t-DARPP regulates phosphatidylinositol-3-kinase-dependent cell growth in breast cancer

<p>Abstract</p> <p>Background</p> <p>Recent reports have shown that t-DARPP (truncated isoform of DARPP-32) can mediate trastuzumab resistance in breast cancer cell models. In this study, we evaluated expression of t-DARPP in human primary breast tumors, and investigated the role of t-DARPP in regulating growth and proliferation in breast cancer cells.</p> <p>Results</p> <p>Quantitative real time RT-PCR analysis using primers specific for t-DARPP demonstrated overexpression of t-DARPP in 36% of breast cancers (13/36) as opposed to absent to very low t-DARPP expression in normal breast tissue (p < 0.05). The mRNA overexpression of t-DARPP was overwhelmingly observed in ductal carcinomas, including invasive ductal carcinomas and intraductal carcinomas, rather than other types of breast cancers. The immunohistochemistry analysis of DARPP-32/t-DARPP protein(s) expression in breast cancer tissue microarray that contained 59 tumors and matched normal tissues when available indicated overexpression in 35.5% of primary breast tumors that were more frequent in invasive ductal carcinomas (43.7%; 21/48). In vitro studies showed that stable overexpression of t-DARPP in MCF-7 cells positively regulated proliferation and anchorage-dependent and -independent growth. Furthermore, this effect was concomitant with induction of phosphorylation of AKT^ser473and its downstream target phospho^ser9GSK3β, and increased Cyclin D1 and C-Myc protein levels. The knockdown of endogenous t-DARPP in HCC1569 cells led to a marked decrease in phosphorylation of AKTs^ser473and GSK3β^ser9. The use of PI3K inhibitor LY294002 or Akt siRNA abrogated the t-DARPP-mediated phosphorylation of AKT^ser473and led to a significant reduction in cell growth.</p> <p>Conclusions</p> <p>Our findings underscore the potential role of t-DARPP in regulating cell growth and proliferation through PI3 kinase-dependent mechanism.</p

Progress in the development of non-​BET bromodomain chemical probes

The bromodomain and extra terminal (BET) family of bromodomains have been the focus of extensive research, leading to the development of many potent, selective chem. probes and recent clinical assets. The profound biol. associated with BET bromodomain inhibition has provided a convincing rationale for targeting bromodomains for the treatment of disease. However, the BET family represents just eight of the at least 56 human bromodomains identified to date. Until recently, there has been significantly less interest in non-​BET bromodomains, leaving a vast area of research and the majority of this new target class yet to be thoroughly investigated. It has been widely reported that several non-​BET bromodomain containing. proteins are associated with various diseases including cancer and HIV. Therefore, the development of chem. probes for non-​BET bromodomains will facilitate elucidation of their precise biol. roles and potentially lead to the development of new medicines. This review summarises the progress made towards the development of non-​BET bromodomain chem. probes to date. In addn., we highlight the potential for future work in this new and exciting area

PRMT1-dependent regulation of RNA metabolism and DNA damage response sustains pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has remained clinically challenging to manage. Here we employ an RNAi-based in vivo functional genomics platform to determine epigenetic vulnerabilities across a panel of patient-derived PDAC models. Through this, we identify protein arginine methyltransferase 1 (PRMT1) as a critical dependency required for PDAC maintenance. Genetic and pharmacological studies validate the role of PRMT1 in maintaining PDAC growth. Mechanistically, using proteomic and transcriptomic analyses, we demonstrate that global inhibition of asymmetric arginine methylation impairs RNA metabolism, which includes RNA splicing, alternative polyadenylation, and transcription termination. This triggers a robust downregulation of multiple pathways involved in the DNA damage response, thereby promoting genomic instability and inhibiting tumor growth. Taken together, our data support PRMT1 as a compelling target in PDAC and informs a mechanism-based translational strategy for future therapeutic development. Statement of significance PDAC is a highly lethal cancer with limited therapeutic options. This study identified and characterized PRMT1-dependent regulation of RNA metabolism and coordination of key cellular processes required for PDAC tumor growth, defining a mechanism-based translational hypothesis for PRMT1 inhibitors

Regulation of β-catenin by t-DARPP in upper gastrointestinal cancer cells

<p>Abstract</p> <p>Background</p> <p>Truncated dopamine and cyclic-AMP-regulated phosphoprotein (t-DARPP) is frequently overexpressed in gastrointestinal malignancies. In this study, we examined the role of t-DARPP in regulating β-catenin.</p> <p>Results</p> <p>The pTopFlash construct that contains multiple TCF/LEF-binding sites was used as a measure of β-catenin/TCF transcription activity. Gastric (AGS, MKN28) and esophageal (FLO-1) adenocarcinoma cancer cell lines that lack t-DARPP expression were utilized to establish stable and transient <it>in vitro </it>expression models of t-DARPP. The expression of t-DARPP led to a significant induction of the pTOP reporter activity, indicative of activation of β-catenin/TCF nuclear signaling. Immunofluorescence assays supported this finding and showed accumulation and nuclear translocation of β-catenin in cells expressing t-DARPP. These cells had a significant increase in their proliferative capacity and demonstrated up-regulation of two transcription targets of β-catenin/TCF: Cyclin D1 and c-MYC. Because phosphorylated GSK-3β is inactive and loses its ability to phosphorylate β-catenin and target it towards degradation by the proteasome, we next examined the levels of phospho-GSK-3β. These results demonstrated an increase in phospho-GSK-3β and phospho-AKT. The knockdown of endogenous t-DARPP in MKN45 cancer cells demonstrated a reversal of the signaling events. To examine whether t-DARPP mediated GSK-3β phosphorylation in an AKT-dependent manner, we used a pharmacologic inhibitor of PI3K/AKT, LY294002, in cancer cells expressing t-DARPP. This treatment abolished the phosphorylation of AKT and GSK-3β leading to a reduction in β-catenin, Cyclin D1, and c-MYC protein levels.</p> <p>Conclusions</p> <p>Our findings demonstrate, for the first time, that t-DARPP regulates β-catenin/TCF activity, thereby implicating a novel oncogenic signaling in upper gastrointestinal cancers.</p

Surface reengineering of RPA70N enables cocrystallization with an inhibitor of the replication protein a interaction motif of ATR interacting protein

Replication protein A (RPA) is the primary single-stranded DNA (ssDNA) binding protein in eukaryotes. The N-terminal domain of the RPA70 subunit (RPA70N) interacts via a basic cleft with a wide range of DNA processing proteins, including several that regulate DNA damage response and repair. Small molecule inhibitors that disrupt these protein-protein interactions are therefore of interest as chemical probes of these critical DNA processing pathways and as inhibitors to counter the upregulation of DNA damage response and repair associated with treatment of cancer patients with radiation or DNA-damaging agents. Determination of three-dimensional structures of protein-ligand complexes is an important step for elaboration of small molecule inhibitors. However, although crystal structures of free RPA70N and an RPA70N-peptide fusion construct have been reported, RPA70N-inhibitor complexes have been recalcitrant to crystallization. Analysis of the P6&lt;sub&gt;1&lt;/sub&gt; lattice of RPA70N crystals led us to hypothesize that the ligand-binding surface was occluded. Surface reengineering to alter key crystal lattice contacts led to the design of RPA70N E7R, E100R, and E7R/E100R mutants. These mutants crystallized in a P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt; lattice that clearly had significant solvent channels open to the critical basic cleft. Analysis of X-ray crystal structures, target peptide binding affinities, and &lt;sup&gt;15&lt;/sup&gt;N-&lt;sup&gt;1&lt;/sup&gt;H heteronuclear single-quantum coherence nuclear magnetic resonance spectra showed that the mutations do not result in perturbations of the RPA70N ligand-binding surface. The success of the design was demonstrated by determining the structure of RPA70N E7R soaked with a ligand discovered in a previously reported molecular fragment screen. A fluorescence anisotropy competition binding assay revealed this compound can inhibit the interaction of RPA70N with the peptide binding motif from the DNA damage response protein ATRIP. The implications of the results are discussed in the context of ongoing efforts to design RPA70N inhibitors. 2013 American Chemical Societ

Discovery of protein-protein interaction inhibitors of replication protein A

Replication protein A (RPA) is a ssDNA binding protein that is essential for DNA replication and repair. The initiation of the DNA damage response by RPA is mediated by protein-protein interactions involving the N-terminal domain of the 70 kDa subunit with partner proteins. Inhibition of these interactions increases sensitivity toward DNA damage and replication stress and may therefore be a potential strategy for cancer drug discovery. Toward this end, we have discovered two lead series of compounds, derived from hits obtained from a fragment-based screen, that bind to RPA70N with low micromolar affinity and inhibit the binding of an ATRIP-derived peptide to RPA. These compounds may offer a promising starting point for the discovery of clinically useful RPA inhibitors. 2013 American Chemical Societ