Med Mol Morphol DOI 10.1007/s00795-013-0064-6

REVIEW

Invasion and metastasis of renal cell carcinoma Shuji Mikami • Mototsugu Oya • Ryuichi Mizuno • Takeo Kosaka • Ken-ichi Katsube • Yasunori Okada

Received: 9 September 2013 / Accepted: 21 October 2013 Ó The Japanese Society for Clinical Molecular Morphology 2013

Abstract Renal cell carcinoma (RCC) represents over 80 % of kidney cancer, and about 30 % of the patients with RCC develop metastasis after the surgery. Invasion of basement membrane (BM) and extracellular matrix (ECM) is an essential event in tumor invasion and metastasis. Matrix metalloproteinases (MMPs), which digest the main components of BM and ECM, are expressed in RCC. Heparanase, which degrades heparan sulfate proteoglycans, is predominantly expressed in high-grade RCCs with a positive correlation with pathological tumor stage and poor prognosis. Bone metastasis is common among the patients with RCC, and increased osteoclastic activity was observed at metastatic sites. Receptor activator of nuclear factor jB ligand (RANKL), which plays an important role in osteoclastogenesis, is predominantly expressed in high-grade This paper was shown in the symposium, ‘‘Molecular and Morphological approach of research on renal cell carcinoma’’, at the Annual Meeting and Scientific Assembly of the 44th Annual Meeting of the Japanese Society for Clinical Molecular Morphology as ‘‘atypia of the renal cell carcinoma, invasion and metastasis’’ and was invited for submission to Medical Molecular Morphology. S. Mikami (&) Division of Diagnostic Pathology, Keio University Hospital, 35 Shinanomachi, Shinjuku-ku, 160-8582 Tokyo, Japan e-mail: [email protected] M. Oya
R. Mizuno
T. Kosaka Department of Urology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, 160-8582 Tokyo, Japan K. Katsube Department of Human Care, Tohto College of Health Science, 4-2-11 Kamishiba, Fukaya, 366-0052 Saitama, Japan Y. Okada Department of Pathology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, 160-8582 Tokyo, Japan

RCC and its expression level is associated with bone metastasis and prognosis. Epithelial-mesenchymal transition (EMT), a switch of epithelial cells to sarcomatoid phenotype, is considered to be critical step during metastasis, and Snail, a major regulator of EMT, is predominantly expressed in high-grade RCC, and high Snail expression is a worse prognostic factor. Accordingly, heparanase, RANKL and Snail may be targets for the development of anti-tumor therapies for RCCs. Keywords Renal cell carcinoma
Metastasis
Heparanase
RANKL
Snail

Introduction Renal cell carcinoma (RCC) is a group of malignancies arising from the epithelium of the renal tubules [1]. RCC is subdivided into different histopathological entities, and clear cell RCC (ccRCC) is the most frequent tumors (approximately 80 % of all cases), followed by papillary RCC (*10 %) and chromophobe RCC (*4 %) [1]. Although RCC can be completely removed by surgery, recurrence is commonly observed during the follow-up. Especially, ccRCC tends to recur frequently, and thus patients with ccRCC have poor prognosis as compared to those with non-ccRCC [2]. Therefore, it is important to elucidate the molecular mechanism of invasion and metastasis of RCC. In this review, we mainly describe the recent data obtained from our studies on our recent researches on invasion and metastasis of ccRCC. Pathological parameters affecting malignant potential of RCC are shown and subsequently, molecular mechanisms of degradation of extracellular matrix (ECM) and basement membrane (BM), which are necessary for invasion and

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metastasis of RCC, are discussed. We also review the molecular mechanism of bone degradation by RCC since bone metastasis is a common and serious complication among the patients with RCC. Finally, epithelial-mesenchymal transition (EMT) in RCC and its association with invasion and metastasis is described.

Pathological parameters affecting prognosis of RCC Pathological prognostic factors contain histological subtype, TNM stage, histological grade, and lymphovascular invasion (Table 1) [1]. As for histological subtypes, patients with ccRCCs generally have poor prognosis as compared to those with non-ccRCCs [2]. Tumor stage as defined in TNM classification is widely accepted as a key prognostic parameter in RCC [1]. Histological grade also represents the major prognostic variable used routinely in localized RCC. Fuhrman et al. [3] developed 4-tier grading system based on nuclear size, shape and content, and the Fuhrman nuclear grading of RCC is the most widely used grading classification. Tumor size has been shown to correspond with higher grade, and associated with malignant potential of RCC [4]. Using advanced imaging techniques such as computed tomography and magnetic resonance imaging, number of small RCC cases, i.e., pathologic T1a (pT1a) cases, is increasing. Many patients with pT1a RCC have a good prognosis, but some patients who have undergone complete tumor resection exhibit local recurrence and/or distant metastasis [5]. In our review of the 338 patients with pT1a RCC, recurrence was observed in 11 patients (3.3 %), and 9 (2.7 %) had metastatic lesions [6]. Statistical analysis showed that the recurrence rate was significantly higher in patients having RCC with Fuhrman nuclear grade 3 or 4 as compared to those with Fuhrman nuclear grade 1 or 2, while no significant correlation was found between tumor recurrence and other clinicopathological parameters including, tumor size, surgical methods, histological subtype and lymphovascular invasion. These findings suggest that high nuclear grade may be one of the most important prognostic factors for predicting tumor recurrence and metastasis after the surgery even in pathologic T1a RCC. Table 1 Prognostic factors for renal cell carcinomas Low risk

High risk

Non-clear

Clear

Primary tumor stage (pT)

pT1, 2

pT3, 4

Lymph node metastasis (pN)

pN0

pN1

Distant metastasis (pM)

pM0

pM1

Fuhrman nuclear grade

G1, 2

G3, 4

Lymphovascular invasion

Negative

Positive

Histological subtype

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Degradation of ECM and BM and its association with invasion and metastasis of RCC Invasion and secondary spread to other organs are characteristic features of malignant tumors and they are the greatest impediment to cancer therapy [7]. Degradation of the ECM and BM, which are the tissue barrier for tumor cells, is a fundamental aspect of cancer cells and an essential event in tumor proliferation and metastasis [8]. Matrix metalloproteinases (MMPs) are a family of zincdependent endopeptidases and may play important roles in tumorigenesis and cancer cell progression because they can digest all the main components of basement membrane and extracellular matrix such as collagens, proteoglycans, laminin, fibronectin, and vitronectin [9]. These are composed of 23 species in humans, two main groups, i.e., secreted type and membrane-anchored type [10, 11]. The prognostic significance of increased MMP expression in several human malignancies has been reported [10]. In RCCs, Kugler et al. [12] demonstrated a strong correlation among increased MMP2, MMP9 expression, and tumor stage using a RT-PCR technique. On the other hand, Lein et al. [13] reported that expression level of MMP9 was increased in both RCC tissues and sera of the patients with RCC, but it was not correlated with tumor type, grade, stage or prognosis. Heparan sulfate proteoglycans (HSPGs), in which heparan sulfate (HS) chains are covalently attached, are the major components of ECM and BM [14]. HSPGs also play a role as receptors for adhesion molecules and growth factors, and therefore, they are implicated in cell adhesion, migration, differentiation and proliferation [15]. Perlecan, one of HSPG, plays as a barrier of the BM and functions as a filter against cationic molecules and macromolecules. This molecule also protects a major matrix component of BM, type IV collagen from proteolytic attack (Fig. 1) [16]. Heparanase activity, which degrades HS chains of HSPGs, had been reported in many metastatic tumors, and its expression was observed in a variety of malignant tumors, showing correlations with malignant phenotype [14]. RTPCR analysis and immunostaining revealed that heparanase is predominantly expressed in ccRCCs as compared to non-ccRCCs, and expression level of heparanase was correlated with pathological tumor stage and distant metastasis [17]. Furthermore, targeting of heparanase mRNA expression in RCC cells (786-O and Caki-2 cells) with small interfering RNA (siRNA) for heparanase effectively inhibited the Matrigel invasion by these cells in vitro [17]. Elevated heparanase expression was a significant and independent predictor of disease-specific survival [17]. Based on these findings, heparanase is considered to play an important role in invasion and metastasis and silencing of the gene might be a potential therapeutic target in

Med Mol Morphol Invading cancer cell Tumor growth

Osteoclastic precursors

Cancer cells

Angiogenesis RANK RANKL OPG

Bone-resorbing factor (PTHrP)

Basement membrane

Perlecan Type IV collagen Perlecan

Growth factor

Osteoclasts

Heparanase MMPs Growth factors stored in Perlecan

Osteoblasts

Invasion through basement membrane

Bone Metastasis to distant organs

Bone absorption

Fig. 1 Degradation of BM by heparanase and MMPs in invasion and metastasis of RCC. Heparanase degrades HS of HSPGs, which play a role in protection of matrix components such as type IV collagen from proteolytic attack. Degradation of HS in the basement membrane causes release of growth factors stored in perlecan leading to tumor cell migration, growth and angiogenesis, which are necessary for invasion and metastasis. Then, cancer cells penetrate the BM by enzymatic degradation of large components such as type IV collagen, and laminin by MMPs

Fig. 2 Mechanism of bone metastasis through vicious cycle. Metastasis of cancer cells to bone triggers a vicious cycle of bone destruction by osteoclastic bone resorption which, in turn, stimulates cancer cell proliferation as follows. Cancer cells release numerous cytokines including parathyroid hormone-related peptide (PTHrP), which promotes the production of RANKL by osteoblasts, resulting in increased binding of RANKL to its receptor RANK, leading to osteoclast activation and bone resorption. Then, growth factors stored in bone are released, and they accelerate tumor growth

ccRCCs. Oligosaccharide-based compounds that inhibit heparanase activity have been recently developed, aiming primarily at halting tumor growth, metastasis and angiogenesis [16].

metastasis of prostate cancer, and blocking the receptor activator of nuclear factor jB (RANK)/RANKL interaction prevents the progression of prostatic carcinoma in bone [20, 21]. In addition, RANKL also triggers the migration of human tumor cells that express RANK [22, 23]. In RCC tissues, RANKL and RANK mRNA expressions are significantly higher in ccRCC tissues as compared to nonneoplastic renal tissues [24]. Immunohistochemically, RANKL and RANK protein are predominantly expressed in high-grade ccRCC as compared to low-grade ccRCC. In contrast, expression of osteoprotegerin (OPG), a decoy receptor for RANKL, in high-grade ccRCC is lower than that in low-grade ccRCC. Furthermore, RANKL and RANK are highly expressed in metastatic RCCs in bone and other organs. Elevated RANKL and RANK expressions are significant predictors of recurrence, bone metastasis and a poor prognosis [24]. In vitro and in vivo analyses have demonstrated that transfection of RANKL cDNA to a ccRCC cell line, Caki-1, accelerates migration of tumor cells, and the effect is inhibited by administration of OPG [24]. All these data suggest that the RANKL– RANK–OPG system is involved not only in the bone metastasis of RCCs but also in metastasis to other organs through the stimulation of cancer cell migration. Clinical trial for denosumab, a fully human monoclonal antibody to RANKL, showed that RANKL inhibition with denosumab provided superior efficacy for prevention of skeletal-related events (SRE) in patients with bone metastases relative to bisphosphonates, the most studied and used antiabsorptive drug in clinical practice [25], and denosumab and bisphosphonates are used as adjunct to systemic targeted therapies to prevent SRE in patients with metastatic RCC to bone [26]. Recently, combination of

Molecular mechanism of bone metastasis of RCC About 30 % of RCC patients develop bone metastasis during the disease course, and bone metastasis can lead to severe bone pain and skeletal complications, finally, resulting in the deaths of patients [18]. Since skeletal damage with bone metastasis is caused mainly by increased osteoclastic activity, it is necessary to elucidate the molecular mechanisms underlying tumor-induced changes in the bone microenvironment. At the site of bone metastasis, accelerated bone remodeling is observed. The ‘vicious cycle’ hypothesis has been proposed to describe how tumor cells interact with bone microenvironment to drive bone destruction and tumor growth in a symbiotic relationship (Fig. 2) [19]. In brief, tumor cells secret various factors, such as parathyroid hormone-related peptide (PTHrP), transforming growth factor-b (TGF-b) and vascular endothelial growth factor, and these factors stimulate osteoblast, resulting in increased production of receptor activator of nuclear factor jB ligand (RANKL). RANKL overexpression leads to increased formation, activation and survival of osteoclasts, causing enhanced bone resorption [19]. Then osteolysis promotes the release of growth factors, such as platelet derived growth factor, bone morphologic protein, TGF-b, from bone, and these factors increase the production of PTHrP and tumor growth [19]. In fact, RANKL expression is up-regulated in bone

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A EMT Metastasis Poor prognosis Renal cell carcinoma with sarcomatoid element

Renal cell carcinoma

B

E-cadherin Snail

Vimentin

Cell adhesion Migration Invasion

MMPs

Fig. 3 Sarcomatoid element arising in RCC (a) and the role of Snail in EMT of RCC (b). During progression of RCC, sarcomatoid element occurs in advanced RCC, and it indicates an aggressive tumor. Morphologically, transition from RCC to sarcomatoid element is considered EMT in RCC (a). In EMT of RCC, Snail represses E-cadherin expression and up-regulates expression of vimentin and MMPs, thereby leading to loss of cell adhesion and enhanced migration and invasion (b)

bisphosphonates and radiation therapy achieved a higher objective response rate and prolonged SRE-free survival than radiotherapy alone in patients with bone metastases from RCC [27].

[34], and in vitro studies suggested that E-cadherin repressor Snail may be involved in EMT of RCC [35–37] In fact, Snail protein is predominantly expressed in high-grade ccRCC tissues and its expression level is associated with primary tumor stage, nuclear grade and presence of sarcomatoid carcinoma [38]. Furthermore, down-regulation of Snail expression in RCC cell line by siRNA led to decreased expression of vimentin, MMP2 and MMP9, and increased expression of E-cadherin together with inhibition of the cell invasion through Matrigel in vitro (Fig. 3). Therefore, the therapeutic approaches based on the use of Snail-specific siRNA may be a new cancer strategy. Acknowledgments This work was supported in part by Grant-inAid for Scientific Research (C) (No. 25460422) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) (S.M.), Grain-in-Aid for Scientific Research (B) (No. 24390374) from MEXT (M.O.), and Grant-in-Aid for Scientific Research (A) (No. 24249022) from MEXT (Y.O.) and Third Term 10-year Strategy for Cancer Control (Y.O.) from the Foundation of Promotion of Cancer Research, and Project for Development of Innovative Research on Cancer Therapeutics (P-Direct) from MEXT (S. M., R.M, T.K. and M.O.). Conflicts of interest None of the authors has any conflicts of interest associated with this study.

References Epithelial-mesenchymal transition (EMT) in RCC During EMT, epithelial cells are converted to the migratory and invasive phenotype, and this process is regarded as a fundamental event during embryogenesis [28]. EMT is also thought to be an important event during malignant tumor progression and metastasis [29]. In advanced RCCs, a sarcomatoid element is commonly observed, and transition from RCC to sarcomatoid element may be regarded as EMT in RCC (Fig. 3). Adhesion protein E-cadherin plays a central role in the process of epithelial morphogenesis, and its expression is down-regulated during progression of malignant epithelial tumors [30]. A hallmark of EMT is the loss of cell adhesion molecule E-cadherin, and several EMT regulators have been identified as E-cadherin repressors [31]. Among them, Snail appears to be a key regulator of EMT as they repress the transcription of E-cadherin, thereby triggering a complete EMT with an acquisition of invasive and tumorigenic properties [31, 32]. Inhibition of Snail function in epithelial cancer cell lines lacking E-cadherin protein restores expression of E-cadherin gene [33]. Snail expression has been detected in many malignant tumors and is associated with invasiveness and metastatic potential of the tumors, suggesting that Snail is a key molecule in the induction of tumor invasion and metastasis [31, 32]. In RCC, loss of E-cadherin expression is associated with metastasis

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