Activation of Grm1 expression by mutated BRaf (V600E) in vitro and in vivo.

Our laboratory previously showed that ectopic expression of Grm1 is sufficient to induce spontaneous melanoma formation with 100% penetrance in transgenic mouse model, TG-3, which harbors wild-type BRaf. Studies identified Grm1 expression in human melanoma cell lines and primary to secondary metastatic melanoma biopsies having wild-type or mutated BRaf, but not in normal melanocytes or benign nevi. Grm1 expression was detected in tissues from mice genetically engineered with inducible melanocyte-specific BRafV600E. Additionally, stable clones derived from introduction of exogenous BRafV600E in mouse melanocytes also showed Grm1 expression, which was not detected in the parental or empty vector-derived cells, suggesting that expression of BRafV600E could activate Grm1 expression. Despite aberrant Grm1 expression in the inducible, melanocyte-specific BRafV600E mice, no tumors formed. However, in older mice, the melanocytes underwent senescence, as demonstrated previously by others. It was proposed that upregulated p15 and TGFß contributed to the senescence phenotype. In contrast, in older TG-3 mice the levels of p15 and TGFß remained the same or lower. Taken together, these results suggest the temporal regulation on the expression of "oncogenes" such as Grm1 or BRafV600E is critical in the future fate of the cells. If BRafV600E is turned on first, Grm1 expression can be induced, but this is not sufficient to result in development of melanoma; the cells undergo senescence. In contrast, if ectopic expression of Grm1 is turned on first, then regardless of wild-type or mutated BRaf in the melanocytes melanoma development is the consequence.

Screening a small molecule library to identify inhibitors of NF-κB inducing kinase and pro-labor genes in human placenta.

The non-canonical NF-κB signaling (RelB/p52) pathway drives pro-labor genes in the human placenta, including corticotropin-releasing hormone (CRH) and cyclooxygenase-2 (COX-2), making this a potential therapeutic target to delay onset of labor. Here we sought to identify small molecule compounds from a pre-existing chemical library of orally active drugs that can inhibit this NF-κB signaling, and in turn, human placental CRH and COX-2 production. We used a cell-based assay coupled with a dual-luciferase reporter system to perform an in vitro screening of a small molecule library of 1,120 compounds for inhibition of the non-canonical NF-κB pathway. Cell toxicity studies and drug efflux transport MRP1 assays were used to further characterize the lead compounds. We have found that 14 drugs have selective inhibitory activity against lymphotoxin beta complex-induced activation of RelB/p52 in HEK293T cells, several of which also inhibited expression of CRH and COX-2 in human term trophoblast. We identified sulfapyridine and propranolol with activity against CRH and COX-2 that deserve further study. These drugs could serve as the basis for development of orally active drugs to affect length of gestation, first in an animal model, and then in clinical trials to prevent preterm birth during human pregnancy.