Global analysis of metastasis-associated gene expression in primary cultures from clinical specimens of clear-cell renal-cell carcinoma

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Int. J. Cancer: 123, 1080–1088 (2008) ' 2008 Wiley-Liss, Inc.

Global analysis of metastasis-associated gene expression in primary cultures from clinical specimens of clear-cell renal-cell carcinoma Xiaojie Tan1, Yujia Zhai1, Wenjun Chang1, Jianguo Hou2, Songqin He1, Liping Lin1, Yongwei Yu3, Danfeng Xu4, Jianru Xiao5, Liye Ma6, Guoping Wang1, Tinghu Cao2 and Guangwen Cao1* 1 Department of Epidemiology, Second Military Medical University, Shanghai, China 2 Department of Urology, The 1st Affiliated Hospital, Second Military Medical University, Shanghai, China 3 Department of Pathology, The 1st Affiliated Hospital, Second Military Medical University, Shanghai, China 4 Department of Urology, The 2nd Affiliated Hospital, Second Military Medical University, Shanghai, China 5 Department of Orthopaedics, The 2nd Affiliated Hospital, Second Military Medical University, Shanghai, China 6 Department of General Surgery, The 1st Affiliated Hospital, Second Military Medical University, Shanghai, China Metastatic clear-cell renal-cell carcinoma (ccRCC) has a poor prognosis and unpredictable course, and there are no molecular markers that reliably predict ccRCC metastasis. In this study, ccRCC specimens from 84 patients were directly cultured in vitro. Primary cultures from 38 of 94 specimens contained more than 90% tumor cells at the fourth passage. After identification by immunostaining, the primary cultures of metastatic and nonmetastatic ccRCC specimens from the age- and gender-matched patients were subjected to cDNA microarray assays. A total of 842 differentially expressed genes with a FDR (false discovery rate) of 4.79% were identified. Pathway analysis and co-occurrence with ‘‘cancer’’, ‘‘metastasis’’ and ‘‘invasion’’ in the literature annotations functionally enriched the 842 genes and provided an indication of the reliability of our microarray assays. Novel genes associated with metastasis were selected based on protein-protein interactions between 205 differentially expressed genes that cooccurred with ‘‘metastasis’’ and those that did not co-occur with ‘‘metastasis’’ on Medline, and the results of co-expression analysis between the co-occurred genes and unpublished genes. FSTL1, AV722783, SLC15A1, DDX17, ORC2L and PKMYT1 were found to be potential ccRCC metastasis-associated novel genes, according to expression patterns in cultures and tumor tissues. Interestingly, the upregulated genes (CAV1, PKMYT1 and ORC2L) were also upregulated and the downregulated genes (FSTL1, GSTM3, CYR61, SLC15A1 and AV722783) were also downregulated in the primary ccRCC specimens compared with expression in adjacent renal tissues in 37 patients. This study has identified new candidate biomarkers and targets for the early diagnosis and treatment of ccRCC metastasis. ' 2008 Wiley-Liss, Inc. Key words: renal-cell carcinoma; clear cell; metastasis; primary culture; gene expression

Renal-cell carcinoma (RCC) accounts for approximately 90295% of neoplasms in the kidney. RCC is more common in men than in women (2:1), and it most often occurs in patients aged 50270 years. The incidence of RCC has been increasing. One-third of patients present with metastatic disease and have a median survival of 7211 months and a 5-year survival of 0210%.1,2 RCC is a pathologically heterogeneous disease, and can be subdivided into clear, papillary, granular, spindle, and mixed cell variants based on cytoplasmic features. Clear-cell RCC (ccRCC) is the most common type of RCC (approximately 80%) and accounts for most cases of metastatic disease.3 There have been no significant improvements in the mortality rate of ccRCC, most likely because currently available therapies for metastatic disease are relatively ineffective. A full understanding of the molecular genetics and signaling pathways involved in the metastatic process of ccRCC is important for early detection of metastasis and the development of innovative treatment options. The prognostic and/or therapeutic potential of several molecular markers of metastatic RCC have been investigated. Carbonic anhydrase IX (CA9), caveolin-1 (CAV1), B7-H1/PD-1, B7-H4, vimentin, CD10, alpha B crystalline (ABC), survivin, vascular Publication of the International Union Against Cancer

cell-adhesion molecule-1, hypoxia-inducible protein 2, adipose differentiation-related protein, cyclin B1, N-cadherin, kallikreins, minichromosome maintenance 2, gemnin and Ki67 have been suggested to be associated with metastasis and poor prognosis of ccRCC.4–19 Immunohistochemistry analysis has most frequently been used to evaluate the diagnostic and prognostic significance of the potential markers in limited numbers of patients with RCC. Unfortunately, there are no widely accepted molecular biomarkers for ccRCC aggressiveness. One of the most likely candidates, CA9, which is a von Hippel-Lindau (VHL)-mediated enzyme expressed in most (>85%) RCC samples, is not an independent prognostic marker. A recent study showed that low CA9 expression levels were not associated with RCC death after adjusting for nuclear grade or coagulative tumor necrosis; furthermore, CA9 expression was reported in extra-renal organs.20 Global gene-expression profiling by cDNA microarray assays can provide insights into the underlying molecular mechanisms of RCC, and lead to the identification of biomarkers for more accurate diagnosis and prognosis, and which could be drug targets for effective therapies. Differences in global gene expression between fresh-frozen RCC tissues and normal renal tissues have been described.21–26 Gene expression changes that resulted in aggressive behavior or metastatic potential have been identified in the primary RCC. A so-called ‘‘metastasis signature’’ derived from primary RCC with different prognosis could be used to classify tumors with and without metastases at the time of surgery.21,22 However, published prognostic gene signatures in RCC have few genes in common. Clinical RCC specimens usually contain some necrotic tissues and nontumor cells such as tumor-infiltrating leukocytes, endothelial cells and fibroblasts, which complicate the global gene-expression analysis. Many microarray assays are necessary to minimize the effects of the necrotic tissues and non-tumor cells in the tumor tissues on gene-expression profiling. To overcome this problem, fresh tumor specimens may be adapted to grow in vitro, as primary cell cultures, to provide homogeneous cellular material for gene-expression profiling. Short-term cultures allow enrichment of RCC cell types from tumor specimens. RCC primary cultures retain the proteomic profile of the corresponding tumor tissues.27,28 Established RCC cell lines are not representative of clin-

This article contains supplementary material available via the Internet at Grant sponsor: National Natural Science Foundation of China; Grant numbers: 30370788, 30571609. Grant sponsor: Shanghai Education Committee, China; Grant number: 08ZZ39. *Correspondence to: Department of Epidemiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People’s Republic of China. Fax: 186-21-25070420. E-mail: [email protected] Received 31 December 2007; Accepted after revision 19 March 2008 DOI 10.1002/ijc.23637 Published online 10 June 2008 in Wiley InterScience (www.interscience.



Mean age (range, yr) Gender (male/female) Stage (tumor-node-metastasis) I(T1, N02x, M0) II(T2, N02x, M0) III(T3, N0212x, Mx) IV(T1b23b, N0212x, M1) Primary tissue samples Metastatic tissue samples Samples for validation Stage (tumor-node-metastasis) I(T1, N0, M0) II(T2, N0, M0) III(T3, Nx, Mx) IV(T1b, Nx, M1) IV(T3b, N0, M1)



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