Volume 131, Issue 3, Pages 830-840 (September 2006) A Transient, EMT-Linked Loss of Basement Membranes Indicates Metastasis and Poor Survival in Colorectal Cancer  Simone Spaderna, Otto Schmalhofer, Falk Hlubek, Geert Berx, Andreas Eger, Susanne Merkel, Andreas Jung, Thomas Kirchner, Thomas Brabletz  Gastroenterology  Volume 131, Issue 3, Pages 830-840 (September 2006) DOI: 10.1053/j.gastro.2006.06.016 Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 1 Clinically relevant loss of the basement membrane during invasion and rebuilding in metastases. Shown is an example of a typical well to moderately differentiated colorectal carcinoma (left half, primary tumor; right half, corresponding liver metastasis). Staining of the laminin α 3 chain (green) marks the basement membrane (BM), double staining includes cytokeratin 18 (red), which marks tumor cells and liver epithelial cells (liver tissue [l] has a slight autoflourescence in green and red). (A and D) Overview of the primary tumor/metastasis; arrows mark the direction of invasion into the gut wall or liver tissue. (B and E) Higher magnifications of central, differentiated tumor areas; attached tumor cells build up tubuli with central lumina (asterisks) and are surrounded by BMs. Of note, metastases show again BMs (E). (C and F) Invasive areas; the BM is successively lost (arrows) of both primary tumor and metastasis, and tumor cells undergo an EMT-like dedifferentiation, finally leading to small clusters or isolated tumor cells (arrowheads) completely lacking any BM. (G) Two types of rectal carcinomas, depending on the phenotype at the invasive front: the upper case invades the muscle (m) in small tumor cell clusters (arrowheads) lacking a BM (iBM-), whereas, in the lower case, the invading clusters are still surrounded by a BM (iBM+). Of note, only the situation at the invasive front is different, the overall differentiation (well- to moderately) was identical, and both types expressed BMs in central areas (serial sections for left, H&E staining; middle, laminin α 3 chain in green; right, double staining including cytokeratin 18 in red) Bar size = 200 μm for A and D and 40 μm for all others. (H) Kaplan–Meier curves for survival and metachronous metastasis. Compared with iBM+ tumors (solid line), the iBM tumors (dotted line) are highly significant correlated with poor 5-year survival (58% vs 93%, respectively) and metachronous distant metastases (48% vs 6%, respectively). X-axis indicates duration in months. Gastroenterology 2006 131, 830-840DOI: (10.1053/j.gastro.2006.06.016) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 2 The lama3a gene is repressed by EMT-associated repressors through E- and Z boxes. (A) Structure of the human lama3a promoter, showing known activator binding elements and potential repressor binding elements, including the newly identified Z-boxes 1 and 2. Repressors, which were shown to bind in the course of this study, are listed above the elements. Scheme is not drawn to scale. (B) Regions of high homology between the human and mouse lama3a promoter region (1000 nucleotides upstream of translation start) are shown as black box. The sequences of this region are shown below, and the conserved repressive elements are boxed; the nucleotides mutated in the course of this study are underlined. All nucleotide numbers are relative to the translational start sites of the human and mouse lama3a genes. (C) Overexpression of the indicated EMT-repressors inhibits the lama3a gene promoter (solid columns) and the control E-cadherin promoter (shaded columns) activity in a dose-dependent manner (amounts in μg transfected DNA) but has no inhibitory effect on the laminin-γ2 chain promoter (open columns). (D) DLD-1 cell clones stably expressing Snail or Slug under the control of a doxycycline-inducible promoter were treated with 1 μg/mL doxycycline (Dox +), which led to a reduction of lama3 mRNA in comparison with corresponding untreated cell clones (Dox −). Relative mRNA expression level was analyzed by real-time RT-PCR. (E) Repressor elements of the lama3a promoter were mutated as indicated by X, and the activity of the mutated reporter constructs was analyzed in NIH 3T3 fibroblasts and the indicated colorectal cancer cell lines, expressing various levels of ZEB1. Z-box 1 (Z1) and Z-box 2 (Z2) have the strongest inhibitory effect, whereas E-box 1 (E1) might even have an activating function. Mutation of Z1, Z2, and E2 activates the promoter in colorectal cancer cell lines, in correlation with the amount of endogenous ZEB1. Shown are activation levels relative to controls. All experiments were done at least in triplicate, and standard deviations of mean values are shown. In (E), the comparable activity of the wild-type lama3 promoter in the different cell lines is indicated by the relative activity of the background vector pGL3basic. Gastroenterology 2006 131, 830-840DOI: (10.1053/j.gastro.2006.06.016) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 3 ZEB1 is a crucial repressor of the lama3a gene and is expressed in invasive tumor cells of colorectal cancer. (A) Gel-shift assay showing binding of recombinant GST-ZEB1 (rZEB) to Z1, Z2, and E2 but not to E1 and mutated Z1 (Z1M). Positive control elements are the μE5-E box from the human Ig heavy chain promoter. Radiolabelled oligos as probes are shown above the lanes. (B) ZEB1 inhibits and dominant negative ZEB1 (dnZEB1) activates the lama3a gene promoter (solid columns) and the E-cadherin promoter (shaded columns) activity in a dose-dependent manner (amounts in μg transfected DNA). (C) SW480 cells were transfected with control siRNA (upper row) or ZEB1-specific siRNA (lower row) oligonucleotides and analyzed 72 hours later. Knockdown of ZEB1 led to a dramatic change of the tumor cell phenotype: Cells adhere to each other, and E-cadherin (red staining) and β-catenin (green staining) localize to adherent junctions in the cell membrane. In contrast, wild-type or control transfected SW480 shows a fibroblastoid phenotype with scattered cells, lacking membranous E-cadherin and β-catenin. A reversal of EMT toward more epithelial differentiation is also indicated at the molecular level as measured by promoter activity and real-time RT-PCR: knockdown of ZEB1 leads to up-regulation of the E-cadherin and the epithelial basement membrane components lama3 and col4a2, whereas expression of the mesenchymal marker vimentin is down-regulated. Of note, also, the γ2 chain of laminin (lamc2) is down-regulated. The insert (upper right corner) shows that β-catenin transcriptional activity is not affected by ZEB1 siRNA transfection when measured in Topflash/Fopflash reporter assay. (D) Protein expression of SW480 clones stable transfected with retroviral shRNA specific for ZEB1 (clones shZ1 and shZ2, reduction of ZEB1 to 11% and 6%, respectively, relative to control clones) is compared with control shRNA clones c1 and c2. Stable knockdown of ZEB1 leads to down-regulation of Vimentin and up-regulation of E-cadherin as well as increased Laminin-5 secretion into the extracellular matrix, which is composed of the subchains Lama3 (∼170 kilodaltons), Lamb3, and Lamc2 (∼150 kilodaltons) and processed Lamc2 (∼105 kilodaltons). The unspecific band is shown as loading control. (E) Example of a well- to moderately differentiated colorectal carcinoma stained in serial sections by immunohistochemistry (specific staining in brown for cytokeratin 18 and ZEB1, nuclear counterstaining in blue) and by flourescence immunohistochemistry (green for laminin; red for cytokeratin) to visualize the BMs. The direction of invasion is indicated by a bold arrow in the overview. The enlarged regions representing central tumor areas and invasive tumor areas are boxed. Stromal cells in both central and invasive regions show strong nuclear staining for ZEB1 (arrowheads). Polarized epithelial tumor cells in central differentiated areas build up tubular structures with central lumina and lack nuclear ZEB1 (arrows) but express laminin and BMs (arrows). In contrast invasive, dedifferentiated tumor cells at the tumor-host interface accumulate cytoplasmic and nuclear ZEB1 (arrows) associated with a loss of surrounding laminin and BMs (arrows). Arrowheads in the lower row mark basement membranes of blood vessels; inserts show higher magnifications of typical tumor cells. Bar size is 200 μm for the overview and 40 μm for the serial sections. Gastroenterology 2006 131, 830-840DOI: (10.1053/j.gastro.2006.06.016) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 4 Model of a transient loss of basement membranes in malignant tumor progression. Transcriptional repressors (ZEB1 in colorectal cancer, others in other cancers) may be activated by not yet identified environmental signals at the tumor host interface and induce a loss of basement membranes and an associated EMT. This stage favors tumor cell dissemination and subsequently metastasis. Down-regulation of ZEB1 in central areas of the primary tumor and metastases allows reformation of basement membranes (BM in green), epithelial redifferentiation (MET), and growth. Gastroenterology 2006 131, 830-840DOI: (10.1053/j.gastro.2006.06.016) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions