Selenium-binding protein 1 is down-regulated in malignant melanoma

Abstract

Selenium-binding protein 1 (SELENBP1) expression is reduced in various epithelial cancer entities compared to corresponding normal tissue and has already been described as a tumor suppressor involved in the regulation of cell proliferation, senescence, migration and apoptosis. We identified SELENBP1 to be down-regulated in cutaneous melanoma, a malignant cancer of pigment-producing melanocytes in the skin, which leads to the assumption that SELENBP1 also functions as tumor suppressor in the skin, as shown by others e.g. for prostate or lung carcinoma. However, in vitro analyses indicate that SELENBP1 re-expression in human melanoma cell lines has no impact on cell proliferation, migration or tube formation of the tumor cells themselves when compared to control-transfected cells. Interestingly, supernatant taken from melanoma cell lines transfected with a SELENBP1 re-expression plasmid led to suppression of vessel formation of HMEC cells. Furthermore, SELENBP1 re-expression alters the sensitivity of melanoma cells for Vemurafenib treatment. The data also hint to a functional interaction of SELENBP1 with GPX1 (Glutathione peroxidase 1). Low SELENBP1 mRNA levels correlate inversely with GPX1 expression in melanoma. The re-expression of SELENBP1 combined with down-regulation of GPX1 expression led to reduction of the proliferation of melanoma cells. In summary, SELENBP1 influences the tumor microenvironment and SELENBP1 action is functionally influenced by GPX1.

Keywords: Grm1 mouse model; glutathione peroxidase 1 (GPX1); malignant melanoma; selenium-binding protein 1 (SELENBP1).

Figure 1. SELENBP1 expression in murine Tg(Grm1) melanoma tissue

(A) SELENBP1 (Sbp1) expression in murine Tg(Grm1) melanoma (n = 2) compared to nevi (n = 2) tissue via RNA-sequencing analysis. (B) Quantitative real-time PCR analysis to calculate mRNA expression in murine nevi (n = 5) and murine melanoma samples (n = 5) (^**p-value: 0.0011). (C) Western blot analysis to detect SelenBP1 on protein level in murine melanoma tissue samples (n = 4) compared to nevi samples (n = 4). GAPDH was used as a loading control. (D) Immunofluorescence analysis of SelenBP1 (green) in murine nevi tissue from the tail (I, II, III) and primary melanomas (IV, V, VI). DAPI was used to visualize the localization of nuclei. All images showed unspecific green staining of the epidermal keratin layer.

Figure 2. SELENBP1 in human melanoma cell lines

(A) SELENBP1 mRNA expression in melanoma cell lines (n = 10) and normal human epidermal melanocytes (NHEM) (n = 8). SELENBP1 expression levels were normalized to β-actin (^*p-value: 0.0228). (B) Western blot analysis for detecting SELENBP1 protein in melanoma cell lines and NHEM. GAPDH served as a loading control. (C) Immunofluorescence staining for SELENBP1 protein using representative melanoma cell lines Mel Juso and Sk-Mel-28 and NHEM. DAPI was used for nuclear detection.

Figure 3. SELENBP1 in vivo in human melanoma (patient)

(A) Expression analysis of SELENBP1 in nevi, melanocytes and keratinocytes. (B) Tissue of normal skin (n = 4), primary melanoma (n = 4) and melanoma metastasis (n = 4) were analyzed for SELENBP1 expression on mRNA level. (C) Geoprofile data sets (GDS1375) showed SELENPB1 mRNA expression in normal skin (NS) and in primary tumor (PT) and metastasis (MET) of melanoma patient. (D) SELENBP1 protein level was analyzed by Western blot analysis. Values below the blot indicate the ratio between SELENBP1 and GAPDH for each sample and were calculated using ImageJ. (E) Immunohistochemical analysis showed SELENBP1 staining in primary melanoma biopsies (n = 5) and melanoma metastases (n = 5) compared to nevi tissue (n = 6). Representative microscopy images were shown for each tissue, as well as H&E staining. (F) Statistical analysis of SELENBP1 immunohistochemistry. Evaluation was performed by a classification into three categories: low, moderate and high SELENBP1 protein expression.

Figure 4. SELENBP1 re-expression in human melanoma cells and cellular mechanisms

(A) Quantitative real-time PCR analysis and (B) Western blot analysis confirmed SELENBP1 (pSBP1) re-expression in Mel Juso and Sk-Mel-28 melanoma cell lines, compared to pcDNA control-transfected cells 24 h after transfection (^**p < 0.01). (C–E) RTCA experiments to analyze alterations in cell attachment, proliferation and migration (ns: not significant). (F) Clonogenic assays for analyzing the impact of SELENBP1 re-expression on self-renewing capacity. (G) Matrigel-based tube formation assays displayed the development of vascular channels after transfection with pSBP1 vector. (H) MSA and H₂O₂ treatment together with pcDNA or SELENBP1 (pSBP1) over-expression, respectively, was analyzed by RTCA (ns: not significant). (I) Supernatant (SN) of melanoma cell lines re-expressing SELENBP1 (pSBP1) and control transfected cells (pcDBNA) was used for cell culture of human dermal microvascular endothelial cells (HMECs). Matrigel-based tube formation assays displayed the vessel formation. (J) RTCA proliferation assay for the melanoma cell line Mel Juso treated with Vemurafenib (5 μM) and transfected with the SELENBP1 expression construct, respectively.

Figure 5. Connection between SELENBP1 and GPX1

(A) Geoprofile data sets (GDS1375) show GPX1 mRNA expression in normal skin (NS), primary tumor (PT) and metastasis (MET) of melanoma patients (^***p < 0.001; ns, not significant). (B) Immunohistochemical analysis showing GPX1 staining in human melanoma samples (n = 10, data bank of proteinatlas.org). Representative microscopy images are shown. Evaluation was performed by classification into three categories: low, medium and high GPX1 protein expression. (C) Western blot analysis of SELENBP1 and GPX1 protein in 18 different melanoma cell lines of different tumor stages (RGP: radial growth phase; VGP: vertical growth phase; PT: primary tumor; MET: metastasis). GAPDH was used as a loading control. (D) Treatment of cells with siRNA against GPX1 and confirmation of successful knock-down by using anti-GPX1 antibody. Analysis of the influence of silenced GPX1 on SELENBP1 expression using an anti-SELENBP1 antibody and analyzing the SELENBP1 expression on mRNA level. (E) Treatment of cells with a re-expression vector (pSBP1) for SELENBP1 and confirmation of successful re-expression by using anti-SELENBP1 antibody. Analysis of the influence of re-expressed SELENBP1 on GPX1 expression using an anti-GPX1 antibody and analyzing the GPX1 expression on mRNA level. (F) RTCA proliferation assay after SELENBP1 re-expression of GPX1 silencing alone and in combination. (G) Clonogenic assays for analyzing the impact of SELENBP1 re-expression and GPX1 knock-down on self-renewing capacity.