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Introduction

Lung cancer is a significant health problem not only in China, but worldwide. According to the information presented in the online database GLOBOCAN 2008 (http://globocan.iarc.fr/factsheet.asp) of the International Agency for Research on Cancer, lung cancer was the most common and most aggressive cancer worldwide in 2008, accounting for 16.5% of the total cancer cases and 22.5% of the total cancer-related deaths in 182 countries. In China, the incidence and mortality rates of lung cancer also rank first among all types of cancer, which were predicted to be 33.5/100,000 and 28.7/100,000, respectively, in 2008 based on the data from 36 cancer registries and the Third National Death Survey (2004–2005)(1).

Approximately 85% of lung cancer cases are non-small cell lung cancer (NSCLC), which includes adenocarcinoma (AD), squamous cell carcinoma (SCC), large cell carcinoma and bronchioloalveolar carcinoma (2). Despite extensive research and clinical efforts, prognosis of NSCLC remains poor. Nevertheless, biomarkers have been recognized to play a critical role in early diagnosis, therapy guidance and prognosis monitoring of NSCLC (3). Over the last two decades, several biomarkers have been identified for NSCLC, but none have been routinely used in clinical settings (4). It is, therefore, urgent to explore more reliable and feasible novel biomarkers for lung cancer.

Adenylate cyclase-associated protein 1 (CAP1) is an actin monomer-binding protein coded by the CAP1 gene (5), which was originally cloned from budding yeast and is located in the downstream of the ras gene (6). Human homology of CAP1 was identified in the early 1990s (7). Both mammal and yeast CAPs interact with actin (8) and play a role in actin turnover (9). Given the critical role for actin filament reorganization in cell migration and the regulatory role for CAP1 in actin filament reorganization (10,11), it is logical to hypothesize that CAP1 may be associated with tumor metastasis. Currently, however, studies on the correlation between CAP1 expression and tumor metastasis are scarce (12). The objective of the present study was to evaluate the potential value of CAP1 in early diagnosis and prognostic prediction of progressive lung cancer.

Materials and methods

Ethical considerations

The study was reviewed and approved by the Institutional Ethics Committee of Tongji University School of Medicine in Shanghai and was conducted in compliance with the Helsinki Declaration. Written informed consent was obtained from all subjects.

Subjects and biopsy specimen collection

Twenty-four lung cancer patients and 6 control subjects with non-neoplastic lung condition(s) who underwent surgical lung resection of neoplastic and non-neoplastic lesions, respectively, at the Shanghai 10th People’s Hospital, China, between 2007 and 2009 were recruited. Cancer types and stages were determined based on results from laboratory tests, X-rays, CT scans, bone scans and MRI scans. Biopsy specimens were collected at surgery, snap-frozen in liquid nitrogen and stored at −80°C until analyses.

Tissue bank specimen acquisition

Tissue blocks of 82 formalin-fixed and paraffin-embedded lung cancer specimens were acquired from the Tissue Bank in the Pathology Department of Tongji University School of Medicine. The documented general demographic data and cancer stages assessed based on the tumor-node-metastasis (TNM) classification system were obtained.

Lung cancer cell line

For in vitro analyses, a low invasive lung cancer cell line 95-C and an invasive lung cancer cell line 95-D were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). For maintenance, cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2 and 95% air.

Real-time PCR

Total RNA was isolated from neoplastic and non-neoplastic lung biopsy specimens using an RNA extraction kit from Isogen (Nippon Gene Co., Ltd., Toyama, Japan). RNA samples were treated with DNase I (Promega Corp., Madison, WI, USA) to remove genomic DNA. First-strand cDNAs were synthesized using a commercial First-Strand cDNA Synthesis kit as instructed by the manufacturer. PCR amplifications of the test gene CAP1 and the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed using, respectively, a pair of primers (forward, 5′-ACT CGC TGC TTG CTG GTC-3′ and reverse: 5′-ATG GGT GCC AAC AAA TCG-3′) designed based on the human CAP1 mRNA sequence (GenBank accession number BT007152) and a pair of primers (forward, 5′-GAA GGT GAA GGT CGG AGTC-3′ and reverse, 5′-CCC GAA TCA CAT TCT CCA AGA A-3) designed based on the human GAPDH cDNA sequence (GenBank access number X01677). All reactions were carried out with the SYBR-Green PCR Core Reagents kit (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). The PCR amplification parameters were: 50°C for 2 min (one cycle), 95°C for 10 min (one cycle), 95°C for 15 sec and 60°C for 1 min (40 cycles). The emission intensity of the SYBR-Green fluorescence was measured in a real-time fashion with the ABI Prism 7700 Sequence Detector from Perkin-Elmer Applied Biosystems. Relative quantitation of CAP1 mRNA abundance was performed using the DataAssist software (Life Technologies, Grand Island, NY, USA).

Western blot analysis

Whole cell and tissues lysates were prepared from cultured 95-C and 95-D cells and biopsy lung specimens, respectively. Following total protein quantitation, the lysates were loaded onto 10% polyacrylamide-SDS gels, separated by electrophoresis and blotted onto N-C membrane blots using a semi-dry transfer system. Blots were incubated with a mouse anti-human CAP1 antibody and a mouse anti-human actin antibody (Sigma, St. Louis, MO, USA) at 4°C overnight. After washing, blots were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 45 min. The immunoreactive signals for both CAP1 and actin were visualized using the ECL system from GE Healthcare UK Ltd. (Buckinghamshire, UK) and subjected to densitometric analyses with the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Relative levels of CAP1 (i.e., after adjustment against actin) were determined based on the densitometric data.

Immunohistochemical and immunocytochemical staining

Lung cancer specimen blocks obtained from the tissue bank were sectioned at 5 μM and mounted onto glass slides. Low and high invasive lung cancer 95-C and 95-D cells were grown on culture slides (BD Biosciences, San Diego, CA, USA) and fixed in 3.7% formaldehyde in PBS. Both tissue sections and cultured cells were incubated with a primary CAP1 antibody at 4°C overnight. After rinsing three times in Tris-buffered saline (0.01 M Tris-HCl pH 7.5, 150 mM NaCl), tissue sections and cultured cells were incubated with FITC-labeled secondary antibody (Invitrogen, Carlsbad, CA, USA) at room temperature for 30 min. After washing, the slides were mounted with a DAPI-containing mounting solution to identify cell nuclei. Fluorescent signals were visualized under an Axiovert 200 microscope and images were captured using an AxioCam CCD camera and the AxioVision software (Leica Microsystems, Wetzlar, Germany). The human peritumoral vascular endothelial cells were used as a positive control.

Staining results were assessed by two pathologists, blind to the specimen identity, independently. The immunoreactive intensity in the test tissue specimens and cultured tumor (95-C and 95-D) cells was graded by the difference against the intensity in endothelial cells in the positive control group: 0, negative; 1 (weak), weaker than epithelial cells; 2 (moderate), the same as epithelial cells; 3 (strong), stronger than epithelial cells. A staining score of 2 or 3 was considered CAP1-positive.

RNA interference

Invasive 95-D cells were seeded onto collagen I-coated 6-well plates at 5×104 cells/well. Following overnight culture, cells were transfected with CAP1 specific siRNA (siCAP1) or non-silencing control siRNA in serum-free RPMI-1640 using Oligofectamine (Invitrogen). The target sequence was AAACCGAGTCCTCAAAGAGTA. Both specific and control siRNA molecules were synthesized by GenePharma Co. (Shanghai, China). Four hours after transfection, an equal volume of medium supplemented with 20% fetal bovine serum (FBS) was added. Transfectants were cultured until further analysis.

Migration assay

Invasive 95-D cells transfected with siCAP or control siRNA were seeded onto the upper chambers of 6-well transwell plates (BD Biosciences, San Jose, CA, USA) in triplicates in RPMI-1640 supplemented with 0.5% FBS. Culture medium containing 10% FBS was added to the bottom chamber. After a 24-h culture, cells on the upper surface of the chamber membrane were removed and those on the lower surface of the membrane were stained by hematoxylin and eosin (H&E). The number of migrated cells was counted under a light microscope (×400).

Statistical analysis

Categorical data expressed as percentage were analyzed by the χ2 test. Correlations between CAP1 expression and various clinicopathological variables was analyzed by multivariate logistic regression analysis. The statistical software SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) was used. Differences were considered significant when P<0.05.

Results

Demographic characteristics

In the present study, 24 lung cancer patients and 6 non-neoplastic control subjects were recruited and 82 tissue bank specimens of lung cancer were included. Of the 24 lung cancer patients, 4 had advanced (metastasis), 10 were of the SCC and 14 of the AD type. The base-line demographic data on the 82 tissue bank cases are presented in Table I.

Table I General demographics and CAP1 immunoreactive signals in the 82 patients whose lung tumor tissue specimens were banked in the institutional tissue bank.

Table I

General demographics and CAP1 immunoreactive signals in the 82 patients whose lung tumor tissue specimens were banked in the institutional tissue bank.

Variables	Total N (%)	CAP1-negative	CAP1-positive	P-value
Age (years)				0.204
≤65	43 (52.4)	15	28
>65	39 (47.6)	19	20
Gender				0.701
Female	27 (32.9)	12	15
Male	55 (67.1)	22	33
Histologic type				0.526
AD	40 (48.8)	18	22
SCC	31 (37.8)	16 (non-AD)	26 (non-AD)
Others	11 (13.4)
Tumor size (T) (cm)				0.231
≤3	37 (45.1)	18	19
>3	45 (54.9)	16	29
Node metastasis (N)				0.01
0–1	42 (51.2)	22	20
2–3	40 (48.8)	12	28
Distant metastasis (M)				0.126
0	63 (76.8)	29	34
1	9 (23.2)	5	14
TNM				0.04
I		17	8
II–IV		17	40

[i] CAP1, cyclase-associated protein 1; AD, adenocarcinoma; SCC, squamous cell carcinoma; TNM, tumor-node-metastasis.

CAP1 mRNA abundance

To assess the association of CAP1 expression with lung cancer type and stage, we first performed real-time PCR to analyze relative CAP1 mRNA abundance in non-neoplastic and lung cancer biopsy specimens. The results are shown in Fig. 1. The relative CAP1 mRNA abundance, expressed as the CAP1/GAPDH ratio, was significantly higher in neoplastic tissues than in control specimens (P=0.028), and in metastatic than in non-metastatic lung cancer specimens (P=0.016). When a comparison was made between histological types, SCC specimens had slightly higher CAP1 mRNA abundance than AD specimens, but the difference failed to reach a statistically significant level (P=0.227).

Figure 1 Relative cyclase-associated protein 1 (CAP1) mRNA levels (i.e., CAP1/GAPDH mRNA ratio) in non-neoplastic lung tissue and lung tumors of different stage and histological type. *P=0.05, comparison made within the same type of bar.

CAP1 protein levels

In addition, mRNA abundance, CAP1 protein levels in biopsy specimens and cultured cells were determined by western blot analysis. Shown in Fig. 2 is an image of a representative blot. When expressed as a densitometric ratio of CAP1 band to β-actin band, the relative CAP1 protein level was significantly elevated in lung cancer patients (0.7527±0.2767) as compared to non-neoplastic control subjects (0.3476±0.1713, P=0.002); it was also significantly elevated in metastatic lung tumors (0.8941±0.1442) as compared to non-metastatic lung tumors (0.4701±0.2647, P=0.002); it was slightly higher in SCC than in AD specimens (0.7440±0.2911 vs. 0.7601±0.2757, P=0.891), but the difference was not significant (P>0.05). In cultured cell lines, the CAP1 protein level was significantly higher in invasive 95-D cells than that in non-invasive 95-C cells (P<0.05).

Figure 2 A representative western blot analysis showing cyclase-associated protein 1 (CAP1) and β-actin bands in indicated biopsy specimens (two representative samples from each of the patient groups) and cultured lung cancer cells.

Correlation between immunoreactive CAP1 signals and cancer metastasis

Immunoreactive CAP1 was detected in the cytoplasm of cancer cells in all 82 tissue bank samples (Fig. 3) and cultured 95-C and 95-D cells and also in the cytoplasm of stromal cells (Fig. 4). Using the signal in the positive control endothelial cells as a reference, the 82 bank samples were classified into a CAP1-negative group, where the tumor staining score was 0 or 1, and a CAP1-positive group, where the tumor staining score was 2 or 3. As shown in Table I, a CAP1-positive status was significantly associated with lymph node metastasis (P=0.04) and TNM stage (P=0.01) as demonstrated by the χ2 test.

Figure 3 Representative sections of pathological lung cancer specimens after immunohistochemical staining. (A) Adenocarcinoma (AD) showing a high percentage of cyclase-associated protein 1 (CAP1)-positive cells; (B) AD showing a low percentage of CAP1-positive cells; (C) squamous cell carcinoma (SCC) showing a high percentage of CAP1-positive cells; (D) SCC showing a low percentage of CAP1-positive cells.

Figure 4 A representative image of non-invasive and invasive 95-C and 95-D lung cancer cells following immunostaining with anti-cyclase-associated protein 1 (CAP1) antibody and nuclear staining with DAPI.

To assess the association of CAP1 expression simultaneously with various demographic variables and tumor properties in lung cancer cases, multivariate logistic regression analysis was performed. These results indicated that the expression level of CAP1 was associated with TNM stage of lung cancer (P=0.01), particularly the lymph node invasion (P=0.04) (Table I).

Reduced migration of invasive lung cancer cells following CAP1 knockdown

To validate the positive correlation between CAP1 and metastasis of lung tumors, we attempted to silence the CAP1 gene in invasive lung cancer 95-D cells with a small interfering RNA (siRNA) molecule specific for human CAP1 and to determine the migration capacity of the CAP1-siRNA carrying cells in vitro. As shown in Fig. 5, the CAP1 gene was adequately silenced by sequence-specific CAP1-siRNA and correspondingly the number of cells that migrated across the transwell membrane was significantly reduced in the CAP1 gene-silenced 95-D cells.

Figure 5 A representative image of invasive 95-D lung cancer cells transfected with the control siRNA and the cyclase-associated protein 1 (CAP1)-specific siRNA respectively after migrating over the transwell membrane in the in vitro cell migration assay. Migrated cells were stained.

Discussion

As a first step toward evaluating the association of CAP1 with histological type and clinical stage of lung cancer, we first assessed the CAP1 gene expression at the mRNA level in 24 lung cancer patients and 6 non-cancer control subjects by real-time PCR. The results clearly demonstrated that the CAP1 gene transcription was significantly elevated in lung cancer patients and the elevation was more pronounced in metastasis (Fig. 1).

Although it is generally assumed that mRNA abundance is predictive of the corresponding protein level, this assumption may not be valid for all human genes. In a study aimed at assessing the abundance of 165 protein spots representing 98 individual genes using 76 lung adenocarcinomas and 9 non-neoplastic lung tissue specimens, marked variation in the mRNA/protein correlation coefficient was found among proteins and between stage I and stage III lung adenocarcinomas (13). To further analyze whether the observed elevation in CAP1 mRNA abundance was correlated with increased CAP1 protein synthesis, we next performed western blot analysis on the CAP1 protein content in biopsy specimens and cultured lung cancer cells. In accordance with the pattern of changes in CAP1 mRNA abundance in various specimens, the CAP1 protein content was significantly higher in cancer than in non-cancer subjects and higher in metastatic than in non-metastatic cancer patients with no difference between SCC and AD (Fig. 2). Since the sample size used in the present study was small and there are no reports in the literature on the CAP1 mRNA/protein correlation coefficient, more thorough studies with larger sample sizes are warranted to validate the concurrent elevation in CAP1 mRNA and protein levels in lung cancer.

To further evaluate the correlation between CAP1 expression and histological type and clinical stage of lung cancer, we performed immunostaining on 82 tissue bank specimens and cultured 95-C and 95-D cells. Despite the lack of reports on intracellular localization of CAP1 protein in lung cancer cells, non-nuclear localization of CAP1 has been observed at the site of rapid actin turnover in non-muscle cells (9). In the present study, stronger immunoreactive CAP1 signals were detected in the perinuclear cytoplasm of lung cancer cells in the biopsy specimens (Fig. 3) and cultured lung cancer cells (Fig. 4). This intracellular localization suggests that CAP1 may be associated with lamellipodium formation and cell migration. Following classification of the 82 tissue bank specimens as CAP1-negative and CAP1-positive, two groups based on their immunoreactive signal densities relative to the density in endothelial cells in the positive control group and a subsequent multivariate regression analysis, the CAP1 immunoreactive signal was positively correlated with metastasis, particularly metastasis to lymph nodes (Table I). Given that lymph node metastasis of lung cancer is associated with poor clinical outcomes (14,15), this finding suggests that immunostaining of the CAP1 protein in lung cancer tissue may provide additional information predictive of patient prognosis.

siRNA-based RNA interference (RNAi) is a post-transcriptional process triggered by the introduction of double-stranded RNA (dsRNA) which leads to gene silencing in a sequence-specific manner (16). As one of the most notable discoveries of the past decade in functional genomics, RNAi has become an important tool for analyzing gene functions in eukaryotes. Using this advanced technology, we successfully silenced the CPA1 gene in invasive lung cancer 95-D cells (Fig. 4), which resulted in a significant reduction in the capacity of 95-D cells to migrate (Fig. 5). This observation further supports a functional role for CAP1 in the process of lung cancer cell metastasis.

At present, the molecular mechanisms underlying the effect of CAP1 on lung cancer cell migration and metastasis are largely unknown. Cofilin is a family of actin-binding proteins which disassembles actin filaments. Cofilin activity is present in the malignant, invasive cancer cells (17) and may function as a factor to regulate cancer cell migration/invasion phenotypes. We previously demonstrated that CAP1 is a downstream target of cofilin (18), suggesting that the CAP1 overexpression-associated lung cancer cell migration and metastasis may involve a cofilin-mediated signaling pathway. Nevertheless, this requires further evaluation.

Acknowledgements

The present study was supported by a research grant from the Shanghai Science Committee (09ZR1424500).

References