sirolimus and Rhabdomyosarcoma--Embryonal

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. The two most common histologic variants are the embryonal and alveolar subtypes. Although successive collaborative group clinical trials have improved survival rates for many RMS patients, the outcome for those patients with metastatic or recurrent disease remains poor. Recent studies have pointed to a possible mesenchymal stem cell as the progenitor for alveolar RMS. Other studies have implicated several cellular mechanisms and pathways being involved in RMS pathogenesis and survival, such as the cyclin-dependent kinase inhibitors, insulin-like growth factor pathway, and the mammalian target of rapamycin pathway, thus providing potential avenues for targeted therapy. Recent clinical trials have tried to improve risk stratification and prediction of clinical outcome based upon clinical or radiographic response to initial therapy and also to determine the role of high-dose chemotherapy with stem cell rescue in high-risk RMS patients.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Child; Child, Preschool; Clinical Trials as Topic; Combined Modality Therapy; Disease Progression; Female; Humans; Male; Mesenchymal Stem Cells; Mice; Neoplasm Metastasis; Neoplasm Staging; Neoplastic Stem Cells; Prognosis; Protein Kinase Inhibitors; Rhabdomyosarcoma, Alveolar; Rhabdomyosarcoma, Embryonal; Risk Factors; Secondary Prevention; Signal Transduction; Sirolimus; Soft Tissue Neoplasms; Survival Rate; Tomography; TOR Serine-Threonine Kinases

In pediatric rhabdomyosarcoma (RMS), elevated Akt signaling is associated with increased malignancy. Here, we report that expression of a constitutively active, myristoylated form of Akt1 (myrAkt1) in human RMS RD cells led to hyperactivation of the mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) pathway, resulting in the loss of both MyoD and myogenic capacity, and an increase of Ki67 expression due to high cell mitosis. MyrAkt1 signaling increased migratory and invasive cell traits, as detected by wound healing, zymography, and xenograft zebrafish assays, and promoted repair of DNA damage after radiotherapy and doxorubicin treatments, as revealed by nuclear detection of phosphorylated H2A histone family member X (γH2AX) through activation of DNA-dependent protein kinase (DNA-PK). Treatment with synthetic inhibitors of phosphatidylinositol-3-kinase (PI3K) and Akt was sufficient to completely revert the aggressive cell phenotype, while the mTOR inhibitor rapamycin failed to block cell dissemination. Furthermore, we found that pronounced Akt1 signaling increased the susceptibility to cell apoptosis after treatments with 2-deoxy-D-glucose (2-DG) and lovastatin, enzymatic inhibitors of hexokinase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), especially in combination with radiotherapy and doxorubicin. In conclusion, these data suggest that restriction of glucose metabolism and the mevalonate pathway, in combination with standard therapy, may increase therapy success in RMS tumors characterized by a dysregulated Akt signaling.

Topics: Animals; Child; Deoxyglucose; DNA Repair; DNA-Activated Protein Kinase; Doxorubicin; Glucose; Glycolysis; Hexokinase; Histones; Humans; Ki-67 Antigen; Lovastatin; Mevalonic Acid; MTOR Inhibitors; Oxidoreductases; Phosphatidylinositol 3-Kinases; Phosphatidylinositols; Proto-Oncogene Proteins c-akt; Rhabdomyosarcoma, Embryonal; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases; Zebrafish

Alveolar rhabdomyosarcoma (RMS) has a much poorer outcome than embryonal RMS. In this study, we found that IGF-I affected the induction of myogenin and cell cycle progression in alveolar RMS cells, but not in embryonal RMS cells. IGF-I enhanced the induction of myogenin protein in alveolar RMS SJ-Rh30 and KP-RMS-MS cells as it did in myoblast C2C12 cells, but not in embryonal RMS RD or KP-RMS-KH cells. IGF-I induction of myogenin protein was blocked by anti-IGF-IR monoclonal antibody alphaIR-3 and the mTOR-specific inhibitor rapamycin. In Rh30mTOR-rr cells, which stably express a rapamycin-resistant mutant mTOR, rapamycin did not inhibit IGF-I induction of myogenin protein. These data suggest that IGF-I induces myogenin in alveolar RMS cells through the IGF-IR/mTOR pathway. In C2C12 cells, IGF-I induces myogenin protein followed by cell cycle arrest leading to myogenic differentiation. IGF-I promoted G1-S cell cycle progression without any signs of terminal differentiation in alveolar RMS cells. On the other hand, IGF-I promoted neither cell cycle arrest nor G1-S cell cycle progression in embryonal RMS cells. In alveolar RMS SJ-Rh30 cells, 4E-BP1, one of two effectors downstream of mTOR, was continuously hyperphosphorylated by IGF-I, whereas in embryonal RMS RD cells, 4E-BP1 was only transiently hyperphosphorylated. These findings suggest that the different effects of IGF-I on myogenin induction and cell cycle progression in alveolar and embryonal RMS cells are due to a difference of phosphorylation status of 4E-BP1. These different responses to IGF-I help to explain immunohistochemical and clinical behavioral differences between alveolar and embryonal RMS.

Topics: Antibiotics, Antineoplastic; Antibodies, Monoclonal; Cell Cycle; Cell Line, Tumor; Humans; Imidazoles; Insulin-Like Growth Factor I; Muscle Development; Myogenin; Protein Biosynthesis; Protein Kinase Inhibitors; Protein Kinases; Pyridines; Receptor, IGF Type 1; Rhabdomyosarcoma, Alveolar; Rhabdomyosarcoma, Embryonal; Sirolimus; TOR Serine-Threonine Kinases

The protein synthetic machinery is activated by diverse genetic alterations during tumor progression in vivo and represents an attractive target for cancer therapy. We show that rapamycin inhibits the induction of transformed foci in vitro by GLI, a transcription factor that functions in the sonic hedgehog-patched pathway in tumors. In control cells, which were nontransformed epithelioid RK3E cells and derivative c-MYC- or RAS-transformed sister cell lines, rapamycin inhibits mTOR and mTOR-dependent activities but increases global protein synthesis, perhaps by activating a feedback mechanism. In GLI-transformed cells, rapamycin inhibits global protein synthesis and turnover and prevents cellular proliferation. In contrast to its effects on protein synthesis, rapamycin affects bromodeoxyuridine incorporation and cell cycle occupancy of GLI cells and control cells to a similar extent. Rare, variant GLI cells isolated by selection in rapamycin are also drug-resistant for protein metabolism and for cell cycle progression through G1. Our results indicate that sensitivity to rapamycin can be induced by a specific oncogene and that inhibition of global protein metabolism is linked to the rapamycin-sensitive phenotype.

Topics: Animals; Antibiotics, Antineoplastic; Cell Cycle; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Genes, myc; Genes, ras; Immunosuppressive Agents; Mice; Mice, Nude; Neoplasm Transplantation; Oncogene Proteins; Phosphotransferases (Alcohol Group Acceptor); Protein Biosynthesis; Protein Kinases; Rhabdomyosarcoma, Alveolar; Rhabdomyosarcoma, Embryonal; Sirolimus; TOR Serine-Threonine Kinases; Trans-Activators; Transcription Factors; Transfection; Transplantation, Heterologous; Zinc Finger Protein GLI1; Zinc Fingers