Advertisement
Defining a role for the homeoprotein Six1 in EMT and mammary tumorigenesis
Derek C. Radisky
Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA.
Address correspondence to: Derek C. Radisky, Department of Cancer Biology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, Florida 32224 USA. Phone: (904) 953-6913; Fax: (904) 953-0277; E-mail: radisky.derek@mayo.edu.
Find articles by Radisky, D. in: JCI | PubMed | Google Scholar
Published August 24, 2009 - More info
J Clin Invest. 2009;119(9):2528–2531. https://doi.org/10.1172/JCI40555.
© 2009 The American Society for Clinical Investigation
Related articles:
Abstract
Six1 is a developmentally regulated homeoprotein with limited expression in most normal adult tissues and frequent misexpression in a variety of malignancies. Here we demonstrate, using a bitransgenic mouse model, that misexpression of human Six1 in adult mouse mammary gland epithelium induces tumors of multiple histological subtypes in a dose-dependent manner. The neoplastic lesions induced by Six1 had an in situ origin, showed diverse differentiation, and exhibited progression to aggressive malignant neoplasms, as is often observed in human carcinoma of the breast. Strikingly, the vast majority of Six1-induced tumors underwent an epithelial-mesenchymal transition (EMT) and expressed multiple targets of activated Wnt signaling, including cyclin D1. Interestingly, Six1 and cyclin D1 coexpression was found to frequently occur in human breast cancers and was strongly predictive of poor prognosis. We further show that Six1 promoted a stem/progenitor cell phenotype in the mouse mammary gland and in Six1-driven mammary tumors. Our data thus provide genetic evidence for a potent oncogenic role for Six1 in mammary epithelial neoplasia, including promotion of EMT and stem cell–like features.
Authors
Erica L. McCoy, Ritsuko Iwanaga, Paul Jedlicka, Nee-Shamo Abbey, Lewis A. Chodosh, Karen A. Heichman, Alana L. Welm, Heide L. Ford
Abstract
Inappropriate activation of developmental pathways is a well-recognized tumor-promoting mechanism. Here we show that overexpression of the homeoprotein Six1, normally a developmentally restricted transcriptional regulator, increases TGF-β signaling in human breast cancer cells and induces an epithelial-mesenchymal transition (EMT) that is in part dependent on its ability to increase TGF-β signaling. TGF-β signaling and EMT have been implicated in metastatic dissemination of carcinoma. Accordingly, we used spontaneous and experimental metastasis mouse models to demonstrate that Six1 overexpression promotes breast cancer metastasis. In addition, we show that, like its induction of EMT, Six1-induced experimental metastasis is dependent on its ability to activate TGF-β signaling. Importantly, in human breast cancers Six1 correlated with nuclear Smad3 and thus increased TGF-β signaling. Further, breast cancer patients whose tumors overexpressed Six1 had a shortened time to relapse and metastasis and an overall decrease in survival. Finally, we show that the effects of Six1 on tumor progression likely extend beyond breast cancer, since its overexpression correlated with adverse outcomes in numerous other cancers including brain, cervical, prostate, colon, kidney, and liver. Our findings indicate that Six1, acting through TGF-β signaling and EMT, is a powerful and global promoter of cancer metastasis.
Authors
Douglas S. Micalizzi, Kimberly L. Christensen, Paul Jedlicka, Ricardo D. Coletta, Anna E. Barón, J. Chuck Harrell, Kathryn B. Horwitz, Dean Billheimer, Karen A. Heichman, Alana L. Welm, William P. Schiemann, Heide L. Ford
Breast cancer progression is a highly orchestrated process that is much more than just acquisition of increased cellular proliferative capacity. The first step in the development of breast cancer, proliferation of epithelial cells into the central lumen, involves the acquisition by the cancer cells of the ability to evade structural and biochemical constraints imposed by cell-cell and cell–extracellular matrix interactions. Progression from early, proliferative disease through ductal carcinoma in situ to invasive breast cancer requires the ability to break down the basement membrane and to interact with the stromal extracellular matrix. The ultimate development of metastatic capability, the most deadly and least treatable stage of breast cancer progression, requires the cancer cells to separate from the parental tumor mass, to intravasate into and subsequently extravasate from the vasculature, and to settle and grow at a distant site. All of these steps, from initial breakdown of tissue structure, through increased invasiveness, and ultimate distribution throughout the body, are characteristic features of epithelial-mesenchymal transition (EMT), a developmental process in which adhesive epithelial cells acquire the motile and migratory characteristics of mesenchymal cells (1). Both developmental and pathologic EMT are associated with dramatic alterations in patterns of gene expression, and recent studies have shown that a number of the transcription factors that regulate developmental EMT, including Snail, Slug, and Twist, are also activated during breast cancer progression (2). Determination that transcription factors involved in early stages of development also become reactivated in tumors provides the possibility of entirely novel targets for therapeutic intervention. However, although there has been considerable recent research effort directed toward understanding how EMT is controlled during breast cancer progression, it is clear that our understanding of this process is still incomplete.
-
Six1 stimulates mammary tumor progression through induction of EMT
Homeobox (Hox) genes encode an extensive family of conserved transcription factors designated as homeoproteins, members of which have been implicated in nearly every step of embryonic development, controlling proliferation, cell death, and morphogenesis. Deregulation of Hox gene expression has been found in many cancer types, and while a direct causative relationship between increased expression and cancer development has not been identified for most Hox genes, those found to be upregulated in cancer are usually associated with poorly differentiated cell types during development (17). Six1 is a homeoprotein that is essential for the development of a number of organs and is upregulated in proliferating precursor populations relative to differentiated adult tissues (18). Six1 was first identified as upregulated in breast cancer cells in 1998 (18). In the decade since, subsequent studies have shown that it may play a critical role in breast cancer development. Six1 shows increased expression in human breast cancer through gene amplification (18, 19), and exogenous overexpression of Six1 in mammary cells is sufficient to induce malignant transformation and chromosomal instability (20). Mechanistic insights come from the observation that overexpression of Six1 leads to abrogation of cell cycle checkpoints (18). The current studies by McCoy et al. (15) and Micalizzi et al. (16) considerably extend these previous investigations by using mouse models to define how Six1 induces EMT to stimulate tumor development and metastasis (Figure 1).
Figure 1Mammary tumor growth and metastasis is induced by overexpression of Six1. (i) Normal mammary epithelium consists of a bilayered epithelial structure of myoepithelial and luminal epithelial cells, surrounded by a continuous basement membrane. (ii) In their study in this issue of the JCI, McCoy et al. (15) show that transgenic mice that express Six1 under control of the mouse mammary tumor virus show induction of mammary ductal hyperplasia and growth of mammary tumors. Micalizzi et al. (16) in their study show that overexpression of Six1 in cultured human mammary epithelial cells leads to induction of EMT through stimulation of the TGF-β receptor and that, following xenograft implantation in immune-compromised mice, Six1-induced EMT facilitates tissue invasion and spread of these cells to the vasculature (iii) and formation of metastases in the lymph and bone (iv).
McCoy et al. (15) present evidence revealing how Six1 may function to promote tumors at the earliest stages of development through the use of new transgenic mice that inducibly express Six1 in mouse mammary epithelial cells. Expression of Six1 led to epithelial hyperplasia and alveolar expansion within the first few weeks. Sustained expression of Six1 led to the formation of aggressive tumors characterized by highly divergent epithelial differentiation that included regions displaying evidence of EMT. Six1 expression was also shown to increase the fraction of epithelial cells expressing mammary stem/progenitor characteristics: isolated primary epithelial cells from Six1-expressing mice showed increased expression of the stem/progenitor cell–associated cell surface markers CD24 and CD29, as well as greatly increased growth as mammospheres, an assay that reflects cell self-renewal. Thus, Six1-induced EMT was associated with increases in the population of stem/progenitor cells and spontaneous tumor growth. Micalizzi et al. (16) used a parallel approach to define how Six1-induced EMT facilitates tumor metastasis using xenograft assays. Overexpression of Six1 in cultured human mammary cells caused immediate morphological EMT, and Six1-overexpressing cells showed a greatly increased propensity for spontaneous metastasis in orthotopic xenografts, as well as substantially greater metastatic capability following intracardiac injection. Analysis of transcriptional alterations in Six1-overexpressing cells revealed the activation of a TGF-β response signature, and inhibition of TGF-β signaling in Six1-overexpressing cells greatly reduced their metastatic capability. Consistent with the concept that Six1 could act to promote metastasis in human tumors, analysis of publicly available datasets showed that increased expression of Six1 was predictive of decreased time to metastasis, relapse, and survival in breast cancer, and of poor prognosis in a number of other cancers.
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)