Selective killing of human cancer cells by targeting a fusion mRNA between CYCLIN D1 and TROP2

e14569 Background: Trop-2 is a calcium signal transducer and a stem cell marker. Trop-2 is widely overexpressed by human cancers and stimulates their growth. A TROP2 mRNA was isolated as post-transcriptionally joined to CYCLIN D1 transcripts, suggesting this as one of the transforming mechanisms of TROP2. Methods: In vitro cell growth assays were utilized to assess the cell growth stimulatory capacity of the chimeric mRNA. Colony assays for growth in soft agarose and tumorigenicity assays in nude mice were utilized to assess for the transforming capacity of the fusion transcript. siRNA constructs were utilized for the stably shut-down of the expression of the CYCLIN D1-TROP2 mRNA. Results: The chimeric mRNA transforms primary cells in vitro and induces aggressive tumor growth in vivo in cooperation with activated RAS. The CYCLIN D1-TROP2 mRNA is expressed by a large fraction of human ovarian, endometrial and gastro-intestinal tumors. The chimera is coexpressed with activated RAS in a subset of tumors, consistent with a cooperative transforming activity. The chimeric mRNA is a bicistronic transcript that independently translates wild type Cyclin D1 and Trop-2 proteins, i.e. it does not generate chimeric, oncogenic proteins. On the other hand, joining to the stable TROP2 mRNA leads to a higher CYCLIN D1 mRNA stability, with inappropriate persistence during the cell cycle and acquisition of transforming capacity. As essentially no normal tissues express the chimeric mRNA, we targeted it for destruction in cancer cells with stably expressed siRNA constructs. Specific targeting led to essential annnihilation of the CYCLIN D1-TROP2 mRNA, in the absence of off-target effects. Silencing of the chimeric mRNA blocked the growth of expressing breast cancer cells. Conclusions: Our findings demonstrate a novel, widespread oncogenic mechanism in human cancers, and open novel avenues for mRNA-targeted anti-cancer therapies. Acknowledgments This work was supported by the the Fondazione of the Cassa di Risparmio della Provincia di Chieti, the Association for the Application of Biotechnology in Oncology (ABO and ABO Project S.p.A., grant no. VE01D0019) and the Marie Curie Transfer of Knowledge Fellowship, contract number 014541. No significant financial relationships to disclose.