Research ArticleHEALTH AND MEDICINE

Dysfunctional MnSOD leads to redox dysregulation and activation of prosurvival AKT signaling in uterine leiomyomas

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Science Advances  04 Nov 2016:
Vol. 2, no. 11, e1601132
DOI: 10.1126/sciadv.1601132
  • Fig. 1 ULMs exhibit increased levels of acetylated MnSOD and 3-NO as well as decreased MnSOD activity.

    (A) Representative images of the TMA containing 60 matched MM and ULM specimens immunostained with MnSOD K122-Ac, MnSOD, and 3-NO antibodies. For each human specimen, two tissue cores were derived from MM and three were derived from ULM. (B) Score frequency distribution of TMA immunostaining. The intensity of well-preserved matched MM/ULM tissue cores was scored numerically as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). Statistically significant differences between normal MM and ULM were evaluated using a χ2 test (***P = 0.0009, ****P < 0.0001; ns, not significant). (C) MnSOD activity was assessed in 13 untreated patient-derived MM and matched ULM cells. Each data point represents the means ± SD of a quadruplicate measurement. Data are represented as fold change to untreated MM cells for each patient (*P < 0.0001; aP = 0.0183; bP = 0.0046; cP = 0.0008; dP = 0.0025; eP = 0.0035; paired t test).

  • Fig. 2 Mitochondrial ROS derived from dysfunctional acetylated MnSOD lead to AKT activation in ULM cells.

    (A) Levels of MnSOD K122-Ac, MnSOD, and pAKT(S473) (pAKT) proteins from cell lysates of untreated MM and ULM cells. pan-AKT (AKT) and actin were used as loading controls. (B and C) ULM and MM cells were exposed to increasing concentrations of PQ or H2O2 for 6 hours, and cellular protein extracts were analyzed by Western blot for pAKT(S473) and AKT. A representative Western blot and the means ± SD of densitometric quantification of three independent experiments are shown [(B) *P < 0.05, ***P < 0.001 versus PQ 0 μM ULM; (C) *P < 0.05, ***P < 0.001 versus H2O2 0 μM ULM; one-way analysis of variance (ANOVA), n = 3]. (D) ULM cells were transiently infected with a lenti-CTR or a lenti–MnSOD K122-R. MnSOD activity (*P = 0.0286; unpaired t test) as well as MnSOD and pAKT protein levels were analyzed, and corresponding densitometric analysis is shown. Data are representative of three patients. (E) ULM cells were treated with 10 μM GC4419 for 6 hours, and pAKT levels were analyzed. AKT was used as loading control. Corresponding densitometric analysis of three independent experiments is shown as means ± SD (**P = 0.0045; unpaired t test, n = 3). (F) ULM and MM cells were treated for 24 hours with increasing doses of PQ or H2O2, and cell viability was measured using WST-1 assay (*P < 0.05, ****P < 0.0001; one-way ANOVA). Results are expressed as means ± SD from three independent experiments (n = 3).

  • Fig. 3 ULM cells are characterized by aberrant MnSOD acetylation and AKT activation under normoxic and hypoxic conditions.

    (A) MitoSOX Red was used to assess mitochondrial superoxide levels in ULM cells under normoxic (20% O2) and hypoxic (2% O2) conditions after 48 hours of incubation time. MitoSOX fluorescence was quantified by analyzing the fluorescence intensity of more than 10 cells for each condition using ImageJ software (****P < 0.0001; one-way ANOVA, n = 3 independent experiments ± SD). RFU, relative fluorescence unit. (B) ULM cells were grown under normoxia and hypoxia for 48 hours, and levels of MnSOD K122-Ac and pAKT(S473) were analyzed. MM cells grown in normoxia were used as control. Hypoxia was verified by detection of HIF-1α. Equal loading was confirmed by anti-AKT. Corresponding densitometric analysis of three independent experiments is shown as means ± SD (*P < 0.05; one-way ANOVA, n = 3). (C and D) MM and ULM cells were grown under both normoxic and hypoxic atmosphere for a total of 48 hours. Twenty-four hours before harvesting, the MnSOD mimetic GC4419 (10 μM) was added to ULM cells. (C) MnSOD activity was assessed as previously described. Data are expressed as means ± SD from three independent experiments (n = 3). (D) pAKT and AKT levels were analyzed by Western blotting, and densitometric analysis of three independent experiments is shown as means ± SD (*P < 0.05, ***P < 0.001, ****P < 0.0001; one-way ANOVA, n = 3).

  • Fig. 4 PTEN activity is reduced in ULM cells as a result of superoxide-dependent oxidation.

    (A) PTEN phosphatase activity was measured in immunoprecipitated PTEN samples from five patient-derived MM and ULM cells using PIP3 as the substrate. Data are represented as fold change to untreated MM cells (paired t test). (B) Protein extracts from untreated MM and ULM cells were analyzed under nonreducing conditions and immunoblotted with anti-PTEN antibody (*P = 0.0162; unpaired t test). (C) ULM cells were treated for 6 hours with 10, 100, 500, and 1000 μM PQ in serum-free medium, and protein extracts were analyzed as described above for oxidized and total PTEN (**P < 0.01, ****P < 0.0001; one-way ANOVA). (D) ULM cells were treated with 100 μM PQ, 10 μM GC4419, or a combination of both drugs for 6 hours. Protein extracts were analyzed under nonreducing conditions and immunoblotted with anti-PTEN antibody (*P < 0.05, **P < 0.01; one-way ANOVA) or under reducing conditions and immunoblotted with pAKT antibody (*P < 0.05, **P < 0.01; one-way ANOVA). For all Western blots in (B) to (D), one representative blot and corresponding densitometric analysis of three independent experiments are shown as means ± SD (n = 3). Anti-actin antibody was used as loading control.

  • Fig. 5 Treatment with the AKT inhibitor MK-2206 leads to superoxide generation in ULM cells.

    (A) Mitochondrial superoxide levels were assessed in ULM cells using MitoSOX Red. ULM cells were treated with vehicle (CTR), various concentrations of MK-2206 (MK; 1, 10, and 25 μM) and 10 μM GC4419 (GC) alone or with 25 μM MK-2206 (GC+MK25) for 6 hours in serum-free media. Representative pictures from three independent experiments are shown. (B) MitoSOX fluorescence was quantified by analyzing the fluorescence intensity of more than 10 cells for each condition using ImageJ software (****P < 0.0001 versus 0 μM MK-2205; ####P < 0.0001 versus 25 μM MK-2205; one-way ANOVA, n = 3 independent experiments ± SD). (C) GSH content was quantified using GSH-Glo reagent. ULM cells were treated at the indicated concentrations of MK-2206 for 24 hours. BSO (10 μM) and 50 μM NAC were used as negative and positive controls, respectively [*P < 0.05, ***P < 0.001, ****P < 0.0001 versus vehicle (dimethyl sulfoxide); one-way ANOVA, n = 3 independent experiments ± SD]. (D) MM and ULM cells were treated with 25 μM MK-2206 alone or in combination with 10 μM GC4419 for 24 hours. Cell viability was determined using WST-1 (**P < 0.01, ****P < 0.0001; one-way ANOVA, n = 3 independent experiments ± SD).

  • Fig. 6 AKT protects ULM cells from oxidative-induced damage.

    (A and B) AKT1, AKT2, and AKT3 were silenced in ULM and MM cells by reverse transfection using siAKT1, siAKT2, and siAKT3. Following AKT knockdown, 50, 100, or 500 μM H2O2 was added to ULM and MM cells for 6 hours, and cell viability was determined using WST-1. Data are shown as means ± SD from three independent experiments (*P < 0.05, **P < 0.01, ****P < 0.0001; one-way ANOVA, n = 3). (C) Proposed working model for the interplay between dysfunctional MnSOD and activation of the AKT pathway and its effects on ULM cell survival. In ULM cells, acetylation of MnSOD impairs its activity, leading to the increase of mitochondrial ROS, which, in turn, activate AKT through oxidative inactivation of PTEN and promote cell survival in the prooxidative ULM microenvironment. The mitochondrion in the figure was taken from the Servier Medical Art database (http://servier.com/Powerpoint-image-bank).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/11/e1601132/DC1

    fig. S1. SIRT3 and iNOS protein levels in ULM.

    fig. S2. Differential expression of MnSOD K122-Ac, MnSOD, and pAKT in MM and ULM cells.

    fig. S3. Overexpression of MnSOD reduces pAKT levels in ULM cells from multiple patients.

    fig. S4. PQ causes PTEN nuclear translocation in ULM cells.

    fig. S5. Different effects of MK-2206 and AKT silencing on ULM cell viability and superoxide generation.

    fig. S6. AKT silencing in ULM and MM cells.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. SIRT3 and iNOS protein levels in ULM.
    • fig. S2. Differential expression of MnSOD K122-Ac, MnSOD, and pAKT in MM and ULM cells.
    • fig. S3. Overexpression of MnSOD reduces pAKT levels in ULM cells from multiple patients.
    • fig. S4. PQ causes PTEN nuclear translocation in ULM cells.
    • fig. S5. Different effects of MK-2206 and AKT silencing on ULM cell viability and superoxide generation.
    • fig. S6. AKT silencing in ULM and MM cells.

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