The Nuclear Effector of Wnt-Signaling, Tcf1, Functions as a T-Cell-Specific Tumor Suppressor for Development of Lymphomas

The HMG-box factor Tcf1 is required during T-cell development in the thymus and mediates the nuclear response to Wnt signals. Tcf1(-/-) mice have previously been characterized and show developmental blocks at the CD4-CD8- double negative (DN) to CD4+CD8+ double positive transition. Due to the blocks in T-cell development, Tcf1(-/-) mice normally have a very small thymus. Unexpectedly, a large proportion of Tcf1(-/-) mice spontaneously develop thymic lymphomas with 50% of mice developing a thymic lymphoma/leukemia at the age of 16 wk. These lymphomas are clonal, highly metastatic, and paradoxically show high Wnt signaling when crossed with Wnt reporter mice and have high expression of Wnt target genes Lef1 and Axin2. In wild-type thymocytes, Tcf1 is higher expressed than Lef1, with a predominance of Wnt inhibitory isoforms. Loss of Tcf1 as repressor of Lef1 leads to high Wnt activity and is the initiating event in lymphoma development, which is exacerbated by activating Notch1 mutations. Thus, Notch1 and loss of Tcf1 functionally act as collaborating oncogenic events. Tcf1 deficiency predisposes to the development of thymic lymphomas by ectopic up-regulation of Lef1 due to lack of Tcf1 repressive isoforms and frequently by cooperating activating mutations in Notch1. Tcf1 therefore functions as a T-cell-specific tumor suppressor gene, besides its established role as a Wnt responsive transcription factor. Thus, Tcf1 acts as a molecular switch between proliferative and repressive signals during T-lymphocyte development in the thymus

Tcf1^−/− mice develop thymic lymphomas.

<p>(A) Heart with thymus and spleen of one Tcf1^+/− and three Tcf1^−/− mice are shown. The number of thymocytes of the mice is from top to bottom: 79, 9, 110, and 355×10⁶ cells. (B) Tcf1^−/− mice are sacrificed at a certain age (8–10, 10–15, 15–18, or >18 wk), and according to the bimodal distribution of the thymus size, mice were considered to have a tumor when the thymus contains >18×10⁶ cells. In total 150 Tcf1^−/− mice were analyzed. The percentage of mice categorized to have a tumor is shown for each age group in the right panel. (C) Histopathology of normal tissue compared to tumors from Tcf1^−/− mice. Paraffin sections of thymus, liver (Tcf1^+/+ and Tcf1^−/−), and heart (Tcf1^−/− only) were stained with hematoxylin and eosin (HE). The HE sections of the Tcf1^−/− mouse show neoplastic cells arranged in cords and sheets in the thymus that infiltrate in the liver and the heart; final magnification, 100×.</p

Blocked Tcf1^−/− thymocytes show high Wnt-signaling in lymphoma development.

<p>(A) Tcf1^−/− mice were crossed to Conductin(Axin2)-LacZ reporter mice and sacrificed at different ages. Dot plots of four representative mice are shown: one control Tcf1^+/− LacZ (age 20 wk), one control Tcf1^−/− LacZ (age 9 wk), and two Tcf1^−/− Axin2-LacZ mice (age 20 wk, Tumor 1+ Tumor 2). Organs were collected and thymocytes were stained for CD4, CD8, CD3, CD44, and CD25 together with FDG to demonstrate Wnt reporter activity in the different thymocyte subsets. The corresponding thymus sizes for the four mice shown are 110×10⁶, 17×10⁶, 39×10⁶, and 175×10⁶ cells, respectively. Expression of Wnt reporter activity is shown per thymocyte subset, DP, ISP, and DN3 for control cells (filled), Tcf1^−/− cells (thin line), and Tcf1^−/− tumor cells (thick line). (B) RNA was isolated of total thymus and the expression level of Lef1 and Hes1 relative to Abl is shown for the control thymus and the two tumor samples as shown in (A). (C) The mean expression levels of Hes1 relative to Abl are shown for small tumors (<i>n</i> = 5) and large tumors (<i>n</i> = 25).</p

Tcf1^−/− lymphomas show deregulated Wnt signaling.

<p>(A) RNA isolated from thymi of 17 different mice was used for microarray analysis. Expression of Lef1, Tcf7, Myc, and Hes1 in the Tcf1^−/− mice without lymphoma (<i>n</i> = 4), Tcf1^+/− mice (control, <i>n</i> = 5) and Tcf1^−/− mice with a lymphoma (Lymphoma, <i>n</i> = 8) is shown. For the Tcf1^−/− lymphoma mice, the phenotype of the tumor is indicated on the horizontal axis. Columns represent independent RNA preparations of the different mice groups. A principal component analysis was performed using Wnt and Notch target genes. A PCA analysis shows clustering of the three groups as well as the effect each of the target genes has on the separation of these groups with samples of Tcf1^+/−, Tcf1^−/−, and Tcf1^−/− with tumors indicated by red, black, and green spheres, respectively. (B) Expression levels of Tcf1, Lef1, Axin2, Cyclin D1, cMyc, Hes1, and Deltex1 as determined by Affymetrix microarray were summarized and normalized using RMA, and the expression relative to Abl was plotted for each sample. Statistical significant differences (<i>p</i><0.05) as determined by Mann–Whitney U test are indicated by an asterisk. (C) A panel of 40 Tcf1^−/− thymic lymphomas and four control thymi (Tcf1^+/−) were analyzed by RQ-PCR. Expression data for Lef1 long (containing the β-catenin interacting domain), <i>Axin2</i>, <i>Deltex1</i>, and <i>Hes1</i> are shown relative to the house keeping gene <i>Abl</i>. Mann–Whitney U tests were performed to calculate the indicated <i>p</i> values.</p

Lack of Tcf1 deregulates the balance between long and short isoforms of Lef1 in Tcf1-deficient lymphomas.

<p>(A) A schematic representation of the long and short isoform of Lef1, depicting the β-catenin binding domain and the HMG-box (DNA binding domain). The ratio of Lef1 long isoform versus Lef1 short isoform was determined using RQ-PCR for 40 lymphomas, four control thymi, and three Tcf1^−/− thymi without tumor. The long versus short ratio for Lef1 at the protein level is depicted for four different Tcf1^−/− thymic tumors (tumor size: 19, 224, 118, 30×10⁶ cells/ml, respectively). (B) Left panel, total Tcf and Lef RNA levels relative to Abl were determined using the data obtained from the Affymetrix data shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001430#pbio-1001430-g003" target="_blank">Figure 3A</a> (Tcf1^+/− total thymus, <i>n</i> = 5). In the middle panel, a Western blot analysis and quantification (right panel) for nuclear protein extracts of total thymocytes (Tcf1^+/+ mouse) is shown. (C) Representative Western blots of two independent experiments (Exp A and Exp B) of sorted thymocyte subsets from two control Tcf1^+/+ mice. Cell populations were sorted into the subsets DN, DP, and SP using lineage markers; CD3, CD4, CD8, CD44, and CD25 and total protein extracts were generated. Antibodies recognizing all isoforms of Tcf1 and Lef1 were used, and β-actin was used a loading control for the Western blots. Quantification of the Tcf and Lef signal of all Western blots was performed by ImageQuant J.</p

Tcf1^−/− thymic lymphomas are phenotypically heterogeneous, malignant, and oligoclonal.

<p>(A) Thymocytes, bone marrow (BM) cells, splenocytes, and lymphocytes obtained from lymph nodes (LN) were stained for CD4, CD8, CD44, and CD25. The CD4/CD8 and CD44/CD25 plot is shown for lineage negative cells for all the different organs. Results are shown for one Tcf1^+/− mouse and three representative Tcf1^−/− mice. (B) Left panel, genomic DNA was prepared from thymocytes derived from a Tcf1^+/− mouse (Lane 1, showing the germline band and a faint pattern of additional bands) and four different Tcf1^−/− mice with thymic lymphomas (DN3 tumor, Lane 2; DP tumors, Lanes 3 and 4) and an ISP tumor (Lane 5). The asterisk indicates the fragment expected for the germ-line (g.l.) TCRβ gene configuration. On the left side, a size marker (M) was included of which the sizes are indicated in the figure (kD). Right panel, genomic DNA was prepared from different organs, T (thymus), B (bone marrow), S (spleen), LN (lymph nodes), and Liv (liver) of one control Tcf1^+/− mouse and four different Tcf1^−/− mice. The phenotype of the lymphoma as determined by FACS analysis is shown for each mouse. The first lane includes a size marker (M) of which the sizes (kD) are indicated in the figure. (C) Tcf1^−/− tumor cells characterized by an intermediate expression of CD3 and CD25 were injected into sublethally irradiated Rag1^−/− mice by tail vein injection. Mice were bled at the time of injection, 4 and 6 wk after injection. The presence of CD3+CD25+ tumor cells in blood is indicated per mouse (<i>n</i> = 8). (D) Six weeks after transfer of the cells, the mice were sacrificed and the thymus, bone marrow, and spleen were analyzed for the presence of tumor cells. Cell suspensions were stained for lineage markers (Mac1, Gr1, B220, Ter119, and NK1.1) and CD4, CD8, CD3, CD44, and CD25. An example of the gating strategy is shown for a bone marrow sample. The percentage of CD3+CD25+ tumor cells is shown per mouse per organ.</p

Tcf1-deficient lymphoma cells depend on Wnt and Notch signaling for their survival.

<p>Several Tcf1^−/− cell lines were established from Tcf1^−/− thymic lymphomas, and all cell lines show Notch1 mutations and a high ratio of Lef1 long over short isoform. (A) The TCF05 cell line (phenotyped as an ISP tumor) was cultured in the absence and presence of the indicated concentrations of γ-secretase inhibitor (DAPT). Percentage of live cells was determined after 24, 48, and 72 h using flow cytometry (AnnexinV/7AAD staining). After 24 and 48 h cell cycle analysis was determined by propidium iodide staining, and the relative expression of Hes1, Deltex, Lef1-L, and Lef1-S was determined. For the cell cycle analysis, one representative example is shown and the mean expression levels relative to Abl are shown of two independent experiments. (B) Three cell lines (Jurkat, TCF05, and TCF07) were cultured for 7 h in the presence or absence of Quercetin (25 µM), and the percentage of live cells was determined by flow cytometry (7AAD/AnnexinV staining). The mean percentage (± SEM) of live cells (7AAD−/AnnV−) is shown for the three cell lines of three independent experiments. (C) The Tcf1^−/− cell line TCF07 was established from a thymic lymphoma characterized as a DN3 tumor with a mutation in exon 34 of Notch1. This cell line and Jurkat cells were transfected with a GFP construct in combination with a control construct (pcDNA3) or a dominant negative Lef1/Tcf1 (pcDNA ΔNTCF) construct (used ratio GFP∶construct, 1∶10). The percentage of viable cells was determined within the transfected (GFP+) cells after 6 h. (D) HEK 293 T cells were transfected with a LEF-1 luciferase reporter plasmid containing Tcf/Lef-responsive elements. The cells were cotransfected with S33-Bcat or pCI (3 µg) or Delta N Tcf1 together with S33 B cat. To control for transfection efficiency, all transfections included the pRLTK-renilla reporter (0.15 µg). Luciferase activities are shown as mean of three independent experiments normalized to renilla activity.</p

Mechanism of lymphomagenesis caused by Tcf1 deficiency.

<p>In the absence of Tcf1, thymocyte development is blocked at several stages (DN1, DN3, and ISP). Arrested thymocytes lack all isoforms of Tcf1, including the repressive isoforms. Loss of these repressive isoforms results in an up-regulation of Lef1, of which the long isoforms are most abundant in the thymus. Subsequently, Lef1 is capable of interacting with β-catenin as a compensatory mechanism, inducing deregulated Wnt signaling as measured by the high expression of <i>Axin2</i> in the vast majority of the lymphomas, forming a pre-leukemic stage. After additional mutations are acquired, of which activating mutation in <i>Notch1</i> frequently occur, and also other oncogenes such as <i>Mef2C</i> and <i>Lmo2</i> are likely candidates to be affected, full-blown lymphoma/leukemia develops.</p

The nuclear effector of Wnt-signaling, Tcf1, functions as a T-cell-specific tumor suppressor for development of lymphomas

The HMG-box factor Tcf1 is required during T-cell development in the thymus and mediates the nuclear response to Wnt signals. Tcf1(-/-) mice have previously been characterized and show developmental blocks at the CD4-CD8- double negative (DN) to CD4+CD8+ double positive transition. Due to the blocks in T-cell development, Tcf1(-/-) mice normally have a very small thymus. Unexpectedly, a large proportion of Tcf1(-/-) mice spontaneously develop thymic lymphomas with 50% of mice developing a thymic lymphoma/leukemia at the age of 16 wk. These lymphomas are clonal, highly metastatic, and paradoxically show high Wnt signaling when crossed with Wnt reporter mice and have high expression of Wnt target genes Lef1 and Axin2. In wild-type thymocytes, Tcf1 is higher expressed than Lef1, with a predominance of Wnt inhibitory isoforms. Loss of Tcf1 as repressor of Lef1 leads to high Wnt activity and is the initiating event in lymphoma development, which is exacerbated by activating Notch1 mutations. Thus, Notch1 and loss of Tcf1 functionally act as collaborating oncogenic events. Tcf1 deficiency predisposes to the development of thymic lymphomas by ectopic up-regulation of Lef1 due to lack of Tcf1 repressive isoforms and frequently by cooperating activating mutations in Notch1. Tcf1 therefore functions as a T-cell-specific tumor suppressor gene, besides its established role as a Wnt responsive transcription factor. Thus, Tcf1 acts as a molecular switch between proliferative and repressive signals during T-lymphocyte development in the thymus.Stemcel biology/Regenerative medicine (incl. bloodtransfusion