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

Abstract

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co-occur with ''metastasis'' on Medline, and the results of co-expression analysis between the co-occurred genes and unpublished genes.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...

Primary Cell Cultures from Human Renal Cortex and Renal-Cell Carcinoma Evidence a Differential Expression of Two Spliced Isoforms of Annexin A3

“…The rate of success in establishing primary cultures was not correlated with clinical-pathological characteristics of neoplastic patients. The growth rate and survival data of our cultures were in agreement with those described by others 33,34 and indicated that RCC cultures have a longer doubling time attributable to differences in cell loss, and probably in growth fraction and in length of cell cycle time as we can hypothesize from literature data. [35][36][37] FACS and immunofluorescence demonstrated that our cortex cultures were essentially composed of epithelial tubular cells of proximal and distal origin, whereas RCC cultures were composed of neoplastic cells derived from proximal tubule cells.…”

A novel functional polymorphism of GSTM3 reduces clear cell renal cell carcinoma risk through enhancing its expression by interfering miR‐556 binding

“…These reports, together with our previously study,7 suggested that GSTM3 might function as a suppressor in tumour progression. Elevated ROS activity also played an important role in cancer cell survival and development 14.…”

“…The fresh surgical specimens of ccRCC from 17 patients were harvested and immediately immersed in ice‐cold PBS containing 1% penicillin/streptomycin and 0.5% glutamine (Beyotime, Shanghai, China). Primary cell culture was performed within 60 min after surgery as previously described 7. As a variation of phenotype might occur at higher passages, we chose the ccRCC cultures at the fourth passage for functional experiments.…”

“…Glutathione‐S‐transferase Mu3 ( GSTM3 ), a GSTM ‐class subunit, was identified as a notably down‐regulated gene. It both down‐regulated in metastatic vs primary ccRCC cells and primary ccRCC cells vs adjacent normal renal tissues 7. We also studied the tumour suppressor role of GSTM3 in the progression of ccRCC and a polymorphism rs1332018 which was significantly associated with the postoperative prognosis of ccRCC 8…”

Differential gene expression profiling of matched primary renal cell carcinoma and metastases reveals upregulation of extracellular matrix genes

“…Owing to tissue availability, genome-wide interrogation of biomarkers associated with metastatic progression and cancerspecific death has primarily been based on observations made in the primary tumor [6][7][8] and not in the more lethal, and more therapeutically relevant metastatic lesion. And, genome-wide studies that have preliminarily interrogated metastatic specimens have done so using small sample sizes [9,10]. Thus, the molecular mechanisms that lead to RCC metastases remain largely unknown and require further study.…”