Bone morphogenetic protein 7 induces mesenchymal-to-epithelial transition in melanoma cells, leading to inhibition of metastasis

Abstract

Bone morphogenetic protein (BMP) 7 counteracts physiological epithelial-to-mesenchymal transition, a process that is indicative of epithelial plasticity in developmental stages. Because epithelial-to-mesenchymal transition and its reversed process mesenchymal-to-epithelial transition (MET) are also involved in cancer progression, we investigated whether BMP7 plays a role in WM-266-4 melanoma cell growth and metastasis. An MTT assay was conducted in WM-266-4 and HEK293T cell lines to show the cell growth inhibition ability of BMP7 and cisplatin. Semiquantitative RT-PCR was used to determine MET in morphologically changed BMP7-treated melanoma cells. MET-induced cells expressed less a basic helix-loop-helix transcription factor (TWIST) in western blot analysis, and we confirm that BMP receptor (Alk2) siRNA transduction could restore TWIST protein expression via blocking of Smad 1, 5 and 8 signaling. Matrigel invasion and cell migration assays were done to investigate the BMP7-induced metastasis inhibition ability. BMP7 treatment only slightly reduced cell growth rate, but induced apparent MET. BMP7 also reduced the invasion and migration ability. Furthermore, BMP7 reduced the resistance of WM-266-4 cells to cisplatin. Collectively, our findings indicate that the metastatis inhibition ability of BMP7 is involved in MET, and that BMP7 could be used as a potential metastasis inhibitor in human melanoma cells.

Figure ;1

Growth curves of WM‐266‐4 and HEK293T cells treated with bone morphogenetic protein (BMP) 7 and cisplatin. (A) BMP7 at 400 ng/mL showed significant growth inhibition of melanoma cells whereas HEK293T cells did not respond even at the maximum concentration tested (*P < 0.015). (B) Both cell lines showed dose‐dependent survival rates in response to cisplatin. The EC₅₀ of cisplatin in the melanoma cell line was approximately 1.5 times higher than in the HEK293T cell line (5.64 and 3.87 μmol/L, respectively). Bars, SD; *P < 0.04.

Figure 2

Mesenchymal‐to‐epithelial transition (MET) induced by bone morphogenetic protein (BMP) 7 in the WM‐266‐4 human melanoma cell line. (A) Cells were treated with various concentrations of BMP7 and cisplatin, and the monolayer morphology was photographed at ×40 magnification 8 days after treatment. Cells of the BMP7‐treated group showed morphological changes resembling cells undergoing MET, transitioning from mesenchymal to epithelial‐like cells in a dose‐responsive manner. In contrast, almost all of the cisplatin‐treated cells died without apparent MET at concentrations of 10 and 100 μmol/L. Scale bar = 50 μm. (B) Actin immunostaining revealed that normal WM‐266‐4 cells have numerous and long cytoplasmic projections (stained strong brown), but the cells under BMP7 100 ng/mL treatment for 10 days show short projections and polygonal cell body. Images are representatives of three replicate experiments. (C) Total RNA was prepared and semiquantitative RT‐PCR was conducted to confirm expression of genes related to BMP7‐responsive receptors. Expression of the BMP7‐specific receptor BMP receptor (Alk2) increased only in the BMP7‐treated group. Expression of Alk6 and noggin increased following BMP7 treatment, but did not show a dose‐dependent response. Expression of Slug, smooth muscle actin (SMA), TWIST, and snail were also decreased in the BMP7‐treated WM‐266‐4 cells. Cisplatin‐treated cells showed decreased levels of Slug mRNA, but not for SMA, TWIST, and snail. The experiments were repeated three times, each with triplicate samples. Semiquantitative RT‐PCR results for (D) Alk2, (E) SMA, and (F) TWIST are shown. P‐values for comparisons in the 95% significant level were analyzed by Dunn’s Multiple Comparison Test. Bars, SD; *P < 0.05.

Figure ;3

BMP receptor (Alk2) gene knockdown abrogated phosphorylation of Smad1/5/8 and a basic helix‐loop‐helix transcription factor (TWIST) inhibition. (A) Western blot analysis shows that bone morphogenetic protein (BMP) 7 treatment phosphorylates Smad1/5/8 in a dose‐dependent manner. Control cells express almost no p‐Smad‐1/5/8, but after 1 h of treatment with BMP7, the cells express p‐Smad1/5/8 increasingly. (B) WM‐266‐4 cells were treated with various concentrations of BMP7 for 8 days and collected for western blotting. TWIST expression was decreased in the 100 and 200 ng/mL BMP7‐treated groups. (C) Two different sequences of siRNA targeting Alk2 (si1 and si2) as well as scrambled sequence siRNA (scr) were transduced twice at 3‐day intervals. Cells were cotreated with BMP7 100 ng/mL for 8 days and the expression levels of Alk2 were examined. Alk2 mRNA expression were decreased in the si1‐ and si2‐transfected groups. (D) Only si1 siRNA inhibited phosphorylation of Smad1/5/8 under BMP7 100 ng/mL treatment. (E) TWIST protein expression decreased in the scrambled siRNA transfection group (scr) but not in the si1‐transfected group.

Figure 4

Bone morphogenetic protein (BMP) 7 inhibited melanoma cell invasion ability. (A) Cells invading through the extracellular basement membrane showed long filopodia‐like mesenchymal cell bodies (left). After invasion, round, non‐attached, single cells were present at the bottom of the Matrigel (right). Scale bar = 50 μm. (B) A 20‐h incubation time allowed complete invasion of most of the treated cells. The remnants covered the upper side of the Matrigel. Photographs were taken at the bottom site at 40 ×  magnification. Scale bar = 50 μm. (C) WM‐266‐4 cells were treated with 100 ng/mL BMP7, 4 μmol/L cisplatin, or both, for 8 days before the invasion assay. The BMP7‐treated group showed lower invaded cell numbers, which were reduced by approximately 65% invaded cell numbers compared with that of the control. Cisplatin did not affect cell invasion ability. The experiments were repeated three times, each with triplicate samples. Cell counts were analyzed using Image Pro Plus software. P‐values for comparisons in the 95% significant level were analyzed by Mann–Whitney two‐tailed test. Bars, SD; *P < 0.018; **P < 0.0042.

Figure 5

Bone morphogenetic protein (BMP) 7 inhibited melanoma cell migration ability and reduced resistance to cisplatin. (A) WM‐266‐4 cells were treated with 100 ng/mL BMP7 or 4 μmol/L cisplatin in 0.1% serum medium for 8 days and grown to 90% confluency. A wound line was made on the monolayer cultures with a microtip, cells were washed twice with PBS, and fresh medium was added. Photographs were taken at 12‐h time points. Migrating cell counts were analyzed using Image Pro Software. BMP7 reduced cell migration ability to <50% of that in the control, whereas cisplatin only slightly inhibited migration ability. Bars, SD. **P < 0.0074; ***P < 0.0006. (B) WM‐266‐4 cells were treated with BMP7 as indicated for 3 days, then with cisplatin at various concentrations for an additional 8 days. Cell resistance to cisplatin gradually decreased with increasing BMP7 concentrations. Bars, SD; *P < 0.005.