The absence of caveolin-1 increases proliferation and anchorage- independent growth by a Rac-dependent, Erk-independent mechanism

Abstract

Anchorage-independent growth (AIG) of cancer cells requires escape from integrin-mediated signals. A protein frequently downregulated in cancer, caveolin-1 (Cav1), mediates integrin control of several growth-regulatory pathways. We report that loss of Cav1 results in faster exit from quiescence and progress through the cell cycle, proliferation without anchorage to substrate, and absence of cyclin D1 downregulation upon serum deprivation or detachment. Surprisingly, this proliferative advantage is independent of Erk-mitogen-activated protein kinase signaling; instead, cyclin expression and cell cycle progression in the absence of Cav1 are driven by increased membrane order and Rac targeting. AIG was induced in Cav1-expressing cells by forced membrane targeting of Rac1 or by inhibiting Cav1-mediated internalization of plasma membrane ordered domains at which Rac1 accumulates. Restoring Rho activity, which is downregulated after loss of Cav1, antagonizes Rac1 and prevents cyclin D1 accumulation after serum starvation or loss of adhesion. Anchorage independence and increased proliferation in Cav1-deficient tumoral and null cells are thus due to an increased fraction of active Rac1 at membrane ordered domains. These results provide insight into the mechanisms regulating growth of cancer cells, which frequently lose Cav1 function.

FIG. 1.

Increased cell proliferation in the absence of Cav1. (a) Flow cytometric analysis of wt and Cav1^−/− MEFs from two different mouse strains (MEF-R [44] and MEF-D [15]) and 3T3L1 fibroblasts before and after shRNA-mediated Cav1 knockdown, showing the percentages of cells in each phase of the cell cycle out of a total of 10,000 cells (left), and analysis of BrdU incorporation after 2-h labeling of wt and Cav1^−/− MEFs-R (right). (b) Western blot analysis of wt and Cav1^−/− MEFs-R using antibodies against cyclin D1, cyclin A, pRb, p130, p107, and γ-tubulin (γ-tub; as a loading control) (left) and Western blot analysis of 3T3L1 fibroblasts before and after Cav1 shRNA-mediated specific knockdown using antibodies against cyclin D1, cyclin A, Cav1, and γ-tubulin (as a loading control) (right). (c) Immunofluorescence staining of wt and Cav1^−/− MEFs-R using an antibody against cyclin D1 and phalloidin to visualize the actin cytoskeleton. Nuclear cyclin D1 mean intensity is represented. At least 30 cells per genotype were analyzed. AU, arbitrary units. (d) Western blot analysis of wt and Cav1^−/− MEFs-R expressing either an empty vector (e) or Cav1 (RC) using antibodies against Cav1, cyclin D1, cyclin A, pRb, and p107. γ-Tubulin was used as a loading control. (e) Flow cytometry analysis of M21L cells expressing either an empty vector or a construct encoding GFP-fused Cav1, showing the percentages of cells in each phase of the cell cycle out of a total of 10,000 cells (left), and GFP-Cav1 expression in adherent M21L melanoma cells (right). Bars (a, c, and e) represent means ± standard errors of the means of three (a and c) or four (e) independent experiments.

FIG. 2.

Cav1 deficiency cooperates in oncogenic transformation of MEFs. (a) Focus formation assay of wt and Cav1^−/− MEFs-R after transfection with the H-Ras oncogene. Total foci formed normalized to wt cells are represented. The ratios of focus formation in transfected cells were 1/1,000 for wt cells and 1/100 for transfected Cav1^−/− cells. Bars represent means ± standard errors of the means of four independent experiments. (b) Micrographs showing focus formation in two independent primary clones (clon) of wt and Cav1^−/− MEFs-R, respectively, after transfection with the H-Ras oncogene or the H-Ras and E1A oncogenes.

FIG. 3.

Faster progression through G₁ and entry into S phase in Cav1^−/− MEFs. (a) Flow cytometric analysis of synchronized wt and Cav1^−/− MEF-R populations at the indicated times after serum stimulation. Graphs show the percentages of cells in each phase of the cell cycle out of a total of 10,000 cells. (b) Western blot analysis of wt and Cav1^−/− MEFs-R after serum stimulation at the indicated time points using antibodies against cyclin D1, cyclin A, pRb, p107, p130, and γ-tubulin (γ-tub; as a loading control). Densitometric analysis of p107 expression normalized to γ-tubulin is indicated (arbitrary units). (c) Immunofluorescence staining of G₀-synchronized populations of wt and Cav1^−/− MEFs-R using antibodies against cyclin D1 and phalloidin to visualize the actin cytoskeleton and accumulation of nuclear cyclin D1. At least 30 cells per genotype were analyzed. AU, arbitrary units. (d) Electrophoretic mobility of pRb and p130 proteins compared to a molecular size standard (m) in wt and Cav1^−/− MEFs-R at the indicated times after serum stimulation. (e) Western blot analysis of p-pRb (pS807/811) in wt and Cav1^−/− MEFs-R at the indicated times after serum stimulation; γ-tubulin was used as a loading control. Bars (a and c) represent means ± standard errors of the means of three independent experiments.

FIG. 4.

Rac1 but not Erk induces cyclin D1 in synchronized Cav1^−/− MEFs. (a) Western blot analysis of Cav1^−/− MEFs using antibodies against the phosphorylated form of Erk (P-Erk), total Erk protein, cyclin D1, and cdk4 (as a loading control) at the indicated times after serum stimulation and in the presence or absence of the specific inhibitor of the p42/44 MAPK pathway, U0126 (10 μM). (b) Rac1 pulldown assay of G₀-synchronized wt and Cav1^−/− MEFs after serum starvation. Proteins bound to GST-p21 binding domain were immunoblotted with an antibody against Rac. Densitometric analysis of the relative activity of Rac1 normalized for whole-cell lysates was performed, and the result is expressed as the ratio to activity in wt MEFs. Bar, mean ± standard error of the mean of four independent experiments. (c) Western blot analysis of wt and Cav1^−/− MEFs after serum stimulation at the indicated time points using antibodies against cyclin D1 and Rac1, with or without infection of adenovirus N17-Rac. Densitometric analysis of cyclin D1 expression normalized to endogenous Rac is indicated (arbitrary units). qPCR for cyclin D1 mRNA in the same samples was performed. Bars, means ± standard deviations of three experiments. (d) Western blot analysis of wt and Cav1^−/− MEFs as in panel c using antibodies against the phosphorylated form of Erk and total Erk protein.

FIG. 5.

Cav1^−/− fibroblasts show AIG. (a) Soft agar assay for colony formation of wt and Cav1^−/− MEFs, Cav1^−/− MEFs reconstituted with Cav1 (RC), and TFs. Colonies were stained with crystal violet and photographed. Shown are numbers of colonies formed in soft agar by wt and Cav1^−/− MEFs and TFs (left graph) and percentages of colony formation by RC MEFs and TFs (right graph). Bars represent means ± standard errors of the means [SEM] of at least three independent experiments. (b) Flow cytometry profiles of wt and Cav1^−/− MEFs-R and -D and 3T3L1 fibroblasts before and after shRNA-mediated Cav1 knockdown, grown in suspension, showing the percentages of cells in each phase of the cell cycle out of a total of 10,000 cells (left) and analysis of BrdU incorporation after 2-h labeling of wt and Cav1^−/− MEFs-R cultured in suspension (right). Bars represent means ± SEM of five independent experiments. (c) Western blot analysis of wt and Cav1^−/− MEFs-R grown in suspension using antibodies against cyclin D1, cyclin A, pRb, p130, p107, and γ-tubulin (γ-tub; as a loading control) (left) and Western blot analysis of 3T3L1 fibroblasts grown in suspension before and after Cav1 shRNA-mediated specific knockdown using antibodies against cyclin D1, cyclin A and γ-tubulin (as a loading control) (right). (d) Immunofluorescence staining of wt and Cav1^−/− MEFs grown in suspension using antibodies against cyclin D1 and phalloidin to visualize the actin cytoskeleton and accumulation of nuclear cyclin D1. Bars represent means ± SEM of three independent experiments after analyzing at least 30 cells per experiment. AU, arbitrary units. (e) Western blot analysis of wt and Cav1^−/− MEF-R grown in suspension and expressing either an empty vector or Cav1 (RC) using antibodies against cyclin D1, cyclin A, pRb, p107, and γ-tubulin. Bars represent means ± SEM of four independent experiments. (f) Flow cytometry profiles of M21L melanoma cells grown in suspension and expressing either an empty vector or a construct encoding GFP-fused Cav1, showing the percentages of cells in each phase of the cell cycle out of a total of 10,000 cells (left), and GFP-Cav1 expression in suspended M21L melanoma cells (right).

FIG. 6.

AIG in Cav1^−/− MEFs is mediated by PI3K-GSK3β but not Erk. (a) Western blot analysis of wt and Cav1^−/− MEFs grown in suspension in the presence or absence of the MEK inhibitor U0126 using antibodies against cyclin D1, cyclin A, and γ-tubulin (γ-tub; as a loading control) and flow cytometry profiles of wt and Cav1^−/− MEFs under the same conditions showing the percentages of cells in each phase of the cell cycle out of a total of 10,000 cells. (b) Western blot analysis of wt and Cav1^−/− MEFs grown in suspension using antibodies against the two phosphorylation sites of Akt required for its full activation (p-Akt pT308 and p-Akt pS473) and total Akt as loading control. (c) Western blot analysis of wt and Cav1^−/− MEFs grown in suspension in the presence of dimethyl sulfoxide (DMSO) or the specific inhibitors of PI3K (LY294002) and GSK3β (LiCl) using antibodies against cyclin D1, cyclin A, and γ-tubulin (as a loading control) and flow cytometry profiles of wt and Cav1^−/− MEFs under the same conditions showing the percentages of cells in each phase of the cell cycle out of a total of 10,000 cells. (d) Western blot analysis of wt and Cav1^−/− MEFs grown in suspension after shRNA-mediated knockdown of GSK3β (left) or Akt (right) using antibodies against GSK3β, Akt, cyclin D1, and γ-tubulin. Bars (a and c) represent means ± standard errors of the means of three independent experiments.

FIG. 7.

Rac activation drives cell cycle progression and AIG. (a) Western blot analysis of Rat1 fibroblasts grown in suspension expressing a constitutively active form of Rac (V12) under the control of a tetracycline-responsive promoter or an empty vector (V8) in the presence or absence of doxycycline using antibodies against cyclin D1 and the myc epitope to detect the induction of V12-Rac expression and against γ-tubulin (tub) as a loading control. (b) G₀/G₁ populations of Rat1-V8 and Rat1-V12 cultured under adhesion conditions or in suspension for 48 h in the absence of doxycycline, as determined by flow cytometry out of a total of 10,000 cells. Bars represent means ± standard errors of the means (SEM) of three independent experiments. (c) Western blot analysis of wt and Cav1^−/− MEFs in suspension, expressing a dominant negative form of Rac (N17-Rac) or an empty vector, using antibodies against cyclin D1, cyclin A, and γ-tubulin (γ-tub). (d) G₀/G₁ populations of wt and Cav1^−/− MEFs grown in suspension, expressing a dominant negative form of Rac (N17-Rac) or an empty vector, as determined by flow cytometry out of a total of 10,000 cells. Bars represent means ± SEM of two independent experiments. (e) Western blot analysis of suspension cultures of Cav1^−/− MEFs transfected with an RNAi oligonucleotide directed against Rac1 or with a scramble siRNA, using antibodies against Rac1, cyclin D1, cyclin A, and γ-tubulin.

FIG. 8.

CEMM/Rac plasma membrane targeting drives cell cycle progression and AIG. (a, left and center) Analysis of BrdU incorporation by immunofluorescence of suspension cultures of wt MEFs after incubation with latex beads either uncoated (U) or coated with CTxB or antibody against TfR. Percentages of BrdU-positive cells are represented. (Right) Western blot analysis of wt MEFs treated as above using antibodies against cyclin D1 and γ-tubulin (γ-tub). Bars represent means ± standard errors of the means (SEM) of two experiments, counting at least 5,000 cells per condition. (b) Immunofluorescence staining of wt MEFs grown in suspension and expressing the membrane-constitutive Rac1 fusion protein myr-HA-Rac using antibodies against Rac1 (top) and HA (bottom), together with Hoechst for nuclear staining. This construct shows enhanced plasma membrane association compared to endogenous Rac. The mean intensity distribution is plotted. AU, arbitrary units. Bars represent means ± SEM of three independent experiments. At least 30 cells per experiment were analyzed. (c) Western blot analysis of wt MEFs grown in suspension and expressing membrane-constitutive Rac1 fusion proteins (myr-HA-Rac or IL-2-HA-Rac) or an empty vector. Antibodies against cyclin D1, cyclin A, and γ-tubulin were used. Results of a densitometric analysis of cyclin D1 and A expression normalized to γ-tubulin are indicated (arbitrary units). (d) G₀/G₁ populations of cells as in panel c, determined by flow cytometry, are represented. Bars represent means ± SEM of five experiments.

FIG. 9.

Rescue of Rho-GTP loading restores cyclin D1 downregulation in both G₀-arrested and suspended Cav1^−/− MEFs. Shown is a Western blot analysis of wt and Cav1^−/− MEFs with or without expression of a constitutively active form of RhoA (V14-Rho). Cav1^−/− MEFs were either serum starved for 72 h (a) or grown under adhesion conditions or in suspension (b). Antibodies against cyclin D1 and γ-tubulin (γ-tub) were used.

FIG. 10.

As a working hypothesis, upon cell detachment from the ECM, Cav1-mediates CEMM endocytosis, leading to the shutdown of several signaling cascades and subsequent cell cycle arrest. In the absence of Cav1, CEMMs cannot be internalized from the plasma membrane, allowing the activation of the Rac, PI3K/Akt, and Erk pathways. On the other hand, absence of Cav1 downregulates Rho activity, which is necessary for Erk effects on cyclin D1 expression and for antagonizing Rac-dependent early cyclin D1 induction. The result is an altered timing of cyclin D1 expression, allowing cell cycle progression in anchorage-independent conditions. Therefore, normal anchorage dependence of growth is regulated by inactivation of adhesive signals upon internalization of CEMMs, which is mediated by Cav1 transport function.