Notch Pathway Inhibition Decreases Pituitary Tumor Growth and Increases Tumor Suppressor Gene Expression in GH3 Xenografted Nude/Nude Mice

ANA MARIA ORNSTEIN; SOFIA PERRONE; LAUTARO ZUBELDIA-BRENNER; SANTIAGO RODRIGUEZ-SEGUI

Orlando

Congreso; The Endocrine Society´s 99 rd Annual Meeting; 2017

Endocrine Society

Notch signaling pathway is involved in a wide group of processes including tumorigenesis and stem cell self-renovation. Nevertheless, its role in pituitary tumor generation has not been systematically addressed. Our principal goal was to evaluate if the pharmacological inhibition of Notch pathways alters somatolactotropic tumor growth, hormone secretion, and suppressor gene expression.700000 GH3 cells (somatolactotropic) were injected subcutaneously in the flank of Nude/Nude mice; after 21 days of growth the tumors were treated ip with DAPT, a gamma secretase inhibitor which interferes with Notch activation (8 mg/kg of body weight). After three weeks of treatment the tumors were extracted, measured and frozen for protein and gene expression. We used the information of existing ChIP-seq data to infer putative Notch1 regulatory regions in pituitary GC cells. These were defined as active enhancers (H3K4me1+/H3K27ac+/Pit1+) which we intersected with Notch1 binding (lifted over from T-ALL). Furthermore, we downloaded and reanalyzed public RNA-seq datasets from human Normal Pituitary and Pituitary Tumour samples and performed GSEA analysis to look for Notch signaling components altered in human pituitary adenomas. These in silico transcriptomic and epigenetic analyses allowed us to choose several tumor suppressors or enhancers of Notch signaling to be evaluated in our experiment: Btg2, Nur4A1, Delta like 1N, Men-1, Zfp35, and Cnot1 Tumor volumes in animals treated with DAPT were in average 42% smaller than in the control group (tumoral mass + SE: 490 + 80,7 and 284+ 67,5 mm2, control and DAPT, n=12 y n=11 respectively, p<0.05). Moreover, the intratumoral prolactin and GH contents were significantly diminished, as were serum prolactin levels at the end of the treatment. The N2ICD (Notch intracellular active domain) product of cleavage of the receptor was significantly diminished in the treated compared to non treated tumors, as was the transcription factor Hes-1, a downstream effector of Notch. Of all the components of Notch signaling tested we found a significant increment in the expression of the tumor suppressors: Btg2 and Cnot1.On the other hand, we tested the effect of DAPT in vitro (1, 5 and 10 uM) and observed lower levels of secreted prolactin in the culture media of treated cells, but no significant changes in the protein levels of HES-1, N2ICD, or PCNA or in the mRNA levels of Hes-1, Notch-1, Notch-2, Cyclin D1, Cyclin D3 at the times tested. Nevertheless, DAPT prevented wound healing in a scratch assay, and inhibited GH3 cell proliferation. These data provide strong evidence of a key role of Notch pathways in GH3 somatolactotropic tumor proliferation and development in vivo.