Use of metabolomic profiling to identify potential markers and mechanism for bladder cancer progression.

Abstract

250 Background: Bladder cancer (BCa) is the second most prevalent urological malignancy and the fourth highest cause of cancer-related death in the United States that is known to be caused by defects in xenobiotic metabolism, which is not well understood. Using unbiased mass spectrometry we report the metabolomic profiles in BCa tissues and demonstrate its biomarker potential as well reveal a possible mechanism regulating altered xenobiotic metabolism in these tumors. Methods: Total metabolome from clinically annotated bladder-derived tissues were examined using mass spectrometry coupled to reverse and aqueous normal phase separation of compounds. Class- specific metabolites were examined in urine specimens for their biomarker potential as well as analyzed using Oncomine Concept Map for alterations in bioprocesses. The latter was validated using a collection of molecular techniques like Q-PCR, immunoblot analysis, methylation assays and use of methyl transferase inhibitor, on bladder-derived cell lines and patients specimens. Results: A total of 2,019 compounds were detected across the 58 bladder-derived specimens, of which 50 named compounds were differential between BCa and its adjacent benign tissue. These included aromatic compounds like aniline, catechols as well as polyamines and S-adenosyl methionine (SAM). A subset of these compounds, were detected in urine and could distinguish BCa from benign, non-muscle-invasive from muscle invasive tumors and delineate patients responding to chemotherapy and TURBT. Bioprocess mapping of BCa-specific metabolome revealed co-enriched concepts describing methylation and cytochrome P450 (CYP) driven xenobiotic metabolism in bladder tumors. The role of methylation in regulation of CYP activity in BCa was confirmed using a combination of Aza-treatment, methylation-specific PCR and bisulphite sequencing on bladder-derived cell lines and tissues. Conclusions: Unbiased metabolomic profiling reveals potential non-invasive metabolic markers for early detection, prognosis and therapeutic response of BCa as well as describes a role for methylation induced silencing of CYP1A1 and 1B1resulting in deficient xenobiotic metabolism in BCa. No significant financial relationships to disclose.