Original article
Up-regulation of TGM2 with ITGB1 and SDC4 is important in the development and metastasis of renal cell carcinoma

https://doi.org/10.1016/j.urolonc.2012.08.022Get rights and content

Abstract

Objective

Tissue transglutaminase (TGM2) up-regulation is involved in the progression and dissemination of carcinomas through β1 integrin (ITGB1) association. Given that TGM2 interaction with syndecan-4 (SDC4) on the cell surface is important in the activation of ITGB1 and integrin-mediated survival signaling, we investigated the roles of TGM2, ITGB1, and SDC4 in the development and metastasis of renal cell carcinoma (RCC).

Material and methods

Expression levels of TGM2, ITGB1, and SDC4 mRNA were analyzed in primary tumor samples (n = 95) and their healthy counterparts in addition to control and RCC epithelial cell lines. TGM2 catalytic activity in 60 randomly selected patient samples was measured by enzyme-linked sorbent plate assay.

Results

TGM2 expression ratio showed a significant 2.9-fold decrease in 67 (70.5%) of the primary RCC tumors (P <0.0001) independent of clinical covariates, including tumor node metastasis (TNM) staging and histopathologic grading. For the remaining 28 (29.5%) tumors, a 1.95-fold increase was recorded in the TGM2 expression levels, which also showed a significant increase in ITGB1 and SDC4 expression levels in 82.6% of the overexpression cases (P <0.001). Up-regulation of TGM2 along with ITGB1 and SCD4 was associated with metastasis and a marked decrease in tumor necrosis. Consistently, RCC cell lines exhibited higher levels of TGM2 expression compared with the control epithelial cell line with a significant up-regulation of ITGB1 and SCD4 recorded for the metastatic lines.

Conclusions

Our findings suggest that TGM2 up-regulation along with ITGB1 and SDC4 plays an important role in the development of RCC tumors and advanced RCC with metastasis.

Introduction

Renal cell carcinoma (RCC) represents 2% to 3% of all cancers and is the most common malignant type of kidney cancer, accounting for 85% of kidney cancers. RCC encompasses different histologic subtypes that include the commonly prevalent clear-cell RCC with more than 70% of all RCC, papillary RCC representing 10% to 15%, chromophobe RCC comprising 5%, and other less common types. Currently, complete resection of the tumor is the only curative treatment of localized RCC tumors. However, about 30% of newly diagnosed patients with RCC already have a metastatic disease, and one-quarter of surgically cured patients would develop metastases subsequently despite chemotherapy, radiotherapy, or systemic therapies [1], [2].

Tumor progression resulting in cancer metastasis is a multistep process, which is dependent on dynamic changes in adhesive and migratory ability of tumor cells. Integrins are the major cell surface receptors mediating cell-to-extracellular matrix adhesion and are involved in the regulation of focal adhesion turnover and survival signaling, which are key events in the process of tumor growth and tumor cell migration [3]. Cooperatively with the integrins, cell adhesion is also organized by cell surface heparan sulfate proteoglycans, syndecans [4]. Of all 4 syndecan genes, syndecan-4 (SDC4) is the only ubiquitously expressed member and functions as an integrin co-receptor in cell adhesion–promoting mitogen-activated protein kinase signaling pathways [4], [5].

Tissue transglutaminase (TGM2) is another ubiquitously expressed protein shown to act as a potential binding partner for integrins and SDC4 [6], [7]. TGM2 is a multifunctional protein, which catalyzes not only calcium-dependent acyl-transfer reactions leading to the formation of stable multiprotein complexes resistant to proteolysis but also calcium-independent GTPase, protein disulfide isomerase, and kinase activities [8]. At the cell periphery, unless inactivated by its binding to fibronectin (FN) or heparan sulfate chains or both, TGM2 was implicated in the formation of a stable and insoluble matrix by cross-linking the deposited matrix proteins [8]. Upon binding to FN, TGM2 is recognized by SDC4 as an adhesion protein and drives RGD-independent cell adhesion and survival signaling in synergy with β1 integrins (ITGB1s) [5], [6], [9].

Although primary tumor cells exhibit decreased TGM2 expression compared with their metastatic counterparts [10], [11], elevated TGM2 expression levels are correlated with an increased metastatic potential in different cancer types, including breast cancer, melanoma, pancreatic adenocarcinoma, and ovarian cancer [11]. Accumulating evidence has implicated a strong correlation between aberrant TGM2 expression and β1/β5 integrins for tumor cell migration, invasion, and drug resistance leading to the development of metastatic phenotype [12], [13].

In the present study, we analyzed the expression levels of TGM2, ITGB1, and SDC4 genes in RCC cell lines and healthy and tumor tissues of 95 patients with RCC to investigate the role of TGM2 in the progression and metastasis of RCC. We showed, for the first time, that elevated expression of TGM2 along with ITGB1 and SDC4 up-regulation increased the risk of RCC metastasis independently from TGM2 activity. Similar results were obtained when established RCC cell lines were investigated for their expression of TGM2, ITGB1, and SDC4.

Section snippets

Patients and tissue specimens

This study comprises 95 patients with renal cancer who underwent radical nephrectomy at the Istanbul Faculty of Medicine, Department of Urology, between 2009 and 2011. The study protocol was approved by the Ethics Committee of Istanbul University and was in accordance with the Helsinki agreement. Histopathologic analysis, classification, staging, and Fuhrman grading of tumors were evaluated according to the previous description in the pathology department of Istanbul University, Istanbul

TGM2, ITGB1, and SDC4 gene expression in relation to clinicopathologic parameters of RCC

Given the increasingly important role of TGM2 in the progression and metastasis of various carcinomas, the basal level of TGM2 mRNA was analyzed in tumor samples from patients with RCC using quantitative real-time PCR. TGM2 was expressed in both tumor and control healthy tissue, and its expression was normalized with respect to 18S rRNA gene expression. Randomly selected paired samples (n = 23) showed no statistically significant differences in TGM2 expression levels when normalized against

Discussion

TGM2, a member of the transglutaminase family, is up-regulated or overactivated in specific clinical and physiologic situations, such as celiac disease, fibrosis, neurodegenerative disorders, and cancer [7]. The observation that reduction in TGM2 gene activity and expression is necessary for the initial progression of primary tumors, whereas high expression of TGM2 in association with ITGB1 leads to increased drug resistance and metastasis is intriguing, as it suggests that TGM2 and ITGB1

References (22)

  • A.M. Belkin

    Extracellular TG2: emerging functions and regulation

    FEBS J

    (2011)
  • Cited by (0)

    This work was partially funded by Grant 109S431 from the Scientific & Technological Research Council of Turkey Health Sciences Division.

    View full text