Statin-Sensitive Akt1/Src/Caveolin-1 Signaling Enhances Oxidative Stress Resistance in Rhabdomyosarcoma

Abstract

Identifying the molecular mechanisms underlying radioresistance is a priority for the treatment of RMS, a myogenic tumor accounting for approximately 50% of all pediatric soft tissue sarcomas. We found that irradiation (IR) transiently increased phosphorylation of Akt1, Src, and Cav1 in human RD and RH30 lines. Synthetic inhibition of Akt1 and Src phosphorylation increased ROS levels in all RMS lines, promoting cellular radiosensitization. Accordingly, the elevated activation of the Akt1/Src/Cav1 pathway, as detected in two RD lines characterized by overexpression of a myristoylated Akt1 form (myrAkt1) or Cav1 (RDCav1), was correlated with reduced levels of ROS, higher expression of catalase, and increased radioresistance. We found that treatment with cholesterol-lowering drugs such as lovastatin and simvastatin promoted cell apoptosis in all RMS lines by reducing Akt1 and Cav1 levels and increasing intracellular ROS levels. Combining statins with IR significantly increased DNA damage and cell apoptosis as assessed by γ histone 2AX (γH2AX) staining and FACS analysis. Furthermore, in combination with the chemotherapeutic agent actinomycin D, statins were effective in reducing cell survival through increased apoptosis. Taken together, our findings suggest that the molecularly linked signature formed by Akt1, Src, Cav1, and catalase may represent a prognostic determinant for identifying subgroups of RMS patients with higher probability of recurrence after radiotherapy. Furthermore, statin-induced oxidative stress could represent a treatment option to improve the success of radiotherapy.

Keywords: Akt1; Src; catalase; caveolin-1; radioresistance; rhabdomyosarcoma.

Figure 1

Involvement of the Akt1, Src, and Cav1 proteins in oxidative stress and radiation response in RMS lines. (A) Quantification of AKT1 and SRC gene expression was performed using an RNA-seq dataset (GSE108022) relative to human FNRMS (n = 66), FPRMS (n = 35), and normal skeletal muscle (n = 5) samples (top panels). Each point represents an individual sample. The mean expression is highlighted by a black horizontal bar. *** p-value < 0.0001; one-way ANOVA test vs. normal skeletal muscle. Correlation between AKT1 and SRC expression was calculated by Pearson’s coefficient (bottom panel). (B) RD and RH30 cells (3 × 10³) were plated in black 96-well plates in triplicates 24 h prior to treatment with the indicated compounds. Quantification of ROS production with the CM-H2DCFDA probe was performed after 24 h (n = 2). Data are mean ± SEM, * p-value < 0.05; ** p-value < 0.001; one-way ANOVA test vs. vehicle-treated cells. (C) RD and RH30 cells (1.5 × 10⁵) were seeded into 60mm dishes. After 24 h, cells were irradiated and collected at the indicated time points for IB (n = 2). Densitometric quantifications are reported in the top graph after tubulin normalization. Data are mean ± SEM, *** p-value < 0.0001; one-way ANOVA test vs. 0 h time point. (D) Subconfluent RD and RH30 cells were pretreated for 2 h with the indicated compounds prior to IR. Cell survival was evaluated by clonogenic assay (n = 3). Data are mean ± SEM, *** p-value < 0.0001; one-way ANOVA test vs. vehicle-treated irradiated cells. Representative images were taken after crystal violet incorporation.

Figure 2

MyrAkt1 signaling drives increased levels of Akt1, Src, Cav1, and catalase, promoting intracellular ROS reduction. (A–C) Control and myrAkt1 cells (1.5 × 10⁵) were seeded into 60 mm dishes. After 48 h of proliferation, cells were processed for IB (n = 2) (A), IF (n = 3) (B), and PCR analyses (n = 2) (C). Densitometric quantifications are reported in the graphs after tubulin or GAPDH normalization. Data are mean ± SEM, ** p-value < 0.001; *** p-value < 0.0001; unpaired Student’s t-test vs. control RD line. Representative images for IF analysis were taken using a fluorescent microscope at 63 X magnification. The scale bar corresponds to 50 µm. (D) After 24 h had elapsed from plating cells (1.0 × 10⁵) into 60 mm dishes, LY294002 and MK2206 or vehicle were provided for up to 72 h before IB analysis (n = 2). Densitometric quantifications are reported in the top graph after tubulin normalization. Data are mean ± SEM, ** p-value < 0.001; *** p-value < 0.0001; one-way ANOVA test vs. vehicle-treated cells. (E,F) Control and myrAkt1 cells (3 × 10³) were plated in black 96-well plates in triplicates 24 h prior to treatment or not with the indicated compounds. Quantification of ROS production with the CM-H2DCFDA probe was performed after 24 h (n = 2). Data are mean ± SEM, * p-value < 0.05; ** p-value < 0.001; *** p-value < 0.0001; unpaired Student’s t-test vs. control RD line (E) or one-way ANOVA test vs. vehicle-treated cells (F).

Figure 3

The expression pattern of Akt1, Src, and Cav1 is conserved in the radioresistant myrAkt1 and RDCav1 lines, and its inhibition promotes radiosensitization. (A,B) IR treatment was performed 24 h after plating control, myrAkt1 and RDCav1 cells (1.5 × 10⁵) in 60 mm dishes. Cells were processed at the indicated time-points for IB (n = 2). To explain the absence of Src and Cav1 bands in the control cells, it should be noted that detection was performed under low exposure conditions to avoid interference with the strong signals arisen from protein homogenates of radioresistant cells. Tubulin was used as loading control. (C,D) Subconfluent myrAkt1 and RDCav1 cells were pretreated for 2 h with the indicated compounds before IR treatment. Cell survival was evaluated by clonogenic assay (n = 3). Data are mean ± SEM, *** p-value < 0.0001; one-way ANOVA test vs. vehicle-treated irradiated cells. Representative images were taken after crystal violet incorporation.

Figure 4

Statins downregulate Akt1 and Cav1 protein levels in myrAkt1 cells and increase ROS production in all RMS lines. (A) Cells were treated for 4 h with statins or vehicle 24 h after plating control and myrAkt1 cells (1.5 × 10⁵) in 60 mm dishes, then processed by IB analysis (n = 2). Densitometric quantifications are reported in the graphs after tubulin normalization. Data are mean ± SEM, ** p-value < 0.001; *** p-value < 0.0001; one-way ANOVA test vs. vehicle-treated cells. (B) Cells (3 × 10³) were plated in black 96-well plates in triplicates. ROS production was evaluated by administering the CM-H2DCFDA probe to all the indicated RMS lines after receiving statins or vehicle at the indicated doses for 24 h (n = 2). Data are mean ± SEM, * p-value < 0.05; ** p-value < 0.001; one-way ANOVA test vs. vehicle-treated cells.

Figure 5

The combination of statins and IR promotes radiosensitization in RMS lines. (A) Subconfluent cells from RD, RH30, myrAkt1, and RDCav1 lines were treated for 2 h with statins or vehicle before IR treatment. Cell survival was evaluated by clonogenic assay (n = 2). Data are mean ± SEM, *** p-value < 0.0001; one-way ANOVA test vs. vehicle-treated irradiated cells. Representative images were taken after crystal violet incorporation. (B) IF analysis of nuclear γH2AX staining in myrAkt1 and RDCav1 cells pretreated for 2 h with statins or vehicle prior to IR and evaluated at the indicated time points using a fluorescent microscope at 63 X magnification. The scale bar corresponds to 50 µm. The quantification is relative to the average number of γH2AX-positive cells counted in ten different fields (n = 2). Data are mean ± SEM, ** p-value < 0.001; *** p-value < 0.0001; one-way ANOVA test vs. vehicle-treated irradiated cells for each time-point. (C) Cells (4 × 10⁴ for myrAkt1 and RDCav1, or 6 × 10⁴ for RD and RH30) were plated in duplicates into 6-well plates. FACS analysis was performed on all RMS lines after treatment with lovastatin or simvastatin and IR for 72 h (n = 2).

Figure 6

The combination of statins and actinomycin D promotes chemosensitization in RMS lines. (A,B) Cells (1.5 × 10³ for myrAkt1 and RDCav1, or 2 × 10³ for RD and RH30) were seeded in triplicates in 96-well plates 24 h prior to treatment with the indicated compounds. Cell survival was evaluated by neutral red assay after 48 h (n = 2). Data are mean ± SEM, * p-value < 0.05; ** p-value < 0.001; *** p-value < 0.0001; one-way ANOVA test vs. vehicle-treated cells. # p-value < 0.05; ## p-value < 0.001; ### p-value < 0.0001; one-way ANOVA test vs. single treatment. (C,D) Cells (4 × 10⁴ for myrAkt1 and RDCav1 or 6 × 10⁴ for RD and RH30) were plated in duplicates into 6-well plates. FACS analysis was performed on all RMS lines after treatment with lovastatin or simvastatin and actinomycin D for 72 h (n = 2).

Figure 7

The Akt1/Src/Cav1 network increases ROS detoxification and DNA repair in RMS cells. Representative scheme of the protein network that facilitates ROS neutralization through increased catalase expression and DNA repair. Statin treatment induces high levels of ROS and radiosensitization by reducing Akt1 and Cav1 activation.