Human Rhabdomyosarcoma Cells Research Articles

Bioassay-guided isolation of cytotoxic constituents of Aframomum melegueta K.Schum. seeds

The seeds of Aframomum melegueta are used extensively in the Nigerian ethnomedicine for the management of cancer. This study therefore aimed at isolating and characterizing its cytotoxic constituents. Methanol extract of the seed was obtained through cold maceration, and it was further partitioned into n – hexane, dichloromethane and ethylacetate. The most active fraction was purified on repeated chromatographic techniques, using vacuum liquid chromatography (VLC), column (CC), and high-performance liquid chromatography (HPLC). The extract, purified fractions and isolated compounds were tested for their cytotoxic activities against the human Rhabdomyosarcoma (RD) and breast (MCF-7) cancer cell lines, using MTT assay. The crude extract and n-hexane fraction were found to be selectively cytotoxic to the cancer cell lines. Bioassay-guided fractionation of the n-hexane fraction led to the isolation of three compounds, which were identified as 6-shogaoal, 6-paradol, and 1-dehydro-6-gingerdione. 6-shogaoal demonstrated the highest cytotoxicity with CC50 values of 0.11 ± 0.02 and 0.25 ± 0.05ìg/mL against RD and MCF-7 cell lines respectively, and these were higher in activity when compared with cyclophosphamide (CC50 = 1.98 ± 0.15 and 0.71 ± 0.7ìg/mL). The presented data validates the ethnomedicinal use of A. melegueta in the treatment of cancer and is also indicative of the potential of 6-shogaol as an anticancer agent.

Expression of oncogenic HRAS in human Rh28 and RMS-YM rhabdomyosarcoma cells leads to oncogene-induced senescence

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. The two predominant histologic variants of RMS, embryonal and alveolar rhabdomyosarcoma (eRMS and aRMS, respectively), carry very different prognoses. While eRMS is associated with an intermediate prognosis, the 5-year survival rate of aRMS is less than 30%. The RMS subtypes are also different at the molecular level—eRMS frequently has multiple genetic alterations, including mutations in RAS and TP53, whereas aRMS often has chromosomal translocations resulting in PAX3-FOXO1 or PAX7-FOXO1 fusions, but otherwise has a “quiet” genome. Interestingly, mutations in RAS are rarely found in aRMS. In this study, we explored the role of oncogenic RAS in aRMS. We found that while ectopic oncogenic HRAS expression was tolerated in the human RAS-driven eRMS cell line RD, it was detrimental to cell growth and proliferation in the human aRMS cell line Rh28. Growth inhibition was mediated by oncogene-induced senescence and associated with increased RB pathway activity and expression of the cyclin-dependent kinase inhibitors p16 and p21. Unexpectedly, the human eRMS cell line RMS-YM, a RAS wild-type eRMS cell line, also exhibited growth inhibition in response to oncogenic HRAS in a manner similar to aRMS Rh28 cells. This work suggests that oncogenic RAS is expressed in a context-dependent manner in RMS and may provide insight into the differential origins and therapeutic opportunities for RMS subtypes.

Anti-proliferative effect of Cistus laurifolius on human cervical adenocarcinoma (Hep2C), human muscle rhabdomyosarcoma (RD), mouse fibrosarcoma (Wehi 164) cell line

In the current study; it was tried to determine antiproliferative activity of extract obtained from Cistus laurifolius leaves on different cancer cell type such as human cervical adenocarcinoma cells (Hep2C), human muscle rhabdomyosarcoma cells (RD), mouse fibrosarcoma cells (Wehi 164) by MTT Assay. The results indicated that Cistus laurifolius inhibited, in a dose-dependent manner, the proliferation of Hep2C, RD, Wehi 164 cells. Maximum anti-proliferatif effects were observed in 1000 µg/ml, 500 µg/ml, 250µg/ml concentrations of extract. These findings suggest that Cistus laurifolius extract could reduce proliferatif effect of cancer cell lines. Their use as potential anticancer agent needs further studies.

Small heat-shock protein HSPB3 promotes myogenesis by regulating the lamin B receptor

One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR.

Sarcoma IL-12 overexpression facilitates NK cell immunomodulation

Interleukin-12 (IL-12) is an inflammatory cytokine that has demonstrated efficacy for cancer immunotherapy, but systemic administration has detrimental toxicities. Lentiviral transduction eliciting IL-12-producing human sarcoma for autologous reintroduction provides localized delivery for both innate and adaptive immune response augmentation. Sarcoma cell lines and primary human sarcoma samples were transduced with recombinant lentivirus engineering expression of human IL-12 (hu-IL-12). IL-12 expressing sarcomas were assessed in vitro and in vivo following implantation into humanized NSG and transgenic human IL-15 expressing (NSG.Tg(Hu-IL-15)) murine models. Lentiviral transduction (LV/hu-IL-12) of human osteosarcoma, Ewing sarcoma and rhabdomyosarcoma cell lines, as well as low-passage primary human sarcomas, engendered high-level expression of hu-IL-12. Hu-IL-12 demonstrated functional viability, eliciting specific NK cell-mediated interferon-γ (IFN-γ) release and cytotoxic growth restriction of spheroids in vitro. In orthotopic xenograft murine models, the LV/hu-IL-12 transduced human sarcoma produced detectable IL-12 and elicited an IFN-γ inflammatory immune response specific to mature human NK reconstitution in the NSG.Tg(Hu-IL-15) model while restricting tumor growth. We conclude that LV/hu-IL-12 transduction of sarcoma elicits a specific immune reaction and the humanized NSG.Tg(Hu-IL-15) xenograft, with mature human NK cells, can define in vivo anti-tumor effects and systemic toxicities. IL-12 immunomodulation through autologous tumor transduction and reintroduction merits exploration for sarcoma treatment.

Enhanced photoluminescence of boron nitride quantum dots by encapsulation within polymeric nanoparticles

Boron nitride quantum dots (BNQDs) have been proposed as probes for bioimaging owing their to outstanding photoluminescent properties, although their hydrophobic nature and strong aggregation tendency in aqueous media limit their application in the biomedical field. In this work, we synthesize BNQDs by a liquid exfoliation–solvothermal process under pressure from boron nitride nanoparticles in N,N-dimethylformamide. The BNQDs display an average size of 3.3 ± 0.6 nm, as measured by transmission electron microscopy, and a (100) crystalline structure. In addition, a quantum yield of 21.75 ± 0.20% was achieved. To ensure complete dispersibility in water and prevent possible elimination by renal filtration upon injection, the BNQDs (20% w/w) are encapsulated within poly(ethylene glycol)-b-poly(epsilon-caprolactone) nanoparticles by a simple and scalable nanoprecipitation method, and hybrid nanocomposite particles with significantly stronger photoluminescence than their free counterparts are produced. Finally, their optimal cell compatibility and bioimaging features are demonstrated in vitro in murine macrophage and human rhabdomyosarcoma cell lines.

A Fusion Transcription Factor-Driven Cancer Progresses to a Fusion-Independent Relapse via Constitutive Activation of a Downstream Transcriptional Target.

Targeted monotherapies usually fail due to development of resistance by a subgroup of cells that evolve into recurrent tumors. Alveolar rhabdomyosarcoma is an aggressive myogenic soft-tissue cancer that is associated with a characteristic PAX3-FOXO1 gene fusion encoding a novel fusion transcription factor. In our myoblast model of PAX3-FOXO1-induced rhabdomyosarcoma, deinduction of PAX3-FOXO1 simulates a targeted therapy that antagonizes the fusion oncoprotein. This simulated therapy results initially in regression of the primary tumors, but PAX3-FOXO1-independent recurrent tumors eventually form after a delay. We report here that upregulation of FGF8, a direct transcriptional target of PAX3-FOXO1, is a mechanism responsible for PAX3-FOXO1-independent tumor recurrence. As a transcriptional target of PAX3-FOXO1, FGF8 promoted oncogenic activity in PAX3-FOXO1-expressing primary tumors that developed in the myoblast system. In the recurrent tumors forming after PAX3-FOXO1 deinduction, FGF8 expression was necessary and sufficient to induce PAX3-FOXO1-independent tumor growth through an autocrine mechanism. FGF8 was also expressed in human PAX3-FOXO1-expressing rhabdomyosarcoma cell lines and contributed to proliferation and transformation. In a human rhabdomyosarcoma cell line with reduced PAX3-FOXO1 expression, FGF8 upregulation rescued oncogenicity and simulated recurrence after PAX3-FOXO1-targeted therapy. We propose that deregulated expression of a PAX3-FOXO1 transcriptional target can generate resistance to therapy directed against this oncogenic transcription factor and postulate that this resistance mechanism may ultimately be countered by therapeutic approaches that antagonize the corresponding downstream pathways. SIGNIFICANCE: In a model of cancer initiated by a fusion transcription factor, constitutive activation of a downstream transcriptional target leads to fusion oncoprotein-independent recurrences, thereby highlighting a novel progression mechanism and therapeutic target.

The Upregulation of a Novel Long Noncoding RNA AK097647 Promotes Enterovirus 71 Replication and Decreases IFN-λ1 Secretion

Background: Enterovirus 71 (EV71) infects millions of children every year in China and has become a challenge to public health. However, there is no effective treatment for EV71 infection. Long noncoding RNAs (lncRNAs) have been found to play various roles in virus replication and infection. Objective: We aimed to explore the role of a novel long noncoding RNA AK097647 (lncRNA-AK097647) during EV71 infection. Methods: To assess the role of lncRNA-AK097647 during EV71 infection, siRNAs were used to silence lncRNA-K097647 expression. RT-qPCR assay and Western blotting were applied to measure the mRNA and protein levels of EV71 VP1 and the phosphorylation of NF-κB. ELISA was used to detect the level of IFN-λ1 expression. Results: The novel lncRNA-AK097647 was upregulated in human rhabdomyosarcoma cells and the blood of hand, foot, and mouth disease patients infected with EV71, as demonstrated by RT-qPCR. Interestingly, RNAi-mediated knockdown of lncRNA-AK097647 dramatically increased the level of IFN-λ1 expression, resulting in the suppression of EV71 replication. In contrast, overexpression of lncRNA-AK097647 decreased the level of IFN-λ1 expression and resulted in increased EV71 replication. In addition, we found that lncRNA-AK097647 could inhibit the phosphorylation of NF-κB. Conclusion: These results suggest a novel mechanism by which EV71 evades the IFN-mediated host antiviral response by increasing lncRNA-AK097647 expression.

Improved plaque assay for human coronaviruses 229E and OC43.

In light of the COVID-19 pandemic, studies that work to understand SARS-CoV-2 are urgently needed. In turn, the less severe human coronaviruses such as HCoV-229E and OC43 are drawing newfound attention. These less severe coronaviruses can be used as a model to facilitate our understanding of the host immune response to coronavirus infection. SARS-CoV-2 must be handled under biosafety level 3 (BSL-3) conditions. Therefore, HCoV-229E and OC43, which can be handled at BSL-2 provide an alternative to SARS-CoV-2 for preclinical screening and designing of antivirals. However, to date, there is no published effective and efficient method to titrate HCoVs other than expensive indirect immunostaining. Here we present an improved approach using an agarose-based conventional plaque assay to titrate HCoV 229E and OC43 with mink lung epithelial cells, Mv1Lu. Our results indicate that titration of HCoV 229E and OC43 with Mv1Lu is consistent and reproducible. The titers produced are also comparable to those produced using human rhabdomyosarcoma (RD) cells. More importantly, Mv1Lu cells display a higher tolerance for cell-cell contact stress, decreased temperature sensitivity, and a faster growth rate. We believe that our improved low-cost plaque assay can serve as an easy tool for researchers conducting HCoV research.

The application of aluminium phthalocyanine AlPs-4-mediated photodynamic therapy against human soft tissue sarcoma (RMS) cell line

The main challenge in the cancer treatment is the on-target drug delivery to the affected cells. Various therapies have been designed to target the affected cells efficiently but still the success is awaited. An iron and cobalt nanocomposite for the effective drug delivery to target cells was designed. The photodynamic effect of anticancer drugs loaded with iron oxide and cobalt ferrite nanomaterials coated with polyvinyl alcohol (PVA) was studied. The iron oxide nanoparticles (IONPs) and cobalt ferrite (CF) NPs without the loaded drugs were characterized by UV, XRD, FTIR, SEM and EDX techniques. The photodynamic effect of the photosensitizer, doxorubicin, and dacarbazine loaded nanomaterials were screened against human rhabdomyosarcoma (RMS) cells after incubation for 3 h, 24 h, and 48 h using MTT assay. The combination of photodynamic therapy (PDT) with chemo drugs is studied over different doses. When RMS cells were exposed to nanomaterials loaded with chemo drugs and PDT alone, it resulted in less cell killing compared to chemo drugs followed by PDT. These results revealed that in the case of combined treatment (combination therapy) the cell viability decreases as compared to individual treatment (monotherapy). The in vitro studies showed positive results which give a new pathway for the in vivo studies.

Synthetic cannabinoid CP-55,940 induces apoptosis in a human skeletal muscle model via regulation of CB1 receptors and L-type Ca2+ channels.

Rhabdomyolysis has been reported in patients who abuse synthetic cannabinoids. However, no studies have yet assessed whether these cases reflect the direct cytotoxicity of synthetic cannabinoids on skeletal muscle, a possibility that the present study sought to address. Specifically, this study investigated the cytotoxicity of the synthetic cannabinoid CP-55,940, a compound that acts equally on both types of cannabinoid receptors (CB1 and CB2), in a human embryonic rhabdomyosarcoma (RD) cell line. Exposure of these cells to CP-55,940 resulted in concentration-dependent decreases in cell viability. These effects were attenuated by pre-incubation with AM251 (30µM), a selective CB1 receptor antagonist, but not by pre-incubation with AM630 (30µM), a selective CB2 receptor antagonist. Following treatment with CP-55,940, RD cells exhibited apoptosis, as indicated by the accumulation of annexin-V, activation of caspase-3, and a loss of the mitochondrial membrane potential. Additionally, CP-55,940 treatment of RD cells led to increases in intracellular Ca2+ levels. CP-55,940-induced cell death was significantly attenuated in the absence of extracellular Ca2+, and was partially decreased by pre-incubation with verapamil (5µM) or diltiazem (5µM), compounds that block the L-type Ca2+ channel. Our results indicate that the cytotoxicity of CP-55,940 towards RD cells (skeletal muscle cells) is mediated by the CB1 receptor, but not by the CB2 receptor. Our results further suggest that calcium influx through the L-type channel may play an important role in the apoptosis induced by these compounds.

Growth inhibition associated with disruption of the actin cytoskeleton by Latrunculin A in rhabdomyosarcoma cells.

Functional genomic screening of KRAS-driven mouse sarcomas was previously employed to identify proliferation-relevant genes. Genes identified included Ubiquitin-conjugating enzyme E2 (Ube2c), Centromere Protein E (Cenpe), Hyaluronan Synthase 2 (Has2), and CAMP Responsive Element Binding Protein 3 Like 2 (Creb3l2). This study examines the expression and chemical inhibition of these candidate genes, identifying variable levels of protein expression and significant contributions to rhabdomyosarcoma (RMS) cell proliferation. Chemical treatment of human and murine RMS cell lines with bortezomib, UA62784, latrunculin A and sorafenib inhibited growth with approximate EC50 concentrations of 15-30nM for bortezomib, 25-80nM for UA62784 and 80-220nM for latrunculin A. The multi-kinase inhibitor sorafenib increased in vitro proliferation of 4 of 6 sarcoma cell lines tested. Latrunculin A was further associated with disruption of the actin cytoskeleton and reduced ERK1/2 phosphorylation. Together, this work advances opportunities for developing therapies to block progression of soft-tissue sarcomas and demonstrates that disruption of the actin cytoskeleton in sarcoma cells by latrunculin A is associated with a reduction in RMS cell growth. (167 words).

Abstract 5001: Barriers to adoptive immunotherapy of pediatric cancer

Abstract As the adoptive immunotherapy with chimeric antigen modified T cells (CAR-T) has not proven effective in pediatric solid tumors, we established a model to define which tumor- and stromal-derived factors must be surmounted to obtain effective treatment. The human alveolar rhabdomyosarcoma cell line RH30 (fusion gene positive) was administered both i.v. and i.m. to NSG mice, and subsequently treated with a FGFR4-specifc CAR-T cells (expressing the M410 binder). Although some response was noted in the i.v. model, the i.m. tumor was entirely resistant. We observed that in both primary tumors and liver metastases RH30 was surrounded with a rich stromal element when CAR-T or control activated T cells were administered. Trichrome and H&E histochemical analysis of i.m. tumors confirmed the presence of a rich collagen-containing stromal element in T cell treated mice. This was not present in untreated tumors. We therefore analyzed cell surface and soluble factors produced by RH30 by RNA array analysis, flow cytometry and ELISA in cultured RH30 alone, or treated with interferon-gamma (IFNG) to model the presence of activated T cells. RH30 expressed transcripts for both canonical and non-canonical chemokines: CCL24, GPI, MIF, CCL26, CCL20, CCL2; as well as the cytokines: IL-15, IL-32, LIF, TGFB1, and CSF1. ELISA confirmed high expression of MIF, TGFB1, CCL24 and LIF. Transcripts for the chemokines CCL8, 9, 10, and 11 were expressed at low or undetectable levels, but were strongly induced by IFNG. And although class I MHC was present, and upregulated in the presence of IFNG, so was PDL1. Surface marker analysis by flow cytometry demonstrated that CD80, CD86 and CXCR2 are absent while the CAR-T targets CD276, EGFR, FGFR4 as well as CXCR4 are present. Flow cytometry also demonstrated consistent expression of PDL2, GAL-9, and CTLA4, and strong induction of HLA class I and PDL1 by IFNG treatment of RH30. We are currently evaluating CRISPR knock-out lines of each soluble factor for effects on gene expression, IFNG-induced marker expression, and tumor stroma in vivo. Our results indicate that in this model of rhabdomyosarcoma, tumor surface expression of glycoproteins that give an immune inhibitory signal, PDL1, PDL2, CTLA4, GAL-9, as well as soluble modulators known to be associated with tumor escape from immune control, MIF, TGFB, and LIF, all must be considered in armoring CAR-T for effective immune destruction of tumor. Moreover, the numerous chemokines produced by tumor alone, as well as induced by IFNG, indicate a complex network for recruitment of local and bone marrow-derived stromal cells that create a physical as well as cellular barrier to adoptive immunotherapy. Citation Format: Rajesh Kumar, Nityashree Shivaprasad, Virginia Hoglund, Cosette LeCeil, Lingyan Wang, Yue Zhang, Dimiter S. Dimitrov, Javed Khan, Rimas J. Orentas. Barriers to adoptive immunotherapy of pediatric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5001.

Abstract 3888: Study of tumor recurrence following loss of PAX3-FOXO1 oncoprotein expression in rhabdomyosarcoma cells

Abstract We previously developed a human alveolar rhabdomyosarcoma (RMS) model using immortalized human myoblasts engineered with constitutive MYCN and doxycycline-inducible PAX3-FOXO1 (P3F) expression constructs. These cells formed primary tumors rapidly when injected into mice fed a doxycycline-containing diet. When doxycycline is discontinued to turn off P3F expression, tumors initially regress, and then recurrent tumors without P3F expression form after a variable delay. We propose that P3F is necessary for initial tumor development but is not needed in later stages due to the accumulation of additional molecular changes. We further hypothesize that these novel changes may result in dysregulated expression of one or more P3F target genes. We derived cell lines from the primary and recurrent tumors and confirmed the expression of P3F in primary tumor-derived (PTD) cells and its absence in recurrent tumor-derived (RTD) cells. By limiting dilution, we generated subclones from RTD cell lines to obtain homogeneous populations of cells with P3F-independent transformation. Microarray analysis revealed that the expression profile of RTD subclones was substantially different from that of PTD cells. However, using untreated parental cells as a reference, we found that RTD and PTD cells shared 92 upregulated genes including 14 P3F transcriptional targets; among these 14 genes, FGF8 showed the highest level of upregulation (>30-fold) in RTD cells. Using a CRISPR-Cas9 approach, FGF8 knockout in these RTD cells impaired transforming capability in focus formation assays and tumorigenicity in vivo. Conversely, FGF8 overexpression in non-transformed MYCN-expressing parental myoblasts induced transformation and tumorigenic capability. In medium transfer studies, conditioned media (CM) from RTD cell cultures or from parental cells overexpressing FGF8 induced in vitro transformation in parental cells, whereas CM from FGF8-knockout RTD cells or parental cells did not induce transformation. To further implicate FGF8, immunodepletion of FGF8 from the RTD conditioned media induced a substantial reduction in transforming activity in these medium transfer studies. To examine the role of FGF8 in PTD cells and human alveolar RMS cells (RH30), we used a similar CRISPR-Cas9 approach to knockout FGF8 expression and similarly found impaired in vitro transformation. Finally, FGF8 overexpression in a spontaneous non-transformed RH30 variant with low P3F expression levels resulted in stimulation of transformation in vitro and tumorigenicity in vivo. Together, our results indicate that FGF8, a P3F transcriptional target, is necessary for the oncogenicity of P3F-positive RMS cells and that dysregulated FGF8 expression leads to the development of P3F-independent recurrent RMS tumors. Citation Format: Salah Boudjadi, Thanh Nguyen, Bishwanath Chatterjee, Puspa Pandey, Wenyue Sun, Frederik Barr. Study of tumor recurrence following loss of PAX3-FOXO1 oncoprotein expression in rhabdomyosarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3888.

Abstract A09: Targeting Hippo-dependent and Hippo-independent regulation of the YAP1 oncoprotein in childhood rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. While outcomes for patients with localized disease have improved, patients with metastatic or recurrent disease continue to do poorly; therefore, new treatments are needed. Previously, we showed that the Src family tyrosine kinase YES1 is upregulated in RMS and necessary for growth, but clinical trials using single-agent dasatinib (Src family kinase inhibitor) have not shown clinical activity in sarcomas. YAP1 (YES-associated protein) is also highly expressed in human RMS tumors, driving cell growth and survival. Thus far, efforts to pharmacologically inhibit YAP1 have been unsuccessful. We hypothesize that a better understanding of the Hippo-dependent and Hippo-independent (through YES1) regulation of YAP1 in RMS may lead to novel therapeutic approaches. Methods: We studied the effect of DNA methyltransferase inhibitors (DNMTi) on cell growth in human RMS cell lines using live cell imaging and analyzed the YES-YAP interaction by proximity ligation assays. We analyzed YES1 and YAP1 signaling with cell fractionation, immunoblots, methylation-specific PCR, qPCR, RNAseq, and genetic loss of function. Results: We demonstrate that DNMTi treatment decreases RMS cell growth and increases apoptosis and differentiation. DNMTi treatment activates canonical Hippo signaling through LATS, shown by a LATS activity biosensor, and increases YAP1 phosphorylation. DNMTi treatment also induces RASSF1 re-expression by promoter demethylation, and the DNMTi-induced growth inhibition can be partially rescued by expression of constitutively active YAPS127A. RNAseq analysis after DNMTi treatment also revealed upregulation of RASSF family members RASSF5 and RASSF6 and downregulation of RASSF4. RASSF4 was previously shown to inhibit MST1 and promote tumorigenesis in the alveolar subtype of RMS. These data suggest the effects of DNMTi treatment are in part due to inhibition of YAP1 in a Hippo-dependent manner. DNMTi treatment also decreases YES1 expression and activation, suggesting DNMTis impact both YES1 and YAP1 activity. Furthermore, YES1 and YAP1 interact in the nucleus of RMS cells and shRNA knockdown of YES1 results in YAP1 cytoplasmic retention and decreased expression of YAP1 target genes, suggesting a role for YES1 in regulating YAP1 cellular localization in a Hippo-independent manner. Lastly, to target both Hippo-dependent and Hippo-independent regulation of YAP1 we combine DNMTi treatment with dasatinib, a YES1 kinase inhibitor. Combination treatment in vitro ablates RMS cell growth and leads to a greater induction of apoptosis than either drug alone. We are currently evaluating this combination strategy in RMS xenografts in mice. Conclusions: Overall, we show that YAP1 is regulated in both Hippo-dependent and Hippo-independent mechanisms in RMS and both mechanisms can be inhibited by a combined treatment of DNMTi and dasatinib, laying the groundwork for future clinical investigations of this novel therapy. Citation Format: Katherine K. Slemmons, Choh Yeung, Josh Baumgart, Amy McCalla, Lee Helman. Targeting Hippo-dependent and Hippo-independent regulation of the YAP1 oncoprotein in childhood rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A09.

Imatinib and Dasatinib Provoke Mitochondrial Dysfunction Leading to Oxidative Stress in C2C12 Myotubes and Human RD Cells

Tyrosine kinase inhibitors (TKIs) can cause skeletal muscle toxicity in patients, but the underlying mechanisms are mostly unclear. The goal of the current study was to better characterize the role of mitochondria in TKI-associated myotoxicity. We exposed C2C12 murine myoblasts and myotubes as well as human rhabdomyosarcoma cells (RD cells) for 24 h to imatinib (1–100 µM), erlotinib (1–20 µM), and dasatinib (0.001–100 µM). In C2C12 myoblasts, imatinib was membrane toxic at 50 µM and depleted the cellular ATP pool at 20 µM. In C2C12 myotubes exposed to imatinib, ATP depletion started at 50 µM whereas membrane toxicity was not detectable. In myoblasts and myotubes exposed to dasatinib, membrane toxicity started at 0.5 µM and 2 µM, respectively, and the ATP drop was visible at 0.1 µM and 0.2 µM, respectively. When RD cells were exposed to imatinib, ATP depletion started at 20 µM whereas membrane toxicity was not detectable. Dasatinib was membrane toxic at 20 µM and depleted the cellular ATP pool already at 0.5 µM. Erlotinib was not toxic in both cell models. Imatinib (20 µM) and dasatinib (1 µM) reduced complex I activity in both cell models. Moreover, the mitochondrial membrane potential (Δψm) was dissipated for both TKIs in myotubes. In RD cells, the Δψm was reduced only by dasatinib. Both TKIs increased mitochondrial superoxide accumulation and decreased the mitochondrial copy number in both cell lines. In consequence, they increased protein expression of superoxide dismutase (SOD) 2 and thioredoxin 2 and cleavage of caspase 3, indicating apoptosis in C2C12 myotubes. Moreover, in both cell models, the mRNA expression of Sod1 and Sod2 increased when RD cells were exposed to dasatinib. Furthermore, dasatinib increased the mRNA expression of atrogin-1 and murf-1, which are important transcription factors involved in muscle atrophy. The mRNA expression of atrogin-1 increased also in RD cells exposed to imatinib. In conclusion, imatinib and dasatinib are mitochondrial toxicants in mouse C2C12 myotubes and human RD cells. Mitochondrial superoxide accumulation induced by these two TKIs is due to the inhibition of complex I and is probably related to impaired mitochondrial and myocyte proliferation.

Beclin1 Binds to Enterovirus 71 3D Protein to Promote the Virus Replication.

Enterovirus 71 (EV71) is the main pathogen causing hand-foot-mouth disease (HFMD) in infants and children, which can also lead to severe neurological diseases and even death. Therefore, understanding the replication mechanism of EV71 is of great significance for the prevention and control of EV71-induced diseases. Beclin1 (BECN1, a mammalian homologue of ATG6 in yeast) is an important core protein for the initiation and the normal process of autophagy in cells. In addition to its involvement in autophagy, Beclin1 has also been reported to play an important role in cancer and innate immune signaling pathways. However, the role of Beclin1 in EV71 replication remains elusive. Here, we primarily found that Beclin1 facilitates EV71 replication in human rhabdomyosarcoma (RD) cells and the autophagy was actually induced, but Beclin1 was not significantly affected at either mRNA level or protein level during early EV71 infection. Further studies discovered that Beclin1 could interacts with EV71 non-structural protein 3D mainly through its evolutionary conserved domain (ECD) and coiled-coiled domain (CCD), thus promoting the replication of EV71 in human rhabdomyosarcoma (RD) cells and human astroglioma (U251) cells. Collectively, we reveal a novel regulatory mechanism associated with Beclin1 to promote EV71 replication, thus providing a potential therapeutic target for the prevention and control of EV71-associated diseases.

Evaluation of Daptomycin-Induced Cellular Membrane Injury in Skeletal Muscle.

Daptomycin, a cyclic lipopeptide antibiotic, has bactericidal activity against Gram-positive organisms and is especially effective against methicillin-resistant Staphylococcus aureus. Although daptomycin causes unique adverse drug reactions such as elevation of creatine phosphokinase or rhabdomyolysis, the detailed mechanisms underlying these adverse drug reactions in skeletal muscle are unclear. This study aimed to elucidate whether daptomycin causes direct skeletal muscle cell toxicity and investigate the relationship between daptomycin exposure and musculoskeletal toxicity. First, we evaluated the relationship between daptomycin exposure and skeletal muscle toxicity. Of the 38 patients who received daptomycin intravenously, an elevation in creatine phosphokinase levels was observed in five. The median plasma trough concentration of daptomycin in patients with elevated creatine phosphokinase levels was significantly higher than that in patients whose creatine phosphokinase levels were within the normal range, suggesting that increased exposure to daptomycin is related to elevation in creatine phosphokinase levels. In an in vitro study using human rhabdomyosarcoma cells, daptomycin reduced cell viability and increased membrane damage. These effects were more marked under hypoxic conditions. A necroptotic pathway seemed to be involved because phosphorylated mixed lineage kinase domain-like protein expression was enhanced following daptomycin exposure, which was significantly enhanced under hypoxic conditions. These findings indicate that daptomycin elicits cytotoxic effects against skeletal muscle cells via the necroptotic pathway, and the extent of toxicity is enhanced under hypoxic conditions.

ATP-Binding Cassette Transporter C4 is a Prostaglandin D2 Exporter in HMC-1 cells

ATP-binding cassette transporter C4 (ABCC4) is associated with multidrug resistance and the regulation of cell signalling. Some prostaglandins (PGs), including: PGE2, PGF2α, PGE3, and PGF3α are known substrates of ABCC4, and are released from some types of cells to exert their biological effects. In the present study, we demonstrate that PGD2 is a novel substrate of ABCC4 using a transport assay based on inside-out membrane vesicles prepared from ABCC4-overexpressing cells. Then, we used two types of cell lines with confirmed ABCC4 mRNA and PGD2 release capacity (human mast cell lines HMC-1 cells and human rhabdomyosarcoma cell lines TE671 cells) to evaluate the contribution of ABCC4. The extracellular levels of PGD2 were unchanged following addition of a selective ABCC4 inhibitor in TE671 cells. Pharmacological inhibition and knockdown of ABCC4 significantly reduced the extracellular levels of PGD2 by at least 53% in HMC-1 cells. Moreover, the extracellular levels of PGD2 decreased by at least 20% using the selective ABCC4 inhibitor in the other mast cell line RBL-2H3 cells. Therefore, our results suggest that ABCC4 functions as a PGD2 exporter in HMC-1 cells.

Imperatorin as a Promising Chemotherapeutic Agent Against Human Larynx Cancer and Rhabdomyosarcoma Cells.

Naturally occurring coumarins are bioactive compounds widely used in Asian traditional medicine. They have been shown to inhibit proliferation, induce apoptosis, and/or enhance the cytotoxicity of currently used drugs against a variety of cancer cell types. The aim of our study was to examine the antiproliferative activity of different linear furanocoumarins on human rhabdomyosarcoma, lung, and larynx cancer cell lines, and dissolve their cellular mechanism of action. The coumarins were isolated from fruits of Angelica archangelica L. or Pastinaca sativa L., and separated using high-performance counter-current chromatography (HPCCC). The identity and purity of isolated compounds were confirmed by HPLC-DAD and NMR analyses. Cell viability and toxicity assessments were performed by means of methylthiazolyldiphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, respectively. Induction of apoptosis and cell cycle progression were measured using flow cytometry analysis. qPCR method was applied to detect changes in gene expression. Linear furanocoumarins in a dose-dependent manner inhibited proliferation of cancer cells with diverse activity regarding compounds and cancer cell type specificity. Imperatorin (IMP) exhibited the most potent growth inhibitory effects against human rhabdomyosarcoma and larynx cancer cell lines owing to inhibition of the cell cycle progression connected with specific changes in gene expression, including CDKN1A. As there are no specific chemotherapy treatments dedicated to laryngeal squamous cell carcinoma and rhabdomyosarcoma, and IMP seems to be non-toxic for normal cells, our results could open a new direction in the search for effective anti-cancer agents.