CEBPD amplification and overexpression in urothelial carcinoma: a driver of tumor metastasis indicating adverse prognosis

Abstract

The molecular aberrations responsible for the progression of urothelial carcinoma (UC) remain largely obscure. To search candidate driver oncogenes in UC, we performed array-based genomic hybridization (aCGH) on 40 UBUC samples. Amplification of 8q11.21 was preferentially identified in patients who developed disease-specific death (53.8%) and distal metastasis (50.0%) but was barely detected in non-eventful cases (3.7% and 0%, respectively). In order to quantify the expression of candidate genes harbored in 8q11.21, laser-capture microdissection coupled with RT-PCR was performed on 32 of the 40 cases submitted to aCGH. With this, we identified CEBPD mRNA expression as most significantly associated with gains of 8q11.21, suggesting amplification-driven expression. By performing CEBPD-specific FISH and immunohistochemistry on 295 UBUCs, we confirmed CEBPD amplification (21.3%) and overexpression (29.8%) were strongly related to each other (p<0.001). Moreover, both were associated with adverse clinicopathologic features and worse outcomes. Furthermore, the clinical significance of CEBPD expression was also confirmed in an independent cohort comprised of 340 UCs from the upper urinary tract. Interestingly, CEBPD knockdown suppressed cell proliferation, migration and, most significantly, cell invasion ability in UC cells. The latter phenotype is attributed to downregulation of MMP2 as identified by RT2 Profiler PCR array. Moreover, expression of CEBPD significantly enhanced MMP2 expression and transcriptional activation by directly binding to its promoter region, as confirmed by promoter reporter assay and chromatin immunoprecipitation assay. Conclusively, CEBPD amplification is a mechanism driving increased mRNA and protein expression that confers aggressiveness in UC through MMP2-mediated cell invasiveness.