Receptor tyrosine kinase gene expression profiling of orbital rhabdomyosarcoma unveils MET as a potential biomarker and therapeutic target.

Receptor tyrosine kinases (RTKs) serve as molecular targets for the development of novel personalized therapies in many malignancies. In the present study, expression pattern of receptor tyrosine kinases and its clinical significance in orbital RMS has been explored. Eighteen patients with histopathologically confirmed orbital RMS formed part of this study. Comprehensive q-PCR gene expression profiles of 19 RTKs were generated in the cases and controls. The patients were followed up for 59.53 ± 20.93 years. Clustering and statistical analysis tools were applied to identify the significant combination of RTKs associated with orbital rhabdomyosarcoma patients. mRNA overexpression of RTKs which included MET, AXL, EGFR was seen in 60-80% of cases; EGFR3, IGFR2, FGFR1, RET, PDGFR1, VEGFR2, PDGFR2 in 30-60% of cases; and EGFR4, FGFR3,VEGFR3 and ROS,IGFR1, EGFR1, FGFR2, VEGFR1 in 10-30% of cases. Immunoexpression of MET was seen in 89% of cases. A significant association was seen between MET mRNA and its protein expression. In all the cases MET gene expression was associated with worst overall survival (P = 0.03).There was a significant correlation of MET mRNA expression with RET, ROS, AXL, FGFR1, FGFR3, PDGFR1, IGFR1, VEGFR2, and EGFR3 genes. Association between MET gene and collective expression of RTKs was further evaluated by semi-supervised gene cluster analysis and Principal component analysis, which showed well-separated tumor clusters. MET gene overexpression could be a useful biomarker for identifying high risk orbital rhabdomyosarcoma patients. Well-separated tumor clusters confirmed the association between MET gene and collective expression of RTK genes. Therefore, the therapeutic potential of multi-kinase inhibitors targeting MET and the 9 other significant RTKs needs to be explored.

EHMT1 promotes tumor progression and maintains stemness by regulating ALDH1A1 expression in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with poor prognosis. Cancer stem cells (CSCs) are seeds for tumor relapse and metastasis. However, pathways that maintain stemness genes are not fully understood. Here, we report that the enzyme euchromatic histone lysine methyltransferase 1 (EHMT1) is expressed in primary and relapse ARMS tumors. EHMT1 suppression impaired motility and induced differentiation in ARMS cell lines and reduced tumor progression in a mouse xenograft model in vivo. RNA sequencing of EHMT1-depleted cells revealed downregulation of ALDH1A1 that is associated with CSCs. Consistent with this, inhibition of ALDH1A1 expression and activity mimicked EHMT1 depletion phenotypes and reduced tumorsphere formation. Mechanistically, we demonstrate that EHMT1 does not bind to the ALDH1A1 promoter but activates it by stabilizing C/EBPß, a known regulator of ALDH1A1 expression. Our findings identify a role for EHMT1 in maintenance of stemness by regulating ALDH1A1 expression and suggest that targeting ALDH+ cells is a promising strategy in ARMS. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Preclinical rationale for entinostat in embryonal rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in the pediatric cancer population. Survival among metastatic RMS patients has remained dismal yet unimproved for years. We previously identified the class I-specific histone deacetylase inhibitor, entinostat (ENT), as a pharmacological agent that transcriptionally suppresses the PAX3:FOXO1 tumor-initiating fusion gene found in alveolar rhabdomyosarcoma (aRMS), and we further investigated the mechanism by which ENT suppresses PAX3:FOXO1 oncogene and demonstrated the preclinical efficacy of ENT in RMS orthotopic allograft and patient-derived xenograft (PDX) models. In this study, we investigated whether ENT also has antitumor activity in fusion-negative eRMS orthotopic allografts and PDX models either as a single agent or in combination with vincristine (VCR). METHODS: We tested the efficacy of ENT and VCR as single agents and in combination in orthotopic allograft and PDX mouse models of eRMS. We then performed CRISPR screening to identify which HDAC among the class I HDACs is responsible for tumor growth inhibition in eRMS. To analyze whether ENT treatment as a single agent or in combination with VCR induces myogenic differentiation, we performed hematoxylin and eosin (H&E) staining in tumors. RESULTS: ENT in combination with the chemotherapy VCR has synergistic antitumor activity in a subset of fusion-negative eRMS in orthotopic "allografts," although PDX mouse models were too hypersensitive to the VCR dose used to detect synergy. Mechanistic studies involving CRISPR suggest that HDAC3 inhibition is the primary mechanism of cell-autonomous cytoreduction in eRMS. Following cytoreduction in vivo, residual tumor cells in the allograft models treated with chemotherapy undergo a dramatic, entinostat-induced (70-100%) conversion to non-proliferative rhabdomyoblasts. CONCLUSION: Our results suggest that the targeting class I HDACs may provide a therapeutic benefit for selected patients with eRMS. ENT's preclinical in vivo efficacy makes ENT a rational drug candidate in a phase II clinical trial for eRMS.

SMYD1 and G6PD modulation are critical events for miR-206-mediated differentiation of rhabdomyosarcoma.

Rhadomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. RMS cells resemble fetal myoblasts but are unable to complete myogenic differentiation. In previous work we showed that miR-206, which is low in RMS, when induced in RMS cells promotes the resumption of differentiation by modulating more than 700 genes. To better define the pathways involved in the conversion of RMS cells into their differentiated counterpart, we focused on 2 miR-206 effectors emerged from the microarray analysis, SMYD1 and G6PD. SMYD1, one of the most highly upregulated genes, is a H3K4 histone methyltransferase. Here we show that SMYD1 silencing does not interfere with the proliferative block or with the loss anchorage independence imposed by miR-206, but severely impairs differentiation of ERMS, ARMS, and myogenic cells. Thus SMYD1 is essential for the activation of muscle genes. Conversely, among the downregulated genes, we found G6PD, the enzyme catalyzing the rate-limiting step of the pentose phosphate shunt. In this work, we confirmed that G6PD is a direct target of miR-206. Moreover, we showed that G6PD silencing in ERMS cells impairs proliferation and soft agar growth. However, G6PD overexpression does not interfere with the pro-differentiating effect of miR-206, suggesting that G6PD downmodulation contributes to - but is not an absolute requirement for - the tumor suppressive potential of miR-206. Targeting cancer metabolism may enhance differentiation. However, therapeutic inhibition of G6PD is encumbered by side effects. As an alternative, we used DCA in combination with miR-206 to increase the flux of pyruvate into the mitochondrion by reactivating PDH. DCA enhanced the inhibition of RMS cell growth induced by miR-206, and sustained it upon miR-206 de-induction. Altogether these results link miR-206 to epigenetic and metabolic reprogramming, and suggest that it may be worth combining differentiation-inducing with metabolism-directed approaches.

PDGFRß reverses EphB4 signaling in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (aRMS) is an aggressive myogenic childhood malignancy, not infrequently presenting as incurable metastatic disease. To identify therapeutic targets, we performed an unbiased tyrosine kinome RNA interference screen in primary cell cultures from a genetically engineered, conditional mouse model of aRMS. We identified ephrin receptor B4 (EphB4) as a target that is widely expressed in human aRMS and that portends a poor clinical outcome in an expression level-dependent manner. We also uncovered cross-talk of this ephrin receptor with another receptor tyrosine kinase, PDGFRß, which facilitates PDGF ligand-dependent, ephrin ligand-independent activation of EphB4 converging on the Akt and Erk1/2 pathways. Conversely, EphB4 activation by its cognate ligand, EphrinB2, did not stimulate PDGFRß; instead, apoptosis was paradoxically induced. Finally, we showed that small-molecule inhibition of both PDGFRß and EphB4 by dasatinib resulted in a significant decrease in tumor cell viability in vitro, as well as decreased tumor growth rate and significantly prolonged survival in vivo. To our knowledge, these results are the first to identify EphB4 and its cross-talk with PDGFRß as unexpected vital determinants of tumor cell survival in aRMS, with EphB4 at the crux of a bivalent signaling node that is either mitogenic or proapoptotic.

Glycogen synthase kinase 3ß represses MYOGENIN function in alveolar rhabdomyosarcoma.

MYOGENIN is a member of the muscle regulatory factor family that orchestrates an obligatory step in myogenesis, the terminal differentiation of skeletal muscle cells. A paradoxical feature of alveolar rhabdomyosarcoma (ARMS), a prevalent soft tissue sarcoma in children arising from cells with a myogenic phenotype, is the inability of these cells to undergo terminal differentiation despite the expression of MYOGENIN. The chimeric PAX3-FOXO1 fusion protein which results from a chromosomal translocation in ARMS has been implicated in blocking cell cycle arrest, preventing myogenesis from occurring. We report here that PAX3-FOXO1 enhances glycogen synthase kinase 3ß (GSK3ß) activity which in turn represses MYOGENIN activity. MYOGENIN is a GSK3ß substrate in vitro on the basis of in vitro kinase assays and MYOGENIN is phosphorylated in ARMS-derived RH30 cells. Constitutively active GSK3ß(S9A) increased the level of a phosphorylated form of MYOGENIN on the basis of western blot analysis and this effect was reversed by neutralization of the single consensus GSK3ß phosphoacceptor site by mutation (S160/164A). Congruently, GSK3ß inhibited the trans-activation of an E-box reporter gene by wild-type MYOGENIN, but not MYOGENIN with the S160/164A mutations. Functionally, GSK3ß repressed muscle creatine kinase (MCK) promoter activity, an effect which was reversed by the S160/164A mutated MYOGENIN. Importantly, GSK3ß inhibition or exogenous expression of the S160/164A mutated MYOGENIN in ARMS reduced the anchorage independent growth of RH30 cells in colony-formation assays. Thus, sustained GSK3ß activity represses a critical regulatory step in the myogenic cascade, contributing to the undifferentiated, proliferative phenotype in alveolar rhabdomyosarcoma (ARMS).

Heparanase-1 and components of the hedgehog signalling pathway are increased in untreated alveolar orbital rhabdomyosarcoma.

BACKGROUND: To assess the activities of heparanase-1 and elements of the hedgehog signalling pathway in alveolar orbital rhabdomyosarcoma. METHODS: Specimens (n = 23) were divided into two groups, those from patients with preoperative chemoradiotherapy or untreated patients; six samples of normal extraocular muscle were used as a normal muscle group. The presence of heparanase-1, patched, smoothened and glioma-associated oncogene homologue-1 protein expression was determined in 23 cases of archival paraffin-embedded alveolar orbital rhabdomyosarcoma after immunohistochemistry. RNA was extracted from three groups of paraffin-embedded specimens and messenger RNA expressions of heparanase-1, smoothened and glioma-associated oncogene homologue-1 compared using nested reverse transcriptase polymerase chain reaction and a limiting dilution analysis. RESULTS: The heparanase-1, patched, smoothened and glioma-associated oncogene homologue-1 protein was expressed in 91.3%, 87.0%, 91.3% and 78.3%, respectively, of the alveolar orbital rhabdomyosarcoma specimens. Untreated rhabdomyosarcoma samples tended to stain intensely, but staining was relatively weak in tissue obtained from the chemoradiotherapy group. The expression levels of heparanase-1, smoothened and glioma-associated oncogene homologue-1 messenger RNA in untreated and chemoradiotherapy groups paralleled that seen with immunology, and there were no significant differences in heparanase-1, smoothened and glioma-associated oncogene homologue-1 messenger RNA levels between the chemoradiotherapy group and the normal muscle group (P > 0.05). However, the messenger RNA in the untreated group were all significantly higher than those in the chemoradiotherapy and normal muscle groups (P < 0.01). CONCLUSIONS: Both heparanase-1 and hedgehog signalling pathway are involved in the pathogenesis of alveolar orbital rhabdomyosarcoma; however, chemotherapy and/or radiotherapy appears to significantly inhibit their upregulation.

Simultaneous targeting of insulin-like growth factor-1 receptor and anaplastic lymphoma kinase in embryonal and alveolar rhabdomyosarcoma: a rational choice.

BACKGROUND: Rhabdomyosarcoma (RMS) is an aggressive soft tissue tumour mainly affecting children and adolescents. Since survival of high-risk patients remains poor, new treatment options are awaited. The aim of this study is to investigate anaplastic lymphoma kinase (ALK) and insulin-like growth factor-1 receptor (IGF-1R) as potential therapeutic targets in RMS. PATIENTS AND METHODS: One-hundred-and-twelve primary tumours (embryonal RMS (eRMS)86; alveolar RMS (aRMS)26) were collected. Expression of IGF-1R, ALK and downstream pathway proteins was evaluated by immunohistochemistry. The effect of ALK inhibitor NVP-TAE684 (Novartis), IGF-1R antibody R1507 (Roche) and combined treatment was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in cell lines (aRMS Rh30, Rh41; eRMS Rh18, RD). RESULTS: IGF-1R and ALK expression was observed in 72% and 92% of aRMS and 61% and 39% of eRMS, respectively. Co-expression was observed in 68% of aRMS and 32% of eRMS. Nuclear IGF-1R expression was an adverse prognostic factor in eRMS (5-year survival 46.9 ± 18.7% versus 84.4 ± 5.9%, p=0.006). In vitro, R1507 showed diminished viability predominantly in Rh41. NVP-TAE684 showed diminished viability in Rh41 and Rh30, and to a lesser extent in Rh18 and RD. Simultaneous treatment revealed synergistic activity against Rh41 and Rh30. CONCLUSION: Co-expression of IGF-1R and ALK is detected in eRMS and particularly in aRMS. As combined inhibition reveals synergistic cytotoxic effects, this combination seems promising and needs further investigation.

Anaplastic lymphoma kinase status in rhabdomyosarcomas.

Rhabdomyosarcoma is a rare soft tissue sarcoma that typically affects children, adolescents, and young adults. Despite treatment via a multidisciplinary approach, the prognosis of advance-stage rhabdomyosarcomas remains poor, and a new treatment strategy is needed. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is a potential target for specific inhibitors. In this study, we investigated 116 rhabdomyosarcomas using a polymer-based ALK immunostaining method and correlated the results with clinicopathological parameters. In addition, we examined ALK status using dual-color fluorescence in situ hybridization, PCR, and sequencing. In immunohistochemical analysis, ALK was detected in 2 (6%) of 33 embryonal rhabdomyosarcomas, 42 (69%) of 61 alveolar rhabdomyosarcomas, and 0 (0%) of 22 other subtypes, including pleomorphic, adult-spindle-cell/sclerosing, and epithelioid variants. Compared with ALK-negative alveolar rhabdomyosarcomas, ALK-positive ones are presented with metastatic spread more frequently and showed a greater extent of myogenin reactivity. Overall survival was not associated with ALK expression. FOXO1 rearrangement was significantly associated with ALK immunoreactivity. The median ALK copy number was greater in ALK-positive tumors than in ALK-negative tumors. Most (93%) cases tested showed no selective increase in the ALK gene dosage. ALK selective amplification and low-level selective gain were noted in one and three cases, respectively. Further, a high-polysomy pattern (≥4 ALK copies in ≥40% of cells) was observed in seven cases. A significant increase in the ALK copy number was exclusive to the ALK-immunopositive cohort, but it was uncommon, accounting for only 30% of the 37 ALK-positive rhabdomyosarcomas. ALK gene rearrangement was not observed in either cohort, while an ALK somatic mutation (I1277T) was found in one ALK-negative embryonal case. Although it remains controversial whether ALK expression without gene rearrangement is therapeutically relevant, this comprehensive analysis may help future studies on the utility of ALK-targeted therapy for patients with rhabdomyosarcoma.

Protein kinase C iota as a therapeutic target in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer exhibiting skeletal-muscle differentiation. New therapeutic targets are required to improve the dismal prognosis for invasive or metastatic alveolar rhabdomyosarcoma. Protein kinase C iota (PKCι) has been shown to have an important role in tumorigenesis of many cancers, but little is known about its role in rhabdomyosarcoma. Our gene-expression studies in human tumor samples revealed overexpression of PRKCI. We confirmed overexpression of PKCι at the mRNA and protein levels using our conditional mouse model that authentically recapitulates the progression of rhabdomyosarcoma in humans. Inhibition of Prkci by RNA interference resulted in a dramatic decrease in anchorage-independent colony formation. Interestingly, treatment of primary cell cultures using aurothiomalate (ATM), which is a gold-containing classical anti-rheumatic agent and a PKCι-specific inhibitor, resulted in decreased interaction between PKCι and Par6, decreased Rac1 activity and reduced cell viability at clinically relevant concentrations. Moreover, co-treatment with ATM and vincristine (VCR), a microtubule inhibitor currently used in rhabdomyosarcoma treatment regimens, resulted in a combination index of 0.470-0.793 through cooperative accumulation of non-proliferative multinuclear cells in the G2/M phase, indicating that these two drugs synergize. For in vivo tumor growth inhibition studies, ATM demonstrated a trend toward enhanced VCR sensitivity. Overall, these results suggest that PKCι is functionally important in alveolar rhabdomyosarcoma anchorage-independent growth and tumor-cell proliferation and that combination therapy with ATM and microtubule inhibitors holds promise for the treatment of alveolar rhabdomyosarcoma.

An adaptive Src-PDGFRA-Raf axis in rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (aRMS) is a very aggressive sarcoma of children and young adults. Our previous studies have shown that small molecule inhibition of Pdgfra is initially very effective in an aRMS mouse model. However, slowly evolving, acquired resistance to a narrow-spectrum kinase inhibitor (imatinib) was common. We identified Src family kinases (SFKs) to be potentiators of Pdgfra in murine aRMS primary cell cultures from mouse tumors with evolved resistance in vivo in comparison to untreated cultures. Treating the resistant primary cell cultures with a combination of Pdgfra and Src inhibitors had a strong additive effect on cell viability. In Pdgfra knockout tumors, however, the Src inhibitor had no effect on tumor cell viability. Sorafenib, whose targets include not only PDGFRA but also the Src downstream target Raf, was effective at inhibiting mouse and human tumor cell growth and halted progression of mouse aRMS tumors in vivo. These results suggest that an adaptive Src-Pdgfra-Raf-Mapk axis is relevant to PDGFRA inhibition in rhabdomyosarcoma.

Histone methyltransferase KMT1A restrains entry of alveolar rhabdomyosarcoma cells into a myogenic differentiated state.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric muscle cancer, which arrested during the process of skeletal muscle differentiation. In muscle myoblast cells, ectopic expression of the histone H3 lysine 9 (H3K9) methytransferase KMT1A blocks differentiation by repressing a myogenic gene expression program. In this study, we tested the hypothesis that activation of a KMT1A-mediated program of transcriptional repression prevents ARMS cells from differentiating. We investigated whether KMT1A represses the expression of differentiation-associated genes in ARMS cells, thereby blocking muscle differentiation. Our results show that expression of KMT1A is induced in human ARMS cancer cell lines when cultured under differentiation-permissible conditions. shRNA-mediated knockdown of KMT1A decreased anchorage dependent and independent cell proliferation and tumor xenograft growth, increased expression of differentiation-associated genes, and promoted the appearance of a terminally differentiated-like phenotype. Finally, shRNA-directed KMT1A knockdown restored the impaired transcriptional activity of the myogenic regulator MyoD. Together, our results suggested that high levels of KMT1A in ARMS cells under differentiation conditions impairs MyoD function, thereby arresting myogenic differentiation in these tumor cells. Thus, targeting KMT1A may be a novel strategy for the treatment of this disease.

Investigation of peroxiredoxin IV as a calpain-regulated pathway in cancer.

Peroxiredoxin IV (Prx IV), a member of the peroxiredoxin family, has been shown to be involved in cell protection against radiation. Peroxiredoxins are also overexpressed and involved in the progression of several tumours. Calpains have been shown to be over-activated in alveolar rhabdomyosarcoma (ARMS). The present study focused on the possible cross-regulations between Prx IV and calpains in ARMS cells. Prx IV abundance was quantified by Western blot analysis in ARMS cells and compared with non-malignant LHCN-M2 cells. Its abundance is quantified in ARMS cells treated or untreated with calpain inhibitors moreover its mRNA expression is also quantified by real-time RT-PCR in these cells. The study showed that Prx IV is overexpressed by five times in ARMS cells when compared to non-malignant myoblasts. Moreover, the inhibition of calpains using chemical inhibitors led to a decrease in Prx IV abundance (64.32 ± 8.25 and 76.79 ± 4.60 for the precursor and secretable forms, respectively, with calpain inhibitor III treatment). It is the first time that a Prx IV calpain-dependent up-regulation is revealed. In summary, calpains may be implied in the tumour phenotype of ARMS cells especially through Prx IV regulation and may, thus, represent a potential therapeutic target to stop progression of ARMS tumour.

Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells.

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies.

Phosphorylation of serine 205 by the protein kinase CK2 persists on Pax3-FOXO1, but not Pax3, throughout early myogenic differentiation.

The myogenic transcription factor Pax3 plays an essential role in early skeletal muscle development and is a key component in alveolar rhabdomyosarcoma (ARMS), a childhood solid muscle tumor. ARMS is characterized by a t(2;13) chromosomal translocation resulting in the fusion of the 5' Pax3 sequences to the 3' FOXO1 sequences to encode the oncogenic fusion protein, Pax3-FOXO1. Posttranslational modifications such as phosphorylation are common mechanisms by which transcription factors are regulated. Consistent with this fact, we demonstrated in a previous report that Pax3 is phosphorylated on Ser205 in proliferating, but not differentiated, primary myoblasts. However, the kinase that mediates this phosphorylation event has yet to be identified. In addition, it is not known whether Pax3-FOXO1 is phosphorylated at this site or how the phosphorylation of the fusion protein changes during early myogenic differentiation. In this report we identify CK2 (formerly termed "casein kinase II") as the kinase responsible for phosphorylating Pax3 and Pax3-FOXO1 at Ser205 in proliferating mouse primary myoblasts. Furthermore, we demonstrate that, in contrast to wild-type Pax3, phosphorylation at Ser205 persists on Pax3-FOXO1 throughout early myogenic differentiation. Finally, we show that Pax3-FOXO1 is phosphorylated at Ser205 in a variety of translocation-containing ARMS cell lines. The results presented in this report not only suggest a possible mechanism by which the disregulation of Pax3-FOXO1 may contribute to tumorigenesis but also identify a novel target for the development of therapies for the treatment of ARMS.

Heparanase activity in alveolar and embryonal rhabdomyosarcoma: implications for tumor invasion.

BACKGROUND: Rhabdomyosarcoma (RMS) is a malignant soft tissue sarcoma of childhood including two major histological subtypes, alveolar (ARMS) and embryonal (ERMS) RMS. Like other human malignancies RMS possesses high metastatic potential, more pronounced in ARMS than in ERMS. This feature is influenced by several biological molecules, including soluble factors secreted by tumor cells, such as heparanase (HPSE). HPSE is an endo-beta-D-glucuronidase that cleaves heparan sulphate proteoglycans. METHODS: We determined HPSE expression by Western blot analysis in ARMS and ERMS cells lines and activity in supernatants by an ELISA assay. Stable HPSE silencing has been performed by shRNA technique in RH30 and RD cell lines and their invasiveness has been evaluated by Matrigel-invasion assay. HPSE activity and mRNA expression have also been quantified in plasma and biopsies from RMS patients. RESULTS: HPSE expression and activity have been detected in all RMS cell lines. Stable HPSE silencing by shRNA technique determined a significant knockdown of gene expression equal to 76% and 58% in RH30 and RD cell lines respectively and induced a less invasive behaviour compared to untreated cells. Finally, we observed that HPSE mRNA expression in biopsies was higher than in foetal skeletal muscle and that plasma from RMS patients displayed significantly more elevated HPSE levels than healthy subjects with a trend to higher levels in ARMS. CONCLUSION: In conclusion, our data demonstrate for the first time HPSE expression and activity in RMS and highlight its involvement in tumor cell invasion as revealed by shRNA silencing. Moreover, HPSE expression in RMS patients is significantly higher with respect to healthy subjects. Further studies are warranted to assess possible relationships between HPSE and clinical behaviour in RMS.

In vitro assessment of novel transcription inhibitors and topoisomerase poisons in rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Current chemotherapy regimes include the topoisomerase II poison etoposide and the transcription inhibitor actinomycin D. Poor clinical response necessitate identification of new agents to improve patient outcomes. METHODS: We assessed the in vitro cytotoxicity (MTT assay) of DNA intercalating agents in five established human RMS cell lines. These include novel classes of transcription inhibitors and topoisomerase poisons, previously shown to have potential as anti-cancer agents. RESULTS: Amongst the former agents, bisintercalating bis(9-aminoacridine-4-carboxamides) linked through the 9-position, and bis(phenazine-1-carboxamides) linked via their side chains, are compared with established transcription inhibitors. Amongst the latter, monofunctional acridine-4-carboxamides related to N-[2-(dimethylamino)ethyl]acridine-4-carboxamide, DACA, are compared with established topoisomerase poisons. CONCLUSIONS: Our findings specifically highlight the topoisomerase poison 9-amino-DACA, its 5-methylsulphone derivative, AS-DACA, and the bis(phenazine-1-carboxamide) transcription inhibitor MLN944/XR5944, currently in phase I trial, as candidates for further research into new agents for the treatment of RMS.

Different telomere maintenance mechanisms in alveolar and embryonal rhabdomyosarcoma.

The activation of a telomere maintenance mechanism (TMM) is crucial for the immortalization of tumor cells. Most human cancers apply telomerase-dependent TMM but some use a mechanism called alternative lengthening of telomeres (ALT). The latter was suggested to be mainly characterizing sarcomas with nonspecific complex karyotypes, whereas telomerase activation is typical of sarcomas generated by specific translocations. In this study, we investigated the TMM and its association with survival in rhabdomyosarcoma (RMS), which is characterized by two major subtypes: one that is harboring a specific translocation (alveolar) and one that has a nonspecific karyotype (embryonal). Telomerase activity (TA), using telomerase repeat amplification protocol (TRAP) assay, and telomere length (TRF), using Southern blotting, were analyzed in tumor samples from 31 patients (16 embryonal and 15 alveolar). Alveolar RMS tumors exhibited no ALT phenotype and the majority presented TA. Some embryonal tumors exhibited an ALT or "ALT-like" phenotype which lacked TA, whereas others expressed telomerase-dependent TMM, and neither TA nor ALT correlated with outcome. The average TRF length of the embryonal tumors was significantly higher than that of the alveolar tumors (10.8 vs. 7.2 kb, P = 0.003). Interestingly, some tumors of both subtypes presented no TMM. These observations suggest that alveolar RMS predominantly use telomerase-dependent TMM, whereas in embryonal tumors both telomerase and ALT may play a role. These findings have important implications for understanding the role of TMM in the development of RMS tumors, and for future designing adapted treatment strategies.

Phosphorylation profiles of protein kinases in alveolar and embryonal rhabdomyosarcoma.

Rhabdomyosarcoma is the most common pediatric soft-tissue sarcoma, which includes two major subtypes, alveolar and embryonal rhabdomyosarcoma. The mechanism of its oncogenesis is largely unknown. However, the oncogenic process of rhabdomyosarcoma involves multi-stages of signaling protein dysregulation characterized by prolonged activation of tyrosine and serine/threonine kinases. To better understand this protein dysregulation, we evaluated the phosphorylation profiles of multiple tyrosine and serine/threonine kinases to identify whether these protein kinases are activated in rhabdomyosarcoma. We applied immunohistochemistry with phospho-specific antibodies to examine phosphorylation levels of selected receptor and non-receptor tyrosine kinases, mammalian target of rapamycin (mTOR), p70S6K, and protein kinase C (PKC) isozymes on alveolar and embryonal rhabdomyosarcoma tissue microarray slides. Our results showed that the phosphorylation levels of these kinases are elevated in some rhabdomyosarcoma tissues compared to normal tissues. Phosphorylation levels of receptor and non-receptor tyrosine kinases are elevated between 26 and 68% in alveolar rhabdomyosarcoma and between 24 and 71% in embryonal rhabdomyosarcoma, respectively, compared to normal tissues. In addition, phosphorylation levels of mTOR and its downstream targets, p70S6K, S6, and 4EBP1, are increased between 50 and 72% in both subtypes of rhabdomyosarcoma. Further, phosphorylation levels of PKCalpha, PKCdelta, PKCtheta, and PKCzeta/lambda are upregulated between 57 and 69% in alveolar rhabdomyosarcoma and between 43 and 72% in embryonal rhabdomyosarcoma. This is the first report to create a phosphorylation profile of tyrosine and serine/threonine kinases involved in the mTOR and PKC pathways of alveolar and embryonal rhabdomyosarcoma. These protein kinases may play roles in the development or tumor progression of rhabdomyosarcomas and thus may serve as novel targets for therapeutic intervention.

Mirk/Dyrk1b mediates cell survival in rhabdomyosarcomas.

Rhabdomyosarcoma is the most common sarcoma in children and is difficult to treat if the primary tumor is nonresectable or if the disease presents with metastases. The function of the serine/threonine kinase Mirk was investigated in this cancer. Mirk has both growth arrest and survival functions in terminally differentiating skeletal myoblasts. Maintenance of Mirk growth arrest properties would cause down-regulation of Mirk in transformed myoblasts. Alternatively, Mirk expression would be retained if rhabdomyosarcoma cells used Mirk survival capability. Mirk expression was significant in 12 of 16 clinical cases of rhabdomyosarcoma. Mirk was detected in each rhabdomyosarcoma cell line examined. Mirk was a functional kinase in each of three rhabdomyosarcoma cell lines, where it proved to be more active than in C2C12 skeletal myoblasts. Mirk mediated survival of the majority of clonogenic rhabdomyosarcoma cells. Knockdown of Mirk by RNA interference reduced the fraction of RD and of Rh30 rhabdomyosarcoma cells capable of colony formation 3- to 4-fold in multiple experiments. Depletion of Mirk induced cell death by apoptosis, as shown by increased numbers of terminal deoxynucleotidyl transferase-mediated nick-end labeling-positive cells and by increased binding of Annexin V. Mirk is a stress-activated kinase that mediates expression of contractile proteins in differentiating myoblasts, but Mirk is not essential for muscle formation in the embryo. It is likely that Mirk also facilitates survival of satellite cell-derived rhabdomyoblasts in regenerating skeletal muscle and aids their differentiation. This survival function is maintained in rhabdomyosarcoma, where Mirk may be a novel therapeutic target.