A high expression ratio of RhoA/RhoB is associated with the migratory and invasive properties of basal-like Breast Tumors

Abstract

Basal-like breast cancer is among the most aggressive cancers and there is still no effective targeted treatment. In order to identify new therapeutic targets, we performed mRNA-Seq on eight breast cancer cell lines. Among the genes overexpressed in basal-like tumors, we focused on the RhoA and RhoB genes, which encode small GTPases known to play a role in the actin cytoskeleton, allowing cells to migrate. qRT-PCR and Western blotting were used for expression studies. Migratory and invasive properties were analysed by wound healing and Boyden chambers assays. Stress fibers formation was evaluated by fluorescent actin labeling. Rho siRNA, small inhibitor Rhosin treatment and BRCA1 transfection were performed to study the role of Rho and BRCA1 proteins. We showed that strong expression of RhoA and low expression of RhoB was associated with the basal-like subtype of breast cancer. Decreasing RhoA expression reduced the migratory and invasive capacities of basal-like cell lines, while decreasing RhoB expression increased these capacities. Rhosin, an inhibitor of RhoA, could also reduce the migration of basal-like cell lines. Rho proteins are involved in the formation of stress fibers, a conformation of the actin cytoskeleton found in migrating cells: inhibition of RhoA expression decreased the formation of these fibers. BRCA1, a gene frequently inactivated in basal-like tumors, appears to play a role in the differential expression of RhoA and RhoB in these tumors, as the restoration of BRCA1 expression in a BRCA1-mutated basal-like cell line decreased expression of RhoA and increased expression of RhoB, resulting in reduced migratory capacity. These results suggest Rho proteins as potential therapeutic targets for basal-like and BRCA1-mutated breast cancer, as migration and acquisition of mesenchymal properties are key functional pathways in these tumors with high metastatic potential.

Keywords: BRCA1; RhoA; RhoB; basal-like breast cancer; triple negative breast cancer.

Figure 1

Rho expression in triple negative breast cancer cell lines. A. mRNA from eight breast cancer cell lines (2 luminal MCF7 and T47D, 1 non tumoral MCF10A and 5 triple negative MDA-MB-231, MDA436, HCC1937, SUM149, SUM1315) were extracted and analysed by mRNA sequencing on the sequencer GS-Flx (Roche-454). Sequence reads were aligned on the human genome (hg19) normalized in reads per kilobase per million mapped reads (RPKM). B. RhoA and RhoB expression in breast cancer cell lines was confirmed by q-RT-PCR. C. Cell proteins were extracted and Western Blot was performed using mouse monoclonal anti-RhoA (Santa Cruz) and rabbit polyclonal anti-RhoB (Santa Cruz) antibodies. Quantification of western blot was performed using ImageJ software and presented in the table under the blots.

Figure 2

Rho expression in triple negative breast tumors. A. RNA was extracted from cryopreserved tumors using AllPrep DNA/RNA kit (Qiagen). Rho expression was analysed by qRT-PCR using TaqMan gene expression inventoried assay. TN: triple negative. Since data is highly non-normal and heteroscedastic, ANOVA was performed after elimination of outliers and log-transformation. There is a statistically significant difference between the three groups as determined by one-way Welch's ANOVA (Welch's F (2, 61.3) = 29.7, p< 0.001). Games-Howell posthoc test shows that the TN cohorts 1 and 2 differ significantly from the luminal cohort (p< 0.001 and p = 0.001 respectively); the difference between the two TN cohorts is not statistically significantly different (p=0.4). B. The Cancer Genome Atlas (TCGA) data are presented as reads per kilobase per million mapped reads (RPKM) values. TNBC: triple negative breast cancers. Again since data is highly non-normal and heteroscedastic, ANOVA was performed after elimination of outliers and log-transformation. There is a statistically significant difference between the two groups as determined by one-way Welch's ANOVA (Welch's F (1, 98) = 118.6, p< 0.001).

Figure 3

Effect of Rho inhibition on migratory and invasive properties of triple negative breast cancer cells. A. MCF7, MDA-MB-231 and HCC1937 cells were transfected with siRNA and migration assay was performed with a Boyden chambers system (8-Am pore size, BD Biosciences). In each cell line, Student t test was made to compare to control siRNA transfected cells. *: p< 0.05; **: p< 0.01. B. MCF7, MDA-MB-231 and HCC1937 cells were transfected with siRNA and cell invasion assay was performed in similar conditions, with Boyden chambers precoated with Matrigel (BD Biosciences). Forty-eight hours later, cells were fixed, stained, and counted. In each cell line, Student t test was made to compare to control siRNA transfected cells. *: p< 0.05. C. MDA-MB-231 and HCC1937 cells were treated with 30 µM of DMSO solubilized Rhosin or with 0.1% DMSO (Control). A wound was made in the confluent cell monolayer by a pipette tip. The ability of cells to migrate into the cleared section was monitored during 24 h. Percentage of migration was defined by three measures of lengthwise migration. In each cell line, Student t test was made to compare to control cells. *: p< 0.05; **: p< 0.01.

Figure 4

Effect of Rho inhibition on actin cytoskeleton of MDA-MB-231 triple negative breast cancer cells. After treatment with siRNA or Rhosin, MDA-MB-231 cells were incubated with 10 µM of Lysophosphatidic Acid for one hour to stimulate the formation of stress fibers. Cells were then fixed, permeabilized and stained with 0.5 µM phalloidin. Finally, cells were mounted with DAPI-Vectashield. A. Example of stained cells. White arrows show stress fibers. B. Photographs of several fields were taken, and the ratio of the number of cells showing stress fibers to the total cell number was performed in triplicate.

Figure 5

Implication of the BRCA1 gene in RHO function in triple negative breast cancer cells. A. Rho expression in SUM1315-LXSN (BRCA1 mutated) and SUM1315-BRCA1 (BRCA1 restored) cells were analyzed by q-RT-PCR. Experiment was performed three times independently. *: p< 0.05; **: p< 0.01 B. SUM1315-LXSN (SL) and SUM1315-BRCA1 (SB) cells were treated with 30 µM of DMSO solubilized Rhosin (Rhosin) or with 0,1% DMSO (Control). A section of a confluent cell monolayer was produced by scratching with a p10 pipette tip. The ability of cells to migrate into the cleared section was monitored during 24 h. Percentage of migration was defined by three measures of lengthwise migration. Student's test between SL and SB cells: *: p< 0.05; **: p< 0.01; Student's test between rhosin treated and control SL cells: ##: p< 0.01; ###: p< 0.001. C. After treatment with Rhosin, SUM1315-LXSN (SL) and SUM1315-BRCA1 (SB) cells were incubated with 10 µM of Lysophosphatidic Acid for one hour to stimulate the formation of stress fibers. Cells were then fixed, permeabilized and stained with 0.5 μM phalloidin. Finally, cells were mounted with DAPI-Vectashield. Photographs of several fields were taken, and the mean ratio of the number of cells showing stress fibers to the total cell number was calculated. This experiment was performed three times independently. Student's test between SL and SB cells: *: p< 0.05; **: p< 0.01; student test between rhosin treated and control SL cells: ##: p< 0.01; ###: p< 0.001.