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. 2017 Jun 20;114(25):E4924-E4933.
doi: 10.1073/pnas.1615730114. Epub 2017 Jun 5.

Mitochondrial Dysfunction Induced by a SH2 Domain-Targeting STAT3 Inhibitor Leads to Metabolic Synthetic Lethality in Cancer Cells

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Free PMC article

Mitochondrial Dysfunction Induced by a SH2 Domain-Targeting STAT3 Inhibitor Leads to Metabolic Synthetic Lethality in Cancer Cells

Davide Genini et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

In addition to its canonical role in nuclear transcription, signal transducer and activator of transcription 3 (STAT3) is emerging as an important regulator of mitochondrial function. Here, we demonstrate that a novel inhibitor that binds with high affinity to the STAT3 SH2 domain triggers a complex cascade of events initiated by interference with mitochondrial STAT3 (mSTAT3). The mSTAT3-drug interaction leads to mitochondrial dysfunction, accumulation of proteotoxic STAT3 aggregates, and cell death. The cytotoxic effects depend directly on the drug's ability to interfere with mSTAT3 and mitochondrial function, as demonstrated by site-directed mutagenesis and use of STAT3 knockout and mitochondria-depleted cells. Importantly, the lethal consequences of mSTAT3 inhibition are enhanced by glucose starvation and by increased reliance of cancer cells and tumor-initiating cells on mitochondria, resulting in potent activity in cell cultures and tumor xenografts in mice. These findings can be exploited for eliciting synthetic lethality in metabolically stressed cancer cells using high-affinity STAT3 inhibitors. Thus, this study provides insights on the role of mSTAT3 in cancer cells and a conceptual framework for developing more effective cancer therapies.

Keywords: OPB-51602; STAT3; mitochondria; small-molecule inhibitor; synthetic lethality.

Conflict of interest statement

Conflict of interest statement: This work was partially funded by Otsuka Pharmaceuticals (Japan), which provided and owns patents on two compounds used in the study, OPB-51602 and OPB-31121.

Figures

Fig. 1.
Fig. 1.
Binding and inhibition of STAT3 signaling by OPB-51602. (A) Proliferation of DU145 and H358 cells incubated for 72 h with OPB-51602 at high (filled symbols) and low (open symbols) cell density. (B) Colony-forming ability of DU145 and H358 cells treated with OPB-51602. *P < 0.01. (C) pTyr705, pSer727, and tSTAT3 in DU145 cells treated with OPB-51602 for 16 h (Upper) or 10 nM for the indicated time (Lower) in CM. (D) Model of the full-length STAT3 (firebrick ribbon) in complex with OPB-51602 (blue). (E) Estimated binding free energy of WT, S636A, V637A, and E638A mutants of the STAT3 SH2D with OPB-51602. Lower section shows molecular simulation of OPB-51602 bound to WT (green) or E638A (blue) SH2D. (F) ITC analysis of OPB-51602 binding to WT STAT3 SH2D. (G) ITC analysis of OPB-51602 binding to STAT3 SH2D mutants (E638A, V637A, and S636A).
Fig. S1.
Fig. S1.
OPB-51602 interferes with STAT3 signaling in cancer cells. (A) Immunoblot analysis of STAT3 and pSTAT3 in H358 and LNCaP cells treated with OPB-51602. (B and C) STAT3 and pSTAT3 in H358 cells treated with OPB-51602 and the indicated compounds for 16 h in FM or CM. (D) STAT3 and pSTAT3 in DU145 cells treated with NVP-BSK805 for 16 h in FM or CM. (E) Amino acids (red) in the OPB-51602 (blue) binding site in the STAT3 SH2D (Upper) and per residue energy decomposition analysis (Lower) of the complex.
Fig. 2.
Fig. 2.
OPB-51602 impairs mitochondrial function. (A) MMP (JC1 staining) in DU145 cells treated with OPB-51602 and OPB-31121 for 2 h in CM. (B) Mitochondrial OCR in control and OPB-51602–treated (100 nM for 2 h) DU145 cells. (C) Mitochondrial shape in DU145 cells treated with OPB-51602 (100 nM) in CM (16 h) or glucose-depleted medium (4 h) and stained with MitoTracker Orange (red) and DAPI (blue). (D) MMP (DIOC6 staining) in isolated mitochondria from DU145 cells treated with OPB-51602 (50 μM) or Valinomycin (100 μM) for 3 h. (E) MMP (DIOC6 staining) in isolated mitochondria from WT and STAT3−/− MEF treated with OPB-51602 for 3 h. (F) Cell viability [sulforhodamine B (SRB) assay] in DU145 and DU145 ρ° cells treated with OPB-51602. *P < 0.01.
Fig. S2.
Fig. S2.
OPB-51602 affects mitochondrial function. (A) MMP (JC1 staining) in H358 cells treated with OPB-51602 and OPB-31121 for 2 h in CM. (B) MMP (JC1 staining) in DU145 cells treated with OPB-51602, OPB-31121 and Valinomycin in FM. (C) ATP level in cells treated with the indicated drugs for 2 h in CM. (D) Lactate production in DU145 and H358 cells treated with OPB-5602, 2-DG (5 mM), and rotenone (10 μM) in FM. (E) ATP production in DU145 and H358 cells treated with OPB-51602 in FM. (F) Glucose uptake in DU145 and H358 cells treated with OPB-51602 for 6 h in FM. (G) Mitochondrial respiratory capacity and ATP production determined in control and OPB-51602 treated (100 nM for 2 h) DU145 cells by OCR measurement with Seahorse analysis shown in Fig. 2B. (H) Seahorse analysis of OCR of DU145 cells untreated (control) or treated (experimental) with OPB-51602 (100 nM) at the indicated time. (I) MMP (JC1 staining) in DU145 and DU-145 ρ° cells. *P < 0.01.
Fig. 3.
Fig. 3.
Altered distribution of STAT3 and proteostasis in response to OPB-51602. (A) Proliferation of immortalized normal (LHS) and Ras-transformed (LHS-Ras) prostate epithelial cells treated with OPB-51602 with or without 2-DG. *P < 0.01. (B) STAT3 and pSTAT3 in H358 cells treated with OPB-51602 in CM or glucose-supplemented CM. (C) STAT3 reporter activity in stably engineered cell lines treated with OPB-51602 for 16 h in complete or glucose-depleted FM (GDM). *P < 0.01. (D) STAT3 and pSTAT3 in cytoplasm, nuclei, and whole-cell lysates from LNCaP treated with OPB-51602 for 16 h. (E) Distribution of STAT3, p62, ubiquitinated proteins, and HDAC6 in cell fractions from H358 cells untreated or treated with OPB-51602 for 16 h.
Fig. S3.
Fig. S3.
Glucose starvation enhances the cellular response to OPB-51602. (A and B) Proliferation of DU145 and H358 cells treated with OPB-51602 in the presence of 2-DG (A) or 2-FDG (B). (C) STAT3 and pSTAT3 in H358 cells treated with OPB-51602 in FM or glucose-depleted FM (GDM). (D) STAT3 and pSTAT3 in DU145 cells treated with OPB-51602 and/or 2-DG (5 mM) for 4 h in FM.
Fig. S4.
Fig. S4.
Intracellular distribution of STAT3 after treatment with OPB-51602. (A) STAT3 and pSTAT3 distribution in cytoplasm and nuclei of DU145 cells untreated or treated with OPB-51602 in CM for the indicated time. (B) STAT3 mRNA in DU145 and H358 cells treated with OPB-51602 in CM. (C) STAT3 and ubiquitinated proteins in DU145 and H358 cells treated with OPB-51602 and PS-341 (10 μM) for the indicated time. (D) STAT3 and ubiquitinated proteins in control and OPB-51602–treated H358 cells lysed in low (LD) or high (HD) detergent buffer. (E and F), Cell fractionation by sucrose gradient centrifugation and analysis of intracellular distribution of STAT3 and pSTAT3 in control and OPB-51602 treated DU145 (E) and H358 (F) cells in CM. (G) Distribution of specific cell compartment markers after cell fractionation of control and OPB-51602–treated DU145 cells. (H) Intracellular distribution of STAT3, pSTAT3, and p62 in DU145 and H358 cells untreated or treated with OPB-51602 for 16 h in FM. (I) Time-lapse fluorescence microscopy determination of the percentage of viable WT and ΔSH2 EGFP-STAT3+ H358 cells and representative images at the indicated time posttransfection. (Magnification: 5×.)
Fig. 4.
Fig. 4.
Induction of STAT3 protein aggregates by OPB-51602. (A) Distribution of EGFP-STAT3 (green) in DU145 and H358 cells untreated or treated with OPB-51602 for 6 h in CM. Nuclei were stained with DAPI (blue). (B) Effect of OPB-51602 on WT and mutated (E638A) EGFP-STAT3. (C and D) WT and truncated variants of EGFP-STAT3 (C) and differential responses to treatment with OPB-51602 (D). (E) Spontaneous aggregation of ΔSH2 EGFP-STAT3 variant in CM cultures. (F) Live fluorescence, phase contrast, and composite images of H358 cells expressing WT or ΔSH2 EGFP-STAT3 at the indicated time points. Arrows indicate the position of EGFP+ cells in phase contrast images.
Fig. 5.
Fig. 5.
Entrapment of p62 in STAT3-rich aggresomes. (A) Coimmunoprecipitation of STAT3 and p62 upon lysis in DISK buffer of control and OPB-51602–treated H358 cells. (B) Distribution of EGFP-STAT3 (green) and Cherry-p62 (red) in cells treated with the indicated drugs for 6 h in CM cultures. (C) Distribution of EGFP-STAT3 (green) and WT or ΔUBA Cherry-p62 (red) in cells treated with OPB-51602 for 6 h in CM. (D) Flow cytometry analysis of autophagy in H358 and DU145 cells treated with OPB-51602 for 6 h in CM. (E) Immunoblot analysis of LC-3 along with STAT3, p62, and ubiquitinated proteins in H358 cells treated with OPB-51602 for the indicated time in CM cultures.
Fig. S5.
Fig. S5.
OPB-51602 affects autophagy in glucose-starved cancer cells. (A) Immunoblot analysis of STAT3, pSTAT3, p62, and ubiquitinated proteins in control (siGL3) and p62 knockdown (sip62) cells with and without treatment with OPB-51602 for 16 h in CM. (B) Flow-cytometry analysis of autophagy in DU145 and H358 cells treated with OPB-51602 and OPB-31121 for 6 h in glucose-rich FM. (C) LC3, STAT3, and ubiquitinated proteins in H358 cells treated with OPB-51602, PS-341 (10 μM), and chloroquine (100 μM) for 16 h in CM. (D) STAT3, p62, and ubiquitinated proteins in DU145 (Upper) H358 (Lower) cells treated with OPB-51602 with or without rapamycin (1 μM) for up to 8 h in CM. (E) Distribution of STAT3 and the indicated proteins in DU145 (Upper) and H358 (Lower) cells treated with OPB-51602 and/or rapamycin (1 μM) for 16 h in CM.
Fig. S6.
Fig. S6.
Mitochondrial dysfunction and STAT3 redistribution in cells treated with STAT3 inhibitors. (A) MMP (JC1 staining) in H358 cells treated with Valinomycin (positive control) and the indicated STAT3 inhibitors in CM. (B) STAT3 and pSTAT3 in DU145 cells treated with STA-21 (50 μM), WP1066 (50 μM), and cryptotanshinone (10 μM) in CM and glucose-supplemented CM. (C) Proliferation of DU145 and H358 cells treated with Stattic or cryptotanshinone alone or in the presence of 2-DG. *P < 0.01. (D) STAT3 and pSTAT3 in DU145 cells treated with STA-21 (Upper) and WP1066 (Lower) in FM or glucose-FM (GFM). (E–H), Intracellular distribution of the indicated proteins in DU145 cells treated with OPB-31121 (E), STA-21 and Stattic (F), CDDO-Me (G), cryptotanshinone and WP1066 (H) in CM.
Fig. S7.
Fig. S7.
Diverse impact on autophagy in response to STAT3 and JAK inhibitors. (A) Flow cytometry analysis of autophagy in DU145 and H358 cells treated with the indicated STAT3 inhibitors for 6 h in CM. (B) Distribution of STAT3 and pSTAT3 in DU145 cells treated with NVP-BSK805 for 16 h.
Fig. 6.
Fig. 6.
Inhibition of tumor growth and impairment of tumor-initiating stem-like cells in vivo. (A and B) STAT3, pSTAT3, and Ki67 in DU145 tumor xenografts after treatment with OPB-51602 (40 mg/kg, daily by mouth) for 3 and 5 d and off treatment for 2 d (5+2). Representative images (A) and IHC quantification (B). (C) Aggresome formation in DU145 tumor xenografts from control and OPB-51602–treated mice (40 mg/kg, daily by mouth) detected by ProteoStat staining (Upper) or p62 immunostaining (Lower). Arrows, large aggregates in drug-treated xenografts. (D) Growth of tumor xenografts of DU145 cells in mice treated with 20 mg⋅kg⋅d–1 of OPB-51602 (n = 5 per group). (E and F), pTyr705 STAT3, pSer727 STAT3, and Ki67 IHC in tumor xenografts of DU145 cells in mice treated with vehicle or 20 mg⋅kg⋅d–1 OPB-51602 for 2 wk. Quantification of pTyr705, pSer727 STAT3 and Ki67 immuno-staining (E) and representative images (F). *P < 0.01.
Fig. S8.
Fig. S8.
In vivo activity of OPB-51602 in mouse tumor xenografts. (A) Tumor growth assessed in control and OPB-51602 treated animals by in vivo bioluminescence imaging after 2 wk of treatment. (B) Tumor weight assessed in control and OPB-51602 treated animals after 2 wk of treatment. (C and D) Fraction of CD44+/CD24 cells (C) and ex vivo tumor-sphere forming ability (D) in cells isolated from control and OPB-51602 treated tumor xenografts after 2 wk of treatment (40 mg/kg, daily by mouth). *P < 0.01.

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