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Originally published In Press as doi:10.1074/jbc.M608857200 on December 26, 2006

J. Biol. Chem., Vol. 282, Issue 8, 5934-5943, February 23, 2007
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Caveolin-1 Tyrosine Phosphorylation Enhances Paclitaxel-mediated Cytotoxicity*

Ayesha N. Shajahan{ddagger}, Aifen Wang{ddagger}, Markus Decker{ddagger}, Richard D. Minshall§, Minetta C. Liu{ddagger}, and Robert Clarke{ddagger}1

From the {ddagger}Department of Oncology, Lombardi Comprehensive Cancer Center, and Departments of Physiology and Biophysics, Georgetown University, College of Medicine, Washington, D. C. 20057 and §Departments of Pharmacology and Anesthesiology, University of Illinois at Chicago, Chicago, Illinois 60612

Received for publication, September 14, 2006 , and in revised form, December 5, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Caveolin-1 (CAV1), a highly conserved membrane-associated protein, is a putative regulator of cellular transformation. CAV1 is localized in the plasmalemma, secretory vesicles, Golgi, mitochondria, and endoplasmic reticulum membrane and associates with the microtubule cytoskeleton. Taxanes such as paclitaxel (Taxol) are potent anti-tumor agents that repress the dynamic instability of microtubules and arrest cells in the G2/M phase. Src phosphorylation of Tyr-14 on CAV1 regulates its cellular localization and function. We report that phosphorylation of CAV1 on Tyr-14 regulates paclitaxel-mediated apoptosis in MCF-7 breast cancer cells. Befitting its role as a multitasking molecule, we show that CAV1 sensitizes cells to apoptosis by regulating cell cycle progression and activation of the apoptotic signaling molecules BCL2, p53, and p21. We demonstrate that phosphorylated CAV1 triggers apoptosis by inactivating BCL2 and increasing mitochondrial permeability more efficiently than non-phosphorylated CAV1. Furthermore, expression of p21, which correlates with taxane sensitivity, is regulated by CAV1 phosphorylation in a p53-dependent manner. Collectively, our findings underscore the importance of CAV1 phosphorylation in apoptosis and suggest that events that negate CAV1 tyrosine phosphorylation may contribute to anti-microtubule drug resistance.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Caveolin-1 (CAV1)2 is a 21-24-kDa protein and the prototype of a family of integral membrane proteins that associate with specific cholesterol- and sphingolipid-rich domains to form the structural foundation of membrane invaginations called caveolae. Caveolae act as sites of signal transduction in various cell types (1). CAV1 is thought to regulate the activity of proteins such as Src kinases, epidermal growth factor tyrosine kinase, Her2/neu (ErbB2) kinase, ERK (extracellular signal-regulated kinase), H-Ras, endothelial nitric-oxide synthase, and G proteins (1, 2) involved in survival pathways. In human breast tumors, CAV1 levels inversely correlate with tumor size (3), and CAV1 expression reduces the growth of mouse mammary tumors and their spontaneous metastasis to lung and bone (4). However, in breast cancer cell culture models, CAV1 is down-regulated in non-invasive human breast cancer cells but upregulated in cells with an invasive phenotype (5-7).

Taxanes are potent anti-tumor agents that function by binding to the beta subunits of tubulin and repressing the dynamic instability of spindles (8, 9), activities that lead to cell cycle arrest in the G2/M phase (10). Taxanes such as paclitaxel (Taxol) or docetaxel (Taxotere) are routinely used in the first-line treatment of metastatic breast, lung, ovarian, and digestive cancers (11). In primary breast cancer, inclusion of taxane in adjuvant chemotherapy reduces the relative risk of recurrence and improves overall survival (12). Acquired resistance through cellular adaptations or mutations in neoplastic cells remains a major problem in chemotherapy. Although taxanes are substrates for ABC transporters, other resistance mechanisms are clearly important (13). Therefore, it is important to improve our understanding of the mechanisms of drug responsiveness and to identify better predictors of drug efficacy.

CAV1 is essential for the formation and movement of caveolae through the cytoplasm along microtubule tracks, and it is localized in the microtubule-organizing center or peri-centrosomal region in Chinese hamster ovary cells (14). These findings suggest an essential relationship between CAV1 and microtubules or microtubule-associated proteins and their function. Treatment with a cytostatic dose of paclitaxel blocks lung cancer cells in G2/M and causes an up-regulation of CAV1, implicating CAV1 in taxane-mediated cell death and perhaps drug resistance (15-18). However, the role of CAV1 function in cell death remains unclear. In macrophages, induction of apoptosis by different apoptotic agents such as simvastatin and camptothecin leads to a large increase in CAV1 expression. As an early event, this increase in CAV1 is independent of caspase activation or DNA fragmentation but is associated with the plasma membrane translocation of phosphatidylserine (19).

Originally identified as a substrate for v-Src (20), CAV1 is phosphorylated on Tyr-14 by c-Src (21). Mounting evidence suggests that phosphorylated CAV1 regulates caveolae formation and function (21-25). The precise role of Src kinase in taxane-mediated cytotoxicity is unclear. Src can increase Taxotere sensitivity by mediating downstream apoptotic events through BCL2 phosphorylation in v-Src-transformed human gall bladder epithelial cells (26). However, in human ovarian cancer cells, an inhibition of Src activity increases paclitaxel-induced cytotoxicity (27). Thus, the precise role of CAV1, and particularly that of phospho-CAV1(Y14), in affecting breast cancer cell responsiveness to taxanes is unknown.

The current study was undertaken to determine whether CAV1 is involved in the cytotoxic and proapoptotic actions of paclitaxel in MCF-7 human breast adenocarcinoma cells. We overexpressed wild type (wt), a phosphorylation-defective CAV1 mutant (Y14F), or empty vector (EV) in MCF-7 cells that normally express low levels of endogenous CAV1. The effects of wtCAV1, Y14F, or EV expression on cell growth, apoptosis, and p53/p21 transcription in response to low dose (10 nM) paclitaxel were analyzed. This study provides novel insights into the function of CAV1 in paclitaxel sensitivity and the role of phosphorylation on Tyr-14 in CAV1-mediated effects on mitochondrial permeability and apoptosis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reagents—MCF-7 cells (obtained from the LCCC Tissue Culture Shared Resource) were cultured in improved minimal essential medium (IMEM; Biofluids, Rockville, MD) supplemented with 5% fetal bovine serum (FBS). Cells were maintained in a humidified atmosphere at 37 °C and 95% air/5% CO2. Paclitaxel was obtained from Sigma and was dissolved in ethanol (which was used as the vehicle control). PP2 was purchased from Calbiochem. All other reagents were obtained from Sigma unless otherwise indicated.

Generation of Stable Cell Lines—MCF-7 cells were grown to 50-60% confluence and transfected with either human wt CAV1 or Tyr-14 -> Phe-14 phosphorylation-deficient mutant (Y14F) in pcDNA6 or EV using the FuGENE 6 transfection reagent (Roche Applied Sciences). Medium was replaced 24 h later with complete growth medium, and the cells were allowed to grow for 5 days. Complete growth medium containing blasticidin S HCl (Invitrogen) (10 µg/ml) was used for stable selection. For all experiments, pooled populations of stable cell lines were used.

Western Blot Analyses—To determine the effects of paclitaxel on CAV1 protein expression, cells were treated with vehicle or 10 nM paclitaxel in FBS-IMEM for 24 h. Controls were treated with vehicle alone (0.02% v/v ethanol). For Western blot analysis, cells were lysed for 30 min at 4 °C in lysis buffer (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 1% Igepal CA-630, 0.1% SDS, 1 mM Na3VO4, 44 µg/ml phenylmethylsulfonyl fluoride) supplemented with Complete Mini protease inhibitor mixture tablets and 1 mM sodium orthovanadate phosphatase inhibitor (Roche Applied Science). Total protein was quantified using the bicinchoninic acid assay (Pierce). Whole cell lysate (20-50 µg) was resolved by SDS-PAGE. The following primary antibodies were used for immunoblotting: monoclonal antibody against phospho-CAV1(Y14) and polyclonal antibody against CAV1 (BD Biosciences); monoclonal cleaved poly(ADP-ribose) polymerase, polyclonal phospho-BCL2(S70), polyclonal p53, polyclonal Src, and phospho-Src(pY418) antibodies (Cell Signaling, Danvers, MA); and monoclonal BCL2 (Stressgen Corp., Ann Arbor, MA); monoclonal beta-tubulin (Sigma); monoclonal p21 (Calbiochem). Equal protein loading of gels was confirmed by immunostaining with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Immunostaining and Confocal Microscopy—Cells grown on coverslips were washed with phosphate-buffered saline and incubated at least 3 h in serum-free and phenol red-free medium. Cells were then fixed, permeabilized, and incubated with primary antibody. Fluorophore conjugates and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Molecular Probes, Inc. (Eugene, OR). Where appropriate, DAPI was added to visualize the nucleus, and non-confocal DAPI images were acquired using Hg lamp excitation and a UV filter set. Confocal microscopy was performed using an Olympus IX-70 confocal microscope with 405-, 488-, and 543-nm excitation lasers. Fluorescence emission was separately detected for each fluorophore in optical sections <1 µm in thickness (pinhole set to achieve 1 Airy unit).

Transcriptional Reporter Assays—Cells were transfected with 0.4 µg of p53 (Panomics, Fremont, CA) and p21 luciferase reporter plasmid (a gift from Dr. Jane Trepel, National Institutes of Health) and 0.1 µg of pCMV-Renilla (Promega, Madison, WI) per well using the FuGENE 6 transfection reagent. The next day, cells were treated with 10 nM paclitaxel for 24 h. Activation of the luciferase constructs was measured using the Dual Luciferase assay kit (Promega). Luciferase values were normalized to Renilla luminescence. Three independent experiments were performed in quadruplicate. Data are presented as the mean ± S.E. for all experiments.

Cell Proliferation Assays—Cells were seeded at a density of 1-2 x 104 cells/well in 24-well plates. For Src inactivation, cells were incubated with 10 µM PP2 for 2-3 h before adding paclitaxel. For small interfering RNA (siRNA)-mediated knock down of CAV1, cells were transiently transfected with CAV1 siRNA for 48 h before adding paclitaxel. To assess paclitaxel-induced growth inhibition, cells were treated with 10 nM paclitaxel (in FBS-IMEM) for 24 h. Cells were then trypsinized, resuspended in phosphate-buffered saline, and counted using a Z1 Single Coulter Counter (Beckman Coulter, Miami, FL). At least three independent experiments were done in sextuplicate. Data were normalized to vehicle-treated cells and are presented as the mean ± S.E. from a representative experiment.

Cell Cycle and Apoptosis Assays—Following treatment of cells with 10 nM paclitaxel in FBS-IMEM for 24 h, cells were fixed in 70% ethanol for 20 min at 4 °C. Cell cycle distribution was measured by fluorescence-activated cell sorting in the Lombardi Comprehensive Cancer Center Flow Cytometry Shared Resource facility. Annexin V and propidium iodide staining was done using an Annexin V-fluorescein isothiocyanate kit (Trevigen, Gaithersburg, MD). Mitochondrial permeability was detected using the ApoAlert mitochondrial membrane sensor kit (Clontech, Mountain View, CA).

Quantitative Real-time Polymerase Chain Reaction—Primer for CAV1 (Hs00971716_m1) and the housekeeping gene ribosomal protein, large, P0 (RPLPO) (Hs99999902_m1) was purchased from Applied Biosystems (Foster City, CA). MCF-7 cells were allowed to grow to 60-70% confluence in 75-cm2 flasks and were then treated with either different concentrations of paclitaxel (0, 10, 100 nM) for 24 h or 10 nM paclitaxel for 0, 24, and 48 h. RNA was extracted using the TRIzol reagent (Invitrogen), cleaned using the RNeasy kit (Qiagen, Valencia, CA), and analyzed by the Agilent Bioanalyzer 2100 (Santa Clara, CA). About 1 µg of DNase I (Invitrogen)-treated RNA was reverse transcribed with SuperScript II reverse transcriptase (Invitrogen) using Oligo(dT)16 (Applied Biosystems). Real-time absolute quantitative PCR for each cDNA sample and a standard curve were established using TaqMan PCR mastermix in the presence of CAV1 primer or the internal control RPLP0 primer. Reactions (10 µl) were run in triplicate in 384-well plates on an ABI Prism 7900 HT sequence detection system using the protocol suggested by the manufacturer. The ratio of CAV1 induction was estimated in comparison with RPLPO expression; data presented are the mean ± S.E.


Figure 1
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  		FIGURE 1.
Paclitaxel induces CAV1 expression in MCF-7 cells. A, quantitative real-time PCR assay was used to measure CAV1 mRNA expression levels in MCF-7 cells following treatment with 10 nM paclitaxel for 24 and 48 h. Data presented are mean ± S.E. of three determinations in which expression levels were determined as a ratio of CAV1:PRLPO (a housekeeping gene). ANOVA, p = 0.004; *, p ≤ 0.05 versus MCF-7 at 0 h. B, Western blot analysis of CAV1 protein expression following treatment with 10 nM paclitaxel for 0 (untreated), 24, or 48 h; representative immunoblot of CAV1 and GAPDH (loading control). C, CAV1 mRNA expression levels in MCF-7 cells following treatment with 0 (vehicle), 10, or 100 nM paclitaxel for 24 h. ANOVA, p = 0.001; *, p ≤ 0.05 versus MCF-7 with 0 nM paclitaxel. D, CAV1 protein levels detected by Western blotting after treatment with 0 (vehicle), 10, or 100 nM paclitaxel for 24 h.

 
Transfection of siRNA—Cells were plated in 12- or 24-well plates in complete medium and allowed to grow to 50% confluence. Approximately, 100 nM p21 siRNA, p53 siRNA (Cell Signaling), CAV1 siRNA (Dharmacon, Lafayette, CO), or their respective control siRNA were transfected using the TransIT-siQUEST (Mirus, Madison, WI) transfection reagent according to the manufacturer's protocol. At 24 h, 10 nM paclitaxel or vehicle was added to the siRNA-transfected cells. Cells were lysed at 48 h post-transfection and subjected to Western blot analysis or cell proliferation assay as described above.

Statistical Analyses—Statistical analyses were performed using the Sigmastat software package (Jandel Scientific, SPSS, Chicago, IL). Where appropriate, protein expression, cell growth, and apoptosis were compared using Student's t test or ANOVA with a post hoc t test for multiple comparisons. Where several groups were compared with the same control, we used Dunnett's test. Differences were considered significant at p ≤ 0.05; all tests were two-tailed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Exposure to Taxane Increased CAV1 Expression in MCF-7 Cells—CAV1 expression is very low but remains detectable in MCF-7 human breast cancer cells. To evaluate the effect of paclitaxel on CAV1 expression, MCF-7 cells were grown to 50-70% confluence and treated with 10 nM paclitaxel for 0, 24, or 48 h or with vehicle alone, 10 nM, or 100 nM paclitaxel for 24 h. Real-time PCR showed a significant increase in CAV1 expression in MCF-7 cells within 24 or 48 h following paclitaxel treatment compared with control (untreated) (Fig. 1A, p ≤ 0.004, one-way ANOVA). Western blot analysis showed a corresponding increase in CAV1 protein within 24 or 48 h (Fig. 1B). Treatment of MCF-7 cells with 10 and 100 nM for 24 h showed a significant increase in both CAV1 transcription (Fig. 1C, p ≤ 0.001, one-way ANOVA) and protein expression (Fig. 1D) compared with controls (vehicle). Thus, in MCF-7 cells, a significant induction of CAV1 occurs within 24 h following treatment with 10 nM paclitaxel. The induction of CAV1 following paclitaxel treatment suggests a role for CAV1 in drug responsiveness. Moreover, attenuating levels of endogenous CAV1 in MCF-7 cells with siRNA correlates with reduction in paclitaxel-induced inhibition of cell growth (Fig. 2, A and B).


Figure 2
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  		FIGURE 2.
CAV1 is required for paclitaxel-induced cell growth inhibition. A, paclitaxel-induced up-regulation of CAV1 expression was inhibited with siRNA in MCF-7 cells. MCF-7 cells were transfected with either control or CAV1 siRNA for 48 h. Next, paclitaxel was added to the transfection medium to attain a final concentration of 10 nM; cells were incubated with paclitaxel for 24 h before Western blot analysis or cell proliferation assay. B, attenuation of CAV1 expression in MCF-7 cells correlated with reduction in paclitaxel-induced inhibition of cell growth. *, p < 0.05 versus control siRNA with vehicle alone. C, incubation of MCF-7 cells with 10 µM PP2 inhibited Src activation as detected by Western blot analysis of phosphor-Src(Y418). Although the level of CAV1 protein expression increased following treatment of MCF-7 cells with 10 nM paclitaxel, phospho-CAV1(Y14) was undetectable using our Western blot analysis protocol (for up to 50 µg of protein loading/gel). Incubation of MCF-7 cells with 10 µM PP2 in addition to 10 nM paclitaxel significantly reduced cell growth inhibition effect of paclitaxel compared with paclitaxel alone (panel D, p ≤ 0.05). *, p < 0.05 versus MCF-7 with paclitaxel alone.

 
Phosphorylation of CAV1 on Tyr-14 by Src is known to regulate CAV1 functions (21-25). To show whether Src inactivation reduces paclitaxel-induced inhibition of cell growth, we used PP2 to inhibit Src activation as detected by phosphorylation on Tyr-418 by Western blot analysis (Fig. 2C). Whereas CAV1 protein expression increases following treatment of MCF-7 cells with 10 nM paclitaxel, phospho-CAV1 (p-CAV1) is undetectable using our Western blot analysis protocol (for up to 50 µg of protein loading/gel). However, incubation of MCF-7 cells with 10 µM PP2 in addition to 10 nM paclitaxel significantly reduces the cell growth inhibition effect of paclitaxel compared with paclitaxel alone (Fig. 2D, p ≤ 0.05).

wtCAV1 Expression Enhanced Paclitaxel-induced Growth Inhibition and Cell Cycle Arrest at G2/M—To establish the functional relevance of CAV1 in paclitaxel sensitivity, we generated MCF-7 cell lines that stably express either the full-length wild-type CAV1 (MCF-7/wtCAV1), Tyr-14 -> Phe phosphorylation-deficient (MCF-7/Y14F) cells, or empty vector (MCF-7/EV) (Fig. 3A). Expression levels of CAV1 in MCF-7/wtCAV1 and MCF-7/Y14F were measured by Western blot analysis using specific antibodies for CAV1 or p-CAV1. CAV1{alpha} contains residues 1-178, whereas CAV1beta contains residues 32-178. Because Tyr-14 is the principal substrate for Src kinase, only CAV1{alpha} undergoes tyrosine phosphorylation (28). CAV1 is thought to interact with Src and inhibit its activation (2). Although not fully inactive, Src kinase activity is decreased in untreated MCF-7/wtCAV1 cells compared with MCF-7/EV and MCF-7/Y14F cells (Fig. 3A).


Figure 3
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  		FIGURE 3.
wtCAV1 increases sensitivity to paclitaxel in MCF-7 cells. Cells were transfected with pcDNA6 plasmid vector containing wild type (wt), phosphorylation-deficient mutant (Y14F), or empty vector (EV) and stably selected. A, Western blot analysis of whole cell lysates from the MCF-7/EV, MCF-7/wtCAV1, and MCF-7/Y14F. Using specific antibodies, phospho-Src(Y418), total Src, phospho-CAV1(Y14), and total CAV1 were detected. GAPDH blot was used as the loading control. Note CAV1 phosphorylation on Tyr-14 was detected only in MCF-7/wtCAV1 cells. B, expression of wtCAV1, but not Y14F, decreased the growth rate of MCF-7 cells. Cells were seeded in quadruplicate in FBS-IMEM and trypsinized and counted at the indicated number of days. *, p ≤ 0.05 versus MCF-7/EV at Days 4, 5, and 6 by Student's t test. C, expression of wtCAV1 increased sensitivity of breast cancer cells to paclitaxel. Cells were cultured in quadruplicate with the indicated concentrations of paclitaxel for 48 h. Data points are the mean of relative proliferation, bars ± S.E. (n = 3).

 
Expression of CAV1 in MCF-7 cells can reduce the rate of proliferation (29). Comparison of growth curves in basal medium shows a significant decrease in the rate of cell proliferation for MCF-7/wtCAV1 cells (p ≤ 0.05) at day 4 through day 6 compared with that for MCF-7/EV cells. In contrast, the difference in rates of proliferation between MCF-7/EV and MCF-7/Y14F cells is not significant (Fig. 3B). Cells were treated with paclitaxel to determine whether CAV1 tyrosine phosphorylation affects growth inhibition by the drug. MCF-7 cells expressing wtCAV1 are more sensitive than cells expressing either EV or Y14F (Fig. 3C). These data suggest that CAV1 phosphorylation on Tyr-14 plays a key role in paclitaxel-induced growth inhibition. Furthermore, the effects of CAV1 on paclitaxel sensitivity are not simply a consequence of changes in the rate of proliferation; cells that have a higher rate of proliferation are generally more sensitive to cell cycle-specific cytotoxic drugs like taxanes (30). Additionally, we did not see any difference in the partitioning of caveolar and intercellular compartments following subcellular fractionation (data not shown) between wtCAV1 or the Y14F mutant-expressing cell under control conditions or following paclitaxel treatment for 24 h. Thus, in terms of paclitaxel sensitivity, caveolar localization is unlikely to account for any differences between MCF-7 cells expressing wtCAV1 and Y14F.

Taxanes stabilize microtubules and block sensitive cells in the G2/M cell cycle phase (31). To determine whether CAV1 expression alters G2/M cell cycle arrest following treatment with paclitaxel, MCF7-EV, MCF7-wtCAV1, or MCF-7/Y14F cells were treated with either vehicle alone or 10 nM paclitaxel for 24 h prior to fluorescence-activated cell sorter analysis of cell cycle distribution (Fig. 4A). MCF-7/Y14F cells exhibited a significant increase (p ≤ 0.05) in the proportion of cells in the S-phase relative to MCF-7/EV. Following treatment with 10 nM paclitaxel, the percentage of cells arrested in the G2/M phase was significantly higher in MCF7-wtCAV1 compared with MCF-7/EV (p ≤ 0.05). In MCF-7/Y14F cells, the percentage of cells in the G2/M phase was comparable with that in MCF-7/EV cells (Fig. 4B). Thus, the taxane-induced decrease in cell proliferation in MCF7-wtCAV1 cells is likely to be a consequence of increased cell cycle arrest in G2/M phase. To assess cellular morphology in vehicle (control) or drug-treated cells, MCF-7/EV, MCF-7/wtCAV1, and MCF-7/Y14F cells were treated with either vehicle or 10 nM paclitaxel for 24 h. Cells were fixed and permeabilized and co-stained for both beta-tubulin and CAV1. In all three cell lines, vehicle-treated cells displayed an organized tubulin network that excludes the nucleus and extends throughout the cytoplasm (Fig. 4C). After 24 h of paclitaxel treatment, microtubule disruption was more distinct in MCF-7 cells expressing wtCAV1 compared with MCF-7 cells expressing EV or Y14F, as evident from clusters of microtubules or asters in the respective cells (Fig. 4D).


Figure 4
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  		FIGURE 4.
wtCAV1 increases the rate of cell cycle arrest in mitosis following paclitaxel treatment. Cells were treated with vehicle (A) or 10 nM paclitaxel (B) in FBS-IMEM for 24 h before cell cycle analysis. Columns, mean for three independent experiments (% total cells); bars, ± S.E. *, p < 0.05 versus MCF-7/EV by Student's t test. Paclitaxel increased the number of cells arrested in mitosis in the presence of wtCAV1. Cells were treated with vehicle (C) or with 10 nM paclitaxel (D), fixed, permeabilized, and stained with anti-beta-tubulin (green) and CAV1 (red) antibody. Confocal images showed very low levels of CAV1 in EV-expressing cells compared with wtCAV1- or Y14F-expressing cells. Cells with wtCAV1 showed a dramatic increase in cells that were arrested in mitosis at 24 h following treatment with 10 nM paclitaxel as visualized by clustering of beta-tubulin. Scale bar, 10 µm.

 
Increased Apoptosis in MCF-7/wtCAV1-expressing Cells in Response to Paclitaxel—Paclitaxel can induce apoptosis in some breast epithelial cells (32). To determine whether CAV1 expression affects apoptosis, we treated MCF-7/EV, MCF-7/wtCAV1, and MCF-7/Y14F cells with 10 nM paclitaxel for 24 h. Apoptosis was detected by flow cytometry after staining for fluorescein isothiocyanate-conjugated Annexin V and for propidium iodide. At 10 nM paclitaxel for 24 h, only 1% of the MCF-7/EV or MCF-7/Y14F cells underwent apoptosis compared with 3% in MCF-7/wtCAV1 cells (Fig. 5A, p ≤ 0.05). Additionally, cells were analyzed for apoptosis by Western blot analysis of anti-poly(ADP-ribose) polymerase cleavage to a 85-89-kDa fragment (Fig. 5B). Paclitaxel-induced poly(ADP-ribose) polymerase cleavage appeared in MCF-7/wtCAV1 within 24 h following drug treatment and was more pronounced at 48 h relative to MCF-7/EV or MCF-7/Y14F cells. Although the poly(ADP-ribose) polymerase antibody (according to the manufacturer) should detect only a single band for cleaved poly(ADP-ribose) polymerase, we detected a nonspecific higher molecular band at ~100-110 kDa that did not accurately reflect levels of full-length poly(ADP-ribose) polymerase in our experiments. These findings suggest that tyrosine phosphorylation of CAV1 accelerates apoptosis in response to paclitaxel treatment and/or could be essential to signaling pathway(s) that are required for the induction of apoptosis.


Figure 5
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  		FIGURE 5.
wtCAV1 enhances apoptosis in MCF-7 cells following paclitaxel treatment. A, at 24 h, apoptosis in 10 nM paclitaxel- or ethanol-treated cells was detected by flow cytometry using fluorescein isothiocyanate-conjugated Annexin V staining. Columns, mean for three independent experiments (% total cells); bars, ± S.E. *, p ≤ 0.05 versus control-treated MCF-7/wtCAV1 cells by Student's t test. B, cells expressing wtCAV1 showed increased poly(ADP-ribose) polymerase cleavage at 24 and 48 h subsequent to 10 nM paclitaxel treatment. GAPDH was used as a loading control.

 
Microtubule disruption following treatment with taxanes increases phosphorylation of BCL2(S70) in the G2/M phase of the cell cycle, abrogating the normal anti-apoptotic function of BCL2 and initiating an apoptotic program in cycling cancer cells (33). To determine whether activation of BCL2 phosphorylation correlates with the degree of apoptosis, Western blot analyses were done with whole cell lysate cells following treatment with vehicle or 10 nM paclitaxel for 24 or 48 h. At 24 h, we detected a significant increase in the level of BCL2(S70) phosphorylation in MCF-7/wtCAV1 cells in comparison with MCF-7/EV cells (p ≤ 0.05). In cells expressing Y14FCAV1, the level of BCL2(S70) phosphorylation remains comparable with cells expressing EV (Fig. 6A). Furthermore, measurement of mitochondrial permeability, which is tightly associated with the release of pro-death molecules into the cytoplasm, is significantly increased in MCF-7/wtCAV1 cells treated with paclitaxel in comparison to cells treated with vehicle alone (Fig. 6B, p ≤ 0.05).

wtCAV1 Regulates p53-dependent p21 Induction following Treatment with Paclitaxel—Low dose (~10 nM) paclitaxel treatment can increase p21 synthesis through a p53-dependent pathway (34, 35), and CAV1 and p53 can induce each other in fibroblasts (36). Inhibition of p53 expression with siRNA in MCF-7/wtCAV1 cells prevents paclitaxel-induced p21 protein expression (Fig. 7, A-C). Thus, in wtCAV1-expressing cells, induction of p21 following paclitaxel treatment is p53-dependent. To determine how expression of CAV1 interferes with p53 activity, we transiently expressed MCF-7/EV, MCF-7/wtCAV1, and MCF-7/Y14F with a p53-responsive promoter-reporter construct. Following 24 h of treatment with paclitaxel, p53-responsive element activation is significantly decreased in MCF-7/wtCAV1-expressing cells relative to vehicle-treated cells (Fig. 7D, p ≤ 0.05); this activation remains unchanged in MCF-7/EV and MCF-7/Y14F cells relative to their respective controls.

MCF-7 cells express a functional p53, and treatment with paclitaxel enhances p53 expression and function as seen in an up-regulation of p21 and changes in cell cycle progression and apoptosis. Induction of p21 confers resistance to the cytotoxic effects of taxanes in MCF-7 cells (37). Transient transfection of a full-length p21 promoter-reporter construct showed that p21 promoter activity in MCF-7/wtCAV1 cells corresponds with p53-responsive element activation under control or drug treatment conditions. In MCF-7/EV and MCF-7/Y14F cells, p21 promoter activity following vehicle or drug treatment remains unchanged, as also occurs with p53 transcriptional activation in these cells (Fig. 7E).

We measured the level of p53 and p21 protein expression in MCF-7/EV, MCF-7/wtCAV1, and MCF-7/Y14F cells. Within 24 and 48 h of drug treatment, p53 and p21 protein levels increased in all the three cell lines (Fig. 8, A and B). However, the increase in MCF-7/wtCAV1 cells was significantly lower at both 24 and 48 h following drug treatment when compared with MCF-7/EV or MCF7-Y14FCAV1 cells (p ≤ 0.05). CAV1 protein expression in both MCF-7/wtCAV1- and MCF-7/Y14F-expressing cells was increased at 48 h following drug treatment. p53 can initiate both cell cycle arrest and apoptosis, and an inhibition of p53-dependent p21 induction may drive cells toward apoptosis (38). These results suggest that in MCF-7/wtCAV1 cells, phosphorylated CAV1 on Tyr-14 regulates induction of p53 and p21 following treatment with paclitaxel and is key in initiating the proapoptotic signal upstream of both p53 and p21.


Figure 6
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  		FIGURE 6.
Paclitaxel treatment of MCF-7/wtCAV1 cells increases BCL2(S70) phosphorylation. A, cells were treated with 10 nM paclitaxel for 24 or 48 h and phosophorylated-BCL2(S70) was detected by immunoblot analysis using a phospho-specific antibody (upper panel). Lower panels, immunoblot analysis of total BCL2 and GAPDH (loading control). B, mitochondrial permeability in MCF-7/wtCAV1 cells is significantly increased compared with control within 24 h following paclitaxel treatment (10 nM). *, p ≤ 0.05 versus control-treated MCF-7/wtCAV1 cells by Student's t test.

 


Figure 7
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  		FIGURE 7.
p53 is required for paclitaxel-induced expression of p21 in MCF-7/EV (A), MCF-7/wtCAV1 (B), and MCF-7/Y14F (C) cells. Cells were transfected with control, p21, or p53 siRNA for 48 h before treatment with 10 nM paclitaxel for 24 h. Induction of p53 and p21 was detected by immunoblot analysis using specific antibodies (top and middle panels). Lower panels, GAPDH immunoblot was used as a loading control. Expression of wtCAV1 and not Y14F-CAV1 regulates p53-dependent up-regulation of p21. Activation of p53-responsive element (D) and p21 promoter (E) in MCF-7/EV, MCF-7/wtCAV1, or MCF-7/Y14FCAV1 cells by luciferase assay. Experiments were done in quadruplicate. The results were normalized to Renilla luminescence and are presented as relative luciferase activity to respective cells under control conditions. *, p ≤ 0.05 versus control-treated MCF-7/wtCAV1 cells by Student's t test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
CAV1 is a versatile protein that has received increased attention in cancer because of its role in cell survival (39). CAV1 proteins form the foundation of cholesterol-rich membrane microdomains called caveolae, within which CAV1 regulates the functions of various signaling proteins (1). Among the multiple cellular events that are regulated by CAV1, regulation of tumorigenesis remains controversial. Alteration or modification of CAV1 expression is common in some transformed cells, and understanding the signaling pathways affected by CAV1 may provide novel insight into new cancer therapies. In the present study, we show that treatment with 10 nM paclitaxel increased CAV1 protein expression in MCF-7 breast cancer cells within 24 h; MCF-7 cells express very low levels of CAV1 under basal conditions. Down-regulation of CAV1 expression with siRNA correlated with decreased paclitaxel-induced growth inhibition. Regulation of CAV1 signaling has been correlated with Src-mediated tyrosine phosphorylation at Tyr-14 (21-25, 40, 41). We show that preventing Src activation with PP2 reduced paclitaxel-induced growth inhibition. We found that expression of wtCAV1, but not the phosphorylation-deficient mutant Y14F, augmented sensitivity to paclitaxel in MCF-7 cells and that tyrosine phosphorylation at Tyr-14 is essential in mediating the molecular events leading to apoptosis.


Figure 8
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  		FIGURE 8.
Paclitaxel-induced increase in p21 expression is regulated by CAV1 Tyr-14 phosphorylation. A, differential induction of p21 in MCF-7 cells expressing EV, wtCAV1, or Y14F was determined by Western blot analysis following treatment of cells with 10 nM paclitaxel for 0, 24, or 48 h. Total protein was analyzed for p53, p21, phospho-CAV1, and total CAV1. GAPDH was used as a loading control. B, graphical representation of p21:GAPDH ratio from three independent experiments. ANOVA, p = 0.005; *, p ≤ 0.05 versus MCF-7/EV.

 
Our results show that expression of wtCAV1, but not Y14F, increases the inhibition of cell growth following treatment with paclitaxel. The inhibition of cell proliferation was associated with an increase in the blockade of cell cycle progression in cells harboring wtCAV1, and this association may explain the reduction in cell growth following paclitaxel treatment. Subsequent to treatment with taxanes, cells arrest in the G2/M-phase due to microtubule stabilization, an event followed by the onset of apoptosis. Thus, our observations imply an interplay between cytoskeletal integrity and nuclear transcriptional events. MCF-7 cells express very low levels of endogenous CAV1 and exhibit both a time- and dose-dependent up-regulation of CAV1 in response to paclitaxel treatment. Because microtubules are involved in the distribution of CAV1 between the Golgi/endoplasmic reticulum system and caveolae (42), interruption of CAV1 delivery by microtubule stabilization following paclitaxel treatment could trigger the cell to increase CAV1 expression. The spatial requirement for CAV1 in taxane-induced cell cycle arrest and subsequent cell death is currently being investigated in our laboratory.

Accumulating evidence suggests a possible role for CAV1 in the initiation of cell death. For example, CAV1 is up-regulated in A549 lung cancer cells in response to paclitaxel (15), and the induction of apoptosis by several agents can significantly increase CAV1 expression in some cells (18). Paclitaxel-induced apoptosis in MCF-7 cells, as shown by Annexin V staining and poly(ADP-ribose) polymerase cleavage, significantly increased in the presence of wtCAV1. Phosphorylation/inactivation of BCL2, as induced by paclitaxel (43, 44), is associated with G2/M cell cycle arrest (45). Increased expression of phospho-BCL2(S70) and mitochondrial permeability in MCF-7/wt-CAV1 cells when compared with MCF-7/Y14F cells imply a role for CAV1 phosphorylation in affecting the mitochondrialdependent, intrinsic apoptosis pathway in paclitaxel-induced cell death.

Clinical studies have shown that taxane-containing regimens are associated with a statistically significant improvement in overall survival compared with nontaxane-containing regimens; however, these regimens are associated with leukopenia and neurotoxicity (46). Considering the heterogeneity among individuals, it is essential to discover molecular biomarkers in breast cancer cells that could help improve the benefits of taxanes by increasing the efficacy-to-toxicity ratio. Tumors that show the highest degree of G2/M phase arrest, BCL2 phosphorylation, and low or absent p21 after drug treatment should exhibit sensitivity to paclitaxel. BCL2, an anti-apoptotic protein, is transcriptionally regulated by factors such as p53 (47). Paclitaxel induces p21 in both p53-wild type and p53-null cells (48). Although CAV1 may be a target in p53-dependent signaling (5, 49), the significance of this interaction in apoptosis is unknown. Inhibition of p53 expression in MCF-7 cells expressing either wtCAV1, Y14F, or EV prevented the induction of p21 following paclitaxel treatment (Fig. 7, A-C). Thus, in cells with a functional p53, the contribution of CAV1 to paclitaxel-mediated cytotoxicity appears to be p53-dependent.

A significant decrease in p53- and p21-luciferase reporter construct activation was observed within 24 h following treatment with paclitaxel in MCF-7/wtCAV1 cells (Fig. 7, D and E). In contrast to its role in cell cycle arrest, p21 can increase cell survival following paclitaxel treatment in MCF-7 cells and inhibition of p21 can increase paclitaxel cytotoxicity in MCF-7 cells (37). Thus, expression of wtCAV1 in MCF-7 cells may increase the rate of apoptosis in MCF-7/wtCAV1 cells by down-regulating p21 expression. To our knowledge, this is the first report showing that phosphorylation of CAV1 at Tyr-14 sensitizes cells to BCL2-mediated apoptosis in a p53/p21-dependent manner.

It is likely that sensitivity as well as resistance to taxanes is dependent on the function of a panel of genes rather than a single gene or pathway. Our data suggest that CAV1 could be an essential component of signal transduction in taxane-mediated cell death. Up-regulation of CAV1 protein following treatment with paclitaxel has prompted investigators to implicate it as a marker for cytotoxicity and cell death in lung cancer cells (15). However, it is unclear how CAV1 regulates cellular events at the onset of apoptosis. Expression of a phosphorylation-deficient Y14F mutant failed to augment sensitivity to paclitaxel when compared with cells expressing wtCAV1 in MCF-7 cells. This observation emphasizes the role of events that regulate phosphorylation of CAV1 in taxane sensitivity. Furthermore, CAV1 acts by regulating paclitaxel-induced apoptosis in a manner that is dependent upon p53/p21 signaling and suppression of BCL2. Thus, this study provides direct experimental evidence of the role of CAV1 and its phosphorylation at Tyr-14 in affecting sensitivity to paclitaxel in p53-wild type breast cancer cells. Further analyses of the involvement of CAV1-mediated signaling in the taxane responsiveness in breast cancer cells are underway in our laboratory and may prove to be important for predicting tumor response to chemotherapy with these drugs.


   FOOTNOTES
 
* This work was supported by Public Health Service Grant R01-CA096483 and Dept. of Defense Grant BC030280 (to R. C.) and Susan G. Komen Breast Cancer Foundation Postdoctoral Fellowship PDF0600477 (to A. N. S.). Technical services were provided by the Flow Cytometry and Cell Sorting, Tissue Culture Core and Microscopy and Imaging Shared Resources Center funded through Public Health Service Award 1P30-CA-51008 (Lombardi Comprehensive Cancer Center support grant). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, College of Medicine, 3970 Reservoir Rd. N.W., NRB W405, Washington, D. C. 20057. Tel.: 202-687-7237; Fax: 202-687-7505; E-mail: clarker{at}georgeotown.edu.

2 The abbreviations used are: CAV1, caveolin-1; EV, empty vector; IMEM, improved minimal essential medium; FBS, fetal bovine serum; GAPDH, glyceraldehyde phosphate dehydrogenase; wt, wild-type; ANOVA, analysis of variance; siRNA, small interfering RNA. Back



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