The objective of this revised NIH renewal application is to continue to explore important signals that mediate the maturation and anti-tumorigenic actions of all-trans retinoic acid (RA) in human teratocarcinomas (TCs) also known as germ cell tumors (GCTs). Our published work reveals that RA induces a neuronal phenotype and non-tumorigenic state in the multipotent human TC cell line NTERA-2 clone D1 (NT2/D1). We discovered through NIH support provided during the prior funding period that the maturation and anti-tumorigenic actions of RA can be uncoupled or dissociated in NT2/D1 cells. This occurs through over-expression of stimulatory growth factors, including transforming growth factor alpha, (TGFalpha) and the flbroblastic growth factor FGF4. This application seeks to build on our preliminary and published work by proposing a novel and direct genetic approach to discover the gene or genes directly mediating the growth inhibitory actions of RA in human TCs. This approach represents an adaptation of the """"""""technical knock-out"""""""" strategy already successfully applied to discover genes mediating growth inhibition by other pathways. The discovery of genes directly mediating the growth inhibitory actions of RA could reveal important cellular pathways activated by RA that propel human TCs to mature and attain a more-growth regulated state. Since retinoids are active anti-tumor agents in diverse human malignancies, understanding those pathways Involved in these clinical responses is important. To achieve these objectives, these specific aims are proposed: (1) To engineer """"""""technical-knock-outs"""""""" with known genes linked to retinoid response to directly evaluate their growth and differentiation effects in human TCs; (2) To engineer """"""""technical knock-out"""""""" vectors using cDNAs from RA-treated or untreated NT2/D1 cells to discover new genes linked to retinoid response in human TCs and (3) To extend this strategy by over- expressing In NT2/D1 cells full-length sense cDNAs prepared from uninduced NT2/D1 cells to learn which species antagonizes the growth inhibitory effects of RA. Using sense and anti-sense vectors for the retinoid receptors RAR alpha, RAR beta, RAR gamma, and RXR alpha themselves, we now provide preliminary data In this revised application that attests to the feasibility of the stated approach to find direct mediators of the RA pathway. The successful completion of these aims will Improve the direct genetic understanding of how RA exerts its Profound anti-tumorigenic actions in human TCs. These studies will help provide mechanistic insights into the actions of RA in a model for differentiation-based therapy of cancer.
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