Role of EPHA3 in colorectal cancer Elena Andretta PhD Thesis, 2015

Role of EPHA3 in colorectal cancer
Elena Andretta
PhD Thesis, 2015

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Role of EPHA3 in colorectal cancer
A Thesis by
Elena Andretta
Submitted to the Faculty of Biosciences
For the Degree of Doctor of Philosophy (Science)
PhD programme in Biochemistry, Molecular Biology and Biomedicine
University Autonoma of Barcelona
Thesis realized in
Molecular Oncology Department
Centre for Molecular Biology and Biochemistry Investigation in Nanomedicine
(CIBBIM-Nanomedicine)-Research Institute of Vall d’Hebron
DIRECTOR TUTOR
Dr. Diego Arango Del Corro Dr. Simó Schwartz Navarro
PhD Student, September 2015
Elena Andretta

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To Umberto and Pia
“Every refutation should be regarded as a great
success; not merely a success of the scientist who
refuted the theory, but also of the scientist who
created the refuted theory and who thus in the first
instance suggested, if only indirectly, the refuting
experiment”. Karl Popper, 1902-1994.

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Index
Introduction………………………………………………………………………………………………………………………………………………………..6
1. ANATOMY AND HYSTOLOGY OF HUMAN INTESTINE .................................................................................... 7
1.1. ANATOMY .......................................................................................................................................................... 8
1.2. HYSTOLOGY ........................................................................................................................................................ 9
2. INTESTINAL CRYPTS: DINAMICS IN HOMEOSTASIS, REGENERETION AND CELLULAR LINEAGE............ 14
2.1 SELF-RENEWAL IN THE INTESTINAL EPITHELIUM ....................................................................................................... 14
2.2 INTESTINAL STEM CELL NICHE ............................................................................................................................... 14
2.3 HOW ARE CRYPTS AND VILLI MADE? SIGNALING AND MORPHOGENETIC PATHWAYS ....................................................... 17
3. CANCER ................................................................................................................................................................ 24
3.1 SIGNALING PATHWAYS IN TUMORS ....................................................................................................................... 24
4. COLORECTAL CANCER ....................................................................................................................................... 27
4.1 CHARACTERISTICS AND CLINICAL CLASSIFICATION ..................................................................................................... 28
4.2 MULTISTEP GENETIC MODEL OF COLORECTAL CANCER : “VOGELGRAM” ...................................................................... 32
4.3 THE DISTINCT PATHS OF COLORECTAL CANCER ......................................................................................................... 33
4.4 SIGNALING PATHWYS AND GENE MUTATIONS IN COLORECTAL CANCER ........................................................................ 35
4.5 COLORECTAL CANCER TREATMENT ........................................................................................................................ 43
5. EPH, AN INTRUIGUING RECEPTOR SUBFAMILY: FROM EMBRIOGENESIS TO TUMOROGENESIS ......... 46
5.1 THE LARGEST RECEPTOR TYROSINE KINASE SUBFAMILY ............................................................................................ 46
5.2 DOMAIN ORGANIZATION OF EPH AND EPHRIN PROTEIN .......................................................................................... 48
5.3 EPH RECEPTOR ACTIVATION ................................................................................................................................ 50
5.4 EPH RECEPTORS AND CELLULAR MECHANISMS: REPULSION VERSUS ADHESION, WHAT DEFINES THE CHOICE? ..................... 51
5.5 EPH RECEPTORS SIGNALING ................................................................................................................................ 55
5.6 CELL POSITIONING, CELL SORTING AND BOUNDARY FORMATION AS CENTRAL MECHANISMS UNDERLYING EPH FUNCTION .... 60
5.7 EPHB/EPHRIN-B SIGNALING IN THE INTESTINAL EPITHELIUM .................................................................................... 61
5.8 DO EPH RECEPTORS PROMOTE OR SUPPRESS TUMORS? ........................................................................................... 65
5.9 EPHB/EPHRIN-B SIGNALING IN COLORECTAL CANCER .............................................................................................. 67
5.10 EPHA3 RECEPTOR ............................................................................................................................................. 71
5.11 EPHA3 IN CANCER ............................................................................................................................................ 71
Aims of studies…………………………………………………………………………………………………………………………………………………78
Material and Methods……………………………………………………………………………………………………………………………………79
Results…………………………………………………………………………………………………………………………………………………………………86
1. GENERATION OF COLON CANCER CELL LINES WITH INDUCIBLE EPHA3 ACTIVITY ..................................................................... 87
2. EPHA3 DOES NOT REGULATE THE GROWTH OF COLON CANCER CELLS .................................................................................. 90
3. CHARACTERIZATION OF THE ROLE OF EPHA3 IN INTESTINAL TUMORIGENESIS USING A KNOCKOUT MOUSE................................. 94
4. REINTRODUCTION OF EPHA3 INTO DEFICIENT COLON CANCER CELLS DOES NOT AFFECT THEIR METASTATIC POTENTIAL ................ 99
5. SURVIVAL OF COLORECTAL CANCER PATIENTS AS A FUNCTION OF EPHA3 TUMOR LEVELS ..................................................... 103

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Discussion………………………………………………………………………………………………………………………………………………………..107
Conclusions……………………………………………………………………………………………………………………………………………………..116
Bibliography…………………………………………………………………………………………………………………………………………………….117
Aknowledgments…………………………………………………………………………………………………………………………………………..136
INDEX OF FIGURES
FIG.1. ANATOMY OF HUMAN GASTROINTESTINAL SYSTEM. ....................................................................................................... 7
FIG.2. ANATOMY AND HISTOLOGY OF THE HUMAN INTESTINE.. ............................................................................................... 13
FIG.3. EPITHELIAL SELF-RENEWAL IN THE INTESTINE. .............................................................................................................. 15
FIG.4. SIGNALING PATHWAYS IN THE SMALL INTESTINE. ......................................................................................................... 18
FIG.5.WNT SIGNALING MECHANISMS ................................................................................................................................. 21
FIG.6. CANCER CELL SIGNALING PATHWAYS AND THE CELLULAR PROCESSES THAT THEY REGULATE. ................................................. 26
FIG.7. AGE-STANDARDISED (WORLD POPULATION) EU MALE AND FEMALE CANCER MORTALITY RATE TRENDS.……………… ................ 27
FIG.8. THE GROWTH FROM POLYP TO METASTATIC TUMOR. .................................................................................................... 29
FIG.9. “VOGELGRAM”. .................................................................................................................................................... 32
FIG.10. ROLE OF Β-CATENIN/TCF IN THE EARLY STAGES OF INTESTINAL TUMORIGENESIS. ............................................................ 37
FIG.11. DIAGRAM OF THE TGF-Β SIGNALING PATHWAY. ........................................................................................................ 39
FIG.12. KRAS/BRAF AND PI3K/AKT PATHWAYS ............................................................................................................... 41
FIG.13. SCHEMATIC REPRESENTATION OF THE MOST FREQUENT MOLECULAR PATHWAYS IN COLORECTAL CANCER, PROGNOSIS AND
TREATMENT RESPONSE. ......................................................................................................................................... 45
FIG.14. THE SCIENTIFIC INTEREST IN EPH-EPHRIN BIOLOGY IS MEASURED BY THE NUMBER OF PUBLICATIONS.. ................................ 47
FIG.15. BINDING INTERACTIONS AND SEQUENCE RELATIONSHIPS OF EPH RECEPTORS AND EPHRINS. ............................................. 47
FIG.16. GENERAL FEATURES OF EPH RECEPTORS AND EPHRINS. ............................................................................................... 49
FIG.17. EPH RECEPTOR ACTIVATION. ................................................................................................................................. 49
FIG.18. REGULATION OF EPH-EPHRIN SIGNALING TO DETERMINE REPULSION OR ADHESION.. ....................................................... 52
FIG.19. EFFECTS OF EPHRIN-A AND EPHA EXPRESSION WITHIN THE CELL MEMBRANE. ................................................................ 54
FIG.20. EXAMPLE OF EPH-EPHRIN COMPLEX DISRUPTION AND CELL DETACHMENT.. ................................................................... 54
FIG.21. EPH RECEPTORS AND SIGNALING CONCEPTS.. ........................................................................................................... 57
FIG.22. EPH RECEPTORS AND TISSUE PATTERNING.. .............................................................................................................. 62
FIG.23. MECHANISMS AND SITES OF ACTION OF EPHRINS AND EPH RECEPTORS DURING REMODELING OF THE VASCULATURE IN MICE. .62
FIG.24. EXPRESSION PATTERN OF EPHB2, EPHB3, AND EPHRIN-B1 IN THE SMALL INTESTINE OF NEONATAL AND ADULT MICE. .......... 64
FIG.25. HYPOTHETICAL ROLE OF THE EPHA2/EPHRINA1 SYSTEM IN SOLID TUMOR CELLS.. .......................................................... 66
FIG.26. SIGNALING OF EPHB4 RECEPTOR IN MAMMARY EPITHELIAL AND BREAST CANCER CELLS.. ................................................. 66
FIG.27. EPHB–EPHRINB INTERACTIONS DURING CRC PROGRESSION. ...................................................................................... 69
FIG.28. EPHB4 AS A PROGNOSTIC MARKER IN COLORECTAL CANCER.. ...................................................................................... 70
FIG.29. EPHB4 INACTIVATION RESULTS IN SHORTER SURVIVAL OF APCMIN/+ MICE.. ...................................................................... 70
FIG.30. EXAMINATION OF WILD TYPE AND EPHA3−/− P0 HEARTS IN MICE.. ............................................................................... 72
FIG.31. LOCATION OF THE CANCER SOMATIC MUTATIONS STUDIED IN EPHA3 DOMAIN STRUCTURE.. ............................................ 76
FIG.32. EPHA3 MUTATIONS REPORTED IN COLORECTAL TUMORS............................................................................................ 77
FIG.33. EPHA3 AND EPHRIN A5 LEVELS IN COLORECTAL CANCER CELL LINES. ............................................................................ 88
FIG.34. INDUCIBLE EPHA3 ACTIVITY IN COLON CANCER CELL LINES. ......................................................................................... 89
FIG.35. EFFECTS OF EPHA3 OVEREXPRESSION ON THE GROWTH OF COLON CANCER CELLS IN VITRO. ............................................. 91
FIG.36. (A, B) EFFECTS OF EPHA3 OVEREXPRESSION ON THE TUMOR GROWTH IN VIVO. (C, D) EPHA3 LEVELS IN XENOGRAFTS OF
COLON CANCER CELL LINES. ..................................................................................................................................... 93