Abstract
Advanced-stage prostate cancer is characterized by osseous metastases whose establishment involves the dynamic interplay of factors and exchange of cellular contents by constituents of the ...tumor bony microenvironment. Among the factors that transport cellular cargoes and facilitate the transmission of signaling complexes for establishment of the bone metastatic lesions are large extracellular vesicles (LEVs), also known as large oncosomes. Information on the contents of LEVs and the mechanisms by which LEVs are formed and regulated is incomplete. In the course of studies aimed at elucidating the mechanisms of bony metastases using the LNCaP-C4-2B prostate cancer progression model, we show that the expression and cellular distribution of nicotinamide riboside: quinone oxidoreductase 2 (NQO2) and glutaminase (GLS, kidney type) both participate in the induction of membrane-localized oxidative stress and the formation of LEVs. The salient features of our findings are the following: The LNCaP model system exhibits gradually increasing oxidative stress levels as it progresses to the bone metastatic derivative C4-2B, while at the same time showing a progressive decrease in the expression level of NQO2. Reverse transcription PCR studies showed that NQO2 mRNA levels were unchanged between LNCaP and its metastatic derivatives C4, C4-2 and C4-2B. As a control, the expression level of the analogous enzyme NADP(H):quinone oxidoreductase 1 (NQO1) was also unchanged. These results suggested the possibility that the NQO2 enzyme in the bone metastatic C4-2B derivative could be localized elsewhere, possibly at an extracellular location. Further studies showed that a fraction of the NQO2 enzyme was associated with the detergent resistant membrane fraction of the C4-2B prostate cancer cells. Molecular modeling studies showed that the NQO2 enzyme had two caveolin-1 and NQO1 enzyme had one caveolin-1 binding sites. While the caveolin-1 binding site of NQO1 is buried inside the molecule and hence inaccessible to caveolin-1, one of the two binding sites available in NQO2 was external and accessible to caveolin-1. We have validated the specific interaction between NQO2 and caveolin-1 using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B cells showed increasing level of expression of the GLS enzyme, suggesting a correlation between increased oxidative stress, glutaminase activity, and metastatic potential. Moreover, we found that the metastatic C4-2B derivative extruded LEVs in which the enzyme NQO2 and caveolae specific caveolin-1 could be visualized as cargo using double immunofluorescence studies. The extrusion of these LEVs could be inhibited by the GLS specific inhibitor BP-TES. Characteristically, curcumin was also found to inhibit the extrusion of these LEVs. This naturally occurring plant-based compound was shown earlier to inhibit prostate cancer bone metastasis in vivo using the C4-2B model. These results indicate a role for the NQO2 enzyme at the membrane level, possibly increasing the oxidative stress in a localized manner by lipid peroxidation. These results also suggest that a glutamine/GLS-mediated metabolic reprogramming in the LNCaP model system as it progresses toward the bone metastatic C4-2B is an integral component of the force driving the metastatic process. Based on the results of our studies, a combinatorial strategy using antimetastatic therapies such as nitrogen-containing bisphosphonates (NBPs) and anti-GLS therapy or, alternatively, a naturally occurring plant compound-based anti-NQO2 and anti-GLS therapy, could be considered for treating advanced prostate cancer patients with bone metastatic complications.
Citation Format: Thambi Dorai, Ankeeta B. Shah, Faith Summers, Rajamma Mathew, Jing Huang, Tze-chen Hsieh, Joseph M. Wu. NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis abstract. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B003.
The molecular mechanisms that govern the maturation of oligodendrocyte lineage cells remain unclear. Emerging studies have shown that N6-methyladenosine (m6A), the most common internal RNA ...modification of mammalian mRNA, plays a critical role in various developmental processes. Here, we demonstrate that oligodendrocyte lineage progression is accompanied by dynamic changes in m6A modification on numerous transcripts. In vivo conditional inactivation of an essential m6A writer component, METTL14, results in decreased oligodendrocyte numbers and CNS hypomyelination, although oligodendrocyte precursor cell (OPC) numbers are normal. In vitro Mettl14 ablation disrupts postmitotic oligodendrocyte maturation and has distinct effects on OPC and oligodendrocyte transcriptomes. Moreover, the loss of Mettl14 in oligodendrocyte lineage cells causes aberrant splicing of myriad RNA transcripts, including those that encode the essential paranodal component neurofascin 155 (NF155). Together, our findings indicate that dynamic RNA methylation plays an important regulatory role in oligodendrocyte development and CNS myelination.
•Oligodendrocyte maturation is accompanied by modifications in m6A mRNA methylation•m6A mRNA methylation is required for oligodendrocyte maturation and CNS myelination•m6A mRNA methylation regulates the transcriptomes of oligodendrocyte lineage cells•Proper neurofascin mRNA splicing in oligodendrocytes requires m6A methylation
Xu et al. show that oligodendrocyte development is associated with dynamic changes in posttranscriptional mRNA methylation. Moreover, they demonstrate that the m6A epigenetic RNA mark has considerable impact on the myelinating cell’s transcriptome and is essential for normal CNS myelination.
The molecular mechanisms that govern the maturation of oligodendrocyte lineage cells remain unclear. Emerging studies have shown that N
-methyladenosine (m
A), the most common internal RNA ...modification of mammalian mRNA, plays a critical role in various developmental processes. Here, we demonstrate that oligodendrocyte lineage progression is accompanied by dynamic changes in m
A modification on numerous transcripts. In vivo conditional inactivation of an essential m
A writer component, METTL14, results in decreased oligodendrocyte numbers and CNS hypomyelination, although oligodendrocyte precursor cell (OPC) numbers are normal. In vitro Mettl14 ablation disrupts postmitotic oligodendrocyte maturation and has distinct effects on OPC and oligodendrocyte transcriptomes. Moreover, the loss of Mettl14 in oligodendrocyte lineage cells causes aberrant splicing of myriad RNA transcripts, including those that encode the essential paranodal component neurofascin 155 (NF155). Together, our findings indicate that dynamic RNA methylation plays an important regulatory role in oligodendrocyte development and CNS myelination.
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