ACTA1 is inhibited by PAX3-FOXO1 through RhoA-MKL1-SRF signaling pathway and impairs cell proliferation, migration and tumor growth in Alveolar Rhabdomyosarcoma
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Abstract Background Alveolar Rhabdomyosarcoma (ARMS) is a pediatric malignant soft tissue tumor with skeletal muscle phenotype. Little work about skeletal muscle proteins in ARMS was reported. PAX3-FOXO1 is a specific fusion gene generated from the chromosomal translocation t (2;13) (q35; q14) in most ARMS. ACTA1 is the skeletal muscle alpha actin gene whose transcript was detected in ARMS. However, ACTA1 expression and regulation in ARMS have not been well investigated. This work aims to explore the expression, regulation and potential role of ACTA1 in ARMS. Results ACTA1 protein was detected in the studied RH30, RH4 and RH41 ARMS cells. ACTA1 was found to be inhibited by PAX3-FOXO1 at transcription and protein levels by employing western blot, luciferase reporter, qRT-PCR and immunofluorescence assays. The activities of ACTA1 gene reporter induced by RhoA, MKL1, SRF, STARS or Cytochalasin D molecule were reduced in the presence of overexpressed PAX3-FOXO1 protein. CCG-1423 is an inhibitor of RhoA-MKL1-SRF signaling, we observed there was a synergistic effect between this inhibitor and PAX3-FOXO1 to suppress ACTA1 reporter activity. Furthermore, PAX3-FOXO1 overexpression decreased ACTA1 protein level and knockdown of PAX3-FOXO1 by siRNA enhanced ACTA1 expression. In addition, both MKL1 and SRF, but not RhoA were also found to be inhibited by PAX3-FOXO1 gene at protein levels and increased once knockdown of PAX3-FOXO1 expression. The association between MKL1 and SRF in cells was decreased accordingly with ectopic expression of PAX3-FOXO1. However, the distribution of MKL1 and SRF in nuclear or cytoplasm fraction was not changed by PAX3-FOXO1 expression. Finally, we showed that ACTA1 overexpression in RH30 cells could inhibit cell proliferation and migration in vitro and impair tumor growth in vivo compared with the control groups. Conclusions ACTA1 is inhibited by PAX3-FOXO1 at transcription and protein levels through RhoA-MKL1-SRF signaling pathway and this inhibition may partially contribute to the tumorigenesis and development of ARMS. Our findings improved the understanding of PAX3-FOXO1 in ARMS and provided a potential strategy for the treatment of ARMS in future.Keywords:
FOXO1
PAX3
Alveolar rhabdomyosarcoma
Ectopic expression
The tumor-specific chromosomal translocation product, PAX3::FOXO1, is an aberrant fusion protein that plays a key role for oncogenesis in the alveolar subtype of rhabdomyosarcoma (RMS). PAX3::FOXO1 represents a validated molecular target for alveolar RMS and successful inhibition of its oncogenic activity is likely to have significant clinical applications. Even though several PAX3::FOXO1 function-based screening studies have been successfully completed, a directly binding small-molecule inhibitor of PAX3::FOXO1 has not been reported. Therefore, we screened small-molecule libraries to identify compounds that were capable of directly binding to PAX3::FOXO1 protein using surface plasmon resonance technology. Compounds that directly bound to PAX3::FOXO1 were further evaluated in secondary transcriptional activation assays. We discovered that piperacetazine can directly bind to PAX3::FOXO1 protein and inhibit fusion protein-derived transcription in multiple alveolar RMS cell lines. Piperacetazine inhibited anchorage-independent growth of fusion-positive alveolar RMS cells but not embryonal RMS cells. On the basis of our findings, piperacetazine is a molecular scaffold upon which derivatives could be developed as specific inhibitors of PAX3::FOXO1. These novel inhibitors could potentially be evaluated in future clinical trials for recurrent or metastatic alveolar RMS as novel targeted therapy options.
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<p>PDF file - 1972K, Supplementary Figure S2. Screen of primary hits for the identification of PAX3-FOXO1 inhibitors.</p>
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<div>Abstract<p>Alveolar rhabdomyosarcoma comprises a rare highly malignant tumor presumed to be associated with skeletal muscle lineage in children. The hallmark of the majority of alveolar rhabdomyosarcoma is a chromosomal translocation that generates the PAX3-FOXO1 fusion protein, which is an oncogenic transcription factor responsible for the development of the malignant phenotype of this tumor. Alveolar rhabdomyosarcoma cells are dependent on the oncogenic activity of PAX3-FOXO1, and its expression status in alveolar rhabdomyosarcoma tumors correlates with worst patient outcome, suggesting that blocking this activity of PAX3-FOXO1 may be an attractive therapeutic strategy against this fusion-positive disease. In this study, we screened small molecule chemical libraries for inhibitors of PAX3-FOXO1 transcriptional activity using a cell-based readout system. We identified the Sarco/endoplasmic reticulum Ca<sup>2+</sup>-ATPases (SERCA) inhibitor thapsigargin as an effective inhibitor of PAX3-FOXO1. Subsequent experiments in alveolar rhabdomyosarcoma cells showed that activation of AKT by thapsigargin inhibited PAX3-FOXO1 activity via phosphorylation. Moreover, this AKT activation appears to be associated with the effects of thapsigargin on intracellular calcium levels. Furthermore, thapsigargin inhibited the binding of PAX3-FOXO1 to target genes and subsequently promoted its proteasomal degradation. In addition, thapsigargin treatment decreases the growth and invasive capacity of alveolar rhabdomyosarcoma cells while inducing apoptosis <i>in vitro</i>. Finally, thapsigargin can suppress the growth of an alveolar rhabdomyosarcoma xenograft tumor <i>in vivo.</i> These data reveal that thapsigargin-induced activation of AKT is an effective mechanism to inhibit PAX3-FOXO1 and a potential agent for targeted therapy against alveolar rhabdomyosarcoma. <i>Mol Cancer Ther; 12(12); 2663–74. ©2013 AACR</i>.</p></div>
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Rhabdomyosarcoma (RMS) is pediatric sarcoma in the soft muscular tissue. One of the two major types of rhabdomyosarcoma is alveolar rhabdomyosarcoma (ARMS). ARMS generally occurs in muscles in the abdomen, arms, and legs. Due to its tendency to grow more rapidly, it is a more aggressive form of the cancer that has a higher risk factor than other forms of RMS. The expression of PAX3‐FOXO1 in ARMS may contribute to the formation of tumors by inducing tumor growth activities or inhibiting tumor suppressing activities. There are many studies investigating targets of PAX3‐FOXO1 and some indicate that PAX3‐FOXO1 binds to long noncoding RNA (lncRNA), although it is not known if this interaction affects the expression of the lncRNA. Nuclear‐enriched autosomal transcript 1 (NEAT1) is a 4kb lncRNA that potentially is bound by PAX3‐FOXO1. Several putative PAX3‐FOXO1 binding sites are present in the DNA fragment of NEAT1 that was identified in a ChIP assay. We are examining the NEAT1 sequence to narrow down the specific site of PAX3‐FOXO1 interaction as well as determining the effect of PAX3‐FOXO1 on NEAT1 expression. The expression of NEAT1 is necessary for differentiation of muscle cells and a change in this expression due to PAX3‐FOXO1 may be linked to ARMS development. Finding the effect of PAX3‐FOXO1 on expression of NEAT1 will help to uncover more information about the mechanisms involved in the development and progression of ARMS. Support or Funding Information NIH BUILD TL4GM118968, NIH BUILD RL5GM118966‐02
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Rhabdomyosarcomas, malignant tumors of mesenchymal origin, are the most common soft tissue sarcomas in children. Of the two subtypes, alveolar tumors (ARMS) portend the worst prognosis. Most ARMS are characterized by a balanced reciprocal chromosomal translocation t(2;13) that fuses the PAX3 to the FOXO1 gene. Expression of the fusion gene is a negative prognostic factor independent of tumor subtype. Despite the overwhelming data implicating the PAX3-FOXO1 chimeric protein in the pathogenesis of ARMS, little is known about its function. To study its function in its endogenous context, myogenic precursor cells were isolated from transgenic mice. These cells express PAX3- FOXO1 under the control of the PAX3 promoter. The absence of any additional genetic lesions enabled us to dissect the effect of PAX3-FOXO1 alone without the contribution of the additional genetic abnormalities in cells derived from tumors. Chapter 1 introduces alvoleolar rhabdomyosarcomas and summarizes current knowledge of PAX3-FOXO1 function. Chapter 2 describes the characterization of PAX3-FOXO1 transgenic myoblasts and details the discovery of a novel mechanisms by which PAX3-FOXO1 regulates p57Kip2 transcription through the degradation of EGR1. Chapter 3 details the regulation of Mdm2 transcription by PAX3-FOXO1 and discusses how this attenuation of TP53 function likely contributes to the relative resistance of ARMS to treatment. Chapter 4 summarizes the progress made in this dissertation and examines future directions
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PAX3-FOXO1 (PAX3-FKHR) is the fusion protein produced by the genomic translocation that characterizes the alveolar subtype of Rhabdomyosarcoma, a pediatric sarcoma with myogenic phenotype. PAX3-FOXO1 is an aberrant but functional transcription factor. It retains PAX3-DNA-binding activity and functionally overlaps PAX3 function while also disturbing it, in particular its role in myogenic differentiation. We herein show that PAX3-FOXO1 interferes with normal FOXO function. PAX3-FOXO1 affects FOXO-family member trans-activation capability and the FOXO-dependent TGF-β response. PAX3-FOXO1 may contribute to tumor formation by inhibiting the tumor suppressor activities which are characteristic of both FOXO family members and TGF-β pathways. The recognition of this mechanism raises new questions about how FOXO family members function.
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The majority of alveolar rhabdomyosarcoma (ARMS) are distinguished through the paired box 3-forkhead box protein O1 (PAX3-FOXO1) fusion oncoprotein, being generated by a 2;13 chromosomal translocation. This fusion-positive ARMS is the most clinically difficult type of rhabdomyosarcoma. The present study characterized four genes [gremlin 1 (GREM1), death-associated protein kinase-1 (DAPK1), myogenic differentiation-1 (MYOD1), and hairy/enhancer-of-split related with YRPW motif-1 (HEY1)] as targets of PAX3-FOXO1.The expression of the four genes, PAX3-FOXO1, and v-myc myelocytomatosis viral-related oncogene, neuroblastoma-derived (avian) (MYCN) was determined in various ARMS cell models and primary tumors. The roles of PAX3-FOXO1 and MYCN expression were examined.Pulse-chase and cycloheximide experiments suggest that GREM1, DAPK1, and MYOD1 are directly regulated by PAX3-FOXO1. PAX3-FOXO1 appears to indirectly down-regulate HEY1 by up-regulating MYCN. Data reveal that the growth-suppressive activity of high PAX3-FOXO1 expression is closely-associated with up-regulation of the GREM1 and DAPK1 tumor-suppressor genes.This study characterized four downstream targets of PAX3-FOXO1 that contribute to the biological activities of growth suppression and myogenic differentiation.
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The chimeric protein PAX3-FOXO1, resulting from a translocation between chromosomes 2 and 13, is the most common genetic aberration in the alveolar subtype of the human skeletal muscle tumor, rhabdomyosarcoma. To understand how PAX3-FOXO1 contributes to tumor development, we isolated and characterized muscle cells from transgenic mice expressing PAX3-FOXO1 under control of the PAX3 promoter. We demonstrate that these myoblasts are unable to complete myogenic differentiation because of an inability to up-regulate p57Kip2 transcription. This defect is caused by reduced levels of the EGR1 transcriptional activator resulting from a direct, destabilizing interaction with PAX3-FOXO1. Neither PAX3 nor FOXO1 share the ability to regulate p57Kip2 transcription. Thus, the breakage and fusion of the genes encoding these transcription factors creates a unique chimeric protein that controls a key cell-cycle and -differentiation regulator.
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<div>Abstract<p>The tumor-specific chromosomal translocation product, PAX3::FOXO1, is an aberrant fusion protein that plays a key role for oncogenesis in the alveolar subtype of rhabdomyosarcoma (RMS). PAX3::FOXO1 represents a validated molecular target for alveolar RMS and successful inhibition of its oncogenic activity is likely to have significant clinical applications. Even though several PAX3::FOXO1 function-based screening studies have been successfully completed, a directly binding small-molecule inhibitor of PAX3::FOXO1 has not been reported. Therefore, we screened small-molecule libraries to identify compounds that were capable of directly binding to PAX3::FOXO1 protein using surface plasmon resonance technology. Compounds that directly bound to PAX3::FOXO1 were further evaluated in secondary transcriptional activation assays. We discovered that piperacetazine can directly bind to PAX3::FOXO1 protein and inhibit fusion protein-derived transcription in multiple alveolar RMS cell lines. Piperacetazine inhibited anchorage-independent growth of fusion-positive alveolar RMS cells but not embryonal RMS cells. On the basis of our findings, piperacetazine is a molecular scaffold upon which derivatives could be developed as specific inhibitors of PAX3::FOXO1. These novel inhibitors could potentially be evaluated in future clinical trials for recurrent or metastatic alveolar RMS as novel targeted therapy options.</p>Significance:<p>RMS is a malignant soft-tissue tumor mainly affecting the pediatric population. A subgroup of RMS with worse prognosis harbors a unique chromosomal translocation creating an oncogenic fusion protein, PAX3::FOXO1. We identified piperacetazine as a direct inhibitor of PAX3::FOXO1, which may provide a scaffold for designing RMS-specific targeted therapy.</p></div>
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Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood muscle sarcoma with a 5-year survival rate of less than 30%. More than 80% of ARMSs harbor a PAX3-FOXO1 fusion transcription factor. However, expression of PAX3-FOXO1 in muscle cells alone is not sufficient and requires the loss of function of Ink4a/ARF to promote malignant proliferation of muscle cells in vitro or initiate ARMS tumor formation in vivo. This prompted us to examine the signaling pathways required to activate the function of PAX3-FOXO1 and to explore the functional interaction between the Ink4a/ARF and PAX3-FOXO1 signaling pathways. Here we report that inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin (a small molecule selective inhibitor of Cdk4/cyclin D1 that we identified in a screen for compounds that inhibit PAX3-FOXO1) led to inhibition of the transcriptional activity of PAX3-FOXO1 in ARMS cell line Rh30. Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1. In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser430. Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells. Our findings indicate that Cdk4 phosphorylates and positively regulates PAX3-FOXO1 and suggest that inhibition of Cdk4 activity should be explored as a promising avenue for developing therapy for ARMS.
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