Introduction

Melanoma is a type of skin cancer that arises from melanocytes. Although melanoma accounts for only approximately 3% of skin cancer cases, its mortality rate (65%) is much higher than that of other types of skin cancer, and epidemiologic estimates of melanoma have identified it as the 20th most common cancer worldwide1,2. Currently, there are several factors influencing the increasing melanoma epidemic, and one of the main causes is the increased exposure of vulnerable populations to ultraviolet radiation (UVR)3,4,5,6,7. It has been reported that the increased incidence and mortality rate of malignant melanoma are positively correlated with increased UV radiation exposure8. The combination of various kinase inhibitors with or without immune checkpoint inhibitors may delay the progression of the disease and reduce the mortality rate of melanoma, and these therapeutic strategies are the future of melanoma treatment9. Unfortunately, the molecular mechanisms of melanoma remain unclear. Exploring the molecular mechanisms that regulate melanoma progression may lead to advances in clinical treatment.

Several reports suggest that the dysregulation of cytokine tumor necrosis factor α (TNFα) production in cells is associated with the development of cancer10,11. TNFα plays a crucial part in cancer therapy by regulating and controlling the expression of TNFAIP8-like 2 (TIPE2) in the protein-8-like protein (TNFAIP8/TIPE) family induced by tumor necrosis factor-α, which mediates inflammation and apoptosis12. TIPE2 represents a new target for cancer immunotherapy, as evidenced by significant tumor growth changes in TIPE2-deficient mice13. Recent papers have reported that the dysregulation of TIPE2 is correlated with the proliferation and migration of tumor cells. For example, adenovirus-mediated TIPE2 repair could dramatically inhibit the migration of gastric cancer cells14. TIPE2 is only weakly expressed in glioma cells, and overexpression of TIPE2 suppresses the migration of U251 cells15. Similarly, TIPE2 suppressed the proliferation and tumorigenesis of breast cancer cells16. As an endogenous inhibitor of Rac1 in primary hepatocellular carcinoma (HCC), TIPE2 effectively suppresses the migration and invasion of HCC17. Moreover, TIPE2 is deemed to be a checkpoint for NK cell maturation and antitumor immunity, and targeting TIPE2 could be a new method for NK cell-based immunotherapy in the future18.

Melanoma is a life-threatening disease with a high mortality rate, so there is an urgent need to thoroughly understand the mechanism underlying the occurrence and development of melanoma and discover new therapeutic targets. The objective of this study was to explore the function of TIPE2 in malignant melanoma. According to our experimental results, overexpression of TIPE2 can suppress the proliferation of melanoma cells. These results demonstrated that TIPE2 plays a considerable role in the development of melanoma.

Merhord

Patients

Melanoma and normal skin tissue specimens were obtained from Xian Biotech Co., Ltd. (Xian, China). There were 38 primary malignant melanoma samples, 10 lymph node metastasis samples, 7 normal skin tissue samples, 2 normal pharyngeal mucosa samples, 2 esophageal mucosa samples, 2 small intestine mucosa samples and 2 lymph node tissue samples. None of the patients received systematic treatment before surgery. Pathological analysis was performed on all specimens. Patient clinical data was gathered from electronic medical records. Informed consents were obtained from all participants. The study was approved by the Institutional Ethics Committee of Zibo Central Hospital. All methods were performed in accordance with the relevant guidelines and regulations. Table 1 showed that TIPE2 expression was substantially linked with melanoma.

Table 1 Relationship between TIPE2 expression and clinical-pathological features in melanoma.
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Cell culture

The metastatic melanoma cell line A375 and the metastatic melanoma cell line SK-MEL-28 were obtained from the Cell Bank of the Chinese Scientific Academy (Shanghai, China). A375 and SK-MEL-28 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, USA) and 1% penicillin‒streptomycin. The cells were cultured in a suitable atmosphere at 37 °C with 5% CO2.

Cell overexpression

LV-TNFAIP8L2 and LV-CON335 lentiviruses were successfully constructed by Shanghai GeneChem Co., Ltd. (Shanghai, China). The culture medium was refreshed every 2–3 days, and only cells in the logarithmic growth phase were chosen for further experiments. For each group, cells were inoculated into 24-well plates at a density of 3 × 105 cells per well, with two wells being used for each group as duplicates. After 72 h of lentivirus infection, the cells were seeded in 10 cm dishes with medium containing antibiotics (puromycin), and the cell survival rate was higher than 90% for one week. A375 and SK-MEL-28 cell lines with stable TIPE2 expression were obtained. Two groups were established for each cell line: the control group and TIPE2 group. The related primer sequences are listed in Table 2. The expression of TIPE2 was verified by RT–qPCR and Western blotting (Fig. 2A).

Table 2 The related primer sequences for RT-PCR.
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Real-time quantitative PCR (RT-qPCR)

Total RNA was extracted from lentivirus-transfected melanoma cells using TRIzol reagent (Invitrogen) following the corresponding instructions. Approximately 5 µg of total RNA was reverse transcribed into first-strand cDNA for each sample. qRT‒PCR was conducted using SYBR Green Master Mix (Tiangen, Beijing, China) and an ABI Prism 7500 System (Life Technologies Corporation, Carlsbad). 18S RNA was used as the internal reference. The expression levels of genes were analyzed using the 2−∆∆CT method.

Western blot analysis

Lentivirus-transfected cells in stable culture were collected and suspended in RIPA lysis buffer (CWBIO, Beijing, China) containing protease inhibitors and phosphatase inhibitors. After incubation on ice for 10 min, the cell suspension (12,000 rpm, 4 °C) was centrifuged for 20 min. Approximately 60 μg of protein was mixed with SDS‒PAGE loading buffer (Beyotime, Shanghai, China), boiled at 100 °C for 5 min, and separated by 10% SDS–polyacrylamide gel electrophoresis. Then, the proteins were transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk for 1 h, followed by overnight treatment with the following primary antibodies at 4 °C: rabbit anti-TIPE2 (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-ERK (1:1000, Cell Signaling, Danvers, MA, USA), rabbit anti-P-ERK (1:1000, Cell Signaling, Danvers, MA, USA), rabbit anti-MRK1 (1:2000, Abcam, Cambridge, MA, USA), rabbit anti-P-MRK (1:2000, Abcam, Cambridge, MA, USA), and mouse anti-GAPDH (1:2000, Santa Cruz Biotechnology, Dallas, USA). After the primary antibody was washed from the membranes, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, USA) and finally the bands were visualized using an ECL chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA).

Cell proliferation, colony formation assay

Lentivirus-transfected cells in stable culture were inoculated into 96-well plates (100 μL/well) at a density of 2000 cells per well and cultured for 0 to 5 days. Cell proliferation was estimated using an Enhanced Cell Counting Kit-8 (Beyotime Biotechnology, Shanghai, China) (CCK-8) that determined daily cell growth. CCK-8 reagent (10 μL) was added to each well and incubated at 37 °C for 1.5 h. The absorbance was measured at 450 nm by a microplate reader (BioTek, Winooski, VT, USA). For the colony formation assays, 400 virally transfected melanoma cells were seeded into each well of a 6-well plate. After 12 days, the cells were fixed with methanol and stained with crystal violet. An Olympus CKX41 (Olympus, Tokyo, Japan) inverted microscope was used to count the number of colonies.

Wound healing assay

The stably cultured cells transfected with lentivirus were seeded in 6-well plates (2 ml/well) at a density of 2 × 105 cells per well, and a monolayer was formed after incubation for 24 h. The cell layer was gently scratched with the tip of a 2 μL pipette. The well was gently rinsed with PBS and the detached cells were removed. DMEM (without FBS) was added to the culture for 24 h. The cell wound width was observed at 0 h and 24 h under a light microscope. Subsequently, the gap distance was quantified by using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the ability of melanoma cells to migrate was analyzed.

In vivo tumorigenesis assay

Male BALB/c nude mice of 4–6 weeks of age were obtained from Shanghai GeneChem Co., Ltd. and raised under the corresponding conditions. A total of 12 nude mice were transfected with the TIPE2 overexpression vector in 200 μl of PBS (TIPE2 group), and 5 × 106 A375 cells were subcutaneously injected into the right armpit of nude mice. Control reagents were subcutaneously injected into the right armpit of nude mice. The tumor size was recorded 1 month later, and the tumor volume was calculated by the following formula: tumor volume = length × width × width × 3.14/6. Approximately 1 month later, all mice were sacrificed by cervical dislocation, and tumor resection, imaging, weighing, and formalin fixation were performed for the follow-up analyses.

Immunohistochemistry (IHC) and immunofluorescence (IF) studies

Mouse tumor tissue was embedded in paraffin, incubated at 4 °C overnight with the appropriate primary antibody, and then stained with an HRP anti-rabbit IgG secondary antibody (Cell Signaling Technology). The following primary and secondary antibodies were used in the immunofluorescence studies using mouse tumor tissue and human tissue samples: rabbit anti-TIPE2 (1:100 dilution; Santa Cruz Biotech, Santa Cruz, CA, USA), rabbit anti-Ki67 (1:300 dilution; Abcam, Cambridge, MA), and rabbit anti-phospho-ERK1/2 (1:100 dilution; Cell Signaling, Danvers, MA, USA). Immunohistochemical results were recorded according to the staining intensity and percentage of positive cells scores. The staining intensity score was defined as follows: 0, no staining; 1, weak; 2, moderate; 3, strong. The percentage of positively stained cells was defined as follows: 0, < 6%; 1, 6–29%; 2, 30–59%; 3, 60–74%; and 4, 75–100%. Adding these two values ​​yielded the final TIPE2 expression results. A score between 0 and 4 was considered low expression, while a score between 5 and 7 was considered high expression.

Data analysis

In this study, GraphPad Prism 8 was used to analyze the data. All experiments were independently conducted at least three times, and each experiment had 3 or more technical replicates expressed as the mean ± standard deviation. Statistically significant differences were verified using Student’s t test. P < 0.05 indicated that the difference was statistically significant.

Result

Melanoma exhibit downregulated TIPE2 expression in patients

To first assess how the TIPE2 might be linked to Melanoma progression, we explored its expression levels in tumor samples from those patients suffering from Melanoma. This analysis revealed that tumor samples exhibited significantly lower TIPE2 expression relative to normal control samples (P < 0.05) (Fig. 1A). In addition, patients with more advanced Melanoma exhibited significantly lower TIPE2 expression than those with less advanced disease (P < 0.05, Fig. 1B). Together these findings suggested the possibility that the TIPE2 is expressed at low levels in Melanoma and may play a role in disease progression.

Fig. 1
figure 1

Melanoma exhibit downregulated TIPE2 expression in patients. (A) Immunohistochemistry was used to assess TIPE2 expression in paired normal and melanoma tissues. (B) Melanoma patients with more advanced disease exhibited lower levels of TIPE2 expression. (C) The prognostic value of TIPE2 in patients with melanoma. High TIPE2 expression was associated with overall survival difference in melanoma patients [HR High = 0.65; p(HR) = 0.0015], see www.kmplot.com. Values represent the mean ± SD; For all panels, (D) Kaplan–Meier survival curves on the TIPE2 level in the TCGA skin cutaneous melanoma database. TIPE2: TNFAIP8-like 2. *P < 0.05.

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Elevated TIPE2 expression correlated with melanoma patient clinical characteristics

To investigate whether TIPE2 affects the prognosis of melanoma patients, We examined it in an online database of prognostic analyses for melanoma patients (KaplanMeier Plotter, www. kmplot.com). [p = 0.15, HR = 0.65] (Fig. 1C). In addition, Kaplan–Meier analysis was performed in 38 patients with TIPE2 mRNA levels classified from the low (n = 26) to the high (n = 12) using the TCGA skin melanoma database. We found that the group with higher TIPE2 mRNA levels had better survival compared to the group with higher TIPE2 mRNA levels (p = 0.020075). (Fig. 1D) These evidences suggest that TIPE2 expression could be a considerable factor affecting the prognosis of patients with melanoma.

Overexpression of TIPE2 in A375 and SK-MEL-28 Cells

We first measured TIPE2 expression in metastatic melanoma cells and lentivirus-transfected melanoma cells by RT‒qPCR and Western blotting. TIPE2 mRNA and protein expression levels were augmented in metastatic melanoma cells transfected with overexpressed TIPE2 compared with lentivirus-free metastatic melanoma cells (P < 0.0001) (Fig. 2A).

Fig. 2
figure 2

TIPE2 overexpression inhibited melanoma cell growth and migration. (A) The lentivirus transfection efficiency was confirmed by Western blotting and qRT-PCR, (B) CCK8 assay of A375 and SK-MEL-28 overexpression cell lines, C cell colony assay of A375 and SK-MEL-28 overexpression cell lines, (D) wound healing assay of A375 and SK-MEL-28 overexpression cell lines. Scale bars: 25/50 μm. TIPE2: TNFAIP8-like 2. n = 3. ***p < 0.001, ****p < 0.0001.

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TIPE2 inhibits proliferation and migration of melanoma cells

To assess the effect of TIPE2 on cell proliferation, we used TIPE2-overexpressing A375 and SK-MEL-28 cells. After lentivirus transfection, TIPE2 protein expression levels in A375 and SK-MEL-28 cells were dramatically increased. Then, cell proliferation was measured by the CCK-8 method. Overexpression of TIPE2 had a more obvious inhibitory effect on cell proliferation over time (P < 0.001) (Fig. 2B). Cells overexpressing TIPE2 showed a dramatic reduction in colony-forming capability when compared with that of control cells. The colony-forming ability of A375 cells transfected with lentivirus was diminished by 68% compared with that of control cells. Similarly, the colony-forming ability capacity of SK-MEL-28 cells transfected with lentivirus was alleviated by 66% compared with that of control cells (P < 0.001) (Fig. 2C). As shown in Fig. 2D, we discovered that overexpression of TIPE2 decreased the wound closure of A375 and SK-MEL-28 cells (P < 0.001).

TIPE2 inhibits melanoma growth in vivo

A375 cells transfected with either the control vector or TIPE2 expression vector were subcutaneously injected into nude mice to induce tumor formation. The experiment demonstrated that the TIPE2 group had significantly reduced tumor volume and weight compared to those of the control group (Fig. 3A–C). These results indicate that overexpression of TIPE2 could significantly suppress the growth of subcutaneous melanoma in vivo. The results were comparable to those of the in vitro experiments. Simultaneously, we examined the expression of Ki-67, a proliferative marker. The expression of Ki-67 in TIPE2-overexpressing melanoma was significantly downregulated (Fig. 3D). Therefore, our results suggest that TIPE2 has a significant inhibitory effect on melanoma growth in vivo.

Fig. 3
figure 3

Overexpression of TIPE2 inhibits melanoma growth in vivo. (A) Representative images of mice 4 weeks after subcutaneous injection of A375 Control and A375 TIPE2 OE cells. (B) After 1 week of inoculation, the tumor volume was measured every 6 days and the tumor growth curve was calculated. (C) Quantification of the average weight of A375 Control and A375 TIPE2 OE tumors. (D) Representative images of Ki67 immunofluorescence staining in A375 Control and A375 TIPE2 OE melanomas, N = 6, *p < 0.05. TIPE2: TNFAIP8-like 2.

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TIPE2 inhibits the activation of MEK/ERK pathway in metastatic melanoma cells

To estimate the potential molecular mechanism by which TIPE2 inhibits the proliferation of melanoma cells in vitro, we conducted RNA sequencing on the A375 cell control group and TIPE2 group. We performed transcriptomic analysis of the A375 cell control group and TIPE2 group (n = 3 for each group). A total of 313 differentially expressed genes were identified, of which 135 genes were upregulated and 178 genes were downregulated. A volcano plot was used to illustrate the differentially expressed genes between the groups (Fig. 4A). The cluster analysis results of the different groups are shown in Fig. 4B. KEGG enrichment analysis was conducted to identify pathways that are associated with the differentially expressed genes (Fig. 4C). By analyzing the transcriptome results, we suggested that MAPK signaling pathway could be the main signaling pathway involved in melanoma cell proliferation. We explored whether the MEK/ERK pathway is the target of TIPE2 action. The results showed that the TIPE2 protein level was significantly augmented in the TIPE2 groups. Moreover, the MEK/ERK signaling pathway was found to be involved in the proliferation and migration of melanoma cells. Consequently, we investigated whether TIPE2 levels impact the expression of MEK and ERK in melanoma cells. As shown in Fig. 4D, there was no dramatic fluctuation in MEK and ERK protein levels between the control group and the TIPE2 group. However, the expression of phosphorylated MEK and ERK was significantly downregulated in TIPE2 A375 and SK-MEL-28 cell groups compared with the control A375 and SK-MEL-28 cell groups, suggesting that TIPE2 negatively regulates the MEK/ERK pathway in melanoma cells. As a result, we believe that TIPE2 mainly reduces the proliferation of melanoma cells through ERK signaling. To explore the relationship between TIPE2 and phosphorylated ERK expression with melanoma tissues, we used immunohistochemical methods to detect TIPE2 and P-ERK expression in melanoma and adjacent paracancerous tissues. As depicted in Fig. 4E and F, TIPE2 exhibited weak staining in primary melanoma and lymph node metastasis tissues. However, it showed strong staining in the adjacent tissue. These findings suggested that TIPE2 was downregulated in the melanoma tissues and that TIPE2 expression was substantially linked with melanoma.

Fig. 4
figure 4

A375 cells were transfected with a control vector or TIPE2 vector, and melanoma cells were screened with puromycin for 1 week for transcriptome analysis. (A) Volcano plot of differentially expressed genes. Upregulated genes are represented by red dots, and downregulated genes are represented by green dots. (B) Cluster analysis of differential gene expression levels. Red indicates a protein-coding gene with relatively high expression, and green indicates a protein-coding gene with relatively low expression, (C) KEGG pathway enrichment analysis, (D) Western blot analysis showed that TIPE2 inhibited the phosphorylation of MEK and ERK, (E) Immunohistochemical analysis showed that p-ERK levels decreased in nude mice with subcutaneous injection of A375 cells stably transfected with TIPE2 compared with control mice, (F) Immunohistochemical analysis showed that P-ERK levels were elevated and TIPE2 expression was downregulated in melanoma tissues compared with adjacent nontumor breast tissue. The experiment was repeated three times with similar results. (N = 3, *p < 0.05), TIPE2: TNFAIP8-like 2; ERK: Extracellular signal-Regulated Kinase.

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TIPE2 had no significant effect on P53 signaling pathway and P13K-AKT signaling pathway in melanoma cell line A375

By analyzing the transcriptome results, we speculated that P53 signaling pathway and P13K-Akt signaling pathway are involved in the main signaling pathways of melanoma cell proliferation. We verified whether the P53 signaling pathway and P13K-AKT signaling pathway were the targets of TIPE2 through WB. The results showed that the protein levels of P53, p-p53, AKT and p-Akt were not statistically significant between control group and TIPE2 group (Fig. 5, P > 0.05). Therefore, we believe that TIPE2 has no significant effect on P53 signaling pathway and P13K-AKT signaling pathway in melanoma cell line A375 and SK-MEL-28.

Fig. 5
figure 5

Relative proteins expression of P53 and P13K-AKT signaling pathway in melanoma cell line (N = 3). TIPE2: TNFAIP8-like 2; p-AKT: phosphorylated AKT.

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TIPE2-mediated melanoma cell viability was significantly reversed by the ERK pathway activators

To determine whether TIPE2 specifically inhibits the viability of melanoma cells by inhibiting ERK signaling, we performed the next step by using an ERK activator (Ro 67–7476). As shown in Fig. 6, TIPE2-overexpressed melanoma cells A375 and SK-MEL-28 were then treated with an ERK pathway activator. The results showed that activation of ERK could restore TIPE2-inhibited melanoma cell viability in melanoma cells A375 (P < 0.05) and SK-MEL-28 (P < 0.001).

Fig. 6
figure 6

TIPE2-mediated melanoma cell viability was significantly reversed by ERK pathway activators (N = 3). TIPE2: TNFAIP8-like 2; ERK: Extracellular signal-Regulated Kinase. *P < 0.05, ***P < 0.001.

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Discussion

The TIPE/TNFAIP8 protein family is involved in immune regulation and tumorigenesis and four members, including TIPE or TNFAIP8 (tumor necrosis factor-α-induced protein 8), TIPE1 (TNFAIP8-like 1), TIPE2 (TNFAIP8-like 2), and TIPE3 (TNFAIP8-like 3) have attracted increasing research attention in recent years19. In the past decade, TIPE2 has been recognized as an important negative regulator of inflammation and immune homeostasis 20. Immune homeostasis is essential for the proper organ functioning, and immune disorders can lead to the development of deadly inflammatory diseases. Numerous studies show that the acquired immune response to cancer is closely related to the inflammatory response21,22. In our vivo experiments, TIPE2-deficient mice were more likely to die of chronic diseases, which was mainly manifested by body weight loss, splenomegaly, leukocytosis, and multiorgan inflammation20. Dysregulation of TIPE2 has been shown to have diverse effects on various cancer types. Recent studies have shown that TIPE2 expression is downregulated in stomach cancer, glioma, breast cancer, and primary liver cancer14,15,16,17. In addition, it was uncovered that the expression levels of TIPE2 in human pancreatic cancer tissues are lower than those in paracancerous tissues, and overexpression of TIPE2 in pancreatic cancer cells could significantly suppress cell proliferation and metastasis and augment apoptosis23. Researchers have confirmed that TIPE2 plays an essential role in regulating the immune system and acts as a protective factor in patients with skin cutaneous melanoma (SKCM). Furthermore, the study suggests that TIPE2 can impact the prognosis of SKCM patients by affecting the cancer stem cell index, which has the characteristics of self-renewal and treatment resistance24,25. According to our evidence, we proved that overexpressed TIPE2 suppressed the proliferation and migration of melanoma cell lines. Compared with that in paracarcinoma tissue, the expression of TIPE2 was substantially downregulated in human melanoma tissues. Furthermore, we found that TIPE2 significantly inhibited the growth of melanoma in vivo. This evidence demonstrated that TIPE2 could act as a suppressor gene in the development and progression of melanoma.

TIPE2 is involved in a variety of signal regulatory pathways, and the regulatory mechanisms of TIPE2 are different in different tissue types. In the field of neurology, researchers have indicated that TIPE2 can regulate phenotypic changes and proinflammatory responses in BV2 cells through the PI3K/AKT and NF-κB signaling pathways, which may contribute to neuroprotection26. Furthermore, the study demonstrated that TIPE2 can inhibit the TAK1/JNK signaling pathway, leading to the suppression of autophagic activity in dendritic cells (DCs)27. TIPE2 has also been shown to positively regulate AKT and P38 levels to suppress the proliferation of breast cancer cells, and tumor volume was notably diminished in mouse models with TIPE2 overexpression28. As a negative regulator of TLR signaling, TIPE2 can restore the control of gastric cancer cell proliferation by triggering the irf4-related signaling cascade 29.

The development and progression of melanoma are linked with multiple genetic alterations30,31,32,33,34,35. Several studies have suggested that the MEK/ERK signaling pathway is involved in the development of melanoma36,37,38. The mitogen-activated protein kinase (MAPK) pathway, a main traditional cell proliferation signaling pathway, consists of three main components: extracellular signal-regulated kinases (Ras/Raf1/MEK/ERK); the c-Jun N-terminal kinase (JNK) family; and MAPK14. Moreover, the ERK/MAPK pathway is currently recognized as one of the most prominent pathways involved in cell proliferation39. Drug research for novel melanoma treatments has concentrated on the inhibition of the MAPK pathway using small molecules, and therapy with BRAF inhibitors combined with MEK inhibitors has significantly improved the treatment response rate and survival rate of melanoma patients. However, long-term use of the drug can result in resistance and reduced efficacy. To overcome this issue, ERK kinase inhibitors are starting to be tested in clinical trials, which may provide new insights for the development of drugs that effectively treat melanoma9. Our evidence indicated that overexpression of TIPE2 considerably suppressed the phosphorylation of MEK and ERK in the MAPK signaling pathway. This finding suggests that TIPE2 could be an important inhibitor of melanoma development by downregulating the MEK/ERK pathway.

Our data indicate that the downregulation of TIPE2 is affiliated with the development of melanoma. TIPE2 inhibits the proliferation and migration of melanoma cells by inhibiting the phosphorylation of ERK. In conclusion, this study shows that TIPE2 could be used as an inhibitor of melanoma and a potential drug target in the future. TIPE2 inhibitors may have a therapeutic effect on melanoma. Since TIPE2 is widely distributed in human tissues and cells, side effects on other human tissues or organs are inevitable. More effort and time should be invested to conduct more in-depth and long-term studies on the value and development of TIPE2-based drugs for melanoma treatment.