Abstract

Simple Summary

   Osteosarcoma is the predominant form of primary bone cancer and
   outcomes for patients with metastatic disease have not improved for
   several decades. Recent genomic and pharmacological studies have
   implicated dysregulated histone deacetylases as druggable targets to
   treat several types of cancers, including osteosarcoma. This study
   aimed to assess the inhibitory effects of 4SC-202, a next-generation
   inhibitor for class I histone deacetylases, on human osteosarcoma cell
   growth in vitro and in vivo. We demonstrated that the anti-tumor effect
   of 4SC-202 involves combined cell-cycle arrest and apoptosis, as well
   as a reduction in cell invasion and migration. Moreover, 4SC-202
   revised the global transcriptome and induced distinct signatures of
   gene expression in vitro. Additionally, 4SC-202 decreased tumor growth
   of established human tumor xenografts in immunodeficient mice in vivo.
   Our study suggests that 4SC-202 may be exploited as a valuable drug to
   promote more effective treatment of patients.

Abstract

   Dysregulation of histone deacetylases (HDACs) is associated with the
   pathogenesis of human osteosarcoma, which may present an epigenetic
   vulnerability as well as a therapeutic target. Domatinostat (4SC-202)
   is a next-generation class I HDAC inhibitor that is currently being
   used in clinical research for certain cancers, but its impact on human
   osteosarcoma has yet to be explored. In this study, we report that
   4SC-202 inhibits osteosarcoma cell growth in vitro and in vivo. By
   analyzing cell function in vitro, we show that the anti-tumor effect of
   4SC-202 involves the combined induction of cell-cycle arrest at the
   G2/M phase and apoptotic program, as well as a reduction in cell
   invasion and migration capabilities. We also found that 4SC-202 has
   little capacity to promote osteogenic differentiation. Remarkably,
   4SC-202 revised the global transcriptome and induced distinct
   signatures of gene expression in vitro. Moreover, 4SC-202 decreased
   tumor growth of established human tumor xenografts in immunodeficient
   mice in vivo. We further reveal key targets regulated by 4SC-202 that
   contribute to tumor cell growth and survival, and canonical signaling
   pathways associated with progression and metastasis of osteosarcoma.
   Our study suggests that 4SC-202 may be exploited as a valuable drug to
   promote more effective treatment of patients with osteosarcoma and
   provide molecular insights into the mechanism of action of class I HDAC
   inhibitors.

   Keywords: osteosarcoma, epigenetics, 4SC-202, domatinostat, histone
   deacetylase inhibitor, xenografts, osteoblast-like differentiation,
   transcriptome, signaling pathways

1. Introduction

   Osteosarcoma (OS) is the most common primary bone cancer that mainly
   occurs in children, adolescents, and young adults. In the past 40
   years, the five-year survival rate has stagnated as standard patient
   care procedures, including multi-agent chemotherapy and surgery, have
   remained relatively unchanged [[44]1]. In addition to the devastating
   side effects and social–emotional consequences of the rigorous
   treatment, additional complications include the need for prostheses
   after surgical amputation [[45]2]. OS metastasis and chemoresistance
   are the key clinical factors leading to a low five-year survival rate
   of approximately 30% in patients with remote involvement
   [[46]3,[47]4,[48]5,[49]6]. Recent studies have shown that tumor
   suppressor and/or oncogene mutations, copy number alterations, fusion
   genes, and epigenetic dysregulation may lead to tumor formation, cancer
   metastasis, and multidrug resistance [[50]7,[51]8,[52]9,[53]10,[54]11].
   These distinct characteristics may pose as vulnerabilities that can be
   exploited as treatment targets. These findings require urgent
   investigations to explore the vulnerabilities outlined above in order
   to develop or repurpose second-line drugs that can improve patient
   survival.

   Epigenetic dysregulation is an evolving hallmark of cancer and results
   from aberrant epigenetic regulators (such as writers, readers, and
   erasers) that modify histone and nonhistone proteins to balance the
   transcription of tumor suppressor genes and oncogenes [[55]12,[56]13].
   Recently, aberrant, or dysregulated, epigenetic regulators are
   overwhelmingly discovered by next-generation sequencing studies in OS
   samples [[57]5,[58]6,[59]14,[60]15,[61]16,[62]17,[63]18,[64]19].
   Targeting dysregulated erasers such as histone deacetylases (HDACs) can
   reinstate the epigenetic homeostasis from an abnormal epigenetic
   landscape and, as a result, they are emerging as druggable targets to
   treat several types of cancers, including osteosarcoma
   [[65]1,[66]6,[67]11,[68]20].

   There are 18 unique human HDAC isoforms, which contain evolutionarily
   conserved catalytic domains that trace back to bacteria and yeast
   [[69]21]. HDACs enzymatically remove acetyl groups (CH3CO-) from
   ε-amino groups of histone lysine residues within multi-protein
   complexes, which enables chromatin condensation and transcriptional
   repression and/or activation, and affects the stability and cellular
   location of non-histone proteins such as master transcription factor
   RUNX2, a key regulator of gene expression in normal bone and OS cells
   [[70]22,[71]23,[72]24]. They can be categorized into four classes
   (I–IV) according to their domain organization, sequence, and functional
   similarity [[73]25]. Class I retains four isoforms (HDACs 1, 2, 3, and
   8), which are primarily located in the nucleus and are broadly
   expressed in most tissues. Class II retains six isoforms (HDACs 4, 5,
   6, 7, 9 and 10), which shuttle between the cytoplasm and nucleus with a
   tissue-restricted pattern of gene expression. Class III retains seven
   isoforms (SIRTs 1–7), which may be located within the nucleus,
   cytoplasm, or mitochondria. Class IV retains one isoform (HDAC11),
   which is the least studied enzyme and shares similarities with the
   catalytic domains of classes I and II. Moreover, the catalytic function
   of classes I, II, and IV, dubbed the “classical” isoforms, depends on a
   cofactor zinc ion (Zn^2+), whereas class III depends on the coenzyme
   nicotinamide adenine dinucleotide (NAD^+) [[74]22]. It is worth noting
   that small chemical inhibitors to HDACs have been designed and
   developed to chelate cofactor Zn^2+ to prevent the accessibility of the
   active site from other complex components near the active site. These
   inhibitors can be classified into two groups. One is those with
   broad-spectrum inhibitory ability to HDACs, collectively named pan-HDAC
   inhibitors (or pan-HDACi), which can block the enzymatic function of
   two or more classes of isoforms. The other is those with an ability to
   selectively block one class or one isoform of HDACs, which are
   collectively named selective HDACi [[75]26].

   In skeletal development, HDACs promote endochondral and intramembranous
   ossification through regulating the expression of crucial genes and
   signaling pathways as well as mediating cell differentiation and
   survival of bone cells including osteoblasts, from which OS tumor
   arises [[76]25,[77]27]. For example, class I HDACs share 45–94% of
   amino acid sequence similarity, resulting in redundant cellular
   functions in development and adult tissue homeostasis. The
   co-expression pattern and overlapping roles of HDAC1 and HDAC2 have
   been reported in several cell types, including podocytes, cardiac, and
   osteoblasts [[78]23,[79]25,[80]28,[81]29,[82]30]. In addition, HDAC1
   and HDAC3 have been shown to physically interact with RUNX2 to regulate
   gene transcription in osteoblasts and chondrocytes [[83]29,[84]31]. The
   significantly elevated expression levels of HDAC1, HDAC2, and HDAC3
   (HDAC1–3) have also been reported in several human OS cell lines and
   primary and metastatic tumor samples [[85]11,[86]32,[87]33]. Therefore,
   the application of a selective inhibitor that can specifically target
   HDAC1-3 may lead to high-quality therapies with better efficacy and
   lower toxicity.

   Since FDA approval of vorinostat, the first HDACi, to treat patients
   with cutaneous T-cell lymphoma in 2006, several pan- and selective HDAC
   inhibitors have been developed and applied to the treatment of
   hematological malignancies [[88]6,[89]34]. Currently, HDACi drugs have
   not been approved for the treatment of solid tumors, including OS
   patients [[90]6]. Additionally, a limited number of OS cases have been
   included in several completed and active clinical trials constructed
   for solid tumors, but no clinical trials have been specifically
   designed to study the disease ([91]https://www.clinicaltrials.gov
   (accessed on 23 March 2021). For example, in a phase I trial study,
   only one OS patient was included and treated with a combination of
   vorinostat and bortezomib, but did not have a response [[92]35]. A
   preclinical study using vorinostat showed modest inhibitory activity in
   OS cells and no objective responses for OS xenografts [[93]36].
   However, many more newly developed HDACi agents have recently been
   studied using established human OS cell lines. According to the
   chemical structures of HDACi, they can be categorized into four groups:
   (a) hydroxamic acids (e.g., vorinostat/SAHA
   [[94]37,[95]38,[96]39,[97]40,[98]41,[99]42,[100]43,[101]44,[102]45],
   AR42 [[103]46], trichostatin A/TSA
   [[104]33,[105]47,[106]48,[107]49,[108]50,[109]51,[110]52,[111]53,[112]5
   4,[113]55], quisinostat [[114]56,[115]57,[116]58], panobinostat/LBH589
   [[117]11,[118]57,[119]58,[120]59,[121]60], abexinostat [[122]61], MC
   1742 [[123]62], and tubacin [[124]63]), (b) short-chain fatty acids
   (e.g., valproate/VPA
   [[125]64,[126]65,[127]66,[128]67,[129]68,[130]69,[131]70,[132]71] and
   butyrate/NaB [[133]72,[134]73]), (c) benzamides (e.g.,
   entinostat/MS-275 [[135]74,[136]75,[137]76,[138]77]), and (d) cyclic
   peptide (e.g., romidepsin/FK228
   [[139]11,[140]58,[141]60,[142]78,[143]79,[144]80,[145]81,[146]82] and
   apicidin [[147]80]). The latter two typically are considered selective
   HDACi. Hence, given the lack of clinical application and the paucity of
   information studying current HDACi in OS, the development and
   investigation of novel HDACi agents, especially selective-HDACi, are
   urgently needed.

   4SC-202 (domatinostat), which is a recently developed small molecule
   that selectively targets class I HDACs, has been studied in several
   types of cancer, including urothelial carcinoma, squamous cell
   carcinoma, myelodysplastic syndrome, cutaneous T-cell lymphoma,
   cholangiocarcinoma, hepatocellular carcinoma, colorectal cancer,
   medulloblastoma, pancreatic cancer, and Merkel cell carcinoma
   [[148]83,[149]84,[150]85,[151]86,[152]87,[153]88,[154]89,[155]90,[156]9
   1,[157]92,[158]93]. Its safety and antitumor activity were established
   in a phase I study as a monotherapy for adult patients with
   hematological malignancies [[159]94]. It is currently being tested in
   several clinical trials in the treatment of patients with hematologic
   malignancies and solid tumors (ClinicalTrials.gov Identifier:
   [160]NCT04874831, [161]NCT04393753, [162]NCT04871594, [163]NCT04133948
   and [164]NCT03812796). Prior to this study, 4SC-202 had not been
   examined for the treatment of OS. In our proof-of-concept study, we
   applied SJSA-1 and hFOB 1.19 cell lines to investigate global
   transcriptomic changes and drug effects of 4SC-202 in vitro and in
   vivo. We chose SJSA-1 because it is an established human OS cell line
   [[165]95] that has been used to examine pan-HDACi drugs including,
   panobinostat and vorinostat [[166]37,[167]46,[168]57,[169]96], whereas
   hFOB 1.19 (hFOB) is an immortalized cell line derived from normal human
   fetal osteoblastic cells [[170]97], which has been frequently applied
   in parallel to examine pan-HDACi including vorinostat, trichostatin A,
   and panobinostat [[171]33,[172]97,[173]98].

2. Materials and Methods

2.1. Cell Culture and Treatment

   For this study all cell lines, including human osteosarcoma SJSA-1
   (CRL-2098) and the human immortalized osteoblast hFOB 1.19 (ATCC,
   CRL-11372), were purchased from the American Type Culture Collection
   (ATCC, Manassas, VA, USA). SJSA-1 and hFOB 1.19 were grown in a
   humidified chamber containing 5% CO[2] and cultured in a growth medium
   (HyClone™ MEM-α medium, Marlborough, MA, USA, SH30265FS) containing 10%
   fetal bovine serum (Fisher Scientific, Waltham, MA, USA, ES009B) and 1%
   HyClone™ penicillin-streptomycin (Cytiva, SV30010). However, the hFOB
   1.19 cell line media also contained 0.3 mg/mL G418 (Fisher Scientific,
   10-131-035) and was maintained at 34 °C, as per ATCC protocol. Cells in
   100 mm tissue culture dishes (Fisher Scientific, 430167) were treated
   with 1 μM 4SC-202 (Adooq Bioscience, Irvine, CA, USA, A14354-25) for 24
   h unless differently stated. The corresponding amount of DMSO (vehicle)
   was used as a control. All assays were performed at 37 °C.

2.2. Western Blot Analysis

   Western blotting analysis was performed as described previously
   [[174]99,[175]100]. Cells were briefly lysed with 1× Laemmli Sample
   Buffer solution (BioRad, Hercules, CA, USA, 1610737), boiled for 5 min
   to 95 °C, and sonicated. Lysates were separated on 4–20% Mini-PROTEAN^®
   TGX™ Precast Protein Gels (BioRad, 4561094) and transferred onto a PVDF
   membrane (BioRad, 1704272) using a semi-dry method of transfer (Bio-Rad
   Trans-blot Turbo system). The transferred blots were probed overnight
   with one of the following primary antibodies: rabbit anti-H3K4Me2
   antibody (Diagenode, Denville, NJ, USA, C15200151), H3K27Ac (Diagenode,
   C15410196), rabbit anti-β-ACTIN (Li-Cor, 926-42210), JAG2 (Cell
   Signaling, #2205, C83A8), NOTCH3 (Proteintech, Chicago, IL, USA,
   55114-1-AP), or NOTCH4 (Abcam, Cambridge, MA, USA, ab184742) primary
   antibodies. After incubation with IRDye 800 goat anti-rabbit (Li-Cor,
   Lincoln, NE, USA, 925-32211) and IRDye 680 goat anti-rabbit (Li-Cor,
   925-68071) secondary antibodies (1:10,000) in 10% adult bovine serum
   blocking buffer was placed on a rocker for 1 h at room temperature.
   After several washes in 1XTBST, the protein signal was visualized on an
   Odyssey imaging system (Li-Cor, Lincoln, NE, USA) and quantified using
   β-actin as a normalizer with the optical density (OD) function of Image
   J software (NIH, Bethesda, MD, USA).

2.3. Cell Viability and Colony Formation Assays

   The cell proliferation and viability assay was performed using a CCK-8
   kit (Dojindo Molecular Technologies, CK04-11, Kumamoto, Japan)
   according to the manufacturer’s instructions and assays were performed
   at least three times. Cells were seeded into a 96-well plate (Fisher
   Scientific, N8010560) at a density of 6250 cells per well as five
   replicates. A black control was also used, which only contained media
   and no cells. Cells were treated with increasing concentrations of
   4SC-202 (or vehicle) for 24, 48, and 72 h, with media and/or treatment
   replacement after 24 and 48 h for the latter time points, respectively.
   hFOB 1.19 was treated with increasing concentrations of 4SC-202 (or
   vehicle) for 72 h with media changes every 24 h. Once the wells reached
   the treatment time point, 10 μL of the CCK-8 was added to each
   condition, including negative controls, and incubated for 1 h at 37 °C
   in 5% CO[2]. The OD at 450 nm of each well was then read using a
   Cytation3. Statistical significance comparing each treated group to the
   control was analyzed using GraphPad software with one-way ANOVA (Holm
   method). For the colony formation assay [[176]99,[177]101], 1000
   cells/mL were seeded into 6-well plates. After attachment to the wells,
   the cells underwent media changes with or without treatment with
   indicated concentrations of 4SC-202 every 2–3 days for about two weeks
   or until visible clonal colonies formed. The wells were washed with
   PBS, fixed with 10% formalin, and stained with 0.5% Crystal Violet
   solution at the endpoint of this study.

2.4. Wound Healing Assay

   To investigate 4SC-202-induced inhibition of cell migration, a
   wound-healing (i.e., in vitro scratch) assay was performed as we
   previously reported [[178]99,[179]100]. Cells were seeded into 6-well
   plates (Fisher Scientific, 353046) and grown to confluence. A vertical
   and horizontal cross-shaped scratch was made using a 2–2000 μL pipette
   tip on the monolayer of confluent cells. Dislodged cells and debris
   were removed by washing the cells three times with PBS. Fresh medium
   containing 1 µM 4SC-202 or DMSO vehicle was added (t = 0), and images
   were taken where the two scratch lines met using an upright Olympus
   IX71 microscope. After the indicated incubation time with either
   vehicle or drug treatment, the same area was photographed again.
   Scratched areas at the initial and final time points were quantified
   using the NIH ImageJ software (Bethesda, MD, USA). The percentage of
   relative wound healing was expressed according to the following
   formula: ((initial scratched area, 4SC-202 added) − (resulting
   scratched area, 4SC-202 added))/((initial scratched area, vehicle
   added) − (resulting scratched area, vehicle added)) × 100.

2.5. Boyden Chamber-Based Cell Migration and Invasion Assays

   The in vitro Boyden migration and invasion assays were performed and
   modified using 8 μm pore-sized cell-culture inserts (Falcon, 08-771-21)
   into wells of a 24-well plate as previously reported [[180]100].
   Briefly, the cells were serum-started overnight with culture media
   containing either 4SC-202 or vehicle. Wells of a 24 well-plate were
   filled with culture medium containing 10% FBS prior to loading the
   cells at a concentration of 4 × 10^4 cells/well into the upper
   compartment of the chamber (i.e., inside the cell-culture insert). For
   the invasion assay, the membrane at the bottom of the insert was coated
   with a layer of 0.2 mg/mL Matrigel overnight prior to the addition of
   the cells (Fisher Scientific, [181]CB354248). After 24 h, the upper
   compartment was washed and fixed in 10% formalin. Cells that traveled
   through the insert remained and were visualized by staining with
   Crystal Violet (Fisher Scientific, C581-25). Any remaining cells on the
   upper surface of the insert were removed with a Q-tip, and the plate
   with inserts was then imaged. The dye from the cells was then extracted
   using 33% acetic acid (Fisher Scientific, A38S-212) and the OD was
   quantified on a Cytation3 at a wavelength of 570 nm. The OD of the
   extracted stain was used to determine the relative number of cells that
   invaded the gel barrier and passed through the insert’s pores.

2.6. Osteoblast Differentiation Assay

   The osteoblast differentiation approach and quantification were
   completed according to the modified procedure described previously
   [[182]100,[183]102]. Cells were cultured to approach confluence in a
   12-well plate (Fisher Scientific, 353043) and then treated with
   appropriate media. The osteoblast differentiation (OB diff) medium used
   contained 50 μM ascorbic acid (Cayman Chemical, Ann Arbor, MI, USA,
   16457), 100 nM dexamethasone (Cayman Chemical, 11015), and 10 mM
   β-glycerophosphate (BGP) (Cayman Chemical, 14405). The assay was
   performed over 14 days, with a medium change every three days. At the
   endpoint, the cells were gently washed, fixed in 10% formalin, and
   stained to visualize calcium deposits with 40 mM Alizarin Red S (ARS)
   pH 4.2 (Sigma, St. Louis, MO, USA, A5533-25G). Images were taken with
   an upright microscope (Olympus, Center Valley, PA, USA, IX71). To
   quantify the staining, ARS was dissolved with 10% (w/v) cetylpyridinium
   chloride in 10 mM sodium phosphate (pH 7.0) on a rocker for
   approximately 3 h. Equal volumes of the extracted solution were
   measured on a Cytation 3 (BioTek, Crawfordsville, IN, USA) at 562 nm.
   Statistical significance comparing among groups was analyzed using
   GraphPad software with two-way ANOVA (Tukey method).

2.7. Analysis of Cell Cycle and Apoptosis by Flow Cytometry

   The cells were seeded into a 10 cm dish, allowed to reach 70%
   confluence, and then treated with DMSO vehicle or 1 µM 4SC-202 for 24
   h. The media and adherent cells were pooled together into a single-cell
   suspension. For cell-cycle analysis, single-cell suspensions were
   pelleted and washed at room temperature twice with 1× phosphate
   buffered saline (PBS), fixed in 66% ethanol for 1 h, then rehydrated in
   1× PBS. In order to ensure only DNA was being measured, the cells were
   incubated for 30 min with RNAse A (Qiagen, Germantown, MD, USA, 19101),
   followed by 1× PBS and 50 µg/mL propidium iodide in the dark. The PI
   was excited at 561 nm, the emission spectrum was detected through a 595
   LP (longpass) mirror and 610/20 bandpass filter, and results were
   generated using a BD Fortessa system (Becton Dickinson, Franklin Lakes,
   NJ, USA). The data were analyzed using FlowJo v10.6 software (FlowJo,
   Ashland, OR, USA). For the apoptosis assay, single-cell suspensions
   were pelleted and washed twice with ice-cold 1× PBS and resuspended in
   500 μL cold 1× binding buffer (25 mM HEPES, 1 mM EDTA, 2% FBS, 1%
   Pen/strep). After the cells were incubated with 5 μL Annexin V-FITC
   (Fisher Scientific, BDB560931) and 50 μg/mL propidium iodide (PI) for
   15 min in the dark, they were analyzed by flow cytometry on a BD
   Fortessa system (Becton Dickinson, Franklin Lakes, NJ, USA). The FITC
   was excited at 488 nm and the emission spectrum was detected through a
   495 LP (longpass) mirror and 530/30 bandpass filter, then analyzed by
   the BD FACSDiva v6.0 software. DMSO- and 4SC-202-treated groups were
   performed in triplicate with the following control groups: PI only,
   Annexin V-FITC only, and treatment only.

2.8. RNA Sequencing, Pathway Analysis, and Data Availability

   Total RNA was isolated using a PureLink^® RNA Mini Kit (ThermoFisher
   Scientific, Waltham, MA, USA, 12183018A) according to the
   manufacturer’s instructions. RNA concentration and purity were measured
   by Thermo Scientific™ NanoDrop™ spectrophotometers. RNA integrity was
   measured on a Bioanalyzer 2100 with RNA 6000 Nano Labchips (Agilent
   Technologies Ireland, Dublin, Ireland). Twelve RNA samples with an RNA
   integrity number >8.0 were used for RNA cleanup, library preparation,
   and sequencing by Novogene according to the procedure and protocols of
   the company (Novogene Corporation, Sacramento, CA, USA). In short,
   according to the manufacturer’s protocol, 1 μg RNA was used for cDNA
   library construction using a NEBNext^® Ul-tra™ II RNA Library Prep Kit
   for Illumina^® (NEB #E7770). The mRNA was enriched with oligo(dT)
   beads, followed by two rounds of purification and random fragmentation
   by adding fragmentation buffer. We used random hexamer primers to
   synthesize the first-strand cDNA, and then added customized
   second-strand synthesis buffer (Illumina), dNTP, RNase H, and DNA
   polymerase I to generate the second strand (ds cDNA). After a series of
   end repair, polyadenylation, and sequencing linker connection, the
   double-stranded cDNA library was completed by size selection and PCR
   enrichment. The 250–350 bp insert library was quantified using a Qubit
   2.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and
   quantitative PCR. We used NGS3K to analyze the size distribution.
   Qualified libraries were sequenced on an Illumina platform using a
   paired-end 150 run (2 × 150 bases). The high-quality reads from 27.5 to
   31.5 million were generated from each library. Paired-end reads were
   aligned to the human genome (GRCh37) and annotated with GENCODE gene
   annotation (v32) using STAR.

   Raw counts were estimated with the option set (--quantMode GeneCounts)
   in STAR. Differential expression (DE) analysis were performed using the
   R [[184]103] package DESeq2 [[185]104]. DE genes were identified based
   on cutoff values of 0.05 for adjusted p-value and/or log2-fold-change.
   To account for gene length bias, gene ontology and KEGG pathway
   enrichment analysis were conducted using R package goseq [[186]105].
   The Benjamini–Hochberg correction for multiple testing in enrichment
   was used and pathways with adjusted p-values less than 0.05 were
   declared significant [[187]106]. The principal component analysis (PCA)
   plots and heatmaps of Euclidean distances among samples are based on
   the expression data and using DESeq2 [[188]104]. Heatmaps of the top
   varying genes in each experiment were produced using the R package
   pheatmap [[189]107]. The number of fragments per kilo base per million
   mapped reads (FPKM) was calculated for each sample to help visualize
   expression patterns for each unigene between the treated and untreated
   samples. Additional functional annotation, pathways, and gene network
   analyses were performed by IPA (Ingenuity Pathways Analysis,
   [190]http://www.ingenuity.com/ (accessed on 11 December 2020) with
   default parameters.

   All RNA-seq experiments were performed in at least three biological
   replicates under each condition. The raw sequence data of RNA-seq
   generated in this study are stored in the National Center for
   Biotechnology Information (NCBI) sequence reading archive database
   (accession numbers: SRR14772115 to SRR14772126). All other data can be
   obtained from the corresponding author upon reasonable request.

2.9. In Vivo Studies

   Athymic nude female mice (stock #002019) were purchased from Jackson
   Laboratory (Bar Harbor, ME, USA). Before transplant, SJSA-1 cells were
   harvested, washed, and resuspended in serum-free MEM-alpha media. Nude
   mice (6–8 weeks old) were anesthetized by isoflurane inhalation (3%).
   Mice were injected subcutaneously to the rear flank with 250 μL of cell
   suspension (5 × 10^6 cells/injection) using a 27G needle (n = 16). Once
   the tumor size reached an average volume of around 60 cubic
   millimeters, the mice were randomly divided into two groups, control
   and treatment, and treated with either vehicle (10% DMSO + 45% PEG400)
   or 4SC-202 (50 mg/kg/d) via intraperitoneal injections daily for 16
   days. Mouse body weight was measured daily. Tumor size was measured
   using digital calipers, and volumes were calculated according to the
   formula, tumor volume (cubic millimeter) = (Width × Width × Length)/2.
   The mice were housed in a specific pathogen-free facility under
   controlled conditions of light, temperature, and humidity.

2.10. Statistical Analysis

   Data were analyzed using Student’s t-test, or one-way (Holm method) or
   two-way ANOVA (Tukey method) accordingly (GraphPad Prism, GraphPad
   Software, Inc., La Jolla, CA, USA). A p-value less than or equal to
   0.05 was considered statistically significant. All data unless
   otherwise specified are expressed as mean ± standard deviation.

3. Results

3.1. HDAC1-3 Have the Highest Expression among All Isoforms in Human OS Cells

   To have a clearer understanding of the relative expression levels of
   class I HDACs, other HDAC isoforms, and the status of gene expression
   in the entire transcriptome of untreated cells, we first performed a
   next-generation RNA sequencing (RNA-seq) analysis. We found that HDAC1,
   HDAC2, and HDAC3 have much higher gene expression than other isoforms
   in SJSA-1 OS cells, as well as hFOB cells ([191]Figure 1A and
   [192]Figure 2A). Previous research showed that 4SC-202 is a selective
   benzamide-type HDACi-targeting class I HDAC ([193]Figure S1A,B) with a
   higher specificity (lower inhibitory constant (Ki) values) for HDAC1
   (14.8 nM), HDAC2 (38.8 nM), and HDAC3 (27.9 nM) than other isoforms and
   lysine-specific histone demethylase 1A (LSD1) (Ki values > 1800 nM)
   [[194]94]. These data prompted us to further examine the potential
   therapeutic effects of this newly developed drug in human OS cells.
   Thus, our data suggest that SJSA-1 together with hFOB are the prominent
   candidate cell lines to examine the repressive consequences of 4SC-202.

Figure 1.

   [195]Figure 1
   [196]Open in a new tab

   Impact of selective pharmacological inhibition of class I histone
   deacetylases with 4SC-202 on human SJSA-1 OS cells. (A) Gene expression
   of 18 histone deacetylases (HDACs) in SJSA-1 cells. Average FPKM (i.e.,
   fragments per kilo base per million mapped reads) values generated from
   RNA-seq analysis of indicated genes from 3 samples treated with DMSO
   vehicle (dark bar) and 3 samples treated with 4SC-202 (grey bar). (B)
   4SC-202 effect on cell proliferation and viability treated with
   increased concentration of 4SC-202 for 24 h. Statistical significance
   comparing each treated group to the control was analyzed using GraphPad
   software with one-way ANOVA (Holm method). (C) Representative Western
   blot analysis of Ac-H3 and β-ACTIN in DMSO-treated and 4SC-202-treated
   conditions in OS cells. (D) Densitometric quantification of the blots
   of protein band intensity of Ac-H3 normalized to housekeeping β-ACTIN
   bands. Number of asterisks indicates level of statistical significance
   between groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <
   0.0001. Data are presented as mean (SD).

Figure 2.

   [197]Figure 2
   [198]Open in a new tab

   Impact of selective pharmacological inhibition of class I histone
   deacetylases with 4SC-202 on human hFOB 1.19 cells. (A) Gene expression
   of 18 histone deacetylases (HDACs) in hFOB 1.19 cells. Average FPKM
   (i.e., fragments per kilo base per million mapped reads) values
   generated from RNA-seq analysis of indicated genes from 3 samples
   treated with DMSO vehicle (dark bar) and 3 samples treated with 4SC-202
   (grey bar). (B) 4SC-202 effect on cell proliferation and viability.
   Statistical significance comparing each treated group to the control
   was analyzed using GraphPad software with one-way ANOVA (Holm method).
   (C) Representative Western blot analysis of Ac-H3 and β-ACTIN at
   DMSO-treated and 4SC-202-treated conditions in bone cells. (D)
   Densitometric quantification of the blots of protein band intensity of
   Ac-H3 normalized to housekeeping β-ACTIN bands. Number of asterisks
   indicates level of statistical significance between groups. * p < 0.05,
   ** p < 0.01, *** p < 0.001. Data are presented as mean (SD).

3.2. 4SC-202 Impairs Human Osteosarcoma Cell Growth and Clonogenicity In
Vitro

   To investigate the anti-proliferative effects of 4SC-202 in OS in
   vitro, we cultured SJSA-1 cells in the presence of different
   concentrations (0.1 to 25 μM) of 4SC-202. Treatment with 4SC-202
   resulted in a dose-dependent inhibitory effect on cell growth at 24 h
   (h) ([199]Figure 1B). Among five concentrations, 1 μM or higher of the
   drug was enough to suppress the cell viability and proliferation in a
   time-dependent fashion (24, 48, and 72 h) ([200]Figure S1C,D).
   Moreover, a similar effect was observed in hFOB 1.19 cells that could
   be attributed to their high proliferation rate ([201]Figure 2B and
   [202]Figure S1E,F). To assess the activity of 4SC-202 in altering
   acetylation and methylation of histone proteins, we cultured the cells
   in the absence or presence of 1 µM 4SC-202 for 24 h and then harvested
   them for Western blotting analysis to detect acetylated histone 3
   (Ac-H3), a substrate of HDACs, and demethylated H3K4Me2, a substrate of
   LSD1. As shown in [203]Figure 1 and [204]Figure 2, Ac-H3 protein
   expression was significantly increased, whereas a change in H3K4Me2
   expression was not detected in SJSA-1 cells ([205]Figure 1C,D and
   [206]Figure S2E,G) or hFOB cells ([207]Figure 2C,D and [208]Figure
   S2F,H). We also observed an increase in H3K27Ac protein level in a
   dose-dependent manner to respond to 4SC-202 treatment in SJSA-1 and
   hFOB cells ([209]Figure S2A–D). Notably, the mRNA levels of class I
   HDACs in the presence of 1 µM 4SC-202 for 24 h were also altered,
   likely due to a compensatory mechanism ([210]Figure 1A and [211]Figure
   2A). Our data suggest that 4SC-202 impairs human osteosarcoma cell
   growth in vitro through the inhibition of the activity of HDACs.

   To examine the long-term effect of 4SC-202 on cell growth and
   clonogenicity in vitro, we performed a colony formation assay, which
   can detect the ability of a single cell to grow into a colony (>50
   cells) by clonal expansion under drug selection. Crystal violet
   staining clearly showed that a 14-day treatment with 4SC-202 at
   concentrations ranging from 1 to 25 μM significantly inhibited
   proliferative SJSA-1 colonies, indicating an inhibitory effect on cell
   growth and clonogenicity ([212]Figure 3A). Notably, we also observed
   remnant cells or small clones (<50 cells), which implies cancer cell
   survival and drug resistance after treatment with 4SC-202 at this
   concentration ([213]Figure 3B). Moreover, the clonogenicity of hFOB
   1.19 cells was also strongly inhibited by 1 μM 4SC-202 treatment for 14
   days ([214]Figure 3A,B). Since clonogenic activity is a sensitive
   indicator of capacity for renewal of cancer stem cells (CSCs) and drug
   resistance, this result implies that HDACs may be required for
   maintenance of CSC numbers and resistance of chemotherapy drugs in OS
   tumor tissues. Altogether, 1 μM 4SC-202 was an effective concentration
   in both cell lines and was therefore applied for all further
   experiments in this study.

Figure 3.

   [215]Figure 3
   [216]Open in a new tab

   Effect of 4SC-202 on colony formation and wound healing. (A)
   Representative images of crystal violet-stained colonies in wells from
   SJSA-1 (top panel) and hFOB 1.19 cells (bottom left). Cells were
   treated with indicated concentrations of 4SC-202 or DMSO vehicle for 14
   days. (B) Representative images of colonies under microscope with 2×
   objective magnification. (C) Representative images of wound healing at
   indicated hours after the mechanical scratch under 10× objective
   magnification from SJSA-1 (top panel) and hFOB 1.19 cells (bottom).
   Left: vehicle, right: treated with 4SC-202. The white and blue lines
   indicate the edges of the wounded area. (D) Quantitative analysis of
   the wound healing area after the scratch. Wound healing area of
   vehicle-treated cells is defined as 100% versus areas of drug-treated
   cells (*** p < 0.001). Data are presented as mean (SD).

3.3. Cell Migration and Invasion In Vitro Are Partially Suppressed by 4SC-202

   To dissect the effects of 4SC-202 on cell migration, we first performed
   a wound-healing assay. After 17 h, 4SC-202-treated SJSA-1 and hFOB 1.19
   cells migrated significantly less than those treated with vehicle DMSO
   control ([217]Figure 3C,D). A Boyden chamber-based cell migration
   system was applied to further examine this phenomenon of migration
   suppression under 4SC-202 treatment. Cells were serum-starved for 16 h
   to minify the contribution from cell proliferation before seeding into
   the trans-well chamber. Quantification of cells able to travel through
   the pores showed an approximate 55% decrease in 4SC-202-treated cells
   compared to vehicle-treated cells ([218]Figure 4A–D). A layer of
   Matrigel was added to the top insert of the chamber to test the cell
   ability to invade through the gel under 4SC-202 treatment. We found
   that 4SC-202 significantly decreased the invasive ability of both
   SJSA-1 and hFOB 1.19 cells by approximately 40% compared to that of the
   cells treated with vehicle ([219]Figure 4A–D). Altogether, this data
   implies that inhibition of class I HDACs by 4SC-202 may suppress tumor
   metastasis in human OS patients.

Figure 4.

   [220]Figure 4
   [221]Open in a new tab

   Effect of 4SC-202 on cell migration and invasion. (A) Representative
   images of crystal violet-stained SJSA-1 cell migration (top panel) or
   invasion (bottom panel) in a trans-well system with or without 4SC-202
   treatment under 10× objective magnification. (B) Representative images
   of crystal violet-stained hFOB 1.19 cell migration (top panel) or
   invasion (bottom panel) in a trans-well system treated with or without
   4SC-202 under 10x objective magnification. (C) Quantification of panel
   A data. (D) Quantification of panel B data. ** p < 0.01, *** p < 0.001.
   Data are presented as mean (SD).

3.4. Cell Cycle Is Arrested at the G2/M-Phase Checkpoint by 4SC-202

   HDACi agents are known inducers of cell-cycle arrest and apoptosis in
   many cancer cell types [[222]108]. A dose-dependent disturbance of
   cell-cycle progression by 4SC-202 was observed in several cancer types
   [[223]83,[224]84,[225]85,[226]93]. To examine the cell-cycle behavior
   in human OS cells, unsynchronized SJSA-1 cells were incubated in the
   absence or presence of 1 μM 4SC-202 for 24 h before propidium iodide
   (PI) staining. Our flow cytometry analysis demonstrated that the
   percentage of cells at the G2/M phase in drug-treated cells
   significantly increased to 87.08% from 21.49% compared to vehicle
   control, whereas cells at the G1 and S phases in 4SC-202-treated cells
   decreased to 6.66% and 6.26%, respectively, and from 41.96% and 36.55%,
   respectively, in control ([227]Figure 5A,B). In parallel, we also
   observed a dramatic blockage of the cell cycle in hFOB cells
   ([228]Figure S3A,B). To further explore the anti-tumor growth effects
   of 4SC-202 in OS cells, we performed flow-cytometric quantification of
   an apoptotic response using annexin V and PI double staining on cells
   treated with the drug for 24 h. Treatment with 4SC-202 significantly
   increased the population of early-phase (Q4 quadrant: annexin V^+ PI^−)
   and late-phase (Q2 quadrant: annexin V^+ PI^+) apoptotic cells to
   10.63% and 10.33%, respectively ([229]Figure 5C,D). Notably, we
   observed no significant cell death (Q1 quadrant: Annexin V^- PI^+) in
   treated SJSA-1 cells compared to the control. Moreover, 4SC-202
   treatment showed a similar drug effect on hFOB cells, although a
   slightly different pattern with an increased population of early- and
   late-phase apoptotic and dead cells to 4.9%, 19.16%, and 9.13%,
   respectively ([230]Figure S3C,D). Collectively, these data indicate
   that cell-cycle arrest and apoptosis induced by 4SC-202 may be
   responsible for the acute anti-proliferative effect of the drug.

Figure 5.

   [231]Figure 5
   [232]Open in a new tab

   Effect of 4SC-202 on the cell-cycle distribution and apoptosis in human
   SJSA-1 OS cells. (A) Representative cell-cycle flow cytometry profiles
   of cells treated with or without 4SC-202 for 24 h. The DNA content of
   cells was analyzed by flow cytometry after staining with propidium
   iodide. (B) Relative cell population quantified data from cell-cycle
   profiles in A. (C) Representative flow cytometry scatter plots of cells
   treated with or without 4SC-202 for 24 h and stained with annexin V
   (positive for apoptotic cells) and propidium iodide. (D) Quantification
   of apoptotic profiles for relative cell population from C. ** p < 0.01.
   Data are presented as mean (SD).

3.5. 4SC-202 Is Incompetent at Initiating and Enhancing Osteogenic
Differentiation of Osteosarcoma Cells into Mineralizing Osteoblast-Like Cells

   Since osteosarcoma cells habitually possess an osteogenic
   differentiation program, we probed the possibility of 4SC-202 alone in
   promoting osteoblast-like differentiation (OB diff) phenotype. As shown
   in the left panels of [233]Figure 6A–C and consistent with our previous
   study [[234]100], SJSA-1 cells cultured with OB diff medium had a
   strong ability to differentiate into mature mineralizing cells to
   produce bone matrix and calcium (positively stained by Alizarin Red S).
   In the absence of OB diff agents, we found that 1 μM 4SC-202 treatment
   was incompetent at inducing osteogenic differentiation for 14 days
   ([235]Figure 6A). Furthermore, with the addition of 1µM of 4SC-202 to
   the OB diff medium, we found that SJSA-1 cells produced similar
   mineralized nodules ([236]Figure 6B) and content of calcium to cells
   treated with OB-diff alone ([237]Figure 6C). These data indicate that
   4SC-202 has little capacity to promote osteogenic differentiation.

Figure 6.

   [238]Figure 6
   [239]Open in a new tab

   Effect of 4SC-202 on osteoblast-like differentiation and
   mineralization. (A) Representative photo of 12-well plate wells stained
   with Alizarin Red S after treatment for two weeks with control media or
   osteoblast differentiation (OB Diff) media or control media + 4SC-202
   or OB Diff media + 4SC-202. (B) Representative images of the plate
   wells in A under 10× objective magnification. (C) Quantitative results
   of Alizarin Red S staining in all 4 groups in A. **** p < 0.0001
   (two-way ANOVA with Tukey method). Data are presented as mean (SD).

3.6. In Vitro 4SC-202 Treatment Revises Global Transcriptomic Profiling and
Induces Distinct Gene Expression Signatures in Human Osteosarcoma Cells

   To assess 4SC-202-induced alterations in global mRNA expression and
   gene signature, we performed whole transcriptomic profiling using an
   unbiased next-generation sequencing approach for SJSA-1 and hFOB cells.
   For this purpose, total RNA was extracted from cells treated for 24 h
   with 1 µM 4SC-202 or 0.1% DMSO and then subjected to shotgun RNA
   sequencing (RNA-seq) analysis. All samples showed an RNA Integrity
   Number (RIN) higher than 9.1 on a bioanalyzer, confirming the RNA
   integrity and quality ([240]Figure S4A,B). Principle component analysis
   of RNA-seq data revealed a marked separation of gene expression changes
   between the control and 4SC-202 treatment groups for each cell line
   (PC1, 97% for both) and a low level of variability among biological
   replicates (in triplicate) (PC2, 1% and 2% for SJSA-1 and hFOB,
   respectively) ([241]Figure S5A,B). Heatmaps of Euclidean distances
   between samples further substantiated the similarity of gene expression
   between samples from the same group (i.e., treated or untreated) as
   well as the differences between samples from different groups (i.e.,
   treated versus untreated) ([242]Figure S6A–C). 4SC-202 treatment
   profoundly changed the global transcriptome of the SJSA-1 and hFOB
   cells, as indicated by the total number of significant differentially
   expressed genes (DEGs) given in [243]Tables S1 and S2. Approximately
   half of DEGs were common to the two cell lines ([244]Figure S5E). Among
   them, the top 20 differentially expressed genes with the smallest
   p-value are labeled with gene symbols ([245]Figure 7A and [246]Figure
   S6A). The top 200 significantly upregulated or downregulated genes in
   the drug-treated groups are highlighted in [247]Figure 7B and
   [248]Figure S6B. Ingenuity pathway analysis (IPA) of the differentially
   expressed genes in the 4SC-202-treated compared to control cells
   highlighted that the human OS cells and transformed osteoblasts shared
   altered canonical signaling pathways, including Wnt/beta-catenin,
   cAMP-mediated signaling, calcium signaling, eNOS signaling, VDR/RXR
   activation, bladder cancer signaling, tumor microenvironment pathway,
   breast cancer regulation by Stathmin1, synaptogenesis signaling,
   pathway regulation of the Epithelial-Mesenchymal Transition (EMT)
   pathway, and the role of osteoblasts, osteoclasts, and chondrocytes in
   rheumatoid arthritis ([249]Figure 7C, [250]Figure S5F and [251]Figure
   S6C, [252]Tables S3 and S4).

Figure 7.

   [253]Figure 7
   [254]Open in a new tab

   Differentially expressed genes and pathways in human OS cells. (A) A
   volcano plot of gene expression from RNA-seq analysis between the
   vehicle control and 4SC-202-treated SJSA-1 OS cells. The top 20
   significantly differentially expressed genes are labeled. (B)
   Unsupervised hierarchical cluster analysis and heatmap representation
   of differentially expressed genes in SJSA-1 cells treated with either 1
   µM 4SC-202 or DMSO for 24 h. Intensity of color indicates expression
   levels (red, high; blue, low). Each column indicates a distinct sample,
   and each row indicates an individual gene. (C) Ingenuity pathways
   analysis (IPA) of cancer canonical signaling pathways associated with
   significantly regulated genes (p < 0.05) in 4SC-202-treated samples
   compared to the vehicle-treated samples.

   On the other hand, we uncovered that the Notch signaling pathway was
   significantly altered only in SJSA-1 cells ([255]Figure 7C). Because
   Notch and Wnt signaling are two of the most deregulated cancer
   signaling pathways in our data, we investigated individual genes that
   were significantly regulated upon 4SC-202 treatment. We found that Wnt
   antagonists WIF1 and FRZB were upregulated but WNT5A was downregulated.
   We also found that JAG2 and NOTCH4 mRNA and protein levels were
   significantly decreased upon the drug treatment ([256]Figure 8B,C).
   Although NOTCH3 mRNA levels were not significantly altered, we found a
   significant decrease in its protein levels upon increasing
   concentrations of 4SC-202 ([257]Figure 8B,C). Interestingly, we
   observed several significantly dysregulated key genes involved in
   cellular processes and cancer treatment, including cell cycle (GADD45A
   and GADD45B in [258]Figure 8D), apoptosis (BCL2L1 and CFLAR in
   [259]Figure 8E), stem-cell renewal and drug resistance (SOX2 and ABCB1
   in [260]Figure 8F), and immunotherapy (MICA and ULBP1 in [261]Figure
   8G). This implies that 4SC-202 induces distinct gene expression
   signatures in treated SJSA-1 OS cells.

Figure 8.

   [262]Figure 8
   [263]Open in a new tab

   Candidate genes regulated by 4SC-202 in human SJSA-1 OS cells. Selected
   differentially expressed genes in (A) the Wnt/ß-catenin signaling
   pathway and (B) the Notch signaling pathway. (C) Representative Western
   blot analysis of JAG2, NOTCH4, NOTCH3, and β-ACTIN in OS cells treated
   with or without 4SC-202. Selected differentially expressed genes in (D)
   cell cycle, (E) apoptosis, (F) cancer stem cells (CSCs) and multidrug
   resistance (MDR), and (G) immunotherapy. Average FPKM values from
   RNA-seq analysis were used. * p < 0.05, ** p < 0.01, *** p < 0.001,
   **** p < 0.0001. Data are presented as mean (SD) of triplicate samples
   treated with or without 4SC-202.

3.7. In Vivo 4SC-202 Treatment Reduces the Tumor Growth of Osteosarcoma in
Mice

   To assess the therapeutic potential of 4SC-202 on established human
   tumor xenografts in immune-compromised mice, we implanted SJSA-1 cells
   into the flanks of athymic nude mice and allowed tumors to grow for
   about two weeks until detectable ([264]Figure 9A). The mice were then
   randomly divided into two groups and treated daily with either vehicle
   or 4SC-202 for 16 days ([265]Figure 9A). Consistent with the observed
   in vitro effects, gross observation of the harvested tumors indicated a
   lessened tumor size in the 4SC-202-treated group ([266]Figure 9B).
   Compared to the vehicle mice, the average tumor mass of the treated
   group was significantly reduced by 70.56%. At the same time, no obvious
   body weight loss or pathological changes were observed during this
   treatment period with this dosage of 50 mg/kg, which was used in a
   previous study in treatment of bile duct cancer [[267]86]. It is also
   worth noting that, consistent with another previous study using the
   SJSA-1 OS model in nude mice [[268]109], no metastasis was observed in
   either group of mice.

Figure 9.

   [269]Figure 9
   [270]Open in a new tab

   4SC-202 reduces the tumor growth of SJSA-1 cell in vivo. (A)
   Illustration of in vivo experimental procedure using a xenograft model
   of nude mice. (B) Photographs of SJSA-1 xenografted tumors at harvest
   from nude mice. (C) Quantification of tumor burden of xenografted
   tumors treated with or without 4SC-202 (n = 7 for vehicle and n = 9 for
   4SC-202 treated mice, *** p < 0.001, data are presented as mean (SD)).
   (D) Proposed future studies on tumor cell behaviors of osteosarcoma.

4. Discussion

   Similar to “oncogene addiction,” the hypothesis of “epigenetic
   vulnerability of cancer cells” is evolving into a new axiom, which has
   been endorsed by increasing evidence from studies on HDACs and HDACi
   [[271]6,[272]20]. Compared with normal cells that have multi-tiered and
   redundant compensating epigenetic pathways or factors, some cancer
   cells mainly rely on specific epigenetic pathways or factors such as
   class I HDACs to sustain the function of key genes to maintain cell
   survival, growth, invasion, metastasis, and drug resistance. This study
   on the use of a selective HDACi, 4SC-202, against HDAC1-3 provides new
   evidence to support this hypothesis in osteosarcoma.

   Previous studies have shown that 4SC-202 has an anti-tumor effect
   against several types of hematologic malignancies and solid tumors
   [[273]83,[274]84,[275]85,[276]86,[277]87,[278]88,[279]89,[280]90,[281]9
   1,[282]92,[283]93]. Our study adds osteosarcoma to this catalog.
   [284]Figure 1C and [285]Figure 2C align with the previous findings that
   4SC-202 effectively targets HDAC1-3 but not LSD1
   [[286]83,[287]94,[288]110]. Consistent with the significantly increased
   expression in primary human osteosarcoma tissues [[289]32,[290]33],
   this study reports higher expression of HDAC1-3 in human SJSA-1 OS
   cells ([291]Figure 1A). A study using pan-HDACi (panobinostat) and a
   HDAC1/2 selective inhibitor (romidepsin) demonstrated that the combined
   functions of HDAC1 and HDAC2 may contribute to the maintenance of
   osteosarcoma growth and metastasis [[292]11]. Other studies using a
   HDAC2 selective inhibitor (CAY10683) and a HDAC1/3 selective inhibitor
   (MS-275) further showed that individual HDAC2 or HDAC3 may also
   contribute to OS behaviors [[293]74,[294]77,[295]111]. Moreover, HDAC1
   may have additional unique roles in OS progression and drug resistance
   [[296]47,[297]53,[298]72]. Our findings support the aforementioned
   studies on the role of HDAC1-3 in OS cell proliferation, invasion,
   metastasis, and cancer stem cell (CSC) maintenance ([299]Figure 9D). If
   the essential roles of HDAC1-3 in OS cells hold true, it is assumed
   that 4SC-202 may supersede the aforementioned pan-HDACi, which have
   systemic toxicity [[300]57], and some selective HDACi with a narrower
   selection, which may have limited clinical utility [[301]81]. Indeed, a
   study showed that the cardiotoxicity by aselective class I HDACi may be
   less than that of pan-HDACi and other selective HDACi due to fewer
   alterations in the expression of heart-specific genes [[302]112].
   However, there are 11 HDACs from classes I, II, and IV. The substrate
   specificity of each HDAC and the requirements for pan-HDACi and/or
   selective HDACi are still contestable. The HDACi that can achieve the
   safest and most effective therapeutic effects still needs to be
   determined. However, some concerns remain in considering the clinical
   use of highly selective HDACi, including 4SC-202, in OS patients given
   the yet unclear roles of HDAC1-3 as putative tumor suppressors in human
   osteosarcoma formation and their well-defined roles in the maintenance
   of in vivo bone mass and homeostasis [[303]32,[304]113]. Nevertheless,
   results from this in vitro and in vivo study using 4SC-202 imply an
   oncogenic role of HDAC1-3 in OS cells and suggest future preclinical
   studies for its combination therapy with other molecular-targeted
   agents such as inhibitors of signaling pathways ([305]Figure 9D).

   Across all HDAC inhibitors, induction of G1/S phase cell cycle arrest
   is dominant over a G2/M arrest [[306]26]. We found that 4SC-202 induced
   G2/M cell cycle arrest and apoptosis in SJSA-1 OS cells, which is
   consistent with the latest studies in other cancer types using this
   agent [[307]83,[308]84,[309]85,[310]93]. A G2/M cell cycle arrest is
   reported to be highly dependent on the upregulation of GADD45s, whereas
   a G1 arrest is considered to be highly dependent on the upregulation of
   p53/CDKN1A [[311]48]. Our finding that 4SC-202 significantly increased
   mRNA levels of GADD45A and GAD45B, whereas p53 expression was
   significantly decreased, provides a potential molecular mechanism of
   action of 4SC-202 ([312]Figure 8D and data not shown). We further
   provide a molecular basis for treatment-induced apoptosis of OS cells
   by showing a decrease in well-known anti-apoptosis genes (BCL2L1 and
   CFLAR/c-FLP) ([313]Figure 8E). This is consistent with previous studies
   that reported that expression of these two genes can be suppressed by
   various HDACi in cancer cells [[314]76,[315]78,[316]82,[317]114].

   Notably, some studies using SJSA-1 OS cells and the non-cancerous cell
   line hFOB as benign control cells found that they are not sensitive to
   a pan-HDACi, SAHA [[318]37,[319]46,[320]98]. In this study, we observed
   a strong inhibition of proliferation, colony formation, invasion,
   migration, cell cycle, and cell survival after treatment with 4SC-202
   in both cell lines at comparable concentrations ([321]Figure 1,
   [322]Figure 2, [323]Figure 3, [324]Figure 4 and [325]Figure 5 and
   [326]Figures S1–S3). In keeping with our observations, this phenomenon
   was reported in 4SC-202-treated urothelial cancer cells and HEK-293
   cells, a urothelial benign control cell line [[327]85]. Indeed, more
   recent studies have shown that both SJSA-1 and hFOB cells are very
   sensitive to the pan-HDACi panobinostat (10–15 nM) [[328]57,[329]59].
   It is not surprising that hFOB cells immortalized by SV40 TAg (large T
   antigen) behave like mesenchymal-stromal cells (MSCs)
   [[330]115,[331]116]. Furthermore, low-dose panobinostat has been shown
   to act predominantly as a potent “differentiating” agent that drives
   terminal osteoblast-like differentiation in OS cells [[332]57].
   Nevertheless, we showed that 4SC-202 cannot initiate and enhance
   osteoblastic differentiation of OS cells, indicating that 4SC-202 and
   panobinostat may have different effects and/or targets on
   osteoblast-like differentiation preprogram ([333]Figure 6). Together,
   our data support the concept that 4SC-202 is a strong inhibitor of
   proliferating cells but may be not a “differentiation agent”.

   Different mechanisms have been proposed for how 4SC-202 exerts its
   effects on tumor growth and survival. These include suppressing
   oncogenic hedgehog-GLI signaling in medulloblastoma cells
   [[334]89,[335]117], inhibiting NF-κB pathway signaling in
   myelodysplastic syndrome cells [[336]84], activating the ASK1/Cyp-D
   mitochondrial pathway in hepatocellular carcinoma cells [[337]92],
   promoting epithelial gene expression of BRD4 and MYC nuclear cofactors
   in pancreatic cells [[338]88], disabling microtubules and thereby
   directly affecting mitotic spindle formation [[339]83], and restoring
   immunogenic HLA class I surface gene expression on Merkel cell
   carcinoma cells [[340]93]. Our findings suggest that 4SC-202 has the
   potential to impair tumor growth and metastasis by directly suppressing
   Wnt and Notch signaling pathways in OS cells ([341]Figure 8C and
   [342]Figure 9A–C). Previously, our study and others have suggested that
   dysregulated Wnt and Notch pathways contribute to osteosarcoma
   initiation, progression, and metastasis
   [[343]5,[344]8,[345]99,[346]109,[347]118,[348]119,[349]120,[350]121].
   Notably, a recent study highlighted the essential roles for HDAC1–3 in
   chromatin regulatory complexes of rhabdomyosarcoma pediatric tumors,
   and that HDACi-induced hyperacetylation disrupts key interactions at
   super enhancers, resulting in decreased transcription at super enhancer
   core regulatory transcription factor genes. It is possible that this is
   a conserved HDACi mechanism, and thus that this is occurring with
   4SC-202-induced hyperacetylation as indicated by protein levels of
   acetylated histone 3 ([351]Figure 1C and [352]Figure 2C, and
   [353]Figure S2). Further studies are needed in order to unravel the
   core regulatory transcription factor circuitry in OS and how 4SC-202
   may deregulate these phenomena.

   Earlier studies using various OS cells found that treatment with a
   pan-HDACi or selective HDACi affects the gene expression of many
   components in Wnt and Notch pathways, such as β-catenin and Notch1
   [[354]41,[355]42,[356]59,[357]60,[358]122,[359]123]. In this study, we
   uncovered that 4SC-202 significantly affects several key components in
   Wnt and Notch pathways by either restoring (e.g., WIF1 and FRZB) or
   suppressing their expression (e.g., WNT5A, JAG2, NOTCH4 and NOTCH3) in
   OS cells ([360]Figure 8A,B). Interestingly, we previously showed that
   WNT5A is highly expressed in most human OS tissues [[361]99]. Other in
   vitro studies also proposed a crucial role of WNT5A in promoting OS
   cell invasiveness and migration [[362]124,[363]125,[364]126,[365]127].
   An earlier study showed that Wnt inhibitory factor 1 (WIF1) is
   epigenetically silenced in human osteosarcoma and that targeted
   disruption in mice accelerates osteosarcomagenesis [[366]120]. These
   studies suggest that the Wnt pathway may be an important target to
   treat OS. Moreover, Jag2 and Notch4 are highly expressed in some murine
   osteosarcomas [[367]109], and NOTCH3 has somatic copy-number
   alterations in about 10% of human osteosarcoma samples [[368]7].
   However, the roles of these Notch genes in osteosarcoma are still
   unclear, although they have been studied in other cancers
   [[369]128,[370]129]. Interestingly, we found that 4SC-202 may
   contribute to NOTCH3 protein degradation but has little effect on its
   mRNA transcription ([371]Figure 8B,C). Indeed, a study showed that
   NOTCH3 acetylation instructs its ubiquitination and proteasome-mediated
   degradation [[372]130], and our previous study showed that Notch
   proteins can form a nuclear complex with HDAC1 in OS cells [[373]131].
   Together, our data suggest that inhibition of Wnt and Notch signaling
   may contribute to 4SC-202′s effect on cell invasion and migration in
   vitro, although our model did not provide direct in vivo evidence
   ([374]Figure 9D).

   Emerging evidence implies that the epigenetic state is associated with
   drug resistance, maintenance of cancer stem cells (CSC), and metastasis
   [[375]132]. This may apply to resistance mechanisms of monotherapy
   using HDACi or in combination with standard-of-care therapy (MAP,
   consisting of methotrexate, adriamycin (doxorubicin), and platinol
   (cisplatin)) for OS patients. Several studies have proposed a mechanism
   of drug resistance using HDACi agents for osteosarcoma in which the
   multidrug resistance protein 1 (MDR1) encoded by the ABCB1 gene is
   upregulated in doxorubicin-resistant OS cells, which are also resistant
   to HDACi agents [[376]80,[377]82]. Consistent with the aforementioned
   studies, we report upregulation of ABCB1 by 4SC-202, implying that
   4SC-202-treated OS cells may survive through this mechanism
   ([378]Figure 8F). Furthermore, similarly to the MAP therapy, certain
   HDACi are powerful agents that kill highly proliferating OS cells but
   fail to suppress CSCs. In contrast, some HDACi may expand SOX2-positive
   CSC population [[379]65]. Interestingly, we found that 4SC-202
   downregulates expression of SOX2 ([380]Figure 8F). So far, the
   mechanism by which HDACi affect the CSC population has not been fully
   studied. However, the inhibitory effect of 4SC-202 on SOX2-positive OS
   cells warrants future research on how type I HDACs regulate the
   maintenance of OS CSC by regulating SOX2 function. Additionally, we
   also found that 4SC-202 upregulates expression of MICA and ULBP1, which
   encode ligands for an activating receptor—NKG2D, expressed on natural
   killer (NK) cells—and stimulate the NK cell-mediated cytotoxicity
   against cancer cells, indicating that 4SC-202 may provide favorable
   immune-modulatory effects on cancer treatment ([381]Figure 8G)
   [[382]77,[383]133]. In summary, our in vitro and in vivo results are
   promising, but further research on the potential effects of 4SC-202
   alone and in combination with other drugs in the context of
   patient-derived xenografts (PDXs), which are assumed to closely
   resemble the original human tumor samples, is necessary and important
   [[384]60,[385]134,[386]135,[387]136]. The potential partners of 4SC-202
   in combination therapy should favor agents that target autophagenesis,
   which promotes cancer cell survival, CSC signaling pathways such as Wnt
   and Notch, drug-resistant genes such as ABCB1, key components of the
   proteasome, and immune cells for immunotherapy.

5. Conclusions

   We have demonstrated that 4SC-202 inhibits osteosarcoma cell growth in
   vitro and in vivo, and advanced our understanding of the key roles of
   HDAC1, HDAC2, and HDAC3 in the biological behaviors of osteosarcoma.
   The anti-tumor effects were featured by combined induction of
   cell-cycle arrest at the G2/M phase, the apoptotic program, and a
   reduction in the invasive and migratory ability of osteosarcoma.
   4SC-202 has little capacity to promote osteogenic differentiation.
   4SC-202 revised the global transcriptome and induced distinct
   signatures of gene expression in osteosarcoma cells in vitro. In vivo,
   4SC-202 decreased tumor growth in established human tumor xenografts in
   immunodeficient mice. Mechanistically, we revealed key targets
   regulated by 4SC-202 that contribute to cell cycle, apoptosis, CSC
   stemness, drug resistance, immunotherapy, and canonical signaling
   pathways associated with the progression and metastasis of
   osteosarcoma. Furthermore, our data provide a rationale for further
   preclinical studies to access the efficacy of 4SC-202 as a second-line
   therapy to improve treatment options for metastatic osteosarcoma.

Acknowledgments