Identification and characterization of 9D7, a novel human protein overexpressed in renal cell carcinoma

Abstract

With the objective of discovering novel tumor-associated antigens of the cancer/testis type, we compared the transcriptional profiles of renal cell carcinoma (RCC) and nontumorous kidney and further screened for genes expressed in RCC and testis, but not other normal tissues. In a first step, a representational difference analysis library consisting of approximately 1,900 RCC cDNA clones was generated. Clones were then spotted onto filters and hybridized with cDNA probes derived from a testis-specific cDNA library, a pool of RCCs and a pool of 10 healthy normal tissues, respectively. Based on strong hybridization signals with both RCC and testis, but not normal tissue probes, 185 clones were sequenced and annotated. Key words: representational difference analysis; transcriptional profiling; renal cell carcinoma; tumor-associated antigen; immunohistochemistryThe discovery of the MAGE family of proteins, based on their recognition by cytotoxic T-lymphocytes (CTL), finally settled a long-standing dispute on the existence of cancer-specific human T cell antigens. 1-7 MAGE genes are expressed in a variety of tumor types, but among normal tissues only in male germ line cells, which, however, lack MHC class I expression and hence the antigenic structures. 8 Subsequently, a number of other genes with similar expression characteristics, usually designated as cancer/ testis (C/T) antigens, were identified. These discoveries were not exclusively based on reactivity with CTLs, but also, as in the case of NY-ESO-1, on reactivity with autologous antibodies, 9,10 or on the comparison of transcript profiles of tumors and normal tissues by representational difference analysis (RDA). 11,12 Examples of antigens identified by the latter approach include the C/T antigens MAGE-1C 13 and LAGE-1. 14 Due to their broad expression in different tumor types, their tumor specificity and their ability to induce both CTL and antibody responses, C/T antigens are considered promising targets for cancer vaccines. Renal cell carcinomas (RCC), however, do not express any of the known C/T antigens at reasonable frequency. RCC seems to be an immunogenic tumor. A number of CTL clones with the capability of lysing both autologous and allogenic RCC cell lines could be raised but the corresponding antigens have not been identified yet. 15 One exception is antigen RU2AS, which is frequently expressed in RCC, but also in normal kidney, testes, bladder and liver. 16 We have set out to systematically search for new C/T antigens of RCC on the basis of their tumor-specific transcriptional profile. To this end we performed RDA between RCC and non-tumorous kidney tissue and selected for candidate antigens on the basis of hybridization with a testis-specific cDNA library. This approach led to the identification of 9D7, a novel human gene overexpressed in RCC. MATERIAL AND METHODS Tissue samples and RNA preparationTumors of the kidney and non-tumorous kidney tissue were obtained from patients who underwent radical nephrectomy. Tumor typing was done according to ...

Tissue-Wide Expression Profiling Using cDNA Subtraction and Microarrays to Identify Tumor-Specific Genes

“…5B). Overexpression of EGLN3 in RCCs was also confirmed by immunohistochemistry (20). Four additional genes, NDGR1, OSF-2, TP73L, and NAT1 were verified in the same way, all exhibiting high agreement between microarray and real-time PCR results.…”

“…Thus far, two splice variants have been demonstrated for EGLN3 (formerly described as 9D7), which recently has been shown to be involved in the regulation of the hypoxia-inducible factor (20,21). By RT-PCR studies using RNA samples derived from normal tissues and tumor samples, we demonstrated that the shorter splice variant is dominant over the longer one in all tissues.…”

Detection of renal cell carcinoma-associated markers via proteome- and other 'ome'-based analyses

“…[41][42][43] Recently, systematic searches for novel tumour antigens of RCC on the basis of their tumour-specific transcriptional profiles have also been performed. Transcriptomes of tumours and normal tissues were assessed by either representational difference analysis (RDA) 44,45 or by microarray technology. 32,[46][47][48][49] More than 1,700 differentially expressed genes/cDNAs have been detected.…”

“…32,[46][47][48][49] Furthermore, 9D7 was identified as a novel gene over-expressed in RCC as well as in other cancers. 45 Besides the differential expression pattern of tumour versus normal cells, there exists a significant distinction in the gene expression profile of tumours obtained from patients with a relatively nonaggressive, compared with a relatively aggressive, form of disease, 47 and of tumours with distinct subtypes. Thus, transcription profiling is a powerful tool for monitoring patients' tumours; this may also have an impact on cancer diagnosis and prognosis and, consequently, on the mode of therapy selected.…”

“…79,80 Using proteomics and/or PROTEOMEX, a number of candidate antigens representing members of the cytoskeletal family, such as cytokeratins -particularly cytokeratin 8cytoskeletal tropomyosin, F-actin capping protein, ª-actin, SM22-AE, stathmin, tubulin-AE, tubulin-â and vimentin, were identified. 45,71 The expression pattern and clinical significance of cytokeratin 8, stathmin and vimentin was further evaluated by immunohistochemistry and/ or Western blot analysis. A heterogeneous expression profile of cytokeratin 8, stathmin and vimentin was demonstrated in the RCC subtypes, suggesting their use as markers for distinguishing between specific RCC subtypes as well as normal renal epithelium.…”