High throughput screening of nuclear receptors identifies NR4A1, a novel tumor suppressor with potential as a therapeutic target in gallbladder cancer

Abstract

AbstractIntroductionGallbladder cancer (GBC) is one of the most common cancer of the hepato-biliary tract, with a strikingly variable incidence and prevalence across different regions of the world. The Indo-Gangetic belt in Northern India is reported to have one of the highest incidences of about 21/100,000. GBC usually goes unnoticed due to the lack of any early symptoms with two third of GBC cases present late at inoperable stages and have very limited treatment options. Nuclear receptors, a family of 48 members are ligand dependent transcription factors. They are of particular interest in cancer research because of their established role in cancer pathogenesis and their excellent druggability that makes them a suitable therapeutic target.MethodologymRNA expression 48 nuclear receptors were assessed in GBC tissue samples (n=13) and chronic cholecystitis tissue samples by Nanostring nCounter. The screening identified orphan receptor NR4A1 to be significantly downregulated in GBC. Western blot were performed to further validate the same. We next interrogated the above findings in 2 different gallbladder cancer cell lines, the highly invasive NOZ and the non-invasive TGBC24TKB. In order to investigate the role of NR4A1 in GBC pathogenesis, NOZ cells were treated with cytosporone B (10µM for 24hours) an agonist of NR4A1. On the other hand NR4A1 was knocked down in TGBC24TKB by siRNA. Expression of different markers of proliferation, invasion and epithelial mesenchymal transition was assessed by qPCR. Cell cycle analysis was done using flow cytometry.ResultsNR4A1 was one of the top differentially expressed (down regulated) nuclear receptors in GBC both in RNA and protein level. Similar finding was observed in highly invasive cell line NOZ in comparison to TGBC24TKB. Cytosporone B treatment led to upregulation of NR4A1, which resulted in reduction of cell migration as evident by delayed wound healing, reduction in invasion with an increase in G0/G1 populations implying a growth arrest. NR4A1 knockdown in TGBC24TKB lead to reduction in G0/G1 fraction and also increase in proliferation markers like mki67.ConclusionNR4A1 in our study acts as a tumor suppressor, loss of which seems to provide a growth and survival advantage to GBC cells. NR4A1 activation by agonist reduced cell proliferation and invasion. We therefore propose NR4A1 as a novel biomarker in GBC with its loss associated with overall poor outcome. Hence its agonists may emerge as a potential candidate for neo-adjuvant therapy for advanced gallbladder cancer.