Introduction: Down syndrome (DS) is the most common chromosomal abnormality in humans and results in an increased risk for hematologic cancers, despite a paradoxically lower rate of adult solid tumors. Epidemiological evidence supports the idea that DS patients have a lower risk for bladder cancer (BCa) in spite of reduced environmental exposures. We sought to evaluate potential protective mechanisms against cancer formation in the Ts65Dn mouse model of DS, and hypothesized Ts65Dn mice would be protected against (BCa) formation and progression. Methods: Tumors were initiated in Ts65Dn and WT mice using N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Ts65Dn or WT animals were given 0.05% BBN for 16 weeks before sacrifice. Bladders were weighed at time of death. For Reactome pathways, Gene Set Enrichment Analysis (GSEA) was performed after RNA-seq. Proliferation was assessed using the hexosaminidase assay on HTB-9, HTB-5 and T24 BCa cells. HTB-9 cells (5x105) xenografts were implanted in the flank of nude mice in 50% Matrigel/saline and allowed to develop over two weeks. When mean tumor sizes per cage reached 50-100mm3 determined using calipers, treatment with 25mg/kg barasertib, i.p., Q1D for 5 days per week was started. Tumors were excised and weighed on the final day. Results: After 16 weeks of 0.05% BBN exposure, Ts65Dn mice had smaller bladder tumors which trended towards lower stage tumors compared to WT mice. Although Ds mice and patients are known to have higher expression of anti-angiogenic proteins as such DSCR1, we did not see a decrease in angiogenesis in the Ds tumors, indicating potentially novel mechanisms of protection. RNA-sequencing indicated pathways associated with the cell cycle were most affected in the Ts65Dn mice, including downregulation of the cell cycle regulator Aurora Kinase B (AURKB). We evaluated drug sensitivity in a set of BCa cell lines using the AURKB specific inhibitor barasertib-HQPA. HTB-9 cells were highly susceptible to barasertib (IC50 ~30-50nM) whereas T24/HTB-5 cells had dramatically higher IC50 values (~50µM). Drug efficacy was assessed by evaluating phosphorylation of the AURKB substrate histone H3. Phospho-histone H3 levels were reduced in all cell lines and inhibition of a known transporter of barasertib using the compound valspodar minimally enhanced barasertib toxicity in T24 cells, indicating the potential for a novel mechanism of resistance. Conclusions: Our results indicate Ts65Dn mice have reduced tumor formation when exposed to BBN. Using Ts65Dn mice as a model may be a means for finding new drug targets as demonstrated by our results targeting AURKB. SOURCE OF Funding: Leo and Anne Albert Institute for Bladder Cancer and Institute for Advancing Medical Innovation at KUMC
