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Clinical Cancer Research Vol. 11, 4646-4652, July 1, 2005
© 2005 American Association for Cancer Research

Review Article

Overexpression of Osteopontin Is Associated with More Aggressive Phenotypes in Human Non–Small Cell Lung Cancer

Zhi Hu1, Dongmei Lin2, Jingsong Yuan1, Ting Xiao1, Husheng Zhang1, Wenyue Sun1, Naijun Han1, Ying Ma1, Xuebing Di1, Meixia Gao1, Jinfang Ma1, Junhang Zhang3, Shujun Cheng1 and Yanning Gao1

Authors' Affiliations: Departments of 1 Etiology and Carcinogenesis and 2 Pathology, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, and 3 Department of Thoracic Surgery, Liaoning Cancer Hospital, Shenyang, P.R. China

Requests for reprints: Yanning Gao, Department of Etiology and Carcinogenesis, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, 17 Panjiayuan Nanli, Beijing 100021, P.R. China. Phone: 86-10-6778-2323; Fax: 86-10-6776-7548; E-mail: yngao{at}pubem.cicams.ac.cn.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The extracellular matrix (ECM) molecule osteopontin is implicated in many pathologic processes, including inflammation, cell proliferation, ECM invasion, tumor progression, and metastasis. The present study evaluated the clinical and biological importance of osteopontin in human lung cancer.

Experimental Design and Results: Tissue microarrays derived from non–small cell lung cancer (NSCLC) patients were analyzed immunohistochemically. Osteopontin protein expression was observed in 64.5% (205 of 318) of primary tumors and 75.5% (108 of 143) of lymph node metastases, but in only 27.9% (12 of 43) of normal-appearing bronchial epithelial and pulmonary tissues. Osteopontin expression was associated with tumor growth, tumor staging, and lymph node invasion. In vitro osteopontin enhanced ECM invasion of NSCLC cells, and an osteopontin antibody abolished this effect. We further analyzed osteopontin levels in circulating plasma derived from 158 patients with NSCLC, 54 patients of benign pulmonary disease, and 25 healthy donors, and found that the median osteopontin levels for the three groups were 319.1, 161.6, and 17.9 ng/mL, respectively.

Conclusions: Overexpression of osteopontin is common in primary NSCLC and may be important in the development and progression of the cancer. Osteopontin levels in the plasma may serve as a biomarker for diagnosing or monitoring patients with NSCLC.

Lung cancer is the leading cause of cancer-related deaths in industrialized countries. It claims >150,000 lives each year in the U.S. alone, exceeding the combined mortality from breast, prostate, and colorectal cancer (1, 2). Despite recent advances in understanding lung cancer biology, the 5-year survival rate for the patients remains <15% (3). For the patients diagnosed with stage IV disease, this figure drops to a mere 1% due to local relapses and distant metastases. Predicting the metastatic behavior of the tumor and eradicating or controlling dissemination of the malignancy remain major clinical challenges to oncologists.

Cancer progression depends on an accumulation of metastasis-supporting genetic modifications and physiologic alterations regulated by cell signaling molecules such as extracellular matrix (ECM) proteins. The latter contribute to interaction among cancer cells and endothelial cells, which play a critical role in the development of local invasion and distant metastasis (4, 5). One such ECM protein is osteopontin. Previous research suggests that osteopontin is up-regulated in a variety of cancers, such as breast, gastric, and colorectal cancers (6, 7). Reports also suggest that some highly metastatic cancer cell lines synthesize abundant osteopontin. For example, the metastatic cell Ca2-5-LT1 expresses osteopontin mRNA at a level nine times higher than that expressed by the nonmetastatic parental cell Rama 37 (8). These findings suggest that osteopontin is a key extracellular molecule involved in tumor development and progression. However, it has not been extensively evaluated as such in lung cancer.

Evidence also suggests that the level of osteopontin is increased in the circulating serum or plasma of patients with ovarian, pancreatic, breast, and head and neck carcinomas (9–13). Thus, it may serve as a biomarker for cancer diagnosis and prognosis. However, only one study has found increased levels of osteopontin in the serum from 20 patients with lung cancer, and the significance of altered osteopontin in this malignant disease is not fully understood (14).

Our previous work suggests that osteopontin is one of the most overexpressed genes in a differential expression cDNA library derived from non–small cell lung cancer (NSCLC).4 The subsequent RT-PCR analysis also indicates that 80.0% of the 35 tumor samples of NSCLC have increased expression of osteopontin mRNA, compared with the corresponding normal lung tissues, which confirms the results of the differential expression library.4 The present study examined the expression of osteopontin in the tumor tissues using an immunohistochemical technique in a comparatively large group of NSCLC patients. The present study also examined protein levels of osteopontin from the circulating plasma from patients with lung cancer and benign pulmonary diseases, as well as from healthy individuals. In addition, the current study used lung cancer cell lines to explore the role(s) of osteopontin in the progression and dissemination of lung cancer.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and tissue samples. For the immunohistochemistry examination, 527 formalin-fixed, paraffin-embedded tissue specimens were obtained as tissue microarrays, which were constructed in the Department of Pathology, Cancer Hospital, Chinese Academy of Medical Sciences. The specimens consisted of 479 lung cancers (including 320 primary tumors of NSCLC and 159 corresponding lymph nodes with metastastic tumors) and 48 control tissues (including 20 samples of normal bronchial epithelium and 28 of normal lung tissue) taken from the same group of NSCLC patients. All the paraffin-embedded tissue specimens were diagnosed and reconfirmed by experienced pathologists. For the lung cancer cases, there were 279 males and 41 females, with ages ranging from 31 to 81 years (mean age, 66 years) at the time of presentation. The patients were clinically staged according to the international tumor-node-metastasis system (15). No patients in this group received any adjuvant systemic therapy.

For ELISA, 249 human plasma samples were obtained from 175 men and 74 women with an average age of 58 years (range, 29-87 years). The donors included 152 cases of NSCLC and 18 cases of SCLC, as well as 79 age- and smoking status-matched controls. The controls were 25 healthy volunteers and 54 patients with benign lung disease. The majority of the latter group comprised 25 cases of advanced pulmonary tuberculosis and 20 of pneumonia; the others were patients with pulmonary granuloma, fibrosis, etc. Peripheral blood (4 mL) was taken prior to treatment by venipuncture and kept in a heparinized tube. Within 30 minutes of blood collection, the samples were centrifuged at 2,000 rpm at 4°C for 10 minutes to separate the plasma and blood cells. The plasma sample was then aliquoted and stored at –80°C.

The use of all of the human samples and the experimental procedures for this study were reviewed and approved by the ethics committee of the Cancer Institute (Hospital), Peking Union Medical College, and Chinese Academy of Medical Sciences.

Cell culture. The adenocarcinoma cell line A549 was purchased from the American Type Culture Collection, Manassas, VA. The PG cell line was obtained from the Department of Pathology, School of Medicine, Beijing University. It was established using the thorax metastases of a large cell lung carcinoma derived from a 65-year-old Chinese male. The cells were cultivated in RPMI 1640 medium (Life Technologies, Grand Island, NY) with 10% FCS, containing 100 units/mL penicillin and 100 µg/mL streptomycin, in a humidified incubator with 5% CO2 at 37°C.

Immunohistochemistry analysis. Immunohistochemical staining was done on 5-µm-thick sections cut from the tissue microarrays. Sections were incubated overnight at 4°C with goat polyclonal antibody against osteopontin (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:100. Biotinylated rabbit anti-goat IgG secondary antibody was applied for 30 minutes, followed by incubation with the avidin-biotin complex with streptavidin. Nuclei were counterstained blue with hematoxylin. The slides were scored by two independent observers. The percentage of the cells with cytoplasmic labeling was recorded from two areas of each specimen, and the labeling intensity was estimated as 1+, 2+ or 3+. The immunohistochemistry results were categorized into two groups: the samples without any labeling, 1+ labeling in <25% cells, and 2+ labeling in <5% cells were considered negative; all the remaining samples were defined as positive.

Construction and transfection of an osteopontin expressive plasmid. The full-length coding sequence of human osteopontin cDNA, amplified by PCR with the primes (forward) 5'-GGG GGT ACC ATG AGA ATT GCA GTG ATT-3' and (reverse) 5'-CCC TCT AGA AAT TGA CCT CAG AAG ATG-3' was cloned into the pcDNA3.1 vector and the resulting plasmid structure was confirmed by sequencing. The A549 cells were then transfected transiently by the recombined osteopontin expression plasmids with LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

In vitro cell invasion assay. This assay was carried out using the BioCoat Matrigel Invasion Chamber with 8 µm size pores (Becton Dickinson, Bedford, MA), according to the manufacturer's instructions. Twenty-four hours after transfection with the osteopontin expression plasmid or the null vector, 0.5 mL of the cell suspension (1 x 105 cells/mL in RPMI 1640) was added to the upper 24-well chambers. For antibody blocking experiments, goat anti-osteopontin polyclonal antibody was added to the cell suspension at 10 µg/mL, whereas the blocking antibody was replaced with bovine serum albumin in the controls. The chambers were incubated for 22 hours (37°C, 5% CO2 atmosphere). After the noninvading cells were removed from the upper surface of the membrane, the invading cells on the lower surface were fixed with 100% methanol, stained with 0.4% crystal violet, and examined under a light microscope.

ELISA for osteopontin in plasma. The osteopontin levels in the plasma were measured with the osteopontin ELISA kit (Immuno-Biological Laboratories, Hokkaido, Japan), which detects human osteopontin when present at ≥5 ng/mL, according to the manufacturer's instructions. Briefly, 1:5 diluted testing samples were incubated in the osteopontin antibody-precoated plate for 1 hour at 37°C. Following washing, 100 µL of labeled osteopontin antibody solution was added into each well and incubated for 30 minutes at 4°C. After washing, tetramethyl benzidine was used as a coloring agent. Testing samples were detected on a plate reader (Model 550 Microplate Reader, Bio-Rad Laboratories, Hercules, CA), and the strength of coloring was proportional to the quantity of osteopontin.

Statistical analysis. The correlation between immunocytochemical labeling of osteopontin and other clinical variables (including histologic grade, nodal status, patient stage, and tumor size) was assessed using the {chi}2 test. The associations between osteopontin staining and the risk of the occurrence and metastasis of NSCLC were estimated by odds risk with a 95% confidence interval. The protein levels of osteopontin in the plasma and the invading cells in invasion assay were evaluated by a one-way ANOVA using the Student-Newman-Keuls procedure for adjustment of multiple pairwise comparisons between groups. All statistical tests were two-sided. Differences with P values <0.05 were considered statistically significant. Calculations were done with the Statistical Package for the Social Sciences version 11.5 (SPSS, Inc., Chicago, IL).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Expression of osteopontin in the tumor tissue of NSCLC. Osteopontin protein expression levels were investigated by immunohistochemical analysis in 527 paraffin-embedded tissue samples presented as tissue microarrays. The immunohistochemistry labeling was successful in 95.6% (504 of 527) of the initially designed samples. The osteopontin-positive label was confined mainly to the cytoplasm, relative to the normal lung tissue (Fig. 1A, B, and C). The osteopontin labeling intensities were highly similar in the two analyzed areas (in the tissue microarrays) taken from each specimen.



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  		Fig. 1. Expression of osteopontin protein in human NSCLC. Immunohistochemical staining with a goat polyclonal antibody against osteopontin on paraffin-embedded tissues arranged in a tissue microarray. A, primary tumor of squamous cell carcinoma of lung. B, metastatic tumor in a lymph node derived from SCC of lung. C, normal lung tissue. Original magnification: x40, x100, and x400.

 
Table 1 shows the number and percentage of osteopontin-positive samples for each group. The osteopontin antibody detected 64.5% (205 of 318) of the samples taken from primary tumors of NSCLC, but only 27.9% of the control samples (bronchial epithelium or pulmonary tissue) were detected with the antibody. The percentage of osteopontin-positive samples in the tumors from stage I, II, and III to IV patients were 47.6%, 63.5% and 78.2%, respectively. In addition, there was a correlation between expressed osteopontin and the size of NSCLC tumors. However, there was no correlation between osteopontin expression and tumor grade for the NSCLCs.


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  		Table 1. Immunocytochemical staining for osteopontin in the samples from tissue microarrays

 
Notably, the percentage of osteopontin expression in the primary tumors with lymph node metastases was significantly different from that in the primary tumors without the metastases (75.3% versus 51.4%), as shown in Table 1. Table 2 shows the expressive status of osteopontin in a group of patients with advanced NSCLC, indicating a consistency between the 143 primary tumors and their matched lymph node metastases, as compared using the McNemar test (P = 0.523).


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  		Table 2. Expressive status of osteopontin in the primary tumors and the corresponding lymph node metastases of the NSCLCs

 
Effect of osteopontin on in vitro ECM invasion in lung cancer cells. Two NSCLC lines, A549 and PG, were chosen for this investigation. PG cells expressed high levels of osteopontin, whereas little endogenous osteopontin was detectable in the A549 cells using RT-PCR (Fig. 2A). A549 cells were successfully transfected with the human osteopontin expression plasmid and then used together with the PG cells to evaluate the effect of osteopontin on ECM invasion with the BioCoat Matrigel Invasion Chamber system.



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  		Fig. 2. Osteopontin-induced ECM invasion in human NSCLC cells. A, expression of endogenous osteopontin in A549 and PG cells detected by RT-PCR. B, A549 cells in the invasion assay: (1) transfected with null vector; (2) transfected with the osteopontin expression plasmid; (3) the osteopontin transfectants treated with the osteopontin antibody; (4) a chart for cell numbers from the three groups of the A549 experiment (**, OPN versus Control, P < 0.05; OPN versus OPN + Ab, P < 0.05). C, PG cells in the invasion assay: (1) transfected with null vector; (2) treated with bovine serum albumin; (3) treated with the osteopontin antibody; (4) a chart for cell numbers from the three groups of the PG experiment (*, Ab versus Control, P < 0.05; Ab versus bovine serum albumin, P < 0.05). B1-3 and C1-3, representative fields on the bottom surfaces of the membrane of the BioCoat Matrigel Invasion Chamber. The number of cells that invaded through the membrane were counted under a light microscope. B4 and C4, average number of cells for three parallel experiments and are means ± SD of values.

 
The A549 cells that expressed exogenous osteopontin protein showed an increased (183%) invasive potential of the ECM compared with control cells' invasive potential. Pretreatment of the cells with the osteopontin antibody attenuated the enhanced ECM invasion (Fig. 2B). Likewise, treatment with the osteopontin antibody drastically suppressed the intrinsically occurring ECM invasion in the PG cells (Fig. 2C).

Levels of osteopontin protein in the circulating plasma from lung cancer patients and controls. Figure 3 shows the plasma samples from patients with lung cancer and controls, categorized into six groups. Detailed data of the results are summarized in Table 3. The median osteopontin level for NSCLC patients was 319.1 ng/mL, which was significantly higher than that for the SCLC (143.1 ng/mL) and that detected in the age- and smoking status-matched controls, including the healthy volunteers (17.9 ng/mL) and the patients with benign pulmonary diseases (161.6 ng/mL). Plasma levels of osteopontin increased with progression of the malignancy. The osteopontin levels in stage I to II and III to IV patients were 296.4 ng/mL and 351.6 ng/mL, respectively; the difference was significant (P < 0.05). On the other hand, no significant difference for the osteopontin levels was observed among the histologic types of NSCLC or the grades of the tumors.



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  		Fig. 3. Levels of osteopontin protein detected in circulating plasma obtained from the six groups. Health, the healthy blood donors; Disease, the patients with pulmonary benign diseases; ADC, adenocarcinoma; SCC, squamous cell carcinoma; Others, including undifferentiated cancer, large cell carcinoma, adenosquamous carcinoma; SCLC, small cell lung cancer. The box plots indicate the levels of osteopontin expressed in individual plasma. The box was bounded above and below by the 75th and 25th percentiles. The horizontal line within the boxes indicates the median level of osteopontin in the plasma. The upper and lower horizontal bars indicate the maximal and minimal levels, respectively. Circles, outliers.

 

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  		Table 3. Osteopontin levels in the plasma from the patients with lung cancer and controls (ng/mL)

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
As an ECM molecule, the multifunctional protein osteopontin is a secreted noncollagenous, sialic acid–rich, glycosylated phosphoprotein that is expressed in a number of cell types including osteoblasts, activated T lymphocytes, macrophages, and some malignant cells (16, 17). This gene is overexpressed in many kinds of solid tumors and can trigger transformation in cultured cells (18). Osteopontin may also induce cell proliferation, migration, and ECM invasion in vitro and be involved in tumor progression and metastasis by interaction with adhesion molecules, integrins {alpha}vß3, {alpha}vß5, and {alpha}vß1, and CD44 variants in an RGD sequence-dependent or -independent manner (19–21). The effects of osteopontin in human lung cancer, however, have not yet been well-defined prior to this study.

The present study immunohistochemically analyzed 318 tumor samples of NSCLC. The results indicate that the expression levels of the osteopontin protein were significantly higher in the tumor tissues than those found in the control tissues (64.5% versus 27.9%, P < 0.001). The data are similar to those of Zhang et al. (22) in 16 cases of SCC and the results of Shijubo et al. (23) in 55 cases of adenocarcinoma. Together, these findings suggest that the overexpression of osteopontin may play an intrinsic role in NSCLC.

In the current study, the correlation between the expression of osteopontin and the tumor size, stage, and grade was investigated separately. Expression of osteopontin increased with the tumor size and the clinical stage. These results suggest that osteopontin enhances the tumor growth of NSCLC, in agreement with previous findings that osteopontin stimulates proliferation of murine melanoma cells and human prostate cancer cells (24, 25) or alternatively, that reduction in osteopontin synthesis by antisense RNA inhibits the tumorigenicity of transformed Rat1 fibroblasts (26).

Further analysis of the primary tumors, with and without corresponding lymph node invasion, showed a very significant difference between osteopontin levels detected from the two groups (P < 0.001). Moreover, it was found that overexpression of osteopontin was remarkably associated with increased risk of lymph node metastasis (odds risk, 2.88; 95% confidence interval, 1.74-4.77; Table 1). These data provide evidence that osteopontin accelerates metastasis in NSCLC. The fact that the expressive status of osteopontin in the invaded lymph nodes resembled that in the matching primary tumors implied that activation of osteopontin may be an early event in NSCLC metastasis and may be a stimulus rather than a concomitant phenomenon.

The migrating ability of cells may be directly linked to tumorigenesis and osteopontin clearly participates in pathways that regulate migration in diverse cell types, including osteoclasts, fibroblasts, macrophages, and tumor cells (27). Although the mechanism of tumor metastasis enhanced by osteopontin is still unclear, in vitro studies with cell lines and experimental animal models have supplied some important clues. For example, osteopontin stimulate cell motility and invasion both in highly invasive (MDA-MB-231) and less invasive (MCF-7) breast cancer cells through activation of nuclear factor {kappa}B and secretion of serine protease urokinase-type plasminogen activator (28, 29).

Previous studies have not investigated the role of osteopontin in lung cancer, although many investigations have examined the role of osteopontin in facilitating invasion by a variety of tumor cell lines (30). The present study explored the effect of osteopontin on migration and invasion of tumor cells by using an in vitro ECM invasion experiment with human NSCLC cell lines, A549 and PG. Along with expression of exogenous osteopontin, the transfected A549 cells acquired a nearly 2-fold ECM invasive potential. Furthermore, a neutralizing antibody against osteopontin was able to block the osteopontin-induced ECM invasion in the A549 cells to an extent which was largely in agreement with that observed in PG cells. As a metastatic NSCLC line, PG cells express high levels of endogenous osteopontin; its intrinsic ability of invasion could also be significantly inhibited by treatment with the antibody. The results from the in vitro invasion assay strongly support the hypothesis that osteopontin can activate the invasive potential of NSCLC cells.

As degradation and remodeling of ECM, including the basement membrane, by proteolytic enzymes are essential steps in the process of cancer invasion and metastasis (31), osteopontin may be critically involved in many aspects of metastasis, ranging from cell proliferation to cell migration and invasion. Furthermore, the present findings are proof in principle that anti-osteopontin intervention may be useful for controlling its malignancy-promoting effects in NSCLC cells, at least in terms of osteopontin-induced cell invasion.

Osteopontin is classified as both a matrix protein and a cytokine, which can be found in the ECM components and many body fluids. Increased protein levels of osteopontin have been detected in the circulating plasma or serum-derived from patients with a number of solid neoplasms (9–13, 32–34). In the circulating blood, osteopontin molecules may contribute to an interaction among cancer cells and endothelial cells, indicating that the expression of osteopontin may regulate cancer invasion, intra- and extravasation, and colonization at distant sites.

In the current study, osteopontin protein levels in the plasma were significantly higher in the NSCLC patients than those observed in patients with SCLC, patients with pulmonary benign diseases, and healthy donors. The osteopontin levels in the plasma of the SCLC were consistent with our findings of immunohistochemistry staining of osteopontin in the tumor tissues,4 suggesting that osteopontin is not critically involved in the aggressive progression of SCLC.

On the other hand, it seems that osteopontin is among the most abundantly expressed proteins in a range of lung diseases such as pulmonary granuloma, fibrosis, and carcinoma (35–38). Koguchi et al. found that osteopontin levels in plasma from patients with tuberculosis are appreciably higher than those in control healthy subjects (39), which is consistent with the results on cases of tuberculosis investigated in this study. The remarkable difference in plasma osteopontin levels exist between NSCLC and pulmonary benign diseases (as shown in Table 3) points to the diagnostic significance of measuring osteopontin plasma levels for the lung cancer.

Furthermore, for NSCLC, osteopontin levels in the plasma were considerably increased in advanced stage (III-IV) relative to low stage (I-II) patients, in accord with the expression of osteopontin in the tumor tissues detected in NSCLC tissue microarrays (see Table 1).

The current findings suggest that the ECM molecule osteopontin plays an important role during development and progression of NSCLC. Up-regulated expression of osteopontin is involved in aggressive phenotypes, such as invasive growth and metastasis, and it might serve as a predictor for dismal prognosis. Increased osteopontin levels in circulating plasma may be useful as a helpful clinical biomarker for diagnosing or monitoring the disease. In addition, the results of the in vitro experiment indicate that osteopontin may be a therapeutic target for the NSCLC patients with a metastatic tendency, as the findings indicate that osteopontin induces ECM invasion directly.


   Acknowledgments
 
We thank Dr. Nan Hu for her technical support, Mr. Ge Huang for his assistance with statistical analysis; and Drs. Beverly E. Griffin and Li Mao for their stimulating discussions and valuable comments about this manuscript.


   Footnotes
 
Grant support: State Key Program of Basic Research (2004CB518707), the National Key Technologies R&D Program (2002BA711A06), and the Specialized Research Fund for the Doctoral Program of Higher Education (20030023005) to Yanning Gao. The National Natural Science Foundation of China (30070836) and the National Key Technologies R&D Program (2002BA711A11) to Shujun Cheng.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 Unpublished data. Back

Received 9/30/04; revised 2/22/05; accepted 3/ 2/05.


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 Abstract
 Materials and Methods
 Results
 Discussion
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