It is becoming increasingly clear that no single marker will have the sensitivity and specificity necessary to be used on its own for diagnosis/prognosis of tumors. Interpatient and intratumor heterogeneity provides overwhelming odds against the existence of such an ideal marker. With this in mind, our laboratory has been applying a long term systematic approach to identify multiple biomarkers that can be used for clinical purposes. As a result of these studies, we have identified and reported several candidate biomarker proteins that are deregulated in bladder cancer. Following the conceptual biomarker development phases proposed by the Early Detection Research Network, we have taken some of the most promising candidate proteins into postdiscovery validation studies, and here we report on the characterization of one such biomarker, the bladder cancer-associated protein (BLCAP), formerly termed Bc10. To characterize BLCAP protein expression and cellular localization patterns in benign bladder urothelium and urothelial carcinomas (UCs), we used two independent sets of samples from different patient cohorts: a reference set consisting of 120 bladder specimens (formalin-fixed as well as frozen biopsies) and a validation set consisting of 2,108 retrospectively collected UCs with long term clinical followup. We could categorize the UCs examined into four groups based on levels of expression and subcellular localization of BLCAP protein and showed that loss of BLCAP expression is associated with tumor progression. The results indicated that increased expression of this protein confers an adverse patient outcome, suggesting that categorization of staining patterns for this protein may have prognostic value. Finally, we applied a combinatorial two-marker discriminator using BLCAP and adipocyte-type fatty acid-binding protein, another UC biomarker previously reported by us, and found that the combination of the two markers correlated more closely with grade and/or stage of disease than the individual markers. The implications of these results in biomarker discovery are discussed.Molecular & Cellular Proteomics 9:161-177, 2010.

14-3-3 proteins comprise a family of highly conserved and broadly expressed multifunctional regulatory proteins that are involved in various cellular processes such as cell cycle progression, cell growth, differentiation, and apoptosis. Transcriptional expression of the isoform of 14-3-3 is frequently impaired in human cancers, including carcinomas of the breast, which has led to the suggestion that this protein might be involved in the neoplastic transformation of breast epithelial cells. Here we report on the analysis of 14-3-3 expression in primary breast tumors using a proteomic approach complemented by immunohistochemical analysis by means of specific antibodies against this isoform. We show that the levels of expression of 14-3-3 were similar in non-malignant breast epithelial tissue and matched malignant tissue with only sporadic loss of expression observed in 3 of the 68 tumors examined. Moreover we show that 14-3-3 immunoreactivity was restricted to epithelial cells and significantly stronger in the myoepithelial cells that line the mammary ducts and lobules. The lack of expression of 14-3-3 in the three breast carcinomas was not associated with high levels of expression of the dominant-negative transcriptional regulator ⌬Np63 or with increased expression of estrogen-responsive finger protein, a ubiquitin-protein ligase (E3) that targets 14-3-3 for proteolysis. Validation of the results was performed retrospectively on an independent clinical tumor sample set using a tissue microarray containing 65 primary tumors. Our data suggest that, contrary to what was previously thought, loss of expression of 14-3-3 protein is not a frequent event in breast tumorigenesis. Molecular & Cellular Proteomics 4: 555-569, 2005.

Bladder cancer is the second most common genitourinary tumor and the fourth most common solid malignancy in Denmark, with an incidence of 1,200 -1,300 patients per year and a mortality rate of ϳ300. It encompasses a large variety of histological heterogeneous tumor types arising predominantly in the epithelium (urothelium) lining of the urinary bladder and the ureters. Tumor types of the urothelium include urothelial carcinomas (UCs), 1 squamous cell carcinomas, adenocarcinomas, as well as other less frequent lesions (1). UCs account for more than 90% of the bladder carcinomas and comprise a wide spectrum of lesions with distinct biological and functional characteristics. Up to 80% of patients with superficial bladder cancer lesions will recur, and of these ϳ25% will progress to invasive disease (2). A major challenge today is to identify the subset of low-grade lesions that may recur and evolve into muscle invasive and subsequently to metastatic disease.Presently, the most reliable prognostic factors for recurrence and progression are grading and staging. These parameters, however, cannot predict with certainty the long-term outcome of the disease, and as a result it is important to devise strategies to identify biomarkers that may predict tumor behavior and clinical outcome. Several prognostic markers have been identified, some of which, like p53 and pRB, have a long-standing association with bladder cancer (3, 4). The product of the retinoblastoma gene (pRB) is a main regulator of cell-cycle progression, while p53 exerts its function as a key DNA checkpoint molecule, triggering growth arrest or apoptotic processes in response to DNA aberrations and cellular stress. pRB and p53 are frequently altered in bladder From the ‡Department

The 14-3-3 proteins constitute a family of highly conserved and broadly expressed multifunctional polypeptides that are involved in a variety of important cellular processes that include cell cycle progression, growth, differentiation, and apoptosis. Although the exact cellular function(s) of 14-3-3 proteins is not fully elucidated, as a rule these proteins act by binding to protein ligands, thus regulating their activity; so far more than 300 cellular proteins have been reported to interact with 14-3-3 proteins. Binding to cognate interacting partners is isoform-specific, but redundancy also exists as several binding peptides can be recognized by all isoforms, and some functions can be carried out by any isoform indistinctly. Moreover by interacting with different ligands in a spatially and temporally regulated fashion the same isoform can play multiple possibly even opposing roles where the resultant cellular outcome will be determined by the integration of the various effects. Although there is a large body of literature on specific aspects of 14-3-3 biology, not much is known on the coordinated aspects of 14-3-3 isoform expression, post-translational modifications, and subcellular localization. To address the question of isoform-specific differences, we carried out a comparative analysis of the patterns of expression, phosphorylation, and subcellular localization of the 14-3-3 ␤, , , , and protein isoforms in transformed human amnion (AMA) cells. To validate as well as broaden our observations we analyzed the occurrence of the various isoforms in a large number of established cell lines and mammary and urothelial tissue specimens. Given the systematic approach we undertook and our application of isoform-discriminating technologies to the analysis of various cellular systems, we expect the data presented in this study to serve as an enabling resource for researchers working with 14-3-3