Identification of new prognostic markers in renal cell carcinoma

The clinical course of renal cell carcinoma (RCC) shows a high
variability. Prognostic markers are essential to enable an
individualized therapeutic strategy. The objective of this study
was the identification of novel independent prognostic markers and
potential therapeutic targets in RCC. The focus was on genes
involved in epithelial-mesenchymal transition (EMT) and cancer stem
cell biology. EMT enhances tumor cell motility and hence plays a
critical role in invasion and metastasis in various carcinomas. A
set of transcription factors acts as master regulators of EMT.
Whether EMT is important for tumor progression in clear cell renal
cell carcinoma (RCC) is unknown. Therefore, EMT-related genes were
selected from the literature, and their role and prognostic
relevance in RCC were analyzed. The known cancer stem cell marker
CXCR4 and the associated TPBG gene were also analyzed in this
project. Additionally, a novel filter strategy was used to analyze
RCC oligonucleotide microarray data for identification of potential
prognostic markers: genes with increasing expression during tumor
progression (normal kidney < primary tumor < metastases) were
selected for outcome analysis because they could be crucial for RCC
biology. Expression of 46 EMT-related genes was analyzed using
oligonucleotide microarrays and gene set enrichment analysis (GSEA)
in tissue samples from normal kidney and G1 and G3 primary RCC, 14
samples each. Expression of selected EMT genes was validated by
real-time polymerase chain reaction (PCR) in normal kidney, primary
RCC and metastases in an independent cohort of 112 patients and
then combined with follow-up data for survival analysis.
Immunohistochemistry, Western blot and flow cytometry were
performed to further examine the expression of CXCR4 and
co-expression of CXCR4 and TPBG on the surface of RCC cells. GSEA
and dChip software were used for microarray data analysis. The EMT
gene set was preferentially expressed in primary tumors compared to
normal tissue (false discovery rate FDR=0.01), but no difference
between G1 and G3 tumors was found. Quantitative RT-PCR showed
down-regulation of critical EMT genes like CDH2 and ZEB1 in
metastases which suggests reversal of EMT during metastasis.
Kaplan-Meier analysis demonstrated a significant better outcome for
patients with low CXCR4, vimentin, fibronectin and TWIST1 mRNA
levels. Multivariate analysis revealed that CXCR4 and vimentin
up-regulation represent independent prognostic markers for poor
cancer-specific survival of RCC patients. The microarray approach
using filtering and further RT-PCR validation of
progression-associated genes revealed that ATAD2, TET3, HELLS and
TOP2A are independent and previously unknown predictors of poor
outcome in RCC patients. Taken together, this study provides strong
evidence that EMT occurs in RCC. Modulation of EMT in RCC,
therefore, might represent a future therapeutic option. Expression
levels of a number of EMT-related genes (like the genes encoding
the cancer stem cell marker CXCR4 and vimentin) could be identified
as independent prognostic markers. Using a novel filtering approach
on array data, additional novel prognostic markers could be
identified. These findings contribute to a better risk
stratification of RCC patients that can support an individualized
and optimized therapeutic strategy.