Pathology – Research and Practice 210 (2014) 822–829
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Original article
Characterization of cathepsin X in colorectal cancer development and progression Doerthe Jechorek a,∗ , Julia Votapek a , Frank Meyer b , Arne Kandulski c , Albert Roessner a , Sabine Franke a a
Department of Pathology, Otto-von-Guericke University of Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany Department of General, Visceral and Vascular Surgery, Otto-von-Guericke University of Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany c Department of Gastroenterology, Hepatology and Infectious Diseases, Otto-von-Guericke University of Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany b
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Article history: Received 21 August 2014 Accepted 22 August 2014 Keywords: Cathepsin X Colorectal cancer Coculture
a b s t r a c t The lysosomal cysteine carboxypeptidase cathepsin X (CTSX), localized predominantly in immune cells, has been associated with the development and progression of cancer. To determine its specific role in colorectal carcinoma (CRC), we analyzed CTSX expression in non-malignant mucosa and carcinoma of 177 patients as well as in 111 adenomas and related it with clinicopathological parameters. Further, the role of CTSX in the adhesion and invasion of the colon carcinoma cell lines HT-29 and HCT116 was investigated in an in vitro culture cell system with fibroblasts and monocytes, reflecting the situation at the tumor invasion front. Epithelial CTSX expression significantly increased from normal mucosa to adenoma and carcinoma, with highest expression levels in high grade intraepithelial neoplasia and in early tumor stages. Loss of CTSX occurred with tumor progression, and correlated with advanced local invasion, lymph node and distal metastasis, lymphatic vessel and vein invasion, tumor cell budding and poorer overall survival of patients with CRC. The subcellular distribution of CTSX changed from vesicular paranuclear expression in the tumor center to submembranous expression in cells of the invasion front. Peritumoral macrophages showed highest expression of CTSX. In vitro assays identified CTSX as relevant factor for cell–cell adhesion and tumor cell anchorage to fibroblasts and basal membrane components, whereas inhibition of CTSX caused increased invasiveness of colon carcinoma cells in mono- and co-culture. In conclusion, CTSX is involved in early tumorigenesis and in the stabilization of tumor cell formation in CRC. The results suggest that loss of CTSX may be needed for tumor cell detachment, local invasion and tumor progression. In addition, CTSX in tumor-associated macrophages indicates a role for CTSX in the anti-tumor immune response. © 2014 Elsevier GmbH. All rights reserved.
Introduction Colorectal cancer (CRC) is among the most common malignancies in western world countries, and despite significantly improved treatment modalities it remains a major cause of cancer mortality in the Western World [1]. The clinical outcome of CRC depends on its efficiency for local invasion and metastasis. Based on their ability to degrade extracellular matrix proteins, cysteine cathepsins, a group of lysosomal cysteine proteases, have been implicated to
Abbreviations: CTSX, cathepsin X; CRC, colorectal cancer; EMT, epithelial mesenchymal transformation; ECM, extracellular matrix. ∗ Corresponding author. Tel.: +49 391 6717953; fax: +49 391 6715818. E-mail address: [email protected] (D. Jechorek). http://dx.doi.org/10.1016/j.prp.2014.08.014 0344-0338/© 2014 Elsevier GmbH. All rights reserved.
play a role in tumor cell invasion, migration and metastasis [2]. For CRC, increased expression and activity mainly of cathepsins B, D and L were reported and are associated with matrix degradation, local tumor spread and prediction of poor prognosis [2]. In this context, the potential of other cysteine cathepsins, such as cathepsin X (CTSX), has been evaluated far less extensively. CTSX expression was thought to be limited to immune cells, such as monocytes, macrophages or dendritic cells [3]. Thus, it is probably not involved in extracellular matrix degradation, but more likely in immune response by regulating proliferation, maturation, migration and adhesion of immune cells as well as phagocytosis and signal transduction [4]. Molecular targets of CTSX exopeptidase activity include the -chain of integrin receptors [5,6], chemokine CXCL-12 [7], bradykinin and kallidin [8] and profilin 1 [9]. Furthermore, CTSX has been reported to bind to cell surface heparin sulfate
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proteoglycans [10], to interact with ␣v3 integrin and ␣v5 integrin [11,12], and to activate 2-integrins [5], thus indicating a major role of CTSX in focal adhesion formation and in the modulation on adhesion to proteins of the extracellular matrix. Recent studies have shown that altered CTSX expression of the tumor epithelium and tumor-associated immune cells is associated with progression and metastasis of several tumors, such as gastric cancer, prostate cancer, hepatocellular carcinoma and malignant melanoma [13–16]. Here, CTSX might be involved in the modulation of adhesive and migration properties of tumor cells by interaction with integrin receptors, thus supporting invasion through the extracellular matrix [17]. Furthermore, CTSX has been suggested to promote tumor progression by bypassing cellular senescence [18] and by inducing an epithelial–mesenchymal transition [15]. Concerning CRC, studies focused mainly on the association of CTSX expression and clinical prognosis [19]. However, the precise function of CTSX in genesis and progression of CRC is still unclear. In the present study, CTSX expression was analyzed in nonmalignant mucosa, adenoma and carcinoma of the colorectum and correlated to clinico-pathological parameters. The role of CTSX in adhesion and invasion of colorectal tumor cells was investigated in an in vitro culture cell system with fibroblasts and monocytes, reflecting the in vivo situation at the tumor invasion front. Materials and methods Patients and tissue samples In this retrospective study, we analyzed specimens of colorectal tissue obtained from surgical resections by partial or complete colectomy, endoscopic mucosectomy and polypectomy between 1995 and 2010, and registered in the archive of the Department of Pathology in Magdeburg, Germany. Tissue collection and performance of this study were in line with the guidelines of the institutional ethical committee, and all patients provided informed consent. The first study group comprised a total of 177 patients with microsatellite-stable sporadic colorectal carcinoma. This group had a median age of 67.3 ± 10.7 (range: 40–91) years, and consisted of 102 males and 75 females. All carcinomas were treated solely by partial colectomy with R0-resection. Carcinomas were classified according to the recent guidelines of the UICC TNM and WHO classification system by two pathologists (D.J., A.R). Distribution of UICC stages, grades of differentiation and localization were as follows: stage I (n = 43), stage II (n = 43), stage III (n = 41), stage IV (n = 50); G1 (n = 25), G2 (n = 118), G3 (n = 34); proximal localization (n = 72), and distal localization (n = 105). Survival data were available for all patients. Further details on the clinico-pathological data of both study groups are given in Table 1. Tumor and paired noncancerous tissue samples were snap frozen in liquid nitrogen, stored at −80 ◦ C for molecular studies and formalin-fixed, paraffin-embedded for immunohistochemical analysis. The second study group comprised 61 male and 50 female patients, ranging in age from 43 to 89 years (median, 65.7 ± 11.3 years) and included formalin-fixed, paraffin-embedded specimens of adenoma with low grade (n = 59) and high grade (n = 38) intraepithelial neoplasia. Tissue microarray design For immunohistochemical analysis, representative tumor regions were selected on H&E-stained slices for inclusion in a tissue array. The arrays were assembled by taking core needle biopsies with a diameter of 0.6 mm from the areas of interest in pre-existing
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Table 1 Clinico-pathological features and immunoreactive scores of colorectal carcinomas investigated by immunohistochemistry for CTSX protein expression. Factor Age 5.5 and were considered CTSXoverexpressing tumors. The 3-, 5-, and 10-year overall survival based on the endpoint of cancer-related death of patients with low or high CTSX expression was 50%, 36% and 16%, or 66%, 52%, and 36%, respectively. Kaplan-Meier curve demonstrated that patients with low CTSX expression had a significantly worse prognosis than those with a CTSX overexpression (p = 0.026) (Fig. 2 C). Strong CTSX-expression in peritumoral macrophages At the tumor–stromal interface of the carcinoma samples, strong immunoreactivity of CTSX was noticed for a group of inflammatory cells of the surrounding stroma. Using double immunofluorescence, CTSX-expressing stromal cells were identified as CD68-positive macrophages (Fig. 3). In carcinomas, CTSX expression of macrophages was extensively stronger than in tumor cells (7.96 ± 2.9 vs. 5.5 ± 3.1; p < 0.001). CTSX-positive macrophages were also found in adenoma mainly surrounding the dysplastic epithelium of the apical parts. Here, expression scores for CTSX of macrophages were higher than for epithelial cells of adenoma (2.97 ± 1.7 vs. 2.13 ± 1.8; p < 0.001), too. Loss of CTSX leads to decreased adhesion and increased invasion To elucidate the role of CTSX in colorectal carcinogenesis and progression, we compared cellular adhesion and invasion of untreated HT-29 cells and HCT116 cells in monoculture and
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Fig. 1. Immunohistochemical analysis for CTSX protein expression in colon adenoma with low grade (A) and high grade (B) intraepithelial neoplasia, in the center (C/D) and the invasion front (E/F) of colorectal carcinoma, in tumor-associated macrophages of the invasion front (G) and in non-tumorous colon mucosa (H). Adenoma and carcinoma cells of the tumor center showed vesicular, partly perinuclearly localized CTSX (*). Tumor glands at the invasion front demonstrated submembranous concentration of CTSX directed to the tumor cell–stroma interface (arrow) and complete loss of CTSX in invasive tumor cell buds (open arrow) (magnification: ×100, ×400).
co-culture with that of tumor cells after CTSX siRNA treatment. Cocultivating tumor cells with human fibroblasts and monocytic cells and applying Matrigel matrix, the in vitro tumor cell culture system approximates the in vivo situation at the invasion front. CTSX siRNA transfected to the tumor cells significantly reduces the expression of CTSX mRNA and protein as described by Krueger et al. [13]. The adhesion of tumor cells treated with siRNA was significantly reduced as compared with untreated cells in monoculture and co-culture (Fig. 2D). Addition of siRNA resulted in a significant reduction of the cell–cell-adhesion of HT-29 cells of 44.4 ± 19% (p = 0.029). The percentage of siRNA-treated HT-29 or HCT 116 cells that adhered to Matrigel decreased significantly to 27.4 ± 1%
and 41.4 ± 8%, respectively (both p < 0.001). Furthermore, CTSXdepleted HT-29 cells showed a 15% decrease in adhesion to fibroblasts (p = 0.125). In contrast, reduced CTSX expression of HCT 116 cells had no effect on adhesion to fibroblasts. Compared to untreated cells, transfection with CTSX siRNA resulted in a highly significant increase of invasive tumor cells in all experimental set-ups (Fig. 2E). The combination of coculture of HCT 116 cells with monocytes and decreased CTSX expression resulted in the greatest number of cells invading through Matrigel (232.4 ± 79%, p = 0.004). Monocultivated HT-29 cells were also highly invasive after siRNA treatment (224.2 ± 65%, p = 0.003). Furthermore, invasion through Matrigel-coated membrane increased
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Fig. 2. Double immunofluorescence of CTSX ((A) FITC) and CD68 ((B) Texas Red) shows CTSX-expressing tumor-associated macrophages ((C) overlay) in colorectal carcinoma (magnification: ×100).
significantly for monocultivated HCT 116 cells, and HT-29 cells cocultivated with macrophages after pretreatment with siRNA (176.2 ± 37%, p = 0.037; 163.5 ± 21, p = 0.004). The invasiveness of HT-29 and HCT 116 tumor cells transfected with siRNA was also increased under coculture with fibroblasts (141.0 ± 17%, p = 0.095; 169.3 ± 49%, p = 0.573). Thus, both adhesion and invasion assay experiments demonstrate that loss of CTSX has significant effects on tumor invasion of colorectal cancer cells. Discussion The overexpression of cysteine cathepsins, in particular cathepsins B, D, and L, has been associated with development and progression in CRC. Based on their ability to degrade extracellular
matrix proteins, they are involved in invasion, migration and metastasis of colorectal tumor cells [2]. Recently, elevated CTSX serum levels were correlated with poorer overall survival for patients with locally resectable CRC. Therefore, CTSX was thought to be a prognostic and predictive marker in CRC [19]. However, there are no studies that give a detailed morphological distribution in tissue of CRC or studies investigating the precise function of CTSX in genesis and progression of CRC. Therefore, in the present study, we analyzed CTSX expression in non-malignant colon mucosa and CRC of 177 patients and 111 colorectal adenomas in relation to clinico-pathological parameters. Furthermore, the role of CTSX in the adhesion and invasion of colorectal tumor cells was investigated in an in vitro culture cell
Fig. 3. CTSX protein expression and its correlation with clinicopathological data and mRNA expression – Immunohistochemical protein expression of CTSX (A), indicated by an immunoreactive score (IRS). Colorectal non-tumorous mucosa, adenoma and carcinoma showed increased expression of CTSX with tumorigenesis. Tumor-associatedmacrophages demonstrated highest expression scores for CTSX. Analysis of CTSX protein and mRNA expression (B) confirmed correlation of both parameters for low- (IRS ≤ 5) and high-expressing (IRS > 5.5) carcinomas (p = 0.036). Data are shown as boxplots representing 25th, 50th, and 75th percentile values and means. Kaplan-Meier plot for survival (C), as a function of CTSX, revealed that the group with low expressing colon carcinomas displayed a significantly poorer clinical outcome than the high expressing group (p = 0.026). In vitro assays – Cellular adhesion (D) and invasion (E) of HT-29 and HCT116 colon carcinoma cells in monoculture and co-culture with 175BR fibroblasts or THP-1 macrophages and after addition of CTSX-specific siRNA. Bar graphs are presented as means ± SD .
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system with fibroblasts and monocytes, reflecting the situation at the tumor invasion front. In general, CTSX expression was up-regulated in colorectal adenoma and carcinoma. However, the highest CTSX levels were observed for early tumor stages but loss of CTSX expression correlated with the clinico-pathological parameters of progressed tumor stages and poor survival. The differences in subcellular und intratumoral distribution of CTSX suggest different functions during tumor development and progression and in different tumor zones. Furthermore, our in vitro studies demonstrate that loss of CTSX is associated with decreased cell adhesion and increased invasion of colon cancer cells. Overexpression of CTSX has already been described for other tumors, such as gastric and prostrate cancer, hepatocellular carcinoma and malignant melanoma [13–16]. Similar to our study, in prostate, prostatic intraepithelial neoplasia and early stages of invasive carcinoma showed equally high expression levels, suggesting that CTSX may be involved in early tumorigenesis [14]. In addition, in a metastatic breast cancer mouse model, loss of CTSX resulted in a delay of early tumor development, demonstrating that CTSX is related to the initial stages of the malignant process and less with tumor progression and metastasis [22]. However, studies on serum levels of CTSX expression have not yet answered the question of whether the cathepsin is involved in early or late stages of CRC [19]. We demonstrated highest expression levels in the central parts of locally restricted or non-metastasized carcinomas, respectively. The most significant increase of CTSX expression was found between adenoma with high grade dysplasia and early invasive pT1 carcinoma reflecting the point of malignant transformation. Furthermore, in adenomas and early tumor stages, CTSX-positive vesicles were mainly accentuated within the perinuclear region, suggesting that CTSX is involved in regulative processes during malignant transformation and early tumor formation. Neoplastic transformation due to CTSX up-regulation might be promoted through bypassing cellular senescence [18], activation of the pro-angiogenic chemokine CXCL-12 [7] or phosphorylation of the IGF-1 receptor [23]. Recently, high expression of proproliferative IGF-1 receptor has been associated with increased risk of CRC [24]. CTSX is also involved in the induction of epithelial–mesenchymal transition [15]. In hepatocellular carcinoma, CTSX was associated with down-regulation of epithelial markers, including E-cadherin, ␣-catenin and -catenin and upregulation of mesenchymal proteins, such as fibronectin, vimentin and N-cadherin, thus contributing to the acquisition of a motile invasive phenotype by the tumor cells [15]. Since CTSX has no transcriptional property and is rather recognized as an ERGolgi-lysosome resident carboxypeptidase [10,25], EMT-related phenotype induced by CTSX overexpression could result from degradation, internalization and lysosomal trafficking of epithelial proteins. In our in vitro assays, CTSX was identified as an important modulator of adhesion and invasion of colorectal tumor cells. Inhibition of CTSX at the mRNA level resulted in reduced adhesion of tumor cells to Matrigel. In addition, adhesion to fibroblast and cell–celladhesion of low-aggressive HT29 was decreased due to loss of CTSX. Our results suggest a role of CTSX in stabilizing tumor cell formation and in the attachment of tumor cells to basal membrane and ECM components. Similar to our results, inhibition of CTSX with siRNA led to reduced adhesion of prostate cancer cells on a fibronectin-coated surface [9] and to impaired migration of gastric cancer cells [13]. In contrast, inhibition of CTSX activity had no impact on the migration of breast cancer cells [3], suggesting that the cathepsin may participate in mechanisms of adhesion and migration, which are not related to proteolysis. These nonproteolytic functions of CTSX may include binding to proteins, such
as integrins [11], which mediate dynamic adhesive cell–cell and cell–ECM interactions and anchor various cytoskeletal and cytoplasmic proteins to the actin cytoskeleton [26]. This leads either to stabilization of cell complexes or to remodeling of the cytoskeleton, formation of focal adhesions and attachment of migrating cells to ECM components [27]. Several studies have demonstrated that the pro- and mature forms of CTSX modulate the function of integrins through specific binding via their integrin-binding motifs RGD (Arg-Gly-Asp) and EGD (Glu-Cys-Asp) [5,6,11,28]. Moreover, CTSX binds cell-surface HSPG [11], which are involved in integrin regulation [29] and are known partners of integrins in focal adhesion formations [30]. In this context, endogenous CTSX is transported to the plasma membrane, accumulates in vesicles at lamellipodia, and is partly associated with the cell surface co-localizing with the relevant integrin subunit [5,11]. Remarkably, in our morphological studies of the tumor invasion zone, we also observed a linear submembranous accumulation of CTSX directed towards the tumor cell–stroma interface. Thus, the intracellular localization of CTSX in cells of the invasion front differs from that in the tumor center, which strengthens the results of our in vitro studies assigning CTSX a role in tumor cell anchorage on ECM. Local invasion requires reduction of intercellular and cell–matrix contacts, dynamic reorganization of the tumor cell cytoskeleton with formation of podia-like extensions, pericellular matrix degradation and oriented migration of the so-called tumor cell buds through ECM [31,32]. Therefore, based on our results on adhesion assays, we hypothesize that loss of CTSX leads to cell contact changes favouring tumor cell invasiveness and tumor spreading. Indeed, performing invasion assays, we demonstrated a significantly increased invasiveness of colon carcinoma cells lacking CTSX. Loss of CTSX did not only affect invasion of tumor cells through Matrigel alone, but also invasion under co-culture with macrophages or fibroblasts. We found decreased CTSX tissue expression in progressed tumor stages and loss of CTSX in the invasion front and in invasive tumor cell buds. Furthermore, loss of CTSX correlated with clinico-pathological factors of progression and poorer survival in patients with CRC. Similar to our data, tumor cells of progressed lung carcinoma and invasive tumor cell lines also showed decreased CTSX expression [3]. Therefore, we presume that CTSX is down-regulated in cells of the invasion front to reach a migrative and invasive phenotype. In contrast to our results, in a recent study of Viˇzin et al. [19], high CTSX serum levels were associated with poor overall survival for patients with locally resectable CRC. Since CTSX is released in large amounts into the circulation by leucocytes and macrophages during immune cell activation and turnover [33], increased serum levels of CTSX in CRC could reflect the overexpression of CTSX in tumor-associated immune cells. Indeed, performing morphological studies of the invasion front, we noticed a strong immunoreactivity of CTSX in macrophages nesting at the tumor–stromal interface of CRC. Similarly to our study, highly CTSX-expressing macrophages in the vicinity of low expressing tumor cells were described in lung carcinoma [3]. Thus, elevated serum levels of CTSX in CRC most likely originate from macrophages that accumulated particularly at the tumor invasive zone. Although CTSX as a carboxypeptidase is unlikely to participate in ECM degradation itself [25], it was shown that CTSX can up-regulate proteins associated with extracellular matrix remodeling, such as MMP2, MMP3 and MMP9 [15]. However, being involved in the activation of the 2 integrin receptors LFA-1 and Mac-1, CTSX most probably regulates transendothelial migration, adhesion to ECM and maturation of macrophages, which leads to accumulation of macrophages at the invasion front [5]. In addition, recruitment of macrophages into the tumor invasion zone could be supported by CTSX-induced processing of the chemokine CXCL-12 [34]. Furthermore, CTSX plays a role in phagocytosis and antigen presentation of macrophages and might be involved in
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anti-tumor immunity [3,5,35]. For example, Mac-1 as a target for CTSX is crucial for FcR-mediated cytotoxity to tumor cells [36]. In summary, our study identified CTSX as adhesion factor in CRC, supporting intratumoral cell–cell stabilization and tumor cell anchorage to ECM. The fact that the cathepsin was most strongly expressed in adenoma and early stages of CRC suggests that CTSX could be involved in early tumorigenesis. Inhibition of CTSX leads to increased invasiveness of colon carcinoma cells in vivo, and loss of CTSX at the invasion front was associated with tumor progression and poorer overall survival of CRC patients. Abundantly expressed CTSX in macrophages at the invasion front of CRC seems to be part of the anti-tumor immune response. Therefore, the influence of CTSX on the development and progression of CRC is likely to be complicated as CTSX functions are different depending on cell type and pathophysiological compartments. Further research is needed to understand its underlying mechanism. Acknowledgements The authors thank all patients and their attending physicians for their participation in this study. We thank H. Wolf, D. Medau, H. Scharfenort, S. Staeck, C. Miethke, C. Kügler and N. Wiest for their reliable technical assistance and B. Wüsthoff for editing the manuscript. References [1] B.K. Edwards, E. Ward, B.A. Kohler, et al., Annual report to the nation on the status of cancer, 1975–2006, featuring colorectal cancer trends and impact of interventions (risk factors, screening, and treatment) to reduce future rates, Cancer 116 (3) (2010) 544–573. [2] D. Kuester, H. Lippert, A. Roessner, et al., The cathepsin family and their role in colorectal cancer, Pathol. Res. Pract. 204 (7) (2008) 491–500. [3] J. Kos, A. Sekirnik, A. Premzl, et al., Carboxypeptidases cathepsins X and B display distinct protein profile in human cells and tissues, Exp. Cell Res. 306 (1) (2005) 103–113. [4] J. Kos, Z. Jevnikar, N. Obermajer, The role of cathepsin X in cell signaling, Cell Adhes. Migr. 3 (2) (2009) 164–166 (Review). [5] N. Obermajer, A. Premzl, T. Zavasnik Bergant, et al., Carboxypeptidase cathepsin X mediates beta2-integrin-dependent adhesion of differentiated U-937 cells, Exp. Cell Res. 312 (13) (2006) 2515–2527. [6] N. Obermajer, U. Repnik, Z. Jevnikar, et al., Cysteine protease cathepsin X modulates immune response via activation of beta2 integrins, Immunology 124 (1) (2008) 76–88. [7] N.D. Staudt, A. Maurer, B. Spring, et al., Processing of CXCL12 by different osteoblast-secreted cathepsins, Stem Cells Dev. 21 (11) (2012) 1924–1935. [8] D.K. Nägler, S. Kraus, J. Feierler, et al., A cysteine-type carboxypeptidase, cathepsin X, generates peptide receptor agonists, Int. Immunopharmacol. 10 (1) (2010) 134–139. ´ Z. Jevnikar, M. Rojnik, et al., Profilin 1 as a target for cathepsin [9] U. Peˇcar Fonovic, X activity in tumor cells, PLoS One 8 (1) (2013) e53918. [10] F.D. Nascimento, C.C. Rizzi, I.L. Nantes, et al., Cathepsin X binds to cell surface heparan sulfate proteoglycans, Arch. Biochem. Biophys. 436 (2) (2005) 323–332. [11] A.M. Lechner, I. Assfalg-Machleidt, S. Zahler, et al., RGD-dependent binding of procathepsin X to integrin alphavbeta3 mediates cell–adhesive properties, J. Biol. Chem. 281 (51) (2006) 39588–39597.
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Original article
Characterization of cathepsin X in colorectal cancer development and progression Doerthe Jechorek a,∗ , Julia Votapek a , Frank Meyer b , Arne Kandulski c , Albert Roessner a , Sabine Franke a a
Department of Pathology, Otto-von-Guericke University of Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany Department of General, Visceral and Vascular Surgery, Otto-von-Guericke University of Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany c Department of Gastroenterology, Hepatology and Infectious Diseases, Otto-von-Guericke University of Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany b
a r t i c l e
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Article history: Received 21 August 2014 Accepted 22 August 2014 Keywords: Cathepsin X Colorectal cancer Coculture
a b s t r a c t The lysosomal cysteine carboxypeptidase cathepsin X (CTSX), localized predominantly in immune cells, has been associated with the development and progression of cancer. To determine its specific role in colorectal carcinoma (CRC), we analyzed CTSX expression in non-malignant mucosa and carcinoma of 177 patients as well as in 111 adenomas and related it with clinicopathological parameters. Further, the role of CTSX in the adhesion and invasion of the colon carcinoma cell lines HT-29 and HCT116 was investigated in an in vitro culture cell system with fibroblasts and monocytes, reflecting the situation at the tumor invasion front. Epithelial CTSX expression significantly increased from normal mucosa to adenoma and carcinoma, with highest expression levels in high grade intraepithelial neoplasia and in early tumor stages. Loss of CTSX occurred with tumor progression, and correlated with advanced local invasion, lymph node and distal metastasis, lymphatic vessel and vein invasion, tumor cell budding and poorer overall survival of patients with CRC. The subcellular distribution of CTSX changed from vesicular paranuclear expression in the tumor center to submembranous expression in cells of the invasion front. Peritumoral macrophages showed highest expression of CTSX. In vitro assays identified CTSX as relevant factor for cell–cell adhesion and tumor cell anchorage to fibroblasts and basal membrane components, whereas inhibition of CTSX caused increased invasiveness of colon carcinoma cells in mono- and co-culture. In conclusion, CTSX is involved in early tumorigenesis and in the stabilization of tumor cell formation in CRC. The results suggest that loss of CTSX may be needed for tumor cell detachment, local invasion and tumor progression. In addition, CTSX in tumor-associated macrophages indicates a role for CTSX in the anti-tumor immune response. © 2014 Elsevier GmbH. All rights reserved.
Introduction Colorectal cancer (CRC) is among the most common malignancies in western world countries, and despite significantly improved treatment modalities it remains a major cause of cancer mortality in the Western World [1]. The clinical outcome of CRC depends on its efficiency for local invasion and metastasis. Based on their ability to degrade extracellular matrix proteins, cysteine cathepsins, a group of lysosomal cysteine proteases, have been implicated to
Abbreviations: CTSX, cathepsin X; CRC, colorectal cancer; EMT, epithelial mesenchymal transformation; ECM, extracellular matrix. ∗ Corresponding author. Tel.: +49 391 6717953; fax: +49 391 6715818. E-mail address: [email protected] (D. Jechorek). http://dx.doi.org/10.1016/j.prp.2014.08.014 0344-0338/© 2014 Elsevier GmbH. All rights reserved.
play a role in tumor cell invasion, migration and metastasis [2]. For CRC, increased expression and activity mainly of cathepsins B, D and L were reported and are associated with matrix degradation, local tumor spread and prediction of poor prognosis [2]. In this context, the potential of other cysteine cathepsins, such as cathepsin X (CTSX), has been evaluated far less extensively. CTSX expression was thought to be limited to immune cells, such as monocytes, macrophages or dendritic cells [3]. Thus, it is probably not involved in extracellular matrix degradation, but more likely in immune response by regulating proliferation, maturation, migration and adhesion of immune cells as well as phagocytosis and signal transduction [4]. Molecular targets of CTSX exopeptidase activity include the -chain of integrin receptors [5,6], chemokine CXCL-12 [7], bradykinin and kallidin [8] and profilin 1 [9]. Furthermore, CTSX has been reported to bind to cell surface heparin sulfate
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proteoglycans [10], to interact with ␣v3 integrin and ␣v5 integrin [11,12], and to activate 2-integrins [5], thus indicating a major role of CTSX in focal adhesion formation and in the modulation on adhesion to proteins of the extracellular matrix. Recent studies have shown that altered CTSX expression of the tumor epithelium and tumor-associated immune cells is associated with progression and metastasis of several tumors, such as gastric cancer, prostate cancer, hepatocellular carcinoma and malignant melanoma [13–16]. Here, CTSX might be involved in the modulation of adhesive and migration properties of tumor cells by interaction with integrin receptors, thus supporting invasion through the extracellular matrix [17]. Furthermore, CTSX has been suggested to promote tumor progression by bypassing cellular senescence [18] and by inducing an epithelial–mesenchymal transition [15]. Concerning CRC, studies focused mainly on the association of CTSX expression and clinical prognosis [19]. However, the precise function of CTSX in genesis and progression of CRC is still unclear. In the present study, CTSX expression was analyzed in nonmalignant mucosa, adenoma and carcinoma of the colorectum and correlated to clinico-pathological parameters. The role of CTSX in adhesion and invasion of colorectal tumor cells was investigated in an in vitro culture cell system with fibroblasts and monocytes, reflecting the in vivo situation at the tumor invasion front. Materials and methods Patients and tissue samples In this retrospective study, we analyzed specimens of colorectal tissue obtained from surgical resections by partial or complete colectomy, endoscopic mucosectomy and polypectomy between 1995 and 2010, and registered in the archive of the Department of Pathology in Magdeburg, Germany. Tissue collection and performance of this study were in line with the guidelines of the institutional ethical committee, and all patients provided informed consent. The first study group comprised a total of 177 patients with microsatellite-stable sporadic colorectal carcinoma. This group had a median age of 67.3 ± 10.7 (range: 40–91) years, and consisted of 102 males and 75 females. All carcinomas were treated solely by partial colectomy with R0-resection. Carcinomas were classified according to the recent guidelines of the UICC TNM and WHO classification system by two pathologists (D.J., A.R). Distribution of UICC stages, grades of differentiation and localization were as follows: stage I (n = 43), stage II (n = 43), stage III (n = 41), stage IV (n = 50); G1 (n = 25), G2 (n = 118), G3 (n = 34); proximal localization (n = 72), and distal localization (n = 105). Survival data were available for all patients. Further details on the clinico-pathological data of both study groups are given in Table 1. Tumor and paired noncancerous tissue samples were snap frozen in liquid nitrogen, stored at −80 ◦ C for molecular studies and formalin-fixed, paraffin-embedded for immunohistochemical analysis. The second study group comprised 61 male and 50 female patients, ranging in age from 43 to 89 years (median, 65.7 ± 11.3 years) and included formalin-fixed, paraffin-embedded specimens of adenoma with low grade (n = 59) and high grade (n = 38) intraepithelial neoplasia. Tissue microarray design For immunohistochemical analysis, representative tumor regions were selected on H&E-stained slices for inclusion in a tissue array. The arrays were assembled by taking core needle biopsies with a diameter of 0.6 mm from the areas of interest in pre-existing
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Table 1 Clinico-pathological features and immunoreactive scores of colorectal carcinomas investigated by immunohistochemistry for CTSX protein expression. Factor Age 5.5 and were considered CTSXoverexpressing tumors. The 3-, 5-, and 10-year overall survival based on the endpoint of cancer-related death of patients with low or high CTSX expression was 50%, 36% and 16%, or 66%, 52%, and 36%, respectively. Kaplan-Meier curve demonstrated that patients with low CTSX expression had a significantly worse prognosis than those with a CTSX overexpression (p = 0.026) (Fig. 2 C). Strong CTSX-expression in peritumoral macrophages At the tumor–stromal interface of the carcinoma samples, strong immunoreactivity of CTSX was noticed for a group of inflammatory cells of the surrounding stroma. Using double immunofluorescence, CTSX-expressing stromal cells were identified as CD68-positive macrophages (Fig. 3). In carcinomas, CTSX expression of macrophages was extensively stronger than in tumor cells (7.96 ± 2.9 vs. 5.5 ± 3.1; p < 0.001). CTSX-positive macrophages were also found in adenoma mainly surrounding the dysplastic epithelium of the apical parts. Here, expression scores for CTSX of macrophages were higher than for epithelial cells of adenoma (2.97 ± 1.7 vs. 2.13 ± 1.8; p < 0.001), too. Loss of CTSX leads to decreased adhesion and increased invasion To elucidate the role of CTSX in colorectal carcinogenesis and progression, we compared cellular adhesion and invasion of untreated HT-29 cells and HCT116 cells in monoculture and
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Fig. 1. Immunohistochemical analysis for CTSX protein expression in colon adenoma with low grade (A) and high grade (B) intraepithelial neoplasia, in the center (C/D) and the invasion front (E/F) of colorectal carcinoma, in tumor-associated macrophages of the invasion front (G) and in non-tumorous colon mucosa (H). Adenoma and carcinoma cells of the tumor center showed vesicular, partly perinuclearly localized CTSX (*). Tumor glands at the invasion front demonstrated submembranous concentration of CTSX directed to the tumor cell–stroma interface (arrow) and complete loss of CTSX in invasive tumor cell buds (open arrow) (magnification: ×100, ×400).
co-culture with that of tumor cells after CTSX siRNA treatment. Cocultivating tumor cells with human fibroblasts and monocytic cells and applying Matrigel matrix, the in vitro tumor cell culture system approximates the in vivo situation at the invasion front. CTSX siRNA transfected to the tumor cells significantly reduces the expression of CTSX mRNA and protein as described by Krueger et al. [13]. The adhesion of tumor cells treated with siRNA was significantly reduced as compared with untreated cells in monoculture and co-culture (Fig. 2D). Addition of siRNA resulted in a significant reduction of the cell–cell-adhesion of HT-29 cells of 44.4 ± 19% (p = 0.029). The percentage of siRNA-treated HT-29 or HCT 116 cells that adhered to Matrigel decreased significantly to 27.4 ± 1%
and 41.4 ± 8%, respectively (both p < 0.001). Furthermore, CTSXdepleted HT-29 cells showed a 15% decrease in adhesion to fibroblasts (p = 0.125). In contrast, reduced CTSX expression of HCT 116 cells had no effect on adhesion to fibroblasts. Compared to untreated cells, transfection with CTSX siRNA resulted in a highly significant increase of invasive tumor cells in all experimental set-ups (Fig. 2E). The combination of coculture of HCT 116 cells with monocytes and decreased CTSX expression resulted in the greatest number of cells invading through Matrigel (232.4 ± 79%, p = 0.004). Monocultivated HT-29 cells were also highly invasive after siRNA treatment (224.2 ± 65%, p = 0.003). Furthermore, invasion through Matrigel-coated membrane increased
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Fig. 2. Double immunofluorescence of CTSX ((A) FITC) and CD68 ((B) Texas Red) shows CTSX-expressing tumor-associated macrophages ((C) overlay) in colorectal carcinoma (magnification: ×100).
significantly for monocultivated HCT 116 cells, and HT-29 cells cocultivated with macrophages after pretreatment with siRNA (176.2 ± 37%, p = 0.037; 163.5 ± 21, p = 0.004). The invasiveness of HT-29 and HCT 116 tumor cells transfected with siRNA was also increased under coculture with fibroblasts (141.0 ± 17%, p = 0.095; 169.3 ± 49%, p = 0.573). Thus, both adhesion and invasion assay experiments demonstrate that loss of CTSX has significant effects on tumor invasion of colorectal cancer cells. Discussion The overexpression of cysteine cathepsins, in particular cathepsins B, D, and L, has been associated with development and progression in CRC. Based on their ability to degrade extracellular
matrix proteins, they are involved in invasion, migration and metastasis of colorectal tumor cells [2]. Recently, elevated CTSX serum levels were correlated with poorer overall survival for patients with locally resectable CRC. Therefore, CTSX was thought to be a prognostic and predictive marker in CRC [19]. However, there are no studies that give a detailed morphological distribution in tissue of CRC or studies investigating the precise function of CTSX in genesis and progression of CRC. Therefore, in the present study, we analyzed CTSX expression in non-malignant colon mucosa and CRC of 177 patients and 111 colorectal adenomas in relation to clinico-pathological parameters. Furthermore, the role of CTSX in the adhesion and invasion of colorectal tumor cells was investigated in an in vitro culture cell
Fig. 3. CTSX protein expression and its correlation with clinicopathological data and mRNA expression – Immunohistochemical protein expression of CTSX (A), indicated by an immunoreactive score (IRS). Colorectal non-tumorous mucosa, adenoma and carcinoma showed increased expression of CTSX with tumorigenesis. Tumor-associatedmacrophages demonstrated highest expression scores for CTSX. Analysis of CTSX protein and mRNA expression (B) confirmed correlation of both parameters for low- (IRS ≤ 5) and high-expressing (IRS > 5.5) carcinomas (p = 0.036). Data are shown as boxplots representing 25th, 50th, and 75th percentile values and means. Kaplan-Meier plot for survival (C), as a function of CTSX, revealed that the group with low expressing colon carcinomas displayed a significantly poorer clinical outcome than the high expressing group (p = 0.026). In vitro assays – Cellular adhesion (D) and invasion (E) of HT-29 and HCT116 colon carcinoma cells in monoculture and co-culture with 175BR fibroblasts or THP-1 macrophages and after addition of CTSX-specific siRNA. Bar graphs are presented as means ± SD .
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system with fibroblasts and monocytes, reflecting the situation at the tumor invasion front. In general, CTSX expression was up-regulated in colorectal adenoma and carcinoma. However, the highest CTSX levels were observed for early tumor stages but loss of CTSX expression correlated with the clinico-pathological parameters of progressed tumor stages and poor survival. The differences in subcellular und intratumoral distribution of CTSX suggest different functions during tumor development and progression and in different tumor zones. Furthermore, our in vitro studies demonstrate that loss of CTSX is associated with decreased cell adhesion and increased invasion of colon cancer cells. Overexpression of CTSX has already been described for other tumors, such as gastric and prostrate cancer, hepatocellular carcinoma and malignant melanoma [13–16]. Similar to our study, in prostate, prostatic intraepithelial neoplasia and early stages of invasive carcinoma showed equally high expression levels, suggesting that CTSX may be involved in early tumorigenesis [14]. In addition, in a metastatic breast cancer mouse model, loss of CTSX resulted in a delay of early tumor development, demonstrating that CTSX is related to the initial stages of the malignant process and less with tumor progression and metastasis [22]. However, studies on serum levels of CTSX expression have not yet answered the question of whether the cathepsin is involved in early or late stages of CRC [19]. We demonstrated highest expression levels in the central parts of locally restricted or non-metastasized carcinomas, respectively. The most significant increase of CTSX expression was found between adenoma with high grade dysplasia and early invasive pT1 carcinoma reflecting the point of malignant transformation. Furthermore, in adenomas and early tumor stages, CTSX-positive vesicles were mainly accentuated within the perinuclear region, suggesting that CTSX is involved in regulative processes during malignant transformation and early tumor formation. Neoplastic transformation due to CTSX up-regulation might be promoted through bypassing cellular senescence [18], activation of the pro-angiogenic chemokine CXCL-12 [7] or phosphorylation of the IGF-1 receptor [23]. Recently, high expression of proproliferative IGF-1 receptor has been associated with increased risk of CRC [24]. CTSX is also involved in the induction of epithelial–mesenchymal transition [15]. In hepatocellular carcinoma, CTSX was associated with down-regulation of epithelial markers, including E-cadherin, ␣-catenin and -catenin and upregulation of mesenchymal proteins, such as fibronectin, vimentin and N-cadherin, thus contributing to the acquisition of a motile invasive phenotype by the tumor cells [15]. Since CTSX has no transcriptional property and is rather recognized as an ERGolgi-lysosome resident carboxypeptidase [10,25], EMT-related phenotype induced by CTSX overexpression could result from degradation, internalization and lysosomal trafficking of epithelial proteins. In our in vitro assays, CTSX was identified as an important modulator of adhesion and invasion of colorectal tumor cells. Inhibition of CTSX at the mRNA level resulted in reduced adhesion of tumor cells to Matrigel. In addition, adhesion to fibroblast and cell–celladhesion of low-aggressive HT29 was decreased due to loss of CTSX. Our results suggest a role of CTSX in stabilizing tumor cell formation and in the attachment of tumor cells to basal membrane and ECM components. Similar to our results, inhibition of CTSX with siRNA led to reduced adhesion of prostate cancer cells on a fibronectin-coated surface [9] and to impaired migration of gastric cancer cells [13]. In contrast, inhibition of CTSX activity had no impact on the migration of breast cancer cells [3], suggesting that the cathepsin may participate in mechanisms of adhesion and migration, which are not related to proteolysis. These nonproteolytic functions of CTSX may include binding to proteins, such
as integrins [11], which mediate dynamic adhesive cell–cell and cell–ECM interactions and anchor various cytoskeletal and cytoplasmic proteins to the actin cytoskeleton [26]. This leads either to stabilization of cell complexes or to remodeling of the cytoskeleton, formation of focal adhesions and attachment of migrating cells to ECM components [27]. Several studies have demonstrated that the pro- and mature forms of CTSX modulate the function of integrins through specific binding via their integrin-binding motifs RGD (Arg-Gly-Asp) and EGD (Glu-Cys-Asp) [5,6,11,28]. Moreover, CTSX binds cell-surface HSPG [11], which are involved in integrin regulation [29] and are known partners of integrins in focal adhesion formations [30]. In this context, endogenous CTSX is transported to the plasma membrane, accumulates in vesicles at lamellipodia, and is partly associated with the cell surface co-localizing with the relevant integrin subunit [5,11]. Remarkably, in our morphological studies of the tumor invasion zone, we also observed a linear submembranous accumulation of CTSX directed towards the tumor cell–stroma interface. Thus, the intracellular localization of CTSX in cells of the invasion front differs from that in the tumor center, which strengthens the results of our in vitro studies assigning CTSX a role in tumor cell anchorage on ECM. Local invasion requires reduction of intercellular and cell–matrix contacts, dynamic reorganization of the tumor cell cytoskeleton with formation of podia-like extensions, pericellular matrix degradation and oriented migration of the so-called tumor cell buds through ECM [31,32]. Therefore, based on our results on adhesion assays, we hypothesize that loss of CTSX leads to cell contact changes favouring tumor cell invasiveness and tumor spreading. Indeed, performing invasion assays, we demonstrated a significantly increased invasiveness of colon carcinoma cells lacking CTSX. Loss of CTSX did not only affect invasion of tumor cells through Matrigel alone, but also invasion under co-culture with macrophages or fibroblasts. We found decreased CTSX tissue expression in progressed tumor stages and loss of CTSX in the invasion front and in invasive tumor cell buds. Furthermore, loss of CTSX correlated with clinico-pathological factors of progression and poorer survival in patients with CRC. Similar to our data, tumor cells of progressed lung carcinoma and invasive tumor cell lines also showed decreased CTSX expression [3]. Therefore, we presume that CTSX is down-regulated in cells of the invasion front to reach a migrative and invasive phenotype. In contrast to our results, in a recent study of Viˇzin et al. [19], high CTSX serum levels were associated with poor overall survival for patients with locally resectable CRC. Since CTSX is released in large amounts into the circulation by leucocytes and macrophages during immune cell activation and turnover [33], increased serum levels of CTSX in CRC could reflect the overexpression of CTSX in tumor-associated immune cells. Indeed, performing morphological studies of the invasion front, we noticed a strong immunoreactivity of CTSX in macrophages nesting at the tumor–stromal interface of CRC. Similarly to our study, highly CTSX-expressing macrophages in the vicinity of low expressing tumor cells were described in lung carcinoma [3]. Thus, elevated serum levels of CTSX in CRC most likely originate from macrophages that accumulated particularly at the tumor invasive zone. Although CTSX as a carboxypeptidase is unlikely to participate in ECM degradation itself [25], it was shown that CTSX can up-regulate proteins associated with extracellular matrix remodeling, such as MMP2, MMP3 and MMP9 [15]. However, being involved in the activation of the 2 integrin receptors LFA-1 and Mac-1, CTSX most probably regulates transendothelial migration, adhesion to ECM and maturation of macrophages, which leads to accumulation of macrophages at the invasion front [5]. In addition, recruitment of macrophages into the tumor invasion zone could be supported by CTSX-induced processing of the chemokine CXCL-12 [34]. Furthermore, CTSX plays a role in phagocytosis and antigen presentation of macrophages and might be involved in
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anti-tumor immunity [3,5,35]. For example, Mac-1 as a target for CTSX is crucial for FcR-mediated cytotoxity to tumor cells [36]. In summary, our study identified CTSX as adhesion factor in CRC, supporting intratumoral cell–cell stabilization and tumor cell anchorage to ECM. The fact that the cathepsin was most strongly expressed in adenoma and early stages of CRC suggests that CTSX could be involved in early tumorigenesis. Inhibition of CTSX leads to increased invasiveness of colon carcinoma cells in vivo, and loss of CTSX at the invasion front was associated with tumor progression and poorer overall survival of CRC patients. Abundantly expressed CTSX in macrophages at the invasion front of CRC seems to be part of the anti-tumor immune response. Therefore, the influence of CTSX on the development and progression of CRC is likely to be complicated as CTSX functions are different depending on cell type and pathophysiological compartments. Further research is needed to understand its underlying mechanism. Acknowledgements The authors thank all patients and their attending physicians for their participation in this study. We thank H. Wolf, D. Medau, H. Scharfenort, S. Staeck, C. Miethke, C. Kügler and N. Wiest for their reliable technical assistance and B. Wüsthoff for editing the manuscript. References [1] B.K. Edwards, E. Ward, B.A. Kohler, et al., Annual report to the nation on the status of cancer, 1975–2006, featuring colorectal cancer trends and impact of interventions (risk factors, screening, and treatment) to reduce future rates, Cancer 116 (3) (2010) 544–573. [2] D. Kuester, H. Lippert, A. Roessner, et al., The cathepsin family and their role in colorectal cancer, Pathol. Res. Pract. 204 (7) (2008) 491–500. [3] J. Kos, A. Sekirnik, A. Premzl, et al., Carboxypeptidases cathepsins X and B display distinct protein profile in human cells and tissues, Exp. Cell Res. 306 (1) (2005) 103–113. [4] J. Kos, Z. Jevnikar, N. Obermajer, The role of cathepsin X in cell signaling, Cell Adhes. Migr. 3 (2) (2009) 164–166 (Review). [5] N. Obermajer, A. Premzl, T. Zavasnik Bergant, et al., Carboxypeptidase cathepsin X mediates beta2-integrin-dependent adhesion of differentiated U-937 cells, Exp. Cell Res. 312 (13) (2006) 2515–2527. [6] N. Obermajer, U. Repnik, Z. Jevnikar, et al., Cysteine protease cathepsin X modulates immune response via activation of beta2 integrins, Immunology 124 (1) (2008) 76–88. [7] N.D. Staudt, A. Maurer, B. Spring, et al., Processing of CXCL12 by different osteoblast-secreted cathepsins, Stem Cells Dev. 21 (11) (2012) 1924–1935. [8] D.K. Nägler, S. Kraus, J. Feierler, et al., A cysteine-type carboxypeptidase, cathepsin X, generates peptide receptor agonists, Int. Immunopharmacol. 10 (1) (2010) 134–139. ´ Z. Jevnikar, M. Rojnik, et al., Profilin 1 as a target for cathepsin [9] U. Peˇcar Fonovic, X activity in tumor cells, PLoS One 8 (1) (2013) e53918. [10] F.D. Nascimento, C.C. Rizzi, I.L. Nantes, et al., Cathepsin X binds to cell surface heparan sulfate proteoglycans, Arch. Biochem. Biophys. 436 (2) (2005) 323–332. [11] A.M. Lechner, I. Assfalg-Machleidt, S. Zahler, et al., RGD-dependent binding of procathepsin X to integrin alphavbeta3 mediates cell–adhesive properties, J. Biol. Chem. 281 (51) (2006) 39588–39597.
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