A Quantitative Proteomic Analysis Uncovers the Relevance of CUL3 in Bladder Cancer Aggressiveness

Laura Grau, Jose L. Luque-Garcia, Pilar González-Peramato, Dan Theodorescu, Joan Palou, Jesus M. Fernandez-Gomez, Marta Sánchez-Carbayo

To identify aggressiveness-associated molecular mechanisms and biomarker candidates in bladder cancer, we performed a SILAC (Stable Isotope Labelling by Amino acids in Cell culture) proteomic analysis comparing an invasive T24 and an aggressive metastatic derived T24T bladder cancer cell line. A total of 289 proteins were identified differentially expressed between these cells with high confidence. Complementary and validation analyses included comparison of protein SILAC data with mRNA expression ratios obtained from oligonucleotide microarrays, and immunoblotting. Cul3, an overexpressed protein in T24T, involved in the ubiquitination and subsequent proteasomal degradation of target proteins, was selected for further investigation. Functional analyses revealed that Cul3 silencing diminished proliferative, migration and invasive rates of T24T cells, and restored the expression of cytoskeleton proteins identified to be underexpressed in T24T cells by SILAC, such as ezrin, moesin, filamin or caveolin. Cul3 immunohistochemical protein patterns performed on bladder tumours spotted onto tissue microarrays (n = 284), were associated with tumor staging, lymph node metastasis and disease-specific survival. Thus, the SILAC approach identified that Cul3 modulated the aggressive phenotype of T24T cells by modifying the expression of cytoskeleton proteins involved in bladder cancer aggressiveness; and played a biomarker role for bladder cancer progression, nodal metastasis and clinical outcome assessment.