Mitochondrial STAT3 supports Ras-dependent oncogenic transformation

Abstract

Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcription factor responsive to cytokine signaling and tyrosine kinase oncoproteins by nuclear translocation when it is tyrosine-phosphorylated. We report that malignant transformation by activated Ras is impaired without STAT3, in spite of the inability of Ras to drive STAT3 tyrosine phosphorylation or nuclear translocation. Moreover, STAT3 mutants that cannot be tyrosine-phosphorylated, that are retained in the cytoplasm, or that cannot bind DNA nonetheless supported Ras-mediated transformation. Unexpectedly, STAT3 was detected within mitochondria, and exclusive targeting of STAT3 to mitochondria without nuclear accumulation facilitated Ras transformation. Mitochondrial STAT3 sustained altered glycolytic and oxidative phosphorylation activities characteristic of cancer cells. Thus, in addition to its nuclear transcriptional role, STAT3 regulates a metabolic function in mitochondria, supporting Ras-dependent malignant transformation.

Figure 1. STAT3 is essential for Ras transformation but not as a transcription factor

(A) Colony formation of wild type (WT) or STAT3-deficient (KO) cells with or without v-Src or H-RasV12 plated in soft agar. (B) Average tumor volume formed by H-RasV12-expressing STAT3-null cells transduced with empty vector (KO), wild type (WT) or Y705F-mutant STAT3 (Y705F) and injected into Balb/c^nu/nu mice (5 mice per group). (C) Average colony formation of H-RasV12-expressing STAT3-deficient cells stably transduced with empty vector (EV), wild type STAT3 (WT), or STAT3 mutants: N-terminal 132 amino acid deletion (ΔN), DNA binding domain VVV461-463AAA (DBD), SH2 domain R609K (SH2), tyrosine phosphorylation site Y705F (YF), serine phosphorylation site S727A and S727D (SA and SD), and the STAT3β isoform (β). (D) Average colony formation of H-RasV12 or v-Src-expressing cells transduced with empty vector (EV), wild type STAT3 (WT), 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 to 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 empty vector (EV) or H-RasV12 (Ras) and transfected with shRNA to STAT3 (shS3) or scrambled control (Scr). (H) Average colony formation of MCF10A cells with and without H-RasV12 and STAT3. Error bars indicate SD.

Figure 2. Localization of STAT3 to mitochondria supports Ras transformation

(A) Fractionation of cells mechanically disrupted in absence of detergent and separated into P100 (plasma membrane), S100 (organelle-free cytosol), and mitochondrial fractions. Samples probed for STAT3, IGF1-R1α, or Bcl-X_L, as indicated. (B) Distribution of STAT3 mutants expressed in H-RasV12-transduced cells. Erk1/2 and Bcl-X_L expression verified fraction purity. (C) Protease-sensitivity of proteins associated with mitochondrial membranes in absence (unt) or presence of proteinase K (+PK) or proteinase K and Triton X-100 (+PK/T). (D) Survival of H-RasV12 expressing cells following glucose depletion. Survival of STAT3-null (KO) or wild type cells expressing H-RasV12 (WT) or STAT3-null cells expressing H-RasV12 reconstituted with empty vector (EV) or STAT3 mutants after growth in low or high glucose medium, as indicated. Asterisks indicate statistically significant differences (p<.05 by student’s t-test) of individual conditions relative to empty vector. (E) Survival of cells exposed to 2% oxygen. (F) Mitochondrially-targeted wild type and mutant STAT3 protein expression. (G) Colony formation by H-RasV12-, (H) v-Src-, and (I, J) N- and K-Ras-expressing cell lines.

Figure 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 wild type (WT) or STAT3-deficient (KO) cells with or without H-RasV12. (B) Mitochondrial membrane potential of H-RasV12-expressing cells with wild type (WT), vector (KO), mitochondrially-targeted wild type (MTS-WT) or S727A-mutant STAT3. Activities of electron transport chain complexes II (C) and V (D) compared between H-RasV12-expressing wild type (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 value <0.05* or <0.001*** by student’s t-test. (E) Lactate dehydrogenase activity in wild type (WT), STAT3-deficient (KO), and H-RasV12-expressing wild type (WT), STAT3-deficient (KO), mitochondrially-targeted wild type (MTS-WT), or mutant STAT3 (MTS-S/A) expressing cells. Results are means of 3 replicates. Error bars indicate SD.