The histone deacetylase inhibitor Suberoylanilide Hydroxamic Acid (SAHA) as a therapeutic agent in rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is an aggressive childhood sarcoma with two distinct subtypes, embryonal (ERMS) and alveolar (ARMS) histologies. More effective treatment is needed to improve outcomes, beyond conventional cytotoxic chemotherapy. The pan-histone deacetylase inhibitor, Suberoylanilide Hydroxamic Acid (SAHA), has shown promising efficacy in limited preclinical studies. We used a panel of human ERMS and ARMS cell lines and xenografts to evaluate the effects of SAHA as a therapeutic agent in both RMS subtypes. SAHA decreased cell viability by inhibiting S-phase progression in all cell lines tested, and induced apoptosis in all but one cell line. Molecularly, SAHA-treated cells showed activation of a DNA damage response, induction of the cell cycle inhibitors p21^Cip1 and p27^Kip1 and downregulation of Cyclin D1. In a subset of RMS cell lines, SAHA promoted features of cellular senescence and myogenic differentiation. Interestingly, SAHA treatment profoundly decreased protein levels of the driver fusion oncoprotein PAX3-FOXO1 in ARMS cells at a post-translational level. In vivo, SAHA-treated xenografts showed increased histone acetylation and induction of a DNA damage response, along with variable upregulation of p21^Cip1 and p27^Kip1. However, while the ARMS Rh41 xenograft tumor growth was significantly inhibited, there was no significant inhibition of the ERMS tumor xenograft RD. Thus, our work shows that, while SAHA is effective against ERMS and ARMS tumor cells in vitro, it has divergent in vivo effects . Together with the observed effects on the PAX3-FOXO1 fusion protein, these data suggest SAHA as a possible therapeutic agent for clinical testing in patients with fusion protein-positive RMS.

Keywords: HDAC inhibitor; PAX3-FOXO; Rhabdomyosarcoma; SAHA; xenograft.

Figure 1.

SAHA decreases RMS cell viability and accumulation. (A) MTT assay assessing viability of ERMS (JR1, RD and Rh36) and ARMS (Rh30 and Rh41) cells in response to 1 μM SAHA, compared to vehicle-treated condition (0.01% DMSO). (B) Total number of RMS cells at the specified timepoints after treatment with 1 μM SAHA compared to vehicle-treated controls (0.01% DMSO). Each point represents the mean of at least 3 experiments. Bars represent standard deviation. Asterisks denote a statistically significant difference (p-value < 0.05).

Figure 2.

SAHA treatment inhibits cell cycle progression and induces apoptosis in RMS cells. (A) Percentage of BrdU-positive cells in the indicated RMS cell lines at 48 hours after treatment with 1 μM SAHA compared to vehicle-treated controls (0.01% DMSO). Each value represents the mean number counted in at least 5 fields, and is the mean of at least 3 independent experiments. (B) Percentage of TUNEL-positive cells in the indicated RMS cell lines at 48 hours after treatment with 1 μM SAHA compared to vehicle (0.01% DMSO). Each value is representative of at least 3 independent experiments, each done in duplicate. Bars represent standard deviation. Asterisks denote a statistically significant difference (p-value < 0.05).

Figure 3.

SAHA induces cell cycle inhibitors and a DNA damage response in RMS cells. Western blot analysis of (A) acetylated histone H4 (AcH4), (B) the indicated cell cycle proteins at 48 hours after treatment with vehicle DMSO control (D) or 1 μM SAHA (S) in the indicated cell lines. (C) Histograms represent the quantification of the western blot bands in the indicated cell lines compared to GAPDH and relative to DMSO from at least 3 independent experiments. Bars represent standard deviation. Asterisks denote a statistically significant difference (p-value < 0.05). (D) Western blot analysis of the DNA damage response protein phospho-H2AX at 48 hours after treatment with vehicle DMSO control (D) or 1 μM SAHA (S) in the indicated cell lines. GAPDH serves as a loading control for all western blots.

Figure 4.

SAHA treatment results in morphologic changes in RMS cells, with a subset showing features of cellular senescence and differentiation. Representative images of RMS cells as observed by light microscopy after (A) 2 and (B) 6 days of treatment with 1 μM SAHA or vehicle (0.01% DMSO), as indicated. Arrowheads indicate flattened cells and arrows indicate elongated and fused morphology. (C) Representative senescence-associated beta-galactosidase (SABG) staining at 6 days of treatment with 1 μM SAHA or control vehicle (0.01% DMSO), as indicated. (D) Percentage of SABG-positive cells in the indicated RMS cell lines 6 days after treatment with 1 μM SAHA compared to vehicle (0.01% DMSO). Each value represents the mean number counted in at least 5 fields, and is the mean of at least 3 independent experiments. Bars represent standard deviation. Asterisks denote a statistically significant difference (p-value < 0.05). (E) Western blot analysis of the senescence markers Lamin B1 and Dec1 in RMS cell lysates after 6 days of treatment with 1 μM SAHA (S) or control vehicle 0.01% DMSO (D), as indicated. GAPDH was used as loading control. Western blots are representative of at least 3 independent experiments. (F) qRT-PCR analysis of MyoD, MyoG and MHC mRNA levels after 48 hours of treatment with 1 μM SAHA or vehicle DMSO control. Gene expression is shown relative to expression of the housekeeping gene GAPDH. Values are representative of at least 3 independent experiments, each done in triplicate. Bars represent standard deviation. Asterisks indicate p-value < 0.05.

Figure 5.

SAHA has prolonged effects after treatment withdrawal and decreases PAX3-FOXO1 protein levels in ARMS cells. (A) Total number of ERMS (JR1, RD and Rh36) and ARMS (Rh30) cells after withdrawal (Off treatment) or not (SAHA) of the 1 μM SAHA treatment at day 6 compared to vehicle-treated controls (0.01% DMSO). Each point represents the mean of at least 3 experiments. Bars represent standard deviation. Asterisks denote a statistically significant difference (p-value < 0.05). Western blot analysis of (B) acetylated histone H4 (AcH4) and (C) the DNA damage response protein phospho-H2AX at day 6 after treatment with 1 μM SAHA (SAHA D6 (+)) and day 12 either after withdrawal of the treatment at day 6 (SAHA D6-12 (-)) or not (SAHA D6-12 (+)) compared to vehicle DMSO control (SAHA D6 (-)) in the indicated cell lines. GAPDH serves as a loading control. (D) Western blot analysis of Pax3-FOXO1 (P3F) at 48 hours after treatment with 1 μM SAHA (S) or vehicle DMSO control (D) in the indicated cell lines; GAPDH serves as a loading control. (E) qRT-PCR analysis of PAX3-FOXO1 mRNA levels in Rh30 and Rh41 cells after 48 hours of treatment with 1 μM SAHA (S) or vehicle DMSO control (D), as indicated. Gene expression is shown relative to expression of the housekeeping gene GAPDH. Values are representative of at least 3 independent experiments, each done in triplicate. Bars represent standard deviation. Asterisks indicate p-value < 0.05.

Figure 6.

SAHA treatment of human RMS xenografts inhibits ARMS tumor growth in vivo and induces cell cycle inhibitors, DNA damage response and differentiation markers in RMS xenografts. Mean tumor volumes (represented as ratio to day 1 of treatment) for (A) Rh41 ARMS and (B) RD ERMS xenografts in immune deficient mice, after treatment with vehicle (CTRL) or SAHA, as indicated. Five to seven mice were used in each cohort at each time point specified. Bars represent standard deviation. Asterisks indicate p-values < 0.05. (C) Representative images of immunohistochemical staining for acetylated Histone 3, in vehicle (DMSO) and SAHA treated RMS xenografts, as indicated. (D) Western blot analysis of xenografts of the indicated RMS tumors, that had been treated with either control vehicle (C) or SAHA (S), for the acetylated histone H4 (AcH4), the cell cycle regulators p27 and p21, phospho-H2AX, and the late myogenic marker Myosin Heavy Chain (MHC); GAPDH was used as loading control. IHC staining and western blots were done at day 21 after tumor harvest.