Transforming Growth Factor-B Suppresses Nonmetastatic Colon Cancer Through Smad4 and Adaptor Protein ELF at an Early Stage of Tumorigenesis

Research Article

Transforming Growth Factor-B Suppresses Nonmetastatic Colon Cancer through Smad4 and Adaptor Protein ELF at an Early Stage of Tumorigenesis

Yi Tang,1 Varalakshmi Katuri,1 Radhika Srinivasan,3 Franz Fogt,4 Robert Redman,2 Girish Anand,5 Anan Said,1 Thomas Fishbein,1 Michael Zasloff,1 E. Premkumar Reddy,6 Bibhuti Mishra,1 and Lopa Mishra1,2

1Laboratory of GI Developmental Biology, Departments of Surgery and Medicine & Lombardi Cancer Center, Georgetown University; 2Department of Veterans Affairs Medical Center, Washington, District of Columbia; Departments of 3Medicine and 4Pathology, University of Pennsylvania, Presbyterian Medical Center; 5Albert Einstein Medical Center and 6Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia, Pennsylvania

Abstract Smad2, and Smad3 phosphorylation at the COOH-terminal serines (7, 8). Subsequent heteromeric complex formation with the L3 loop Although transforming growth factor-B (TGF-B) is both a region phosphoserine-binding pockets of Smad4 facilitates nuclear suppressor and promoter of tumorigenesis, its contribution to translocation and TGF-h target gene activation (9–11). Adaptor early tumor suppression and staging remains largely un- proteins are required for functional specificity and Smad known. In search of the mechanism of early tumor suppres- modulation. In previous studies, we have shown that ELF, a h- sion, we identified the adaptor protein ELF, a B-spectrin from spectrin, is a crucial adaptor protein in TGF-h signaling and is stem/progenitor cells committed to foregut lineage. ELF required for Smad3 and Smad4 localization and signaling (12, 13). activates and modulates Smad4 activation of TGF-B to confer This finding was interesting because h-spectrins are major cell polarity, to maintain cell architecture, and to inhibit dynamic scaffold molecules involved in generating functionally epithelial-to-mesenchymal transition. Analysis of development distinct membrane protein domains, conferring cell polarity and of colon cancer in (adult) elf +/À/Smad4+/À, elf +/À, Smad4+/À, regulating endocytic traffic (14, 15). and gut epithelial cells from elf À/À mutant mouse embryos Originally described for its transforming capability, TGF-h is also pinpoints the defect to hyperplasia/adenoma transition. a growth inhibitor in epithelial tissues as it is both a suppressor and Further analysis of the role of ELF in human colorectal cancer promoter of tumorigenesis. It has been suggested that nearly all confirms reduced expression of ELF in Dukes’ B1 stage tissues colon cancers, pancreatic cancers, and gastric carcinomas have (P < 0.05) and of Smad4 in advanced colon cancers (P < 0.05). mutations inactivating some component of TGF-h signaling from This study indicates that by modulating Smad 4, ELF has a key h B T RII frameshift mutations with microsatellite instability to role in TGF- signaling in the suppression of early colon mutations in Smad4, Smad2, or an as yet untested component of cancer. (Cancer Res 2005; 65(10): 4228-37) the TGF-h signaling pathway (16–19). Genetic studies in mice have provided strong models and further evidence for the role of TGF-h Introduction in tumor suppression in early stages. Tgf -hÀ/À/Rag2À/À mutant The transforming growth factor-h (TGF-h) signaling pathway mice that live to adulthood rapidly develop colon cancer by 5 has been known to play an important role in gastrointestinal months of age, preceded by precancerous lesions with inflammation D716 epithelial cell homeostasis; cell differentiation, proliferation, and and hyperplasia (20). Smad4 deficiency in the Apc mouse migration; and modulation of gastrointestinal cancers (1–3). The increases adenoma size and promotes cancer progression (2) and +/À enlarging TGF-h superfamily comprises >40 members, which Smad4 mutant mice develop gastric polyps and carcinomas (17). include TGF-h1, TGF-h2, and TGF-h3; bone morphogenetic To understand the role of ELF in Smad4 tumor suppression, we +/À +/À proteins; activins; Nodal; Lefty-1; Lefty-2; anti-Mu¨llerian hormone; intercrossed elf mutant mice with Smad4 mutant mice and and other growth/differentiation factors (4, 5). Despite the diverse determined a genetic basis for ELF/Smad4 interaction in early and complex responses they elicit, the basic signaling cascade of colorectal cancers. It was possible that, in addition to its TGF-h is composed of TGF-h type I and type II transmembrane involvement in TGF-h signaling, ELF, as a h-spectrin, could be serine/threonine kinase receptors (ThRI and ThRII), and the important in conferring cell polarity and maintaining cell cellular response is controlled by intracellular signaling proteins, architecture. Support for this hypothesis comes from embryonic À/À Smads (6). day 11.5 (E11.5) elf mutant embryos that display a profoundly TGF-h ligand binding results in phosphorylation at Gly-Ser (GS) abnormal gut phenotype, with flattened gut epithelial cells and loss in the cytoplasmic tail domain of ThRI by ThRII, activation of of villi. We then investigated the abnormal gut phenotype and determined that the distribution of cell polarization markers dependent upon h-spectrin, such as E-cadherin, Na+-K+-ATPase, Note: Y. Tang and V. Katuri contributed equally to this work. and microtubule associated protein-2 (MAP-2), was altered. We Supplementary data for this article are available at Cancer Research Online (http:// found that the cellular polarization through abnormal distribution cancerres.aacrjournals.org/). + + Requests for reprints: Lopa Mishra, Laboratory of GI Developmental Biology, of proteins, such as Na -K -ATPase, could be restored by ELF À/À Departments of Medicine and Surgery, Lombardi Cancer Center and Georgetown expression in elf mutant cells. However, alterations in MAP-2 University, Med/Dent Building, NW 210-12, 3900 Reservoir Road, Northwest, expression were not associated with aberrant expression of tubulin Washington, DC 20007. Phone: 202-687-5707; Fax: 202-687-0992; E-mail: [email protected]. or microtubule function. Finally, we extrapolated our findings of I2005 American Association for Cancer Research. abnormal cellular architecture and early colon cancer suppression

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to human studies. A strikingly reduced expression of ELF alone was added to cover the entire tissue on the slide, and we monitored color seen in Dukes’ B1 stage tissues (P < 0.05) and with a concomitant development under the microscope. After rinsing in distilled water for loss of Smad4 expression in advanced colon cancers (Dukes’ D 2 minutes, we counterstained with modified Harris hematoxylin solution stage, P < 0.05). Our results indicate that ELF maintains cellular (Sigma) for 1 minute followed by a rinse in distilled water for 3 minutes. polarization by localization of a specific subset of proteins and is Sections were dehydrated by passage through graded alcohol concen- trations and finally xylene. Coverslips were mounted using DPX (Sigma) involved in preservation of cell architecture, the disruption of before observation. which may be a key to early colon cancer development. Cell polarity assays. Antibodies to Na+-K+-ATPase, vimentin, E- cadherin, and tubulin were used for immunohistologic characterization of the gut in elf À/À and wild-type mice as described above. In transient Materials and Methods transfection assays, elf À/À and wild-type MEFs were seeded at a density of Generation of elf +/À/Smad4+/À mice. Elf À/À mice die predominantly 2 Â 105 cells/well in six-well dishes. They were then transfected with a at E11.5. Mice heterozygous for the elf mutation (elf +/À) are normal and construct that contains the full-length of elf or vector DNA alone (3 Agof fertile. Elf +/À mice were intercrossed with Smad4+/À mice to generate DNA per well). For localization studies, transfections with pEYFP-Mem elf +/À/Smad4+/À mutants to analyze the onset of colon adenomas. Elf +/ (encoding GAP-43, which contains a signal for posttranslational palmitoy- À/Smad4+/À mutations were maintained on a mixed 129SvEv/Black Swiss lation of cysteines 3 and 4 that targets membranes), pEYFP-Nuc (encoding a background. The presence of mutations was monitored by use of the gene with three copies of the nuclear localization signal of the SV40 large PCR as described previously (13, 17). antigen fused at its COOH terminus), and pECFP-Golgi (encoding the NH2- Confocal laser scanning immunofluorescence microscopy. Colocal- terminal human h-1,4-galactosyltransferase that helps in targeting the ization studies were done with antibodies against ELF and Smad4 on fusion protein to the trans-medial region of the Golgi apparatus) were used. normal gastric tissues, wild-type mouse gastric antral cells, or mouse Transfected cells were washed 2Â with DMEM after 12 to 18 hours and embryonic fibroblasts (MEF). Monoclonal mouse and polyclonal goat and then treated with 5 Ag/mL TGF-h and incubated for an additional 24 hours. rabbit primary antibodies were visualized with tetramethyl rhodamine All experiments were repeated at least thrice and similar results were isothiocyanate–conjugated goat secondary rabbit IgG or FITC-conjugated obtained each time. Cells were then fixed and analyzed by confocal goat anti-mouse IgG or cyanine (Cy5). The samples were analyzed with a microscopy as above. Bio-Rad Radiance 2000 confocal microscope (Bio-Rad, Cambridge, MA) Luciferase assays. Elf À/À and elf +/+ MEFs were plated 1 day before with an ILT model 5470 K laser (Ion Laser Technology, Salt Lake City, UT). transfection, in 12-well plates, at a density of 1.5 Â 105 to 2.5 Â 105 cells per Digital images were analyzed using Metamorph (Universal Imaging, well in DMEM medium (10% fetal bovine serum, 1% P/S, 1% L-glutamine). Downingtown, PA) and figures were prepared using Adobe Photoshop. For TGF-h response assays, the cells were transfected with p3TP-lux (1.5 Generation of mouse embryo–derived fibroblasts. Mouse embryo– Ag) in controls and in cells treated with placitaxel (a microtubule- derived fibroblasts harboring the null allele elf as well as wild-type, Elf stabilizing agent; Sigma) or nocodazole (a microtubule-disrupting agent; knockout (elf À/À), and elf wild-type (elf +/+) MEFs were derived as previously Sigma). The cells were subsequently incubated for 18 hours with or without described (13). Briefly, embryos E14.5 were tritrurated in 0.25% trypsin/ TGF-h1 (100 pmol/L). In mutant rescue assays, the elf +/+ and elf À/À cells 1 mmol/L EDTA and genotyped as previously described. The lines were were transfected with a construct that contains the full-length of elf or propagated in DMEM supplemented with 10% fetal bovine serum, 100 units/ vector DNA alone and subsequently treated with TGF-h1 (100 pmol/L). mL penicillin, and 50 Ag/mL streptomycin to establish wild-type and elf À/À Protein normalized luciferase activity in cell lysates was measured in a TD- fibroblasts that were cultured over multiple passages to obtain sufficient 20/20 Luminometer (Turner Designs, Sunnyvale, CA) by using substrate cells to perform the experiments. The fibroblasts used for the experiments prepared in accordance with Promega luciferase assay system. All assays were at passages 3 to 25. Three different elf À/À and wild-type fibroblast lines were carried out in duplicate or triplicate. were tested in different experiments and the results obtained were also TGF-h–responsive regions of the human PAI-1 promoter contain DNA independent of passage number. Representative data are shown. elements homologous to the consensus binding site for the transcription Histology and immunohistochemical staining. Colon carcinomas factor AP1. The p3TP-Lux luciferase reporter is a well-described and widely detected in mutant mice were fixed in 10% neutral buffered formalin and used artificial promoter construct that was empirically designed to have paraffin-embedded, and later, H&E staining was used to confirm histologic maximal responsiveness to TGF-h. Interestingly, using a variety of TGF-h– characterization. An indirect immunoperoxidase procedure was used for dependent luciferase gene reporters, it has been found that only Smad3 can immunohistochemical localization of ELF and Smad4 proteins in colorectal directly interact with Smad binding elements found in the promoters of cancer tissue samples. Serial, sagittal sections of colorectal cancer tissues many TGF-h–responsive genes. Thus, Smad3 activates transcription of the were immersed in xylene to remove paraffin, then dehydrated in graded 3TP-Lux, a reporter that contains elements of the PAI-l promoter. alcohol, and rinsed in 1Â PBS. Endogenous peroxide was quenched using Functional effects of reduced Smad3 expression are seen in our model as 3% hydrogen peroxide (Sigma, St. Louis, MO). Nonspecific binding sites reduced basal and TGF-h1–stimulated levels of luciferase expression using were blocked using 1 mL PBS containing 5% goat serum and 1 mg/mL the TGF-h1–responsive reporter construct 3TP-Lux in the elf null- bovine serum albumin (BSA). The sections were incubated overnight at 4jC phenotype cells compared with control cells. in a humidor with primary antibodies, monoclonal antibody to Smad4 Cancer specimens. Formalin-fixed and paraffin-embedded colorectal linker region (Zymed, San Francisco, CA), and peptide-specific antibody to cancer and colon specimens were obtained from the Department of ELF (13) diluted to 2.5 to 5 Ag/mL in 1Â PBS containing 1 mg/mL BSA. Pathology, University of Pennsylvania Medical Center-Presbyterian. Twenty- Primary antibodies to Na+-K+-ATPase, vimentin, E-cadherin, and ankyrin one colorectal cancers collected from patients with varying grades and (Santa Cruz Biotechnology, Santa Cruz, CA) were used to for cell stages of colorectal cancer, identified by the Dukes’ classification, were polarization studies. In addition, to examine cell proliferation and analyzed for ELF and Smad4 expression. All the specimens were collected apoptosis in mouse colon tissues, primary antibodies of anti-p-histone after colectomy. Tissues were collected randomly at various zones of cancer H3 (Ser10), a mitosis maker (Upstate, Charlottesville, VA), and anti-active manifestation, including the rectum, and the ascending, transverse, caspase 3 (Promega, Madison, WI) were used. All further steps were carried descending, and sigmoid colon. Tumor grade was determined by histology out at room temperature. Four 5-minute rinses with 1Â PBS followed each and markers, such as carcinoembryonic antigen (CEA; Table 1). In staging successive step. The sections were then incubated with peroxidase- the tumors, Dukes’ classification B1, C1, and D stages represent dysplasia, conjugated secondary antibodies (Jackson Immunoresearch Laboratories, adenomas, and invasive carcinomas, respectively. Two independent blinded West Grove, PA) as previously described (13). The secondary antibody was pathologists evaluated the tumors used in the study. The control samples of diluted in 1Â PBS containing 1% goat serum. After rinses, 200 to 500 ALof normal colon tissue used in the present investigation were taken from the the insoluble peroxidase substrate 3,3V-diaminobenzidine (Sigma) was borders of the surgical specimens. www.aacrjournals.org 4229 Cancer Res 2005; 65: (10). May 15, 2005

Table 1. Clinical classification of 21 colorectal cancer tissues used in the study

Dukes’ stage CEA Metastatic Histology

D 3.9 0 Moderately diffused carcinoma B1 <1.0 1 Moderate to well-diffused carcinoma B1 3.1 1 Moderately diffused adenocarcinoma C1 2.7 CT na Adenocarcinoma B1 1.2 1 Moderately diffused invasive adenocarcinoma B1 5 1 Moderately diffused invasive adenocarcinoma B1 1.3 1 Two invasive moderately diffused adenocarcinoma B1 3.5 1 Moderately diffused adenocarcinoma C1 3.5 CT na Invasive moderately diffused adenocarcinoma D 907 0 Moderate to poorly diffused adenocarcinoma and metastasis to liver D 12 0 Moderately diffused invasive adenocarcinoma B1 <1.0 1 Moderately diffused adenocarcinoma C1 3.1 1 Invasive adenocarcinoma C1 3.5 1 Invasive adenocarcinoma B1 1.6 1 Moderately diffused adenocarcinoma D 2,915 0 Moderately diffused adenocarcinoma with metastasis to the ovary C2 1.7 1 Moderately diffused adenocarcinoma D 5,814 0 Invasive moderately diffused adenocarcinoma N/A No cancer 1 Tubular adenoma–high-grade dysplasia B1 na 0 Moderately diffused adenocarcinoma N/A No cancer 0 No cancer in this segment of the bowel B1 na 0 Moderately diffused adenocarcinoma

Abbreviations: N/A, not applicable; na, not available; CT, computed tomography scan.

Statistical analysis. Global v2 test was used to test the hypothesis that the colonic mucosa. Increased cell proliferation was observed in the coefficient of each variable was equal to 0. Tissue sample sets of elf +/À/Smad4+/À colon tissues (Figs. 2C, arrows,andS1C), immunohistochemical data were compared with assess the significance. A compared with normal colon tissues (Figs. 2A and S1A), when P value <0.05 was required for statistical significance, and all tests were using immunochemical labeling by the anti-p-Histone H3 (Ser10) two-sided. All tests were done with SPSS 10.1 software (SPSS, Inc., antibody (a mitosis marker; ref. 21). This indicated that colonic Chicago, IL). epithelial cell proliferation was stimulated by the disruption of elf. Loss of response to TGF-h signaling in elf +/À/Smad4+/À cells Results could indicate that colonic epithelial cell apoptosis may also be Elf +/À/Smad4+/À intercrosses establish the synergistic role altered in the elf +/À/Smad4+/À mutants. We further examined of ELF and Smad4 in colorectal cancers. To determine the role epithelial apoptosis in the colonic tissue with anticaspase 3 of ELF and Smad4 in colorectal cancer, we intercrossed elf +/À and labeling. In wild-type control mice, apoptosis was noted in crypt Smad4+/À mice. Out of 19 elf +/À/Smad4+/À mice, 3 developed epithelial cells (Figs. 2D, arrows, and S1D) but few apoptotic cells colon adenomas as early as 8 to 13 months of age (Fig. 1A) and were seen in elf +/À, Smad4+/À, and elf +/À/Smad4+/À mutant 7 almost all of them developed gastric tumors at 12 months of age, 1 colonic epithelium (Figs. 2F and S1F). Therefore, we observed whereas in Smad4+/À heterozygotes late onset carcinomas were significantly decreased apoptotic cells in elf +/À/Smad4+/À colon seen (17). In elf +/À, late-onset hepatocellular cancers are seen. In tissues (Figs. 2F and S1F) compared with normal colon tissues contrast, we did not find any colorectal polyps, tumors, and (Figs. 2D, arrows, and S1D), when using the apoptotic marker, adenomas in either elf +/À or Smad4+/À mice at the age of 8 to 12 antiactive caspase 3. The suppressed apoptosis indicates that elf months. H&E staining of the colon adenoma sections revealed may be important in the TGF-h induction of apoptosis in crypt aberrant crypts with loss of normal cellular structure in elf +/À/ epithelial cells and may contribute to the epithelial cell hyperplasia Smad4+/À mice (Fig. 1B and C) compared with normal wild-type in the elf +/À/Smad4+/À colonic mucosa. colon tissues (Fig. 1D and E). Interestingly, we also noted Loss of polarity in elf À/À gut epithelial cells. We first aberrations in expression patterns of vimentin (Fig. 1F-K). investigated abnormalities in intestinal epithelial cell morphology Increased cell proliferation and suppressed apoptosis in in the elf À/À mutant embryos, then analyzed the role of ELF/ elf +/À/Smad4+/À colonic mucosa. The occurrence of precancer- TGF-h in organelle formation and gut epithelial cell polarity. ous colonic polyps in these double heterozygotes compared with H&E-stained sections showed gut epithelial cells to be severely none in the elf +/À or Smad4+/À colons suggests that disruption of flattened with loss of villi in E11.5 elf À/À mutant embryos. elf, in addition to disruption of Smad4, results in hyperplasia of Further visualization of plasma membrane, Golgi, and nuclei by transfection of pEYFP-Mem, pECFP-Golgi, and pEYFP-Nuc (Clontech, Palo Alto, CA) into elf À/À mutant MEFs revealed all 7 7 Unpublished observations. three to be normal. Strikingly, we observed a marked distortion

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Figure 1. Histologic analysis of colon polyps in wild-type and elf +/À/Smad4+/À mice. A, macroscopic analysis of colon tumor development in elf +/À/Smad4 +/À mice. B and C, H&E-stained sections of colon polyps in elf+/À/Smad4 +/À mice. D and E, H&E-stained sections of normal wild-type colon tissues. Immunostaining with vimentin in E11.5 wild-type (F) and elf À/À mutant (G) gut tissues. Immunohistochemical detection of E-cadherin in E11.5 wild-type (H) and elf À/À mutant (I) gut tissues, adult wild-type normal colon tissues (J), and elf +/À/Smad4 +/À mutant colon tissues (K).

in expression patterns of Na+-K+-ATPase, MAP-2, and actin with K and S2). Na+-K+-ATPase labeling showed irregular and punctate increased vimentin, decreased E-cadherin, but normal ankyrin B intracellular localization, and it seemed absent at the apical and G expression in the elf À/À and decreased E-cadherin (brush border) plasma membrane in the elf +/À/Smad4+/À colonic expression in the elf +/À/Smad4+/À gut epithelial cells (Figs. 1F- mucosa and aberrantly localized in elf À/À cells (Fig. 3C and E) www.aacrjournals.org 4231 Cancer Res 2005; 65: (10). May 15, 2005

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research compared with normal, wild-type control, as well as Smad4+/À exclude abnormalities in microtubule function as a cause of the colonic mucosa (Fig. 3A, B, and D). observed phenotype seen in elf À/À mutant embryos, we analyzed To further explore the role of ELF in cell polarization and to microtubule distribution and function in wild-type and elf À/À investigate the possibility of rescuing localization of Na+-K+-ATPase mutant MEFs and in embryos. Immunofluorescence confocal signaling in the elf À/À mutants by rescuing Na+-K+-ATPase microscopy determined that the subcellular distribution of h- membrane localization through restoration of ELF activity, we tubulin is unaltered in the mutant embryos.7 Furthermore, neither transiently transfected full-length elf into the elf À/À mutant MEFs a microtubule-stabilizing agent (placitaxel) nor a microtubule- (transfection efficiency of 43.8 F 2.5 %; Fig. 3D-F, arrows). disrupting agent (such as nocodazole) corrected TGF-h signaling Correction of Na+-K+-ATPase localization at the membrane by in elf À/À mutant fibroblasts (Fig. 4F). Lack of response to TGF-h transient transfection of elf À/À MEFs with full-length elf was stimulation in mutant cells suggests that microtubule modulation documented by confocal immunofluorescent microscopy (Fig. 3F; of Smads may be less relevant and secondary to ELF spectrins. the frequency of correction being 41 F 1.8%). These data suggest This was further supported by transient transfection of the that a functional ELF spectrin with inherent dynamic stability that reporter construct p3TP-Lux, into wild-type and elf À/À cultured is responsive to environmental cues may represent a key regulatory MEFs. When we treated transfected wild-type MEFs with TGF-h1, element for Na+-K+-ATPase modulation. the luciferase activity induced was seven to eight times that of Microtubules have been shown to modulate TGF-h–induced elf À/À MEFs (Fig. 3H). However, in MEFs derived from elf À/À Smad signaling (22). We noted an aberrant expression of MAP-2 mouse embryos, TGF-h1–dependent induction of p3TP-Lux was in elf À/À mutant embryonic and in the elf +/À/Smad4+/À colonic abolished, as it was in vector controls, which indicated that the mucosa adult gut tissue (Fig. 4B and E) compared with normal TGF-h1 response needs ELF (Fig. 3H). In mutant rescue assays, wild-type embryonic and adult elf control tissues (Fig. 4A and C). restoration of elf in elf À/À-cultured MEFs dramatically induced a Interestingly, both h-spectrin and MAP-2 have been shown to be 7- to 8-fold increase in luciferase activity, almost as in the wild important for microtubule bundling and function (23, 24). To type (elf +/+).

Figure 2. Cell proliferation and apoptosis in wild-type, Smad4 +/À, and elf +/À/ Smad4 +/À colonic mucosa. Immunohistochemical detection of mitotic cells in normal wild-type (A), Smad4 +/À (B), and elf +/À/Smad4 +/À (C) mutant colon tissues. D to F, identification of apoptotic cells in normal wild-type, Smad4 +/À, and elf +/À/Smad4 +/À mutant colon tissues, using antiactive caspase 3 antibody. Arrows, mitotic (B and C) and apoptotic cells (D and E).

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Figure 3. ELF rescues loss of polarity and Na+-K+-ATPase expression in elf À/À mutants. A to C, immunohistochemical labeling of wild-type (A), Smad4 +/À (B), and elf +/À/Smad4 +/À (C) mutant colon tissues with anti–Na+-K+-ATPase. D, immunofluorescent confocal microscopy showing normal Na+-K+-ATPase distribution (rhodamine) in wild-type MEFs (arrow) transfected with pcDNA3.1 DNA only. E, Na+-K+-ATPase expression is decreased and aberrant in elf À/À MEFs. F, Na+-K+-ATPase expression is rescued by overexpression of ELF (arrow). G, Western blots show ELF expression in elfÀ/À MEFs transfected with a construct containing the full-length of elf cDNA (lane 3) restored back to its endogenous level, when compared with the ELF expression in both wild-type (lane 1) and elfÀ/À (lane 2) MEFs transfected with pcDNA3.1 only. H, mutant rescue assays demonstrating restoration of TGF-h response in elf À/À MEFs on transfection with full-length elf. I, schematic representation of the role of ELF in the TGF-h-Smad signaling pathway.

ELF localization in human colon cancers. ELF expression was intense labeling and 14.2% showed moderate labeling (P V 0.05) of observed along the tubular crypts of Leiberku¨hn in the mucosa of the basal crypts (Tables 1 and 2). The lamina propria separating the control colon tissues (Fig. 5A). Strong labeling was observed at the crypts with loose connective tissue, capillaries, and strands of base of the crypt and a gradient of moderate to intense labeling in smooth muscle did not label strongly for ELF. Labeling for ELF in the apical regions. ELF expression was mostly cytoplasmic. The colorectal cancer tissue samples showed reduction or loss of muscularis mucosa beneath the deep end of the crypts also expression in aberrant crypt foci (Fig. 5B and C). In Dukes’ B1, an exhibited faint labeling (Fig. 5A). Of the normal colons, 66% showed early stage of cancer, ELF expression was reduced and abnormal www.aacrjournals.org 4233 Cancer Res 2005; 65: (10). May 15, 2005

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research compared with that of controls, which suggested a down-regulation moderate labeling and 23.8% showed intense labeling; almost 57.1% of this gene in early neoplasia (Fig. 5B; Table 2). Abnormal ELF exhibited disruption of ELF. About 24% and 15% of carcinomas expression was also observed in the epithelial outpockets from the showed moderate and intense labeling for ELF, respectively (P V basal crypts that form adenomatous polyclonal polyps, an 0.05). ELF expression was strikingly absent in 9 of 10 B1 and 2 of 4 obligatory step for tumor progression. In adenomas, 14.2% showed C1 cancers (Fig. 5B and C; Tables 1 and 2). Aberrant or reduced

Figure 4. Loss of polarity in Smad4 +/À and elf +/À/Smad4 +/À colon tissues. Immunohistochemical detection of MAP-2 in E11.5 wild-type (A) and elf À/À mutant (B) gut tissues. Loss of polarized expression of MAP-2 is also observed in Smad4 +/À (D) and, to a greater extent, in elf +/À/Smad4 +/À (E) colon tissues when compared with normal wild-type tissues (C). F, analysis of the TGF-h response in control elf +/+ and elf À/À cell lines treated with placitaxel or nocodozole under transient transfection conditions.

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ELF expression or loss of it was observed in almost all B1 colon We observed loss of Smad4 expression in B1, C1, and D tumors, cancers. In C1 and D1 late-stage tumors, we observed loss of as well as marked loss of expression in the late stages (Dukes’ D expression in the crypt epithelial cells and a simultaneous positive cancers; Fig. 5E and F). In other types of tumors, 42.8% of adenoma labeling in the stromal tissue. had moderate staining, whereas 52.3% of carcinomas showed loss Smad4 localization in relation to ELF expression in human of Smad4 expression (P V 0.05). colon cancers. In the control colorectal tissues, we observed labeling of Smad4 in the basal one third of the crypts of Leiberku¨hn Discussion similar to ELF labeling (Fig. 5A and D). Prominent ELF and Smad4 Adult elf +/À/Smad4+/À mutant mice bred onto a 129SvEv/Black expression with an increased staining at the base of the crypt was Swiss background develop cancerous tumors of the colon. Here, we an important correlative feature observed (Fig. 5A and D). A high show that ELF suppresses early events in colon cancer formation. level of expression was seen in the submucosa and muscularis These studies are similar to findings from Tgf -h1À/À mice, in which mucosa at the deep end of the crypts but not in the lamina propria the tumor suppressor activity of TGF-h was not directed at cell that separates the crypts (Fig. 5D). Of the normal tissues, 28% proliferation, suppression of inflammation or maintenance of showed moderate labeling, whereas 20% and 38% showed intense genetic stability, or via regulation of adenomatous polyposis coli and loss of labeling, respectively (P V 0.05; Tables 1 and 2). In these (APC) levels (2). Progression of tumors from adenomatous to in situ normal samples, labeling was mostly in the cytoplasm. and invasive carcinomas may result from an inability to maintain

Figure 5. Immunohistochemical analysis of human colorectal cancers. A, ELF immunostaining in normal human colon tissue. Labeling at the base of the crypts of Leiberku¨hn and at the tips is observed. B, ELF expression in Dukes’ B1 is significantly reduced or lost in adenomatous polyps. C, a marked decrease in ELF expression in Dukes’ D tumors. D, anti-Smad4-labeled normal colon controls. E and F, reduced or loss of Smad4 expression in the basal crypts of B1 and D stage tumors, respectively. G, negative control (normal colonic tissue with secondary antibody only).

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Table 2. Immunohistochemical labeling results for 21 colorectal cancers

Cancer type Labeling for

ELF Smad4

++ + À NA P ++ + À NA P

Normal 14 3 4 À 0.0043 9 8 3 1 0.0401 Adenoma 5 3 11 2 0.011 6 9 3 3 0.0504 Carcinoma 4 6 10 1 0.016 2 4 11 4 0.0304

NOTE: Data obtained were statistically significant at P V 0.05. ++, intense; +, moderate; À, loss of or reduced labeling. Abbreviation: NA, not available.

normal tissue architecture (20). Spectrins are involved in the in the elf h-spectrin mutant phenotype resembles the Drosophila h- generation of cell polarity and protein sorting (23–26); hence, it is spectrin mutant, which in turn is reminiscent of the Drosophila conceivable that abnormalities in spectrin function could result in labial phenotype, particularly in the gut (36). Control of the the partial or complete loss of cellular polarity, a characteristic homeotic gene labial is dependent upon extracellular gradients of feature of tumor cells (27). wingless and decapentaplegic (the Drosophila homologue of TGF-h Spectrins are key proteins involved in the support of general during embryogenesis; ref. 37), which suggests that the relationship membrane integrity, stabilization of cell-to-cell interactions, axonal between elf and TGF-h is important for gut epithelial cell formation growth, and the formation of the sarcoplasmic reticulum (24, 28). and is conserved through evolution. They are also known to be involved in the generation of cell Here, we show that loss of ELF expression correlates with B1 polarity and protein sorting (23, 29, 30). Spectrins create a colon cancers and that a strict correlation exists between ELF and multifunctional scaffold on which membrane proteins, cytoplasmic Smad4 expression in the normal epithelial cells of the crypts of signaling molecules, and structural elements are organized in Leiberku¨hn (Fig. 5A and D). Previous coimmunoprecipitation distinct domains; in this way, the general cytoarchitecture and studies indicate an interaction between ELF and Smad4 that is tissue integrity of cells are maintained (24, 30). essential for TGF-h–mediated gene expression in mutant mice, as h 8 Resistance to TGF- is commonly associated with late events in well as in human gastric cancers (13). 2 Smad4 expression is tumorigenesis and probably is secondary to inactivating mutations prominent in normal colonic crypts with typically diffuse epithelial in ThRII, Smad2, or Smad4. Our recent studies that show that the staining. Stronger labeling is observed in the villus apex and the disruption of TGF-h signaling by inactivation of the adaptor bottom one third of the crypts. Loss of Smad4 expression in protein h-spectrin, encoded by elf (14, 31), results in Smad4 advanced colorectal tumors substantiates earlier reports and localization and activation (13). Hence, aberrations in ELF further supports the role of Smad4 as a gastrointestinal tumor expression should also affect the otherwise normal downstream suppressor and a potential marker in colorectal cancer (38). events leading to abrogation of Smad4 activity. Studies have The basal crypt region of the colonic epithelial cells is known to established the role of Smad4 as a tumor suppressor, and failure of harbor stem cells, which proliferate and migrate toward the villus Smad4 expression has been associated with advanced stage while differentiating into cell types (39). On reaching the villus apex, disease, the presence of lymph node metastasis, and a significant cells become apoptotic and are shed into the gut lumen. These shorter overall survival (31, 32). The essential and adaptor protein repetitive cycles of proliferation, differentiation, and shedding are ELF, a h-spectrin, may therefore play a similar role in suppressing events that maintain the integrity of normal colonic epithelia. gastrointestinal tumors. Expression of ELF and Smad4 in normal colon, particularly in the Loss of h-spectrins, such as ELF, an important protein necessary basal crypt region, suggests a functional role via the TGF-h signaling for maintaining the structural integrity of epithelial cells, may be pathway. As the cells reach the midcrypt region, ELF activity is pivotal for epithelial cell integrity and maintenance of tissue reduced along with cell differentiation. Absence of ELF expression, architecture in the early stages. Establishment of spatial coordinates especially in the basal crypts of B1 and C1 tumors, suggests an during differentiation of polarized cells involves a positional cue inactivation of the TGF-h signaling pathway via the abrogation of from cadherins that results in targeting of h-spectrin to a discrete Smad4 functions. These events may modulate the differentiation plasma membrane domain (33). The spectrin tetramer is then able to signals at the stem cell compartment (basal crypt) whereby the capture and stabilize additional membrane interacting proteins, proliferative stage is maintained and may lead to tumor formation. + + such as ankyrin and the basolateral Na -K -ATPase, to form the Interestingly, compared with Smad4, ELF expression was characteristic profile of a polarized membrane domain (34, 35). To significantly lost or reduced in Dukes’ B1 cancers, especially in facilitate simultaneous reception and transmission of positional the basal crypts correlating with loss of differentiation and the information, one of the two binding sites on the tetramer for ankyrin onset of colonic neoplasia. With further invasiveness and would allow for interaction with a cell adhesion molecule for a metastasis of the tumors, both ELF and Smad4 expression was positional clue, whereas the second ankyrin allows for acquisition of these studies indicate the requirement of h-spectrin for this + + interaction. The lack of polarized distribution of the Na -K -ATPase 8 Unpublished observations.

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diminished or lost particularly in the bottom third compartment potentially closely links the Wnt signaling to TGF-h signaling and this may serve as a prognostic factor for a poor outcome. Our pathways (43, 44). With this scenario, it will be interesting to findings suggest that in normal colon samples, after TGF-h delineate the cross-talk between signaling pathways that involves stimulation, ELF interacts with Smad4 in the cytoplasm and ELF and the key players in colorectal carcinogenesis, such as Wnt localizes to the nucleus for transcriptional control. Aberrant signaling. In view of the present findings, we suggest that loss of nuclear and cytoplasmic labeling of ELF in B1, C1, and D tumors ELF potentially contributes to the events that lead to onset of also suggests mislocalization of ELF, which ablates TGF-h–induced colorectal cancer and ELF probably plays an important role in transcriptional response. tumor suppressor mechanisms in colon cancer that can be Indeed, h-spectrins and ELF bind to E-cadherin via the h- recognized as a potential early marker in colorectal carcinoma. catenin (40), an important mediator of the Wnt signaling pathway (41) that has a central role in colorectal carcinoma, controlling the switch between proliferation and differentiation in intestinal Acknowledgments epithelial cells (42). Disruption of the G1 phase of the cell cycle Received 12/22/2004; revised 2/23/2005; accepted 3/2/2005. blocks a genetic program that is physiologically active in the Grant support: Sally Funderberg award, NIH grants RO1 DK56111 (L. Mishra) and proliferative compartment of colon crypts. Regulation of intracel- RO1 DK58637 (B. Mishra), and Veterans Affairs Merit Award (L. Mishra). h The costs of publication of this article were defrayed in part by the payment of lular -catenin signaling through APC, originally identified from page charges. This article must therefore be hereby marked advertisement in familial adenomatous polyposis patient studies, and by p53 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Transforming Growth Factor-β Suppresses Nonmetastatic Colon Cancer through Smad4 and Adaptor Protein ELF at an Early Stage of Tumorigenesis

Yi Tang, Varalakshmi Katuri, Radhika Srinivasan, et al.

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