Tzu-Ling Cheng, Marc Symons*, Tzuu-Shuh Jou§
Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine
No. 7, Chung-Shan S. Road Taipei, 100 Taiwan
* Center for Oncology and Cell Biology, North Shore-LIJ Research Institute, 350 Community Drive, 11030, Manhasset, NY, USA
§To whom reprint should be addressed.
Tel: 8862-23123456 ext.7258 Fax: 8862-23709820
E-mail: [email protected]
Manuscript Information: 41 pages of text, and 8 figures in the paper.
Running Title: Cdc42 modulates anoikis
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ABSTRACT
Ras family small GTPases play a critical role in malignant transformation, and Rho subfamily members contribute significantly to this process. Anchorage-independent growth and the ability to avoid detachment-induced apoptosis (anoikis) are hallmarks of transformed epithelial cells. In this study, we have demonstrated that constitutive activation of Cdc42 inhibits anoikis in Madin Darby canine kidney (MDCK) epithelial cells. We showed that activated Cdc42 could stimulates the ERK, JNK and p38 MAPK pathways in suspension condition; however, inhibition of these signalling does not affect Cdc42-stimulated cell survival. However, we demonstrated that iInhibition of phosphatidylinositol 3-kinase (PI3K) pathway however abolishesd the protective effect of Cdc42 on anoikis. Taking advantage of a double regulatory expression system, we also showed that Cdc42-stimulated cell survival in suspension condition is, at least in part, mediated by Rac1. The consequence of Rac1 activation initiates aWe also provide evidence for a positive regulatory feedback loop between involving Rac1 and PI3K. In addition, we show that the survival functions of both constitutively active Cdc42 and Rac1 GTPases are abrogated by Latrunculin B, an actin filament-depolymerizing agent, implying an important role for the actin cytoskeleton in mediating survival signaling activated by Cdc42 and Rac1. Together, our results suggest indicate a role for Cdc42 in anchorage-independent survival of epithelial cells. We also propose conclude that this survival function depends on a positive feedback loop involving Rac1
and PI3K.
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Key Words: Anoikis, apoptosis, Rac1, Cdc42, PI3 kinase, Akt
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INTRODUCTION
The interaction between cells and the surrounding matrix is a major determinant of cellular behavior, modulating gene expression, cell growth and differentiation, cell migration, and overall tissue architecture [1]. Anchorage-dependent survival is also an important consequence of cell-matrix interaction [2]. Epithelial cells, endothelial cells, and muscle cells undergo programmed cell death when they are deprived of the contact with extracellular matrix [3].
Apoptosis induced by disruption of the interaction between epithelial cells and extracellular matrix has been termed as "anoikis", which means homelessness in Greek [4]. Anoikis plays an essential role in regulating tissue homeostasis in normal epithelial tissues. When keratinocytes and colonic epithelial cells migrate from the dividing basal layer toward the outer lining layer [5], the cells lose the ability to divide and eventually exofoliate from the monolayer. Anoikis also regulates involution of mammary glands [6], and is an important step in the first cavitation of embryogenesis [7]. Anoikis also modulates many pathological conditions. An important characteristic of transformed cells is the loss of anchorage-dependent growth control, thereby disrupting an essential surveillance mechanism that prevents cells from colonizing elsewhere when they are detached from their normal residence [8] [9]. The capability of escaping anoikis regulation is thus a critical step in oncogenesis.
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Initially identified as major players relaying the signalling from lipid and growth factor components in serum to actin cytoskeleton organization, Rho family GTPases have been demonstrated to regulate a large number of biological processes in response to cell-cell and cell-substratum adhesion [10, 11]. Rho family members regulate distinct actin cytoskeleton-based structures; namely, Cdc42 induces filopodia, Rac1 stimulates lamellipodia formation, while RhoA regulates the formation of stress fibers and focal adhesions [12-14]. There is considerable cross talk between members of the Rho family, the details of which appear to depend on the cell type and observation conditions [15]. Notably however, in most circumstances, Cdc42 appears to act upstream of Rac1 [14].
Rho family GTPases play an important role in cell transformation [16]. Expression of constitutively active (hydrolysis-defective) Rac1 in Rat1 fibroblasts induces serum- and anchorage-independent growth and is tumorigenic in nude mice [17]. Cdc42 regulates anchorage-independent growth and dominant negative Cdc42 N17 inhibits Ras focus formation and anchorage-independent growth [18]. We have also shown that constitutively active Rac1 protects MDCK cells from anoikis, while dominant negative Rac1 potentiates anoikis in MDCK cells [19]. In this paper, we address the role of Cdc42 in the regulation of anoikis, and examine its connection to survival signalling that is stimulated by Rac1.
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Experimental procedures
Plasmids
Coding sequences expressing constitutively active and dominant negative Cdc42 were amplified by PCR using pCMVneoMYC-Cdc42V12 and pCMVneoMYC-Cdc42N17 (gifts of Dr.
Arie Abo and Matt Hart, Onyx Pharmaceuticals) as a template respectively. The PCR primers were designed so that the amplified products were tagged with a 5' EcoRI and a 3' XhoI site. The PCR products were cloned into the EcoRI and XhoI restricted pCMV-Tag2B (Stratagene) to tag a FLAG epitope at their amino terminals, and resulted in two intermediate plasmids, pCF-Cdc42V12 and pCF-Cdc42N17. The FLAG-tagged Cdc42V12 coding sequence was released from pCF- Cdc42V12 by NotI and XhoI digestion, and cloned into similarly restricted vector, pIND(SP1), to generate pISF-Cdc42V12. By a similar strategy, pISF-Cdc42N17 was also made. pISF-Cdc42V12 and pISF-Cdc42N17 were confirmed to express FLAG-tagged constitutively active or dominant negative Cdc42 in an ecdysone responsive manner by immunoblotting and immunofluorescence after they were transiently transfected into MDCK and HEK293 cell clones which had been stably transfected with heterodimeric ecdysone receptors system [20]. pVgRXR (Invitrogen) is used to express the heterodimeric ecdysone receptors and confer zeocin (Invitrogen) resistance during drug selection. All the engineered plasmids were made according to standard molecular biological techniques and were confirmed by DNA nucleotide sequencing.
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Stable cell lines construction
Madin-Darby canine kidney (MDCK) cells were grown in DMEM containing 10% fetal bovine serum at 37oC in a humidified atmosphere containing 5% CO2. Transfection was performed using lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. Our ultimate goal is to establish dual regulatory cell lines expressing different small GTPase genes independently under tetracycline and ecdysone inducible systems. Therefore, we started with a well characterized MDCK cell line expressing dominant negative Rac1 (Rac1N17) gene under tetracycline repressible system [21, 22], and established ecdysone inducible expression system by zeocin selection and a FACS enrichment strategy [20] in this cell line which we named Rac1N17.1. We co-transfected 6 x 105 Rac1N17.1 cells in a p-35 well with 3.8 µg pISF-Cdc42V12 or pISF-Cdc42N17 respectively and 0.2 µg pUB-Bsd (Invitrogen), which carried blasticidin-S selection marker. Six hours after transfection, the cells were trypsinized and splitted onto 6 p-100 wells with the addition of 20 ng/ml of doxycycline to suppress the expression of the originally established (stably integrated) Rac1N17 transgene. The day after transfection, cell culture medium was replete with 10 µg/ml of blasticidin-S for selection of stably transfected clones. Individual candidate clones expressing FLAG-tagged Cdc42V12 or Cdc42N17 were identified by immunofluorescence and Western blotting using anti-FLAG antibody (M2) from Sigma. In parallel, empty pIND(SP1) vector was cotransfected and selected in the same way to establish mock transfectant control (used to generate data described in Figure 1C). To induce the transgene, MDCK clones expressing the mutant Cdc42 genes were added with the indicated
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concentration of ponasterone (Stratagene), and an aliquot of cells were added with the same volume of vehicle (alcohol) as a control. To establish MDCK cell line expressing constitutively active PI3K mutant in the background of tetracycline repressible Rac1N17 transgene, we followed the same transfection and selection protocol as described above except we co-transfected 6 x 105 Rac1N17.1 cells in a p-35 well with 3.8 µg plasmid expressing CMV promoter driven PI3K constitutively active mutant (p110*) and 0.2 µg pVgRXR (Invitrogen), which carried zeocin selection marker.
Antibodies and inhibitors
Immunoblotting was performed with one of the following primary antibodies: rabbit anti-Cdc42 and anti-p110 catalytic domain of PI3K (Santa Cruz), rabbit anti-total ERK p42/44, rabbit anti-phosphorylated ERK p42/44 (T202/Y204), rabbit anti-total and anti- phosphorylated JNK, rabbit anti-total and anti- phosphorylated JNK, anti- phosphorylated GSK-3 (the above antibodies were from Cell Signaling Technology), mouse anti Rac1 (UBI), anti-myc (purified from 9E10 hybridoma) or anti-FLAG tag (M2, Sigma). The secondary antibody used for Western blotting analyis was either an anti-rabbit or anti-mouse HRP-linked antibody at 1:3000 dilution (Amersham). The following small molecule inhibitors were dissolved in DMSO and used for dissecting Cdc42V12 mediated survival signalling pathway taken by suspended cells: MEK inhibitor U0126 (Promega), JNK inhibitor SP600125 (Tocris), p38 inhibitor SB203580, PI3K inhibitor LY294002 (Calbiochem), and Latrunculin B (Calbiochem). When above inhibitors were
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used, the same volume of DMSO was also added to the control wells.
Western blot analysis
Adherent or suspended cells were washed in PBS, and cell extracts were prepared by lysing cells in boiling SDS sample buffer (2% SDS, 150 mM NaCl, 62.5 mM Tris-HCl (pH 6.8), 10%
glycerol, 50 mM dithiothreitol, 0.01% bromophenol blue). The protein samples were separated by SDS-PAGE and transferred to nitrocellulose membrane (Schleicher & Schuell) and the membranes were blocked in 5% non-fat milk in PBST (0.1% Tween-20 in PBS) followed by immunoblotting analysis. Blots were developed with ECL reagent (Amersham-Pharmacia Biotech) for Western blotting analysis of total lysate or SuperSignal West Femto substrate (Pierce) for GST-PAK pull-down assay.
P38 and PI 3-kinase activity assay
Immobilized phospho-p38 (Thr180/Tyr182) monoclonal antibody (Cell Signaling
Technology) and immobilized phospho-Akt (Ser473) monoclonal antibody (1G1) (Cell Signaling
Technology) were used to determine p38 and Akt activities of MDCK cells in suspension.
Extracts were made from 6 x106 cells that were induced to express transgenes for 24 hours under
adherent condition and further cultured in suspension for 2 hours. The cells were then extracted
using lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton, 2.5
mM sodium pyrophosphate, 1 mM β-glycerol phosphsate, 1 mM Na3VO4, and 1 µg/ml leupeptin)
supplied by the manufacturer. Immunoprecipitation of activated p38 and Akt were carried out
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overnight and for 3 hours at 4oC respectively. Immunoprecipitates of activated Akt were washed
twice with lysis buffer and twice with kinase buffer (25 mM Tris pH 7.5, 5 mM β-glycerol
phosphsate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2). Then kinase reactions were carried
out at 30oC for 30 minutes in the presence of 200 µM ATP and 1 µg of GSK-3 to assay PKB
activity. Reactions were terminated by adding 2X SDS sample buffer and the boiled samples were
loaded onto a SDS-PAGE gel. Immunoprecipitates of activated p38 were washed three times with
lysis buffer and 2X SDS sample buffer was added before loading onto a SDS-PAGE gel. Western
blotting analysis was processed as described above and incubated with rabbit anti-phosphorylated
GSK-3 or anti-phosphorylated p38 antibody to assess the activities of Akt and p38 respectively.
p38 activity assay
Immobilized phospho-p38 (Thr180/Tyr182) monoclonal antibody (Cell Signaling
Technology) was used to determine p38 activities of MDCK cells in suspension. Extracts were
made from 6 x106 cells that were induced to express transgenes for 24 hours under adherent
condition and further cultured in suspension for 2 hours. The cells were then extracted using lysis
buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton, 2.5 mM sodium
pyrophosphate, 1 mM β-glycerol phosphsate, 1 mM Na3VO4, and 1 µg/ml leupeptin) supplied by
the manufacturer. Immunoprecipitation of activated p38 was carried out overnight at 4oC.
Immunoprecipitates of activated p38 were washed three times with lysis buffer and 2X SDS
sample buffer was added before loading onto a SDS-PAGE gel. Western blotting analysis was
processed as described above and incubated with rabbit anti-phosphorylated p38 antibody to
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assess the activities of p38.
Akt activity assay
Immobilized phospho-Akt (Ser473) monoclonal antibody (1G1) (Cell Signaling
Technology) was used to determine Akt activities of MDCK cells in suspension. Extracts were
made from 6 x106 cells that were induced to express transgenes for 24 hours under adherent
condition and further cultured in suspension for 2 hours. The cells were then extracted using lysis
buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton, 2.5 mM sodium
pyrophosphate, 1 mM β-glycerol phosphsate, 1 mM Na3VO4, and 1 µg/ml leupeptin) supplied by
the manufacturer. Immunoprecipitation of activated Akt were carried out for 3 hours at 4oC.
Immunoprecipitates of activated Akt were washed twice with lysis buffer and twice with kinase
buffer (25 mM Tris pH 7.5, 5 mM β-glycerol phosphsate, 2 mM DTT, 0.1 mM Na3VO4, and 10
mM MgCl2). Then kinase reactions were carried out at 30oC for 30 minutes in the presence of 200 µM ATP and 1 µg of GSK-3. Reactions were terminated by adding 2X SDS sample buffer and the
boiled samples were loaded onto a SDS-PAGE gel. Western blotting analysis was processed as
described above and incubated with rabbit anti-phosphorylated GSK-3 to assess the activities of
Akt.
Immunofluorescence mMicroscopy
The procedure for morphological studies was the same as the one published previously [21].
The mouse anti-FLAG antibody (M2, Sigma) was used at 1 ng/ml for indirect immunofluorescence.
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Anoikis induction and DNA fragmentation assays.
Mutant GTPases expressing MDCK cells were induced to express transgenes by adding the indicated concentration of ponasterone (for ecdysone inducible transgenes) or removing doxycycline (for tetracycline repressible transgenes) for 24 hours as a monolayer and subsequently trypsinized and cultured in suspension on ultra low attachment plates (Costar) at a density of 5 x104 cells/ ml for 16-18 hours. Cells were then processed for assessing the level of DNA fragmentation using the Cell Death ELISA kit (quantifying histone-associated DNA fragments) using the protocol suggested by the manufacturer (Roche Molecular Biomedicals). Lysates assayed were equivalent to 2.5 x104 cells. Each condition contained at least four independent samples. Results were representative of at least three independent experiments, and shown as the means with S.E.M.
Cell survival assay
10,000 MDCK cells expressing Cdc42V12 and control cells in 200 µl of culture medium
were maintained in suspension condition for 18 hours. Then, 40 µl of a modified MTT reagent,
MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
mixed with an electron coupling reagent (phenazine ethosulfate) from Promega, was added to the
well with gentle pipetting. After incubation in the incubator at 37oC for 60 minutes, absorbance at
490 nm was recorded. In the meantime, 10,000 control cells were detached from plastic dish and
immediately processed for similar assay. Results were representative of three independent
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experiments, and shown as the means with S.E.M
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Rac1 activation assay
The procedure of preparing GST-PAK fusion protein and processing cellular lysates to perform the pull down assay is essentially as described before [23]. In brief, the p21-binding domain (PBD) of p21-activated kinase 1 (PAK1) was fused with glutathione-S-transferase to make a recombinant protein (GST-PBD). The GST-PBD fusion protein was then expressed, purified, and coupled to glutathione-sepharose beads (Amersham-Pharmacia Biotech). One twentieth of the clarified cell lysate was immunoblotted for the corresponding GTPase specific antibody to confirm the presence of equal loading, while the rest of the lysate was then incubated for 60 min at 4°C with 40µg GST-PBD coupled to glutathione-sepharose beads for pull down assay.
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