Mesothelin promotes brain metastasis of non-small cell lung cancer by activating MET

doi:10.21203/rs.3.rs-3664228/v1

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Research Article

Mesothelin promotes brain metastasis of non-small cell lung cancer by activating MET

https://doi.org/10.21203/rs.3.rs-3664228/v1

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published 03 Apr, 2024

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Brain metastasis (BM) is common among cases of advanced non-small cell lung cancer (NSCLC) and is the leading cause of death for these patients. Mesothelin (MSLN), a tumor-associated antigen expressed in many solid tumors, has been reported to be involved in the progression of multiple tumors. However, its potential involvement in BM of NSCLC and the underlying mechanism remain unknown. In this study, we found that MSLN expression was significantly elevated in both serum and tumor tissue samples from NSCLC patients with BM and correlated with a poor clinical prognosis. Our in vitro and ex vivo experiments demonstrated that MSLN significantly enhanced the brain metastatic abilities of NSCLC cells, especially blood–brain barrier (BBB) extravasation. Mechanistically, we found that MSLN facilitated the expression and activation of MET through the c-Jun N-terminal kinase (JNK) signaling pathway, which allowed tumor cells to disrupt tight junctions and the integrity of the BBB and thereby penetrate the barrier. Intriguingly, our in vivo experiments indicated that drugs targeting MSLN (anetumab) and MET (crizotinib) effectively blocked the development of BM and prolonged the survival of mice, with anetumab showing a superior therapeutic effect compared with crizotinib. In conclusion, our results demonstrate that MSLN plays a critical role in BM of NSCLC by modulating the JNK/MET signaling network and thus, provides a potential novel therapeutic target for preventing BM in NSCLC patients.

MSLN

NSCLC

brain metastasis (BM)

blood–brain barrier (BBB)

MET

Lung cancer is one of the most lethal and aggressive malignancies. Histologically, lung cancer is categorized as small cell lung cancer and non-small cell lung cancer (NSCLC), with NSCLC accounting for approximately 85% of the total incidence of lung cancer [1]. Metastasis is the leading cause of death among lung cancer patients, with approximately 50% of lung cancer patients eventually developing brain metastasis (BM) [2]. Despite major advances in methods for lung cancer diagnosis and treatment in recent years, the median survival of lung cancer patients with BM remains only 4–6 months, and thus, treatment of this cancer poses a great challenge to clinicians [3]. The main reason for this high mortality rate is that the existing diagnostic and treatment methods for lung cancer patients with BM are very limited. Therefore, the identification of new targets for lung cancer treatment is urgently needed to improve the prognosis of these lung cancer patients and reduce lung cancer mortality.

The development of BM is a complex, multi-step process that involves tumor cells invading the brain parenchyma through the blood–brain barrier (BBB), entering the brain microenvironment, and sending signals critical to their survival and continued growth. The BBB, which is composed of a network of closely opposed endothelial cells in the cerebral capillaries and characterized by the presence of continuous tight junctions (TJs), serves as a barrier between the peripheral circulatory system and neural tissue, controlling brain homeostasis and blocking entry of neurotoxins and pathogens into the central nervous system [4]. The “opening” of TJs by tumor cells in order to penetrate the BBB and enter the brain microenvironment is the decisive rate-limiting step in the development of BM. However, the underlying mechanisms remain largely unknown.

Mesothelin (MSLN) is a glycophosphatidylinositol (GPI)-anchored glycoprotein that in recent years, has been found to be overexpressed in many types of solid tumors, including lung, esophageal, pancreatic, gastric, biliary tract, endometrial, thymic, colon and breast cancers [5]. Studies have shown that abnormal MSLN expression promotes malignant transformation of tumors as well as tumor cell invasiveness by causing functional changes related to tumor cell proliferation, apoptosis, and metastasis [6–8]. However, it remains unclear whether MSLN is involved in BM of NSCLC.

In the present study, we aimed to investigate the role of MSLN in BM of NSCLC and to reveal the underlying mechanism. We first measured MSLN expression in both serum and tumor tissue samples from NSCLC patients with BM and then investigated the relationship between MSLN expression and clinical prognosis. Next we performed in vitro and ex vivo experiments to determine the effect of MSLN on the metastatic abilities of NSCLC cells, especially their ability to penetrate the BBB. To elucidate the mechanism by which MSLN influences BM of NSCLC, we performed studies to identify signaling pathways in NSCLC cells that were activated by MSLN upregulation. Finally, we tested the effects of administration of a drug targeting MSLN or the related signaling pathway on the development of BM and survival in a mouse model of NSCLC.

Increased MSLN expression correlates with BM of NSCLC

To identify proteins potentially involved in the BM of NSCLC, we employed two NSCLC cell lines, PC9 (EGFR^Dexon19 mutation) and H2030 (K-ras^G12C mutation) cells, to develop a high-brain metastatic subpopulation (PC9-BrM and H2030-BrM, Fig. 1A) and performed further proteomics analyses in PC9 and PC9-BrM cells to characterize the protein expression profile found in BM in our previous work[11, 12]. Our results showed that the expression of MSLN was significantly up-regulated in the protein profiling (Fig. 1B), and the increased MSLN expression was verified by western blotting in both PC9-BrM and H2030-BrM cells compared to the respective parental cells (Fig. 1C). We also measured MSLN expression in a normal human bronchial epithelial cell line (BEAS2B) and in five NSCLC cell lines and found that MSLN was not detected in non-cancerous BEAS2B cells but was clearly expressed in the NSCLC cell lines characterized by aggressive metastatic capacities, such as in H460 cells (Fig. S1), which is regarded as a highly metastatic lung cancer cell line [13].

We next examined the expression of MSLN in clinical samples. Serum samples were collected from 160 patients including 113 patients with NSCLC, 23 with primary brain tumors (PBTs) and 24 healthy controls (HC). The 113 NSCLC patients included 22 cases of early lung cancer (ELC), 25 cases with bone metastasis (BoM), 22 cases with liver metastasis (LM) and 44 cases with lung cancer brain metastasis (LCBM). No significant differences were observed between the subgroups in this cohort with respect to age or sex (Table S2). ELISA results showed that compared with the control groups, the serum MSLN level was significantly increased in LCBM patients (Fig. 1D). We further performed IHC staining on surgical specimens from 36 patients with ELC (BM-, primary tissue) and 34 patients with LCBM (BM+, secondary tissue). The results showed that MSLN expression was significantly higher in the tissues of the BM + group than in those of the BM- group (Fig. 1E). Analysis of the GEPIA public database (http://gepia.cancer-pku.cn/) showed that high MSLN expression in NSCLC patients was significantly associated with low survival (n = 478, Fig. 1F). Together these findings indicate that MSLN is involved in malignant progression of lung cancer and may play an important role in promoting BM in NSCLC.

MSLN promotes the migration and invasion of NSCLC brain metastatic cells in vitro

It is well known that tumor cells undergo epithelial–mesenchymal transition (EMT) to become aggressive and migratory, and this is an important event in tumor metastasis and tumor progression [14]. Our previous study found that the brain metastatic cell line PC9-BrM exhibits a mesenchymal-like phenotype and strong ability to migrate and invade [11], as do H2030-BrM cells as demonstrated in this study (Fig. S2). Some studies have concluded that MSLN plays an important role in tumor migration and invasion [15, 16]. Hence, we hypothesized that MSLN plays a role in the process of BM of NSCLC. In our experiments, knockdown of MSLN significantly reduced the migratory and invasive capacity of brain metastatic lung cancer cells (PC9-BrM and H2030-BrM), whereas overexpression of MSLN increased the migratory and invasive capacity of the parental cells (PC9 and H2030, respectively), as evidenced by the wound healing and Transwell assays (Fig. 2A-C, Fig. S3). These results suggest that MSLN significantly promotes the migration and invasion of NSCLC brain metastatic cells. In addition, we examined the expression of EMT markers by western blotting and found that knockdown of MSLN reversed the mesenchymal phenotype of PC9-BrM cells based on increased E-cadherin expression and decreased N-cadherin and Slug expression (Fig. 2D), while overexpression of MSLN facilitated EMT of PC9 cells (Fig. 2E).

Because the expression of MMP family members is essential for tumor progression and metastasis [15–17], we further analyzed whether MMPs are involved in the MSLN-induced enhanced aggression of brain metastatic cells. We found that MMP7, rather than the more widely studied MMP2/9, was regulated by MSLN. The level of cellular and secretory MMP-7 detected by western blotting and ELISA was significantly decreased upon MSLN deletion in PC9-BrM cells and was increased with MSLN overexpression in PC9 cells (Fig. 2D-F). In summary, our data indicate that MSLN plays a promotive role in the enhanced migration and invasion of NSCLC brain metastatic cells.

MSLN helps brain metastatic cells penetrate the BBB by degrading inter-endothelial TJs

The BBB can restrict the invasion of many pathogens, and therefore, the crossing of the BBB by tumor cells is a critical step in BM [18]. We found that the highly brain metastatic cells were more capable of penetrating an endothelial cell layer than were the parental cells (Fig. S4A) [11]. To further investigate the role of MSLN in BM, we examined the trans-endothelial cell migration ability of brain metastatic cells with or without MSLN interference, using an in vitro BBB model and an ex vivo bionic BBB microfluidic chip model established previously [9]. The in vitro BBB model was established using Transwell chambers coated with hBMVECs and primary human astrocytes (HA-1800), while the ex vivo bionic BBB model was established on a well-designed microfluidic chip where HA-1800 cells were introduced into the brain parenchyma chamber and hBMVECs were introduced into the vascular channels for co-culture with HA-1800 cells under the dynamic flow shear force (Fig. 3A). The results showed that silencing MSLN resulted in a significant decrease in the ability of brain metastatic lung cancer cells to penetrate the endothelium (Fig. 3B, Fig. S4B). Consistently, trans-endothelial migration was significantly increased among parental cells overexpressing MSLN (Fig. 3C, Fig. S4C). These results suggest that MSLN helps metastatic lung cancer cells to cross the BBB. TJs, the key structures that maintain the barrier function of the BBB, mainly consist of ocludin, JAMs, claudins, zonula occludens (ZO), and calmodulin (VE-cadherin), which form a junctional complex to maintain the stability of the barrier. Disruption of the junctional complex leads to the loss of cell–cell contacts and the formation of cellular gaps, which creates the opportunity for tumor cells to cross the BBB [18].

We next investigated whether MSLN in lung cancer cells affected the expression of these junctional complex proteins by hBMVECs. We treated hBMVEC monolayers for 24 h with conditioned medium obtained over a 24-h period from the indicated tumor cells and then observed the distribution of VE-cadherin, JAM-A and claudin-5 expression by immunofluorescence imaging. The image results showed that the distributions of VE-cadherin, JAM-A and claudin-5 became discontinuous and vastly diminished in hBMVEC monolayers treated with conditioned medium derived from tumor cells with relatively high expression of MSLN (Fig. 3D, E). We further quantified the expression of TJ complex proteins by western blotting. Consistent with the immunofluorescence observations, we found that conditioned medium from tumor cells with MSLN knockdown did not induce as much degradation of the cadherin, JAM-A and claudin-5 expression patterns among hBMVEC monolayers (Fig. 3F). Conversely, the degradation of these endothelial junction proteins was significantly increased after treatment of hBMVECs with conditioned medium from MSLN-overexpressing parental cells (Fig. 3G). These results indicate that MSLN promotes the ability of brain metastatic cells to penetrate the BBB by destroying TJ complexes.

Disruptive effect of MSLN on TJs is dependent on MET expression

Studies have shown that MET plays a biological role in the BM of many tumor cells [19, 20], including the ability of the cells to cross the BBB [21]. We assessed the co-expression of MSLN and MET in lung cancer specimens from BM-negative early-stage lung cancer patients (n = 25) and BM-positive patients (n = 25) and found that MSLN and MET expression levels were both elevated in lung cancer tissues of BM-positive lung cancer patients and showed a closely correlation (Fig. 4A-C). In lung cancer cell lines, western blot analysis further confirmed that the expression levels of MET and phosphorylated MET (p-MET) decreased with knockdown of MSLN, whereas overexpression of MSLN promoted the cellular expression levels of MET and p-MET (Fig. 4D-E, Fig. S5A). To determine whether MET is involved in MSLN-regulated tumor cell penetration of the BBB, we knocked out MET in PC9-BrM cells with siRNA (Fig. 4F) and then overexpressed MSLN in PC9-BrM cells in which we had previously knocked down MSLN (PC9-BrM-SH2) (Fig. 4G). In vitro and ex vivo trans-BBB assays showed that MET knockdown inhibited the ability of PC9-BrM cells to penetrate the BBB, whereas MET overexpression relieved the suppression of the trans-BBB ability of PC9-BrM-SH2 cells (Fig. 4H, Fig. S5B). We further evaluated expression of the junctional proteins of the BBB after treatment of hBMVECs with conditioned medium from the indicated metastatic cells. The results showed that MET knockdown inhibited the ability of brain metastatic cells to degrade TJ complexes, whereas overexpression of MET significantly enhanced the TJ complex degrading capacity of PC9-BrM-SH2 cells (Fig. 4I, J). Taken together, these data suggest that the effect of MSLN on tumor cell penetration of the BBB is dependent on MET.

MSLN regulates the expression and phosphorylation of MET through the JNK signaling pathway in brain metastatic cells

Previous studies reported that activation of JNK promotes NSCLC metastasis by activating MMPs [22]. In addition, enhancement of JNK signaling promotes activation of MMP-9, which further promotes the degradation of TJ proteins and leakage of the BBB [23]. To further determine the potential mechanisms underlying the effect of MSLN on MET expression, we detected JNK activity and MET expression in PC9-BrM cells transfected with MSLN-targeted shRNA or treated with a JNK inhibitor, JNK-IN-8, or a MET inhibitor, crizotinib. Western blot analysis demonstrated that both knockdown of MSLN and JNK-IN-8 treatment led to inhibited expression and phosphorylation of MET along with suppression of JNK activity in brain metastatic cells, whereas overexpression of MSLN significantly enhanced JNK activity in these cells (Fig. 5A-B, F, Fig. S5A, C). Notably, JNK-IN-8 treatment did not affect the MSLN expression but did efficiently attenuate MET activation in lung cancer cells overexpressing MSLN, whereas crizotinib had no effect on MSLN expression and JNK signaling (Fig. 5C-D, E-G). Taken together, these results suggest that MSLN regulates the expression and phosphorylation of MET through the JNK signaling pathway in brain metastatic cells.

Anetumab and crizotinib treatments therapeutically inhibit BM in vivo

A previous study demonstrated that anetumab can specifically target MSLN-positive tumors and inhibit tumor growth in subcutaneous and orthotopic xenograft models [24], and another study reported that anetumab has preliminary anti-tumor activity in patients with MSLN-positive solid tumors in a phase I study [25]. The MET inhibitor crizotinib is widely used in the clinical treatment of lung cancer patients, including those with BM [26]. We further evaluated and compared the therapeutic efficacies of MSLN- or MET-targeting therapies in an in vivo preclinical BM model. For establishment of the mouse models, PC9-BrM cells (control) or PC9-BrM cells with MSLN knockdown (shMSLN) were introduced into nude mice by intracardiac injection. On the third day after inoculation, anetumab (0.2 mg/kg, once per week for 5 weeks, intravenously [i.v.]) and crizotinib (5 mg/kg, every 2 days for 4 weeks, intraperitoneally [i.p.]) were administrated separately or jointly to the mice inoculated with PC9-BrM cells. An in vivo BBB leakiness assay was performed by intravenous injection of Texas Red-Dextran (70,000 MW), DyLight 488-Lycopersicon Esculentum Lectin (LEL) on the 10th day. Dextran was used as an indicator of BBB leakiness while LEL was used to label the BBB. The diffused dextran indicated the impared BBB in mice injected by PC9-BrM cells (Control) while the dextran diffusion was significantly suppressed once MSLN and MET are targeted separately or jointly (Fig. 6A). As evidenced by the regular weekly bioluminescence images acquired from the 30th day after inoculation of cancer cells, it was found that anetumab, crizotinib and genetic silencing of MSLN all significantly inhibited the occurrence of BM in vivo and prolonged the survival of mice (Fig. 6B-D). In addition, therapies that targeted MSLN (either pharmacologically or genetically) seemed to offer superior therapeutic effects compared with the MET-targeting inhibitor. However, combined treatment with anetumab and crizotinib did not result in prolonged survival of the mice, with most mice dying within 20 days without any secondary metastases. We speculated that the combination of the two drugs might have had a toxicological effect that shortened the survival period of the mice. Overall, our in vivo results suggest that therapies targeting MSLN exhibited remarkable therapeutic efficacy for inhibiting the BM.

Currently, effective treatment options for BM are very limited, resulting in a poor prognosis for patients with BM. At present, the main tumor types that produce BM are lung cancer, breast cancer and melanoma. Among them, lung cancer is the most common, and BM is the main cause of death among lung cancer patients. The underlying mechanisms mediating lung cancer BM remain unclear due to the unique intracranial microenvironment, which creates highly complex and selective conditions for the invasion of lung cancer cells into the brain. Therefore, there is an urgent need to uncover the molecular mechanism underlying BM from lung cancer in support of the development of new therapeutic strategies to improve the prognosis of these patients.

Given the heterogeneity of primary tumors, only a few cells can successfully form BM. To study this metastatic cell population, we established two cell lines (PC9-BrM, H2030-BrM) that were selectively metastatic to the brain based on the methods of isolation of cells with tissue-metastatic tropism. We found that MSLN expression was significantly increased via proteomics analysis of the BM subgroup PC9-BrM cell line and its parent PC9 cell line constructed in the early stage of our research [27], and this increased expression of MSLN was verified by western blot analysis. In clinical samples, we found that MSLN was specifically highly expressed in the serum and tissues of patients with BM from NSCLC and was associated with poor prognosis.

MSLN, a cell surface glycoprotein, is highly expressed in many tumors and known to promote malignant progression [28–32], while it is found at very low levels in normal human tissues [33, 34]. MSLN has been reported to activate nuclear factor kappa B (NF-κB) in pancreatic cancer, which leads to increased production of the inflammatory factor interleukin (IL)-6 and, at the same time, enhances IL-6/IL-6R transduction signaling pathways, resulting in enhanced proliferation of pancreatic cancer cells [35]. Also, MSLN expression in tumors was shown to be associated with a significant increase in microvessel density in newly formed pancreatic metastatic tissue [36]. Another study demonstrated that MSLN knockdown significantly inhibits in vitro cell adhesion, migration, and invasion (critical steps necessary for metastasis) and also reverses EMT and attenuates stem cell properties among cancer cells [30]. Additionally, binding of MSLN to the mucin MUC16 was found to significantly enhance the motility and invasion of pancreatic cancer cells by selectively inducing the expression of MMP-7 [32]. As MSLN is positively expressed in many malignant tumors, it can have multiple biological functions in the malignant progression of tumors and can be considered as a valid indicator of tumor progression. In the present study, we demonstrated that MSLN overexpression facilitated EMT of lung cancer cells and promoted their expression and secretion of MMP7, thereby increasing the migratory and invasive capacities of PC9-BrM cells.

Tumor cell penetration of the BBB is the rate-limiting step in BM [37, 38]. To study the pathology of BM, we applied a common Transwell model as well as our constructed multi-organ microfluidic chip to study tumor cell extravasation through the BBB. The "BBB" constructed on the chip mimics the physiological microenvironment in terms of structural integrity and barrier function and allows real-time visualization of the entire process of tumor BM, which is not possible with the Transwell system or animal models [9]. Our experiments with both models demonstrated that MSLN promotes the crossing of the BBB by NSCLC cells. Destruction of the BBB is necessary for the migration of tumor cells across the endothelium and is achieved via degradation of brain endothelial cell junction proteins. We treated brain endothelial cells with conditioned medium from brain metastasis cells and found that MSLN knockdown resulted in the inhibition of VE-cadherin, JAM-A and claudin-5 degradation among brain endothelial cells and thereby significantly reduced the number of tumor cells migrating across the endothelial layer.

From the results of the present study, we propose a working model in which MSLN promotes lung cancer cell crossing of the BBB by regulating MET expression through the JNK signaling pathway to enhance the ability of lung cancer cells to disrupt TJs between endothelial cells, thereby achieving BBB penetration for BM (Fig. 6E). JNK belongs to the mitogen-activated protein kinase (MAPK) family and is involved in a variety of cellular processes including cell proliferation, apoptosis, angiogenesis, differentiation, metastasis, invasion and inflammation through the activation of a large number of nuclear and non-nuclear molecules [39–41]. Previous studies have confirmed that sustained abnormal function of JNK contributes to tumorigenesis, particularly metastasis and invasion in many cancers [42–45]. In colon cancer cells, JNK was found to regulate cancer progression and metastasis through EMT. Another study demonstrated that chemokine ligand 7 (CCL7) interacts with chemokine receptor 3 (CCR3) to promote cell proliferation, invasion and migration via the ERK and JNK signaling pathways [44]. In addition, significant upregulation of JNK and p-JNK in PC3 and 22RV1 cells was shown to play an important role in stimulating cell migration and invasion [46]. This evidence suggests that JNK plays an important role in tumor invasion and metastasis.

In multiple malignancies, MET overexpression is associated with tumorigenesis, metastasis and a poor overall prognosis. MET signaling has been reported to be essential for the growth and metastasis of gastric, kidney and breast cancers [47–49] and to promote the expression of stem cell-associated genes in pancreatic cancer [50]. In breast cancer patients, brain metastatic cells expressing high levels of activated MET induce angiogenesis through CXCL1 and IL-8 secretion and specifically promote BM [51]. Increased plasma soluble Met (sMet) concentration were associated with lower overall survival in NSCLC patients [52], supporting the results of other studies showing that MET overexpression and amplification are associated with poor prognosis in NSCLC patients [53–56]. Furthermore, MET knockdown was found to significantly reduce the incidence of BM from lung cancer cells in vitro [57]. Our results showed that MET was the key effector for MSLN/JNK signaling to promote tumor cells penetration of the BBB. However, the specific mechanism by which MET contributes to the destruction of TJs needs to be clarified in further studies. Crizotinib, an FDA-approved small molecule inhibitor of the ALK, MET and ROS1 tyrosine kinases for advanced NSCLC [58–61], has shown satisfactory antitumor activity [62]. In the present study, we found that crizotinib significantly inhibited the occurrence of BM in vivo and prolonged the survival of mice. In addition, a recent study reported sensitivity to crizotinib-targeted therapy in patients with BM from NSCLC with concomitant activation of MET receptors and ALK fusion genes [63]. Together these data support the potential of crizotinib as an effective targeted therapy for NSCLC patients with BM.

Given that MSLN expression is rather limited in most normal tissues but is highly elevated in lung adenocarcinoma tumors and involved in the malignant progression of the tumors, MSLN is a potential target for antigen-specific therapy [64]. The main strategies for targeting MSLN currently include tumor vaccines, antibody-based therapies. and chimeric antigen receptor T-cell (CAR-T) therapies. The combination of the bacteria-based vaccine CRS-207, an attenuated form of a Listeria monocytogenes vector that overexpresses human MSLN, with pemetrexed/cisplatin chemotherapy provided objective disease control in unresectable malignant pleural mesothelioma and induced significant clinical outcomes, suggesting that tumor vaccines may be potential candidates for cancer therapy [65]. Anetumab ravtansine (ARav) is a novel antibody–drug conjugate that is currently undergoing clinical trials for several malignancies that express MSLN. The antibody binds MSLN with high affinity and induces internalization of DM4 (the conjugate combines with ravtansine). Once inside the cell, the SPDB (N-succinimidyl 4-(2-pyridyldithio)) junction is cleaved [66] and DM4 binds to microtubule proteins, disrupting microtubule dynamics and thereby inhibiting cell division and proliferation. In vivo, ARav showed potent antitumor activity against MSLN-expressing mesothelioma, pancreatic and ovarian xenografts from cancer patients [67]. Among immunotherapies, CAR-T therapy is considered one of the most promising new approaches for cancer treatment. CAR-T cells are engineered T cells that produce an artificial T receptor targeting a specific protein. To date, fourth-generation CARs favor the secretion of cytokines (including IL-12 and IL-15) and thus strongly influence the immune components of the tumor microenvironment [68]. Preclinical studies in a mouse model of metastatic pancreatic adenocarcinoma demonstrated that CAR-T cells targeting MSLN can induce tumor cytotoxicity and eradicate lung metastases [69, 70]. In an in situ mouse model of mesothelioma, local intrapleural injection of CAR-T cells targeting MSLN produced potent anti-tumor activity that correlated with their proliferation and persistence after 200 days [71]. Similar results were recently reported in a preclinical model of gastric cancer following peritumor injection of CAR-T cells targeting MSLN [72]. In the present study, we found that anetumab reduced the incidence of lung cancer BM and was superior to crizotinib, effectively prolonging the survival of mice. These results provide strong support for the further investigation of MSLN-targeted therapy for patients with BM from lung cancer.

In conclusion, our study provides evidence that MSLN promotes MET expression via the JNK signaling pathway, which then helps tumor cells degrade TJs of the BBB, thereby promoting the development of BM. According to our findings, MSLN can be used not only as a biomarker for the diagnosis and prognosis of lung cancer, but also as an effective target in therapies for patients with BM in the future.

Cell lines and cell culture

Human bronchial epithelial cells (16HBE cells), human lung microvascular endothelial cells (hPMECs), human lung fibroblasts (HFL1 cells), human monocyte cells (THP-1 cells) and PC9-BrM cells were obtained from the laboratory of Dr. Wang [9]. Normal bronchial epithelial cells (BEAS-2B cells) and lung cancer cell lines (H1299, H2030, PC9, H1975, H460, and H226 cells) were purchased from the Chinese Academy of Medical Sciences (Beijing, China). The brain metastatic lung cancer cell line H2030-BrM was generated by injecting H2030 cells into the left ventricle of immunodeficient mice and isolating the metastatic cells from harvested areas of brain metastases. Human brain microvascular endothelial cells (hBMVECs) and human astrocytes (HA-1800) were purchased from Sciencell (Sciencell, USA) and cultured in the appropriate medium recommended by the manufacturer. The above cell types were cultured at 37°C in humidified air with 5% CO₂. The different cell types were authenticated by short tandem repeat profiling and tested for mycoplasma contamination.

SDS-PAGE and western blot analysis

Cells were lysed in a mixture of radioimmunoprecipitation assay (RIPA) protein lysis buffer (Thermo Scientific) containing the protease inhibitor phenylmethylsulfonyl fluoride (PMSF, Beyotime) and a phosphatase inhibitor cocktail (Sigma-Aldrich) at 4°C for 45 min. The BCA Protein Quantification Kit (Thermo Fisher Scientific) was used to measure protein concentrations. Proteins (30 µg/lane) were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels and transferred to nitrocellulose membranes (Millipore, USA). Each membrane was blocked with protein-free rapid blocking buffer (EpiZyme, Shanghai) for 15 min and then incubated overnight at 4°C with the following antibodies: anti-E-cadherin (1:500, 13-1700, Invitrogen), anti-N-cadherin (1:200, sc-393933, Santa Cruz Biotechnology), anti-Slug (1:500, ab27568, Abcam), anti-matrix metalloproteinase 7 (MMP7, 1:1000, ab205525, Abcam), anti-MSLN (1:3000, ab133489, Abcam), anti-glyceraldehyde phosphate dehydrogenase (GAPDH, 1:5000, 10494-1-AP, Proteintech), anti-cleaved poly(ADP-ribose) polymerase (PARP, 1:1000, #5625, Cell Signaling Technology), anti-cleaved caspase-3 (1:1000, #9661, Cell Signaling Technology), anti-vascular endothelial (VE)-cadherin (1:1000, ab205336, Abcam), anti-junctional adhesion molecule (JAM)-A (1:1000, ab269948, Abcam), anti-claudin 5 (1:1000, ab131259, Abcam), anti-phosphorylated (p)-MET (1:5000, ab68141, Abcam), anti-Met (ab51067, 1:5000, Abcam), anti-p-JNK (1:1000, #4668, Cell Signaling Technology), and anti-t-JNK (1:1000, #9252, Cell Signaling Technology). The next day, the membranes were washed with Tris-buffered saline with Tween 20 (TBST) for 8 min at room temperature twice and then incubated with secondary antibodies (1:5000, SA00001-2, Proteintech; 1:5000, SA00001-1, Proteintech) for 1 h at room temperature. The membranes were then washed three times with TBST for 8 min each, followed by scanning and visualization of the immunoreactivity by enhanced chemiluminescence (ECL, Advansta). Protein expression in three independent experiments was quantified using ImageJ software (National Institutes of Health, USA).

Clinical samples

Surgical specimens and serum samples were collected from the Second Hospital of Dalian Medical University following a standard operation procedure. Written informed consent was obtained from all participants. This study was approved by the Ethics Review Committee of the Second Hospital of Dalian Medical University.

Enzyme-linked immunosorbent assay (ELISA)

Target protein concentrations were measured using a Human MMP7 ELISA Kit (Elabscience) and a Human MSLN ELISA Kit (Omnimabs). We followed the manufacturer’s instructions and measured the optical density (OD) of the solution in each well at 450 nm. Finally, the protein concentration in each sample was obtained by comparison with the standard curve.

Immunohistochemistry (IHC) staining and scoring

Surgical specimens were embedded in paraffin and sectioned for ICH analysis. The tissue sections were dewaxed, hydrated, and rinsed in running water for 10 min. The sections were then soaked in boiling sodium citrate antigen repair solution for 20 min before addition of endogenous peroxidase blocker followed by dropwise addition of normal goat serum working solution for blocking. Solutions of anti-MSLN antibody (1:100, Proteintech, 66404-1-Ig) and anti-c-MET antibody (1:150, Proteintech, 25869-1-AP) were added for incubation overnight at 4°C. The following day, biotin-labeled secondary antibody was added dropwise followed by dropwise addition of HRP-labeled streptavidin working solution. The tissue sections were then stained with DAB, rinsed with tap water, stained with hematoxylin for 20 s, and rinsed again before being dehydrated, cleared, and sealed. IHC images were quantitatively assessed and automatically scored using the IHC Profiler open source plugin[10].

Establishment of stable overexpression and knockdown cell lines

PC9 and PC9-BrM cells were transfected with viral vectors for MSLN overexpression and knockdown, respectively, according to the manual for lentivirus use (Shanghai Genechem Co., Ltd.), and the overexpressed or knockdown cells were screened with puromycin (1 µg/ml) for 1 week. The target sequences of MSLN shRNA1 and shRNA2 were 5'- GGAUGAGCUCUACCCACAATT-3' and 5'-CUUGCUUUCCAGAACAUGATT-3', respectively. PC9-BrM cells were transfected with c-MET-specific small interfering RNAs (siRNAs) (siRNA-1: 5'- GCCUGAAUGAUGAUGACAUUCUTT-3' and siRNA-2:5') using Lipofectamine 2000 - GCUGGUGGCACUUUACUUATT − 3') or control siRNA (GenePharma) for 48 h. MSLN knockdown in PC9-BrM cells (PC9-BrM-SH2) was achieved by transfection of the cells with c-MET plasmid or negative control plasmid using Lipofectamine 2000 for 48 h, after which the cells were screened with G418 for 1 week. The efficiency of overexpression or knockdown was assessed by western blotting.

Wound healing assays

Cells were inoculated in 6-well plates, and once they reached confluency, wounds were created using a sterile 100-µl pipette tip. The cells were washed in suspension with phosphate-buffered saline (PBS) and imaged. After 24 h of incubation in serum-free medium, the healing process of cells migrating to cover the wound area was observed microscopically and imaged. Cellular wound healing rates were analyzed using ImageJ software.

Transwell migration and invasion assays

Cell suspensions with a cell density of 15–20×10⁵ cells/ml in 200 µl of serum-free medium were added to Transwell chambers. For invasion experiments, the Transwell membrane was wrapped in advance with a matrix gel (BD, USA). The lower chamber was then spiked with medium containing 20% fetal bovine serum and incubated for 24 h. The cells were then fixed in 4% paraformaldehyde for 20 min and stained with crystal violet for 20 min. The stromal gel and cells were removed from the Transwell chamber layer with a cotton swab and photographed under a microscope to observe and count the cells. The data were analyzed using ImageJ software.

Trans-endothelial assays

hBMVECs (1×10⁵) and HA1800 cells (1×10⁵) were inoculated in the upper and bottom wells of a Transwell chamber and cultured until complete monolayers had formed. Brain metastatic cells (4×10⁵) in medium containing 1% serum were inoculated in the top inserts, and 500 µl of medium with 20% serum was added to the bottom chamber. After 24 h, the green fluorescent protein (GFP)-labeled BM cells that had invaded via the membrane were photographed on a fluorescent microscope for counting.

Trans-BBB assays on a microfluidic chip

A bionic multi-organ microfluidic chip that allows real-time visual monitoring of the entire BM process was fabricated as previously described [11]. Briefly, tumor cells were edited to stably express green fluorescent protein (GFP), while hBMVECs were labeled red with the Cell Tracker^™ CM-Dil dye (Invitrogen, USA) according to the manufacturer’s instructions. After the biomimetic “lung” organ and “brain” organ were constructed, tumor cells were introduced to the upstream “lung” organ to allow the occurrence of BM. The trans-BBB events were then observed using on inverted fluorescent microscope. The observation starting time was designated as the time when the first cell reached the downstream vascular channel along with the fluid, and the images were captured after 36 h.

Generation of conditioned medium

Tumor cells were incubated in serum-free medium on 6-well plates for 24 h. After 24 h, tumor cell supernatant samples were centrifuged and filtered to remove cellular debris. The collected conditioned medium was then stored at − 80℃ until further use.

Immunofluorescence (IF) staining

For immunofluorescent staining, hBMVECs were washed three times with PBS, fixed in 4% paraformaldehyde, and permeabilized in 0.1% Triton X-100 solution (Sigma, USA) for 10 min. For blocking, 3% BSA solution was added for 30 min, and then cells were incubated with primary antibody (anti-JMA-A, Abcam; anti-VE-cadherin and anti-claudin 5, Invitrogen; 1:50 dilution) overnight at 4°C. After three washes with PBS, the cells were incubated in solution of fluorescein isothiocyanate (FITC)-labeled secondary antibody (1:100 dilution; Proteintech, USA) at room temperature. Cell nuclei were stained with 1 µg/ml DAPI (40,6-diamidino-2-phenylindole; Sigma, USA) for 10 min at room temperature. Images were obtained using a confocal microscope (Leica TCS SP5II, Germany).

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Total RNA was extracted from different groups of cells and reverse transcribed to cDNA using the One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311, Transgen Biotech, Beijing, China) according to the manufacturer’s protocol. The relative levels of MSLN mRNA transcripts, c-MET mRNA transcripts, and GAPDH transcripts were quantified by qRT-PCR using Top Green qPCR SuperMix (AQ131, Transgen Biotech) and the following specific primers. The primer sequences were: h-MSLN-F 5'-CTGGAAGCCTGCGTGGAT-3′ and h-MSLN-R 5'-CCAGGTGCTGGATCACAGACT-3′; h-MET-F 5'- TCCAGGCAGTGCAGCATGTA-3′ and h-MET-R 5'-TCAAGGATTTCACAGCACAGTGA-3′; h-GAPDH-F 5'-CATGAGAAGTATGACAACAGCCT-3′ and h GAPDH-R 5'-AGTCCTTCCACGATACCAAAGT-3′. All data were analyzed by the 2^-ΔΔCt method.

Animal study

Four-week-old female BALB-c-nu mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). The PC9 cell line stably expressing GFP-luciferase fusion protein was constructed [11], cultured and collected in a cell suspension of 10⁷ cells/ml. After the mice were anesthetized, 100 µl of cell suspension was injected into the left ventricle of each mouse. After retro-orbital injection of D-Luciferin (150 mg/kg body weight; Promega, USA) at the indicated time points, images of mouse tumor metastases were acquired using the IVIS Spectrum Xenogen instrument (PerkinElmer, USA). In vivo imaging software (version 2.50) was used to analyze the bioluminescence images. All animal experiments were performed in accordance with a protocol approved by the Animal Protection and Use Committee of Dalian Medical University.

BBB leakiness assay

Mice were injected in the tail vein with 100mg/kg Texas Red dextran (70,000 MW, Thermo Fisher Scientific, D1864). After 3 hours, mice were injected in the tail vein with 10 mg/kg DyLight 488-Lycopersicon Esculentum Lectin (LEL) (Thermo Fisher Scientific, L32470). After 10 minetes, each mouse was anaesthetised and perfused with ice PBS until there was no blood, followed by 4% paraformaldehyde for 3–5 min. Brain tissue was extracted and immersed in 30% sucrose overnight. Tissue cryosections with 6 µm thick were stained with DAPI (Solarbio) and images were obtained with an Leica TCS SP5II confocal microscopy (Leica). Three random areas of each section were collected and three sections of each brain were examined.

Statistical analysis

The data are expressed as the mean ± standard deviation (SD). The data were plotted using GraphPad Prism 8.0 software and then statistically analyzed using SPSS 19.0 software. To identify statistical differences between groups, the data were compared among experimental groups using analysis of variance (ANOVA), t-test or chi-square test. Statistical significance was defined by P < 0.05.

Acknowledgements

We appreciate the support of the Clinical Medical Research Center of Liaoning Province and the Dalian Respiratory Protection Engineering Center Laboratory.

Author’s contributions

S.X, W.D and W.L designed and analyzed all experiments. S.X, W.D and W.L performed cell assays, animal experiments, IHC staining assays and write the manuscript. M.X performed the microfluidic chip assays. M.L collected the clinical samples. M.T and S.W. provided protocols and technical input. M.L. helps the tissue staining. Q.W, W.L and E.L conceived and supervised the project, and revised the manuscript. All authors read and approved the submitted manuscript.

Ethics approval statement

Animal study was approved by the Animal Ethics Review Committee of Dalian Medical University. Clinical study was approved by the Ethics Review Committee of the Second Hospital of Dalian Medical University.

Competing interests

The authors declare no competing interests.

Funding

This work was supported by a grant from the National Natural Science Foundation of China (No. 81972916, 82103054 and 82027805), Liaoning Revitalization Talents Program (XLYC2002013), and the Science and Technology Innovation Foundation of Dalian (2020JJ25CY018), Dalian Science and Technology Talent Innovation Support Plan (2022RQ037), Beijing Natural Science Foundation (7232020), Dalian Science and Technology Innovation Fund (2021JJ12SN42).

Availability of data and material

All data are available from the Prof. Qi Wang upon reasonable request.

Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA. Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc. 2008;83:584–94.
Sorensen JB, Hansen HH, Hansen M, Dombernowsky P. Brain metastases in adenocarcinoma of the lung: frequency, risk groups, and prognosis. J Clin Oncol. 1988;6:1474–80.
Cheng H, Perez-Soler R. Leptomeningeal metastases in non-small-cell lung cancer. Lancet Oncol. 2018;19:e43–e55.
Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier. Neurobiol Dis. 2010;37:13–25.
Morello A, Sadelain M, Adusumilli PS. Mesothelin-Targeted CARs: Driving T Cells to Solid Tumors. Cancer Discov. 2016;6:133–46.
Servais EL, Colovos C, Rodriguez L, Bograd AJ, Nitadori J, Sima C, Rusch VW, Sadelain M, Adusumilli PS. Mesothelin overexpression promotes mesothelioma cell invasion and MMP-9 secretion in an orthotopic mouse model and in epithelioid pleural mesothelioma patients. Clin Cancer Res. 2012;18:2478–89.
Kachala SS, Bograd AJ, Villena-Vargas J, Suzuki K, Servais EL, Kadota K, Chou J, Sima CS, Vertes E, Rusch VW, Travis WD, Sadelain M, Adusumilli PS. Mesothelin overexpression is a marker of tumor aggressiveness and is associated with reduced recurrence-free and overall survival in early-stage lung adenocarcinoma. Clin Cancer Res. 2014;20:1020–8.
Bharadwaj U, Marin-Muller C, Li M, Chen C, Yao Q. Mesothelin overexpression promotes autocrine IL-6/sIL-6R trans-signaling to stimulate pancreatic cancer cell proliferation. Carcinogenesis. 2011;32:1013–24.
Liu W, Song J, Du X, Zhou Y, Li Y, Li R, Lyu L, He Y, Hao J, Ben J, Wang W, Shi H, Wang Q. AKR1B10 (Aldo-keto reductase family 1 B10) promotes brain metastasis of lung cancer cells in a multi-organ microfluidic chip model. Acta Biomater. 2019;91:195–208.
Varghese F, Bukhari AB, Malhotra R, De A. IHC Profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples. PLoS ONE. 2014;9:e96801.
Liu W, Song J, Du X, Zhou Y, Li Y, Li R, Lyu L, He Y, Hao J, Ben J, Wang W, Shi H, Wang Q. AKR1B10 (Aldo-keto reductase family 1 B10) promotes brain metastasis of lung cancer cells in a multi-organ microfluidic chip model. Acta Biomater. 2019;91:195–208.
Liu W, Zhou Y, Duan W, Song J, Wei S, Xia S, Wang Y, Du X, Li E, Ren C, Wang W, Zhan Q, Wang Q. Glutathione peroxidase 4-dependent glutathione high-consumption drives acquired platinum chemoresistance in lung cancer-derived brain metastasis. Clin Transl Med. 2021;11:e517.
Navab R, Liu J, Seiden-Long I, Shih W, Li M, Bandarchi B, Chen Y, Lau D, Zu YF, Cescon D, Zhu CQ, Organ S, Ibrahimov E, Ohanessian D, Tsao MS. Co-overexpression of Met and hepatocyte growth factor promotes systemic metastasis in NCI-H460 non-small cell lung carcinoma cells. Neoplasia. 2009;11:1292–300.
Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer. 2009;9:265–73.
Chen SH, Hung WC, Wang P, Paul C, Konstantopoulos K. Mesothelin binding to CA125/MUC16 promotes pancreatic cancer cell motility and invasion via MMP-7 activation. Sci Rep. 2013;3:1870.
Chang MC, Chen CA, Chen PJ, Chiang YC, Chen YL, Mao TL, Lin HW, Lin WH, Chiang WF, Cheng. Mesothelin enhances invasion of ovarian cancer by inducing MMP-7 through MAPK/ERK and JNK pathways. Biochem J. 2012;442:293–302.
Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell. 2010;141:52–67.
Custodio-Santos T, Videira M, Brito MA. Brain metastasization of breast cancer. Biochim Biophys Acta Rev Cancer. 2017;1868:132–47.
Xing F, Liu Y, Sharma S, Wu K, Chan MD, Lo HW, Carpenter RL, Metheny-Barlow LJ, Zhou X, Qasem SA, Pasche B, Watabe K. Activation of the c-Met Pathway Mobilizes an Inflammatory Network in the Brain Microenvironment to Promote Brain Metastasis of Breast Cancer. Cancer Res. 2016;76:4970–80.
Breindel JL, Haskins JW, Cowell EP, Zhao M, Nguyen DX, Stern DF. EGF receptor activates MET through MAPK to enhance non-small cell lung carcinoma invasion and brain metastasis. Cancer Res. 2013;73:5053–65.
Choi YP, Lee JH, Gao MQ, Kim BG, Kang S, Kim SH, Cho NH. Cancer-associated fibroblast promote transmigration through endothelial brain cells in three-dimensional in vitro models. Int J Cancer. 2014;135:2024–33.
Ochieng JK, Kundu ST, Bajaj R, Leticia Rodriguez B, Fradette JJ, Gibbons DL. MBIP (MAP3K12 binding inhibitory protein) drives NSCLC metastasis by JNK-dependent activation of MMPs, Oncogene, 39 (2020) 6719–6732.
Liao B, Geng L, Zhang F, Shu L, Wei L, Yeung PKK, Lam KSL, Chung SK, Chang J, Vanhoutte PM, Xu A, Wang K, Hoo RLC. Adipocyte fatty acid-binding protein exacerbates cerebral ischaemia injury by disrupting the blood-brain barrier. Eur Heart J. 2020;41:3169–80.
Golfier S, Kopitz C, Kahnert A, Heisler I, Schatz CA, Stelte-Ludwig B, Mayer-Bartschmid A, Unterschemmann K, Bruder S, Linden L, Harrenga A, Hauff P, Scholle FD, Muller-Tiemann B, Kreft B, Ziegelbauer K. Anetumab ravtansine: a novel mesothelin-targeting antibody-drug conjugate cures tumors with heterogeneous target expression favored by bystander effect. Mol Cancer Ther. 2014;13:1537–48.
Hassan R, Blumenschein GR Jr., Moore KN, Santin AD, Kindler HL, Nemunaitis JJ, Seward SM, Thomas A, Kim SK, Rajagopalan P, Walter AO, Laurent D, Childs BH, Sarapa N, Elbi C, Bendell JC. First-in-Human, Multicenter, Phase I Dose-Escalation and Expansion Study of Anti-Mesothelin Antibody-Drug Conjugate Anetumab Ravtansine in Advanced or Metastatic Solid Tumors. J Clin Oncol. 2020;38:1824–35.
Costa DB, Shaw AT, Ou SH, Solomon BJ, Riely GJ, Ahn MJ, Zhou C, Shreeve SM, Selaru P, Polli A, Schnell P, Wilner KD, Wiltshire R, Camidge DR, Crino L. Clinical Experience With Crizotinib in Patients With Advanced ALK-Rearranged Non-Small-Cell Lung Cancer and Brain Metastases. J Clin Oncol. 2015;33:1881–8.
Liu W, Zhou Y, Duan W, Song J, Wei S, Xia S, Wang Y, Du X, Li E, Ren C, Wang W, Zhan Q, Wang Q. Glutathione peroxidase 4-dependent glutathione high-consumption drives acquired platinum chemoresistance in lung cancer-derived brain metastasis. Clin Translational Med. 2021;11:e517.
Chang M-C, Chen C-A, Chen P-J, Chiang Y-C, Chen Y-L, Mao T-L, Lin H-W, Lin Chiang W-H, Cheng W-F. Mesothelin enhances invasion of ovarian cancer by inducing MMP-7 through MAPK/ERK and JNK pathways. Biochem J. 2012;442:293–302.
Cheng WF, Huang CY, Chang MC, Hu YH, Chiang YC, Chen YL, Hsieh CY, Chen CA. High mesothelin correlates with chemoresistance and poor survival in epithelial ovarian carcinoma. Br J Cancer. 2009;100:1144–53.
He X, Wang L, Riedel H, Wang K, Yang Y, Dinu CZ, Rojanasakul Y. Mesothelin promotes epithelial-to-mesenchymal transition and tumorigenicity of human lung cancer and mesothelioma cells. Mol Cancer. 2017;16:63.
Okła K, Surówka J, Frąszczak K, Czerwonka A, Kaławaj K, Wawruszak A, Kotarski J, Wertel I. Assessment of the clinicopathological relevance of mesothelin level in plasma, peritoneal fluid, and tumor tissue of epithelial ovarian cancer patients. Tumour Biology: the Journal of the International Society For Oncodevelopmental Biology and Medicine. 2018;40:1010428318804937.
Chen S-H, Hung W-C, Wang P, Paul C, Konstantopoulos K. Mesothelin binding to CA125/MUC16 promotes pancreatic cancer cell motility and invasion via MMP-7 activation. Sci Rep. 2013;3:1870.
Morello A, Sadelain M, Adusumilli PS. Mesothelin-Targeted CARs: Driving T Cells to Solid Tumors. Cancer Discov. 2016;6:133–46.
Chang K, Pai LH, Batra JK, Pastan I, Willingham MC. Characterization of the antigen (CAK1) recognized by monoclonal antibody K1 present on ovarian cancers and normal mesothelium. Cancer Res. 1992;52:181–6.
Bharadwaj U, Marin-Muller C, Li M, Chen C, Yao Q. Mesothelin overexpression promotes autocrine IL-6/sIL-6R trans-signaling to stimulate pancreatic cancer cell proliferation. Carcinogenesis. 2011;32:1013–24.
Avula LR, Rudloff M, El-Behaedi S, Arons D, Albalawy R, Chen X, Zhang X, Alewine C. Mesothelin Enhances Tumor Vascularity in Newly Forming Pancreatic Peritoneal Metastases. Mol Cancer Res. 2020;18:229–39.
Jia W, Martin TA, Zhang G, Jiang WG. Junctional adhesion molecules in cerebral endothelial tight junction and brain metastasis. Anticancer Res. 2013;33:2353–9.
Steeg PS, Camphausen KA, Smith QR. Brain metastases as preventive and therapeutic targets. Nat Rev Cancer. 2011;11:352–63.
Gkouveris I, Nikitakis NG. Role of JNK signaling in oral cancer: A mini review. Tumour Biology: the Journal of the International Society For Oncodevelopmental Biology and Medicine. 2017;39:1010428317711659.
Dhillon AS, Hagan S, Rath O, Kolch W. MAP kinase signalling pathways in cancer. Oncogene. 2007;26:3279–90.
Bubici C, Papa S. JNK signalling in cancer: in need of new, smarter therapeutic targets. Br J Pharmacol. 2014;171:24–37.
Li H, Qiu Z, Li F, Wang C. The relationship between MMP-2 and MMP-9 expression levels with breast cancer incidence and prognosis. Oncol Lett. 2017;14:5865–70.
Bai Y, Yang H, Zhang G, Hu L, Lei Y, Qin Y, Yang Y, Wang Q, Li R, Mao Q. Inhibitory effects of resveratrol on the adhesion, migration and invasion of human bladder cancer cells. Mol Med Rep. 2017;15:885–9.
Fu H, Hu Z, Wen J, Wang K, Liu Y. TGF-beta promotes invasion and metastasis of gastric cancer cells by increasing fascin1 expression via ERK and JNK signal pathways. Acta Biochim Biophys Sin (Shanghai). 2009;41:648–56.
Kwon GT, Cho HJ, Chung W-Y, Park K-K, Moon A, Park JHY. Isoliquiritigenin inhibits migration and invasion of prostate cancer cells: possible mediation by decreased JNK/AP-1 signaling. J Nutr Biochem. 2009;20:663–76.
Si-Tu J, Cai Y, Feng T, Yang D, Yuan S, Yang X, He S, Li Z, Wang Y, Tang Y, Ye C, Li Z. Upregulated circular RNA circ-102004 that promotes cell proliferation in prostate cancer. Int J Biol Macromol. 2019;122:1235–43.
Corso S, Migliore C, Ghiso E, De Rosa G, Comoglio PM, Giordano S. Silencing the MET oncogene leads to regression of experimental tumors and metastases. Oncogene. 2008;27:684–93.
Schmidt L, Duh FM, Chen F, Kishida T, Glenn G, Choyke P, Scherer SW, Zhuang Z, Lubensky I, Dean M, Allikmets R, Chidambaram A, Bergerheim UR, Feltis JT, Casadevall C, Zamarron A, Bernues M, Richard S, Lips CJ, Walther MM, Tsui LC, Geil L, Orcutt ML, Stackhouse T, Lipan J, Slife L, Brauch H, Decker J, Niehans G, Hughson MD, Moch H, Storkel S, Lerman MI, Linehan WM, Zbar B. Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nat Genet. 1997;16:68–73.
Ponzo MG, Lesurf R, Petkiewicz S, O'Malley FP, Pinnaduwage D, Andrulis IL, Bull SB, Chughtai N, Zuo D, Souleimanova M, Germain D, Omeroglu A, Cardiff RD, Hallett M, Park M. Met induces mammary tumors with diverse histologies and is associated with poor outcome and human basal breast cancer. Proc Natl Acad Sci USA. 2009;106:12903–8.
Li C, Wu J-J, Hynes M, Dosch J, Sarkar B, Welling TH, Pasca M, di Magliano DM. Simeone, c-Met is a marker of pancreatic cancer stem cells and therapeutic target, Gastroenterology, 141 (2011).
Xing F, Liu Y, Sharma S, Wu K, Chan MD, Lo H-W, Carpenter RL, Metheny-Barlow LJ, Zhou X, Qasem SA, Pasche B, Watabe K. Activation of the c-Met Pathway Mobilizes an Inflammatory Network in the Brain Microenvironment to Promote Brain Metastasis of Breast Cancer. Cancer Res. 2016;76:4970–80.
Gao H-F, Li A-N, Yang J-J, Chen Z-H, Xie Z, Zhang X-C, Su J, Lou N-N, Yan H-H, Han J-F, Wu Y-L. Soluble c-Met Levels Correlated With Tissue c-Met Protein Expression in Patients With Advanced Non-Small-Cell Lung Cancer. Clin Lung Cancer. 2017;18:85–91.
Peters S, Adjei AA. MET: a promising anticancer therapeutic target. Nat Rev Clin Oncol. 2012;9:314–26.
Awad MM, Oxnard GR, Jackman DM, Savukoski DO, Hall D, Shivdasani P, Heng JC, Dahlberg SE, Jänne PA, Verma S, Christensen J, Hammerman PS, Sholl LM. MET Exon 14 Mutations in Non-Small-Cell Lung Cancer Are Associated With Advanced Age and Stage-Dependent MET Genomic Amplification and c-Met Overexpression. J Clin Oncology: Official J Am Soc Clin Oncol. 2016;34:721–30.
Engelman JA, Jänne PA. Mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer. Clin Cancer Research: Official J Am Association Cancer Res. 2008;14:2895–9.
Turke AB, Zejnullahu K, Wu Y-L, Song Y, Dias-Santagata D, Lifshits E, Toschi L, Rogers A, Mok T, Sequist L, Lindeman NI, Murphy C, Akhavanfard S, Yeap BY, Xiao Y, Capelletti M, Iafrate AJ, Lee C, Christensen JG, Engelman JA, Jänne PA. Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. Cancer Cell. 2010;17:77–88.
Breindel JL, Haskins JW, Cowell EP, Zhao M, Nguyen DX, Stern DF. EGF receptor activates MET through MAPK to enhance non-small cell lung carcinoma invasion and brain metastasis. Cancer Res. 2013;73:5053–65.
Shaw AT, Kim D-W, Nakagawa K, Seto T, Crinó L, Ahn M-J, De Pas T, Besse B, Solomon BJ, Blackhall F, Wu Y-L, Thomas M, O'Byrne KJ, Moro-Sibilot D, Camidge DR, Mok T, Hirsh V, Riely GJ, Iyer S, Tassell V, Polli A, Wilner KD, Jänne PA. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N Engl J Med. 2013;368:2385–94.
Kwak EL, Bang Y-J, Camidge DR, Shaw AT, Solomon B, Maki RG, Ou S-HI, Dezube BJ, Jänne PA, Costa DB, Varella-Garcia M, Kim W-H, Lynch TJ, Fidias P, Stubbs H, Engelman JA, Sequist LV, Tan W, Gandhi L, Mino-Kenudson M, Wei GC, Shreeve SM, Ratain MJ, Settleman J, Christensen JG, Haber DA, Wilner K, Salgia R, Shapiro GI, Clark JW. A.J. Iafrate, Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer, N Engl J Med, 363 (2010) 1693–1703.
Ou S-HI, Kwak EL, Siwak-Tapp C, Dy J, Bergethon K, Clark JW, Camidge DR, Solomon BJ, Maki RG, Bang Y-J, Kim D-W, Christensen J, Tan W, Wilner KD, Salgia R, Iafrate AJ. Activity of crizotinib (PF02341066), a dual mesenchymal-epithelial transition (MET) and anaplastic lymphoma kinase (ALK) inhibitor, in a non-small cell lung cancer patient with de novo MET amplification. J Thorac Oncol. 2011;6:942–6.
Bergethon K, Shaw AT, Ou S-HI, Katayama R, Lovly CM, McDonald NT, Massion PP, Siwak-Tapp C, Gonzalez A, Fang R, Mark EJ, Batten JM, Chen H, Wilner KD, Kwak EL, Clark JW, Carbone DP, Ji H, Engelman JA, Mino-Kenudson M, Pao W, Iafrate AJ. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncology: Official J Am Soc Clin Oncol. 2012;30:863–70.
Camidge DR, Otterson GA, Clark JW, Ignatius Ou S-H, Weiss J, Ades S, Shapiro GI, Socinski MA, Murphy DA, Conte U, Tang Y, Wang SC, Wilner KD. Villaruz, Crizotinib in Patients With MET-Amplified NSCLC. J Thorac Oncol. 2021;16:1017–29.
Wang P, Xiao P, Ye Y, Liu P, Han L, Dong L, She C, Yu J. Rapid response of brain metastasis to crizotinib in a patient with KLC1-ALK fusion and MET gene amplification positive non-small cell lung cancer: a case report. Cancer Biol Med. 2017;14:183–6.
Kachala SS, Bograd AJ, Villena-Vargas J, Suzuki K, Servais EL, Kadota K, Chou J, Sima CS, Vertes E, Rusch VW, Travis WD, Sadelain M, Adusumilli PS. Mesothelin overexpression is a marker of tumor aggressiveness and is associated with reduced recurrence-free and overall survival in early-stage lung adenocarcinoma. Clin Cancer Research: Official J Am Association Cancer Res. 2014;20:1020–8.
Hassan R, Alley E, Kindler H, Antonia S, Jahan T, Honarmand S, Nair N, Whiting CC, Enstrom A, Lemmens E, Tsujikawa T, Kumar S, Choe G, Thomas A, McDougall K, Murphy AL, Jaffee E, Coussens LM, Brockstedt DG. Clinical Response of Live-Attenuated, Listeria monocytogenes Expressing Mesothelin (CRS-207) with Chemotherapy in Patients with Malignant Pleural Mesothelioma. Clin Cancer Research: Official J Am Association Cancer Res. 2019;25:5787–98.
Staudacher AH, Brown MP. Antibody drug conjugates and bystander killing: is antigen-dependent internalisation required? Br J Cancer. 2017;117:1736–42.
Golfier S, Kopitz C, Kahnert A, Heisler I, Schatz CA, Stelte-Ludwig B, Mayer-Bartschmid A, Unterschemmann K, Bruder S, Linden L, Harrenga A, Hauff P, Scholle F-D, Müller-Tiemann B, Kreft B, Ziegelbauer K. Anetumab ravtansine: a novel mesothelin-targeting antibody-drug conjugate cures tumors with heterogeneous target expression favored by bystander effect. Mol Cancer Ther. 2014;13:1537–48.
Qin L, Zhao R, Li P. Incorporation of functional elements enhances the antitumor capacity of CAR T cells. Exp Hematol Oncol. 2017;6:28.
He J, Zhang Z, Lv S, Liu X, Cui L, Jiang D, Zhang Q, Li L, Qin W, Jin H, Qian Q. Engineered CAR T cells targeting mesothelin by piggyBac transposon system for the treatment of pancreatic cancer. Cell Immunol. 2018;329:31–40.
Sun Q, Zhou S, Zhao J, Deng C, Teng R, Zhao Y, Chen J, Dong J, Yin M, Bai Y, Deng H, Wen J. Engineered T lymphocytes eliminate lung metastases in models of pancreatic cancer. Oncotarget. 2018;9:13694–705.
Adusumilli PS, Cherkassky L, Villena-Vargas J, Colovos C, Servais E, Plotkin J, Jones DR, Sadelain M. Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity. Sci Transl Med. 2014;6:261ra151.
Lv J, Zhao R, Wu D, Zheng D, Wu Z, Shi J, Wei X, Wu Q, Long Y, Lin S, Wang S, Wang Z, Li Y, Chen Y, He Q, Chen S, Yao H, Liu Z, Tang Z, Yao Y, Pei D, Liu P, Zhang X, Zhang Z, Cui S, Chen R, Li P. Mesothelin is a target of chimeric antigen receptor T cells for treating gastric cancer. J Hematol Oncol. 2019;12:18.

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Mesothelin promotes brain metastasis of non-small cell lung cancer by activating MET

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

Increased MSLN expression correlates with BM of NSCLC

MSLN promotes the migration and invasion of NSCLC brain metastatic cells in vitro

MSLN helps brain metastatic cells penetrate the BBB by degrading inter-endothelial TJs

Disruptive effect of MSLN on TJs is dependent on MET expression

Anetumab and crizotinib treatments therapeutically inhibit BM in vivo

Discussion

Materials and methods

Cell lines and cell culture

SDS-PAGE and western blot analysis

Clinical samples

Enzyme-linked immunosorbent assay (ELISA)

Immunohistochemistry (IHC) staining and scoring

Establishment of stable overexpression and knockdown cell lines

Wound healing assays

Transwell migration and invasion assays

Trans-endothelial assays

Trans-BBB assays on a microfluidic chip

Generation of conditioned medium

Immunofluorescence (IF) staining

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Animal study

BBB leakiness assay

Statistical analysis

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1