Karoline J. Briegel

Dendritic cell progenitor is transformed by a conditional v-Rel estrogen receptor fusion protein v-ReIER

A conditional v-Rel estrogen receptor fusion protein, v-RelER, causes estrogen-dependent but otherwise unaltered v-rel-specific transformation of chicken bone marrow cells. Here, we demonstrate that such v-relER-transformed cells exhibit B lymphoid determinants in line with earlier studies on v-rel-transformed cells. However, following inactivation of v-RelER oncoprotein activity by administration of an estrogen antagonist, cells differentiate into antigen-presenting dendritic cells as judged by several morphological and functional criteria. Additionally, under yet different culture conditions, v-relER cells differentiate into cells resembling polymorphonuclear neutrophils. Our studies therefore suggest that the conditional v-RelER, and probably also the authentic v-Rel, transform a common progenitor for neutrophils and dendritic cells.

Molecular characterization of TGFβ-induced epithelial-mesenchymal transition in normal finite lifespan human mammary epithelial cells

Epithelial-mesenchymal transition (EMT) is a morphogenetic program essential for embryonic development and wound healing, but can adversely cause fibrosis and metastatic cancer progression when deregulated. Here, we established a model of efficient EMT induction in normal finite lifespan human mammary epithelial cells (HMEC) using transforming growth factor beta (TGFbeta). We demonstrate that EMT in HMEC occurs in three distinctive phases that are governed by a hierarchy of EMT-activating transcription factors (TFs). Loss of epithelial cell polarity (ZO-1), and acquisition of mesenchymal marker (Vimentin, Fibronectin) expression are immediate-early events, whereas switching from E-cadherin to N-cadherin protein expression occurs only after EMT-like morphological changes become apparent. The kinetics of TF induction suggests that ZEB1 and SNAIL mediate early EMT induction reinforced by ZEB2, while GOOSECOID and FOXC2 may play a role in EMT maintenance. TWIST and SLUG were not significantly induced in this system. Furthermore, we show for the first time that normal HMEC acquire a CD44(+)/CD24(-/low) stem cell phenotype during a third phase of EMT that is characterized by maximum TF expression levels. Our results may have important implications for understanding potential changes that might occur in normal breast epithelium under pathological conditions triggering elevated TGFbeta levels.

The Embryonic Transcription Cofactor LBH Is a Direct Target of the Wnt Signaling Pathway in Epithelial Development and in Aggressive Basal Subtype Breast Cancers

ABSTRACT L imb-bud and heart (LBH) is a novel key transcriptional regulator of vertebrate development. However, the molecular mechanisms upstream of LBH and its role in adult development are unknown. Here we show that in epithelial development, LBH expression is tightly controlled by Wnt signaling. LBH is transcriptionally induced by the canonical Wnt pathway, as evident by the presence of conserved functional T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) binding sites in the LBH locus and rapid β-catenin-dependent upregulation of endogenous LBH by Wnt3a. In contrast, LBH induction by Wnt/β-catenin signaling is inhibited by Wnt7a, which in limb development signals through a noncanonical pathway involving Lmx1b. Furthermore, we show that LBH is aberrantly overexpressed in mammary tumors of mouse mammary tumor virus (MMTV)-Wnt1-transgenic mice and in aggressive basal subtype human breast cancers that display Wnt/β-catenin hyperactivation. Deregulation of LBH in human basal breast cancer appears to be Wnt/β-catenin dependent, as DKK1 and Wnt7a inhibit LBH expression in breast tumor cells. Overexpression studies indicate that LBH suppresses mammary epithelial cell differentiation, an effect that could contribute to Wnt-induced tumorigenesis. Taken together, our findings link LBH for the first time to the Wnt signaling pathway in both development and cancer and highlight LBH as a potential new marker for therapeutically challenging basal-like breast cancers.

Hedgehog-Producing Cancer Cells Respond to and Require Autocrine Hedgehog Activity

A number of Smoothened (SMO) pathway antagonists are currently undergoing clinical trials as anticancer agents. These drugs are proposed to attenuate tumor growth solely through inhibition of Hedgehog (HH), which is produced in tumor cells but acts on tumor stromal cells. The pivotal argument underlying this model is that the growth-inhibitory properties of SMO antagonists on HH-producing cancer cells are due to their off-target effects. Here, we show that the tumorigenic properties of such lung cancer cells depend on their intrinsic level of HH activity. Notably, reducing HH signaling in these tumor cells decreases HH target gene expression. Taken together, these results question the dogma that autocrine HH signaling plays no role in HH-dependent cancers, and does so without using SMO antagonists.

The T Box Transcription Factor TBX2 Promotes Epithelial-Mesenchymal Transition and Invasion of Normal and Malignant Breast Epithelial Cells

The T box transcription factor TBX2, a master regulator of organogenesis, is aberrantly amplified in aggressive human epithelial cancers. While it has been shown that overexpression of TBX2 can bypass senescence, a failsafe mechanism against cancer, its potential role in tumor invasion has remained obscure. Here we demonstrate that TBX2 is a strong cell-autonomous inducer of the epithelial-mesenchymal transition (EMT), a latent morphogenetic program that is key to tumor progression from noninvasive to invasive malignant states. Ectopic expression of TBX2 in normal HC11 and MCF10A mammary epithelial cells was sufficient to induce morphological, molecular, and behavioral changes characteristic of EMT. These changes included loss of epithelial adhesion and polarity gene (E-cadherin, ß-catenin, ZO1) expression, and abnormal gain of mesenchymal markers (N-cadherin, Vimentin), as well as increased cell motility and invasion. Conversely, abrogation of endogenous TBX2 overexpression in the malignant human breast carcinoma cell lines MDA-MB-435 and MDA-MB-157 led to a restitution of epithelial characteristics with reciprocal loss of mesenchymal markers. Importantly, TBX2 inhibition abolished tumor cell invasion and the capacity to form lung metastases in a Xenograft mouse model. Meta-analysis of gene expression in over one thousand primary human breast tumors further showed that high TBX2 expression was significantly associated with reduced metastasis-free survival in patients, and with tumor subtypes enriched in EMT gene signatures, consistent with a role of TBX2 in oncogenic EMT. ChIP analysis and cell-based reporter assays further revealed that TBX2 directly represses transcription of E-cadherin, a tumor suppressor gene, whose loss is crucial for malignant tumor progression. Collectively, our results uncover an unanticipated link between TBX2 deregulation in cancer and the acquisition of EMT and invasive features of epithelial tumor cells.

Congenital heart disease reminiscent of partial trisomy 2p syndrome in mice transgenic for the transcription factor Lbh

Partial trisomy 2p syndrome includes a spectrum of congenital heart disease (CHD) that is characterized by complex malformations of the outflow and inflow tracts, defects in cardiac septation, heart position, as well as abnormal ventricular development. Lbh (limb-bud and heart) is a novel, highly conserved putative transcriptional regulatory protein, which displays a unique spatiotemporal gene expression pattern during early mouse heart development. Here we show that human LBH maps to chromosome 2p23, a genomic region related to CHD in partial trisomy 2p syndrome. Remarkably, transgenic overexpression of Lbh in mice throughout the embryonic myocardium from a cardiomyocyte-specific promoter of the cardiac ankyrin repeat protein gene (Carp/Ankrd1) models CHD reported in humans with partial trisomy 2p syndrome. The malformations in Carp-Lbh transgenic mice reflect impaired pulmonary outflow tract valvulogenesis, cardiac septation, inflow tract morphogenesis, as well as abnormalities in ventricular cardiomyocyte growth. Furthermore, we demonstrate that overexpression of Lbh in cultured mammalian cells represses the synergistic activity of key cardiac transcription factors, Nkx2.5 and Tbx5, leading to reduced activation of the common target gene, Anf (Nppa). Strikingly, reduced levels of Anf expression were also observed in embryonic day 9.5 Carp-Lbh transgenic mice. Thus, repression of Nkx2.5 and Tbx5-mediated gene expression by deregulated Lbh may account in part for the cardiac anomalies observed in these mice. Our findings implicate LBH as a candidate gene for CHD associated with partial trisomy 2p syndrome and suggest an important role of Lbh in transcriptional control during normal cardiogenesis.

Hedgehog Signaling Regulates Bladder Cancer Growth and Tumorigenicity

The role of Hedgehog (HH) signaling in bladder cancer remains controversial. The gene encoding the HH receptor and negative regulator PATCHED1 (PTCH1) resides on a region of chromosome 9q, one copy of which is frequently lost in bladder cancer. Inconsistent with PTCH1 functioning as a classic tumor suppressor gene, loss-of-function mutations in the remaining copy of PTCH1 are not commonly found. Here, we provide direct evidence for a critical role of HH signaling in bladder carcinogenesis. We show that transformed human urothelial cells and many urothelial carcinoma cell lines exhibit constitutive HH signaling, which is required for their growth and tumorigenic properties. Surprisingly, rather than originating from loss of PTCH1, the constitutive HH activity observed in urothelial carcinoma cell lines was HH ligand dependent. Consistent with this finding, increased levels of HH and the HH target gene product GLI1 were found in resected human primary bladder tumors. Furthermore, on the basis of the difference in intrinsic HH dependence of urothelial carcinoma cell lines, a gene expression signature was identified that correlated with bladder cancer progression. Our findings therefore indicate that therapeutic targeting of the HH signaling pathway may be beneficial in the clinical management of bladder cancer.

Embryonic transcription factors in human breast cancer

Growing evidence suggests that breast cancer cells often reactivate latent developmental programs in order to efficiently execute the multi‐step process of tumorigenesis. This review focuses on key transcriptional regulators of embryonic development that are deregulated in breast cancer and discusses the molecular mechanisms by which these proteins control carcinogenesis. Reminiscent of their function during development, embryonic transcription factors regulate changes in gene expression that promote tumor cell growth, cell survival and motility, as well as a morphogenetic process called epithelial‐mesenchymal transition (EMT), which is implicated in both breast metastasis and tumor recurrence. Because of their pivotal roles in breast tumor progression, these factors represent valuable new biomarkers for breast cancer detection as well as promising new targets for anti‐invasive drugs. IUBMB Life, 58: 123‐132, 2006

Biophysical Characterization Reveals Structural Disorder in the Developmental Transcriptional Regulator LBH

Limb-bud and heart (LBH) is a key transcriptional regulator in vertebrates with pivotal roles in embryonic development and human disease. Herein, using a diverse array of biophysical techniques, we report the first structural characterization of LBH pertinent to its biological function. Our data reveal that LBH is structurally disordered with no discernable secondary or tertiary structure and exudes rod-like properties in solution. Consistent with these observations, we also demonstrate that LBH is conformationally flexible and thus may be capable of adapting distinct conformations under specific physiological contexts. We propose that LBH is a member of the intrinsically disordered protein (IDP) family, and that conformational plasticity may play a significant role in modulating LBH-dependent transcriptional processes.

Identification and Analysis of Escherichia coli Ribonuclease E Dominant-Negative Mutants

The Escherichia coli (E. coli) ribonuclease E protein (RNase E) is implicated in the degradation and processing of a large fraction of RNAs in the cell. To understand RNase E function in greater detail, we developed an efficient selection method for identifying nonfunctional RNase E mutants. A subset of the mutants was found to display a dominant-negative phenotype, interfering with wild-type RNase E function. Unexpectedly, each of these mutants contained a large truncation within the carboxy terminus of RNase E. In contrast, no point mutants that conferred a dominant-negative phenotype were found. We show that a representative dominant-negative mutant can form mixed multimers with RNase E and propose a model to explain how these mutants can block wild-type RNase E function in vivo.