Inhibition of RAF1 kinase activity restores apicobasal polarity and impairs tumour growth in human colorectal cancer.
Colorectal cancer (CRC) remains one of the leading causes of cancer-related death. Novel therapeutics are urgently needed, especially for tumours with activating mutations inKRAS(∼40%). Here we investigated the role of RAF1 in CRC, as a potential, novel target.



Colonosphere cultures were established from human tumour specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. The role of RAF1 was tested by generating knockdowns (KDs) using three independent shRNA constructs or by using RAF1-kinase inhibitor GW5074. Clone-initiating and tumour-initiating capacities were assessed by single-cell cloning and injecting CRC cells into immune-deficient mice. Expression of tight junction (TJ) proteins, localisation of polarity proteins and activation of MEK-ERK pathway was analysed by western blot, immunohistochemistry and immunofluorescence.



KD or pharmacological inhibition of RAF1 significantly decreased clone-forming and tumour-forming capacity of all CRC cultures tested, includingKRAS-mutants. This was not due to cytotoxicity but, at least in part, to differentiation of tumour cells into goblet-like cells. Inhibition of RAF1-kinase activity restored apicobasal polarity and the formation of TJs in vitro and in vivo,without reducing MEK-ERK phosphorylation. MEK-inhibition failed to restore polarity and TJs. Moreover, RAF1-impaired tumours were characterised by normalised tissue architecture.



RAF1 plays a critical role in maintaining the transformed phenotype of CRC cells, including those with mutatedKRAS. The effects of RAF1 are kinase-dependent, but MEK-independent. Despite the lack of activating mutations in RAF1, its kinase domain is an attractive therapeutic target for CRC.
Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands.
Tijana
T
Borovski
Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands.
Thomas T
TT
Vellinga
Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands.
Jamila
J
Laoukili
Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York City, New York, USA.
Evan E
EE
Santo
Department of Hematology, Erasmus Medical Center, Rotterdam, The Netherlands.
Szabolcs
S
Fatrai
Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands.
Susanne
S
van Schelven
Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands.
Andre
A
Verheem
Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands.
Dieuwke L
DL
Marvin
Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands.
Inge
I
Ubink
Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands.
Inne H M
IHM
Borel Rinkes
Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands.
Onno
O
Kranenburg
D000230:Adenocarcinoma / Q000188:drug therapy / Q000235:genetics*; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D016764:Cell Polarity / Q000187:drug effects / Q000235:genetics; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics*; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D009368:Neoplasm Transplantation; D010636:Phenols / Q000494:pharmacology; D010766:Phosphorylation; D061251:Primary Cell Culture; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors* / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D019108:Tight Junctions; D014407:Tumor Cells, Cultured
D007211:Indoles; D010636:Phenols; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D019908:Proto-Oncogene Proteins c-raf; D020930:MAP Kinase Kinase Kinases; C489251:5-iodo-3-((3,5-dibromo-4-hydroxyphenyl)methylene)-2-indolinone
ACTIN CYTOSKELETON; CELL ADHESION; CELL SIGNALLING; COLORECTAL CANCER