tgf-b signaling pathway inactivation and cell cycle deregulation in the development of gastric...

8/2/2019 TGF-b Signaling Pathway Inactivation and Cell Cycle Deregulation in the Development of Gastric Cancer Role of th

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TGF- b signaling pathway inactivation and cell cycle deregulationin the development of gastric cancer: Role of the b-spectrin, ELF

Sang Soo Kim a,1 , Kirti Shetty a,1 , Varalakshmi Katuri a, Krit Kitisin a , Hye Jung Baek a ,Yi Tang a , Blair Marshall a , Lynt Johnson a, Bibhuti Mishra a , Lopa Mishra a,b, *

a Laboratory of Cancer Genetics, Digestive Diseases, and Developmental Molecular Biology, Department of Surgery, Medicine,Lombardi Cancer Center, Georgetown University, Washington, DC, USA

b Department of Veterans Affairs, Washington, DC, USA

Received 18 March 2006Available online 19 April 2006

Abstract

We have shown that loss of ELF, a stem cell adaptor protein, disrupts TGF- b signaling through Smad3 and Smad4 localization.Notably elf +/ /smad4 +/ mice develop gastric cancer presenting this as an important model for analyzing molecular event in gastric car-cinogenesis. To gain further insight into the functional role of ELF in gastric cancer suppression, we carried out a detailed character-ization of cell cycle events leading to gastric tumorigenesis. elf / cells and elf +/ /smad4 +/ mice demonstrate a marked alterationof cell cycle regulators, such as Cdk4, K-Ras, and p21. Levels of Cdk4 increased compared to normal controls, suggesting loss of ELF results in functional abnormalities in cell cycle regulation. We further demonstrate that the elf / MEFs show a disruption of G1/S cell cycle transition and a signicant reduction in senescence. Thus, in response to ELF deciency, the abnormalities of G 1/S check-point and senescence contribute their increment of susceptibility to malignant transformation.

2006 Published by Elsevier Inc.

Keywords: ELF; Gastric cancer; Smad4; Cell cycle; Senescence

Gastric carcinoma is a leading cause of cancer-relateddeaths worldwide. With approximately 755,000 new casesdiagnosed each year, it ranks second in incidence only tolung cancer. Most gastric cancers are diagnosed at anadvanced stage, contributing to a dismal 5-year survivalrate of under 20% [13]. The factors that govern progres-sion from gastric epithelial cell hyperplasia through dyspla-

sia to in situ carcinoma and invasive disease remain poorlyunderstood and are thought to involve multiple pathways.Helicobacter pylori is one of the most prominent reasons

in gastric oncogenic activation by the cytotoxic-associatedantigen A (cag A), which is known to bind signaling mole-cules such as erb2, c-met, and zo-1. Infection with H. pylori

perturbs several signaling pathways thus promoting gastriccarcinogenesis [4]. Germ line mutations in E-cadherin hasbeen one of the rst few genes clearly implicated in diffusetype of gastric cancer. Subsequently oncogenic activationof b-catenin (1727%) in the differentiated type, K-Ras(up to 18%) in diffuse and differentiated types, as well asamplication of c-erbB2 or c-met in approximately 10%

of both cancer types have been identied. Among tumorsuppressors, APC mutations although frequent in gastricadenoma are rarely found in gastric cancers. Similarly,mutations in p16 and p53 are seen in up to 20% of cases.Microsatellite instability (MSI) is present in up to 33% of gastric tumors [5], and is in turn associated with more fre-quent frameshift mutations of TGF RII, BAX, andhMSH3 genes [6]. Other involved molecules are CD-44,c-erb2, tie-1, c-met, and more recently, Smad4 [7].

Recent genetic studies using mouse knockouts havedemonstrated a strong role for the TGF- b signaling

0006-291X/$ - see front matter 2006 Published by Elsevier Inc.

doi:10.1016/j.bbrc.2006.03.236

* Corresponding author. Fax: +1 202 687 0992.E-mail addresses: [email protected] , [email protected]

(L. Mishra).1 These authors contributed equally to the work.

www.elsevier.com/locate/ybbrcBiochemical and Biophysical Research Communications 344 (2006) 12161223

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pathway in gastric carcinogenesis. The TGF- b familyencompasses a number of ubiquitous growth and differen-tiation factors. These proteins control cell fate by regulat-ing differentiation, proliferation, migration, andapoptosis. Their signaling mechanism is mediated throughtype I and II receptors, transmembrane serine/threonine

kinases, and transmitted to Smads, specic intracellularmediators. Activation of Smads results in nuclear translo-cation and activation of gene expression [810]. Vertebratespossess at least nine Smad proteins falling into three func-tional classes: (i) Receptor activated Smads (R-Smads):Smad1, Smad2, Smad3, Smad5, and Smad8; (ii) Co-medi-ator Smads: Smad4 and Smad10; and (iii) InhibitorySmads: Smad6 and Smad7. Smad2 and Smad4 are knowntumor suppressors in humans, and inactivation of Smad4 isa common aberration noted in human gastrointestinaltumors [1113]. The pleiotrophic effects of Smad proteinsand their interactions with various transcriptional factors,adaptors, and ubiquitinators. Among these, Runx tran-scriptional activators play a prominent role in suppressinggastric cancer. Runx3-null mice exhibit gastric hyperplasia,and approximately half of human cancers lack Runxexpression due to hemizygous deletion and DNA methyla-tion of the Runx promoter [14,15].

In addition, the activities of Smads can be modulated byadaptors such as embryonic liver fodrin (ELF), lamin orSmad anchor for receptor activation (SARA), as well asfunctional interactions with multiple signal transductionpathways [16,17]. These adaptor proteins play a criticalrole in localizing Smad and facilitating the functions of TGF- b . ELF is a key protein involved in endodermal

stem/progenitor cell commitment to foregut lineage [17].It is also crucial for protein sorting, cell adhesion, andthe development of a polarized differentiated epithelial cell.In prior work, we have demonstrated that TGF- b triggersphosphorylation and association of ELF, a b-spectrin, withSmad3 and Smad4 resulting in nuclear translocation [16].elf +/

mice display a phenotype similar to mice with com-pound haploinsufficiency at smad2 and smad3 loci. Disrup-tion of elf gene expression may be an important modulatorof TGF- b inactivation. An examination of elf +/ andelf +/

/smad4 +/

mice for tumor development revealed that40% of elf +/ mice developed tumors of varying etiology.This tumor incidence was comparable to that seen insmad4 +/ mice (45%). Interestingly, 90% of elf +/ /smad4 +/

double heterozygous mutants developed tumors,suggesting a cooperative interaction between ELF andSmad4 leading to enhanced tumorigenesis [18,19].

Inhibition of cell proliferation is central to the TGF- bresponse in epithelial, endothelial, hematopoietic, neural,and certain types of mesenchymal cells. Escape from thisresponse is a hallmark of many cancer cells. TGF- b caninduce anti-proliferative gene responses at any point duringthe division cycle. These responses are effective at inhibitingcell cycle progression primarily during G 1. Once a cellbecomes committed to executing DNA replication in late

G1, the division cycle will proceed for the most part unde-

terred by TGF- b until the cell enters G 1 again followingmitosis, at which point the cell cycle will arrest [20]. Themechanism of TGF- b mediated growth arrest at the G 1phase in epithelial cells includes the inhibition of cyclinD, E, and A mRNA expressions [2123], a reduction of cyclin-dependent kinase 4 (Cdk4) synthesis, and a down-

regulation of Cdk2 kinase activity [24,25]. Additionally,p21cip1 , a Cdk inhibitor, has been reported to be a potentialcellular mediator in TGF- b induced cell cycle arrest [26 28]. The induction or redistribution of these molecules byTGF- b effectively suppresses the activity of the G 1 cyclin/Cdk complex.

Epistatistically increased gastric tumors in elf +/ /smad4 +/

mutant mice present an ideal model for furtheranalysis of the genetic cooperation between ELF andSmad4. In this study, we sought to demonstrate changesin cell proliferation and expression of proteins involvedin cell cycle regulation, such as Cdk4, Ras, and p21 inelf

/ cells and elf +/ /smad4 +/ mice. Interestingly, wefound that loss of ELF upregulates the level of these pro-teins in cultured cells and hyperplastic tissues. We furtherdemonstrate that elf / MEFs show impairment of transi-tion at the G 1/S cell cycle checkpoint and a signicantreduction of senescence. Our results indicate that the lossof ELF, a modulator of the TGF- b pathway, leads toderegulation of cell cycle control and senescence. The pres-ence of a prominent gastric tumor phenotype in the elf +/ /smad4 +/

with the signicant disruption of key cell cycleregulation indicates a strong tumor suppressor role forELF and Smad4 in gastric carcinogenesis.

Materials and methods

Mouse maintenance and analysis. Mice were monitored at least twice aweek for possible symptoms related to tumor formation. At autopsy, theesophagus and stomach were bisected and attened during xation orwere expanded with xative by clamping the adjacent duodenum/esoph-agus and infusing the lumen with a syringe. They were then cleaned of blood, food, and feces, xed overnight in 10% buffered formalin, pro-cessed, and embedded in paraffin. Animal care was in accordance withinstitutional guidelines and under approved animal care protocols.

MEFs generation and culture. MEFs were derived from E14.5 embryosgenerated from intercrosses between elf +/ mice. The comparisonsbetween wild type and mutant cells were performed between littermates.To immortalize the wild type and mutant MEFs, the cells were seeded

1 106

cells in a 10-cm dish and left untouched (except for the mediumchange every 3 day) for at least a month. Individual colonies were pickedand sub-cultured sequentially through 24-well, 12-well, 6-well, and 10-cmplates.

G 1 /S cell cycle analysis. For G 1/S checkpoint analysis, MEFs plated 1day before were synchronized at G 0 by incubation for 4 days in starvationmedium containing 0.5% fetal bovine serum (FBS). The synchronized cellswere released by changing medium to complete medium containing 15%FBS. After harvesting the cells by trypsin treatment, the cells were xed by70% ethanol, stained by propidium iodide, and analyzed by using FAC-SCalibur owcytometer (BD Biosciences). Three embryos from differentlittermates representing each genotype were used. Data are represented asthe average of three experiments. The DNA contents and cell cycle of MEFs were analyzed by CellQuest (BD Biosciences).

Cell senescence analysis. MEFs were plated as 5 104 MEF cells per 1-

well chamber slide (Becton Dickinson) for 3 day, and then processed for

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acidic b-galactosidase activity. In brief, cells were washed, xed 5 min in1 mM MgCl 2, 0.5% glutaraldehyde in PBS, washed, and incubated at37 C with freshly prepared senescence staining solution: 1 mg/ml 5-bro-mo-4-chloro-3-indolyl b-D-galactoside/40 mM citric acid/sodium phos-phate (pH 6.0)/5 mM potassium ferrocyanide/5 mM potassiumferricyanide/150 mM NaCl/2 mM MgCl 2.

Histology, immunohistochemical staining, and Western blotting. Sec-tions of hyperplastic gastric tissue (5 l m thickness) were immersed inxylene to remove paraffin, then dehydrated in graded alcohol, andrinsed in PBS. Endogenous peroxide was quenched using 3% hydrogenperoxide (Sigma). Non-specic binding sites were blocked using PBScontaining 3% goat serum and 3% BSA. The sections were incubatedovernight at 4 C in a humid chamber with primary antibody. Thesections were then incubated with peroxidase-conjugated secondaryantibody (Jackson Immunoresearch Laboratories). Between reagentchanging, tissues were extensively washed by PBS with 0.1% Tween 20.The insoluble peroxidase substrate, DAB (Sigma), was added to coverthe entire tissue on the slide, and color development was monitoredunder the microscope. After rinsing in distilled water for 2 min,counterstaining was performed with modied Harris hematoxylin (Sig-ma) for 2 min followed by a rinse in distilled water for 3 min. Sectionswere dehydrated by passage through graded alcohol concentrations andnally xylene. Coverslips were mounted using DPX (Fluka) beforeobservation. Western blot analysis was carried out according to stan-dard procedures using ECL detection (Amersham). The following pri-mary antibodies were used: ELF (against residues 214) Cdk4, cyclinD1, K-Ras, p21, Smad4, b-actin (Santa Cruz), Mdm2 (Calbiochem),and p53 (Oncogen Research Products). Horseradish peroxidase-conju-gated donkey anti-rabbit or sheep anti-mouse antibodies (JacksonImmuno Research) were used as secondary antibodies.

Statistical analysis. Global v 2 test was used to test the hypothesis thatthe coefficient of each variable was equal to 0. Sample sets of data werecompared to assess the signicance. A P value


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comparing attened wild type. To see whether this chang-ing of cell shape is related with the senescence, we per-formed the X-gal stain in several different passages of MEFs ( Fig. 3). At P3 passage, statistically signicantreduced X-gal positive population of elf / MEFs indi-cates that loss of ELF prevents proper senescence, leadingto the accumulation of defective cells which are more sus-ceptible to tumorigenesis.

Loss of ELF leads to an alteration of oncogenic and cell

cycle proteins

To provide further insight into the progression totumorigenesis, we generated immortalized cell lines fromthe primary MEFs and examined the expression patternof regulatory protein by Western blot ( Fig. 4A). elf mutantcells showed a higher expression of Cdk4 (183%) andSmad4 (570%), indicating abnormal cell cycle arrest andover-compensation of the TGF- b signaling pathway,respectively, during immortalization. In order to nd outthe relationship between loss of ELF and gastric malignan-cy, we examined the expressions of ELF, p53, and p21 inhuman cancer cell lines of gastric origin ( Fig. 4B). Amongthe seven gastric cell lines, loss of ELF was found in twocell lines with stabilized but non-functional p53 which can-not transactivate the expression of p21. These results sug-gested that loss of ELF may require the inactivation of p53 pathway in the immortalization process to overcomep53-dependent tumor protection system including G 1/Scheckpoint and senescence.

To explore our hypothesis that cell cycle deregulationoccurs due to loss of ELF, we examined the expression of Cdk4 and cyclin D1, G 1/S checkpoint proteins, in hyper-plastic gastric tissue of elf +/ /smad4 +/ mice (Fig. 4C).Immunohistochemical analysis showed an increased

expression of Cdk4 and cyclin D1 proteins in the gastric

tissue of elf +/ /smad4 +/ mice compared to the wild type.These results indicate that escaping from TGF- b growthinhibition results in increased cyclin D1 and activation of Cdk4 kinase activity. Next, to examine whether thesetumors contain common genetic alterations or different sec-ondary genetic events, we analyzed the expression of Mdm2 and K-Ras. Increased expression of Mdm2 andK-Ras was seen in elf +/ /smad4 +/ gastric tissue com-pared to wild type controls. In summary, our results sug-gest that a mutation in the elf locus enables the cells to

undergo faster entry into the S-phase of cell cycle, confer-ring resistance to senescence and apoptotic stimuli and sus-ceptibility to transformation.

Discussion

The TGF- b pathway occupies a central position in thesignaling networks that control growth, differentiation,and apoptosis [23]. TGF- b induces inhibition of cell cycleprogression during the G 1 phase through the control of Cdks. In mammalian cells, tightly regulated cyclins-Cdksact sequentially during the G 1/S transition and are requiredfor cell cycle progression through this period. Loss of TGF- b responsiveness results in deregulated cellulargrowth and is believed to be a crucial step in the develop-ment of various tumors, including gastric cancer [29]. Wehave previously noted a higher rate of gastric proliferationin elf +/ /smad4 +/ mutant mice, suggesting that the dis-ruption of TGF- b signaling noted in elf -decient miceresults in inhibition of growth arrest [18]. Our preliminaryexperiments show that elf +/ /smad4 +/ and smad4 +/

mice exhibit a tendency to develop tumors as a functionof age. Importantly, smad4 +/ mice are a well-establishedmodel for gastric carcinoma. smad4 +/ mice develop fewertumors at a later time point (45% at 1214 months) than

those of elf +/

smad4+/

animals (90% at 710 months).

Fig. 2. Loss of ELF alters of G 1/S arrest of cell cycle. (A) Representative histograms of FACS showing DNA content of synchronized and released wildtype (WT) or elf / (MT) MEFs. When releasing, cells were also treated with nocodazole (100 ng/ml) to prevent the continuous cycling of cells. Graphsrepresent percentages of cells distribution in indicated stages of cell cycle in ow cytometry analysis. (B) Western blot analysis of p21 in type (WT) orelf

/ (MT).



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Fig. 3. Senescence phenotype in elf / MEFs. Photographs of acidic b-galactosidase activity staining of wild type (A) and elf / (B) MEFs in passage 3.Arrows indicate the senescence-resistance cells. (C) To provide a quantitative comparison, X-gal stained passage 1 (P1) and passage 3 (P3) of wild type(WT) or elf / (MT) MEFs were counted. Statistically signicant differences ( P > 0.01) are indicated by asterisks.

Fig. 4. Loss of ELF leads to an alteration of oncogenic and cell cycle proteins in vitro and in vivo. Protein expression patterns in immortalized MEFs (A)and cancer cell lines (B). (C) Loss of elf increase in cyclin D1, Cdk4, Mdmd2, and K-Ras in elf +/ /smad4 +/ mutant mice, as compared to wild type

controls. Arrows indicate the positive signals.

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Our present work seeks to further elucidate the contribu-tion of ELF in the enhanced susceptibility of the elf +/ /smad4 +/

mice to the development of gastric tumors anddetermine the nature of secondary events that lead totumor formation.

The mechanisms whereby TGF- b arrests the cell cycle

have been studied mainly in epithelial cells, such as minklung epithelial cells and human keratinocytes, focusing onthe regulation of G 1 cyclin-dependent kinases. In minklung epithelial cells, TGF- b treatment induces inhibitionof Cdk4 synthesis and Cdk2 inactivation and subsequentG1 arrest [30]. In HaCaT cells, TGF- b induces growtharrest through the downregulation of cell-cycle regulators,including cyclin E, cyclin A, Cdk2, and Cdk4 [31]. Severalcyclin-dependent kinase (Cdk) inhibitors have been impli-cated in TGF- b induced cell cycle arrest. TGF- b binds tocyclin D/Cdk4, consequently releasing the p27 proteinsfrom Cdk4, which then binds to Cdk2, blocking its activity[3234]. Thus, sequential action results in inhibition of thecyclin-dependent kinases, which prevent progression of theG1 phase of the cell cycle. The Cdk inhibitor p21 has alsobeen implicated in TGF- b mediated growth inhibition inmany different cell types. Expression of p21 is induced byTGF- b through the transcription factors, Sp-1 and Sp-3,thus inhibiting the kinase activity of cyclin E/Cdk2 [33].However, the fact that there exist multiple cell cycle regula-tors involved in TGF- b action prompts several questionsrelating to their actual roles in a certain cell type, whetherthey act via redundancy or in a coordinate manner, andwhether their relative importance is largely common or dif-ferent depending on cell type.

Our study specically examines the role of ELF, a mod-ulator of TGF- b signaling,in the dysregulation of cell cycleevents leading to gastric cancer. Our results demonstrate amarkedly higher expression of Cdk4 in elf /cells andelf +/

/smad4 +/ mice, implying that TGF- b induced G 1arrest has been disrupted by the loss of ELF. The expres-sion of p21 was also markedly decreased in elf / cellseven in the presence of TGF- b . This is consistent with pre-vious data in which p21 was demonstrated to be a majordownstream target of TGF- b in gastric cancer cell lines[35]. TGF- b treatment of SNU16 gastric cancer cells induc-es expression of p21 with subsequent inhibition of G 1-cyclin-associated Cdks [35]. Inhibition of G 1-Cdk enzymat-ic activities results in under phosphorylation of p130, thatbinds to transcription factors such as the E2F family.Upregulation of p21 was shown to be sufficient to arrestthe cells in G1 phase [35]. Furthermore, inhibition of p21by anti-sense RNA results in the abrogation of sensitivityto TGF- b induced growth arrest [35]. Thus loss of thep21 gene or failure of p21 induction may contribute toTGF- b resistance in gastric carcinoma cells.

Senescence is a programmed cellular response in normalcells, the induction of which depends on the accumulatednumber of cell divisions [36]. Unlike cells undergoing apop-tosis, senescent cells have a large and at morphology,

express acidic b-galactosidase, and show a permanent cell

cycle G1 phase arrest [37]. Senescent cells contain an acti-vated p53 transcription factor [38,39] and elevated levelsof p21 [40]. The elevated level of p53 in response to multi-ple stress signals results in an increase in the transcriptionof the p21 gene. The p21 protein is a general inhibitor of CDKs, which phosphorylate Rb and allow G 1 to S-phase

progression [41,42]. In addition, p21 protein also inhibitsPCNA-mediated DNA replication, a mechanism by whichp21 functions as a growth inhibitor independent of itsCDK inhibitor activity [43,44]. Taken together, theseresults indicate the possibility that the G 1/S cell cyclecheckpoints are critical controls for senescence. Our resultsdemonstrated that the loss of ELF causes reduced senes-cence, and decreases the ability to abolish defective cells,leading to an accumulation of mutated cells that are moresusceptible to tumorigenesis.

Our data suggest that loss of ELF also results in com-mon genetic alterations or a group of diverse secondarygenetic events such as stabilization of p53 and inductionof oncogenic Ras . p53 and K-Ras genes are frequentlymutated and stabilized in cancer [45,46]. K-Ras mutationsare seen up to 28% of gastric carcinomas [4749]. Ourstudy demonstrates increased expression of p53 andK-Ras in elf / cells and elf +/ /smad4 +/ gastric tissue,respectively. p21 is a downstream effector of activatedK-Ras, and downregulation of p21 in elf mutated tissuemay induce the development of tumors.

In summary, we have demonstrated abnormalities in theexpression of several cell-cycle proteins in elf / cells andthe gastric tissues of elf +/ /smad4 +/ mutant mice. Wealso demonstrate that the loss of ELF leads to the abnor-

mality of G 1/S checkpoint and senescence. This, in turn,then leads to cancer development in mutant mice. Our datasuggest that the disruptions in TGF- b signaling mediatedby the loss of ELF result in profound and previously unrec-ognized interactions with cell-cycle regulation. We are thusprovided with intriguing and potentially useful insightsinto the tumor biology of gastric cancers, and in the futuremay be able to utilize these cell cycle proteins in the earlydiagnosis and targeted therapeutics of these potentiallylethal cancers.

Acknowledgments

This work was supported by NIH Grants RO1CA106614A (L.M.), RO1 DK56111 (L.M.), RO1DK58637 (B.M.), VA Merit Award (L.M.), and R. Robertand Sally D. Funderburg Research Scholar (L.M.).

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