FGF2 promotes metastasis of uveal melanoma cells via store-operated calcium entry

Abstract

Uveal melanoma (UM), the most common primary intraocular malignancy in adults, is highly metastatic and associated with dismal prognosis. Fibroblast growth factor 2 (FGF2) has been shown to induce cell proliferation and angiogenesis of melanoma and other malignancies. However, the expression of FGF2 in UM and its effects on melanoma cell migration are not well known. In this study, we found FGF2 expression was related to UM histological subtype and presence of metastasis. In vitro experiments showed that FGF2 treatment caused increased horizontal and vertical migration and F-actin cytoskeleton assembly as well as decreased adhesive activity of MUM2B cells, together with increased intracellular calcium concentration and expression of ORAI1 and STIM1 - two key regulatory proteins of store-operated calcium entry (SOCE). The mouse xenograft model showed that MUM2B cells with FGF2 stimulation grew into larger tumor masses and were prone to metastasis. In addition, the SOCE inhibitor 2-aminoethoxydiphenyl borate (2-APB) reversed all of these effects of FGF2. Finally, human UM samples and mouse xenograft model samples were used to confirm the correlation of FGF2 with ORAI1 and STIM1 expression. Taken together, our study suggests that FGF2 promotes metastasis of UM via SOCE.

Keywords: FGF2; ORAI1; STIM1; metastasis; store-operated calcium entry; uveal melanoma.

Figure 1

FGF2 expression in human uveal melanoma tissue samples. (A) Representative images of negative FGF2 expression in a spindle cell subtype sample and positive FGF2 expression in an epithelioid subtype sample (immunohistochemical staining, 100×); (B) FGF2 expression was higher in lung metastatic foci than in the primary foci from the same patient (immunohistochemical staining, 100×).

Figure 2

The effect of FGF2 on intracellular Ca²⁺ concentration and expression of store-operated calcium entry-regulated proteins. (A) FGF2 stimulation elevated intracellular calcium concentration, and 2-APB reversed this effect (left); (B) FGF2 stimulation increased the expression of STIM1 and ORAI1 of MUM2B cells, and 2-APB reversed this effect (left). Relative amounts of protein expression of STIM1 and ORAI1 compared with β-actin (right) (*P<0.05 between FGF2-treated cells and the control cells, **P<0.05 between FGF2/2-APB-treated cells and the FGF2-treated cells).

Figure 3

2-APB impairs increased horizontal and vertical migration and decreased adhesion of MUM2B cells caused by FGF2. (A) Images of wound healing assay at 0, 12, and 24 h (100×) (left). Migration distances at different times in the wound healing assay (right); (B) Cells invading through the Boyden chamber were stained (200×) (left). The numbers of invading cells were counted in five predetermined fields (right); (C) The numbers of bound cells were counted in five high magnification (200×) fields (*P<0.05 between FGF2-treated cells and the control cells, **P<0.05 between FGF2/2-APB-treated cells and the FGF2-treated cells).

Figure 4

2-APB impairs cytoskeleton assembly of MUM2B cells caused by FGF2. (Immunofluorescent staining of F-actin, Scale bar: 50 μm). Abbreviation: DAPI, diamidino-phenyl-indole.

Figure 5

2-APB inhibits in vivo tumor growth, metastasis caused by FGF2 and decreases the expression of STIM1 and ORAI1 in a MUM2B xenograft mouse model. (A) Xenograft tumor volumes are monitored over time (upper). Representative xenograft tumors on Day 30 post injection (lower); (B) Mice injected with cells with FGF2 stimulation showed lung metastasis and vascular invasion (H&E staining, 200×); (C) Representative images of the expression of FGF2, STIM1, and ORAI1 in xenograft tumor tissues (immunohistochemical staining, 400×).

Figure 6

FGF2 expression was concomitant with the expression of STIM1 and ORAI1 in human uveal melanoma tissue samples. Representative images of the expression of FGF2, STIM1, and ORAI1 in FGF2-negative and -positive groups (immunohistochemical staining, 200×) (left). Percentages of STIM1 or ORAI1 negative and positive expression in the FGF2-negative and FGF2-positive groups (right).