Claudin‐11 Enhances Invasive and Metastatic Abilities of Small‐Cell Lung Cancer Through MT1‐MMP Activation

Abstract

ABSTRACT

Small‐cell lung cancer (SCLC) is an aggressive tumor characterized by the frequent development of distant metastases. This study aimed to explore the mechanism of SCLC metastasis using an originally developed orthotopic transplantation model with DMS273 cells. An analysis of G3H cells, a highly metastatic subline of DMS273 cells, revealed that claudin‐11 promotes the invasive and metastatic ability of the cells. Further analysis revealed that membrane type 1‐matrix metalloproteinase (MT1‐MMP), which degrades a wide range of extracellular matrix components, was coprecipitated with claudin‐11. Gelatin zymography revealed that claudin‐11 enhanced MT1‐MMP activity, and MT1‐MMP silencing suppressed the invasive and metastatic ability of G3H cells. Moreover, in MT1‐MMP silencing DMS273 cells, the enhancement of invasion and metastatic potential induced by CLDN11 overexpression was abolished. These results demonstrate that claudin‐11 enhances the invasive capacity of the cells by activating MT1‐MMP, which promotes metastatic formation in the orthotopic transplantation model. Additionally, claudin‐11 expression was detected in SCLC tumor samples, and higher expression of CLDN11 correlated with poor prognosis in patients with SCLC. These findings suggest that the claudin‐11/MT1‐MMP axis plays an important role in SCLC pathogenesis.

Abbreviations

• MMP
• matrix metalloproteinase
• SCLC
• small cell lung cancer
• T1-MMP
• membrane type 1-matrix metalloproteinase

Introduction

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with a 5-year overall survival rate of 5%–10% and is highly metastatic [1–3]. Thus, the development of novel treatment strategies for preventing metastasis is necessary. Clarifying the mechanism of SCLC metastasis is crucial to developing novel treatment strategies. A new orthotopic transplantation model using the human SCLC cell line DMS273 was developed for further studies on SCLC metastasis [4]. This orthotopic model can be used to analyze all steps of cancer metastasis. In this model, metastatic foci were found in distant tissues, such as bone and brain, as observed in patients with SCLC. Therefore, this model is an effective tool for investigating SCLC metastasis. Interferon-induced transmembrane protein 1 (IFITM1) was identified as a prometastatic factor in SCLC using this model [5].

Claudin-11 is a member of the claudin family, which has four transmembrane domains and is a crucial component of tight junctions [6–8]. Claudin-11 is expressed mainly in the central nervous system (CNS) myelin, inner ear, and Sertoli cells of the testes under normal physiological conditions [9–11]. Particularly, claudin-11 constitutes approximately 7% of the total CNS myelin protein and is the third most abundant protein in CNS myelin [12]. Claudin-11 knockout mice exhibit hindlimb weakness, severe deafness, and hypogonadism due to tight junction dysfunction in these tissues [11–13]. In cancer, hypermethylation of CLDN11 and downregulation of its expression are associated with increased cell motility and invasiveness in gastric cancer and nasopharyngeal carcinoma cells [14, 15]. Additionally, claudin-11 suppression by siRNA enhances glioblastoma stem-like cell growth [16]. On the contrary, claudin-11 expression is induced by the epithelial–mesenchymal transition transcription factor Snail, and claudin-11 promotes SRC activation and collective migration in squamous cell carcinoma [17]. Therefore, claudin-11, like other claudins [18, 19], can play a role in cancer suppression and promotion. However, its role in SCLC remains unknown.

Membrane type 1-matrix metalloproteinase (MT1-MMP, also known as MMP14) has been identified as a tumor-specific proMMP-2 activator [20] and plays an important role in cancer progression and metastasis [21, 22]. MT1-MMP expression is associated with poor prognosis in many cancers, including SCLC [23, 24]. In this study, we aimed to identify the mechanism of SCLC metastasis using the orthotopic transplantation model with DMS273 cells and found that claudin-11 enhances the invasive and metastatic abilities of the cells through MT1-MMP activation.

Materials and Methods

In Vitro Growth Assay

For the in vitro cell growth assay, cells were seeded in normal 96-well plates (SUMILON, Tokyo, Japan) at a density of 1 × 10³ cells/well in 100 μL of Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum. After 72 h of culture, cell growth was determined using the MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay.

Matrigel Invasion Assay

An analysis of in vitro invasion abilities was performed using a Transwell chamber culture system (Becton, Dickinson & Company, Franklin Lakes, NJ, USA) as described previously with some modifications [4, 5]. The upper chambers were placed on 24-well culture plates filled with 0.75 mL of the conditioned medium of G3H cells [4]. The cells dissociated with 0.5 mM ethylenediaminetetraacetic acid were suspended in 0.5 mL of serum-free DMEM and added to the upper chamber (2 × 10⁴ cells in the control chamber; 1 × 10⁵ cells in the Matrigel chamber). After 22 h of culture, the migrated cells on the lower surface of the filter of the upper chambers were fixed in 2.5% glutaraldehyde and stained with 4′,6-diamidino-2-phenylindole dihydrochloride. The migrated cells were counted using a fluorescence microscope (Leica Microsystems) at × 100 magnification, and the average number of cells in the four fields was taken for each well. Percentage invasion was calculated with the following equation: (Mean number of cells invading through the Matrigel chamber) × 0.2 × 100/(Mean number of cells migrating through the control chamber).

Plasmid Construction and Lentivirus Preparation

For shRNA expression, an SV40 promoter-driven puromycin-expressing lentivirus vector based on pLenti6/V5-GW/lacZ (Invitrogen). An shRNA expression cassette was inserted into the defective U3 region of the 3′ long terminal repeat of the vector. The target sequences of shRNAs were as follows: shCLDN11#1, 5′-GACTTTATGCAATAAATAACA-3′; shCLDN11#2, 5′-CCTTCAAGGTCATTTACTTGT-3′; and shLacZ, 5′-GCAGTTATCTGGAAGATCAGG-3′. The CSII-CMV-MCS-IRES2-Bsd vector, kindly provided by H. Miyoshi (RIKEN Tsukuba Institute), was used for cDNA expression. Claudin-11 cDNA was amplified from a cDNA pool of DMS273 cells by PCR with a 3 × FLAG-tag or HA-tag sequence-flanked 5′-primer and cloned into CSII-CMV-MCS-IRES2-Bsd at the NheI and XhoI sites. The C-terminally 17 aa truncated (del191-) and the Y191F/Y192F (YY/FF) claudin-11 mutants were also generated by PCR and cloned accordingly into the CSII-CMV-MCS-IRES2-Bsd vector. FLAG-tagged wild-type MT1-MMP cDNA was amplified from pSG5 vector-based constructs [25] and cloned into CSII-CMV-MCS-IRES2-Bsd at the NheI and EcoRI sites. VSV-G pseudotyped replication-defective lentiviruses were prepared as described previously [5].

Statistical Analysis

Statistical analysis was performed using the Student's t-test, Fisher's exact test, and Wilcoxon rank-sum test. A p value < 0.05 indicated statistical significance.

Other Methods

Additional methods are described in Document S1.

Results

Claudin-11 Is Highly Expressed in the Potent Metastatic Subline of DMS273 Cells

An orthotopic SCLC metastatic model was established using the human SCLC cell line DMS273, and G3H, a highly metastatic subline, was isolated from a bone metastatic focus of this model [4]. The G3H subline exhibited enhanced metastatic potential in the orthotopic model compared with parental DMS273-GFP cells (Figure 1a). The in vitro phenotype, including growth rate and invasiveness, was investigated. The results showed that G3H has a higher invasive ability than DMS273-GFP (Figure 1b).

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Total RNA from both cells was extracted, and the samples were subjected to mRNA microarray analysis to establish the mRNA expression profiles of G3H cells and parental DMS273-GFP cells (Figure 1c). This analysis identified 90 probes demonstrating a > 3-fold change (p < 0.01), with 57 probes upregulated and 33 probes downregulated in G3H cells compared with DMS273-GFP cells (Figure 1c). Among the upregulated genes, claudin-11 was of particular interest because of the absence of literature on its role in SCLC. Claudin-11 has been implicated in cancer progression in squamous cell carcinoma [17], indicating its potential role in SCLC.

RT-qPCR analysis of claudin-11 was performed to validate the mRNA microarray analysis results. The analysis revealed a significant increase in claudin-11 mRNA levels, approximately 2.5-fold, in G3H cells compared with parental DMS273-GFP cells (Figure 2a). Furthermore, Western blot analysis confirmed elevated expression of claudin-11 at the protein level in G3H cells (Figure 2b). These findings indicate that claudin-11 is highly expressed in the metastatic G3H subline compared to parental DMS273-GFP cells at both the mRNA and protein levels.

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Subsequently, the subcellular localization of claudin-11 in G3H cells was examined using immunofluorescence. Claudin-11 was predominantly located in the plasma membrane at intercellular boundaries and partially colocalized with the tight junction protein ZO-1 (Figure 2c). Additionally, immunohistochemistry of tumors from the SCLC orthotopic metastasis model using G3H cells revealed clear claudin-11 staining on the plasma membrane in both orthotopic and metastatic tumors (Figure 2d). These findings indicate that claudin-11 is expressed in tumors from the orthotopic metastasis model.

Increased Claudin-11 Expression Is Associated With Poor Prognosis in Patients With SCLC

Next, claudin-11 expression in human lung cancer cell lines and patients with SCLC was investigated. Western blot analysis of eight human lung cancer cell lines (4 SCLC cell lines: DMS273, DMS53, DMS114, and NCI-H69 and 4 non-SCLC (NSCLC) cell lines: A549, NCI-H23, NCI-H460, and NCI-H226) revealed that claudin-11 was detectable in three of the four SCLC cell lines but was not detected in the 4 NSCLC cell lines (Figure 3a). Claudin-11 expression in patients with SCLC was evaluated with a commercially available human SCLC tissue array (Figure 3b), which contained a total of 81 SCLC lung tumors from 45 patients. Claudin-11 protein was detected in > 30% of SCLC tumors but was not detected in normal lung tissues.

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Analyzed the clinical expression profile data (GSE69091) from the cBioPortal database () was performed (Figure 3d) [26]. In this dataset, claudin-11 mRNA expression data in lung tumors and overall survival data from 77 patients with SCLC were available. The analysis revealed that some patients with SCLC exhibited high expression of CLDN11 mRNA. Patients with strong CLDN11 expression showed a significant decrease in overall survival compared with those with low/negative CLDN11 expression (p = 0.0425, log-rank test; Figure 3d). Furthermore, another clinical expression profiling dataset (GSE43346) was analyzed [27]. In this study, 23 patients with SCLC were classified based on the expression levels of several genes into two groups: Group L, which had low JUB and high GRP expression, and Group H, which had high JUB and low GRP expression. Group L showed shorter overall survival than Group-H [27]. Additionally, CLDN11 expression was higher in Group L than in Group H (p < 0.01, student's t-test, Figure 3e). Thus, claudin-11 is expressed in part in lung tumors of patients with SCLC, and its expression correlates with poor prognosis.

Claudin-11 Enhances the Invasive Ability and Promotes the Formation of Metastatic Tumors in DMS273 Cells

CLDN11-overexpressing cells were established using the parental DMS273-GFP cell line to investigate the role of claudin-11 in the model (Figure 4a). These CLDN11-overexpressing cells exhibited similar growth rates in vitro and notably increased the invasive ability compared with the control vector cells (Figure 4b,c). In the orthotopic model, CLDN11-overexpressing cells showed a higher incidence of metastasis and a higher number of organs positive for metastasis than the control cells (Figure 4d). However, the orthotopic tumor growth rates of both cell types were similar (Figure 4e). The effect of CLDN11 overexpression on lung metastasis formation was assessed using an experimental metastasis model with tail vein injection in nude mice (Figure 4f). CLDN11-overexpressing cells exhibited significantly more nodules than the control vector cells (Wilcoxon rank-sum test, p < 0.01). Additionally, the subcutaneous tumor growth capacity of CLDN11-overexpressing and control vector cells was similar (Figure 4g). These findings indicate that CLDN11 overexpression enhances the invasion ability and distant metastatic formation. However, it does not affect the tumor growth of DMS273-GFP cells.

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Next, we generated stable CLDN11-silenced G3H cells using shRNA (Figure 5a,b). CLDN11-silenced cells showed in vitro 2D growth rates similar to control cells (Figure 5c). CLDN11-silenced cells showed a decreased invasive ability compared with control shRNA-transfected cells (p < 0.05, student's t-test, Figure 5d). In the orthotopic model, CLDN11-silenced cells demonstrated significantly lower metastatic incidence (p < 0.005, Wilcoxon rank sum-test, Figure 5e) and fewer organs positive for metastasis (p < 0.05, Wilcoxon rank-sum test, Figure 5e). However, the orthotopic tumor growth rates of the two cell types were similar (Figure 5f). Therefore, CLDN11 silencing suppresses the invasive and distant metastatic abilities of G3H cells. The findings of both shRNA silencing and overexpression experiments indicate that claudin-11 enhances the invasive ability and promotes the formation of metastatic tumors in DMS273 cells.

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Claudin-11 Interacts With and Stimulates MT1-MMP

To further investigate the underlying mechanisms by which claudin-11 enhances the invasive ability and metastatic formation of DMS273 cells, immunoprecipitation experiments were performed to explore claudin-11-interacting proteins using FLAG-claudin-11-overexpressing DMS273-GFP cells. MT1-MMP plays an important role in invasion and metastasis in various cancers [21, 24], and certain claudins have been reported to stimulate MT1-MMP-mediated proMMP-2 activation [28]. Therefore, whether MT1-MMP could be coprecipitated with FLAG-claudin-11 was examined. Endogenous MT1-MMP was coprecipitated with FLAG-claudin-11 (Figure 6a). Under our experimental conditions, endogenous SRC protein, a reported claudin-11 interacting protein in squamous cell carcinoma cells [17], was coprecipitated with FLAG-claudin-11 (Figure 6a). Furthermore, when endogenous MT1-MMP in G3H cells was immunoprecipitated with an MT1-MMP antibody, coprecipitation of endogenous claudin-11 was observed (Figure 6b).

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Next, a gelatin zymography assay was used to determine the effect of claudin-11 on MT1-MMP activity. The level of active MMP2 was significantly higher in CLDN11-overexpressing DMS273-GFP cells than in control vector-transfected cells, and this activation was diminished with the MMP inhibitor BB94 treatment (Figure 6c). In contrast, CLDN11-knockdown in G3H cells had no effect on MT1-MMP protein levels but decreased active MMP2 levels (Figure 6d,e). Additionally, the interaction between claudin-11 and MT1-MMP in HEK293T cells was assessed by cotransfecting HA-CLDN11 and FLAG-MT1-MMP cDNAs and performing immunoprecipitation with anti-FLAG antibodies. Coprecipitation of HA-claudin-11 with FLAG-MT1-MMP was observed (Figure 6f). Gelatin zymography in cotransfected HEK293T cells showed that claudin-11 stimulates MT1-MMP activity (Figure 6g). These results demonstrate that claudin-11 interacts with and stimulates MT1-MMP.

Furthermore, the amount of MT1-MMP on the cell surface in cotransfected HEK293T cells was measured using a water-soluble biotin labeling compound. Cell surface MT1-MMP was more abundant in CLDN11-cotransfected cells than in those without CLDN11 transfection (Figure 6h).

Claudin-11-MT1-MMP Axis Contributes to the Invasive and Metastatic Potential of DMS273 Cells

Whether MT1-MMP affects the invasive and metastatic abilities of G3H cells was investigated. In the Matrigel invasion assay, MT1-MMP-silenced G3H cells exhibited significantly lower invasive activity than control cells (p < 0.01, student's t-test, Figure 7a). In the orthotopic model, MT1-MMP-silenced cells showed significantly fewer organs positive for metastasis than control cells (p < 0.05, Wilcoxon rank sum-test, Figure 7b). However, the orthotopic tumor growth rates were similar between the two cell types (data not shown). Next, we examined whether claudin-11 enhances invasion and metastasis even in MT1-MMP-silenced DMS273-GFP cells (Figure 7c). In cells expressing control shRNA, CLDN11 overexpression significantly enhanced invasive capacity. However, this effect was abolished in MT1-MMP-silenced cells (Figure 7d). No significant difference was observed in the in vitro proliferative potential of these cells (data not shown). In the orthotopic metastasis model, CLDN11 overexpression failed to enhance metastatic potential in the MT1-MMP-silenced DMS273-GFP cells (Figure 7e,f). These findings suggest that MT1-MMP is essential for claudin-11-induced invasion and metastasis in DMS273 cells.

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To further investigate the interaction between claudin-11 and MT1-MMP, two mutants of the C-terminal cytoplasmic domain of claudin-11 with 3 × FLAG-tag (one with two phosphorylatable tyrosines (Tyr191 and Tyr192) substituted with phenylalanine (YY/FF) and the other with a deletion mutant of amino acids 191–206 (del191-)) were generated (Figure 8a). Overexpression of wild-type claudin-11 and the mutants did not significantly affect the amount of MT1-MMP protein in the DMS273-GFP cells (Figure 8b). Immunocytochemistry using an anti-FLAG antibody revealed that the YY/FF mutant, similar to the wild type, predominantly localized at cell boundaries, whereas the del191- mutant exhibited reduced localization to these regions (Figure 8c). Immunoprecipitation analysis revealed that endogenous MT1-MMP proteins were coprecipitated with the YY/FF mutant claudin-11 similarly to wild-type claudin-11, but to a lesser extent with the del191- mutant (Figure 8d). Consistent with the immunoprecipitation results, the YY/FF mutant stimulated MT1-MMP-mediated proMMP-2 activation similar to the wild type, whereas the del191- mutant did not (Figure 8e). Thus, the C-terminal 191–206 aa of claudin-11 is important for the activation of MT1-MMP, whereas the phosphorylatable tyrosines in this region are not.

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The effects of overexpression of these mutants in DMS273-GFP cells on their Matrigel invasive ability were analyzed. Both mutants failed to enhance invasion (p < 0.05, student's t-test, Figure 8f). These results suggest that the enhancement of invasion by claudin-11 requires not only MT1-MMP activation but also additional events involving the phosphorylation at Tyr191/192.

Discussion

The results showed higher claudin-11 expression in the potent metastatic subline G3H cells than in the parental cells. Additionally, claudin-11 enhanced the invasive and metastatic abilities of DMS273 cells. Furthermore, claudin-11 expression correlated with poor prognosis in patients with SCLC, indicating its involvement in SCLC pathogenesis, particularly in frequent metastasis.

Previous studies have explored claudin-11 function in various cancers. In gastric and nasopharyngeal cancers, hypermethylation of the promoter region and reduced mRNA expression were noted in cancerous regions compared to non-cancerous regions [14, 15]. Claudin-11 has been reported to suppress cell proliferation in glioblastoma stem-like cells [16]. Conversely, Li et al. showed that claudin-11 promotes collective migration and contributes to the progression of head and neck squamous cell carcinoma (HNSCC) [17]. Thus, similar to other members of the claudin family, claudin-11 can act as a tumor suppressor and promoter [18, 19]. Our results clearly indicate that claudin-11 enhances metastatic formation in a xenograft model of the human SCLC cell line DMS273.

MT1-MMP was identified as a coprecipitating protein with claudin-11 (Figure 6a,b,f). Increased tumoral expression of MT1-MMP has been reported to be a negative prognostic factor for the survival of patients with SCLC [23, 29]. Claudin-11 enhances MT1-MMP-mediated proMMP-2 activation (Figure 6c,d,g) and promotes the invasive and metastatic ability in DMS273 cells but does not in MT1-MMP-silenced cells (Figure 7d,f). These results indicate that claudin-11 enhances the invasive and metastatic ability of DMS273 cells by enhancing MT1-MMP activity. Similarly, Claudins-1, −2, −3, and −5 stimulate pro-MMP-2 activation by MT1-MMP in HEK293T cells [28], and claudin-1 activates MT1-MMP and MMP-2 in human oral squamous cell carcinoma cells [30]. Claudins are classified as classical or non-classical based on amino acid sequences [18], and claudin-1,-2,-3, and -5 are classical claudins. In contrast, claudin-11 is a non-classical claudin and the first of its kind to activate MT1-MMP.

A deletion mutant of the intracellular C-terminus domain of claudin-11 (del191-) exhibited partial co-precipitation with MT1-MMP and showed a reduced ability to enhance MT1-MMP activity and the invasive potential of DMS273 cells (Figure 8d–f). This mutant showed reduced localization at cell boundaries compared to the wild type (Figure 8c), which may underlie its diminished MT1-MMP stimulation. In HEK293T cells, wild-type claudin-11 overexpression increased MT1-MMP localization on the cell surface (Figure 6h). A possible hypothesis from these results is that claudin-11 may stimulate MT1-MMP activity by facilitating its localization to the cell surface. However, further investigation is required to elucidate the precise molecular mechanism.

Within the deleted region of the del191- mutant, two phosphorylated tyrosine residues (Y191/Y192) were identified [17]. The nonphosphorylatable claudin-11 mutant (Y191F/Y192F) interacted with and activated MT1-MMP but did not enhance the invasive ability of DMS273-GFP cells (Figure 8d–f), indicating that additional events are required for the enhanced invasive ability induced by claudin-11. Tyrosine-phosphorylated claudin-11 has been reported to activate SRC kinase and p190RhoGAP in HNSCC [17]. These results are similar to those of our study, which showed coimmunoprecipitation of SRC with claudin-11 in DMS273-GFP cells (Figure 6a), indicating that SRC signaling activation might be crucial for the enhanced invasive ability induced by claudin-11.

High claudin-11 expression levels have been reported to correlate with poor prognosis in patients with HNSCC, colon cancer, ovarian cancer, and breast cancer [17, 31]. Immunostaining of a tissue microarray of lung tumors in SCLC revealed claudin-11 expression in some patients with SCLC (Figure 3b,c). Analysis of the mRNA expression and overall survival data of 77 patients with SCLC (GSE69091) revealed an association between higher claudin-11 mRNA expression and shorter survival (Figure 3d). Additionally, elevated claudin-11 expression correlated with poor prognosis in a Japanese study of patients with SCLC (Figure 3e) [27]. These findings suggest that claudin-11 is a tumor-promoting factor in SCLC and a potential prognostic marker. Given that increased MT1-MMP expression is a negative prognostic factor for the survival of patients with SCLC [23], the claudin-11 –MT1-MMP axis has a significant impact on patient survival.

In conclusion, this study demonstrated that claudin-11 stimulated MT1-MMP activity, contributing to the invasive and metastatic potential of DMS273 cells. In the broader context of SCLC, other mechanisms may also be involved. For instance, claudin-11 facilitates the collective migration and circulating tumor cell formation in HNSCC [17], suggesting a possible role in SCLC. Further studies are needed to comprehensively elucidate the functions of claudin-11 in SCLC.

Author Contributions

Shuichi Sakamoto: conceptualization, data curation, formal analysis, funding acquisition, investigation, writing – original draft. Hiroyuki Inoue: investigation. Takahisa Takino: formal analysis, investigation, methodology, writing – review and editing. Yasuko Kohda: investigation. Junjiro Yoshida: investigation. Shun#x2010;ichi Ohba: investigation. Ihomi Usami: investigation. Takeshi Suzuki: supervision, writing – review and editing. Manabu Kawada: supervision, writing – review and editing. Masanori Hatakeyama: supervision, writing – review and editing.

Acknowledgments

We thank Dr. H. Miyoshi (RIKEN) for providing the CSII-EF-MCS-Bsd vector. We also thank Professor Y. Hayakawa (Toyama University), Dr. S. Takagi (JFCR), and Dr. K. Moriwaki (OMPU) for their helpful discussions.

Ethics Statement

Approval of the research protocol by an institutional review board: Animal experiments were approved by the Institute Committee for Animal Experiments at the Institute of Microbial Chemistry and that it conforms to the provisions of the Declaration of Helsinki.

Consent

The authors have nothing to report.

Conflicts of Interest

Masanori Hatakeyama is the Editor-in-Chief of Cancer Science. The others have no conflicts of interest.

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