m6A modification negatively regulates translation by switching mRNA from polysome to P-body via IGF2BP3.

In the cytoplasm, mRNAs are dynamically partitioned into translating and non-translating pools, but the mechanism for this regulation has largely remained elusive. Here, we report that m6A regulates mRNA partitioning between polysome and P-body where a pool of non-translating mRNAs resides. By quantifying the m6A level of polysomal and cytoplasmic mRNAs with m6A-LAIC-seq and m6A-LC-MS/MS in HeLa cells, we observed that polysome-associated mRNAs are hypo-m6A-methylated, whereas those enriched in P-body are hyper-m6A-methylated. Downregulation of the m6A writer METTL14 enhances translation by switching originally hyper-m6A-modified mRNAs from P-body to polysome. Conversely, by proteomic analysis, we identify a specific m6A reader IGF2BP3 enriched in P-body, and via knockdown and molecular tethering assays, we demonstrate that IGF2BP3 is both necessary and sufficient to switch target mRNAs from polysome to P-body. These findings suggest a model for the dynamic regulation of mRNA partitioning between the translating and non-translating pools in an m6A-dependent manner.

Oncofetal IGF2BP3-mediated control of microRNA structural diversity in the malignancy of early-stage lung adenocarcinoma.

The nature of microRNA (miRNA) dysfunction in carcinogenesis remains controversial because of the complex connection between miRNA structural diversity and biological processes. Here, we found that oncofetal IGF2BP3 regulates the selective production of a subset of 3'-isoforms (3'-isomiRs), including miR-21-5p and Let-7 family, which induces significant changes in their cellular seed occupancy and structural components, establishing a cancer-specific gene expression profile. The D-score, reflecting dominant production of a representative miR-21-5p+C (a 3'-isomiR), discriminated between clinical early-stage lung adenocarcinoma (LUAD) cases with low and high recurrence risks, and was associated with molecular features of cell cycle progression, epithelial-mesenchymal transition pressure, and immune evasion. We found that IGF2BP3 controls the production of miR-21-5p+C by directing the nuclear Drosha complex to select the cleavage site. IGF2BP3 was also involved in the production of 3'-isomiRs of miR-425-5p and miR-454-3p. IGF2BP3-regulated these three miRNAs are suggested to be associated with the regulation of p53, TGF-ß, and TNF pathways in LUAD. Knockdown of IGF2BP3 also induced a selective upregulation of Let-7 3'-isomiRs, leading to increased cellular Let-7 seed occupancy and broad repression of its target genes encoding cell cycle regulators. The D-score is an index that reflects this cellular situation. Our results suggest that the aberrant regulation of miRNA structural diversity is a critical component for controlling cellular networks, thus supporting the establishment of a malignant gene expression profile in early stage LUAD.

Optimized infrared photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (IR-PAR-CLIP) protocol identifies novel IGF2BP3-interacting RNAs in colon cancer cells.

The conserved family of RNA-binding proteins (RBPs), IGF2BPs, plays an essential role in posttranscriptional regulation controlling mRNA stability, localization, and translation. Mammalian cells express three isoforms of IGF2BPs: IGF2BP1-3. IGF2BP3 is highly overexpressed in cancer cells, and its expression correlates with a poor prognosis in various tumors. Therefore, revealing its target RNAs with high specificity in healthy tissues and in cancer cells is of crucial importance. Photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) identifies the binding sites of RBPs on their target RNAs at nucleotide resolution in a transcriptome-wide manner. Here, we optimized the PAR-CLIP protocol to study RNA targets of endogenous IGF2BP3 in a human colorectal carcinoma cell line. To this end, we first established an immunoprecipitation protocol to obtain highly pure endogenous IGF2BP3-RNA complexes. Second, we modified the protocol to use highly sensitive infrared (IR) fluorescent dyes instead of radioactive probes to visualize IGF2BP3-crosslinked RNAs. We named the modified method "IR-PAR-CLIP." Third, we compared RNase cleavage conditions and found that sequence preferences of the RNases impact the number of the identified IGF2BP3 targets and introduce a systematic bias in the identified RNA motifs. Fourth, we adapted the single adapter circular ligation approach to increase the efficiency in library preparation. The optimized IR-PAR-CLIP protocol revealed novel RNA targets of IGF2BP3 in a human colorectal carcinoma cell line. We anticipate that our IR-PAR-CLIP approach provides a framework for studies of other RBPs.

KIAA1429/VIRMA promotes breast cancer progression by m6 A-dependent cytosolic HAS2 stabilization.

N6 -methyladenosine (m6 A), the most abundant internal modification in eukaryotic mRNA, plays important roles in many physiological and pathological processes, including the development and progression of cancer. RNA modification by m6 A is regulated by methyltransferases, demethylases, and m6 A-binding proteins that function in large part by regulating mRNA expression and function. Here, we investigate the expression of m6 A regulatory proteins in breast cancer. We find that expression of KIAA1429/VIRMA, a component of the m6 A methyltransferase complex, is upregulated in breast cancer tissue and correlates positively with poor survival. KIAA1429/VIRMA is mislocalized to the cytosol of breast cancer tissues and cell lines, and shRNA-mediated knockdown inhibits breast cancer cell proliferation, migration, and invasion. Mechanistically, KIAA1429/VIRMA is shown to bind to the m6 A-dependent RNA-binding protein insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), leading to recruitment and stabilization of m6 A-modified hyaluronan synthase 2 (HAS2) mRNA. HAS2 mRNA and KIAA1429/VIRMA mRNA levels correlate positively in breast cancer tissues, suggesting that the KIAA1429/VIRMA-IGF2BP3-HAS2 axis promotes breast cancer growth and contributes to poor prognosis.

Atractylodes macrocephala III suppresses EMT in cervical cancer by regulating IGF2BP3 through ETV5.

Atractylodes macrocephala III (ATL III), with anti-inflammatory and antitumor effects, is the main compound of Atractylodes macrocephala. Whether ATL III has an effect on cervical cancer and the specific mechanism are still unclear. Here, we investigated the effects of ATL III on cervical cancer cells at different concentrations and found that ATL III downregulates insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), which was found to be highly expressed in cervical cancer tissue by RNA-Seq. In this study, we found that ATL III promotes apoptosis and regulates epithelial-mesenchymal transition (EMT) in cervical cancer cells (HeLa and SiHa cells) and that IGF2BP3 is a common target gene of ATL III in HeLa and SiHa cells. The expression level of IGF2BP3 in cervical cancer cells was proportional to their migration and invasion abilities. This was verified by transfection of cells with a small interfering RNA and an IGF2BP3 overexpression plasmid. After ATL III treatment, the migration and invasion abilities of cervical cancer cells were obviously reduced, but these effects were attenuated after overexpression of IGF2BP3. In addition, the transcription factor IGF2BP3 was predicted by the JASPAR system. After intersection with our sequencing results, we verified the promotional effect of ETV5 (ETS translocation variant 5) on IGF2BP3 and found that ALT III inhibited ETV5. In general, our research showed that ATL III inhibits the migration and invasion of cervical cancer cells by regulating IGF2BP3 through ETV5.

Exosomal circSIPA1L3-mediated intercellular communication contributes to glucose metabolic reprogramming and progression of triple negative breast cancer.

BACKGROUND: Breast cancer is the most common malignant tumor, and metastasis remains the major cause of poor prognosis. Glucose metabolic reprogramming is one of the prominent hallmarks in cancer, providing nutrients and energy to support dramatically elevated tumor growth and metastasis. Nevertheless, the potential mechanistic links between glycolysis and breast cancer progression have not been thoroughly elucidated. METHODS: RNA-seq analysis was used to identify glucose metabolism-related circRNAs. The expression of circSIPA1L3 in breast cancer tissues and serum was examined by qRT-PCR, and further assessed its diagnostic value. We also evaluated the prognostic potential of circSIPA1L3 by analyzing a cohort of 238 breast cancer patients. Gain- and loss-of-function experiments, transcriptomic analysis, and molecular biology experiments were conducted to explore the biological function and regulatory mechanism of circSIPA1L3. RESULTS: Using RNA-seq analysis, circSIPA1L3 was identified as the critical mediator responsible for metabolic adaption upon energy stress. Gain- and loss-of-function experiments revealed that circSIPA1L3 exerted a stimulative effect on breast cancer progression and glycolysis, which could also be transported by exosomes and facilitated malignant behaviors among breast cancer cells. Significantly, the elevated lactate secretion caused by circSIPA1L3-mediated glycolysis enhancement promoted the recruitment of tumor associated macrophage and their tumor-promoting roles. Mechanistically, EIF4A3 induced the cyclization and cytoplasmic export of circSIPA1L3, which inhibited ubiquitin-mediated IGF2BP3 degradation through enhancing the UPS7-IGF2BP3 interaction. Furthermore, circSIPA1L3 increased mRNA stability of the lactate export carrier SLC16A1 and the glucose intake enhancer RAB11A through either strengthening their interaction with IGF2BP3 or sponging miR-665, leading to enhanced glycolytic metabolism. Clinically, elevated circSIPA1L3 expression indicated unfavorable prognosis base on the cohort of 238 breast cancer patients. Moreover, circSIPA1L3 was highly expressed in the serum of breast cancer patients and exhibited high diagnostic value for breast cancer patients. CONCLUSIONS: Our study highlights the oncogenic role of circSIPA1L3 through mediating glucose metabolism, which might serve as a promising diagnostic and prognostic biomarker and potential therapeutic target for breast cancer.

Association of the m6 A reader IGF2BP3 with tumor progression and brain-specific metastasis in breast cancer.

BACKGROUND: This study aimed to determine the role of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), an N6 -methyladinosine reader, in the progression and distant metastasis of breast cancer. METHODS: IGF2BP3 expression was assessed in 152 pairs of breast cancer and adjacent normal tissue (ANT) by real-time quantitative polymerase chain reaction and in 561 cases of breast cancer and 163 cases of ANT by immunohistochemistry. Survival curves were estimated using the Kaplan-Meier method and then compared statistically using the log-rank test. The prognostic role of IGF2BP3 was determined by Cox regression analysis. RESULTS: Analysis of public gene data sets revealed that IGF2PB3 predicted distant metastasis in breast cancer and was highly correlated with brain metastasis. In the clinical retrospective cohort, the positive rate of IGF2BP3 increased gradually with breast cancer progression. Positive IGF2BP3 expression was related to poor distant metastasis-free survival (DMFS, p = .030) and Cox regression analysis identified IGF2BP3 as an independent risk factor for DMFS (hazard ratio, 1.876; 95% confidence interval, 1.128-3.159; p = .019). Positive IGF2BP3 expression was markedly related to breast cancer brain metastasis (p = .011) but not to lung and bone metastasis. Moreover, patients with IGF2BP3-positive brain metastasis had lower survival than patients with IGF2BP3-negative brain metastasis (p = .041). Gene expression profiling results indicated that high IGF2BP3 expression was associated with the PD-1 checkpoint pathway, HER2-HER3 signaling, and epithelial-mesenchymal transition. CONCLUSIONS: IGF2BP3 may serve as a novel predictive biomarker and a potential therapeutic target for breast cancer brain metastasis, which warrants further investigation. PLAIN LANGUAGE SUMMARY: As an m6 A reader, IGF2BP3 is dysregulated and implicated in various cancers but its role in breast cancer has not been fully clarified. In this study, we found that IGF2BP3 was upregulated in breast cancer and IGF2BP3 expression increased gradually during breast cancer progression. IGF2BP3 expression exerted no effect on the overall survival and breast cancer-specific survival of breast cancer patients; however, IGF2BP3-positive patients were more likely to develop distant metastasis than IGF2BP3-negative patients. In addition, IGF2BP3 was associated with brain-specific metastasis in breast cancer patients. These findings warrant further investigation because they provide a rationale for novel predictive or therapeutic approaches.

IGF2BP3 boosts lactate generation to accelerate gastric cancer immune evasion.

The CD8+ T cells mediated antitumor immunity plays a critical function on gastric cancer (GC) immunotherapy. However, the mechanism of N6-methyladenosine (m6A) and lactate in GC immune microenvironment are still unclear. Here, present research investigated the role of Insulin like growth factor II mRNA binding protein 3 (IGF2BP3) in GC and its in-depth mechanisms in the antitumor immunity. Data illustrated that high IGF2BP3 level was associated to GC poor prognosis and tumor infiltration. Functional assays demonstrated that IGF2BP3 overexpression could promote the lactate accumulation, and impair the CD8+ T cells' antitumor immunity activity in co-culture system. Correspondingly, IGF2BP3 silencing enhanced the CD8+ T cells' antitumor immunity activity towards co-cultured GC cells. Mechanistically, IGF2BP3 could bind the m6A site on LDHA mRNA, thereby promoting its mRNA stability. Rescue assays elucidated that IGF2BP3/LDHA axis impaired the CD8+ T cells antitumor immunity by triggering lactate excess tumor microenvironment. In conclusion, our findings demonstrate that IGF2BP3 impairs the CD8+ T cells antitumor immunity by targeting LDHA/lactate axis, providing a novel therapeutic insight for GC immunotherapy.

Circ_0098823 binding with IGF2BP3 regulates DNM1L stability to promote metastasis of hepatocellular carcinoma via mitochondrial fission.

Hepatocellular carcinoma (HCC) is highly metastatic and invasive. CircRNA participates in gene regulation of multiple tumor metastases, but little is known whether it is a bystander or an actual player in HCC metastasis. We aim to explore the molecular mechanisms of novel circRNAs in HCC metastasis. RT-qPCR was used to detect the expression of 13 circRNAs derived by the ERBB3 gene. The function of circ_0098823 and DNM1L in HCC cells were estimated by CCK-8, transwell assays, flow cytometry, electron microscope, and in vivo experiments. RNA binding protein of circ_0098823 was confirmed by RNA pull-down, mass spectrometry, and RNA immunoprecipitation. The expression of DNM1L after IGF2BP3 knockdown was detected by RT-qPCR and western blot. Circ_0098823 was significantly up-regulated both in HCC tissues and HGF induced cell lines. Circ_0098823 overexpression significantly enhanced proliferation, migration, and invasion but decreased apoptosis of HCC cells, particularly promoted mitochondrial fission. Compared with the control group, the tumors in the circ_0098823 knockdown mice were significantly smaller and lighter. Circ_0098823 silencing suppressed DNM1L expression, a key molecule for fission, which enhanced proliferation, migration and invasion, and inhibited apoptosis of HCC cell. IGF2BP3 was a binding protein of circ_0098823. The expression and mRNA stability of DNM1L were down-regulated by IGF2BP3 knockdown. IGF2BP3 knockdown significantly alleviated the excessive migration, invasion and apoptosis of HCC cells caused by circ_0098823 overexpression. This study uncovered a novel circ_0098823 with tumor-promoting effect, and the mechanism by which circ_0098823 participates in HCC progression through IGF2BP3-guided DNM1L. Our study broadens molecular understanding of HCC progression.

LINC00942 inhibits ferroptosis and induces the immunosuppression of regulatory T cells by recruiting IGF2BP3/SLC7A11 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignant tumor with a high recurrence rate and a poor prognosis. Long intergenic nonprotein coding RNA 942 (LINC00942) is reported to be related to ferroptosis and the immune response in HCC and serves as an oncogene in various cancers. This research aimed to explore the contribution of LINC00942 in HCC progression. Functional assays were used to evaluate the functional role of LINC00942 in vitro and in vivo. Mechanistic assays were conducted to assess the association of LINC00942 with insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) and solute carrier family 7 member 11 (SLC7A11) and the regulatory pattern of LINC00942 in HCC cells. LINC00942 was found to exhibit upregulation in HCC tissue and cells. LINC00942 facilitated HCC cell proliferation, suppressed ferroptosis, and converted naive CD4+ T cells to inducible Treg (iTreg) cells by regulating SLC7A11. Furthermore, SLC7A11 expression was positively modulated by LINC00942 in HCC cells. IGF2BP3 was a shared RNA-binding protein (RBP) for LINC00942 and SLC7A11. The binding between the SLC7A11 3' untranslated region and IGF2BP3 was verified, and LINC00942 was found to recruit IGF2BP3 to promote SLC7A11 mRNA stability in an m6A-dependent manner. Moreover, mouse tumor growth and proliferation were inhibited, and the number of FOXP3+CD25+ T cells was increased, while ferroptosis was enhanced after LINC00942 knockdown in vivo. LINC00942 suppresses ferroptosis and induces Treg immunosuppression in HCC by recruiting IGF2BP3 to enhance SLC7A11 mRNA stability, which may provide novel therapeutic targets for HCC.

IGF2BP3 promotes glutamine metabolism of endometriosis by interacting with UCA1 to enhances the mRNA stability of GLS1.

BACKGROUND: Insulin like growth factor II mRNA binding protein 3 (IGF2BP3) has been implicated in numerous inflammatory and cancerous conditions. However, its precise molecular mechanisms in endometriosis (EMs) remains unclear. The aim of this study is to examine the influence of IGF2BP3 on the occurrence and progression of EMs and to elucidate its underlying molecular mechanism. METHODS: Efects of IGF2BP3 on endometriosis were confrmed in vitro and in vivo. Based on bioinformatics analysis, RNA immunoprecipitation (RIP), RNA pull-down assays and Fluorescent in situ hybridization (FISH) were used to show the association between IGF2BP3 and UCA1. Single-cell spatial transcriptomics analysis shows the expression distribution of glutaminase 1 (GLS1) mRNA in EMs. Study the effect on glutamine metabolism after ectopic endometriotic stromal cells (eESCs) were transfected with Sh-IGF2BP3 and Sh-UCA1 lentivirus. RESULTS: Immunohistochemical staining have revealed that IGF2BP3 was upregulated in ectopic endometriotic lesions (EC) compared to normal endometrial tissues (EN). The proliferation and migration ability of eESCs were greatly reduced by downregulating IGF2BP3. Additionally, IGF2BP3 has been observed to interact with urothelial carcinoma associated 1 (UCA1), leading to increased stability of GLS1 mRNA and subsequently enhancing glutamine metabolism. Results also demonstrated that IGF2BP3 directly interacts with the 3' UTR region of GLS1 mRNA, influencing its expression and stability. Furthermore, UCA1 was able to bind with c-MYC protein, stabilizing c-MYC mRNA and consequently enhancing GLS1 expression through transcriptional promotion. CONCLUSION: These discoveries underscored the critical involvement of IGF2BP3 in the elevation and stability of GLS1 mRNA in the context of glutamine metabolism by interacting with UCA1 in EMs. The implications of our study extended to the identification of possible therapeutic targets for individuals with EMs.

The G allele of SNP rs3922 reduces the binding affinity between IGF2BP3 and CXCR5 correlating with a lower antibody production.

Effective vaccines that function through humoral immunity seek to produce high-affinity antibodies. Our previous research identified the single-nucleotide polymorphism rs3922G in the 3'UTR of CXCR5 as being associated with nonresponsiveness to the hepatitis B vaccine. The differential expression of CXCR5 between the dark zone (DZ) and light zone (LZ) is critical for organizing the functional structure of the germinal center (GC). In this study, we report that the RNA-binding protein IGF2BP3 can bind to CXCR5 mRNA containing the rs3922 variant to promote its degradation via the nonsense-mediated mRNA decay pathway. Deficiency of IGF2BP3 leads to increased CXCR5 expression, which results in the disappearance of CXCR5 differential expression between DZ and LZ, disorganized GCs, aberrant somatic hypermutations, and reduced production of high-affinity antibodies. Furthermore, the affinity of IGF2BP3 for the rs3922G-containing sequence is lower than that for the rs3922A counterpart, which may explain the nonresponsiveness to the hepatitis B vaccination. Together, our findings suggest that IGF2BP3 plays a crucial role in the production of high-affinity antibodies in the GC by binding to the rs3922-containing sequence to regulate CXCR5 expression.

METTL3-mediated m6A methylation of SLC38A1 stimulates cervical cancer growth.

The objective of this study was to better characterize the role of the glutamine transporter SLC38A1 in cervical cancer and explore the underlying mechanisms. Data from public databases and clinical cervical cancer tissue samples were used to assess the expression of SLC38A1 and its prognostic significance. Immunohistochemical staining, qRT-PCR, and Western blotting were used to evaluate the expression of relevant genes and proteins. Cell viability, cell cycle, apoptosis, and intracellular glutamine content were measured using CCK-8, flow cytometry, and biochemical assays. Additionally, the RNA immunoprecipitation (RIP) assay was used to examine the impact of METTL3/IGF2BP3 on the m6A modification of the SLC38A1 3'UTR. Both cervical cancer specimens and cells showed significantly increased expression of SLC38A1 and its expression correlated with an unfavorable prognosis. Knockdown of SLC38A1 inhibited cell viability and cell cycle progression, induced apoptosis, and suppressed tumor growth in vivo. Glutaminase-1 inhibitor CB-839 reversed the effects of SLC38A1 overexpression. METTL3 promoted m6A modification of SLC38A1 and enhanced its mRNA stability through IGF2BP3 recruitment. Moreover, METTL3 silencing inhibited cell viability, cell cycle progression, intracellular glutamine content, and induced apoptosis, but these effects were reversed by SLC38A1 overexpression. In conclusion, METTL3-mediated m6A methylation of SLC38A1 stimulates cervical cancer progression. SLC38A1 inhibition is a potential therapeutic strategy for cervical cancer.

ALKBH5-Mediated m6A Modification of XBP1 Facilitates NSCLC Progression Through the IL-6-JAK-STAT3 Pathway.

The X-box-binding protein 1 (XBP1) is an important transcription factor during endoplasmic reticulum stress response, which was reported as an oncogene in non-small cell lung cancer (NSCLC) tumorigenesis and development. However, the regulatory mechanism of XBP1 expression in NSCLC progression was less reported. N6-methyladenosine (m6A) RNA modification is an emerging epigenetic regulatory mechanism for gene expression. This study aimed to investigate the regulatory role of the m6A modification in XBP1 expression in NSCLC. We identified XBP1 as a downstream target of ALKBH5-mediated m6A modification in A549 and PC9 cells. Knockdown of ALKBH5 increased the m6A modification and the stability of XBP1 mRNA, while overexpression of ALKBH5 had the opposite effect. Furthermore, IGF2BP3 was confirmed to be a reader of XBP1 m6A methylation and to enhance the stability of XBP1 mRNA. Additionally, IGF2BP3 knockdown significantly reversed the increase in XBP1 stability mediated by ALKBH5 depletion. In vivo and in vitro experiments demonstrated that ALKBH5/IGF2BP3 promotes the proliferation, migration, and invasion of NSCLC cells by upregulating XBP1 expression. In addition, we also showed that XBP1 promoted NSCLC cell proliferation, migration, and invasion by activating IL-6-JAK-STAT3 signaling. Our research suggested that ALKBH5-mediated m6A modification of XBP1 facilitates NSCLC progression through the IL-6-JAK-STAT3 pathway.

Multi-omics analysis unveils the predictive value of IGF2BP3/SPHK1 signaling in cancer stem cells for prognosis and immunotherapeutic response in muscle-invasive bladder cancer.

BACKGROUND: Muscle invasive bladder cancer (MIBC) is a life-threatening malignant tumor characterized by high metastasis rates, poor prognosis, and limited treatment options. Immune checkpoint inhibitors (ICIs) targeting PD-1 and PD-L1 represent an emerging treatment for MIBC immunotherapy. However, the characteristics of patients likely to benefit from immunotherapy remain unclear. METHODS: We performed single-cell mass cytometry (CyTOF) analysis of 179,483 single cells to characterize potential immunotherapy-related cancer stem cells (CSCs)-like populations in the tumor microenvironment of 38 MIBC tissues. The upregulated expression of IGF2BP3 in CD274 + ALDH + CSC-like cells, which was associated with poor clinical prognosis, was analyzed by bulk RNA-sequencing data from an in-house cohort. The functional role of IGF2BP3 was determined through cell proliferation, colony formation, cell apoptosis and sphere formation assays. The regulation of SPHK1 expression by IGF2BP3 was  investigated using methylated RNA immunoprecipitation sequencing (MeRIP-seq) and bulk RNA-sequencing (bulk RNA-seq). We further utilized single-nucleus RNA sequencing (snRNA-seq) data from 67,988 cells of 25 MIBC tissues and single-cell RNA sequencing (scRNA-seq) data from MIBC patient-derived organoids to characterize the molecular features of bladder cancer cells co-expressing IGF2BP3 and SPHK1. Spatial transcriptomics (ST) and co-detection by indexing (CODEX) analysis were used to describe the spatial distribution and interactions of IGF2BP3 + SPHK1 + bladder cancer cells and immune cells. RESULTS: A subset of CD274 + ALDH + CSC-like cells was identified, associating with immunosuppression and low survival rates in MIBC patients. IGF2BP3, an m6A reader gene, was found to be upregulated in the CD274 + ALDH + CSC-like cell population and linked to poor clinical prognosis in MIBC. Knockout of IGF2BP3 dramatically promoted cell apoptosis and reduced cell proliferation in T24 cells. By integrating MeRIP-seq and bulk RNA-seq analyses, we identified SPHK1 served as a substrate for IGF2BP3 in an m6A-dependent manner. Further snRNA-seq, scRNA-seq, ST, and CODEX analysis revealed a closer topographical distance between IGF2BP3 + SPHK1 + bladder cancer cells and exhausted CD8 + T cells, providing one explanation for the superior response to immunotherapy in IGF2BP3 + SPHK1 + bladder cancer cells-enriched patients. Finally, an ICI-associated signature was developed based on the enriched genes of IGF2BP3 + SPHK1 + bladder cancer cells, and its potential ability to predict the response to immunotherapy was validated in two independent immunotherapy cohort. CONCLUSIONS: Our study highlighted the critical involvement of the IGF2BP3/SPHK1 signaling in maintaining the stemness of CSCs and promoting MIBC progression. Additionally, these findings suggested that the IGF2BP3/SPHK1 signaling might serve as a biomarker for prognosis and immunotherapy response in MIBC.

Licochalcone A decreases cancer cell proliferation and enhances ferroptosis in acute myeloid leukemia through suppressing the IGF2BP3/MDM2 cascade.

Licochalcone A (Lico A), a naturally bioactive flavonoid, has shown antitumor activity in several types of cancers. However, few studies have focused on its effect on acute myeloid leukemia (AML). Cell viability and colony formation potential were detected by CCK-8 assay and colony formation assay, respectively. Cell cycle distribution and apoptosis were assessed by flow cytometry. Ferroptosis was assessed by measuring reactive oxygen species (ROS), lipid ROS, malondialdehyde (MDA), and glutathione (GSH). Protein expression levels were determined by immunoblotting and immunohistochemistry (IHC), and mRNA expression was detected by real-time qPCR. The m6A modification of MDM2 mRNA was verified by methylated RNA immunoprecipitation (MeRIP) assay, and the interaction of IGF2BP3 and MDM2 mRNA was analyzed by RIP assay. Actinomycin D was used to evaluate mRNA stability. The efficacy of Lico A in vivo was examined by a murine xenograft model. Lico A suppressed cell proliferation and induced ferroptosis in MOLM-13 and U-937 in vitro, and slowed the growth of xenograft tumors in vivo. IGF2BP3 was highly expressed in human AML specimens and cells, and Lico A suppressed IGF2BP3 expression in AML cells. Lico A exerted the anti-proliferative and pro-ferroptosis effects by downregulating IGF2BP3. Moreover, IGF2BP3 enhanced the stability and expression of MDM2 mRNA through an m6A-dependent manner. Downregulation of IGF2BP3 impeded AML cell proliferation and enhanced ferroptosis via repressing MDM2. Furthermore, Lico A could affect the MDM2/p53 pathway by downregulating IGF2BP3 expression. Lico A exerts the anti-proliferative and pro-ferroptosis activity in AML cells by affecting the IGF2BP3/MDM2/p53 pathway, providing new evidence for Lico A as a promising agent for the treatment of AML.

IGF2BP3 suppresses ferroptosis in lung adenocarcinoma by m6A-dependent regulation of TFAP2A to transcriptionally activate SLC7A11/GPX4.

Ferroptosis is recently discovered as an important player in the initiation, proliferation, and progression of human tumors. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) has been reported as an oncogene in multiple types of cancers, including lung adenocarcinoma (LUAD). However, little research has been designed to investigate the regulation of IGF2BP3 on ferroptosis in LUAD. qRT-PCR and western blot were used to measure the mRNA and protein expression of IGF2BP3 and transcription factor AP-2 alpha (TFAP2A). CCK-8 assay was performed to determine cell viability. DCFH-DA and C11-BODIPY staining were used to detect the levels of intracellular reactive oxygen species (ROS) and lipid ROS. The corresponding assay kits were used to analyze the levels of malondialdehyde (MDA) and glutathione (GSH). SRAMP website and m6A RNA immunoprecipitation (Me-RIP) were used to predict and confirm the m6A modification of TFAP2A. RIP experiments were conducted to confirm the binding of IGF2BP3 and TFAP2A. RNA stability assay was performed using actinomycin D. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter experiments were performed to confirm the interaction between TFAP2A and cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11) or glutathione peroxidase 4 (GPX4). Mice xenotransplant model was also constructed to explore the effect of IGF2BP3 on LUAD tumor growth and ferroptosis. IGF2BP3 and TFAP2A were both highly expressed in LUAD. IGF2BP3 or TFAP2A knockdown induced ferroptosis by aggravating erastin-induced cell viability suppression, increasing the production of intracellular ROS, lipid ROS, and MDA, and decreasing GSH synthesis, GSH/GSSG ratio, and cystine uptake. Mechanistically, IGF2BP3 stabilized TFAP2A expression via m6A modification. Moreover, sh-IGF2BP3-mediated ferroptosis was significantly abated by TFAP2A overexpression. Furthermore, TFAP2A binds to the promoters of SLC7A11 and GPX4 to promote their transcription. Also, IGF2BP3 depletion suppressed LUAD tumor growth by inducing ferroptosis in mice. IGF2BP3 suppresses ferroptosis in LUAD by m6A-dependent regulation of TFAP2A to promote the transcription of SLC7A11 and GPX4. Our findings suggest that targeting IGF2BP3/TFAP2A/SLC7A11/GPX4 axis might be a potential therapeutic choice to increase ferroptosis sensitivity in LUAD.

IGF2BP3 stabilizes SESN1 mRNA to mitigate oxidized low-density lipoprotein-induced oxidative stress and endothelial dysfunction in human umbilical vein endothelial cells by activating Nrf2 signaling.

Atherosclerosis (AS) represents a prevalent initiating factor for cardiovascular events. Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) is an oncofetal RNA-binding protein that participates in cardiovascular diseases. This work aimed to elaborate the effects of IGF2BP3 on AS and the probable mechanism by using an oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) model. Results indicated that IGF2BP3 expression was declined in the blood of AS patients and ox-LDL-induced HUVECs. IGF2BP3 elevation alleviated ox-LDL-provoked viability loss, apoptosis, oxidative DNA damage and endothelial dysfunction in HUVECs. Moreover, IGF2BP3 bound SESN1 and stabilized SESN1 mRNA. Furthermore, SESN1 interference reversed the impacts of IGF2BP3 overexpression on the apoptosis, oxidative DNA damage and endothelial dysfunction of ox-LDL-challenged HUVECs. Additionally, the activation of Nrf2 signaling mediated by IGF2BP3 up-regulation in ox-LDL-treated HUVECs was blocked by SESN1 absence. Collectively, SESN1 stabilized by IGF2BP3 might protect against AS by activating Nrf2 signaling.

N6-methyladenosine modification of OIP5-AS1 promotes glycolysis, tumorigenesis, and metastasis of gastric cancer by inhibiting Trim21-mediated hnRNPA1 ubiquitination and degradation.

BACKGROUND: Opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) has been demonstrated to play vital roles in development and progression of tumors such as gastric cancer (GC). However, the detailed molecular mechanism of OIP5-AS1 has not been completely elucidated. Our study aimed to investigate the role and the epigenetic regulation mechanism of OIP5-AS1 in GC. METHODS: OIP5-AS1 expression in GC tissues was detected by RT-qPCR. Loss- and gain-of-function experiments were conducted to assess the biological function of OIP5-AS1 in vitro and in vivo. The interaction of OIP5-AS1 with insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) or heterogeneous nuclear nucleoprotein A1 (hnRNPA1) was verified by bioinformatics analysis, RNA pull-down assays, and RNA immunoprecipitation assays. RESULTS: In this study, we identified that OIP5-AS1 is specifically overexpressed in GC tumor tissues and cell lines and correlated with a poor prognosis. The loss of OIP5-AS1 suppressed the proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and glycolysis of GC cells, but the ectopic expression of OIP5-AS1 had the opposite impact. Meanwhile, knockdown of OIP5-AS1 inhibited tumor growth in patient-derived xenograft models, as well as repressed tumor metastasis. Mechanistically, IGF2BP3 could bind to OIP5-AS1 by N6-methyladenosine (m6A) modification sites on OIP5-AS1, thereby stabilizing OIP5-AS1. Moreover, OIP5-AS1 prevented Trim21-mediated ubiquitination and degradation of hnRNPA1, stabilizing hnRNPA1 protein and promoting the malignant progression of GC by regulating PKM2 signaling pathway. CONCLUSIONS: In conclusion, this study highlighted that OIP5-AS1 is an oncogenic m6A-modified long non-coding RNA (lncRNA) in GC and that IGF2BP3/OIP5-AS1/hnRNPA1 axis may provide a potential diagnostic or prognostic target for GC.

Hypoxia increases the biogenesis of IGF2BP3-bound circular RNAs.

BACKGROUND: Insulin-like Growth Factor 2 Binding Protein 3 (IGF2BP3) promotes cancer migration and invasion by binding to several coding and non-coding RNAs. Hypoxia stimulates tumor progression by upregulating Hypoxia Inducible Factors and downstream signaling. Quaking (QKI) gene, which is upregulated in hypoxia and promotes epithelial to mesenchymal transition (EMT), induces circular RNAs. Therefore, the axis between IGF2BP3, QKI, circular RNAs and their respective host genes under hypoxia was studied. METHODS AND RESULTS: Several IGF2BP3-bound circular RNAs were previously identified in HepG2. There were 13 circRNAs originating from 8 host genes bound to IGF2BP3. We confirmed their binding to IGF2BP3 in U87MG using an RNA Immunoprecipitation assay. MALAT1, an oncogenic lncRNA was also found to be associated with IGF2BP3. Three adherent cell lines expressing high levels of IGF2BP3 viz., HeLa, HepG2 and U87MG were cultured under normoxia (20%O2) and hypoxia (<0.2%O2) for 48-168 h. Expression of IGF2BP3, QKI, EMT markers, IGF2BP3-bound circRNAs and their host mRNAs expression were assessed by quantitative real-time PCR (qRT-PCR) in both normoxia and hypoxia. The hypoxia markers viz., VEGF and CA9 were upregulated in all the cell lines in hypoxia at all time points along with an increase in SNAIL. We found 6 genes, viz., PHC3, CDYL, ANKRD17, ARID1A, NEIL3 and FNDC3B with increased expression both at the mRNA and circRNA level indicating their synergistic role in tumor initiation. Overall, we found that circRNA to mRNA expression was observed to be increased for most of the genes and time points of hypoxia in all the cell lines. IGF2BP3 and QKI were also upregulated in hypoxia indicating their role in circRNA biogenesis and stability. CONCLUSION: Our data implies that hypoxia augments circRNA biogenesis which might subsequently play a role in tumor progression.