Dataset Information

Concurrent targeting of MAP3K3 and BRD4 by miR-3140-3p overcomes acquired resistance to BET inhibitors in neuroblastoma cells.

ABSTRACT: Neuroblastoma (NB) harboring MYCN amplification is a refractory disease with a poor prognosis. As BRD4, an epigenetic reader belonging to the bromodomain and extra terminal domain (BET) family, drives transcription of MYCN in NB cells, BET inhibitors (BETis) are considered useful for NB therapy. However, clinical trials of BETis suggested that early acquired resistance to BETis limits their therapeutic benefit. MicroRNAs are small non-coding RNAs that mediate post-transcriptional silencing of target genes. We previously identified miR-3140-3p as a potent candidate for nucleic acid therapeutics for cancer, which directly targets BRD4. We demonstrated that miR-3140-3p suppresses tumor cell growth in MYCN-amplified NB by downregulating MYCN and MYC through BRD4 suppression. We established BETi-acquired resistant NB cells to evaluate the mechanism of resistance to BETi in NB cells. We revealed that activated ERK1/2 stabilizes MYCN protein by preventing ubiquitin-mediated proteolysis via phosphorylation of MYCN at Ser62 in BETi-acquired resistant NB cells, thereby attenuating the effects of BETi in these cells. miR-3140-3p efficiently downregulated MYCN expression by directly targeting the MAP3K3-ERK1/2 pathway in addition to BRD4 suppression, inhibiting tumor cell growth in BETi-acquired resistant NB cells. This study suggests that miR-3140-3p has the potential to overcome resistance to BETi in NB.

SUBMITTER: Liu C

PROVIDER: S-EPMC8253920 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Targeting the epigenome to modulate gene expression programs driving cancer development has emerged as an exciting avenue for therapeutic intervention. Pharmacological inhibition of the bromodomain and extraterminal (BET) family of chromatin adapter proteins has proven effective in this regard, suppressing growth of diverse cancer types mainly through downregulation of the c-MYC oncogene, and its downstream transcriptional program. While initially effective, resistance to BET inhibitors (BETi) typically occurs through mechanisms that reactivate MYC expression. We have previously shown that lung adenocarcinoma (LAC) is inhibited by JQ1 through suppression of FOSL1, suggesting that the epigenetic landscape of tumor cells from different origins and differentiation states influences BETi response. Here, we assessed how these differences affect mechanisms of BETi resistance through the establishment of isogenic pairs of JQ1 sensitive and resistant LAC cell lines. We found that resistance to JQ1 in LAC occurs independent of FOSL1 while MYC levels remain unchanged between resistant cells and their JQ1-treated parental counterparts. Furthermore, while epithelial-mesenchymal transition (EMT) is observed upon resistance, TGF-β induced EMT did not confer resistance in JQ1 sensitive LAC lines, suggesting this is a consequence, rather than a driver of BETi resistance in our model systems. Importantly, siRNA knockdown demonstrated that JQ1 resistant cell lines are still dependent on BRD4 expression for survival and we found that phosphorylation of BRD4 is elevated in resistant LACs, identifying casein kinase 2 (CK2) as a candidate protein mediating this effect. Inhibition of CK2, as well as downstream transcriptional targets of phosphorylated BRD4-including AXL and activators of the PI3K pathway-synergize with JQ1 to inhibit BETi resistant LAC. Overall, this demonstrates that the mechanism of resistance to BETi varies depending on cancer type, with LAC cells developing JQ1 resistance independent of MYC regulation, and identifying CK2 phosphorylation of BRD4 as a potential target to overcome resistance in this cancer.

Project description:BACKGROUND:The oncogene MYCN is critical for tumorigenesis of several types of cancers including neuroblastoma. We previously reported that miR-506-3p repressed MYCN expression in neuroblastoma cells. However, the mechanism underlying such regulation was undetermined since there is no miR-506-3p target site in MYCN 3'UTR. METHODS:By a systematic investigation combining microarray, informatics and luciferase reporter assay, we identified that the transcriptional factor pleiomorphic adenoma gene-like 2 (PLAGL2) is a direct target of miR-506-3p that mediates its regulation on MYCN expression. Using CHIP-PCR and luciferase reporter assay, we validated the transcriptional regulation of MYCN by PLAGL2 and we further demonstrated the transcriptional regulation of PLAGL2 by MYCN. We examined the function of PLAGL2 in regulating neuroblastoma cell fate by cell viability assay, colony formation and Western blotting of differentiation markers. We examined the effect of retinoic acid, the differentiation agent used in neuroblastoma therapy, on miR-506-3p, PLAGL2 and MYCN expressions by quantitative PCR and Western blots. We investigated the clinical relevance of PLAGL2 expression by examining the correlation of tumor PLAGL2 mRNA levels with MYCN mRNA expression and patient survival using public neuroblastoma patient datasets. RESULTS:We found that miR-506-3p directly down-regulated PLAGL2 expression, and we validated a PLAGL2 binding site in the MYCN promoter region responsible for promoting MYCN transcription, thereby establishing a mechanism through which miR-506-3p regulates MYCN expression. Conversely, we discovered that MYCN regulated PLAGL2 transcription through five N-Myc-binding E-boxes in the PLAGL2 promoter region. We further confirmed the reciprocal regulation between endogenous PLAGL2 and MYCN in multiple neuroblastoma cell lines. Moreover, we found that PLAGL2 knockdown induced neuroblastoma cell differentiation and reduced cell proliferation, and combined knockdown of PLAGL2 and MYCN showed a synergistic effect. More strikingly, we found that high tumor PLAGL2 mRNA levels were significantly correlated with high MYCN mRNA levels and poor patient survival in neuroblastoma patients. Furthermore, we found that retinoic acid increased expression of miR-506-3p and repressed expression of MYCN and PLAGL2. CONCLUSIONS:Our findings altogether suggest that the interplay network formed by PLAGL2, MYCN and miR-506-3p is an important mechanism in regulating neuroblastoma cell fate, determining neuroblastoma prognosis, and mediating the therapeutic function of retinoic acid.

Dataset Information

Concurrent targeting of MAP3K3 and BRD4 by miR-3140-3p overcomes acquired resistance to BET inhibitors in neuroblastoma cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets