In normal epithelial tissues, the multifunctional cytokine transforming growth factor-β (TGF-β) acts as a tumor suppressor through growth inhibition and induction of differentiation whereas in advanced cancers, TGF-β promotes tumor progression through induction of tumor invasion, neoangiogenesis, and immunosuppression. The molecular mechanisms through which TGF-β shifts from a tumor suppressor to a tumor enhancer are poorly understood. We now show a role for the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in repressing the protumorigenic effects of TGF-β. The TGF-β effector SMAD3 inducibly interacts with PTEN on TGF-β treatment under endogenous conditions. RNA interference (RNAi) suppression of PTEN expression enhances SMAD3 transcriptional activity and TGF-β–mediated induction of SMAD3 target genes whereas reconstitution of PTEN in a null cancer cell line represses the expression of TGF-β–regulated target genes. Targeting PTEN expression through RNAi in a PTEN wild-type cell line increases TGF-β–mediated invasion but does not affect TGF-β–mediated growth inhibition. Reconstitution of PTEN expression in a PTEN-null cell line blocks TGF-β–induced invasion but does not modulate TGF-β–mediated growth regulation. These effects are distinct from Akt and Forkhead family members that also interact with SMAD3 to regulate apoptosis or proliferation, respectively. Pharmacologic inhibitors targeting TGF-β receptors and phosphatidylinositol 3-kinase signaling downstream from PTEN cooperate to block TGF-β–mediated invasion. Thus, the loss of PTEN expression in human cancers may contribute to a role for TGF-β as a tumor enhancer with specific effects on cellular motility and invasion. (Cancer Res 2005; 65(24): 11276-81)
Transforming growth factor-β (TGF-β) regulates a diverse set of biological activities, including proliferation, apoptosis, differentiation, motility, extracellular matrix deposition, and angiogenesis (1). The breadth of cellular responses is derived from the hundreds of genes transcriptionally regulated by TGF-β. TGF-β initiates cellular effects through the activation of serine/threonine kinase receptors that phosphorylate and activate the intracellular mediators SMAD2 and SMAD3 (2–4) to promote or inhibit the transcription of target genes (4–6). The ability of TGF-β to modulate gene expression partially depends on interactions with other signaling pathways. Recent reports highlight the importance of interactions between SMAD3 and components of phosphatidylinositol 3-kinase (PI3K) signaling, a prosurvival pathway activated in many cancers. Akt directly binds SMAD3, sequestering SMAD3 in the cytoplasm and inhibiting TGF-β-induced apoptosis (7, 8), whereas FOXO family members interact with SMAD3 to activate transcription of p21WAF1/CIP1, but not plasminogen activator inhibitor-1 (PAI-1; ref. 9). Phosphatase and tensin homologue (PTEN) is a tumor suppressor deleted or mutated in many solid tumors, including glioblastomas (10). As PTEN antagonizes the effects of PI3K on phosphoinositide phosphorylation, the loss of PTEN permits the constitutive activation of signaling downstream from PI3K (10) to increase cellular resistance to apoptosis and tumor cell invasion. We report a novel mechanism through which PTEN could prevent invasion during cancer progression: PTEN can functionally interact with SMAD3 to negatively regulate TGF-β signaling and prevent TGF-β–mediated invasion.
Materials and Methods
Retroviral expression. Indicated sense sequences for PTEN RNA interference (RNAi) were cloned into pSuperRetropuro vector (OligoEngine, Seattle, WA). Short hairpin RNA 1 (shRNA1) human PTEN bp 700 to 722: 5′-CGGGAAGACAAGTTCATGTACTT-3′. shRNA2 human PTEN bp 319 to 339: 5′-GATCTTGACCAATGGCTAAGT-3′. pBabe-bleo vector containing PTEN, C124G, or G129E phosphatase-dead mutants was provided by Frank Furnari (Ludwig Institute for Cancer Research, San Diego, CA).
Immunoprecipitation. Endogenous lysates, collected from HaCaT cells treated with 100 pmol/L TGF-β for 15 minutes, were exposed to α-PTEN or α-matrix metalloproteinase 7 (Santa Cruz Biotechnology, Santa Cruz, CA) and protein A/G beads (Amersham, Piscataway, NJ). In overexpression studies, hemaglutinin (HA)- or Flag-tagged constructs were expressed in 293T cells using FuGENE 6 (Roche, Indianapolis, IN) and immunoprecipitated using α-HA (Santa Cruz Biotechnology).
Western blotting. Cell lysates were prepared as previously described (11) using α-SMAD2, α-SMAD4 (Zymed, Carlsbad, CA), α-SMAD3, α-PTEN, α-HA, α-PAI-1, α-p21, α-c-myc (Santa Cruz Biotechnology), α-Flag (Stratagene, La Jolla, CA), α-pAkt, α-Akt (Cell Signaling, Beverly, MA), and α-tubulin (Sigma, St. Louis, MO).
Luciferase reporter assay. Luciferase assays were done as previously described (11) with the addition of 100 pmol/L TGF-β or buffer 24 hours after transfection.
Thymidine incorporation assay. Thymidine incorporation assays were done as previously described (11) with addition of TGF-β and/or buffer 20 hours before thymidine addition.
Migration and invasion assay. For HaCaT, pretreated cells were wounded and treated with the indicated drug, DMSO, 100 pmol/L TGF-β, or buffer overnight. For U87MG, 25,000 pretreated cells were plated in serum-free media in the upper chamber of Matrigel-coated wells or uncoated control chambers (BD Biosciences, Bedford, MA). The lower chamber contained 1% serum with or without TGF-β. The ratio of cells invading through Matrigel to cells migrating through the control chamber determined the percentage of invading cells.
TGF-β induces SMAD3 and PTEN interaction. Based on prior reports showing physical and functional interactions between members of the TGF-β and PI3K signaling pathways, we hypothesized a physical or functional role for the PI3K pathway inhibitor PTEN in the regulation of TGF-β signaling. To determine if PTEN could physically interact with TGF-β effector SMADs, we did coimmunoprecipitation experiments in HaCaT cells, a human keratinocyte cell line with intact TGF-β responses and wild-type PTEN expression. Endogenous PTEN interacted with endogenous SMAD3 and, less significantly, with SMAD2 in a TGF-β-responsive manner (Fig. 1A). The structural basis of the interaction between SMAD3 and PTEN was further determined in overexpression studies. PTEN inducibly coimmunoprecipitated with full-length or NH2-terminal SMAD3 (Fig. 1B), suggesting that PTEN binds to the Mad homology 1 (MH1) domain of SMAD3 that contains the DNA-binding domain. Although PTEN did not interact with the SMAD3 COOH terminus or linker region (Fig. 1B), TGF-β-induced phosphorylation of the COOH-terminal SSxS sequence is likely important for conformational changes in SMAD3 to permit PTEN binding as the nonphosphorylated COOH-terminal MH2 domain interacts with the NH2-terminal MH1 domain (2). Indeed, a SMAD3 mutant in which COOH-terminal serines were mutated to alanine, mimicking nonphosphorylated SMAD3, did not inducibly interact with PTEN (Fig. 1C). These data suggest that TGF-β–mediated phosphorylation of COOH-terminal serines induces a conformational change in SMAD3 to permit NH2-terminal binding to PTEN.
The phosphatase activity of PTEN is located on the NH2 terminus (12, 13) whereas phosphorylation of the COOH terminus of PTEN regulates stability and binding to other proteins (14, 15). In overexpression experiments, TGF-β induced an interaction between SMAD3 and the NH2 terminus of PTEN (amino acids 1-274) despite reduced PTEN protein stability on deletion of the COOH terminus (Fig. 1D). Although SMAD3 bound to PTEN in or near its phosphatase domain, the interaction did not require the phosphatase activity of PTEN as SMAD3 inducibly interacted with PTEN mutants that lack total phosphatase (C124G) or lipid phosphatase (G129E) activities (data not shown; ref. 16). The addition of the extreme PTEN COOH terminus to the NH2 terminus of PTEN (the Δ274-346 mutant) prevented the induction of SMAD3 binding by TGF-β (Fig. 1D), indicating that the COOH terminus of PTEN regulates SMAD3 binding. Furthermore, a PTEN mutant lacking COOH-terminal phosphorylation sites, A5, bound strongly to SMAD3 (data not shown), showing that phosphorylation of the COOH tail of PTEN negatively regulates the interaction with SMAD3. Thus, SMAD3 and PTEN physically associate in both NH2 termini with regulation by COOH termini of each protein.
RNAi to PTEN increases transcription of TGF-β-responsive promoters. To determine the functional significance of PTEN expression on TGF-β/SMAD3–mediated transcription, we targeted PTEN expression with stable expression of shRNA directed against PTEN with two different shRNA sequences that decreased PTEN expression by ∼75% and 99%, respectively (Fig. 2A). In HaCaT, shRNA targeting of PTEN expression augmented TGF-β transcriptional activation of multiple TGF-β-responsive promoters (Fig. 3A and B and Supplementary Fig. S1A and B). Transcriptional activity of the TGF-β-responsive 3TP-luc reporter was induced by TGF-β in an inverse relationship to PTEN expression levels (Fig. 3A). Targeting PTEN expression additionally enhanced TGF-β–mediated transcription of a reporter containing concatamerized SMAD3 DNA binding elements (SBE; Supplementary Fig. S1A; ref. 3). Expression of SMAD3 in cells expressing an SBE reporter induced transcription in a dose-dependent manner whereas decreased PTEN expression enhanced this effect (Fig. 3B and Supplementary Fig. S1B). Together, theses results show that PTEN represses TGF-β- and SMAD3-mediated transcriptional activation.
RNAi to PTEN enhances transcription of a promoter repressed by TGF-β. To determine if the effects of PTEN on TGF-β–mediated transcription were limited to activation, we evaluated the activity of a promoter repressed by TGF-β (4). Loss of PTEN expression enhanced the basal activity of a reporter with concatamerized TGF-β inhibitory elements (TIE; Fig. 3C). Whereas targeting PTEN expression did not abolish the transcriptional repression of TIE sequences by TGF-β, TGF-β did not repress transcription to basal levels in the absence of PTEN (Fig. 3C).
Reconstitution of PTEN, but not of phosphatase-dead mutant PTEN, blocks TGF-β-responsive promoters. In experiments reciprocal to those in HaCaT cells, we reconstituted the expression of wild-type PTEN or phosphatase-dead PTEN mutants in the PTEN-null human U87MG glioma cell line and evaluated the ability of SMAD3 to induce transcription of an SBE reporter. Wild-type PTEN inhibited SMAD3-dependent transcription whereas the expression of C124G and G129E mutant PTEN proteins did not (Fig. 3D). Reconstitution of wild-type PTEN expression in U87MG cells also inhibited 3TP-luc in a dose-dependent manner (data not shown). Together, these data show that PTEN inhibits SMAD3-dependent transcription through a mechanism requiring PTEN phosphatase activity although the phosphatase activity itself is not required for the PTEN-SMAD3 interaction.
PTEN expression does not prevent TGF-β-induced SMAD3 nuclear accumulation. As SMAD3 and PTEN interact on both functional and physical levels, we sought to elucidate the mechanism by which PTEN inhibits SMAD3-dependent transcription. SMAD3-mediated transcription is regulated by its COOH-terminal phosphorylation and translocation to the nucleus; thus, we examined the effect of PTEN expression on this pathway. RNAi suppression of PTEN expression in HaCaT cells did not enhance TGF-β-induced SMAD3 nuclear accumulation (Fig. 2C) or COOH-terminal phosphorylation of SMAD2 or SMAD3 (Fig. 2D). Restoration of wild-type PTEN expression in U87MG cells also did not prevent TGF-β–mediated SMAD3 nuclear translocation or COOH-terminal phosphorylation (data not shown). Although other PI3K pathway members (specifically Akt) regulate SMAD3 transcription by cytoplasmic sequestration (7, 8), our data show that PTEN regulates SMAD3 through a distinct mechanism.
RNAi to PTEN alters TGF-β-regulated c-myc and PAI-1 expression. As TGF-β modulates cellular behaviors through transcriptional regulation of target genes, we examined the effects of PTEN on the ability of TGF-β to regulate expression of several proteins that critically regulate cell behavior. TGF-β potently induces the expression of several cyclin-dependent kinase inhibitors, including p21WAF1/CIP1 (5), while repressing c-myc expression (4) to decrease cell cycle progression. TGF-β induced p21WAF1/CIP1 and repressed c-myc expression in HaCaT cells (Fig. 2A) that are potently growth inhibited by TGF-β (Fig. 4A). In contrast to FOXO family member effects on TGF-β signaling, the loss of PTEN expression did not alter the ability of TGF-β to induce p21WAF1/CIP1 expression (Fig. 2A). shRNA targeting of PTEN expression in HaCaT elevated basal c-myc expression but did not completely block TGF-β–mediated repression of c-myc expression (Fig. 2B), similar to our results in transcriptional assays (Fig. 3C).
In reciprocal experiments, reconstitution of wild-type or mutant PTEN expression in U87MG cells did not alter p21WAF1/CIP1 or c-myc expression (Fig. 2B). The failure of TGF-β to induce p21WAF1/CIP1 or repress c-myc expression in U87MG cells shows two molecular mechanisms through which this glioma cell line is resistant to TGF-β–mediated growth inhibition. Furthermore, the restoration of PTEN did not reestablish TGF-β–mediated control of these cell cycle–related target genes (Fig. 2B).
To evaluate TGF-β regulation of an extracellular matrix protein implicated in tumor cell migration and invasion, we determined the effects of targeting PTEN expression on the ability of TGF-β to regulate PAI-1 expression. Although PAI-1 inhibits proteases involved in invasion, PAI-1 effects are complex as PAI-1 may positively regulate invasion in a cell type/state–dependent manner (17). Indeed, PAI-1 expression is an independent marker of poor prognosis for glioblastomas, a highly invasive tumor (18). TGF-β induced PAI-1 protein expression in both HaCaT and U87MG (Fig. 2A and B), showing that TGF-β–mediated regulation of the extracellular matrix is maintained in these cell lines. RNAi to PTEN in HaCaT cells increased the efficacy with which TGF-β induced PAI-1 protein expression (Fig. 2A). In the reciprocal experiment, reconstititution of wild-type, but not phosphatase-dead PTEN, expression in U87MG cells blocked TGF-β–mediated PAI-1 expression (Fig. 2B). Thus, PTEN expression inversely correlates with the ability of TGF-β to induce PAI-1.
Loss of PTEN expression alone is not sufficient to prevent TGF-β–mediated growth inhibition. Changes in TGF-β–mediated gene expression observed in the presence and absence of PTEN could modulate cellular behaviors, such as proliferation or invasion, which are important in cancer. In HaCaT cells, the elevation of c-myc expression detected with RNAi targeting of PTEN (Fig. 2A) did not increase basal cell proliferation or prevent TGF-β–mediated growth inhibition (Fig. 4A), likely due to the potent TGF-β–mediated induction of p21WAF1/CIP1 (Fig. 2A). Similarly, reconstitution of PTEN expression in U87MG cells did not enhance TGF-β–mediated growth inhibition (Supplementary Fig. S1C). Together, these data show that restoration of PTEN expression in PTEN-null cancer cells does not reestablish the potent growth inhibitory effects of TGF-β observed in epithelial cells and that loss of PTEN expression does not prevent TGF-β–mediated growth inhibition during cancer progression.
PTEN represses TGF-β–mediated cellular migration and invasion. Both TGF-β and PTEN regulate tumor cell invasion. We therefore examined the effects of PTEN expression on TGF-β mediated cellular motility/invasion. The effects of decreased PTEN expression on HaCaT cell migration were determined in a wound closure assay because these cells did not invade through the artificial extracellular matrix of the Matrigel assay (data not shown). RNAi to PTEN specifically altered cell migration in response to TGF-β but did not effect basal migration of HaCaT cells (Fig. 4B). Similarly, reconstitution of wild-type PTEN expression in U87MG cells blocked TGF-β–mediated invasion in the Matrigel invasion assay (Fig. 4C). Together, these results confirm a functional interaction between PTEN and SMAD3 on TGF-β transcriptional targets with a biological consequence for TGF-β–mediated invasion.
Targeting TGF-β and mammalian target of rapamycin signaling with pharmacologic inhibitors prevents TGF-β–mediated migration. As we showed that antagonism of PTEN through RNAi increased TGF-β–mediated migration, mutation or loss of PTEN during cancer progression may promote proinvasive TGF-β signals in addition to effects of PI3K activation. During cancer progression, the balance of TGF-β effects on tumors shifts towards tumor progression as growth inhibition/differentiation responses are lost. We previously showed that a novel type I TGF-β receptor inhibitor, SB-431542, decreased glioma cell migration (11). Based on this prior work and our new understanding of the interaction between PTEN and TGF-β signaling, we investigated the effects of a combination of TGF-β receptor and PI3K pathway inhibitors. In parallel to our results showing minimal effect of PTEN on TGF-β–mediated proliferation, SB-431542 and rapamycin (sirolimus), an inhibitor of the PI3K downstream target mammalian target of rapamycin, did not cooperate to reduce cell proliferation although both agents inhibited basal proliferation of U87MG cells (data not shown). In contrast, the combination of SB-431542 and rapamycin blocked TGF-β–mediated migration more effectively than either monotherapy in HaCaT cells (Fig. 4D). Together, our results suggest that there may be a therapeutic benefit for the specific combination of SB-431542 and rapamycin as well as a potential benefit for other small-molecule inhibitor combinations that target both TGF-β and PI3K signals.
The dichotomous effects of TGF-β in cancer are now well appreciated but poorly understood. TGF-β–mediated growth inhibition and regulation of differentiation are important for suppressing early cancers whereas TGF-β–mediated extracellular matrix deposition promotes invasion in late cancers (1). As the mutation and deletion of the PTEN tumor suppressor gene occurs in many advanced cancers (12) in which TGF-β functions to promote tumor progression, we hypothesize that loss of PTEN contributes to this shift in TGF-β activity. The loss of PTEN alone does not permit escape from TGF-β-mediated growth inhibition but may facilitate cell proliferation induced by mutations in TGF-β-independent pathways. Further, the loss of PTEN may increase expression of PAI-1 and other proteins that contribute to tumor invasion in TGF-β-rich microenvironments. However, this novel role for PTEN in the control of tumor cell migration and invasion has yet to be shown in vivo. We are working to address the limitations of cell culture models through the use of genetically engineered mouse models of cancer.
The precise mechanism through which loss of PTEN elevates proinvasive TGF-β/SMAD3 signals remains under investigation. Our data show that the phosphatase activity of PTEN is required to inhibit TGF-β-mediated transcription. PTEN may dephosphorylate SMAD3 at sites other than the COOH terminus to regulate its activity. Other SMAD3 phosphorylation sites are already known to be regulated by mitogen-activated protein kinases and cyclin-dependent kinases (19, 20). As PTEN does not alter SMAD3 nuclear translocation, the mechanism through which PTEN regulates SMAD3/TGF-β activity is distinct from that of other PI3K signal transduction members. Furthermore, the PTEN/SMAD3 interaction broadens our knowledge of PI3K pathway components in TGF-β signaling and introduces a new level of complexity for interactions between these two major signal transduction pathways in cancer.
Most solid cancers develop from the dysregulation of multiple pathways. Therefore, the successful targeting of a single molecular pathway as with imatinib mesylate for chronic myelogenous leukemia is unlikely to induce widespread tumor responses: effective molecular targeted therapies will require targeting two or more molecular pathways in combination. In this study, we further validate the functional interactions between two important pathways involved in the pathogenesis of multiple cancers. Our results suggest that targeting both the TGF-β receptor and targets downstream of PI3K may benefit patients through the reduction of cancer cell proliferation and migration. Further, expression and activation of individual pathway components could partially determine cancer sensitivity to other pathway inhibitors. For example, disruption of Akt/PI3K activities may not completely reverse the effects of PTEN loss on TGF-β signaling but PTEN loss could be a marker for tumors with strong responses to anti-TGF-β therapies. Further study of the mechanisms of PTEN and other PI3K family member interactions with TGF-β signal transduction may both contribute to a greater understanding of their function in cancer and create new therapeutic opportunities.
Note: A.B. Hjelmeland is a Paul Brazen/American Brain Tumor Association Fellow. J.N. Rich is a Damon Runyon-Lilly Clinical Investigator and a Sidney Kimmel Cancer Foundation Translational Scholar.
Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Grant support: Pediatric Brain Tumor Foundation of the United States (J.N. Rich), Accelerate Brain Cancer Cure (J.N. Rich), Southeastern Brain Tumor Foundation (A.B. Hjelmeland), American Heart Association (C.D. Kontos), and NIH grants NS047409, P50 CA108786 (J.N. Rich), and HL70165 (C.D. Kontos).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.