Ephrin type-B receptor 3 (EPHB3) is a member of the largest family of receptor tyrosine kinases, Eph [1
]. Eph receptors are subdivided into classes A and B based on sequence homology and binding affinity for their membrane-bound ligands [2
]. EPHA receptors bind glycosylphosphatidylinositol-anchored ephrin-A ligands and EPHB receptors bind transmembrane ephrin-B ligands [3
]. EPHB/ephrin-B interactions are key regulators in diverse physiological and pathological processes associated with development and disease of different organ systems [4
]. In particular, a wide variety of cancer cells express EPHB receptors and their cancer-related activities are complex and intriguing; their roles can be either tumor suppressive or oncogenic depending on the type of cancer and the cellular context [1
The roles of EPHB3 in colorectal cancer (CRC) development have been characterized using CRC mouse models. EPHB3 is normally expressed by cells in the stem cell niche at the base of intestinal crypts, where EPHB3 and its homologue EPHB2 generate and maintain the architecture of the villus–crypt axis through their interaction with ephrin-B ligands [7
]. The loss of EPHB3 accelerates colorectal tumorigenesis and prompts the formation of invasive adenocarcinoma in Apc Min/+
mice, suggesting that EPHB3 suppression represents a critical step in CRC progression [8
]. Moreover, EPHB2 and EPHB3-induced compartmentalization can restrict the spread of CRC cells [9
]. EPHB3 is downregulated in advanced stages of human CRC [10
], which suggests that EPHB3 functions as a tumor suppressor in this context. However, due to the complex functions and bidirectional signaling of the EPH/ephrin system, this hypothesis needs to be verified with a large cohort of human CRC samples. Indeed, despite all the strong evidence supporting a tumor suppressive role for EPHB3 in mouse studies, Xaun et al. recently reported that EPHB3 is an independent prognostic factor for poor survival in CRC patients [11
]. Similarly, there are conflicting data regarding the effect of EPHB3 on the progression of non-small cell lung cancer [12
While EPHB2 expression is consistently associated with better survival of CRC patients [14
], the prognostic value of EPHB3 in CRCs has not been well characterized. Additionally, no studies have comprehensively assessed the changes in EPHB3 expression from benign to malignant tumors of the colorectum. In this study, we sought to quantify EPHB3 expression in a variety of precancerous lesions and numerous CRC samples, and to determine its prognostic significance. Furthermore, we analyzed alterations in the expression of EPHB3 during various stages of CRC progression: adenoma-to-carcinoma transformation, tumor budding, and lymph node metastasis. We also evaluated EPHB3 expression in a colitis-associated cancer (CAC) model, and its effects on cancer growth and migration were assessed using CRC cell lines.
2. Materials and Methods
We collected 610 formalin-fixed, paraffin-embedded (FFPE) CRC tissue samples between 2004 and 2006 from the archives of the Department of Pathology at Seoul National University Hospital (SNUH) (Seoul, Korea). Information including patient gender and age; tumor location, histological type, and level of differentiation; evidence of lympho-vascular invasion; American Joint Committee on Cancer/International Union against Cancer (AJCC/UICC) cancer stage (7th edition); time of death; tumor recurrence; and duration of follow-up were obtained by reviewing the clinical and pathological reports. With regard to tumor location, proximal colon was defined as proximal to the splenic flexure (cecum, ascending and transverse colon) and distal colon was defined as distal to the splenic flexure (splenic flexure, descending, sigmoid colon, and rectum). Tumor budding was defined as a single tumor cell or a cluster of <5 tumor cells at the invasive margins. Fifty-nine CRC samples were obtained from the Department of Pathology at Jeju National University Hospital (JNUH) (Jeju, Korea). Twenty-two of the CRC tumors arose from pre-existing adenomas and 37 were ulcerofungating CRCs with lymph node metastases. The histopathologic features of the CRCs were determined by three gastrointestinal pathologists (J.M.B. and G.H.K. assessed the SNUH samples and B.G.J. assessed the JUNH samples). Additionally, 32 paired, fresh colorectal cancer tissues and matched normal tissues were provided by the Jeju National University Hospital Biobank, a member of the National Biobank of Korea, for which informed consent was obtained from all participants. All procedures were conducted in accordance with the 1975 Helsinki Declaration, revised in 2013. The study was approved by the Institutional Review Board of SNUH (C-1502-029-647) and (JNUH 2017-06-029), respectively.
2.2. DNA Isolation
FFPE CRC tissue samples (n = 610) were retrieved from the Pathology archives of the SNUH. Genomic DNA isolation was performed as follows: cancer areas (cancer cells > 70% of selected area) were microdissected with surgical blades from 10 μm-thick, unstained tissues. The tissues were digested in lysis buffer (100 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), 0.05 mg/mL tRNA and 1 mg/mL proteinase K) at 55 °C for 48 h, followed by a 10-min incubation at 95 °C to inactivate the proteinase K. The DNA was stored at −20 °C.
2.3. Microsatellite Instability (MSI) Analysis
The genomic DNA of CRCs was subjected to MSI analysis using the fluorescent multiplex PCR method with five NCI-recommended microsatellite markers (BAT25, BAT-26, D5S346, D17S250, and D2S123). The MSI status of each CRC sample was classified as either MSI-high (≥2 unstable markers of 5), MSI-low (1 unstable marker of 5), or microsatellite stable (no unstable markers).
2.4. DNA Methylation Analysis
DNA analysis for the determination of CpG island methylator phenotype (CIMP) status was carried out as previously described [17
]. Sodium bisulphite modification of genomic DNA samples was performed for all 1133 CRC specimens. The quantitative measurement of the promoter CpG island methylation of eight CIMP marker genes (CRABP1, CACNA1G, CDKN2A (p16), IGF2, MLH1, NEUROG1, RUNX3, and SOCS1) was performed using MethyLight assay, methylation specific real-time PCR. A CIMP-high tumor was defined when having five or more hypermethylated markers, a CIMP-low tumor was defined when having one to four hypermethylated markers, and a CIMP-negative tumor was defined as having no hypermethylated markers. A hypermethylated CpG island locus was defined when the percentage of the methylated reference (PMR) value was >4. The MethyLight assay for each CIMP marker gene was repeated independently three times, and promoter hypermethylation was defined as a PMR value >4 observed in at least two of three experiments.
2.5. KRAS/BRAF Mutation Analysis
KRAS/BRAF mutation analysis was performed as previously described [17
]. KRAS codon 12 and 13 mutations, and BRAF codon 600 mutations were detected using PCR-restriction fragment length polymorphism and direct sequencing techniques. Among the 788 CRC samples, 39 and 81 were excluded from the KRAS and BRAF mutation analyses, respectively, due to insufficient DNA quantity.
2.6. Mice and Induction of CAC
Induction of CAC in mice was carried out as previously described [18
]. Briefly, 5- to 6-week-old C57BL/6 male mice were purchased from OrientBio (Seongnam, Korea) and maintained at the Animal Research Facility at Jeju National University School of Medicine under pathogen-free conditions. Mice (n
= 32) were intraperitoneally injected with azoxymethane (AOM) (7.4 mg/kg body weight) (Sigma-Aldrich, St Louis, MO, USA) dissolved in phosphate-buffered saline (PBS). After AOM administration, the mice were subjected to 3-week cycles of 1 week with 1% dextran sulfate sodium (DSS treatment) (MP Biomedicals, Santa Ana, CA, USA) added to their drinking water, and 2 weeks of drinking water without DSS (recovery period). The protocol was the same for the control group (n
= 27) except they received no AOM injections on day 0. Some mice were euthanized prior to DSS treatment and others were euthanized after DSS treatment. Colon tissues were then harvested. Upon opening the colon, the mucosal surface was observed, and tumors were counted. A 1-cm piece of the distal colon was bisected longitudinally; half was stored in RNAlater®
stabilization solution (Ambion, Austin, TX, USA) for real-time PCR analysis and the other half was fixed in a 4% paraformaldehyde neutral buffer solution for histologic examination. Animal experiments were performed in accordance with the institutional guidelines of the Jeju National University for animal use and care.
2.7. Tissue Microarray (TMA) Construction
Thirteen TMAs containing 770 CRCs from the SNUH were constructed and histologically evaluated. In brief, the perimeters of representative tumor areas were marked for each sample. Core tissue biopsies (2 mm in diameter) were extracted from each paraffin block (donor blocks) and arranged on a new recipient paraffin block (tissue array block) using a trephine apparatus (SuperBioChips Laboratories, Seoul, Korea). For CRCs from the JNUH, two TMAs containing 24 pairs of adenoma and carcinoma areas and 15 TMAs containing 182 CRCs were generated using 4 mm core tissue biopsies [19
]. For ulcerofungating CRCs, both superficial and invading areas were included, and, if present, lymph node metastases were as well. In addition, three TMAs from CAC mouse model animals were constructed as previously described [18
2.8. Immunohistochemistry (IHC) and Evaluation
IHC and interpretation was carried out as previously described [15
]. IHC was performed on 4 μm TMA sections using a BOND-MAX automated immunostainer and a Bond Polymer Refine Detection kit (Leica Microsystems, Wetzlar, Germany) according to the manufacturer’s instructions. The primary antibodies used were anti-EPHB3 (Novus Biologicals, Littleton, CO, USA; 1B3) and anti-β-catenin (Novocastra Laboratories, Newcastle, UK; 17C2). EPHB3 expression was assessed at the tumor cell membrane. Histo-scores (H-scores; range: 0–300) were obtained by multiplying the intensity score (0 = negative; 1 = weak; 2 = moderate; 3 = strong) and the percentage of positive tumor cells (range: 0–100%). For statistical analyses, TMA sections with H-scores < 40 were defined as negative, and those with H-scores > 40 were defined as positive. β-catenin staining was considered as positive when more than 10% of the tumor cell showed strong nuclear positivity.
2.9. RNA Extraction and Quantitative Real-Time PCR
Total RNA was extracted from 30 paired fresh CRC tissues and matched normal colon tissues with TRIZOL (Invitrogen, Carlsbad, CA, USA). Complementary DNA was generated by reverse transcription using the GoScript reverse transcription system (Promega, Madison, WI, USA). Subsequently, to determine the expression of target genes, real-time PCR was performed with Premix EX Taq (Takara Bio, Shiga, Japan) in a StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA). The cycling conditions were as follows: initial denaturation for 30 s at 95 °C, followed by 40 cycles of 95 °C for 1 s and 60 °C for 5 s. The TaqMan gene expression assays (Applied Biosystems) used were as follows: Hs00177903-m1 (EPHB3), Hs00362096-m1 (EPHB2), Hs00197437 (OLFM4), Hs00394267_m1 (LRIG1), Hs00173664_m1 (LGR5), Hs01075864_m1 (CD44), Hs01009250-m1 (PROM1/CD133), Hs002379687_s1 (CD24), Hs00233455_m1 (CD166), and Hs0275899_g1 (GAPDH). GAPDH served as the endogenous control.
2.10. Colon Cancer Cell Lines
Ten colon cancer cell lines (DLD1, HT29, HCT116, HCT15, SW620, LOVO, SW480, KM12C, KM12L4, and KM12SM) were obtained from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in MEM, DMEM, RPMI1640, or L15 medium (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco), and maintained in a humidified incubator with 5% CO2 at 37 °C.
The anti-EPHB3 antibody was purchased from Novus Biologicals and anti-β -actin antibody was purchased from Abcam (Cambridge, UK). Anti-EPHB2 was purchased from R&D systems (Minneapolis, MN, USA). The anti-caspase-3, anti-β-catenin, anti-SLUG, anti-AKT, anti-ERK, anti-phospho-AKT, anti-phospho-ERK, anti-cleaved PARP, anti-BAX, and anti-BIM antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-mouse IgG-HRP and anti-rabbit IgG-HRP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
2.12. Western Blot Analysis
Cellular proteins were extracted using lysis buffer (iNtRON Biotechnology, Seongnam, Korea) and proteins were quantitated using BCA protein assay kits (Pierce, Rockford, IL, USA). Cell lysates were run on a 10% SDS-polyacrylamide gel, and proteins were transferred to a PVDF membrane (Millipore Corporation, Bedford, MA, USA) and blocked with 5% non-fat dry milk in PBS-Tween-20 (0.1%, v/v) for 1 h. The membranes were then probed with specific primary antibodies. After overnight incubation at 4 °C and washing with TBS containing 0.1% Tween-20, the membranes were incubated for 1 h with corresponding secondary antibodies. The target proteins were visualized with an Alliance-Mini.HD9 chemiluminescence documentation system (UVItec Cambridge, UK).
2.13. Transfection of EPHB3 and siRNA
Full-length cDNA encoding EPHB3 (pCMV6-EPHB3) was purchased from Origene (Rockville, MD, USA). Cells (1 × 106 cells/well) were seeded in a 6-well plate and transfected with 5 μg of pCMV-EPHB3, control vector, pCMV-EGFP, or EPHB3 siRNA pool (Dharmacon, Lafyette, CO, USA) using the Invitrogen Neon transfection system. Twenty-four hours post-transfection, the cells were subjected to proliferation and migration assays, RNA was extracted for real-time PCR, and protein was extracted for Western blot analysis. All experiments were carried out independently at least twice.
2.14. Proliferation Assay and Colony Formation Assay
For the proliferation assay, 5 × 103 cells/well counted by LUNA-II (Logos Biosystems, Gyeonggi-do, Korea) were seeded in the wells of a 96-well plate and cultured at 37 °C. After adding 10 μL of Cell Counting Kit-8 reagent (Dojindo, Kumamoto, Japan) to each well and incubating for 1 h, optical density was measured at 450 nm in an automatic microplate reader (Thermo Labsystems, Rockford, IL, USA). For the colony formation assay, 5 × 103 cells were seeded in a 60 mm culture dish and cultured for 2–3 weeks until distinguishable colonies appeared. Colonies were fixed with 70% methanol and stained with 0.01–0.1% crystal violet. All experiments were performed in triplicate and repeated at least twice independently.
2.15. Migration Assay
For the migration assay we used 24-well transwell culture plates with inserts (8 μm pore size) (BD Bioscience, San Diego, CA, USA). We seeded 2 × 105 cells in 300 μL serum-free medium into the upper chambers, and 500 μL medium with 10% FBS into the lower chambers. After 24 h, cells that remained on the top surface of the transwell insert were carefully wiped away with a cotton swab. The cells that migrated through the pores to the lower surface of the insert were fixed in methanol for 10 min, stained with crystal violet, and counted 1 h later. All experiments were carried out independently at least 2–3 times.
2.16. Statistical Analysis
Statistical analyses were performed using SPPSS statistical software version 18.0 (SPSS, Chicago, IL, USA) and Prism version 5.0 (GraphPad Software, San Diego, CA, USA). The association between EPHB3 positivity and clinico-pathological characteristics were assessed using Fisher’s exact test or Pearson’s chi-square test. Between-group comparisons of the real-time PCR data were performed using Student’s t-test. The correlations between EPHB3 and candidate cancer stem cell markers were evaluated using the Spearman correlation test. Survival analysis was performed using the Kaplan–Meier and log-rank method. To identify independent prognostic factors, we performed multivariate analyses using the Cox proportional hazards model. A p-value < 0.05 was considered statistically significant.
In this study, we investigated the expression profile of EPHB3 in various precancerous lesions and in human CRCs. Although EPHB3 expression in CRCs has been reported by previous studies, to our knowledge, this is the most comprehensive study to demonstrate the alterations of EPHB3 expression throughout CRC development and progression. Using a large cohort of CRC patients for whom we had tumoral histopathological and molecular data, we found that EPHB3 expression is associated with improved clinical outcomes. This is consistent with its role as a tumor suppressor, controlling cellular positioning and restricting tumor cell motility [9
In accordance with its role as a stem cell-related gene in the intestinal crypts, the expression of EPHB3
mRNA positively correlated with multiple intestinal stem cell (ISC) markers including EPHB2, OLFM4,
D), but not with LGR5
. In addition, EPHB3 protein expression closely correlated with that of its homologue, EPHB2 (Figure 6
B). These results may imply that close associations among ISC signature genes represent a stem cell hierarchy that is maintained during colorectal carcinogenesis. As CD44
only showed a positive correlation with EPHB3
among the candidate CSC markers we examined, it would be interesting to explore the interplay between CD44 and EPHB3 and its effect on cancer stem cell biology in CRCs in future studies.
Different molecular pathways contribute to the development of CRCs including chromosomal instability (CIN), MSI, and CIMP. MSI-high CRCs usually occur in the proximal colon. They have a high BRAF
mutation rate, and they are characterized by lymphocytic infiltration, mucin secretion, and poor differentiation [26
]. Interestingly, EPHB3 positivity was significantly higher in MSI-high CRCs than in MSI-low or negative ones (Table 2
). We also found that mucin production and lymphocytic infiltration was significantly higher in EPHB3-positive CRCs, and although it did not reach statistical significance, EPHB3-positive CRCs appeared more frequently in the proximal colon than in the distal (Table 1
). These findings suggest that the MSI-high phenotype may have an impact on EPHB3 expression. Inconsistent with this hypothesis, we found no association between EPHB3 and BRAF mutations, and lower EPHB3 positivity in poorly differentiated CRCs, which makes it more complicated to characterize the link between MSI and EPHB3 in CRCs.
In the normal small and large intestines, EPHB3-positive cells were restricted to the crypt bases, supporting the notion that EPHB3 may be an ISC marker. In HPs and SSAs, EPHB3 was expressed at the base of the crypts, whereas in TSAs and TAs, EPHB3 expression was no longer confined to the crypt bases. Notably, in TSAs, EPHB3 expression was frequently observed in ECF as spots in which a group of cells with stem cell-like phenotypes exist and expand. Compared with other lesions, TAs exhibited a much stronger, diffuse EPHB3 expression. This may be because most conventional TAs develop through upregulated Wnt signaling due to an APC
gene mutation, and EPHB3 is a direct target of Wnt signaling [7
]. However, when comparing the expression of EPHB3 and nuclear β-catenin, which reflects aberrant Wnt activation, in CRCs, no significant association was observed. The independent functioning of the EPHB3 and Wnt signaling pathways in CRCs may be due to epigenetic regulation or the involvement of other pathways. The transcriptional enhancement of EPHB3 is influenced by the intestinal stem-cell regulator, ASCL2, MAP kinase, and Notch signaling, in addition to Wnt/ β-catenin signaling. In fact, elevated Notch activity in CRCs leads to enhancer dysfunction and EPHB3 silencing [26
]. Additionally, in gastric cancers EZH2, a methyltransferase and the core catalytic subunit of polycomb repressive complex 2, may mediate the epigenetic suppression of EPHB3 [27
We found that enhanced EPHB3 expression in adenoma significantly decreased during the transformation to carcinoma, and it declined further when cancer cells invaded into the muscular layers. These findings indicate that EPHB3 expression increases during the early stages of tumorigenesis but is suppressed as tumors progress. This biphasic expression pattern has been reported for other intestinal stem cell markers such as LGR5 and EPHB2 [15
]. In addition, EPHB3 expression was substantially reduced in the budding cells at the invasive front of tumors. As E-cadherin is frequently downregulated in budding cells and is a crucial step that promotes tumor invasion and metastasis [29
], we investigated whether EPHB3 influences E-cadherin expression and vice versa. Notably, E-cadherin suppression by siRNAs in DLD1 cells led to the downregulation of EPHB3, but it had no effect on EPHB2 expression. Additionally, EPHB3 suppression led to the decline of E-cadherin expression. Since E-cadherin and EPHB3 are tumor suppressors, the concomitant reduction of these two proteins in budding cells at the invasive tumor front may significantly contribute to the migration of these cancer cells. Regarding the association of EPHB3 with the epithelial-to-mesenchymal transition (EMT), Ronsch et al. demonstrated that SNAIL1, an EMT regulator, silences EPHB3 by disabling a transcriptional enhancer element to facilitate the EMT in several CRC cell lines [30
]. However, whether EMT regulators are typically involved in the budding of CRCs is controversial because recent studies using immunohistochemistry and RNA in situ hybridization on human CRC samples neglected to find the expression of any EMT transcription factors by cancer cells at the invasive front of CRC tumors [19
]. Further studies are required to identify the molecular mechanisms (including signaling pathways) that govern EPHB3 silencing at the invasive tumor fronts.
To investigate the underlying mechanism of EPHB3 as an effective prognostic marker in CRC patients, we transfected DLD1 cells with an EPHB3 expression plasmid and found that overexpression of EPHB3 attenuated the growth and migration of these cells. In addition, upon EPHB3 transfection, phospho-ERK levels decreased significantly but phospho-AKT levels were not altered. In addition, cleaved caspase 3 and PARP levels increased, indicating that the MAPK pathway may be involved in the EPHB3-induced growth suppression by enhancing apoptosis, which suggests a pro-apoptotic role for EPHB3. On the other hand, in non-small cell lung cancers, EPHB3 overexpression leads to decreased AKT activity, which suppresses tumor cell migration and metastasis [13
]. These data indicate that the mechanism of EPHB3-mediated tumor suppression may differ by cancer type. Additionally, since BAX levels remained the same and BIM expression decreased only slightly upon EPHB3 transfection, it is unlikely that EPHB3-induced programmed cell death is mediated by the intrinsic apoptotic pathway and the Bcl-2 family of proteins.