Volume 135, Issue 3, Pages 899-906 (September 2008) B-RafV600E Cooperates With Alternative Spliced Rac1b to Sustain Colorectal Cancer Cell Survival  Paulo Matos, Carla Oliveira, Sérgia Velho, Vânia Gonçalves, Luís Teixiera da Costa, Mary Pat Moyer, Raquel Seruca, Peter Jordan  Gastroenterology  Volume 135, Issue 3, Pages 899-906 (September 2008) DOI: 10.1053/j.gastro.2008.05.052 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Over expression of Rac1b in colorectal tumors and cell lines. (A) Expression of endogenous Rac1b in normal colon mucosa and 3 colorectal cell lines was detected by RT-PCR (top). Western blot analysis using an anti-Rac1 antibody (middle) and a CRIB-domain pull-down assay (bottom) demonstrate the amount of total vs active GTP-bound Rac1 and Rac1b in the cell lines. Their KRAS and BRAF genotypes are indicated. (B) RT-PCR analysis of Rac1b expression in representative colorectal tumors from freshly frozen (T1–T5) or paraffin-embedded tumors (T6–T7) and correlation with the indicated KRAS and BRAF genotypes. (C) Correlation between Rac1b over expression and the KRAS and BRAF genotypes in 45 primary colorectal tumors, 13 of which existed as paired tumor/mucosa samples. Expression levels of Rac1b were quantified by real-time PCR as described in Materials and Methods. Below the graph, the percentage values are given, as well as the number of positive cases vs the total tumor number in parentheses. KRAS mutations were in codons 12 or 13, and of the eleven mutated BRAF alleles nine were V600E and two were K601E, both oncogenic.12,29 Gastroenterology 2008 135, 899-906DOI: (10.1053/j.gastro.2008.05.052) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Specific interference with Rac1b and B-RafV600E expression decreases survival of colorectal tumor cells. (A) Specificity of the small interfering RNA oligonucleotides (siRNAs) for the depletion of Rac1b and mutant B-B-RafV600E. (Top) SW480 colorectal cells expressing either Myc-Rac1-wt or Myc-Rac1b-wt were transfected with either control (siCtrl) or one of 2 Rac1b-specific oligos (siRac1b′A or ′B), as indicated. (Bottom) SW480 colorectal cells expressing either GFP-B-Raf-wt or GFP-B-B-RafV600E were transfected with either control (siCtrl) or a mutant-specific siBRafVE oligo,23 as indicated. Cells were lysed after 24 hours and analyzed by Western blot with either anti-tubulin (loading control) or anti-tag (target protein levels) antibodies. Note that the 2 Rac1b siRNAs efficiently repressed Myc-Rac1b expression without affecting the Myc-Rac1 protein and that the siBRafVE oligo strongly decreased the expression of the mutant protein without depleting the wild-type B-Raf protein. (B) Specific depletion of endogenous Rac1b and B-B-RafV600E in HT29 colorectal cells. Cells were transfected with the indicated siRNAs and analyzed after 24 hours by Western blot with the indicated antibodies for the expression levels of either B-Raf or Rac1b. Endogenous Rac1b protein was depleted by approximately 70%, whereas B-B-RafV600E depletion led to a decrease of approximately 50% in the amount of endogenous B-Raf protein because HT29 cells are heterozygous for the B-B-RafV600E mutation. Transcript depletion from the mutant B-Raf allele reached 80% in HT29 cells as documented by ARMS RT-PCR (bottom panels) using the amplification of RNA Pol2 as internal reference. Note that the simultaneous knockdown of Rac1b and B-B-RafV600E reached depletion efficiencies equivalent to those of the individual knockdowns. Also, the depletion of B-B-RafV600E shows no effect on the expression level of Rac1b protein (or of its transcripts; data not shown). As further controls, the colorectal cells SW480 (do not express Rac1b) and Caco2 (express Rac1b but no B-B-RafV600E) were transfected with the indicated siRNAs and analyzed after 24 hours by Western blot with either anti-tubulin (loading control) or anti-Rac1b antibodies. (C–F) Depletion of Rac1b and B-B-RafV600E affects colorectal tumor cell viability. (C) HT29 cells were transfected with the indicated siRNAs and viable cells counted microscopically after 24 and 48 hours. (D) Representative phase contrast images taken 48 hours after treatment with the indicated siRNAs. (E) Depletion of Rac1b as in (C) and images as in (D), but in Caco-2 and SW480 colorectal tumor cells. Shown are mean values of 3 independent experiments, with ≥1000 cells counted at each time point for each sample. Bars indicate standard deviation. Note that SW480 cells, which do not express Rac1b, are not affected in their viability. Gastroenterology 2008 135, 899-906DOI: (10.1053/j.gastro.2008.05.052) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Depletion of Rac1b and B-B-RafV600E impairs cell-cycle progression and promotes apoptosis in colorectal tumor cells. (A) HT29 cells or (B) SW480 and Caco2 cells were transfected with the indicated siRNAs, pulse-labeled with bromodeoxyuridine and S-phase cells counted microscopically. In a parallel Western blot analysis, the cyclin D1 and β-tubulin (loading control) levels were determined. BrdU-positive HT29 cells decreased from 38% to 23% (1.6-fold) upon depletion of Rac1b, to 21% (1.8-fold) after depletion of B-B-RafV600E, and to 12% upon simultaneous suppression of both proteins (3.3-fold). Depletion of Rac1b in Caco2 cells produced a 1.6-fold (from 42% to 28%) decrease in BrdU incorporation, but had no effect on SW480 cells. (C) HT29 cells or (D) SW480 and Caco2 cells were transfected with the indicated siRNAs and the number of apoptotic cells determined after 48 hours with the TUNEL assay. In a parallel Western blot analysis the caspase-cleaved PARP and β-tubulin (loading control) levels were determined. Note the synergistic 7.8-fold increase in apoptosis upon combined depletion of Rac1b and B-B-RafV600E. Depletion of Rac1b also increased cell death in Caco2 cells. (E, F) Representative fluorescence images of TUNEL-stained (E) HT29 or (F) SW480 and Caco2 cells taken 48 hours after treatment with the indicated siRNAs. All graphs show mean values of 3 independent experiments, with ≥1000 cells counted at each time point for each sample. Bars indicate standard deviation. Gastroenterology 2008 135, 899-906DOI: (10.1053/j.gastro.2008.05.052) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Synergistic effect of B-B-RafV600E and Rac1b on fibroblast transformation. (A) The synergistic activities of B-B-RafV600E and Rac1b were tested in a focus formation assay following transfection of NIH 3T3 cells with the indicated GFP-tagged constructs. After cell fixation, foci were microscopically checked for the presence of a green fluorescent signal, then stained and macroscopically counted. Shown are mean values of 4 independent experiments together with photographs of representative dishes. Bars indicate standard deviation. Note the synergy when B-B-RafV600E and wild-type Rac1b are simultaneously expressed. (B) Western blot showing the expression levels of the indicated GFP-tagged proteins after transfection into NIH 3T3 cells. Gastroenterology 2008 135, 899-906DOI: (10.1053/j.gastro.2008.05.052) Copyright © 2008 AGA Institute Terms and Conditions