Mitochondria undergo fission and fusion to maintain homeostasis, and tumors exhibit the dysregulation of mitochondrial dynamics. We recently demonstrated that ectopic HRasG12V promotes mitochondrial fragmentation and tumor growth through Erk phosphorylation of the mitochondrial fission GTPase Dynamin-related protein 1 (Drp1). However, the role of Drp1 in the setting of endogenous oncogenic KRas remains unknown. Here, we show that Drp1 is required for KRas-driven anchorage-independent growth in fibroblasts and patient-derived pancreatic cancer cell lines, and it promotes glycolytic flux, in part through the regulation of hexokinase 2 (HK2). Furthermore, Drp1 deletion imparts a significant survival advantage in a model of KRas-driven pancreatic cancer, and tumors exhibit a strong selective pressure against complete Drp1 deletion. Rare tumors that arise in the absence of Drp1 have restored glycolysis but exhibit defective mitochondrial metabolism. This work demonstrates that Drp1 plays dual roles in KRas-driven tumor growth: supporting both glycolysis and mitochondrial function through independent mechanisms.

The activation of G-protein-coupled receptors (GPCRs) leads to the activation of mTORC2 in cell migration and metabolism. However, the mechanism that links GPCRs to mTORC2 remains unknown. Here, using Dictyostelium cells, we show that GPCR-mediated chemotactic stimulation induces hetero-oligomerization of phosphorylated GDP-bound Rho GTPase and GTP-bound Ras GTPase in directed cell migration. The Rho-Ras hetero-oligomers directly and specifically stimulate mTORC2 activity toward AKT in cells and after biochemical reconstitution using purified proteins in vitro. The Rho-Ras hetero-oligomers do not activate ERK/MAPK, another kinase that functions downstream of GPCRs and Ras. Human KRas4B functionally replace Dictyostelium Ras in mTORC2 activation. In contrast to GDP-Rho, GTP-Rho antagonizes mTORC2-AKT signaling by inhibiting the oligomerization of GDP-Rho with GTP-Ras. These data reveal that GPCR-stimulated hetero-oligomerization of Rho and Ras provides a critical regulatory step that controls mTORC2-AKT signaling.

Mitochondria are dynamic organelles that constantly fuse and divide to maintain their proper morphology, which is essential for their normal functions. Energy production, a central role of mitochondria, demands highly folded structures of the mitochondrial inner membrane (MIM) called cristae and a dimeric phospholipid (PL) cardiolipin (CL). Previous studies identified a number of factors involved in mitochondrial dynamics, crista formation, and CL biosynthesis, yet it is still enigmatic how these events are interconnected and cooperated. Here, we first report that mitochondrial fusion-division dynamics are important to maintain CL abundance. Second, our genetic and biochemical analyses revealed that intra-mitochondrial PL transport plays an important role in crista formation. Finally, we show that simultaneous defects in MIM fusion and intra-mitochondrial PL transport cause a drastic decrease in crista structure, resulting in CL depletion. These results expand our understanding of the integrated functional network among the PL transport, crista formation, and CL biogenesis.

Oncogenic KRas activates mitochondrial fission through Erk-mediated phosphorylation of the mitochondrial fission GTPase Drp1. Drp1 deletion inhibits tumorigenesis of KRas-driven pancreatic cancer, but the role of mitochondrial dynamics in other Ras-driven malignancies is poorly defined. Here we show that in vitro and in vivo growth of KRas-driven lung adenocarcinoma is unaffected by deletion of Drp1 but is inhibited by deletion of Opa1, the GTPase that regulates inner membrane fusion and proper cristae morphology. Mechanistically, Opa1 knockout disrupts cristae morphology and inhibits electron transport chain (ETC) assembly and activity, which inhibits tumor cell proliferation through loss of NAD+ regeneration. Simultaneous inactivation of Drp1 and Opa1 restores cristae morphology, ETC activity, and cell proliferation indicating that mitochondrial fission activity drives ETC dysfunction induced by Opa1 knockout. Our results support a model in which mitochondrial fission events disrupt cristae structure, and tumor cells with hyperactive fission activity require Opa1 activity to maintain ETC function.

Dysfunctional mitochondria are removed via multiple pathways, such as mitophagy, a selective autophagy process. Here, we identify an intracellular hybrid mitochondria-lysosome organelle (termed the mitochondria-lysosome-related organelle [MLRO]), which regulates mitochondrial homeostasis independent of canonical mitophagy during hepatocyte dedifferentiation. The MLRO is an electron-dense organelle that has either a single or double membrane with both mitochondria and lysosome markers. Mechanistically, the MLRO is likely formed from the fusion of mitochondria-derived vesicles (MDVs) with lysosomes through a PARKIN-, ATG5-, and DRP1-independent process, which is negatively regulated by transcription factor EB (TFEB) and associated with mitochondrial protein degradation and hepatocyte dedifferentiation. The MLRO, which is galectin-3 positive, is reminiscent of damaged lysosome and could be cleared by overexpression of TFEB, resulting in attenuation of hepatocyte dedifferentiation. Together, results from this study suggest that the MLRO may act as an alternative mechanism for mitochondrial quality control independent of canonical autophagy/mitophagy involved in cell dedifferentiation.

Impaired mitochondrial respiratory activity contributes to the development of insulin resistance in type 2 diabetes. Metformin, a first-line antidiabetic drug, functions mainly by improving patients' hyperglycemia and insulin resistance. However, its mechanism of action is still not well understood. We show here that pharmacological metformin concentration increases mitochondrial respiration, membrane potential, and ATP levels in hepatocytes and a clinically relevant metformin dose increases liver mitochondrial density and complex 1 activity along with improved hyperglycemia in high-fat- diet (HFD)-fed mice. Metformin, functioning through 5' AMP-activated protein kinase (AMPK), promotes mitochondrial fission to improve mitochondrial respiration and restore the mitochondrial life cycle. Furthermore, HFD-fed-mice with liver-specific knockout of AMPKα1/2 subunits exhibit higher blood glucose levels when treated with metformin. Our results demonstrate that activation of AMPK by metformin improves mitochondrial respiration and hyperglycemia in obesity. We also found that supra-pharmacological metformin concentrations reduce adenine nucleotides, resulting in the halt of mitochondrial respiration. These findings suggest a mechanism for metformin's anti-tumor effects.