An essential role for p120-catenin in Src- and Rac1-mediated anchorage-independent cell growth - PubMed (original) (raw)
An essential role for p120-catenin in Src- and Rac1-mediated anchorage-independent cell growth
Michael R Dohn et al. J Cell Biol. 2009.
Abstract
p120-catenin regulates epithelial cadherin stability and has been suggested to function as a tumor suppressor. In this study, we used anchorage-independent growth (AIG), a classical in vitro tumorigenicity assay, to examine the role of p120 in a different context, namely oncogene-mediated tumorigenesis. Surprisingly, p120 ablation by short hairpin RNA completely blocked AIG induced by both Rac1 and Src. This role for p120 was traced to its activity in suppression of the RhoA-ROCK pathway, which appears to be essential for AIG. Remarkably, the AIG block associated with p120 ablation was completely reversed by inhibition of the downstream RhoA effector ROCK. Harvey-Ras (H-Ras)-induced AIG was also dependent on suppression of the ROCK cascade but was p120 independent because its action on the pathway occurred downstream of p120. The data suggest that p120 modulates oncogenic signaling pathways important for AIG. Although H-Ras bypasses p120, a unifying theme for all three oncogenes is the requirement to suppress ROCK, which may act as a gatekeeper for the transition to anchorage independence.
Figures
Figure 1.
shRNA knockdown of p120 in MDCK cells. (A) Lysates from parental MDCK cells and two representative p120 knockdown monoclonal cell lines, MDCK–p120i-1 and MDCK–p120i-2 cells, were analyzed by Western blotting for levels of p120, E-cadherin, N-cadherin, β-catenin, α-catenin, desmoglein, and occludin. Tubulin levels were determined as a loading control. (B) MDCK and MDCK–p120i-1 cells plated on glass coverslips were stained for p120, E-cadherin, or ZO-1 as described in Materials and methods. Nuclei were visualized with Hoechst stain. Bar, 10 µm.
Figure 2.
p120 loss has no effect on Rac1-, Src-, or H-Ras–induced morphology in two dimensions. (A–C) MDCK and MDCK–p120i-1 cells infected with DA-Rac1 (A), DA-Src (B), or DA–H-Ras (C) were plated on glass coverslips. Infected cells were visualized with GFP. Actin and nuclei were visualized with phalloidin and Hoechst staining, respectively. Bars, 10 µm.
Figure 3.
p120 is required for Rac1- and Src-induced AIG. (A) MDCK, MDCK–p120i-1, and MDCK–p120i-2 cells with and without DA-Rac1, DA-Src, or DA–H-Ras were plated in soft agar as described in Materials and methods. (B) MDCK (closed shapes) and MDCK–p120i-1 (open shapes) cells with and without DA-Rac1, DA-Src, or DA–H-Ras were seeded at 104cells per well in 6-well dishes, and at the times indicated, cells were trypsinized and counted. Error bars represent standard deviation from three independent experiments. Bar, 0.5 mm.
Figure 4.
Inhibition of ROCK restores Rac1- and Src-induced AIG in the absence of p120. (A) MDCK and MDCK–p120i-1 cells plated on glass coverslips were serum starved (0.1% FBS) overnight and stained for actin and vinculin as described in Materials and methods. Nuclei were visualized with Hoechst stain. (B) MDCK and MDCK–p120i-1 cells were serum starved overnight and left untreated or treated with 10 ng/ml HGF for 30 min. Cell lysates were analyzed for RhoA GTPase activity by rhotekin-binding assay as described in Materials and methods, and the results from three separate experiments are depicted graphically. (C) MDCK and MDCK–p120i-1 cells with and without DA-Rac1 or DA-Src were plated in soft agar in the absence or presence of 10 µM Y-27632 as described in Materials and methods. (D) Cell lysates from MDCK and MDCK–p120i-1 cells with and without DA-Rac1, DA-Src, or DA–H-Ras were analyzed by Western blotting for levels of Ser-3–phosphorylated cofilin and total cofilin. Phosphocofilin levels were normalized to total cofilin levels from three separate experiments and depicted graphically (*, P <0.003; **, P <0.04; ***, P <0.0002). (B and D) Error bars represent standard deviation from three independent experiments. Bars: (A) 10 µm; (C) 0.5 mm.
Figure 5.
Direct inhibition of cofilin inhibits Rac1-, Src-, and H-Ras–induced AIG. (A) MDCK and MDCK–p120i-1 cells expressing DA-Rac1, DA-Src, or DA–H-Ras with and without murine LIMK1 were plated in soft agar as described in Materials and methods. (B) Lysates from MDCK and MDCK–p120i-1 cells with and without murine LIMK1 or cofilin shRNA were analyzed by Western blotting for levels of LIMK, Ser-3–phosphorylated cofilin, and total cofilin. Tubulin levels were determined as a loading control. Phosphocofilin levels were normalized to total cofilin levels and depicted graphically. (C) MDCK cells expressing DA-Rac1, DA-Src, or DA–H-Ras with and without cofilin shRNA were plated in soft agar as described in Materials and methods. Bars, 0.5 mm.
Figure 6.
DA cofilin is not sufficient to rescue the AIG block induced by p120 ablation in Rac1- and Src-transformed cells. (A) Lysates from MDCK, MDCK–p120i-1, and MDCK–p120i-1 cells expressing DA cofilin(S3A)-GFP were analyzed by Western blotting for levels of p120, cofilin(S3A)-GFP, and total cofilin. Tubulin levels were determined as a loading control. (B) Cells in A stably expressing DA-Rac1 or DA-Src were plated in soft agar as described in Materials and methods. Bar, 0.5 mm.
Figure 7.
E-cadherin knockdown suppresses AIG induced by Rac1 but not Src. (A) Lysates from MDCK cells with and without p120 shRNA or E-cadherin shRNA were analyzed by Western blotting for levels of p120 and E-cadherin. Tubulin levels were determined as a loading control. (B and C) MDCK and MDCK/E-cadherin shRNA cells stably expressing DA-Rac1 or DA-Src were plated in soft agar in the presence or absence of the ROCK inhibitor Y-27632 (B) as described in Materials and methods, and the percentage of colony formation relative to wild-type MDCK cells was calculated for each oncogene (C). Error bars represent standard deviation from three independent experiments. Bar, 0.5 mm.
Figure 8.
p120-dependent and -independent control of AIG via inhibition of ROCK. (A) Under normal conditions, cell proliferation is dependent on both growth factors (GFs; serum dependence) and adhesion to a solid substrate (anchorage dependence). Anchorage dependence is mediated primarily by integrins, whereas most oncogenes, including activated variants of Src, Rac1, and H-Ras, are defined in part by their ability to permit or induce AIG (B). Oncogenes are thought to circumvent anchorage dependence by constitutive activation of downstream survival and proliferation signals (e.g., MAPK and PI-3K) normally generated by integrins. Our model suggests that inhibition of ROCK is also essential. For Rac1 and Src, p120 is required and appears to function via suppression of RhoA–ROCK. H-Ras also suppresses ROCK but utilizes a different pathway that bypasses p120. The suppression of ROCK leads to the activation of cofilin, which is necessary but not sufficient for AIG, suggesting that other ROCK activities also play a role. The exact mechanisms underlying these events are not yet clear but likely involve rearrangement of the actin cytoskeleton and/or regulation of contractility. For normal cells, the effects of RhoA activity are context dependent. In soft agar, RhoA generates contractility and promotes anoikis, whereas on stiff matrices (e.g., plastic), RhoA participates in mechanical tension-based feedback loops that promote growth. According to our data, transformed cells circumvent RhoA-, actin-, and/or tension-dependent cell death via suppression of ROCK.
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