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Science 324 (5935): 1713-1716

Copyright © 2009 by the American Association for the Advancement of Science

Mitochondrial STAT3 Supports Ras-Dependent Oncogenic Transformation

Daniel J. Gough1,*, Alicia Corlett1,*,{dagger}, Karni Schlessinger1,{ddagger}, Joanna Wegrzyn2, Andrew C. Larner2, and David E. Levy1,§

1 Department of Pathology and New York University Cancer Institute, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA.
2 Department of Biochemistry and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA.


Figure 1
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Fig. 1. STAT3 is essential for Ras transformation but not as a transcription factor. (A) Colony formation of WT or STAT3-deficient (KO) cells with or without v-Src or H-RasV12 plated in soft agar. EV, empty vector. (B) Average tumor volume formed by H-RasV12–expressing STAT3-null cells transduced with EV (KO), WT, or Y705F-mutant STAT3 (Y705F) and injected into Balb/cnu/nu mice (5 mice per group). (C) Average colony formation of H-RasV12–expressing STAT3-deficient cells stably transduced with EV, WT STAT3, or STAT3 mutants. {Delta}N, N-terminal 132–amino acid deletion; DBD, DNA binding domain VVV461-463AAA; SH2, SH2 domain R609K; YF, tyrosine phosphorylation site Y705F; SA, serine phosphorylation site S727A; SD, serine phosphorylation site S727D; and β, the STAT3β isoform. (D) Average colony formation of H-RasV12– or v-Src–expressing cells transduced with EV, WT STAT3, or STAT3 with a mutated nuclear localization signal (NLS). (E) Depletion of H-Ras or STAT3 protein by shRNA in T24 human bladder carcinoma cells compared with scrambled shRNA control (Scr). (F) Average colony formation of T24 cells depleted for STAT3 or H-Ras. (G) Expression of H-Ras and STAT3 in human breast epithelial MCF10A cells transduced with EV or H-RasV12 (Ras) and transfected with shRNA to STAT3 (shS3) or Scr. (H) Average colony formation of MCF10A cells with and without H-RasV12 and STAT3. Error bars indicate SD.

 

Figure 2
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Fig. 2. Localization of STAT3 to mitochondria supports Ras transformation. (A) Fractionation of cells mechanically disrupted in the absence of detergent and separated into P100 (plasma membrane), S100 (organelle-free cytosol), and mitochondrial fractions. Samples probed for STAT3, IGF1-R1{alpha}, or Bcl-XL, as indicated. (B) Distribution of STAT3 mutants expressed in H-RasV12–transduced cells. Erk1/2 and Bcl-XL expression verified fraction purity. (C) Protease-sensitivity of proteins associated with mitochondrial membranes in absence (Unt) or presence (+PK) of proteinase K or of proteinase K and Triton X-100 (+PK/T). (D) Survival of H-RasV12–expressing cells after glucose depletion. Survival of STAT3-null (KO) or WT cells expressing H-RasV12 (WT) or STAT3-null cells expressing H-RasV12 reconstituted with EV or STAT3 mutants after growth in low or high glucose medium, as indicated. Asterisks indicate statistically significant differences (P < 0.05 by Student’s t test) of individual conditions relative to EV. (E) Survival of cells exposed to 2% oxygen. (F) Mitochondrially targeted WT and mutant STAT3 protein expression. (G) Colony formation by H-RasV12–, (H) v-Src–, and (I and J) N– and K-Ras–expressing cell lines. Error bars indicate SD.

 

Figure 3
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Fig. 3. Mitochondrial STAT3 augments electron transport chain activity. (A) Mitochondrial membrane potential measured with TMRE and recorded as mean fluorescence intensity (MFI) by flow cytometry in WT or STAT3-deficient (KO) cells with or without H-RasV12. (B) Mitochondrial membrane potential of H-RasV12–expressing cells with WT, vector (KO), mitochondrially targeted WT (MTS-WT), or S727A-mutant STAT3. Activities of electron transport chain complexes II (C) and V (D) compared between H-RasV12–expressing WT or STAT3-deficient (KO) cells. Unit activity of individual complexes normalized to citrate synthase activity from equivalent numbers of mitochondria. Statistical significance with *P < 0.05 or ***P < 0.001 by Student’s t test. (E) Lactate dehydrogenase activity in WT and STAT3-deficient (KO) and in H-RasV12–expressing WT, STAT3-deficient (KO), MTS-WT–, or mutant STAT3 (MTS-S/A)–expressing cells. Results are means of three replicates. Error bars indicate SD.

 


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