Gene expression is extensively reprogrammed in cancer. These dysregulated genes include subpopulations called RNA regulons that are coordinately regulated by posttranscriptional mechanisms at the RNA level and control key features of tumor aggressiveness. To understand the molecular consequences of dysregulated RNA regulons in cancer, the goal of this exploratory, high-risk/high-reward R21 proposal is to develop a zinc finger-directed RNA-cleaving agent to suppress RNA regulons that are upregulated in many tumors. Our prototype links the tandem zinc finger (TZF) domain from tristetraprolin (TTP) to the endoribonuclease RNase4 (R4). In cells, the chimeric TZF-R4 protein is expected to bind and rapidly degrade mRNA substrates of TTP, but our design will allow substrate specificity to be systematically modified. The TTP TZF domain was chosen for our prototype because it has been evolutionarily selected to target an RNA regulon containing AU-rich elements (AREs), which includes many mRNAs that encode regulators of the cell cycle, angiogenesis, and metastasis. Furthermore, TTP expression or activity is frequently suppressed in human cancers; in particular, low TTP levels in breast tumors are associated with poor patient prognosis. This proposal is aimed at providing the ?proof of concept? that a TZF-R4 chimera can function as a guided RNA degradation system in cancer cells to suppress a pro-tumorigenic RNA regulon and attenuate associated tumor cell phenotypes. Purified TZF-R4 shows selective RNA recognition and cleavage in vitro, and suppressed two known TTP substrate mRNAs when transiently transfected into cells. Furthermore, TZF-R4 dramatically slows cell proliferation when expressed in a clonal, stably-transfected breast cancer cell line. Building upon these key preliminary results, two specific aims will be pursued. First, we will use transcriptome- wide approaches to define the RNA regulon that is targeted and destabilized by TZF-R4 when stably expressed in aggressive breast cancer cell models and demonstrate that TZF-R4 suppresses multiple mRNA targets more efficiently than current technologies. Second, we will assess the impact of TZF-R4 on breast cancer cell proliferation, stemness, invasion, migration, and in vivo tumor development. Several future applications of this technology are envisioned, including: (i) discovery tools for characterizing RNA-mediated biological pathways, (ii) developing methods for promoting uptake of purified TZF-R4 into cells, bypassing transfection and opening possibilities for direct in vivo administration of this reagent, (iii) restoration or augmentation of TTP function to suppress tumor aggressiveness or inflammatory signaling, and (iv) expanding the specificity of the TZF-R4 platform by altering its RNA-targeting specificity. Strategies to broaden the scope include the iterative or combinatorial modification of the TZF moiety and substitution of other RNA-binding domains to `guide' the chimeric protein, creating a tunable family of targeted ribonucleases with long-term impact.
This project will develop a new method to suppress expression of gene subpopulations by delivering an endoribonuclease to mRNA substrates via a tandem zinc finger (TZF) RNA-binding domain. Our initial prototype will target mRNAs recognized by the RNA-binding protein tristetraprolin (TTP), which are aberrantly stabilized in a wide variety of cancers owing to suppression or deactivation of endogenous TTP, but our design will allow substrate specificity to be systematically modified. This technology has the long-term potential to be utilized as a discovery tool for dissecting RNA-mediated biological pathways involved in cancer and other disease states, as well as a putative anti-cancer and/or anti-inflammatory strategy.