mGPDH Deficiency leads to melanoma metastasis via induced NRF2

Abstract

Oxidative stress critically influences carcinogenesis and the progression of melanoma, and aggressive malignant melanoma activity is due to its high metastatic ability. Some findings in several cancer cell lines have indicated that mGPDH, a component of the mitochondrial respiratory chain, also modulates oxidative stress. However, the role of mGPDH in melanoma remains elusive. Here, we report that the mGPDH protein level is decreased in human skin melanoma compared to normal skin and decreased in metastatic melanoma compared to primary melanoma. Our in vivo and in vitro experiments indicated that mGPDH depletion accelerated melanoma migration and invasion without affecting proliferation or apoptosis. Mechanistically, we found elevated NRF2 protein levels in human skin melanoma and mGPDH-knockout (ko) metastatic xenografts in the lungs of nude mice. Moreover, in A375 melanoma cells, the loss of mGPDH-induced NRF2 expression but did not affect NRF2 protein degradation. Additionally, melanoma metastasis induced by the loss of mGPDH was rescued by the further down-regulation of NRF2 in vivo and in vitro. Consistently, mGPDH overexpression (oe) depressed NRF2 expression and attenuated the malignant properties of melanoma cells. In conclusion, our findings suggest that mGPDH suppresses melanoma metastasis by inhibiting NRF2 and downstream oxidative signals, highlighting the therapeutic potential of mGPDH for melanoma treatment.

Keywords: NRF2; mGPDH; melanoma; metastasis.

FIGURE 1

Decreased mGPDH expression in primary and metastatic melanoma samples. A, mGPDH protein levels and WB grey value quantification in skin melanoma tissue and adjacent skin tissue from 3 patients (n = 3). B‐D, mGPDH protein expression in a commercial melanoma tissue array (ME2082c, Biomax) was detected by IHC. The tissue array contained tissue from 101 patients with primary malignant melanoma (diagnosed with stage I in 6 cases, II in 55 cases, III in 8 cases and IV in 2 cases; T1 in 3 cases, T2 in 11 cases, T3 in 4 cases and T4 in 53 cases; and N0 in 63 cases, N1 in 7 cases and N2 in 1 case; containing 45 skin, 16 recta, 6 eye and 6 soft tissue samples) and 53 metastatic malignant melanoma tissues (lymphatic metastasis). B, Representative IHC images of mGPDH in primary and metastatic melanoma tissue sections. Scale bar, 50 μm. C, The distribution of mGPDH IHC scores for the tissue sections from the primary and metastatic melanoma groups. IHC scores: ‐: 0‐2; +: 2‐4; ++: 4‐8; +++: 8‐12. D, The frequencies of tissue sections with high mGPDH expression (IHC score≥4) and low mGPDH expression (IHC score<4) in the primary and metastatic melanoma groups. In A, the p‐value was derived from Student's t test; In D, the P‐Values were derived from one‐tailed Fisher's exact tests. *P < 0.05

FIGURE 2

mGPDH silencing induced melanoma cell metastasis in vitro and in vivo. A, RT‐PCR validation of the knockdown efficacy of siRNA targeting mGPDH. B, Transwell migration and Matrigel invasion assays of A375 cells transfected with control siRNA or mGPDH siRNA. Scale bar, 200 μm. C, Results of cell quantification by Transwell migration and Matrigel invasion assays (n = 3). D‐G, Luciferase sh‐control shRNA (Sh‐ko‐control) and mGPDH‐ko shRNA (Sh‐ko‐mGPDH) were transfected into A375 cells to construct stable melanoma cell lines. The cells (2 million) were injected into nude mice via the tail vein, and melanoma cell metastasis was observed in vivo. Transfected A375 cell lines were continuously cultured for 8 wk to ensure consistency with the timing of the in vivo experiment. D. Relative mRNA expression (n = 3). E, Protein levels of mGPDH were detected by intensity quantification. F, G, In vivo metastasis assays. At 8 wk post‐injection of the stable cells, melanoma cell metastasis in nude mice was assessed by bioluminescence imaging, and the photon flux ratio was quantified (n = 5‐7 mice per group). In A, C, D and G, the P‐values were derived from Student's t tests. *P < 0.05, **P < 0.01, ***P < 0.001

FIGURE 3

mGPDH silencing activated NRF2. A, B, The protein and the mRNA levels of mGPDH and NRF2 signalling pathway in A375 cells without or with mGPDH siRNA treatment were detected by WB analysis and RT‐PCR (n = 3). C, NRF2 protein half‐life (t_1/2) in A375 cells transduced with mGPDH siRNA was determined by pulse‐chase assay and immunoblotting. A375 cells were transfected with control siRNA and mGPDH siRNA for 48 h, after which cycloheximide (50 μmol/L) was administered to block protein synthesis. D. Protein levels of NRF2 and HO‐1 in skin melanoma tissue and adjacent skin tissue from patients were determined by intensity quantification (n = 3). E. Lung tissues of melanoma metastasis from nude mice injected with Luciferase‐control shRNA (Sh‐ko‐control)/mGPDH‐ko shRNA (Sh‐ko‐mGPDH) A375 cells were harvested. Representative images of lungs and their corresponding tissue sections stained with H&E are shown. Scale bars, 500 μm (gross morphology) and 200 μm (H&E staining). The expression of mGPDH, NRF2 and HO‐1 was detected by IHC, as shown in the right three images. Scale bar, 200 μm. The P‐Values were derived from Student's t tests. *P < 0.05, **P < 0.01, ***P < 0.001

FIGURE 4

Down‐regulation of NRF2 rescued mGPDH loss‐induced melanoma metastasis. Control shRNA, mGPDH‐ko shRNA (Sh‐ko‐mGPDH) and/or NRF2 ‐ko shRNA (Sh‐ko‐NRF2) were transfected into A375 cells to construct the following 3 stable cell lines: Sh‐control, Sh‐ko‐mGPDH and Sh‐ko‐mGPDH Sh‐ko‐NRF2 A375 cells. These cell lines were cultured for 8 wk. A, WB analysis was used to detect mGPDH and NRF2 expression, and the intensity was quantified in the three cell lines. B, C, Cell migration and invasion were evaluated and quantified by Transwell migration and Matrigel assays, respectively (n = 3). Scale bar, 200 μm. D, In in vivo metastasis assays, nude mice were injected with the three stable cell lines. D, E, After 8 wk, melanoma cell metastasis in the nude mice was assessed by bioluminescence imaging and quantified as the photon flux ratio (n = 5 mice per group). F. Lung images and H&E staining for the three groups. Scale bars, 500 μm and 200 μm. The P‐Values were derived from one‐way ANOVA with the Newman‐Keuls multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

FIGURE 5

mGPDH‐overexpression alleviated melanoma metastasis. A‐D, A375 cells were transfected with a vector (Oe‐vector) or mGPDH‐overexpression plasmid (Oe‐mGPDH). A, Relative protein levels were detected by WB analysis, and the intensity was quantified. B, Transwell migration (upper) and Matrigel invasion assays (lower). Scale bar, 200 μm. C, D, Relative results of cell quantification by Transwell migration and Matrigel invasion assays (n = 3). E‐I, Luciferase‐control vector shRNA (Sh‐oe‐vector) or luciferase‐overexpression mGPDH shRNA (Sh‐oe‐mGPDH) was transfected into the A375 cell line to construct stable cell lines. These cells were intravenously injected into nude mice, and melanoma cell metastasis was observed for 8 wk in vivo. E, The relative mRNA expression of mGPDH was detected (n = 3). F, Transfected A375 cell lines were consistently cultured in vitro for 8 wk, and then mGPDH and NRF2 protein levels were detected. G‐I, In vivo metastasis assays. G, H, At 8 wk post‐injection of the stable cell lines, melanoma cell metastasis in the nude mice was assessed by bioluminescence imaging and quantified (n = 4 mice per group). I, Metastatic lung tissue images and H&E staining of the two groups. Scale bars, 500 μm and 200 μm. The p‐values were derived from Student's t tests. ***P < 0.001, **P < 0.01