Mfn2-mediated mitochondrial fusion promotes autophagy and suppresses ovarian cancer progression by reducing ROS through AMPK/mTOR/ERK signaling

Abstract

Mitochondrial dynamics are balanced fission and fusion events that regulate mitochondrial morphology, and alteration in these events results in mitochondrial dysfunction and contributes to many diseases, including tumorigenesis. Ovarian cancer (OC) cells exhibit fragmented mitochondria, but the mechanism by which mitochondrial dynamics regulators contribute to OC is considerably less clear. Here, we elucidated the potential role of Mfn2-mediated mitochondrial fusion in OC and present evidence that genetic or pharmacological activation of Mfn2 leads to mitochondrial fusion and reduces ROS generation, which correlates with reduced cell proliferation, invasion, migration, and EMT in OC cells. Also, increased mitochondrial fusion promotes the F-actin remodeling, reduces lamellipodia formation, and thus reduces EMT. Increased expression of Mfn2 triggers AMPK, promotes autophagy, reduces ROS, and suppresses OC progression by downregulating the p-mTOR (2481 and 2448) and p-ERK axis. OC patients with higher Mfn2 expression have better survival than those with lower Mfn2 levels. Our findings demonstrate that restoration of Mfn2-mediated mitochondrial fusion suppressed OC progression and suggest that this process could be a potential strategy in OC treatment.

Keywords: AMPK; Autophagy; EMT; Mfn2; Ovarian cancer; ROS.

Fig. 1

Mfn2 expression modulating cell proliferation in human OC cells. Genetic or pharmacological activation of Mfn2 reduces OC cell proliferation. We performed silencing or overexpression of Mfn2 to study cell proliferation of OC cells. A and B Immunoblotting analysis demonstrated the increased expression of Mfn2 by leflunomide (50 µM for 24 h) and reduction of Drp1 by mDivi-1 (20 µM for 24 h) in OC cells. Ai and Bi The Western blot data were quantified using Image J software and normalized to the loading control. C Representative confocal images showed mitochondrial morphology after leflunomide and mDivi-1 treatment. Cells were incubated with MitoTracker green (100 nM in reduced medium), and images were taken randomly from different regions. Scale bar: 10 µm. D and E Graphs representing the relative cell proliferation analyzed by MTT assay after treatment with leflunomide and mDivi-1. Increased expression of Mfn2 by leflunomide significantly suppressed the OC cells proliferation. F and G Western blot analysis confirmed the Mfn 1 and 2 expression after Mfn2 silencing or overexpression in OC cells. After overexpression and silencing of Mfn2, there are no changes in Mfn1 expression. Fi and Gi The Western blot data were quantified using Image J software and normalized to the loading control. H and I Representative confocal images displayed mitochondrial morphology after Mfn2 overexpression and silencing in the A2780 and SKOV-3 cells. Cells were incubated with MitoTracker green (100 nM in reduced medium), and images were taken randomly from different regions. Scale bar: 25 µm and zoom 8 µm. J and K Graphs display the relative cell proliferation analyzed by MTT assay after Mfn2 overexpression and silencing in OC cells. Mfn2 overexpression notably reduced the proliferation of both OC cells. L and M Graphs display the relative cell proliferation analyzed by MTT assay after Mfn2 silencing in OC cells with or without leflunomide treatment. Leflunomide treatment notably reduced the proliferation in Mfn2 silenced A2780 and OVCAR-3 cells. N and Ni Immunoblotting analysis and their quantification demonstrated that leflunomide treatment reduced PCNA and Cyclin D1 expression in A2780 cells. O, Oi, P and Pi Immunoblotting analysis and their quantification by image J showed that Mfn2 overexpression reduces OC cell proliferation by reducing PCNA and Cyclin D1 expression in OC cells. Data are represented as mean ± SD. Differences among groups were considered significant *p < 0.05, **p < 0.01, and ***p < 0.001, ns non-significant (p > 0.05). Mfn2 O/E Mfn2 overexpression, si small interfering RNAs, PCNA proliferating cell nuclear antigen

Fig. 2

A Mfn2 depletion increasing OC cell migration and invasion. A and B Microscopic images of two OC cells invaded the ECM layer after Mfn2 silencing and overexpression in cells compared with control. After Mfn2 silencing, increased invasion in OC cells was noted, whereas Mfn2 overexpression reduces the invasion in both cell types. Images represented here were captured in 10 X magnification. Ai and Bi Quantification of cells invaded the extracellular matrix and reached the bottom of the invasion chamber using image J software. C Increased expression of Mfn2 by leflunomide (50 uM for 24 h) and downregulation of Drp1 by mDivi-1 (20 uM for 24 h) caused reduced migration in A2780 cells. Ci The percentage of the reduced area or wound closure in captured images was calculated using the Image J after leflunomide and mDivi-1 treatment in cells at each time point. D and E Microscopic images of wound closure after Mfn2 silencing and overexpression in two OC cells. Di and Ei The percentage of a reduced area or wound closure in the captured images was calculated using Image J after Mfn2 overexpression and silencing in two OC cells at each time point. Reducing Mfn2 expression increases the cell migration of both OC cells. Mfn2 O/E Mfn2 overexpression, si small interfering RNAs. Data are represented as mean ± SD. Differences among groups were considered significant *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 3

Mfn2 expression modulating EMT and actin remodeling in OC cells. Activation of Mfn2 by leflunomide (50 uM for 24 h) and downregulation of Drp1 by mDivi-1 (20 uM for 24 h) regulate EMT markers in SKOV-3 cells. A Pharmacological activation of Mfn2 increased the E-cadherin expression. Ai Quantification of Western blot using image J and normalized to the loading control. B and C Mfn2 expression was silenced and overexpressed in two OC cells. Immunoblotting analysis and their quantification (Bi and Ci) showed increased E-cadherin, and reduced vimentin expression after Mfn2 overexpression in two OC cells. The Mfn2-silenced cells group displayed a reduced E-cadherin and increased vimentin expression in two OC cells. D Mfn2 expression modulates F-actin remodeling in A2780 cells. Silencing Mfn2 in cells results in increased stress fibers and F-actin polymerization, whereas increased Mfn2 expression reduces stress fibers and lamellipodia formation. Representative images were taken by confocal microscopy. Scale bar: 8 µm; white arrows indicates elongated and increased stress fibers/lamellipodia. E Immunofluorescence study confirmed the negative correlation between Mfn2 and vimentin expression. After overexpression and silencing of Mfn2 in both cell types, cells were stained with DAPI, Mfn2, and vimentin in A2780 cells. Mfn2-overexpressed cells displayed reduced vimentin expression. Representative images were taken by confocal microscopy. Scale bar: 10 µm. F Immunofluorescence study confirmed the positive correlation between overexpressed Mfn2 and E-cadherin expression. Mfn2-overexpressed and -silenced cells were stained with DAPI, Mfn2, and E-cadherin in OVCAR-3 cells. Silencing Mfn2 reduced the E-cadherin expression and overexpressed Mfn2 displayed increase in E-cadherin expression in OVCAR-3 cells. Representative images were taken by confocal microscope in each group. Scale bar 10 µm. Mfn2 O/E Mfn2 overexpression, si small interfering RNAs. Data are represented as mean ± SD. Differences among groups were considered significant *p < 0.05, **p < 0.01, and ***p < 0.001, ns non-significant (p > 0.05)

Fig. 4

Mfn2 overexpression inducing mitochondrial fusion and promotes autophagy. We silenced and overexpressed Mfn2 separately in three OC cell lines and performed a Western blot to examine the potential correlation of Mfn2 expression and autophagic markers (Beclin-1 ATG5, LC3-I/II). A and B Mfn2 overexpression in two OC cell lines was positively correlated with ATG5, Beclin-1, and LC3-II expression. In contrast, silencing Mfn2 reduces the expression of autophagic markers. Ai and Bi Quantification of Western blot using Image J software and normalized to the loading control. C and D A2780 and SKOV-3 cells were treated with leflunomide (50 uM) and mDivi-1 (20 uM) for 24 h. Western blot analysis showed increased LC3-II, Beclin1, and ATG5 and reduced p62 expression in both cell lines. Ci and Di Quantification of Western blot using Image J software and normalized to the loading control. E and F The immunofluorescence study confirmed the positive correlation between Mfn2 and LC3-I/II expression. Cells after Mfn2 overexpression and silencing stained with DAPI, Mfn2, and LC3 in A2780 and OVCAR-3 cells. An increased expression of Mfn2 exhibited an increased expression of LC3, and in contrast, reduced Mfn2 showed reduced LC3 expression in Mfn2-silenced cells compared to control. Representative images were taken by confocal microscope in each group. Scale bar 10 µm. All experiments were performed in triplicates. Mfn2 O/E Mfn2 overexpression, si small interfering RNAs. Data are represented as mean ± SD. Differences among groups were considered significant *p < 0.05, **p < 0.01, and ***p < 0.001, ns non-significant (p > 0.05)

Fig. 5

Activation of Mfn2 modulating mitochondrial ROS generation in OC cells. A and B Confocal microscopic analysis of mitochondrial ROS generation after leflunomide (50 uM) and mDivi-1 (20 uM) treatment for 24 h in both cell types. Leflunomide and mDivi-1 treatment reduced ROS generation compared to control. Scale bar 75 µm. C and D After Mfn2 silencing and overexpression, cells were stained with MitoROS (5 uM) dye in OVCAR-3 and A2780 cells, and representative images were taken by confocal microscopy to measure superoxide production in mitochondria of live cells. Scale bar 10 and 25 µm. Mfn2 overexpression decreases ROS generation, and silencing of Mfn2 displayed increased ROS generation in both cell types. E and F The graph shows the relative ATP production in OC cells after Mfn2 silencing and overexpression in both cell types. Each group was normalized to the control ATP production. Data are represented as mean ± SD. Differences among groups were considered significant with *p < 0.05, **p < 0.01, and ***p < 0.001, ns non-significant (p > 0.05). Mfn2 O/E Mfn2 overexpression, si small interfering RNAs

Fig. 6

Mfn2-mediated mitochondrial fusion modulating the AMPK/mTOR signaling pathway. Whole cell lysates of overexpressed Mfn2, silenced Mfn2, and parental A2780 (A) and OVCAR-3 (B) cells were prepared and examined for expression of Mfn2, Mfn1, AMPK, mTOR activities by Western blot with Mfn1, Mfn2, p-mTOR (S2481 and 2448), p-ERK, and p-AMPK and β-Actin antibodies. Blots were stripped and reprobed with Total mTOR, AMPK, and ERK antibodies. Expression of p-AMPK is increased in overexpressed Mfn2 group in both cell lines, and silenced Mfn2 cells inhibit p-AMPK. Overexpressed Mfn2 in cells reduced p-mTOR and p-ERK, while after silencing Mfn2, cells increased the p-mTOR and p-ERK levels in both cell types. Ai and Bi Quantification of Western blot using Image J software and normalized to the loading control. C and D A2780 and SKOV-3 cells were treated with leflunomide (50 uM) and mDivi-1 (20 uM) for 24 h. Western blot analysis showed increased p-AMPK and reduced p-mTOR (S2481 and 2448), p-ERK expression in both OC cells. Ci and Di Quantification of Western blot using Image J software and normalized to the loading control. E and F Western blot analyses for protein levels of Mfn1, Mfn2, p-mTOR (S2481 and 2448), p-AMPK, p-ERK, β-Actin, Total mTOR, AMPK, and ERK in two OC cells after transient transfection with Mfn2 siRNA and overexpression plasmid separately, followed by treatment with 100 uM H₂O₂ for 12 h as indicated. Ei and Fi Quantification of Western blot using Image J software and normalized to the loading control. G and Gi Western blot analysis and quantification for p-mTOR (S2481 and 2448) and p-ERK, mTOR, ERK, and β-Actin in OVCAR-3 cells after transient transfection with Mfn2 siRNA, followed by treatment with 20 mM NAC for 12 h. Data are represented as mean ± SD. Differences among groups were considered significant *p < 0.05, **p < 0.01, and ***p < 0.001, ns non-significant (p > 0.05)

Fig. 7

Mfn2 expression correlating with survival of OC patients. A Kaplan–Meier (KM) plot progression-free survival (PFS) of OC patients with higher and lower expression of Mfn2 (Logrank p value = 0.018). Median survival (low 18.3 months; high 21 months). B KM plot for PFS of OC patients undergoing chemotherapy treatment with platin drug with higher and lower expression of Mfn2 (Logrank p value = 0.0027). Median survival (low 17.77 months; high 23.24 months) C KM plot for PFS of OC patients undergoing chemotherapy treatment with paclitaxel drug with higher and lower expression of Mfn2 (Logrank p value = 0.00011). Median survival (low 15.77 months; high 22 months). D KM plot for PFS of OC patients undergoing chemotherapy treatment with taxol drug with higher and lower expression of Mfn2 (Logrank p value = 0.18). Median survival (low 16 months; high 17 months). E Schematic portraying the outcome of increased Mfn2-mediated mitochondrial fusion on the OC cell survival and the underlying mechanism. Figure was generated with BioRender