Rac1b co-expression abrogates B-Raf-V600E- induced SA-β-gal accumulation
PT-Rac1b
endo-Rac1b
0 24 48 72 Time (h)
0.25μM Shield
NT 96
-Rac1b
NCM460
HT
29
Co
11
5
PT
BR
afV
E
PT
-Rac
1b
PT
-Rac
1b
+P
T-B
Ra
fVE
PT
-Ctr
l
0.25μM Shield 72 h
-Tubulin
-B-Raf
-Rac1b
B
(A) NCM460 cells were transfected with inducible ProteoTuner (pT)- Rac1b construct and Rac1b expression was induced for 96 h. Cells maintained Rac1b expression levels comparable to those of the cancer cell lines HT29 and Co115 for 72 h. (B) Western blot showing that individual or combined expression of PT-B-Raf-V600E and PT-Rac1b in NCM460 cells reached equivalent fusion protein levels after 72 h of induction with Shield. (C) Senescence-associated β-galactosidase (SA-β-gal) activity observed in NCM460 cells under the transfection conditions described in (B). Note that co-expression of Rac1b abrogates the B-Raf-V600E induced senescence activity. Statistically significant differences (p<0.05) of data from Mock versus BRafVE are indicated as ** and for BRafVE versus BRafVE+Rac1b as ¥.
0
1
2
3
4
Mock Rac1b BRAF VE BRAF VE + Rac1b
Fo
ld t
o c
on
tro
l
**
¥
NCM460
A
C
Expression of B-Raf-V600E in non-transformed colonocytes induces
cellular senescence but does not alter Rac1b levels
0
1
2
3
4
5
Vehicle 0,25μM Shield
pT-BRaf V600E
Fold
to
co
ntr
ol (
ctrl
)
12 h 36 h 70 h
0.25μM Shield
< pT-BRaf VE
< endo-BRaf
-Rac1b
-Tubulin
-BRaf
HT29 (MSS) Co115 (MSI) NCM460 NCM460
A
B
(A) Colon cancer cells HT29 and Co115, transfected with either B-Raf-V600E- specific or control siRNA oligonucleotides. Following 48 h, the resulting expression levels of B-Raf and Rac1b proteins were analyzed by Western blot. Note that specific reduction in the accumulation of the oncogenic B-Raf variant had no effect on Rac1b levels. NCM460 transfected with either a GFP-B-Raf-V600E vector for 48 h or with an inducible ProteoTuner (pT)-B-Raf-V600E fusion protein. Again, no changes in Rac1b protein levels were observed after the indicated incubation periods. (B) Senescence-associated β-galactosidase (SA-β-gal) activity observed in NCM460 cells transfected with pT-B-Raf-V600E following 70 h of incubation with control solvent or Shield1 for transgene induction.
B-Raf-V600E but not Rac1b increase the expression of OIS-associated cell-cycle
inhibitor genes
1/8 1/4
1/2
NM
po
ol
INK4A (p16)
CDKN2B (p15)
ARF (p14)
Pol2
BR
afV
E
Rac
1b
Rac
1b
+
BR
afV
E
CDKN1A (p21)
Ctrl
1/1
-0,4
0,0
0,4
0,8
1,2
1,6
Ctrl Rac1b BRafVE Rac1b+ BRafVE
Log2
(fo
ld t
o c
on
tro
l) p21-Cip1
p15-INK4B
p14-ARF **
**
§
**
** ¥
1/1 1/9
1/3 HT
29
Cac
o2
Co
11
5
Mu
co
sa
NCM460
TP53
Pol2
A
B
C
(A) Semi-quantitative RT-PCR to document expression of the p53 tumor suppressor gene in NCM460 cells. RNA polymerase II transcript (Pol2) used as internal control gene. B) Semi-quantitative RT-PCR analysis of the indicated cell-cycle inhibitory genes following individual or combined expression of PT-B-Raf-V600E and PT-Rac1b in NCM460 cells for 70 h. As positive control for p16INK4A amplification, a pool of cDNA from normal colon mucosa (NM pool) was included. (C) Real-time PCR quantification of the samples described in (B). Note that Rac1b overexpression led to incomplete reversion of the B-Raf-V600E-induced upregulation of p14, p15 and p21 transcript levels. Statistically significant differences (p<0.05) of measured values compared to Rac1b samples are indicated as **, for (BRafVE+Rac1b) versus BRafVE or Rac1b alone as §, and for (BRafVE+Rac1b) versus BRafVE but not Rac1b as ¥.
Rac1b overexpression antagonizes B-Raf-V600E-induced cell cycle inhibitor expression at the protein level
NCM460 cells were transfected with the indicated pT-based inducible constructs and lysed after 24, 48 and 72 h of Shield treatment. (A) Samples were analyzed by SDS-PAGE and Western blot using the indicated antibodies. (B) Densitometric analysis of band intensities to quantify the effects of B–Raf-V600E and Rac1b at the indicated time point (dt) on the indicated concentration of cell cycle-inhibitory proteins (d[protein]). Note that the p14, p15 and p21 protein levels were significantly downregulated by Rac1b overexpression.
-1,5
-1,0
-0,5
0,0
0,5
1,0
1,5
Ctrl Rac1b BRafVE Rac1b+ BRafVE
Log2
(d
[pro
tein
]/d
t)
p21Cip Protein
24 h
48 h
72 h
-1,5
-1,0
-0,5
0,0
0,5
1,0
1,5
Ctrl Rac1b BRafVE Rac1b+ BRafVE
Log2
(d
[Pro
tein
]/d
t)
p15INK4B Protein
24 h
48 h
72 h
-2,0
-1,5
-1,0
-0,5
0,0
0,5
1,0
Ctrl Rac1b BRafVE Rac1b+ BRafVE
Log2
(d
[mR
NA
]/d
t)
p14ARF Protein
24 h
48 h
72 h
B
BR
afV
E
Rac
1b
Rac
1b
+
BR
afV
E
Ctr
l
BR
afV
E
Rac
1b
Rac
1b
+
BR
afV
E
Ctr
l
BR
afV
E
Rac
1b
Rac
1b
+
BR
afV
E
Ctr
l
24 h 48 h 72 h
-Rac1b
-Tubulin
-B-Raf
-p21Cip
-p15INK4A
-p14ARF
A
Mutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that overexpression of splice variant Rac1b occurs in around 80% of colorectal tumors carrying a mutation in BRAF. Using both BRaf-V600E-directed RNAi and overexpression we demonstrate that this mutation does not directly lead to Rac1b overexpression, indicating the latter as an independent event during tumor progression. Nonetheless, we observed that expression of oncogenic BRaf-V600E in non-transformed colonocytes (NCM460 cell line) increased both the transcript and protein levels of p14ARF, p15INK4b and p21CIP1 and led to increased expression of β-galactosidase, all indicators of OIS induction. Interestingly, whereas the protein levels of these markers were reduced upon Rac1b overexpression, the levels of their respective transcripts remained unchanged. Importantly, the co-expression of Rac1b with B-Raf-V600E reverted the OIS phenotype, reducing the expression levels of the cell-cycle inhibitors and β-galactosidase to those of control cells. These data identify increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.
-PEst-OE/BIA/UI4046/2011 - Ciência 2007 - SFRH/BPD/94322/2013
Andreia Henriques1,2 Patrícia Barros1,2 Paulo Matos2,3 and Peter Jordan1,2
1 Department of Human Genetics, National Health Institute Doutor Ricardo Jorge, Lisbon, Portugal;
2 BioFIG - Centre for Biodiversity, Functional and Integrative Genomics, Faculty of Sciences, University of Lisbon; 3 Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon.
- In a normal colonocyte model, the expression of oncogenic mutant B-Raf-V600E leads to oncogene-induced senescence.
- Co-expression of Rac1b counteracts this phenotype by suppressing the upregulation of p14ARF, p15INK4b and p21CIP.
- Overexpression of Rac1b, which correlates with mutation in BRAF in colorectal tumors, is proposed as a mechanism
to escape from OIS.
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