Targeting Mitochondrial COX-2 Enhances Chemosensitivity via Drp1-Dependent Remodeling of Mitochondrial Dynamics in Hepatocellular Carcinoma

Abstract

Mitochondria are highly dynamic organelles and undergo constant fission and fusion, which are both essential for the maintenance of cell physiological functions. Dysregulation of dynamin-related protein 1 (Drp1)-dependent mitochondrial dynamics is associated with tumorigenesis and the chemotherapeutic response in hepatocellular carcinoma (HCC). The enzyme cyclooxygenase-2 (COX-2) is overexpressed in most cancer types and correlates with a poor prognosis. However, the roles played by the translocation of mitochondrial COX-2 (mito-COX-2) and the interaction between mito-COX-2 and Drp1 in chemotherapeutic responses remain to be elucidated in the context of HCC. Bioinformatics analysis, paired HCC patient specimens, xenograft nude mice, immunofluorescence, transmission electron microscopy, molecular docking, CRISPR/Cas9 gene editing, proximity ligation assay, cytoplasmic and mitochondrial fractions, mitochondrial immunoprecipitation assay, and flow cytometry analysis were performed to evaluate the underlying mechanism of how mito-COX-2 and p-Drp1Ser616 interaction regulates the chemotherapeutic response via mitochondrial dynamics in vitro and in vivo. We found that COX-2 and Drp1 were frequently upregulated and confer a poor prognosis in HCC. We also found that the proportion of mito-COX-2 and p-Drp1Ser616 was increased in HCC cell lines. In vitro, we demonstrated that the enhanced mitochondrial translocation of COX-2 promotes its interaction with p-Drp1Ser616 via PTEN-induced putative kinase 1 (PINK1)-mediated Drp1 phosphorylation activation. This increase was associated with higher colony formation, cell proliferation, and mitochondrial fission. These findings were confirmed by knocking down COX-2 in HCC cells using CRISPR/Cas9 technology. Furthermore, inhibition of Drp1 using pharmacologic inhibitors (Mdivi-1) or RNA interference (siDNM1L) decreased mito-COX-2/p-Drp1Ser616 interaction-mediated mitochondrial fission, and increased apoptosis in HCC cells treated with platinum drugs. Moreover, inhibiting mito-COX-2 acetylation with the natural phytochemical resveratrol resulted in reducing cell proliferation and mitochondrial fission, occurring through upregulation of mitochondrial deacetylase sirtuin 3 (SIRT3), which, in turn, increased the chemosensitivity of HCC to platinum drugs in vitro and in vivo. Our results suggest that targeting interventions to PINK1-mediated mito-COX-2/p-Drp1Ser616-dependent mitochondrial dynamics increases the chemosensitivity of HCC and might help us to understand how to use the SIRT3-modulated mito-COX-2/p-Drp1Ser616 signaling axis to develop an effective clinical intervention in hepatocarcinogenesis.

Hepatocyte-Derived Prostaglandin E2-Modulated Macrophage M1-Type Polarization via mTOR-NPC1 Axis-Regulated Cholesterol Transport from Lysosomes to the Endoplasmic Reticulum in Hepatitis B Virus x Protein-Related Nonalcoholic Steatohepatitis

“…Western blotting analysis was performed as previously described [ 50 ]. According to the standard procedures commonly used in the laboratory, detection operations were performed: gel preparation, electrophoresis, transfer, blocking, primary antibody incubation, secondary antibody incubation, and luminescence development.…”

Intracellular antibody targeting HBx suppresses invasion and metastasis in hepatitis B virus‐related hepatocarcinogenesis via protein phosphatase 2A‐B56γ‐mediated dephosphorylation of protein kinase B

“…Bioinformatics analysis was performed as described previously. 19 Briefly, volcano plots and heatmaps were drawn based on the obtained differential expression genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to identify pathways significantly affected by the hepatocarcinogenesis process of DEGs.…”

“…HE and IHC assays were performed as described previously. 19 Briefly, the sections were rehydrated and stained with HE after dewaxing. The IHC sections were incubated with primary antibodies (anti‐HBx, anti‐AKT, anti‐MMP2, anti‐MMP9, and anti‐B56γ), and images were taken by an inverted microscope (Nikon, Tokyo, Japan).…”

“…Microarray data (GSE83148) and RNA sequencing (GSE94660) on mRNA expression were screened from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). Bioinformatics analysis was performed as described previously 19 . Briefly, volcano plots and heatmaps were drawn based on the obtained differential expression genes (DEGs).…”

The Role and Therapeutic Perspectives of Sirtuin 3 in Cancer Metabolism Reprogramming, Metastasis, and Chemoresistance

“…Chemotherapeutic agents, such as doxorubicin, cisplatin, oxaliplatin, and epirubicin, could inhibit the expression of SIRT3 in tumor cells. In hepatocellular carcinoma cells, SIRT3 sensitizes hepatocellular carcinoma cells to chemotherapy drugs via regulating mitochondrial cyclooxygenase-2 (mito-COX-2)/Drp1 with phosphorylation at serine 616 (p-Drp1 Ser616 ) and glutathione S-transferase pi 1 (GSTP1)/c-Jun N-terminal kinase (JNK) signaling pathway ( 97 , 98 ). In lung cancer, SIRT3 enhances the sensitivity of lung cancer cells to cisplatin by deacetylating mutant p53 in small-cell lung cancer and elevating the FOXO3α/chromatin licensing and DNA replication factor 1 (CDT1) axis in lung cancer ( 99 , 100 ).…”