Expert Opinion on Investigational Drugs

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Histone deacetylase inhibitors in oral squamous cell carcinoma treatment Jason Tasoulas, Constantinos Giaginis , Efstratios Patsouris, Evangelos Manolis & Stamatios Theocharis To cite this article: Jason Tasoulas, Constantinos Giaginis , Efstratios Patsouris, Evangelos Manolis & Stamatios Theocharis (2015) Histone deacetylase inhibitors in oral squamous cell carcinoma treatment, Expert Opinion on Investigational Drugs, 24:1, 69-78, DOI: 10.1517/13543784.2014.952368 To link to this article: https://doi.org/10.1517/13543784.2014.952368

Published online: 12 Sep 2014.

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Review

Histone deacetylase inhibitors in oral squamous cell carcinoma treatment 1.

Introduction

2.

Histone deacetylases

3.

HDACs and cancer

4.

HDCAs in OSCC development

Jason Tasoulas, Constantinos Giaginis†, Efstratios Patsouris, Evangelos Manolis & Stamatios Theocharis †

and progression 5.

HDACis in OSCC treatment

6.

Conclusion

7.

Expert opinion

University of the Aegean, School of Environment, Department of Food Science and Nutrition, Myrina, Greece

Introduction: The involvement of the histone deacetylases (HDACs) family in tumor development and progression is well demonstrated. HDAC inhibitors (HDACis) constitute a novel, heterogeneous family of highly selective anticancer agents that inhibit HDACs and present significant antitumor activity in several human malignancies, including oral squamous cell carcinoma (OSCC). Areas covered: This review summarizes the current research on the anticancer activity of HDACis against OSCC. The review also presents the molecular mechanisms of HDACis action and the existing studies evaluating their utilization in combined therapies of OSCC. Expert opinion: The currently available data support evidence that HDACis may provide new therapeutic options against OSCC, decreasing treatment side effects and allowing a more conservative therapeutic approach. Future research should be focused on in vivo and clinical evaluation of their utilization as combined therapies or monotherapies. Before HDACis can be brought into clinical practice as treatment options for OSCC, further evaluation is needed to determine their optimal dosage, the appropriate duration of treatment and whether they should be used in combination or as stand-alone therapeutics. Keywords: anticancer therapy, histone deacetylases, histone deacetylase inhibitors, oral squamous cell carcinoma Expert Opin. Investig. Drugs (2015) 24(1):69-78

1.

Introduction

Oral cancer (OC) is the sixth most common cancer worldwide, and its incidence is estimated around 275,000 new cases every year [1]. Over the recent decades, the OC prevalence increased in Europe (especially in France and Hungary) as also in Japan [1,2]. Additionally, epidemiological data indicate a decrease of patients’ average age, attributed to increased tobacco smoking and alcohol drinking prevalence [2-4]. Despite the significant reduction of mortality rates in a large amount of different cancer types, the 5-year survival rate of OC patients does not exceed 50% and has remained relatively unchanged for the past three decades [2-4]. The ineffectiveness of the current treatment options and the high mortality rates are mainly attributed to the fact that most of OC patients are diagnosed at advanced disease stage [2-4]. Moreover, the aggressive treatment required to improve cure rates for advanced lesions is associated with increased morbidity [2-4]. In this aspect, the development of novel anticancer substances, such as histone deacetylase inhibitors (HDACis) and their potential introduction in the clinical practice for OC treatment, is strongly recommended in order for cure rates to be improved. Oral squamous cell carcinoma (OSCC) represents the most frequent malignancy of the oral cavity, and according to the WHO, 90% of oral malignant neoplasms are 10.1517/13543784.2014.952368 © 2015 Informa UK, Ltd. ISSN 1354-3784, e-ISSN 1744-7658 All rights reserved: reproduction in whole or in part not permitted

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Article highlights. .

. .

.

Histone deacetylases (HDACs) are crucial regulators of cell proliferation, differentiation and apoptosis in various hematological and solid malignancies including oral squamous cell carcinoma (OSCC). HDAC inhibitors (HDACis) can exert anticancer functions by both HDAC related and nonrelated mechanisms. In vitro studies have suggested that HDACis can decrease the dosage of the less selective, toxic agents commonly used in OSCC therapy (i.e., cisplatin, gefitinib) and achieve comparable or even higher antiproliferative results when administered as combined therapy. Evaluating the interaction of HDACs and novel HDACis in animal models and clinical trials is a strong prerequisite to presume the optimal dosage, the duration and the exact utilization of HDACis in combined therapies or monotherapies of OSCC.

This box summarizes key points contained in the article.

squamous cell carcinomas (SCC) [5]. OSCC is an invasive epithelial neoplasm arising from the stratified epithelium of oral mucosa with an increased metastatic potential, especially on the regional lymph nodes [5]. Buccal mucosa, tongue’s anterior two-thirds, hard and soft palate, gingiva and mouth’s floor are the most common sites of OSCC arising [5]. Tobacco and alcohol usage is associated with de novo p53gene mutations, and they are considered as the main cause of oral malignancies [6]. The presence of high risk-HPV type 16 in oral and oropharyngeal SCCs combined with the fact that E6 and E7 HPV-oncogenic proteins deregulate the cell cycle may reflect an HPV involvement in OSCC [7,8]. Furthermore, published data imply an association between poor oral health (i.e., periodontal disease) and OSCC, which is strengthened by the fact that oral and oropharyngeal SCC‘s prevalence is significantly higher in low socio-economic class population [9]. Current therapeutic approaches of OSCC are based on the surgical management combined with radiotherapy and/or chemotherapy (cisplatin and 5-fluorouracil) [10,11]. However, the nonselective cytotoxicity of these therapies limits their effectiveness, increases side effects and indicates the emergency for novel treatment options [10,11]. In this respect, HDACis are agents that exert anticancer activity in vitro and in vivo, leading to clinical trials evaluating their efficacy in multiple cancer types [12-15]. Notably, two HDACis are currently approved by the U.S. Food and Drug Administration, vorinostat and romidepsin, both with indications for cutaneous T-cell lymphoma (CTCL) [16]. In the last few years, several in vitro and in vivo studies have demonstrated that HDACis can also exert anticancer action against OSCC, supporting evidence for their potential utility in combined therapeutic strategies with classical chemotherapeutic or DNA-demethylating agents. In this aspect, the present review aimed to provide an overview of the HDACis family of 70

substances and their role in OSCC treatment. The molecular mechanisms of HDACis anticancer action and the existing studies evaluating HDACis utilization in combined therapies of OSCC are also discussed. 2.

Histone deacetylases

DNA is wrapped around a histone octamer (H2A, H2B, H3, H4) creating nucleosomes; the combination of which with nonhistone proteins create chromatin, the structural complex of eukaryotic DNA [17-19]. The rate of DNA compaction permits or blocks gene expression and regulates the cellular phenotype [17-19]. Epigenetic modification on histone N-tails and the nonhistone proteins can alter the DNA-compaction level and affect the gene expression. These post-translational modification systems are also called ‘the histone code’ (i.e., methylation/demethylation) [17-19]. The histone code enzymes that acetylate lysine residues’ e-amino group on histones are named histone acetyltransferases (HATs) and the enzymes that reverse this action are named histone deacetylases (HDACs) [20]. Four classes of eukaryotic HDACs with different cellular localization, cofactors and functions exist (Table 1) [21-24]. The balance between HATs and HDACs is crucial for cell’s normal migration, differentiation, proliferation and apoptosis, whereas any disturbance of this balance may lead to genes overexpression or underexpression related to cancer development [18,25]. Moreover, HATs and HDACs are also capable of affecting the cell procedures indirectly, interacting with nonhistone proteins (i.e., p53) [20]. Based on microarray analyses revealing that HDAC activity affects only 2 -- 5% of the expressed genes, it has been suggested that this indirect mechanism of HDACs action is more important for tumorigenesis than histone deacetylation [26]. 3.

HDACs and cancer

HDACs have been considered as crucial regulators of cell proliferation, differentiation and apoptosis in various hematological and solid malignancies. In fact, aberrant deacetylation of histones by enhanced HDAC activity in human tumors has been shown to lead to conformational changes within nucleosome, which results in transcriptional repression of genes involved in differentiation and negative regulation of cell proliferation, migration and metastasis [27,28]. In the last few years, HDACs and especially class I members have been shown to be overexpressed in many human malignancies, including lung, gastric, colorectal, breast, ovarian, endometrial, pancreatic, prostate, brain and renal cell carcinomas as also in hematological malignancies, being associated with crucial clinicopathological parameters for patients’ management and prognosis [29]. Notably, most of the above studies suggested that HDACs overexpression was directly associated with dedifferentiation, enhanced proliferation and invasion, advanced disease stage and poor prognosis [29].

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Histone deacetylase inhibitors in oral squamous cell carcinoma treatment

Table 1. Members of the HDAC family. Class

Name (OMIM number)

Cytogenetic location [21]

Cellular localization

Cofactor

1p34.1 6q21 5q31.3 Zp13.1 2q37.3 17q21.31 12q13.11 7p21.1

Nuclear

Zn2+

Nuclear

Zn2+

Function

[21]

I

IIa

IIb

III

IV

HDAC1 HDAC2 HDAC3 HDAC8 HDAC4 HDAC5 HDAC7 HDAC9

(601241) (605164) (605166) (300269) (605314) (605315) (606542) (606543)

HDAC6 (300272)

Xp11.23

HDAC10 (608544) SIRT1 (604479) SIRT2 (604480) SIRT3 (604481) SIRT4 (604482) SIRT5 (604483) SIRT6 (606211) SIRT7 (606212) HDAC11

22q13.31-33 10q21.3 19q 11p15.5 12q 6p23 19p13.3 17q25.3 3p25.2

Nuclear; cytoplasmic

Zn2+

Nuclear; cytoplasmic; mitochondrial

NAD

Cytoplasmic

Zn2+

Regulation of the cell survival [21,23]; chemosensitivity of cancer cells absence results to defective DNA-repairing mechanisms and increases lethality [21-23] Muscle cell differentiation [22]

Electrical activity-dependent regulation of gene expression in skeletal muscle cells [24] Deacetylation of HSP-90, HSP-90-based chaperone protein complexes; cell adhesion; mobility; autophagy [22] Deacetylation of HSP-90 and VEGFR [22] Unclear on mammalian cells [22]

Immune system regulation via association with IL10 expression [22]

HDAC: Histone deacetylase.

HDAC1 and 3 overexpression was implicated in cervical cancer development [20], whereas a study of 140 colorectal cancer samples reported overexpressed class I HDACs (HDACs 1, 2, 3) [30]. HDAC2 and HDAC3 expression was significantly higher in less differentiated tumors, being correlated with negative hormone receptor status in breast carcinoma. Additionally, high HDAC2 expression was significantly associated with HER2 overexpression and the presence of nodal metastasis, whereas HDAC1 was highly expressed in hormone receptor positive tumors [31]. HDAC1, 2 and 3 expression was associated with tumor grade, and HDAC2 expression had an impact on patient survival in hepatocellular carcinoma [32]. Enhanced HDAC1 expression was identified as an independent predictor of poor prognosis in lung adenocarcinoma patients [33]. High HDAC2 levels were associated with lymphatic tumor spread and lower tumor differentiation grade in esophageal adenocarcinoma. Moreover, for neoadjuvanttreated tumors, there was a trend of correlation with high pretherapeutic HDAC2 expression and tumor regression after chemotherapy [34]. HDAC1, 2 and 3 were expressed at high levels in ovarian and endometrial carcinoma. In addition, high-level expression of all three HDACs was associated with poor prognosis in ovarian endometrioid carcinomas [35]. HDAC1, 2 and 3 were highly expressed in prostate carcinoma, and HDAC2 expression was associated with shorter PSA relapse time after radical prostatectomy [36]. Enhanced HDAC1, 2, 4 and 6 expression was more frequently observed in malignant compared with benign thyroid lesions, being

associated with crucial clinicopathological parameters for patients’ management and prognosis [37]. Increased levels of HDAC8 were present in neuroblastoma, being associated with more advanced tumor stage and poorer patients’ prognosis [38]. High levels of HDACs class IIa were also observed in breast and colorectal cancer [39,40]. Enhanced HDAC7 and HDAC9 expression was associated with poor prognosis in childhood acute lymphoblastic leukemia [41]. HDAC5 and HDAC9 were elevated in medulloblastoma and overexpression of HDAC10 was observed in hepatocellular carcinoma [42,43]. Paradoxically, whereas the most comprehensive data so far documented that HDACs expression was associated with poorer prognosis, elevated HDAC6 levels were correlated with better prognosis in breast cancer [44]. These conflicting data about HDAC6 relation with different cancer types reflect the complicated role of HDACs on cellular functions.

HDCAs in OSCC development and progression

4.

In vitro, several HDACs were related with OSCC, but their exact role remains to be elucidated. JHU-029, JHU-011 head and neck SCC cell lines, HO1N1, KYSE-30 and KYSE-150 esophageal SCC cell lines and FaDU hypopharyngeal SCC cell lines were evaluated with immunohistochemistry and mass spectrometry and an association of HDAC1 and HDAC2 with the DNp63a (p63 isoform), an important

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Table 2. HDACis classified by chemical structure. Chemical structure Hydroxamates

Cyclic peptides

Benzamides

Aliphatic fatty acids

Name

HDAC class target

Suberoylanilide hydroxamic acid (Vorinostat) Trichostatin A LBH589 (Panobinostat) Depsipeptide-FK228 (Romidepsin) Apicidin MS-275 (Entinostat) MGCD0103 (Mocetinostat) Valproic acid Sodium butyrate (S)-HDAC42

Pan inhibitor

Class I and II Class I and II Class I Class I and II Class I Class I Class I and II Class I and II Class I and II

HDAC: Histone deacetylase; HDACis: Histone deacetylase inhibitors.

mediator of apoptotic pathway, was established [45]. More specifically, HDAC1 and HDAC2-p63 complexes bound to transcription promoter PUMA suppressed the transcription of bcl-2 apoptotic genes and allowed SCC-cell survival [45]. In vitro studies in Ca9-22, Cal-27, HSC-3, SAS, TW2.6 HNSCC cell lines via western blotting, wound-healing assay, as well as in vivo studies in mice, supported evidence that HDAC2 may modify and stabilize HIF-1a, which can increase the migration and invasion of OSCC cells, thereby increasing the tumor’s aggressiveness [46]. Another study examining the HDAC6 mRNA and proteins levels, by RT-PCR and western blot analysis, respectively, in OSCC-derived cell lines and normal cells, supported evidence for an association between HDAC6 levels, tumor stage and OSCC aggressiveness [47]. There are also several data supporting a potential implication of HDACs in oral tumor development and progression. High HDAC2 expression was shown to correlate with advanced stage, larger tumor size, lymph node metastasis and poor prognosis in 93 OSCC patients [48]. HDAC2 nuclear staining increased significantly from epithelial dysplasia to OSCCs. According to this study, it was shown for the first time that HDAC protein overexpression may be a frequent event in OSCC and could be used as a prognostic factor [48]. Additionally, a recent study by our group conducted on 49 mobile tongue SCC specimens demonstrated that HDACs may participate in the formation and progression of mobile tongue SCC, reinforcing their possible use as biomarkers as also the therapeutic utility of HDACis in mobile tongue SCC chemoprevention and treatment [49]. In fact, enhanced HDAC1 expression was significantly associated with poor histological grade of differentiation and presence of lymph node metastases. Intense HDAC1 staining intensity was significantly associated with stromal infiltration reaction and shape of tumor invasion. Moreover, intense HDAC2 staining intensity was significantly associated with the presence of 72

muscular invasion and depth of invasion. Notably, mobile tongue SCC patients with increased HDAC1 expression presented shorter overall and disease-free survival compared with those with decreased HDAC1 expression [49]. HDAC6 upregulation was evident in both mRNA and protein levels of primary OSCCs [47]. Among the clinical variables analyzed, the clinical tumor stage was found to be associated with the HDAC6 expression levels. In fact, a significant difference in the HDAC6 expression levels between the early- (stage I and II) and advanced-stage (stage III and IV) tumors was recorded [47]. In a recent study, the expression levels of SIRT1, a class III HDAC member, were assessed by immunohistochemistry on specimens from 437 consecutive HNSCC patients, whereas acetylated histone status and p53 expression were also examined [50]. SIRT1 expression predominated in older patients with absence of lymph node metastasis and early clinical stage [50]. It was also positively correlated with the expression of acetylated histone H3K9 and H4K16, but not with that of p53. Multivariate analysis identified SIRT1 expression as an independent and good indicator of patients’ prognosis [50]. 5.

HDACis in OSCC treatment

HDACis represent a heterogeneous family of substances, consisting of hydroxamates, cyclic peptides, benzamides and aliphatic fatty acids (Table 2). The HDACis family members are either natural products or semifactitial derivatives of natural products [51]. These agents can block the HDAC activity and affect the DNA package level. They create more relaxed chromatin structures and induce gene expression either by targeting straight to the histone related activity of HDACs or by inhibiting the deacetylation of transcription factors (TFs) and consequently increasing TF’s activity [52]. The currently available studies evaluating the antitumor effects of HDACis in OSCC are summarized in Table 3. Paradoxically, the antitumor activity of HDACis has been linked not only with gene-expression induction but also with gene-expression suppression. Cyclin D1 gene, which induces cell proliferation and participates in the NF-kB tumorigenic pathway, was downregulated in HDACi-treated JB6 mouse cells [53]. Specifically, the HDACi thrichostatin A (TSA) inhibited the NF-kB pathway, preventing p65 dimer binding to NF-kB sites and p65 binding to the cyclin D1 promoter and obstacled cyclin D1 gene transcription [53]. In addition, the HDACis TSA and sodium butyrate (NaB) decreased ErbB2 levels, which are elevated in 30% of breast cancers and relate with tumors resistant to chemotherapeutic agents [54,55]. Both agents inhibited ErbB2 expression from the amplified gene, whereas TSA was also reported to disrupt mature ErbB2 transcripts and induce their degradation [54,55]. Hydroxamates Suberoylanilide hydroxamic axid (SAHA) (Vorinostat) is a nonselective HDACi, and since October 2006 it is the first FDA-approved HDACi for mono- or combined-therapy 5.1

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Histone deacetylase inhibitors in oral squamous cell carcinoma treatment

Table 3. The currently available studies evaluating the antitumor effects of HDACis in OSCC. In vitro/clinical study in OSCC

Name (family)

Outcome

Mechanism

SAHA (hydroxamates)

Inhibition of cell proliferation, migration, invasion and induced apoptosis in HNSCC Chemosensitization on cisplatin; apoptosis induction Apoptosis; synergic action with cisplatin Chemosensitization on cisplatin

Modulation of ErbB receptor expression; reversion of EMT

+/-

Bruzzese et al. (2011) [56]

RE stress mechanisms

+/-

Suzuki et al. (2009) [57]

CytC, p53 and caspase-3 related apoptosis Decreasing lysosomal pH; increased cathepsin activity and reduced LAMP-2 level Unknown

+/-

Shen et al. (2007) [58]

+/-

Eriksson et al. (2013) [60]

+/-

De Schutter et al. (2009) [59]

Unknown

+/-

De Schutter et al. (2009) [59]

TSA (hydroxamates)

LBH589 (hydroxamate)

MGCD0103 (benzamide) MS-275 (benzamide) DepsipeptideFK228 (cyclic peptides) Apicidin (cyclic peptide) Valproic acid (aliphatic fatty acid)

NaB (aliphatic fatty acid) (S)-HDAC42 (aliphatic fatty acid)

Radiosensitization; increased radiation-induced cell cycle arrest Radiosensitization; increased radiation-induced cell cycle arrest Cell death Radiosensitization; increased radiation-induced cell cycle arrest Chemosensitization on cisplatin; apoptosis induction Regulation of transcription; cell cycle control Apoptosis; autophagy

Inhibition of cell proliferation; synergistic with 5-Aza-dC-VPA-ATRA growth inhibition Radiosensitization

Increased Ad-HSVtk infectivity Inhibition of cell proliferation Tumor growth suppress

Author

Prystowsky et al. (2009) [61]

G2/M arrest; inhibition of mitotic genes expression Unknown

+/-

De Schutter et al. (2009) [59]

G1/S arrest

+/-

Sato et al. (2006) [69]

Induction of p21Waf1/Cip1; decreased Ki67 staining

-/+

Sato et al. (2006) [69]

" p21WAF1/Cip1 levels causing G2/M arrest " LC3-II, ATG5, AVOs " p21; terminal differentiation markers expression; cellular senescence induction Induction of H3 and H4 hyperacetylation; increased levels of DSB; decreased Rad51 expression Increased CAR expression in host cells " p27Kip1 G1 phase arrest Caspase-dependent apoptosis; downregulation of phosphor-Akt, cyclin D1, CDK6

+/-

Haigentz et al. (2012) [64], Han et al. (2000) [67]

+/-

Gan et al. (2012) [71]

+/-

Shoji et al. (2012) [70]

+/-

Kothari et al. (2010) [72]

+/-

Gong et al. (2010) [73]

+/-

Bai et al. (2011) [74]

CAR: Coxsackievirus and adenovirus receptor; DSB: Double-strand break; EMT: Epithelial--mesenchymal transition; HDACis: Histone deacetylase inhibitors; NaB: Sodium butyrate; OSCC: Oral squamous cell carcinoma; SAHA: Suberoylanilide hydroxamic axid; TSA: Thrichostatin A; VPA: Valproic acid.

against CTLC [16]. SAHA in combination with the EGFR tyrosine kinase inhibitor gefitinib induced synergistic inhibition of proliferation, migration and invasion, as well as induction of apoptosis in HNSCC cells [56]. SAHA enhanced the antitumor effect of gefitinib by modulating ErbB receptor expression and reverting the epithelial--mesenchymal transition (EMT) from the mesenchyme to the epithelial phenotype via E-cadherin and ErbB3 upregulation and vimentin, EGFR

and ErbB2 downregulation [56]. Moreover, a strong, antiproliferative and synergistic action of SAHA and gefitinib was noted [56]. It was also shown that SAHA can increase apoptosis rate, inducing endoplasmic reticulum stress mechanisms in HSC-3 OSCC cells [57]. Using MTT and TUNEL assays, a synergistic cisplatin-SAHA effect was demonstrated, with SAHA to increase cells’ chemosensitivity to cisplatin [57]. The synergistic effect with cisplatin was also established in Tca8113 and KB

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cell lines treated with cisplatin/SAHA. MTS, colony formation, TUNEL and flow cytometry assay revealed an increased rate of CytC, p53 and caspase-3 related apoptosis when a cisplatin/ SAHA combination was applied [58]. TSA and LBH589 (Panobinostat) have been shown to inhibit HDAC classes I and II, and LB4589 has been subjected to clinical evaluation against CTCL in Phase III trials. A study in SQD9, SCC61, Cal27 and SC179 cell lines reported higher levels of radiotherapy effectiveness when combined with TSA, LBH589 or MGCD0103 [59]. The results were extracted after treatment with sulfhorodamine B by BrdU assays, western blotting and RT-PCR, but the exact mechanism of action was not elucidated. Moreover, in vitro pretreatment with TSA before cisplatin chemotherapy in UT-SCC-77 OSCC cells revealed significantly better results than monotherapy [60]. The mechanism for chemosensitization on cisplatin included decrease in the lysosomal pH and LAMP-2 levels and increase in the activity of cathepsins [60]. In vitro studies conducted on LBH589-treated OSCC cell lines of hypopharynx (FaDu) and oral cavity (CAL-27, SCC-15, UM-SCC-1 and UM-SCC-47) by the use of flow cytometry and cDNA microarrays revealed upregulation of p21 and induction of G2/M arrest and cell death [61]. Global RNA expression studies following treatment of the HNSCC cell line FaDu with LBH589 revealed downregulation of genes required for chromosome congression and segregation (SMC2L1), sister chromatid cohesion (DDX11) and kinetochore structure (CENP-A, CENP-F and CENP-M) [61]. These LBH589-induced changes in gene expression coupled with the downregulation of MYC and BIRC5 (survivin) provided a plausible explanation for the early mitotic arrest and cell death observed [61]. Notably, when LBH589-induced changes in gene expression were compared with gene expression profiles of 41 primary HNSCC samples, many of the genes that were downregulated by LBH589 showed increased expression in primary HNSCC, suggesting that some HNSCC patients may respond to treatment with LBH589 [61]. Interestingly, a recent study showed that SAHA and TSA inhibited the growth of two CD44+ cancer stem-like HNSCC cell lines, inducing apoptosis and cell cycle arrest [62]. Both HDACis were also capable of altering the cancer stem cell phenotype in HNSCC, raising the possibility that they may have therapeutic potential for cancer stem cells of HNSCC [62]. Moreover, TSA was shown to reduce the number of cancer stem cells and inhibit clonogenic sphere formation in HNSCC cell lines [63]. Interestingly, TSA induced EMT in HNSCC cells, accumulation of BMI-1, an oncogene associated with tumor aggressiveness, and expression of the vimentin mesenchymal marker. These data supported substantial evidence that HDAC inhibition may constitute a novel strategy to disrupt the population of cancer stem cells in head and neck tumors in order to create a homogeneous population of cancer cells with biologically defined signatures and predictable behavior [63]. 74

Cyclic peptides Depsipeptide-FK228 (romidepsin), a natural HDACi isolated from Chromobacterium violaceum, is the second FDAapproved HDACi for CTCL treatment and the first HDACi in Phase II clinical trials on HNSCC patients [64]. Patients’ assessment was performed by radiographic examination, and the exact mechanism of action was assessed using immunohistochemistry, DNA microarray analyses and DNA methylation studies [64]. This study documented that depsipeptide-FK228 induced p21waf1/Cip1 expression and decreased cell proliferation (Ki67 levels), resulting in transcription regulation and cell cycle control [64]. Single-agent depsipeptide exerted limited activity for the treatment of HNSCC, but it effectively achieved tumor-associated HDAC inhibition. Thus, the authors proposed that further evaluation of other HDACis in combination with active therapies may be justified [64]. Although depsipeptide was unable to stand as a monotherapy, it exerted significant synergistic action, in vitro to androgen-independent prostate PC-3, DU145 and C4-2 cancer cell lines, when combined with the nonspecific docetaxel , in a dose similar to the FDA-approved dose for CTCL [65]. Despite the fact that depsipeptide was proven incapable to confront OSCC and other solid tumors as monotherapy, it was able to restore the normal cell cycle, supporting evidence that in the future it may be utilized in combined therapies against OSCC. In addition, depsipeptide increased the effectiveness of gene therapy with adenoviruses against p53 in esophageal Tn and TE2 SCC cell lines [66]. Apicidin is a natural cyclic tetra-peptide isolated from Fusarium spp., which presents significant antitumor effects due to its antiproliferative and differentiation-inducing activity on human cancer cells. The antiproliferative effects were attributed to apicidin-induced increased levels of p21WAF1/Cip1 and gelsolin, which causes G2/M phase cell cycle arrest [67]. Specifically, apicidin upregulated p21WAF1, leading to G2/M arrest and inducing apoptosis in OSCC cell lines [67]. Moreover, apicidin-treated YD-8 and YD-10B OSCC cell lines assessed by MTT and Trypan blue assay, DAPI staining and flow cytometry presented elevated LC3-II levels and G2/M phase cell cycle arrest, increased apoptotic cell death and elevated autophagy levels [68]. 5.2

Benzamides Both MS-275 and MGCD0103, two benzamide analogs, were shown to inhibit HDACs class I. MS-275 (Entinostat) achieved decreased dose of cisplatin when used in a combined anticancer therapy, in vitro, against HSC-3 OSCC cell line. The action mechanism included G1/S arrest and apoptosis [69]. Considering the amount of cisplatin’s side effects, such as nephrotoxicity and myelosuppression, it was emphasized the fact that it achieved the same antitumor results with lower drug doses [69]. Pretreatment of MGCD0103 and 5-aza20-deoxycytidine (5-Aza-dC) was used before radiotherapy against OSCC cell lines; however, the results did not 5.3

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Histone deacetylase inhibitors in oral squamous cell carcinoma treatment

demonstrate better radiosensitization effect compared with single-5-Aza-dC pretreatment [59]. Aliphatic fatty acids Valproic acid (VPA) is used in clinical practice for the treatment of bipolic and seizure disorders. Moreover, it is an HDACi of classes I and IIa and its activity has recently been evaluated against HNSCC. In fact, VPA was shown to exert dose-dependent radionsensitizing ability by chromatin decondensation with histone hyperacetylation and downregulation of Rad51 in TE9, TE10, TE11 and TR14 esophageal SCC cell lines [70]. VPA radiosensitization was accompanied by an increase in gH2AX levels, indicating the presence of doublestrand breaks (DSBs) and a decrease in Rad51 expression, a DSB repair protein. However, the IC50 dose for radiosensitization was significantly higher (1.02 -- 2.15 mM) compared with the safe VPA concentrations (0.4 mM) [70]. Furthermore, another study reported that VPA as monotherapy or in combination with other anticancer agents (5-Aza-dC, ATRA) induced cell differentiation and tumor growth inhibition in ORL-48, ORL-156, ORL-166, ORL-215, ORL-295N and ORL-310N HNSCC cell lines [71]. MTT and clonogenic assays were used to identify increased p21-levels and terminal differentiation markers in the VPA-treated cells [71]. VPA’s anticancer role was also demonstrated in combination with gene therapy using a recombined adenovirus treatment [72]. VPA increased coxsackievirus and adenovirus receptor expression, in vitro (NT8e cell line), as well as, in vivo, in a HNSCC xenograft mouse model and consequently increased Ad-HSVthymidine kinase effectiveness, which was low due to HDAC activity [72]. According to this study, VPA was considered as one of the most promising HDACis for future anticancer treatment against OSCC and HNSCC [72]. NaB is another aliphatic fatty acid with HDAC inhibitory activity against classes I and IIa members. The effect of NaB on inhibition of human Tca8113 OSCC cells was investigated by immunohistochemistry and flow cytometry [73]. NaB significantly inhibited the proliferation of Tca8113 cells in a timeand dose-dependent manner. Tca8113 cells treated with NaB at the concentration of 2 and 4 mmol/L were arrested in G0/ G1 phase [73]. NaB treatment led to considerable upregulation of p27(Kip1) expression. Thus, it was speculated that NaB treatment may inhibit the growth of OSCC cell line, in vitro, and induce cell cycle arrest, which may probably be attributed to the increased p27 protein expression [73]. In addition, antitumor activity of a phenyl-butyrate-based HDACi named (S)-HDAC42 was demonstrated, in vitro, against Ca922, SAS and HSC-3 OSCC cell lines, as well as, in vivo, in xenograft mouse models [74]. (S)-HDAC42 downregulated phospho-Akt, cyclin D1 and cyclin-dependent kinase 6 and increased p21 and p27 expression. Also, NF-k B pathway was blocked and reactive oxygen species were activated. Notably, (S)-HDAC42 achieved higher antiproliferative effects against OSCC cell lines compared with SAHA and its tumor-suppressing ability was remarkable [74].

Moreover, (S)-HDAC42 exhibited high potency in suppressing OSCC tumor growth in a Ca922 xenograft nude mouse model [74].

5.4

6.

Conclusion

HDACis constitute an achievement of molecular medicine. They can reduce tumor burden by inducing cell cycle arrest and inhibiting cell proliferation, as well as by inducing cell differentiation and apoptosis. Selective toxicity and limited side effects are demanded and usually absent features on anticancer therapy. HDACis are well-tolerated drugs, and the demanded activity and effectiveness can be achieved with micromolar or nanomolar concentrations. Malignant cell lines were 10 times more sensitive than healthy cells, suggesting that HDACis present selective toxicity, affecting mainly malignant cells. HDACis are also able to decrease the doses of more toxic therapeutic agents when combined in therapy, and therefore they can limit the side effects of other anticancer agents. Both in vitro and in vivo data and ongoing clinical trials have recently revealed that HDACis could be used against different solid tumors and hematological malignancies, consisting one of the most promising classes of new anticancer agents. Already two of them, a hydroxamate (SAHA) and a cyclic peptide (romidepsin), are clinically approved for CTCL treatment. As for OSCC, accumulated knowledge exists from experimental data, supporting evidence that HDAC overexpression may be associated with tumor development and progression in OSCC and HDACis could be considered as potential anticancer agents for OSCC treatment. However, HDACis effectiveness against OSCC remains to be confirmed in clinical trials. Research data imply that the currently available HDACis cannot stand as monotherapy against OSCC. However, they exert important antitumor activity on combined therapies, acting synergistically with the other therapeutic elements and even lowering the doses demanded for adequate tumor growth inhibition. Notably, in vitro studies evaluating novel HDACis, such as (S)-HDAC42, are encouraging for the future of OSCC treatment, exerting higher antitumor activity compared with the existent HDACis. 7.

Expert opinion

HDACis exert antitumor activity in HDAC overexpressionrelated OSCC tumors, causing cell cycle arrest and inducing apoptosis via several molecular mechanisms. The effects are restricted to neoplastic cells, and these agents seem to be highly selective. They are commonly targeting one or more classes of HDACs, but some members are affecting only a few HDACs family members. In vitro studies have demonstrated a synergistic action of HDACis with other therapeutic agents. HDACi can decrease the dosage of the less selective, toxic agents commonly used in OSCC therapy (i.e., cisplatin, gefitinib) and achieve comparable or even higher

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antiproliferative effects when administered as combined therapy. These results are promising rendering HDACis as potential anticancer agents with lower frequency and intensity of side effects. However, the radio- or chemosensitization effect is not universal. Some agents are not capable of increasing OSCC radio- or chemosensitivity, whereas novel HDACis can achieve even higher antiproliferative effects compared with the family’s most investigated members. It is of note that most of the currently existing data are derived from in vitro studies, whereas the studies in living organisms are limited. This fact creates the need to examine the interaction of HDACs and HDACis in animal models and clinical trials in order to presume the efficiency, the optimal dosage and duration and the appropriate role of HDACis in OSCC therapy. Thus far, these agents seem to present clinical utility only as cofactors of the main therapy, lowering the dosage and the respective adverse effects. Novel and more effective HDACis could be utilized as monotherapy in the future. Despite the progress in understanding the HDACs and HDACis’ function, there are a lot of features to be clear in order to interpret the data of the ongoing research in the field. HDACs are involved in the formation and the progression of OSCC, but the current knowledge base has limited data regarding the role and the molecular mechanism of each HDAC in this human malignancy. The outcome of the Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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Affiliation Jason Tasoulas1, Constantinos Giaginis†1,2 MSc PhD, Efstratios Patsouris1, Evangelos Manolis1 & Stamatios Theocharis1 † Author for correspondence 1 Associate, National and Kapodistrian University of Athens, Medical School, First Department of Pathology, Athens, Greece 2 Assistant Professor of Human Physiology, University of the Aegean, School of Environment, Department of Food Science and Nutrition, 2 Mitropoliti Ioakeim Street, Myrina, Lemnos 81400, Greece Tel: +30 22540 83117; Fax: +30 22540 83109; E-mail: [email protected]