Tetraspanin CD151 regulates transforming growth factor beta signaling: implication in tumor metastasis

Abstract

Tetraspanin CD151 is associated with laminin-binding integrins and controls tumor cell migration and invasion. By analyzing responses of breast cancer cells to various growth factors, we showed that depletion of CD151 specifically attenuates transforming growth factor beta1 (TGFbeta1)-induced scattering and proliferation of breast cancer cells in three-dimensional Matrigel. CD151-dependent cell scattering requires its association with either alpha3beta1 or alpha6 integrins, but it is independent of the recruitment of CD151 to tetraspanin-enriched microdomains. We also found that CD151 regulates the compartmentalization of TGF-beta type I receptor (TbetaRI/ALK-5) and specifically controls the TGFbeta1-induced activation of p38. In contrast, signaling leading to activation of Smad2/3, c-Akt, and Erk1/2 proteins was comparable in CD151(+) and CD151(-) cells. Attenuation of TGFbeta1-induced responses correlated with reduced retention in the lung vascular bed, inhibition of pneumocyte-induced scattering of breast cancer cells in three-dimensional Matrigel, and decrease in experimental metastasis to the lungs. These results identify CD151 as a positive regulator of TGFbeta1-initiated signaling and highlight the important role played by this tetraspanin in TGFbeta1-induced breast cancer metastasis.

Figure 1

CD151 regulates responses of MDA-MB-231 breast cancer cells to TGFβ1. A, E. Cells were embedded into growth factor reduced Matrigel as described in Materials and Methods and grown for 8 days without (top panel) or in the presence of TGFβ1 (10ng/ml). Presented photographs of representative fields. Scale bar represents 50μm. B. The time-course of cellular responses to TGFβ1 in Matrigel. Growth of cells in 3-D Matrigel was analysed as described in (A). Scale bar represents 50μm. C. Quantification of TGFβ1-induced changes in morphology of the colonies in 3-D Matrigel. More than 50 colonies were analysed for each of the cell lines in each of three separate experiments. Colonies were defined as having a “compact phenotype” when no individual cell borders could be discerned within the colony. Ratio of average number of “compact” colonies for CD151-positive and CD151-depleted MDA-MB-231 was calculated for each experiment. Data are presented as mean of ratios ± standard deviation. D. Measurements of cell proliferation in the presence of TGFβ1. Cell proliferation was measured using Alamar Blue by a spectrofluorometric method. Data represent mean ±SD of three experiments (each in triplicate). AFU, Arbitrary Fluorescence Units.

Figure 2

The association with integrins is critical for CD151-dependent cellular responses to TGFβ1. Growth of cells in 3-D Matrigel (A) and quantification of cellular responses (B) were performed as described in the legend to Figure 1. C, D. MDA-MB-231, MDA-MB-231/CD151rec, MDA-MB-231/CD151-QRD, MDA-MB-231/CD151palm, MDA-MB-231/α3β1^low, MDA-MB-231/α6^lowβ1/β4 and MDA-MB-231/α3β1^lowα6^lowβ1/β4 cells were plated in 3-D Matrigel and grown in presence of TGFβ1 as described in Figure 1. Scale bar represents 50μm.

Figure 3

The effect of CD151 depletion on TGFβ1-induced signaling. A, B Serum-starved MDA-MB-231/CD151(+) and MDA-MB-231/CD151(−) cells were incubated with TGFβ1 (10ng/ml) for indicated time intervals and then scraped into Laemmli buffer. Proteins were resolved in 10% SDS-PAGE under reduced conditions. The proteins were transferred to a nitrocellulose membrane and probed with indicated Abs. C. Densitometric analysis of activation p38 (i.e. measurements of phospho-p38) for the results shown in Fig.3A. Measurements were done for three independent experiments. A.D.U. – arbitrary densitometry units. D. The role of CD151 in the membrane compartmentalisation of TβRI. Cell lysates from MDA-MB-231/CD151(+) and MDA-MB-231/CD151(−) were fractionated in gradient of sucrose as described in the Materials and Methods. Equal volumes of each fraction were resolved in 10% SDS-PAGE. Distribution of proteins in the gradient fractions was assessed by Western blotting using specific antibody Abs. The α2–, α3– and α6–panels represent western blots detecting corresponding integrin subunits.

Figure 4

Depletion of CD151 attenuated pulmonary metastasis. A. The areas occupied by MDA-MB-231/CD151(+) and MDA-MB-231/CD151(−) metastatic lesions were measured as described in Material and Methods and plotted relative to the total lung area (%). Error bars, SEM. B. Numbers of lesions counted in lung sections used for the morphometric assessment the lesions shown in (A). Error bars, SEM.

Figure 5

Depletion of CD151 diminishes acute retention of MDA-MB-231 in the lung vascular bed. BCECF – labelled CD151-positive and CD151-negative MDA-MB-231 were introduced via the carotid artery into anaesthetised mice and monitored intravitally. Numbers adherent within the hepatic sinusoidal and pulmonary capillaries were quantified in 7 and 10 random static fields of view, respectively. A. Shown photographs of representative fields. Note, a significant reduction in recruitment of labelled CD151-negative MDA-MB-231 cells in the lungs (right panel) as compared to the control, MDA-MB-231/CD151(+) cells (left panel). B. Quantification of the results from 5 independent experiments. Note, depletion of CD151 decreased retention of the cells in the lungs (top histogram) but not within the liver (bottom histogram). Error bars, SEM.

Figure 6

Contribution of pneumocytes and TGFβ1 to growth of MDA-MB-231 cells in 3-D Matrigel. Cells embedded in 3-D Matrigel were grown above the monolayer of HPL1D cells in the absence (A) or presence of 10μM SB431542 (B) as described in the legend to Figure 1. Presented photographs of representative fields. Scale bar represents 50μm.