The tetraspanin CD151 is required for Met-dependent signaling and tumor cell growth

Abstract

CD151, a transmembrane protein of the tetraspanin family, is implicated in the regulation of cell-substrate adhesion and cell migration through physical and functional interactions with integrin receptors. In contrast, little is known about the potential role of CD151 in controlling cell proliferation and survival. We have previously shown that β4 integrin, a major CD151 partner, not only acts as an adhesive receptor for laminins but also as an intracellular signaling platform promoting cell proliferation and invasive growth upon interaction with Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF). Here we show that RNAi-mediated silencing of CD151 expression in cancer cells impairs HGF-driven proliferation, anchorage-independent growth, protection from anoikis, and tumor progression in xenograft models in vivo. Mechanistically, we found that CD151 is crucially implicated in the formation of signaling complexes between Met and β4 integrin, a known amplifier of HGF-induced tumor cell growth and survival. CD151 depletion hampered HGF-induced phosphorylation of β4 integrin and the ensuing Grb2-Gab1 association, a signaling pathway leading to MAPK stimulation and cell growth. Accordingly, CD151 knockdown reduced HGF-triggered activation of MAPK but not AKT signaling cascade. These results indicate that CD151 controls Met-dependent neoplastic growth by enhancing receptor signaling through β4 integrin-mediated pathways, independent of cell-substrate adhesion.

FIGURE 1.

Generation of CD151-deficient cells. A431 and A549 carcinoma cells were transduced with lentiviral vectors to express shRNA sequences targeting CD151 transcript (shCD151) or with empty lentiviral vectors (con). CD151 silencing was verified by real-time quantitative PCR (A) as well as by immunoblotting (IB) (B). The expression of β-actin was checked by Western blotting on total lysates to provide a loading control. IP, immunoprecipitation. C, the co-immunoprecipitation of β1 and β4 integrins with CD151 was assayed by Western blotting in control and CD151-deficient A431 cells. tot. lysate, total lysate.

FIGURE 2.

CD151 is required for cell growth response induced by HGF. A, cell viability was assessed in control (con) and CD151-deficient cells by MTT assays at two distinct time points (see “Materials and Methods” for details). Values in the graphs indicate the amount of reaction product, revealed by absorbance at 570 nm (average of two independent experiments performed in duplicate +S.E.). a.u., arbitrary units. B, HGF-induced cell proliferation was assessed by growth curves (see “Materials and Methods” for details). Cell staining with crystal violet was eventually revealed by absorbance at 562 nm. Three independent experiments were performed in triplicate with consistent results (**, p < 0.001; *, p < 0.05). con n.t., control untreated.

FIGURE 3.

CD151 mediates anchorage-independent growth and cell survival induced by HGF. A, A549 cells were grown in soft agar, in the presence or absence of HGF, to test their ability to survive and proliferate without anchorage to the extracellular matrix. After 3 weeks, living cells were stained with tetrazolium salts and scored by evaluating absorbance at 570 nm (see “Materials and Methods” for details). Values shown in graphs are means +S.E. of three independent experiments performed in triplicate, normalized to non-stimulated control cells; *, p < 0.05. a.u., arbitrary units; n.t., untreated; n.s., not significant; con, empty lentiviral vectors. B, A549 cells were grown in suspension in poly-HEMA-coated dishes to induce anoikis. After 72 h, cell viability was assessed by the MTT assay (see “Materials and Methods”). HGF induced cell survival in control cells, whereas this response was lost in CD151-deficient cells and partially rescued by CD151 re-expression (re-expr.) (quantitative PCR analysis of CD151 expression in the different cell lines is shown in supplemental Fig. 2). Data shown are the means +S.E. of three independent experiments performed in triplicate. Statistical significance was established by globally comparing the three experimental conditions by one-way analysis of variance test; **, p < 0.0001. Moreover, Student's t test analysis of paired samples indicated statistical significance of shCD151 and CD151-re-expression condition versus control, with p < 0.001 for both cases (not shown).

FIGURE 4.

CD151 is required for HGF-dependent tumorigenesis in vivo. A549 cells, either control or carrying a HGF autocrine loop, were transduced with shRNA-expressing constructs to knock down CD151 levels (see expression analysis in supplemental Fig. 3) and then injected subcutaneously in nude mice to score their tumorigenic potential. Tumor volume was periodically measured, indicating that although HGF autocrine signaling induced striking tumor growth, this effect was almost completely blunted in CD151-deficient cells. Two independent experiments were performed, including five mice per each experimental group, showing consistent results. Significance was assessed by Student's t test analysis on the complete series of values of the four experimental groups, revealing that only control (con) tumors expressing HGF were statistically different from any other condition; **, p < 0.005.

FIGURE 5.

CD151 knockdown subverts HGF-induced signaling in cancer cell lines. A, control (con) and CD151-deficient A549 cells were starved 48 h and treated with 50 ng/ml HGF for the indicated times (or left untreated). Tyrosine-phosphorylated (P-Tyr) Met was detected by immunoprecipitation (IP) followed by immunoblotting (IB) (top row). Moreover, total protein cell lysates were probed to detect activated Akt (Phospho-Akt), total Akt (tot. Akt), activated MAPK/Erk (Phospho-Erk), and total MAPK/Erk (total Erk). CD151 knockdown impaired HGF-induced MAPK activation but was without significant effect on Met and Akt activation. n.s., not significant. B, A549 cells were serum-starved for 48 h, put in suspension, and stimulated or not with HGF (50 ng/ml for 15 min). Erk activation and total Erk were revealed with appropriate antibodies. CD151 knockdown hampers Erk activation in HGF-stimulated cells, in the absence of cell-substrate adhesion. C, hyperactivation of RAS by ectopic overexpression of K-RAS^G12V or re-expression of CD151 enhanced HGF-dependent soft agar growth of CD151-deficient cells. A549 cells were grown in soft agar in the presence of 50 ng/ml HGF. Foci of viable cells were eventually scored as in Fig. 3A. Data shown are the means +S.E. of two independent experiments performed in triplicate; **, p < 0.001; *, p < 0.01. a.u., arbitrary units.

FIGURE 6.

CD151 mediates β4 integrin phosphorylation by Met. A549 and A431 cells (control (con) and CD151-depleted) were serum-starved for 48 h and treated or not with HGF 50 ng/ml for 20 min. CD151 knockdown inhibits HGF-induced β4 integrin tyrosine phosphorylation. IP, immunoprecipitation; IB, immunoblotting. n.s., not significant.

FIGURE 7.

CD151 mediates the formation of a ternary complex coupling β4 integrin with Met. A, CD151 was immunopurified from different lysates of GTL16 tumor cells (upon protein solubilization with either Brij 97 or Nonidet P-40 detergent (NP-40)), and immunocomplexes were analyzed by Western blotting with specific antibodies to detect associated receptor molecules. Both β4 integrin and Met were selectively found in association with CD151. Specificity controls were provided by protein immunoprecipitation (IP) with a non-related antibody (nrs). IB, immunoblotting. B, β4 integrin and Met were efficiently co-immunoprecipitated in control GTL16 and A549 cells, whereas this association was strikingly reduced in CD151-deficient cells. Comparable expression levels of Met and β4 integrin in both cell lines were confirmed by immunoblotting. Two independent shRNA sequences were used to knock down CD151 expression in A549 cells (see ”Materials and Methods“ for details and supplemental Fig. 4 for validation). C, the expression of β4 integrin was silenced in GTL-16 cells by using shRNA targeting β4 (bottom panels). CD151 was immunopurified from different cell lysates, and immunocomplexes were analyzed by Western blotting with antibodies specific for Met and β4 integrin (upper panels), as in panel A. CD151-Met interaction takes place even in the absence of β4 expression. nrAb, non-related antibody. D, GTL-16 cells were treated or not with 10 mm methyl-β-cyclodextrin (MβCD) for the indicated times. CD151 was immunoprecipitated from cell lysates, and immunocomplexes were analyzed by Western blotting with antibodies specific for the β4 integrin and the Met receptor (as above). Methyl-β-cyclodextrin treatment disrupted Met-CD151-β4 complexes. n.t., untreated.

FIGURE 8.

CD151 is required for efficient Gab1-Grb2 coupling in the Met signaling cascade. CD151-deficient and CD151-proficient A549 cells were transiently transfected with Gab1 and stimulated with 50 ng/ml HGF for 15 min. Gab1 was precipitated from cell lysates, and the immunocomplexes were probed with the indicated antibodies by Western blotting. CD151 down-modulation impairs HGF-induced association of Gab1 with Grb2 in A549 cells. IP, immunoprecipitation; IB, immunoblotting; Con, empty lentiviral vectors; n.s., not significant.