Protein phosphatase 2A-B56γ-Drp1-Rab7 signaling axis regulates mitochondria-lysosome crosstalk to sensitize the anti-cancer therapy of hepatocellular carcinoma.

Mitochondria-lysosome crosstalk is an intercellular communication platform regulating mitochondrial quality control (MQC). Activated dynamin-related protein 1 (Drp1) with phosphorylation at serine 616 (p-Drp1Ser616) plays a critical role in mitophagy-dependent cell survival and anti-cancer therapy for hepatocellular carcinoma (HCC). However, the underlying mechanisms that p-Drp1Ser616 involved in regulating mitochondria-lysosome crosstalk and mediating anti-HCC therapy remain unknown. HCC cells and mouse xenograft models were conducted to evaluate the relationship between p-Drp1Ser616 and Ras-associated protein 7 (Rab7) and the underlying mechanism by protein phosphatase 2A (PP2A)-B56γ regulating mitophagy via dephosphorylation of p-Drp1Ser616 in HCC. Herein, we found that Drp1 was frequently upregulated and was associated with poor prognosis in HCC. Mitochondrial p-Drp1Ser616 was a novel inter-organelle tethering protein localized to mitochondrion and lysosome membrane contact sites (MCSs) via interaction with Rab7 to trigger an increase in the mitochondria-lysosome crosstalk, resulting in PINK1-Parkin-dependent mitophagy and anti-apoptosis in HCC cells under the treatment of chemotherapy drugs. Moreover, we demonstrate that B56γ-mediated direct dephosphorylation of p-Drp1Ser616 inhibited mitophagy and thus increased mitochondria-dependent apoptosis. Overall, our findings demonstrated that activation of B56γ sensitizes the anti-cancer effect of HCC chemoprevention via dephosphorylated regulation of p-Drp1Ser616 in inhibiting the interaction between p-Drp1Ser616 and Rab7, which may provide a novel mechanism underlying the theranostics for targeting intervention in HCC.

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

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.