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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.
Evidence has accumulated indicating that only a minority of cancer cells with stem cell properties, cancer stem cells (CSCs), are responsible for the maintenance and growth of tumors.[1-3] These CSC subpopulations show a capacity for high tumorigenicity, self-renewal, and differentiation. To date, human CSCs have been identified, purified, and characterized in a variety of tumors.[4-8] In squamous cell carcinoma of the head and neck (SCCHN), since Prince et al. first demonstrated that the purified CD44+ population possessed the properties of CSCs, various cell surface markers, including CD133 and ALDH1, have been identified for the isolation of CSCs.[10-12] Moreover, numerous studies have demonstrated that conventional cancer treatments are potentially ineffective against CSCs, which are subsequently responsible for tumor relapse and distant metastases.[13-15] Therefore, the development of novel therapeutic strategies to overcome treatment resistance of CSCs is urgently needed.
Recent studies have demonstrated that not only genetic but also epigenetic changes, including DNA methylation and histone modifications, play an essential role in cancer development.[16, 17] Of histone modifications, acetylation and deacetylation affect the chromatin structure and gene expression, and they are regulated by the antagonistic activities of two enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). The aberrant expression of HDACs has been found in a variety of malignancies, which can alter gene transcription and enhance cell proliferation, indicating that HDACs are a promising target for cancer therapy. To date, several HDAC inhibitors (HDACi) have been developed and used in clinical trials for the treatment of cancers, and have been shown to induce differentiation, growth arrest, or apoptosis in tumor cells.[18, 19] In addition to these known anti-tumor effects, since HDACi are involved in the acetylation of non-histone proteins, the functions of various transcription factors, including p53, STAT3, and NFκB, are also modulated.[20-22] Thus, HDACi have multiple biologic effects, and therefore, their diverse effects have not been fully elucidated.
To explore the possibility of targeting epigenetics changes for treatment against CSCs, we investigated the effects of two HDACi, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), on SCCHN cell lines. In previous studies, we and others have demonstrated that a small population possessing CSC properties was increased by culturing in serum-free medium (SFM) with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).[23-25] Using these culture conditions, we first examined the sensitivity of CD44+ cancer stem-like cells to HDACi. We then focused on whether the CSC properties are altered by treatment with HDACi. Interestingly, the expression of cancer stem cell markers, CD44 and ABCG2, on SCCHN cell lines was decreased by HDACi treatment. Furthermore, HDACi have demonstrated not only downregulation of the mRNA expression levels of stemness-related genes and sphere formation, but a synergistic effect with chemotherapeutic agents. Thus, our data revealed that HDACi may have therapeutic potential for CSCs in SCCHN.
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In previous studies, we reported that a subpopulation of CD44+ cells enriched under serum-free medium culture conditions possessed not only a marked capacity for forming tumor spheres, proliferation, migration, and invasion, but also resistance to apoptosis-inducing stimuli, including chemotherapeutic agents, as compared with that of CD44- cells.[23, 26] Based on these findings, we here investigated the possibility of HDACi for the treatment of CSCs in SCCHN, and we made the following important observations of CD44 + enriched SCCHN cell lines: (i) Two HDACi, SAHA and TSA, showed cytotoxic activity, induction of apoptosis, and cell cycle arrest as reported in other malignancies; (ii) HDACi were able to alter CSC phenotypes: decrease CD44 and ABCG2 expression, suppress sphere formation, decrease stemness gene expression, and inhibit EMT; and (iii) HDACi had synergistic effects in combination with chemotherapeutic agents.
In general, HDACi induce hyperacetylation of core histone proteins, resulting in relaxation of the chromatin structure, promoting access to transcription factors, and changing gene expression. In addition to such epigenetic effects, HDACi also induces the reversible acetylation of non-histone proteins, including transcription factors, DNA repair enzyme, and signal transduction mediators. So far, it has been demonstrated that as many as 7–10% of expressed genes are altered by treatment with HDACi using cDNA arrays.[31-33] Although the multiple mechanisms mediated by HDACi have not been completely elucidated, it has been shown that the main anti-cancer effects of HDACi are the induction of apoptosis and differentiation, and cell cycle arrest in G1 or G2/M. In our experiments, two HDACi, SAHA and TSA, showed cytotoxic activity, induction of apoptosis, and cell cycle arrest to SCCHN cell lines, confirming that these HDACi also act on SCCHN cells, as demonstrated in other tumor cells. With regard to the cell cycle arrest induced by HDACi in tumor cells, Richon et al. have demonstrated that low concentrations of HDACi predominantly induce G1 arrest, while high concentrations induce both G1 and G2/M arrest. Under our experimental conditions, HSC-2 treated with HDACi showed predominantly G2/M arrest rather than G1 arrest. On the other hand, KUMA-1 treated with TSA showed similar findings; however, treatment with SAHA induced cell cycle arrest in both G1 and G2/M. Besides the concentrations, various factors, such as exposure time, types of HDACi, and types of cell lines, affect cell cycle effects.
In addition to these anti-cancer effects, HDACi have been demonstrated to have multiple biological activities, including the induction of differentiation, inhibition of angiogenesis, and immunomodulatory activity. Interestingly, HDACi suppressed various CSC properties of CD44+-enriched SCCHN cell lines. Self-renewal is one of the well-known properties of CSCs and is regulated by interactions among a variety of stem cell regulators. Histone deacetylase inhibitors suppressed the expression of Bmi1, Notch, Nanog, and Oct-4, which are essential for stem cell self-renewal. Downregulation of each gene at the concentration used in this study was relatively modest; however, the suppression of multiple genes may lead to a strong inhibiting effect on self-renewal capacity. Indeed, tumor sphere formations reflecting self-renewal capacity were effectively inhibited by treatment with HDACi.
In this study, inhibition of CD44 and ABCG2 expression on tumor cells by treatment with HDACi are of particular importance. It has been shown that these molecules are not only cell surface markers of CSCs, but are also responsible for maintenance of the CSC phenotype. CD44 is a multifunctional transmembrane glycoprotein receptor that binds to hyaluronan. It has been shown that CD44-hyaluronan interaction induces signaling events, which promote tumor cell proliferation, migration, invasion, angiogenesis, and metastases.[35, 36] In fact, Bourguignon et al. have demonstrated that hyaluroan-CD44 interaction activates cancer stem markers, Nanog, Oct-4, and Sox2, and leads to multidrug transporter, ABCB1 (MDR1) gene expression. ABCB1 is related to the efflux of chemotherapeutic agents, including doxorubicin and paclitaxel, and its overexpression induces the acquisition of chemotherapy resistance. Meanwhile, ABCG2 is another well-known multidrug resistant protein, which effluxes not only chemotherapeutic agents, but also Hoechst 33342. In a variety of cases, the cells expressing ABCG2 appear as side population (SP) cells in a Hoechst-based flow cytometry profile, and SP cells have also been identified as stem/progenitor cells.[38, 39] The SP cells obtained from freshly isolated tumor cells and tumor cell lines enrich the cell with cancer stem cell phenotypes as compared with the corresponding non-SP cells. Thus, the inhibition of CD44 and/or ABCG2 expression on tumor cells is closely related to loss of stem cell properties and chemotherapy resistance. Contrary to our findings, To et al. have demonstrated that ABCG2 expression in some tumor cell lines was upregulated after treatment with another HDACi, depsipeptide, where ABCG2 promoter has a reproducible pattern of histone modification due to HDACi. Further studies are required to elucidate these differences.
Evidence has accumulated indicating that induction of EMT, which promotes invasion and metastases, plays an important role in the acquisition of CSC properties in a variety of cancers.[41-44] In SCCHN, Chen et al. have shown that ALDH+ putative CSCs enriched from SCCHN cell lines using an anchorage-independent culture technique increased EMT-related marker expression. In our studies, upon treatment with HDACi, E-cadherin, which acts as a master regulator of EMT, was upregulated, and the mRNA expression level of TGF-β, which is known to be an important inducer of EMT was downregulated, indicating that HDACi acts to inhibit EMT characteristics in CD44 + SCCHN cell lines. So far, SAHA has been shown to have reverted the mesenchymal phenotype by inducing E-cadherin and downregulating vimentin in SCCHN. Similarly, in renal epithelial cells, TSA has been able to prevent TGF-β1-induced ENT. Conversely, several studies have shown that in prostate cancer cells and endometrial adenocarcinoma cells, HDACi could induce EMT.[48, 49] The EMT phenomenon is regulated by a complex network consisting of a variety of EMT-related molecules, including epithelial and mesenchymal-related factors, and therefore such controversies may occur depending on the type of markers tested. Additionally, culture and treatment conditions and the type of cell line in each experiment vary, and therefore the clinical utility of HDACi should proceed carefully. Indeed, in a phase I trial of SAHA, a partial response was observed in a patient with laryngeal carcinoma; however, in a phase II trial, SAHA could not show any efficacy as defined by tumor responses.
Taken together, our studies indicated at least cell cycle arrest, downregulation of stemness-related genes, decreased CD44 and ABCG2 expression, and inhibition of EMT, and thereby HDACi would lead to decreased drug resistance. Based on such pleiotropic effects, two HDACi combined with cisplatin or docetaxel would show synergistic effects. Our data suggest the further possible use of HDACi in combination with chemotherapy, radiotherapy, and/or immunotherapy. Currently, to overcome the treatment resistance of CSCs, various approaches, such as immunotherapy, molecularly targeted drugs, and gene therapy are being developed. Better understanding of epigenetic mechanisms as well as interplay among epigenetic factors may provide new insights for developing new pharmacological strategies.