Transforming Growth Factor-β Promotes Survival of Mammary Carcinoma Cells through Induction of Antiapoptotic Transcription Factor DEC1

Transforming growth factor-β (TGF-β) signaling facilitates tumor growth and metastasis in advanced cancer. In the present study, we identified differentially expressed in chondrocytes 1 (DEC1, also known as SHARP2 and Stra13) as a downstream target of TGF-β signaling, which promotes the survival of breast cancer cells. In the mouse mammary carcinoma cell lines JygMC(A) and 4T1, the TGF-β type I receptor kinase inhibitors A-44-03 and SB431542 induced apoptosis of cells under serum-free conditions. Oligonucleotide microarray and real-time reverse transcription-PCR analyses revealed that TGF-β induced DEC1 in these cells, and the increase of DEC1 was suppressed by the TGF-β type I receptor kinase inhibitors as well as by expression of dominant-negative TGF-β type II receptor. Overexpression of DEC1 prevented the apoptosis of JygMC(A) cells induced by A-44-03, and knockdown of endogenous DEC1 abrogated TGF-β–promoted cell survival. Moreover, a dominant-negative mutant of DEC1 prevented lung and liver metastasis of JygMC(A) cells in vivo. Our observations thus provide new insights into the molecular mechanisms governing TGF-β–mediated cell survival and metastasis of cancer. [Cancer Res 2007;67(20):9694–703]

Apoptosis is an important mechanism of negative regulation of cancer development and metastasis (1). During the process of metastasis, apoptosis occurs through various mechanisms; after the detachment of tumor cells from the extracellular matrix and the neighboring cells at primary tumor sites, forms of cell death known as anoikis and amorphosis occur. In the bloodstream, cell death occurs through immune surveillance and/or destruction by mechanical stress. At sites of secondary metastasis, cell death occurs after extravasation during the phase of formation of micrometastasis. These findings suggest that the metastatic potential of cancer cells is closely associated to their resistance to apoptosis.

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates the growth, differentiation, and apoptosis of various types of cells. TGF-β transduces signals through two distinct serine-threonine kinase receptors, termed type I (TβR-I) and type II (TβR-II). TβR-I is activated by TβR-II on ligand binding and transduces signals through various proteins, of which Smad proteins are the major signal transducers for TGF-β (2, 3). Activated TβR-I phosphorylates receptor-regulated Smads (R-Smads; i.e., Smad2 and Smad3), which interact with common mediator Smad (Smad4) and translocate to the nucleus. Nuclear Smad complexes bind to various transcription factors and transcriptional coactivators and regulate transcription of target genes. Transcriptional corepressors, including c-Ski and SnoN, inhibit TGF-β signaling through interaction with Smad complexes. Inhibitory Smads, including Smad6 and Smad7, are induced by TGF-β and bone morphogenetic proteins (BMP), bind to type I receptors, and prevent phosphorylation of R-Smads, resulting in inhibition of TGF-β family signaling.

TGF-β exerts both inhibitory and stimulatory effects on the progression of tumors (4, 5). In early stages of carcinogenesis, TGF-β serves as a tumor suppressor through inhibition of cell growth. Thus, mutations of signal components of TGF-β, including TβR-II and Smad4, have been reported to be responsible for progression of certain gastrointestinal tumors (6, 7). However, some tumor cells escape the growth inhibition induced by TGF-β, and TGF-β facilitates the progression and metastasis of tumors in advanced stages of cancer. TGF-β induces epithelial-to-mesenchymal transdifferentiation (EMT) in mammary epithelial cells. TGF-β also acts on the tumor microenvironment, where it stimulates angiogenesis and tissue fibrosis and causes local and systemic immunosuppression, leading to progression and metastasis of tumors.

TGF-β induces apoptosis of various types of cells, and the mechanisms of TGF-β–induced apoptosis have been extensively studied. Smad proteins play critical roles in execution of TGF-β–induced death in certain types of cells (8). Several genes regulated by Smad transcriptional complexes have been reported to be involved in TGF-β–mediated apoptosis, including DAP-kinase, SHIP, and GADD45β (8). In addition, certain molecules including the adaptor protein Daxx have been reported to be directly activated by TβR-II and to induce cell death through activation of c-Jun NH₂-terminal kinase (9). Most of these molecules regulate the expression of members of the Bcl-2 family and activate various caspases. On the other hand, it is becoming evident that TGF-β also promotes cell survival under certain conditions (8). TGF-β has been shown to promote cell survival through activation of the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, which plays a key role in mediating cell survival downstream of tyrosine kinase signaling, including that mediated by insulin-like growth factor or platelet-derived growth factor (PDGF).

Several studies have revealed recently that targeting of TGF-β signaling in breast cancer cells by various strategies, such as use of soluble TβR-II antagonist, neutralizing TGF-β antibody, or small-molecule type I receptor kinase inhibitors, can prevent cancer metastasis (10–13). Consistent with this, we showed previously that systemic gene transfer of Smad7 inhibits the metastasis of mouse mammary carcinoma JygMC(A) cells in an in vivo experimental model (14). Thus, TGF-β is considered one of the molecular targets in the treatment of breast cancer metastasis. However, little is known about the downstream signaling pathways of TGF-β involved in the process of metastasis of breast cancer.

Here, we present the first evidence that the roles played by TGF-β signaling in survival of breast cancer cells are mediated by induction of differentially expressed in chondrocytes 1 (DEC1, also known as SHARP2 and Stra13). DEC1 is a basic helix-loop-helix transcription factor, which is frequently overexpressed in certain cancers, including breast carcinomas (15). Correlation between the expression of DEC1 and tumor grade in breast cancer has been reported (16). We found that TGF-β induces DEC1 and prevents apoptosis of mouse mammary carcinoma cells. In addition, we show that a dominant-negative mutant of DEC1 (dnDEC1) prevents lung and liver metastasis of breast cancer cells in vivo. Our observations thus provide new insight into the molecular mechanisms governing TGF-β–mediated survival of tumors and may aid the development of new strategies for cancer therapy.

Cell culture and reagents. Mouse mammary carcinoma 4T1 cells and mouse mammary epithelial NMuMG cells were obtained from American Type Culture Collection. 4T1 cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. NMuMG cells were maintained in DMEM containing 10% FBS, 10 μg/mL insulin, and antibiotics. Mouse mammary carcinoma JygMC(A) cells were cultured as described previously (14). Cells were grown in a 5% CO₂ atmosphere at 37°C. The TβR-I kinase inhibitor used in the present study, A-44-03, is a dihydrochloride salt form of A-77-01 (17). SB431542 was obtained from Sigma. LY294002 and STI571 (imatinib mesylate/Gleevec) were purchased from Calbiochem and Novartis Pharma, respectively. The contents of one capsule of STI571 were dissolved in 17 mL of distilled water, centrifuged, filtered, and used as 10 mmol/L stock solution (18).

Apoptosis assays. For detection of cytosolic DNA ladder formation, both floating cells and adherent cells were collected and lysed with a lysis buffer [20 mmol/L Tris-HCl (pH 7.5), 10 mmol/L EDTA, 0.5% Triton X-100]. Cell extracts were incubated with 0.1 mg/mL RNase A and 0.2 mg/mL proteinase K at 42°C for 1 h. DNA was purified by standard phenol-chloroform extraction and ethanol precipitation. Dry DNA pellets were then resuspended in TE containing 0.2 mg/mL RNase A, and samples were electrophoretically separated on 2% agarose gel containing 0.01% ethidium bromide. For terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assays, cells were fixed in 4% paraformaldehyde. After permeabilization in PBS containing 0.1% Triton X-100 and 0.1% sodium citrate, reagents of the In situ Cell Death Detection kit (TMR red, Roche Diagnostics) were added. The nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence was examined using an IX71 microscope (Olympus) and measured by the Integrated Intensity Program of MetaVue (Molecular Devices Corp.).

Quantitative real-time reverse transcription-PCR. Total RNAs were extracted using Trizol reagent (Invitrogen). First-strand cDNAs were synthesized using the SuperScript First-Strand Synthesis System (Invitrogen) with oligo(dT) primers. Quantitative real-time reverse transcription-PCR (RT-PCR) was done as described previously (13). The primer sequences were as follows: mouse DEC1: 5′-GAAGCACGTGAAAGCATTGACA (forward) and 5′-CCCGACAAATCACCAGCTTG (reverse); mouse plasminogen activator inhibitor-1 (PAI-1): 5′-CCACAAAGGTCTCATGGACCAT (forward) and 5′-TGAAAGTGTTGTGCCCTCCAC (reverse); and mouse hypoxanthine phosphoribosyltransferase 1 (HPRT1): 5′-CTGGTTAAGCAGTACAGCCCCA (forward) and 5′-GGTCCTTTTCACCAGCAAGCT (reverse). All samples were run in duplicate in each experiment. Values were normalized to mouse HPRT1.

Immunoblotting. Cells were lysed after various treatments with NP40 lysis buffer [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% NP40, 1% aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride]. The supernatants were measured for protein concentrations, and those with equal amounts of total proteins were applied to 8.5% SDS-gel electrophoresis, followed by semidry transfer of the proteins to Pall FluoroTtrans W membrane (Pall Life Sciences). Nonspecific binding of proteins to the membrane was blocked by incubation in TBS-T buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.1% Tween 20] containing 5% skim milk. Separated proteins were immunoblotted with anti-Flag antibody (M2; Sigma), anti–phospho-Smad2 (Ser^465/467) antibody (Cell Signaling Technology), or anti-Smad2/3 antibody (BD Transduction Laboratories). Detection of immunoblotted proteins was done by enhanced chemiluminescence.

Construction of recombinant adenoviruses and adenoviral infection. Recombinant E1-deleted adenoviral vectors carrying cDNAs encoding β-galactosidase (LacZ) reporter gene, Smad6, Smad7, and c-Ski were described previously (19). cDNAs encoding Flag-tagged dominant-negative TβR-II (dnTβR-II), which lacks the intracellular domain of TβR-II, and Flag-tagged human DEC1 were subcloned into the pAxCAwtit cassette cosmid (Takara). Each cosmid carrying the expression unit and adenovirus DNA terminal protein complex was cotransfected into E1 transcomplemental cell line 293 cells. The recombinant adenoviruses generated by homologous recombination were isolated. For adenoviral infection, 5 × 10⁵ of JygMC(A) cells per well in six-well plates were infected with adenovirus vectors at 30 to 300 plaque-forming units per cell.

Microarray analysis. mRNAs were extracted from JygMC(A) cells treated without or with TGF-β3 and those infected with Ad-LacZ or Ad-Smad7. Total RNAs were used to prepare cDNA and conduct oligonucleotide microarray analysis using GeneChip Mouse Genome 430 2.0 Array (Affymetrix) according to the manufacturer's instructions. FileMaker Pro software (Filemaker, Inc.) was used for statistical analysis.

RNA interference and oligonucleotides. Stealth small interfering RNA (siRNA) duplex oligoribonucleotides against mouse DEC1 were synthesized by Invitrogen. JygMC(A) cells were transfected in the presence of 150 pmol of either siRNA or control siRNA in a 500 μL volume with 7.5 μL LipofectAMINE RNAiMAX reagent (Invitrogen) per well of a six-well plate according to the manufacturer's protocols. To confirm knockdown of DEC1, cells were harvested 24 h after siRNA transfection and subjected to quantitative real-time RT-PCR.

Generation of JygMC(A) cells stably expressing dnDEC1. To establish JygMC(A) cells that stably express the dnDEC1 (Jyg-dnDEC1), a human DEC1 mutant lacking the basic region was cloned into pCAG-IRES-Puro expression vector (14). The pCAG-Flag-dnDEC1-IRES-Puro plasmid was introduced into JygMC(A) cells using LipofectAMINE 2000 reagent (Invitrogen). Stable clones were obtained by puromycin (Sigma) selection (8 μg/mL) in the culture medium, and several clones were then isolated by limiting dilution. JygMC(A) cells stably expressing the empty vector pCAG-empty-IRES-Puro (Jyg-empty) were used as a control.

In vivo experiment using JygMC(A) cells stably expressing dnDEC1. To investigate in vivo tumor growth and metastasis of parental JygMC(A), Jyg-empty, or Jyg-dnDEC1, a mouse experimental model of metastasis was used. All animal procedures were done in the animal experiment laboratory of the Japanese Foundation for Cancer Research (JFCR) according to the guidelines proposed by the Science Council of Japan. Female BALB/c nu/nu mice (4 weeks of age) were purchased from Charles River Japan. Mice were maintained under specific pathogen-free conditions. Parental JygMC(A), Jyg-empty, or Jyg-dnDEC1 cells (10⁷ cells) were xenografted into the mammary fat pad of each mouse (n > 6 mice per group). Primary tumor growth and metastases were examined as described previously (14). Statistical differences to controls were validated by the two-sided Student's t test. P < 0.05 was considered significant.

Antiapoptotic effects of exogenous and endogenous TGF-β on murine normal epithelial and breast cancer cells. Murine breast carcinoma JygMC(A) cells undergo cell death when cultured in low concentrations of serum. Interestingly, TGF-β promoted the survival of the JygMC(A) cells under these conditions, and the low-molecular-weight TβR-I kinase inhibitor A-44-03 further enhanced the death of JygMC(A) cells (Fig. 1A). Regulation of cell survival by TGF-β signaling was further investigated by DNA ladder formation and TUNEL assay. DNA ladder formation was observed under serum-free conditions, but not in the presence of 10% serum (Fig. 1B). TGF-β suppressed DNA ladder formation, whereas A-44-03 enhanced it under these conditions, indicating that inhibition of TGF-β signaling leads to cell death by apoptosis in JygMC(A) cells. Apoptosis of JygMC(A) cells was also observed in a TUNEL assay under serum-free conditions, and the cell survival–promoting effect of TGF-β was confirmed (Fig. 1C and D). Another low-molecular-weight TβR-I kinase inhibitor SB431542, which is less potent than A-44-03 (17), also induced apoptosis of JygMC(A) cells, although 10 μmol/L SB431542 was required to induce their apoptosis in the presence of 1 ng/mL TGF-β3 (Supplementary Fig. S1A and B). These findings suggest that exogenous TGF-β promotes the survival of JygMC(A) cells and that endogenous TGF-β produced by JygMC(A) cells functions as a prosurvival factor for these cells.

To determine whether the survival-promoting effect of TGF-β is limited to JygMC(A) cells, we examined TGF-β–mediated survival of other mammary epithelial cells (i.e., murine breast cancer 4T1 cells and murine normal epithelial NMuMG cells). Similar to JygMC(A) cells, A-44-03 induced apoptosis of 4T1 cells (Fig. 2A and B). Although A-44-03 failed to significantly induce apoptosis of NMuMG cells, TGF-β potently enhanced their survival. It is important to note that Smad2 was significantly phosphorylated in the absence of exogenous TGF-β in JygMC(A) cells and 4T1 cells, but only very weakly in NMuMG cells (Fig. 2C, compare lane 2 and lane 5 with lane 8). These findings suggest that cell survival mediated by autocrine TGF-β signaling may be related to tumor growth and metastasis.

Roles of PDGF receptor and Akt signaling in the promotion of cell survival by TGF-β. To elucidate the mechanisms by which TGF-β promotes the survival of JygMC(A) cells, we did cDNA microarray analysis. Smad7 inhibits TGF-β family signaling through direct binding to activated type I receptors as well as by other mechanisms (20). The Smad7 gene was infected into JygMC(A) cells by an adenoviral vector, and genes regulated by TGF-β signals were determined with the Affymetrix GeneChip Mouse Genome 430 2.0 Array (Supplementary Table S1). The results of the microarray analysis were confirmed by quantitative real-time RT-PCR analysis. Among 59 genes regulated by TGF-β, we were interested in the Pdgfb gene (encoding PDGF-B chain) because it has been reported to mediate TGF-β–induced proliferation of glioma cells, and epigenetic regulation of the human PDGFB gene is closely related to the prognosis of glioblastoma (21). We confirmed by real-time RT-PCR that expression of PDGF-B mRNA was induced by TGF-β and suppressed by A-44-03 (Supplementary Fig. S2A).

Because it has been reported that TGF-β induces cell survival by activation of PI3K-Akt signaling (8), we examined whether an inhibitor of PDGF receptor signaling (STI571) or one of Akt signaling (LY294002) inhibits the promotion of cell survival by TGF-β under serum-free conditions. Although STI571 and LY294002 induced apoptosis of JygMC(A) cells in the absence of TGF-β, neither of them was able to induce apoptosis of JygMC(A) cells in the presence of exogenous TGF-β (Supplementary Fig. S2B and C). Although the combination of STI571 and LY294002 strongly induced apoptosis of JygMC(A) cells in the absence of TGF-β, it induced their apoptosis only weakly in the presence of TGF-β (Supplementary Fig. S2B and C).

Promotion of the survival of JygMC(A) cells by TGF-β is mediated by DEC1. Because PDGF receptor and PI3K-Akt signaling may not be the major signaling pathways involved in TGF-β–mediated cell survival of JygMC(A) cells, we further analyzed the genes regulated by TGF-β in JygMC(A) cells. Among the 59 genes regulated by TGF-β, we found that mRNA for Dec1 (also termed basic helix-loop-helix domain containing, class B2) was strongly suppressed by Smad7 in the presence of serum (Supplementary Table S1). We confirmed the effects of TGF-β and A-44-03 on the transcription of DEC1 mRNA in the presence or absence of 10% serum (Fig. 3A). Quantitative real-time RT-PCR analysis revealed that A-44-03 suppressed the transcription of DEC1 mRNA in JygMC(A) cells in the presence and absence of 10% serum. Although TGF-β induced the transcription of DEC1 mRNA only weakly in the presence of 10% serum, significant increase in DEC1 mRNA by TGF-β was detected in the absence of serum in JygMC(A) cells. SB431542 also suppressed the expression of DEC1 mRNA in serum-free conditions (Supplementary Fig. S1C). We also assessed the expression of DEC1 in serum-free conditions using other mammary epithelial cells (Fig. 3A). Similar to JygMC(A) cells, 4T1 cells exhibited modest induction of DEC1 mRNA by TGF-β and strong suppression of expression of it by A-44-03. Although TGF-β strongly induced DEC1 mRNA in NMuMG cells, significant suppression of DEC1 mRNA expression by A-44-03 could not be observed because the basal level of expression of DEC1 mRNA in NMuMG cells was very low. It should be noted that these profiles of expression of DEC1 mRNA correlated closely with the pattern of apoptosis induced by A-44-03 treatment, as shown in Fig. 2B. TGF-β has also been shown to promote cell survival in human breast cancer MDA-MB-231 cells (22). In agreement with this finding, induction of DEC1 mRNA by TGF-β was also observed in MDA-MB-231 cells (Supplementary Fig. S3).

We next examined the effect of DEC1 on the apoptosis induced by A-44-03 by infection of adenovirus carrying DEC1 cDNA. Expression of DEC1 protein after adenovirus infection was confirmed by immunoblot analysis (Fig. 3B). A-44-03 induced apoptosis of JygMC(A) cells in the absence of serum, and neither control adenovirus carrying the LacZ gene nor stimulation by TGF-β significantly affected the apoptosis induced by A-44-03 (Fig. 3C and D). However, infection of adenovirus carrying DEC1 resulted in potent suppression of apoptosis of the cells induced by A-44-03. These findings suggested that the promotion of the survival of JygMC(A) cells by TGF-β is mediated by DEC1.

Promotion of survival of JygMC(A) cells by TGF-β is mediated in Smad-dependent fashion. To determine whether TGF-β prevents apoptosis of JygMC(A) cells in Smad-dependent fashion and whether the expression of DEC1 is indeed mediated by the TGF-β signaling pathway, we examined the effects of adenoviruses carrying various negative regulators of TGF-β signaling on TGF-β–mediated cell survival. Among the negative regulators of TGF-β signaling, c-Ski binds to Smad complexes and suppresses their transcriptional activity as a transcriptional corepressor, whereas dnTβR-II binds to TGF-β but fails to transduce intracellular signals and acts as a dominant-negative inhibitor of Smad-dependent and non-Smad pathways (20, 23). Smad7 inhibits both TGF-β and BMP signaling, whereas Smad6 inhibits BMP but not TGF-β signaling efficiently (20). As shown in Fig. 4A, infection of these adenoviruses resulted in efficient expression of transfected proteins in JygMC(A) cells. Analysis of the expression of a TGF-β target gene, PAI-1, revealed that dnTβR-II, Smad7, and c-Ski effectively suppressed TGF-β signaling, whereas Smad6 failed to do so (Fig. 4B). Apoptosis of JygMC(A) cells was determined by TUNEL assay in the absence or presence of TGF-β under serum-free conditions (Fig. 4C). dnTβR-II, Smad7, and c-Ski, but not Smad6, induced apoptosis of JygMC(A) cells in the presence and absence of TGF-β. Because c-Ski inhibits TGF-β signaling through interaction with Smad2/3 and Smad4, these findings suggest that TGF-β induces survival of JygMC(A) cells in Smad-dependent fashion. We also confirmed that expression of DEC1 is suppressed by dnTβR-II as well as by Smad7 and c-Ski (Fig. 4D), indicating that the expression of DEC1 is mediated by TGF-β signaling.

Silencing endogenous DEC1 abolishes the prosurvival effect of TGF-β. Because forced expression of DEC1 rescued the induction of apoptosis of JygMC(A) cells by A-44-03, we further examined the role of endogenous DEC1 in TGF-β–promoted survival of JygMC(A) cells by the siRNA method. As shown in Fig. 5A, we efficiently knocked down endogenous DEC1 mRNA in the cells by transfection of siRNA targeting DEC1. Interestingly, reduction of DEC1 mRNA expression resulted in significant increase in the number of apoptotic cells in the absence or presence of TGF-β under serum-free conditions (Fig. 5B and C). These findings suggest that the promotion of cell survival by TGF-β is dependent on DEC1, which is transcriptionally induced by endogenous TGF-β.

dnDEC1 induces apoptosis of JygMC(A) cells and inhibits their metastasis. Acquisition of resistance to apoptosis is thought to be related to the metastatic phenotype of cancer cells (1). Because DEC1 seemed to act as an important regulator of survival of breast cancer cells, we examined whether the DEC1-mediated survival signal is involved in the process of metastasis. We used a mutant of DEC1 lacking the basic region (dnDEC1), which acts in dominant-negative fashion (24). JygMC(A) cells, which stably express dnDEC1, were established and two stable clones, termed Jyg-dnDEC1#1 and Jyg-dnDEC1#2, were used for further investigations (Fig. 6A,, top). As expected, both Jyg-dnDEC1#1 cells and Jyg-dnDEC1#2 cells underwent apoptotic cell death in serum-starved conditions (Fig. 6A , bottom). Furthermore, overexpression of the dnDEC1 mutant induced apoptosis even in the presence of TGF-β, suggesting that dnDEC1 effectively antagonizes TGF-β–mediated cell survival.

Finally, we investigated the growth and metastatic potential of Jyg-dnDEC1 cells in in vivo experiments. After s.c. inoculation into nude mice and formation of primary tumors, JygMC(A) cells metastasize to lung and liver within 3 weeks (14). Growth of the primary tumors and metastases to lung and liver in mice bearing parental JygMC(A), Jyg-empty, and Jyg-dnDEC1 cells are shown in Fig. 6B, to D. Growth of the primary tumors did not differ significantly between the mice bearing parental JygMC(A), Jyg-empty, and Jyg-dnDEC1 cells (Fig. 6B). However, the lungs and livers in the mice bearing Jyg-dnDEC1#1 or Jyg-dnDEC1#2 weighed less than those in mice bearing parental JygMC(A) or Jyg-empty (Fig. 6D,, top). Intriguingly, stable expression of dnDEC1 in JygMC(A) cells produced few metastatic nodules in the lungs and liver (Fig. 6C  and D, bottom). These findings indicate that DEC1 contributes to the metastasis of JygMC(A) cells by promoting cell survival without affecting primary tumor growth in vivo.

In the present study, we showed that serum starvation induced apoptosis of JygMC(A) cells and that suppression of endogenous TGF-β signaling by the TβR-I kinase inhibitors strongly enhanced this apoptosis, suggesting that autocrine TGF-β signaling in JygMC(A) cells supports their own survival.

Autocrine TGF-β induces survival of breast cancer cells. Although it has been reported that TGF-β causes apoptosis in certain types of cells (8), apoptosis is induced by exogenous TGF-β at much higher concentrations than those required for inhibition of cell growth. Lei et al. (22) reported that disruption of autocrine TGF-β signaling by the ectopic expression of a soluble TGF-β type III receptor induces apoptosis of human breast cancer MDA-MB-231 cells. Their findings indicate that TGF-β, acting in autocrine fashion, supports the survival of cancer cells, consistent with our findings. In normal mammary epithelial NMuMG cells, which exhibited low basal TGF-β signaling activity, a cell survival–promoting effect of TGF-β was observed with the addition of exogenous TGF-β, whereas A-44-03 failed to significantly enhance apoptosis (see Fig. 2A–C). By contrast, in breast cancer JygMC(A) cells and 4T1 cells, phosphorylation of Smad2 was observed even in the absence of TGF-β stimulation (see Fig. 2C), and A-44-03 potently induced apoptosis of these cells. We measured the amounts of TGF-βs but could not detect active forms of TGF-βs in the conditioned medium of these cells.⁶

Apparently, latent TGF-β is activated on the surface of cells and immediately binds to the receptors, preventing the detection of active TGF-β in the conditioned medium. Thus, during the progression of cancer, cancer cells might gain the ability to activate TGF-β signaling in autocrine fashion, and this property might protect them from various apoptotic stimuli, enabling their survival.

TGF-β has been reported to act as a prosurvival factor via Akt/protein kinase B signaling (22, 25, 26). Moreover, TGF-β has been reported to induce the expression of PDGF in certain types of cells, which may lead to activation of PI3K-Akt signaling. We reported previously that TGF-β enhances the growth of MG63 osteosarcoma cells through induction of PDGF-A and that this growth-stimulatory activity of TGF-β is abolished by treatment with STI571/Gleevec (18). In addition, TGF-β was shown to stimulate the proliferation of glioblastoma cells via induction of PDGF-B, which may be related to the poor prognosis of human glioma (21). However, in the present study, we showed that pharmacologic inhibition of PDGF receptor signaling by STI571 or that of PI3K-Akt signal by LY294002 failed to induce apoptosis of JygMC(A) cells in the presence of exogenous TGF-β and that the combination of ST1571 and LY294002 only partially antagonized TGF-β–induced cell survival. These findings suggest that survival signals other than PI3K-Akt must be present in these cells.

TGF-β induces survival of breast cancer cells through DEC1. We further analyzed DNA microarray data to identify the downstream molecule(s) involved in TGF-β–mediated cell survival and found that TGF-β induces the expression of DEC1, which may antagonize serum deprivation–induced apoptosis. DEC1, also known as SHARP2 and Stra13, is widely expressed in most normal tissues (27, 28) and associated with developmental events in many cells and regulation of circadian rhythms (29–31). DEC1 has been suggested to serve as a downstream target of TGF-β (32). Separate from TGF-β signaling, DEC1 is also induced in response to hypoxia (33). DEC1 is overexpressed in various cancers, including breast cancer, colorectal cancer, pancreatic cancer, non–small cell lung cancer, and oligodendroglioma (15, 34–37). Moreover, a significant correlation between DEC1 expression and grade in breast carcinomas has been reported (16). Although DEC1 has been reported to antagonize serum deprivation–induced apoptosis of colon carcinoma (38), the role of DEC1 in TGF-β–induced cell survival has not been elucidated. In the present study, we found that levels of expression of DEC1 in the breast cancer JygMC(A) cells and 4T1 cells were much higher than that in the normal mammary epithelial NMuMG cells and that DEC1 plays a critical role in TGF-β–mediated survival of breast carcinoma cells, strongly suggesting that expression of DEC1 is important for cancer progression.

DEC1 was originally identified as a basic helix-loop-helix transcription factor (39). DEC1 binds to CACGTG E-boxes (24, 32) and represses the transcription of many target genes (30, 40, 41). In the present study, we showed that dnDEC1, which competes with endogenous DEC1 for DNA binding, induces apoptosis of JygMC(A) cells and prevents metastasis, suggesting that TGF-β–induced DEC1 prevents apoptosis of JygMC(A) cells at the transcriptional level. Li et al. (42) reported that the antiapoptotic protein survivin is a target of DEC1 and suggested that survivin is responsible for the survival of cells induced by DEC1. However, our microarray analysis data indicated that survivin is not induced by TGF-β in JygMC(A) cells, and we were unable to identify other well-known apoptosis-related genes. Identification of the transcriptional target(s) of DEC1 responsible for the survival of breast cancer cells is thus required in the future.

DEC1 is involved in the metastasis of breast cancer induced by TGF-β signaling. We have reported previously that systemic gene transfer of Smad7 inhibits the metastasis of JygMC(A) cells through induction of EMT and suppression of cell migration (14). In the present study, we showed that suppression of the DEC1-mediated survival signal caused apoptosis, whereas forced expression of DEC1 did not affect the migration of cells (data not shown). dnDEC1 thus prevented the metastasis of JygMC(A) cells through loss of resistance to apoptosis and not through reduction of cancer cell motility.

Several molecules have been reported to be involved in the metastasis induced by TGF-β. Blockade of TGF-β signaling by dnTβR-II results in decrease in the secretion of parathyroid hormone–related protein and prevention of bone metastasis (43). Multiple secreted and cell surface proteins, including CTGF and interleukin 11, have been shown to coordinately regulate the osteolytic metastasis of breast cancers (44). In addition to these secreted and cell surface molecules, the homeodomain transcription factor CUTL1/CDP/Cux-1 was shown to be induced by TGF-β and to affect the motility and invasion of breast carcinomas (45). Yang et al. (46) also reported that the transcription factor Twist induces EMT and plays an essential role in the metastasis of breast carcinoma. In our microarray experiment (Supplementary Table S1), however, only CTGF, but not other candidate genes, was significantly regulated by TGF-β signaling. Metastases are induced in multiple steps, and different molecules may be involved in organ-specific metastases (47). Our findings suggest that DEC1 may participate in the process of metastasis of breast cancers through induction of cell survival.

In conclusion, the findings of the present study show that activation of endogenous TGF-β signaling in cancer cells is in certain conditions important for their survival and metastasis. TGF-β mediates survival signals through its downstream target DEC1, suggesting that suppression of DEC1 function may be a novel strategy for treatment of lung and liver metastasis of breast cancer.

Grant support: Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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.

We thank Dr. H. Azuma (Osaka Medical College, Takatsuki, Japan) for the JygMC(A) cell line; Drs. Y. Hamashima, T. Kajimoto, and M. Node (Kyoto Pharmaceutical University, Kyoto, Japan) for A-44-03; Dr. S. Horie (Teikyo University, Tokyo, Japan) for valuable discussion; and Dr. H. Meguro (University of Tokyo, Tokyo, Japan) for microarray analysis; M. Takahata, N. Kaneniwa, E. Kobayashi, and Y. Yuuki for technical assistance; and all members of the Biochemistry Laboratory of the JFCR.