The utility of tetraspanin CD9 as a biomarker for metastatic clear cell renal cell carcinoma

doi:10.1016/j.bbrc.2016.02.008

Biochemical and Biophysical Research Communications

Volume 471, Issue 1, 26 February 2016, Pages 21-25

https://doi.org/10.1016/j.bbrc.2016.02.008 Get rights and content

Highlights

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The contributions of tetraspanin CD9 to renal cell carcinoma (RCC) metastasis are inconclusive.
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Cell lines and human biopsies were used to understand the contribution of CD9 to metastasis.
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Upon knockdown of CD9, RCC cells obtained a more metastatic phenotype.
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E-cadherin was repressed and Snail, Twist1, and Zeb1 were elevated in the absence of CD9.
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Using CD9 along with E-cadherin as a biomarker helps predict type and metastatic potential of RCC.

Abstract

The use of tetraspanin CD9 as a biomarker for renal cell carcinomas (RCC) has been explored with minor conclusions. Identification of a biomarker that not only distinguishes between the different types of renal cell carcinomas, but also predicts the metastatic potential of these tumors would significantly advance diagnosis and prognosis of kidney cancers. We utilized established cell lines to better understand the contribution of CD9 to the metastatic potential of clear cell renal cell carcinomas, and then applied our findings to the TCGA database and immunohistochemical analysis of human samples based on tumor grading to determine the utility of CD9 as a biomarker for RCC. Clear cell renal cell carcinoma (ccRCC) cell expression of tetraspanin CD9 was compared to normal kidney cells and found to be elevated. Upon knockdown of CD9, ccRCC cells obtained a more metastatic phenotype. We found E-cadherin expression to be repressed and the endothelial to mesenchymal transition markers Snail, Twist1, and Zeb1 to be elevated upon CD9 knockdown. Upon observing these gene expression changes in the TCGA database and in 10 cases, we found that CD9 and E-cadherin expression was lowered in higher grade ccRCC tumors. There was a significant correlation between CD9 and either E-cadherin, Snail, or Zeb1 in these tumors. Collectively, using tetraspanin CD9 in tandem with E-cadherin as a biomarker in renal cell carcinoma will help to not only distinguish between types, but also predict the metastatic potential of RCC.

Introduction

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults and accounts for 90–95% of neoplasms arising from the kidney [1]. Treatment-resistance, poor patient prognosis, and the difficulty to discriminate between different types of RCC highlights the need for predictive and distinctive biomarkers [2]. The most common histologic pattern of RCC is clear cell (ccRCC), which represents over 75% of RCC cases and exhibits the worst prognosis [3]. The traditional tumor, nodes, and metastasis (TNM) system on its own offers limited prognostic ability due to substantial differences in the biological behavior of renal tumors classified in the same stage [4]. While several genes have been reported to distinguish the subtypes of RCC, very few have been reported to predict the aggressiveness of the disease [5]. Therefore, biomarkers for RCC that can not only distinguish localized tumors but predict high risk for recurrence and metastasis are necessary in developing effective treatment options.

The role of the tetraspanins as metastatic suppressors in numerous types of cancers has been extensively studied; however, limited research has examined the association between tetraspanins and RCC. CD9 is of particular interest in the 33-member family of tetraspanins because the majority of research has demonstrated reduced CD9 expression in advanced and aggressive metastatic tumors and the suppression of motility in vitro and tumor metastasis in vivo after ectopically overexpressing CD9 [6]. CD9 is expressed in human kidney tissues and demonstrated differential staining in between four types of RCCs [7]. In combination with vimentin, the lack of CD9 expression was used distinguish clear cell from chromophobe RCC [8]. Another group revealed elevated CD9 expression in approximately half of ccRCC cases, but was unable to determine any correlation between CD9 and tumor grade or stage [9]. These studies hint at the useful relationship between CD9 expression and ccRCC diagnosis or prognosis. Herein, we specifically examined the expression and contribution of CD9 to ccRCC using human cell lines and human tissue samples, further defined CD9 as a potential biomarker of ccRCC, and allude to the potential contribution of CD9 to metastasis.

Section snippets

Reagents and antibodies

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA were purchased from Gibco (Grand Island, NY). A murine monoclonal antibody specific for the second extracellular loop of CD9 (mAb7) was previously generated and described in our laboratory [10]. Anti-E-cadherin, anti-Zeb1 and anti-Snail antibodies were from Cell Signaling Technology (Beverly, MA), and Anti-Twist1 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). All other

Results and discussion

Previous research linking CD9 and ccRCC is inconsistent. One group demonstrated CD9 histologic staining in about half of ccRCC cases examined [9] and another group found a lack of CD9 expression in ccRCCs [8]. To determine the role CD9 plays in ccRCC, we first measured CD9 expression on the cell surface of a normal kidney cell line (HK-2) and three ccRCC cell lines (Caki, SK-RC-04, and SK-RC-17) (Fig. 1A). Interestingly, all ccRCC cell lines displayed increased and near equivalent expression of

Financial support

University of Tennessee Health Science Center Vascular Biology Center of Excellence.

Conflicts of interest

None of the authors declare any conflicts of interest.

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The utility of tetraspanin CD9 as a biomarker for metastatic clear cell renal cell carcinoma

Highlights

Abstract

Introduction

Section snippets

Reagents and antibodies

Results and discussion

Financial support

Conflicts of interest

Cancer Treat. Rev.

Urol. Oncol.

Hum. Pathol.

Pathol. Res. Pract.

J. Biol. Chem.

Renal-cell carcinoma

N. Engl. J. Med.

Epidemiology and staging of renal cell carcinoma

Semin. Interv. Radiol.

Molecular markers for early detection of renal carcinoma: investigative approach

Clin. cancer Res. Off. J. Am. Assoc. Cancer Res.

Tetraspanin proteins promote multiple cancer stages

Nat. Rev. Cancer