IGFBP-3 can either inhibit or enhance EGF-mediated growth of breast epithelial cells dependent upon the presence of fibronectin

Abstract

Progression of breast cancer is associated with remodeling of the extracellular matrix, often involving a switch from estrogen dependence to a dependence on EGF receptor (EGFR)/HER-2 and is accompanied by increased expression of the main binding protein for insulin-like growth factors (IGFBP-3). We have examined the effects of IGFBP-3 on EGF responses of breast epithelial cells in the context of changes in the extracellular matrix. On plastic and laminin with MCF-10A normal breast epithelial cells, EGF and IGFBP-3 each increased cell growth and together produced a synergistic response, whereas with T47D breast cancer cells IGFBP-3 alone had no effect, but the ability of EGF to increase cell proliferation was markedly inhibited in the presence of IGFBP-3. In contrast on fibronectin with MCF-10A cells, IGFBP-3 alone inhibited cell growth and blocked EGF-induced proliferation. With the cancer cells, IGFBP-3 alone had no effect but enhanced the EGF-induced increase in cell growth. The insulin-like growth factor-independent effects of IGFBP-3 alone on cell proliferation were completely abrogated in the presence of an EGFR, tyrosine kinase inhibitor, Iressa. Although IGFBP-3 did not affect EGFR phosphorylation [Tyr(1068)], it was found to modulate receptor internalization and was associated with activation of Rho and subsequent changes in MAPK phosphorylation. The levels of fibronectin and IGFBP-3 within breast tumors may determine their dependence on EGFR and their response to therapies targeting this receptor.

FIGURE 1.

IGFBP-3 modulation of EGF-induced growth of breast epithelial cells. MCF-10A (A, plastic; B, laminin) and T47D (C, plastic; D, laminin) were dosed with EGF (0–25 ng/ml), IGFBP-3 (100 ng/ml), or a combination of each for a further 48 h. Cell proliferation was assessed using tritiated thymidine incorporation. Graphs represent the mean of three experiments that were each performed in triplicate.

FIGURE 2.

IGFBP-3 modulation of EGF-induced growth of breast epithelial cells on fibronectin. MCF-10A (A) and T47D (B) cells were dosed with EGF (0–25 ng/ml), IGFBP-3 (100 ng/ml), or a combination of each for a further 48 h. Cell proliferation was assessed using tritiated thymidine incorporation. Graphs represent the mean of three experiments that were each performed in triplicate.

FIGURE 3.

SPD modulation of EGF-induced growth of breast epithelial cells. MCF-10A (A and C) and T47D (B and D) cells were dosed with EGF (0–25 ng/ml), SPD (10 ng/ml), or a combination of each for a further 48 h on either plastic (A and B) or fibronectin (0.25 μg/ml) (C and D). Cell proliferation was assessed by cell counting. Graphs represent the mean of three experiments that were each performed in triplicate.

FIGURE 4.

The effects of IGFBP-3 on cell growth involve the EGF receptor. A, MCF-10A cells were dosed with EGF (10 ng/ml) or Iressa (1 μm) or a combination of the two for 4 h. Cells were then lysed and processed for p-EGFR followed by total EGFR to show comparable levels of protein loaded. The blot is representative of experiments repeated three times (arbitrary optical density units for A: CT = 0.31, Iressa = 0, EGF = 0.97; EGF + Iressa = 0.19). CT, control. B, MCF-10A cells were dosed with IGFBP-3 (100 ng/ml) or SPD (5 and 10 ng/ml) in the presence or absence of Iressa (1 μm) for 24 h. Cell proliferation was assessed using tritiated thymidine incorporation. The graph represents the mean of three experiments that were each performed in triplicate. C, MCF-10A cells were dosed with IGFBP-3 (100 ng/ml) with or without Iressa (1 μm) for 15 min. Cells were lysed as in A and immunoblotted for p-P42/44MAPK followed by ERK-2 as a loading control. The graph shows the percentage change in OD of three representative blots. D, MCF10A and T47D cells were dosed with EGF (10 min) or IGFBP-3 (10 min to 8 h), E, MCF10A and T47D cells were dosed with EGF in the presence or absence of IGFBP-3. D and E were processed and assessed for HER2 and/or EGFR phosphorylation as in A. F–I, MCF-10A and T47D cells were treated as in E for 1 h on either plastic (A and B, n = 3 for each) or on fibronectin (C and D, n = 2 for each) and then lysed and processed to assess the internalization of the EGF receptor (as described under “Experimental Procedures”). Representative blots showing changes in levels of EGFR inside the cell (I) or on the cell surface (E) are shown for each experiment. We also show total levels of EGFR (where 1 = SFM, 2 = EGF, 3 = IGFBP-3, 4 = EGF+IGFBP-3) together with GAPDH as a loading control. We used MHC class 1 as a loading control for the external fraction and GAPDH for the internal component. The graphs show the percentage change in ODs between internal EGFR compared with external EGFR corrected for their respective controls. J and K, MCF-10A cells were dosed with IGFBP-3 (100 ng/ml; 5 min) and processed for Rho activation as described under “Experimental Procedures.” The graph represents the mean of three separate experiments. L, MCF-10A cells were treated with IGFBP-3 (100 ng/ml) with and without Y-27632 (5 μm) for 24 h and assessed for tritiated thymidine incorporation. The graph represents the mean of three experiments that were each performed in triplicate.

FIGURE 4.

The effects of IGFBP-3 on cell growth involve the EGF receptor. A, MCF-10A cells were dosed with EGF (10 ng/ml) or Iressa (1 μm) or a combination of the two for 4 h. Cells were then lysed and processed for p-EGFR followed by total EGFR to show comparable levels of protein loaded. The blot is representative of experiments repeated three times (arbitrary optical density units for A: CT = 0.31, Iressa = 0, EGF = 0.97; EGF + Iressa = 0.19). CT, control. B, MCF-10A cells were dosed with IGFBP-3 (100 ng/ml) or SPD (5 and 10 ng/ml) in the presence or absence of Iressa (1 μm) for 24 h. Cell proliferation was assessed using tritiated thymidine incorporation. The graph represents the mean of three experiments that were each performed in triplicate. C, MCF-10A cells were dosed with IGFBP-3 (100 ng/ml) with or without Iressa (1 μm) for 15 min. Cells were lysed as in A and immunoblotted for p-P42/44MAPK followed by ERK-2 as a loading control. The graph shows the percentage change in OD of three representative blots. D, MCF10A and T47D cells were dosed with EGF (10 min) or IGFBP-3 (10 min to 8 h), E, MCF10A and T47D cells were dosed with EGF in the presence or absence of IGFBP-3. D and E were processed and assessed for HER2 and/or EGFR phosphorylation as in A. F–I, MCF-10A and T47D cells were treated as in E for 1 h on either plastic (A and B, n = 3 for each) or on fibronectin (C and D, n = 2 for each) and then lysed and processed to assess the internalization of the EGF receptor (as described under “Experimental Procedures”). Representative blots showing changes in levels of EGFR inside the cell (I) or on the cell surface (E) are shown for each experiment. We also show total levels of EGFR (where 1 = SFM, 2 = EGF, 3 = IGFBP-3, 4 = EGF+IGFBP-3) together with GAPDH as a loading control. We used MHC class 1 as a loading control for the external fraction and GAPDH for the internal component. The graphs show the percentage change in ODs between internal EGFR compared with external EGFR corrected for their respective controls. J and K, MCF-10A cells were dosed with IGFBP-3 (100 ng/ml; 5 min) and processed for Rho activation as described under “Experimental Procedures.” The graph represents the mean of three separate experiments. L, MCF-10A cells were treated with IGFBP-3 (100 ng/ml) with and without Y-27632 (5 μm) for 24 h and assessed for tritiated thymidine incorporation. The graph represents the mean of three experiments that were each performed in triplicate.

FIGURE 5.

IGFBP-3 affects EGF-induced activation of p44/42 MAPK in breast epithelial cells. Cells were dosed EGF (0–25 ng/ml) or IGFBP-3 (100 ng/ml) or in combination for 5, 15, 30, and 120 min, lysed, and probed for p44/42 MAPK and also GAPDH to show equal loading of protein. Representative Western immunoblots for p44/42 MAPK and GAPDH are shown for MCF-10A cells (A), T47D cells (B), and Hs578T cells (C). The bottom panel in each figure shows the mean densitometry values of three separate experiments upon which statistical analysis was performed and included in the results.

FIGURE 6.

IGFBP-3 affects EGF-induced activation of p44/42 MAPK in breast epithelial cells on fibronectin. Cells were dosed EGF (0–25 ng/ml) or IGFBP-3 (100 ng/ml) or in combination for 5, 15, 30, and 120 min, lysed, and probed for p44/42 MAPK and also GAPDH to show equal loading of protein. Representative Western immunoblots for p44/42 MAPK and GAPDH are shown for MCF-10A cells (A), T47D cells (B), and Hs578T cells (C). The bottom panel in each figure shows the mean densitometry values of three separate experiments upon which statistical analysis was performed and included in the results.