Erk2 phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth

Abstract

Ras is mutated in up to 30% of cancers, including 90% of pancreatic ductal adenocarcinomas, causing it to be constitutively GTP-bound, and leading to activation of downstream effectors that promote a tumorigenic phenotype. As targeting Ras directly is difficult, there is a significant effort to understand the downstream biological processes that underlie its protumorigenic activity. Here, we show that expression of oncogenic Ras or direct activation of the MAPK pathway leads to increased mitochondrial fragmentation and that blocking this phenotype, through knockdown of the mitochondrial fission-mediating GTPase Drp1, inhibits tumor growth. This fission is driven by Erk2-mediated phosphorylation of Drp1 on Serine 616, and both this phosphorylation and mitochondrial fragmentation are increased in human pancreatic cancer. Finally, this phosphorylation is required for Ras-associated mitochondrial fission, and its inhibition is sufficient to block xenograft growth. Collectively, these data suggest mitochondrial fission may be a target for treating MAPK-driven malignancies.

Figure 1. Ras-induced mitochondrial fission is required for tumor growth

(A) Mitochondrial morphologies of HEK-TtH cells or HEK-TtH cells stably expressing HRas^G12V plus scramble or Drp1 shRNA. Red: MitoTracker Red; Blue: DAPI. Scale Bar = 20μm. (B) Immunoblot of Flag-HRas^G12V and Drp1 in cells visualized in (A). GAPDH = Loading control. (C) Quantitation of mitochondrial morphologies observed in cells described in (A). n>50 cells, blindly scored by 3 people, 3 independent experiments; Error bar: S.E.M of mean percentages from 1 representative experiment. (D) HEK-TtH cells expressing vector or HRas^G12V were transfected with mito-dsRed and mito-PA-GFP. mito-PA-GFP was activated by a 405-nm laser pulse in a 4μm region of interest (white box) then green fluorescence was tracked over a 1 hour time course. (E–G) HEK-TtH cell expressing HRas^G12V and an shRNA targeting either scramble control or Drp1 were injected into mice and tumor volume (E) was measured over time. Tumors were removed at day 17 to be photographed (F) and weighed (G). n=5 tumors per cell line; error bars: S.E.M. of mean tumor volume (E) or tumor weight (G). ** Two-tailed student t-test, p=0.00749 (E) or p=0.00242 (G).

Figure 2. Erk2 phosphorylates Drp1 Serine 616

(A) Alignment of the consensus Erk2 target sequence with amino acids 612–620 of human Drp1 (isoform 1) and the corresponding sequence from the indicated species. (B–C) Recombinant, active GST-Erk2^R67S was incubated with either GST alone, GST-Drp1^518–736 or GST-Drp1^{518–736, S616A} in the presence of γ³²P-ATP (B) or ATP (C) and resolved by SDS-PAGE. Drp1 phosphorylation was detected by autoradiography (B) or immunoblot (A). (D–J) Phosphorylation of Drp1 (P616) and Erk1/2 (Y202/T204) were monitored by immunoblot in the following cells: (D) HEK-TtH cells grown in serum-free DMEM supplemented with 10μM Mek inhibitor PD325901 for 16 hrs, then supplemented with 10% FBS over an 8-hour time course; (E) HEK-TtH cells supplemented with 10% FBS and treated with 0.78–200nM of PD325901 for 8 hrs. (F) HEK-TtH cells stably expressing HRas^G12V treated with 0.78–200nM of PD325901 for 8 hrs; (G) HEK-TtH cells transfected with increasing amounts of Raf-22W; (H) HeLa cells transfected with increasing amounts of Raf-22W in the presence of DMSO or PD325901; (I) HEK-TtH cells transfected with increasing amounts of MEK-DD; (J) HeLa cells transfected with increasing amounts of active MEK-DD in the presence of DMSO or PD325901; Tom20, CoxIV, GAPDH: Loading Controls.

Figure 3. Activation of Ras or MAPK signaling leads to Mek-dependent mitochondrial fragmentation

(A) Mitochondrial morphologies of HEK-TtH cells stably expressing mito-YFP and HRas^G12V and treated with either DMSO or 200nM PD325901 for 24 hours. Green: mito-YFP; Blue: DAPI. Scale Bar = 20μm. (B) Quantitation of mitochondrial morphologies observed in cells described in (A). n>50 cells, blindly scored by 5 people, 3 independent experiments; Error bar: S.E.M of mean percentages from 1 representative experiment. (C–F) Mitochondrial morphologies of HEK-TtH cells transfected with mito-YFP plus either vector, Raf-22W or Mek-DD and treated with either DMSO or 200nM PD325901 for 24 hours as indicated. Green: mito-YFP; Blue: DAPI. Scale Bar = 20μm; Quantitation of mitochondrial morphologies: n>50 cells, blindly scored by 5 people, 3 independent experiments; Error bar: S.E.M of mean percentages from 1 representative experiment.

Figure 4. Patient-derived pancreatic cancer cell lines are characterized by Mek-dependent Drp1 phosphorylation and mitochondrial fragmentation

(A) Immunoblot analysis of phospho-Drp1 (P616) and Drp1 in a panel of 8 patient-derived pancreatic cell lines. CoxIV: Loading Control. (B–C) Immunoblot analysis of MPanc96 cells treated with 200nM PD325901 over a timecourse of 12 hours (C) or treated with 0.78–200nM for 8 hours (D). Graph: p-Drp1 levels normalized to total Drp1 levels; n=3, Error bars: S.E.M. Two-tailed student t-test comparing treatment to control, **p<0.01; *p<0.05. (D) Mitochondrial morphology of 8 patient-derived pancreatic cancer cell lines. Green: anti-Tom20; Blue: DAPI. Scale Bar = 20μm. (E) Mitochondrial morphology of MPanc96 cells treated with DMSO or 200nM PD325901 for 48hrs. Green: anti-Tom20; Blue: DAPI. Scale Bar = 20μm. Graph: quantitation of mitochondrial morphologies. n>50 cells, blindly scored by 5 people, 3 independent experiments; Error bar: S.E.M of mean percentages from 1 representative experiment. (F–H) BxPC3 cells expressing scramble or Drp1 shRNA were analyzed by immunoblot (F) then injected into mice and tumor volume was measured over time (G). Tumors were excised and analyzed by immunoblot (H). Blot represents first 7 tumors for each cell type (red boxes). Tubulin, GAPDH: Loading controls. Error bars: S.E.M. of mean tumor volume. ** Two-tailed student t-test, p<0.004.

Figure 5. Drp1 S616 is phosphorylated in human pancreatic ductal adenocarcinoma

(A) Three serial sections were cut from formalin-fixed, paraffin-embedded sections from 12 pancreatic ductal adenocarcinomas and stained with Hematoxylin and Eosin (H&E) or antibodies against phospho-Drp1 (S616) and phospho-Erk1/2 (T202/Y204). Representative images of colocalization are shown from 6 tumors. Scale Bar = 100μm (IHC). (B) The levels of phospho-Drp1 (S616) and phospho-Erk1/2 (T202/Y204) staining, as well as the degree of co-localization, were determined for each of 12 pancreatic ductal adenocarcinomas examined. (C) Additional sections were cut from two of the tumors (7, 11) and stained with an anti-mitochondria antibody (MTC02) to detect mitochondria (red). Blue: DAPI. Scale Bar = 20μm.

Figure 6. Drp1 Serine 616 is required for Ras-induced mitochondrial fission and Ras-induced tumor growth

(A) HEK-TtH cells were engineered to express Flag-HRas^G12V plus an shRNA targeting either scramble or Drp1 and rescued with either vector, Flag-Drp1^WT or Flag-Drp1^S616A. (B) The mitochondrial morphologies were analyzed. Green: anti-Tom20; Blue: DAPI. (C) Mitochondrial morphologies were quantified in: n>50 cells, blindly scored by 5 people, 3 independent experiments; Error bar: S.E.M of mean percentages from 1 representative experiment. Scale Bar = 20μm. (D–E) Cells were injected into mice and tumor volume measured over time (D). Tumor volumes at day 17 are shown in (E). n=10 tumors per cell line; error bar: S.E.M. of mean tumor volume. Two-tailed student t-test, **p<0.001; *p<0.05; n.s.=p>0.15.