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1230 | CANCER DISCOVERYOCTOBER 2014 www.aacrjournals.org Active CREB1 Promotes a Malignant TGFb2 Autocrine Loop in Glioblastoma Laura Rodón 1 , Alba Gonzàlez-Juncà 1 , María del Mar Inda 1 , Ada Sala-Hojman 1 , Elena Martínez-Sáez 2 , and Joan Seoane 1,3,4 RESEARCH ARTICLE ABSTRACT In advanced cancer, including glioblastoma, the TGFβ pathway acts as an oncogenic factor. Some tumors exhibit aberrantly high TGFβ activity, and the mechanisms underlying this phenomenon are not well understood. We have observed that TGFβ can induce TGFβ2, generating an autocrine loop leading to aberrantly high levels of TGFβ2. We identified cAMP-responsive element–binding protein 1 (CREB1) as the critical mediator of the induction of TGFβ2 by TGFβ. CREB1 binds to the TGFB2 gene promoter in cooperation with SMAD3 and is required for TGFβ to activate transcription. Moreover, the PI3K–AKT and RSK pathways regulate the TGFβ2 autocrine loop through CREB1. The levels of CREB1 and active phosphorylated CREB1 correlate with TGFβ2 in glioblastoma. In addition, using patient-derived in vivo models of glioblastoma, we found that CREB1 levels deter- mine the expression of TGFβ2. Our results show that CREB1 can be considered a biomarker to stratify patients for anti-TGFβ treatments and a therapeutic target in glioblastoma. SIGNIFICANCE: TGFβ is considered a promising therapeutic target, and several clinical trials using TGFβ inhibitors are generating encouraging results. Here, we discerned the molecular mechanisms respon- sible for the aberrantly high levels of TGFβ2 found in certain tumors, and we propose biomarkers to predict the clinical response to anti-TGFβ therapies. Cancer Discov; 4(10); 1230–41. ©2014 AACR. See related commentary by Wotton, p. 1123. 1 Vall d Hebron Institute of Oncology (VHIO), Barcelona, Spain. 2 Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron University Hospital, Barcelona, Spain. 3 Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain. 4 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). Corresponding Author: Joan Seoane, Vall d’Hebron Institute of Oncology, Psg. Vall d’Hebron 119-129, 08035 Barcelona, Spain. Phone: 34-93-489- 4167; Fax: 34-93-489-4015; E-mail: [email protected] doi: 10.1158/2159-8290.CD-14-0275 ©2014 American Association for Cancer Research. on May 17, 2021. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from Published OnlineFirst August 1, 2014; DOI: 10.1158/2159-8290.CD-14-0275

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Page 1: Active CREB1 Promotes a Malignant TGF b2 Autocrine Loop in ... · 1230 | CANCER DISCOVERYOCTOBER 2014 Active CREB1 Promotes a Malignant TGF b2 Autocrine Loop in Glioblastoma Laura

1230 | CANCER DISCOVERY�OCTOBER 2014 www.aacrjournals.org

Active CREB1 Promotes a Malignant TGFb2 Autocrine Loop in Glioblastoma Laura Rodón 1 , Alba Gonzàlez-Juncà 1 , María del Mar Inda 1 , Ada Sala-Hojman 1 , Elena Martínez-Sáez 2 , and Joan Seoane 1,3,4

RESEARCH ARTICLE

ABSTRACT In advanced cancer, including glioblastoma, the TGFβ pathway acts as an oncogenic

factor. Some tumors exhibit aberrantly high TGFβ activity, and the mechanisms

underlying this phenomenon are not well understood. We have observed that TGFβ can induce TGFβ2,

generating an autocrine loop leading to aberrantly high levels of TGFβ2. We identifi ed cAMP-responsive

element–binding protein 1 (CREB1) as the critical mediator of the induction of TGFβ2 by TGFβ. CREB1

binds to the TGFB2 gene promoter in cooperation with SMAD3 and is required for TGFβ to activate

transcription. Moreover, the PI3K–AKT and RSK pathways regulate the TGFβ2 autocrine loop through

CREB1. The levels of CREB1 and active phosphorylated CREB1 correlate with TGFβ2 in glioblastoma.

In addition, using patient-derived in vivo models of glioblastoma, we found that CREB1 levels deter-

mine the expression of TGFβ2. Our results show that CREB1 can be considered a biomarker to stratify

patients for anti-TGFβ treatments and a therapeutic target in glioblastoma.

SIGNIFICANCE: TGFβ is considered a promising therapeutic target, and several clinical trials using TGFβ

inhibitors are generating encouraging results. Here, we discerned the molecular mechanisms respon-

sible for the aberrantly high levels of TGFβ2 found in certain tumors, and we propose biomarkers to

predict the clinical response to anti-TGFβ therapies. Cancer Discov; 4(10); 1230–41. ©2014 AACR.

See related commentary by Wotton, p. 1123.

1 Vall d Hebron Institute of Oncology (VHIO), Barcelona, Spain. 2 Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron University Hospital, Barcelona, Spain. 3 Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain. 4 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain.

Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).

Corresponding Author: Joan Seoane, Vall d’Hebron Institute of Oncology, Psg. Vall d’Hebron 119-129, 08035 Barcelona, Spain. Phone: 34-93-489-4167; Fax: 34-93-489-4015; E-mail: [email protected]

doi: 10.1158/2159-8290.CD-14-0275

©2014 American Association for Cancer Research.

on May 17, 2021. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst August 1, 2014; DOI: 10.1158/2159-8290.CD-14-0275

Page 2: Active CREB1 Promotes a Malignant TGF b2 Autocrine Loop in ... · 1230 | CANCER DISCOVERYOCTOBER 2014 Active CREB1 Promotes a Malignant TGF b2 Autocrine Loop in Glioblastoma Laura

OCTOBER 2014�CANCER DISCOVERY | 1231

INTRODUCTION

Work over the past decade has shown that the TGFβ path-

way is a therapeutic target in advanced tumors. Step by step,

the oncogenic response to TGFβ is being elucidated, evidenc-

ing that TGFβ promotes tumor progression in a complex and

pleiotropic manner. A potent immune suppressor, TGFβ at

high levels facilitates tumor growth through the inhibition

of the anticancer immune response. On the other hand,

TGFβ has been shown to promote proliferation, angiogen-

esis, and invasion through the induction of metalloproteases,

to enhance the self-renewal of cancer-initiating cells (CIC),

and to mediate metastasis due in part to the induction of an

epithelial-to-mesenchymal transition ( 1–5 ).

The oncogenic response to TGFβ has prompted the design

of therapies based on the blockade of the TGFβ signal ( 6, 7 ).

Several inhibitors of the TGFβ pathway are being developed

in clinical trials, and some patients are benefi tting from anti–

TGFβ-based therapies ( 8, 9 ). However, we are still unable to

predict which patients will respond to the inhibition of the

TGFβ pathway. The identifi cation of biomarkers of response

to treatment is mandatory to stratify the patients who should

be treated with inhibitors of TGFβ. Importantly, high TGFβ

activity confers poor prognosis and, in this sense, one hypoth-

esis to be considered is that the aberrantly high TGFβ activity

found in some tumors might confer a selective advantage to

the tumor. Hence, the inhibition of the TGFβ signal in those

tumors could lead to a therapeutic effect. It is then crucial

to identify and study the characteristics of tumors that show

aberrantly high TGFβ activity.

The TGFβ ligands (TGFβ1, TGFβ2, and TGFβ3) bind

and activate a heterodimeric complex formed by the TβRII

and the TβRI and initiate an intracellular signaling cascade

through the phosphorylation (p) of the specifi c receptor-

regulated SMADs (R-SMAD) SMAD2 and SMAD3. The

phosphorylation of R-SMADs facilitates their binding to

SMAD4, and the SMAD complex shuttles to the nucleus,

where it regulates gene expression ( 10 ). Importantly, TGFβ is

a pleiotropic cytokine, and the response to the TGFβ signal

depends on the cellular context. The basis of the pleiotropic

response to TGFβ resides, in part, in the fact that SMADs

have low affi nity for DNA and they cooperate with other tran-

scription factors to bind gene promoters. Hence, the presence

or the transcriptional activity of the SMAD cofactors is what

determines the specifi c genes that are activated by TGFβ in a

particular cell ( 1 , 10 , 11 ).

Glioma is the most frequent primary tumor of the brain,

and glioblastoma (GBM , a grade 4 glioma) is the most aggres-

sive of human tumors, with virtually no effi cient therapies

( 12–15 ). We and others have recently shown that the TGFβ

pathway has a key role in GBM. The TGFβ pathway is aber-

rantly active in some GBM tumors and, moreover, elevated

p-SMAD2 (used as a readout of TGFβ activity) correlates

with poor prognosis in patients with GBM ( 2 , 16 ). However,

on May 17, 2021. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from

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Rodón et al.RESEARCH ARTICLE

not much is known about the molecular mechanisms under-

lying the abnormally high levels of TGFβ activity. Interest-

ingly, no genomic alterations have been identifi ed in GBM

components of the TGFβ pathway that could explain the

hyperactivity of TGFβ found in some tumors ( 1 ). The stroma

(infl ammatory cells in particular) is one of the sources of

TGFβ ( 17 ). Moreover, recent work from our group has shown

that the amplifi cation of the deubiquinating enzyme USP15 gene is responsible for the stabilization of the TβRI and

hyperactivation of the TGFβ pathway ( 18 ). Still, USP15 gene

amplifi cation (present in only 2% of GBMs) and stroma-

derived TGFβ do not account for the large proportion of

GBM tumors that present high TGFβ activity.

Here, we aimed to understand the molecular mechanisms

responsible for the increased activity of TGFβ in GBM. We

observed that TGFβ can induce the expression of TGFβ2,

thus generating an autocrine loop that leads to the accumu-

lation of TGFβ2, ending in a hyperactivation of the TGFβ

signal.

We went on to discern the mechanisms underlying the

TGFβ autocrine loop and identifi ed the cAMP-responsive

element–binding protein 1 (CREB1) as a critical mediator and

a SMAD cofactor in the induction of TGFβ2 by TGFβ. CREB1

belongs to the CREB/activating transcription factor (ATF)

family of transcription factors and is activated by means of

phosphorylation of its Ser133 residue by different kinases,

including AKT and ribosomal S6 kinase (RSK) ( 19–22 ). CREB

factors promote tumorigenesis in many cancers, including

non–small cell lung carcinoma, acute myelogenous leukemia,

and glioma ( 19 , 23–27 ). Interestingly, the CREB/ATF fam-

ily of transcription factors has been shown to regulate and

cooperate with SMADs in the transcriptional regulation of

multiple genes ( 28–31 ).

RESULTS TGFb1 and TGFb2 Are Highly Expressed in GBM and Confer Poor Prognosis

First, we decided to address whether the high activity of the

TGFβ pathway present in some GBM tumors was due to the

overproduction of the TGFβ ligands. Through the analysis

of public databases [Oncomine and Repository for Molecu-

lar Brain Neoplasia Data (REMBRANDT)], we analyzed the

mRNA levels of the TGFβ ligands, and we observed that the

levels of TGFB1 and TGFB2 mRNA were higher in GBM than

in normal brain tissue, while TGFB3 was expressed at similar

levels in GBM and in normal brain ( Fig.  1A ). Interestingly,

high levels of TGFB1 or TGFB2 conferred poor prognosis in

patients with GBM, and that was not the case for TGFB3 ( Fig. 1B ). On the basis of these results, we decided to discern

the molecular mechanisms involved in the overproduction of

TGFB2 mRNA.

Because the TGFB2 gene is not targeted for amplifi cation

in GBM (data not shown, The Cancer Genome Atlas), we

quickly discarded the possibility that TGFB2 mRNA overpro-

duction was due to an increase in gene dosage and decided

to focus on the transcriptional regulation of TGFB2 . We

observed that TGFB2 mRNA was induced by TGFβ1 in a GBM

cell line (LN229), whereas TGFB1 and TGFB3 mRNA levels

were not affected by TGFβ1 treatment ( Fig.  2A ). Moreover,

the effect of TGFβ on TGFB2 was not specifi c to the ligand

because TGFβ1, TGFβ2, and TGFβ3 induced the expression

Figure 1.   Expression of TGFβ ligands in GBM. A, expression of TGFB1 , TGFB2 , and TGFB3 mRNA in healthy brain tissue and GBM was examined in the Oncomine database. B, Kaplan–Meier curves showing the overall survival of patients with TGFB 1 , TGFB 2 , and TGFB 3 mRNA levels upregulated the indicated fold. Statistical signifi cance was assessed by the log-rank test. Data were obtained from the REMBRANDT program from the National Cancer Institute.

44

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OCTOBER 2014�CANCER DISCOVERY | 1233

TGFβ2 Autocrine Loop RESEARCH ARTICLE

of TGFB2 ( Fig. 2B ). As expected, the induction of the mRNA

levels resulted in an induction of TGFβ2 protein secretion, as

observed through ELISA ( Fig. 2C ). In addition, the induction

of TGFβ2 by TGFβ was blunted by the treatment with the

TβRI inhibitor LY-2109761 ( Fig. 2D ). This indicated that in

LN299 cells an autocrine loop was generated where any TGFβ

ligand induced TGFβ2 secretion and, in turn, TGFβ2 could

induce its own expression. We decided to evaluate whether

this phenomenon was unique for LN299 cells and analyzed

several cell lines (including GBM and non-GBM cell lines) for

the presence of the TGFβ2 loop. We observed that TGFβ1 did

not induce TGFβ2 in all GBM cell lines tested (e.g., A172),

and, on the other hand, the TGFβ2 loop was also present in

some non-GBM cell lines, such as HACAT, MCF-7, SUM-159,

and MDA-MB-231 ( Fig. 2E ).

CREB1 Regulates the Induction of TGFb2 by TGFb We went on to discern the molecular mechanisms

involved in the regulation of TGFβ2 by TGFβ and studied

the TGFB2 gene promoter region because our previous

data showed that the regulation was at the transcriptional

level. Looking for conserved regions among different ani-

mal species through sequence alignment, we found two

conserved SMAD-binding elements (SBE) surrounding a

cAMP response element (CRE) described to support CREB1

binding ( Fig.  3A ). CREB1 has been shown to interact and

cooperate with SMADs in the transcriptional activation of

certain genes ( 30, 31 ). This suggested that CREB1 cooper-

ates with SMADs to activate TGFB2 gene transcription. To

assess this hypothesis, we performed RNAi experiments

using independently designed shRNAs and siRNAs target-

ing CREB1 . Knockdown of CREB1 decreased the induction

of TGFB2 by TGFβ, indicating that CREB1 is necessary for

the regulation of TGFB2 transcription by TGFβ ( Fig. 3B and

C ). To further validate this result, we decided to develop an

alternative experimental approach using an endogenous

repressor of CREB1 called inducible cAMP early repressor

( ICER; ref. 32 ). Overexpression of ICER impaired the ability

Figure 2.   TGFβ induces TGFβ2 expression in GBM and non-GBM cell lines. A, qRT-PCR of TGFB 1 , TGFB 2 , and TGFB 3 in LN229 cells treated with TGFβ1 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, qRT-PCR of TGFB 2 in LN229 cells treated with TGFβ1, TGFβ2, and TGFβ3 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in culture supernatant from LN229 cells treated with TGFβ for 72 hours. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGF b 2 in LN229 cells treated with TGFβ1 and/or the TβRI inhibitor (TβRI inh.) LY-2109761 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. E, qRT-PCR of TGFB 2 in GBM and non-GBM cell lines treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD.

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Rodón et al.RESEARCH ARTICLE

of TGFβ to induce TGFβ2 ( Fig.  3D ). These results indi-

cated that CREB1 is required for the TGFβ autocrine loop.

To prove that CREB1 binds to the identifi ed CRE in the

proximal region of the TGFB2 promoter, we performed chro-

matin immunoprecipitation (ChIP) experiments. p-CREB1

and SMAD2 and/or SMAD3 bound to the proximal region

of the TGFB2 promoter, and the interaction was increased in

response to TGFβ ( Fig. 4A ). Moreover, we performed reporter

assays using a reporter construct containing the proximal

−77/+64 region of the TGFB2 promoter upstream of the luci-

ferase gene. A reporter with a mutation in the CRE-binding site

was also engineered. TGFβ mildly but consistently induced

the TGFB2 reporter. However, TGFβ was not able to induce

the transcriptional activity of the mutated version of the

TGFB2 reporter ( Fig. 4B ). We decided to immunoprecipitate

endogenous p-CREB1 and, interestingly, found that endog-

enous SMAD3, but not SMAD2, interacted with p-CREB1

in response to TGFβ ( Fig.  4C ). To further assess the role

of SMAD2 and SMAD3 in the process, we independently

knocked down SMAD2 and SMAD3 using RNAi. Knock-

down of SMAD2 did not affect the induction of TGFβ2 by

TGFβ, whereas knockdown of SMAD3 repressed the TGFβ2

autocrine loop ( Fig.  4D ). Together, our results showed that

p-CREB1 formed a complex with SMAD3 to bind the CRE

and the SBEs at the proximal region of the TGFB2 promoter

and induced the transcriptional activation of the promoter.

PI3K and RSK Regulate the TGFb2 Autocrine Loop through CREB1

CREB1 transcriptional activity is dependent on CREB1

phosphorylation at Ser133 by many different serine/threo-

nine protein kinases, including AKT and p90 RSK ( 19–22 ).

Phosphorylation at Ser133 is required for CREB1 binding

to the transcriptional coactivator CREB1-binding protein,

enabling the CREB1-dependent activation of transcription

( 33, 34 ). We reasoned that if CREB1 transcriptional activity

Figure 3.   CREB1 regulates the autocrine induction of TGFβ2 by TGFβ. A, nucleotide sequence of the proximal region of the TGFB 2 promoter. The SBEs and CREB1 site (CRE) are indicated relative to the transcription start site. ClustalW sequence alignment for 3 animal species [ Homo sapiens ( H.s .), Pan troglodytes ( P.t. ), and Mus musculus ( M.m .)] shows the conservation of the binding sites. B, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an shRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an siRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGFβ2 in LN229 cells expressing ICER treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. The molecular weights are shown.

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Control siCREB1

TGFβ – +– +

Control ICERTGFβ – +– +

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TGFβ2 Autocrine Loop RESEARCH ARTICLE

is required for the TGFβ2 autocrine loop, the signaling

pathways that regulate CREB1 activity should also affect

the induction of TGFβ2 by TGFβ. To validate this hypoth-

esis, we performed a pharmacologic approach using selec-

tive inhibitors of the PI3K–AKT pathway (LY-294002) and

RSK (BI-D1870). As expected, CREB1 phosphorylation was

reduced upon treatment with PI3K and RSK inhibitors,

indicating that CREB1 activity was regulated by the PI3K–

AKT and the RSK pathways ( Fig. 5A and B ). In cells treated

with PI3K or RSK inhibitors, TGFβ was not able to induce

TGFB2 mRNA at the same level as in control cells ( Fig. 5A

and B ). Moreover, both PI3K and RSK inhibitors impaired

TGFβ2 protein secretion induced by TGFβ as measured

by ELISA ( Fig.  5C and D ). Interestingly, the effect of the

inhibitor was more pronounced on the secretion of TGFβ2

than on the mRNA level of TGFB2 , possibly indicating a

putative regulation of the TGFβ loop at the level of pro-

tein translation. Further work is required to validate this

hypothesis.

TGFb2 Expression Correlates with CREB1 Protein Levels in Patient Tumors

We then hypothesized that if CREB1 is required for the

TGFβ2 autocrine loop, tumors with high levels of TGFβ2

might present elevated levels of CREB1 and p-CREB1.

Through the analysis of the REMBRANDT database, we

observed a positive correlation between TGFB2 and CREB1 mRNA levels in human GBM samples ( Fig.  6A ). Interest-

ingly, there were no tumors with high TGFB2 and low CREB1 (see the upper left quadrant of Fig.  6A ), showing the rel-

evance of the CREB1 transcription factor for the expression

of TGFB2 . The same analysis was performed in the case of

TGFB1 , and no correlation was observed between TGFB1 and

CREB1 levels ( Fig.  6B ). We then addressed whether TGFβ2

protein levels correlated with the levels of phosphorylated

CREB1 by performing an IHC analysis of a collection of

GBM samples. As expected, a positive correlation between

TGFβ2 and p-CREB1 staining was observed ( Fig. 6C ). Exam-

ples of high and low TGFβ2, p-CREB1–expressing tumors

are shown. Interestingly, using the same collection of sam-

ples, we determined that p-CREB1 also correlated with

p-SMAD2 levels (Supplementary Fig.  1). Again using the

REMBRANDT database, we observed that the patient popu-

lation with tumors expressing high TGFB2 and CREB1 had

a signifi cantly shorter overall survival (OS) than the rest

of the patients, indicating that combined upregulation of

p-CREB1 and TGFβ2 confers poor prognosis in patients

with GBM ( Fig. 6D ).

Figure 4.   CREB1 binds at the proximal region of the TGFB2 promoter and induces its transcriptional activation. A, ChIP of p-CREB1 and SMAD2/3 in LN229 cells treated with TGFβ for 1 hour. A qRT-PCR is shown with specifi c primers for the TGFB2 promoter. Fold enrichment is shown relative toIgG; ***, P < 0.005; *, P < 0.05 using the Student t test; data, mean ± SD. B, luciferase assay in LN229 transfected with the (−77/+64) wild-type (WT) or (−77/+64) mutCRE TGFB2 luciferase reporter constructs and treated with TGFβ for 32 hours; *, P < 0.05 using the Student t test; data, mean ± SD. C, immunoprecipitation (IP) of p-CREB1 in LN229 cells treated with TGFβ for 1 hour and immunoblot analysis of SMAD2/3. Asterisk, an unrelated band; arrowhead, SMAD3 band. D, qRT-PCR of TGFB2 and immunoblot analysis of the indicated proteins in LN229 cells expressing an siRNA targeting SMAD2 or SMAD3 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control; ***, P < 0.005 using the Student t test; data, mean ± SD.

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Rodón et al.RESEARCH ARTICLE

Figure 5.   PI3K and RSK regulate the TGFβ-mediated induction of TGFβ2 through CREB1. A, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with TGFβ for 3 hours and the PI3K inhibitor (inh) LY-294002 for 24 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with increasing amounts of the RSK inhibitor BI-D1870 for 24 hours and TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with TGFβ for 48 hours and the PI3K inhibitor for 72 hours. *, P < 0.05, using the Student t test; data, mean ± SD. D, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with the RSK inhibitor BI-D1870 for 72 hours and TGFβ for 48 hours. *, P < 0.05, using the Student t test; data, mean ± SD.

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Figure 6.   TGFβ2 correlates with CREB1 expression in GBM patient tumors. A and B, graphs showing the correlation between CREB1 and TGFB 1 (B) or TGFB 2 (A) mRNA levels in patient GBM tumor samples. Data obtained from the REMBRANDT database. A Spearman test was used, and the correlation coeffi cient (ρ) and the two-tailed P value are shown. C, graph showing the correlation between p-CREB1 and TGFβ2 protein levels in tissue microarrays (TMA) from patient GBM samples. Not all spots were evaluable in all stainings. A Spearman test was used, and the correlation coeffi cient (ρ) and the two-tailed signifi cance are shown. Representative images from the TMAs are shown; scale bar, 50 μm. D, Kaplan–Meier curves showing the OS of patients with TGFB2 mRNA levels upregulated ≥3-fold and CREB1 mRNA levels upregulated ≥2-fold. Statistical signifi cance was assessed by the log-rank test. Data obtained from the REMBRANDT database.

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TGFβ2 Autocrine Loop RESEARCH ARTICLE

CREB1 Regulates the Levels of TGFb2 in Patient-Derived Xenograft Models

We then decided to address the relevance of the CREB1-

dependent TGFβ2 autocrine loop in patients. To this end, we

used a patient-derived xenograft (PDX) model based on the

orthotopic inoculation using stereotaxis of freshly obtained

patient-derived tumor cells in the brains of immunocompro-

mised mice ( Fig.  7A ). The tumors generated in mice repro-

duce the same histopathologic characteristics and oncogenic

mutations as the tumor of the patient ( 4 ). We identifi ed two

patients who showed elevated expression levels of CREB1 and

TGFβ2 in their tumors. Neurospheres from both patients

were infected with lentivirus targeting CREB1, and cells were

inoculated in mice. Tumors originated from the cells express-

ing the shRNA targeting CREB1 , expressed lower levels of

CREB1 and, importantly, lower levels of TGFβ2 ( Fig.  7B ),

further demonstrating that CREB1 protein levels are crucial

for the expression of high levels of TGFβ2 in GBM.

Interestingly, mice inoculated with neurospheres expressing

shRNAs targeting CREB1 generated smaller and fewer tumors

than control neurospheres (Supplementary Fig. 2). Moreover,

the OS of these mice was longer than that of control mice

( Fig. 7C ). This is in agreement with our previous work in which

we showed that TGFβ regulates the ability of neurospheres to

initiate tumors ( 3, 4 ) and, moreover, the two selected models

respond to TGFβ receptor inhibitors (unpublished observa-

tions), indicating that in those two tumors the regulation of

the TGFβ activity is relevant for cancer progression.

DISCUSSION

The oncogenic function of the TGFβ pathway is progres-

sively being elucidated. In advanced tumors, TGFβ can induce

proliferation, invasion, angiogenesis, and immunosuppres-

sion ( 1, 2 ). In addition, TGFβ can increase the self-renewal

capacity of a cell population with stem-cell characteristics

called CICs ( 3–5 ). Because of its role in oncogenesis, TGFβ

is considered a therapeutic target, and several clinical trials

using anti-TGFβ agents are now under clinical development

( 6, 7 ). However, one of the crucial questions still remaining is

how to predict in which tumors TGFβ acts as an oncogenic

factor and, thus, which patients should be treated with anti-

TGFβ compounds.

Work from our laboratory and others has shown that

tumors present diverse levels of TGFβ activity. In particular

in GBM, we observed that some tumors show aberrantly

high TGFβ activity and this correlated with poor prognosis

( 16 ). The presence of extremely high TGFβ activity in the

most-aggressive tumors suggests that TGFβ may confer a

selective advantage to the tumor and, hence, the inhibition

of the TGFβ pathway might exhibit an antitumoral effect.

But why are there tumors with such high TGFβ activity?

Interestingly, the answer does not reside in the presence of

activating genomic alterations of components of the TGFβ

pathway as it happens in many other oncogenic pathways. In

fact, in GBM, the components of the TGFβ signal are seldom

mutated, and no gene amplifi cations of the TGFβ ligands

Figure 7.   CREB1 regulates TGFβ2 expression in PDX models. A, scheme showing the experimental procedure. B, IHC of p-CREB1 and TGFβ2 from mouse tumors 60 days after inoculation with neurospheres expressing shRNAs targeting CREB1 and control shRNAs; scale bar, 50 μm (C). Kaplan–Meier survival curves from mice in B. D, the TGFβ2 malignant autocrine loop. In GBM, TGFβ collaborates with the PI3K and RSK pathways through a CREB1–SMAD3 transcriptional complex to induce TGFβ2 expression. This leads to the generation of an autocrine loop and accumulation of TGFβ2 in the tumor, hyperactivation of TGFβ, and tumor progression.

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CREB1

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Rodón et al.RESEARCH ARTICLE

nor hyperactive mutations in the TGFβ receptors have been

described ( 1 ).

To discern the molecular mechanisms underlying the

increased TGFβ activity in GBM, we specifi cally focused on

TGFβ2. We observed that TGFβ induced TGFβ2 and that

in turn TGFβ2 induced its own expression. This generated a

self-feeding autocrine loop, leading to extremely high levels

of TGFβ2. However, the autocrine loop was not present in all

the cell lines tested, and hence the TGFβ2 response to TGFβ

depended on the cellular context. This is a typical character-

istic of the TGFβ signaling pathway. The specifi c responses

to TGFβ are determined by the characteristics of the cell that

receives the signal. Frequently, the pleiotropic response to

TGFβ is determined by the SMAD transcription complex.

SMADs have low affi nity for DNA, and they cooperate with

other transcription factors to bind to specifi c gene promoters.

Thus, the presence of a certain cofactor is what determines

the transcriptional response to TGFβ ( 10, 11 ). In the case

of the induction of TGFB2 by TGFβ, we identifi ed CREB1

as the SMAD cofactor. CREB1 interacts with SMAD3, not

SMAD2, binds to the TGFB2 promoter, and is required for the

TGFβ-dependent induction of TGFB2 . Still further studies are

required to understand why CREB1 is expressed in certain

tumors and not others. Most likely, the state of differentia-

tion of the tumor cell is what determines the expression of

CREB1.

We corroborated our data by analyzing human tumors.

A correlation between CREB1 or p-CREB1 and TGFβ2 was

observed and, more importantly, no tumor expressing low

levels of CREB1 presented high TGFB2 . This was not the case

for TGFβ1. Importantly, patients with tumors expressing

high levels of CREB1 and TGFB2 had a shorter OS than the

rest of the patients. Moreover, CREB1 transcriptional activity

is induced upon phosphorylation by many different protein

kinases that in turn are regulated by oncogenic signaling

pathways, such as the PI3K–AKT and the RSK pathways

( 19 , 21 , 22 ). This implies that to establish a TGFβ2 autocrine

loop, the tumor cell has to express CREB1 and, at the same

time, exhibit an active PI3K or RSK pathway to acquire a high

level of phosphorylated CREB1 ( Fig.  7D ). In this sense, we

observed that p-CREB1 correlated with TGFβ2 in patients

with GBM. Interestingly, although functional direct cross-

talk between the PI3K and TGFβ pathways has been described

in breast cancer ( 35 ), we did not observe a strong modulation

of TGFβ activity by the PI3K–AKT pathway and vice versa,

indicating that direct PI3K–TGFβ cross-talk is not present in

our GBM cells.

To functionally validate the relevance of CREB1 in the

regulation of TGFβ2 expression and its autocrine loop, we

decided to use in vivo models that recapitulate the human

tumor as faithfully as possible. Instead of using established

cell lines that are adapted to grow in vitro and, hence, diverge

from real tumors, we decided to use PDXs in which freshly

obtained GBM samples are stereotactically inoculated in

the brain of immunocompromised mice ( 4 ). Through loss-

of-function studies in two PDX models in which TGFβ2

and CREB1 were highly expressed, we demonstrated that

CREB1 expression was required for TGFβ2 expression and,

hence, showed that our proposed mechanism of regulation

of TGFβ2 levels is relevant in vivo in patient-derived models.

The TGFβ pathway is considered a therapeutic target in

GBM, and TGFβ inhibitors are currently under clinical develop-

ment, showing promising results ( 8, 9 ). Right now, it is crucial

to be able to predict which patients may benefi t from the inhi-

bition of the TGFβ pathway. Biomarkers to stratify patients to

be treated with inhibitors of TGFβ are needed. It is reasonable

to hypothesize that tumors with high TGFβ activity might have

better chances to respond to TGFβ inhibition, because high

TGFβ activity confers poor prognosis and provides a selective

advantage to the tumor. Our work sheds light on the molecu-

lar mechanisms underlying the aberrantly high levels of TGFβ2

found in tumors. The transcription factor CREB1 is crucial for

the generation of a malignant autocrine TGFβ2 loop, and hence

CREB1 expression/phosphorylation levels might be considered

a biomarker of response to anti-TGFβ treatments. Moreover,

because CREB1 has been described as a putative therapeutic

target ( 36, 37 ), our results suggest that compounds against

CREB1 could be effective anticancer agents due to their effect

on the oncogenic levels of TGFβ2.

METHODS Plasmids

The CREB1 short hairpin sequence used was 5′-GAGAGAG

GTCCGTCTAATG-3′, and the lentiviral knockdown sequences

targeting CREB1 were (i) 5′-ACCAACAAATGACAGTTCA-3′ and

(ii) 5′-TGAACTGTCATTTGTTGGT-3′ (Open Biosystems). Knock-

down of CREB1, SMAD2, and SMAD3 by siRNA was performed by

transfection of SmartPool siRNA (Dharmacon). Silencer Negative

Control No. 1 siRNA (Dharmacon) was used as negative control.

The TGFB 2 promoter constructs comprise a genomic DNA fragment

spanning bases −77 to +63 of TGF b 2 (relative to the transcriptional

start codon) cloned into pGL3-basic vector with or without a CREB1-

binding site mutation (CGTCAC to TGGCAC; ref. 38 ).

Antibodies and Reagents Specifi c antibodies against p-SMAD2, SMAD2, p-AKT, AKT,

p-CREB1, CREB1 (Cell Signaling Technology), TGFβ2, CREM (Santa

Cruz Biotechnology), and Tubulin (Sigma) were used for immunoblot

analysis and IHC. Antibodies against SMAD2/3 (Upstate), ChIPAb +

Phospho-CREB1 (Ser133; Millipore), and rabbit IgG (Upstate) were

used for ChIP and IP.

The treatments used were as follows: TGFβ1, TGFβ2, TGFβ3

(100 pmol/L; R&D Systems), PI3K inhibitor LY-294002 (10 μmol/L;

Merck Millipore), TβRI inhibitor LY-2109761 (Tocris), and RSK

inhibitor BI-D1870 (5 μmol/L; Axon Medchem BV).

Cell Culture GBM neurospheres were generated as described previously ( 4 , 39 ).

Briefl y, tumor samples were processed within 30 minutes after surgical

resection. Minced pieces of human GBM samples were digested with

collagenase I (200 U/mL; Sigma) and DNase I (500 U/mL; Sigma) in

PBS for 2 hours at 37°C with constant vigorous agitation. The single-

cell suspension was fi ltered through a 70-mm cell strainer (BD Falcon)

and washed with PBS. Finally, cells were resuspended and subsequently

cultured in neurosphere medium. The neurosphere medium consisted

of Neurobasal medium (GIBCO) supplemented with B27 (GIBCO),

L -glutamine (GIBCO), penicillin/streptomycin, and growth factors

(20 ng/mL EGF and 20 ng/mL FGF2; PeproTech). Human GBM speci-

mens were obtained from the Vall d’Hebron Hospital (Barcelona, Spain).

The clinical protocol was approved by the Vall d’Hebron Institutional

Review Board, with informed consent obtained from all subjects. The

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OCTOBER 2014�CANCER DISCOVERY | 1239

TGFβ2 Autocrine Loop RESEARCH ARTICLE

293T, A172, U373, U251, and LN229 cell lines were obtained from the

ATCC and cultured in DMEM supplemented with 10% FBS. Cell lines

were passaged for less than 6 months following resuscitation and were

not authenticated. Transient transfections were carried out using either

the calcium phosphate transfection method or Lipofectamine 2000

(Invitrogen). Lysates were collected 48 to 72 hours after transfection.

RNA Extraction , Retrotranscription , and Quantitative Real-Time PCR

Cells were seeded in 60-mm plates at 70% confl uency. After the

described treatments, cultured cells were disrupted in lysis buffer

from the RNeasy Mini Kit (Qiagen), and mRNA was purifi ed follow-

ing the manufacturer’s instructions. mRNA was quantifi ed, and 300

to 800 ng of mRNA from each sample was retrotranscribed using the

High Capacity cDNA Reverse Transcription Kit (Applied Biosystems)

following the product indications. After cDNA synthesis, quantita-

tive real-time PCR (qRT-PCR) was performed. All qRT-PCR was

performed using TaqMan probes from Applied Biosystems, accord-

ing to the manufacturer’s recommendations. Reactions were carried

out in an ABI 7900 sequence detector (PerkinElmer) and results were

expressed as fold change calculated by the C t method relative to the

control sample. GAPDH and POLR2A were used as internal normali-

zation controls.

Western Blot Analysis Cells were lysed in RIPA buffer supplemented with protease inhibi-

tors (Roche). Whole-cell extracts were quantifi ed using the BCA Protein

Assay Kit (Pierce) and were then separated on 7% to 12% SDS-PAGE

gels and transferred to polyvinylidene difl uoride membranes. Mem-

branes were blocked with 5% milk and probed with specifi c antibodies.

Blots were then incubated with a horseradish peroxidase–linked sec-

ond antibody and developed with chemiluminescence.

ELISA For the quantitative determination of TGFβ2 protein levels

secreted to the media, we used the Human TGFβ2 Quantikine

ELISA Kit (R&D Systems) following the manufacturer’s specifi ca-

tions. Supernatant from serum-starved cells was collected 72 hours

after the start of the indicated treatments.

siRNA Transfection LN229 cells were seeded and transfected with 100 pmol/L of siRNAs

against SMAD2 or SMAD3 using Lipofectamine 2000 (Invitrogen).

Silencer Negative Control No. 1 siRNA was used as a negative control.

Retroviral Infections The 293T Phoenix-Ampho cells were transfected using the calcium

phosphate transfection method with the retroviral vectors pLNCX2,

pLNCX2-ICER, pRetroSuper, and pRetroSuper-sh CREB1 . After

16 hours, medium was replaced, and recombinant retrovirus was

harvested for an additional 24 hours. For infection, medium con-

taining recombinant retrovirus was added to LN229 cells. Polybrene

(Sigma) was added at a concentration of 8 μg/mL. Phoenix-Ampho

cells were incubated in fresh medium for an additional 8 hours; after

this time, the LN229 cells were washed, and the medium containing

recombinant retrovirus from Phoenix-Ampho cells was added. This

process of infection was repeated two more times every 8 hours. Two

days later, antibiotic selection was added to the medium [puromycin

(1 μg/mL), neomycin (0.5 μg/mL), and higromycin (100 μg/mL)].

Lentiviral Infections The 293T cells were transfected using the calcium phosphate trans-

fection method with pMD2.G-enveloping plasmid, psPAX.2-packag-

ing plasmid, and either pGIPZ or pGIPZ-sh CREB1 . After 16 hours,

medium was replaced with neurosphere medium with 5 mmol/L of

sodium butyrate, and recombinant lentivirus was harvested for an

additional 24 hours. For infection, medium containing recombinant

lentivirus was added to previously dissociated neurospheres. Poly-

brene (Sigma) was added at a concentration of 8 μg/mL. Following

16 hours of incubation, the neurospheres were washed and incubated

in fresh neurosphere medium. The 293T cells were incubated in fresh

neurosphere medium containing sodium butyrate (5 mmol/L) for

24 hours, and a second round of neurosphere infection was repeated

as previously described.

Luciferase Assays Cells were transfected with different TGFB2 promoter reporter

constructs and pRL-TK Renilla luciferase plasmid (Promega) using

Lipofectamine 2000. After a 16-hour incubation at 37°C, cells were

treated with TGFβ for a further 32 hours. Luciferase counts were

measured using a Sirius Luminometer (Berthold).

The fi refl y luciferase activity was normalized with renilla luciferase

activity. Data are represented as relative activity (compared with basal

promoter activity) and are expressed as the mean ± SD of triplicates

from a representative experiment.

Tissue Microarrays and Immunohistochemical Staining For tissue microarray (TMA) generation, three 0.6-mm cores were

taken from separate areas, and each one was arrayed into recipient

blocks in a 1-mm–spaced grid.

Formalin-fi xed, paraffi n-embedded tissue sections were depar-

affi nized and hydrated. Antigen retrieval was performed using pH

6 Citrate Antigen Retrieval Solution (DAKO). Peroxidase blocking

was done with 3% H 2 O 2 at room temperature for 10 minutes. For

TGFβ2, slides were incubated with a blocking solution (10% nor-

mal goat serum, 2% BSA) for 1 hour at room temperature. TGFβ2

antibody (Santa Cruz Biotechnology; sc-90) was used at a 1:500

dilution, p-CREB1 (Cell Signaling Technology; 9198) was used at

a 1:100 dilution, and p-SMAD2 (Cell Signaling Technology; 3108)

was used at a 1:250 dilution. A human-specifi c anti-Nestin antibody

was used at 1:200 dilution, to determine tumor area. As a detection

system, EnVision FLEX + (DAKO) was used following the manu-

facturer’s instructions and developed with freshly prepared 0.05%

3′,3-diaminobenzidine tetrahydrochloride. Finally, the slides were

counterstained with hematoxylin, dehydrated, and mounted. Positive

and negative controls were performed in each run. The quantifi cation

of the staining was expressed as H score. The H score was determined

by the formula: 3 × percentage of strong staining + 2 × percentage of

moderate staining + percentage of weak staining, giving a range of 0

to 300 for the H scores.

Chromatin Immunoprecipitation LN229 cells were grown to 70% confl uence, serum starved for

16 hours, and cultured in the presence or absence of TGFβ for 1 hour.

Cells were trypsinized and crosslinked in 1% formaldehyde for 10 min-

utes at room temperature. Crosslinking was quenched with a glycine

solution (0.125 mol/L) for 5 minutes in formaldehyde, and cells were

washed twice with PBS. Pelleted cells were lysed in 1 mL ChIP buffer

(1 volume of SDS buffer with 0.5 volumes of Triton buffer), and soni-

cated in a Bioruptor (Diagenode). Soluble material was quantifi ed by

Bradford assays, and p-CREB1 and SMAD2/3 were immunoprecipi-

tated from 1,000 μg of protein. Antibodies were incubated overnight

with the chromatin in a 500 μL volume. Immunocomplexes were

recovered with 30 μL of a protein A/G bead slurry. Immunoprecipi-

tated material was washed three times with a low-salt buffer and once

with a high-salt buffer. DNA complexes were decrosslinked in 100 μL

decrosslink buffer (1% SDS and 100 mmol/L NaHCO 3 ) at 65°C for

3 hours, and DNA was then eluted in 100 μL of water using a PCR

purifi cation kit (Qiagen). DNA (2 μL) was used for each qRT-PCR

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Rodón et al.RESEARCH ARTICLE

reaction with SYBR green (Roche). Antibodies against SMAD2/3

(Upstate), ChIPAb + Phospho-CREB1 (Ser133; Millipore), and rabbit

IgG (Upstate) were used for ChIP. The proximal TGFB 2 promoter

primer set used spans −84 to −18 relative to the transcriptional start site.

Immunoprecipitation LN229 cells were grown to 70% confl uence, serum starved for 16

hours, and cultured in the presence or absence of TGFβ1 for 1 hour.

Cells were washed with cold PBS and lysed with RIPA. The protein

lysate was precleared and incubated overnight at 4°C on a rotator

in IP Buffer (0.25 mol/L NaCl, 0.1% NP40, and 50 mmol/L Hepes

pH7.3) with the p-CREB1 antibody (Millipore) or normal rabbit IgG.

Then, 30 μL of protein A/G PLUS-Agarose (Santa Cruz Biotechnol-

ogy) was added and incubated for 1 hour at 4°C on a rotator. Samples

were washed fi ve to six times with IP buffer and resolved by SDS–

PAGE, as described previously.

Intracranial Tumor Assay All mouse experiments were approved by and performed according

to the guidelines of the Institutional Animal Care Committee of the

Vall d’Hebron Research Institute in agreement with the European

Union and national directives. The cells were stereotactically inocu-

lated into the corpus striatum of the right brain hemisphere (1 mm

anterior and 1.8 mm lateral to the bregma; 2.5 mm intraparenchy-

mal) of 9-week-old NOD/SCID mice (Charles River Laboratories).

Mice were euthanized when they presented neurologic symptoms or

a signifi cant loss of weight.

Statistical Analyses Student t tests were performed for statistical analyses. Data in all

graphs are represented as means ± SD of biologic triplicates. A Spear-

man correlation test was used to analyze the relationships between

CREB1, p-CREB1, and TGFβ2.

Disclosure of Potential Confl icts of Interest No potential confl icts of interest were disclosed.

Authors’ Contributions Conception and design: L. Rodón, J. Seoane

Development of methodology: L. Rodón, M. del Mar Inda,

A. Sala-Hojman

Acquisition of data (provided animals, acquired and managed

patients, provided facilities, etc.): A. Gonzàlez-Juncà, A. Sala-Hojman

Analysis and interpretation of data (e.g., statistical analysis,

biostatistics, computational analysis): A. Gonzàlez-Juncà, M. del

Mar Inda, A. Sala-Hojman, E. Martínez-Sáez, J. Seoane

Writing, review, and/or revision of the manuscript: L. Rodón,

A. Gonzàlez-Juncà, M. del Mar Inda, A. Sala-Hojman, E. Martínez-Sáez,

J. Seoane

Administrative, technical, or material support (i.e., reporting or

organizing data, constructing databases): A. Sala-Hojman

Study supervision: J. Seoane

Grant Support This work has been supported by a European Research Council

grant (ERC 205819), Asociación Española contra el Cancer (AECC),

Josef Steiner Cancer Research Foundation, and grant FIS (FI11/00198).

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.

Received March 14, 2014; revised July 28, 2014; accepted July 28,

2014; published OnlineFirst August 1, 2014.

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2014;4:1230-1241. Published OnlineFirst August 1, 2014.Cancer Discovery   Laura Rodón, Alba Gonzàlez-Juncà, María del Mar Inda, et al.   Glioblastoma

2 Autocrine Loop inβActive CREB1 Promotes a Malignant TGF

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