DEDD Interacts with PI3KC3 to Activate Autophagy and Attenuate Epithelial–Mesenchymal Transition in Human Breast Cancer

Published OnlineFirst June 19, 2012; DOI: 10.1158/0008-5472.CAN-11-3832

Cancer Molecular and Cellular Pathobiology Research

Qi Lv, Wei Wang, Jianfei Xue, Fang Hua, Rong Mu, Heng Lin, Jun Yan, Xiaoxi Lv, Xiaoguang Chen, and Zhuo-Wei Hu

Abstract Epithelial-to-mesenchymal transition (EMT), a crucial developmental program, contributes to cancer invasion and metastasis. In this study, we show that death-effector domain-containing DNA-binding protein (DEDD) attenuates EMT and acts as an endogenous suppressor of tumor growth and metastasis. We found that expression levels of DEDD were conversely correlated with poor prognosis in patients with breast and colon cancer. Both in vitro and in vivo, overexpression of DEDD attenuated the invasive phenotype of highly metastatic cells, whereas silencing of DEDD promoted the invasion of nonmetastatic cells. Via direct interaction with the class III PI-3- kinase (PI3KC3)/Beclin1, DEDD activated autophagy and induced the degradation of Snail and Twist, two master regulators of EMT. The DEDD–PI3KC3 interaction led to stabilization of PI3KC3, which further contributed to autophagy and the degradation of Snail and Twist. Together, our ﬁndings highlight a novel mechanism in which the intracellular signaling protein DEDD functions as an endogenous tumor suppressor. DEDD expression therefore may represent a prognostic marker and potential therapeutic target for the prevention and treatment of cancer metastasis. Cancer Res; 72(13); 3238–50. 2012 AACR.

Introduction obtaining self-renewal and tumor-initiating ability (8–10). Metastasis is the spread of cancer cells to other parts of the Hence, once undergoing EMT, cancer cells acquiring invasive body and metastatic tumor is primarily responsible for most properties can detach from primary tumor tissue and enter the cancer deaths (1). Despite the mechanism of tumor metastasis surrounding stroma and circulation, creating a favorable tissue remains most enigmatic in the field of cancer biology, new microenvironment for the cancer cell colonization in distant concepts and mechanistic insights into the process of tumor tissue (11, 12). metastasis have emerged from recent advances (2, 3). For The death-effector domain-containing DNA-binding protein instance, the epithelial-to-mesenchymal transition (EMT), an (DEDD) is a member of family of death effector domain- essential cell-biological program during embryonic develop- containing proteins that comprise 7 proteins including FADD, ment, plays a critical role in the regulation of tumor progres- c-FLIP, Caspase-8/10, and DEDD1/2 (13). DEDD plays an sion and metastasis (4–6). In contrast to the temporally and important role in CD95-mediated apoptosis in response to a spatially precise regulation of EMT during development, EMT the activation of TNF- through activating caspase-3 in cyto- in tumor progression and metastasis may be droved by auton- plasm or caspase-6 in nucleus as a scaffold protein (14). Recent omous oncogenic activation or inactivation of signaling mole- studies found that DEDD can suppress the activity of Cdk1/ cules with or without additional stimulation (7). Moreover, cyclin B1 complexes and keep S6K1 activity, suggesting that recent studies show that the activation of EMT can promote DEDD participates in the regulation of cell cycle and inhibits – noncancer stem cells (CSC) to enter into a CSCs-like state: cell cell mitosis (15 17). Although other members of DED-containing proteins such as FADD have been implicated in a diverse array of diseases such as Huntington's disease, Type II diabetes, Authors' Affiliation: Molecular Immunology and Cancer Pharmacology autoimmune disease, arthritis, and cancer (18), there is no Groups, State Key Laboratory of Bioactive Substance and Function of direct evidence to show whether DEDD involves in the regu- Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, PR China lation of tumor development, growth, invasion, and metastasis. Using a yeast 2-hybrid screening system, we have recently Note: Supplementary data for this article are available at Cancer Research fi Online (http://cancerres.aacrjournals.org/). identi ed a number of molecules, including DEDD and TRB3, physically interacting with TGF-b1 signal molecule SMAD3 to Corresponding Author: Zhuo-Wei Hu, Institute of Materia Medica, Chi- b nese Academy of Medical Sciences & Peking Union Medical College, attenuate or enhance TGF- 1/Smad3-mediated cancer cell Beijing 100050, PR China. Phone: 86-10-8316-5034; Fax: 86-10-8316- invasion or EMT (19, 20). 5034; E-mail: [email protected] In this study, we therefore hypothesized that DEDD might doi: 10.1158/0008-5472.CAN-11-3832 play a role in the regulation of tumor metastasis through 2012 American Association for Cancer Research. regulation of EMT. We found that the expression levels of

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DEDD correlate conversely with the poor prognosis of breast weeks after injection, all mice were killed and the number of and colon cancers. Having discovered the functional asso- surface metastases per lung was determined under a dissecting ciation of DEDD expression with cancer progression and microscope. metastasis, we study the potential mechanism responsible for the DEDD-mediated antimetastatic effect. Our studies Analysis of tumor growth suggest that DEDD can function as an endogenous suppres- Female BALB/C-nu mice were injected with MCF-7 cells or sor of tumor growth and metastasis through regulating the MDA-MB-231 cells into the 4th mammary fat pad from ﬂank. stability of critical autophagy modulators to maintain autop- Tumor growth was monitored externally using vernier calipers hagy activation and promote the degradation of master EMT for 48 to 52 days. More details are provided in Supplementary inducers. Methods.

Materials and Methods Results Cell culture DEDD expression negatively correlates with poor Breast cancer and other cancer cell lines were purchased prognosis of breast and colon cancers from the American Type Culture Collection (ATCC) and main- To determine the potential role of DEDD in the regulation of tained as described (21–24). 293ET cells were obtained from tumor growth and metastasis, relationship between the the Cell Culture Center of Peking Union Medical College expression levels of DEDD and the metastatic grade of cancers (PUMC, China). All of the cell lines were authenticated by the was determined using cancer tissue microarrays with known PUMC using the AmpF/STR Identifiler Kit (Applied Biosys- clinical follow-up records, containing 60 cases ductal breast tems) and the ATCC fingerprint database. The last cell authen- carcinoma specimens (Fig. 1A, top) and 60 cases colon ade- tication was tested at December 2011. nocarcinoma specimens (Fig. 1B, top) were analyzed. Tissues were scored and classified (low or high DEDD staining) on the Plasmids basis of the intensity of DEDD labeling and percentage of Flag-DEDD was a gift from Dr. Marcus E. Peter, University DEDD-positive tumor cells. Among these specimens, Kaplan– of Chicago, USA (25). HA-FADD was a gift from Dr. Sug Hyung Meier survival analysis of ductal breast carcinoma specimens Lee, The Catholic University of Korea, South Korea (26). The and colon adenocarcinoma specimens revealed a correlation constructs pcDNA6-HA-PI3KC3, pCMV6-Entry-Flag-Beclin1, between the lower DEDD expression and reduced overall pGEX-4T-1-DEDD, pCMV2-Flag-Snail/Twist, and pGL6-E-cad- survival times (P < 0.05; Fig. 1A and B, bottom). In addition, herin-luc were generated by PCR and confirmed by sequencing. the expression of DEDD was examined in 6 breast epithelial cancer cell lines, including 3 nonmetastatic cancer cell lines DEDD-silencing cells and 3 metastatic cancer cell lines (Fig. 1C). The expression of Three shRNAs were designed on the basis of the DEDD DEDD could be detected in metastatic and nonmetastatic sequence (NM_032998) identified with siRNA Target Finder cancer cell lines but the metastatic cancer cells expressed (Ambion). Stable cell lines were selected hygromycin. A single much lower level of DEDD than the nonmetastatic cancer cells clone was amplified by a dilution cloning technique. did. The identical results were also observed in several cancer cell lines derived from liver, colon, pancreas, and prostate DEDD-overexpressing cells cancers (Fig. 1D). These data suggest that the expression level Human DEDD cDNA was cloned into the pCDNA 3.1 expres- of DEDD negatively correlates with the metastatic phenotypes sion vector (Invitrogen) and transfected into MDA-MB-231 of tumor and may be used as a prognosis marker for these cells. Stable cell lines were selected by G418. A single clone was cancers. amplified by a dilution cloning technique. DEDD inhibits metastatic phenotypes of cancer cells Tissue microarray slides and immunohistochemistry We investigated whether the expression level of DEDD Human breast or colon cancer tissue microarray slides (US regulated invasive phenotypes of breast cancer cells by Biomax) were detected as previously described (27). expressing short hairpin RNAs (shRNA) to knock down DEDD expression in nonmetastatic MCF-7 cells or by Chemoinvasion assay stably overexpressing DEDD in metastatic MDA-MB-231 Assays were conducted following previous report (28) as in cells. The shRNAs from 3 different DEDD sequences Supplementary Methods. (shRNA1, shRNA2, or shRNA3) expressed in MCF-7 cells reduced DEDD expression by 50%, 90%, and 84%, respec- Soft agar assay tively (Supplementary Fig. S1A), compared with the cells Soft agar assay were carried out as previously described expressing a control shRNA whose sequence did not (29). match any known human gene. Overexpressing DEDD in MDA-MB-231 by transfecting pCDNA3.1-DEDD signifi- Analysis of metastasis cantly increased the expression of DEDD compared with Female BALB/C-nu mice were injected with MCF-7 cells or the cells transfected with pCDNA3.1-control vector (Sup- MDA-MB-231 cells according to a previous study (30). Nine plementary Fig. S1B).

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Figure 1. The expression levels of DEDD conversely correlate with the poor prognosis of breast and colon cancers. A and B, DEDD levels in breast (A) and colon (B) tumor tissues (top; scale bars, 40 mm) and Kaplan–Meier plots (bottom) of overall survival of 60 patients with ductal breast carcinomas and colon adenocarcinomas stratiﬁed by DEDD expression levels. Tissues were scored as low or high DEDD staining for all tumor cells. C and D, DEDD levels were detected by immunoblotting (IB) in the nonmetastatic or metastatic breast cancer cell lines (C) and other tissue-derived cancer cell lines (D). Data are representative of 3 independent assays with identical results.

Silencing DEDD expression in nonmetastatic MCF-7 cells increased the proliferation (Supplementary Fig. S2A) and resulted in the acquisition of invasive ability as indicated by inhibited the TNF-a–induced apoptosis in MCF-7 cells (Sup- Transwell invasion assay (Fig. 2A). Consistently, depletion of plementary Fig. S2B). Major differences in cell morphology had DEDD stimulated colony formation of these cells in soft agar, been observed between cells expressing control shRNA and indicating that silencing DEDD increases anchorage-depen- cells expressing DEDD shRNAs grown on Matrigel. The former dent growth (Fig. 2B). Moreover, knocking down of DEDD exhibited a cobble-stone-like morphology, the border of cells

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and Matrigel was sharp; although the latter exhibited a cobble- agar (Fig. 2B). Overexpressing DEDD also decreased prolifer- stone-like morphology too, the border of cells showed a spike- ation but promoted apoptosis of MDA-MB-231 cells (Supple- like structure, data suggesting that MCF-7 cells silencing mentary Fig. S2A and S2B). Moreover, cells expressing empty DEDD acquire a metastatic phenotype (Fig. 2C, left). Because vector exhibited a spindle-like fibroblastic morphology but DEDD shRNA2 was more efficient to silence DEDD expression, DEDD-overexpressing cells had a cobble-stone-like morphol- it was chosen for the subsequent studies. In contrast, over- ogy as showed by 3-dimensional Matrigel culture (Fig. 2C, expressing DEDD significantly reduced the invasive capacity of right). Interestingly, prolonged growth time or increased MDA-MB-231 cells as indicated in Transwell invasion assay cell number did not enhance the invasive capacity of (Fig. 2A), and prevented colony formation of these cells in soft MCF-7-control shRNA cells or MDA-MB-231-DEDD cells

Figure 2. Ectopic expression of DEDD inhibits but depletion of DEDD promotes tumor cell migration, invasion, and the anchorage- independent growth. A, the invasive properties of DEDD shRNA MCF-7 and pcDNA3.1-DEDD MDA-MB-231 cells together with their controls were analyzed by Transwell invasion assay (magnification, 200). Data are presented as mean SEM of 3 independent assays. B, representative photographs indicating soft agar colony formation were taken at 20 days after culture of the indicated cells (top) and summary of 3 independent assays (bottom) are presented as mean SEM. C, Data are phase-contrast micrographs of MCF-7 (left, magnification, 400) and MDA-MB-231 cells (right, magnification, 100) cultured on Matrigel. , P < 0.01.

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Figure 3. Cancer cells overexpressing DEDD loss but cancer cells silencing DEDD acquires the capacity of tumor growth and metastasis. A and, B, MDA-MB-231 cells (A) or MCF-7 cells (B) were i.v. injected into male nude mice. Representative lungs and hematoxylin and eosin staining of lung sections were presented to show metastases in lungs (scale bar, 100 mm). The metastatic nodules are indicated with yellow arrows. The number of metastatic nodules is presented as mean SEM (n ¼ 6). C, MDA-MB-231 cells were injected into the fat pads of nude mice. Photographs of representative mice and tumors are shown, along with H&E staining of tumor (T) and normal (N) breast tissue (left, scale bar, 100 mm). Each data point of the growth curve is mean SEM of primary tumors (right). D, MCF-7 cells were injected into the fat pads of nude mice. Photographs of representative tumors (left) and the growth curve (right) are shown. Data are presented as mean SEM (n ¼ 6); , P < 0.01.

(Supplementary Fig. S2C and S2D), indicating that the regu- of large metastatic nodules in the lungs. In contrast, the latory effect of DEDD expression level on the invasive capacity number and volume of metastatic nodules in the lung of mice is not a simple growth-dependent phenomenon. injected with MDA-MB-231-DEDD cells were signiﬁcantly reduced as compared with that injected with MDA-MB-231- DEDD inhibits tumor metastasis and growth control vector cells (Fig. 3A and B). These results indicate that We next evaluated the in vivo effects of DEDD-changing expression level of DEDD correlates inversely with the meta- expression on tumor metastasis and growth. Control shRNA static ability of breast cancer cells. MCF-7 and DEDD shRNA MCF-7 cells were injected into the To determine the effect of DEDD expression level on tumor lateral tail vein of 6-week-old athymic mice. Nine weeks after growth, the DEDD-silencing or DEDD-overexpressing breast injection, injection of mice with DEDD shRNA MCF-7 cells but cancer cells were implanted under the 4th mammary fat pads not with control shRNA MCF-7 cells resulted in the formation of athymic nude mice. Comparing with mice implanted with

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Figure 4. Overexpressing DEDD inhibits but silencing DEDD promotes EMT. A, expression of E-cadherin and vimentin were detected by immunoﬂuorescence staining (scale bar, 35 mm). B, the expression of epithelial or mesenchymal markers was detected by Western blotting (IB) in MCF-7 cells and MDA-MB-231 cells. C, left, silencing DEDD increased the expression of Snail and Twist in MCF-7 cells; overexpression of DEDD reduced the expression of Snail and Twist in MDA-MB-231 cells (right). D, the mRNA levels of Snail and Twist were detected by quantitative PCR (qPCR). Data are presented as mean SEM of 3 independent assays. E, luciferase activity of E-cadherin (cad) reporter gene was detected in MCF-7 cells and MDA-MB-231 cells. Data are presented as the mean SEM of 3 independent assays; , P < 0.05.

MDA-MB-231-control vector cells, which formed large tumors (10, 11). To assess the potential roles of DEDD in the regulation within 52 days (Fig. 3C), the mice implanted with the MDA-MB- of EMT, we examined the expression of EMT-associated mar- 231-DEDD cells showed a significant reduction in the tumor kers in the presence or absence of DEDD expression. We found growth, even no tumors were observed in some mice. Also, that DEDD shRNA MCF-7 cells showed spindle-like fibroblastic silencing DEDD significantly promoted the tumor growth in morphology, one of the main characteristics of EMT (data no mice implanted with MCF-7 cancer cells containing DEDD shown). The expression of epithelial marker E-cadherin was shRNA compared with mice implanted with MCF-7 cancer significantly reduced in these cells (Fig. 4A and B, left). In cells containing control shRNA (Fig. 3D). These results indicate contrast, the expression of mesenchymal markers Vimentin that the expression level of DEDD correlates negatively with and a-SMA was dramatically upregulated in these cells (Fig. 4A the tumor growth of breast cancer. and B, left). However, overexpression of DEDD in MDA-MB-231 cells led to an increase in the expression of epithelial marker E- DEDD inhibits EMT by downregulating the expression of cadherin and a decrease in the expression of mesenchymal Snail and Twist markers vimentin and a-SMA (Fig. 4A and B, right). To verify EMT, a process by which tumor-associated epithelial cells the universal effects of DEDD on EMT, DEDD was knock- obtain mesenchymal features and result in a reduced cell–cell downed in a nonmetastatic colon cancer cell line SW480 by contact and increased motility, has a critical role in metastasis DEDD shRNA. We found that the expression of E-cadherin was

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significantly reduced but the expression of Vimentin was degradation of Snail/Twist does not depend on the ubiqui- increased in SW480 DEDD shRNA cells (Supplementary Fig. tin-proteasome system. S3A). Taken together, our studies indicate that DEDD is a Autophagy is another intracellular degradation system for negative EMT modulator. the majority of proteins and some organelles (37). To verify A number of factors, particularly transcriptional factors whether autophagy-lysosome system participated in the reg- Snail and Twist, play critical role in the regulation of EMT ulation of DEDD-mediated degradation of Snail/Twist, GFP- (31–34). We examined the expression of Snail and Twist in the LC3 and Flag-Snail or Flag-Twist were cotransfected into MCF- presence or absence of DEDD. The expression of Snail and 7 cells, and the cellular localization of LC3 and Snail/Twist was Twist was very low in the control shRNA MCF-7 cells. Silencing observed by immunofluorescence. We found that LC3 was DEDD in MCF-7 cells significantly upregulated the expression colocalized with Snail/Twist in the presence or absence pro- of Snail and Twist (Fig. 4C). In contrast, overexpression of tein synthesis inhibitor cycloheximide (Fig. 5C; Supplementary DEDD significantly decreased the expression of Snail and Twist Fig. S4C). Furthermore, inhibiting autophagy by 3-MA could in MDA-MB-231 cells (Fig. 4C). However, DEDD–knocking significantly attenuate the degradation of Snail/Twist because down or DEDD-overexpressing cancer cells did not change a similar degradation rate was observed in MCF-7-control the amounts of Snail and Twist mRNAs compared with their shRNA and MCF-7-DEDD shRNA cells (Fig. 5D; Supplementary controls as indicated by quantitative PCR analysis (Fig. 4D). Fig. S4D). Thus, activation of the autophagy-lysosome system Because Snail and Twist negatively regulate the expression of participates in the degradation of Snail/Twist, and this process epithelial marker E-cadherin, the upregulated Snail and Twist is DEDD dependent. expression in DEDD-silencing cells resulted in a suppressive promoter activity of E-cadherin as indicated by luciferase DEDD activates autophagy modulating the expression of reporter assays (Fig. 4E). In contrast, the downregulated PI3KC3 expression of Snail and Twist in DEDD-overexpressing cells Because we had found that DEDD-mediated degradation of resulted in an enhanced promoter activity of E-cadherin in Snail/Twist, we examined if altering the expression level of compared with the control vector cells (Fig. 4E). DEDD directly activate autophagic signal molecules. We found that silencing or overexpressing DEDD did not change the DEDD induces the degradation of Snail and Twist by expression of autophagic signal molecules such as Akt, p-Akt, activating the autophagy–lysosome degradation system p-mTOR in MCF-7 or MDA-MB-231 cells (Fig. 6A). However, Because the expression level of DEDD did not significantly the expression of class III PI-3-kinase (PI3KC3) was suppressed change the expression of Snail/Twist mRNAs (Fig. 4D), we in MCF-7-DEDD shRNA cells compared with MCF-7-control examined the potential effect of DEDD-changing expression on shRNA cells. In contrast, the expression of PI3KC3 was the stability of Snail and Twist. The pCMV2-Flag-snail or increased in DEDD-overexpressing MDA-MB-231 cells com- pCMV2-Flag-twsit expression vector was transiently trans- pared with the cells with the empty vector. Also, Beclin1 fected into the MCF-7-control shRNA or MCF-7-DEDD shRNA expression was upregulated in DEDD-overexpressing MDA- cells. pEGFP-N1 vector expressing EGFP protein was used as MB-231 cells (Fig. 6A). control to normalize the experimental variation between LC3 and p62/SQSTM are 2 indicators reflecting the effectual different time points. We found that the half-life of Flag-Snail autophagy flux (38). We found that alternating DEDD expres- was 5.7 hours in MCF-7-control shRNA cells but the degrada- sion did not change the expression level of LC3 I/II. However, tion of Flag-Snail was restrained and the half-life was signif- silencing DEDD in MCF-7 cells significantly increased the icantly prolonged to 10 hours in the MCF-7-DEDD shRNA cells expression and aggregation of p62 but overexpressing DEDD (Fig. 5A). Similarly, depletion of DEDD inhibited the degrada- in MDA-MB-231 cells reduced the expression and aggregation tion of Twist and the half-life of Twist was extended from 6.2 of p62 (Fig. 6A and B). These results suggest that the enhanced hours in MCF-7-control shRNA cells to 9.7 hours in MCF-7- expression of DEDD induces but the reduced expression of DEDD shRNA cells (Supplementary Fig. S4A). DEDD attenuates autophagy activation. Previous work indicated the degradation of Snail and Twist PI3KC3 and Beclin1 are 2 critical components of the PI3KC3 depends on the ubiquitin-proteasome degradation pathway core complex, which plays a central role in the activation of (35, 36), we thus examined if the inhibition of proteasome autophagy (39, 40). To examine the effect of PI3KC3 and changed the DEDD-mediated degradation of Snail/Twist. Flag- Beclin1 on the DEDD-mediated EMT, different concentrations Snail was transfected into MCF-7-control shRNA or MCF-7- of PI3KC3-siRNA pools or Beclin1-siRNA pools were trans- DEDD shRNA cells. The cells were then treated with protein fected into MCF-7-control shRNA cells. The different amounts synthesis inhibitor cycloheximide and proteasome inhibitor of pCDNA6-HA-PI3KC3 or pCMV6-Flag-Beclin1 expressing MG132 to block de novo synthesis and the ubiquitin-protea- vector were also transfected into MCF-7-DEDD shRNA cells. some degradation of Snail. We found that the Flag-Snail's We found that silencing of PI3KC3 or Beclin1 could increase degradation in MCF-7-control shRNA cells was much rapid the expression of Snail and Twist, downregulate the expression than that in MCF-7-DEDD shRNA cells (Fig. 5B). The identical of E-cadherin, and inhibit the degradation of p62 in a concen- result was also observed in MCF-7 cells transfected with Flag- tration-dependent manner (Fig. 6C and D, left). In contrast, the Twist (Supplementary Fig. S4B). However, treatment of cells overexpression of PI3KC3 or Beclin1 could decrease the with proteosome inhibitor MG132 did not change Snail/Twist expression of Snail/Twist, increase the expression of E-cad- degradation. These data indicate that the DEDD-mediated herin, and the degradation of p62 in a concentration-

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Figure 5. Depletion of DEDD stabilizes Snail through attenuating the autophagy–lysosome degradation system. A and B, MCF-7 cells were cotransfected with pCMV2-Flag-Snail and pEGFP-N1 vector. Cells were treated with 20 mmol/L of cycloheximide (CHX; A) or CHX plus 10 mmol/L of MG132 (B) for the indicated times. Flag-Snail and EGFP were detected by immunoblotting (IB) with indicated antibodies. C, LC3-GFP (green) and Flag-snail (red) were cotransfected into the MCF-7 cells containing control shRNA and incubated for 36 hours. The cells were treated with 20 mmol/L of CHX for the indicated times. The cell nuclei were stained with DAPI (scale bar, 35 mm). The arrows indicate the colocalization of LC3 and Snail. D, cells were handled as described in A and treated with 20 mmol/L of CHX and 10 mmol/L of 3-methyladenine (3-MA) for the indicated times. Cell extracts were prepared and immunoblotting was carried out as described in A. DAPI, 40,6-diamidino-2-phenylindole.

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Figure 6. DEDD regulates autophagy through modulating the expression of PI3KC3. A, the expression of autophagy- associated proteins was detected by immunoblotting with indicated antibodies. B, the expression of p62 was detected by immunoﬂuorescence staining. The green signal indicates the staining of corresponding protein and the blue signal represents the nuclear DNA staining by DAPI (scale bar, 35 mm). C and D, left, MCF-7- control shRNA cells were transfected with the indicated concentrations of PI3KC3-siRNA (C) or Beclin1-siRNA (D); right, MCF-7-DEDD shRNA cells were transfected with the indicated concentrations of pCDNA6-HA- PI3KC3 (C) or pCMV2-Flag- Beclin1 (D). Protein lysates were analyzed by immunoblotting with the indicated antibodies. DAPI, 40,6-diamidino-2-phenylindole.

dependent manner (Fig. 6C and D, right). These data indicate acted directly with the complex to regulate autophagy. We that PI3KC3 and Beclin1 participate in the DEDD-mediated found that DEDD interacted physically with PI3KC3 and regulation of EMT by promoting the autophagy-associated Beclin1, as showed by co-precipitated of Flag-DEDD with degradation of Snail and Twist. endogenous PI3KC3 or Beclin1 (Fig. 7A), which was con- ﬁrmed by glutathione S-transferase pull-down assay (Fig. DEDD stabilizes PI3KC3 by interacting with PI3KC3/ 7B), and by immunoprecipitation of endogenous DEDD with Beclin1 PI3KC3 or Beclin1 (Fig. 7C). To determine whether the The function or activity of PI3KC3/Beclin1 complex can interaction of DEDD with PI3KC3 and Beclin1 have effect be regulated by several signal molecules such as UVRAG, on the formation of core complex, the interaction of PI3KC3 Bcl-2, and Rubicon (39, 41). We wondered if DEDD inter- with Beclin1 was determined by co-immunoprecipitation in

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Figure 7. DEDD stabilizes PI3KC3 through a physical interaction with PI3KC3/Beclin1. A–C, DEDD interacts with PI3KC3 and Beclin1 was conﬁrmed by semi-endogenous co-immunoprecipitation (A), glutathione S-transferase (GST) pull- down (B), and endogenous co- immunoprecipitation (C). D, whole- cell extracts of MDA-MB-231 cells were immunoprecipitaed (IP) and blotted (IB) with indicated antibodies. E, MCF-7 cells were transfected with pCDNA6-HA-PI3KC3 and treated with 20 mmol/L of cycloheximide (CHX) for the indicated times. Cell extracts were prepared and detected by Western blotting with indicated antibodies. F, schematic diagram illustrates the role of DEDD in the regulation of metastatic phenotypes. HA, hemagglutinin; MW, molecular weight.

MDA-MB-231 cells containing empty or DEDD-expressing Discussion vector. Overexpression of DEDD signiﬁcantly enhanced the Our recent work indicates that DEDD attenuates TGF-b1/ interaction between PI3KC3 and Beclin1 (Fig. 7D). We Smad3 signaling through interacting with transcription factor further found that silencing DEDD promoted the degrada- Smad3 to suppress the TGF-b1/Smad3-mediaed invasion in tion of PI3KC3 and the half-life of PI3KC3 was reduced from cancer cells (19). In this study, we have showed that the ex- 9.2 to 3.3 hours (Fig. 7E). A similar result was also observed pression level of DEDD in breast and colon cancers correlates in SW480 control shRNA and DEDD shRNA cells (Supple- conversely with the poor prognosis of breast and colon cancers: mentary Fig. S3B). However, silencing DEDD did not signif- the lower is DEDD expression in beast and colon cancers, the icantly change the half-life of Beclin1 (data not shown). higher is the cancer-induced death rate because the expression Taken together, DEDD supports the stability of PI3KC3 level of DEDD decides the metastatic phenotypes of the cancer through interacting with PI3KC3/Beclin1. cells, including migration, invasion, growth, and metastasis

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(1, 5). Indeed, in metastatic MAD-MB-231 cells losing DEDD required to decipher the precise role and mechanism for expression, ectopic expression of DEDD stabilizes and activates antimetastatic roles of DEDD. the PI3KC3 through physically interacting with this protein, Our studies indicate that the alterations in the expression leading to the autophagy-lysosome dependent degradation of level of DEDD regulate the EMT through DEDD interacting Snail and Twist, 2 master inducers of EMT, to attenuate the with the PI3KC3/Beclin1 complex, which plays central role in EMT process in these cancer cells. In contrast, in nonmeta- the initiation and maturation of autophagosomes (39, 40). The static MCF-7 cells normally expressing DEDD, silencing DEDD DEDD's effect on the stability of PI3KC3/Beclin1 complex expression promotes the degradation and inactivation of the seems to be achieved through DEDD interacting with PI3KC3 PI3KC3, leading to an attenuation of autophagy activity and the and stabilizes this protein. Indeed, we find that DEDD can autophagy-lysosome–dependent degradation pathway. Hence, interact with PI3KC3 or Beclin1, respectively. Ectopic expres- accumulation and activation of Snail and Twist in these cancer sion of DEDD results in an increase in the expression of PI3KC3 cells promote the activation of the EMT process. Indeed, and Beclin1 in MDA-MB-231 cancer cells, whereas knockdown restoring DEDD expression in metastatic MAD-MB-231 breast of DEDD results in a decrease in the expression of PI3KC3 and cells results in a loss of metastatic phenotype whereas knock- Beclin1 in MCF-7 cancer cells. However, silencing DEDD down of DEDD expression in the nonmetastatic MCF-7 breast expression results in a marked decrease in the degradation cells results in the acquisition of the most central traits of the half-life of PI3KC3 but not Beclin1 (Fig. 7), indicating that the CSCs: invasive/metastatic and tumor-initiating ability (1–3, 7). DEDD–PI3KC3 interaction plays a crucial role in maintaining Thus, our studies indicate that DEDD is critically involved in the the stability of the PI3KC3/Beclin1 complex. Thus, alterations negative regulation of cancer progression and metastasis in the expression of PI3KC3 result in either activation or through maintaining autophagy activity to suppress the EMT inactivation of autophagy-dependent degradation of Snail/ (summarized in Fig. 7F). Twist and other positive EMT markers. Despite we do not Metastasis is a complex and multistep process, which has to know the precise mechanism how DEDD interacts with be divided into 2 phases, namely, physical translocation of a PI3KC3 to maintain the stability of this protein and why loss tumor cell from the primary tumor to a distant tissue to seed of DEDD expression promotes the degradation of PI3KC3, and colonization of disseminated tumor cell in the tissue (4–6). recent work indicate that DEDD induces a diversity of cell Recent researches indicate that EMT is not only involved in the biologic activity through interacting with several key molecules 2-phase events in cancer to promote metastasis, but also to attenuate or support the stability of these proteins in the participates in the regulation of tumor progression (42). The cells (19, 46). Thus, a key question to be addressed is: what molecular and cellular mechanisms for the regulation of EMT program is responsible for shutdown or turn-on of DEDD have been emerged from these studies and, particularly, many expression under the physiologic or pathologic situations. positive inducers and signal pathways for the induction of the Although targeting the activity of E-cadherin repressors EMT have been identified (5, 42). For example, a mechanistic such as Snail/Twist to attenuate EMT may be promising linkage between the Snail/Twist-induced EMT and CSC-like therapeutic strategy against tumor metastasis and progres- behaviors has been indicated for the understanding of prome- sion, these EMT inducers are transcription factors and difficult tastatic role of this EMT inducer because these cancer cells to access or target (5). Our discovery, DEDD interacting with on the EMT process acquire CSC-like traits (7). Our current PI3KC3/Beclin1 to promote the autophagy-dependent degra- studies provide evidence to show that the alteration in the dation of Snail and Twist, may provide mechanistic insights expression levels of DEDD regulates the EMT and results in into developing novel therapeutic strategy against tumor loss or acquisition of metastatic phenotypes, thereby deter- metastasis and progression. Currently, a wide range of selective mining the process of tumor progression and metastasis. PI3K inhibitors has been tested in preclinical studies and some Interestingly, a very recent study by Mori and colleagues report have entered clinical trials in oncology. Unveiling the Vps34 that DEDD deficiency results in a defect in uterine decidua- structure (47) and discovery of selective PI3KC3 inhibitor (48) lization and infertile because of reducing Akt expression, may shed the new light on the perspective anticancer targets. indicating that DEDD is indispensable for the establishment However, because of the complexity of PI3K signaling path- of an adequate uterine environment to support early pregnan- ways, developing an effective anticancer therapy may be cy in mice (43). This finding is consistent and support to the difficult. Particularly, the potential therapeutic effects of phar- fact that DEDD is involved in the regulation of embryos macologic Vps34 modulation may well be context-dependent, developmental programs including EMT (19). However, it and thus there could perhaps be a need for biomarkers for needs to point out, previous work indicated that DEDD can patient selection. For this perception, our current finding may enter nucleus to inhibit rDNA transcription (25), regulate cell provide mechanistic insights into design of chemical tools and cycle, and inhibit cell mitosis to reduce cell and body size via potential drugs to activate or antagonize this key molecule modulation of rRNA synthesis (17), attenuate protein synthe- involved in vesicular trafficking and autophagy. sis, and promote apoptosis (44, 45). These growth-impeding In summary, our current studies show that DEDD is an or apoptosis-promoting roles of DEDD may also contribute endogenous suppressor of tumor growth and metastasis in to the DEDD-induced antimetastatic role. Taken together, human breast cancer and the expression level of DEDD decides these studies support a notion that tumor metastatic ability the phenotypes of cancer cells in migration, invasion, growth, may be an innate property shared by the bulk of cells present metastasis, and maybe resistance to the chemotherapy early in a developing tumor mass (1). Hence, further study is because it negatively regulates the EMT. As tumor metastasis

3248 Cancer Res; 72(13) July 1, 2012 Cancer Research

DEDD-Activated Autophagy against Metastasis

is a devastative disease, our findings suggest that the expres- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Q. Lv, J. Xue, Z.-W. Hu sion level of DEDD can be detected and used as the prognostic Writing, review, and/or revision of the manuscript: Q. Lv, F. Hua, X. Chen, marker of cancers and therapeutic target for the prevention Z.-W. Hu and treatment of cancer progression and metastasis. There- Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): Q. Lv fore, additional insight into the regulatory role of DEDD– Study supervision: Z.-W. Hu PI3KC3 interaction in autophagy and EMT may ultimately serve as an entry point to translate modifiers of these inter- Grant Support actions into clinical endpoints. This work was supported by grants from National Major Basic Research Program of China (973: #2006CB503808), National Natural Science Foundation of Disclosure of Potential Conflicts of Interest China (81030056, 30973557, 81101595), and Creation of Major New Drugs No potential conflicts of interest were disclosed. (2009ZX09301-003-13, 2009ZX09301-003-9-1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Authors' Contributions advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this Conception and design: Q. Lv, Z.-W. Hu fact. Development of methodology: Q. Lv, Z.-W. Hu Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Q. Lv, W. Wang, J. Xue, F. Hua, R. Mu, Received November 28, 2011; revised March 16, 2012; accepted April 6, 2012; H. Lin, J. Yan, X. Lv published OnlineFirst June 19, 2012.

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3250 Cancer Res; 72(13) July 1, 2012 Cancer Research

DEDD Interacts with PI3KC3 to Activate Autophagy and Attenuate Epithelial−Mesenchymal Transition in Human Breast Cancer

Qi Lv, Wei Wang, Jianfei Xue, et al.

Cancer Res 2012;72:3238-3250. Published OnlineFirst June 19, 2012.

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