E. J. Rellinger1, S. Lee1, J. Qiao1, B. T. Craig1, K. Kim1, C. V. Romain1, D. H. Chung1 1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA
Introduction: High-risk group neuroblastoma (NB) patients have a poor prognosis with ~50% overall survival. Nearly half of high-risk NBs demonstrate MYCN amplification; however, conventional drug discovery strategies have had limited success in targeting this transcription factor. The bromodomain and extraterminal (BET) family of proteins coactivates transcription by interacting with histones. BET inhibitors, such as JQ1, have recently been shown to block NB proliferation and induce apoptosis in vitro and halt tumor formation in in vivo murine models by inhibiting MYCN signaling. Our laboratory has characterized the role of gastrin-releasing peptide (GRP) signaling in NB tumor initiation, progression, and metastasis. Its cognate receptor, GRP-R, modulates expression of N-myc, along with PI3K/AKT and ERK. As such, we set out to delineate the effects of JQ1 treatment on GRPR signaling and assess the phenotypic effects of this epigenetic regulator in NBs.
Methods: Cell viability was determined using CCK-8 assay. Immunoblotting was performed to assess for N-myc, GRP-R, p-AKT, p-ERK expression after JQ1 treatment in NB cells. Tumor sphere formation in serum-free media was used to select for stem-like cancer cells. Tumor sphere formation and expression of neural and stem cell markers served as measures of JQ1 efficacy. We used a subcutaneous murine xenograft model to determine the effects of intraperitoneal JQ1 administration on tumor progression. At harvest, tumor size and immunohistochemistry were our primary measures of tumor progression and differentiation.
Results: JQ1 inhibited cell growth in both MYCN-amplified and MYCN-nonamplified NBs; n-myc expression was decreased in MYCN-amplified cell lines (Fig. A; *p < 0.05 vs. control). Notably, JQ1 blocked the expression of GRP-R and its downstream pathways, PI3K/AKT and ERK, highlighting that BET inhibitors act on both MYCN-dependent and MYCN-independent signaling pathways (Fig. B). We next selected for purported tumor-initiating cells utilizing an in vitro sphere assay and found that JQ1 diminished sphere formation, decreased expression of stem cell markers, and induced neural differentiation. JQ1 was shown to decrease tumor size and induced neural differentiation in our xenograft model.
Conclusion: BET inhibitor downregulates NB cell proliferation, induces apoptosis, and promotes neural differentiation irrespective of MYCN copies, further implicating its therapeutic potential in the treatment of high-risk group of NBs.
