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Low-dose LBH589 treatment induces cell cycle arrest and senescence. Heatmap representation of differentially expressed genes enriched in functional groups associated with (a) cell cycle regulation, (b) senescence-associated secretory phenotype, and (c) apoptosis. Each column represents a distinct sample.
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Histone deacetylase inhibitors (HDACi) were identified nearly four decades ago based on their ability to induce cellular differentiation. However, the clinical development of these compounds as cancer therapies has focused on their capacity to induce apoptosis in hematologic and lymphoid malignancies, often in combination with conventional cytotoxi...
Citations
... The differentiation potential of HDACi, such as panobinostat, vorinostat or trichostatin A has been described in normal adipocyte development, mesenchymal stem cells, and undifferentiated myeloid malignancies [20][21][22]. Moreover, our previous work with panobinostat in osteosarcoma and extracranial rhabdoid tumour demonstrate the ability of sustained low-dose HDACi to inhibit tumour growth and drive multi-lineage cellular differentiation in solid malignancies [23,24]. ...
... Upon reaching a bioluminescence intensity of 1 × 10 6 , mice were randomly assigned to receive vehicle control or 5 mg/kg panobinostat daily via intraperitoneal injection. We previously demonstrated that this dose is tolerable and efficacious in both osteosarcoma and extracranial malignant rhabdoid tumour models [23,24]. Vehicle control-treated mice demonstrated progressive tumour growth and a median survival of 21 days (range 14-23 days) following commencement of treatment (Figure 5a,b). ...
... ATRT is an aggressive malignancy primarily affecting infants and young children with poor outcomes, highlighting the urgent need for improved therapeutic strategies. As an emerging class of anticancer therapy, HDACi has been explored in a large range of hematological and solid tumours [23,24,59]. Here, we show that sustained, low-dose panobinostat treatment inhibits tumour growth and drives neuronal differentiation in ATRT. ...
Atypical teratoid rhabdoid tumour (ATRT) is a rare but highly aggressive undifferentiated solid tumour arising in the central nervous system and predominantly affecting infants and young children. ATRT is exclusively characterized by the inactivation of SMARCB1, a member of the SWI/SNF chromatin remodelling complex that is essential for the regulation of large sets of genes required for normal development and differentiation. Histone deacetylase inhibitors (HDACi) are a promising anticancer therapy and are able to mimic the normal acetylation functions of SMARCB1 in SMARCB1-deficient cells and drive multilineage differentiation in extracranial rhabdoid tumours. However, the potential efficacy of HDACi in ATRT is unknown. Here, we show that human ATRT cells are highly responsive to the HDACi panobinostat and that sustained treatment leads to growth arrest, increased cell senescence, decreased clonogenicity and induction of a neurogenesis gene-expression profile. Furthermore, in an orthotopic ATRT xenograft model, continuous panobinostat treatment inhibits tumour growth, increases survival and drives neuronal differentiation as shown by the expression of the neuronal marker, TUJ1. Collectively, this preclinical study supports the therapeutic potential of panobinostat-mediated differentiation therapy for ATRT.
... Our findings support the aforementioned studies on the role of HDAC1-3 in OS cell proliferation, invasion, metastasis, and cancer stem cell (CSC) maintenance ( 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 [57], and some selective HDACi with a narrower selection, which may have limited clinical utility [81]. Indeed, a study showed that the cardiotoxicity by aselective class I HDACi may be ...
... Our findings support the aforementioned studies on the role of HDAC1-3 in OS cell proliferation, invasion, metastasis, and cancer stem cell (CSC) maintenance ( 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 [57], and some selective HDACi with a narrower selection, which may have limited clinical utility [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 [112]. ...
... 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 [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) [57,59]. It is not surprising that hFOB cells immortalized by SV40 TAg (large T antigen) behave like mesenchymal-stromal cells (MSCs) [115,116]. ...
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.
... 96,97 However, studies have also found that low doses of LBH589 can induce terminal differentiation and irreversible mitotic arrest, but not cell death, in committed osteogenic progenitors. 98 Treatment with panobinostat upregulates osteogenic differentiation genes, including RUNX2, ALPL, BMP4 and SPP1. ...
For multicellular organisms, it is essential to produce a variety of specialized cells to perform a dazzling panoply of functions. Chromatin plays a vital role in determining cellular identities, and it dynamically regulates gene expression in response to changing nutrient metabolism and environmental conditions. Intermediates produced by cellular metabolic pathways are used as cofactors or substrates for chromatin modification. Drug analogues of metabolites that regulate chromatin-modifying enzyme reactions can also regulate cell fate by adjusting chromatin organization. In recent years, there have been many studies about how chromatin-modifying drug molecules or metabolites can interact with chromatin to regulate cell fate. In this review, we systematically discuss how DNA and histone-modifying molecules alter cell fate by regulating chromatin conformation and propose a mechanistic model that explains the process of cell fate transitions in a concise and qualitative manner.
... Six week old male NOD/SCID mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). First, 1 × 10 7 U-2 OS/luc2 cells (200 µL of 1: 1 mixed cell suspension and matrigel) were subcutaneously injected into the left flanks of mice to establish a U-2 OS xenograft animal model [21]. A total of 20 mice were randomly divided into 2 groups after tumors reached 100 mm 3 , including non-treated control (CTRL, 0.1% DMSO) and regorafenib (10 mg/kg/day by gavage for 14 days). ...
... Our current results also presented that both PD98059 (MEK/ERK pathway inhibitor) and miltefosine (AKT inhibitor) both inhibit the expression of tumor progression-associated proteins (VEGF, MMP-9, Cyclin-D1, C-FLIP, MCL-1, and XIAP) in osteosarcoma U-2 OS cells in vitro ( Figure 4F,G). In our previous studies, we indicated that regorafenib inhibited ERK and AKT phosphorylation in HCC in vitro and in vivo [9,21]. In this present study, we also proved that regorafenib reduces protein levels of pERK and pAKT (Ser473) in osteosarcoma U-2 OS cells and MG-63 cells ( Figure 4C,D). ...
Osteosarcoma is the most common type of bone cancer. Multimodality treatment involving chemotherapy, radiotherapy and surgery is not effective enough to control osteosarcoma. Regorafenib, the oral multi-kinase inhibitor, has been shown to have positive efficacy on disease progression delay in chemotherapy resistant osteosarcoma patients. However anti-cancer effect and mechanism of regorafenib in osteosarcoma is ambiguous. Thus, the aim of this study is to investigate the efficacy and molecular mechanism of regorafenib on osteosarcoma in vitro and in vivo. Human osteosarcomas U-2 OS or MG-63 were treated with regorafenib, miltefosine (protein kinase B (AKT) inhibitor), or PD98059 (mitogen-activated protein/extracellular signal-regulated kinase (MEK) pathway inhibitor) for 24 or 48 h. Cell viability, apoptotic signaling transduction, tumor invasion, expression of tumor progression-associated proteins and tumor growth after regorafenib treatment were assayed by MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, transwell assay, Western blotting assay and in vivo animal experiment, respectively. In these studies, we also indicated that regorafenib suppressed cell growth by prompting apoptosis of osteosarcoma cells, which is mediated through inactivation of ERK and AKT signaling pathways. After regorafenib treatment, downregulation of related genes in invasion (vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9)), proliferation (CyclinD1) and anti-apoptosis (X-linked inhibitor of apoptosis protein (XIAP), myeloid cell leukemia-1 (MCL-1), and cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (C-FLIP)) were found. Moreover, upregulation of caspase-3 and caspase-8 cleavage were also observed. In sum, we suggest that regorafenib has potential to suppress osteosarcoma progression via inactivation of AKT and ERK mediated signaling pathway.
... HDAC inhibitors (HDACi) are of potential therapeutic benefit in pediatric patients. Indeed, the potential for low dose, continuous exposure to panobinostat to induce terminal differentiation in pediatric pre-clinical models of pediatric cancer has now been well established [16][17][18]. HDACi may also act synergistically with current therapies. HDACi cause apoptosis of human neuroblastoma cells in vitro with an enhanced inhibitory effect when combined with retinoic acid [19,20]. ...
... Indeed, a chemical screen in patient derived DIPG cell cultures has confirmed the HDACi panobinostat as being a promising therapeutic strategy for this class of tumors [27]. This, coupled with the published data on panobinostat promoting differentiation in pediatric tumors [16][17][18], provides an attractive therapeutic option. ...
Purpose:
This was an open label, phase I (3 + 3 design), multi-centre study evaluating panobinostat in pediatric patients with refractory solid tumors.
Methods:
Primary endpoints were to establish MTD, define and describe associated toxicities, including dose limiting toxicities (DLT) and to characterize its pharmacokinetics (PK). Secondary endpoints included assessing the anti-tumour activity of panobinostat, and its biologic activity, by measuring acetylation of histones in peripheral blood mononuclear cells.
Results:
Nine patients were enrolled and treated with intravenous panobinostat at a dosing level of 15 mg/m2 which was tolerated. Six were evaluable for adverse events. Two (33%) patients experienced Grade 3-4 thrombocytopenia, 1 (17%) experienced Grade 3 anemia, and 2 (33%) experienced Grade 3 neutropenia. Grade 4 drug related pain occurred in 2 (33%) of the patients studied. Two (33%) patients experienced a Grade 2 QTcF change (0.478 ± 0.006 ms). One cardiac DLT (T wave changes) was reported. PK values for 15 mg/m2 (n = 9) dosing were: Tmax 0.8 h, Cmax 235.2 ng/mL, AUC0-t 346.8 h ng/mL and t1/2 7.3 h. Panobinostat significantly induced acetylation of histone H3 and H4 at all time points measured when compared to pre-treatment samples (p < 0.05). Pooled quantitative Western blot data confirmed that panobinostat significantly induced acetylation of histone H4 at 6 h (p < 0.01), 24 h (p < 0.01) and 28-70 h (p < 0.01) post dose.
Conclusion:
A significant biological effect of panobinostat, measured by acetylation status of histone H3 and H4, was achieved at a dose of 15 mg/m2. PK data and drug tolerability at 15 mg/m2 was similar to that previously published.
... Interestingly, the use of bone cement loaded with histone deacetylase inhibitors has shown to be associated with a lower rate of metastasis and a better survival in patients affected by osteosarcoma and chondrosarcoma [36]. Panobinostat has also shown to cause differentiation at low dosage leading to reduction of tumour growth in osteosarcoma xenografts [37]. ...
Osteosarcoma is an aggressive cancer with a poor long term prognosis. Neo-adjuvant poly-chemotherapy followed by surgical resection remains the standard treatment, which is restricted by multi-drug resistance. If first-line therapy fails, disease control and patient survival rate drop dramatically. We aimed to identify alternative apoptotic mechanisms induced by the histone deacetylase inhibitor panobinostat in osteosarcoma cells.
Saos-2, MG63 and U2-OS osteosarcoma cell lines, the immortalized human osteoblast line hFOB and the mouse embryo osteoblasts (MC3T3-E1) were treated with panobinostat. Real time viability and FACS confirmed the cytotoxicity of panobinostat. Cell stress/death related factors were analysed by RT-qPCR and western blot. Cell morphology was assessed by electron microscopy.
10 nM panobinostat caused cell viability arrest and death in all osteosarcoma and osteoblast cells. P21 up-regulation was observed in osteosarcoma cells, while over-expression of p73 was restricted to Saos-2 (TP53−/−).
Survivin and Bcl-2 were suppressed by panobinostat. Endoplasmic reticulum (ER) stress markers BiP, CHOP, ATF4 and ATF6 were induced in osteosarcoma cells. The un-spliced Xbp was no further detectable after treatment.
Autophagy players Beclin1, Map1LC3B and UVRAG transcripts over-expressed after 6 hours. Protein levels of Beclin1, Map1LC3B and p62 were up-regulated at 72 hours. DRAM1 was stable. Electron micrographs revealed the fragmentation and the disappearance of the ER and the statistically significant increase of autophagosome vesiculation after treatment.
Panobinostat showed a synergistic suppression of survival and promotion of cell death in osteosarcoma cells. Panobinostat offers new perspectives for the treatment of osteosarcoma and other malignant bone tumours.
... Other epigenetic modifiers, such as inhibitors of histone deacetylase (HDAC) and histone methyltransferase (HMT), were subsequently investigated for inducing senescence (Supplementary Table 1). The Class I and II HDAC inhibitors were characterized, such as sodium butyrate in gynecologic cancer cells, human fibrosarcoma HT1080 cells and human or mouse fibroblast [118,[140][141][142][143][144][145], saha (vorinostat) in human colon cancer HCT116 cells and human breast cancer MCF-7, MDA-MB-231 and MBA-MD-468 cells [146], LBH589 (panobinostat) in osteosarcoma cells [147], 4phenylbutyric acid in human breast cancer MCF-7 cells [148] and valproic acid in medulloblastomas, supratentorial primitive neuroectodermal tumor cells and human hepatocarcinoma cells [149,150], etc. Other HDAC inhibitors, including trichostatin A and MS-275, were characterized in human diploid fibroblasts and mesenchymal stem cells, respectively [89,118,140,143,151]. ...
Background:
Recently, the chemotherapeutic drug-induced cellular senescence is considered per se a promising anti-cancer approach. This drug-induced senescence, which shows both similar, and different hallmarks from replicative senescence and oncogene-induced senescence, was regarded as a key determinant of tumor response to chemotherapy in vitro and in vivo. To date, plenty of effective chemotherapeutic drugs that evoke senescence in cancer cells have been reported. The targets of these drugs differ substantially from each other, as well as from DNA damage response, telomerase activity inhibition through senescence-related CDK, p53 and Rb signaling pathways.
Objective:
The purpose of this review is to summarize these senescence-targeting small-molecular drugs and remark their specific traits and corresponding mechanisms that offer novel perspectives in cancer therapy.
Method:
We collected information and data from manuscripts, publications and online database.
Result:
In this review, 50 senescence-targeting small-molecular drugs were summarized with emphasis on their molecular structures, targets/mechanisms as well as the effects for inducing senescent phenotype in certain cancer cell lines.
Conclusion:
To date, plenty of effective small-molecular chemotherapeutic drugs were exploited to evoke cellular senescence in cancer therapy through different mechanisms and pathways. Insight into the mechanisms and signaling pathways of these senescence-targeting chemotherapeutic drugs will facilitate the successful treatment of cancers in clinic.
... n = 5-10, **P < 0.01 unpaired ttest. h A heat map depicting differentially expressed genes from preimmortal wild type (WT), EsrCre and Hic1KO MEFs 48 h following treatment with tamoxifen, performed by the Australia Genome Research Facility (Melbourne, VIC, Australia) using the MouseWG-6 v2.0 Expression BeadChip (Illumina, San Diego, CA, USA) as previously described [69]. Detailed bioinformatic methods are described in Supplementary Information. ...
Hypermethylated-in-Cancer 1 (Hic1) is a tumor suppressor gene frequently inactivated by epigenetic silencing and loss-of-heterozygosity in a broad range of cancers. Loss of HIC1, a sequence-specific zinc finger transcriptional repressor, results in deregulation of genes that promote a malignant phenotype in a lineage-specific manner. In particular, upregulation of the HIC1 target gene SIRT1, a histone deacetylase, can promote tumor growth by inactivating TP53. An alternate line of evidence suggests that HIC1 can promote the repair of DNA double strand breaks through an interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex. Using a conditional knockout mouse model of tumor initiation, we now show that inactivation of Hic1 results in cell cycle arrest, premature senescence, chromosomal instability and spontaneous transformation in vitro. This phenocopies the effects of deleting Brca1, a component of the homologous recombination DNA repair pathway, in mouse embryonic fibroblasts. These effects did not appear to be mediated by deregulation of Hic1 target gene expression or loss of Tp53 function, and rather support a role for Hic1 in maintaining genome integrity during sustained replicative stress. Loss of Hic1 function also cooperated with activation of oncogenic KRas in the adult airway epithelium of mice, resulting in the formation of highly pleomorphic adenocarcinomas with a micropapillary phenotype in vivo. These results suggest that loss of Hic1 expression in the early stages of tumor formation may contribute to malignant transformation through the acquisition of chromosomal instability.
... 8 Growing evidence from in vitro and in vivo animal studies has demonstrated that histone deacetylase inhibitors exert their anti-tumor activity in osteosarcoma both as a single drug 10 and acting synergistically with other anticancer agents, [11][12][13][14] sensitize cancer cells to radiation, 15 increase natural killer cellmediated cytotoxicity 16 as well as inducing cell differentiation. 17 These experimental results suggest the probable future use of histone deacetylase inhibitors in osteosarcoma. However, little is known about the mechanisms of histone deacetylases in carcinogenesis and the expression patterns of histone deacetylase isoforms in actual human osteosarcoma tissues. ...
Epigenetic aberrations are recognized as having pivotal roles in cancer etiology and progression. Histone deacetylases are among the most studied epigenetic modulators in various cancer types. The expression levels of class I histone deacetylase isoforms 1, 2, and 3 in patient-derived primary osteosarcoma cells (6 cases) was investigated, comparing them to normal bone graft-derived osteoblasts (6 cases) using the immunoblotting technique. Expression profiles of histone deacetylases in high-grade osteosarcoma tissue of 89 patients were examined and their association with clinicopathologic parameters and the patient survival was evaluated. Histone deacetylases were immunohistochemically stained on formalin-fixed paraffin-embedded biopsied tissue. Primary osteosarcoma cells expressed higher levels of histone deacetylase 1 and histone deacetylase 2, but lower levels of histone deacetylase 3 compared to benign osteoblasts. Overall, 82, 99, and 93% of 89 osteosarcomas showed nuclear expression of the histone deacetylase isoforms 1, 2, and 3, respectively. Low levels of histone deacetylase 1 were significantly associated with a high Enneking stage (P=0.014) and the presence of initial metastasis (P=0.040), while low levels of histone deacetylase 3 were significantly correlated with age >15 years (P=0.026). Univariate survival analysis found significantly shorter survival in the patients with a high Enneking stage (P<0.001), axial location (P=0.009), presence of initial metastasis (P<0.001), low-histone deacetylase 1 expression (P=0.038), and low-all-histone deacetylases expression (P=0.016). Multivariate survival analysis showed that only axial location (P=0.011) and low-all-histone deacetylases expression (P=0.039) were independent prognostic factors. In subgroup analysis of stage IIB patients (n=45), only axial location and low-all-histone deacetylases expression were associated with shorter survival in both univariate and multivariate analysis (axial location, P=0.008 and 0.010; low-all-HDACs, P=0.013 and 0.038, respectively). Low levels of all-histone deacetylases expression were significantly associated with advanced disease status and short survival. These findings may be a guide to future use of histone deacetylase inhibitors in osteosarcoma patients.
... Inhibition of HDACs can lead to cell cycle arrest, apoptosis and, in some cases, differentiation. Panobinostat, a broad spectrum HDAC inhibitor, has been approved for use in multiple myeloma and a number of pre-clinical studies have shown efficacy for HDAC inhibitors against osteosarcoma either as a single agent or in combination with other therapies [51][52][53]. Recently, a combination of panobinostat with the proteasome inhibitor, carfilzomib has been shown to be highly synergistic in promoting apoptosis of multiple osteosarcoma cell lines [54]. ...
The survival rates for patients with osteosarcoma has remained almost static for the past three decades. Current standard of care therapy includes chemotherapies such as doxorubicin, cisplatin, and methotrexate along with complete surgical resection and surgery with or without ifosfamide and etoposide for relapse, though outcomes are hoped to be improved through clinical trials. Additionally, increased understanding of the genetics, signaling pathways and microenvironmental factors driving the disease has led to the identification of promising agents and potential paths towards translation of an exciting array of novel targeted therapies. Here, we review the mechanism of action of these emerging therapies and how, with clinical translation, they can potentially improve the survival rates for osteosarcoma patients in the near future.
