Alveolar rhabdomyosarcoma (ARMS) is a devastating pediatric disease driven by expression of the oncogenic fusion gene PAX3-FOXO1A. In this study, we report overexpression of the nuclear receptor NR4A1 is rhabdomyosarcomas which is sufficient to drive high expression of PAX3-FOXO1A there. RNAi-mediated silencing of NR4A1 decreased expression of PAX3-FOXO1A and its downstream effector genes. Similarly, cell treatment with the NR4A1 small molecule antagonists 1,1-bis(3-indolyl)-1-(p-hydroxy or p-carbomethoxyphenyl)methane (C-DIM) decreased PAX3-FOXO1A. Mechanistic investigations revealed a requirement for the NR4A1/Sp4 complex to bind GC-rich promoter regions to elevate transcription of the PAX3-FOXO1A gene. In parallel, NR4A1 also regulated expression of β1-integrin which with PAX3-FOXO1A contributed to tumor cell migration that was blocked by C-DIM/NR4A1 antagonists. Taken together, our results provide a preclinical rationale for the use of NR4A1 small molecule antagonists to treat ARMS and other rhabdomyosarcomas driven by PAX3-FOXO1A.

Diabetes mellitus (DM), a multifaceted metabolic disorder if not managed properly leads to secondary complications. Diabetic peripheral neuropathy (DPN) is one such complication caused by nerve damage that cannot be reversed but can be delayed. Recently, diabetes patients are using dietary supplements, although there remains a general skepticism about this practice. Curcumin (CUR), one such supplement can help prevent underlying low-grade inflammation in diabetes, but it is plagued by poor oral bioavailability. To better understand the role of bioavailability in clinical outcomes, we have tested double-headed nanosystems containing curcumin (nCUR) on DPN. Because CUR does not influence glucose levels, we have also tested the effects of nCUR combined with long-acting subcutaneous insulin (INS). nCUR with or without INS alleviates DPN at two times lower dose than unformulated CUR, as indicated by qualitative and quantitative analysis of the hind paw, sciatic nerve, spleen, and L4−6 spinal cord. In addition, nCUR and nCUR+INS preserve hind paw nerve axons as evident by the Bielschowsky silver stain and intraepidermal nerve fibers (IENF) density measured by immunofluorescence. The mechanistic studies further corroborated the results, where nCUR or nCUR+INS showed a significant decrease in TUNEL positive cells, mRNA expression of NLRP3, IL-1β, and macrophage infiltration while preserving nestin and NF200 expression in the sciatic nerve. Together, the data confirms that CUR bioavailability is proportional to clinical outcomes and INS alone may not be one of the solutions for DM. This study highlights the potential of nCUR with or without INS in alleviating DPN and warrants further investigation.

<p>Cell proliferation studies. (A) C2C12 cells were transfected with siNR4A1 or treated with 15 muM DIM-C-pPhCO₂Me (24 hr), and effects on cell number were determined. (B) Rh30 and Rh18 cells were transfected with siCtl or siNR4A1 {plus minus} TGFβ (5 ng/ml) and cell numbers were counted. * Significantly (p<0.05) decreased.</p>

<p>Antibodies, oligonucleotides and primers.</p>

<p>Role of Sp1, p300 and CBP in PAX3-FOXO1A expression. ARMS cells were transfected with siSp1 (A), sip300 (B) and siCBP (C) for 72 hr, and whole cell lysates were analyzed by western blots.</p>