p21 interacts with Smad3 and modulates TGFb induced transcriptional activity and downstream genes involved in cell invasion It has been previously shown that cytoplasmic p21 regu lates actin cytoskeleton that through binding and inhibiting ROCK1, resulting in decreased phosphorylation of actin depolymerizing protein cofilin and increased cell migra tion in NIH3T3 fibroblasts and HeLa cells. Therefore, we examined the phosphorylation and total protein expression levels of cofilin in breast cancer cells in response to TGFb. As shown in Figure S7A, TGFb has no effect on the phosphorylation of cofilin. As cytoplasmic p21 contributes to regulate cofilin, we then examined the localization of p21 under the stimulation of TGFb. Treatment with TGFb caused accumulation of p21 in the nucleus in a time dependent manner.

This suggests that TGFb induced and p21 driven cell migration and invasion in human breast cancer cells are not mediated through the ROCK/ LIMK/cofilin pathway. Besides its function as a cell cycle regulator, p21 has also been shown to interact with multiple transcription factors to selectively inhi bit or induce expression of sets of genes involved in dis tinct biological functions, such as mitosis, DNA repair, survival and ECM components. Thus, we investi gated whether p21 could interact with the Smad pro teins to regulate the TGFb pro invasive effects. Smad/ p21 interactions were analyzed by co immunoprecipita tion studies in HEK293 and SCP2 cells co transfected with myc Smad2, myc Smad3 and flag p21.

As shown in Figure 6A, while we could not detect any ligand induced association between Smad2 and p21, we found TGFb to clearly induce complex formation between Smad3 and p21 in the two cell lines. To assess the impact of the p21/Smad3 interaction on TGFb signaling, we then examined the effect of p21 on TGFb induced Smad3 activity. As shown in Figure 6B, we found that knocking down p21 did not affect TGFb induced Smad3 phos phorylation. However, using a TGFb/Smad transcrip tional reporter construct, we found that p21 is required for TGFb induced Smad transcriptional activity. Indeed, as shown in Figure 6C, p21 gene silen cing abolished TGFb induced luciferase activity of the Smad reporter construct. Conversely, CAGA12 luc activity was markedly potentiated in SCP2 cells overex pressing p21 in response to TGFb.

These results indi cate that TGFb induces a complex formation between p21 and Smad3 and that while p21 does not affect the earlier stages of Smad3 activation, it is required for TGFb mediated Smad transcrip tional activity. We next performed gene profiling experiments in par ental and p21 deficient SCP2 cells, using transiently Carfilzomib transfected p21 siRNA as well as stably transfected p21 shRNA. Our arbitrary cutoff was set up at a minimum of two fold induction. This led us to identify multiple p21 dependent TGFb target genes, among which were selected those known to be associated with the tumor metastasis process.