Clinicopathological significance of N-cadherin and VEGF in advanced gastric cancer brain metastasis and the effects of metformin in preclinical models

  • Authors:
    • Kyong-Hwa Jun
    • Jung Eun Lee
    • Se Hoon Kim
    • Ji-Han Jung
    • Hyun‑Joo Choi
    • Young Il Kim
    • Hyung-Min Chin
    • Seung-Ho Yang
  • View Affiliations

  • Published online on: August 10, 2015     https://doi.org/10.3892/or.2015.4191
  • Pages: 2047-2053
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Abstract

Gastric cancer is the second most common cause of cancer-related death worldwide. Although brain metastasis is a rare complication of gastric cancer, no standard therapy for gastric cancer brain metastasis has been established. We attempted to identify biological markers that predict brain metastasis, and investigated how to modulate such markers. A case-control study of patients newly diagnosed with gastric cancer who had developed brain metastasis during follow-up, was conducted. These patients were compared with patients who had advanced gastric cancer but no evidence of brain metastasis. Immunohistochemistry was used to analyze the expression of E-cadherin, N-cadherin, MSS1, claudin-3, claudin-4, Glut1, clusterin, ITGB4, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and p53. The expression of VEGF tended to be higher in the case group (33.3 vs. 0%, p=0.055). Median survival was significantly correlated with vascular invasion (12 vs. 33 months, p=0.008) and N-cadherin expression (36 vs. 12 months, p=0.027). We also investigated the effects of metformin in tumor-bearing mouse models. VEGF expression was decreased and E-cadherin increased in the metformin‑treated group when compared with the control group. The expression of the mesenchymal marker MMP9 was decreased in the metformin-treated group. Brain metastasis of advanced gastric cancer was associated with the expression of VEGF. Metformin treatment may be useful for modulating the metastatic capacity by reducing VEGF expression and blocking epithelial-to-mesenchymal transition.

Introduction

Gastric cancer is the fourth most common malignancy and the second most common cause of cancer-related death worldwide (1). As a result of early detection, developments in surgical techniques and the development of anticancer agents, the survival of patients with gastric cancer has improved. However, the prognosis of advanced gastric cancer is still poor. While a 5-year survival rate of patients with stage I is more than 96%, that of patients with stage IV is less than 20% (2). Central nervous system (CNS) metastasis is a rare complication of gastric cancer, occurring in 0.16–0.69% of gastric cancer patients, and its clinical features, treatment outcomes and prognostic factors remain unclear (3).

Numerous molecular events are involved in metastatic progression, such as loss of cellular adhesion, detachment of tumor cells from the original sites, degradation of the extracellular matrix, migration of tumor cells, angiogenesis, and implantation of tumor cells at distant sites (4). The transmembrane glycoproteins E- and N-cadherin play a role in calcium-dependent cell-to-cell adhesion, and reduced E-cadherin expression is associated with gastric and colon cancer progression and metastasis (5,6). MSS1 is a nuclear-encoded mitochondrial GTPase, and silencing of MSS1 gene expression is associated with neuroinflammation (7). Claudin-3 and -4 are major structural molecules of the tight junctions that link epithelial cells (8). They are associated with worse malignancy grades, not only in terms of size and invasiveness, yet also in potential metastatic ability and patient outcome. Malignant cells show increased glucose uptake in vitro and in vivo, and this is facilitated by glucose transporters (Gluts) (9). The expression of Glut1 has been described in several malignancies, including those of the esophagus, colon and brain, and Glut1 expression is associated with tumor aggressiveness and survival in gastric cancer (10). Clusterin expression is upregulated in many malignancies, including head and neck, breast and prostate cancers (11). The function of clusterin in cell reconstruction contributes to the occurrence and progression of cancer (12). ITGB4 is an adhesion receptor for lamins and plays a key role in the progression of various carcinomas via its ability to orchestrate key signal transduction events and promote cell motility (13). Vascular endothelial growth factor (VEGF) is one of the most important factors promoting vascularization, and it plays a role in both physiological and malignant conditions (14). Several studies have reported that VEGF is associated with lymph node metastasis in gastric cancer. Epidermal growth factor receptor (EGFR) is a transmembrane receptor that contributes to many processes involved in cell survival and proliferation and also inhibits apoptosis, which leads to cancer development (15). Preusser et al evaluated the expression of EGFR immunohistochemically in tissue samples of gastroesophageal cancer brain metastasis, which they proposed could be treated by agents targeting EGFR (16). p53 is a nuclear phosphoprotein involved in regulating cell proliferation by inhibiting the G1 to S phase progression of the cell cycle, and it is frequently mutated in a variety of human cancers (17). The present study evaluated the expression of E- and N-cadherin, MSS1, claudin-3 and -4, Glut1, clusterin, ITGB4, VEGF, EGFR and p53 in surgical specimens of gastric cancer with and without brain metastasis and analyzed their correlations with clinicopathological features and survival.

Metformin is an oral biguanide introduced in the 1950 for the treatment of type 2 diabetes (18). A recent epidemiologic survey found that metformin had also significant effects on tumorigenesis (19). It was also reported that metformin inhibits human gastric cancer cell proliferation and tumor growth, possibly by suppressing cell cycle-related molecules (20). Candidate markers associated with brain metastasis were investigated in tumor-bearing mouse models treated with metformin to evaluate its use in preventing or targeting brain metastasis therapeutically.

Materials and methods

Patients

Nine samples of gastric adenocarcinoma with brain metastasis were acquired from St. Vincent's Hospital, The Catholic University of Korea from June 1997 to September 2009. Two of the nine patients underwent craniotomies to resect metastatic brain lesions. Seven patients were diagnosed with brain metastasis using brain magnetic resonance imaging (MRI), four of whom showed positive cytology in the cerebrospinal fluid (CSF). Thirteen samples of advanced gastric adenocarcinoma without brain metastasis were also included.

The study protocol was approved by the Institutional Review Board of St. Vincent's Hospital, The Catholic University of Korea. The stages of all patients were evaluated in accordance with the guidelines of the Japanese Classification of Gastric Carcinoma (21). The surgical treatment comprised gastric resection, bypass procedures or laparotomy alone. All patients were followed for a median of 12.5 (range 3–80) months. Patient data were obtained from our own and the Korea Central Cancer Registry database. Survival time was measured from the date of the initial surgery to that of death or last follow-up. Patients who died as a result of surgery or from other causes were excluded from the present study.

Construction of the tissue microarray block

Formalin-fixed paraffin-embedded tissues were obtained from the subjects. Using hematoxylin and eosin (H&E)-stained slides, a representative tumor site was chosen, and the site corresponding to the confirmed tumor site in the paraffin block was marked. Areas with necrosis, hemorrhage or artifacts were excluded. Single-core biopsy specimens 2 mm in diameter were taken from the representative regions (Seongkohn Trader's Co, Seoul, Korea), placed on a trimethylamine (TMA) mold with 60-pores, and re-embedded with paraffin. The TMA blocks were prepared as 4-µm-thick sections and stained with H&E. The tissues were then examined to determine whether an appropriate tumor site had been selected.

Immunohistochemistry

We performed immunohistochemical staining using tissue microarray sections and a BenchMark XT autostainer (Ventana Medical Systems, Tucson, AZ, USA), according to the manufacturer's protocol. The respective antibodies used for immunohistochemistry of E- and N-cadherin, MSS1, claudin-3 and -4, Glut1, clusterin, ITGB4, VEGF, EGFR and p53 were 36/E-cadherin (1:50; BD Biosciences, Rockville, MD, USA), N-cadherin rabbit monoclonal antibody (1:100; Enzo Life Sciences, Plymouth Meeting, PA, USA), ab78161 (1:25), ab15102 (1:50) (both from Abcam, Cambridge, MA, USA), mouse monoclonal antibody to claudin-4 (1:100; Young in Frontier, Seoul, Korea), ab14683 (1:200; Abcam), mouse monoclonal antibody to clusterin (1:50; Young in Frontier), ITGB4 rabbit monoclonal antibody (1:15; Abgent, San Diego, CA, USA), mouse monoclonal antibodies to VEGF (1:100), EGFR (1:100) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) and ab61256 (1:100; Abcam). Ki-67 p53, bcl-2 and Bax were detected using clone PP-67 (1:100; Abcam). Clone images were acquired using an Olympus BX41 microscope with a DP72 digital camera (both from Olympus, Tokyo, Japan).

The immunostained slides were examined under light microscopy by one of the authors (S.H. Kim). The immunohistochemical results were scored semi-quantitatively using a four-point scale: 0, no immunoreaction; 1+, faint or equivocal immunoreaction in <10% of cells; 2+, unequivocal, strong immunoreaction in <30% of cells; and 3+, unequivocal, strong immunoreaction in >30% of cells. Tumors with 1+, 2+ or 3+ expression were interpreted as positive, while tumors with no expression were interpreted as negative.

Inoculation of intracranial cancer cells and experimental design

The human gastric adenocarcinoma MK28 cell line was provided by one of the authors (S.H. Kim) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) under routine tissue culture conditions. Four-weeks-old male athymic nude mice (Central Laboratory Animals, Seoul, Republic of Korea) were used. The nude mice were anesthetized using an intraperitoneal (i.p.) injection of 12 mg/kg xylazine (Rompun; Cutter Laboratories, Shawnee, KS, USA) and 30 mg/kg ketamine (Ketalar; Parke-Davis, Morris Plains, NJ, USA). The mice were then stereotactically inoculated with 1×106 MK28 cells into the right frontal lobe (2 mm lateral and 1 mm anterior to the bregma at a depth of 2.5 mm from the skull) using a sterile Hamilton syringe fitted with a 26-gauge needle (Hamilton, Reno, NV, USA) and a microinfusion pump (Harvard Apparatus, Holliston, MA, USA).

The experimental design is shown in Fig. 1. Each group contained five mice initially. Mice in the treatment group were treated with metformin (2 mg/25 g/day) via i.p. injection for 14 days, 1 week after being inoculated intracranially with MK28 cells. Mice in the control and treatment groups were euthanized 8 weeks after the intracranial inoculation, and tumor specimens were obtained for reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. All experiments were approved by the Ethics Committee of the St. Vincent's Hospital, The Catholic University of Korea.

Reverse transcription-PCR

Total RNA from all specimens was extracted using an RNeasy Mini kit (Qiagen), and 1 µg was reverse-transcribed using RT-premix (M-Biotech, Seoul, Korea). RT-PCR was performed on cDNA samples using a DNA Thermal Cycler (Bio-Rad) with Go Taq Green Master Mix (Promega, Madison, WI, USA), RNase-free water and the following primers: E-cadherin 5′-AAGTGACCGATGATGATGCC-3′ (forward), and 5′-CTTCTCTGTCCATCTCAGCG-3′ (reverse); and 18S rRNA 5′-CGCGGTTCTATTTTGTTGGT-3′ (forward), and 5′-AGTCGGCATCGTTTATGGTC-3′ (reverse). Expression of MMP9, N-cadherin and VEGF was assessed by RT-PCR using the following primers: MMP9 5′-AGTTTGGTGTCGCGGAGCAC-3′ (forward), and 5′-TACATGAGCGCTTCCGGCAC-3′ (reverse); N-cadherin 5′-GCCACCATATGACTCCCTCTTAGT-3′ (forward), and 5′-CAGAAAACTAATTCCAATCTGAAA-3′ (reverse); and VEGF 5′-CCAGCGAAGCTACTGCCGTCCA-3′ (forward), and 5′-ACAGCGCATCAGCGGCACAC-3′ (reverse). The RT-PCR products were separated on 1.2% agarose gels containing ethidium bromide and were visualized with ultraviolet light.

Western blot analysis

Total proteins in the tumor specimens were extracted using PRO-PREP Protein Extraction Solution (Intron Biotechnology, Gyeonggi-Do, Korea) according to the manufacturer's instructions. Western blot analysis was performed to confirm expression using antibodies recognizing the specific epitopes. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes and detected with antibodies to E-cadherin (BD Biosciences, Franklin Lakes, NJ, USA), MMP9 (Abcam), VEGF (Santa Cruz Biotechnology), N-cadherin (Abcam) and β-actin (Sigma-Aldrich, St. Louis, MO, USA). After washing, the membrane was incubated with anti-mouse IgG or anti-rabbit IgG, horseradish peroxidase (HRP)-linked secondary antibody (Cell Signaling Technology, Danvers, MA, USA) or anti-goat IgG HRP-antibody derived from rabbit (Sigma-Aldrich). Immunoreactivity was detected using the ECL chemiluminescence system (Thermo, Rockford, IL, USA) and quantified using an imaging densitometer (Model LAS 4000 mini; GE Healthcare Bio-Sciences AB Hercules, Japan). The density of each band was quantified using Quantity One software (Bio-Rad Laboratory, Hercules, CA, USA).

Statistical analysis

The distributions of patient characteristics were compared between the study and control groups using the Wilcoxon rank-sum and Fisher's exact tests for continuous and discrete variables, respectively. Differences in the immunohistochemical results between groups were analyzed using the Chi-square test. Overall survival was analyzed using the Kaplan-Meier method, and survival data were compared using a log-rank test. A p-value of <0.05 was considered to indicate a statistically significant result.

Results

Patient characteristics

The characteristics of the present study population are listed in Table I. There were no significant differences in age, gender, histology type, tumor size, stage, vascular invasion or perineural invasion between the case and control groups. Lymphatic invasion was significantly higher in the control group than the case group (92.3 vs. 44.4%, p=0.023). The median interval between the diagnosis of stomach cancer and the detection of brain metastasis was 9 (range 1–77) months.

Table I

Characteristics of the patients.

Table I

Characteristics of the patients.

ParametersCase groupControl groupP-value
Gender>0.05
 Men710
 Women23
Age (years)>0.05
 Median (range)74 (45–82)71 (43–81)
Differentiation>0.05
 Well01
 Moderate44
 Poor57
 Mucinous01
Size>0.05
 Mean ± SD5.0±1.86.7±2.5
Stage>0.05
 I10
 II02
 III74
 IV17
Lymphatic invasion44.40%92.30%0.023
Vascular invasion44.40%38.50%>0.05
Neural invasion33.30%69.20%0.192
Follow-up (months)>0.05
 Median (range)12 (4–80)18 (3–36)
Immunohistochemistry

The immunohistochemical staining results for each protein are presented in Table II. E-cadherin had the strongest expression in both groups (100 vs. 69.2%). EGFR expression was lowest in the case group (0%), while the expression levels of ITGB4 and VEGF were lowest in the control group (0%). No significant differences in biomarker expression were detected between cases and controls. However, VEGF expression tended to be higher in the case group (33.3 vs. 0%, p=0.055). The immunoreactivity of N-cadherin and VEGF is shown in Fig. 2.

Table II

Immunostaining.

Table II

Immunostaining.

BiomarkerPositive
P-value
Case (%)Control (%)
E-cadherin10069.20.115
N-cadherin66.746.2>0.05
MSS166.738.5>0.05
Claudin 344.423.1>0.05
Claudin 455.623.1>0.05
Glut 133.330.8>0.05
Clusterin44.438.5>0.05
ITGB422.20>0.05
VEGF33.300.055
EGFR07.7>0.05
p5366.753.9>0.05

[i] Data are expressed as a percentage of cases. VEGF, vascular endothelial growth factor; EGFR, epidermal growth factor receptor.

Survival analysis

Survival analyses with respect to the clinicopathological variables and biomarker expression profiles using the Kaplan-Meier method are summarized in Table III and IV. N-cadherin expression (36 vs. 12 months, p=0.027) and vascular invasion (12 vs. 33 months, p=0.008) were significantly correlated with the median survival.

Table III

Analysis of immunostaining for overall survival.

Table III

Analysis of immunostaining for overall survival.

Median survival (months) Positivity vs. negativityP-value
E-cadherin20 vs. 8>0.05
N-cadherin36 vs. 120.027
MSS120 vs. 16>0.05
Claudin 332 vs. 16>0.05
Claudin 432 vs. 16>0.05
Glut 120 vs. 33>0.05
Clusterin16 vs. 32>0.05
VEGF16 vs. 20>0.05
EGFR20 vs. 32>0.05
p5316 vs. 36>0.05

[i] VEGF, vascular endothelial growth factor; EGFR, epidermal growth factor receptor.

Table IV

Analysis of clinical factors for overall survival.

Table IV

Analysis of clinical factors for overall survival.

Median survival (months)P-value
Age (<70 vs. ≥70)18 vs. 32>0.05
Tumor size (mm)(<50 vs. ≥50)16 vs. 33>0.05
M stage (0 vs. 1)32 vs. 12>0.05
Lymphatic invasion (yes vs. no)20 vs. NR>0.05
Vascular invasion (yes vs. no)12 vs. 330.008
Neural invasion (yes vs. no)16 vs. 330.061
Brain metastasis (yes vs. no)16 vs. 20>0.05

[i] VEGF, vascular endothelial growth factor; EGFR, epidermal growth factor receptor; NR, not reported.

Modulation of VEGF and N-cadherin by metformin in tumor-bearing mice

As shown in Fig. 3, the inoculation of MK28 cells was found in the mouse brain. The level of VEGF mRNA was decreased in the treatment group compared with the controls. E-cadherin was increased in the treatment group, while N-cadherin did not alter after administration of metformin. To investigate changes in epithelial-to-mesenchymal transition (EMT), expression of the mesenchymal marker MMP9 was assessed. The MMP9 expression was decreased in the metformin-treated group compared with the controls. These findings are compatible with the western blotting results (Fig. 4).

Discussion

The present study evaluated the correlations between clinicopathological findings and survival in brain metastasis of advanced gastric cancers. The overall survival of all 22 patients with advanced gastric cancer was influenced by vascular invasion and expression of N-cadherin. Our results suggested a role for VEGF expression in gastric cancer brain metastasis. It has been reported that elevated VEGF expression contributes to the ability of breast and prostate cancer cells to metastasize to the brain in nude mice (22,23). Angiogenic factors expressed by metastatic cancers are potent mediators of angiogenesis and vascular permeability in brain metastasis. High VEGF levels in CSF have a very high specificity for leptomeningeal metastasis (24). The role of angiogenesis in brain metastasis is not clear. Bevacizumab monoclonal antibody to VEGF and sunitinib, a multi-kinase inhibitor that affects the VEGF receptor are in clinical trials for treating brain metastasis of breast and lung cancer (25).

Generally, the loss of E-cadherin and the gain of N-cadherin indicate the conversion of tumor cells into a metastatic phenotype; nevertheless, we could not find any correlation between brain metastasis and E- or N-cadherin expression. In 146 gastric cancer patients, including 84 patients with stage I, the 5-year survival rate of the N-cadherin-positive group was significantly worse than that of the N-cadherin-negative group (58 vs. 78%) (26). By contrast, in our series of advanced gastric cancers, the survival of patients with positive N-cadherin immunoreactivity was longer than that of those with negative immunoreactivity, while E-cadherin expression did not correlate with survival. This discrepancy could have arisen since the number of patients we enrolled was very small, and early-stage gastric cancers were excluded from our series.

Of the nine patients with brain metastasis, resection and radiation therapy were performed in two, radiation only in two, intrathecal methotrexate chemotherapy in one and four were conservatively treated. In 19 cases of cytologically confirmed leptomeningeal carcinomatosis, the median survival was 4–8 weeks (27,28). Other authors have described a longer median survival of ~13 months for patients who underwent surgical resection of brain metastasis followed by radiotherapy (29,30). Since the systemic disease was already advanced and multiple lesions were not accessible surgically, it is not difficult to choose aggressive multidisciplinary treatment. The overall survival of patients with and without brain metastasis did not differ in our series of advanced gastric cancers. However, since no standard therapy for gastric cancer brain metastasis has been established, it is important to investigate the predictors of brain metastasis and develop a preventive strategy for gastric cancer brain metastasis. We investigated modulation of the expression of VEGF, N- and E-cadherin, and MMP9 in mouse models of brain metastasis that had been inoculated with gastric cancer cells transcranially.

The invasion and metastasis of solid tumors require EMT for tumor cells to invade and metastasize (31). During EMT, non-motile, polarized epithelial cells embedded via cell-cell junctions in a cell collective dissolve their cell-cell junctions and transform into individual, non-polarized, motile and invasive mesenchymal cells. Previously, we reported that the reduced expression of molecules involved in EMT, such as E-cadherin, could be used to identify patients at high risk for developing brain metastasis of non-small cell lung cancer (32). In the present study, we suggest that VEGF expression is associated with brain metastasis of advanced gastric cancer. Metformin inhibited VEGF expression in MCF-7 breast cancer cells via the AMP-activated protein kinase pathway (33). Metformin inhibits both angiogenesis and the metastatic spread of various cancer cells by preventing EMT (34,35). In tumor-bearing mouse models, metformin administration decreased the expression of VEGF and MMP9. In addition, the expression of E-cadherin was increased, although that of N-cadherin did not significantly change. In mouse models, the dose of metformin administered to the mice (2 mg/25 g/day) is in the same range as that administered to diabetic patients (3 g/75 kg/day) (36). These findings suggest that metformin may prevent or attenuate the EMT process and cell invasiveness.

In conclusion, brain metastasis in advanced gastric cancer was associated with the expression of VEGF. Metformin treatment affects the metastatic capacity of gastric cancers by reducing VEGF expression and blocking EMT.

Acknowledgments

The authors wish to acknowledge the financial support of the St. Vincent's Hospital, Research Institute of Medical Science Foundation (SVHR-2012-06).

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Online ISSN:1791-2431

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Spandidos Publications style
Jun K, Lee JE, Kim SH, Jung J, Choi HJ, Kim YI, Chin H and Yang S: Clinicopathological significance of N-cadherin and VEGF in advanced gastric cancer brain metastasis and the effects of metformin in preclinical models. Oncol Rep 34: 2047-2053, 2015
APA
Jun, K., Lee, J.E., Kim, S.H., Jung, J., Choi, H., Kim, Y.I. ... Yang, S. (2015). Clinicopathological significance of N-cadherin and VEGF in advanced gastric cancer brain metastasis and the effects of metformin in preclinical models. Oncology Reports, 34, 2047-2053. https://doi.org/10.3892/or.2015.4191
MLA
Jun, K., Lee, J. E., Kim, S. H., Jung, J., Choi, H., Kim, Y. I., Chin, H., Yang, S."Clinicopathological significance of N-cadherin and VEGF in advanced gastric cancer brain metastasis and the effects of metformin in preclinical models". Oncology Reports 34.4 (2015): 2047-2053.
Chicago
Jun, K., Lee, J. E., Kim, S. H., Jung, J., Choi, H., Kim, Y. I., Chin, H., Yang, S."Clinicopathological significance of N-cadherin and VEGF in advanced gastric cancer brain metastasis and the effects of metformin in preclinical models". Oncology Reports 34, no. 4 (2015): 2047-2053. https://doi.org/10.3892/or.2015.4191