Skip Navigation


Carcinogenesis Advance Access originally published online on July 17, 2007
Carcinogenesis 2007 28(11):2321-2327; doi:10.1093/carcin/bgm159
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
28/11/2321    most recent
bgm159v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (16)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Chow, J. Y.C.
Right arrow Articles by Carethers, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chow, J. Y.C.
Right arrow Articles by Carethers, J. M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

RAS/ERK modulates TGFß-regulated PTEN expression in human pancreatic adenocarcinoma cells

Jimmy Y.C. Chow1, Khai T. Quach1, Betty L. Cabrera1, Jennifer A. Cabral1, Stayce E. Beck1,2 and John M. Carethers1,3,4,*

1 Division of Gastroenterology, Department of Medicine
2 Biomedical Sciences Program
3 Rebecca and John Moores Comprehensive Cancer Center, University of California, San Diego, MC 0063, 9500 Gilman Drive, La Jolla, CA 92093-0063, USA
4 VA San Diego Healthcare System, San Diego, CA 92161, USA

* To whom correspondence should be addressed. Tel: +858 534 3320; Fax: +858 534 3338; Email: jcarethers{at}ucsd.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-ß might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFß and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFß surface receptors. Cells were treated with TGFß1 and separated into cytosolic/nuclear fractions for western blotting with phospho-SMAD2, SMAD 2, 4 phospho-ATP-dependent tyrosine kinases (Akt), Akt and PTEN antibodies. PTEN mRNA levels were assessed by reverse transcriptase–polymerase chain reaction. The MEK1 inhibitor, PD98059, was used to block the downstream action of oncogenic K-RAS/ERK, as was a dominant-negative (DN) K-RAS construct. TGFß increased phospho-SMAD2 in both cytosolic and nuclear fractions. PD98059 treatment further increased phospho-SMAD2 in the nucleus of both pancreatic cell lines, and DN-K-RAS further improved SMAD translocation in K-RAS mutant CAPAN cells. TGFß treatment significantly suppressed PTEN protein levels concomitant with activation of Akt by 48 h through transcriptional reduction of PTEN mRNA that was evident by 6 h. TGFß-induced PTEN suppression was reversed by PD98059 and DN-K-RAS compared with treatments without TGFß. TGFß-induced PTEN expression was inversely related to cellular proliferation. Thus, oncogenic K-RAS/ERK in pancreatic adenocarcinoma facilitates TGFß-induced transcriptional down-regulation of the tumor suppressor PTEN in a SMAD4-independent manner and could constitute a signaling switch mechanism from growth suppression to growth promotion in pancreatic cancers.

Abbreviations: Akt, ATP-dependent tyrosine kinases; DN, dominant negative; PTEN, phosphatase and tensin homolog deleted on chromosome 10; PI3K, phosphatidylinositol-3-kinase; PCR, polymerase chain reaction; TGF, transforming growth factor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Pancreatic cancer has the highest death rate to incident ratio of cancers in the USA (1). The pathogenesis of pancreatic cancer is complex and seems to involve environmental influences and genetic alterations.

Transforming growth factor (TGF)-ß1 over-expression is associated with poor survival in patients with pancreatic cancer (2). It often suppresses early tumorigenesis but later enhances tumor progression (3–5). Although TGFß1 exerts an inhibitory effect on cancer cell growth in vitro, it also stimulates the synthesis of the extracellular matrix and has been implicated in the regulation of cell differentiation, angiogenesis, immunosuppression and fibrosis (6).

TGFß signals through a heterotetrameric receptor complex comprising type II receptors and type I receptors, both serine/threonine kinases (7), followed by a canonical SMAD-dependent signaling cascade (8). Several studies have shown that expression of dominant-negative (DN) transforming growth factor-ß receptor type 2 constructs in mice lead to enhanced mammary tumorigenesis with or without carcinogens (9–11). Moreover, results obtained from conditional ablation of SMAD4 in mouse mammary epithelium (12) confirm the importance of signaling through the TGFß/SMAD pathway during tumor initiation and progression as suggested in earlier reports of enhanced colon tumorigenesis in SMAD4-null mouse models (13,14). These effects, however, are not limited to the mammary gland and colon since DN transforming growth factor-ß receptor type 2 expression in the lung and skin also resulted in enhanced carcinogen-induced tumorigenesis (11,15).

The genetic profile of pancreatic cancer suggests contribution from both oncogenes and tumor suppressor genes. The tumor suppressor SMAD4 is deleted from >64% of pancreatic cancers (16,17), removing this TGFß signaling molecule from the majority of these tumors. K-RAS is mutated in >90% of pancreatic cancers, although the presence of oncogenic K-RAS is not specific to cancer as it can also be found in benign pancreatic lesions (18,19). Numerous reports have described a correlation between RAS transformation and deficient TGFß responsiveness, particularly with regards to TGFß anti-mitogenic responses. RAS transformation of lung, intestinal, liver or mammary epithelial cells confers resistance to growth inhibition by TGFß (20,21). Kretzschmar et al. (22) showed that oncogenic RAS inhibits the TGFß signal transduction pathway at the level of SMAD by RAS/ERK-induced phosphorylation of clusters of four Ser/Thr-Pro sites in the linker region of SMAD2 and SMAD3 and preventing accumulation of SMAD2 and SMAD3 in the nucleus and inhibiting TGFß signaling.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), also known as MMAC1/TEP1, is localized to chromosome 10q23 (23,24). It is a dual specificity phosphatase and antagonizes phosphoinositide-3-kinase (PI3K)/ATP-dependent tyrosine kinases (Akt) signaling pathway (25), and thus plays a functional role in cell cycle arrest and apoptosis (26,27). Description of germ line mutations and deletions of PTEN in two hereditary cancer predisposition diseases, Cowden Disease and the Bannayan–Riley–Ruvalcaba syndrome (28–31), point to a role of PTEN as a tumor suppressor gene in the pathogenesis of both benign and malignant growth. PTEN is one of the most frequently mutated proteins in a variety of cancers (23,24,32), but PTEN mutations rarely occur in pancreatic cancer (33). PTEN expression has been shown to be regulated by TGFß1 in keratinocytes (34), and PTEN mRNA levels were also reduced in a model of TGFß1 over-expressing transgenic mice that develop pancreatic fibrosis (35). Reduction of PTEN mRNA levels in pancreatic cancer cells following TGFß1 treatment has also been reported (35). Although PTEN is not found mutated in pancreatic cancers, the reduction of its expression may give pancreatic cells an additional growth advantage.

Our present study focused on whether TGFß modulates PTEN expression in SMAD4-null pancreatic cancer cells and whether that modulation was influenced by oncogenic K-RAS. This investigation may provide clues to the conversion of TGFß function from that of a tumor suppressor in early carcinogenesis to one of a tumor promoter in more mature cancers. Both SMAD4 loss and K-RAS activation are typical findings in pancreatic cancers. We found that TGFß reduces PTEN expression in the absence of SMAD4, and oncogenic K-RAS and downstream effectors potentiate TGFß-suppressed PTEN expression. Inhibition of K-RAS or its downstream effectors functionally ‘switched on’ PTEN expression where it may promote tumor suppression.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Materials and reagents
PD98059, a MEK inhibitor, was purchased from PharMingen (San Diego, CA). A solution of PD98059 in dimethyl sulfoxide (Sigma, St Louis, MO) was prepared and used after diluting with medium for each assay. Dimethyl sulfoxide was also used as a blank control. DN construct of K-RAS was a generous gift from Dr Rik Derynck (University of California, San Francisco, CA).

Cell cultures
BxPC-3 and CAPAN-1 cells were obtained from American Type Culture Collection and they were maintained in RPMI and Dulbecco's modified Eagle medium (Gibco BRL, Gaithersburg, MA), respectively, supplemented with 10% fetal calf serum (Gibco BRL), without any antibiotics in an incubator at 37°C and 5% CO2. To analyze the effect of TGFß1 on PTEN expression and cell proliferation, pancreatic cancer cell lines were grown to 70–80% confluency in the corresponding medium containing 10% fetal bovine serum. Afterwards, cells were washed twice in phosphate-buffered saline, starved for 30 min in serum-free medium and finally treated for 24 and 48 h with 10 ng/ml TGFß1 or medium alone.

Cell growth assay
For determination of cell number, 30 000 human pancreatic carcinoma cells per well were seeded onto six-well plates (Nunc, Wiesbaden, Germany), and incubated overnight in complete growth medium (RPMI 1640 + 10% fetal bovine serum). Complete medium was replaced with serum-depleted medium with or without TGFß and/or inhibitors. Cell numbers from each well were determined every 24 h for 3 days. Up to 12 independent experiments were performed.

Transfection of the CAPAN-1 cells
One day before transfection, exponentially growing cells were trypsinized, and 1–2 x 106 cells were plated onto 10 cm Petri dishes. Cells were then transfected the next day with the DN K-RAS construct or empty vector. The DN construct is known to suppress activated RAS by blocking RAS guanine nucleotide exchange factors (36). Transfection was carried out using Transfast (Promega, Madison, WI). Briefly, plasmid DNA was mixed with serum-free medium, followed by transfection reagent at the charge ratio of 1:1. The mixture was allowed to react for 10–15 min, and it was transferred to the cells to be transfected. One hour after transfection, complete medium was overlaid on top of the cells and allowed to incubate for 48 h.

Total RNA extraction and semi-quantative reverse transcriptase–polymerase chain reaction
Total RNA extraction from the control and TGFß-treated cells was conducted using Trizol reagent (Invitrogen Corporation, Carlsbad, CA). Cells grown on six-well plate were lysed with the reagent (1 ml per well). Lysates were combined with chloroform, mixed and the pellets precipitated with isopropanol and 75% ethanol and air-dried. Total RNA was then extracted and re-suspended in water. Two micrograms of total RNA were reversed transcribed into cDNA and amplified by polymerase chain reaction (PCR) for PTEN and SMAD4 expression (SuperScript II, Invitrogen Corporation). Briefly, following inactivation at 65°C for 10 min, 1 µl of the reaction mixture were incubated in buffer containing 0.2 mM concentrations of dATP, dCTP, dGTP, dTTP, 0.2 µM concentrations each of oligonucleotide primers, 3 mM MgCl2 and a 10x buffer consisting of 200 mM Tris–HCl (pH 8.0), 500 mM KCl and 1 U Taq polymerase. PCR primers were designed to amplify a region spanning from nucleotides 117 to 741 of the PTEN gene (forward strand, 5'-CAGAAAGACTTGAAGGCGTAT-3' and reverse strand, 5'-AACGGCTGAGGGAACTC-3'), as described previously (37). The 624 bp fragment contains an NsiI restriction site allowing to distinguish this region from the PTEN pseudogene (37). PCR primers used to amplify SMAD4 were forward strand, 5'-AACGTTAGCTGTTGTTTTTCAC-3'and reverse strand, 5'-AGAGTATGTGAAGAGATGGAG-3'. The conditions for PCR reactions of PTEN were as follows: denaturation at 95°C for 3 min and 35 cycles of 94°C for 30 s, 55°C for 30 s and 74°C for 4 min. Primers used for GAPDH were used as a loading control (forward strand, 5'-ACCACAGTCCATGCCATCAC-3' and reverse strand, 5'-TCCACCACCCTGTTGCTGTA-3').

Nuclear–cytoplasmic fractionation, western blotting analysis and immunoprecipitation
Cytoplasmic- and nuclear-enriched fractions were prepared by scraping 5 x 106 cells in 1.5 ml of hypotonic lysis buffer consisting of 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.9), 2.5 mM MgCl2, 0.2 mM ethylenediaminetetraacetic acid, 0.5 mM dithiothreitol, 40 mM NaF, 4 mM ß-glycerophosphate, 0.2% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM benzamidine HCl and 2 µg/ml each aprotinin and leupeptin. Cells suspension was maintained on ice for 20 min and inverted several times. The nuclear fraction (P1) was isolated by centrifugation of the suspension at 3500g for 5 min, followed by washing in 0.5 ml of hypotonic buffer. The supernatant, representative of cytoplasmic fraction (S1), was added with NaCl to 0.14 M final concentration and clarified by 10 min centrifugation at 10 000g. Nuclear proteins were extracted from nuclear fraction by lysis in 0.3 ml hypertonic lysis buffer (50 mM Tris–HCl, 0.14 M NaCl, 0.25 M KCl, 1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 0.5 mM dithiothreitol, plus protease and phosphatase inhibitors), followed by 1 h rotation at 4°C, and clarified by 10 min centrifugation at 10 000g. Respectively, 50 µg of cytoplasmic and 30 µg of nuclear proteins were analyzed by western blotting and probed to specific antibodies. Phospho-SMAD2, phospho-Akt, ERK1/2 and phospho-ERK1/2 1:1000 (Cell Signaling Technology, Danvers, MA); SMAD2 1:1000 (BD Transduction Laboratories, San Diego, CA); SMAD4 (1 µg/ml Upstate Biotechnology, Lake Placid, NY); Akt 1:200 and PTEN 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA); {alpha}-tubulin, histone H1 1:500 (Santa Cruz Biotechnology) and GAPDH 1:5000 (Ambion, Austin, TX) antibodies were utilized.

For total lysates, cells were washed three times with ice-cold phosphate-buffered saline, and the cells lysed using total lysis buffer (150 mM NaCl, 10 mM Tris–HCl, pH 7.8, 1 mM ethylenediaminetetraacetic acid, 0.5% Triton X-100, 1 mM sodium orthovanadate) containing protease inhibitors (1 µg/ml leupeptin and 100 µg/ml phenylmethylsulfonyl fluoride). Cells were then incubated at 4°C for 30 min with constant shaking, then scraped into microcentrifuge tubes and centrifuged at 12 000g for 15 min to remove insoluble material. The protein content in each sample was determined and adjusted. For immunoprecipitation, lysates were incubated with the immunoprecipitating antibody for 1 h at 4°C, followed by another 1 h incubation with protein A–agarose again at 4°C. Pellets were treated with 2x loading buffer (50 mM Tris, pH 6.8, 2% sodium dodecyl sulfate, 100 mM dithiothreitol, 0.2% bromophenol blue, 20% glycerol) and boiled for 5 min. Loading buffer supernatants from immunoprecipitation studies or cell lysates were then re-suspended in 2x gel loading buffer, boiled for 5 min and then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (9% polyacrylamide). Resolved proteins were transferred overnight at 4°C onto a polyvinylidene difluoride membrane (Millipore Corporate, Billerica, MA), blocked with a 5% solution of skim milk for 30 min at room temperature, followed by further incubation with monoclonal antibodies to specific antibodies. After washing with phosphate-buffered saline with 1% Tween, the secondary antibody was applied to the membrane. After washing with PBS with 1% Tween, the membrane was treated with a chemiluminescent solution according to manufacturer's instructions and exposed to X-ray film. Densitometric analysis of the blots was performed using an AlphaImager digital imaging system (Alpha Innotech Corporation, San Leandro, CA).

Data analysis
All data are expressed as the means for a series of experiments ± SEM. Data were analyzed by either one-way analysis of variance followed by Student–Newman–Keul's post hoc test or by Student's t-tests for unpaired samples using Instat 3.0 (San Diego, CA). P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
TGFß mediates SMAD2 activation and nuclear translocation in SMAD4-null and ERK activated BxPc-3 and CAPAN-1 cells
TGFß1 signaling usually requires SMAD4 together with SMAD2 activation, and nuclear translocation to transmit the TGFß1-SMAD-responsive signaling event. To assess if TGFß1 could mediate SMAD2 translocation in SMAD4-null cells, we treated BxPc-3 and CAPAN-1 cells with TGFß, and observed SMAD compartmentalization using western blots after cytoplasmic/nuclear separation. As shown in Figure 1, TGFß1 (10 ng/ml) caused activation and translocation of SMAD2 in WT-K-RAS BxPc-3 pancreatic cancer and oncogenic K-RAS CAPAN-1 cells. To address if activated RAS/ERK affected TGFß-SMAD signaling, we inhibited MEK with the pharmacologic inhibitor, PD98059, which is activated downstream of K-RAS, and inhibited K-RAS itself with a DN-K-RAS construct. Pre-treatment of CAPAN-1 cells with PD98059 or with transfection with the DN-RAS construct (Figure 2A), or pre-treatment of BxPc-3 cells with PD98059 (Figure 2B) in the presence of TGFß markedly improved SMAD2 activation and translocation. Note that SMAD4 protein was not detectable which further confirmed that the cells are SMAD4 null (Figure 1). Thus, inhibition of the RAS/ERK pathway markedly improved translocation of SMADs in pancreatic cancer cells, indicating a modulatory role by RAS on SMAD2 translocation, even in the absence of SMAD4.


Figure 1
View larger version (43K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Effects of TGFß treatment on subcellular trafficking of SMAD2 in BxPc-3 and CAPAN-1 cells. Cells were treated with or without TGFß (10 ng/ml) for 24 h. The cells were then lysed and separated into nuclear and cytoplasmic fractions. Note the appearance of phospho-SMAD2 in the nuclear and cytosolic fractions. Tubulin and histone H1 blots indicate purity of the fractions. SMAD4 is not detectable in either cell line, confirming their SMAD4-null status. T84 colon cancer cells were electrophoresed separately as a positive control for SMAD4 expression.

 


Figure 2
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Effect of RAS/ERK inhibition and TGFß treatment on subcellular trafficking of SMAD2 in CAPAN-1 and BxPc-3 cells. Cells were either pretreated with or without PD98059 (50 µM) for 1 h (A and B), or transiently transfected with a DN construct of K-RAS (A) 48 h before treatment with TGFß. Cell lysates were blotted with anti-phospho-SMAD2, tubulin and histone antibodies. Note that PD98059 and DN-K-RAS transfection improved TGFß-induced p-SMAD2 nuclear translocation. Tubulin served as a loading control and indicated purity of the cytoplasmic fractions, whereas histone served as a loading control and purity of the nuclear fractions.

 
TGFß treatment suppresses PTEN expression in SMAD4-null pancreatic cells
We examined the effects of TGFß upon expression of PTEN in SMAD4-null pancreatic cancer cells. Both BxPC-3 and CAPAN-1 cells expressed abundant PTEN mRNA as determined by semi-quantitative reverse transcriptase–PCR (Figure 3). We observed minimal reduction of PTEN mRNA levels after 3 and 6 h of TGFß1 treatment as compared with the untreated control. However, beginning at 6 h in BxPC-3 cells and by 24 h incubation with TGFß1, a significant reduction of amplifiable PTEN mRNA was observed in both pancreatic cancer cell lines that recovered to some degree by 48 h after treatment (Figure 3). To verify the PTEN mRNA findings, we then examined the steady-state levels of PTEN protein. At the protein level in BxPc-3 cells, PTEN protein was also present and unchanged from controls through 24 h after TGFß1 treatment (Figure 4A). However, PTEN protein expression was reduced by 48 h after treatment in BxPc-3 (Figure 4A) and CAPAN-1 cells (not shown, but 48 h data shown in Figure 6B), following the reduced PTEN mRNA expression at 24 h. Therefore, TGFß1 appears to mediate suppression of PTEN mRNA and protein in a SMAD4-independent manner. PTEN function antagonizes PI3K function, and one of the effectors of PI3K, Akt (25). To assess whether the TGFß-induced reduction in PTEN expression coincides with an imbalance in function of PI3K, we determined the activation of phospho-Akt at the same time PTEN protein is suppressed. As shown in Figure 4B, the suppression of PTEN by TGFß is simultaneous with Akt activation, demonstrating functional loss of PTEN.


Figure 3
View larger version (33K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. TGFß treatment down-regulates PTEN transcription. CAPAN-1 and BxPc-3 cells were treated with a single dose of TGFß (10 ng/ml), and transcription of PTEN was assessed by semi-quantitative reverse transcriptase–PCR. Cells were lysed by Trizol reagents and mRNA was extracted. PTEN mRNA is markedly reduced by 24 h, with subsequent recovery of transcription by 48 h after treatment with TGFß. Thus, PTEN is down-regulated transiently at the transcriptional level by 24 h after a single TGFß treatment. GAPDH was used as loading control.

 


Figure 4
View larger version (30K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. TGFß treatment reduces PTEN protein and activates Akt in SMAD4-null BxPc-3 cells. (A) Cell lysates were blotted with an anti-PTEN antibody. Note that PTEN protein is reduced by ~50% at 48 h after a single TGFß dose (10 ng/ml). Data shown are mean ± SEM of four experiments. *P < 0.05 versus control of 48 h. (B) The identical treatment condition in (A) also activated Akt at 48 h in BxPc3 cells. The data shown are representative of three experiments.

 
Modulation of TGFß-induced PTEN suppression by RAS/ERK constitutes an ‘on/off’ switch
As the RAS/ERK pathway is activated in pancreatic cancers, we assessed if RAS/ERK modulates the TGFß-induced PTEN suppression observed in the SMAD4-null cell lines. PD98059, the MEK inhibitor, had no effect on amplifiable, semi-quantitative PTEN mRNA alone in CAPAN-1 (Figure 5A) and had no statistical significance to the small observed increase in PTEN mRNA in BxPc-3 cells with 50 µM PD98059 (Figure 5B). Treatment with PD98059, however, rescued TGFß-induced PTEN mRNA suppression (Figure 5A and B). Similarly at the protein level, PD98059 had no effect on PTEN protein alone (Figure 6A). However, PD98059 reversed TGFß-induced PTEN protein suppression (Figure 6A). Additionally, CAPAN-1 cells transfected with DN-K-RAS and treated with TGFß for 48 hreversed the TGFß-induced PTEN suppression (Figure 6B). Thus, RAS/ERK appears to facilitate TGFß-induced PTEN suppression as blockage of ERK or RAS restores PTEN expression to pre-treatment levels. Here, RAS/ERK can ‘switch’ PTEN off through TGFß signaling when activated and restore PTEN to ‘on’ through TGFß signaling when RAS/ERK is inactivated.


Figure 5
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. PD98059 reverses TGFß-induced PTEN suppression at the transcriptional level. We treated both SMAD4-null CAPAN-1 (A) and BxPc-3 (B) cells with the MEK1 inhibitor PD98059 and performed semi-quantitative reverse transcriptase–PCR for PTEN mRNA at 24 h after TGFß treatment. PD98059 had little effect on the basal levels of PTEN mRNA (A and B, lanes 2 and 3). However, MEK inhibition rescued TGFß-induced PTEN transcriptional suppression in a dose-dependent manner (A and B, lanes 5 and 6). Thus, by blocking MEK/ERK activation, downstream of K-RAS activation, PTEN transcription can be switched back ‘on’ in the presence of TGFß treatment. Data shown are means ± SEM of three to four experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus control; {dagger}{dagger}P < 0.01 and {dagger}{dagger}{dagger}P < 0.001 versus TGFß treatment alone.

 


Figure 6
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. RAS/ERK inhibition reverses TGFß-induced PTEN protein suppression in pancreatic cancer cells. (A) We treated ERK-activated BxPc-3 cells with PD98059 in the presence or absence of TGFß and performed western blotting. Note that pretreatment of cells with PD98059 1 h before TGFß treatment significantly reversed the suppressive effect of TGFß on PTEN protein expression (lane 4 versus lane 2). GAPDH was used as a loading control. (B) RAS/ERK-activated CAPAN-1 cells were transfected with 3 µg of DN-K-RAS followed by TGFß treatment. Cells were then lysed 48 h after TGFß treatment and analyzed by western blotting for PTEN protein and GAPDH protein as a loading control. Note that direct K-RAS inhibition coupled with TGFß treatment yielded the greatest expression of PTEN protein, as K-RAS inhibition reversed TGFß-induced PTEN protein suppression (lane 6 versus lane 4). Data shown are means ± SEM of five experiments. *P < 0.05 versus control and {dagger}P < 0.05 versus TGFß treatment alone.

 
TGFß-induced pancreatic cancer cell proliferation parallels PTEN expression and modulation by RAS/ERK
We assessed the biological consequences of modulation of TGFß-induced PTEN suppression in the SMAD4-null pancreatic cancer cell lines. Cells were incubated with TGFß at concentrations shown to suppress PTEN expression both at protein and mRNA levels. As shown in Figure 7, addition of TGFß for 72 h significantly affects cell growth. TGFß treatment causes pancreatic cell proliferation when compared with cells that were not treated with TGFß. We then examined the effect of ERK by inhibiting its upstream inducer MEK1 using PD98059, using three increasing concentrations for inhibition. As shown in Figure 7, PD98059 (12.5 µM to 50 µM) caused a dose-dependent reduction in cell growth with TGFß treatment. With increasing blockade of MEK1/ERK, TGFß-induced proliferation was converted to TGFß-induced growth suppression. Thus, the RAS/ERK pathway contributes to the ability of SMAD4-null pancreatic cancer cells to proliferate. RAS/ERK also modulates TGFß-induced proliferation, which is paralleled by modulation of TGFß-induced PTEN expression.


Figure 7
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7. Effect of TGFß and RAS/ERK inhibition on BxPc-3 cell growth. BxPC-3 cells were plated in complete medium (supplemented with 10% fetal bovine serum). Cells were then grown in serum-free medium (serum depleted) in control medium, in medium containing 10 ng/ml TGFß alone, or TGFß in the presence of 12.5–50 µM PD98059. Cell number was determined on day 3. Data shown are means ± SEM of 12 independent assays and normalized to controls. *P < 0.05 and **P < 0.01 versus cells without TGFß treatment; {dagger}P < 0.05, {dagger}{dagger}P < 0.01 and {dagger}{dagger}{dagger}P < 0.001 versus cells with TGFß but no PD98059 inhibitor treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Our investigation links two commonly affected signaling pathways in pancreatic cancer in the regulation of the tumor suppressor PTEN. PTEN mRNA suppression by TGFß was first described in keratinocytes when PTEN was originally cloned (34), but the mechanisms for TGFß-induced PTEN regulation had not been examined. On the other hand, PTEN expression blocked TGFß-mediated cell invasion whereas PTEN absence increases TGFß-induced invasion (38). The effects might be mediated by SMAD3, as this intracellular signaling molecule inducibly interacts with PTEN (38). PTEN is commonly affected in multiple cancers, often inactivated by genetic (23,24,33) or epigenetic (39) events during the pathogenesis of a particular cancer. This does not appear to be the case in pancreatic adenocarcinoma. PTEN protein expression in pancreatic cancers has previously been shown via immunohistochemistry to be reduced (35), but not due to genetic events (33). Here, we demonstrate that (i) TGFß suppresses PTEN expression in SMAD4-null cells at the transcriptional level with subsequent reduction in protein expression and simultaneous increase in phospho-Akt; (ii) RAS/ERK signaling interferes with TGFß-SMAD nuclear translocation, and SMAD2 translocation is improved with inhibition of RAS/ERK activity; (iii) RAS/ERK signaling facilitates TGFß-induced PTEN suppression and PTEN expression is restored with inhibition of RAS/ERK; and (iv) proliferation events are inversely related to expression of the tumor suppressor PTEN as regulated by TGFß and modulated by RAS/ERK.

Taken together, our findings have a number of implications in pancreatic adenocarcinoma. First, we implicate PTEN, as regulated by TGFß, as a controller of cellular proliferation in pancreatic cancer. Thus, PTEN through ligand regulation, in addition to genetic and epigenetic events, contributes to cancer growth. Regulation of PTEN by TGFß is complex as this regulation occurs in SMAD4-null cells. As depicted in Figure 8, we speculate that there are at least two intracellular pathways by which TGFß regulates PTEN. With TGFß-mediated SMAD-dependent signaling, a well-characterized suppressor pathway in epithelial cells, we suggest that this cascade enhances PTEN expression. In our pancreatic cell models studied, PTEN expression is lessened by (i) loss of SMAD4, which is well-described to facilitate translocation of SMAD2 into the nucleus; and (ii) interference of SMAD2 translocation by activated RAS/ERK, which has been suggested to phosphorylate the linker region of SMAD2/3 and prohibits its translocation to the nucleus (22). Because SMAD2 translocation was improved with blockage of RAS/ERK, as well as restoration of TGFß-induced PTEN expression, SMAD2 may have nuclear effects that are independent of the presence of SMAD4, and this has been suggested in the literature (40,41).


Figure 8
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8. Schematic summarizing our working model. Pancreatic cancer cells are often SMAD4 null, and thus TGFß utilizes signaling pathways to regulate PTEN expression in a SMAD4-independent manner. We hypothesize that TGFß-induced SMAD-dependent signaling restores PTEN expression, and speculate that PTEN is suppressed via non-SMAD pathways. As most pancreatic cells have activated RAS/ERK because of oncogenic mutations, TGFß-SMAD-induced PTEN transcription is inhibited (A). When activated RAS/ERK is blocked, TGFß-induced PTEN transcription is resumed (B). Thus, in a SMAD4-null environment, activated RAS/ERK acts as a ‘switch’ to convert TGFß signaling functionally from a tumor suppressor to tumor promoter via modulation of PTEN expression.

 
Second, we also speculate the presence of a TGFß-mediated SMAD-independent pathway, which down-regulates PTEN and facilitates cellular growth. Our data strongly suggest the existence of such a pathway because TGFß mediates PTEN suppression in the absence of SMAD4 and in the presence of activated RAS/ERK, both of which attenuate SMAD2 nuclear translocation. Thus, SMAD-dependent signaling is blocked, which is the case in most human pancreatic cancers, while TGFß still has cellular effects. There are a number of reports that indicate that TGFß can have dual roles: growth suppression and, in the right scenario, induce growth proliferation. In many cases, TGFß's role in cellular proliferation is demonstrated in metastasis, which represents mature cancers with the accumulated pathway derangements. In pancreatic cancers, TGFß ligand is often up-regulated, and this is believed to be due to loss of negative feedback regulation by SMAD-dependent signaling (35). The increased amounts of ligand could help increasingly signal through a proliferative-driven SMAD-independent pathway through down-regulation of PTEN favoring unbalanced mitogenic PI3K signaling. Recently, a second mechanism has been demonstrated that reduces PTEN levels after TGFß stimulation. TGFß signaling induces dephosphorylation of PTEN, reducing its stability in the cytoplasm (42). Thus, there appears to be at least two mechanisms, reduced PTEN expression and reduced PTEN stability, to decrease PTEN protein levels to increase the pancreatic cancer cell's ability to proliferate with TGFß signaling.

Third, RAS/ERK, which is activated in over 90% of pancreatic cancers, modulates PTEN expression through TGFß signaling. The regulation appears to be through blocking SMAD translocation, as previously suggested in the literature (22). However, K-RAS mutations, in particular, are one of the earliest events in pancreatic cancer pathogenesis, and oncogenic mutations are seen in benign pancreatic conditions such as pancreatitis (18,19). Because RAS/ERK activation is a potent inhibitor of SMAD signaling, TGFß-induced PTEN regulation is likely affected at a very early stage in pancreatic carcinogenesis.

In summary, we indicate that PTEN may be a key tumor suppressor involved in pancreatic tumorigenesis. Unlike other tumors in which PTEN is inactivated by a variety of genetic or epigenetic mechanisms, PTEN expression is ligand regulated by TGFß signaling. The TGFß-induced PTEN expression may be determined by either or both of SMAD-dependent and SMAD-independent signaling pathways. When SMAD-dependent signaling is blocked by oncogenic K-RAS or activated ERK, TGFß induces down-regulation of PTEN coupled with growth proliferation of pancreatic cells. When RAS/ERK activation is blocked, SMAD2 nuclear translocation is restored (even without SMAD4), TGFß-induced PTEN down-regulation is reversed and cellular proliferation is reduced to non-TGFß-induced levels. We suggest that RAS/ERK activation ‘switches’ the balance of the dual roles of TGFß in favor of proliferation when PTEN is turned off, and the switch can be reversed with blockage of RAS/ERK to turn PTEN expression on reducing cellular proliferation. This regulatory mechanism could potentially be controlled pharmacologically.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
United States Public Health Service (K01-DK073090 to J.Y.C., T32-HL07212 to S.E.B., CA90231 and DK067287 to J.M.C.); National Pancreas Foundation; VA Research Service.


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

  1. Warshaw AL, et al. Pancreatic carcinoma. N. Engl. J. Med. (1992) 326:455–465.[Web of Science][Medline]
  2. Friess H, et al. Enhanced expression of transforming growth factor beta isoforms in pancreatic cancer correlates with decreased survival. Gastroenterology (1993) 105:1846–1856.[Web of Science][Medline]
  3. Derynck R, et al. TGF-beta signaling in tumor suppression and cancer progression. Nat. Genet. (2001) 29:117–129.[CrossRef][Web of Science][Medline]
  4. Pasche B. Role of transforming growth factor beta in cancer. J. Cell. Physiol. (2001) 186:153–168.[CrossRef][Web of Science][Medline]
  5. Roberts AB, et al. The two faces of transforming growth factor beta in carcinogenesis. Proc. Natl Acad. Sci. USA (2003) 100:8621–8623.[Free Full Text]
  6. Massague J. TGFbeta signaling: receptors, transducers, and Mad proteins. Cell (1996) 85:947–950.[CrossRef][Web of Science][Medline]
  7. Massague J, et al. Transcriptional control by the TGF-beta/Smad signaling system. EMBO J. (2000) 19:1745–1754.[CrossRef][Web of Science][Medline]
  8. ten Dijke P, et al. New insights into TGF-beta-Smad signalling. Trends Biochem. Sci. (2004) 29:265–273.[CrossRef][Web of Science][Medline]
  9. Siegel PM, et al. Transforming growth factor beta signaling impairs Neu-induced mammary tumorigenesis while promoting pulmonary metastasis. Proc. Natl Acad. Sci. USA (2003) 100:8430–8435.[Abstract/Free Full Text]
  10. Gorska AE, et al. Transgenic mice expressing a dominant-negative mutant type II transforming growth factor-beta receptor exhibit impaired mammary development and enhanced mammary tumor formation. Am. J. Pathol. (2003) 163:1539–1549.[Abstract/Free Full Text]
  11. Bottinger EP, et al. Expression of a dominant-negative mutant TGF-beta type II receptor in transgenic mice reveals essential roles for TGF-beta in regulation of growth and differentiation in the exocrine pancreas. EMBO J. (1997) 16:2621–2633.[CrossRef][Web of Science][Medline]
  12. Li W, et al. Squamous cell carcinoma and mammary abscess formation through squamous metaplasia in Smad4/Dpc4 conditional knockout mice. Development (2003) 130:6143–6153.[Abstract/Free Full Text]
  13. Takaku K, et al. Intestinal tumorigenesis in compound mutant mice of both Dpc4 (Smad4) and Apc genes. Cell (1998) 92:645–656.[CrossRef][Web of Science][Medline]
  14. Takaku K, et al. No effects of Smad2 (madh2) null mutation on malignant progression of intestinal polyps in Apc(delta716) knockout mice. Cancer Res. (2002) 62:4558–4561.[Abstract/Free Full Text]
  15. Amendt C, et al. Expression of a dominant negative type II TGF-beta receptor in mouse skin results in an increase in carcinoma incidence and an acceleration of carcinoma development. Oncogene (1998) 17:25–34.[CrossRef][Web of Science][Medline]
  16. Hahn SA, et al. Homozygous deletion map at 18q21.1 in pancreatic cancer. Cancer Res. (1996) 56:490–494.[Abstract/Free Full Text]
  17. Hahn SA, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science (1996) 271:350–353.[Abstract]
  18. Carethers JM, et al. Neoplasia of the Gastrointestinal Tract. In: Textbook of Gastroenterology—Yamada T, Alpers DH, Kaplowitz N, Laine L, Owyang C, Powell DW, eds. (2003) Philadelphia, PA: Lippincott-Raven Publishers. 557–583. 4th edition.
  19. Laghi L, et al. Common occurrence of multiple K-RAS mutations in pancreatic cancers with associated precursor lesions and in biliary cancers. Oncogene (2002) 21:4301–4306.[CrossRef][Web of Science][Medline]
  20. Schwarz LC, et al. Loss of growth factor dependence and conversion of transforming growth factor-beta 1 inhibition to stimulation in metastatic H-ras-transformed murine fibroblasts. Cancer Res. (1988) 48:6999–7003.[Abstract/Free Full Text]
  21. Filmus J, et al. Development of resistance mechanisms to the growth-inhibitory effects of transforming growth factor-beta during tumor progression. Curr. Opin. Oncol. (1993) 5:123–129.[Medline]
  22. Kretzschmar M, et al. A mechanism of repression of TGFbeta/Smad signaling by oncogenic Ras. Genes. Dev. (1999) 13:804–816.[Abstract/Free Full Text]
  23. Li J, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science (1997) 275:1943–1947.[Abstract/Free Full Text]
  24. Steck PA, et al. Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat. Genet. (1997) 15:356–362.[CrossRef][Web of Science][Medline]
  25. Maehama T, et al. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. (1998) 273:13375–13378.[Abstract/Free Full Text]
  26. Di Cristofano A, et al. The multiple roles of PTEN in tumor suppression. Cell (2000) 100:387–390.[CrossRef][Web of Science][Medline]
  27. Simpson L, et al. PTEN: life as a tumor suppressor. Exp. Cell. Res. (2001) 264:29–41.[CrossRef][Web of Science][Medline]
  28. Liaw D, et al. Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat. Genet. (1997) 16:64–67.[CrossRef][Web of Science][Medline]
  29. Marsh DJ, et al. Germline mutations in PTEN are present in Bannayan-Zonana syndrome. Nat. Genet. (1997) 16:333–334.[CrossRef][Web of Science][Medline]
  30. Huang SC, et al. Genetic heterogeneity in familial juvenile polyposis. Cancer Res. (2000) 60:6882–6885.[Abstract/Free Full Text]
  31. Carethers JM. Unwinding the heterogeneous nature of hamartomatous polyposis syndromes. JAMA (2005) 294:2498–2500.[Free Full Text]
  32. Dong JT, et al. PTEN/MMAC1 is infrequently mutated in pT2 and pT3 carcinomas of the prostate. Oncogene (1998) 17:1979–1982.[CrossRef][Web of Science][Medline]
  33. Sakurada A, et al. Infrequent genetic alterations of the PTEN/MMAC1 gene in Japanese patients with primary cancers of the breast, lung, pancreas, kidney, and ovary. Jpn. J. Cancer Res. (1997) 88:1025–1028.[CrossRef][Web of Science]
  34. Li DM, et al. TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta. Cancer Res. (1997) 57:2124–2129.[Abstract/Free Full Text]
  35. Ebert MP, et al. Reduced PTEN expression in the pancreas overexpressing transforming growth factor-beta 1. Br. J. Cancer (2002) 86:257–262.[CrossRef][Web of Science][Medline]
  36. Feig LA, et al. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol. Cell. Biol. (1988) 8:3235–3243.[Abstract/Free Full Text]
  37. Sano T, et al. Differential expression of MMAC/PTEN in glioblastoma multiforme: relationship to localization and prognosis. Cancer Res. (1999) 59:1820–1824.[Abstract/Free Full Text]
  38. Hjelmeland AB, et al. Loss of phosphatase and tensin homologue increases transforming growth factor beta-mediated invasion with enhanced SMAD3 transcriptional activity. Cancer Res. (2005) 65:11276–11281.[Abstract/Free Full Text]
  39. Goel A, et al. Frequent inactivation of PTEN by promoter hypermethylation in microsatellite instability-high sporadic colorectal cancers. Cancer Res. (2004) 64:3014–3021.[Abstract/Free Full Text]
  40. Ijichi H, et al. Smad4-independent regulation of p21/WAF1 by transforming growth factor-beta. Oncogene (2004) 23:1043–1051.[CrossRef][Web of Science][Medline]
  41. Fink SP, et al. TGF-beta-induced nuclear localization of Smad2 and Smad3 in Smad4 null cancer cell lines. Oncogene (2003) 22:1317–1323.[CrossRef][Web of Science][Medline]
  42. Vazquez F, et al. Phosphorylation of the PTEN tail regulates protein stability and function. Mol. Cell. Biol. (2000) 20:5010–5018.[Abstract/Free Full Text]
Received March 23, 2007; revised July 2, 2007; accepted July 3, 2007.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
S. J. Assinder, Q. Dong, H. Mangs, and D. R. Richardson
Pharmacological Targeting of the Integrated Protein Kinase B, Phosphatase and Tensin Homolog Deleted on Chromosome 10, and Transforming Growth Factor-{beta} Pathways in Prostate Cancer
Mol. Pharmacol., March 1, 2009; 75(3): 429 - 436.
[Abstract] [Full Text] [PDF]

Home page
 GutHome page
M Mimeault and S K Batra
Recent progress on normal and malignant pancreatic stem/progenitor cell research: therapeutic implications for the treatment of type 1 or 2 diabetes mellitus and aggressive pancreatic cancer
Gut, October 1, 2008; 57(10): 1456 - 1468.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
J. Y. C. Chow, H. Dong, K. T. Quach, P. N. Van Nguyen, K. Chen, and J. M. Carethers
TGF-{beta} mediates PTEN suppression and cell motility through calcium-dependent PKC-{alpha} activation in pancreatic cancer cells
Am J Physiol Gastrointest Liver Physiol, April 1, 2008; 294(4): G899 - G905.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
T. Tamguney and D. Stokoe
New insights into PTEN
J. Cell Sci., December 1, 2007; 120(23): 4071 - 4079.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
28/11/2321    most recent
bgm159v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (16)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Chow, J. Y.C.
Right arrow Articles by Carethers, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chow, J. Y.C.
Right arrow Articles by Carethers, J. M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?