NR4A1 Antagonists Target the Oncogenic PAX3-FOXO1A Gene and Induce Interleukin -24 in Rhabdomyosarcoma (RMS)
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Rhabdomyosarcoma (RMS) is a pediatric cancer for which common therapeutics include cytotoxic chemotherapeutics that result in adverse health outcomes later in adulthood. Previous studies have found NR4A1 expression contributes to the oncogenicity of several solid tumors and this study aims to determine if the established NR4A1-dependent mechanisms are observed in RMS. The orphan nuclear receptor NR4A1 is expressed in tumors from RMS patients and RMS cell lines. NR4A1 knockdown/antagonism also decreases the expression of growth promoting/pro-survival genes, inhibits mTOR signaling and induce oxidative stress. 1,1-Bis(3-indolyl)-1-(p-substituted phenyl)methane (C-DIM) analogs are NR4A1 ligands in RMS cells and inhibit RMS cell growth in vitro and in vivo. We also report overexpression of the nuclear receptor NR4A1 in rhabdomyosarcoma that is sufficient to drive high expression of the oncogenic PAX3-FOXO1A protein. RNAi for NR4A1 or C-DIM treatment decreased expression of PAX3-FOXO1A and its downstream effector genes. NR4A1 also regulated expression of β1-integrin, along with PAX3-FOXO1A, contributed to tumor cell migration that was blocked by C-DIM/NR4A1 antagonists. RNASeq studies using NR4A1 knockdown and antagonism and PAX3- FOXO1A antagonism in RMS cells revealed several genes that are commonly iii regulated by PAX3-FOXO1A and NR4A1, including the tumor suppressor-like factor interleukin-24 (IL-24). Studies show IL-24 expression is repressed in ARMS tumors and PAX3-FOXO1A positive tumors and transfection of ARMS cells with siPAX3-FOXO1A or siNR4A1, or treatment with C-DIM/NR4A1 antagonist result in IL-24 induction. In cells cotransfected with siPAX3-FOXO1A and siIL-24 or in IL-24 KO Rh30 cells, effects of siPAX3-FOXO1A were significantly abrogated. These data show that the oncogenic activity of PAX3- FOXO1A was due to, in part, IL-24 repression. Therefore, IL-24 induction contributes to the anti-carcinogenic actions observed upon PAX3-FOXO1A suppression. Previous studies have demonstrated the ability of C-DIMs to inhibit NR4A1-dependent pathways, yet these compounds exhibit low serum half-life, making them unsuitable for therapeutic applications. The current studies using selected p-hydroxyphenyl-substituted analogs (second-generation C-DIMs) show up to 10 times more potent than the parent compound in vitro and in vivo. This new class of potent NR4A1 antagonists is being developed for future clinical applications for treating RMS, a cancer which has a poor prognosis with current treatments.
Lacey, Alexandra Dianne (2017). NR4A1 Antagonists Target the Oncogenic PAX3-FOXO1A Gene and Induce Interleukin -24 in Rhabdomyosarcoma (RMS). Doctoral dissertation, Texas A & M University. Available electronically from