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The Extracellular Matrix Protein TGFBI Induces Microtubule Stabilization and Sensitizes Ovarian Cancers to Paclitaxel
Ahmed, Ahmed Ashour
Mills, Anthony D.
Ibrahim, Ashraf E.K.
Temple, Jillian
Blenkiron, Cherie
Vias, Maria
Massie, Charlie E.
Iyer, N. Gopalakrishna
McGeoch, Adam
Crawford, Robin
Nicke, Barbara
Downward, Julian
Swanton, Charles
Bell, Stephen D.
Earl, Helena M.
Laskey, Ronald A.
Caldas, Carlos
Brenton, James D.
text
article
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Cancer cell
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10.1016/j.ccr.2007.11.014
urn:nbn:nl:kb-1427973328896
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S1535-6108(07)00338-8
10.1016/j.ccr.2007.11.014
Elsevier Inc.
Figure1
Loss of TGFBI Is Sufficient to Induce Paclitaxel Resistance
(A) Volcano plot shows log fold change in gene expression in the paclitaxel-resistant cell line SKOV-3TR compared to the sensitive parental line SKOV-3 and plotted against the likelihood of differential expression. Note that negative log2 expression ratios indicate underexpression in SKOV-3TR. Data points represent the probability value for differential gene expression and data shown is from four replicate experiments.
(B) Relative expression levels of TGFBI in different cell lines using real time PCR.
(C) Immunocytochemistry of stained sections from embedded cell pellets using anti-TGFBI antibody. Scale bars, 10 m.
(D) Western blotting of culture medium from SKOV-3TR, mock-transfected SKOV-3, and TGFBI siRNA-transfected SKOV3-K cell lines probed with anti-TGFBI antibody.
(E) Effect of stable KD of TGFBI (SKOV3-A, SKOV3-K, and SKOV3-AK) on paclitaxel-induced apoptosis measured by FITC-annexin V and 7-AAD staining at 48 hr following paclitaxel treatment (150, 300, 600, 1200, and 2000 nM) compared to SKOV-3, mock-transfected SKOV-3 (mtSKOV3), and SKOV-3TR cells. Filled triangle indicates increasing paclitaxel dose across each group of bars.
(F) Effect of stable KD of TGFBI on caspase 3/7 activation 48 hr following paclitaxel treatment.
(G and H) Transient TGFBI-KD in OVCAR3 and TR175 lines induces paclitaxel resistance. Caspase 3/7 activation was estimated 48 hr following transfection using either a pool of 4 siRNAs targeting TGFBI or nontargeting scrambled controls (sc). OVCAR3 cells (G) or TR175 cells (H) were treated with paclitaxel for 48 hr. Immunoblot confirming knockdown of TGFBI protein is shown in (G). Error bars show mean SD.
Figure2
Loss of TGFBI Causes Defective Paclitaxel-Induced Microtubule Polymerization
(AF) Overexpression of TGFBI in SKOV-3TR by pCSMT-TGFBI sensitizes microtubules to the polymerizing effect of paclitaxel. Arrowheads indicate paclitaxel-induced bundles (PIBs). Green, tubulin; blue, Dapi-stained DNA. Scale bars, 10 m.
(G) Paclitaxel induces Glu-tubulin formation. Cells were serum starved for 24 hr then treated with paclitaxel in serum-free medium (SFM) at 0, 4, 16, 75, 300, and 1200 nM concentrations for 1 hr. Lysates were collected for fluorescence immuno-blotting with anti-Glu-tubulin and anti-alpha tubulin. Bars represent the fold increase in Glu-tubulin fluorescence intensity values normalized for alpha-tubulin intensity values. Filled triangle indicates increasing paclitaxel dose across each group of bars.
(H) A fluorescence immunoblot of soluble (sol) and insoluble (insol) tubulin fractions.
(I) SKOV-3TR and SKOV3-K cells have increased soluble tubulin. Graph shows the percentages of soluble tubulin in relation to total tubulin in the different cell lines. Quantitative measurements were performed using fluorescence immunoblotting with anti-alpha tubulin antibody as described in Supplemental Experimental Procedures. Results shown are from two independent experiments. Horizontal bars indicate median values.
Figure3
Loss of TGFBI Induces Mitotic Abnormalities
(A and B) Stable KD of TGFBI in SKOV-3 cells results in abnormal mitotic spindle formation and centrosome amplification. Magenta, tubulin; blue, Dapi-stained DNA; yellow, gamma tubulin.
(C) Proportion of abnormal mitotic cells at 930 days in SKOV-3TR, stable knockdown, and mock transfected (mt) cell line pools.
(D) Proportion of interphase cells showing centrosome amplification. Number of cells counted is shown above corresponding bars.
(E) Proportion of abnormal mitotic cells after 48 hr following transient knockdown of TGFBI using a pool of four siRNAs. Error bars show mean SD.
Figure4
TGFBI Induces Microtubule Stabilization
(A) Model of TGFBI induction of microtubule stabilization.
(B) Microtubule stabilization demonstrated with anti-Glu tubulin antibody. SKOV-3 cells were plated for 90 min on rTGFBI or polylysine-coated glass slides (20 g/ml) before immunofluorescence. Scale bars, 10 m.
(C) Cells were adhered to polylysine, fibronectin, or rTGFBI-coated wells (20 g/ml) for 90 min before lysates were collected for immunoblotting using anti-phosphorylated FAK (P397). Also shown are immunoblots for lysates of cells treated in suspension with or without paclitaxel at 3 M.
(D) Percentages of SKOV-3 cells showing Glu-tubulin following adhesion to rTGFBI, fibronectin, or polylysine.
(E) 48 hr following transfection using either FAK siRNA or nontargeting siRNAs, SKOV-3 cells were plated on rTGFBI coated glass slides for 90 min and the percentage of cells showing Glu-tubulin formation was estimated by immunofluorescence.
(F) SKOV-3 cells were either treated with the Rho A inhibitor C3 toxin in SFM or with SFM alone for 4 hr before plating on rTGFBI-coated glass slides and estimation of Glu-tubulin formation. Error bars show mean SD.
Figure5
rTGFBI Sensitizes Resistant Cells to the Effect of Paclitaxel
(A) Cells were adhered to rTGFBI-coated wells (20 g/ml) or noncoated wells for 24 hr prior to paclitaxel treatment for 48 hr. Shown is the percentage of apoptotic cells as measured using FITC-annexin V and 7-AAD staining.
(B) Cells were serum starved for 24 hr then treated with paclitaxel in serum-free medium (SFM) at 0, 4, 16, 75, 300, and 1200 nM concentrations for 1hour before lysates were collected for fluorescence immunoblotting using anti-Glu-tubulin and anti-alpha tubulin. Bars represent the fold increase inglu-tubulin fluorescence intensity values normalized for alpha tubulin intensity values. Filled triangle indicates increasing paclitaxel dose across each group of bars.
(C) The slope and 95% confidence intervals for the linear regression of Glu-tubulin formation following paclitaxel treatment in SKOV-3 and mtSKOV3 (SKOV3-WT), SKOV3-A and SKOV3-K (SKOV3-KD), and SKOV3-KD following plating on rTGFBI.
(D) SKOV-3TR cells were either plated on plastic or rTGFBI as in (A) and either treated with paclitaxel alone (SKOV3-TR) or with paclitaxel and verapamil 3.3 M (TR V and TR V rTGFBI) for 48 hr before caspase 3/7 activity was estimated. Also shown is the data for the parental sensitive line (SKOV-3) plated on plastic.
(E and F) A2780 Cells (E) or PE0188 cells (F) were either plated on plastic or on rTGFBI (20 g/ml) before paclitaxel treatment for 48 hr. Shown is the fold increase in caspase 3/7 activity.
(G) Cells were either pretreated with 50 g/ml of rTGFBI in SFM or SFM alone for 2 hr followed by paclitaxel treatment for 1 hr, washing, and incubation in full media for 48 hr. Shown is the percentage of apoptotic cells measured by FITC-annexin V and 7-AAD staining. ArTGFBIanti-alphaVbeta3; SKOV3-A cells pretreated with anti-alphaVbeta3 in SFM before treatment with rTGFBI and paclitaxel, ArTGFBIFAK-KD; SKOV3-A cells were transfected with siRNA targeting FAK 48 hr prior to rTGFBI and paclitaxel treatment, ArTGFBIC3-toxin; SKOV3-A cells were pretreated with the Rho A inhibitor, C3 toxin, in SFM for 4 hr before the application of rTGFBI and paclitaxel. Error bars show mean SD.
Figure6
The ECM Protein TGFBI Sensitizes Ovarian Carcinoma Cells In Vivo to the Effect of Paclitaxel
Correlation between expression of TGFBI and ECM genes in (A) CTCR-OV01 study and (B) independent ovarian cancer data set from Spentzos etal. (2005). Graphs show proportion of ECM-related genes increasing as a function of coexpression with TGFBI. (C) TGFBI expression in pretreatment biopsies of advanced ovarian carcinoma using real-time PCR. Resistant cases (n 5; magenta) are compared to sensitive cases (n 11; green). (D) Three-dimensional reconstruction of Z stack images obtained following double immunofluorescent staining of a representative ovarian carcinoma tissue section. Arrowheads indicate amplified centrosomes in a single cell confirmed by examination in all three planes. Anti- tubulin, green; chromosomal DNA, Hoechst 33258 red. Scale bar, 10 m. (E) Centrosome amplification is associated with paclitaxel resistance. Centrosome counting was performed on samples from which adequate frozen tissue was available (n 10; samples common between [C] and [E] are indicated by squares). In (C) and (E), horizontal bars indicate median values. (F) Immunohistochemistry for TGFBI of representative posttreatment ovarian cancer sample. Paclitaxel-induced morphological changes colocalize with focal TGFBI expression (brown staining, green box 1) but not in areas of low TGFBI expression (magenta box 2). Scale bars for main subfigure and boxes, 500 m and 50 m, respectively.
Figure7
Proposed Model of Modulation of Paclitaxel Resistance by TGFBI via Effects on Microtubule Stability
Article
The Extracellular Matrix Protein TGFBI Induces Microtubule Stabilization and Sensitizes Ovarian Cancers to Paclitaxel
Ahmed Ashour
Ahmed
1
3
5
Anthony D.
Mills
4
Ashraf E.K.
Ibrahim
1
Jillian
Temple
1
3
Cherie
Blenkiron
3
Maria
Vias
3
Charlie E.
Massie
3
N. Gopalakrishna
Iyer
3
Adam
McGeoch
4
Robin
Crawford
5
Barbara
Nicke
6
Julian
Downward
6
Charles
Swanton
6
Stephen D.
Bell
4
Helena M.
Earl
3
Ronald A.
Laskey
4
Carlos
Caldas
2
3
James D.
Brenton
1
3
james.brenton@cancer.org.uk
1
Functional Genomics of Drug Resistance Laboratory, Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
2
Breast Cancer Functional Genomics Laboratory, Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
3
Department of Oncology, Hutchison/MRC Research Centre, Hills Road, Cambridge, CB2 0XZ, UK
4
MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge, CB2 0XZ, UK
5
Gynaecological Oncology Regional Centre, Box 242, Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Hills Road, Cambridge CB2 0QQ, UK
6
Signal Transduction Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
Corresponding author
Published: December 10, 2007
Summary
The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability.
CELLCYCLE
CHEMBIO
CELLBIO
SIGNIFICANCE
Extracellular matrix (ECM) proteins such as fibronectin induce resistance to chemotherapy via activation of intracellular survival pathways. We now show that the ECM protein TGFBI mediates specific sensitization to paclitaxel by inducing stabilization of microtubules via integrin-mediated signaling pathways. Analysis of paclitaxel-treated ovarian cancers from a prospective clinical trial shows TGFBI protein expression in areas of paclitaxel-induced cytotoxicity. Bioinformatic analysis of three microarray expression data sets from 223 ovarian and breast cancer samples show that TGFBI expression is tightly coregulated with other genes that induce paclitaxel sensitization such as THBS1. These data show that paclitaxel response can be modulated by ECM proteins and raise the prospect of improving the therapeutic index of taxanes via manipulation of these proteins and their downstream signaling pathways.
Introduction
Taxanes are microtubule-stabilizing drugs that have been extensively used as effective chemotherapeutic agents in the treatment of solid tumors (McGuire etal., 1996; Sandler etal., 2006). However, taxane resistance limits clinical utility to approximately 50% of patients with breast or ovarian cancer. Identification of mechanisms of taxane resistance that are therapeutically accessible is, therefore, required to improve treatment.
Paclitaxel, a prototype taxane, stabilizes microtubule polymers leading to mitotic arrest and apoptosis (Schiff etal., 1979; Ibrado etal., 1998; Scatena etal., 1998). General mechanisms of drug resistance, including overexpression of the ABC/MDR transporter family of proteins (Peer etal., 2004), delayed G2/M transition (Tan etal., 2002), defective mitotic checkpoints (Anand etal., 2003), and alterations in apoptosis regulation (Huang etal., 1997), may alter paclitaxel sensitivity. More specifically, alterations of microtubules induce severe taxane resistance (Zhang etal., 1998; Gonalves etal., 2001; Alli etal., 2002; Wang etal., 2004). These include -tubulin mutations which may decrease paclitaxel binding to microtubules (Giannakakou etal., 1997). Alternatively, factors that increase the ratio of unstable to stable microtubules induce profound taxane resistance. This may occur by mutations in nonpaclitaxel binding sites, alterations in tubulin isoforms (Gonalves etal., 2001; Barlow etal., 2002; Hari etal., 2006), overexpression of -III tubulin (Mozzetti etal., 2005), and overexpression of the microtubule-associated protein stathmin (Alli etal., 2002).
Cells contain subsets of stable and dynamic microtubules that are functionally distinct (Gundersen etal., 1984), and there is strong evidence that extracellular stimuli regulate microtubule stability. Serum starvation or loss of direct cell contact results in loss of microtubule stabilization, while fibronectin-mediated adhesion or treatment of cells with lysophosphatidic acid or TGF induce microtubule stability (Cook etal., 1998; Palazzo etal., 2004; Gundersen etal., 1994). Whether extracellular matrix (ECM) modulation of microtubule stability may alter paclitaxel sensitivity has been, to date, unknown.
We describe here the identification of TGFBI (transforming growth factor beta induced) as an ECM protein that induces microtubule stabilization and modulates paclitaxel sensitivity in vitro and in patients receiving paclitaxel therapy.
Results
TGFBI Is Significantly Underexpressed in an Ovarian Cancer Cell Line Model of Paclitaxel Resistance
To identify genes associated with the acquisition of paclitaxel resistance, we studied the ovarian cancer cell line SKOV-3TR, which was derived from SKOV-3 by prolonged and repeated exposure to increasing doses of paclitaxel (Duan etal., 1999). We compared the expression profiles of the parent and resistant lines using cDNA microarrays (
Figure1A) after confirming that SKOV-3 and SKOV-3TR were isogenic by short tandem repeats genotyping (data not shown). Examination of the most differentially expressed genes showed several candidates previously identified in chemotherapy response including CP, SOD2 (Ueta etal., 2001), HMGA1 (Huang etal., 1994; Kasparkova etal., 2003), and CXCL1 (Taxman etal., 2003) (Figure1A; Table S1 in the Supplemental Data available with this article online).
The most underexpressed gene in the taxane-resistant line was transforming growth factor beta induced (TGFBI; also known as Big-h3, -ig H3, and keratoepithelin), which is an extracellular matrix protein whose secretion is induced by TGF1 stimulation (Skonier etal., 1992). Its functions include cell adhesion to the ECM and integrin-mediated signaling (Jeong and Kim, 2004; Billings etal., 2002). Quantitative real-time PCR confirmed striking underexpression (>1000 fold) of TGFBI (Figure1B). This underexpression was maintained after growing the cells without paclitaxel for several months (data not shown). Immunocytochemistry using anti-TGFBI antibody on paraffin-embedded cell blocks and immunoblotting of conditioned media from the two cell lines showed very low secretion of TGFBI protein (Figures 1C and 1D) from the resistant SKOV-3TR cells.
Loss of TGFBI Is Sufficient to Induce Paclitaxel Resistance
To examine the effect of loss of TGFBI expression on paclitaxel sensitivity, stable transfected cell lines expressing short interfering RNAs (siRNA) against TGFBI were generated from the parental SKOV-3 line. Two vectors targeting 21 bp sequences at nucleotides 810 or 1318 of the TGFBI coding sequence were transfected independently or together to generate stable cell line pools SKOV3-A, SKOV3-K, and SKOV3-AK, respectively. Effective knockdown (KD) of TGFBI mRNA and protein was achieved in all three lines (Figures 1B and 1D and data not shown).
The effect of paclitaxel treatment on these cells was examined. Early apoptosis was measured after 48 hr of paclitaxel exposure by flow cytometry of cells stained with FITC-annexin V and 7-AAD (Wang etal., 1998). Cell lines that lacked TGFBI (SKOV-3TR and TGFBI-KD cells) showed a significantly lower percentage of paclitaxel-induced apoptosis (p < 0.001, one-way ANOVA) in contrast to control SKOV-3 cells (Figure1E). Caspase 3/7 activation 48 hr following paclitaxel treatment was significantly reduced in SKOV-3TR and TGFBI-KD cells (p < 0.001, two-way ANOVA) (Figure1F) as was caspase 3 cleavage (FigureS1A).
Analysis of 9 ovarian and 11 breast cancer cell lines showed that TGFBI expression was significantly lower in resistant (n 7) cells (p 0.027, two-way ANOVA), and this was independent of cell type (Figures S1B and S1C). Importantly, transient knockdown of TGFBI using a pool of four siRNAs in four additional ovarian cancer cell lines, OVCAR3 (Figure1G), TR175 (Figure1H), 1847 (FigureS1D), and PE01 (FigureS1E), resulted in significant resistance to paclitaxel-induced caspase activation (p < 0.001, two-way ANOVA). These results were confirmed using at least two individual siRNAs in each cell line (data not shown).
SKOV-3TR cells have accumulated multiple paclitaxel resistance mechanisms during in vitro selection (Lamendola etal., 2003). For example, expression profiling of SKOV-3TR shows upregulation of MDR1 and ABCB6 (Duan etal., 2005; data not shown). The intensity values of a fluorescent derivative of paclitaxel were lower in SKOV-3TR compared to parental SKOV-3 and could be corrected using the MDR1 inhibitor verapamil (FigureS2A).However, induction of paclitaxel resistance in SKOV-3 cells by specific downregulation of TGFBI strongly suggested that TGFBI was an important component of the resistance shown in SKOV-3TR.
To investigate how TGFBI modulated response to paclitaxel, we analyzed common resistance mechanisms using TGFBI-KD cells. Intracellular intensity of fluorescent paclitaxel was not different after TGFBI-KD (FigureS2A). In addition, apoptosis induction using UV, cisplatin, or nocodazole treatment was not significantly different between TGFBI-KD and parental cells (FigureS2B). These data show that paclitaxel resistance arising from specific loss of TGFBI is not caused by nonspecific alterations in apoptotic or multi-drug-resistance pathways.
Abnormal mitotic checkpoints have previously been shown to confer paclitaxel resistance (Anand etal., 2003). Mitotic SKOV-3 cells lacking TGFBI showed a slight increase in prophase (FigureS2C), but no significant difference in the duration of mitotic progression was observed when measured by time-lapse imaging of individual cells (p 0.1, one-way ANOVA, FigureS2D). A characteristic feature of overriding mitotic checkpoints is persistence of checkpoint proteins following metaphase (Anand etal., 2003). FigureS2E shows no significant difference (p 0.4, Chi Square test) between KD and control cell lines in the proportion of postmetaphase cells showing persistent cyclin B1 staining. BubR1, a mitotic checkpoint protein expressed prior to anaphase, is thought to sense tension across the spindle when chromosomes are aligned in the metaphase plate (Nicklas, 1997). Correct sensing of spindle tension leads to degradation of BubR1 and anaphase transition. Spindle poisons such as nocodazole and paclitaxel abolish tension leading to persistent expression of BubR1 (Logarinho etal., 2004). Cells with abnormal BubR1 function progress to anaphase in spite of exposure to spindle poisons. The expression of BubR1 in wild-type, KD, and SKOV-3TR cells was normal following nocodazole treatment indicating that sensing of spindle tension was not impaired (FigureS2F). Delayed cell-cycle transition has been shown to confer paclitaxel resistance (Tan etal., 2002). There was no difference in cell-cycle profiles of nonsynchronized TGFBI-KD or SKOV-3TR cells as compared to mock-transfected SKOV-3 (data not shown). To test cell-cycle profiles in synchronized cells, we used HeLa cells as they express comparable levels of TGFBI to those of SKOV-3 (data not shown) and are a well characterized cell-cycle model with intact mitotic checkpoints and are sensitive to paclitaxel (Anand etal., 2003). FigureS2G shows that transient knockdown of TGFBI had no effect on cell-cycle progression in HeLa cells synchronized in G1 by double thymidine block.
TGFBI Restores Paclitaxel-Induced Tubulin Polymerization
Microtubule bundle formation has been described as the hallmark of paclitaxel exposure in vivo (Schiff and Horwitz, 1980) and is lost in resistant cells (Giannakakou etal., 1997). Indeed, SKOV-3 cells treated with 2 M paclitaxel for 24 hr showed striking paclitaxel-induced bundles (PIBs) in interphase cells (
Figures 2A and 2B and FigureS3A) and severe failure of mitotic spindle organization. Mitotic cells showed a prometaphase-like state where chromosomes formed ring-like structures around centrally radiating microtubule fibers, which we termed mitosis-like wheels (MLWs) (FigureS3B). In contrast, a minority of SKOV-3TR cells showed PIBs (2.4% versus 93%) or MLWs (1.7% versus 92%) (Figures 2C and 2D and FigureS3A). Importantly, SKOV-3TR cells transfected with a myc-tagged TGFBI expression plasmid (pCSMT-TGFBI) showed a modest increase (12% versus 3.7%) in the proportion of PIBs and MLWs following paclitaxel treatment (p < 0.001, two-sided t test) (Figures 2E and 2F and FigureS3A).
Paclitaxel induces microtubule stabilization, and posttranslational modifications of tubulin, such as detyrosination or acetylation, accumulate in these stable microtubules. Detyrosination exposes Glu residues at the carboxy terminus of alpha-tubulin that can be detected using antibodies and, therefore, used as a marker of microtubule stability (Gundersen etal., 1984). Cells lacking TGFBI (SKOV-3TR, SKOV3-K) showed impaired paclitaxel-induced microtubule stabilization as evidenced by decreased Glu-tubulin formation following paclitaxel treatment (Figure2G). Similarly, transient TGFBI-KD of SKOV-3 using pooled siRNAs resulted in intermediate reduction of paclitaxel induced Glu-tubulin (Figure2G). These data suggest that selective loss of TGFBI induces paclitaxel resistance at the level of microtubules. Alteration of microtubule function leading to paclitaxel resistance is associated with increased ratios of soluble to insoluble intracellular tubulin (Gonalves etal., 2001; Barlow etal., 2002; Hari etal., 2006). Consistent with this, SKOV-3TR and SKOV3-K cells showed increased soluble tubulin compared to parental SKOV-3 cells (Figures 2H and 2I).
TGFBI Silencing Results in Increased Mitotic Abnormalities in Cancer Cell Lines
Tight regulation of tubulin dynamics is crucial for normal completion of mitosis, and alterations in microtubules that induce taxane resistance can cause geometric deformities in the mitotic spindle (Gonalves etal., 2001; Kline-Smith and Walczak, 2004). Mitotic cells from SKOV-3TR had a significantly higher proportion of abnormal mitosis compared to control SKOV-3 cells (14% versus 1.9%, respectively; p < 0.001, two-way ANOVA) (
Figure3C). To confirm that loss of TGFBI was sufficient to induce mitotic abnormalities, we examined the effects of TGFBI knockdown on mitosis in SKOV3-K and SKOV3-A cells. Strikingly, abnormal mitotic figures were observed including monopolar spindle formation, multiple centrosomes with multipolar spindles (Figure3A), and abnormal spindle architecture (Figure3B) in stably transfected cells 930 days following transfection. Control cells transfected with empty vector and examined in parallel at the same time points did not show significant mitotic abnormalities (Figure3C). In addition, interphase cells showed a significant increase in the proportion of cells with centrosome amplification (Figure3D). These phenotypes were also observed 48 hr after transient TGFBI-KD of HeLa, 1847, and OVCAR3 cell lines and also in SKOV-3 using A810 and K1318 constructs, although lower proportions of abnormal mitoses were seen (Figure3E and data not shown).
To examine whether direct colocalization of TGFBI with microtubules or centrosomes could explain these findings, we characterized the subcellular localization of carboxy-terminal tagged TGFBI (with green fluorescent protein or myc epitopes). Tagged-TGFBI localized in the Golgi apparatus and in intracellular vesicles along microtubule fibers and accumulated at cellular protrusions consistent with secretion (FigureS4). No tagged-TGFBI was seen in the nucleus of interphase cells and there was no localization in mitosis to spindle poles, spindle fibers, or condensed chromosomes.
rTGFBI Protein Promotes Cell Adhesion and Microtubule Stabilization
Integrin-mediated adhesion of cells to fibronectin induces microtubule stabilization (Palazzo etal., 2004), suggesting that ECM and paclitaxel might have additive effects on microtubules. The stabilizing effect of fibronectin requires integrin-mediated focal adhesion kinase (FAK) activation and Rho A (Palazzo etal., 2004). TGFBI is known to mediate adhesion also in an integrin-dependent manner (Billings etal., 2002; Nam etal., 2003; Jeong and Kim, 2004; Park etal., 2004). This suggested a model where TGFBI could modulate paclitaxel sensitivity via microtubule stabilizing effects (
Figure4A). rTGFBI promoted adhesion of SKOV-3 cells, and this effect was partially antagonized by pretreating cells with a blocking antibody to alphaVbeta3 integrin (Figures S5A and S5B) (p < 0.001, two-sided t test).
Figure4B shows that cells plated on rTGFBI showed a significant increase in Glu-tubulin formation indicating microtubule stabilization (Gundersen etal., 1984; Palazzo etal., 2004). In addition, adhesion of cells to rTGFBI, or fibronectin, induced phosphorylation of FAK (Figure4C). Attachment to fibronectin, but not to the integrin-independent adhesion peptide polylysine (PL), also showed Glu-tubulin formation (Figure4D). To test whether FAK was required for microtubule stabilization by rTGFBI, we knocked down FAK in SKOV-3 cells using siRNAs, and these cells showed a significant decrease in microtubule stabilization following adhesion to rTGFBI (Figure4E) (p<0.001, two-sided t test). To test whether Rho A was also required for rTGFBI microtubule stabilization, Rho A was specifically inactivated using the cell permeable inhibitor C3 toxin. This resulted in a significant decrease in microtubule stabilization following adhesion to rTGFBI (Figure4F) (p < 0.001, two-sided t test). These data confirmed that rTGFBI induced microtubule stabilization and that this required intact FAK and Rho A signaling. Extension of these experiments to nonmalignant cells using NIH 3T3 fibroblasts also showed that rTGFBI induced Glu-tubulin formation and FAK phosphorylation (FigureS5C). In addition, inactivation of Rho in NIH 3T3 cells using a dominant-negative Rho A expression construct significantly reduced the rTGFBI-mediated stabilization of microtubules (FigureS5D).
rTGFBI Protein Sensitizes TGFBI-KD Cells to Paclitaxel in an Integrin-Dependent Manner
To test whether extracellular TGFBI was able to sensitize cells to the effect of paclitaxel, SKOV3-A and SKOV3-K cells were plated on rTGFBI-coated wells. This significantly increased paclitaxel-induced apoptosis (p < 0.001, two-way ANOVA) (
Figure5A). Importantly, KD of TGFBI significantly reduced the slope of the dose response curve for paclitaxel-induced Glu-tubulin formation (KD cells slope 1.3, 95% C. I. 0.62.1 versus SKOV-3 control cells slope 2.8, 95% C. I. 2.23.4) (Figures 5B and 5C). Plating KD cells on rTGFBI restored the slope of Glu-tubulin formation (slope 2.4, 95% C. I. 1.63.1) (Figures 5B and 5C and FigureS5E). SKOV-3TR cells plated on rTGFBI and treated with paclitaxel showed only a modest increase in apoptosis (data not shown), which we attributed to lower intracellular paclitaxel concentrations (FigureS2A). Increasing the level of intracellular paclitaxel by inhibiting paclitaxel efflux using verapamil significantly increased the sensitizing effect of rTGFBI in SKOV-3TR (Figure5D). Verapamil also increased formation of stable microtubules in SKOV-3TR cells plated on rTGFBI following treatment with paclitaxel (Figure5B). Control SKOV-3 cells plated on rTGFBI without paclitaxel treatment did not show increased caspase 3/7 activity (data not shown). We then tested whether rTGFBI was able to sensitize other paclitaxel-resistant ovarian cancer cells that had not been selected in vitro for resistance (see also FigureS1B). Figures 5E and 5F show that rTGFBI significantly increased paclitaxel-induced caspase activation in A2780 and PE0188 cells (p < 0.001, two-way ANOVA).
Incubation of TGFBI-KD cells in serum-free media conditioned with rTGFBI also resulted in a significant reversal of paclitaxel resistance (p < 0.001 one-way ANOVA) (Figure5G and FigureS5F). To test whether this sensitization was integrin dependent, we pretreated SKOV3-K cells with anti-alphaVbeta3 blocking antibody before exposure to rTGFBI-conditioned media. This pretreatment significantly reduced paclitaxel-induced apoptosis (p < 0.001, one-way ANOVA), confirming that rTGFBI binding to alphaVbeta3 was required for paclitaxel sensitization (Figure5G and FigureS5F). To examine whether microtubule stabilization was required for rTGFBI-mediated paclitaxel sensitization, we blocked Rho and FAK pathways downstream of rTGFBI-integrin binding. Pretreatment with C3 toxin of SKOV3-KD cells for 4 hr before paclitaxel treatment decreased the sensitizing effect of rTGFBI (Figure5G and FigureS5F). Similarly, transient knockdown of FAK using a single siRNA blocked the sensitizing effect of rTGFBI in Figure5G and FigureS5F.
FAK induces proliferation and survival in cancer cells, and therefore, downregulation of FAK was expected to sensitize cells to chemotherapy (Judson etal., 1999; Sood etal., 2004). However, FAK is required for ECM-induced microtubule stabilization (Palazzo etal., 2004), and our data suggested that loss of FAK might also induce paclitaxel resistance. We have recently shown using a RNAi screen for all kinases (in which, therefore, TGFBI was not included) that loss of FAK (PTK2) and its family member PTK2B induced paclitaxel resistance in A549 non-small-cell lung carcinoma and HCT116 colon cancer cells, respectively (Swanton etal., 2007). Transient knockdown of FAK in SKOV-3 and breast cancer MDA-MB-231 cells (defined as paclitaxel sensitive, FigureS1B) using independent siRNAs also induced significant resistance to paclitaxel-mediated apoptosis (p < 0.001, two-way ANOVA) (FigureS5G and data not shown). In addition, inhibition of Rho A resulted in resistance to paclitaxel induced apoptosis (FigureS5H). These results show that perturbing components of pathways involved in microtubule stability is sufficient to induce paclitaxel resistance.
TGFBI Is Tightly Coexpressed with Fibronectin
In parallel with the in vitro work, we carried out a prospective randomized clinical trial (CTCROV01) specifically designed to examine the molecular response to carboplatin and paclitaxel monotherapy in patients with advanced ovarian cancer. Tumor samples were collected prior tothe start of neoadjuvant therapy and at subsequent interval debulking surgery (Supplemental Data and FigureS6A).
As TGFBI is an integral component of the ECM, we investigated whether TGFBI expression correlated with the expression of other ECM transcripts that signal via integrins. We performed a correlation analysis between the expression of TGFBI and all other informative genes on the array (n 3426), across all samples derived from patients treated with paclitaxel (n 20), and ranked the genes according to their level of correlation with TGFBI. Strikingly, 18 out of the 20 top ranked genes were ECM-related genes (Table S2). Notably, these genes included fibronectin (FN1) (r 0.89, Pearson correlation), collagen 5A1 (COL5A1) (r 0.83, Pearson correlation), and thrombospondin-2 (THBS2) (r 0.88, Pearson correlation). THBS1 is a homolog of THBS2 and was recently found to induce paclitaxel sensitivity through extracellular signaling (Lih etal., 2006). There was a significantly positive correlation between the probability of a gene being ECM-related and its degree of coexpression with TGFBI (r 0.89, Pearson correlation; p < 0.001, linear regression) (
Figure6A).
To confirm this finding in external data sets, we analyzed published expression microarray data from ovarian and breast cancers (Spentzos etal., 2005; Naderi etal., 2007). Coregulation of TGFBI with ECM genes was reproduced with striking similarity (Figure6B and data not shown). Importantly, the top ranked coexpressed genes in these independent sets consistently included fibronectin and THBS2. THBS1 was also highly ranked in the Spentzos ovarian cancer data set (data not shown).
TGFBI Is Underexpressed in Paclitaxel-Resistant Primary Ovarian Carcinoma Tissues
To determine whether TGFBI expression was associated with clinical paclitaxel resistance, we analyzed microarray expression data from twenty patients randomized to paclitaxel monotherapy in CTCR-OV01. Patients who showed paclitaxel resistance, as indicated by CA125 monitoring, had a significantly lower expression of TGFBI (p 0.046, Wilcoxon test; 3.9% false discovery rate). RNA from 16/20 samples was available for real-time PCR. Patients who showed no paclitaxel response had a significantly lower expression of TGFBI (p 0.0087, n 16, Wilcoxon test) in their pretreatment samples compared to those who responded (Figure6C).
Centrosome Amplification Is Present in Paclitaxel-Resistant Ovarian Cancer Samples
As loss of TGFBI resulted in spindle deformities and centrosome amplification, we examined whether centrosome amplification correlated with paclitaxel resistance in clinical samples. To accurately count centrosomes in samples from the clinical study, we used three-dimensional reconstructions of confocal images from 20 m tissue sections. Adequate amounts of tumor tissue were available in 10 pretreatment samples. Reconstructed images were scored by rotating the image to count the number of centrosomes associated with each nucleus (Figure6D and Movie S1). Using this method, a total number of 2376 nuclei were examined in 10 cancer samples, and counting was performed blind to the clinical outcome of patients. We found a significantly higher (p 0.019, Wilcoxon test) proportion of cells with centrosome amplification in paclitaxel-resistant patients (7.6%) compared to paclitaxel responders (3.9%) (Figure6E).
Paclitaxel-Induced Cell Death in Ovarian Cancer Samples Colocalizes with Areas of High TGFBI Protein Expression
We next examined ovarian cancer samples taken after paclitaxel treatment from the CTCR-OV01 study using immunohistochemistry for TGFBI. Sections of ovarian cancer tissue revealed morphological changes typical of paclitaxel-induced cytotoxicity, including cytoplasmic vacuolation and multinucleation (Seiler etal., 2004), and these changes were localized to areas of high TGFBI staining (Figure6F and Figures S6B and S6C). TGFBI labeling was absent in the ECM around neighboring cells lacking paclitaxel-induced changes and in areas of necrosis (Figure6F and FigureS6D). Positive TGFBI staining was significantly associated with evidence of paclitaxel-induced cytotoxicity (n 14, p 0.0035, Chi square test with Monte-Carlo simulation). Importantly, focal TGFBI staining was present in pretreatment samples; however cytoplasmic vacuolation and multinucleation were not seen (FigureS6E).
Discussion
Our results suggest that the ECM protein TGFBI modulates paclitaxel response via regulation of microtubule stability. Acetylated and detyrosinated microtubules define a stable subpopulation of microtubule polymers that resist depolymerization (Gundersen etal., 1984). This population is less dynamic compared to unstable microtubules and, therefore, less likely to contribute to the proportion of depolymerized tubulin in the cell (
Figure7). As paclitaxel primarily targets polymerized microtubules, an increase in the proportion of unstable microtubules induces paclitaxel resistance (Gonalves etal., 2001; Barlow etal., 2002; Hari etal., 2006). We show that extracellular TGFBI stabilized microtubules and increased sensitivity to paclitaxel. Conversely, selective loss of TGFBI by KD resulted in mitotic spindle abnormalities and paclitaxel resistance. These findings mimic previous results from cell lines with intracellular alterations of tubulin that also caused paclitaxel resistance and mitotic instability (Gonalves etal., 2001; Martello etal., 2003). TGFBI was originally identified as induced by TGF stimulation in adenocarcinoma cells (Skonier etal., 1992), and our results may explain how TGF induces microtubule stabilization in serum-starved fibroblasts (Gundersen etal., 1994). Recombinant TGFBI induced microtubule stabilization that was dependent on integrin-mediated FAK and Rho signaling. The exact mechanisms of microtubule stabilization downstream of FAK and Rho remain unknown but may include mDIA1 or inactivation of the microtubule associated protein stathmin (Palazzo etal., 2004; Baldassarre etal., 2005).
The relationship between ECM proteins and drug resistance is likely to be complex. Previous studies have shown that ECM proteins induced resistance to DNA-damaging drugs such as cisplatin and etoposide, and decreased apoptosis was associated with 1-integrin-mediated activation of PI3K and AKT (Sethi etal., 1999; Hodkinson etal., 2006). In contrast, the ECM protein THBS1 sensitizes pancreatic cancer cells specifically to taxane-induced apoptosis in a CD47-dependent manner (Lih etal., 2006). Here, we show that TGFBI is tightly coexpressed with THBS1 in ovarian and breast cancers. It is possible that the two proteins may cooperate to induce taxane sensitization through activating CD47-driven pathways (THBS1) and integrin-mediated microtubule stabilization (TGFBI). The finding that the protein expression ofTGFBI in tissue samples treated with paclitaxel monotherapy, specifically colocalized with areas of taxane-induced cytotoxicity suggests that TGFBI, and possibly other ECM proteins, modulate taxane effects in vivo. Our data extend previous reports and suggests that the specific molecular targets of individual drugs are likely to determine whether the effect of the ECM is agonistic or antagonistic with chemotherapy.
Importantly the findings reported here have potentially significant clinical implications. First, TGFBI protein expression is lost in a third of primary ovarian cancers (A.A.A., A.E.K.I., and J.D.B., unpublished data) and TGFBI has previously been shown to be methylated and downregulated in lung cancer (Shao etal., 2006; Zhao etal., 2006). Future studies should correlate the expression of TGFBI with taxane response to test the value of TGFBI as a predictive biomarker. Second, in ovarian cancer, FAK is overexpressed, plays a role in regulating invasion and metastasis, and is associated with poor clinical outcome (Judson etal., 1999; Sood etal., 2004). FAK also regulates tumor growth either directly, through activation of ERK-dependent pathways (Hecker etal., 2002), or indirectly through inducing angiogenesis (Sheta etal., 2000). Downregulation of FAK using siRNAs results in inhibited growth and metastasis of pancreatic cancer (Duxbury etal., 2004) and could, therefore, be attractive therapeutically. However, previous studies have demonstrated that FAK is required for adhesion-dependent microtubule stabilization (Palazzo etal., 2004). In the current study, loss of FAK or Rho A blocked microtubule stabilization and paclitaxel sensitization by rTGFBI. Importantly, and in contrast to previous publications (Halder etal., 2005), our results show that loss of FAK results in increased resistance to taxanes, and this was confirmed using independent siRNAs and experimental studies (Swanton etal., 2007). As FAK is low or absent in one-third of patients with ovarian cancer (Sood etal., 2004), it could be used as a marker to identify patients who may not benefit from paclitaxel treatment. Furthermore, although downregulation of FAK may also induce growth inhibition, our data argue against combining FAK inhibitors with paclitaxel treatment.
We describe here a mechanism of specific paclitaxel sensitization via induction of microtubule stabilization by the ECM protein TGFBI. Furthermore, we show that expression of TGFBI in ovarian and breast tumors is tightly coregulated with other ECM proteins that either induce microtubule stabilization or paclitaxel sensitization. The effectiveness of taxanes in improving survival in ovarian cancer remains controversial (International Collaborative Ovarian Neoplasm Group, 2002), and TGFBI could be used as a biomarker for selecting patients for taxane therapy. In addition, as TGFBI is an ECM protein, activating peptides or antibodies that mimic its action may be a strategy for modulation of response to taxane chemotherapy.
Experimental Procedures
Cambridge Translational Cancer Research Ovary 01 Study
The details of this study are described in Supplemental Experimental Procedures. The study was approved by the Cambridge Local Research Ethics Committee (LREC). All patients gave written informed consent prior to participation.
Cell Culture
Cell lines were obtained from Cell Services (Cancer Research UK London Research Institute) and were maintained in RPMI 140 medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin and incubated at 37C and 5% CO2. SKOV-3TR cells were maintained in 0.3 M paclitaxel.
Apoptosis Assays, Annexin V Binding, and Flow Cytometric Analyses
Cells were grown in 6-well plates to 80% confluence before paclitaxel, nocodazole, cisplatin, or verapamil treatment (Sigma). For rTGFBI pretreatment experiments, adherent cells in 24-well tissue culture plates were incubated with rTGFBI at 50 g/ml in serum-free media (SFM) for 2 hr followed by paclitaxel treatment for 1 hr to make up the required concentrations. Cells were then washed with PBS and incubated with normal media for 48 hr. For integrin-blocking experiments, attached cells were incubated with the mouse monoclonal antibody anti-alphaVbeta3 (Chemicon) for 1 hr (1 in 100 dilution) in SFM. Rho A inhibition in SKOV-3 cells was achieved by treating attached cells with 2g/ml cell permeable C3 transferase recombinant protein (Cytoskeleton) in SFM for 4 hr prior to paclitaxel treatment for 1 hr. Early apoptosis was estimated using apoptosis detection kit (R & D systems) and 7-AAD (Molecular Probes) following the manufacturers' instructions and as previously described (Wang etal., 1998). Flow cytometric analysis was performed with a LSR II flow cytometer (BD bioscience) and analyzed with the FACSDiva software (BD bioscience).
Caspase 3 and 7 Assays
We first confirmed the optimum number of cells and linear range for paclitaxel dose response for the assay. Cells were plated at 1 104 cells per well in a 384-well plate in 20 l volume overnight before paclitaxel was added in 10 l volume to reach the final indicated concentrations. Caspase 3/7 activity was estimated 48 hr following treatment by adding 30 l of the Caspase-Glo 3/7 Assay reagent (Promega). Luminescence was read following at least 1 hr of incubation on a luminescence plate reader (Infinite M200, Tecan) using the i-control software (Tecan).
Acknowledgments
This work was supported by Cancer Research UK (CR-UK) and the Medical Research Council (MRC). A.A.A. and C.S. hold CR-UK Clinician Scientist Fellowships. C.B. was funded by the National Translational Cancer Research Network (NTRAC). N.G.I. was a recipient ofa National Medical Research Council (Singapore) Medical Research Fellowship. We thank our patients and members of the Gynaecological Oncology Multidisciplinary Team at Cambridge University Hospitals NHS Foundation Trust for their participation in the CTCR-OV01 clinical study. We thank Drs. Shin-Ichi Ohnuma, Daniel F. Schorderet, and Ching Yuan for gifts of reagents; Dr. Andrew E. Teschendorff for sharing unpublished data; and Lysa Baginsky and John Brown for expert technical assistance. We are also grateful to Drs. Fanni Gergely, Masashi Narita, and Natalie Thorne for helpful discussions.
Supplemental Data
The Supplemental Data include Supplemental Experimental Procedures, five supplemental figures, two supplemental tables, and one supplemental movie and can be found with this article online at http://www.cancercell.org/cgi/content/full/12/6/514/DC1/.
Supplemental Data
Document S1. Supplemental Experimental Procedures, Five Supplemental Figures, and Two Supplemental Tables
Movie S1. Three-Dimensional Reconstruction and Rotation Is Required for Accurate Counting of Centrosomes
Three-dimensional (3D) reconstruction and rotation of Z stack images obtained following double immunofluorescent staining of a representative ovarian carcinoma tissue section. Reconstructed images were scored by rotating the image to count the number of centrosomes associated with each nucleus. Anti- tubulin, green; chromosomal DNA, Hoechst 33258 red.
Accession Numbers
The microarray data tables are available from the Gene Expression Omnibus (GEO) at http://www.ncbi.nlm.nih.gov/geo/. Series numbers, GSE2627 and GSE9455 for cell lines and CTCR-OV01 study, respectively.
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Boyd
T.A.
Libermann
S.A.
Cannistra
Unique gene expression profile based on pathologic response in epithelial ovarian cancer
J. Clin. Oncol.
23
2005
7911
7918
Swanton etal., 2007
C.
Swanton
M.
Marani
O.
Pardo
P.H.
Warne
G.
Kelly
E.
Sahai
F.
Elustondo
J.
Chang
J.
Temple
A.A.
Ahmed
Regulators of mitotic arrest and ceramide metabolism are determinants of sensitivity to paclitaxel and other chemotherapeutic drugs
Cancer Cell
11
2007
1
15
Tan etal., 2002
M.
Tan
T.
Jing
K.H.
Lan
C.L.
Neal
P.
Li
S.
Lee
D.
Fang
Y.
Nagata
J.
Liu
R.
Arlinghaus
Phosphorylation on tyrosine-15 of p34(Cdc2) by ErbB2 inhibits p34(Cdc2) activation and is involved in resistance to taxol-induced apoptosis
Mol. Cell
9
2002
993
1004
Taxman etal., 2003
D.J.
Taxman
J.P.
MacKeigan
C.
Clements
D.T.
Bergstralh
J.P.
Ting
Transcriptional profiling of targets for combination therapy of lung carcinoma with paclitaxel and mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor
Cancer Res.
63
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Ueta etal., 2001
E.
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T.
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T.
Osaki
Mn-SOD antisense upregulates in vivo apoptosis of squamous cell carcinoma cells by anticancer drugs and gamma-rays regulating expression of the BCL-2 family proteins, COX-2 and p21
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94
2001
545
550
Wang etal., 1998
T.H.
Wang
H.S.
Wang
H.
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P.
Giannakakou
J.S.
Foster
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J.
Wimalasena
Microtubule-interfering agents activate c-Jun N-terminal kinase/stress-activated protein kinase through both Ras and apoptosis signal-regulating kinase pathways
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273
1998
4928
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Y.
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S.
Veeraraghavan
F.
Cabral
Intra-allelic suppression of a mutation that stabilizes microtubules and confers resistance to colcemid
Biochemistry
43
2004
8965
8973
Zhang etal., 1998
C.C.
Zhang
J.M.
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E.
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W.N.
Hait
The role of MAP4 expression in the sensitivity to paclitaxel and resistance to vinca alkaloids in p53 mutant cells
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16
1998
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Y.
Zhao
M.
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T.K.
Hei
Loss of Betaig-h3 protein is frequent in primary lung carcinoma and related to tumorigenic phenotype in lung cancer cells
Mol. Carcinog.
45
2006
84
92
CCELL
833
S1535-6108(07)00338-8
10.1016/j.ccr.2007.11.014
Elsevier Inc.
Article
The Extracellular Matrix Protein TGFBI Induces Microtubule Stabilization and Sensitizes Ovarian Cancers to Paclitaxel
Ahmed Ashour
Ahmed
1
3
5
Anthony D.
Mills
4
Ashraf E.K.
Ibrahim
1
Jillian
Temple
1
3
Cherie
Blenkiron
3
Maria
Vias
3
Charlie E.
Massie
3
N. Gopalakrishna
Iyer
3
Adam
McGeoch
4
Robin
Crawford
5
Barbara
Nicke
6
Julian
Downward
6
Charles
Swanton
6
Stephen D.
Bell
4
Helena M.
Earl
3
Ronald A.
Laskey
4
Carlos
Caldas
2
3
James D.
Brenton
1
3
∗
james.brenton@cancer.org.uk
1
Functional Genomics of Drug Resistance Laboratory, Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
2
Breast Cancer Functional Genomics Laboratory, Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
3
Department of Oncology, Hutchison/MRC Research Centre, Hills Road, Cambridge, CB2 0XZ, UK
4
MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge, CB2 0XZ, UK
5
Gynaecological Oncology Regional Centre, Box 242, Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Hills Road, Cambridge CB2 0QQ, UK
6
Signal Transduction Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
∗
Corresponding author
Published: December 10, 2007
Summary
The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability.
CELLCYCLE
CHEMBIO
CELLBIO
KBOA0000000000096
2015-01-21T04:49:15
S300.5
S300
S1535-6108(07)00338-8
10.1016/j.ccr.2007.11.014
CCELL
1535-6108
833
FLA
NON-CRC
UNLIMITED
CRUK
2007-12-10T00:00:00Z
15356108/v12i6/S1535610807003388/main.xml
130503
MAIN
JA 5.0.2 ARTICLE
FULL-TEXT
15356108/v12i6/S1535610807003388/main.assets/gr6.jpg
146543
IMAGE-DOWNSAMPLED
15356108/v12i6/S1535610807003388/main.assets/gr4.jpg
76817
IMAGE-DOWNSAMPLED
15356108/v12i6/S1535610807003388/main.assets/gr3.jpg
51574
IMAGE-DOWNSAMPLED
15356108/v12i6/S1535610807003388/main.assets/gr2.jpg
68761
IMAGE-DOWNSAMPLED
15356108/v12i6/S1535610807003388/main.assets/gr1.jpg
92114
IMAGE-DOWNSAMPLED
15356108/v12i6/S1535610807003388/main.assets/gr7.gif
4957
IMAGE-DOWNSAMPLED
15356108/v12i6/S1535610807003388/main.assets/gr5.gif
23613
IMAGE-DOWNSAMPLED
15356108/v12i6/S1535610807003388/main.assets/gr7.sml
2418
IMAGE-THUMBNAIL
15356108/v12i6/S1535610807003388/main.assets/gr5.sml
2991
IMAGE-THUMBNAIL
15356108/v12i6/S1535610807003388/main.assets/gr6.sml
7660
IMAGE-THUMBNAIL
15356108/v12i6/S1535610807003388/main.assets/gr4.sml
4736
IMAGE-THUMBNAIL
15356108/v12i6/S1535610807003388/main.assets/gr3.sml
4557
IMAGE-THUMBNAIL
15356108/v12i6/S1535610807003388/main.assets/gr2.sml
6451
IMAGE-THUMBNAIL
15356108/v12i6/S1535610807003388/main.assets/gr1.sml
5087
IMAGE-THUMBNAIL
15356108/v12i6/S1535610807003388/main.assets/mmc2.mov
1495787
VIDEO
15356108/v12i6/S1535610807003388/main.assets/mmc1.pdf
1946317
APPLICATION
15356108/v12i6/S1535610807003388/main.pdf
1600584
MAIN
1.4
DISTILLED OPTIMIZED BOOKMARKED
15356108/v12i6/S1535610807003388/main.raw
59328
S1535-6108(07)X0074-6
CCELL
1535-6108
12
6
20071206
493
586
S1535-6108(07)00338-8
10.1016/j.ccr.2007.11.014
514
527
main.pdf
PDF
1.4
local
1427973328896
collectiebehoudsniveau 1
2015-04-01T15:03:34.103+02:00
local
1427973329408
0
SHA-512
065a671906c4b5b9da7156fb44a691b37020578d484fce130ed54c8bc62f5e60619f81666e4c0929835a0139af30f1eb8f30d6bc18aca6abf9a90864cf0b0eda
java.security.MessageDigest
16243
not checked
metadata.xml
local
1427973329409
0
SHA-512
3c0c8ee5460ddb5d2455f20bda708c1bbb4a1f8abcf546c77e0b3c3a90b53423b98cad1e6fd0e4ea8254625feb2d1bf279ff07e33c7a3dd3ec23da97a3c7c212
java.security.MessageDigest
2418
not checked
gr7.sml
local
1427973329410
0
SHA-512
7355dcb444c29b4ea821979d2266e72e6257524a39c2faced8ca07fe6bba34cfb316364eb79d2788fde53941facf3f122ecfd072f3578a03848b33563efcc344
java.security.MessageDigest
76817
not checked
gr4.jpg
local
1427973329411
0
SHA-512
b30a0429c8d3e072f46e1a7f5f7b444d00bc389e2874bc74e700879eb627bdc0783bb1cee61694985192d4a0541a67fcd0ff8c463ae17ca4d19150cf13cdfc2c
java.security.MessageDigest
5087
not checked
gr1.sml
local
1427973329412
0
SHA-512
fcc8a2b354fe23d7acebb13ff0b8da29dad970010fe960c7b87e6999c5f32415876fffcd7cfab9eb2871197ae595f2719ecc380bc407c7fe70337322043f7475
java.security.MessageDigest
68761
not checked
gr2.jpg
local
1427973329413
0
SHA-512
925a25aa9715c17a09ad6a2ac0f3d5f845ef888f9b32041b2fe823ad7107b62a4e99f74317607e5412f68b80cb559f01cac9298e2b3b146593282aa74b0974ba
java.security.MessageDigest
51574
not checked
gr3.jpg
local
1427973329414
0
SHA-512
2731db355d5ea4ce81ff43e8cb15db4a1a260c897e3a2b7f510c7881dec33e0e951d6ce8336ec563ad4a1312f47441660f9a72e207a8f64c4883a0b584cb92ce
java.security.MessageDigest
7660
not checked
gr6.sml
local
1427973329415
0
SHA-512
50ad2ff1ca0b5871b0100a625d2f33d16a80981168424a5a38c3402b01ca024b8bcd9e7604d4b1373e2ec1cd7e950f141546cc49775cec36e2eb6ec730341bc0
java.security.MessageDigest
92114
not checked
gr1.jpg
local
1427973329416
0
SHA-512
6495253372cef2ae9a3e4d38c4de99a0d54cf8e2e89b9e369973e2162a5602b798d59a255611bd6d8ecd2ec658aa90a02cce6848ebc72f86421cb78cbd5c71fb
java.security.MessageDigest
1495787
not checked
mmc2.mov
local
1427973329417
0
SHA-512
077b794ad3043bb6795693834a3a047017b634c6f8e825b727662f62f64ac9bb4090539223f8d24c9da2b3a65ce6af0899db64d34bc987c31f281069ba036507
java.security.MessageDigest
4957
not checked
gr7.gif
local
1427973329418
0
SHA-512
b40bcfe37a1617bf862ebd8cec52afd5f4d18262870d5317d39f630f542cdac9c46799bf90b68de1844d2fe2a83186a9babcbe83b414e54ed31c2652418881e9
java.security.MessageDigest
6451
not checked
gr2.sml
local
1427973329419
0
SHA-512
7b0b3aa5446eee869147abe890ab7b4e6460a88a0625b7a2c0c5c3177eadc1280413c7dcddf1b0821c0e21383537eed7f4d75c1904dd116ee8e4546fc9580688
java.security.MessageDigest
146543
not checked
gr6.jpg
local
1427973329420
0
SHA-512
5b7809b16fa6bb144fffeaf04219990c6562e25558714ca95001d850aaa4b59b06ceec05dbb8abdb45fc9e9f7b517f4cd9b791e4a9158796e69944e5b828b937
java.security.MessageDigest
1946317
not checked
mmc1.pdf
local
1427973329421
0
SHA-512
339c76f24a3135f9074b23e275afbd2be99d32ba4c97a4b2fe47841e824311d1696f467940309485e853fa706183a9f331e9200798f7b057fb1195c9a00d7dbf
java.security.MessageDigest
23613
not checked
gr5.gif
local
1427973329422
0
SHA-512
1f7df6a5c17005db217f8033ad5a01224e54a7c819c7512c9ac8da32956157cbce46baea0168ea16b6687faaedf1d3644657ba30f8f675f45113278f1a63eacd
java.security.MessageDigest
2991
not checked
gr5.sml
local
1427973329423
0
SHA-512
6f6d08e5976004bfd27094f0b81e83fbabea28860c138c88ce3cb69342d3440fa2706ccda45b27ddf6c52f825385d8acd0fee52783c3cde1bbe1e9a5f9093112
java.security.MessageDigest
4557
not checked
gr3.sml
local
1427973329424
0
SHA-512
ae67c52ad581fe55ef71de4fe4859a7ac31cc2d8fab7f62b251f7937ebe22dfbf6a68df87b5c9e5f4c785d27cec04a40707953079de45ad73139902103d58348
java.security.MessageDigest
4736
not checked
gr4.sml
local
1427973329425
0
SHA-512
875d8c1cdd5a07d5118b47d1dc24db61f9c6e2f82273ea9b994242acb7f5671f9f9fd193b7255f0c30cc20e7332b1e72484f39fd42118ef9aad190f1763ac452
java.security.MessageDigest
59328
not checked
main.raw
local
1427973329426
0
SHA-512
55078e12ed001fa016e270befbfa84ec2115de61594dbb8fdcb33e55efc9b33237d121b87066a262d38f128f2537c973051b86605f27c7c1e536dfbc43218796
java.security.MessageDigest
1600584
Adobe Acrobat Document
1.4
DIAS
48
DIAS tentative identification
main.pdf
local
1427973329427
0
SHA-512
e9f5f554ee4488b4e84466d18bef625604d6b4109854943fb95cf801fba6b345af357b715b311a07e94b6aac20a1d58f90a650028144eb25b6e1a6f361f9ae12
java.security.MessageDigest
130503
not checked
main.xml
free
00001
KB-agent-id
1
supplier
KB-owner-id
00001
KB-agent-id
1
Elsevier
organization
Elsevier Inc.
ingestion
2015-04-01T15:03:34.103+02:00
Connector
software
Digitaal Magazijn release 1.5
ejournals_esp_1
streamprofile
ingestion2015-04-02T05:41:38.151+01:00Generic IngestsoftwareDigitaal Magazijn release 1.5