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

Abstract

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-beta might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFbeta 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 TGFbeta surface receptors. Cells were treated with TGFbeta1 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. TGFbeta 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. TGFbeta 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. TGFbeta-induced PTEN suppression was reversed by PD98059 and DN-K-RAS compared with treatments without TGFbeta. TGFbeta-induced PTEN expression was inversely related to cellular proliferation. Thus, oncogenic K-RAS/ERK in pancreatic adenocarcinoma facilitates TGFbeta-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.

Fig. 1

Effects of TGFβ treatment on subcellular trafficking of SMAD2 in BxPc-3 and CAPAN-1 cells. Cells were treated with or without TGFβ (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.

Fig. 2

Effect of RAS/ERK inhibition and TGFβ 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 TGFβ. Cell lysates were blotted with anti-phospho-SMAD2, tubulin and histone antibodies. Note that PD98059 and DN-K-RAS transfection improved TGFβ-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.

Fig. 3

TGFβ treatment down-regulates PTEN transcription. CAPAN-1 and BxPc-3 cells were treated with a single dose of TGFβ (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 TGFβ. Thus, PTEN is down-regulated transiently at the transcriptional level by 24 h after a single TGFβ treatment. GAPDH was used as loading control.

Fig. 4

TGFβ 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 TGFβ 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.

Fig. 5

PD98059 reverses TGFβ-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 semiquantitative reverse transcriptase–PCR for PTEN mRNA at 24 h after TGFβ treatment. PD98059 had little effect on the basal levels of PTEN mRNA (A and B, lanes 2 and 3). However, MEK inhibition rescued TGFβ-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 TGFβ treatment. Data shown are means ± SEM of three to four experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus control; ††P < 0.01 and †††P < 0.001 versus TGFβ treatment alone.

Fig. 6

RAS/ERK inhibition reverses TGFβ-induced PTEN protein suppression in pancreatic cancer cells. (A) We treated ERK-activated BxPc-3 cells with PD98059 in the presence or absence of TGFβ and performed western blotting. Note that pretreatment of cells with PD98059 1 h before TGFβ treatment significantly reversed the suppressive effect of TGFβ 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 TGFβ treatment. Cells were then lysed 48 h after TGFβ treatment and analyzed by western blotting for PTEN protein and GAPDH protein as a loading control. Note that direct K-RAS inhibition coupled with TGFβ treatment yielded the greatest expression of PTEN protein, as K-RAS inhibition reversed TGFβ-induced PTEN protein suppression (lane 6 versus lane 4). Data shown are means ± SEM of five experiments. *P < 0.05 versus control and †P < 0.05 versus TGFβ treatment alone.

Fig. 7

Effect of TGFβ 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 TGFβ alone, or TGFβ 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 TGFβ treatment; †P < 0.05, ††P < 0.01 and †††P < 0.001 versus cells with TGFβ but no PD98059 inhibitor treatment.

Fig. 8

Schematic summarizing our working model. Pancreatic cancer cells are often SMAD4 null, and thus TGFβ utilizes signaling pathways to regulate PTEN expression in a SMAD4-independent manner. We hypothesize that TGFβ-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, TGFβ-SMAD-induced PTEN transcription is inhibited (A). When activated RAS/ERK is blocked, TGFβ-induced PTEN transcription is resumed (B). Thus, in a SMAD4-null environment, activated RAS/ERK acts as a ‘switch’ to convert TGFβ signaling functionally from a tumor suppressor to tumor promoter via modulation of PTEN expression.