Expression and role of adrenomedullin in renal tumors and value of its mRNA levels as prognostic factor in clear‐cell renal carcinoma

Abstract

Antiangiogenic therapies are used for advanced clear-cell renal carcinomas (cRCC), but without curative possibilities, underlining the need for new therapeutic targets. Adrenomedullin (AM), a multifunctional peptide, is highly expressed in several tumors and plays an important role in angiogenesis and tumor growth through its receptors: calcitonin receptor-like receptor/receptor activity-modifying protein 2 and 3 (CLR/RAMP2 and CLR/ RAMP3). In this study, real-time quantitative reverse-transcription-PCR showed AM mRNA levels were higher in cRCC and in chromophobe renal carcinomas (chRCC) than in normal renal tissue. Interestingly, AM mRNA expression in cRCC correlated strongly with VEGF-A mRNA expression. Immunohistochemically, AM, CLR and RAMP2 were localized in the carcinomatous epithelial compartment of cRCC. Interestingly, RAMP3 immunostaining was found only in the inflammatory cells that infiltrated tumors, suggesting a cross talk between tumor cells and the microenvironment. We also observed that cRCC cells BIZ and 786-O expressed and secreted AM into the culture medium. In vitro, exogenous AM treatment stimulated cell proliferation, migration and invasion, indicating the cell can respond to AM. The action of AM was specific and was mediated by the CLR/RAMP2 and CLR/ RAMP3 receptors. Clinical data showed the prognostic value of AM. High AM mRNA levels were associated with an increased risk of relapse after curative nephrectomy for cRCC. These findings highlight the implication of the AM pathway in the metastatic process and the prognostic relevance of AM in cRCC and point to a potential new therapeutic target. ' UICCKey words: renal cell carcinoma; adrenomedullin; CLR; RAMP2/ RAMP3 Renal-cell carcinoma (RCC) is the most common type of kidney cancer and accounts for about 85% of malignant renal tumors 1,2 and 2% of all adult human cancers. 2 Most RCC are classified as clear cell RCC (cRCC); the other subtypes include mainly chromophobe cell renal carcinoma (chRCC) and papillary renal carcinoma (pRCC). Resection of primary cRCC can be curative when the disease is localized. Distant metastases, however, remain the main cause of treatment failure and death.

Adrenomedullin and calcitonin gene-related peptide receptors in endocrine-related cancers: opportunities and challenges

“…RAMP3 mRNA expression is elevated in prostate carcinoma cells as opposed to those from prostate hypertrophy (Mazzocchi et al 2004). In a study of renal tumours, RAMP3 elevation was reported in inflammatory cells associated with the tumour, whereas RAMP2 was found in the tumour cells themselves (Deville et al 2009). This is consistent with a complex role of AM and its receptors, acting directly on the tumour itself and on its environment.…”

“…This suggests that the antagonist was able to reduce the activity of endogenous AM produced by the tumour or local blood vessels (Ishikawa et al 2003). Moreover, antibodies to the AM 1 and AM 2 receptors inhibited migration and capillary tube formation of endothelial cells and the growth of human tumour xenografts but it is important to note that the specificity of these antibodies has not been properly reported (Fernandez-Sauze et al 2004, Kaafarani et al 2009). …”

“…Where information is available, it is the AM 1 receptor that is most often implicated in the direct effects on growth and survival of tumour cells. RAMP2 is expressed by clear cell renal carcinoma cells, hepatocellular carcinoma cells and pancreatic tumour cells (Keleg et al 2007, Park et al 2008, Deville et al 2009; it is also expressed in a number of endocrinerelated and other cancers (Table 1). In some cases there is also expression of RAMP3, making it difficult to know which receptor is mediating the effects of AM; however, neither renal nor pancreatic cancer cells express RAMP3.…”

Adrenomedullin Blockade Suppresses Growth of Human Hormone–Independent Prostate Tumor Xenograft in Mice

“…(7,23). Human umbilical venous smooth muscle cells (HUVSMC; ScienCell Research Laboratories, Clinisciences,) were cultured in smooth muscle cell (SMC) growth medium M199 with 20% FBS to confluence.…”

“…To assess programmed cell death, tissue sections were evaluated using mAb F7-26 to detect single-strand DNA (ssDNA; Eurobio AbCys). Staining was carried out as detailed previously (17,23).…”