NETO2 promotes melanoma progression via activation of the Ca<sup>2+</sup>/CaMKII signaling pathway

doi:10.1007/s11684-022-0935-0

Front. Med.

2023, Vol. 17

Issue (2) : 263-274 https://doi.org/10.1007/s11684-022-0935-0

RESEARCH ARTICLE

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

Susi Zhu^1,^2,^3,^4,⁵, Xu Zhang^1,^2,^3,⁴, Yeye Guo^1,^2,^3,⁴, Ling Tang^1,^2,^3,^4,⁵, Zhe Zhou^1,^2,^3,⁴, Xiang Chen^1,^2,^3,⁴(

), Cong Peng^1,^2,^3,⁴(

)

¹. Department of Dermatology, Xiangya Hospital, Central South University, Changsha 41000, China
². Hunan Key Laboratory of Skin Cancer and Psoriasis, Changsha 41000, China
³. Hunan Engineering Research Center of Skin Health and Disease, Changsha 41000, China
⁴. National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha 41000, China
⁵. Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 41000, China

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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 Ca²⁺/CaMKII signaling pathway

Corresponding Author(s): Xiang Chen,Cong Peng

Just Accepted Date: 01 December 2022 Online First Date: 07 February 2023 Issue Date: 26 May 2023

Cite this article:

Susi Zhu,Xu Zhang,Yeye Guo, et al. NETO2 promotes melanoma progression via activation of the Ca²⁺/CaMKII signaling pathway[J]. Front. Med., 2023, 17(2): 263-274.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-022-0935-0
https://academic.hep.com.cn/fmd/EN/Y2023/V17/I2/263

Fig.1 NETO2 overexpression in melanoma tissues correlated with melanoma prognosis. (A) Relative NETO2 mRNA expression in normal skin, nevi, and melanoma tissues; data were obtained from the GEO datasets GSE46517 and GSE3189. (B) Relative NETO2 mRNA expression in normal and melanoma tissues; data were obtained from GEPIA (TCGA). (C) IHC analyses of NETO2 protein levels in a human melanoma tissue microarray (left). Scale bar, 50 μm. The IHC score for NETO2 in benign and malignant and metastasic melanoma tissues (right). (D) The Kaplan–Meier overall and disease-free survival curves for melanoma patients correlated with NETO2 expression; data were obtained from GEPIA. The horizontal lines in all dot plots represent the means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig.2 NETO2 promoted melanoma cell proliferation in vitro. (A) NETO2 expression in PIG1 cells and melanoma cell lines. GAPDH was used as a loading control. (B) SK-Mel-5 and SK-Mel-28 cells that had been infected with shMOCK or shNETO2 lentivirus. (C) Cell proliferation was assessed after NETO2 knockdown in SK-Mel-5 and SK-Mel-28 cells with CCK-8 assays. (D) Colony-formation capability was assessed in SK-Mel-5 and SK-Mel-28 cells with NETO2 knockdown; the number of clones of SK-Mel-5 and SK-Mel-28 cells was quantified. (E) WM35 cells were infected with control or NETO2-expressing lentivirus. (F) CCK-8 assays were conducted in NETO2-overexpressing WM35 cells. (G) Colony-formation capability was assessed in NETO2-overexpressing WM35 cells; the number of WM35 cell colonies was quantified. The data (means ± SEMs) were obtained from triplicate experiments. **P < 0.01, ***P < 0.001.

Fig.3 NETO2 promoted the growth of melanoma tumors in vivo. (A,B) Representative images of nude mice injected with differentially treated SK-Mel-28 cells (infected with shMOCK, shNETO2#2, or shNETO2#3) and their corresponding tumors. (C) Tumor size was analyzed quantitatively. (D,E) Immunohistochemical staining of Ki-67, P-CaMKII, and P-CREB in xenograft tumors; scale bar, 50 µm. The data (means ± SEMs) were obtained from triplicate experiments. **P < 0.01.

Fig.4 NETO2 promotes melanoma cell migration and invasion by inducing MMPs. (A) Wound healing assay in SK-Mel-5 and SK-Mel-28 cells expressing NETO2 and control shRNA, respectively; quantified data from wound healing assays at 0, 24, and 48 h are shown. (B,C) Transwell migration and invasion assays with NETO2 knockdown melanoma cells. (D) Western blot analysis of MMP2 and MMP9 levels in melanoma cells infected with shNETO2 lentivirus. (E) Wound healing assay in WM35 cells expressing NETO2 (pLVX-NETO2) or the control (pLVX-IRES). Quantitative analysis of the wounded areas of WM35 cells at 0, 12, and 24 h. (F) Migration (12 h) and invasion (24 h) assays in WM35 cells overexpressing NETO2. (G) Western blot analysis of MMP2 and MMP9 levels in melanoma cells infected with pLVX-NETO2 lentivirus; band intensities were quantified using ImageLab. The data (means ± SEMs) were obtained from triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig.5 NETO2 activates the Ca²⁺/CaMKII pathways. (A,B) Flow cytometry detection of calcium ions in SK-Mel-5 and SK-Mel-28 cells expressing NETO2 or control shRNA, respectively. (C) Western blot analysis of CaMKII, P-CaMKII, CREB, P-CREB, and GAPDH in SK-Mel-5 and SK-Mel-28 cells infected with shNETO2 lentivirus; band intensities were quantified using ImageLab. (D,E) Flow cytometry detection of calcium ions in WM35 cells infected with NETO2 lentivirus. (F) Western blot analysis of CaMKII, P-CaMKII, CREB, P-CREB, and GAPDH in WM35 cells infected with NETO2-expressing lentivirus; band intensities were quantified using ImageLab. The data (mean ± SEM) were obtained from triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig.6 Inhibition of CaMKII antagonized NETO2-mediated proliferation, migration, and invasion of melanoma cells. (A) Western blot analysis of NETO2, P-CaMKII, P-CREB, and MMP2 levels in NETO2-overexpressing melanoma cells with or without 10 μmol/L KN93 treatment; band intensities were quantified using ImageLab. (B) CCK-8 assays were conducted to analyze the effects of KN93 on melanoma cell proliferation. (C) Colony-formation capability was assessed in WM35 cells with or without 10μmol/L KN93 treatment. (D,E) Wound healing and Transwell assays were used to analyze melanoma cell migration and invasion, respectively. The data (mean ± SEM) were obtained from triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig.7 Schematic diagram depicting the NETO2-mediated induction of the CaMKII/CREB pathway.

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