Patrick J. Miller

Identification of serine 205 as a site of phosphorylation on Pax3 in proliferating but not differentiating primary myoblasts

Pax3, a member of the paired class homeodomain family of transcription factors, is essential for early skeletal muscle development. Previously, others and we have shown that the stability of Pax3 is regulated on a post‐translational level. Evidence in the literature and from our laboratory suggests that phosphorylation, a common form of regulation, may play a role. However, at present, the sites of Pax3 phosphorylation are not known. We demonstrate here the first evidence that Pax3 exists as a phosphoprotein in proliferating mouse primary myoblasts. Using an in vitro kinase assay, deletion, and point mutant analysis, we conclusively identify Ser205 as a site of phosphorylation. The phosphorylation of Ser205 on endogenously expressed Pax3 was confirmed in vivo using antibodies specific for phosphorylation at Ser205. Finally, we demonstrate for the first time that the phosphorylation status of endogenous Pax3 changes rapidly upon the induction of myogenic differentiation. The presence of phosphorylation in a region of Pax3 important for mediating protein–protein interactions, and the fact that phosphorylation is lost upon induction of differentiation, allow for speculation on the biological relevance of phosphorylation.

FOXO1 stimulates ceruloplasmin promoter activity in human hepatoma cells treated with IL-6

FOXO1, a member of the winged-helix family of transcription factors, is a ubiquitously expressed protein involved in regulating a variety of cellular processes including glucose homeostasis, apoptosis, cell cycle control, muscle differentiation, and angiogenesis. In addition to these biological functions, FOXO1 is a key player in the oxidative stress response by stimulating the expression of metal-containing anti-oxidant proteins such as manganese superoxide dismutase, selenoprotein P, and catalase. Evidence in the literature suggests that FOXO1 may also be capable of regulating the expression of the anti-oxidant protein Ceruloplasmin (Cp), a six-copper-containing protein synthesized and secreted mainly by the liver. In the present report, we demonstrate that FOXO1 stimulates Cp promoter activity in conjunction with the cytokine IL-6. Through deletional analysis and in vitro binding studies, we determine the DNA sequence responsible for the FOXO1-dependent regulation of the Cp proximal promoter. Finally, we demonstrate that FOXO1 is capable of enhancing the expression of endogenous Cp in human hepatic carcinoma cells treated with IL-6. These results allow us to identify FOXO1 as a regulator of Cp expression to promote the anti-oxidant pathway in response to IL-6 signaling.

Identification of CK2 as the kinase that phosphorylates Pax3 at Ser209 in early myogenic differentiation.

The myogenic transcription factor Pax3, a member of the paired class homeodomain family of transcription factors, plays an essential role in early skeletal muscle development. We previously demonstrated that Pax3 is phosphorylated at three specific residues (Ser201, Ser205, and Ser209) and that the pattern of phosphorylation at these sites changes throughout early myogenesis. Further, we demonstrated that the protein kinase CK2 phosphorylates Pax3 at Ser205 and that this phosphorylation event is required for the subsequent phosphorylation of Ser201 by GSK3β. However, the kinase that phosphorylates Pax3 at Ser209 has yet to be identified. In the present work we use standard purification methods and in vitro biochemical analyses to provide solid evidence identifying the protein kinase CK2 as phosphorylating Pax3 at Ser209. Further, we qualitatively demonstrate that the phosphorylation of Pax3 at Ser209 by CK2 is enhanced when Ser205 is previously phosphorylated. Taken together, our results allow us to propose a mechanism to describe the ordered phosphorylation of Pax3 throughout early myogenesis.

Comparative transcriptomic analysis reveals the oncogenic fusion protein PAX3-FOXO1 globally alters mRNA and miRNA to enhance myoblast invasion

Rhabdomyosarcoma, one of the most common childhood sarcomas, is comprised of two main subtypes, embryonal and alveolar (ARMS). ARMS, the more aggressive subtype, is primarily characterized by the t(2;13)(p35;p14) chromosomal translocation, which fuses two transcription factors, PAX3 and FOXO1 to generate the oncogenic fusion protein PAX3-FOXO1. Patients with PAX3-FOXO1-postitive tumors have a poor prognosis, in part due to the enhanced local invasive capacity of these cells, which leads to the increased metastatic potential for this tumor. Despite this knowledge, little is known about the role that the oncogenic fusion protein has in this increased invasive potential. In this report we use large-scale comparative transcriptomic analyses in physiologically relevant primary myoblasts to demonstrate that the presence of PAX3-FOXO1 is sufficient to alter the expression of 70 mRNA and 27 miRNA in a manner predicted to promote cellular invasion. In contrast the expression of PAX3 alters 60 mRNA and 23 miRNA in a manner predicted to inhibit invasion. We demonstrate that these alterations in mRNA and miRNA translate into changes in the invasive potential of primary myoblasts with PAX3-FOXO1 increasing invasion nearly 2-fold while PAX3 decreases invasion nearly 4-fold. Taken together, these results allow us to build off of previous reports and develop a more expansive molecular model by which the presence of PAX3-FOXO1 alters global gene regulatory networks to enhance the local invasiveness of cells. Further, the global nature of our observed changes highlights the fact that instead of focusing on a single-gene target, we must develop multi-faceted treatment regimens targeting multiple genes of a single oncogenic phenotype or multiple genes that target different oncogenic phenotypes for tumor progression.

Acquisition of an oncogenic fusion protein is sufficient to globally alter the landscape of miRNA expression to inhibit myogenic differentiation

The differentiation status of tumors is used as a prognostic indicator, with tumors comprised of less differentiated cells exhibiting higher levels of aggressiveness that correlate with a poor prognosis. Although oncogenes contribute to blocking differentiation, it is not clear how they globally alter miRNA expression during differentiation to achieve this result. The pediatric sarcoma Alveolar Rhabdomyosarcoma, which is primarily characterized by the expression of the PAX3-FOXO1 oncogenic fusion protein, consists of undifferentiated muscle cells. However, it is unclear what role PAX3-FOXO1 plays in promoting the undifferentiated state. We demonstrate that expression of PAX3-FOXO1 globally alters the expression of over 80 individual miRNA during early myogenic differentiation, resulting in three primary effects: 1) inhibition of the expression of 51 miRNA essential for promoting myogenesis, 2) promoting the aberrant expression of 43 miRNA not normally expressed during myogenesis, and 3) altering the expression pattern of 39 additional miRNA. Combined, these changes are predicted to have an overall negative effect on myogenic differentiation. This is one of the first studies describing how an oncogene globally alters miRNA expression to block differentiation and has clinical implications for the development of much needed multi-faceted tumor-specific therapeutic regimens.

Acquisition of an oncogenic fusion protein serves as an initial driving mutation by inducing aneuploidy and overriding proliferative defects.

While many solid tumors are defined by the presence of a particular oncogene, the role that this oncogene plays in driving transformation through the acquisition of aneuploidy and overcoming growth arrest are often not known. Further, although aneuploidy is present in many solid tumors, it is not clear whether it is the cause or effect of malignant transformation. The childhood sarcoma, Alveolar Rhabdomyosarcoma (ARMS), is primarily defined by the t(2;13)(q35;q14) translocation, creating the PAX3-FOXO1 fusion protein. It is unclear what role PAX3-FOXO1 plays in the initial stages of tumor development through the acquisition and persistence of aneuploidy. In this study we demonstrate that PAX3-FOXO1 serves as a driver mutation to initiate a cascade of mRNA and miRNA changes that ultimately reprogram proliferating myoblasts to induce the formation of ARMS. We present evidence that cells containing PAX3-FOXO1 have changes in the expression of mRNA and miRNA essential for maintaining proper chromosome number and structure thereby promoting aneuploidy. Further, we demonstrate that the presence of PAX3-FOXO1 alters the expression of growth factor related mRNA and miRNA, thereby overriding aneuploid-dependent growth arrest. Finally, we present evidence that phosphorylation of PAX3-FOXO1 contributes to these changes. This is one of the first studies describing how an oncogene and post-translational modifications drive the development of a tumor through the acquisition and persistence of aneuploidy. This mechanism has implications for other solid tumors where large-scale genomics studies may elucidate how global alterations contribute to tumor phenotypes allowing the development of much needed multi-faceted tumor-specific therapeutic regimens.

Isolation of putative FOXO1 genomic elements using an improved in vitro technique to isolate genomic regulatory sequences

The regulation of gene expression drives many biological processes and alterations in normal regulation are integral in the development of the diseased state. Therefore, the ability to screen genomic DNA for direct targets of DNA binding proteins (DNA-BP) would provide valuable information about the mechanisms underlying these processes. At present chromatin immunoprecipitation (ChIP) and its variants are the primary methods for identifying regulatory elements. However, some DNA-BPs, such as the winged-helix transcription factor FOXO1, are difficult to ChIP thereby detracting from the use of this technique as a nonbiased screen to isolate regulatory sequences. In this report we use an improved in vitro method to Pull Out Regulatory Elements (PORE), which uses purified protein with a stable genomic library to isolate regulatory elements directly bound by a DNA-BP, to identify putative FOXO1 genomic regulatory sequences. We first validate this technique using two known DNA-BP (FOXO1 and Pax3) by demonstrating their ability to bind and amplify identified promoter elements when present in a genomic DNA context or when present in the context of our stable genomic library. Subsequent use of this technique with FOXO1 isolated regulatory elements associated with several genes known to be regulated in a FOXO1-dependent manner and multiple genes whose biological functions are consistent with the known biological functions of FOXO1 proving that the in vitro PORE is a valuable and easy to use alternative to ChIP for the isolation of genomic regulatory elements.

Abstract A62: Phosphorylation of Ser205 by the protein kinase CK2 persists on Pax3‐FOXO1, but not Pax3, throughout 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, which is associated with a four year survival rate of only 17%, is most commonly 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. More recently it was demonstrated that Pax3‐FOXO1 is also phosphorylated at unidentified sites in non‐physiologically relevant cells. Despite this information, it is not known whether Pax3‐FOXO1 is phosphorylated at Ser205 or how the phosphorylation of the fusion protein changes during myogenic differentiation. In this report, we use standard in vitro kinase assays to 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 use standard Western blot analysis to demonstrate that in contrast to wild‐type Pax3, phosphorylation at Ser205 persists on Pax3‐throughout early myogenic differentiation. Finally, we use standard Western blot analysis to 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 identifies a novel target for the development of therapies to be used for the treatment of ARMS. Citation Information: Cancer Res 2009;69(23 Suppl):A62.