiency. Our findings reveal that USP47 directs m6A-dependent metabolic programs to orchestrate Treg homeostasis and suggest novel approaches for selective immune modulation in cancer and autoimmune diseases by targeting of USP47.

Retraction Note: Therapeutic potential of targeting IRES-dependent c-myc translation in multiple myeloma cells during ER stress.

Retraction Note: Therapeutic potential of targeting IRES-dependent c-myc translation in multiple myeloma cells during ER stress.

Total ginsenosides promotes intestinal epithelial proliferation via affecting polyamine-mediated HuR on post-transcriptional control

Ginseng, as a functional food, is widely used worldwide because of its various benefits. This study aims to elucidate the potential effects of total ginsenosides (TG) in intestinal mucosal restitution. Establishing IEC-6 cell proliferation inhibition model by culturing with 5 mmol/L DL-α-difluoromethylornithine (DFMO, a polyamine inhibitor) for 48 h. The intestinal injury mice model was induced by 3% Dextran sodium sulfate (DSS) for 7 d. TG reversed the cell growth inhibition, regulated cell cycle and the decrease in c-Myc protein expression caused by DFMO, without affecting mRNA expression; TG increased translation efficiency and half-life of c-Myc mRNA, suggesting that TG promoted cell proliferation through affecting translation of c-Myc. Furthermore, TG could prevent the decrease of human antigen R (HuR)/c-Myc mRNA, p-HuR and p-Chk2 expression caused by DFMO, as well as reduce the expression of HuR in cytoplasm while increasing the expression in nucleus, indicating that TG influenced post-transcriptional control of c-Myc through polyamine-mediated HuR pathway for intestinal mucosal restitution. Panax ginseng aqueous extract (PGE) prevented intestinal mucosal injury induced by DSS(improved clinical symptoms and restored colon integrity) through enhancing HuR and c-Myc levels. Our results may provide a scientific basis for ginseng as a potential functional food ingredient and assistance therapeutic agent to prevent intestinal injury.

Oncogenic PKA signaling increases c-MYC protein expression through multiple targetable mechanisms.

Genetic alterations that activate protein kinase A (PKA) are found in many tumor types. Yet, their downstream oncogenic signaling mechanisms are poorly understood. We used global phosphoproteomics and kinase activity profiling to map conserved signaling outputs driven by a range of genetic changes that activate PKA in human cancer. Two signaling networks were identified downstream of PKA: RAS/MAPK components and an Aurora Kinase A (AURKA)/glycogen synthase kinase (GSK3) sub-network with activity toward MYC oncoproteins. Findings were validated in two PKA-dependent cancer models: a novel, patient-derived fibrolamellar carcinoma (FLC) line that expresses a DNAJ-PKAc fusion and a PKA-addicted melanoma model with a mutant type I PKA regulatory subunit. We identify PKA signals that can influence both de novo translation and stability of the proto-oncogene c-MYC. However, the primary mechanism of PKA effects on MYC in our cell models was translation and could be blocked with the eIF4A inhibitor zotatifin. This compound dramatically reduced c-MYC expression and inhibited FLC cell line growth in vitro. Thus, targeting PKA effects on translation is a potential treatment strategy for FLC and other PKA-driven cancers.

MiR-542-5p targets c-myc and negates the cell proliferation effect of SphK1 in intestinal epithelial cells.

Intestinal epithelial barrier defects occur commonly during a variety of pathological conditions, though their underlying mechanisms are not completely understood. Sphingosine-1-phosphate (S1P) has been shown to be a critical regulator of proliferation and of maintenance of an intact intestinal epithelial barrier, as is also sphingosine kinase 1 (SphK1), the rate-limiting enzyme for S1P synthesis. SphK1 has been shown to modulate its effect on intestinal epithelial proliferation through increased levels of c-myc. We conducted genome-wide profile analysis to search for differential microRNA expression related to overexpressed SphK1 demonstrating adjusted expression of microRNA 542-5p (miR-542-5p). Here, we show that miR-542-5p is regulated by SphK1 activity and is an effector of c-myc translation that ultimately serves as a critical regulator of the intestinal epithelial barrier. miR-542-5p directly regulates c-myc translation through direct binding to the c-myc mRNA. Exogenous S1P analogs administered in vivo protect murine intestinal barrier from damage due to mesenteric ischemia reperfusion, and damaged intestinal tissue had increased levels of miR-542-5p. These results indicate that miR-542-5p plays a critical role in the regulation of S1P-mediated intestinal barrier function, and may highlight a novel role in potential therapies.

Kindlin-2 enhances c-Myc translation through association with DDX3X to promote pancreatic ductal adenocarcinoma progression.

Rationale: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive solid tumor, with extremely low survival rates. Identifying key signaling pathways driving PDAC progression is crucial for the development of therapies to improve patient response rates. Kindlin-2, a multi-functional protein, is involved in numerous biological processes including cell proliferation, apoptosis and migration. However, little is known about the functions of Kindlin-2 in pancreatic cancer progression in vivo. Methods: In this study, we employ an in vivo PDAC mouse model to directly investigate the role of Kindlin-2 in PDAC progression. Then, we utilized RNA-sequencing, the molecular and cellular assays to determine the molecular mechanisms by which Kindlin-2 promotes PDAC progression. Results: We show that loss of Kindlin-2 markedly inhibits KrasG12D-driven pancreatic cancer progression in vivo as well as in vitro. Furthermore, we provide new mechanistic insight into how Kindlin-2 functions in this process, A fraction of Kindlin-2 was localized to the endoplasmic reticulum and associated with the RNA helicase DDX3X, a key regulator of mRNA translation. Loss of Kindlin-2 blocked DDX3X from binding to the 5'-untranslated region of c-Myc and inhibited DDX3X-mediated c-Myc translation, leading to reduced c-Myc-mediated glucose metabolism and tumor growth. Importantly, restoration of the expression of either the full-length Kindlin-2 or c-Myc, but not that of a DDX3X-binding-defective mutant of Kindlin-2, in Kindlin-2 deficient PDAC cells, reversed the inhibition of glycolysis and pancreatic cancer progression induced by the loss of Kindlin-2. Conclusion: Our studies reveal a novel Kindlin-2-DDX3X-c-Myc signaling axis in PDAC progression and suggest that inhibition of this signaling axis may provide a promising therapeutic approach to alleviate PDAC progression.

RNA Deadenylation Subunit CNOT3 Promotes Myeloid Leukemia By Driving Translation of Oncogenic Targets

While somatic alterations in genetic and epigenetic mechanisms in tumorigenesis have been studied extensively, how processes that affect post-transcriptional and translational regulation impact leukemia development is much less well developed. To identify novel regulators of post-transcriptional and translation control in AML, we surveyed several genome-wide CRISPR screens and observed that a number of subunits in the RNA deadenylation CNOT complex were highly ranked among genes essential for survival of human and mouse leukemia cells. The CCR4-NOT (CNOT) complex is one of two major multi-subunit polyA deadenylation complexes which mediate the shortening of the poly(A) tails. The CNOT complex was found to be recruited to m6A transcripts to mediate mRNA degradation. An involvement of the CNOT complex with RNA methylation, mRNA degradation and translation suggest a role of CNOT in bridging these processes to mediate complex gene expression regulation in pathogenesis of AML. Among the subunits of CNOT complex, we found that the highest ranked CNOT3 adaptor subunit demonstrated elevated expression in human AML cell lines (7/9 AML cell lines) and in primary AML patients (8/11 primary samples) assayed by immunoblots. Intracellular staining analysis further demonstrated that CNOT3 is highly expressed in leukemia stem cells-enriched CD34+ compartments in 13 out of 17 tested primary AML samples. Notably, high CNOT3 mRNA was associated with a poor prognosis in 3 independent AML patient cohorts i.e. TCGA-AML, Beat-AML and AMLCG 1999. Depletion of CNOT3 by shRNA-mediate knockdown and CRISPR/Cas9-mediated knockout in a panel of genetically diverse human leukemia cells (MOLM13, MV4-11, HL-60, NB4 and OCI-AML3) resulted in strong inhibition of cell growth, increased myeloid differentiation and apoptosis and significantly delayed leukemia development in vivo (median survival of 42 days, control versus 93.5 days, sh#1 and "not reached", sh#2). CNOT3 ablation also reduced survival and colony forming ability of primary AML patient cells. On the other hand, loss of CNOT3 function in normal cord-blood derived CD34+ (CB-CD34+) robustly induced myeloid differentiation without causing cell death, suggesting a potential therapeutic window to target CNOT3 in leukemia cells. In addition, overexpression of CNOT3 promoted cell growth in both CB-CD34+ and leukemia cells. Taken together, the data strongly indicates that CNOT3 promotes leukemogenesis. To uncover the mechanisms underpinning CNOT3 function in leukemia cells, we employed a CRISPR/Cas9 saturation domain mutagenesis screen by tiling the entire CNOT3 gene with sgRNAs. We identified the NOT box in the C-term domain of CNOT3 to be most critical for AML cell viability. In addition, truncation of the NOT box in CNOT3 abrogated its ability to drive proliferation in leukemia cells. This suggests a functional dependence of leukemia cells on CNOT3 interaction with the deadenylation complex via this domain. Transcriptomic and proteomic profiling of leukemia cells depleted of CNOT3 revealed an increase in p53 pathways, IFN-g immune response and downregulation of genes in G2/M checkpoint and MYC- target genes. We further observed a marked decrease in c-MYC protein upon ablation of CNOT3. We also noted the presence of c-MYC mRNA with longer poly(A) tails in CNOT3 depleted cells. However, there is no significant alternation in total abundancy and half-life of c-MYC mRNA, indicating a post-transcriptional regulation of c-MYC protein expression. In fact, depletion of CNOT3 significantly reduced the abundancy of c-MYC mRNA in the polysome-enriched and actively translated mRNAs, indicating that CNOT3 regulates translation of c-MYC. Immunoprecipitation of CNOT3 and mass spectrometry analysis of co-precipitated proteins captured proteins involved in protein synthesis including translation elongation factors and ribosomal proteins, suggesting a functional interaction of CNOT3 with the translation machinery. Overall, we uncovered a critical role of the CCR4-NOT deadenylation complex in myeloid leukemia and nominated CNOT3 as a potential target for AML therapy.

Asparagine Bioavailability Regulates the Translation of MYC oncogene in Lymphoid Malignancies

Non-essential amino acids are becoming increasingly important in cancer metabolism because of their roles beyond protein translation. These include providing precursors for macromolecule biosynthesis, serving as substrates for epigenetic modifications, regulating redox states, and modulating cell signaling. Acute lymphoblastic leukemia (ALL) is one such example where asparagine is shown to be conditionally essential for cancer growth and proliferation. Exploiting this metabolic vulnerability, L-Asparaginase (ASNase) has been used in the clinic to successfully treat ALL patients through depleting the circulating asparagine. Our investigation into the mechanism of resistance to ASNase in ALL cells yielded a striking correlation between asparagine bioavailability and c-MYC protein levels. c-MYC is a key transcription factor and has been shown to regulate the transcription of more than 10% of genes, including many metabolic genes, to meet the demands of tumor growth and progression. In addition, it has a well-documented role in normal hematopoiesis and hematological malignancies, including ALL, where high c-MYC levels have been shown to be a marker for leukemic initiation and growth. Using asparagine auxotrophic ALL cells that do not express asparagine synthetase (ASNS), the asparagine biosynthesis enzyme, we found that c-MYC protein levels were acutely depleted when asparagine was withdrawn from the tissue culture medium. However, c-MYC protein levels were sustained when ASNS cDNA was re-introduced into these cells. These results indicate the possibility of a reciprocal association between metabolism and a critical oncogene MYC. We also discovered that asparagine depletion inhibited the translation of c-MYC without changing its mRNA expression or altering the rate of protein degradation. Mechanistic Target of Rapamycin Complex 1 (mTORC1) and General Controlled Nonderepressible 2 (GCN2) are the two central molecular components involved in amino acid sensing and translation control. Asparagine starvation notably caused a decrease in global protein synthesis, mediated by suppression of mTORC1 and simultaneous activation of GCN2. Our studies using small molecule inhibitors and polysome profiling showed that repression of c-MYC mRNA translation is not due to the activation of the GCN2 pathway or a consequence of global translation suppression. In addition, both the '5' and '3' untranslated regions (UTRs) were not required for this inhibitory effect on c-MYC mRNA translation during asparagine depletion. Our results suggest that while asparagine depletion causes a defect in global translation at the initiation step, its effect on MYC mRNA translation is possibly due to elongation stalling. While ASNase can efficiently deplete circulating asparagine, its clinical applications are limited to ALL due to its low-level expression of ASNS. However, aspartate, a critical substrate for asparagine biosynthesis, can be pharmacologically targeted using electron transport chain (ETC) inhibitors. Our studies in ASNase-resistant ALL and lymphomas showed that while asparagine depletion alone did not affect c-MYC protein expression, co-treatment with ETC inhibitors (phenformin) showed a significant decrease in c-MYC protein levels. We extended this observation to test the synergy of ASNase and phenformin to treat an orthotopic MYC-driven B-cell lymphoma model. While either of the single treatments only modestly reduced the tumor burden, the double treatment showed the most significant reduction in the tumor burden, which correlated with a significant reduction of c-MYC protein expression. These results suggest that perturbing the asparagine biosynthesis pathway can be clinically employed to inhibit c-MYC protein expression and improve therapeutic outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Synergistic Anti-Tumor Effect on Novel Combination of Selinexor with Azacitidine in AML By Targeting XPO1/eIF4E/C-MYC Signaling

Background: Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy with high mortality and poor outcome. Highly effective therapy options are always needed for AML. Selinexor (KPT-330) is an oral bioactive selective CRM1 (XPO1) inhibitor, and the clinical trial showed the combination of KPT-330 over standard induction therapy benefits much for AML patients. Azacitidine (AZA), a DNA hypomethylation agent, can induce cell apoptosis and exhibit potential therapeutic efficacy against AML. However, it has not yet determined the anti-tumor effect of KPT-330 with AZA in AML. Here we examined the anti-leukemia effect and potential mechanism of KPT-330 combined with AZA in AML cells to provide evidence for further clinical trials. Methods: A total of 64 AML patients’ BM samples and 64 normal control from healthy volunteers were obtained from Zhongda Hospital Southeast University, in which the 2 patients’ samples were further treated with indicated drugs in vitro. Cell Counting Kit-8 (CCK-8) was used for cell viability assay, Annexin-V/PI staining following flow cytometry analysis for apoptosis, and qPCR and Western Blot for gene expression in AML cell lines and patient samples. CalcuSyn analysis was performed for a synergistic effect. RNA-seq was performed in U937 cells treated with 0.7uM AZA for 48 hrs, and the significantly differentially expressed genes (DEGs) were identified and gene set enrichment analysis (GSEA) was performed. Results: Cell proliferation arrest and apoptosis were significantly observed in U937 and MV4-11 cells treated with the combination of KPT-330 with AZA compared to single drug control (data not shown). Then, we further explored the synergistic effect of the combination in patients’ samples. Results showed that cell growth arrest and apoptosis significantly increased in primary samples from two de novo AML patients compared to the single drug controls, and CalcuSyn analysis showed a synergistic effect of the combination on cell growth arrest (Fig. 1A &1B). To understand the mechanisms underlying the synergy, RNA-seq analysis was performed in U937 cells treated by 0.7uM AZA and 4655 DEGs were identified. XPO1, eIF4E, and MYC are in the top DEGs upon AZA treatment, and GSEA analysis indicated MYC_TARGETS and apoptosis pathway are enriched in DEGs (Fig. 1C &1D ). Moreover, the mRNA level of XPO1 and eIF4E was increased in our AML patient cohort compared with healthy controls (Fig. 1E), and the high expression of XPO1 or elF4E are associated with poor outcome (Fig. 1F). XPO1 is the key component of nuclear-exporting machinery; elF4E, the cargo of XPO1 is translation initiation factor and plays a critical role in translational machinery, which regulates the C-MYC translation. It is also reported that AZA regulates the transcription of C-MYC by affecting methylation. We observed a combination of KPT-330 with AZA significantly down-regulated the mRNA and protein level of oncogene eIF4E and C-MYC in two of the patients’ samples compared to single drug controls (Fig. 1G &1H ). Also, C-MYC knock-down was not only significantly increased the apoptosis in U937 cells compared to scramble shRNA (shCTL) control, but also significantly enhanced the combination-mediated apoptosis compared to that of single drug control in U937 cells (Fig. 1I). these data indicated that XPO1/eIF4E/C-MYC signaling plays critical roles in the combination-mediated anti-leukemia, and the mechanistic model is summarized in Fig.1J. Conclusions: Our data for the first time showed the synergistic effect of a novel combination of KPT-330 with AZA on cell growth arrest and apoptosis in AML patients and also identified the underlying mechanism by targeting XPO1/eIF4E/C-MYC signaling. Our results also highlighted the feasibility of the clinical trial for the combination in the therapy of AML patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tumor suppressor mediated ubiquitylation of hnRNPK is a barrier to oncogenic translation

Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) is a multifunctional RNA binding protein (RBP) localized in the nucleus and the cytoplasm. Abnormal cytoplasmic enrichment observed in solid tumors often correlates with poor clinical outcome. The mechanism of cytoplasmic redistribution and ensuing functional role of cytoplasmic hnRNPK remain unclear. Here we demonstrate that the SCFFbxo4 E3 ubiquitin ligase restricts the pro-oncogenic activity of hnRNPK via K63 linked polyubiquitylation, thus limiting its ability to bind target mRNA. We identify SCFFbxo4-hnRNPK responsive mRNAs whose products regulate cellular processes including proliferation, migration, and invasion. Loss of SCFFbxo4 leads to enhanced cell invasion, migration, and tumor metastasis. C-Myc was identified as one target of SCFFbxo4-hnRNPK. Fbxo4 loss triggers hnRNPK-dependent increase in c-Myc translation, thereby contributing to tumorigenesis. Increased c-Myc positions SCFFbxo4-hnRNPK dysregulated cancers for potential therapeutic interventions that target c-Myc-dependence. This work demonstrates an essential role for limiting cytoplasmic hnRNPK function in order to maintain translational and cellular homeostasis.

Matrine, a potential c-Myc inhibitor, suppresses ribosome biogenesis and nucleotide metabolism in myeloid leukemia.

Previous studies have shown that matrine, a natural compound extracted from the herb Sophora flavescens, has a good anti-leukemia effect, but its key target and mechanism remains unclear. Here, we found that only c-Myc could respond rapidly to matrine treatment in three myeloid leukemia cell lines, and matrine inhibited both transcription and translation of c-Myc. Ribosome biogenesis and nucleotide metabolism, the key downstream of c-Myc, were significantly suppressed after matrine treatment. Therefore, our results confirmed that matrine is a special c-Myc inhibitor which suppresses ribosome biogenesis and nucleotide metabolism by inhibiting c-Myc in myeloid leukemia. This study provides scientific basis for the development of matrine derivatives to c-Myc-driven cancers.

Mechanisms of miR-3189-3p-mediated inhibition of c-MYC translation in triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the lack of estrogen receptor, progesterone receptor, and HER2. Our lab previously characterized miR-3189-3p as a microRNA with potent anti-cancer activity against glioblastoma. Here, we hypothesized a similar activity in TNBC cells. As miR-3189-3p is predicted to target a variety of RNA binding proteins, we further hypothesized an inhibitory effect of this miRNA on protein synthesis.MethodsMDA-MB-231 and MDA-MB-468 cells were used to investigate the effect of miR-3189-3p on cell proliferation, migration, and invasion. TGCA database was used to analyze the expression of miR-3189-3p, c-MYC, 4EPB1, and eIF4E in breast cancer. Western blotting and RT-qPCR assays were used to assess the expression of selected proteins and RNAs after transfections.ResultsAlthough c-MYC is not a predicted gene target for miR-3189-3p, we discovered that c-MYC protein is downregulated in miRNA-treated TNBC cells. We found that the downregulation of c-MYC by miR-3189-3p occurs in both normal growth conditions and in the absence of serum. The mechanism involved the direct inhibition of eIF4EBP1 by miR-3189-3p. Additionally, we found that miR-3189-3p could negatively affect cap-independent translation mediated by internal ribosome entry sites (IRES) or by m6A. Finally, miR-3189-3p sensitized TNBC cells to doxorubicin.ConclusionOverall, results indicated that miR-3189-3p exerts its anti-tumor activity through targeting translational regulatory proteins leading to an impairment in c-MYC translation, and possibly other oncogenic factors, suggesting that miR-3189-3p, alone or in combination, could be a valuable therapeutic approach against a malignancy with few treatment options.

Localization of a TORC1-eIF4F translation complex during CD8+ T cell activation drives divergent cell fate

Activated CD8+ T lymphocytes differentiate into heterogeneous subsets. Using super-resolution imaging, we found that prior to the first division, dynein-dependent vesicular transport polarized active TORC1 toward the microtubule-organizing center (MTOC) at the proximal pole. This active TORC1 was physically associated with active eIF4F, required for the translation of c-myc mRNA. As a consequence, c-myc-translating polysomes polarized toward the cellular pole proximal to the immune synapse, resulting in localized c-myc translation. Upon division, the TORC1-eIF4A complex preferentially sorted to the proximal daughter cell, facilitating asymmetric c-Myc synthesis. Transient disruption of eIF4A activity at first division skewed long-term cell fate trajectories to memory-like function. Using a genetic barcoding approach, we found that first-division sister cells often displayed differences in transcriptional profiles that largely correlated with c-Myc and TORC1 target genes. Our findings provide mechanistic insights as to how distinct Tcell fate trajectories can be established during the first division.

Critical Role for Cap-Independent c-MYC Translation in Progression of Multiple Myeloma.

Dysregulated c-myc is a determinant of multiple myeloma progression. Translation of c-myc can be achieved by an mTOR-mediated, cap-dependent mechanism or a cap-independent mechanism where a sequence in the 5'UTR of mRNA, termed the internal ribosome entry site (IRES), recruits the 40S ribosomal subunit. This mechanism requires the RNA-binding factor hnRNP A1 (A1) and becomes critical when cap-dependent translation is inhibited during endoplasmic reticulum (ER) stress. Thus, we studied the role of A1 and the myc IRES in myeloma biology. A1 expression correlated with enhanced c-myc expression in patient samples. Expression of A1 in multiple myeloma lines was mediated by c-myc itself, suggesting a positive feedback circuit where myc induces A1 and A1 enhances myc translation. We then deleted the A1 gene in a myc-driven murine myeloma model. A1-deleted multiple myeloma cells demonstrated downregulated myc expression and were inhibited in their growth in vivo. Decreased myc expression was due to reduced translational efficiency and depressed IRES activity. We also studied the J007 inhibitor, which prevents A1's interaction with the myc IRES. J007 inhibited myc translation and IRES activity and diminished myc expression in murine and human multiple myeloma lines as well as primary samples. J007 also inhibited tumor outgrowth in mice after subcutaneous or intravenous challenge and prevented osteolytic bone disease. When c-myc was ectopically reexpressed in A1-deleted multiple myeloma cells, tumor growth was reestablished. These results support the critical role of A1-dependent myc IRES translation in myeloma.

BZW1 Facilitates Glycolysis and Promotes Tumor Growth in Pancreatic Ductal Adenocarcinoma Through Potentiating eIF2α Phosphorylation

Pancreatic ductal adenocarcinoma (PDAC) is characterized by severe metabolic stress due to fibrosis and poor vascularization. BZW1 is an eIF5-mimic protein involved in tumorigenesis and progression. The aim of this study was to investigate the role of BZW1 in metabolic stress resistance in PDAC. BZW1 expression was evaluated in human PDAC tissue microarray and PDAC cells. Glycolysis regulation of BZW1 and its correlation with glycolysis-related genes was analyzed. Tumor growth, cell proliferation, and apoptosis were evaluated in mice xenograft tumors and patient-derived organoids. The results of bioinformatic screening identified that BZW1 was 1 of the top 3 genes favorable for tumor progression in PDAC. The analysis of our cohort confirmed that BZW1 was overexpressed in human PDAC tissues compared with nontumor tissues, and its abnormal expression was correlated with large tumor size and poor prognosis. BZW1 promoted cell proliferation and inhibited apoptosis in both mouse xenograft models and PDAC-derived organoids via facilitating glycolysis in the oxygen-glucose-deprivation condition. Mechanically, BZW1 served as an adaptor for PKR-like endoplasmic reticulum (ER) kinase (PERK), facilitated the phosphorylation of eIF2α, promoted internal ribosome entry site-dependent translation of HIF1α and c-Myc, and thereby boosted the Warburg effect. In organoid-based xenografts with high BZW1 levels, both the PERK/eIF2α phosphorylation inhibitor GSK2606414 and ISRIB significantly suppressed tumor growth and prolonged animal survival. BZW1 is a key molecule in the internal ribosome entry site-dependent translation of HIF1α/c-Myc and plays crucial roles in the glycolysis of PDAC. BZW1 might serve as a therapeutic target for patients with pancreatic cancer.