MS-275 (Entinostat) Promotes Radio-Sensitivity in PAX3-FOXO1 Rhabdomyosarcoma Cells

Abstract

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. About 25% of RMS expresses fusion oncoproteins such as PAX3/PAX7-FOXO1 (fusion-positive, FP) while fusion-negative (FN)-RMS harbors RAS mutations. Radiotherapy (RT) plays a crucial role in local control but metastatic RMS is often radio-resistant. HDAC inhibitors (HDACi) radio-sensitize different cancer cells types. Thus, we evaluated MS-275 (Entinostat), a Class I and IV HDACi, in combination with RT on RMS cells in vitro and in vivo. MS-275 reversibly hampered cell survival in vitro in FN-RMS RD (RASmut) and irreversibly in FP-RMS RH30 cell lines down-regulating cyclin A, B, and D1, up-regulating p21 and p27 and reducing ERKs activity, and c-Myc expression in RD and PI3K/Akt/mTOR activity and N-Myc expression in RH30 cells. Further, MS-275 and RT combination reduced colony formation ability of RH30 cells. In both cell lines, co-treatment increased DNA damage repair inhibition and reactive oxygen species formation, down-regulated NRF2, SOD, CAT and GPx4 anti-oxidant genes and improved RT ability to induce G2 growth arrest. MS-275 inhibited in vivo growth of RH30 cells and completely prevented the growth of RT-unresponsive RH30 xenografts when combined with radiation. Thus, MS-275 could be considered as a radio-sensitizing agent for the treatment of intrinsically radio-resistant PAX3-FOXO1 RMS.

Keywords: DNA damage; HDACs; MS-275; pediatric cancers; radiotherapy; rhabdomyosarcoma; soft tissue sarcoma.

Figure 1

MS-275 induces reversible cell growth arrest in FN-RMS and FP-RMS cells. (A) Dose of MS-275 able to reduce by 50% the cell survival of RD (Left) and RH30 (Right) cell lines was identified treating the cells for 24 h with increasing concentrations of the drug. Cell viability was measured by Trypan Blue dye exclusion test. Results represent the mean values of three independent experiments ± SD. (B,C) Effect of MS-275 IC₅₀ on cell number of adherent (B) and floating (C) RD (Left) and RH30 (Right) cells: after four days of treatment the drug was washed out or not and counts were performed for further 6 days. Surviving cells were counted using Trypan blue dye exclusion test. Results represent the mean values of four independent experiments ± SD. (D) Effect of MS-275 on cell viability, measured as metabolic activity by an MTT assay, of RD (Left) and RH30 (Right) cells treated as in (B,C). Results represent the mean values of four independent experiments ± SD. Statistical significance: *** p ≤ 0.001 vs. Untreated cells; ^$ p ≤ 0.05, ^$$ p ≤ 0.01, ^$$$ p ≤ 0.001 Washout vs. No Washout.

Figure 2

MS-275 reversibly reduces class I and IV HDACs expression and activity in FN-RMS and FP-RMS cells. (A) HDAC1, 2, 3, 8 and 11 transcript levels in RD (Left) and RH30 (Right) cells treated for 4 days with MS-275 (IC₅₀) followed by 24 h of drug washout. Transcript levels were measured by qRT-PCR assays and GAPDH mRNA was used as endogenous control. The relative mRNA expression levels are presented as the average fold changes in treated tumor cell lines vs. untreated cells, set at 1. (B) HDACs activity in RD (Left) and RH30 (Right) cells treated as in (A) was measured by enzymatic assay and presented as the average fold changes. Results represent the mean values of three independent experiments ± SD. Statistical significance: * p ≤ 0.05, *** p ≤ 0.001 vs. Untreated cells; ^$$$ p ≤ 0.001 Wash Out vs. No Wash Out. (C) Cellular morphology of RMS cells, RD (Left) and RH30 (Right), untreated or treated with MS-275 (IC₅₀) for 4 days was analyzed under light microscope at 200X magnification.

Figure 3

MS-275 affects cell cycle distribution, induces PARP1 but not caspase cell death cascades and promotes necrosis. (A) FACS analysis performed on RD (Left) and RH30 (Right) untreated or treated for 4 days with MS-275 (IC₅₀). Data (Up) showing the percentage of cells in each cell cycle phase representing the mean value of three independent experiments. (Down) a representative of three independent experiments is shown. (B) Cell lysates from RD (Left) and RH30 (Right) cells treated for 24 h and 4 days with MS-275 (IC₅₀) were analyzed by immunoblotting with specific antibodies for the indicated proteins; GAPDH expression was used as a loading control. Histograms of densitometric analysis (Down) represent the mean values of three independent experiments ± SD. Statistical significance: ** p ≤ 0.01, *** p ≤ 0.001 vs. Untreated cells.

Figure 4

MS-275 induces PARP1 but not Caspase cell death cascades and promotes necrosis in FP-RMS cell lines. (A) Annexin V analysis performed on RD (Left) and RH30 (Right) cells untreated or treated for 24 h with MS-275 IC₅₀. Histograms (RD left, RH30 right) showing the percentage of necrotic and apoptotic cells representing the mean value of three independent experiments. A FACS plot representative of three independent experiments is shown (RD left, RH30 right). (B) Cell lysates from RD (Left) and RH30 (Right) cells treated for 24 h and 2 days were analyzed by immunoblotting with specific antibodies for indicated proteins; GAPDH expression was used as a loading control. Histograms of densitometric analysis (RD left, RH30 right) represent the mean values of three independent experiments ± SD. (C) RD (Left) and RH30 (Right) cells treated as in (B) were analyzed for Caspase 3 activity by a specific assay. Histograms represent the mean values of three independent experiments ± SD. Statistical significance: *** p ≤ 0.001 vs. Untreated cells.

Figure 5

MS-275 induces PARP1 activation. (A) Cell lysates from RD (Left) and RH30 (Right) cells treated with MS-275 IC₅₀ for 6 h, 24 h and 2 days were analyzed by immunoblotting with specific antibodies for the indicated proteins; GAPDH expression was used as loading control of samples. Histograms of densitometric analysis (RD left, RH30 right) represent the mean values of three independent experiments ± SD. (B) RD (Left) and RH30 (Right) cells treated as in (A) were analyzed for PARP1 activity by a specific assay. Histograms represent the mean values of three independent experiments ± SD. Statistical significance: *** p ≤ 0.001 vs. Untreated cells.

Figure 6

MS-275 radiosensitizes FP-RMS cell lines. (A) Colony formation assay of RD (Left) and RH30 (Right) treated with MS-275 IC₅₀, RT alone or with the combination. Three hours after RT (4 Gy), cells were seeded at low concentrations for colony assays. Colony forming efficiency was calculated by crystal violet absorbance after 14 days of MS-275 treatment; RD (Left) and RH30 (Right). The lower panel shows representative pictures of colonies. Results represent the mean values ± SD of three independent experiments. (B) Cell lysates from RD (Left) and RH30 (Right) treated as in (A) were analyzed 6 h after irradiation by immunoblotting with specific antibodies for the indicated proteins. GAPDH expression was used as the loading control. Histograms of densitometric analysis (Left) represent the mean values of three independent experiments ± SD. (C) Mitochondrial super-oxide anion production of RD (Left) and RH30 (Right) treated as in (A) was assessed by MitoSox Red staining, half-hour (0.5), 6 or 12 h after RT. Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. Untreated cells; ^$$$ p ≤ 0.001 vs. RT; ^### p ≤ 0.001 vs. MS-275.

Figure 7

Pre-treating RMS with MS-275 promotes RT-induced apoptosis. (A) Annexin V analysis performed after 24 h of irradiation (4 Gy) on RD (Left) and RH30 (Right) cells untreated or treated with MS-275 IC₅₀. Histograms (Up) show the percentage of necrotic and apoptotic cells representing the mean value of three independent experiments. Representative FACS plots (Down) of three different experiments. (B) FACS analysis performed on RD (left) and RH30 (right) treated as in (A). Histograms show the percentage of cells in the cell cycle phases and represent the mean value of four independent experiments. The lower panel shows a representative FACS plot of one of three independent experiments. Statistical significance: ** p ≤ 0.01, *** p ≤ 0.001 vs. Untreated cells; ^$$ p ≤ 0.01, ^$$$ p ≤ 0.001 vs. RT; ^## p ≤ 0.01, ^### p ≤ 0.001 vs. MS-275.

Figure 8

MS-275 counteracts the ability of RT to activate the HR-mediated DSBs repair pathway in RH30 and inhibits anti-oxidant molecular response in both RMS cell lines. (A) RD (Left) and RH30 (Right) were treated with MS-275 (IC₅₀) and RT (4 Gy) alone or pre-treated (24 h) with MS-275 and then irradiated and values of three independent experiments ± SD. (B) Gene expression of antioxidant enzymes superoxide dismutase (SOD-2), catalase (CAT), glutathione peroxidase (GPx)-4 and nuclear factor erythroid 2 p45-related factors (NRF2) were investigated by qRT-PCR, 12 h after RT in cells treated as in (A). The gene expression was reported as fold change vs untreated conditions reported equal to 1. Histograms are representative of three independent experiments performed in triplicate. Statistical significance: *** p ≤ 0.001 vs. Untreated cells; ^$$$ p ≤ 0.001 vs. RT; ^### p ≤ 0.001 vs. MS-275.

Figure 9

Effects of MS-275 combined or not with irradiation on in vivo tumor growth. The diagram above all the figures resumes how the experiment was performed. (A) Growth curve of tumor volumes from xenografted RD and RH30 cell lines, untreated, MS-275-treated, irradiated (RT), MS-275-pre-treated and irradiated (RT + MS-275). Tumor volumes were evaluated as described in methods and represent the mean ± SEM of 8 mice per group. The graphs show the sequential treatments of xenografted mice started when tumors reached a volume of approximately 0.5 cm³. Results represent the mean values ± SD. Statistical significance: ** p ≤ 0.01, *** p ≤ 0.001 vs. Untreated mice; ^$$$ p ≤ 0.001 vs. RT-treated mice; ^### p ≤ 0.001 vs. MS-275-treated mice. (B) Tumor weights mice injected with RD (Left) and RH30 (Right) and treated with MS-275 and RT alone or in combination. (C) Kaplan–Meier estimates for rates of progression for untreated, MS-275, RT, or MS-275 + RT combination in RMS-derived tumors.