NETO2 promotes melanoma progression via activation of the Ca²⁺/CaMKII signaling pathway

Abstract

Melanoma is the most aggressive cutaneous tumor. Neuropilin and tolloid-like 2 (NETO2) is closely related to tumorigenesis. However, the functional significance of NETO2 in melanoma progression remains unclear. Herein, we found that NETO2 expression was augmented in melanoma clinical tissues and associated with poor prognosis in melanoma patients. Disrupting NETO2 expression markedly inhibited melanoma proliferation, malignant growth, migration, and invasion by downregulating the levels of calcium ions (Ca²⁺) and the expression of key genes involved in the calcium signaling pathway. By contrast, NETO2 overexpression had the opposite effects. Importantly, pharmacological inhibition of CaMKII/CREB activity with the CaMKII inhibitor KN93 suppressed NETO2-induced proliferation and melanoma metastasis. Overall, this study uncovered the crucial role of NETO2-mediated regulation in melanoma progression, indicating that targeting NETO2 may effectively improve melanoma treatment.

Keywords

melanoma / neuropilin and tolloid-like 2 / Ca ²⁺/CaMKII signaling pathway

1 Introduction

Calcium ion (Ca²⁺), a signaling ion, is an intracellular second messenger that plays a crucial and ubiquitous role in intracellular signaling and regulates several cellular functions, such as cell proliferation, migration, and transcription factor activation [1,2]. The cytosolic Ca²⁺ under resting conditions is 100 nmol/L, while the extracellular Ca²⁺ concentration is substantially high at approximately 2 mmol/L [3]. Previous studies have shown that Ca²⁺ signaling is highly activated in melanoma, promoting its proliferation and invasion [4,5]. Melanoma is the deadliest form of skin cancer, and its incidence is progressively increasing worldwide [6]. Patients diagnosed early with melanoma can be cured by surgical resection. However, patients with advanced or metastatic melanoma respond poorly to conventional treatments, such as surgery, radiotherapy, and chemotherapy, which is a crucial cause of melanoma-related death and leads to tumor lesion recurrence [7]. Therefore, revealing the detailed mechanism of melanoma is urgently necessary to spur the development of novel therapeutic strategies for this malignancy.

Neuropilin and tolloid-like 2 (NETO2) is a single-pass integral membrane protein that contains two CUB (Complement Clr/Cls, Uegf, Bampl) domains and one extracellular domain [8]. In 2009, through the combination of proteomic methods with immunoprecipitation and mass spectrometry, NETO2 was identified as an interaction partner of neuronal kainate receptors that accelerates the low density lipoprotein type A module (LDLa) recovery of kainate receptors after desensitization and increases the opening probability and time of the channel [9,10]. Previous reports have shown that NETO2 is associated with neuropsychiatric disorders and the regulation of emotional behavior [11,12]. An increasing number of studies have recently shown that NETO2 plays a key role in human malignant tumors. NETO2 promotes the progression of osteosarcoma and esophageal and gastric cancer via activation of the PI3K/AKT signaling pathway [13–15]; it also promotes pancreatic cancer progression by activating the STAT3 signaling pathway [16]. However, the exact mechanism of NETO2 in melanoma must be explored.

Calmodulin-dependent protein kinases (CaMKs) are multifunctional serine/threonine kinases; among which, CaMKII is the core protein and the most necessary sensor and regulator in Ca²⁺ signaling [17]. The considerable role of CaMKII in cancer progression has been previously reported; although a few studies have reported on the role of CaMKII in melanoma progression and development, its precise role in these processes remains unclear [18,19]. Several studies have also shown that CaMKII directly or indirectly participates in the phosphorylation and transcriptional activation of cyclic adenosine monophosphate response element binding (CREB) [20,21]. In addition, a previous study showed that NETO2 enhanced kainate receptor-mediated Ca²⁺ influx in HEK293 cells [22]. Thus, we hypothesized that NETO2 is involved in the progression of melanoma by activating the Ca²⁺-dependent CaMKII/CREB pathway.

In this study, we first demonstrated that NETO2 is overexpressed in melanoma and associated with the survival rate of melanoma patients. We further demonstrated that NETO2 promotes melanoma cell proliferation and migration. We then demonstrated that NETO2 increases the intracellular calcium concentration, which results in the increased phosphorylation of CaMKII and CREB. Herein, we provide novel and cogent evidence of the relationship between melanoma progression and the NETO2 gene and suggest a feasible therapeutic target for melanoma.

2 Materials and methods

2.1 Antibodies and reagents

Antibodies against NETO2 (ab171651) and MMP9 (ab76003) were purchased from Abcam (Cambridge, MA, USA); antibodies against MMP2 (10373-2-AP) and GAPDH (6004-1-IG) were obtained from Proteintech (Wuhan, China); antibodies against CREB (9197s) and P-CREB (9198s) were obtained from Cell Signaling Technology (CST, Beverly, MA, USA); and antibodies against CaMKII (YT0623) and P-CaMKII (YP0279) were purchased from ImmunoWay (Plano, TX, USA). KN93 phosphate was purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA).

2.2 Cell culture, DNA plasmids, and lentiviral infection

The SK-Mel-5, SK-Mel-28, A375, WM35, and HEK293T cell lines were purchased from the American Type Culture Collection. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C and 5% CO₂. The human melanocyte line PIG1 was generated as described in our previous study [23]. Lentiviruses carrying NETO2 shRNA or the NETO2 sequence for its overexpression were generated as previously described [24]. The NETO2 shRNA lentiviral plasmid was purchased from Jikai Gene Chemical Co., Ltd. (Shanghai, China). The NETO2 plasmid was obtained from Sino Biological (Beijing, China) and subsequently ligated into the lentiviral vector pLVX-IRES-Puro. The melanoma cells were infected with lentivirus and a complete medium at a ratio of 1:1.

2.3 Bioinformatics analysis

NETO2 mRNA expression data from tumors and normal tissues were obtained from the GSE46517 and GSE3189 datasets from the NCBI GEO database. Correspondingly, the online tool GEPIA was used to analyze the NETO2 expression data and overall and disease-free survival rates in patients with melanoma.

2.4 Western blot

Cells were harvested in cold PBS and lysed in RIPA buffer containing a protease inhibitor (1 mmol/L) and phosphatase inhibitor (1 mmol/L) mixture (Bimake, Shanghai, China). The protein concentration was detected by the BCA protein assay reagent (Bimake, Shanghai, China). Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membranes were blocked for 1 h with 5% nonfat milk and then incubated with the diluted appropriate primary and secondary antibodies. The protein bands were visualized with an imaging analysis system (Bio-Rad, Hercules, CA, USA).

2.5 Proliferation and colony formation assays

Cells (2 × 10³ cells/well) were seeded in 96-well plates and cultured for 24, 48, and 72 h. Cell proliferation was measured by Cell Counting Kit-8 assay (CCK8, Selleck Chemicals, Houston, TX, USA) following the manufacturer’s protocols. For the colony formation assay, the cells (1 × 10³ cells/well) were seeded into 6-well plates. The culture medium was then changed, and the cells were cultured for two weeks. Next, colonies were fixed in 4% formaldehyde and stained with 0.1% crystal violet. The colonies were then counted and analyzed.

2.6 Migration and invasion assays

For the migration assays, cells were seeded at 8 × 10⁵ in 6-well plates and incubated overnight at 37 °C, and linear scratch wounds were created using 10 μL pipette tips. The plates were washed with PBS twice to remove the non-adhered cells, and photographs were taken at different time points (0, 12, 24, and 48 h). For the Transwell assay, 5 × 10⁴ cells were seeded into an 8 μm pore chamber (Corning Incorporated) with or without Matrigel (BD Biosciences, CA) in 100 μL of FBS-free medium, and medium containing 30% FBS was placed in the bottom chamber as an attractant. After incubation for 12 or 24 h, the cells were fixed in 4% paraformaldehyde and stained with crystal violet. Images were taken, and the number of migrated/invaded melanoma cells was determined using an inverted microscope.

2.7 Intracellular Ca²⁺ measurement

The fluorescent dye Fluo-4 AM (Beyotime Biotechnology, Haimen, Jiangsu, China) was used to monitor the cytosolic Ca²⁺ concentration in melanoma cells according to the manufacturer’s instructions. Briefly, the cells were maintained with 200 μL of Fluo-4 AM (5 µM) and incubated at 37 °C for 30 min. Then, the cells were washed with PBS thrice and detected via flow cytometry.

2.8 Xenograft tumor model

BALB/c-nu mice (4–6 weeks old) purchased from Hunan SJA Laboratory Animal Co., Ltd. were randomly assigned to three groups (n = 7). SK-Mel-28 cells (shMOCK, shNETO2#2, shNETO2#3) were resuspended in cold serum-free DMEM at a density of 5 × 10⁶ cells/0.1 mL and subcutaneously inoculated into the right flanks of the nude mice. Tumor growth was monitored three times a week using calipers, and tumor volume was calculated as length × width × height × 0.52 (mm). On day 26, the mice were sacrificed, and tumor tissues were harvested. These tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned at a thickness of 5 μm, and subjected to immunohistochemical analysis. Animals were cared for in accordance with the “3R” principle of experimental animal ethics, and xenograft tumor models were established under the approval of the Ethics Committee of Xiangya Hospital at Central South University.

2.9 Immunohistochemistry

A human melanoma tissue array (DC-Mel21020) containing 56 primary melanomas, 23 metastatic melanomas, 18 nevi, and 2 skin tissues was obtained from Alenabio Biotechnology (Beijing, China). Tumor tissues from xenograft mice were embedded in paraffin blocks and subjected to immunohistochemistry experiments performed following standard procedures.

2.10 Statistical analyses

GraphPad Prism 8.0 software (GraphPad Prism Software Inc., La Jolla, CA, USA) was used for statistical analyses. The results are presented as the mean values ± SDs and were analyzed by Student’s t-test. Survival rates were compared using the log-rank test. Differences with a P value < 0.05 were considered statistically significant.

3 Results

3.1 NETO2 overexpression in melanoma tissues correlated with melanoma prognosis

We first analyzed the expression profile of NETO2 in melanoma datasets from NCBI GEO, Oncomine, and TCGA databases. Analysis of the Talantov melanoma dataset showed that the mRNA expression of NETO2 was significantly upregulated in melanoma tissues compared with normal skin tissues (Fig. S1A). This result was confirmed with the GSE46517, GSE3189, and SKCM datasets (Fig.1 and 1B). Next, immunohistochemical (IHC) staining showed that NETO2 staining was more intense in malignant melanomas tissues (P < 0.05) and metastatic melanoma tissues (P < 0.001) than in benign tissues (Fig.1). Additionally, we used a public database to analyze the relationship between NETO2 expression and the prognosis of melanoma patients. High NETO2 in melanoma patients was associated with reduced overall and disease-free survival (Fig.1 and S1B). Overall, these findings suggest that NETO2 is frequently upregulated in melanoma tissues and might play an important role in melanoma progression.

3.2 NETO2 promoted the proliferation of melanoma cells in vitro

We determined the expression of NETO2 in four different melanoma cell lines (WM35, A375, SK-Mel-28 and SK-Mel-5) and normal melanocytes (PIG1) to further clarify its function in melanoma. NETO2 protein and mRNA expression were significantly increased in the four melanoma cell lines compared with the normal melanocytes (Fig.2 and S2A). As NETO2 was preferentially expressed in SK-Mel-5 and SK-Mel-28 cells, we then investigated the functional significance of NETO2 in these melanoma cell lines by using two distinct shRNAs targeting NETO2. Lentivirus-mediated expression of shNETO2#2 or shNETO2#3 markedly reduced NETO2 protein levels in SK-Mel-5 and SK-Mel-28 cells (Fig.2 and S2B). We found that disruption of NETO2 expression significantly inhibited melanoma proliferation, as measured by CCK-8 assays (Fig.2), and reduced colony formation based on plate colony formation assays (Fig.2). By contrast, WM35 cells showed low levels of NETO2 expression and were used to establish a model of NETO2 overexpression (Fig.2 and 2E). CCK-8 and plate colony formation assays showed that NETO2 respectively enhanced the proliferation and colony-forming capability of WM35 cells (Fig.2 and 2G). In addition, we observed that depletion of NETO2 upregulated the level of BAX expression and downregulated the level of BCL2 expression in SK-Mel-5 and SK-Mel-28 cells, while the opposite results were obtained in WM35 cells overexpressing NETO2 (Fig. S2C). Overall, these results indicate that NETO2 is essential for melanoma cell proliferation.

3.3 Silencing NETO2 suppressed melanoma growth in vivo

NETO2 promoted melanoma proliferation in vitro. Thus, we then examined the impact of NETO2 disruption on cell proliferation in melanoma-derived xenografts. SK-Mel-28 cells expressing shNETO2 (shNETO2#2 or shNETO2#3) or shMOCK were transplanted into nude mice by hypodermic injection. The control (shMOCK) and shNETO2#2 groups formed tumors after injection, while the tumor formation rate in the shNETO2#3 group was zero (Fig.3). The mice were sacrificed 26 days after implantation, and the subcutaneous tumor volumes were significantly decreased in the shNETO2 group compared with the control group (Fig.3 and 3C). We found that cell proliferation was markedly reduced in melanoma xenografts derived from SK-Mel-28 cells expressing shNETO2 compared with control tumors, as demonstrated by Ki67 immunohistochemistry (Fig.3 and 3E). Collectively, these data demonstrate that disruption of NETO2 expression potently suppressed melanoma growth, indicating that NETO2 is critical for maintaining the tumorigenic potential of melanoma in vivo.

3.4 NETO2 promoted the migration and invasion of melanoma cells

The most important property of melanoma is its potent capacity to metastasize. Thus, we investigated the function of NETO2 in the migration and invasion of melanoma. SK-Mel-5 and SK-Mel-28 cells transduced with lentiviruses expressing NETO2 or control shRNA were seeded into 6-well plates and monitored for 48 h after wounding. At 24 and 48 h, the wound closure rate of SK-Mel-5 and SK-Mel-28 cells expressing NETO2 shRNA was markedly lower than that of those expressing control shRNA (Fig.4). Similarly, NETO2 depletion reduced migration and invasion, as demonstrated by Transwell assays (Fig.4 and 4C). In addition, Western blot analysis showed that NETO2 knockdown reduced MMP2 and MMP9 protein levels in melanoma cells (Fig.4). Conversely, we observed that NETO2 promoted wound healing, cell migration, and invasion in WM35 cells (Fig.4 and 4F). NETO2 overexpression in WM35 cells induced the expression of MMP2 and MMP9 (Fig.4). These data suggest that NETO2 is involved in the invasion and migration of melanoma cells.

3.5 NETO2 activates the calcium signal pathway in melanoma

Researchers previously found that NETO2 is involved in mediating calcium influx in normal cells [22]. We investigated the possible correlation of NETO2 with intracellular calcium elevation in melanoma cells through Fluo-4 AM to elucidate the molecular mechanisms through which NETO2 inactivation inhibits the malignant progression of melanoma. The flow cytometry results showed that the intracellular calcium concentration significantly decreased after the knockdown of NETO2 in SK-Mel-5 and SK-Mel-28 cells compared with the control groups (Fig.5 and 5B). We then detected the levels of calcium signal related proteins in melanoma cells expressing shNETO2. The phosphorylation of CaMKII and CREB was abolished but not that of FAK or PKA/PKC when the expression of NETO2 was blocked (Fig.5 and S3A). In addition, silencing NETO2 significantly reduced the population of cells positive for P-CaMKII and P-CREB in mouse tumor tissues, as shown by immunohistochemistry (Fig.3 and 3E). By contrast, overexpression of NETO2 dramatically increased the concentration of intracellular calcium (Fig.5 and 5E) and phosphorylation of CaMKII and CREB (Fig.5) in WM35 cells. Collectively, these results demonstrate that NETO2 positively regulates the calcium signaling axis, thus mediating the malignant phenotype of melanoma.

3.6 Inhibition of CaMKII/CREB activity by the CaMKII inhibitor KN93 suppressed the NETO2-driven melanoma phenotype

The expression of CaMKII and CREB was significantly affected by NETO2. Thus, we then explored the possible impact of the pharmacologic inhibition of CaMKII activity by a small-molecule inhibitor named KN93 on NETO2-driven melanoma cell proliferation, invasion and migration. Western blot analysis confirmed that KN93 treatment reduced P-CaMKII protein levels but had no effect on NETO2 expression in WM35 cells (Fig.6). Further analysis showed that NETO2 overexpression induced MMP2 and P-CREB expression, which could be reversed by KN93 (Fig.6). KN93 also reversed the increased cell viability and colony formation of melanoma cells induced by NETO2 overexpression (Fig.6 and 6C). In addition, wound healing and Transwell assays showed that KN93 abrogated the changes in migration and invasion in NETO2 overexpressing cells (Fig.6 and 6E). These data indicate that NETO2 promotes the proliferation and metastasis of melanoma through the CaMKII/CREB signaling pathway.

4 Discussion

The present study shows that NETO2 was upregulated in clinical tissues of patients with melanoma. High NETO2 expression in melanoma tissues was linked to poor survival prognosis. Functionally, NETO2 significantly increased cell viability and metastasis via activation of the CaMKII/CREB signaling pathway (Fig.7). To the best of our knowledge, this is the first study to reveal the roles and mechanisms of NETO2 in melanoma.

Although recent studies have suggested the abnormality of NETO2 expression in human malignancies, the underlying mechanism remains unclear. Calcium triggers and regulates physiologic cellular processes essential for cell proliferation, movement, survival, and death. Previous studies have demonstrated that the regulation of Ca²⁺ homeostasis in adipocytes is a key signal linking metabolic stress to inflammation and metabolic deterioration in obesity [25]. Furthermore, the Ca²⁺ microdomain is the earliest signaling event observed upon T cell activation [26]. Inhibiting intracellular Ca²⁺ overload in cardiac microvascular endothelial cells by sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA) improves mitochondrial quality control and alleviates cardiac microvascular ischemia-reperfusion injury [27]. A growing evidence also revealed that Ca²⁺ homeostasis and signaling are altered in tumor cells [28]. Mookerjee-Basu et al. showed that EGFR4 suppresses Ca²⁺ responses and promotes anticancer immunity by regulating KCa3.1 and Kv1.3 expression, respectively [29]. SYT4 promoted dendrite extension and melanogenesis in alpaca melanocytes by regulating Ca²⁺ influx via TRPM1 channels [30]. Ca²⁺ signaling strictly relies on store- and receptor-operated calcium channels, such as ORAI1, STIM1, and members of the TRP family of ion channels [31,32]. NETO2, an auxiliary subunit of kainate-type glutamate receptors (KARs), regulates KAR trafficking and gating via its interaction with KARs at multiple sites [33]. In addition, a previous study showed that the KAR-mediated modulation of transmission is reliant on external Ca²⁺ entry via Ca²⁺-permeant KARs [34]. Therefore, we speculate that NETO2 regulates the concentration of calcium in melanoma cells by interacting with KARs or other calcium channel proteins. However, the specific mechanism must be verified by further experiments.

Disruption of Ca²⁺ homeostasis facilitates the formation of malignant phenotypes. A transient increase in cytoplasmic Ca²⁺ activates a series of important calcium-dependent cellular events, including the CaMK, FAK, and PKA pathways [35,36]. Excessive elevation of Ca²⁺ concentration impairs tumor cell migration by activating CaMKII. Zhou et al. found that Yap promotes the regulation of HCC migration by regulating the mitogen/SERCA/CaMKII pathway [37]. In our work, manipulation of the NETO2 gene significantly regulated protein phosphorylation and the highly malignant melanoma phenotype via the Ca²⁺/CaMKII pathways rather than the FAK or PKA signaling pathway. The Ca²⁺/CaMKII pathway plays a vital role in the carcinogenesis of various cancers. Chang et al. demonstrated that CaMKII regulates the expression and phosphorylation of c-FLIP and modulates apoptosis in melanoma cells [18]. In addition, Richa et al. suggested that the NETO2 C terminus is phosphorylated at Ser409 by CaMKII and PKA [38]. Specifically, KN93, an inhibitor of CaMKII, did not change the expression of NETO2 or inhibit the increase in calcium concentration induced by NETO2 in the current study (Fig. S4A). However, the relationship between NETO2 and CaMKII requires further investigation.

CREB, as a transcription factor, regulates diverse cellular responses, including proliferation, survival, metastasis, and differentiation [39]. Previous studies have shown that the activation of CAMKII participates in CREB phosphorylation [40], while studies on CaMKII/CREB have primarily focused on chronic pain. A study showed that CaMKII mediates malignancy in apicidin-persistent liver cancer cells via CREB signaling pathway activation [21]. In addition, Jie et al. showed that GLA significantly reduced the capability of the CREB-AP1 protein complex to bind the promoters of MMP2 in osteosarcoma cells [41]. MMPs are key proteases that are involved in tumor invasion and metastasis [42,43]. Previous studies indicated that NETO2 could decrease the invasion of esophageal squamous cell carcinoma cells by inhibiting the PI3K/Akt/Snail or PI3K/Akt/MMP2 pathway [13]. This result supports the idea of a strong molecular link between NETO2 and matrix metalloproteinases. In addition, we detected changes in MMP9 protein levels in melanoma cells wherein NETO2 was silenced or overexpressed. Thus, we showed that NETO2 inhibits melanoma cell migration and invasion by modulating MMP2 and MMP9, respectively.

In this work, we showed that NETO2 could promote the invasion and metastasis of melanoma cells by respectively regulating the expression of MMP2 and MMP9; mechanistically, this process may be mediated through increased levels of intracellular calcium, which further activate the CaMKII/CREB signaling pathway. Overall, NETO2, which facilitates melanoma progression, can act as a potential therapeutic target and may be conducive to determining the diagnosis and prognosis of melanoma.

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