Heide L. Ford

A Mechanism of Cyclin D1 Action Encoded in the Patterns of Gene Expression in Human Cancer

Here we describe how patterns of gene expression in human tumors have been deconvoluted to reveal a mechanism of action for the cyclin D1 oncogene. Computational analysis of the expression patterns of thousands of genes across hundreds of tumor specimens suggested that a transcription factor, C/EBPbeta/Nf-Il6, participates in the consequences of cyclin D1 overexpression. Functional analyses confirmed the involvement of C/EBPbeta in the regulation of genes affected by cyclin D1 and established this protein as an indispensable effector of a potentially important facet of cyclin D1 biology. This work demonstrates that tumor gene expression databases can be used to study the function of a human oncogene in situ.

The miR-106b-25 cluster targets Smad7, activates TGF-β signaling, and induces EMT and tumor initiating cell characteristics downstream of Six1 in human breast cancer

The role of TGF-β signaling in tumorigenesis is paradoxical: it can be tumor suppressive or tumor promotional, depending on context. The metastatic regulator, Six1, was recently shown to mediate this switch, providing a novel means to explain this elusive ‘TGF-β paradox’. Herein, we identify a mechanism by which Six1 activates the tumor promotional arm of TGF-β signaling, via its ability to upregulate the miR-106b-25 microRNA cluster, and further identify a novel function for this cluster of microRNAs. Although expression of the miR-106b-25 cluster is known to overcome TGF-β-mediated growth suppression via targeting p21 and BIM, we demonstrate for the first time that this same cluster can additionally target the inhibitory Smad7 protein, resulting in increased levels of the TGF-β type I receptor and downstream activation of TGF-β signaling. We further show that the miR-106b-25 cluster is sufficient to induce an epithelial-to-mesenchymal transition and a tumor initiating cell phenotype, and that it is required downstream of Six1 to induce these phenotypes. Finally, we demonstrate a significant correlation between miR-106b, Six1, and activated TGF-β signaling in human breast cancers, and further show that high levels of miR-106b and miR-93 in breast tumors significantly predicts shortened time to relapse. These findings expand the spectrum of oncogenic functions of miR-106b-25, and may provide a novel molecular explanation, through the Six1 regulated miR-106b-25 cluster, by which TGF-β signaling shifts from tumor suppressive to tumor promoting.

The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-β signaling

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-beta signaling in human breast cancer cells and induces an epithelial-mesenchymal transition (EMT) that is in part dependent on its ability to increase TGF-beta signaling. TGF-beta 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-beta signaling. Importantly, in human breast cancers Six1 correlated with nuclear Smad3 and thus increased TGF-beta 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-beta signaling and EMT, is a powerful and global promoter of cancer metastasis.

Six1 expands the mouse mammary epithelial stem/progenitor cell pool and induces mammary tumors that undergo epithelial-mesenchymal transition

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.

TRAIL receptor-targeted therapeutics: Resistance mechanisms and strategies to avoid them

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors are attractive therapeutic targets in cancer because agents that activate these receptors directly induce tumor cell apoptosis and have low toxicity to normal tissues. Consequently, several different drugs that target these receptors (recombinant TRAIL and various agonistic antibodies that activate one of the two TRAIL receptors) have been developed and are being tested in human clinical trials. However, in vitro and in vivo data suggest that resistance to these agents may limit their clinical effectiveness. In this review, we discuss recent findings about some of the ways these resistance mechanisms arise, potential biomarkers to identify TRAIL resistance in patients (Six1, GALNT14, XIAP, certain microRNAs) and potential ways to circumvent resistance and resensitize tumors.

Cell cycle-regulated phosphorylation of the human SIX1 homeodomain protein.

Human SIX1 (HSIX1) is a member of the Six class of homeodomain proteins implicated in muscle, eye, head, and brain development. To further understand the role of HSIX1 in the cell cycle and cancer, we developed an HSIX1-specific antibody to study protein expression at various stages of the cell cycle. Our previous work demonstrated that HSIX1 mRNA expression increases as cells exit S phase and that overexpression of HSIX1 can attenuate a DNA damage-induced G2 cell cycle checkpoint. Overexpression of HSIX1 mRNA was observed in 44% of primary breast cancers and 90% of metastatic lesions. Now we demonstrate that HSIX1 is a nuclear phosphoprotein that becomes hyperphosphorylated at mitosis in both MCF7 cells and in Xenopus extracts. The pattern of phosphorylation observed in mitosis is similar to that seen by treating recombinant HSIX1 with casein kinase II (CK2) in vitro. Apigenin, a selective CK2 inhibitor, diminishes interphase and mitotic phosphorylation of HSIX1. Treatment of MCF7 cells with apigenin leads to a dose-dependent arrest at the G2/M boundary, implicating CK2, like HSIX1, in the G2/M transition. HSIX1 hyperphosphorylated in vitro by CK2 loses its ability to bind the MEF3 sites of the aldolase A promoter (pM), and decreased binding to pM is observed during mitosis. Because CK2 and HSIX1 have both been implicated in cancer and in cell cycle control, we propose that HSIX1, whose activity is regulated by CK2, is a relevant target of CK2 in G2/M checkpoint control and that both molecules participate in the same pathway whose dysregulation leads to cancer.

Six1 Overexpression in Ovarian Carcinoma Causes Resistance to TRAIL-Mediated Apoptosis and Is Associated with Poor Survival

Tumorigenesis can arise from inappropriate activation of developmental genes in mature tissues. Here, we show that the developmental regulator Six1 is overexpressed in ovarian carcinoma cell lines (OCC) compared with normal ovarian surface epithelium. As observed in other cancers, Six1 overexpression in OCC leads to increased A-type cyclin expression and increased proliferation. In addition, Six1 overexpression renders OCC resistant to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis, and Six1 knockdown in the TRAIL-resistant SKOV3 ovarian carcinoma line dramatically sensitizes the cells to TRAIL. Because inactivation of the TRAIL response has been linked to metastasis, and because antibodies and recombinant ligand that activate the TRAIL pathway are currently in clinical trials against ovarian carcinoma, we screened normal ovarian and carcinoma specimens for Six1 mRNA. Six1 was overexpressed in 50% of the early-stage (stage I) and 63% of the late-stage (stages II, III, and IV) ovarian carcinomas examined, with late-stage carcinomas expressing approximately 3-fold higher Six1 mRNA levels on average compared with early-stage tumors. Importantly, in patients with late-stage disease, high Six1 expression was associated with significantly shortened survival (P = 0.0015). These data suggest that Six1 may contribute to ovarian epithelial carcinogenesis by simultaneously increasing proliferation and decreasing TRAIL-mediated apoptosis and imply that Six1 may be an important determinant of TRAIL therapy response that should be considered in patient selection for TRAIL-related clinical trials.

Epithelial–mesenchymal transition in development and cancer

The epithelial-mesenchymal transition (EMT) is a critical developmental process from the earliest events of embryogenesis to later morphogenesis and organ formation. EMT contributes to the complex architecture of the embryo by permitting the progression of embryogenesis from a simple single-cell layer epithelium to a complex three-dimensional organism composed of both epithelial and mesenchymal cells. However, in most tissues EMT is a developmentally restricted process and fully differentiated epithelia typically maintain their epithelial phenotype. Recently, elements of EMT, specifically the loss of epithelial markers and the gain of mesenchymal markers, have been observed in pathological states, including epithelial cancers. Analysis of the molecular mechanisms of this oncogenic epithelial plasticity have implicated the inappropriate expression and activation of developmental EMT programs, suggesting that cancer cells may reinstitute properties of developmental EMT including enhanced migration and invasion. Thus, in the context of cancer, an EMT-like process may permit dissemination of tumor cells from the primary tumor into the surrounding stroma, setting the stage for metastatic spread. Consistent with this hypothesis, activation of these developmental EMT programs in human cancer correlates with advanced disease and poor prognosis. This review will focus on the current knowledge regarding developmental EMT pathways that have been implicated in cancer progression.

Chapter 5 The Six Family of Homeobox Genes in Development and Cancer

The homeobox gene superfamily encodes transcription factors that act as master regulators of development through their ability to activate or repress a diverse range of downstream target genes. Numerous families exist within the homeobox gene superfamily, and are classified on the basis of conservation of their homeodomains as well as additional motifs that contribute to DNA binding and to interactions with other proteins. Members of one such family, the Six family, form a transcriptional complex with Eya and Dach proteins, and together these proteins make up part of the retinal determination network first identified in Drosophila. This network is highly conserved in both invertebrate and vertebrate species, where it influences the development of numerous organs in addition to the eye, primarily through regulation of cell proliferation, survival, migration, and invasion. Mutations in Six, Eya, and Dach genes have been identified in a variety of human genetic disorders, demonstrating their critical role in human development. In addition, aberrant expression of Six, Eya, and Dach occurs in numerous human tumors, and Six1, in particular, plays a causal role both in tumor initiation and in metastasis. Emerging evidence for the importance of Six family members and their cofactors in numerous human tumors suggests that targeting of this complex may be a novel and powerful means to inhibit both tumor growth and progression.

Gene Amplification Is a Mechanism of Six1 Overexpression in Breast Cancer

The Six1 homeoprotein plays a critical role in expanding progenitor populations during normal development via its stimulation of proliferation and inhibition of apoptosis. Overexpression of Six1 is observed in several tumor types, suggesting that when expressed out of context, Six1 may contribute to tumorigenesis by reinstating properties normally conveyed on developing cells. Indeed, Six1 contributes to tumor cell proliferation both in breast cancer and in rhabdomyosarcomas, in which it is also implicated in metastasis. Whereas Six1 overexpression has been reported in several tumor types, the mechanism responsible for its overexpression has not previously been examined. Here we show that a change in gene dosage may contribute to Six1 mRNA overexpression. Significant Six1 gene amplification and overrepresentation occurs in numerous breast cancer cell lines as compared with normal mammary epithelial cells, and the changes in gene dosage correlate with increased Six1 mRNA levels. Of 214 human infiltrating ductal breast carcinomas examined for Six1 gene dosage, 4.7% show Six1 amplification/overrepresentation, and tumors that exhibit an increase in Six1 gene dosage overexpress Six1 mRNA. These data implicate Six1 gene amplification/overrepresentation as a mechanism of Six1 mRNA overexpression in human breast cancer.