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High EPHB2 mutation rate in gastric but not endometrial tumors with microsatellite instability

Abstract

The EPH/EFN family of receptor tyrosine kinases regulates cell adhesion and migration and has an important role in controlling cell positioning in the normal intestinal epithelium. Inactivation of EPHB2 has recently been shown to accelerate tumorigenesis in the colon and rectum, and we have previously demonstrated frequent frameshift mutations (41%) in an A9 coding microsatellite repeat in exon 17 of EPHB2 in colorectal tumors with microsatellite instability (MSI). In this study, we extended these analyses to extracolonic MSI cancers, and found frameshift EPHB2 mutations in 39% (25/64) of gastric tumors and 14% (8/56) of endometrial tumors. Regression analysis of these EPHB2 mutation data on the basis of our previously proposed statistical model identified EPHB2 as a selective target of frameshift mutations in MSI gastric cancers but not in MSI endometrial carcinomas. These results suggest a functional role for EPHB2 in gastric tumor progression, and emphasize the differences between the tumorigenic processes in MSI gastrointestinal and endometrial cancer.

Main

The receptor tyrosine kinase (RTK) EPHB2 is emerging as an important tumor suppressor gene in colorectal cancer (Alazzouzi et al., 2005; Batlle et al., 2005). EPHB2 is a member of the largest family of RTKs. EPH receptors and their Ephrin (EFN) ligands regulate numerous developmental processes, particularly in the vasculature and nervous system (Henkemeyer et al., 1996; Gerety et al., 1999). At the cellular level, interactions between EPH receptors and EFN ligands mediate cytoskeleton organization, cell migration and attachment to the substrate in several cell types such as neurons, endothelial cells and enterocytes (Holder and Klein, 1999; Elowe et al., 2001; Batlle et al., 2002).

Inactivation of EPHB2 has been shown to accelerate tumorigenesis initiated by APC mutations in the colon and rectum of APCMin/+ mice and reduced EPHB2 expression has been observed in colorectal tumors compared to the normal epithelium and premalignant lesions (Batlle et al., 2005; Guo et al., 2006). We have recently reported that 41% of colorectal tumors with MSI acquire somatic mutations in an A9 repeat in exon 17 of EPHB2 (Alazzouzi et al., 2005).

MSI occurs in 10–20% of colorectal, gastric and endometrial tumors as the result of defects in mismatch repair mechanism, and it is revealed as frequent insertions and deletions in mono- and dinucleotide DNA tracks known as microsatellites. Inactivation of key genes containing coding microsatellites through the accumulation of these frameshift mutations is believed to contribute to the tumorigenic process in MSI tumors. Whereas mutation in some genes such as the TAF1B and BAX occur at high frequency in colorectal, gastric and endometrial tumors, other genes, such as TGFBR2, show significant differences in the incidence of mutations depending on the tumor type (Duval et al., 2002; Woerner et al., 2003). Differences in the mutation frequency of these MSI target genes between tumor types are believed to reveal differences in the pathways that are important for the tumorigenic process in different organs, and can provide valuable insight into differential oncogenic processes (Duval et al., 2002).

In this study, we used single-strand conformation polymorphism (SSCP), direct automated sequencing and fragment analysis to investigate the incidence of mutations in the A9 track of EPHB2 (Accession number NM_017449) in a panel of 64 gastric and 56 endometrial MSI tumors.

We found frameshift mutations in the A9 mononucleotide repeat in EPHB2 in 25 of the 64 MSI gastric cases investigated (39.1%; Table 1 and Figure 1a and b). No associations were found between EPHB2 mutations and other clinicopathological factors such as age, sex, degree of differentiation, localization (proximal, middle or distal) and TNM stage. EPHB2 has been shown to be a direct transcriptional target of the transcriptional complex TCF/β-catenin, a signaling cascade that regulates proliferation and differentiation of the epithelial layer of the gastrointestinal tract (Batlle et al., 2002). Consistent with this, among 76 different human normal tissues recently investigated, EPHB2 expression was highest in the intestinal and gastric mucosa (Lugli et al., 2005). However, EPHB2 activity inhibits gastrointestinal tumor formation, and therefore inactivation of this tyrosine kinase confers tumor cells a growth advantage (Batlle et al., 2005; Guo et al., 2006). In good agreement with this, we have previously shown that 41% of the tumors of the colon and rectum displaying MSI have mutations in the A9 track of EPHB2 (Table 1) (Alazzouzi et al., 2005). Although normal epithelial cells of the stomach express high levels of EPHB2, over 85% of the gastric tumors show reduced or absent EPHB2 expression, suggesting that inactivation of this kinase in the stomach may contribute to gastric tumor formation and progression (Lugli et al., 2005). In this study, we used our previously published statistical model (Woerner et al., 2003) and performed an improved regression analysis using mutation frequencies of more than 1700 coding and non-coding repeat tracts within nearly 300 genes (Woerner, manuscript in submission), to demonstrate that the mutation frequency observed in EPHB2 in MSI gastric tumors is significantly above what could be expected by chance in an A9 repeat (Figure 2a). The high frequency of EPHB2 mutations in gastric tumors suggests that these mutations are clonally selected and confer gastric tumor cells a growth advantage.

Table 1 Mutation frequency in EPHB2 A9 track in different tumor types with microsatellite instability
Figure 1
figure1

EPHB2 mutations in gastric and endometrial MSI tumors. The A9 repeat in exon 17 of the larger EPHB2 transcript (Accession number NM_017449) and flanking genomic DNA sequence were PCR amplified in 64 gastric and 56 endometrial MSI tumor samples and mutations in this fragment were identified by fragment analysis, direct automated sequencing and/or SSCP, as described previously (Alazzouzi et al., 2005). (a and b) Direct automated sequencing of the A9 track in EPHB2 showing representative examples of gastric tumors with wild-type and mutant EPHB2, respectively. (c and d) Fragment analysis showing representative examples of endometrial tumors with wild-type and mutant EPHB2, respectively (red: tumor sample; blue: control normal sample; black: size standards; the size of the different fragments are shown in base pairs; arrows indicate the A9 → A8 mutation). The MSI status of these tumors was characterized as described previously (Yamamoto et al., 1999; Woerner et al., 2003; Bilbao et al., 2006). Informed consent for genetic analysis of the tumor sample was obtained from each patient, according to the Human Investigations and Ethical Committee – approved research proposal in the corresponding Institution.

Figure 2
figure2

EPHB2 mutation frequency in gastric and endometrial MSI tumors. Mutation frequency data of more than 1700 coding and non-coding mononucleotide repeat tracts (MNRs) from MSI gastric (a) and endometrial (b) tumors was used to perform a nonlinear regression analysis (Woerner et al., 2003). The x axis represents the MNR length and the y axis the mutation frequency. Each MNR is represented by a filled gray circle. The mean mutation frequency (black solid line) and two prediction lines (twofold standard error, gray dashed lines) are shown. Repeats outside the region delimited by the two prediction lines are likely to represent genes under positive or negative selection pressure. The A9 EPHB2 repeat was above the upper prediction line for MSI gastric cancer (a) but not for MSI endometrial cancer (b), suggesting that EPHB2 mutations confer gastric (but not endometrial) tumor cells a growth advantage. Statistical analysis was performed using the R software environment version 2.1.1 (www.R-project.org) in combination with the software library nls2 (version 2003.1) for non-linear regression (Huet et al., 2003). Basic mathematical improvements compared to the original model will be reported elsewhere (Woerner et al., manuscript in submission).

In contrast, we found mutations in the A9 track of EPHB2 in only eight of the 56 endometrial MSI tumors investigated (14.3%; Table 1 and Figure 1c and d). No associations were found between EPHB2 mutations and other clinicopathological factors such as age, grade, myometrial invasion and histological type (endometroid or non-endometroid). The incidence of EPHB2 mutations in the A9 repeat in MSI endometrial tumors is therefore substantially lower than in gastric (and colorectal) MSI tumors. Endometrial tumors exhibit an overall lower mutational load in microsatellite repeats than gastric (and colorectal) tumors (Schwartz et al., 1999; Woerner et al., 2003), thus complicating direct comparison of the respective mutation frequencies in EPHB2 (see Figure 2). However, even when these differences are taken into account, the regression model used revealed that the EPHB2 mutation incidence observed in endometrial tumors is not significantly above the frequency of mutations expected by chance in an A9 repeat (see Figure 2b), suggesting that EPHB2 mutations do not confer a growth advantage to endometrial tumor cells, but rather accumulate as the result of a stochastic process characteristic of non-selective repeats. However, functional analysis will be necessary to unequivocally confirm the potential oncogenic role of EPHB2 both in gastric and endometrial tumors.

In conclusion, in this study, we show that EPHB2 mutations are common in gastric (39.1%) but not in endometrial (14.3%) tumors displaying MSI. These results suggest that EPHB2 may have tumor suppressor activities in gastric tumors as well as in colorectal tumors, and highlight the existence of differences in the tumorigenic pathways leading to gastrointestinal and endometrial cancer.

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Acknowledgements

This study was partly funded by grants from the Spanish Fondo de Investigaciones Sanitarias (FIS PI051394) and from the FMM to DA; Deutsche Krebshilfe Grant (Kn) to JG and SW; Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to HY and KI, POCTI/SAU-OBS/56921/2004 da Fundação para a Ciência e a Tecnologia of Portugal to RS, NIH, Grant number R37 CA63585 to MP; and ICIC, Fondo de Investigaciones Sanitarias (FIS-ISCiii-RTICCC), Fundación Canaria de Investigación y Salud (FUNCIS) and Dirección General de Universidades del Gobierno de Canarias to N D-C and JC D-C, grant from Fundação para a Ciência e a Tecnologia, Portugal to AMF and Grants-in-Aid for Cancer Research and for the Third Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare of Japan to HY and KI. VD is supported by a predoctoral fellowship from the Vall d'Hebron Research Institute.

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Davalos, V., Dopeso, H., Velho, S. et al. High EPHB2 mutation rate in gastric but not endometrial tumors with microsatellite instability. Oncogene 26, 308–311 (2007). https://doi.org/10.1038/sj.onc.1209780

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Keywords

  • EPHB2
  • cancer
  • microsatellite instability
  • colorectal
  • stomach
  • endometrium

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