TBX2 induces epithelial-mesenchymal transition (EMT) in breast epithelial cells.

<p>(A) Bright field images (40x magnification) of murine HC11 mammary epithelial cells stably transfected with pCDNA3 (+vector) or pCDNA3-TBX2 (+TBX2) expression plasmids showing morphological changes in TBX2-expressing HC11. (B) Western blot analysis using whole cell lysates from HC11+vector and HC11+TBX2 cells shows TBX2-induced downregulation of epithelial (Epi) and upregulation of mesenchymal (Mes) marker proteins. E-cad  =  E-cadherin; ß-cat  =  ß-catenin; N-cad  =  N-cadherin; Vim  =  Vimentin. Actin (ß-actin) was used as loading control. Fold changes in protein levels quantified by densitometry and normalized to Actin values are shown. (C) Bright field images (40X magnification) of human MCF10A mammary epithelial cells stably expressing pCDNA3 or pCDNA3-TBX2 reveal mesenchymal transformation of MCF10A+TBX2 cells. (D) Western Blot analysis shows that ectopic expression of TBX2 in MCF10A cells prompts a switch of EMT marker expression. (E) Immunofluorescence analysis of TBX2 (red) and EMT marker expression (40X magnification) shows loss of membrane-associated expression of epithelial (green) with a reciprocal gain of mesenchymal (red) marker expression in HC11+TBX2 cells as compared to HC11+vector control cells. Nuclei were stained with Hoechst 33258 (blue). (F) qPCR analysis comparing <i>Tbx2</i> and EMT marker expression in HC11+vector and HC11+TBX2 cells. E-cad  =  <i>E-cadherin</i>; ß-cat  =  <i>ß-catenin</i>; ZO1 =  <i>zona occludens 1;</i> Dsp  =  <i>Desmoplakin</i>; N-cad  =  <i>N-cadherin</i>; Mmp3 =  <i>matrix metalloprotease 3</i>. Values were normalized to <i>Gapdh</i>. Fold changes as compared to vector control cells are shown. Error bars represent the mean ± SEM (n = 3; Student <i>t</i>-test). <i>P</i> values are indicated.</p

Proposed model for TBX2 regulation of EMT.

<p>TGFß signaling, or amplification of human chromosomal region 17q23 lead to aberrant TBX2 expression in differentiated breast epithelial cells, which normally lack TBX2 expression. TBX2 directly represses transcription of the epithelial differentiation marker <i>E-cadherin</i> and downregulates other epithelial markers. In contrast, TBX2 induces expression of mesenchymal markers, thereby resulting in EMT and invasion of normal and malignant breast epithelial cells.</p

Knockdown of TBX2 reduces pulmonary metastasis of human MDA-MB-435 breast carcinoma cells.

<p>(A) Representative images of lungs harvested from athymic <i>nu/nu</i> Nude mice forty days after tail vein injection with MDA-MB-435 tumor cell clones expressing either control non-target shRNA (shCtrl) or TBX2-specific shRNA (shTBX2). Top panel: India ink staining of lungs shows the absence of surface lung metastases in mice injected with shTBX2-expressing MDA-MBA-435 tumor cells (Magnification 7x). Only the control group produced macroscopic lung nodules (asterixes) and an elevated number of micrometastases (white arrows). Bottom panel: H&E stained paraffin-sections of representative lungs from each study group (Magnification: 40X). Dotted lines highlight lung metastases in the control group. (B) Quantification of total lung metastasis burden in the same sets of mice as in (A). Average numbers of lung surface metastases are shown; white column  =  mean of 5 control mice analyzed: black column  =  mean of 10 mice injected with two MDA-MB435-shTBX2 tumor cell clones. Data represent the mean ± S.D. (n≥5; Student <i>t</i>-test). (C) Inhibition of TBX2 does not significantly affect cell proliferation of MDA-MB-435 tumor cells. Equal numbers of control non-target shRNA and shTBX2-expressing cells were grown under sub-confluent conditions and counted every 3 days over a 9-day period. Error bars represent the mean ± S.D. (n = 3; Student <i>t</i>-test). (D) qPCR showing that stable knockdown of TBX2 does not significantly alter <i>p21</i> mRNA expression levels in MDA-MB-435 tumor cells. Values were normalized to <i>GAPDH</i> and fold changes compared to the shRNA control group are shown. Error bars represent the mean ± SEM (n = 3; Student <i>t-</i>test). NS  =  not significant.</p

Knockdown of TBX2 in metastatic human breast cancer cell lines abrogates tumor cell invasion.

<p>(A, B) Inhibition of TBX2 significantly reduces cell motility rates of (A) MDA-MB-435 tumor cells in <i>in vitro</i> scratch assays, and (B) of MDA-MB-157 breast cancer cells in Transwell migration assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041355#s4" target="_blank">Methods</a>). Data represent the mean ± S.D. (n = 3; ANOVA test). (<b>C</b>) MDA-MB-435 cells stably expressing TBX2-specific shRNA (+shTBX2) grow as non-invasive spheres in three-dimensional (3D) Matrigel, whereas MDA-MB-435 control tumor cells expressing non-target shRNA (+shCtrl) grow as spheroids that invade the surrounding extracellular matrix. (D, E) Transwell matrigel assays showing that knockdown of TBX2 significantly reduces invasion rates of (<b>D</b>) MDA-MB-435, and (E) MDA-MB-157 breast tumor cells. Data represent mean ± S.D. (n = 3; Student’s <i>t</i>-test). P values are indicated.</p

TBX2 bind to the <i>E-cadherin</i> promoter <i>in vivo and</i> represses <i>E-cadherin</i> transcription.

<p>(A) Schematic of the <i>E-cadherin/CDH1</i> promoter depicting the location of potential TBX2 binding sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041355#pone.0041355-Redmond1" target="_blank">[10]</a>: half T-site (black box) and InR  =  Initiator element (hatched box), and of ChIP primers used in (B). (B) ChIP analysis shows <i>in vivo</i> binding of exogenous TBX2 to the most proximal region of the endogenous <i>E-cadherin</i> gene promoter in HC11 mammary epithelial cells. DNA derived from sheared chromatin fragments from HC11+vector and HC11+TBX2 was immunoprecipitated with two antibodies specific to TBX2 (#1 =  Millipore AB4147; #2 =  SC-17880x), an antibody specific to acetyl Histone 3, or normal rabbit IgG and quantified by semi-quantitative PCR. As a control, <1% of input chromatin was used in the PCR analysis. The bar graph on the bottom panel shows a quantification of the TBX2-specific ChIPs for the proximal (−131/+61) <i>E-cadherin</i> promoter as a function of the percentage of input chromatin. (C) Transient reporter assays of HC11 cells transiently co-transfected with a human <i>E-Cadherin</i> promoter (−108 to +125) luciferase reporter construct (pEcad-luc) in combination with pCDNA3 vector (basal) or increasing concentrations of pCDNA3-TBX2 (+TBX2), as indicated. One representative experiment of n = 3 biological replicates is shown; P-value: *<i>p<0.05</i> (triplicate samples; Student <i>t</i>-test).</p

<i>TBX2</i> overexpression in primary human breast tumors is correlated with reduced metastasis-free survival.

<p>Kaplan Meier analysis demonstrates that <i>TBX2</i> mRNA overexpression is associated with shortened recurrence-free survival (A) in a meta-analysis of six combined published microarray datasets comprising 1107 primary human breast tumors <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041355#pone.0041355-Sims1" target="_blank">[57]</a>, and (B) in an NKI study of 295 women with early-stage invasive breast carcinomas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041355#pone.0041355-vandeVijver1" target="_blank">[66]</a>. In both of these datasets, the optimal cut-point value of <i>TBX2</i> expression was used to divide the samples into high (above median; green) and low (below median; blue) <i>TBX2</i> expression. A batch correction was performed on the six-study set (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041355#s4" target="_blank">Methods</a>). High <i>TBX2</i> expression is particularly associated with poor prognosis in estrogen receptor-positive (ER+) breast tumors (right panels). ER status was determined by immunohistochemistry (IHC). P values are indicated.</p

TBX2 imparts a mesenchymal phenotype on human breast cancer cells.

<p>(A) Western blot analysis shows efficient TBX2 knockdown in human MDA-MBA-435 tumor cells stably expressing TBX2-specific shRNA (shTBX2) as compared to cells expressing non-target shRNA (shCtrl). Actin was used as loading control. (B) Depletion of TBX2 in MDA-MB-435 tumor cells leads to a loss of the mesenchymal morphology characteristic for this breast carcinoma cell line. Representative images of high-density cell cultures are shown (40X magnification). (C) Inhibition of TBX2 in human MDA-MB-157 breast carcinoma cells through transient transfection with TBX2-targeted siRNAs (siTBX2) induces a ‘cobblestone’-like epithelial cell morphology. In contrast, MDA-MB-157 cells transiently transfected with scrambled siRNA control (siCtrl) exhibit a profound ‘spindle-like’ mesenchymal phenotype. Representative images of high-density cultures (40x magnification) of tumor cells three days post siRNA transfection are shown. (D, E) qPCR analysis of <i>TBX2</i> and EMT marker expression in (D) MDA-MB-435 cells expressing shCtrl or shTBX2, and in (E) MDA-MB-157 cells 3 days post transfection with siCtrl or siTBX2. TBX2 knockdown leads to an upregulation of epithelial adhesion and tight junction genes (E-cad  =  <i>E-cadherin</i>; ZO1 =  <i>zona occludens 1</i>), whereas it results in loss of mesenchymal marker expression: N-cad  =  <i>N-cadherin</i>; VIM  =  <i>Vimentin</i>; FN1 =  <i>Fibronectin</i>, and MMP3 =  <i>matrix metalloprotease 3</i>. Values were normalized to <i>GAPDH</i> and fold changes compared to the respective control groups are shown. Data represent the mean ± SEM (n = 3; Student <i>t</i>-test); p-values: <i>*p<0.05;</i> **<i>p = 0.003</i>; ***<i>p<0.001.</i> (F) Immunofluorescence analysis (40X magnification) confirms the re-expression of epithelial (green) E-cadherin, ß-catenin, and ZO1, and the loss and/or mislocalization of mesenchymal (red) markers (N-cadherin, Vimentin) in TBX2-depleted MDA-MB-435 cells. Nuclei were stained with Hoechst 33258 (blue).</p

Endogenous TBX2 is induced during TGFß-mediated EMT of primary human mammary epithelial cells (HMEC).

<p>(A) Bright field (40x magnification) and immunofluorescence images (63X magnification) of primary HMEC treated with 5 ng/ml TGFß1 (+TGFß) for 12 days as compared to untreated control cells (-TGFß). TGFß induces EMT-like morphological changes and nuclear expression of TBX2 (red). Nuclei were stained with Hoechst 33258 (blue). (B) qPCR analysis shows a time course analysis of <i>TBX2</i> mRNA induction in TGFß–treated HMEC in comparison to changes in epithelial <i>E-cadherin</i> (E-cad) and mesenchymal <i>Vimentin</i> (Vim) expression. Values were normalized to <i>GAPDH</i> mRNA and represent fold changes as compared to control untreated HMEC at the indicated time points. Error bars represent SEM of each sample in triplicates. (C) Western blot analysis of TBX2 and EMT marker expression in untreated HMEC (−) and in HMEC treated with TGFß for 12 days. E-cad  =  E-cadherin; ß-cat  =  ß-catenin; Vim  =  Vimentin; N-cad  =  N-cadherin; Epi =  epithelial; Mes  =  mesenchymal markers. Actin  =  ß-actin was used as loading control. Densitometric quantification of fold changes in protein levels normalized to Actin values is shown.</p

TBX2 promotes migration and invasion of mammary epithelial cells.

<p>(A, B) <i>In vitro</i> ‘scratch’ assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041355#s4" target="_blank">Methods</a>) monitoring the migration of (A) murine HC11 and (B) human MCF10A cells stably expressing pCDNA3 vector (+vector) or pCDNA3-TBX2 (+TBX2) over a period of 24–32 hours (h). Representative bright field images of cells (10x magnification) are shown in the left panel. Right panel: statistical evaluation of the distance between the two borders (dotted lines; left panels) at different time points after ‘scratch’ (n = 3; ANOVA test). (C, D) Transwell matrigel invasion assays show a significantly increased ability of TBX2-expressing HC11 (C) and MCF10A cells (D) to invade through a matrigel layer (n = 3, Student’s <i>t</i>-test). The mean ± S.D. is shown. P values are indicated.</p

Overexpression of TBX2 in normal breast epithelial cell lines results in reduced expression of <i>E-cadherin</i> and knockdown of TBX2 in breast carcinoma cell lines leads to an increase of <i>E-cadherin</i> expression.

<p>Overexpression of TBX2 in normal breast epithelial cell lines results in reduced expression of <i>E-cadherin</i> and knockdown of TBX2 in breast carcinoma cell lines leads to an increase of <i>E-cadherin</i> expression.</p