MCP-1 overexpressed in tuberous sclerosis lesions acts as a paracrine factor for tumor development

Abstract

Patients with tuberous sclerosis complex (TSC) develop hamartomatous tumors showing loss of function of the tumor suppressor TSC1 (hamartin) or TSC2 (tuberin) and increased angiogenesis, fibrosis, and abundant mononuclear phagocytes. To identify soluble factors with potential roles in TSC tumorigenesis, we screened TSC skin tumor-derived cells for altered gene and protein expression. Fibroblast-like cells from 10 angiofibromas and five periungual fibromas produced higher levels of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein than did fibroblasts from the same patient's normal skin. Conditioned medium from angiofibroma cells stimulated chemotaxis of a human monocytic cell line to a greater extent than conditioned medium from TSC fibroblasts, an effect blocked by neutralizing MCP-1-specific antibody. Overexpression of MCP-1 seems to be caused by loss of tuberin function because Eker rat embryonic fibroblasts null for Tsc2 (EEF Tsc2(-/-)) produced 28 times as much MCP-1 protein as did EEF Tsc2(+/+) cells; transient expression of WT but not mutant human TSC2 by EEF Tsc2(-/-) cells inhibited MCP-1 production; and pharmacological inhibition of the Rheb-mTOR pathway, which is hyperactivated after loss of TSC2, decreased MCP-1 production by EEF Tsc2(-/-) cells. Together these findings suggest that MCP-1 is an important paracrine factor for TSC tumorigenesis and may be a new therapeutic target.

Figure 1.

CD68-positive cells are more abundant in TSC skin tumors than in normal-appearing skin from a patient with TSC. (A) In sections of normal-appearing skin, cells staining for CD68 (brown) are sparse in the dermis. Bar, 65 μm. (B) In sections of a periungual fibroma, there are dilated vessels and fibrosis and a more cellular stroma. Many stromal cells stain positive for CD68. Bar, 65 μm. (C) CD68-positive cells are located near vessels among CD68-negative, fibroblast-like cells. Bar, 15 μm. Similar results were observed in four angiofibromas and four periungual fibromas from six patients.

Figure 2.

Cells cultured from TSC skin tumors express the fibroblast marker HSP47 but not CD68. The cytoplasm of TSC fibroblasts (A) and angiofibroma cells (B) stain positive for HSP47 (green), whereas U937 cells (C), a human monocytic cell line, are negative. In contrast, the cytoplasm of TSC fibroblasts (D) and angiofibroma cells (E) are negative for CD68, and the cytoplasm of U937 cells (F) stains positive (green). Nuclei fluoresce blue with DAPI. Bar, 15 μm. Similar results were observed in cells from two other patients.

Figure 3.

Measurement of cytokines in culture supernatants reveals increased production of MCP-1 by angiofibroma cells. Fibroblasts from normal-appearing skin (NL) or angiofibroma cells (AF) from seven patients were incubated in 1% FBS/DMEM for 24 h, and supernatants were collected for cytokine measurement using ELISA. Lines connect paired samples for each patient. The inset shows the data as a box plot. *, P = 0.018.

Figure 4.

MCP-1 production is stimulated by FBS. Paired cultures of angiofibroma cells (AF) and TSC fibroblasts (NL) from two patients were seeded at 10,000 cells/well in 96-well plates, in DMEM containing 0%, 1%, 2%, or 10% FBS (1% BSA was added to the 0% FBS condition). After a 12-h incubation, MCP-1 concentration in the medium was measured by ELISA (pg/ml), and expressed per total cellular ATP (luminescence units) as an indication of cell number. Results are means ± SD of values from triplicate wells. Fresh medium with 10% FBS does not contain detectable MCP-1.

Figure 5.

MCP-1 produced by TSC tumor cells is chemotactic for monocytes. Conditioned medium (CM), from TSC fibroblasts (closed bars) or angiofibroma cells (open bars), CM plus control antibody, or CM plus anti-MCP-1 antibody was added to the bottom chamber of cell migration plates. THP-1 cells (a human monocytic cell line) were added to the top chamber. Cells that migrated through 8-μm pores to the feeder tray after a 2-h incubation were lysed and detected by CyQuant GR dye that exhibits enhanced fluorescence upon binding cellular nucleic acids. Results are geometric means ± SD of values from three separate migration chambers. *, P = 0.019 and **, P = 0.007 as compared with CM from TSC fibroblasts. Conditioned medium from angiofibroma cells or TSC fibroblasts contained 1640 and 60 pg/ml MCP-1, respectively. Similar results were observed in three separate experiments.

Figure 6.

EEF Tsc2 ^−/− fibroblasts produced more MCP-1 than normal (EEF Tsc2 ^+/+) fibroblasts. Culture supernatants were collected after incubating EEF cells for the indicated time, and MCP-1 in the medium was measured by ELISA. Results are means ± SD of triplicate supernatants.

Figure 7.

Transfection of WT but not mutant TSC2 into EEF Tsc2 ^−/− cells inhibits MCP-1 production. EEF Tsc2 ^−/− cells were transfected with the indicated amounts of human TSC2 constructs and/or empty vector. In the last lane, EEF Tsc2^+/+ cells were transfected with empty vector. After transfection, cells were maintained in DMEM with 10% FBS overnight before the culture medium was replaced with DMEM containing 2% FBS; then incubation continued for an additional 24 h. The cells were harvested, and tuberin expression was detected by Western blot. Culture supernatants were collected, and MCP-1 release into the medium was measured by ELISA. Results are the mean ± SD of three separate wells. M1, mutation G294E; M2, mutation I365del; P1, polymorphism M286V; P2, polymorphism R367Q. *P, < 0.01 compared with empty vector control. Similar results were obtained in three separate experiments.

Figure 8.

MCP-1 production by EEF Tsc2 ^−/− cells is inhibited by rapamycin, FTI-277, and LY294002. Rat MCP-1 was measured in culture supernatants by ELISA after incubating equal numbers of cells 24 h in serum-free medium without or with inhibitor at the indicated concentration. MCP-1 concentration was expressed per total secreted protein at the end of the incubation and is reported as the mean ± SD of triplicate wells. *, P < 0.01 compared with control. Similar results were obtained in three separate experiments.