Trihydrophobin 1 Interacting Protein As Well As The Relationship Between The Sensitivity Of The Trihydrophobin 1 Breast Cancer Cells To Chemotherapy Drugs Research

Human Papilloma virus E6-associated protein (E6-AP), which is known as an E3ubiquitin ligase, mediates ubiquitination and subsequent degradation of a series of cellularproteins. In this paper, we identify here tfihydrophobin 1 (TH1), an integral subunit of thehuman negative transcription elongation factor (NELF) complex, as a novel E6-APinteraction protein and a target of E6-APmediated degradation. Overexpression of E6-APresults in degradation of TH1 in a dose-dependent manner, whereas knock-down ofendogenous E6-AP elevates the TH1 protein level. TH1 protein turnover is substantiallyfaster, compared to controls, in cells that overexpressed E6-AP. Wild-type E6-APpromotes the ubiquitination of TH1, while a catalytically inactive point mutant of E6-APabolishes its ubiquitination. Furthermore, in vitro ubiquitination assay also demonstratesthat TH1 can be ubiquitinated by E6-AP. The degradation is blocked by treatment withproteasome inhibitor MG132. Herein, we provide strong evidence that TH1 is a specificsubstrate that is targeted for degradation through E6-AP-catalyzed polyubiquitination. The transcriptional activity of the androgen receptor (AR) modulated by positive ornegative regulators plays critical roles in controlling the growth and survival of prostatecancer cells. In this study, we report that TH1 is a potent transcriptional repressor tosuppress AR activity. Knock-down of TH1 expression augment AR transcriptionalactivation. The attenuation of AR activity by TH1 is in a physical interaction dependentmanner, and TH1 N-terminal LXXLL motif is indispensable. TH1 can interact with bothtransactivation domain and Ligand binding domain of AR, and interferes withintramolecular interaction, hence decreases the stability of AR. Using chromatinimmunoprecipitation (ChIP) analyses, we show that TH1 dynamically associates with ARat the active androgen-responsive prostate-specific antigen (PSA) promoter in LNCaPcells. In agreement with these findings, TH1 overexpression blocks the PSA geneexpression in LNCaP cells. Taken together, these results indicate that TH1 is a novelregulator to control the duration and magnitude of AR transcriptional activation, and maybe directly involved in AR-related developmental, physiological, and pathologicalprocesses. Cancer develops when the balance between cell proliferation and cell death isdisrupted, and the ensuing aberrant proliferation leads to tumor growth. We report in thispaper that depletion of TH1 by small hairpin RNA sensitizes the breast cancer cell lines tochemotherapy drugs induced apoptosis. Furthermore, knock-down of TH1 expression cancause cell cycle G2/M phase arrest, inhibit the proliferation of two human breast cancercell lines: estrogen-dependent, ER-positive T47D and estrogen-independent, ER-negativeMDA-MB-231. We also determined the phenotypic effects of TH1 knock-downT47D-derived tumors and control T47D-derived tumors in nude mice. TH1 knock-downT47D-derived tumors showed 60% and 70% reduction in tumor volume and weightrespectively. As a proliferation inhibitor, p21 expression is elevated in TH1 knock-downversus parental control cells. Further study indicates that over-expression of TH1 proteininhibits the promoter activity of p21, hence reduce both mRNA and protein level of p21.Knock-down of p21 expression using siRNA in TH1 knock-down cells counteracts cellcycle G2/M phase arrest and the chemotherapy drugs induced apoptosis. These resultssuggest that TH1 may functions as a novel regulator in chemotherapy drugs inducedapoptosis by regulating the expression of p21 in breast cancer cell lines.