Abstract
OBJECTIVE To investigate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and vascular endothelial growth factor (VEGF) in lung carcinomas, and to clarify their roles in carcinoma progression.
METHODS Expression of EMMPRIN and VEGF was examined with tissue microarrays (TMAs) of lung carcinomas (n = 181), and their suppression in adjacent normal lung samples (n = 40) were determined by immunohistochemistry. The results were compared with clinicopathological findings for the same tumors.
RESULTS Both EMMPRIN and VEGF were occasionally expressed in pseudostratified columnar epithelium and frequently in lung carcinomas. Histologically, EMMPRIN and VEGF displayed higher levels in large (LCC) cell carcinomas than adenocarcinoma (AD), squamous (SQ) and small cell carcinomas (SCC) (P < 0.05). EMMPRIN was more highly expressed in SQ as compared with AD (P < 0.05), while the converse was true for VEGF (P < 0.05). Binding was generally more intense for EMMPRIN in samples from male compared to female patients (P < 0.05), whereas the latter tended to exhibit more VEGF expression (P < 0.05). Positive associations of VEGF expression with the TNM stage and amounts of EMMPRIN were noted in the lung carcinomas (P < 0.05).
CONCLUSION EMMPRIN and VEGF possibly contribute to physiological repair of normal lung and histogenesis of lung carcinoma. Both proteins might be involved in the molecular basis for differences in the incidence of lung carcinoma between men and women.
keywords
Introduction
Growth of solid tumors depends on angiogenesis, a complex and multi-step process which facilitates metastasis formation by increasing the likelihood of tumor cells entering the blood circulation as well as providing nutrients and oxygen for growth at the metastatic site[1]. Critical steps during tumor angiogenesis include the outgrowth of endothelial cells from preexisting capillary vessels, their migration from parental vessels, degradation of the extracellular matrix (ECM) and vascular tube formation, which is enhanced by vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and extracellular matrix metalloproteinase inducer (EMMPRIN)[1,2].
Structurally, EMMPRIN (also named as CD147 or basigin) is a glycosylated cell surface transmembrane protein which belongs to the immunoglobulin superfamily. It is composed of two immunoglobulin domains in the extracellular region, a single transmembrane domain and a short cytoplasmic domain containing 39 amino acids[3,4]. EMMPRIN has a broad tissue distribution with expression on activated T cells, differentiated macrophages, and other normal tissues. However, its expression is elevated in numerous cancers, which increases the potential for tumor invasion by inducing MMP synthesis of the surrounding stromal cells, including membrane type 1 and type 2 MMP, and the endogenous activators of MMP-2[3-12]. Recently in vivo evidence has indicated that EMMPRIN might stimulate tumor angiogenesis by elevating VEGF and MMP expression in the neighboring fibroblasts and epithelial cells via microvesicles in a paracrine manner[13,14].
VEGF is a homodimeric glycoprotein with a molecular weight of approximately 45 kDa. It promotes angiogenesis by directly binding to receptors on vascular epithelial cells, resulting in several responses: i) maintaining the viability of immature vasculature by inducing anti-apoptotic signals; ii) providing mitogenic and survival stimuli for endothelial cells; iii) inducing chemotaxis and expression of proteinases[15]. Taken together, it is suggested that EMMPRIN and VEGF have a major influence on invasion and metastasis of malignant tumors.
Lung carcinoma ranks as the leading cause of cancer mortality in the majority of the industrialized countries[16]. Tumorigenesis and progression of lung carcinoma is a multistage process with a multifactorial etiology, involving various gene-environmental interactions. Our study is the first investigation of EMMPRIN expression in lung carcinoma along with examination of VEGF expression in adjacent normal lung tissue. The results were related to the clinicopathological parameters of the tumors to clarify the roles these proteins may have in stepwise development of lung carcinomas.
Patients and Methods
Subjects
Lung carcinomas (n = 181) and normal lung tissues (n = 40) were collected from our affiliated hospital and the Kouseiren Takaoka Hospital between 1993 and 2006. The patients with lung carcinoma were 121 men and 60 women (a range of 40~90 years, mean = 67.8 years). Among them, 65 cases had tumors accompanied with lymph node metastasis. All patients provided consent for use of their tumor tissue for clinical research, and our University Ethical Committee approved the research protocol. Follow-up on all patients was conducted by consulting their case documents and connecting with them via telephone.
Pathology
All tissues were fixed in 4% neutralized formaldehyde, embedded in paraffin and sliced into 4 μm sections. The sections were stained with hematoxylin and eosin (H&E) to confirm their histological diagnosis which was classified according to WHO criteria[17]. The staging for each lung carcinoma was evaluated according to the UICC system for the extent of tumor spread[18]. Pleural invasion, lymphatic and venous invasion, and lymph node metastasis were determined as well.
Tissue microarray (TMA)
Representative areas of solid tumors were identified in H&E-stained sections of the selected tumor cases. Two 2 mm diameter or one 5 mm tissue core per donor block was punched out and transferred to a recipient block with a maximum of 48 cores using a Tissue Microarrayer (AZUMAYA KIN-1, Japan). Four μm-thick sections were consecutively sliced from the recipient block and transferred to poly-lysine-coated glass slides. H&E staining was performed on the TMA to confirm the tumor tissue character (Fig.1a).
H&E staining of TMA of lung carcinomas (a). Note EMMPRIN localization in the membrane and/or cytoplasm of pseudostratified columnar epithelium (d, weak), lung carcinoma (f&g, strong), but not type I, II alveolar cells (b). VEGF is localized in the cytoplasm of pseudostratified columnar epithelium (e, weak) and lung carcinomas (h&i, strong), but not type II alveolar cells (c).
Immunohistochemistry
Consecutive sections were deparaffinized with xylene, dehydrated with alcohol, and subjected to antigen retrieval by heating in a target-retrieval solution (TRS, DAKO, Carpinteria, USA) for 15 min in a microwave oven (Oriental rotor Lmt. Co. Japan). Bovine serum albumin (5%) was then applied for 1 min to prevent non-specific binding. The sections were incubated with primary antibodies for 15 min, then treated with the antimouse or anti-rabbit Envison-PO (DAKO, USA) antibodies for 15 min. All the incubations were performed in a microwave oven to allow intermittent heating as described previously[19]. After each treatment, the slides were washed with TBST (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) 3 times for 1min. Mouse anti-EMMPRIN (Novo Castra, Newcastle upon Tyne, UK; 1:50) and rabbit anti-VEGF (LAB VISION, Fremont, USA; ready to use) antibodies were employed for the detection of the respective proteins. Binding sites were visualized with 3,3’-diaminobenzidine (DAB). After counterstaining with Mayer’s hematoxylin, the sections were dehydrated, cleared and mounted. Omission of the primary antibody and usage of non-immune IgG in identical protein concentrations to the EMMPRIN and VEGF antibodies were used as a negative control, and appropriate positive controls were utilized as recommended by the manufacturers.
Evaluation of immunohistochemistry
The immunoreactivity to EMMPRIN was localized in the cytoplasm and membrane and VEGF in the cytoplasm (Fig.1). One hundred cells were randomly selected and counted from 5 representative fields of each section blindly by 3 independent observers (Takano Y, Takahashi H and Zheng H). The positive percentage of counted cells was graded semi-quantitatively according to a 4-tier scoring system: negative (-), 0~5%; weakly positive (+), 6%~25%; moderately positive (++), 26%~50%; and strongly positive (+++), 51%~100%.
Statistical analysis
Statistical evaluation was performed using the Spearman correlation test to analyze the rank data. P <0.05 was considered as statistically significant. SPSS 10.0 software was employed to analyze all data.
Results
As shown in Fig.1, both EMMPRIN and VEGF were occasionally expressed in the cytoplasm of the pseudostratified columnar epithelium, and frequently in lung carcinomas. Histologically, EMMPRIN and VEGF displayed higher expression in large cell carcinoma (LCC) than adenocarcinoma (AD), squamous (SQ) or small cell carcinomas (SCC) (P < 0.05). EMMPRIN was highly expressed in SQ, compared with AD (P < 0.05), while it was the converse for VEGF (P < 0.05). There was more EMMPRIN in tissues from male than female patients (P < 0.05), whereas it was the converse for VEGF (P < 0.05). EMMPRIN was expressed more in the lung carcinoma with lymph node metastasis or high staging, but the difference was not statistically significant (P > 0.05). It is worth noting the positive association of VEGF expression with the TNM staging and EMMPRIN expression in lung carcinomas (P < 0.05, Tables 1, 2).
Relationship between EMMPRIN expression and clinicopathological parameters for lung carcinomas.
Relationship between VEGF expression and clinicopathological parameters for lung carcinomas.
Discussion
The microenvironment of the tumor-host interface is involved in many processes impacting on tumor progression, including angiogenesis, growth, invasion and metastasis of tumors. The cross-talk interaction between a tumor and stromal cells participates in tumor-immune escape, spreading and angiogenesis, which is conducted by a number of soluble and membrane molecules, including EMMPRIN and VEGF[12,20]. In the present study, it was found that EMMPRIN and VEGF were positively expressed in stratified squamous epithelium of normal lung tissue. Gabison et al.[21] also reported EMMPRIN to be predominantly expressed in corneal epithelium, but markedly elevated in the anterior stroma of ulcerated corneas. VEGF can be released by MMPs or heparinases to remodel tissue and repair healing because of induction of VEGF mRNA following injury[15]. Therefore, we believe that EMMPRIN and VEGF are possibly involved in stromal remodeling and lung epithelial repair in a soluble form. In lung carcinomas, the positive rate of EMMPRIN reaches a high 75.7%, similar to reports of ovary tumor, renal and oral carcinomas[10,11]. We also found that up-regulated EMMPRIN was involved in the tumorigenesis and progression of gastric carcinoma, possibly by regulating angiogenesis or degradation of tenascin[22,23]. Experimental over-expression of EMMPRIN in relatively less aggressive carcinoma cells lines results in an ability to form large and malignant tumors with a more invasive phenotype in nude mice[6]. These findings suggest that high EMMPRIN expression plays an essential role in lung carcinoma.
Histologically, LCC showed more expression of EMMPRIN and VEGF than other histological types of lung carcinoma. Additionally, EMMPRIN expression was higher in SQ than AD, while it was the converse for VEGF. From our data, it was concluded that LCC belongs to a specific type of lung carcinoma, with high expression of angiogenic factors, such as EMMPRIN and VEGF. The differential expression of both markers appears to be the molecular basis of histogenesis of SQ and AD. On the other hand, there was more EMMPRIN expression in tumors from male compared to female, whereas it was the converse for VEGF. According to the literature, the genetic background of men and women determines their differences in morbidity and histological types of lung carcinoma[24]. Our data indicate that EMMPRIN and VEGF possibly belong to differential gender genetic factors for males and females.
Elevated EMMPRIN and VEGF expression has also been shown to correlate with progression of various malignancies[4,8,11]. Our results suggested that EMMPRIN expression was higher in lung carcinoma associated with lymph node metastasis or advanced stage, but the differences were not significant. A large body of evidence indicates that increased EMMPRIN can stimulate the synthesis and secretion of VEGF and MMP in surrounding stromal cells, which is closely linked to angiogenesis, invasion and metastasis[5,13]. Yang et al.[25] found that EMMPRIN expression rendered breast carcinoma cells resistant to anoikis, a form of apoptosis, mediated by down-regulation of the pro-apoptotic BH3-only protein, Bim, through a MAP kinase-dependent pathway. Marieb et al.[26] documented that up-regulated EMMPRIN expression stimulates hyaluronan production to participate in anchorage-independent growth of cancer cells by elevating hyoluronan synthases. The data suggested that EMMPRIN possibly contributes to metastasis and progression of lung carcinoma in some sense by regulating apoptosis, proliferation, angiogenesis and degradation of the ECM.
The expression of VEGF by tumor cells is also potentiated by common genetic events that lead to malignant transformation, such as those that cause aberrant mitogenesis and resistance to apoptosis. VEGF also plays an integral part in tumor growth not only by direct induction of angiogenesis, but also by mediation of the secretion of enzymes involved in degrading the ECM[15]. VEGF binding to its receptor on tumor cells will assist in their proliferation and other malignant events, which are closely linked to tumor progression[27].
In the present study, we found that VEGF expression was positively correlated with TNM staging. However its high expression in tumors associated with lymph node metastasis displayed no statistical significance. These findings suggested that VEGF may be a good marker to indicate malignant behavior of lung carcinoma. Tang et al.[28] found that in tumor cells, EMMPRIN regulates VEGF production via the PI3K-Akt pathway. The positive relationship between VEGF and EMMPRIN expression provided in vivo evidence for the inductive effect of EMMPRIN on VEGF expression in tumors.
In conclusion, high expression of EMMPRIN and VEGF plays an important role in lung carcinomas. Differential expression of both markers was involved in the histogenesis of the lung carcinomas studied, and LCC is a specific carcinoma with rich angiogenic factors. EMMPRIN and VEGF had a significant influence on the progression of lung carcinoma possibly by induction of tumor angiogenesis by tumor stromal interaction.
Acknowledgements
We particularly would like to thank Tokimasa Kumada and Hideki Hatta for their technical help, and Yukari Inoue for her secretarial assistance.
Footnotes
The work was partially supported by the Natural Scientific Foundation of China (No.30600286), Shenyang Outstanding Scholar Foundation, Japanese Smoking Research Foundation, Shenyang Outstanding Talent Foundation, and Liaoning BaiQianwan Talent Program.
- Received May 12, 2008.
- Accepted June 16, 2008.
- Copyright © 2008 by Tianjin Medical University Cancer Institute & Hospital and Springer
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