Supplementary Methods and Materials from The Transcriptional Repressor ZEB1 Promotes Metastasis and Loss of Cell Polarity in Cancer

Supplementary Figure 2 from The Transcriptional Repressor ZEB1 Promotes Metastasis and Loss of Cell Polarity in Cancer

Supplementary Table 1 from Expression of L1-CAM and ADAM10 in Human Colon Cancer Cells Induces Metastasis

Supplementary Figure Legends 1-3 from The Transcriptional Repressor ZEB1 Promotes Metastasis and Loss of Cell Polarity in Cancer

Recent evidence shows that a sub-population of Wnt/beta-catenin target genes is specifically induced in different tissue contexts. FZD7 is a putative Wnt/beta-catenin target gene and although it is highly expressed in well-differentiated colorectal cancer tumour cells, its expression is decreased in de-differentiated tumour cells at the invasive front despite elevated Wnt/beta-catenin signalling in this area. This variable expression of FZD7 implicates additional regulation by the microenvironment; however, this has not been investigated. To begin to elucidate the role of extracellular matrix in regulating FZD7 expression, we generated a FZD7 promoter reporter and analysed FZD7 promoter activity in colorectal cancer cells grown on different matrices. We demonstrate that the FZD7 promoter is regulated by beta-catenin in colorectal cancer cells and observed decreased promoter activity in cells grown on fibronectin but not collagen I or collagen IV. Thus, expression of FZD7 in colorectal cancer may be regulated by fibronectin in the microenvironment.

Gregory PA, Bert AG, Paterson EL, et al. (Hanson Institute and Division of Human Immunology, Institute of Medical and Veterinary Science, Adelaide, Australia). The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 2008;10:593–601.

The transcriptional activator beta-catenin is the key mediator of the canonical Wnt signaling pathway. However, beta-catenin does not itself bind DNA, but functions via interaction with T-cell factor (TCF)/ lymphoid-enhancing factor (LEF) transcription factors. These proteins contain a high-mobility group (HMG) box that binds DNA in a sequence-specific manner. Thus, in the case of active Wnt signaling, beta-catenin activates, in cooperation with proteins of the TCF/LEF family, the expression of a wide variety of genes. To date, the list of established Wnt targets is far from complete. The establishment of plasmids harbouring reporter genes under control of the native promoter sequences provides a tool to validate novel putative Wnt targets by directly quantifying the beta-catenin-dependent activation of each specific gene. In this chapter, we describe how to generate such reporter plasmids using the MMP7 promoter as an example.

The embryonic programme ‘epithelial–mesenchymal transition’ (EMT) is thought to promote malignant tumour progression. The transcriptional repressor zinc‐finger E‐box binding homeobox 1 (ZEB1) is a crucial inducer of EMT in various human tumours, and was recently shown to promote invasion and metastasis of tumour cells. Here, we report that ZEB1 directly suppresses transcription of microRNA‐200 family members miR‐141 and miR‐200c, which strongly activate epithelial differentiation in pancreatic, colorectal and breast cancer cells. Notably, the EMT activators transforming growth factor β2 and ZEB1 are the predominant targets downregulated by these microRNAs. These results indicate that ZEB1 triggers an microRNA‐mediated feedforward loop that stabilizes EMT and promotes invasion of cancer cells. Alternatively, depending on the environmental trigger, this loop might switch and induce epithelial differentiation, and thus explain the strong intratumorous heterogeneity observed in many human cancers.

AbstractInvasion and metastasis are the hallmarks of malignant tumor progression and the main cause of death in cancer. The embryonic program “epithelial-mesenchymal transition” (EMT) is thought to trigger invasion by allowing tumor cell dissemination. Here, we describe that the EMT-inducing transcriptional repressor ZEB1 promotes colorectal cancer cell metastasis and loss of cell polarity. Thereby, ZEB1 suppresses the expression of cell polarity factors, in particular of Lgl2, which we found reduced in colorectal and breast cancers. We further show that retention of Lgl2 expression is critical for the epithelial phenotype and that its loss might be involved in metastasis. Thus, by linking EMT, loss of polarity, and metastasis, ZEB1 is a crucial promoter of malignant tumor progression. [Cancer Res 2008;68(2):537–44]

Background & Aims: Inappropriate activation of beta-catenin in adult tissues is associated with a wide variety of cancers, especially in the digestive tract. Classic transgenic and knockout murine models in which beta-catenin is activated in large fields of cells have provided experimental support in favor of a role for this molecule in tumorigenesis. However, these models do not reproduce the sporadic nature of the majority of human cancers, beginning with the activation of an oncogene at random in a single cell. Methods: We used the "hit and run" strategy to generate a mouse model in which the expression of an activated form of beta-catenin occurs sporadically in vivo. Results: Sporadic, multifocal lesions were observed in the stomach of 3% of mice aged 8 months and older. These lesions were associated with loss of Sonic hedgehog (Shh), and a causal relationship between beta-catenin activation and Shh inhibition was established in gastric cells in vitro. No lesion was detected in the intestine or in the liver. In addition, one third of female mutant mice developed benign perimammary papillomas. Mutant mice were also hypersensitive to chemically induced premalignant skin lesions. Conclusions: These results challenge the view that activation of beta-catenin induces malignant cancerogenesis, because they show in mice that sporadically activated beta-catenin is sufficient for tumor initiation, yet without further malignant progression, and that it sensitizes cells to environmental hits. This model represents a powerful tool to investigate the interplay between genetic and environmental factors in tumor progression.

Malignant progression of colorectal carcinomas is characterized by an epithelial-mesenchymal transition (EMT)-like de-differentiation of the invading tumor cells. However a re-differentiation towards an epithelial phenotype, resembling a mesenchymal-epithelial transition (MET), is detectable in metastases. This indicates that malignant progression is based on dynamic processes, which can not be explained solely by irreversible genetic alterations, but must be additionally regulated by the tumor environment. The main oncoprotein in colorectal cancer is the Wnt-pathway effector beta-catenin, which in most cases is overexpressed due to mutations in the adenomatous polyposis coli (APC) tumor suppressor. EMT of tumor cells is associated with a nuclear accumulation of the transcriptional activator beta-catenin, which is reversed in metastases. Nuclear beta-catenin is involved in two fundamental processes in embryonic development: EMT and stem cell formation. Accumulating data demonstrate that aberrant nuclear expression of beta-catenin can also confer these two abilities to tumor cells, indicating the crucial role of aberrant Wnt-signaling for malignant tumor progression.

Abstract L1-CAM, a neuronal cell adhesion receptor, is also expressed in a variety of cancer cells. Recent studies identified L1-CAM as a target gene of β-catenin-T-cell factor (TCF) signaling expressed at the invasive front of human colon cancer tissue. We found that L1-CAM expression in colon cancer cells lacking L1-CAM confers metastatic capacity, and mice injected in their spleen with such cells form liver metastases. We identified ADAM10, a metalloproteinase that cleaves the L1-CAM extracellular domain, as a novel target gene of β-catenin-TCF signaling. ADAM10 overexpression in colon cancer cells displaying endogenous L1-CAM enhanced L1-CAM cleavage and induced liver metastasis, and ADAM10 also enhanced metastasis in colon cancer cells stably transfected with L1-CAM. DNA microarray analysis of genes induced by L1-CAM in colon cancer cells identified a cluster of genes also elevated in a large set of human colon carcinoma tissue samples. Expression of these genes in normal colon epithelium was low. These results indicate that there is a gene program induced by L1-CAM in colon cancer cells that is also present in colorectal cancer tissue and suggest that L1-CAM can serve as target for colon cancer therapy. [Cancer Res 2007;67(16):7703–12]

BACKGROUND & AIMS Loss of the basement membrane (BM) is considered an important step toward tumor malignancy. However, the BM is still expressed in most typical colorectal adenocarcinomas; nevertheless, these tumors can invade and develop metastases. The aim of this study was to investigate the role, mechanisms, and clinical relevance of BM turnover in malignant colorectal cancer (CRC) progression. METHODS Expression of BM components and their transcriptional regulation and clinical relevance were investigated in human CRCs and cell lines. RESULTS Our data show new aspects in BM turnover in CRCs with impact on malignant tumor progression: (1) The BM is still expressed in the main tumor mass of most colorectal adenocarcinomas, but selectively lost at invasive regions of the tumor in many cases. (2) Selective loss of the BM at the invasive front has high clinical and tumor biologic relevance for distant metastasis and survival. (3) The BM is reexpressed in metastases, indicating that its loss is transient and regulated by environmental factors. (4) This transient loss is not only due to proteolytic breakdown but to a down-regulated synthesis and linked to an epithelial-mesenchymal transition (EMT) in tumor cells, and, thereby, zinc-finger-enhancer protein 1 (ZEB1) is the crucial transcriptional repressor of BM components in CRCs. CONCLUSIONS A transient BM loss at the invasive front is correlated with increased distant metastasis and poor patient survival, indicating its tumor biologic relevance and usefulness as a prognostic marker. Targeting ZEB1 might be a promising therapeutic option to prevent metastasis.

The dissemination of tumour cells is the prerequisite of metastases and is correlated with a loss of epithelial differentiation and the acquisition of a migratory phenotype, a hallmark of malignant tumour progression. A stepwise, irreversible accumulation of genetic alterations is considered to be the responsible driving force. But strikingly, metastases of most carcinomas recapitulate the organization of their primary tumours. Although current models explain distinct and important aspects of carcinogenesis, each alone can not explain the sum of the cellular changes apparent in human cancer progression. We suggest an extended, integrated model that is consistent with all aspects of human tumour progression - the 'migrating cancer stem (MCS)-cell' concept.