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Inhibition of RAF1 kinase activity restores apicobasal polarity and impairs tumour growth in human colorectal cancer
  1. Tijana Borovski1,
  2. Thomas T Vellinga1,
  3. Jamila Laoukili1,
  4. Evan E Santo2,
  5. Szabolcs Fatrai3,
  6. Susanne van Schelven1,
  7. Andre Verheem1,
  8. Dieuwke L Marvin1,
  9. Inge Ubink1,
  10. Inne H M Borel Rinkes1,
  11. Onno Kranenburg1
  1. 1Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands
  2. 2Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York City, New York, USA
  3. 3Department of Hematology, Erasmus Medical Center, Rotterdam, The Netherlands
  1. Correspondence to Professor Onno Kranenburg, Cancer Center, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3584CX, The Netherlands; o.kranenburg{at}


Background and aim Colorectal cancer (CRC) remains one of the leading causes of cancer-related death. Novel therapeutics are urgently needed, especially for tumours with activating mutations in KRAS (∼40%). Here we investigated the role of RAF1 in CRC, as a potential, novel target.

Methods 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.

Results KD or pharmacological inhibition of RAF1 significantly decreased clone-forming and tumour-forming capacity of all CRC cultures tested, including KRAS-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.

Conclusions RAF1 plays a critical role in maintaining the transformed phenotype of CRC cells, including those with mutated KRAS. 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.


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