Research ArticleExperimental Studies
Open Access

Impact of SMAD2 and MET Expression on Lymph Node Metastasis of HER2-positive Gastric Cancer Cells

GEN TSUJIO, MASAKAZU YASHIRO, TAKASHI SAKUMA, RIKA AOYAMA, KOJI MARUO, YURIE YAMAMOTO and KIYOSHI MAEDA
Anticancer Research October 2023, 43 (10) 4359-4364; DOI: https://doi.org/10.21873/anticanres.16631

Abstract

Background/Aim: Intra-tumoral heterogeneity, which is frequently found in various types of cancers, has been suggested to play an important role in cancer progression and metastasis. The findings of our previous study suggested that p-SMAD2 and c-MET signaling might play important roles in the progression to lymph node metastasis of HER2-positive gastric cancer. In this study, we confirmed the effect of SMAD2/MET signaling in the progression of HER2-positive gastric cancer in an animal model. Materials and Methods: NCI-N87 cells over-expressing ERBB2, SMAD2, MET were used. To confirm the role of SMAD2 and MET expression on lymph node metastasis of gastric cancer, we orthotopically injected NCI-N87 cells with or without the knockdown of both SMAD2 and MET into the gastric walls of BALBc nude mice. Results: The number of metastatic lymph nodes was significantly smaller in the knockdown group compared to that in the control group. However, there was no significant difference in gastric tumor size between the two groups. Conclusion: SMAD2 and MET signaling might play important roles specifically in the progression to lymph node metastasis of HER2-positive gastric cancer. c-MET and SMAD2 may be useful targets for preventing lymph node metastasis in patients with HER2-positive gastric cancer.

Key Words:

The intra-tumoral heterogeneity of cancer-associated molecules has been reported to be associated with malignant tumor progression such as chemoresistance and distant metastasis in various types of carcinomas (1-6). In particular, HER2-positive gastric cancer has been reported to show intra-tumoral heterogeneity of HER2 expression ranging from 4.8% to 75.4% (7-11). Various frequencies of the intra-tumoral heterogeneity of HER2 expression have been reported, especially in diffuse type of gastric cancer (10). However, few studies have reported the correlation between intra-tumoral heterogeneity patterns and distant metastasis in HER2-positive gastric cancer (12). HER2 protein over-expression is present in about 20% of gastric cancer cases (13). In HER2-positive gastric cancer, even with para-aortic lymph node metastasis, an effect of anti-HER2 therapy (trastuzumab) is expected (14). On the other hand, HER2-positive gastric cancer patients respond well to trastuzumab at first, but a considerable portion ultimately acquires resistance to this therapy. It has been reported that co-amplification or co-over-expression of other receptor tyrosine kinases (RTKs) besides HER2, such as EGFR, c-MET, and FGFR2, may result in trastuzumab resistance (12). It is hoped that determining the mechanism of the resistance to anti-HER2 therapy will lead to a new, more effective strategy for this disease.

We previously demonstrated a correlation between p-SMAD2 (SMAD2), c-MET (MET) and HER2 (ERBB2) expression and the status of LN metastasis in gastric cancer according to clustering analysis, and revealed that p-SMAD2 and c-MET signaling have an important role in the progression of HER2-positive gastric cancer to LN metastasis (15). Here, we sought to confirm the significance of SMAD2 and MET expression in the progression of HER2-positive gastric cancer in an animal model. The hope was that in vivo confirmation of our findings might help unravel the mechanism of LN metastasis and thereby point the way to new molecular target therapies for HER2-positive gastric cancer.

Materials and Methods

Cell lines. We used six human gastric cancer cell lines and one human gastric fibroblast cell line. NCI-N87, NUGC-4 and OE19 are HER2-positive gastric cancer cell lines whereas MKN45, NUGC-3 and GCIY are HER2-negative gastric cancer cell lines. NCI-N87 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). OE19 was purchased from the European Collection of Authenticated Cell Cultures (Porton Down, UK). MKN45 and NUGC-3 were purchased from the Japanese Collection of Research Bioresources (Osaka, Japan). GCIY was purchased from the RIKEN BioResource Center (Ibaraki, Japan). The fibroblast cell line CAF-128 was established from a patient with type-2 poorly differentiated gastric cancer. CAF-128 was primary-cultured from the tumoral wall of a gastric cancer. The fibroblasts from the third to the fifth passage in culture were used and confirmed by αSMA staining, as previously reported (16). Gastric cancer cells and fibroblasts were incubated in Dulbecco’s modified Eagle’s medium (DMEM; Nikken, Kyoto, Japan) containing 10% fetal bovine serum (Nichirei, Tokyo, Japan), 100 IU/ml penicillin (Wako, Osaka, Japan) and 0.5 mmol/l sodium pyruvate (Wako) at 37°C, 21% oxygen and 5% carbon dioxide.

Quantitative reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated from each cell line using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), and the quality of RNA was analyzed using NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). RT-PCR was performed on an ABI Prism 7000 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol using TaqMan Fast Advanced Master Mix (2×) and TaqMan probes (20×) (Thermo Fisher Scientific). The total reaction volume was 20 μl. The amplification parameters were set at 95°C for 3 s and 60°C for 30 s (40 cycles total). The mRNA level of each gene was normalized by the internal control HPRT1 mRNA.

siRNA for knockdown of SMAD2 and MET. SMAD2 and MET siRNA were transfected into NCI-N87 cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s protocol. The knockdown efficacy was evaluated forty-eight hours after transfection.

Animal models. The effect of SMAD2 and MET on LN metastasis of orthotopic gastric cancer was examined using 4-week-old female BALB/c nude mice (Nihon CLEA, Tokyo, Japan). All experiments were performed according to the standard guidelines for animal experiments of Osaka Metropolitan University Medical School. The orthotopic injection technique was performed using a previously reported method (17). The mouse abdomen was opened via a midline incision under aseptic conditions, and the stomach was exteriorized. Then mice were divided into two groups, a SMAD2/MET knockdown group and a control group. Into the stomachs of the former, we injected NCI-N87 cells in which SMAD2 and MET had been knocked down via transfection with 7.5 nM of SMAD2 and MET siRNA; into the control group we injected unaltered NCI-N87 cells (in which HER2, SMAD2 and MET had been shown to be over-expressed). Specifically, NCI-N87 cells (5×106 cells in 100 μl of DMEM per mouse) were injected into the subserous layer of the middle of the stomach using a 500-μl syringe and 30-G needle. A cotton swab was pressed against the injection site to prevent tumor cell leakage into the peritoneal cavity. Subsequently, the stomach was returned to the peritoneal cavity, and the peritoneum and skin were sutured.

At 10 weeks after tumor cell injection, the mice were sacrificed with an overdose of sevoflurane. Part of each orthotopic tumor and accompanying LNs were fixed in 10% formalin, embedded in paraffin and sectioned for staining and immunohistochemistry. Then we counted the number of metastatic LNs of each mouse. The largest (a) and smallest (b) diameters of the tumors were measured under the microscope with HE staining, and tumor size was calculated using the equation V=a×b2×1/2.

Immunohistochemical staining. Bond Oracle™ HER2 IHC System (Leica Biosystems, Newcastle Upon Tyne, UK) was used for HER2 staining, according to the manufacturer’s instructions. Immunohistochemical staining for c-MET (1:200; Santa Cruz Biotechnology, Dallas, TX, USA) and p-SMAD2 (1:2,000; Chemicon International, Temecula, CA, USA) was performed as follows. First, we performed deparaffinization and slides were heated. After blocking endogenous peroxidase activity, the samples were incubated with an antibody for 1 h at room temperature. Then, the samples were incubated with the biotinylated secondary antibody. The samples were treated with streptavidin-peroxidase reagent, and counterstained with Mayer’s hematoxylin.

Ethics approval and concept to participate. This study was approved by the Medical Ethics Committee of Osaka Metropolitan University (approval no. 924). Patients provided written informed consent, and ethical approval was obtained from the institutional review boards of Osaka Metropolitan University (reference no. 912). This retrospective study was conducted in accordance with the principles of the Declaration of Helsinki.

Statistical analysis. Gene expression data and in vivo data are expressed as the means±standard errors, and significant differences were analyzed using the unpaired Student’s t-test. JMP 13 software (SAS Institute Japan, Tokyo, Japan) was used for the analyses.

Results

Expression level of ERBB2, SMAD2, MET and TGFBR1 in gastric cancer cells. ERBB2, SMAD2, MET and TGFBR1 expression in gastric cancer cells were compared with those in CAF-128 cells. ERBB2 mRNA was over-expressed in OE-19, NUGC-4 and NCI-N87 cells. SMAD2 mRNA was over-expressed in NCI-N87 cells. MET mRNA was over-expressed in MKN45, NUGC-4 and NCI-N87 cells. TGFBR1, part of the upstream signaling pathway of SMAD2, was over-expressed in GCIY and NCI-N87 cells. Thus, NCI-N87 cells were identified as over-expressing all four genes implicated in cancer progression: ERBB2, SMAD2, MET and TGFBR1 (Figure 1A).

Figure 1.
Figure 1.

mRNA expression. A) ERBB2, SMAD2, MET, and TGFBR1 mRNA expression in each cell line. RNA expression level in each gastric cancer cell line relative to RNA expression level in CAF-128 cells. ERBB2 is over-expressed in NCI-N87, NUGC-4 and OE-19 cells. SMAD2 is over-expressed in NCI-N87 cells. MET is over-expressed in NCI-N87, NUGC-4 and MKN45 cells. TGFBR1, part of the upstream pathway of SMAD2, is over-expressed in NCI-N87 and GCIY cells. NCI-N87 is the only cell line in which ERBB2, SMAD2, MET and TGFBR1 are all over-expressed. B) SMAD2, MET, and TGFBR1 mRNA expression in NCI-N87 cells with or without SMAD2 siRNA and MET siRNA. SMAD2 siRNA significantly knocked down SMAD2 expression in NCI-N87cells (p=0.005). MET siRNA significantly knocked down MET expression in NCI-N87 cells (p<0.001).

Knockdown of both SMAD2 and MET. Since NCI-N87 cells over-express ERBB2, SMAD2, and MET, we used them to examine the effect of SMAD2 siRNA on the orthotopic tumor and metastatic lymph modes. SMAD2 and MET expression were significantly decreased following transfection with SMAD2 siRNA (p=0.005) or MET siRNA (p<0.001) (Figure 1B).

Effect of SMAD2 siRNA and MET siRNA on the orthotopic tumor of HER2-positive gastric cancer cells and metastatic lymph modes in vivo. To examine the impact of SMAD2 and MET expression on LN metastasis of HER2-positive gastric cancer in vivo, an orthotopic tumor model was generated by injecting NCI-N87 cells in nude mice. Figure 2A shows macroscopic findings of gastric cancer developed by the orthotopic inoculation of HER2-positive NCI-N87 cells with (knockdown) and without (control) SMAD2 and MET siRNA treatment. Gastric tumors were found at the antrum of the gastric wall in both the control and SMAD2/MET knockdown groups. In the control group, involvement of numerous LNs was detected around the stomach. In contrast, only a few LNs were found around the stomach of the SMAD2/MET knockdown group (Figure 2A).

Figure 2.
Figure 2.

Effect of SMAD2 siRNA and MET siRNA on the orthotopic tumor and metastatic lymph nodes in a mouse model. A) Macroscopic findings in mice with orthotopic gastric cancers generated following injection of NCI-N87 cells. In both groups, thickening of the gastric wall from the body to the antrum is observed (arrow). In the control group, numerous LNs around the stomach (arrowhead) are observed. In the SMAD2/MET knockdown group, only a few LNs around the stomach are observed. B) Difference in the number of metastatic lymph nodes and size of primary orthotopic gastric cancer. The number of metastatic lymph nodes is significantly lower in the knockdown group than in the control group (p=0.004). No statistically significant between-group difference in the size of the primary orthotopic gastric tumor was found (p=0.808). C) Immunohistochemical staining for mouse orthotopic gastric cancers generated following injection of NCI-N87 cells. In the control group, mouse orthotopic gastric cancers stained HER2-positive, p-SMAD2-positive and c-MET-positive. In the SMAD2/MET knockdown group, mouse orthotopic gastric cancers stained HER2-positive, p-SMAD2-weak and c-MET-weak.

The number of metastatic LNs was determined using HE stain, and the size of the primary gastric tumor was measured. The average number of metastatic LNs was 4.8 in the control group and 1.0 in the SMAD2/MET knockdown group. The average tumor size of the primary orthotopic gastric cancer was 0.66 cm3 in the control group and 0.76 cm3 in the SMAD2/MET knockdown group. The number of metastatic LNs in the SMAD2/MET knockdown group was significantly small compared to that in the control group (p=0.004). However, there was no significant difference in the size of the primary orthotopic gastric tumors between the control and SMAD2/MET knock-down groups (p=0.808) (Figure 2B).

Figure 2C shows microscopic findings of the orthotopic tumor and metastatic LNs in vivo. The control orthotopic tumors introduced via NCI-N87 cells were identified as moderately differentiated using H&E staining and positive for HER2, p-SMAD2 and c-MET. The orthotopic tumors introduced by NCI-N87 cells treated with SMAD2 and MET siRNA showed weak p-SMAD2 and c-MET expression compared to those of the control group while HER2 expression was the same as in the control group (Figure 2C).

Discussion

In this study, NCI-N87 cells showed expression of SMAD2, MET, and ERBB2. The knockdown of SMAD2 and MET expression was found to suppress LN metastasis of mouse orthotopic gastric cancers but did not affect the primary tumor size. These findings suggest that SMAD2 and MET signaling might be associated with LN metastasis, but they do not affect the growth of the primary tumor in HER2-positive gastric cancer. The correlation between HER2 signaling and SMAD2 and MET signaling might play an important role for the malignant progression of HER2-positive gastric cancer. In a previous study, we suggested a correlation between p-SMAD2 (SMAD2), c-MET (MET) and HER2 (ERBB2) expression and LN metastasis in gastric cancer according to clustering analysis (15). In this study, we were able to confirm these results in an animal study.

c-MET is known as a hepatocyte growth factor (HGF) receptor. It is a dimeric transmembrane polypeptide consisting of an α-chain and a β-chain (18). HGF signaling pathway has been implicated in tumor progression, angiogenesis and metastasis. It has been reported that c-MET is frequently expressed in HER2-positive gastric cancer (19-21). MET amplification has been suggested to be associated with resistance against HER2 therapy in gastric cancer (22, 23). c-MET might be a potent tumoral lymphangiogenesis factor, which stimulates LN metastasis as previously reported (24, 25).

SMAD2 is phosphorylated following activation of TGF-β1/TGFβ1 receptor signaling and is involved in tumor progression (26-28). In breast cancer, HER2 and the TGFβ1/Smad signaling pathway are correlated and have synergistic effects in cancer progression (29, 30). It has also been reported that, in gastric and kidney cancers, the TGF-β1 pathway induces VEGF-C, which contributes to tumoral lymphangiogenesis around the tumor (31, 32).

As discussed above, HGF/c-MET and TGF-β/SMAD signaling pathways have been shown to be correlated with LN metastasis (24, 25, 31, 32). Our study clarified that HGF/c-MET and TGF-β/SMAD pathways may play important roles in inducing LN metastasis in HER2-positive gastric cancer, resulting in the poor prognosis found in our previous study (15). These findings may help elucidate the mechanism of LN metastasis and identify new target molecules for HER2-positive gastric cancer drug development. The expression of SMAD2 and c-MET may also serve as predictive factors for LN metastasis in patients with HER2-positive gastric cancer. Combination therapy with a c-MET inhibitor or a SMAD2 inhibitor with a HER2 inhibitor may be a useful treatment to prevent cases of HER2-positive gastric cancer from progressing to LN metastasis.

In conclusion, SMAD2 and MET expression in HER2-positive gastric cancer might play important roles in LN metastasis. This study may contribute to the development of a novel targeted drug therapy for patients with HER2-positive gastric cancer.

Footnotes

  • Authors’ Contributions

    GT and MY designed and performed the experiments and co-wrote the manuscript. GT and MY contributed equally. TS, RA, KM, YY, and KM collected the tumor specimens and contributed to the in vitro experiments. KM suggested and co-designed the study. All Authors read and approved the final manuscript.

  • Conflicts of Interest

    The Authors declare that they have no competing interests in relation to this study.

  • Funding

    This study was funded in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS KAKENHI, nos. 18H02883 and 21H03008). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

  • Received July 16, 2023.
  • Revision received August 3, 2023.
  • Accepted August 4, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Impact of SMAD2 and MET Expression on Lymph Node Metastasis of HER2-positive Gastric Cancer Cells
GEN TSUJIO, MASAKAZU YASHIRO, TAKASHI SAKUMA, RIKA AOYAMA, KOJI MARUO, YURIE YAMAMOTO, KIYOSHI MAEDA
Anticancer Research Oct 2023, 43 (10) 4359-4364; DOI: 10.21873/anticanres.16631
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