American Association for Cancer Research
00085472can161921-sup-168924_1_supp_3778423_bb14xb.doc (12.78 MB)

Supplementary Figure 1 through 7 and Supplementary Tables 1 through 6 and 8 through 14, and Supplementary Materials and Methods from Somatic Ephrin Receptor Mutations Are Associated with Metastasis in Primary Colorectal Cancer

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posted on 2023-03-31, 01:01 authored by Lucy Mathot, Snehangshu Kundu, Viktor Ljungström, Jessica Svedlund, Lotte Moens, Tom Adlerteg, Elin Falk-Sörqvist, Verónica Rendo, Claudia Bellomo, Markus Mayrhofer, Carme Cortina, Magnus Sundström, Patrick Micke, Johan Botling, Anders Isaksson, Aristidis Moustakas, Eduard Batlle, Helgi Birgisson, Bengt Glimelius, Mats Nilsson, Tobias Sjöblom

Supplementary Fig. 1. Luciferase reporter assay reveals downregulation of BMP pathway activity upon overexpression of mutant BMPR2 Supplementary Fig. 2. Genes found in the largest number of significant outcomes when comparing mutation rate between metastatic and non-metastatic patients Supplementary Fig. 3. In situ mutation detection in FF colon tumour sections Supplementary Fig. 4. Expression levels of EPHB1 in DLD-1 cell lines overexpressing EPHB1 and its four mutant versions Supplementary Fig. 5. EPHB2 interacts with ephrinB1 ligand used as a positive control for the compartmentalisation experiment Supplementary Fig. 6. Detections of four non-synonymous mutations at the transcript level by Sanger sequencing and real time growth analysis of cell lines used in the compartmentalisation experiment Supplementary Fig. 7. The EPHB1 kinase inhibitor dasatinib abrogates the compartmentalisation phenotype of EPHB1 cells co-cultured with ephrinB1 ligand expressing cells Supplementary Table 1. Probe sequences for in situ mutation detection Supplementary Table 2. Probe performance for in situ mutation detection Supplementary Table 3. LNA primers for reverse transcription in situ Supplementary Table 4. Fluorescently labelled detection probes for RCPs Supplementary Table 5. Genes and corresponding length of the coding regions included in this study Supplementary Table 6. Patient-matched tumour and normal samples Supplementary Table 8. Comparison of pathway mutation rates in metastatic vs non-metastatic samples Supplementary Table 9. Functional annotation of mutated genes unique to metastatic patients Supplementary Table 10. Somatic mutations in Ephrin receptor genes Supplementary Table 11. Mean read depth of Eph receptor tyrosine kinases Supplementary Table 12. Prediction scores for amino acid conservation and effect of point mutations Supplementary Table 13. Mutation counts for Eph receptor mutations in situ Supplementary Table 14. Primer sequences for Sanger sequencing mutant constructs. Supplementary Materials and Methods


Swedish Cancer Foundation

Uppsala-Umeå Comprehensive Cancer Consortium

European Union's Seventh Framework Programme

Swedish Foundation for Strategic Research

VINNOVA (Companion diagnostic initiative) and the Innovative Medicines Initiative (IMI) Joint Undertaking



The contribution of somatic mutations to metastasis of colorectal cancers is currently unknown. To find mutations involved in the colorectal cancer metastatic process, we performed deep mutational analysis of 676 genes in 107 stages II to IV primary colorectal cancer, of which half had metastasized. The mutation prevalence in the ephrin (EPH) family of tyrosine kinase receptors was 10-fold higher in primary tumors of metastatic colorectal than in nonmetastatic cases and preferentially occurred in stage III and IV tumors. Mutational analyses in situ confirmed expression of mutant EPH receptors. To enable functional studies of EPHB1 mutations, we demonstrated that DLD-1 colorectal cancer cells expressing EPHB1 form aggregates upon coculture with ephrin B1 expressing cells. When mutations in the fibronectin type III and kinase domains of EPHB1 were compared with wild-type EPHB1 in DLD-1 colorectal cancer cells, they decreased ephrin B1–induced compartmentalization. These observations provide a mechanistic link between EPHB receptor mutations and metastasis in colorectal cancer. Cancer Res; 77(7); 1730–40. ©2017 AACR.

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