Epithelial-mesenchymal transition (EMT) has been implicated in promoting breast cancer invasion and metastasis. Recent studies have shown that this important process is also responsible for the generation of breast cancer stem cells (CSCs), which are resistant to conventional therapy and contribute to tumor metastasis and relapse. Independent of these findings, mutations in BRCA1/2, TP53 and PTEN have emerged as high- penetrance susceptibility genes and are clinically relevant for the determination of breast cancer risk and prognosis. Among these genes, PTEN inactivation promotes both EMT and CSC enrichment. However, the underlying molecular mechanisms behind these activities are largely unknown. In our genetic analysis of PTEN functions in epithelial morphogenesis, we have identified Abi1, a key component of the WAVE regulatory complex (WRC), as a new substrate for PTEN. Protein interaction studies have further demonstrated a novel interaction between Abi1 and Smarcc1 of the SWI/SNF chromatin remodeling complex, which links PTEN to SWI/SNF-mediated epigenetic regulation of cancer. PTEN dephosphorylates Abi1 and consequently downregulates Abi1 and Smarcc1. Overexpression of Abi1 or Smarcc1 in human mammary epithelial cells promotes EMT and enhances stem cell activity. Based on these findings, we hypothesize that PTEN loss induces EMT and CSC enrichment through Abi1 and Smarcc1 in breast cancer. To test this hypothesis, we propose two specific aims. In Aim 1, the role of Abi1 in PTEN loss-induced EMT, CSC enrichment, tumorigenicity and metastasis will be analyzed by gain- and loss-of-function approaches in breast cancer cells and a mouse xenograft mammary fat pad model. In addition, we will delete one or both Abi1 alleles in PTEN- null mouse mammary glands to determine if Abi1 elevation mediates PTEN loss-induced breast tumor formation. To elucidate the signaling events downstream of PTEN-Abi1, we will focus on the interaction of Abi1 with WRC components and Abl kinase and their role in the EMT and CSC enrichment. In Aim 2, we will map the binding sites that mediate the Abi1-Smarcc1 interaction by site-directed mutagenesis. We will then analyze the functional consequences of the Abi1-Smarcc1 interaction in SWI/SNF complex assembly and chromatin remodeling activity at the promoters of the EMT-inducing transcription factors. The impact on EMT and CSC activity will be analyzed both in breast cancer cells and mouse xenografts. Furthermore, we will test the hypothesis that elevated Smarcc1 competes with Smarcc2 in the SWI/SNF complex and thus induces EMT and CSC activity by overexpressing Smarcc2 and knocking down Smarcc1 in PTEN-deficient breast cancer cells. Successful completion of the proposed studies will lay the foundation for developing therapeutic strategies targeting EMT and CSCs in PTEN-deficient breast cancer. It will also shed light on the new PTEN- Abi1-Smarcc1 pathway.
|Effective start/end date||4/1/16 → 3/31/18|
- National Institutes of Health (NIH)