Cooperative publication accepted in Cancer Research

article accepted in Cancer Research:

B-Raf inhibitors induce epithelial differentiation in BRAF-mutant colorectal cancer cells.

Herr R ¹, Köhler M ¹, Andrlova H ², Weinberg F ³, Möller Y ⁴, Halbach S ⁵, Lutz L ⁶, Mastroianni J ⁷, Klose M ⁸, Bittermann N ⁹, Kowar S ⁸, Zeiser R ¹⁰, Olayioye MA ⁴, Lassmann S ¹¹, Busch H ¹², Boerries M ¹², Brummer T ¹³.

¹ZBSA, University of Freiburg.
²Department of Hematology and Oncology, University Medical Center.
³Institute of Molecular Medicine and Cell Research, Albert-Ludiwgs-University Freiburg.
⁴Institute of Cell Biology and Immunology, University of Stuttgart.
⁵Biology, Institute of Molecular Medicine and Cell Research (IMMZ).
⁶University Medical Centre, Department of Pathology, Albert-Ludwigs-University.
⁷University Medical Centre, Department of Hematology and Oncology, Albert-Ludwigs-University.
⁸Institute of Molecular Medicine, Albert-Ludwigs-University.
⁹Institute of Clinical Pathology, University Medical Centre.
¹⁰Hematology/Oncology, University of Freiburg Medical Center.
¹¹Institute of Surgical Pathology, University Medical Center.
¹²Institute of Molecular Medicie, Albert-Ludwigs-University Freiburg.
¹³Institute of Molecular Medicine and Cell Research, University of Freiburg tilman.brummer@zbsa.de.

Abstract

BRAF mutations are associated with aggressive, less-differentiated and therapy-resistant colorectal carcinoma (CRC). However, the underlying mechanisms for these correlations remain unknown. To understand how oncogenic B-Raf contributes to carcinogenesis, in particular to aspects other than cellular proliferation and survival, we generated three isogenic human CRC cell line models in which we can dynamically modulate the expression of the B-RafV600E oncoprotein. Doxycyclin-inducible knockdown of endogenous B-RafV600E decreases cellular motility and invasion in conventional and three-dimensional (3D) culture, while it promotes cell-cell contacts and induces various hallmarks of differentiated epithelia. Importantly, all these effects are recapitulated by B-Raf (PLX4720, vemurafenib, dabrafenib) or MEK inhibitors (trametinib). Surprisingly, loss of B-RafV600E in HT29 xenografts does not only stall tumor growth, but also induces glandular structures with marked expression of Cdx-2, a tumor suppressor and master transcription factor of intestinal differentiation. By performing the first transcriptome profiles of PLX4720-treated 3D cultures of HT29 and Colo-205 cells, we identify several upregulated genes linked to epithelial differentiation and effector functions, such as claudin-1, a Cdx-2 target gene encoding a critical tight junction component. Thereby, we provide a mechanism for the clinically observed correlation between mutant BRAF and the loss of Cdx-2 and claudin-1. PLX4720 also suppressed several metastasis-associated transcripts that have not been implicated as targets, effectors or potential biomarkers of oncogenic B-Raf signaling so far. Together, we identify a novel facet of clinically applied B-Raf or MEK inhibitors by showing that they promote cellular adhesion and differentiation of CRC cells.