Positive Feedback Loop Between Mitochondrial Fission And Cytosolic Calcium Signaling Is Essential For HCC Metastasis

[Background]Mitochondria are cellular organelles well known for their important functions, including production of ATP, reactive oxygen species?ROS? and many biosynthetic intermediates, regulation of and Ca²⁺ signaling, and contribution to proliferation and apoptosis. In a majority of cell types, mitochondria continually change their shape through the combined actions of fission and fusion in response to cellular energy demands and environmental challenges. Dynamin related protein 1?Drp1? and mitochondrial fission 1?FIS1? are major proteins controlling mitochondrial fission, whereas mitofusins?MFN1 and MFN2? and optic atrophy 1?OPA1? are mainly required for mitochondrial fusion. Recently, cumulative evidence is beginning to reveal the close links between cancers and unbalanced mitochondrial dynamics. Several studies have reported that the expression of mitochondrial fission/fusion proteins such as Drp1, MFN1 and MFN2 is dysregulated in human cancers of lung, bladder and breast and the disruption of mitochondrial network exhibits a considerable effect on the apoptosis of cancer cells. Moreover, a significant association has been observed between the clinical prognosis of lung cancer patients and abnormalities in proteins that govern mitochondrial dynamics. Our previous study has demonstrated that mitochondrial fission was frequently increased in HCC tissues due to upregualtion of Drp1 and downregulaiton of MFN1. Furthermore, we have found that increased mitochondrial fission promotes autophagy and survival of HCC cells mainly by ROS-mediated coordinated regulation of NF-?B and p53 pathways.Ca²⁺ is a ubiquitous cellular signal and impact nearly every aspect of cellular life. In non-excitable cells, the predominant Ca²⁺ influx mechanism is store-operated calcium entry?SOCE?, which is mainly mediated by STIM1, which serves as a Ca²⁺ sensor in ER, and Orai1, which is an essential pore-forming component of the SOCE channel in the cell membrane. Changes in cytosolic Ca²⁺ can be “decoded” by the cells, which allows the ubiquitous calcium signal to specifically regulate cellular processes, especially migration and metastasis of cancer cells.To date, whether mitochondrial dynamics can modulate the global cytosolic Ca²⁺ signaling remains largely unknown. Moreover, whether deregulation of mitochondrial dynamics affects cell migration and metastasis in HCC remains to be determined.[Purpose ]1. To investigate whether there is a regulatory loop between mitochondria fusion and cytoplasmic calcium signal; 2. To explore the molecular mechanisms of regulatory loop between mitochondria fusion and cytoplasmic calcium signal; 3. To investigate the mechanism of the exchange regulatory loop between mitochondria fusion and cytoplasmic calcium signal regulating liver cancer metastasis.[Methods] 1. The mitochondrial specific dye Mitotracker Green and cytoplasmic calcium ion specific dye Fluo-4 were used to detect the interactive relationship between the regulation of mitochondrial dynamic equilibrium and cytosolic calcium signals; 2. Western blot and q PCR technology were used to detect the the protein and mRNA levels of mitochondrial fission and fusion molecules and related signaling pathways; 3. Flow cytometry were used to confirm that mitochondrial fission and fusion can regulate cytoplasmic ROS production; 4. Immunohistochemical techniques were used to detect the relevance between mitochondrial dynamic molecules and STIM1?the regulatory molecules of SOCE?;5. Immunofluorescence and phalloidin staining technique were used to detect the impact of mitochondrial dynamics on cell adhesion and lamellipodia formation; 6. Transwell assay were use to detect the impact of mitochondrial fission and fusion on invasive ability of HCC cells; 7. Luciferase reporter assay were used to detect the roles of the cis-regulatory elements of Drp1 and FIS1 promoter.; 8. Potential transcription factors, which may bind to the above-identified critical regions in both promoters, were predicted by the Jaspar program; 9. The orthotopical nude mice model were used to detect the effect of mitochondrial fission on the metastasis of HCC ceslls in vivo; 10. HE staining were used to assess intrahepatic and lung metastasis of primary HCC with different mitochondrial dynamic status;[ Results ] Our data showed that increased mitochondrial fission promoted the cytosolic calcium concentration [Ca²⁺]c and serum-stimulated calcium oscillation in HCC cells. We further found that mitochondrial fission-mediated cytosolic calcium signaling was dependent on ROSactivated NF-?B pathways, which facilitated the expression of STIM1 and subsequent storeoperated calcium entry?SOCE?. Interestingly, we also demonstrated that sustained increase in [Ca²⁺]c promoted mitochondrial fission by up-regulating expression of Drp1 and FIS1 via transcription factors NFATC2 and c-Myc, respectively. Moreover, the positive feedback loop between mitochondrial fission and cytosolic calcium signaling significantly promoted HCC cell migration. Also, increased mitochondrial fission promotes metastasis of HCC cells in vivo and was associated with poor relapse-free survival. Importantly, treatment with mitochondrial division inhibitor-1 significantly decreased cytosolic calcium signaling in HCC cells and had a potential treatment effect for HCC metastasis in vivo.[Conclusiom]Our findings demonstrate that increased mitochondrial fission plays a critical role in regulation of cytosolic calcium homeostasis and HCC cell metastasis, which provides a strong evidence for this process as drug target in HCC treatment.