Apoptosis In Rhabdomyosarcoma Cells Research Articles

Pharmacological EZH2 inhibition combined with retinoic acid treatment promotes differentiation and apoptosis in rhabdomyosarcoma cells

BackgroundRhabdomyosarcomas (RMS) are predominantly paediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (< 30%), highlighting a need for novel therapies to improve outcomes. Differentiation therapeutics are agents that induce differentiation of cancer cells from malignant to benign. The histone methyltransferase, Enhancer of Zeste Homolog 2 (EZH2) suppresses normal skeletal muscle differentiation and is highly expressed in RMS tumours.ResultsWe demonstrate combining inhibition of the epigenetic modulator EZH2 with the differentiating agent retinoic acid (RA) is more effective at reducing cell proliferation in RMS cell lines than single agents alone. In PAX3-FOXO1 positive RMS cells this is due to an RA-driven induction of the interferon pathway resulting in apoptosis. In fusion negative RMS, combination therapy led to an EZH2i-driven upregulation of myogenic signalling resulting in differentiation. In both subtypes, EZH2 is significantly associated with enrichment of trimethylated lysine 27 on histone 3 (H3K27me3) in genes that are downregulated in untreated RMS cells and upregulated with EZH2 inhibitor treatment. These results provide insight into the mechanism that drives the anti-cancer effect of the EZH2/RA single agent and combination treatment and indicate that the reduction of EZH2 activity combined with the induction of RA signalling represents a potential novel therapeutic strategy to treat both subtypes of RMS.ConclusionsThe results of this study demonstrate the potential utility of combining EZH2 inhibitors with differentiation agents for the treatment of paediatric rhabdomyosarcomas. As EZH2 inhibitors are currently undergoing clinical trials for adult and paediatric solid tumours and retinoic acid differentiation agents are already in clinical use this presents a readily translatable potential therapeutic strategy. Moreover, as inhibition of EZH2 in the poor prognosis FPRMS subtype results in an inflammatory response, it is conceivable that this strategy may also synergise with immunotherapies for a more effective treatment in these patients.

CD44 and CD221 directed magnetic cubosomes for the targeted delivery of helenalin to rhabdomyosarcoma cells

Confining chemotherapy to tumour sites by means of active targeting nanoparticles (NPs) may increase the treatment effectuality while reducing potential side effects. Cubosomes are one of the next-generation drug delivery nanocarriers by virtue of their biocompatibility and bioadhesion, sizeable payload encapsulation and high thermostability. Herein, an active tumour targeting system towards rhabdomyosarcoma (RMS) cells was evaluated. Cubosomes were loaded with helenalin (a secondary metabolite from Arnica plants), which we have previously shown to induce apoptosis in RMS cells. The functionalization of the cubosomes was accomplished to enable binding to membrane receptors and translocation under a magnetic field. RMS cells overexpress CD44 and CD221 on their membrane surface and, therefore, hyaluronic acid (HA, a ligand for CD44) and antibodies (Abs) against CD221 were coupled to cubosomes via electrostatic attraction and the thiol-Michael reaction, respectively. Magnetization of the cubic phase NPs was achieved by embedding superparamagnetic iron oxide NPs (SPIONPs) into the cubic matrix. Single-function and multi-function cubosomes had Im3m cubic phase structures with well-organized lattice patterns. Conjugation with 2% HA or anti-CD221 half Abs and/or 1% SPIONPs showed significantly higher uptake into RMS cells compared to unfunctionalized cubosomes. CD44 and CD221 directed magnetic (triple-function) cubosomes were capable of internalizing into RMS cells in an energy-independent mechanism. Helenalin-laden triple functionalized cubosomes showed limited impact on the viability of control fibroblast cells, while they induced a high degree cytotoxicity against RMS cells. Profound tumour cell death was observed in both two-dimensional (2D) culture and three-dimensional (3D) tumour spheroids.

Mechanical Confinement and DDR1 Signaling Synergize to Regulate Collagen-Induced Apoptosis in Rhabdomyosarcoma Cells.

Fibrillar collagens promote cell proliferation, migration, and survival in various epithelial cancers and are generally associated with tumor aggressiveness. However, the impact of fibrillar collagens on soft tissue sarcoma behavior remains poorly understood. Unexpectedly, this study finds that fibrillar collagen‐related gene expression is associated with favorable patient prognosis in rhabdomyosarcoma. By developing and using collagen matrices with distinct stiffness and in vivo‐like microarchitectures, this study uncovers that the activation of DDR1 has pro‐apoptotic and of integrin β1 pro‐survival function, specifically in 3D rhabdomyosarcoma cell cultures. It demonstrates that rhabdomyosarcoma cell‐intrinsic or extrinsic matrix remodeling promotes cell survival. Mechanistically, the 3D‐specific collagen‐induced apoptosis results from a dual DDR1‐independent and a synergistic DDR1‐dependent TRPV4‐mediated response to mechanical confinement. Altogether, these results indicate that dense microfibrillar collagen‐rich microenvironments are detrimental to rhabdomyosarcoma cells through an apoptotic response orchestrated by the induction of DDR1 signaling and mechanical confinement. This mechanism helps to explain the preference of rhabdomyosarcoma cells to grow in and metastasize to low fibrillar collagen microenvironments such as the lung.

Selective BH3 mimetics synergize with BET inhibition to induce mitochondrial apoptosis in rhabdomyosarcoma cells.

BH3 mimetics are promising novel anticancer therapeutics. By selectively inhibiting BCL-2, BCL-xL, or MCL-1 (i.e. ABT-199, A-1331852, S63845) they shift the balance of pro- and anti-apoptotic proteins in favor of apoptosis. As Bromodomain and Extra Terminal (BET) protein inhibitors promote pro-apoptotic rebalancing, we evaluated the potential of the BET inhibitor JQ1 in combination with ABT-199, A-1331852 or S63845 in rhabdomyosarcoma (RMS) cells. The strongest synergistic interaction was identified for JQ1/A-1331852 and JQ1/S63845 co-treatment, which reduced cell viability and long-term clonogenic survival. Mechanistic studies revealed that JQ1 upregulated BIM and NOXA accompanied by downregulation of BCL-xL, promoting pro-apoptotic rebalancing of BCL-2 proteins. JQ1/A-1331852 and JQ1/S63845 co-treatment enhanced this pro-apoptotic rebalancing and triggered BAK- and BAX-dependent apoptosis since a) genetic silencing of BIM, BAK or BAX, b) inhibition of caspase activity with zVAD.fmk and c) overexpression of BCL-2 all rescued JQ1/A-1331852- and JQ1/S63845-induced cell death. Interestingly, NOXA played a different role in both treatments, as genetic silencing of NOXA significantly rescued from JQ1/A-1331852-mediated apoptosis but not from JQ1/S63845-mediated apoptosis. In summary, JQ1/A-1331852 and JQ1/S63845 co-treatment represent new promising therapeutic strategies to synergistically trigger mitochondrial apoptosis in RMS.

Co-targeting MCL-1 and ERK1/2 kinase induces mitochondrial apoptosis in rhabdomyosarcoma cells

The RAS/MEK/ERK genetic axis is commonly altered in rhabdomyosarcoma (RMS), indicating high activity of downstream effector ERK1/2 kinase. Previously, we have demonstrated that inhibition of the RAS/MEK/ERK signaling pathway in RMS is insufficient to induce cell death due to residual pro-survival MCL-1 activity. Here, we show that the combination of ERK1/2 inhibitor Ulixertinib and MCL-1 inhibitor S63845 is highly synergistic and induces apoptotic cell death in RMS in vitro and in vivo. Importantly, Ulixertinib/S63845 co-treatment suppresses long-term survival of RMS cells, induces rapid caspase activation and caspase-dependent apoptosis. Mechanistically, Ulixertinib-mediated upregulation of BIM and BMF in combination with MCL-1 inhibition by S63845 shifts the balance of BCL-2 proteins towards a pro-apoptotic state resulting in apoptosis induction. A genetic silencing approach reveals that BIM, BMF, BAK and BAX are all required for Ulixertinib/S63845-induced apoptosis. Overexpression of BCL-2 rescues cell death triggered by Ulixertinib/S63845 co-treatment, confirming that combined inhibition of ERK1/2 and MCL-1 effectively induces cell death of RMS cells via the intrinsic mitochondrial apoptotic pathway. Thus, this study is the first to demonstrate the cytotoxic potency of co-inhibition of ERK1/2 and MCL-1 for RMS treatment.

Protein Arginine Methyltransferase (PRMT) Inhibitors-AMI-1 and SAH Are Effective in Attenuating Rhabdomyosarcoma Growth and Proliferation in Cell Cultures.

Rhabdomyosarcoma (RMS) is a malignant soft tissue cancer that develops mostly in children and young adults. With regard to histopathology, four rhabdomyosarcoma types are distinguishable: embryonal, alveolar, pleomorphic and spindle/sclerosing. Currently, increased amounts of evidence indicate that not only gene mutations, but also epigenetic modifications may be involved in the development of RMS. Epigenomic changes regulate the chromatin architecture and affect the interaction between DNA strands, histones and chromatin binding proteins, thus, are able to control gene expression. The main aim of the study was to assess the role of protein arginine methyltransferases (PRMT) in the cellular biology of rhabdomyosarcoma. In the study we used two pan-inhibitors of PRMT, called AMI-1 and SAH, and evaluated their effects on proliferation and apoptosis of RMS cells. We observed that AMI-1 and SAH reduce the invasive phenotype of rhabdomyosarcoma cells by decreasing their proliferation rate, cell viability and ability to form cell colonies. In addition, microarray analysis revealed that these inhibitors attenuate the activity of the PI3K-Akt signaling pathway and affect expression of genes related to it.

The novel dual BET/HDAC inhibitor TW09 mediates cell death by mitochondrial apoptosis in rhabdomyosarcoma cells.

Targeting the epigenome of cancer cells with the combination of Bromodomain and Extra Terminal (BET) protein inhibitors and histone deacetylase (HDAC) inhibitors has shown synergistic antitumor effects in several cancer types. In this study, we investigate the antitumor potential of the novel dual BET/HDAC inhibitor TW09 in rhabdomyosarcoma (RMS) cells. TW09 reduces cell viability, suppresses long-term clonogenic survival and induces cell death in RMS cells in a dose-dependent manner. Compared to BET/HDAC co-inhibition using JQ1 and MS-275, TW09 induces similar cell death at equimolar concentrations and regulates BET and HDAC target proteins (e.g. c-MYC, H3 acetylation). Mechanistic studies revealed that TW09 upregulates BIM, NOXA, PUMA and BMF, while downregulating BCL-XL, leading to proapoptotic rebalancing of BCL-2 proteins. This results in BAK and BAX activation and caspase-dependent apoptosis, since individual genetic silencing of BIM, NOXA, PUMA, BMF, BAK or BAX, overexpression of BCL-2 or the caspase inhibition with zVAD.fmk all rescue JQ1/BYL719-induced cell death. In conclusion, TW09 shows potent antitumor activity in RMS cells in vitro by inducing mitochondrial apoptosis and may represent a promising new therapeutic option for the treatment of RMS.

Co-inhibition of BET proteins and PI3Kα triggers mitochondrial apoptosis in rhabdomyosarcoma cells.

Remodeling transcription by targeting bromodomain and extraterminal (BET) proteins has emerged as promising anticancer strategy. Here, we identify a novel synergistic interaction of the BET inhibitor JQ1 with the PI3Kα-specific inhibitor BYL719 to trigger mitochondrial apoptosis and to suppress tumor growth in models of rhabdomyosarcoma (RMS). RNA-Seq revealed that JQ1/BYL719 co-treatment shifts the overall balance of BCL-2 family gene expression towards apoptosis and upregulates expression of BMF, BCL2L11 (BIM), and PMAIP1 (NOXA) while downregulating BCL2L1 (BCL-xL). These changes were confirmed by qRT-PCR and western blot analysis. Ingenuity pathway analysis (IPA) of RNA-Seq data followed by validation qRT-PCR and western blot identified MYC and FOXO3a as potential transcription factors (TFs) upstream of the observed gene expression pattern. Immunoprecipitation (IP) studies showed that JQ1/BYL719-stimulated increase in BIM expression enhances the neutralization of antiapoptotic BCL-2, BCL-xL, and MCL-1. This promotes the activation of BAK and BAX and caspase-dependent apoptosis, as (1) individual silencing of BMF, BIM, NOXA, BAK, or BAX, (2) overexpression of BCL-2 or MCL-1 or (3) the caspase inhibitor N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (zVAD.fmk) all rescue JQ1/BYL719-induced cell death. In conclusion, co-inhibition of BET proteins and PI3Kα cooperatively induces mitochondrial apoptosis by proapoptotic re-balancing of BCL-2 family proteins. This discovery opens exciting perspectives for therapeutic exploitation of BET inhibitors in RMS.

Bayesian Modeling Identifies PLAG1 as a Key Regulator of Proliferation and Survival in Rhabdomyosarcoma Cells.

We recently developed a novel computational algorithm that incorporates Bayesian methodology to identify rhabdomyosarcoma disease genes whose expression level correlates with copy-number variations, and we identified PLAG1 as a candidate oncogenic driver. Although PLAG1 has been shown to contribute to other type of cancers, its role in rhabdomyosarcoma has not been elucidated. We observed that PLAG1 mRNA is highly expressed in rhabdomyosarcoma and is associated with PLAG1 gene copy-number gain. Knockdown of PLAG1 dramatically decreased cell accumulation and induced apoptosis in rhabdomyosarcoma cells, whereas its ectopic expression increased cell accumulation in vitro and as a xenograft and promoted G1 to S-phase cell-cycle progression. We found that PLAG1 regulates IGF2 expression and influences AKT and MAPK pathways in rhabdomyosarcoma, and IGF2 partially rescues cell death triggered by PLAG1 knockdown. The expression level of PLAG1 correlated with the IC50 of rhabdomyosarcoma cells to BMS754807, an IGF receptor inhibitor. IMPLICATIONS: Our data demonstrate that PLAG1 contributes to proliferation and survival of rhabdomyosarcoma cells at least partially by inducing IGF2, and this new understanding may have the potential for clinical translation.

MicroRNA-410-3p upregulation suppresses proliferation, invasion and migration, and promotes apoptosis in rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is one of the most common types of soft tissue sarcoma in children; however, the pathogenesis of RMS is unclear. MicroRNAs (miRs) are involved in the development and progression of RMS. The role of miR-410-3p in RMS cell invasion, migration, proliferation and apoptosis, and its possible mechanism were investigated in the current study. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to detect the expression of miR-410-3p in RMS tissues and cells. In addition, the present study investigated the expression levels of molecules associated with the epithelial-mesenchymal transition (EMT), including E-cadherin, N-cadherin, Slug and Snail, and apoptotic factors, including Bcl-2-associated X protein (bax), cleaved-caspase 3, cleaved poly (ADP-ribose) polymerase (PARP), p53 and Bcl-2. Cell Counting Kit-8, terminal deoxynucleotidyl transferase dUTP nick end labeling and Transwell assays were conducted to determine the functional roles of miR-410-3p. Exogenous expression of miR-410-3p inhibited RMS cell invasion, migration and proliferation, induced apoptosis, suppressed the expression of Snail, Slug, N-cadherin and Bcl-2, and increased the expression of E-cadherin, bax, cleaved-caspase 3, cleaved PARP and p53. In summary, it was proposed that miR-410-3p overexpression suppressed invasion, migration and proliferation, downregulated the expression of EMT-associated molecules, and promoted apoptosis and the expression of apoptotic factors in RMS cells. Therefore, miR-410-3p may serve as a novel tumor suppressor gene in RMS, and could possess diagnostic and therapeutic potentials for the treatment of RMS.

Erratum] OBP‑801, a novel histone deacetylase inhibitor, induces M‑phase arrest and apoptosis in rhabdomyosarcoma cells.

Subsequent to the publication of the above article, the authors have realized that errors were introduced into Fig.4 at the typesetting stage. Essentially, in Fig.4B, the P‑value should have read as "P=0.13" (not as 0.013), and in Fig4D, the labels for OBP- and OBP+ were set the wrong way around. The correct version of Fig.4, as originally submitted, is shown opposite. The Editor apologizes to the authors for introducing these errors into their figure, and to the readership for any inconvenience caused. [the original article was published in Oncology Reports 41: 643-649, 2019; DOI: 10.3892/or.2018.6813.

OBP‑801, a novel histone deacetylase inhibitor, induces M‑phase arrest and apoptosis in rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer of musculoskeletal origin. Despite multidisciplinary approaches, such as surgical resection, irradiation, and intensive chemotherapy, adopted for its treatment, the prognosis of patients with high‑risk RMS remains poor. Thus, molecularly targeted therapies are required to improve patient survival and minimize side effects. Histone deacetylases (HDACs) modify transcription by deacetylation of the lysine residues in chromatin histone tails and several non‑histone proteins. HDAC inhibitors, classes of compounds targeted to various HDAC proteins, are being studied for their roles in several types of cancers in a rigorous manner. This study aimed to investigate the potential of a novel HDAC inhibitor, OBP‑801, as a therapeutic agent for the treatment of RMS. We used 8 RMS cell lines in this study. Protein expression patterns, cell proliferation, cell cycle status, and apoptosis in RMS cells after OBP‑801 treatment invitro were investigated. We also studied the antitumor activity of OBP‑801 in an invivo xenograft mouse model. We observed cell cycle arrest at the M‑phase and apoptosis in all RMS cell lines after exposure to pharmacological levels of OBP‑801 for 24h. Immunofluorescence staining revealed that OBP‑801 may induce mitotic catastrophe via chromosome misalignment and reduced survivin expression, ultimately leading to apoptosis. Our results demonstrated that the novel HDAC inhibitor OBP‑801 was an effective inhibitor of RMS cell line proliferation and may be a potent therapeutic option for RMS.

Co-targeting of BET proteins and HDACs as a novel approach to trigger apoptosis in rhabdomyosarcoma cells

Histone acetylation marks exert essential functions in regulating gene expression. These marks are written by histone acetyltransferases (HATs), removed by histone deacetylases (HDACs) and read by e.g. BET proteins. While BET inhibitors are promising new anticancer drugs, little is yet known about their antitumor activity in rhabdomyosarcoma (RMS). We therefore investigated the efficacy of the prototypic BET inhibitor JQ1 alone or in combination with other epigenetic modifiers, namely HDAC inhibitors (HDACIs). Here, we discover a synergistic interaction of the panBET inhibitor JQ1 together with various HDACIs, i.e. Quisinostat (JNJ-26481585), Vorinostat (SAHA), Entinostat (MS-275) and Panobinostat (LBH589), inducing apoptosis in RMS cells, whereas JQ1 as single agent exhibits little cytotoxicity. Calculation of combination index (CI) confirmed the synergism of this combination. Importantly, JQ1 and JNJ-26481585 act in concert to suppress colony formation and to trigger apoptosis in an in vivo model. Mechanistic studies revealed that combination of JQ1 and JNJ-26481585 cooperatively upregulates BIM and BMF, while downregulating BCL-xL. This shifted ratio of pro- and antiapoptotic BCL-2 proteins engages activation of BAX and BAK and increases caspases-3 and -7 activity. Individual silencing of BIM or NOXA, overexpression of BCL-2 or MCL-1 as well as addition of the caspase inhibitor zVAD.fmk significantly rescue JQ1/JNJ-26481585-induced apoptosis. Thus, co-targeting of histone acetylation by concomitant inhibition of HDAC and BET proteins synergistically induces mitochondrial apoptosis by shifting the ratio of pro- and antiapoptotic BCL-2 proteins towards apoptosis. These findings indicate that combinatorial use of BET and HDACIs may represent a promising new strategy for the treatment of RMS.

Concomitant epigenetic targeting of LSD1 and HDAC synergistically induces mitochondrial apoptosis in rhabdomyosarcoma cells

The lysine-specific demethylase 1 (LSD1) is overexpressed in several cancers including rhabdomyosarcoma (RMS). However, little is yet known about whether or not LSD1 may serve as therapeutic target in RMS. We therefore investigated the potential of LSD1 inhibitors alone or in combination with other epigenetic modifiers such as histone deacetylase (HDAC) inhibitors. Here, we identify a synergistic interaction of LSD1 inhibitors (i.e., GSK690, Ex917) and HDAC inhibitors (i.e., JNJ-26481585, SAHA) to induce cell death in RMS cells. By comparison, LSD1 inhibitors as single agents exhibit little cytotoxicity against RMS cells. Mechanistically, GSK690 acts in concert with JNJ-26481585 to upregulate mRNA levels of the proapoptotic BH3-only proteins BMF, PUMA, BIM and NOXA. This increase in mRNA levels is accompanied by a corresponding upregulation of BMF, PUMA, BIM and NOXA protein levels. Importantly, individual knockdown of either BMF, BIM or NOXA significantly reduces GSK690/JNJ-26481585-mediated cell death. Similarly, genetic silencing of BAK significantly rescues cell death upon GSK690/JNJ-26481585 cotreatment. Also, overexpression of antiapoptotic BCL-2 or MCL-1 significantly protects RMS cells from GSK690/JNJ-26481585-induced cell death. Furthermore, GSK690 acts in concert with JNJ-26481585 to increase activation of caspase-9 and -3. Consistently, addition of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) significantly reduces GSK690/JNJ-26481585-mediated cell death. In conclusion, concomitant LSD1 and HDAC inhibition synergistically induces cell death in RMS cells by shifting the ratio of pro- and antiapoptotic BCL-2 proteins in favor of apoptosis, thereby engaging the intrinsic apoptotic pathway. This indicates that combined treatment with LSD1 and HDAC inhibitors is a promising new therapeutic approach in RMS.

Role of autophagy in temozolomide‐induced apoptosis in Rhabdomyosarcoma cells

Rhabdomyosarcoma (RMS) is a muscle‐derived tumor and is the most common pediatric soft tissue sarcoma representing 5% of all childhood cancers. RMS is a major clinical problem in pediatric oncology. Treatment of RMS with the oral alkylating agent temozolomide (TMZ), alone or in combination with other drugs, has recently received considerable interest. However, the mechanism of action of TMZ remains unclear. The aim of this investigation was to determine if autophagy modulates TMZ‐induced cell death in RMS cell lines. MTT assay and Nicoletti flow cytometry analysis were used to measure cell death and apoptosis in SJCRH30 (RH30) human RMS cells and the non‐transformed mouse myoblast cell line C2C12, following TMZ treatment (100 μM). We monitored autophagy using transmission electron microcopy (autophagosome and autophagolysosome formation) and immunoblotting (LC3 lipidation, Atg15‐12 conjugation, p62 degredation). The effect of bafilomycin A1 on TMZ‐induced cytotoxicity was also evaluated. We showed that TMZ decreased the viability of RMS cells in a dose/time‐dependent manner and induced accumulation of sub‐G1 cell population, representing apoptotic cells. Interestingly, TMZ induced apoptosis by 17‐fold in the RH30 cells, but only increased apoptosis by 2.7‐fold in C2C12 cells. In RH30 cells, TMZ decreased the expression of antiapoptotic proteins Bcl‐XL and Mcl‐1, and increased the expression of the death gene Nix. Moreover, we showed that TMZ altered biochemical markers of autophagy, such as LC3 lipidation, Atg5–12 conjugation, and p62 degradation, and induced morphological evidence autophagy, including accumulation of autophagosomes and autophagolysosome, determined by transmission electron. Finally, treatment of RMS cells with the autophagy flux inhibitor (Bafilomycin A1, 4 and 6 nM) had no significant effect TMZ‐induced cell death in either cell line. In conclusion, our investigation showed that TMZ induced simultaneous autophagy and apoptosis in both RH30 and C2C12 cells; however, cell death induction by TMZ does not appear to be dependent on Bafilomycin A1 inhibited processes, such as autophagosome‐lysosome fusion or autolysosome acidification.

Arsenic trioxide induces Noxa-dependent apoptosis in rhabdomyosarcoma cells and synergizes with antimicrotubule drugs

The prognosis of metastatic or relapsed rhabdomyosarcoma (RMS) is poor, highlighting the need of new treatment options. In the present study, we evaluated the in vitro efficacy of arsenic trioxide (ATO) in RMS, a FDA-approved drug used in pediatric leukemia. Here, we report that ATO exerts antitumor activity against RMS cells both as single agent and in combination with microtubule-targeting drugs. Monotherapy with ATO reduces cell viability, triggers apoptosis and suppresses clonogenic survival of RMS cells, at least in part, by transcriptional induction of the proapoptotic BH3-only protein Noxa. siRNA-mediated knockdown of Noxa significantly rescues ATO-mediated cell death, demonstrating that Noxa is required for cell death. Also, ATO suppresses endogenous Hedgehog (Hh) signaling, as it significantly reduces Gli1 transcriptional activity and expression levels of several Hh target genes. Furthermore, we identify synergistic induction of apoptosis by ATO together with several antimicrotubule agents including vincristine (VCR), vinblastine and eribulin. The addition of the broad-range caspase inhibitor zVAD.fmk or overexpression of the antiapoptotic protein Bcl-2 significantly reduce ATO/VCR-induced cell death, indicating that the ATO/VCR combination triggers caspase-dependent apoptosis via the mitochondrial pathway. In summary, ATO exerts antitumor activity against RMS, especially in combination with antimicrotubule drugs. These findings have important implications for the development of novel therapeutic strategies for RMS.

Combined chemical–genetic approach identifies cytosolic HSP70 dependence in rhabdomyosarcoma

Cytosolic and organelle-based heat-shock protein (HSP) chaperones ensure proper folding and function of nascent and injured polypeptides to support cell growth. Under conditions of cellular stress, including oncogenic transformation, proteostasis components maintain homeostasis and prevent apoptosis. Although this cancer-relevant function has provided a rationale for therapeutically targeting proteostasis regulators (e.g., HSP90), cancer-subtype dependencies upon particular proteostasis components are relatively undefined. Here, we show that human rhabdomyosarcoma (RMS) cells, but not several other cancer cell types, depend upon heat-shock protein 70 kDA (HSP70) for survival. HSP70-targeted therapy (but not chemotherapeutic agents) promoted apoptosis in RMS cells by triggering an unfolded protein response (UPR) that induced PRKR-like endoplasmic reticulum kinase (PERK)-eukaryotic translation initiation factor α (eIF2α)-CEBP homologous protein (CHOP) signaling and CHOP-mediated cell death. Intriguingly, inhibition of only cytosolic HSP70 induced the UPR, suggesting that the essential activity of HSP70 in RMS cells lies at the endoplasmic reticulum-cytosol interface. We also found that increased CHOP mRNA in clinical specimens was a biomarker for poor outcomes in chemotherapy-treated RMS patients. The data suggest that, like human epidermal growth factor receptor 2 (HER2) amplification in breast cancer, increased CHOP in RMS is a biomarker of decreased response to chemotherapy but enhanced response to targeted therapy. Our findings identify the cytosolic HSP70-UPR axis as an unexpected regulator of RMS pathogenesis, revealing HSP70-targeted therapy as a promising strategy to engage CHOP-mediated apoptosis and improve RMS treatment. Our study highlights the utility of dissecting cancer subtype-specific dependencies on proteostasis networks to uncover unanticipated cancer vulnerabilities.

JNJ-26481585 primes rhabdomyosarcoma cells for chemotherapeutics by engaging the mitochondrial pathway of apoptosis

Rhabdomyosarcoma (RMS) is a common soft-tissue sarcoma in childhood with a poor prognosis, highlighting the need for new treatment strategies. Here we identify a synergistic interaction of the second-generation histone deacetylase inhibitor (HDACI) JNJ-26481585 and common chemotherapeutic drugs (i.e. Doxorubicin, Etoposide, Vincristine, Cyclophosphamide and Actinomycin D) to trigger apoptosis in RMS cells. Importantly, JNJ-26481585/Doxorubicin cotreatment also significantly suppresses long-term clonogenic survival of RMS cells and tumor growth in vivo in a preclinical RMS model. Mechanistically, JNJ-26481585/Doxorubicin cotreatment causes upregulation of the BH3-only proteins Bim and Noxa as well as downregulation of the antiapoptotic proteins Mcl-1 and Bcl-xL. These changes in the ratio of pro- and antiapoptotic Bcl-2 proteins contribute to JNJ-26481585/Doxorubicin-mediated apoptosis, since knockdown of Bim or Noxa significantly inhibits cell death. Also, JNJ-26481585 and Doxorubicin cooperate to stimulate activation of Bax and Bak, which is required for JNJ-26481585/Doxorubicin-induced apoptosis, since silencing of Bax or Bak protects against apoptosis. Consistently, overexpression of Bcl-2 significantly reduces JNJ-26481585/Doxorubicin-mediated apoptosis. JNJ-26481585/Doxorubicin cotreatment leads to caspase activation and caspase-dependent apoptosis, since the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) rescues cells from apoptosis. In conclusion, the second-generation HDACI JNJ-26481585 cooperates with chemotherapeutics to engage mitochondrial apoptosis in RMS cells, demonstrating that JNJ-26481585 represents a promising strategy for chemosensitization of RMS.

Preclinical evaluation of perifosine as a potential promising anti-rhabdomyosarcoma agent.

Rhabdomyosarcoma (RMS) is a highly malignant and metastatic pediatric cancer that arises from the skeletal muscle. Recent studies have identified an important role of AKT signaling in RMS progression. In the current study, we investigated the activity of perifosine, an oral alkylphospholipid AKT inhibitor, against human RMS cells (RD and Rh-30 lines) both in vivo and in vitro, and studied the underlying mechanisms. We showed that perifosine significantly inhibited RMS cell growth in concentration- and time-dependent manners. Meanwhile, perifosine induced dramatic apoptosis in RMS cells. At the signaling level, perifosine blocked AKT activation, while inducing reactive oxygen species (ROS) production as well as JNK and P38 phosphorylations in RMS cells. Restoring AKT activation by introducing a constitutively active-AKT (CA-AKT) only alleviated (not abolished) perifosine-induced cytotoxicity in RD cells. Yet, the ROS scavenger N-acetyl cysteine (NAC) as well as pharmacological inhibitors against JNK (SP-600125) or P38 (SB-203580) suppressed perifosine-induced cytotoxicity in RMS cells. Thus, perifosine induces growth inhibition and apoptosis in RMS cells through mechanisms more than just blocking AKT. In vivo, oral administration of perifosine significantly inhibited growth of Rh-30 xenografts in severe combined immunodeficient (SCID) mice. Our data indicate that perifosine might be further investigated as a promising anti-RMS agent.

Abstract 28: Deregulation of NAC complex inhibits muscle differentiation and blocks apoptosis in rhabdomyosarcoma cells

Abstract Rhabdomyosarcoma (RMS) is a common malignant soft tissue sarcoma in children with limited treatment options and high failure rate during standard therapy. New therapeutic targets or strategies are therefore in high demand. The NAC complex performs many diverse biological functions, and the deregulation of its subunits has been correlated with many cancers. We sought to understand the function of NAC complex in normal muscle cells and rhabdomyosarcoma cells. With western blot, quantitative real time PCR (qRT-PCR), chromatin immunoprecipitation and immunostaining analysis, we found that the muscle specific subunit of NAC complex, skNAC, which is the alternatively spliced isoform of NACα, was downregulated in RMS cells. In normal cells, skNAC shuttled from the cytoplasm to the nucleus upon differentiation. We also showed that skNAC associated with muscle specific promoters together with BTF3 in differentiated cells, and this association is dependent on the expression of BTF3. The expression of skNAC was regulated by the splicing factor SRPK3, a serine arginine protein kinase. Overexpression of SPRK3 induced skNAC expression and muscle differentiation. We also detected the deregulation of another NAC complex subunit, NACβ (also known as BTF3). We found that BTF3 is highly overexpressed and localized in the nucleus in both RMS cell lines and human RMS tumor samples compared with normal samples. Depletion of BTF3 induced apoptosis as detected by the strong cleavage of PARP and caspase 3. BTF3 depletion also decreased the cell viability in RMS by a cologenic assay. We also confirmed that BTF3 downregulation promoted muscle gene expression and induced muscle differentiation in RMS cells. However, BTF3 played a different role in normal cells. With the deletion of BTF3 in C2C12 cells, no apoptosis signal was detected and cell differentiation was inhibited, which suggested that a precise level of BTF3 is important for normal cell function. In conclusion, we found that the NAC complex subunits, skNAC and BTF3, were deregulated in RMS cells. The restoration of skNAC by SRPK3 overexpression rescued muscle differentiation. BTF3 functioned as an anti-apoptosis factor in RMS cells and depletion of BTF3 induced apoptosis and promoted differentiation. With no apoptosis effect in normal cells, BTF3 could serve as a cancer specific therapeutic target in RMS cells. Citation Format: Meiling Zhang, Judith Davie. Deregulation of NAC complex inhibits muscle differentiation and blocks apoptosis in rhabdomyosarcoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 28. doi:10.1158/1538-7445.AM2015-28