piperidines and Rhabdomyosarcoma--Alveolar

PARP inhibitors (PARPi) are used in a wide range of human solid tumours but a limited evidence is reported in rhabdomyosarcoma (RMS), the most frequent childhood soft-tissue sarcoma. The cellular and molecular effects of Olaparib, a specific PARP1/2 inhibitor, and AZD2461, a newly synthesized PARP1/2/3 inhibitor, were assessed in alveolar and embryonal RMS cells both as single-agent and in combination with ionizing radiation (IR).. Cell viability was monitored by trypan blue exclusion dye assays. Cell cycle progression and apoptosis were measured by flow cytometry, and alterations of specific molecular markers were investigated by, Real Time PCR, Western blotting and immunofluorescence experiments. Irradiations were carried out at a dose rate of 2 Gy (190 UM/min) or 4 Gy (380 UM/min). Radiosensitivity was assessed by using clonogenic assays.. Olaparib and AZD2461 dose-dependently reduced growth of both RH30 and RD cells by arresting growth at G2/M phase and by modulating the expression, activation and subcellular localization of specific cell cycle regulators. Downregulation of phospho-AKT levels and accumulation of γH2AX, a specific marker of DNA damage, were significantly and persistently induced by Olaparib and AZD2461 exposure, this leading to apoptosis-related cell death. Both PARPi significantly enhanced the effects of IR by accumulating DNA damage, increasing G2 arrest and drastically reducing the clonogenic capacity of RMS-cotreated cells.. This study suggests that the combined exposure to PARPi and IR might display a role in the treatment of RMS tumours compared with single-agent exposure, since stronger cytotoxic effects are induced, and compensatory survival mechanisms are prevented.

Topics: Apoptosis; Blotting, Western; Cell Division; Cell Line, Tumor; Cell Survival; Child; DNA Damage; Dose-Response Relationship, Drug; Flow Cytometry; Fluorescent Antibody Technique; Histones; Humans; Isoenzymes; Phthalazines; Piperazines; Piperidines; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Radiation Tolerance; Radiation, Ionizing; Real-Time Polymerase Chain Reaction; Rhabdomyosarcoma, Alveolar; Rhabdomyosarcoma, Embryonal

Alveolar rhabdomyosarcoma (ARMS) is a muscle-derived childhood tumor characterized by production of oncogenic PAX3/7-FOXO1 chimeric transcription factors. While downstream targets of the PAX3-FOXO1 oncoprotein in ARMS have been defined, the functional relevance of these targets is unclear. Here, we show that upregulation of the cannabinoid receptor 1 (Cnr1/Cb1) by PAX3-FOXO1 in mouse primary myoblasts and ARMS cell lines, contributes to PAX3-FOXO1 phenotypes, both in vivo and in vitro. In primary myoblasts, Cnr1 was dispensable for PAX3-FOXO1 to mediate cell proliferation, differentiation, or transformation; however, Cnr1 function was essential to increase the invasive capacity conferred by PAX3-FOXO1 overexpression in these cells. Genetic or pharmacologic abrogation of Cnr1 inhibited the enhanced basement membrane invasion induced by PAX3-FOXO1. Cnr1 loss by either route also dramatically reduced lung metastasis formation. Taken together, our findings strongly implicate Cnr1 as a novel tractable target to inhibit ARMS invasion and metastasis.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p16; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Myoblasts; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms, Experimental; Oncogene Proteins, Fusion; Paired Box Transcription Factors; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Reverse Transcriptase Polymerase Chain Reaction; Rhabdomyosarcoma, Alveolar; Tumor Suppressor Protein p14ARF