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. 2013 Nov;104(11):1468-75.
doi: 10.1111/cas.12271. Epub 2013 Oct 1.

Alteration of cancer stem cell-like phenotype by histone deacetylase inhibitors in squamous cell carcinoma of the head and neck

Kazuaki Chikamatsu  1 , Hiroki Ishii, Takaaki Murata, Koichi Sakakura, Masato Shino, Minoru Toyoda, Katsumasa Takahashi, Keisuke Masuyama
Affiliations

Alteration of cancer stem cell-like phenotype by histone deacetylase inhibitors in squamous cell carcinoma of the head and neck

Kazuaki Chikamatsu et al. Cancer Sci. 2013 Nov.

Abstract

Recent progression in the understanding of stem cell biology has greatly facilitated the identification and characterization of cancer stem cells (CSCs). Moreover, evidence has accumulated indicating that conventional cancer treatments are potentially ineffective against CSCs. Histone deacetylase inhibitors (HDACi) have multiple biologic effects consequent to alterations in the patterns of acetylation of histones and are a promising new group of anticancer agents. In this study, we investigated the effects of two HDACi, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), on two CD44+ cancer stem-like cell lines from squamous cell carcinoma of the head and neck (SCCHN) cultured in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. Histone deacetylase inhibitors inhibited the growth of SCCHN cell lines in a dose-dependent manner as measured by MTS assays. Moreover, HDACi induced cell cycle arrest and apoptosis in these SCCHN cell lines. Interestingly, the expression of cancer stem cell markers, CD44 and ABCG2, on SCCHN cell lines was decreased by HDACi treatment. In addition, HDACi decreased mRNA expression levels of stemness-related genes and suppressed the epithelial-mesencymal transition phenotype of CSCs. As expected, the combination of HDACi and chemotherapeutic agents, including cisplatin and docetaxel, had a synergistic effect on SCCHN cell lines. Taken together, our data indicate that HDACi not only inhibit the growth of SCCHN cell lines by inducing apoptosis and cell cycle arrest, but also alter the cancer stem cell phenotype in SCCHN, raising the possibility that HDACi may have therapeutic potential for cancer stem cells of SCCHN.

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Figures

Figure 1
Figure 1
Effects of suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) on growth of squamous cell carcinoma of the head and neck (SCCHN) cell lines. HSC‐2 and KUMA‐1 were cultured in serum‐free medium supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Trypsinized cells were plated in 96‐well flat‐bottomed plates, incubated overnight, and then exposed to various concentrations of histone deacetylase inhibitors (HDACi) for 48 h. Cell viability was assessed using an MTS assay as described in Materials and Methods. Representative data are shown from at least three experiments performed in triplicate.
Figure 2
Figure 2
Flow cytometry analysis of apoptosis in squamous cell carcinoma of the head and neck (SCCHN) cell lines, HSC‐2 and KUMA‐1. Tumor cells were incubated for 72 h and then treated with suberoylanilide hydroxamic acid (SAHA) (4 μM) or trichostatin A (TSA) (1 μM). After a further 48 h, the cells were harvested and analyzed for apoptosis as described in Materials and Methods.
Figure 3
Figure 3
Effect of suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) on sphere formation of squamous cell carcinoma of the head and neck (SCCHN) cell lines, HSC‐2 and KUMA‐1. Cells were seeded at a density of 1 × 103 cells/mL in 24‐well ultra‐low attachment plates. After 7 days' culture, spheres were counted. Asterisks indicate a significant difference in the number of spheres (*P < 0.05 and **P < 0.01).
Figure 4
Figure 4
Cell cycle distribution in squamous cell carcinoma of the head and neck (SCCHN) cell lines, HSC‐2 and KUMA‐1. Cells were incubated with histone deacetylase inhibitors (HDACi) for 48 h and analyzed by flow cytometry as described in Materials and Methods. (a) Representative diagrams of cell cycle distribution in cells treated with HDACi. (b) Analyses were performed in triplicate and are expressed as bar graphs.
Figure 5
Figure 5
Suppression of CD44 and ABCG2 expression on squamous cell carcinoma of the head and neck (SCCHN) cell lines, HSC‐2 and KUMA‐1, treated with histone deacetylase inhibitors (HDACi). Flow cytometry, immunoblotting, and real‐time quantitative RT‐PCR were performed as described in Materials and Methods. Treatment with each HDACi for 48 h decreased the expression level of CD44 and ABCG2 in both flow cytometric analysis (a) and immunoblotting assay (b). CD44 and ABCG2 mRNA expression levels were also lower in SCCHN cell lines treated with HDACi (c). HSC‐2 did not obviously detect ABCG2 expression. MFIR, mean fluorescence intensity ratio.
Figure 6
Figure 6
Downregulation of stemness genes in squamous cell carcinoma of the head and neck (SCCHN) cell lines, HSC‐2 and KUMA‐1. Total RNA was extracted from SCCHN cells treated with HADCi, and the expression level of BMI1, Notch, Nanog, and Oct‐4 was detected by real‐time quantitative RT‐PCR as described in Materials and Methods.
Figure 7
Figure 7
Histone deacetylase inhibitors (HDACi) suppress epithelial‐mesenchymal transition (EMT)‐related molecules in squamous cell carcinoma of the head and neck (SCCHN) cell lines. (a) Cell lysates from HSC‐2 and KUMA‐1, untreated or treated with HDACi were immunoblotted with anti‐E‐cadherin. (b) Real‐time quantitative RT‐PCR was performed to detect transforming growth factor‐β (TGF‐β) mRNA levels on SCCHN cell lines, HSC‐2 and KUMA‐1 treated with HDACi. GAPDH (glyceraldehyde 3‐phosphate dehydrogenase) gene expression was evaluated as a control for mRNA normalization.
Figure 8
Figure 8
Effects of combined histone deacetylase inhibitors (HDACi) (suberoylanilide hydroxamic acid [SAHA] and trichostatin A [TSA]) and chemotherapeutic agents (docetaxel and cisplatin) on squamous cell carcinoma of the head and neck (SCCHN) cell lines, HSC‐2 and KUMA‐1, assessed by MTS assay. The effect of combined treatment was analyzed at the 50% growth inhibition rate. Isoborogram of the interactions between SAHA and docetaxel (a), SAHA and cisplatin (b), TSA and docetaxel (c), and TSA and cisplatin (d), respectively.
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