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. 2009 Nov 26;28(47):4133-46.
doi: 10.1038/onc.2009.271. Epub 2009 Sep 14.

A Wnt kinase network alters nuclear localization of TCF-1 in colon cancer

R Najdi  1 , A Syed, L Arce, H Theisen, J-Ht Ting, F Atcha, A V Nguyen, M Martinez, R F Holcombe, R A Edwards, J L Marsh, M L Waterman
Affiliations

A Wnt kinase network alters nuclear localization of TCF-1 in colon cancer

R Najdi et al. Oncogene. .

Abstract

Constitutive activation of the Wnt/beta-catenin pathway has been implicated as the primary cause of colon cancer. However, the major transducers of Wnt signaling in the intestine, T-cell factor 1 (TCF-1) and TCF-4, have opposing functions. Knockout of TCF-4 suppresses growth and maintenance of crypt stem cells, whereas knockout of TCF-1 leads to adenomas. These phenotypes suggest that TCF-4 is Wnt-promoting, whereas TCF-1 acts like a tumor suppressor. Our study of TCF expression in human colon crypts reveals a mechanistic basis for this paradox. In normal colon cells, a dominant-negative isoform of TCF-1 (dnTCF-1) is expressed that is equally distributed between nuclear and cytoplasmic compartments. In colon cancer cells, TCF-1 is predominantly cytoplasmic. Localization is because of active nuclear export and is directed by an autocrine-acting Wnt ligand that requires Ca2+/calmodulin-dependent kinase II (CaMKII) activity for secretion and a downstream step in the export pathway. TCF-4 remains nuclear; its unopposed activity is accompanied by downregulation of dnTCF-1 and increased expression of full-length isoforms. Thus, the dnTCF-1 and TCF-4 balance is corrupted in cancer by two mechanisms, a Wnt/CaMKII kinase signal for nuclear export and decreased dnTCF-1 expression. We propose that dnTCF-1 provides homeostatic regulation of Wnt signaling and growth in normal colon, and the alterations in nuclear export and promoter usage contribute to aberrant Wnt activity in colon cancer.

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Conflict of interest statement

Conflict of Interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
TCF-1 is localized in the cytoplasm in colon cancer. Immuno-fluorescent detection of TCF-1 and TCF-4 in paraffin-embedded sections of tumor and normal matched human colon tissue. Antibody staining is green. Nuclei are stained with DAPI (false colored here as red to allow visualization). In the merged images, co-localization of antibody and DAPI is yellow. Control experiments show that in normal colon tissues, (a) β-catenin is cytoplasmic while in colon cancer, (b) β-catenin is nuclear and cytoplasmic thus validating the tissue integrity for these studies and confirming elevated Wnt signaling in the tumor. TCF-4 is predominantly nuclear in both normal and colon cancer tissue (c and d). TCF-1 is both nuclear and cytoplasmic in normal colon tissue (e), but TCF-1 is cytoplasmic and absent from nuclei in colon cancer (f). Scale bar is 20 μm.
Figure 2
Figure 2
TCF-1 isoforms in normal colon and colon cancer. (a) Western blots of DLD-1, Colo320 and SW480 colon cancer cell lines probed with antibodies that detect all forms of TCF-1 reveal FL-TCF-1B as the major form in colon cancer. (b) Western blots of DLD-1 colon cancer cells probed with an antibody that detects all forms of TCF-4 confirm that TCF-4 is expressed as a full-length E-tail isoform. (c) Schematic showing the predicted sizes of FL-TCF-4E (64 KDa), FL-TCF-1B (48 KDa) and dnTCF-1E (48 KDa) and the antibody locations on TCF-1 and TCF-4 (d) Crypt epithelial cells were isolated and purified from normal colon tissue of three different patients. Western blots of colon cancer cell line extracts and the crypt epithelial cell extracts were probed with two antibodies. One antibody detects all forms of TCF-1 and another detects E tail-containing isoforms of TCF-1. The merged signals from the two antibodies confirm that FL-TCF-1B is the major form in colon cancer and that dnTCF-1E is the major form in normal colon.
Figure 3
Figure 3
TCF-1 is exported from the nucleus in response to CaMKII via a CRM1 dependent mechanism. The colon cancer cell lines DLD-1, Colo320 and SW480 were stained for endogenous TCF-1 (red). F-actin was stained with Phalloidin (green) to illuminate the cytoplasm and nuclei were stained with DAPI (blue). (a) TCF-1 is nuclear and cytoplasmic in DLD-1 cells. (c) TCF-1 is cytoplasmic in Colo320 cells. (b) and (d) TCF-1 localizes to the nucleus of both DLD-1 and Colo320 cells after a 6-hour treatment with the CRM1 inhibitor Leptomycin B. (e) TCF-1 is predominantly nuclear in mock transfections of SW480 cells. (f) TCF-1 is exported to the cytoplasm in SW480 cells expressing CaMKII (T286D). (g) When CaMKII (T286D) is co-expressed with kinase dead TAK1 (K63W) and NLK (K155M), TCF-1 remains nuclear. Scale bar is 20 μm. (h) Transfection of activated CaMKII (T286D) in SW480 cells lowers Wnt signaling levels by 50–70% as measured by luciferase reporters.
Figure 4
Figure 4
Levels of activated CaMKII correlate with TCF-1 export level in colon cancer. DLD-1, Colo320 and SW480 cells were stained for the phospho-T286 modification of CaMKII (Green). F- actin staining is red and nuclear staining is blue. (a) and (b) DLD-1 and Colo320 cells express high levels of activated CaMKII while SW480 cells do not (c). Scale bar is 20 μm. (d) Western blots of colon cancer cell extracts probed for phosphorylated, active CaMKII show detectable levels in DLD-1 and Colo320 cells, but not in SW480 cells.
Figure 5
Figure 5
TCF-1 export is regulated by secreted ligand. Media from SW480 cells that had been mock-transfected or transfected with a plasmid containing constitutively active CaMKII (T286D) for 48 hours, or RPMI media, or media from DLD-1 cells was transferred to newly seeded SW480 cells and these cells were stained for endogenous TCF-1 (red). F-Actin staining is green and nuclear staining is blue. (a) In cells with mock-transfected media, TCF-1 is nuclear. (b) Cytoplasmic TCF-1 is observed in cells treated with conditioned media from CaMKII (T286D)-transfected cells. (c) TCF-1 is nuclear in cells grown in RPMI media, but cytoplasmic in cells treated with conditioned media from DLD-1 cells (d).
Figure 6
Figure 6
Wnt signaling regulates TCF-1 export. SW480 cells were transfected with a plasmid containing constitutively active CaMKII (T286D) alone or with expression plasmids for SFRP and stained for TCF-1 (red). (a) TCF-1 is predominantly nuclear in mock transfections of SW480 cells. (b) TCF-1 is exported to the cytoplasm in SW480 cells expressing CaMKII (T286D). Expression of SFRP1 (c), SFRP3 (d), or SFRP4 (e) block CaMKII’s ability to trigger TCF-1 export. DLD-1 cells were either mock-transfected or transfected with a plasmid containing SFRP4 and stained for TCF-1 (red). (f) TCF-1 is nuclear and cytoplasmic in mock-transfected DLD-1 cells, but is restricted to the nucleus in SFRP4-transfected DLD-1 cells (g). Media from SW480 cells that had been transfected with a plasmid containing constitutively active CaMKII (T286D) for 48 hours was transferred to newly seeded SW480 cells or Dkk-1 transfected SW480 cells and these cells were stained for TCF-1 (red). (h) Cytoplasmic TCF-1 is observed in cells treated with conditioned media from CaMKII (T286D)-transfected cells. (i) Dkk-1 blocks the conditioned media’s potential to trigger TCF-1 export. Scale bar is 20 μm.
Figure 7
Figure 7
TCF-1 export is an auto-activating loop. Media from SW480 cells that had been mock-transfected (a) or transfected with a plasmid containing constitutively active CaMKII (T286D) for 48 hours (b) was transferred to newly seeded SW480 cells and treated with 20μM (c) or 60μM (d) of the CaMKII inhibitor, KN-93 for 6 hour. (c) and (d) Varying KN-93 concentrations block the conditioned media from triggering TCF-1 export.
Figure 8
Figure 8
Working Model. (a) In normal colon, dnTCF-1E is expressed in the nuclei of cells where it counteracts the actions of FL-TCF-4E and preserves a balance between the signals that promote growth and those that promote differentiation. (b) In colon cancer, export of dnTCF-1E protein predominates. Export is mediated by CRM1 in response to activated Ca2+/Calmodulin dependent Kinase II (CaMKII) and TAK-1-NLK kinases. Export requires Wnt ligand action, a soluble factor secreted in response to activated CaMKII since SFRP proteins and Dkk-1 can block the export pathway. CaMKII actions is also required downstream of the Wnt ligand, suggesting that the export pathway functions as an auto-activating loop. (c) FL-TCF-1B is expressed and dnTCF-1E is under-expressed to further skew the balance towards the growth-promoting signals in colon cancer.
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