Overexpression of the HER2͞Neu protooncogene has been linked to the progression of breast cancer. Here we demonstrate that the growth of prostate cancer LNCaP cells can also be increased by the stable transfection of HER2͞Neu. Using AG879, a HER2͞Neu inhibitor, and PD98059, a MAP kinase inhibitor, as well as MAP kinase phosphatase-1 (MPK-1), in the transfection assay, we found that HER2͞Neu could induce prostate-specific antigen (PSA), a marker for the progression of prostate cancer, through the MAP kinase pathway at a low androgen level. Reporter assays and mammalian two-hybrid assays further suggest this HER2͞Neu-induced androgen receptor (AR) transactivation may function through the promotion of interaction between AR and AR coactivators, such as ARA70. Furthermore, we found this HER2͞Neu 3 MAP kinase 3 AR-ARAs 3 PSA pathway could not be blocked completely by hydroxyf lutamide, an antiandrogen used in the treatment of prostate cancer. Together, these data provide a novel pathway from HER2͞Neu to AR transactivation, and they may represent one of the reasons for the PSA re-elevation and hormone resistance during androgen ablation therapy in prostate cancer patients.

Whereas several apoptosis-related proteins have been linked to the antiapoptotic effects of Akt serine-threonine kinase, the search continues to explain the Akt signaling role in promoting cell survival via antiapoptotic effects. Here, we demonstrate that Akt phosphorylates the androgen receptor (AR) at Ser-210 and Ser-790. A mutation at AR Ser-210 results in the reversal of Akt-mediated suppression of AR transactivation. Activation of the phosphatidylinositol-3-OH kinase͞Akt pathway results in the suppression of AR target genes, such as p21, and the decrease of androgen͞AR-mediated apoptosis, which may involve the inhibition of interaction between AR and AR coregulators. Together, these findings provide a molecular basis for cross-talk between two signaling pathways at the level of Akt and AR-AR coregulators that may help us to better understand the roles of Akt in the androgen͞AR-mediated apoptosis.

RhoA GTPase plays a crucial role in numerous biological functions and is linked to cancer metastasis. However, the understanding of the molecular mechanism responsible for RhoA transcription is still very limited. Here we show that RhoA transcription is orchestrated by the Myc/Skp2/Miz1/p300 transcription complex. Skp2 cooperates with Myc to induce RhoA transcription by recruiting Miz1 and p300 to the RhoA promoter independently of SCF-Skp2 E3 ligase activity. Deficiency of this complex results in impairment in RhoA expression, cell migration, invasion, and breast cancer metastasis, recapitulating the phenotypes observed in RhoA knockdown, and RhoA restoration rescues the defect in cell invasion. Strikingly, the overexpression of Myc/Skp2/Miz1 complex is found in metastatic human cancers and correlated with RhoA expression. Our study provides great insight into how oncogenic Skp2 and Myc coordinate to induce RhoA transcription and establishes a novel SCF-Skp2 E3 ligase-independent function for oncogenic Skp2 in transcription and cancer metastasis.

Androgen receptor (AR) is a hormone-activated transcriptional factor that can bind to androgen response elements and that regulates the transcription of target genes via a mechanism that presumably involves cofactors. We report here the cloning of a novel AR coactivator ARA55 using a yeast two-hybrid system. ARA55 consists of 444 amino acids with the predicted molecular mass of 55 kDa and its sequence shows very high homology to mouse hic5, a TGF-␤1-inducible gene. Yeast and mammalian two-hybrid systems and co-immunoprecipitation assays all prove ARA55 can bind to AR in a liganddependent manner. Transient transfection assay in prostate cancer DU145 cells further demonstrates that ARA55 can enhance AR transcriptional activity in the presence of 1 nM dihydrotestosterone or its antagonists such as 100 nM 17␤-estradiol or 1 M hydroxyflutamide. Our data also suggest the C-terminal half of ARA55, which includes three LIM motifs, is sufficient to interact with AR. Northern blot and polymerase chain reaction quantitation showed ARA55 can be expressed differently in normal prostate and prostate tumor cells. Together, our data suggests that ARA55 may play very important roles in the progression of prostate cancer by the modulation of AR transactivation. The androgen receptor (AR)1 is a member of the steroid receptor (SR) superfamily and plays an important role in male sexual differentiation and prostate cell proliferation (1). The well conserved DNA binding domain (DBD) within AR has two zinc finger structures that are involved in DNA binding. The C-terminal region of the AR, including the hinge region and the ligand-binding domain, is responsible for the functions of dimerization and androgen binding. The N-terminal region is involved in the transcriptional activation of AR.The discovery of transcriptional interference/squelching of SRs provided the concept of the existence of transcriptional cofactors that mediate SR function (2, 3). Recently, several putative cofactors (either coactivators or corepressers) for SRs have been identified and characterized (4,5). Further studies of the interaction of SRs with these cofactors suggested that these SR-cofactor complexes play essential roles for the regulation of SRs target gene transcription by interaction with general transcription factors and the remodeling of chromatin (4, 5).The in vivo significance of these cofactors and their relationship to diseases, however, remains unclear. Recently, an estrogen receptor coactivator, AIB1, was identified with higher expression in ovarian cancer cell lines and breast cancer cells than in other cell lines tested (6), implying that increased expression of cofactors might be involved in some hormoneresponsive tumors. The question whether cofactors of AR, the major promoter of prostate tumor growth, can also play vital roles for the maintenance of androgen-dependent status is thus of vital interest.Here we report for the isolation and characterization of a novel AR coactivator, ARA55, which can bind to wild type AR (wtAR) and mutant AR (m...

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway controls several important biological functions, such as cell growth regulation, apoptosis, and migration. However, the way in which PI3K/Akt controls androgen receptor (AR)-mediated prostate cancer cell growth remains unclear and controversial. Here Prostate cancer is the second leading cause of cancer-related death among men in the United States. The normal prostate and prostate cancers at early stages require androgen for growth and survival. In addition to androgen signaling, which plays an essential role in survival of prostate cancer, the phosphatidylinositol 3-kinase (PI3K) 1 /Akt pathway represents another important survival signal for prostate cancer cells. It appears that these two pathways can compensate for each other in growth regulation of prostate cancer LNCaP cells, because androgen treatment can rescue cells from apoptosis induced by application of PI3K inhibitors (1). Furthermore, activation of the PI3K/Akt pathway protects cells from apoptosis induced by serum starvation and androgen deprivation (2).Recent rapid progress of the PI3K/Akt signal pathway studies, as well as its influence on the androgen receptor (AR)-mediated prostate cancer growth, has resulted in many exciting yet controversial results. Here we address these controversial results by summarizing Akt-AR-related results and provide new data, as well as possible explanations for the distinct roles of the PI3K/Akt pathway in AR-mediated prostate cancer growth. Particular emphases will be: 1) Akt suppresses versus induces AR activity, 2) Akt phosphorylation sites on AR protein, and 3) promotion of AR degradation by the PI3K/Akt pathway. EXPERIMENTAL PROCEDURESReagents-pCDNA3 cAkt (3) and mutant AR S210A/S790A were described previously (4). pCDNA3-PTEN was a gift from Dr. Charles L. Sawyers, and pGEX-KG-PTEN was from Dr. Frank B. Furnari. Insulinlike growth factor-1 (IGF-1) and LY294002 was from Calbiochem. 5␣-Dihydrotestosterone (DHT), doxycycline (Dox), and cycloheximide were from Sigma. The anti-AR polyclonal antibody, NH27, was produced as described previously (3). The mouse monoclonal PTEN and prostatespecific antigen (PSA) antibodies and the goat polyclonal ␤-actin antibody were from Santa Cruz Biotechnology. The mouse monoclonal Akt and phospho-Akt (Ser 473 ) antibodies were purchased from Cell Signaling.Cell Culture and Transfections-DU145, PC-3, and COS-1 cell lines were maintained in Dulbecco's minimum essential medium containing penicillin (25 units/ml), streptomycin (25 g/ml), and 10% fetal calf serum (FCS). LNCaP cells were maintained in RPMI 1640 with 10% FCS. Transfections were performed using SuperFect TM according to standard procedures (Qiagen).Luciferase Reporter Assays-Luciferase reporter assay was as described previously with some modifications (5). The cells were transfected with plasmids in 10% charcoal-stripped serum (CSS) medium for 16 h and then treated with ethanol or 10 nM DHT for 16 h. The cells were lysed, and luciferase activity was detected by the dua...

The information of protein targets and small molecule has been highly valued by biomedical and pharmaceutical research. Several protein target databases are available online for FDA-approved drugs as well as the promising precursors that have largely facilitated the mechanistic study and subsequent research for drug discovery. However, those related resources regarding to herbal active ingredients, although being unusually valued as a precious resource for new drug development, is rarely found. In this article, a comprehensive and fully curated database for Herb Ingredients’ Targets (HIT, http://lifecenter.sgst.cn/hit/) has been constructed to complement above resources. Those herbal ingredients with protein target information were carefully curated. The molecular target information involves those proteins being directly/indirectly activated/inhibited, protein binders and enzymes whose substrates or products are those compounds. Those up/down regulated genes are also included under the treatment of individual ingredients. In addition, the experimental condition, observed bioactivity and various references are provided as well for user's reference. Derived from more than 3250 literatures, it currently contains 5208 entries about 1301 known protein targets (221 of them are described as direct targets) affected by 586 herbal compounds from more than 1300 reputable Chinese herbs, overlapping with 280 therapeutic targets from Therapeutic Targets Database (TTD), and 445 protein targets from DrugBank corresponding to 1488 drug agents. The database can be queried via keyword search or similarity search. Crosslinks have been made to TTD, DrugBank, KEGG, PDB, Uniprot, Pfam, NCBI, TCM-ID and other databases.

Androgen receptor (AR) is a member of the steroid receptor superfamily that may require coactivators for proper or maximal transactivation. Using a yeast twohybrid screening followed by mammalian cell analyses, we identified a novel ligand-dependent AR-associated protein, ARA54, which consists of 474 amino acids with a molecular mass of 54 kDa. We demonstrated that ARA54 might function as a preferential coactivator for AR-mediated transactivation in human prostate cancer DU145 cells. Interestingly, our data also showed that ARA54 could significantly enhance the transcriptional activity of LNCaP mutant AR (ARt877a) but not wild type AR or another mutant AR (ARe708k) in the presence of 10 nM 17␤-estradiol or 1 M hydroxyflutamide. These results imply that both ARA54 and the positions of the AR mutation (877 versus 708) might contribute to the specificity of AR-mediated transactivation. Our findings further demonstrated that the C-terminal domain of ARA54 can serve as a dominant negative inhibitor and exogenous full-length ARA54 can reverse this squelching effect on AR transcriptional activity. Co-expression of ARA54 with other AR coactivators, such as ARA70 or SRC-1, showed additive stimulation of AR-mediated transactivation, which indicates that these cofactors may function individually as AR coactivators to induce AR target gene expression. Through our findings, we have identified and characterized a novel AR coactivator, ARA54, which may play an important role in the AR signaling pathway in human prostate.

Context: Recent studies suggest that miR-146b deregulation in papillary thyroid carcinoma (PTC) was associated with advanced tumor characteristics. However, the influence of miR-146b expression on the prognosis of PTC remains unknown. We sought to correlate tumor expression levels of miR-146b with the prognosis of a previously reported PTC cohort and reveal the underlying mechanisms via a PTC cell line model. Methodology:Expression levels of miR-146b were assessed via quantitative real-time PCR in 71 cases of PTC with distinct clinico-pathogenetic characteristics. All patients were classified into the disease-free or active disease group, based on their medical records at the end of the follow-up period. In vitro gain-of-function experiments were performed in a BCPAP human papillary thyroid cancer cell line model, which harbored the homozygous mutation of BRAF. BCPAP cells were transfected with a mimic-miR-146b and nonspecific microRNA (miRNA) control to determine whether miR-146b overexpression promotes cell migration and invasion. Proliferation assay, colony formation assay, and chemotherapy-induced apoptosis were also determined. Results:Multivariate logistic regression analysis demonstrated advanced tumor stage, presence of cervical lymph node metastasis, and miR-146b expression were independent risk factors for poor prognosis in PTC. Patients with higher miR-146b expression levels had significantly poorer overall survival compared with those with lower miR-146b levels. The associated hazard ratio was 3.92 (95% confidence interval, 1.73-8.86, log-rank P Ͻ .05). Overexpression of miR-146b significantly increased cell migration and invasiveness. Furthermore, miR-146b also increased resistance to chemotherapy-induced apoptosis. Conclusions:Our results suggest that miR-146b is a novel prognostic factor of PTC. Furthermore, in vitro functional studies provided the mechanistic explanation for miR-146b in tumor aggressiveness. These results enhance understanding of the molecular mechanisms involved in tumor aggressiveness in PTC, provide new prognostic biomarkers, and ultimately offer new leads for developing therapies for PTC. (J Clin Endocrinol Metab 98: E196 -E205, 2013)