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Osteopontin is associated with decreased apoptosis and αv integrin expression in lung adenocarcinoma
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Osteopontin is associated with decreased apoptosis and ␣v integrin expression in lung adenocarcinoma a ˇ Christophe Stemberger , Koviljka Matuˇsan-Ilijaˇs b , Manuela Avirovic´ b , c Ljiljana Bulat-Kardum , Aldo Ivanˇcic´ d , Nives Jonjic´ b , Ksenija Luˇcin b,∗ a
Department of Clinical Cytology, Clinical Hospital Center Rijeka, Rijeka, Croatia Department of Pathology, Rijeka University School of Medicine, Rijeka, Croatia c Department of Internal Medicine, Clinical Hospital Center Rijeka, Rijeka, Croatia d Department of Thoracovascular Surgery, Clinical Hospital Center Rijeka, Rijeka, Croatia b
a r t i c l e
i n f o
Article history: Received 29 May 2013 Received in revised form 12 July 2013 Accepted 14 July 2013 Available online xxx Keywords: Lung carcinoma Osteopontin ␣v Integrin Tissue array analysis Immunohistochemistry Apoptosis
a b s t r a c t Osteopontin (OPN) is a glycoprotein involved in invasion, progression and metastasis of many carcinomas. It contains several functional domains including binding sites for ␣v integrins, cell surface molecules playing a major role in mediating cell migration and adhesion. The aim of the study was to evaluate the expression of osteopontin in human non-small cell lung cancer (NSCLC) and to determine its possible prognostic significance as well as relation to apoptosis and ␣v integrin expression. We analyzed 111 surgically resected NSCLC for immunohistochemical expression of OPN and ␣v integrin. OPN expression was compared to apoptotic rate and clinicopathological parameters such as tumor size, histological grade, lymph node status, pT, and TNM stage. Apoptotic rate was measured by TUNEL staining method. OPN expression in NSCLC was significantly higher in lung adenocarcinomas (AC) then in squamous cell carcinomas (p < 0.001). There was no correlation between OPN expression and clinicopathological parameters. The level of OPN expression in AC was associated with decreased apoptotic activity of tumor cells (p = 0.006), and correlated with ␣v integrin expression (p = 0.048), particularly in low stage tumors (p = 0.013). Prolonged tumor cell survival in lung AC due to OPN and ␣v integrin overexpression may have an impact on tumor progression and resistance to therapy. © 2013 Elsevier GmbH. All rights reserved.
Introduction Lung cancer is one of the most common human malignancies worldwide and a major cause of death in many developed countries. Surgical resection continues to play an important role in the treatment of this disease, especially during the early stages. However, many patients with presumed localized disease have occult metastases at the time of diagnosis and suffer from disease recurrence. Since the current clinicopathological staging system is unreliable in predicting the outcome of the disease, new biomarkers are needed for disease monitoring and predicting prognosis of patients with lung cancer, as well as for planning new treatment strategies. One of the markers recently linked to cancer development, progression and metastasis in many types of human tumors is a multifunctional protein named osteopontin (OPN). OPN is a member of the small integrin binding ligand N-linked glycoproteins (Bellahcène et al., 2008). It is involved in a variety of
∗ Corresponding author at: Department of Pathology, Rijeka University School of ´ Branchetta 20, 51 000 Rijeka, Croatia. Medicine, Brace E-mail address: [email protected] (K. Luˇcin).
physiological and pathological processes, including invasion, progression and metastasis of cancer (Wai and Kuo, 2008). It is synthesized by a variety of cell types including fibroblasts, osteoblasts, osteocytes, dendritic cells, macrophages, smooth muscle cells, endothelial cells and some epithelial cells. The main functional domains of OPN consist of an RGD motif, which binds to ␣v and ␣51 integrins, and a thrombin cleaved epitope SVVYGLR (OPN-R, a non-RGD domain), which binds to integrin receptors ␣41, ␣91, and ␣94, present on a number of immune cells such as macrophages, neutrophils and T lymphocytes. The hyaluronan receptor, CD44, has also been identified as a receptor for OPN. Binding of OPN to these cell surface receptors stimulates cell adhesion, migration, and specific signaling functions resulting in proliferation and increased cell survival. It has been shown that increased OPN expression levels correlate with enhanced malignancy and poor prognosis in several malignant tumors, including non-small cell lung carcinoma (NSCLC) (Weber et al., 2010). Moreover, it has been shown that OPN has value as a clinical tumor progression marker (Weber et al., 2010). Considerable evidence suggests that OPN participates in lung carcinogenesis, but the mechanisms involved in lung cancer progression are largely unknown (Boldrini et al., 2005; Donati et al., 2005; Hu et al., 2005; Blasberg et al., 2010; Jin et al.,
0065-1281/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.acthis.2013.07.009
ˇ Please cite this article in press as: Stemberger C, et al. Osteopontin is associated with decreased apoptosis and ␣v integrin expression in lung adenocarcinoma. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.07.009
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2012). OPN has long been known to be able to bind to some cell surface integrins, which provide clues to the possible functional role of OPN in cancer. From animal studies it has been reported that the integrinbinding RGD domain of human osteopontin promotes lung carcinoma growth and metastasis through activation of survival pathways, while cell–cell adhesion was not affected (Courter et al., 2010). Integrins are transmembrane proteins that mediate interactions between adhesion molecules on adjacent cells and/or the extracellular matrix (ECM) (Giancotti and Ruoslahti, 1999). They have diverse roles in several biological processes including cell migration during development and wound healing, cell differentiation and apoptosis. Their activities can also regulate the metastatic and invasive potential of tumor cells. They exist as heterodimers consisting of alpha and beta subunits. These various integrins recognize the arginine-glycine-aspartic acid (RGD) peptide sequence found in many ECM proteins, including osteopontin. The current study was undertaken to investigate the expression of osteopontin in resected human NSCLC in order to assess its prognostic significance, relation to cell survival and to the expression of ␣v integrins. We show that OPN is overexpressed in NSCLC, with lung AC showing higher level of OPN overexpression compared to SCC. In adenocarcinoma, OPN overexpression was associated with prolonged cellular survival and correlated with ␣v integrins. A better understanding of the implications of OPN in lung tumorigenesis might facilitate development of new therapeutic regimens to benefit patients with these malignancies. Materials and methods Tumor samples
Table 1 Osteopontin expression in relation to clinicopathological characteristics of nonsmall cell lung carcinoma patients. Parameter
Gender Female Male Age Years; median (range) Tumor location Right upper lobe Left upper lobe Right lower lobe Left lower lobe Right middle lobe Tumor size (cm) ≤3 >3 N status N0 N1 pT 1 2+3 TNM stage I II + III Histological type Adenocarcinoma Squamous cell carcinoma Large cell carcinoma Histological grade 1+2 3 a *
We examined 111 consecutive lobectomy specimens with NSCLC diagnosis confirmed at the Department of Pathology, University of Rijeka, from January 2001 to December 2011. Clinicopathological characteristics are shown in Table 1. The age of patients ranged from 42 to 85 years (mean 63.5 years), among which 80 were male and 31 female. None of the patients had previous or synchronous cancer. The surgically resected specimens were fixed in 10% buffered formalin and embedded in paraffin. All sections were stained with hematoxylin and eosin (H&E). The slides were reviewed and classified according to WHO (2004) diagnostic criteria, with adenocarcinoma, squamous cell carcinoma and large cell carcinoma as the three main types. The histological types of cancer included 52 adenocarcinomas, 49 squamous cell carcinomas and 10 large cell carcinomas. The tumors were staged according to UICC criteria (2010). The study was approved by the Ethics Committee of Clinical Hospital Center Rijeka and Rijeka University School of Medicine. Tissue microarray (TMA) construction Hematoxylin and eosin-stained sections of tumors were used to mark tumor areas of interest. Four tissue cores, each 2 mm in diameter, were placed into a recipient paraffin block using a manual tissue arrayer (Alphelys, Plaisir, France). Normal liver tissue was used for orientation. Cores were spaced at intervals of 0.5 mm in the x and y axes. One section from each TMA block was stained with hematoxylin and eosin for morphological assessment. Serial sections were cut from TMA blocks for immunohistochemical and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) staining. Five m thick sections were placed on adhesive glass slides (Capillary Gap Microscope Slides, 75 m, Code S2024, DakoCytomation, Glostrup, Denmark), left to dry at 37 ◦ C overnight and stored in the dark at +4 ◦ C.
#
N (%)
OPN H-scorea (mean ± SD)
p value*
31 (28) 80 (72) 64 (42–85) 26 (23) 33 (30) 21 (19) 23 (21) 8 (7) 43 (39) 67 (61)
132 ± 64 125 ± 60
0.551
79 (71) 32 (29)
126 ± 61 132 ± 63
0.599
71 (64) 40 (36)
137 ± 61 123 ± 62
0.270
63 (57) 48 (43)
134 ± 60 120 ± 63
0.225
52 (47) 49 (44) 10 (9)
150 ± 58 104 ± 57 134 ± 59
68 (61) 43 (39)
160 ± 63 127 ± 59
<0.001#
0.052
Histoscore. Student’s t-test. p value – adenocarcinoma versus squamous cell carcinoma.
Immunohistochemistry Tumor samples were processed for immunohistological analysis to determinate OPN and ␣v integrin expression. Following deparaffinization in xylene and rehydration in alcohols, heat-induced epitope retrieval was achieved. Antigen retrieval for anti-OPN antibody (clone OPN3, Novocastra, Newcastle upon Tyne, UK, dilution 1:100) was achieved by immersing slides in Tris-EDTA buffer (pH 9.0) and boiling for 10 min in a water bath. For ␣v integrin staining (clone P2W7; Santa Cruz Biotechnology, Santa Cruz, CA, USA, dilution 1:25), we employed antigen retrieval using proteinase K (DakoCytomation, Glostrup, Denmark). Following blockage of non-specific antibody binding, sections were incubated with primary antibodies against OPN and ␣v integrin human epitopes. Immunohistochemistry was performed with Dako Autostainer Plus (DakoCytomation Colorado Inc., Fort Collins, CO, USA) according to the manufacturer’s protocol, using the Envision peroxidase procedure. All reagents for automated immunohistochemistry were purchased from DakoCytomation (ChemMate TM Envision HRP detection Kit K5007). Negative control slides were prepared by substituting the primary antibody with Dako ChemMate antibody diluent. Slides were counterstained with Mayer’s hematoxylin, dehydrated and mounted. TUNEL The TUNEL method was used to detect DNA strand breaks generated during apoptotic cell death. Following deparaffinization in xylene and rehydration in alcohols, heat-induced epitope retrieval was achieved by immersing slides in Tris-EDTA buffer (pH 9.0) and boiling for 5 min in a water bath. Slides were allowed to cool
ˇ Please cite this article in press as: Stemberger C, et al. Osteopontin is associated with decreased apoptosis and ␣v integrin expression in lung adenocarcinoma. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.07.009
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during 20 min, and then pre-incubated with normal bovine serum (3%) for 20 min. For TUNEL – alkaline phosphatase staining, In Situ Cell Death Detection Kit (Cat. No. 11684809910, Roche Applied Science, Indianapolis, IN, USA) was used at 37 ◦ C in the dark. Terminal deoxynucleotidyl transferase was diluted 1:5 using TUNEL Dilution Buffer (Cat. No. 1966006; Roche, Mannheim, Germany). Sections were incubated with TUNEL reaction mixture for 60 min. Subsequently, alkaline phosphatase-conjugated sheep antibody was added for 30 min followed by fast red chromogen (Fast Red tablets, Cat. No. 1496549; Roche, Mannheim, Germany) for 20 min. Slides were counterstained with Mayer’s hematoxylin, dehydrated and mounted. Two negative control slides were prepared by substituting TUNEL reaction mixture with nucleotide mixture and phosphate buffered saline, respectively. Positive controls were TUNEL positive cells displaying morphological features of apoptosis. Evaluation of immunohistochemistry and TUNEL staining Two observers, blinded to clinical data, evaluated the staining scores independently. The mean values of the OPN staining intensity were graded semi-quantitatively using a histological score (H-score) according to the following equation: H-score = Pxi1–3 , where i is the intensity of staining with a value of 1, 2 or 3 (mild, moderate and strong staining), and P is the percentage of cells stained with particular intensity, varying between 1% and 100%. For statistical purpose, OPN and ␣v integrin H-score was divided into low and high group, with a mean H-score of 150 and H-score of 150, used as cut off value, respectively. Avoiding necrotic areas, the percentage of apoptotic cells was evaluated by counting positive staining of 1000 tumor cells in tumor areas with the highest density of positive cells using a high power objective, and was determined as apoptotic index (AI). Positive apoptotic bodies and positive extracellular chromatin fragments were considered as positive staining. Statistical analysis Statistical analysis was performed using Statistica 7 software (StatSoft, Inc., Tulsa, OK, USA). The mean values of continuous data with normal distribution (OPN H-score) were compared by Student’s t-test. For variable without normal distribution (apoptotic activity) non-parametric tests were used. A difference between two groups was calculated using the Mann Whitney test, while Kruskal–Wallis test was used for comparing the quality scores between 4 groups. The correlation between two continuous variables (OPN and ␣v integrin) was measured by Pearson’s correlation test. Statistical differences with p value less than 0.05 were considered significant. Results Expression of OPN in normal lung tissue and NSCLC In normal lung tissue OPN was observed in epithelial cells of the bronchial mucous glands and as a diffuse cytoplasmic staining in alveolar macrophages. Bronchial and alveolar epithelial cells were generally negative or showed focal, faint positivity, which was more intense along the luminal surface. In lung carcinoma, OPN staining was observed in the cytoplasm of tumor cells in the form of granules of various sizes and staining intensity. The staining pattern was heterogeneous and ranged from staining of small number of tumor cells with low intensity (Fig. 1A) to strong and diffuse positivity throughout the TMA sections (Fig. 1C). There were few OPN negative TMAs, but none of examined tumors was completely OPN negative. The OPN H-score values are shown in Table 1. The mean OPN H-score for the whole NSCLC group was 128 (range 1.5–290).
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OPN staining was higher in AC (mean 150, range 5–160) compared to SCC (mean 104, range 1.5–234) and this was statistically significant (p < 0.001). The OPN H-score for large cell carcinoma was in between (mean 130, range 30–205).
Expression of OPN in relation to clinicopathological parameters When OPN H-score was compared to usual clinicopathological parameters, we could not find any significant associations in the NSCLC group. The results are shown in Table 1. We only found a trend toward a significant association between higher OPN level and lower histological grade (p = 0.052). There was no significant difference in the level of OPN expression between small (<3 cm) and large (>3 cm) tumors (p = 0.551), between lymph node negative and positive tumors (p = 0.599), between pT1 and pT2/3 tumors (p = 0.27), and between TNM stage 1 and stage 2/3 tumors (p = 0.225). We obtained similar results when AC and SCC were analyzed separately (Table 2). There was no difference in the level of OPN expression with regard to tumor size (p = 0.638 and 0.8, respectively), lymph node status (p = 0.51 and 0.861, respectively), pT status (0.666 and 0.425, respectively), and TNM stage (p = 0.152 and 0.448, respectively. Also, when only major histological subtypes where considered, without large cell subtype, a trend toward the association between higher OPN expression and lower histological grade was lost (p = 0.578 and p = 0.31, respectively).
Expression of OPN in relation to apoptotic index Apoptotic activity of tumor cells for the whole NSCLC group ranged from 0% to 8%, mean 1.5%. There was no statistically different apoptotic activity between NSCLC tumors with low or high OPN expression (p = 0.66, Table 3). However, when analyzed separately, we found an association between OPN expression and apoptotic activity of tumor cells in adenocarcinoma. Statistically higher apoptotic activity was observed in tumors with low OPN expression and vice versa (p = 0.006, Fig. 2). In SCC there was only a trend toward the association between OPN and apoptotic activity, although in the opposite direction (p = 0.08).
Expression of ˛v integrins in normal lung tissue and NSCLC In normal lung tissue there was no expression of ␣v integrins. In lung carcinoma, ␣v integrin showed a membranous staining pattern (Fig. 1E and F). The staining of fibroblasts, endothelial cells, lymphocytes and macrophages was also observed. The mean value of membranous ␣v integrin H-score for all NSCLC sample was 132 (range 0–283). AC showed lower ␣v integrin expression compared to SCC (mean 97; range 0.2–225, and mean 176, range 3–283, respectively). The level of ␣v integrin expression was statistically different between two major histological types (p < 0.001) (data not shown).
Correlation of OPN and ˛v integrin expression There was no correlation between OPN and ␣v integrin expression when considering all NSCLC samples (Pearson correlation test, r = −0.08, p = 0.4), as well as SC samples (r = −0.06, p = 0.66). In the AC group we found a weak correlation between OPN and ␣v integrin (r = 0.28, p = 0.048), and a strong correlation in the group of patients with lower clinical stage AC (r = 0.45, p = 0.013). The results are shown in Fig. 3.
ˇ Please cite this article in press as: Stemberger C, et al. Osteopontin is associated with decreased apoptosis and ␣v integrin expression in lung adenocarcinoma. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.07.009
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Fig. 1. Immunohistochemical and TUNEL staining in lung adenocarcinoma. (A) Adenocarcinoma sample with weak cytoplasmic OPN staining followed by (B) high apoptotic activity of tumor cells. (C) Adenocarcinoma sample with strong OPN staining and (D) low apoptotic activity of tumor cells. (E) ␣v integrin expression with faint apical membranous staining and (F) strong membranous ␣v integrin expression.
Association between OPN, ˛v integrin expression and apoptosis
6 Median 25%-75% Min-Max
Apoptotic activity
5
4
3
2
When the AC tumor samples were divided into four groups based on a level of OPN and ␣v integrin expression (both positive, both negative or only one marker positive) and compared to the apoptotic index, we found a significant difference between groups (Fig. 4). Namely, the highest apoptotic rate was observed in the group of AC samples that were negative for both proteins, while in the group of tumors that were positive for both markers, the apoptotic rate had the lowest value (Kruskal Wallis test, p < 0.01).
1
Discussion 0
-1
Low
High
Osteopontin expression Fig. 2. Osteopontin expression in lung adenocarcinoma according to apoptotic activity of tumor cells: low apoptotic activity is associated with high OPN expression and vice versa (Mann Whitney test, p = 0.006).
In the current study we showed the upregulation of OPN in NSCLC and different OPN expression among major NSCLC subtypes. Also, we showed an association between the higher level of OPN expression and lower apoptotic rate in lung adenocarcinoma, which correlated with the ␣v integrin expression. There are several reports on the role of OPN in lung carcinoma progression however, the results are somehow contradictory
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Table 2 Osteopontin expression in relation to clinicopathological characteristics of adenocarcinoma and squamous cell carcinoma patients. Adenocarcinoma Parameter Gender Female Male Age Years; median (range) Tumor location Right upper lobe Left upper lobe Right lower lobe Left lower lobe Right middle lobe Tumor size ≤3 >3 N status N0 N1 pT 1 2+3 TNM stage I II + III Histological grade 1+2 3 a *
Squamous cell carcinoma OPN H-scorea (mean ± SD)
N%
p value*
N%
17 (33) 35 (67)
9 (18) 40 (82)
64 (42–85)
64 (44–78)
11 (21) 17 (33) 8 (15) 13 (25) 3 (6)
10 (20) 16 (33) 8 (16) 10 (21) 5 (10)
OPN H-scorea (mean ± SD)
p value*
22 (43) 29 (57)
155 ± 64 147 ± 60
0.638
17 (35) 32 (65)
107 ± 63 103 ± 55
0.8
38 (73) 14 (27)
147 ± 59 159 ± 58
0.51
34 (69) 15 (31)
105 ± 57 102 ± 59
0.861
21 (41) 30 (59)
155 ± 61 148 ± 58
0.666
16 (33) 33 (67)
113 ± 59 99 ± 57
0.425
30 (59) 21 (41)
161 ± 60 137 ± 63
0.152
28 (57) 21 (43)
109 ± 56 97 ± 59
0.448
40 (77) 12 (23)
153 ± 60 142 ± 53
0.578
28 (57) 21 (43)
111 ± 52 94 ± 63
0.31
Histoscore. Student s t-test.
and difficult to compare. Zhang et al. (2001) showed predominant OPN expression in NSCLC compared to small cell lung cancer, with higher OPN expression in SCC compared to AC. Higher levels of OPN have been shown to correlate with higher grade (Jin et al., 2012; Wang et al., 2012), higher clinical stage, lymph node metastasis (Hu et al., 2005; Jin et al., 2012; Wang et al., 2012), and patient’s survival (Schneider et al., 2004; Boldrini et al., 2005; Donati et al., 2005). Furthermore, it has been recognized as one of the critical prometastatis genes for lung cancer (Wang et al., 2012). In our study, OPN expression did not correlate with clinical prognostic variables, and the same was shown in some other reports (Donati et al., 2005; Mack et al., 2008; Rud et al., 2012). It could be explained by the method we used, i.e. TMA sections may not be as representative as whole tissue sections for correlations with clinical parameters. Also, the scoring system is different, and also the antibody that was used. On the contrary, TMA proved to be a useful tool for analyzing the simultaneous expression of various proteins in the particular population of tumor cells, as well as for comparing proliferative or apoptotic activity with the particular level of OPN protein expression. Also, there are contradictory reports regarding the association between OPN expression and histological grades. We showed higher levels of OPN expression in lower histological grade, as shown by others (Donati et al., 2005; Frey et al., 2007). However, the opposite relationship (Boldrini et al., 2005; Jin
et al., 2012) or the lack of association (Hu et al., 2005) was also reported. Although a large body of evidence suggests that OPN participates in NSCLC progression, the mechanisms by which it exerts its protumoral role are still unknown. Shijubo et al. (2003) have reported that coexpression of OPN and VEGF is closely associated with angiogenesis and poor prognosis in stage 1 lung adenocarcinoma. Cui et al. (2009) showed that OPN is involved in the formation of malignant pleural effusion in lung cancer by promoting VEGF secretion. In this regard, from animal studies it has been shown that OPN plays a crucial role for tumor growth and angiogenesis of human lung cancer cells in vivo by interacting with ␣vb3 integrin (Cui et al., 2007). Thus, targeting the interaction between OPN and ␣vb3 integrin may be effective in lung cancer treatment. In vitro osteopontin enhanced extracellular matrix invasion of NSCLC cells, and an osteopontin antibody abolished this effect (Hu et al., 2005). Also, it has been shown that OPN induces NSCLC invasion and expression of MMP2 and MMP9 (Ni et al., 2009), increases migration of lung cancer cells via activation of ␣vb3 integrin and NF-kB-dependent pathway (Fong et al., 2009). In this study we showed that inhibition of apoptosis may be one of mechanisms by which OPN mediates lung AC progression. Adenocarcinoma samples with high levels of OPN expression showed low apoptotic ratio and vice versa. Interestingly, it was not true for
Table 3 The association between osteopontin expression and apoptotic activity in non-small cell lung carcinoma. Apoptotic index (median, range) OPN expression*
Non small cell lung carcinoma
Adenocarcinoma
Squamous cell carcinoma
Low High p value**
1.5 (0–5) 1.5 (0–8) 0.66
2.0 (0–5) 0.9 (0–3) 0.006
1.2 (0–4) 1.8 (0–6) 0.08
* **
Mean used as cut off value. Mann-Whitney test.
ˇ Please cite this article in press as: Stemberger C, et al. Osteopontin is associated with decreased apoptosis and ␣v integrin expression in lung adenocarcinoma. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.07.009
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6
6
A
5
250
4
Apoptotic activity
300
αV integrin expression
200
150
Median 25%-75% Min-Max
3
2
100
1
50
0
0
-1 low/ low
-50 -50
0
50
100
150
200
250
240 220 200
αV integrin expression
180 160 140 120 100 80 60 40 20 0
0
50
100
150
200
250
300
200
250
300
Osteopontin expression
C 240 220 200 180
αv integrin expression
high/ high
Fig. 4. Association between apoptotic activity of tumor cells and OPN-␣v integrin coexpression in lung adenocarcinoma: apoptotic rate is highest in the group of tumors that were negative for both proteins, and lowest in the group of tumors that were positive for both markers (Kruskal Wallis test, p < 0.01).
B
160 140 120 100 80 60 40 20 0 -20 -50
low/ high
300
Osteopontin expression
-20 -50
high/ low
Osteopontin / αv integrin coexpression
0
50
100
150
Osteopontin expression
Fig. 3. Correlation between immunohistochemical OPN and ␣v integrin expression in (A), NSCLC (p = 0.4); (B) lung adenocarcinoma (p = 0.048), and (C) stage 1 lung adenocarcinoma (Pierson’s correlation test, p = 0.013).
SCC, where also the lack of correlation with ␣v integrins was found. This points to the difference between the two major histological NSCLC types, which were historically considered as a single tumor, and even today they are not always analyzed as separate entities. From in vitro and animal studies, OPN is known as a survival factor that mediates cell apoptosis under certain pathological stresses (Stendal et al., 2004). OPN inhibits apoptosis in endothelial cells (Scatena et al., 1998), melanocytes (Geissinger et al., 2002) and cardiac fibroblasts (Zohar et al., 2004). However, little is known about the prosurvival role of OPN in human carcinoma tissue. In our previous study we showed an association between OPN expression, decreased apoptosis and NF-kB activation in renal-cell carcinoma (Matuˇsan-Ilijaˇs et al., 2011). Interestingly, in the work of Frey et al. (2007) an association between OPN expression and NF-kB activation was found in lung adenocarcinoma that could have the role in protecting cells from apoptosis. In the work of Polat et al. (2013) the influence of OPN silencing on survival and migration of lung carcinoma cells was shown. Previous studies have shown that OPN may prevent apoptosis through interaction with integrins (Scatena et al., 1998; Song et al., 2008; Zhao et al., 2008) and CD44 variants (Lin and Yang-Yen, 2001; Stendal et al., 2004). The downstream signaling pathways seem to differ and depend both on which receptors are engaged and on the cell type. Courter et al. (2010) showed in a mouse tumor model that the RGD domain of OPN mediates lung carcinoma growth and metastasis mainly through decreased apoptosis. Similar findings were noted in vitro, where wild type OPN, but not mutant RGD, protected cells from apoptosis triggered by hypoxia and serum starvation. This indicates a possible role of ␣v integrin receptors in downstream signaling, leading to apoptosis inhibition. In in vitro and animal studies of hepatocellular carcinoma it has been shown that OPN depletion decreased the expression of ␣v, 1 and 3 integrins (Zhao et al., 2008). Our results on the correlation between OPN and ␣v integrins in adenocarcinoma tumor samples are consistent with this finding. This correlation was particularly pronounced in early stage tumors, pointing to the possible role of OPN-␣v integrin interaction in the development of metastasis in patients who presented with localized disease. Whereas cancer is most often thought of as a disease that is caused by uncontrolled proliferation, apoptosis shares an equally, if not a more important role, in cancer progression and metastasis (Townson et al., 2003). Metastasis is a multistep process during which tumor cells are subjected to shear forces and immunological
ˇ Please cite this article in press as: Stemberger C, et al. Osteopontin is associated with decreased apoptosis and ␣v integrin expression in lung adenocarcinoma. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.07.009
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attack. Stimuli which prevent tumor cell apoptosis, not only allow tumor cells to complete a metastatic cascade and reach secondary tumor sites, but may also confer them with resistance to conventional anticancer therapy. In this regard, in an in vitro study OPN has been shown to induce chemoresistance by inhibiting apoptosis (Pang et al., 2011). Moreover, early clinical data also suggest that chemotherapy resistance in NSCLC may be increased with increased expression of OPN (Stewart, 2010). These results suggest that it could be reasonable to target OPN and/or its receptors in order to improve the outcome of lung carcinoma patients. Blocking of OPN activity is still in an experimental phase of investigation (Shevde et al., 2010), while ␣v integrins are already being targeted in clinics (Cox et al., 2010). In conclusion, our data show the difference between major NSCLC types with regard to the levels of OPN overexpression, association with apoptotic activity of tumor cells and with ␣v integrin expression. OPN overexpression in lung AC is associated with increased tumor cell survival and paralleled by increased expression of ␣v integrins. Thus, lung adenocarcinoma patients may benefit from targeting OPN and/or ␣v integrin receptor. Acknowledgements This work was supported by the Ministry of Science, Education, and Sports of the Republic of Croatia (grant 062-0620095-0078). ˇ We thank Mrs. Helga Frketic´ and Mr. Ozren Stanfel for their excellent technical support. References Bellahcène A, Castronovo V, Ogbureke KU, Fisher LW, Fedarko NS. Small integrin-binding ligand N-linked glycoproteins (SIBLINGs): multifunctional proteins in cancer. Nat Rev Cancer 2008;8:212–26. Blasberg JD, Goparaju CM, Pass HI, Donington S. Lung cancer osteopontin isoforms exhibit angiogenic functional heterogeneity. J Thor Cardiovasc Surg 2010;139:1587–93. Boldrini L, Donati V, Dell’Omodarme M, Prati MC, Faviana P, Camacci T, et al. Prognostic significance of osteopontin expression in early stage non-small cell lung cancer. Br J Cancer 2005;93:453–7. Courter D, Cao H, Kwok S, Kong C, Banh A, Kuo P, et al. The RGD domain of human osteopontin promotes tumor growth and metastasis through activation of survival pathways. PLoS ONE 2010;5(3):e9633. Cox D, Brennan M, Moran N. Integrins as therapeutic targets: lessons and opportunities. Nat Rev Drug Discov 2010;9:804–20. Cui R, Takahashi F, Ohashi R, Gu T, Yoshioka M, Nishio K, et al. Abrogation of the interaction between osteopontin and ␣v3 integrin reduces tumor growth of human lung cancer cells in mice. Lung Cancer 2007;57:302–10. Cui R, Takahashi F, Ohashi R, Yoshioka M, Gu T, Tajima K, et al. Osteopontin is involved in the formation of malignant pleural effusion in lung cancer. Lung Cancer 2009;63:368–74. Donati V, Boldrini L, Dell Omodarme M, Prati MC, Faviana P, Cammaci T, et al. Osteopontin expression and prognostic significance in non-small cell lung cancer. Clin Cancer Res 2005;11:6459–65. Fong YC, Liu SC, Huang CY, Li TM, Hsu SF, Kao ST, et al. Osteopontin increases lung cancer cell migration via activation of the ␣v3 integrin/FAK/Akt and NF-kB-dependent pathway. Lung Cancer 2009:263–70. Frey AB, Wali A, Pass H, Lonardo F. Osteopontin is linked to p65 and MMP-9 expression in pulmonary adenocarcinoma but not in malignant pleural mesothelioma. Histopathology 2007;50:720–6.
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Geissinger E, Weisser C, Fischer P, Schartl M, Wellbrock C. Autocrine stimulation by osteopontin contributes to antiapoptotic signalling of melanocytes in dermal collagen. Cancer Res 2002;62:4820–8. Giancotti FG, Ruoslahti E. Integrin signalling. Science 1999; 285(5430):1028–32. Hu Z, Lin D, Yuan J, Xiao T, Zhang H, Sun W, et al. Overexpression of osteopontin is associated with more aggressive phenotypes in human non-small cell lung cancer. Clin Cancer Res 2005;11:4646–52. Jin Y, Tong D, Chen J, Feng Z, Yang J, Shao C, et al. Overexpression of osteopontin, ␣v3 and Pim-1 associated with prognostically important clinicopathologic variables in non-small cell lung cancer. PLoS ONE 2012;7(10):e48575. Lin YH, Yang-Yen HF. The osteopontin-CD44 survival signal involves activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. J Biol Chem 2001;276:46024–30. Mack P, Redman MW, Chansky K, Williamson SK, Farneth NC, Lara PN Jr, et al. Lower osteopontin plasma levels are associated with superior outcomes in advanced non-small cell lung cancer patients receiving platinum-based chemotherapy: SWOG study S0003. J Clin Oncol 2008;26:4766–71. – c´ G, Hadˇzisejdic´ Matuˇsan-Ilijaˇs K, Damante G, Fabbro D, Dordevi I, Grahovac M, et al. Osteopontin expression correlates with nuclear factor-B activation and apoptosis downregulation in clear cell renal cell carcinoma. Pathol Res Pract 2011;207:104–10, 15. Ni Q, Ji H, Zhao Z, Fan X, Endostar Xu C. a modified endostatin inhibits non small cell lung cancer in vitro invasion through osteopontin-related mechanism. Mol Cell Pharm 2009;614: 1–6. Pang H, Cai L, Yang Y, Chen X, Sui G, Zhao C. Knockdown of osteopontin chemosensitizes MDA-MB-231 cells to cyclophosphamide by enhancing apoptosis through activating p38 MAPK pathway. Cancer Biother Radiopharm 2011;26:165–73. Polat B, Wohlleben G, Katzer A, Djuzenova CS, Technau A, Flentje M. Influence of osteopontin silencing on survival and migration of lung cancer cells. Strahlenther Onkol 2013;189:62–7. Rud AK, Lund-Iversen M, Berge G, Brustugun OT, Solberg SK, Mælandsmo G, et al. Expression of S100A4, ephrin-A1 and osteopontin in non-small lung cancer. BMC Cancer 2012;12: 333–42. Scatena M, Almeida M, Chaisson ML, Fausto N, Nicosia RF, Giachelli CM. NF-kappa mediates ␣v3 integrin-induced endothelial cell survival. J Cell Biol 1998;141:1083–93. Schneider S, Yochim JM, Brabender J, Uchida K, Danenberg KD, Metzger R, et al. Osteopontin but not osteonectin messenger RNA expression is a prognostic marker in curatively resected non-small cell lung cancer. Clin Cancer Res 2004;10: 1588–96. Shevde LA, Das S, Clark DW, Samant RS. Osteopontin: an effector and an effect of tumor metastasis. Curr Molecular Med 2010;10:71–81. Shijubo N, Kojima H, Nagata M, Ohchi T, Suzuki A, Abe S, et al. Tumor angiogenesis of non-small cell lung cancer. Microsc Res Tech 2003;60:186–98. Song G, Ming Y, Mao Y, Bao S, Ouyang G. Osteopontin prevents curcumin-induced apoptosis and promotes survival through Akt activation via ␣v3 integrins in human gastric cancer cells. Exp Biol Med 2008;233:1537–45. Stendal T, Borset M, Sundan A. Role of osteopontin in adhesion, migration, cell survival and bone remodeling. Exp Oncol 2004;26:179–84. Stewart DJ. Tumor and host factors that may limit efficacy of chemotherapy in non-small cell and small cell lung cancer. Crit Rev Oncol Hematol 2010;75:173–234.
ˇ Please cite this article in press as: Stemberger C, et al. Osteopontin is associated with decreased apoptosis and ␣v integrin expression in lung adenocarcinoma. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.07.009
G Model ACTHIS-50750; No. of Pages 8 8
ARTICLE IN PRESS C. Sˇ temberger et al. / Acta Histochemica xxx (2013) xxx–xxx
Townson JL, Naumov GN, Chambers AF. The role of apoptosis in tumor progression and metastasis. Curr Mol Med 2003;3: 631–42. Wai PY, Kuo PC. Osteopontin: regulation in tumor metastasis. Cancer Metastasis Rev 2008;10:3–18. Wang XM, Li J, Yan MX, Liu L, Jia DS, Geng Q, et al. Integrative analyses identify osteopontin, LAMB3 and ITGB1 as critical prometastatic genes for lung cancer. PLoS ONE 2012;8(2):e5571. Weber GF, Lett GS, Haubein NC. Osteopontin is a marker for cancer aggressiveness and patient survival. Br J Cancer 2010;103:861–9.
Zhang J, Takahashi K, Takahashi F, Shimizu K, Ohsita F, Kameda Y, et al. Differential osteopontin expression in lung cancer. Cancer Lett 2001;171:215–22. Zhao J, Dong L, Wu G, Xu D, Chen J, Li K, et al. Down-regulation of osteopontin suppresses growth and metastasis of hepatocellular carcinoma via induction of apoptosis. Gastroenterology 2008;135:956–68. Zohar R, Zhu B, Liu Sodek J, McCulloch CA. Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase3-independent pathway. Am J Physiol Heart Circ Physiol 2004;287:1730–9.
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Osteopontin is associated with decreased apoptosis and ␣v integrin expression in lung adenocarcinoma a ˇ Christophe Stemberger , Koviljka Matuˇsan-Ilijaˇs b , Manuela Avirovic´ b , c Ljiljana Bulat-Kardum , Aldo Ivanˇcic´ d , Nives Jonjic´ b , Ksenija Luˇcin b,∗ a
Department of Clinical Cytology, Clinical Hospital Center Rijeka, Rijeka, Croatia Department of Pathology, Rijeka University School of Medicine, Rijeka, Croatia c Department of Internal Medicine, Clinical Hospital Center Rijeka, Rijeka, Croatia d Department of Thoracovascular Surgery, Clinical Hospital Center Rijeka, Rijeka, Croatia b
a r t i c l e
i n f o
Article history: Received 29 May 2013 Received in revised form 12 July 2013 Accepted 14 July 2013 Available online xxx Keywords: Lung carcinoma Osteopontin ␣v Integrin Tissue array analysis Immunohistochemistry Apoptosis
a b s t r a c t Osteopontin (OPN) is a glycoprotein involved in invasion, progression and metastasis of many carcinomas. It contains several functional domains including binding sites for ␣v integrins, cell surface molecules playing a major role in mediating cell migration and adhesion. The aim of the study was to evaluate the expression of osteopontin in human non-small cell lung cancer (NSCLC) and to determine its possible prognostic significance as well as relation to apoptosis and ␣v integrin expression. We analyzed 111 surgically resected NSCLC for immunohistochemical expression of OPN and ␣v integrin. OPN expression was compared to apoptotic rate and clinicopathological parameters such as tumor size, histological grade, lymph node status, pT, and TNM stage. Apoptotic rate was measured by TUNEL staining method. OPN expression in NSCLC was significantly higher in lung adenocarcinomas (AC) then in squamous cell carcinomas (p < 0.001). There was no correlation between OPN expression and clinicopathological parameters. The level of OPN expression in AC was associated with decreased apoptotic activity of tumor cells (p = 0.006), and correlated with ␣v integrin expression (p = 0.048), particularly in low stage tumors (p = 0.013). Prolonged tumor cell survival in lung AC due to OPN and ␣v integrin overexpression may have an impact on tumor progression and resistance to therapy. © 2013 Elsevier GmbH. All rights reserved.
Introduction Lung cancer is one of the most common human malignancies worldwide and a major cause of death in many developed countries. Surgical resection continues to play an important role in the treatment of this disease, especially during the early stages. However, many patients with presumed localized disease have occult metastases at the time of diagnosis and suffer from disease recurrence. Since the current clinicopathological staging system is unreliable in predicting the outcome of the disease, new biomarkers are needed for disease monitoring and predicting prognosis of patients with lung cancer, as well as for planning new treatment strategies. One of the markers recently linked to cancer development, progression and metastasis in many types of human tumors is a multifunctional protein named osteopontin (OPN). OPN is a member of the small integrin binding ligand N-linked glycoproteins (Bellahcène et al., 2008). It is involved in a variety of
∗ Corresponding author at: Department of Pathology, Rijeka University School of ´ Branchetta 20, 51 000 Rijeka, Croatia. Medicine, Brace E-mail address: [email protected] (K. Luˇcin).
physiological and pathological processes, including invasion, progression and metastasis of cancer (Wai and Kuo, 2008). It is synthesized by a variety of cell types including fibroblasts, osteoblasts, osteocytes, dendritic cells, macrophages, smooth muscle cells, endothelial cells and some epithelial cells. The main functional domains of OPN consist of an RGD motif, which binds to ␣v and ␣51 integrins, and a thrombin cleaved epitope SVVYGLR (OPN-R, a non-RGD domain), which binds to integrin receptors ␣41, ␣91, and ␣94, present on a number of immune cells such as macrophages, neutrophils and T lymphocytes. The hyaluronan receptor, CD44, has also been identified as a receptor for OPN. Binding of OPN to these cell surface receptors stimulates cell adhesion, migration, and specific signaling functions resulting in proliferation and increased cell survival. It has been shown that increased OPN expression levels correlate with enhanced malignancy and poor prognosis in several malignant tumors, including non-small cell lung carcinoma (NSCLC) (Weber et al., 2010). Moreover, it has been shown that OPN has value as a clinical tumor progression marker (Weber et al., 2010). Considerable evidence suggests that OPN participates in lung carcinogenesis, but the mechanisms involved in lung cancer progression are largely unknown (Boldrini et al., 2005; Donati et al., 2005; Hu et al., 2005; Blasberg et al., 2010; Jin et al.,
0065-1281/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.acthis.2013.07.009
ˇ Please cite this article in press as: Stemberger C, et al. Osteopontin is associated with decreased apoptosis and ␣v integrin expression in lung adenocarcinoma. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.07.009
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2012). OPN has long been known to be able to bind to some cell surface integrins, which provide clues to the possible functional role of OPN in cancer. From animal studies it has been reported that the integrinbinding RGD domain of human osteopontin promotes lung carcinoma growth and metastasis through activation of survival pathways, while cell–cell adhesion was not affected (Courter et al., 2010). Integrins are transmembrane proteins that mediate interactions between adhesion molecules on adjacent cells and/or the extracellular matrix (ECM) (Giancotti and Ruoslahti, 1999). They have diverse roles in several biological processes including cell migration during development and wound healing, cell differentiation and apoptosis. Their activities can also regulate the metastatic and invasive potential of tumor cells. They exist as heterodimers consisting of alpha and beta subunits. These various integrins recognize the arginine-glycine-aspartic acid (RGD) peptide sequence found in many ECM proteins, including osteopontin. The current study was undertaken to investigate the expression of osteopontin in resected human NSCLC in order to assess its prognostic significance, relation to cell survival and to the expression of ␣v integrins. We show that OPN is overexpressed in NSCLC, with lung AC showing higher level of OPN overexpression compared to SCC. In adenocarcinoma, OPN overexpression was associated with prolonged cellular survival and correlated with ␣v integrins. A better understanding of the implications of OPN in lung tumorigenesis might facilitate development of new therapeutic regimens to benefit patients with these malignancies. Materials and methods Tumor samples
Table 1 Osteopontin expression in relation to clinicopathological characteristics of nonsmall cell lung carcinoma patients. Parameter
Gender Female Male Age Years; median (range) Tumor location Right upper lobe Left upper lobe Right lower lobe Left lower lobe Right middle lobe Tumor size (cm) ≤3 >3 N status N0 N1 pT 1 2+3 TNM stage I II + III Histological type Adenocarcinoma Squamous cell carcinoma Large cell carcinoma Histological grade 1+2 3 a *
We examined 111 consecutive lobectomy specimens with NSCLC diagnosis confirmed at the Department of Pathology, University of Rijeka, from January 2001 to December 2011. Clinicopathological characteristics are shown in Table 1. The age of patients ranged from 42 to 85 years (mean 63.5 years), among which 80 were male and 31 female. None of the patients had previous or synchronous cancer. The surgically resected specimens were fixed in 10% buffered formalin and embedded in paraffin. All sections were stained with hematoxylin and eosin (H&E). The slides were reviewed and classified according to WHO (2004) diagnostic criteria, with adenocarcinoma, squamous cell carcinoma and large cell carcinoma as the three main types. The histological types of cancer included 52 adenocarcinomas, 49 squamous cell carcinomas and 10 large cell carcinomas. The tumors were staged according to UICC criteria (2010). The study was approved by the Ethics Committee of Clinical Hospital Center Rijeka and Rijeka University School of Medicine. Tissue microarray (TMA) construction Hematoxylin and eosin-stained sections of tumors were used to mark tumor areas of interest. Four tissue cores, each 2 mm in diameter, were placed into a recipient paraffin block using a manual tissue arrayer (Alphelys, Plaisir, France). Normal liver tissue was used for orientation. Cores were spaced at intervals of 0.5 mm in the x and y axes. One section from each TMA block was stained with hematoxylin and eosin for morphological assessment. Serial sections were cut from TMA blocks for immunohistochemical and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) staining. Five m thick sections were placed on adhesive glass slides (Capillary Gap Microscope Slides, 75 m, Code S2024, DakoCytomation, Glostrup, Denmark), left to dry at 37 ◦ C overnight and stored in the dark at +4 ◦ C.
#
N (%)
OPN H-scorea (mean ± SD)
p value*
31 (28) 80 (72) 64 (42–85) 26 (23) 33 (30) 21 (19) 23 (21) 8 (7) 43 (39) 67 (61)
132 ± 64 125 ± 60
0.551
79 (71) 32 (29)
126 ± 61 132 ± 63
0.599
71 (64) 40 (36)
137 ± 61 123 ± 62
0.270
63 (57) 48 (43)
134 ± 60 120 ± 63
0.225
52 (47) 49 (44) 10 (9)
150 ± 58 104 ± 57 134 ± 59
68 (61) 43 (39)
160 ± 63 127 ± 59
<0.001#
0.052
Histoscore. Student’s t-test. p value – adenocarcinoma versus squamous cell carcinoma.
Immunohistochemistry Tumor samples were processed for immunohistological analysis to determinate OPN and ␣v integrin expression. Following deparaffinization in xylene and rehydration in alcohols, heat-induced epitope retrieval was achieved. Antigen retrieval for anti-OPN antibody (clone OPN3, Novocastra, Newcastle upon Tyne, UK, dilution 1:100) was achieved by immersing slides in Tris-EDTA buffer (pH 9.0) and boiling for 10 min in a water bath. For ␣v integrin staining (clone P2W7; Santa Cruz Biotechnology, Santa Cruz, CA, USA, dilution 1:25), we employed antigen retrieval using proteinase K (DakoCytomation, Glostrup, Denmark). Following blockage of non-specific antibody binding, sections were incubated with primary antibodies against OPN and ␣v integrin human epitopes. Immunohistochemistry was performed with Dako Autostainer Plus (DakoCytomation Colorado Inc., Fort Collins, CO, USA) according to the manufacturer’s protocol, using the Envision peroxidase procedure. All reagents for automated immunohistochemistry were purchased from DakoCytomation (ChemMate TM Envision HRP detection Kit K5007). Negative control slides were prepared by substituting the primary antibody with Dako ChemMate antibody diluent. Slides were counterstained with Mayer’s hematoxylin, dehydrated and mounted. TUNEL The TUNEL method was used to detect DNA strand breaks generated during apoptotic cell death. Following deparaffinization in xylene and rehydration in alcohols, heat-induced epitope retrieval was achieved by immersing slides in Tris-EDTA buffer (pH 9.0) and boiling for 5 min in a water bath. Slides were allowed to cool
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during 20 min, and then pre-incubated with normal bovine serum (3%) for 20 min. For TUNEL – alkaline phosphatase staining, In Situ Cell Death Detection Kit (Cat. No. 11684809910, Roche Applied Science, Indianapolis, IN, USA) was used at 37 ◦ C in the dark. Terminal deoxynucleotidyl transferase was diluted 1:5 using TUNEL Dilution Buffer (Cat. No. 1966006; Roche, Mannheim, Germany). Sections were incubated with TUNEL reaction mixture for 60 min. Subsequently, alkaline phosphatase-conjugated sheep antibody was added for 30 min followed by fast red chromogen (Fast Red tablets, Cat. No. 1496549; Roche, Mannheim, Germany) for 20 min. Slides were counterstained with Mayer’s hematoxylin, dehydrated and mounted. Two negative control slides were prepared by substituting TUNEL reaction mixture with nucleotide mixture and phosphate buffered saline, respectively. Positive controls were TUNEL positive cells displaying morphological features of apoptosis. Evaluation of immunohistochemistry and TUNEL staining Two observers, blinded to clinical data, evaluated the staining scores independently. The mean values of the OPN staining intensity were graded semi-quantitatively using a histological score (H-score) according to the following equation: H-score = Pxi1–3 , where i is the intensity of staining with a value of 1, 2 or 3 (mild, moderate and strong staining), and P is the percentage of cells stained with particular intensity, varying between 1% and 100%. For statistical purpose, OPN and ␣v integrin H-score was divided into low and high group, with a mean H-score of 150 and H-score of 150, used as cut off value, respectively. Avoiding necrotic areas, the percentage of apoptotic cells was evaluated by counting positive staining of 1000 tumor cells in tumor areas with the highest density of positive cells using a high power objective, and was determined as apoptotic index (AI). Positive apoptotic bodies and positive extracellular chromatin fragments were considered as positive staining. Statistical analysis Statistical analysis was performed using Statistica 7 software (StatSoft, Inc., Tulsa, OK, USA). The mean values of continuous data with normal distribution (OPN H-score) were compared by Student’s t-test. For variable without normal distribution (apoptotic activity) non-parametric tests were used. A difference between two groups was calculated using the Mann Whitney test, while Kruskal–Wallis test was used for comparing the quality scores between 4 groups. The correlation between two continuous variables (OPN and ␣v integrin) was measured by Pearson’s correlation test. Statistical differences with p value less than 0.05 were considered significant. Results Expression of OPN in normal lung tissue and NSCLC In normal lung tissue OPN was observed in epithelial cells of the bronchial mucous glands and as a diffuse cytoplasmic staining in alveolar macrophages. Bronchial and alveolar epithelial cells were generally negative or showed focal, faint positivity, which was more intense along the luminal surface. In lung carcinoma, OPN staining was observed in the cytoplasm of tumor cells in the form of granules of various sizes and staining intensity. The staining pattern was heterogeneous and ranged from staining of small number of tumor cells with low intensity (Fig. 1A) to strong and diffuse positivity throughout the TMA sections (Fig. 1C). There were few OPN negative TMAs, but none of examined tumors was completely OPN negative. The OPN H-score values are shown in Table 1. The mean OPN H-score for the whole NSCLC group was 128 (range 1.5–290).
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OPN staining was higher in AC (mean 150, range 5–160) compared to SCC (mean 104, range 1.5–234) and this was statistically significant (p < 0.001). The OPN H-score for large cell carcinoma was in between (mean 130, range 30–205).
Expression of OPN in relation to clinicopathological parameters When OPN H-score was compared to usual clinicopathological parameters, we could not find any significant associations in the NSCLC group. The results are shown in Table 1. We only found a trend toward a significant association between higher OPN level and lower histological grade (p = 0.052). There was no significant difference in the level of OPN expression between small (<3 cm) and large (>3 cm) tumors (p = 0.551), between lymph node negative and positive tumors (p = 0.599), between pT1 and pT2/3 tumors (p = 0.27), and between TNM stage 1 and stage 2/3 tumors (p = 0.225). We obtained similar results when AC and SCC were analyzed separately (Table 2). There was no difference in the level of OPN expression with regard to tumor size (p = 0.638 and 0.8, respectively), lymph node status (p = 0.51 and 0.861, respectively), pT status (0.666 and 0.425, respectively), and TNM stage (p = 0.152 and 0.448, respectively. Also, when only major histological subtypes where considered, without large cell subtype, a trend toward the association between higher OPN expression and lower histological grade was lost (p = 0.578 and p = 0.31, respectively).
Expression of OPN in relation to apoptotic index Apoptotic activity of tumor cells for the whole NSCLC group ranged from 0% to 8%, mean 1.5%. There was no statistically different apoptotic activity between NSCLC tumors with low or high OPN expression (p = 0.66, Table 3). However, when analyzed separately, we found an association between OPN expression and apoptotic activity of tumor cells in adenocarcinoma. Statistically higher apoptotic activity was observed in tumors with low OPN expression and vice versa (p = 0.006, Fig. 2). In SCC there was only a trend toward the association between OPN and apoptotic activity, although in the opposite direction (p = 0.08).
Expression of ˛v integrins in normal lung tissue and NSCLC In normal lung tissue there was no expression of ␣v integrins. In lung carcinoma, ␣v integrin showed a membranous staining pattern (Fig. 1E and F). The staining of fibroblasts, endothelial cells, lymphocytes and macrophages was also observed. The mean value of membranous ␣v integrin H-score for all NSCLC sample was 132 (range 0–283). AC showed lower ␣v integrin expression compared to SCC (mean 97; range 0.2–225, and mean 176, range 3–283, respectively). The level of ␣v integrin expression was statistically different between two major histological types (p < 0.001) (data not shown).
Correlation of OPN and ˛v integrin expression There was no correlation between OPN and ␣v integrin expression when considering all NSCLC samples (Pearson correlation test, r = −0.08, p = 0.4), as well as SC samples (r = −0.06, p = 0.66). In the AC group we found a weak correlation between OPN and ␣v integrin (r = 0.28, p = 0.048), and a strong correlation in the group of patients with lower clinical stage AC (r = 0.45, p = 0.013). The results are shown in Fig. 3.
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Fig. 1. Immunohistochemical and TUNEL staining in lung adenocarcinoma. (A) Adenocarcinoma sample with weak cytoplasmic OPN staining followed by (B) high apoptotic activity of tumor cells. (C) Adenocarcinoma sample with strong OPN staining and (D) low apoptotic activity of tumor cells. (E) ␣v integrin expression with faint apical membranous staining and (F) strong membranous ␣v integrin expression.
Association between OPN, ˛v integrin expression and apoptosis
6 Median 25%-75% Min-Max
Apoptotic activity
5
4
3
2
When the AC tumor samples were divided into four groups based on a level of OPN and ␣v integrin expression (both positive, both negative or only one marker positive) and compared to the apoptotic index, we found a significant difference between groups (Fig. 4). Namely, the highest apoptotic rate was observed in the group of AC samples that were negative for both proteins, while in the group of tumors that were positive for both markers, the apoptotic rate had the lowest value (Kruskal Wallis test, p < 0.01).
1
Discussion 0
-1
Low
High
Osteopontin expression Fig. 2. Osteopontin expression in lung adenocarcinoma according to apoptotic activity of tumor cells: low apoptotic activity is associated with high OPN expression and vice versa (Mann Whitney test, p = 0.006).
In the current study we showed the upregulation of OPN in NSCLC and different OPN expression among major NSCLC subtypes. Also, we showed an association between the higher level of OPN expression and lower apoptotic rate in lung adenocarcinoma, which correlated with the ␣v integrin expression. There are several reports on the role of OPN in lung carcinoma progression however, the results are somehow contradictory
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Table 2 Osteopontin expression in relation to clinicopathological characteristics of adenocarcinoma and squamous cell carcinoma patients. Adenocarcinoma Parameter Gender Female Male Age Years; median (range) Tumor location Right upper lobe Left upper lobe Right lower lobe Left lower lobe Right middle lobe Tumor size ≤3 >3 N status N0 N1 pT 1 2+3 TNM stage I II + III Histological grade 1+2 3 a *
Squamous cell carcinoma OPN H-scorea (mean ± SD)
N%
p value*
N%
17 (33) 35 (67)
9 (18) 40 (82)
64 (42–85)
64 (44–78)
11 (21) 17 (33) 8 (15) 13 (25) 3 (6)
10 (20) 16 (33) 8 (16) 10 (21) 5 (10)
OPN H-scorea (mean ± SD)
p value*
22 (43) 29 (57)
155 ± 64 147 ± 60
0.638
17 (35) 32 (65)
107 ± 63 103 ± 55
0.8
38 (73) 14 (27)
147 ± 59 159 ± 58
0.51
34 (69) 15 (31)
105 ± 57 102 ± 59
0.861
21 (41) 30 (59)
155 ± 61 148 ± 58
0.666
16 (33) 33 (67)
113 ± 59 99 ± 57
0.425
30 (59) 21 (41)
161 ± 60 137 ± 63
0.152
28 (57) 21 (43)
109 ± 56 97 ± 59
0.448
40 (77) 12 (23)
153 ± 60 142 ± 53
0.578
28 (57) 21 (43)
111 ± 52 94 ± 63
0.31
Histoscore. Student s t-test.
and difficult to compare. Zhang et al. (2001) showed predominant OPN expression in NSCLC compared to small cell lung cancer, with higher OPN expression in SCC compared to AC. Higher levels of OPN have been shown to correlate with higher grade (Jin et al., 2012; Wang et al., 2012), higher clinical stage, lymph node metastasis (Hu et al., 2005; Jin et al., 2012; Wang et al., 2012), and patient’s survival (Schneider et al., 2004; Boldrini et al., 2005; Donati et al., 2005). Furthermore, it has been recognized as one of the critical prometastatis genes for lung cancer (Wang et al., 2012). In our study, OPN expression did not correlate with clinical prognostic variables, and the same was shown in some other reports (Donati et al., 2005; Mack et al., 2008; Rud et al., 2012). It could be explained by the method we used, i.e. TMA sections may not be as representative as whole tissue sections for correlations with clinical parameters. Also, the scoring system is different, and also the antibody that was used. On the contrary, TMA proved to be a useful tool for analyzing the simultaneous expression of various proteins in the particular population of tumor cells, as well as for comparing proliferative or apoptotic activity with the particular level of OPN protein expression. Also, there are contradictory reports regarding the association between OPN expression and histological grades. We showed higher levels of OPN expression in lower histological grade, as shown by others (Donati et al., 2005; Frey et al., 2007). However, the opposite relationship (Boldrini et al., 2005; Jin
et al., 2012) or the lack of association (Hu et al., 2005) was also reported. Although a large body of evidence suggests that OPN participates in NSCLC progression, the mechanisms by which it exerts its protumoral role are still unknown. Shijubo et al. (2003) have reported that coexpression of OPN and VEGF is closely associated with angiogenesis and poor prognosis in stage 1 lung adenocarcinoma. Cui et al. (2009) showed that OPN is involved in the formation of malignant pleural effusion in lung cancer by promoting VEGF secretion. In this regard, from animal studies it has been shown that OPN plays a crucial role for tumor growth and angiogenesis of human lung cancer cells in vivo by interacting with ␣vb3 integrin (Cui et al., 2007). Thus, targeting the interaction between OPN and ␣vb3 integrin may be effective in lung cancer treatment. In vitro osteopontin enhanced extracellular matrix invasion of NSCLC cells, and an osteopontin antibody abolished this effect (Hu et al., 2005). Also, it has been shown that OPN induces NSCLC invasion and expression of MMP2 and MMP9 (Ni et al., 2009), increases migration of lung cancer cells via activation of ␣vb3 integrin and NF-kB-dependent pathway (Fong et al., 2009). In this study we showed that inhibition of apoptosis may be one of mechanisms by which OPN mediates lung AC progression. Adenocarcinoma samples with high levels of OPN expression showed low apoptotic ratio and vice versa. Interestingly, it was not true for
Table 3 The association between osteopontin expression and apoptotic activity in non-small cell lung carcinoma. Apoptotic index (median, range) OPN expression*
Non small cell lung carcinoma
Adenocarcinoma
Squamous cell carcinoma
Low High p value**
1.5 (0–5) 1.5 (0–8) 0.66
2.0 (0–5) 0.9 (0–3) 0.006
1.2 (0–4) 1.8 (0–6) 0.08
* **
Mean used as cut off value. Mann-Whitney test.
ˇ Please cite this article in press as: Stemberger C, et al. Osteopontin is associated with decreased apoptosis and ␣v integrin expression in lung adenocarcinoma. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.07.009
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6
6
A
5
250
4
Apoptotic activity
300
αV integrin expression
200
150
Median 25%-75% Min-Max
3
2
100
1
50
0
0
-1 low/ low
-50 -50
0
50
100
150
200
250
240 220 200
αV integrin expression
180 160 140 120 100 80 60 40 20 0
0
50
100
150
200
250
300
200
250
300
Osteopontin expression
C 240 220 200 180
αv integrin expression
high/ high
Fig. 4. Association between apoptotic activity of tumor cells and OPN-␣v integrin coexpression in lung adenocarcinoma: apoptotic rate is highest in the group of tumors that were negative for both proteins, and lowest in the group of tumors that were positive for both markers (Kruskal Wallis test, p < 0.01).
B
160 140 120 100 80 60 40 20 0 -20 -50
low/ high
300
Osteopontin expression
-20 -50
high/ low
Osteopontin / αv integrin coexpression
0
50
100
150
Osteopontin expression
Fig. 3. Correlation between immunohistochemical OPN and ␣v integrin expression in (A), NSCLC (p = 0.4); (B) lung adenocarcinoma (p = 0.048), and (C) stage 1 lung adenocarcinoma (Pierson’s correlation test, p = 0.013).
SCC, where also the lack of correlation with ␣v integrins was found. This points to the difference between the two major histological NSCLC types, which were historically considered as a single tumor, and even today they are not always analyzed as separate entities. From in vitro and animal studies, OPN is known as a survival factor that mediates cell apoptosis under certain pathological stresses (Stendal et al., 2004). OPN inhibits apoptosis in endothelial cells (Scatena et al., 1998), melanocytes (Geissinger et al., 2002) and cardiac fibroblasts (Zohar et al., 2004). However, little is known about the prosurvival role of OPN in human carcinoma tissue. In our previous study we showed an association between OPN expression, decreased apoptosis and NF-kB activation in renal-cell carcinoma (Matuˇsan-Ilijaˇs et al., 2011). Interestingly, in the work of Frey et al. (2007) an association between OPN expression and NF-kB activation was found in lung adenocarcinoma that could have the role in protecting cells from apoptosis. In the work of Polat et al. (2013) the influence of OPN silencing on survival and migration of lung carcinoma cells was shown. Previous studies have shown that OPN may prevent apoptosis through interaction with integrins (Scatena et al., 1998; Song et al., 2008; Zhao et al., 2008) and CD44 variants (Lin and Yang-Yen, 2001; Stendal et al., 2004). The downstream signaling pathways seem to differ and depend both on which receptors are engaged and on the cell type. Courter et al. (2010) showed in a mouse tumor model that the RGD domain of OPN mediates lung carcinoma growth and metastasis mainly through decreased apoptosis. Similar findings were noted in vitro, where wild type OPN, but not mutant RGD, protected cells from apoptosis triggered by hypoxia and serum starvation. This indicates a possible role of ␣v integrin receptors in downstream signaling, leading to apoptosis inhibition. In in vitro and animal studies of hepatocellular carcinoma it has been shown that OPN depletion decreased the expression of ␣v, 1 and 3 integrins (Zhao et al., 2008). Our results on the correlation between OPN and ␣v integrins in adenocarcinoma tumor samples are consistent with this finding. This correlation was particularly pronounced in early stage tumors, pointing to the possible role of OPN-␣v integrin interaction in the development of metastasis in patients who presented with localized disease. Whereas cancer is most often thought of as a disease that is caused by uncontrolled proliferation, apoptosis shares an equally, if not a more important role, in cancer progression and metastasis (Townson et al., 2003). Metastasis is a multistep process during which tumor cells are subjected to shear forces and immunological
ˇ Please cite this article in press as: Stemberger C, et al. Osteopontin is associated with decreased apoptosis and ␣v integrin expression in lung adenocarcinoma. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.07.009
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attack. Stimuli which prevent tumor cell apoptosis, not only allow tumor cells to complete a metastatic cascade and reach secondary tumor sites, but may also confer them with resistance to conventional anticancer therapy. In this regard, in an in vitro study OPN has been shown to induce chemoresistance by inhibiting apoptosis (Pang et al., 2011). Moreover, early clinical data also suggest that chemotherapy resistance in NSCLC may be increased with increased expression of OPN (Stewart, 2010). These results suggest that it could be reasonable to target OPN and/or its receptors in order to improve the outcome of lung carcinoma patients. Blocking of OPN activity is still in an experimental phase of investigation (Shevde et al., 2010), while ␣v integrins are already being targeted in clinics (Cox et al., 2010). In conclusion, our data show the difference between major NSCLC types with regard to the levels of OPN overexpression, association with apoptotic activity of tumor cells and with ␣v integrin expression. OPN overexpression in lung AC is associated with increased tumor cell survival and paralleled by increased expression of ␣v integrins. Thus, lung adenocarcinoma patients may benefit from targeting OPN and/or ␣v integrin receptor. Acknowledgements This work was supported by the Ministry of Science, Education, and Sports of the Republic of Croatia (grant 062-0620095-0078). ˇ We thank Mrs. Helga Frketic´ and Mr. Ozren Stanfel for their excellent technical support. References Bellahcène A, Castronovo V, Ogbureke KU, Fisher LW, Fedarko NS. Small integrin-binding ligand N-linked glycoproteins (SIBLINGs): multifunctional proteins in cancer. Nat Rev Cancer 2008;8:212–26. Blasberg JD, Goparaju CM, Pass HI, Donington S. Lung cancer osteopontin isoforms exhibit angiogenic functional heterogeneity. J Thor Cardiovasc Surg 2010;139:1587–93. Boldrini L, Donati V, Dell’Omodarme M, Prati MC, Faviana P, Camacci T, et al. Prognostic significance of osteopontin expression in early stage non-small cell lung cancer. Br J Cancer 2005;93:453–7. Courter D, Cao H, Kwok S, Kong C, Banh A, Kuo P, et al. The RGD domain of human osteopontin promotes tumor growth and metastasis through activation of survival pathways. PLoS ONE 2010;5(3):e9633. Cox D, Brennan M, Moran N. Integrins as therapeutic targets: lessons and opportunities. Nat Rev Drug Discov 2010;9:804–20. Cui R, Takahashi F, Ohashi R, Gu T, Yoshioka M, Nishio K, et al. Abrogation of the interaction between osteopontin and ␣v3 integrin reduces tumor growth of human lung cancer cells in mice. Lung Cancer 2007;57:302–10. Cui R, Takahashi F, Ohashi R, Yoshioka M, Gu T, Tajima K, et al. Osteopontin is involved in the formation of malignant pleural effusion in lung cancer. Lung Cancer 2009;63:368–74. Donati V, Boldrini L, Dell Omodarme M, Prati MC, Faviana P, Cammaci T, et al. Osteopontin expression and prognostic significance in non-small cell lung cancer. Clin Cancer Res 2005;11:6459–65. Fong YC, Liu SC, Huang CY, Li TM, Hsu SF, Kao ST, et al. Osteopontin increases lung cancer cell migration via activation of the ␣v3 integrin/FAK/Akt and NF-kB-dependent pathway. Lung Cancer 2009:263–70. Frey AB, Wali A, Pass H, Lonardo F. Osteopontin is linked to p65 and MMP-9 expression in pulmonary adenocarcinoma but not in malignant pleural mesothelioma. Histopathology 2007;50:720–6.
7
Geissinger E, Weisser C, Fischer P, Schartl M, Wellbrock C. Autocrine stimulation by osteopontin contributes to antiapoptotic signalling of melanocytes in dermal collagen. Cancer Res 2002;62:4820–8. Giancotti FG, Ruoslahti E. Integrin signalling. Science 1999; 285(5430):1028–32. Hu Z, Lin D, Yuan J, Xiao T, Zhang H, Sun W, et al. Overexpression of osteopontin is associated with more aggressive phenotypes in human non-small cell lung cancer. Clin Cancer Res 2005;11:4646–52. Jin Y, Tong D, Chen J, Feng Z, Yang J, Shao C, et al. Overexpression of osteopontin, ␣v3 and Pim-1 associated with prognostically important clinicopathologic variables in non-small cell lung cancer. PLoS ONE 2012;7(10):e48575. Lin YH, Yang-Yen HF. The osteopontin-CD44 survival signal involves activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. J Biol Chem 2001;276:46024–30. Mack P, Redman MW, Chansky K, Williamson SK, Farneth NC, Lara PN Jr, et al. Lower osteopontin plasma levels are associated with superior outcomes in advanced non-small cell lung cancer patients receiving platinum-based chemotherapy: SWOG study S0003. J Clin Oncol 2008;26:4766–71. – c´ G, Hadˇzisejdic´ Matuˇsan-Ilijaˇs K, Damante G, Fabbro D, Dordevi I, Grahovac M, et al. Osteopontin expression correlates with nuclear factor-B activation and apoptosis downregulation in clear cell renal cell carcinoma. Pathol Res Pract 2011;207:104–10, 15. Ni Q, Ji H, Zhao Z, Fan X, Endostar Xu C. a modified endostatin inhibits non small cell lung cancer in vitro invasion through osteopontin-related mechanism. Mol Cell Pharm 2009;614: 1–6. Pang H, Cai L, Yang Y, Chen X, Sui G, Zhao C. Knockdown of osteopontin chemosensitizes MDA-MB-231 cells to cyclophosphamide by enhancing apoptosis through activating p38 MAPK pathway. Cancer Biother Radiopharm 2011;26:165–73. Polat B, Wohlleben G, Katzer A, Djuzenova CS, Technau A, Flentje M. Influence of osteopontin silencing on survival and migration of lung cancer cells. Strahlenther Onkol 2013;189:62–7. Rud AK, Lund-Iversen M, Berge G, Brustugun OT, Solberg SK, Mælandsmo G, et al. Expression of S100A4, ephrin-A1 and osteopontin in non-small lung cancer. BMC Cancer 2012;12: 333–42. Scatena M, Almeida M, Chaisson ML, Fausto N, Nicosia RF, Giachelli CM. NF-kappa mediates ␣v3 integrin-induced endothelial cell survival. J Cell Biol 1998;141:1083–93. Schneider S, Yochim JM, Brabender J, Uchida K, Danenberg KD, Metzger R, et al. Osteopontin but not osteonectin messenger RNA expression is a prognostic marker in curatively resected non-small cell lung cancer. Clin Cancer Res 2004;10: 1588–96. Shevde LA, Das S, Clark DW, Samant RS. Osteopontin: an effector and an effect of tumor metastasis. Curr Molecular Med 2010;10:71–81. Shijubo N, Kojima H, Nagata M, Ohchi T, Suzuki A, Abe S, et al. Tumor angiogenesis of non-small cell lung cancer. Microsc Res Tech 2003;60:186–98. Song G, Ming Y, Mao Y, Bao S, Ouyang G. Osteopontin prevents curcumin-induced apoptosis and promotes survival through Akt activation via ␣v3 integrins in human gastric cancer cells. Exp Biol Med 2008;233:1537–45. Stendal T, Borset M, Sundan A. Role of osteopontin in adhesion, migration, cell survival and bone remodeling. Exp Oncol 2004;26:179–84. Stewart DJ. Tumor and host factors that may limit efficacy of chemotherapy in non-small cell and small cell lung cancer. Crit Rev Oncol Hematol 2010;75:173–234.
ˇ Please cite this article in press as: Stemberger C, et al. Osteopontin is associated with decreased apoptosis and ␣v integrin expression in lung adenocarcinoma. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.07.009
G Model ACTHIS-50750; No. of Pages 8 8
ARTICLE IN PRESS C. Sˇ temberger et al. / Acta Histochemica xxx (2013) xxx–xxx
Townson JL, Naumov GN, Chambers AF. The role of apoptosis in tumor progression and metastasis. Curr Mol Med 2003;3: 631–42. Wai PY, Kuo PC. Osteopontin: regulation in tumor metastasis. Cancer Metastasis Rev 2008;10:3–18. Wang XM, Li J, Yan MX, Liu L, Jia DS, Geng Q, et al. Integrative analyses identify osteopontin, LAMB3 and ITGB1 as critical prometastatic genes for lung cancer. PLoS ONE 2012;8(2):e5571. Weber GF, Lett GS, Haubein NC. Osteopontin is a marker for cancer aggressiveness and patient survival. Br J Cancer 2010;103:861–9.
Zhang J, Takahashi K, Takahashi F, Shimizu K, Ohsita F, Kameda Y, et al. Differential osteopontin expression in lung cancer. Cancer Lett 2001;171:215–22. Zhao J, Dong L, Wu G, Xu D, Chen J, Li K, et al. Down-regulation of osteopontin suppresses growth and metastasis of hepatocellular carcinoma via induction of apoptosis. Gastroenterology 2008;135:956–68. Zohar R, Zhu B, Liu Sodek J, McCulloch CA. Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase3-independent pathway. Am J Physiol Heart Circ Physiol 2004;287:1730–9.
ˇ Please cite this article in press as: Stemberger C, et al. Osteopontin is associated with decreased apoptosis and ␣v integrin expression in lung adenocarcinoma. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.07.009