Intrinsically disordered proteins (IDPs) that lack a unique 3D structure and comprise a large fraction of the human proteome play important roles in numerous cellular functions. ProstateAssociated Gene 4 (PAGE4) is an IDP that acts as a potentiator of the Activator Protein-1 (AP-1) transcription factor. HomeodomainInteracting Protein Kinase 1 (HIPK1) phosphorylates PAGE4 at S9 and T51, but only T51 is critical for its activity. Here, we identify a second kinase, CDC-Like Kinase 2 (CLK2), which acts on PAGE4 and hyperphosphorylates it at multiple S/T residues, including S9 and T51. We demonstrate that HIPK1 is expressed in both androgendependent and androgen-independent prostate cancer (PCa) cells, whereas CLK2 and PAGE4 are expressed only in androgendependent cells. Cell-based studies indicate that PAGE4 interaction with the two kinases leads to opposing functions. HIPK1-phosphorylated PAGE4 (HIPK1-PAGE4) potentiates c-Jun, whereas CLK2-phosphorylated PAGE4 (CLK2-PAGE4) attenuates c-Jun activity. Consistent with the cellular data, biophysical measurements (small-angle X-ray scattering, single-molecule fluorescence resonance energy transfer, and NMR) indicate that HIPK1-PAGE4 exhibits a relatively compact conformational ensemble that binds AP-1, whereas CLK2-PAGE4 is more expanded and resembles a random coil with diminished affinity for AP-1. Taken together, the results suggest that the phosphorylation-induced conformational dynamics of PAGE4 may play a role in modulating changes between PCa cell phenotypes. A mathematical model based on our experimental data demonstrates how differential phosphorylation of PAGE4 can lead to transitions between androgen-dependent and androgen-independent phenotypes by altering the AP-1/androgen receptor regulatory circuit in PCa cells.intrinsic disorder | androgen resistance | prostate cancer | PAGE-4 | phenotypic heterogeneity C ontrary to conventional wisdom that structure defines protein function (1), it is now increasingly evident that a large fraction of the human proteome is composed of intrinsically disordered proteins (IDPs) lacking rigid 3D structure (2-4). IDPs exist as conformational ensembles that are highly malleable, facilitating their interactions with multiple partners. These interactions are "wired" to form scale-free networks (5, 6) that represent the main conduit of information flow in the cell. Furthermore, the organization and properties of such protein interaction networks are evolutionarily conserved, underscoring their functional significance (7).By occupying hub positions in such networks, IDPs play critical roles in many biological processes, such as transcription, translation, and signaling (8, 9). IDPs also participate in higher order phenomena, such as regulation of the cell division cycle (10-12), circadian rhythmicity (13, 14), and phenotypic plasticity (15, 16). Recent evidence suggests that several IDPs can act in a prion-like manner to create protein-based molecular memories that drive the emergence and inheritance of biological traits (17), furt...

Point-of-care echocardiography using a miniaturized echocardiographic platform substantially improved the detection of important cardiovascular pathology compared with PE. Use of this device by a cardiovascular specialist with training in echocardiography as a routine adjunct to PE appears to be useful.

Regorafenib, a novel multikinase inhibitor, has recently demonstrated overall survival benefits in metastatic colorectal cancer (CRC) patients. Our study aimed to gain further insight into the molecular mechanisms of regorafenib and to assess its potential in combination therapy. Regorafenib was tested alone and in combination with irinotecan in patient-derived (PD) CRC models and a murine CRC liver metastasis model. Mechanism of action was investigated using in vitro functional assays, immunohistochemistry and correlation with CRC-related oncogenes. Regorafenib demonstrated significant inhibition of growth-factor-mediated vascular endothelial growth factor receptor (VEGFR) 2 and VEGFR3 autophosphorylation, and intracellular VEGFR3 signaling in human umbilical vascular endothelial cells (HuVECs) and lymphatic endothelial cells (LECs), and also blocked migration of LECs. Furthermore, regorafenib inhibited proliferation in 19 of 25 human CRC cell lines and markedly slowed tumor growth in five of seven PD xenograft models. Combination of regorafenib with irinotecan significantly delayed tumor growth after extended treatment in four xenograft models. Reduced CD31 staining indicates that the antiangiogenic effects of regorafenib contribute to its antitumor activity. Finally, regorafenib significantly delayed disease progression in a murine CRC liver metastasis model by inhibiting the growth of established liver metastases and preventing the formation of new metastases in other organs. In addition, our results suggest that regorafenib displays antimetastatic activity, which may contribute to its efficacy in patients with metastatic CRC. Combination of regorafenib and irinotecan demonstrated an increased antitumor effect and could provide a future treatment option for CRC patients.What's new?Regorafenib is a multikinase inhibitor with antiangiogenic activity recently approved in the US and in Europe for the treatment of metastatic colorectal cancer in patients who failed previous therapies. Here, a research team led by Bayer Pharma AG, the discoverer of the drug, confirms inhibition of key mediators of angiogenesis and lymphangiogenesis (VEGFR2 and VEGFR3) as the potential antiangiogenic mechanism of action of the drug. Regorafenib further inhibited growth of established and prevented formation of new liver metastases, and in combination with the chemotherapeutic drug irinotecan led to significant tumor growth delay in four patient-derived colorectal cancer xenograft models. The authors speculate that combination treatments including regorafenib may provide novel therapeutic opportunities for patients with therapy-resistant colorectal cancer.

A PCR method for uniform amplification of a mixture of DNA templates differing in GC content is described using the two enzyme approach (Klentaql and P fu DNA polymerase) and a combination of DMSO and betaine. This method was applied to amplify the CGG repeat region from the fragile X region.Development of PCR for the amplification of nucleic acids has revolutionalized several fields of molecular biology. Since the introduction of this method several parameters of the system have been modified to amplify longer nucleic acid fragments (Foord and Rose 1994, and references within). A variety of DNA polymerases have been used for PCR because of their improved thermal stability (Lawyer et al. 1993) and editing functions (Lundberg et al. 1991;Mattila et al. 1991).Use of a two-enzyme approach (Klentaq I and Pfi~ DNA polymerase) has enabled the amplification of fragments up to 35 kb in length (Barnes 1994;Cheng et al. 1994).Several reagents have been shown to facilitate DNA strand separation either because they disrupt base pairing [e.g., dimethylsulfoxide (DMSO), formamide] or isostabilize DNA [e.g., tetraalkylammonium (TAA) salts such as tetramethylammonium chloride (TMAC), tetraethylammonium chloride, and N,N,N-trimethylglycine (betaine)] (Melchior and yon Hippel 1973; Rees et al. 1993). Some of these reagents have been included in PCR reactions to amplify GCrich templates. Recently, it has been shown that at lower concentrations TMAC can enhance the amplification of AT-rich DNA fragments, and at higher concentrations it can inhibit enzyme amplification (Chevet et al. 1995). However, templates with high GC contents are variably amplifiable or completely unamplifiable even with the addition of DMSO, formamide, glycerol, TMAC, or 7-deazaguanosine triphosphate (7cdGTP), which lowers the melting temperature of the substituted DNA (McConlogue et al. 1988;Bookstein et al. 1990;Hung et al. 1990;Sarkar et al. 1990;Pomp and Medrano 1991). In spite of all the advances described above there is no single method that has been developed that could amplify to a similar extent all DNA templates in a heterogeneous mixture.There are a number of PCR applications (e.g., construction of cDNA library from a small number of cells, representational differential analysis of two different genomic DNA, or cDNAs from two different cell types) for which it would be highly desirable to have a method whereby a mixture of DNA fragments of similar length (0.5-10 kb) can be amplified with equal efficiency regardless of base composition. Therefore, we explored amplification by using isostabilizing quarternary amines that did not inhibit the DNA polymerases. Betaine is a natural amino acid analog and has many useful properties such as osmoprotection of Escherichia coli cells, stabilization of proteins against thermal denaturation, and isostabilization of DNA (Csonka 1989;Santoro et al. 1992; Rees et al. 1993). However, use of betaine as a reagent for amplification of GC-rich templates has not been explored.In this report we studied the efficiency of ...

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Regorafenib is an orally administered inhibitor of protein kinases involved in tumor angiogenesis, oncogenesis, and maintenance of the tumor microenvironment. Phase III studies showed that regorafenib has efficacy in patients with advanced gastrointestinal stromal tumors or treatment‐refractory metastatic colorectal cancer. In clinical studies, steady‐state exposure to the M‐2 and M‐5 metabolites of regorafenib was similar to that of the parent drug; however, the contribution of these metabolites to the overall observed clinical activity of regorafenib cannot be investigated in clinical trials. Therefore, we assessed the pharmacokinetics and pharmacodynamics of regorafenib, M‐2, and M‐5 in vitro and in murine xenograft models. M‐2 and M‐5 showed similar kinase inhibition profiles and comparable potency to regorafenib in a competitive binding assay. Inhibition of key target kinases by all three compounds was confirmed in cell‐based assays. In murine xenograft models, oral regorafenib, M‐2, and M‐5 significantly inhibited tumor growth versus controls. Total peak plasma drug concentrations and exposure to M‐2 and M‐5 in mice after repeated oral dosing with regorafenib 10 mg/kg/day were comparable to those in humans. In vitro studies showed high binding of regorafenib, M‐2, and M‐5 to plasma proteins, with unbound fractions of ~0.6%, ~0.9%, and ~0.4%, respectively, in murine plasma and ~0.5%, ~0.2%, and ~0.05%, respectively, in human plasma. Estimated free plasma concentrations of regorafenib and M‐2, but not M‐5, exceeded the IC50 at human and murine VEGFR2, suggesting that regorafenib and M‐2 are the primary contributors to the pharmacologic activity of regorafenib in vivo.

The process of metastasis is highly complex 1. In the case of breast cancer, there are frequently long timespans between cells leaving the primary tumour and growth of overt metastases 2, 3. Possible reasons for disease indolence and subsequent transitioning back to aggressive growth include interplay with myeloid and fibroblastic cells in the tumour microenvironment and ongoing immune surveillance 4-6. However, the signals causing actively growing cells to enter into an indolent state, and enabling them to survive for extended periods of time, are not well understood. In this work, we reveal how the behaviour of indolent breast cancer cells in the lung is determined by their interactions with alveolar epithelial cells, in particular AT1 cells. This crosstalk promotes the formation of fibronectin (FN) fibrils by indolent cells that drive integrindependent pro-survival signals. Combined in vivo RNA sequencing and drop-out screening identified Secreted frizzled-related protein 2 (Sfrp2) as a key mediator of this interaction. Sfrp2 is induced in breast cancer cells by signals emanating from lung

Biologically active natural products can be regarded as evolutionary selected and biologically validated starting points in structural space for the development of compound libraries. For libraries designed and synthesized around a given natural product, a higher hit rate and the identification of biologically relevant hits can be expected, justifying a probably higher investment in the development of the corresponding syntheses. This approach requires the development of complex multistep reaction sequences on the solid phase. Employing the protein phosphatase Cdc25 inhibitor dysidiolide as an example, we demonstrate that this goal can be achieved successfully. The reaction sequences developed led to dysidiolide analogues in overall 8-12 linear steps with the longest sequence on the solid support amounting to up to 11 sequential transformations. The desired products were obtained in overall yields ranging from 6% to 27% and in multimilligram amounts starting from 100 mg of resin. The transformations applied include a variety of very different reaction types widely used in organic synthesis (i.e., an asymmetric cycloaddition employing a removable chiral auxiliary, different organometallic transformations, olefination reactions, different oxidation reactions, acidic hydrolyses, and a nucleophilic substitution). Biological investigation of the eight dysidiolide analogues synthesized showed that they inhibit Cdc25C in the low micromolar range with the IC(50) value varying by a factor of 20 and that they display considerable and differing biological activities in cytotoxicity assays employing different cancer cell lines.