Tarea 3. Identificación de las variables a considerar en los escenarios y su representación

Based on the positive correlation between KSR1 and BRCA1 expressions in the clinical samples and the tumour growth inhibitory role of KSR1, we then examined the effect of KSR1 on BRCA1 protein and whether its tumour suppressive action is BRCA1-dependent.

3.2.5.1 KSR1 affects the protein abundance of BRCA1 but not the mRNA levels

We first studied the effect of KSR1 on BRCA1 mRNA expression. As shown in Figure 37A, overexpression of KSR1 in MCF7 cells had no effect on BRCA1 mRNA levels after 24 hours transient transfection. However, our western blotting showed that KSR1 overexpression increased BRCA1 protein abundance after 24 and 48 hours of transfection (Figure 37B). We then checked whether KSR1 has the same effect in other breast cancer cell lines. MDA231 and ZR75-1 cells were transfected with pCMV6-vector or pCMV6-KSR1 for 24 hours. Consistently, similar results showed that BRCA1 protein expression was elevated after KSR1 up-regulation in both MDA231 and ZR75-1 cells (Figure 37C).

We then went on to check the effect of KSR1 silencing on BRCA1 protein expression. Firstly, MCF7 cells were transfected with sicontrol (siCT) or siKSR1 for 72 hours. Western blotting was subsequently performed to measure the protein abundance of BRCA1. As shown in

Figure 37D, KSR1 silencing resulted in a decrease of BRCA1 protein. Likewise, consistent

results were observed in other two breast cancer cell lines, MDA231 and ZR75-1, which showed that BRCA1 protein was down-regulated upon knockdown of KSR1 (Figure 37E). These data support that KSR1 affects the protein abundance of BRCA1 but not its mRNA levels.

146

Figure 37 Effect of KSR1 on BRCA1 mRNA/protein levels.

A. MCF7 cells were transfected with either pCMV6 vector or pCMV6-KSR1 plasmids for 24

hours. RNA was extracted and RT-qPCR was performed to measure BRCA1 gene expression. B. MCF7 cells were transfected with either pCMV6 vector or pCMV6-KSR1 plasmids for 24 or 48 hours. The protein expression levels of BRCA1 were assessed by western blotting using indicated antibodies. C. MDA231 or ZR75-1 cells were transiently transfected with either pCMV6 or pCMV6-KSR1 plasmids for 24 hours. The protein expression levels of BRCA1 were assessed by western blotting. D. For knock-down of KSR1, MCF7 cells were transfected with either control siRNA (siCT) or siKSR1 for 72 hours. BRCA1 abundance was assessed by western blotting. E. MDA231 or ZR75-1 cells were transfected with either control siRNA (siCT) or siKSR1 for 72 hours. BRCA1 abundance was assessed by western blotting. Representative blots are shown here. Experiments were repeated at least three times.

147

3.2.5.2 KSR1 stabilizes BRCA1 through reducing BRCA1 ubiquitination

Based on our results above showing that KSR1 affects the protein expression of BRCA1, we next decided to examine whether KSR1 is involved in BRCA1 degradation. MCF7 cells were transfected with either pCMV6 vector or pCMV6-KSR1 plasmids for 24 hours followed by treatment with different proteasome inhibitors MG115 or MG132 (10 µM, 6h), which can impede BRCA1 degradation through its ubiquitination. Subsequently, BRCA1 protein expression was measured by western blotting. Our results showed that the effect of KSR1 overexpression on BRCA1 protein levels was compromised in the presence of MG115 and MG132, as the fold change of BRCA1 abundance in the absence of proteasome inhibitors was 2.4 and subsequently declined to 1.5 (MG132) or 1.6 (MG115) (Figure 38). This indicated that KSR1 might affect BRCA1 stability partly by regulating BRCA1 degradation and ubiquitination, although an alternative mechanism other than protection from ubiquitin- mediated degradation is possible.

Therefore, we sought to investigate the effect of KSR1 overexpression on BRCA1 ubiquitination. As shown in Figure 39A, a ubiquitination assay revealed that ubiquitinated BRCA1 was reduced upon overexpression of KSR1 in MCF7 cells. In order to show whether this happens in other breast cancer cells, we performed the same ubiquitination assay in MDA231 cells where similar result was also observed (Figure 39B). In summary, our data Figure 38 Effect of KSR1 overexpression on BRCA1 protein levels in the presence of proteasome inhibitors.

MCF7 cells were transiently transfected with pCMV6 vector or pCMV6-KSR1 plasmids for 24 hours, followed by treatment with MG115 or MG132 (10 µM, 6h). BRCA1 protein levels were then assessed by western blotting with specific antibodies as indicated. Representative blots are shown here. Experiments were repeated at least three times.

148

demonstrates that KSR1 appears to stabilize BRCA1 through reducing ubiquitination of BRCA1.

3.2.5.3 The inhibitory effect of KSR1 on tumour growth is through BRCA1

Based on the fact that BRCA1 is a well-established tumour suppressor in breast cancer and the described link herein between KSR1 and BRCA1, we suspected that KSR1 acts as an inhibitory factor through BRCA1 in this setting. Therefore, we examined whether the tumour suppressive action of KSR1 is dependent on BRCA1. In vitro 3D Matrigel and soft agar assays were performed to examine tumour formation after different combinations of KSR1 and BRCA1 expression levels.

As shown in Figure 40A (upper panel) and Figure 40C, 3D Matrigel assay showed that comparing with MCF7-vector cells, MCF7-KSR1 cells formed much smaller size acini (lane

1 vs 3), which is consistent to our previous finding. Furthermore, the tumour inhibitory effect

Figure 39 Effect of KSR1 on BRCA1 ubiquitination.

A. MCF7 or B. MDA231 cells were transfected with either pCMV6 vector or pCMV6-KSR1

plasmids together with Myc-Ubiquitin as indicated for 48 hours and followed by treatment with MG132 for 6 hours. Subsequently, protein samples were extracted and immunoprecipitation was then performed with IgG or specific anti-BRCA1 antibody. Western blotting was implemented to measure the Myc-tagged ubiquitinated BRCA1 using anti-Myc antibody. Representative blots are shown here. Experiments were repeated at least three times.

149

of KSR1 was abolished when BRCA1 protein was silenced, as 3D Matrigel demonstrated that the colonies recuperated in this setting (lane 3 vs 4). Similarly, in the soft agar assay (Figure

40B, lower panel; Figure 40D), our results revealed that the size of colonies derived from

MCF7-KSR1 cells was significantly smaller than the counterparts formed by MCF7-vector cells. Moreover, when BRCA1 protein was deleted, the colonies generated by MCF7-KSR1 cells were recovered (lane 3 vs 4). These results strongly support our hypothesis that the inhibitory effect of KSR1 on breast tumour growth is through BRCA1.

Figure 40 BRCA1 is required for the tumour suppressive effect of KSR1.

A. 3-D Matrigel overlay assay (upper panel). MCF7-vector or MCF7-KSR1 stable cells were

transfected with either siCT or siBRCA1 for 72 hours and then embedded in Matrigel and cultured for 6 or 7 days in 10% FCS DMEM supplemented with 2% Matrigel. Phase contrast images of representative acini structures were taken in the end of the experiment. Scale bar, 50 µm. B. Soft agar growth assay (lower panel). After 72 hours transfection with either siCT or siBRCA1, MCF7- parental or MCF7-KSR1 cells were incubated for 14 days in soft agar. Optical sections of representative colonies are shown. Scale bar, 50 µm. C. Acini or D. Colonies (~30 per condition) were measure by Axiovert 100 MetaMorph as described before and normalized to MCF7-vector siCT group. Bar chart shows data of three independent experiments, mean ± SD (**P < 0.01 and ***P < 0.01; P value determined by Student's t-test).

150

3.2.6 KSR1 regulates BRCA1 stability via elevated BARD1 abundance and