Mutation or misexpression of homeobox genes is common in neoplastic disease (1
), but a causal role for these genes in tumorigenesis has been a subject of debate. Recently, a role for homeobox genes in tumorigenesis has been strongly supported by the demonstration that several homeobox genes play an active role in breast and other cancers (37
). Because homeobox genes regulate developmental programs that coordinate many different cell behaviors important in embryogenesis, it is not surprising that their misexpression in differentiated tissues can result in the acquisition of numerous tumor-promoting properties such as proliferation, migration, invasion, and survival.
The Six1 homeobox gene plays an important role in the development of multiple different tissues by regulating proliferation and survival (2
). We have previously shown that Six1 is overexpressed in breast cancer (4
), where it stimulates proliferation, causes genomic instability, and transforms immortalized human mammary epithelial cells (38
). In addition, we have previously reported that Six1-mediated upregulation of cyclin A1 is critical for the ability of Six1 to increase proliferation of breast cancer cells (5
). In the present study, we identified Six1-mediated alterations in the expression of a number of genes included in the TβRS. We further showed that Six1 overexpression in MCF7 cells led to activation of the TGF-β pathway, a previously unreported target pathway of Six1. Together, these data demonstrate that Six1, in addition to upregulating cyclin A1, also increases TGF-β signaling in mammary carcinoma cells.
The TGF-β pathway plays a dual role in tumor progression. Evidence from numerous experimental systems has defined a tumor suppressive role for TGF-β based upon the growth-inhibitory activity of TGF-β in normal cells and early neoplastic lesions (11
). However, in later stages of tumorigenesis, resistance to the growth-inhibitory effects permits the unmasking of pro-metastatic functions. In breast cancer patients, the TβRS has been correlated with metastasis (13
), suggesting an association between increased TGF-β signaling and advanced disease. Based on its established pro-proliferative activity in mammary carcinoma cells coupled with its activation of TGF-β signaling, Six1 expression has the potential to selectively promote the pro-metastatic activity of TGF-β while antagonizing its growth-inhibitory function. Therefore, in the context of Six1 expression, TGF-β signaling may be a potent contributor to tumor progression.
TGF-β signaling is believed to play a role in breast cancer progression by stimulating metastatic spread, in part through induction of EMT (12
). Treatment of numerous cell lines with TGF-β induces EMT, and the effects of TGF-β include the dissolution of adherens junctions and tight junctions, increased migration and invasion, regulation of epithelial and mesenchymal markers, and alteration of cell matrix adhesion (23
). In our studies, in addition to increased TGF-β signaling we observed numerous features of EMT in both MCF7 mammary carcinoma cells and in the nontransformed mammary epithelial cell line MCF12A, indicating a general role for Six1 in induction of an EMT in mammary epithelial cell lines. Consistent with these results, analysis of MCF7-Six1–derived tumors displayed both increased cytoplasmic E-cadherin and nuclear Smad3 compared with MCF7-Ctrl tumors, confirming our in vitro observations in vivo. While our study is the first to describe a role for Six1 in oncogenic EMT, a recent Six1/Six4
double-knockout study in mice does suggest a possible role for the Six family proteins in regulating developmental EMT during the delamination of migratory muscle precursor cells from the dermomyotome and subsequent migration to the limb bud (22
). In addition, the highly related family member Six2 is critical for maintaining the mesenchymal progenitor cell population in the developing kidney, and loss of Six2 results in premature and ectopic differentiation of mesenchymal cells into epithelia (39
). Together, these data implicate the Six family of homeoproteins as critical regulators of EMT in both developmental and oncogenic contexts.
The role of TGF-β signaling in EMT is well established. Our study shows that inhibition of the TGF-β signaling reversed elements of Six1-induced EMT. These data demonstrate that Six1-induced EMT is, at least in part, dependent on TGF-β signaling. Interestingly, inhibition of TGF-β signaling was unable to completely reverse Six1-induced EMT, indicating that Six1 activates additional pathways responsible for inducing elements of EMT, or alternatively that the cells exhibit a more permanent EMT that is no longer dependent on increased TGF-β signaling (31
). In support of the involvement of additional EMT-promoting pathways, Six1 has been implicated in the Sonic hedgehog pathway (40
), the Notch pathway (41
), and the Wnt/β-catenin pathway during normal development (42
), all of which can contribute to EMT (19
Recent evidence has convincingly established a role for EMT in metastatic dissemination and tumor progression (16
). The loss of epithelial properties and simultaneous acquisition of mesenchymal properties permits detachment from the neighboring cells, invasion through the underlying basement membrane, movement into the surrounding stroma, and eventual intravasation into local blood or lymphatic vessels, setting the stage for metastatic spread (15
). We demonstrate, for what we believe is the first time, in an animal model that Six1 overexpression not only activates TGF-β signaling and induces EMT, but that it also promotes lymph node metastasis and, on rare occasion, bone metastasis, of breast cancer cells from an orthotopic site. This is remarkable, as one of the commonly cited drawbacks of xenograft and other mouse models of cancer is that metastatic cells preferentially colonize the lungs and usually fail to colonize other sites common for human breast cancer, including the lymph nodes, liver, bone, and brain (43
). Indeed, considerable effort has been directed toward developing xenograft models that can be used to study bone metastasis, as most human-derived cells lines do not metastasize to the bone when grown orthotopically in mice (44
). In addition, many orthotopic xenograft models used to study metastasis debulk the primary tumors to allow more time for distant metastases to develop. The fact that Six1 is sufficient to induce metastasis to lymph nodes and bone within the time span of primary tumor growth suggests that it is a powerful regulator of the metastatic process. However, while the orthotopic model implicates Six1 in the early stages of metastasis, it does not adequately assess whether Six1 contributes to the later stages of metastasis, including extravasation and distant site colonization, stages in which regulators of EMT and TGF-β signaling are involved (13
). Therefore, we also tested the role of Six1 in inducing experimental metastasis using an intracardiac injection model. We demonstrate that intracardiac injection of MCF7-Six1 cells significantly increased metastatic burden and decreased overall survival when compared with injection of MCF7-Ctrl cells. Together these results demonstrate that Six1 is not only a major regulator of lymphatic metastasis, but that it also contributes to later stages of metastasis, potentially by increasing survival in the bloodstream, extravasation, or proliferation at a distant site.
Importantly, we demonstrate that TGF-β signaling is a critical mediator of Six1-induced experimental metastasis. Interestingly, in the intracardiac injection model, Six1 regulates the TGF-β response in a manner that is consistent with the notion that Six1 expression antagonizes the tumor-suppressive activity of TGF-β and mediates a switch to its pro-metastatic activity. In mice injected with the MCF7-Ctrl cells, inhibition of TGF-β signaling significantly decreased survival while increasing the metastatic burden, as would be expected if this signaling pathway was acting in a tumor-suppressive manner. In contrast, in the mice injected with Six1-expressing cells, inhibition of TGF-β signaling decreased their metastatic burden and significantly increased their survival, suggesting that in vivo Six1 may be important for promoting the pro-metastatic effects of TGF-β while inhibiting its tumor-suppressive effects. Consistent with this hypothesis, Six1 has been previously shown to increase proliferation of breast cancer cells (5
), which would antagonize the well-established growth-inhibitory and tumor-suppressive activity of TGF-β (11
). In combination, the ability of Six1 to activate TGF-β signaling while attenuating its growth-inhibiting effects would preferentially activate the pro-metastatic activity of TGF-β, including EMT.
As is the case with many cancers, breast cancer metastasis represents the most deadly stage of tumor progression and is currently the least treatable stage. An understanding of the underlying mechanisms of the metastatic process will provide the opportunity for targeted therapeutics and also for better screening of breast cancer patients most at risk for spread. Our findings that overexpression of Six1 mRNA in human breast cancers is significantly associated with shortened time to metastasis and to relapse, and with decreased survival, reinforces the role of Six1 in human breast cancer progression. These clinical findings, coupled with our experimental and clinical findings that Six1 activates TGF-β signaling, that Six1 and nuclear Smad3 immunoreactivity correlate in human breast tumors, and that Six1 induces EMT and promotes metastatic spread in immunocompromised mice, establish Six1 as a viable candidate for future studies as a potential drug target in breast cancer. Additionally, our results show that the pro-metastatic properties of Six1 may not be confined specifically to breast cancer. On the contrary, in a surprisingly large number of cancers, Six1 expression is significantly associated with disease progression or poor outcome. Thus, we propose that Six1 is a global regulator of tumor metastasis, at least in part due to its ability to increase TGF-β signaling and induce EMT.