Twist2 Contributes to Breast Cancer Progression by Promoting an Epithelial–Mesenchymal Transition and Cancer Stem-Like Cell Self-Renewal

Oncogene (2011) 30, 4707–4720 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Twist2 contributes to breast cancer progression by promoting an epithelial–mesenchymal transition and cancer stem-like cell self-renewal

X Fang1,4, Y Cai1,4, J Liu1, Z Wang1,QWu2, Z Zhang2, CJ Yang3, L Yuan1 and G Ouyang1

1State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen, China; 2Department of Breast Surgery, The First Afﬁliated Hospital of Xiamen University, Xiamen, China and 3College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China

The epithelial to mesenchymal transition (EMT) is a typic changes, lose expression of E-cadherin and other highly conserved cellular programme that has an components of epithelial cell junctions, adopt a me- important role in normal embryogenesis and in cancer senchymal cell phenotype and acquire motility and invasion and metastasis. We report here that Twist2, a invasive properties that allow them to migrate through tissue-speciﬁc basic helix-loop-helix transcription factor, the extracellular matrix. EMT is triggered by several is overexpressed in human breast cancers and lymph node extracellular signals, including components of the metastases. In mammary epithelial cells and breast cancer extracellular matrix and growth factors, and is mediated cells, ectopic overexpression of Twist2 results in morpho- by the activation of EMT transcription factors such as logical transformation, downregulation of epithelial Twist1, Snai1, Slug, ZEB1 and ZEB2 (Maeda et al., markers and upregulation of mesenchymal markers. 2005; Thiery and Sleeman, 2006; Ouyang et al., 2010). Moreover, Twist2 enhances the cell migration and Accumulating evidence suggests that aberrant activation colony-forming abilities of mammary epithelial cells and of the EMT developmental programme contributes to breast cancer cells in vitro and promotes tumour growth tumour invasion, metastatic dissemination and acquisi- in vivo. Ectopic expression of Twist2 in mammary epithelial tion of therapeutic resistance (Yang et al., 2004; Yang cells and breast cancer cells increases the size and number of and Weinberg, 2008; Ruan et al., 2009; Thiery et al., their CD44high/CD24low stem-like cell sub-populations, 2009; Singh and Settleman, 2010; Hanahan and Wein- promotes the expression of stem cell markers and enhances berg, 2011). the self-renewal capabilities of stem-like cells. In addition, The Twist proteins Twist1 and Twist2 (also named exogenous expression of Twist2 leads to constitutive Dermo-1) are highly conserved basic helix-loop-helix activation of STAT3 (signal transducer and activator of (bHLH) transcription factors that are structurally transcription 3) and downregulation of E-cadherin.Thus, characterised by a conserved domain, which contains a the overexpression of Twist2 may contribute to breast stretch of basic amino acids adjacent to two amphi- cancer progression by activating the EMT programme and pathic a-helices that are separated by an interhelical enhancing the self-renewal of cancer stem-like cells. loop. Twist1 is a regulator of embryonic development Oncogene (2011) 30, 4707–4720; doi:10.1038/onc.2011.181; that participates in EMT during differentiation of the published online 23 May 2011 mesoderm in Drosophila and in neural crest migration in vertebrates (Thisse et al., 1987; Chen and Behringer, Keywords: Twist2; epithelial–mesenchymal transition; 1995). Twist1 is overexpressed in various human solid cancer stem cell; breast cancer; metastasis tumours and is involved in tumour invasion and metastasis. Ectopic overexpression of Twist1 remark- ably enhances the invasive and metastatic capacities of Madin–Darby canine kidney epithelial cells, human Introduction mammary epithelial cells and breast cancer cells by inducing EMT (Yang et al., 2004; Ansieau et al., 2008; The epithelial to mesenchymal transition (EMT) is a Ansieau et al., 2010). Twist2, which was cloned as an well-coordinated developmental programme that has a E12-binding partner using a yeast two-hybrid screen, very important role in the development of the mesoderm shows more than 90% identity with Twist1 in the bHLH from the epithelium during embryogenesis (Acloque and carboxy-terminal domains and regulates a series of et al., 2009; Kalluri and Weinberg, 2009). During the developmental processes in embryogenesis (Li et al., EMT process, epithelial cells undergo dramatic pheno- 1995). During mouse embryogenesis, the expression pattern of Twist2 extensively overlaps, but is distinct Correspondence: Dr G Ouyang, State Key Laboratory of Stress Cell from, that of mouse Twist1. Twist2 is predominantly Biology, School of Life Sciences, Xiamen University, Xiamen 361005, expressed in a subset of mesodermal- and ectodermal- China. derived tissues and may function as a potent negative E-mail: [email protected] 4These authors contributed equally to this work. regulator of gene expression during differentiation of a Received 11 September 2010; revised and accepted 15 April 2011; subset of mesenchymal cell lineages. Twist2 represses the published online 23 May 2011 transcription of the myogenic bHLH protein MyoD. Twist2 promotes EMT and stem cell self-renewal X Fang et al 4708 In addition, Twist2 inhibits osteoblast maturation transcription factors and osteo-/chondrogenic-inductive and maintains cells in a preosteoblast phenotype (Lee factors in the early stages of cell fate determination in et al., 2000; Gong and Li, 2002; Franco et al., 2011). human mesenchymal stem cells (Isenmann et al., 2009). Similar to Twist1, Twist2 overrides oncogene-induced However, the functional signiﬁcance of Twist2 in human premature senescence by promoting EMT in human breast cancer and in regulating CSC stemness as well as epithelial cells (Ansieau et al., 2008). Twist2 is also the underlying cellular and molecular mechanisms are involved in p12CDK2ÀAp1-induced EMT of hamster cheek poorly studied. pouch carcinoma-I cells (Tsuji et al., 2008). Twist2 In this study, we demonstrate that Twist2 is a potent mRNA is overexpressed in a large variety of human inducer of EMT. In human mammary epithelial cells primary tumours; however, the transcriptional expres- and breast cancer cells, overexpression of Twist2 sion pattern of Twist2 differs from that of Twist1 in upregulates characteristic mesenchymal markers, down- some of these cancers (Ansieau et al., 2008). These regulates epithelial markers and promotes cell migra- differences suggest that Twist1 and Twist2 may have tion. Moreover, Twist2 increases the abundance of overlapping but distinct roles in different sets of CD44high/CD24low sub-populations and promotes self- tumours. renewal of stem-like cells. Furthermore, Twist2 increases Recently, Mani et al. (2008) demonstrated that Twist1 the colony-forming activity of mammary epithelial cells transforms non-tumourigenic, immortalised human and breast cancer cells in vitro and promotes tumour- mammary epithelial cells into mesenchymal-like cells igenesis in vivo. Taken together, these data suggest that and dedifferentiates HER2/neu-infected human mam- Twist2 contributes to breast cancer progression by mary epithelial cells into CD44high/CD24low cancer stem- promoting EMT and cancer stem-like cell self-renewal. like cells via EMT. Cancer stem cells (CSCs), which are also known as tumour initiating cells or tumourigenic cells, are cell populations in tumour tissues with stem Results cell-like properties (Al-Hajj et al., 2003; Brabletz et al., 2005; Bao et al., 2006; Mani et al., 2008; Gupta et al., Twist2 is overexpressed in breast cancer 2009a). Twist1 induces a breast CSC phenotype by Although the Twist2 gene is overexpressed (more than directly reducing the level of CD24 (Vesuna et al., 2009). twofold) in only 3.6% of breast cancer tissue samples Yin et al. (2010) reported that Twist1 may be an compared with healthy breast tissue, the protein level of important regulator of stemness in epithelial ovarian Twist2 in human breast cancer remains unknown cancer cells. The EMT-derived cells, which are induced (Ansieau et al., 2008). We therefore measured the by Twist1 in human mammary epithelial cells, exhibited protein levels of Twist2 in human breast cancer samples, the potential for multilineage differentiation similar to consisting of 33 specimens of primary breast cancer mesenchymal stem cells (Battula et al., 2010). Twist2, tissue, 12 specimens of matched non-cancer breast tissue similar to Twist1, is a potential mediator of mesench- and 8 specimens of matched breast metastatic tumours ymal stem cell self-renewal and lineage commitment. In from the lymph nodes, using immunohistochemistry addition, Twist2 may function to regulate critical (Figure 1). Of the 33 primary breast cancer tissue

Figure 1 Twist2 is overexpressed in human breast cancer, whereas E-cadherin is downregulated. Pictures from one representative case are presented. (a) Most non-cancer breast tissues were negative for Twist2 staining. (b) A total of 31 (93.9%) specimens of primary breast cancer tissue were positive for Twist2 staining. Twist2 was distributed in both the cytoplasm and the nucleus in most of the primary breast cancer tissue specimens (28/31). (c) All eight specimens of breast metastatic tumours were Twist2 positive. Twist2 was distributed in both the cytoplasm and the nucleus in most cases (six out of eight). (d) Non-cancer breast tissues were strongly positive for E-cadherin staining. Compared with the matched non-cancer breast tissues, the levels of E-cadherin in six of eight specimens of primary breast cancer tissues (e) and breast metastatic tumours from lymph node (f) were decreased.

Oncogene Twist2 promotes EMT and stem cell self-renewal X Fang et al 4709 specimens, 31 (93.9%) were positive for Twist2. Among expressing cells, whereas the mesenchymal markers these 31 Twist2-positive samples, 28 (90.3%) showed fibronectin, N-cadherin and vimentin were notably positive for Twist2 in both the cytoplasm and the upregulated (Figure 2d). In addition, real-time reverse nucleus, whereas Twist2 was primarily located in the transcriptase–PCR analysis demonstrated a gene ex- nucleus in the other 3 cases. Interestingly, all eight breast pression pattern consistent with EMT, including metastatic tumour specimens were Twist2 positive. E-cadherin repression and the concomitant induction Twist2 was distributed in both the cytoplasm and the of Vimentin, N-cadherin, Fibronectin, transforming nucleus in six of these samples, whereas Twist2 was growth factor-b1(TGF-b1), Twist1, Snai1, Slug, ZEB1 mostly localised in the nucleus in the other two samples. and ZEB2 (Figures 2e and f; Po0.05). To further In contrast, most non-cancer breast tissue samples examine whether Twist2 overexpression induced an (8/12) were negative for Twist2 staining, whereas the EMT phenotype in human breast cancer cells, we used other 4 non-cancer breast tissues samples were positive the low-tumourigenic human breast cancer cell line for the staining of Twist2, which was mainly located in MCF-7 to establish Twist2-expressing cells (Figure 2g). the nucleus. These data indicate that Twist2 may be The MCF-7/Twist2 cells showed a mesenchymal-like involved in tumourigenesis and metastasis of human morphology (Figure 2h), a decreased level of E-cadherin breast cancer. and b-catenin, and an increase in fibronectin and The expression of E-cadherin correlates to the N-cadherin expression in dense culture (Figure 2i). invasive potential of several types of human cancer Twist2 was distributed in both the nucleus and the including breast cancer. To determine whether a cytoplasm in MCF-7/Twist2 cells (Figure 2j). correlation existed between the expression patterns of Analysis using immunofluorescence staining further Twist2 and E-cadherin in human breast cancer, we also revealed that vimentin (Figure 3a) and fibronectin analysed E-cadherin expression using immunochemical (Figure 3b) were strongly induced in Twist2-expressing staining in the eight surgical specimens of human breast MCF-10A cells. In contrast, levels of E-cadherin, which cancer tumour tissues with lymph node metastases showed membrane localisation in vector cells, were mentioned above. Compared with the matched non- dramatically decreased in MCF-10A/Twist2 cells cancer breast tissues, we found that six of eight cases of (Figure 3c). b-Catenin was modestly downregulated breast tumour tissues and their matched six lymph node and translocated into the cytoplasm in MCF-10A/ metastatic specimens displayed decreased E-cadherin. Twist2 cells (Figure 3d). Ectopic expression of Twist2 Among these six cases, five cases showed strong positive in MCF-7 cells also decreased the levels of E-cadherin staining for Twist2 in the cytoplasm and nucleus (Figure 3e) and b-catenin (Figure 3f). (Figure 1). These results suggest that a reverse correla- To extend our analysis to a model with more tion may exist between the expression patterns of Twist2 physiological relevance, we used an MCF-10A three- and E-cadherin in human breast cancer progression. dimensional cell culture model to test some markers of EMT using immunofluorescence. The expression of E- cadherin, which is a central component of cell–cell Twist2 induces EMT in mammary epithelial cells and adhesion junctions, was dramatically downregulated, breast cancer cells whereas vimentin was sharply upregulated in the acinar On the basis of our observations that E-cadherin may be structures formed by Twist2-overexpressing MCF-10A involved in the functions of Twist2 in human breast cells compared with vector-infected cells (Figure 4a). We cancer progression, we next investigated whether Twist2 also investigated whether Twist2 alters epithelial cell was capable of inducing the EMT in human mammary polarity. Unlike monolayer cultures, mammary epithe- epithelial cells. We used MCF-10A cells to generate lial cells grown in three dimensions recapitulate various stable cell lines that constitutively expressed either Flag- features of in vivo breast epithelium, such as apicobasal tagged Twist2 or empty vectors by retroviral infection. polarisation (Debnath et al., 2003). To analyse changes MCF-10A cells are immortalised, non-transformed in polarity, we used immunofluorescence to visualise the human mammary epithelial cells that lack endogenous orientation of the Golgi apparatus in acini using the cis- Twist2 expression. Stable ectopic expression of Twist2 in Golgi protein GM130. In addition, we examined the MCF-10A cells was confirmed using as anti-Flag distribution of a6 integrin, which is a basal marker. In antibody and western blotting analysis (Figure 2a). vector-cell-derived acini, the apical portion of the Golgi Twist2 was located in both the nuclei and the cytoplasm, was always oriented towards the lumen. However, but mainly in the nuclei of MCF-10A/Twist2 cells as Twist2-expressing cell-derived acini showed a modest shown by immunofluorescence staining (Figure 2b). disruption in Golgi orientation; some of the Golgi Compared with vector-infected cells, Twist2-expressing apparatus was oriented towards the basal surface, which cells underwent a striking morphological change, which was rarely seen in control acini (Figure 4b). Consistent entailed a transformation from a cobblestone-like with these results, a6 integrin, which was located on the epithelial morphology to an elongated fibroblast-like basal surface of acini formed by vector cells, was morphology when the cells were plated at either low or depolarised in acini derived from Twist2-expressing cells high densities (Figure 2c). Western blot analysis of (Figure 4c). In addition, Twist2-expressing cell-derived MCF-10A cells that were grown in dense cultures acini elicited excess proliferation and nearly filled showed a relatively moderate downregulation of the luminal space with an increased proliferation marker epithelial markers E-cadherin and b-catenin in Twist2- Ki-67 (Figure 4d) and a decreased apoptotic marker

Oncogene Twist2 promotes EMT and stem cell self-renewal X Fang et al 4710

Oncogene Twist2 promotes EMT and stem cell self-renewal X Fang et al 4711 activated caspase 3 (Figure 4e). Collectively, these breast cancer cells in vitro. To further examine whether observations demonstrate that the ectopic expression Twist2 expression impacted tumour growth in vivo,we of Twist2 promotes an EMT phenotype in human subcutaneously injected the same number of Twist2- mammary epithelial cells and breast cancer cells, expressing or control MCF-7 cells into nude mice. As indicating that Twist2 is a potent inducer of EMT. shown in Figure 5e, Twist2 overexpression markedly enhanced the subcutaneous tumour growth over the control group after 4 weeks, and the volumes of the Twist2 enhances cell migration and colony formation tumours in the nude mice injected with Twist2-expres- in vitro and promotes tumour growth in vivo sing cells were larger than those of the control group. The EMT is characterised by increased cell motility and Taken together, our results indicate that Twist2 has is also a critical step in tumour progression. Given the prooncogenic activity and functions to promote tumour mesenchymal phenotype of MCF-10A/Twist2 cells, we growth both in vitro and in vivo. hypothesised that ectopic Twist2 expression enhances cell motility as a consequence of the EMT process. To test this hypothesis, we examined whether Twist2 Overexpression of Twist2 in MCF-10A and MCF-7 expression affects the motility of MCF-10A cells in a cells increases their CD44high/CD24low progenitor wound healing assay. Confluent cell monolayers of sub-population and promotes stem-like cell self-renewal vector- and Twist2-expressing cells were wounded with a As noted earlier, EMT generates cells with stem cell-like pipette tip. After 16 h, Twist2-expressing cells migrated properties (Mani et al., 2008). However, the functional away from the wound edge and showed wound closure, significance of Twist2 in breast CSCs remains unex- whereas the wound of vector-transfected cells did not plored. We determined whether stable expression of heal (Figure 5a). These results suggest that increased Twist2 induced such stem cell-like phenotypes in human Twist2 expression promotes cell motility. To investigate mammary epithelial cells and breast cancer cells. whether Twist2 enhances the colony-forming ability of Representative stem cell markers were analysed by MCF-10A cells in vitro, we seeded 200 cells in triplicate western blot. As shown in Figure 6a, compared with wells of six-well plates for a colony-forming assay. After the vector control, Twist2 expression caused an increase 10 days of culture, Twist2-expressing cells formed in the protein levels of the stem cell markers CD44, colonies that were more numerous and larger than Bmi-1 and Sox2. To test the self-renewal ability of those of the vector-transfected cells (Figure 5b). MCF-10A/Twist2 cells, we turned to a well-charac- MCF-10A cells are immortalised, non-transformed terised tumoursphere culture model to examine the human mammary epithelial cells, and do not show capacity for mammosphere formation in non-adherent capabilities of transforming and tumour growth even serum-free medium. Mammosphere formation assays of with ectopic overexpression of oncogene K-Ras, H- Twist2-expressing MCF-10A cells revealed an increase RasV12 or LBX1 (Ansieau et al., 2008; Yu et al., 2009). in the size and number of mammospheres compared Therefore, we used MCF-7 cells ectopically expressing with vector-transduced cells (Po0.05; Figures 6b and c). Twist2 to determine the effects of Twist2 expression on To further explore the role of Twist2 in stem cell cell migration and colony-forming activity in vitro and phenotypes in human breast cancer, we performed flow on tumourigenicity in nude mice. Wound healing assays cytometric analysis to identify CD44high/CD24low popu- showed that Twist2-expressing cells showed greater lations, which is a phenotype associated with normal migration potential than vector cells (Figure 5c). breast stem cells and potential breast cancer progenitors. Twist2-expressing MCF-7 cells formed more and larger Flow cytometric analysis demonstrated that the propor- colonies than the vector cells in six-well plates after 20 tion of CD44high/CD24low cells was 7.50% in the vector days of incubation (Figure 5d). These data suggest that control but reached 35.65% in MCF-10A/Twist2 cells Twist2 enhances the transformative activity of human (Po0.01; Figure 6d). We further investigated the effect

Figure 2 Ectopic overexpression of Twist2 in human mammary epithelial cells and breast cancer cells results in epithelial– mesenchymal transition (EMT). (a) MCF-10A cells constitutively expressing either Flag-tagged Twist2 or empty vector were generated by retroviral infection with a retroviral pBABE-puro and confirmed by western blot analysis using specific antibodies against Twist2 and the Flag tag. (b) Analysis using immunofluorescence reveals that Twist2 is located in both the nuclei and the cytoplasm but mainly in the nuclei of MCF-10A/Twist2 cells. (c) Twist2 induced mesenchymal morphology in MCF-10A cells. Cells were plated at low (top row) or high (bottom row) densities. Cells were monitored for morphological changes using phase-contrast microscopy. ( Â 100.) (d) Western blot analysis was used to evaluate EMT markers in cell lysates. Ectopic overexpression of Twist2 in MCF-10A cells increases the expression of the mesenchymal markers fibronectin, N-cadherin and vimentin but decreases the expression of the epithelial markers E-cadherin and b-catenin. (e) Relative levels of mRNAs encoding E-cadherin, vimentin, N-cadherin and fibronectin in MCF-10A cells expressing vector or Twist2 were determined by real-time reverse transcriptase–PCR. GAPDH mRNA was used to normalise variability in template loading. Error bars represent the mean±s.d. of triplicate experiments. *Po0.05. (f) Twist2 increases the transcription of other known EMT inducers in MCF-10A cells. Real-time reverse transcriptase–PCR analysis showed the relative mRNA expression levels of known EMT inducers in MCF-10A cells expressing vector or Twist2. GAPDH mRNA was used to normalise variability in template loading. Error bars represent the mean±s.d. of triplicate experiments. *Po0.05. (g) MCF-7 cells that constitutively express either Flag-tagged Twist2 (Twist2) or empty vector (vector) were generated by retroviral infection and confirmed by immunoblotting analysis. (h) Twist2 induces mesenchymal morphology in MCF-7 cells. ( Â 100.) (i) Twist2 induces changes in the expression of EMT markers in MCF-7 cells. The expression of epithelial and mesenchymal markers was analysed by western blotting. (j) Analysis using immunofluorescence reveals the nuclear and cytoplasm localisation of Twist2 in MCF-7/Twist2 cells.

Oncogene Twist2 promotes EMT and stem cell self-renewal X Fang et al 4712

Figure 3 Overexpression of Twist2 induces EMT marker alterations in MCF-10A (a–d) and MCF-7 (e, f) cells in two-dimensional culture. Twist2 induces the expression of vimentin (a) and ﬁbronectin (b), downregulates the expression of E-cadherin (c) and modestly alters the localisation of b-catenin (d) in MCF-10A cells. Ectopic expression of Twist2 in MCF-7 cells decreases the levels of E-cadherin (e) and b-catenin (f).

of Twist2 overexpression on the stemness of cancer Twist2 represses E-cadherin promoter activity stem-like cells in human breast cancer cells. As shown in Members of the bHLH family of transcription factors Figure 6e, Twist2 overexpression upregulated the dimerise to form heterodimers, which in turn bind the E- expression of stem cell markers CD44, Bmi-1 and box consensus sequence (CANNTG) in GC-rich pro- Sox2. The number and size of mammospheres was moter regions to regulate a diverse set of genes (Li et al., increased in MCF-7/Twist2 cells (Po0.01) (Figures 6f 1995; Gong and Li, 2002). To determine whether the and g). Moreover, Twist2 augmented the proportion of decreased expression of E-cadherin observed in Twist2- CD44high/CD24low cancer stem-like cells from 2.43% in overexpressing cells was the consequence of transcrip- MCF-7/vector cells to 15.04% in MCF-7/Twist2 cells tional downregulation, MCF-10A/vector and MCF- (Po0.01; Figure 6h). These data demonstrate that the 10A/Twist2 cells were transiently transfected with a overexpression of Twist2 can endow human mammary reporter plasmid containing the luciferase gene epithelial cells and breast cancer cells with a stem under the control of an 887 bp fragment of the cell-like phenotype. human E-cadherin promoter (from À680 to þ 207;

Oncogene Twist2 promotes EMT and stem cell self-renewal X Fang et al 4713

Figure 4 Overexpression of Twist2 induces EMT marker alterations in MCF-10A cells in three-dimensional culture. In acinar structures formed by vector- and Twist2-expressing MCF-10A cells, Twist2 downregulates the expression of E-cadherin, upregulated the expression of vimentin (a), alters the orientation of polar proteins GM130 (b) and a6 integrin (c), increases the level of proliferation marker Ki-67 (d) and decreases the level of apoptotic marker activated caspase 3 (e).

Fernando et al., 2010). As shown in Figure 7a, reporter active in more than 50% of primary breast tumours and activity was signiﬁcantly repressed in MCF-10A/Twist2 tumour-derived cell lines. Levels of STAT3, which is cells compared with control cells (Po0.01). These phosphorylated at tyrosine residue 705 (Y705), are results show that Twist2 directly or indirectly regulates frequently elevated in breast carcinomas (Lo et al., E-cadherin transcription in these cells. 2007). A previous report has shown that STAT3 activates Twist1 expression by binding to its promoter (Cheng et al., 2008). In addition, ectopic Twist1 STAT3 (signal transducer and activator of transcription expression in MCF-7 cells induces constitutive STAT3 3) is constitutively active in Twist2-expressing cells activation (Sullivan et al., 2009). To determine the Constitutive activation of STAT3 and its downstream relationship exists between Twist2 and STAT3, we targets are involved in multiple steps of tumourigenesis examined the expression and localisation of STAT3 from initiation to progression. STAT3 is constitutively that was phosphorylated at Y705 in MCF-10A/Twist2

Oncogene Twist2 promotes EMT and stem cell self-renewal X Fang et al 4714

Figure 5 Twist2 enhances cell migration and the colony formation of MCF-10A and MCF-7 cells in vitro and promotes tumour growth in vivo.(a) Twist2 expression in MCF-10A cells promotes cell migration in a wound-healing assay. ( Â 100.) (b) Twist2- expressing MCF-10A cells form more and larger colonies compared with the empty vector-expressing cells in six-well plates. (c) Twist2 enhances MCF-7 cell migration in a wound-healing assay. ( Â 100.) (d) Twist2-expressing MCF-7 cells form more and larger colonies compared with vector cells in six-well plates. (e) Twist2 promotes tumour growth in vivo. Eight female BALB/c nude mice, which were aged 4–6 weeks, were randomly divided into two groups. MCF-7 vector- or Twist-expressing cells (5 Â 106) were subcutaneously injected into BALB/c nude mice.

cells using western blot analysis. As shown in Figure 7b, MCF-10A/Twist2 cells, whereas no obvious pSTAT3 phosphorylated STAT3 (Y705) was upregulated in was observed in the vector-transfected cells (Figure 7c). Twist2-expressing cells compared with that in the We further examined the expression and localisation of controls. However, we were unable to find another pSTAT3 (Y705) in human breast cancer cells by activated form of STAT3, pSTAT3 (S727), in either the immunoblotting and immunofluorescence. Ectopic ex- vector- or the Twist2-transfected cells (data not shown). pression of Twist2 also constitutively activated STAT3 We also used immunofluorescence to confirm the (Figure 7d) and the activated pSTAT3 (Y705) was expression and localisation of phosphorylated STAT3 mainly distributed in the nuclei of MCF-7/Twist2 cells (Y705) in MCF-10A/Twist2 cells. Analysis using im- (Figure 7e). These results indicate that STAT3 activity is munofluorescence revealed that STAT3 was constitu- associated with Twist2 function in mammary epithelial tively activated and mainly located in the nuclei of cells and breast cancer cells.

Oncogene Twist2 promotes EMT and stem cell self-renewal X Fang et al 4715

Figure 6 Ectopic overexpression of Twist2 in mammary epithelial cells (a–d) and breast cancer cells (e–h) increases their CD44high/ CD24low progenitor sub-population and promotes stem-like cell self-renewal. Twist2 upregulated the expression of the stem cell markers CD44, Bmi-1 and Sox2 in MCF-10A (a) and MCF-7 (e) cells. Twist2 increases the size and number of mammospheres in MCF-10A (b, c) and MCF-7 (f, g) cells *Po0.05, **Po0.01. Twist2 increases the size of the CD44high/CD24low progenitor sub-population in MCF-10A (d) and MCF-7 (h) cells. Flow cytometry was used to analyse CD44high/CD24low sub-populations in the cells expressing either empty vector or Twist2. The percentages of the mean CD44high/CD24low population are indicated.

Discussion lial cells progressively develop into hyperplasia, carcinoma in situ, invasive cancer and metastasis. Twist2 has Breast carcinogenesis is a multistep and multipath been shown to be involved in some human primary disease process during which normal mammary epithe- tumours; however, little is known about the functional

Oncogene Twist2 promotes EMT and stem cell self-renewal X Fang et al 4716

Figure 7 Ectopic Twist2 expression signiﬁcantly downregulates E-cadherin promoter activity and constitutively activates STAT3. Luciferase reporter activity is signiﬁcantly repressed in MCF-10A/Twist2 cells compared with MCF-10A/vector cells (**Po0.01) (a). STAT3 is constitutively activated and primarily localised to the nuclei in MCF-10A/Twist2 (b, c) and MCF-7/Twist2 (d, e) cells, whereas no obvious pSTAT3 (Y705) is observed in empty vector-transfected cells.

signiﬁcance of Twist2 in human breast cancer. In this efﬁciently regulate the multistep process of tumourigen- study, our results demonstrate that Twist2 is over- esis. EMT is a highly coordinated developmental expressed in human breast cancers and lymph node process during embryogenesis and a pathological feature metastases and promotes an EMT phenotype in human in tumourigenesis (Acloque et al., 2009; Kalluri and mammary epithelial cells and breast cancer cells, Weinberg, 2009; Hanahan and Weinberg, 2011). Devel- indicating that Twist2 may function as a potent driver opmental EMT regulators including Twist1, Twist2, of EMT during breast cancer development. Of note, Snai1, Slug and Six1, as well as developmental signalling there is a huge discrepancy between the percentage of pathways, including TGF-b and Wnt/b-catenin, are Twist2-positive human primary breast tumours assessed aberrantly activated in cancers and correlate with poor by immunohistochemistry, which detected the protein clinical outcomes. Recently, EMT inducers or regula- level of the Twist2 gene in this report, and the tors, such as Twist1, Snai1, TGF-b, ZEB1, YB-1, LBX1 percentage of human breast samples harbouring an and Six1, have been shown to induce well-differentiated overexpression of Twist2 mRNA levels (more than cells and cancer cells to form populations with stem cell- twofold), which was measured by real-time quantitative like characteristics via promoting EMT, indicating that PCR analysis (Ansieau et al., 2008). We also observed there is a cross-talk between the EMT programme and that the Twist2 mRNA level and the Twist2 protein the pathways involved in regulating stemness in stem expression in two cases of human breast cancer tissue cells (Mani et al., 2008; Morel et al., 2008; Evdokimova samples and their matched non-cancer and lymph node et al., 2009; Iliopoulos et al., 2009; McCoy et al., 2009; metastatic specimens are not always correlated (data not Polyak and Weinberg, 2009; Wellner et al., 2009; Yu shown). Further work will be necessary to characterise et al., 2009; Ouyang et al., 2010; Singh and Settleman, the discrepancy between the mRNA level and the 2010). In this study, we demonstrated that Twist2 has a protein expression of Twist2 gene in breast cancer critical role in EMT and is involved in reprogramming progression. differentiated cells and cancer cells into stem-like Increasing evidence suggests that cancer cells cells. The ectopic expression of Twist2 in mammary often reactivate latent developmental programmes to epithelial cells and breast cancer cells increases the

Oncogene Twist2 promotes EMT and stem cell self-renewal X Fang et al 4717 CD44high/CD24low stem-like cell sub-population and the On phosphorylation of a tyrosine residue, activated self-renewal capability of these stem-like cells. This STAT3 translocates to the nucleus and regulates the finding was further confirmed by the increased expres- transcription of numerous target genes (Levy and sion of stem cell markers such as CD44, Bmi-1 and Sox2 Darnell, 2002). STAT3 activation has an important role in Twist2-expressing cells. Our study supports a link in TGF-b-mediated EMT and mediates epidermal between the EMT programme and the gain of epithelial growth factor-/epidermal growth factor receptor-in- stem cell-like properties. duced EMT by increasing the expression of Twist1 Of all the EMT markers regulated by Twist2, (Yang et al., 2006; Lo et al., 2007). Moreover, as a E-cadherin is considered a hallmark of epithelial cells. prosurvival pathway, STAT3 signalling is critical for E-cadherin is a central adhesion molecule of cell–cell breast cancer stem-like cell maintenance (Zhou et al., adhesion junctions that is essential for the formation 2007). In this study, we found that STAT3 was and maintenance of the epithelial cell phenotype. Loss constitutively active and mainly located in the nuclei of E-cadherin, which is consistently observed at sites of of Twist2-expressing mammary epithelial cells and EMT in embryonic development and tumourigenesis, breast cancer cells. Therefore, our current data suggest has wide-ranging transcriptional and functional con- that STAT3 may be involved in Twist2-induced EMT sequences for human breast epithelial cells (Peinado and stem-like cell self-renewal in human mammary et al., 2007; Onder et al., 2008). In a previous study, epithelial cells and breast cancer cells. short hairpin RNA-mediated knockdown of E-cadherin In summary, Twist2 contributes to breast cancer triggered EMT and resulted in the acquisition of a progression by promoting the activation of EMT and by mesenchymal phenotype and increased CSC activity in enhancing the self-renewal of cancer stem-like cells. The HMLER breast cancer cells (Gupta et al., 2009b). critical role of Twist2, along with Twist1, Snai1, TGF-b, Moreover, the E-cadherin promoter is frequently re- YB-1 and LBX1, in promoting EMT and stem cell self- pressed directly or indirectly by transcription factors renewal indicates that cancer cells often reactivate latent such as Twist1, Snai1, Slug, ZEB1, ZEB2, FOXC2, developmental programmes to efficiently regulate the KLF8 and E47, thereby disrupting the polarity of multistep process of tumourigenesis. These EMT epithelial cells and maintaining a mesenchymal pheno- inducers or regulators are often kept silent in adults to type (Yang and Weinberg, 2008; Thiery et al., 2009; maintain epithelial homeostasis and integrity. There- Ouyang et al., 2010). Our results show that Twist2 fore, knowledge of the molecular details of the directly or indirectly binds to the promoter of physiology and pathology of EMT and its function in E-cadherin and downregulates the expression of regulating the stemness of stem cells may facilitate the E-cadherin. This result indicates that Twist2 may promote development of new biomarkers for cancer detection as EMT and generate mesenchymal cells with stemness by well as more specific and potent anti-invasive drugs. down-egulating the expression of E-cadherin. The polycomb group protein Bmi-1 may be involved in Twist2-induced repression of E-cadherin expression. Materials and methods In this study, we demonstrated that the expression of stem cell marker Bmi-1 is increased in Twist2-expressing Cell culture and generation of cell lines human mammary epithelial cells and breast cancer cells. Human mammary epithelial cell line MCF-10A, breast cancer Bmi-1, a member of the polycomb-repressive complex 1, cell line MCF-7, packaging cell line GP2-293 and retroviral is commonly deregulated in various tumours, such as vector pBABE-puro were generous gifts from Professor breast cancer, and has a critical role in maintaining self- Kunxin Luo (Department of Molecular and Cell Biology, renewal in normal and malignant human mammary University of California, Berkeley, CA, USA). MCF-10A cells stem cells (Dimri et al., 2002; Liu et al., 2005, 2006). were maintained as described (Debnath et al., 2003). MCF-7 Bmi-1 inhibits phosphatase and tensin homologue, and GP2-293 cells were cultured in Dulbecco’s modied Eagle’s induces EMT in human nasopharyngeal epithelial cells, media supplemented with 10% fetal bovine serum. The human and is involved in the regulation of self-renewal and Twist2-Flag complementary DNA was cloned into a pBABE- puro expression vector. Cell lines expressing either Twist2-Flag differentiation of stem cells (Song et al., 2009b). A (MCF-10A/Twist2 and MCF-7/Twist2) or empty vector recent report showed that Twist1 directly regulated (MCF-10A/vector and MCF-7/vector) were generated by Bmi-1. Bmi-1-containing polycomb-repressive complex retroviral infection. Briefly, the GP2-293 packaging cells were directly represses E-cadherin expression. Bmi-1 and Twist1 grown to 60% confluency and co-transfected with 5 mgof are mutually essential to promote EMT and tumour- pBABE-puro or pBABE-puro-Twist2-Flag and 5 mgof initiating capability of human head and neck squamous pCMV-VSVG using the Lipofectamine 2000 (Invitrogen, cell carcinoma cells (Yang et al., 2010). Together with our Carlsbad, CA, USA). The viral supernatants were collected findings, Bmi-1 may be involved in Twist2-induced at 48 h after transfection and spun at 3000 g for 3 min to repression of E-cadherin expression. Further investigation remove cells and debris. The supernatant was transferred to a is required to understand the role of Bmi-1 in Twist2- new tube and filtered through a 0.45 mm filter. For retroviral infection of cells, MCF-10A or MCF-7 cells were plated at induced downregulation of E-cadherin. B105 cells per well on a six-well plate. After 24 h, the medium Constitutive activation of STAT3 has been observed was removed and 2 ml of the culture supernatant, which in a number of human epithelial malignancies, including contained retroviral vectors and polybrene (Sigma-Aldrich, tumours of the prostate, breast, lung, head and neck, St Louis, MO, USA) at a final concentration of 8 mg/ml, was brain and pancreas (Bromberg, 2002; Chan et al., 2004). added to the wells. After 24 h of infection, the culture medium

Oncogene Twist2 promotes EMT and stem cell self-renewal X Fang et al 4718 with viral supernatant was removed and replaced with fresh ATTC-30; GAPDH,forward:50-ACCCAGAAGACTGTG medium containing 2 mg/ml of puromycin for selection. MCF-10A GATGG-30,reverse:50-TCTAGACGGCAGGTCAGGTC-30. and MCF-7 cells that constitutively expressed either Flag-tagged Twist2 or empty vector were confirmed by western blot analysis Generation of E-cadherin reporter construct and luciferase with specific antibodies against Twist2 and the Flag tag. reporter assay Genomic DNA was isolated from MCF-10A cells and the Western blot analysis E-cadherin promoter was amplified using the following primers: Western blotting was performed as described previously (Song forward, 50-(BglII) CAAAAAATTAGGCTGCTAGCTC-30 et al., 2009a). Cell lysates were subjected to SDS–polyacryla- and reverse, 50-(HindIII) AATGCGTCCCTCGCAAGTCA-30. mide gel and immunoblot analysis with antibodies to Twist2 MCF-10A/vector or MCF-10A/Twist2 cells were seeded in six- (Abnova, Taipei, Taiwan), Flag (Sigma-Aldrich), E-cadherin, well plates at 2.5 Â 104 cells per well. After 24 h, cells were b-catenin, N-cadherin, fibronectin, GM130, CD44, CD24 (BD transfected with 2 mg E-cadherin promoter luciferase plasmid in Biosciences, Bedford, MA, USA), vimentin, Sox2 (R&D triplicate. After 48 h of transfection, cell lysates were prepared Systems, Minneapolis, MN, USA), STAT3, pSTAT3 to measure luciferase activity. (Tyr705), pSTAT3 (Ser727), active (cleaved) caspase 3 (Cell Signaling Technology, Danvers, MA, USA), a6 integrin Wound healing assay (Millipore, Billerica, MA, USA), Ki-67 (Zymed, San Francis- Cells were seeded in 6-cm dishes at a density of 5 105.At24h co, CA, USA), Bmi-1 and b-actin (Millipore). Â later, a wound was incised in the central area of the confluent culture, followed by careful washing to remove detached cells Immunofluorescence and the addition of fresh medium. Phase contrast images of the Cells were seeded on coverslips in six-well plates. After 24 h, wounded area were recorded using an inverted microscope at the cells were washed with phosphate-buffered saline (PBS) indicated time. twice, permeabilised in 0.1% Triton X-100 and fixed with 4% paraformaldehyde in PBS for 20 min. The paraformaldehyde Flow cytometry M was quenched with three washes in 100 m glycine in PBS. high low Coverslips were permeabilisated and then blocked for 1 h in Identification of CD44 /CD24 cells was performed using blocking buffer (PBS containing 10% newborn calf serum, 1% monoclonal anti-CD44-fluorescein isothiocyanate (clone G44- bovine serum albumin and 0.02% Triton X-100). After being 26) and anti-CD24-PE (clone ML5) antibodies (BD Bios- blocked, coverslips were incubated with antibodies in staining ciences). Cells were labelled and CD44/CD24 markers were buffer (blocking buffer lacking bovine serum albumin) over- analysed using a FACSCalibur flow cytometer (BD Bios- night at 4 1C. Coverslips were washed three times with staining ciences). buffer and then incubated with Alexa 488-conjugated and/or Alexa 546-conjugated secondary antibodies (Invitrogen) for Mammosphere culture 1 h at room temperature. Coverslips were then washed twice Mammosphere culture was performed as described in Dontu with 10% fetal bovine serum/0.02% Triton X-100 in PBS and et al. (2003) with a slight modification. Briefly, to induce stained with Hoechst 33 258. Finally, coverslips were washed sphere formation, cell cultures were dissociated to single cells three times with PBS and one time with water before being by 0.05% trypsin–EDTA solution and plated into 24-well mounted on slides. Immunofluorescence was observed with a ultra-low attachment plates (Corning Life Sciences, Lowell, Leica confocal microscope (Leica, Solms, Germany). MA, USA) at a density of 4000 viable cells/ml. Cells were grown in a serum-free Dulbecco’s modied Eagle’s medium/F12 Real-time reverse transcriptase–PCR medium supplemented with B27 (Invitrogen), 20 ng/ml Total cellular RNA was prepared using TRIzol reagent epidermal growth factor, 20 ng/ml basic fibroblast growth (Invitrogen) and the expression levels of E-cadherin, Vimentin, factor, 4 mg/ml heparin (Sigma-Aldrich) and 1% methyl N-cadherin, Fibronectin, TGF-b1, Twist1, Snai1, Slug, ZEB1 cellulose. The mammospheres were cultured for 7–10 days. and ZEB2 mRNAs were determined by real-time reverse transcriptase–PCR using SYBR Green. Data shown are Three-dimensional culture assay normalised to GAPDH expression and represent the average Cells were grown in three-dimensional culture on a layer of of three repeated experiments. Primer sequences were as growth factor-reduced Matrigel (BD Biosciences) in eight-well follows. E-cadherin, forward: 50-TGCCCAGAAA ATGAA chamber slides. Indirect immunofluorescence staining and analysis AAAGG-30, reverse: 50-GTGTATGTGGCAATGCGTTC-30; were performed as outlined previously (Debnath et al., 2003). Vimentin, forward: 50-GAGAACTTTGCCGTTGAAGC-30, reverse: 50-GCTTCCTGTAGGTGGCAATC-30; N-cadherin, forward: 50-ACAGTGGCCACCTACAAAGG-30, reverse: 50- Colony formation assay CCGAGATGGGGTTGATAATG-30; Fibronectin, forward: Cells were seeded in six-well plates at a density of 200 cells/well 50-CAGTGGGAGACCTCGAGAAG-30, reverse: 50-TCCCT and maintained in complete medium for 10–20 days. After CGGAACATCAGAAAC-30; TGF-b1, forward: 50-CAGAAA most of the colonies had expanded to more than 50 cells, the TACAGCAACAATTCCTGG-30, reverse: 50-TTGCAGTGT cells were washed with PBS, fixed in methanol for 15 min and GTTATCCCTGCTGTC-30; Twist1, forward: 50-GGAGT stained with crystal violet for 15 min. The plates were then CCGCAGTCTTACGAG-30, reverse: 50-TCTGGAGGACCT photographed, and the colonies were counted. At least three GGTAGAGG-30; Snai1, forward: 50-CCTCCCTGTCAGAT independent experiments were carried out for each assay. GAGGAC-30, reverse: 50-CCAGGCTGAGGTATTCCTTG- 30; Slug, forward: 50-GGGGAGAAGCCTTTTTCTTG-30, Tumour growth assay reverse: 50-TCCTCATGTTTGTGCAGGAG-30; ZEB1,for- To measure tumourigenicity in vivo in nude mice, an MCF-7/ ward: 50-AACCCAACTTGAACGTCACA-30, reverse: 50-ATTA vector or MCF-7/Twist2 cell suspension (5 Â 106 cells/ml) was CACCCAGACTGCGTCA-30; ZEB2,forward:50-TTCCTGG mixed with an equal volume of Matrigel (BD Biosciences) and GCTACGACCATAC-30, reverse: 50-TGTGCTCCATCAAGCA subcutaneously injected (200 ml total) into 4- to 6-week-old

Oncogene Twist2 promotes EMT and stem cell self-renewal X Fang et al 4719 female BALB/c nude mice. These eight mice were treated Statistical analysis weekly with 20 mlofa10À2 M ethanolic solution of E2 applied The results of the experimental studies were expressed as the to the skin of the neck. Mice were killed at 4 weeks after mean±s.d. Statistical differences were analysed by Student’s t- injection and examined for the growth of subcutaneous test. Po0.05 was regarded as significant. tumours. All animals were used in accordance with institutional guidelines, and the current experiments were approved by the Use Committee for Animal Care and performed in the Conflict of interest Cancer Research Centre, Xiamen University, China. The authors declare no conflict of interest.

Immunohistochemical staining of breast cancer specimens Tissue sections from 33 specimens of primary breast cancer, 12 Acknowledgements specimens of matched non-cancer breast tissue and 8 specimens of matched breast metastatic tumours from the lymph We thank Professor Kunxin Luo (Department of Molecular node were fixed in 10% formaldehyde and embedded in and Cell Biology, University of California, Berkeley), paraffin. Sections were then cut and stained using immuno- Professor Shideng Bao (Stem Cell Biology and Regenerative histochemistry as previously described (Bao et al., 2004). Medicine, Lerner Research Institute, Cleveland Clinic) and Briefly, paraffin-embedded tissue sections were processed for reviewers for critical comments. This work was supported by antigen retrieval by heating the sections in 10 mM sodium grants from the National Nature Science Foundation of China citrate (pH 6.0) at 95 1C for 20 min. Sections were immunos- (no. 30871242, 31071302), National Basic Research Program tained with the monoclonal antibodies anti-Twist2 (1:200) or of China (no. 2009CB941601, 2010CB732402), the Funda- anti-E-cadherin (1:200). The immunostaining was performed mental Research Funds for the Central Universities (no. with an ABC staining system (Santa Cruz Biotechnology, 2010121095), the Science Planning Program of Fujian Pro- Santa Cruz, CA, USA) using an avidin-biotinylated-peroxidase vince (2009J1010), the Outstanding Young Science Founda- detection method. Tissue procurement was approved by the tion of Fujian Province (no. 2010J06013) and the 985 Project institutional review board of Xiamen University. grant from Xiamen University.

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