While endogenous LBX1 mRNA level is not detectable in parental MCF-10A cells, it is expressed in several breast cancer cell lines (Supplemental Fig. S3), including a metastatic cell line MDA-MB-231 (Fig. 1D; Supplemental Fig. S3), which has mesenchymal cell characteristics, including a strong migratory ability. To test whether constitutive LBX1 expression in MDA-MB-231 cells contributes to their migratory phenotype, we knocked down endogenous LBX1 using lentivirus-driven shRNA constructs (shLBX1). Three shRNA constructs were identified as producing efficient knockdown of endogenous LBX1 mRNA, as confirmed by quantitative RT–PCR (qRT–PCR) (Fig. 1E). Inhibition of LBX1 expression in MDA-MB-231 cells using each of the three shRNA constructs resulted in significantly reduced cell migration in transwell assays (P < 0.05), whereas no effect was observed with shRNA against luciferase (shLuc) (Fig. 1F). Moreover, the LBX1 knockdown constructs also resulted in increased E-cadherin expression (for two shRNA constructs) and reduced fibronectin expression (for one shRNA construct) (Supplemental Fig. S4). Thus, suppression of LBX1 in MDA-MB-231 cells partially reverts the EMT process. In addition to MDA-MB-231 cells, overexpression of LBX1 also causes similar mesenchymal morphological changes and an increase in fibronectin in the luminal type breast cancer cell line T47D (Supplemental Fig. S5).

To address the potential relationship between LBX1 and other transcription factors and signaling molecules implicated in EMT, we tested whether LBX1 itself enhances the expression of known EMT inducers. LBX1 dramatically increased endogenous mRNA levels of transforming growth factor β2 (TGFB2), Snail1, ZEB1, and ZEB2 in MCF-10A cells, up to eightfold to 10-fold higher than vector-infected cells (Fig. 2A). No detectable change was observed in the level of TWIST1 mRNA. To test for a direct transcriptional effect on the promoters of the responsive EMT inducers, we transiently transfected luciferase reporter constructs containing the presumed proximal promoters of ZEB1 and Snail1 into HEK293 cells. Compared with the empty vector, LBX1 induced increased promoter activity of threefold to fourfold and fivefold to sixfold for ZEB1 and Snail1, respectively (Fig. 2B). We then used chromatin immunoprecipitation (ChIP) assays to determine whether LBX1 binds in vivo to the promoter region of these putative transcriptional targets. For each of the four potential target genes ZEB1, ZEB2, Snail1, and TGFB2, we used five overlapping primer sets, covering 2-kb regions upstream of the transcription start sites, to PCR-amplify genomic sequences from immunoprecipitated formalin cross-linked chromatin. For all four candidates, a promoter sequence containing the presumed LBX1-binding sequence was readily amplified with one or more sets of primers from the LBX1 immunoprecipitate, but not from IgG control (Fig. 2C). The full set of ChIP analyses for all five ZEB1 and TGFB2 promoter fragments, performed in duplicate, is shown in Supplemental Figure S6. To evaluate whether these EMT inducers play an important role downstream from LBX1, we performed individual and combination siRNA knockdown experiments in LBX1-expressing cells (Supplemental Fig. S7). Suppression of ZEB1, ZEB2, Snail1, and TGFB2 together, but not individually, significantly inhibited LBX1-induced migration and partially reverted expression of EMT marker. Taken together, these results suggest that LBX1 may be a direct transcriptional activator of ZEB1, ZEB2, Snail1, and TGFB2, four critical factors that have been implicated in the regulation of EMT.

Mammary epithelial cells that have undergone EMT have been shown recently to have an increased CD44^high/CD24^low population, a phenotype linked to both normal breast stem cells and potential breast cancer progenitors (Al-Hajj et al. 2003; Dontu et al. 2003; Sleeman et al. 2006). This is associated with an increased capacity for mammosphere formation, an anchorage-independent growth characteristic correlated with pluripotent progenitors (Mani et al. 2008). To test the effect of LBX1 on these features of breast cancer “stemness,” we compared the effect of LBX1 on MCF-10A cells with that of the prototype EMT regulators Snail1 and Twist1. Flow cytometric analysis of LBX1-expressing cells demonstrated a 10-fold increase in the CD44^high/CD24^low population, compared with vector-transduced cells (P < 0.001), comparable with the effect of Snail1 and Twist1 (Fig. 3A). Breast progenitor cell populations have been defined most carefully in the human mammary epithelial cell (HMEC) assay (Elenbaas et al. 2001; Mani et al. 2008). Consistent with our observations in MCF-10A cells, we detected a significant increase in the CD44^high/CD24^low population within LBX1-expressing HMECs (Fig. 3B). Mammosphere formation assays also showed an increase in both the size and number of mammospheres in LBX1-expressing cells, in both MCF-10A cells and HMECs (Figs. 3C,D; Supplemental Fig. S9). Conversely, suppression of endogenous LBX1 in MDA-MB-231 breast cancer cells led to reduced growth in the specialized mammosphere media, but not under standard in vitro culture conditions (Supplemental Fig. S10). The bFGF- and EGF-supplemented, serum-free mammosphere media has been shown to selectively enhance proliferation of stem cell-like subpopulation of cancer cells (Lee et al. 2006). Collectively, these results indicate that LBX1 triggers expression of markers and functional characteristics associated with breast cancer progenitors, a feature recently defined for inducers of EMT (Mani et al. 2008).

Twist proteins have been shown recently to cooperate with activation of the Ras pathway in promoting even more profound characteristics of EMT and initiation of tumorigenesis (Ansieau et al. 2008). To investigate the functional properties of LBX1 in a tumorigenesis model, we coexpressed it with an activated form of HRAS (HRAS^V12) in MCF-10A cells. Cell morphology in LBX1 + HRAS^V12-infected cells was dramatically different than seen with either construct alone, with prominent elongated spindle-shaped cells (Supplemental Fig. S11). Analysis of mesenchymal and epithelial cell markers showed a cooperative effect of LBX1 and HRAS^V12 in inducing characteristic features of EMT (Fig. 4A): N-cadherin induction was higher in LBX1 + HRAS^V12 cells relative to either LBX1 alone or HRAS^V12 alone, and most striking was the complete loss of epithelial markers E-cadherin and ZO-1 in LBX1 + HRAS^V12 cells. Thus, these two constructs appear to work together in inducing a more complete EMT phenotype. We further tested their ability to cooperate in generating tumors following subcutaneous inoculation into nude mice (Fig. 4B). None of four mice injected with MCF-10A cells expressing vector, LBX1, or HRAS^V12 alone developed tumors at 3 mo. In contrast, four out of four mice inoculated with cells expressing LBX1 + HRAS^V12 cells gave rise to tumors within 1 mo. LBX1 + HRAS^V12 cells demonstrated even more migratory activity in transwell assays than cells expressing either construct alone (Fig. 4C). Similar cooperative effect with LBX1 was also evident with ErbB2, a growth factor receptor commonly overexpressed in human breast cancer (Fig. 4C; Supplemental Fig. S12). For instance, in three-dimensional (3D) Matrigel culture, dramatic differences were evident: LBX1-expressing MCF-10A cells formed smaller acinar structures than vector-infected cells, whereas HRAS^V12-expressing cells formed larger 3D structures with preserved boundaries and failure to invade into the Matrigel (Fig. 4D). Remarkably, MCF-10A cells expressing both LBX1 and HRAS^V12 showed a prominent invasive phenotype, forming multiple cellular protrusions extending into the Matrigel (Fig. 4D). The invasive phenotype displayed by cells coexpressing LBX1 and ErbB2 was further evident in Matrigel/collagen 3D assay (Fig. 4D), which highlights the ability of cancer cells to invade through collagen (Xiang and Muthuswamy 2006). Taken together, these results demonstrate potent cooperation between LBX1 and characteristic oncogenes in promoting EMT and invasive properties correlated with tumorigenesis.

Although LBX1 is physiologically expressed during neural and muscle differentiation, database screening indicated that its expression is more prevalent in breast cancer than in cancers of neural or muscle origin. Indeed, RNA in situ hybridization for LBX1 in 14 human breast cancers demonstrated high-level expression in nine tumors, but not in surrounding normal breast epithelium (Supplemental Fig. S13). Interestingly, in one breast tumor of the triple-negative subtype (negative for estrogen receptor [ER], progesterone [PR], and Her2), which had adjacent foci of histologically high-grade and low-grade tumor, LBX1 expression was only present within the invasive lesion (Fig. 5A). To address a possible correlation between LBX1 expression and other known prognostic markers in breast cancer, we interrogated a large published data set, including 251 clinically annotated cases of breast cancer for which RNA microarray and protein immunohistochemistry (IHC) data are available (Miller et al. 2005). LBX1 expression in these breast cancers was significantly correlated with advanced tumor grade (grade III) (P = 0.0023), mutant p53 status (P = 0.00056), negative ER (P = 0.038), and PR status (P = 0.0023) (Fig. 5B). The correlation between LBX1 expression and adverse prognostic markers was also evident when analyzing p53 mutant-like tumors defined by their microarray expression profile analysis (P = 2.1e − 06). Since no ERBB2/HER2 protein expression data are available for this breast cancer data set, we analyzed mRNA expression of ERBB2/HER2, together with the IHC-verified ER/PR status, to define the subset of cancers characterized as ER/PR/HER2 “triple negative.” Clinically, this breast cancer subset is known to be associated with higher tumor grades, poor response to chemotherapy, and an unfavorable prognosis. Indeed, higher LBX1 mRNA levels were present in triple-negative tumors (P = 0.0017). Taken together, these data suggest that aberrant expression of the developmentally regulated EMT inducer LBX1 is associated with more invasive subtypes of human breast cancer.

Cells (1 × 10⁵) were plated without EGF on 8-μm pore size Transwell filters (Corning) in 3D medium as described (Debnath et al. 2003). Assays were stained and quantified after cells migrated for 48 h. For MDA-MB-231 cells, 0.3 × 10⁵ cells were used in the assay for 24 h.