Sendurai A. Mani

Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways.

Loss of the epithelial adhesion molecule E-cadherin is thought to enable metastasis by disrupting intercellular contacts-an early step in metastatic dissemination. To further investigate the molecular basis of this notion, we use two methods to inhibit E-cadherin function that distinguish between E-cadherins cell-cell adhesion and intracellular signaling functions. Whereas the disruption of cell-cell contacts alone does not enable metastasis, the loss of E-cadherin protein does, through induction of an epithelial-to-mesenchymal transition, invasiveness, and anoikis resistance. We find the E-cadherin binding partner beta-catenin to be necessary, but not sufficient, for induction of these phenotypes. In addition, gene expression analysis shows that E-cadherin loss results in the induction of multiple transcription factors, at least one of which, Twist, is necessary for E-cadherin loss-induced metastasis. These findings indicate that E-cadherin loss in tumors contributes to metastatic dissemination by inducing wide-ranging transcriptional and functional changes.

Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes

The epithelial-to-mesenchymal transition (EMT) produces cancer cells that are invasive, migratory, and exhibit stem cell characteristics, hallmarks of cells that have the potential to generate metastases. Inducers of the EMT include several transcription factors (TFs), such as Goosecoid, Snail, and Twist, as well as the secreted TGF-β1. Each of these factors is capable, on its own, of inducing an EMT in the human mammary epithelial (HMLE) cell line. However, the interactions between these regulators are poorly understood. Overexpression of each of the above EMT inducers up-regulates a subset of other EMT-inducing TFs, with Twist, Zeb1, Zeb2, TGF-β1, and FOXC2 being commonly induced. Up-regulation of Slug and FOXC2 by either Snail or Twist does not depend on TGF-β1 signaling. Gene expression signatures (GESs) derived by overexpressing EMT-inducing TFs reveal that the Twist GES and Snail GES are the most similar, although the Goosecoid GES is the least similar to the others. An EMT core signature was derived from the changes in gene expression shared by up-regulation of Gsc, Snail, Twist, and TGF-β1 and by down-regulation of E-cadherin, loss of which can also trigger an EMT in certain cell types. The EMT core signature associates closely with the claudin-low and metaplastic breast cancer subtypes and correlates negatively with pathological complete response. Additionally, the expression level of FOXC1, another EMT inducer, correlates strongly with poor survival of breast cancer patients.

Slug and Sox9 cooperatively determine the mammary stem cell state

Regulatory networks orchestrated by key transcription factors (TFs) have been proposed to play a central role in the determination of stem cell states. However, the master transcriptional regulators of adult stem cells are poorly understood. We have identified two TFs, Slug and Sox9, that act cooperatively to determine the mammary stem cell (MaSC) state. Inhibition of either Slug or Sox9 blocks MaSCxa0activity in primary mammary epithelial cells. Conversely, transient coexpression of exogenous Slug and Sox9 suffices to convert differentiated luminal cells into MaSCs with long-term mammary gland-reconstituting ability. Slug and Sox9 induce MaSCs by activating distinct autoregulatory gene expression programs. We also show that coexpression of Slug and Sox9 promotes the tumorigenic and metastasis-seeding abilities of human breast cancer cells and is associated with poor patient survival, providing direct evidence that human breast cancer stem cells are controlled by key regulators similar to those operating in normal murine MaSCs.

Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers

The metastatic spread of epithelial cancer cells from the primary tumor to distant organs mimics the cell migrations that occur during embryogenesis. Using gene expression profiling, we have found that the FOXC2 transcription factor, which is involved in specifying mesenchymal cell fate during embryogenesis, is associated with the metastatic capabilities of cancer cells. FOXC2 expression is required for the ability of murine mammary carcinoma cells to metastasize to the lung, and overexpression of FOXC2 enhances the metastatic ability of mouse mammary carcinoma cells. We show that FOXC2 expression is induced in cells undergoing epithelial-mesenchymal transitions (EMTs) triggered by a number of signals, including TGF-β1 and several EMT-inducing transcription factors, such as Snail, Twist, and Goosecoid. FOXC2 specifically promotes mesenchymal differentiation during an EMT and may serve as a key mediator to orchestrate the mesenchymal component of the EMT program. Expression of FOXC2 is significantly correlated with the highly aggressive basal-like subtype of human breast cancers. These observations indicate that FOXC2 plays a central role in promoting invasion and metastasis and that it may prove to be a highly specific molecular marker for human basal-like breast cancers.

The Epithelial-to-Mesenchymal Transition and Cancer Stem Cells: A Coalition Against Cancer Therapies

During cancer progression, some cells within the primary tumor may reactivate a latent embryonic program known as epithelial-to-mesenchymal transition (EMT). Through EMT, transformed epithelial cells can acquire the mesenchymal traits that seem to facilitate metastasis. Indeed, there is accumulating evidence that EMT and mesenchymal-related gene expression are associated with aggressive breast cancer subtypes and poor clinical outcome in breast cancer patients. More recently, the EMT program was shown to endow normal and transformed mammary epithelial cells with stem cell properties, including the ability to self-renew and efficiently initiate tumors. This link between EMT and stem cells may have numerous implications in the progression of breast tumors. The EMT process may facilitate the generation of cancer cells with the mesenchymal traits needed for dissemination as well as the self-renewal properties needed for initiation of secondary tumors. Breast cancer stem cells are resistant to many conventional cancer therapies, which can promote tumor relapse. Therefore, the generation of cancer stem cells by EMT may promote the development of refractory and resistant breast tumors. The purpose of this review is to summarize the findings related to EMT and stem cells in cancer progression and therapy resistance.

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk.

Epithelial Mesenchymal Transition Traits in Human Breast Cancer Cell Lines Parallel the CD44hi/CD24lo/- Stem Cell Phenotype in Human Breast Cancer

We review here the recently emerging relationship between epithelial-mesenchymal transition (EMT) and breast cancer stem cells (BCSC), and provide analyses of published data on human breast cancer cell lines, supporting their utility as a model for the EMT/BCSC state. Genome-wide transcriptional profiling of these cell lines has confirmed the existence of a subgroup with mesenchymal tendencies and enhanced invasive properties (‘Basal B’/Mesenchymal), distinct from subgroups with either predominantly luminal (‘Luminal’) or mixed basal/luminal (‘Basal A’) features (Neve et al. Cancer Cell, 2006). A literature-derived EMT gene signature has shown specific enrichment within the Basal B subgroup of cell lines, consistent with their over-expression of various EMT transcriptional drivers. Basal B cell lines are found to resemble BCSC, being CD44highCD24low. Moreover, gene products that distinguish Basal B from Basal A and Luminal cell lines (Basal B Discriminators) showed close concordance with those that define BCSC isolated from clinical material, as reported by Shipitsin et al. (Cancer Cell, 2007). CD24 mRNA levels varied across Basal B cell lines, correlating with other Basal B Discriminators. Many gene products correlating with CD24 status in Basal B cell lines were also differentially expressed in isolated BCSC. These findings confirm and extend the importance of the cellular product of the EMT with Basal B cell lines, and illustrate the value of analysing these cell lines for new leads that may improve breast cancer outcomes. Gene products specific to Basal B cell lines may serve as tools for the detection, quantification, and analysis of BCSC/EMT attributes.

Epithelial-mesenchymal transition and cancer stem cells: a dangerously dynamic duo in breast cancer progression

Aberrant activation of a latent embryonic program - known as the epithelial-mesenchymal transition (EMT) - can endow cancer cells with the migratory and invasive capabilities associated with metastatic competence. The induction of EMT entails the loss of epithelial characteristics and the de novo acquisition of a mesenchymal phenotype. In breast cancer, the EMT state has been associated with cancer stem cell properties including expression of the stem cell-associated CD44+/CD24-/low antigenic profile, self-renewal capabilities and resistance to conventional therapies. Intriguingly, EMT features are also associated with stem cells isolated from the normal mouse mammary gland and human breast reduction tissues as well as the highly aggressive metaplastic and claudin-low breast tumor subtypes. This has implications for the origin of these breast tumors as it remains unclear whether they derive from cells that have undergone EMT or whether they represent an expansion of a pre-existing stem cell population that expresses EMT-associated markers to begin with. In the present review, we consider the current evidence connecting EMT and stem cell attributes and discuss the ramifications of these newly recognized links for our understanding of the emergence of distinct breast cancer subtypes and breast cancer progression.

Endothelial Cells Promote the Colorectal Cancer Stem Cell Phenotype through a Soluble Form of Jagged-1

We report a paracrine effect whereby endothelial cells (ECs) promote the cancer stem cell (CSC) phenotype of human colorectal cancer (CRC) cells. We showed that, without direct cell-cell contact, ECs secrete factors that promoted the CSC phenotype in CRC cells via Notch activation. In human CRC specimens, CD133 and Notch intracellular domain-positive CRC cells colocalized in perivascular regions. An EC-derived, soluble form of Jagged-1, via ADAM17 proteolytic activity, led to Notch activation in CRC cells in a paracrine manner; these effects were blocked by immunodepletion of Jagged-1 in EC-conditioned medium or blockade of ADAM17 activity. Collectively, ECs play an active role in promoting Notch signaling and the CSC phenotype by secreting soluble Jagged-1.

Cell type-specific DNA methylation patterns in the human breast

Cellular identity and differentiation are determined by epigenetic programs. The characteristics of these programs in normal human mammary epithelium and their similarity to those in stem cells are unknown. To begin investigating these issues, we analyzed the DNA methylation and gene expression profiles of distinct subpopulations of mammary epithelial cells by using MSDK (methylation-specific digital karyotyping) and SAGE (serial analysis of gene expression). We identified discrete cell-type and differentiation state-specific DNA methylation and gene expression patterns that were maintained in a subset of breast carcinomas and correlated with clinically relevant tumor subtypes. CD44+ cells were the most hypomethylated and highly expressed several transcription factors with known stem cell function including HOXA10 and TCF3. Many of these genes were also hypomethylated in BMP4-treated compared with undifferentiated human embryonic stem (ES) cells that we analyzed by MSDK for comparison. Further highlighting the similarity of epigenetic programs of embryonic and mammary epithelial cells, genes highly expressed in CD44+ relative to more differentiated CD24+ cells were significantly enriched for Suz12 targets in ES cells. The expression of FOXC1, one of the transcription factors hypomethylated and highly expressed in CD44+ cells, induced a progenitor-like phenotype in differentiated mammary epithelial cells. These data suggest that epigenetically controlled transcription factors play a key role in regulating mammary epithelial cell phenotypes and imply similarities among epigenetic programs that define progenitor cell characteristics.