Liver metastasis is dependent on bidirectional interactions between cancer cells and the microenvironment of the liver. TGF-?, released from cancer cells and other cells within the liver, induces activation of hepatic stellate cells (HSCs) into tumor-promoting myofibroblasts (MFs). The long-term goal of our program is to define mechanisms governing HSC activation and develop strategies to target HSCs. In this application, we focus on E1A binding protein p300 (p300), an acetyltransferase with a poorly defined role in HSCs and amenable to inhibition by small molecules. Our Preliminary Data demonstrate that (1) p300 resides in both cytoplasm and nucleus of HSCs, (2) p300 knockdown (KD) or pharmacological inhibition inhibits nuclear translocation of SMADs and MF activation of HSCs, (3) TGF-?1 induces a cytoplasmic p300/Importin7/8/SMADs complex and a nuclear p300/SMADs/TAZ complex, leading to the expression of a fibrogenic gene set including ?-smooth muscle actin, and (4) in a tumor/HSC coimplantation mouse model, p300 KD impairs paracrine effects of HSCs on promoting tumor growth. These novel findings have led to the Central Hypothesis that p300 promotes HSC activation and a prometastatic liver microenvironment by facilitating TGF-?-induced nuclear translocation of SMADs and transcription of TGF-? target genes.
Three Specific Aims are proposed to test the hypothesis.
Aim 1 focuses on how p300 assembles a cytoplasmic p300/Importin7/8/SMADs complex to promote TGF-?-stimulated nuclear transport of SMADs. In vitro and cell- based studies, p300 mutants and a p300 inhibitor C646 will be used to test that p300 phosphorylation by TGF?1, the nuclear localization signal and acetyltransferase activity of p300 are required for TGF?1-stimulated p300/Importin7/8/SMADs interactions, SMAD nuclear transport and MF activation of HSCs.
Aim 2 studies epigenetics by which the p300/SMADs/TAZ complex regulates MF activation of HSCs. We will test the hypothesis that TGF?1 induces TAZ acetylation by p300 and suppresses TAZ ubiquitination, thereby stabilizing TAZ and the p300/SMADs/TAZ complex. Additionally, a focused study (ChIP assays) and a complementary genome-wide analyzing approach (ChIP sequencing) will be used to interrogate how the p300/SMADs/TAZ complex facilitates TGF?-induced gene transcription.
Aim 3 focuses on in vivo role of p300 for HSC activation and liver metastasis. Conditional p300 knockout mice, portal vein tumor implantation and in vivo tumor imaging technique will be used to study if p300 knockout in MF/activated-HSCs or pharmacological inhibition of p300 abrogates liver metastatic growth in mice. Our studies will yield novel mechanistic insights into MF activation of HSCs and help identify new targets and interventions to prevent and treat metastatic liver disease.
Liver metastasis is a leading cause of cancer-related death worldwide. Under cancer invasion of the liver, hepatic stellate cells (HSCs) are activated into myofibroblasts, which in turn, promote cancer cell implantation and growth in the liver. This proposal focuses on how p300 acetyltransferase promotes TGF-?1-mediated activation of HSCs into tumor-promoting myofibroblasts. The results will significantly advance our understanding of HSC biology and help identify new therapeutic targets to suppress tumor/HSC interactions and liver metastasis.
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