Because cancer stem cells appear generally to be resistant to conventional chemotherapy, the link between EMT and “stemness” provides additional support for the idea that EMT contributes to lethality. We initiated the present study to directly test the hypothesis that EMT markers could identify the subset of muscle-invasive tumors displaying aggressive clinical behavior. In human bladder cancer cell lines, we observed a direct correlation between E-cadherin and p63 expression, and an inverse correlation between these markers and Zeb-1, Zeb-2, and vimentin, indicating they cluster into unique “epithelial” and “mesenchymal” subsets. Our data confirm that mesenchymal markers are expressed almost exclusively by muscle-invasive tumors, the subset of bladder cancer with worse outcome. Interestingly, our data also demonstrate that within this muscleinvasive subset, tumors retaining p63, display significantly worse survival than those muscle-invasive tumors displaying a more typical “mesenchymal” phenotype. We discuss EX 527 possible explanations for the link between p63 and poor survival outcome. Previous studies concluded that loss of p63 is associated with shorter survival in patients with bladder cancer. In contrast, our results suggest that maintenance of p63 expression is associated with adverse outcomes in patients with muscle-invasive bladder cancer. On the surface our results appear to contradict these other studies. However, we suspect this apparent discrepancy is due in part to the inclusion of superficial cancers displaying uniformly good survival characteristics in the earlier analyses as opposed to our current focus on their impact in muscle-invasive disease. As a group, the most superficial bladder cancers have excellent long-term survival, and uniformly express high levels of E-cadherin and p63. On the other hand, loss of p63 is restricted to a subset of the muscle-invasive tumors, and muscleinvasive disease is typically associated with worse clinical outcomes as compared to superficial, non-invasive cancer. In addition, the commercial antibody that is most commonly used to measure p63 in tissue sections cannot distinguish the two major p63 isoforms, so the relationship between DN p63 expression and poor outcome in muscle-invasive bladder cancers could not be recognized in previous IHC-based studies that employed this reagent. We attempted to confirm our mRNA-based results using the 4A4 on tissue microarrays and the results were inconclusive. Consistent with this conclusion, while our manuscript was being prepared for submission Cordon-Cardo’s group generated an anti-p63 antibody that is specific for the DN isoforms and used it on an independent cohort of muscle-invasive tumors to show that DN p63 protein levels also identify a lethal subset of cancers, whereas their results with the 4A4 antibody were inconclusive. Ultimately prospective studies will be required to rigorously test the clinical significance of these findings. Work performed over the past 5 years has renewed interest in the role of EMT in cancer progression and metastasis. One influential study demonstrated that isogenic metastatic variants of a murine breast cancer cell line overexpressed the E-cadherin repressor, Twist, and that elevated Twist expression was required for metastasis.