B-Raf(V600E) cooperates with alternative spliced Rac1b to sustain colorectal cancer cell survival.
OBJECTIVE
In colorectal tumors, activating BRAF mutations occur alternative to KRAS oncogenic mutations, but in cell culture possess a much lower transforming capacity. Rac1b, a hyperactive Rac1 spliced variant, is over expressed in some colorectal tumors and activates the transcription factor nuclear factor-kappaB, which initiates a transcriptional response that promotes cell cycle progression and inhibits apoptosis. The aim of this study was to determine whether Rac1b overexpression is associated with B-Raf(V600E) in primary colorectal tumors and whether a functional cooperation between these 2 proteins exists in colorectal cells with a wild-type KRAS genotype.


METHODS
Screening of BRAF and KRAS mutations by direct sequencing and Rac1b mRNA expression analysis by quantitative real-time polymerase chain reaction were conducted in 74 samples (13 normal colonic mucosa, 45 primary colorectal tumors, and 16 colorectal cancer [CRC] cell lines). RNA interference and focus formation assays were used to assess the cooperation between Rac1b and B-Raf(V600E) in cancer cell viability.


RESULTS
Rac1b overexpression and B-Raf(V600E) are significantly associated in primary colorectal tumors (P = .008) and colorectal cell lines. The simultaneous suppression of both proteins dramatically decreased CRC cell viability through impaired cell-cycle progression and increased apoptosis.


CONCLUSIONS
Our data demonstrate that Rac1b and B-Raf(V600E) functionally cooperate to sustain colorectal cell viability and suggest they constitute an alternative survival pathway to oncogenic K-Ras. These results reveal a novel molecular characteristic of colon tumors containing B-Raf mutations and should help in defining novel targets for cancer therapy.
Matos
Paulo
P
Centre of Human Genetics, National Health Institute Dr Ricardo Jorge, Lisbon, Portugal.
Oliveira
Carla
C
Velho
Sérgia
S
Gonçalves
Vânia
V
da Costa
Luís Teixiera
LT
Moyer
Mary Pat
MP
Seruca
Raquel
R
Jordan
Peter
P
D017398:Alternative Splicing; D002453:Cell Cycle / Q000235:genetics; D016923:Cell Death / Q000235:genetics; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D034622:RNA Interference; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D020830:rac1 GTP-Binding Protein / Q000235:genetics*
C107953:RAC1 protein, human; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020830:rac1 GTP-Binding Protein