Dissociation of EphB2 signaling pathways mediating progenitor cell proliferation and tumor suppression

Abstract

Signaling proteins driving the proliferation of stem and progenitor cells are often encoded by proto-oncogenes. EphB receptors represent a rare exception; they promote cell proliferation in the intestinal epithelium and function as tumor suppressors by controlling cell migration and inhibiting invasive growth. We show that cell migration and proliferation are controlled independently by the receptor EphB2. EphB2 regulated cell positioning is kinase-independent and mediated via phosphatidylinositol 3-kinase, whereas EphB2 tyrosine kinase activity regulates cell proliferation through an Abl-cyclin D1 pathway. Cyclin D1 regulation becomes uncoupled from EphB signaling during the progression from adenoma to colon carcinoma in humans, allowing continued proliferation with invasive growth. The dissociation of EphB2 signaling pathways enables the selective inhibition of the mitogenic effect without affecting the tumor suppressor function and identifies a pharmacological strategy to suppress adenoma growth.

Figure 1. EphB Signaling Regulates Two Separate Transcriptional Programs

(A–C) Differentially expressed mRNAs 12h after inhibiting EphB signaling by an ephrin-B2-Fc injection cluster into two separate programs, cell cycle (blue frame in B) and cell organization (yellow frame in B), by gene ontology classification. Significant gene ontology terms are indicated by red frame. Genes listed in (A) and (C) belong to the indicated gene ontology terms. n=4 mice in each group. (D and E) Quantitative representation of expression levels (mean+SEM) of selected genes (names marked red in A and C) implicated in cell cycle (D) or cytoskeletal (E) regulation relative to Fc injected animals.

Figure 2. EphB2 kinase activity is required for its mitogenic effect but is dispensable for cell positioning

(A) Schematic depiction of wild type EphB2 and modified receptors. (B) Quantification of cell proliferation in colon crypts in adult mice with different EphB receptor modifications. The number of proliferating cells in each mouse line was normalized to its wild type control, giving the proliferation index. Kinase activity is required for the mitogenic effect of EphB2, whereas the PDZ domain-binding C-terminus is redundant. Broken lines indicate the levels for wild type and EphB2: EphB3 double null mice. (C) Quantification of the distance of Paneth cells to the crypt base relative to wild type mice. The intracellular domain of EphB2 is required, but both kinase activity and the PDZ domain-binding C-terminus are redundant, for cell positioning. Broken lines indicate the levels for wild type and EphB2 +/+; EphB3 −/− mice. Representative images for the cell proliferation and migration analyses are shown in the supplemental material. n=3–8 mice in each group, except for the EphB2 K661RΔVEV994/ K661RΔVEV994; EphB3 −/− mice where n=2. Data are represented as mean+SEM. * = p≤ 0.05, ** = p≤ 0.01, *** = p≤ 0.001, compared to EphB2 +/+; EphB3−/−, Student's t-test.

Figure 3. EphB mediated cell positioning is conveyed by phosphatidylinositol 3-kinase

(A and B) Ls174t cells plated on a confluent mosaic of 293t cells and 293t cells expressing ephrin-B2 and GFP are preferentially found on the 293t wild type cells. Ls174t cells exposed to increasing concentrations of the PI3K inhibitor LY294002 loose their preference for wild type 293t cells in a dose-dependent manner. (C and D) In vivo administration of LY294002 results in increasing displacement of Paneth cells from 3 to 7 days compared to animals administered with vehicle. PI3K inhibitor results in cell displacement to a similar degree as ephrin-B2-Fc, and administration of both LY294002 and ephrin-B2-Fc together does not further accentuate this phenotype. (E and F) Injection of ephrin-B2-Fc significantly decreases proliferation in colon crypts, where as LY294002 administration does not. Treatment of the two inhibitors together does not further reduce proliferation as compared to the ephrin-B2-Fc only treatment. n=3 mice in each group. Data are represented as mean+SEM. * = p≤ 0.05, ** = p≤ 0.01, *** = p≤ 0.001, Student's t-test. Scale bars=60 μm in B, 10 μm in D and 15μm in F.

Figure 4. EphB signaling does not affect β-catenin mediated transcription

(A–D) Inhibition of EphB signaling by an injection of ephrin-B2-Fc in TOPGAL mice results in a significant reduction in Ki67 mRNA levels (p=0.02, Student's t-test), whereas β-gal mRNA levels (p=0.59) and the number of β-gal expressing cells (p=0.30) are unaltered. Data are represented as mean+SEM. n=3 mice in each group. (E) Microarray analysis reveals little effect on the expression levels of β-catenin target genes in colon 3 or 12 hours after injection of ephrin-B2-Fc compared to control Fc protein. * = p≤ 0.05. Scale bar = 10 μm.

Figure 5. Cyclin D1 is required for the mitogenic effect of EphB

(A and B) Western blot analysis of cyclin D levels in the colon after inhibition of EphB signaling with ephrin-B2-Fc compared to vehicle (PBS). (C and D) mRNA levels for cyclin D family members are not altered after an injection of ephrin-B2-Fc as assayed by qRT-PCR (C, at 12 hours after ephrin-B2-Fc injection) or microarray analysis (D). (E and F) Western blot analysis of cyclin D levels in the colon in wild type (Wt) mice and in mice with a modified (F620D/F620D) constitutively active EphB2 receptor. (G and H) Cyclin D1 levels are too low to be detected by immunohistochemistry in wild type mice, but are readily detected in cell nuclei in colon crypts in EphB2 F620D/F620D mice. (I) Cyclin D1 positive cells are detected in the stroma of wild type small intestine (arrowheads). (J and K) Quantification of Ki67-immunoreative cells in crypts of the small intestine and colon reveals a similar degree of reduction in the number of proliferating cells in cyclin D1 −/− mice and in animals receiving an ephrin-B2-Fc injection (24h prior to analysis) compared to wild type animals receiving control Fc protein. Administration of ephrin-B2-Fc does not reduce proliferation further in cyclin D1 −/− mice. (L–O) Ki67-immiunoreactive cells in the crypts of the small intestine. n=3 mice in each group, except for n=2 in some cyclin D1 −/− groups. Data are represented as mean+SEM. Scale bar=10 μm in G and 30μm in I. ** = p≤0.01, *** = p≤0.001, Student's t test.

Figure 6. Proliferation, but not migration, is mediated by Abl

(A and B) Electroporation of Abl shRNA in intestinal explants leads to a reduction in the number of electroporated cells (GFP expressing) incorporating BrdU. (C and D) Proliferation in the Abl mutant mouse is reduced in both small intestine and colon. (E and F) Gleevec (100mg/kg) leads to a decrease in cyclin D1 protein levels in the colon detected by Western blot analysis. (G) Administration of Gleevec to TOPGAL mice leads to a 50% reduction in Ki67 transcript, without affecting the expression of β-galactosidase (p=0.87). (H and I) Inhibiting Abl with Gleevec or ephrin-B2-Fc reduces cell proliferation in the small intestine and colon to a similar extent, but there is no additive effect. (J) Gleevec does not affect cell proliferation in colon crypts in cyclin D1 −/− mice. (K) Analysis of EphB2 F620D/F620D mice with constitutively active EphB2 receptors reveal an increase in the number of BrdU positive cells when compared to wild type littermates. Administration of Gleevec to wild type and EphB2 F620D/F620D mice results in a suppression of proliferation to the same level in both genotypes. (L–Q) Paneth cells, visualized by lysozyme (Lys)-immunoreactivity, are displaced in EphB2; EphB3 double null mutant animals. Inhibition of EphB signaling with ephrin-B2-Fc also results in displaced Paneth cells 3 days after an injection. In contrast, animals receiving Gleevec for three consecutive days as well as Abl −/− mice show no evidence of Paneth cell displacement. n=3–6 mice in each group, except for n=2 cyclin D1 −/− mice receiving Gleevec. Data are represented as mean+SEM. * = p≤ 0.05, ** = p≤ 0.01, *** = p≤ 0.001, Student's t-test. Scale bar = 10 μm.

Figure 7. Dissociation of EphB signaling pathways during tumor progression

(A–C) Inhibition of EphB signaling with ephrin-B2-Fc or administration of Gleevec in APC^min mice reduces the number of BrdU-incorporating cells in untransformed crypts in the small intestine (A) and colon (B) as well as in adenomas (C). (D–F) Adenomas in APC^min mice display high levels of cyclin D1 compared to the surrounding tissue (D). Inhibition of EphB signaling with ephrin-B2-Fc (E) or Gleevec (F) in APC^min mice leads to reduced cyclin D1 levels in adenomas. Boxed areas are shown in higher magnification. (G) Analysis of protein levels by Western blot in human colon adenoma and carcinoma samples. All values were normalized to neighboring untransformed tissue from the same patient. EphB2 receptor expression is high in adenomas. At the transition to carcinoma, EphB2 is down regulated and the proliferation marker PCNA and cyclin D1 are maintained independently of EphB expression. (H) The ratio of cyclin D1/EphB2 protein changes at the transition from colon adenoma to carcinoma and there is no significant further change during the progression to more advanced UICC cancer stages. (I) Ls174t cells expressing EphB2 and EphB3 receptors display a significant increase in cyclin D1 levels after being plated together with 293t cells expressing ephrin-B2, as compared to Ls174t cells plated together with wild type 293t cells. KM12 cells, expressing no or low levels of EphB receptors, do not up regulate cyclin D1 when plated together with ephrin-B2 expressing cells. (J–L) HT-29 and KM12 cells treated with Gleevec for 48 hours display a reduced number of BrdU positive cells as compared to cells treated with vehicle. Gleevec administration also results in reduced levels of cyclin D1 in both cell lines. (M and N) HT-29 cells transfected with shRNA against Abl with GFP display significantly reduced levels of cyclin D1 after 24 hours, as compared to cells transfected with GFP only. (O and P) Administration of EGF (PD153035) or IGF (AEW-541) receptor inhibitor to HT-29 or KM12 colon carcinoma cell lines results in reduced proliferation in both cell lines, whereas PD135035 only affects cyclin D1 in HT-29 cells and AEW-541 only in KM12 cells. Data are represented as mean+SEM. n=3–4 mice in each group. All in vitro experiments were made in triplicate. Scale bar=50 μm. * = p≤0.05, ** = p≤0.01, *** = p≤0.001, Student's t test.