Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell's sensitivity to Hsp90 inhibition.     

Laser Doppler Flare Imaging and Quantitative Thermal Thresholds Testing Performance in Small and Mixed Fiber Neuropathies

IntroductionSmall fiber neuropathy might be a part of typical mixed small and large fiber neuropathy, or a distinct entity, affecting exclusively small nerve fibers.ObjectivesExplore the utility of small nerve fiber testing in patients with clinical presentation suggesting small fiber neuropathy, with and without evidence for concomitant large fiber neuropathy.MethodsPatients attending the neuromuscular clinic from 2012 to 2015 with a clinical presentation suggesting small nerve fiber impairment, who had Laser Doppler flare imaging (LDI_Flare) and quantitative thermal testing (QTT) were evaluated for this study. Patients with clinical or electrophysiological evidence for concomitant large fiber neuropathy were not excluded.ResultsThe sensitivities of LDI_Flare, cooling and heat threshold testing were 64%, 36%, and 0% respectively for clinically highly suggestive small fiber neuropathy, 64%, 56%, and 19% respectively for mixed fiber neuropathy, and 86%, 79%, and 29% respectively for diabetic mixed fiber neuropathy.DiscussionLDI_Flare and cooling thresholds testing are non-invasive small nerve fiber testing modalities, with moderate performance in patients with small and mixed fiber neuropathy, and excellent performance in diabetic mixed fiber neuropathy.     

Antiangiogenic Arming of an Oncolytic Vaccinia Virus Enhances Antitumor Efficacy in Renal Cell Cancer Models

Oncolytic vaccinia viruses have shown compelling results in preclinical cancer models and promising preliminary safety and antitumor activity in early clinical trials. However, to facilitate systemic application it would be useful to improve tumor targeting and antitumor efficacy further. Here we report the generation of vvdd-VEGFR-1-Ig, a targeted and armed oncolytic vaccinia virus. Tumor targeting was achieved by deletion of genes for thymidine kinase and vaccinia virus growth factor, which are necessary for replication in normal but not in cancer cells. Given the high vascularization typical of kidney cancers, we armed the virus with the soluble vascular endothelial growth factor (VEGF) receptor 1 protein for an antiangiogenic effect. Systemic application of high doses of vvdd-VEGFR-1-Ig resulted in cytokine induction in an immunocompromised mouse model. Upon histopathological analysis, splenic extramedullary hematopoiesis was seen in all virus-injected mice and was more pronounced in the vvdd-VEGFR-1-Ig group. Analysis of the innate immune response after intravenous virus injection revealed high transient and dose-dependent cytokine elevations. When medium and low doses were used for intratumoral or intravenous injection, vvdd-VEGFR-1-Ig exhibited a stronger antitumor effect than the unarmed control. Furthermore, expression of VEGFR-1-Ig was confirmed, and a concurrent antiangiogenic effect was seen. In an immunocompetent model, systemic vvdd-VEGFR-1-Ig exhibited superior antitumor efficacy compared to the unarmed control virus. In conclusion, the targeted and armed vvdd-VEGFR-1-Ig has promising anticancer activity in renal cell cancer models. Extramedullary hematopoiesis may be a sensitive indicator of vaccinia virus effects in mice.In 2002 renal cell cancer accounted for more than 200,000 cases and 100,000 deaths worldwide (33). Unfortunately, chemotherapy, radiotherapy, and immunotherapy yield low response rates (9, 17) in this cancer type. Thus, prognosis for patients is poor, especially when the disease is metastatic, as median survival is only 8 months (19). Although recently approved drugs, such as sorafenib, sunitinib, temsirolimus, and bevacizumab, have provided additional tools for treatment of renal cell cancer (7), they are usually not curative, and thus new treatment approaches are needed.Oncolytic vaccinia viruses are promising agents for cancer treatment and have shown compelling results in preclinical tumor models (40, 42, 45). Moreover, good safety and preliminary evidence of antitumor efficacy were seen in phase 1 clinical trials (22, 26, 32). Vaccinia virus has a strong oncolytic effect due to its fast replication cycle (45) and a high innate tropism to cancer tissue (34). Tumor targeting can be further improved by deleting vaccinia virus genes that are necessary for replication in normal cells but not in cancer cells. For example, deletions of either thymidine kinase (TK) or vaccinia virus growth factor (VGF) or both have been shown to reduce pathogenicity compared to wild-type virus (3, 5, 27). To enhance antitumor potency, oncolytic vaccinia viruses can be armed with therapeutic transgenes, such as immunostimulatory factors (26) or suicide genes (14, 16, 35). With regard to kidney cancer, an arming approach with antiangiogenenic molecules seems logical, considering the high vascularization characteristic of renal tumors (20).Vascular endothelial growth factor (VEGF) is a major player in tumor angiogenesis and is highly expressed in renal cell cancers (29). VEGF binds to the fms-like-tyrosine kinase receptor (flt-1 or VEGFR-1) and kinase domain region receptor (KDR or VEGFR-2) with high affinity (13). The soluble vascular endothelial growth factor receptor 1-Ig fusion protein (VEGFR-1-Ig) used in this study is derived from the membrane-bound VEGFR-1 and binds human and murine VEGF without inducing vascular endothelial cell mitogenesis (31). Blocking VEGF with this or closely related molecules has been shown to inhibit tumor growth in several cancer models (18, 21, 25, 39).Although tumor cell selective replication can be enhanced by deletion of TK and/or VGF to reduce pathogenicity (3, 5, 27), high doses of attenuated vaccinia virus may increase serum cytokine concentrations which parallel the onset of toxic events, as seen with other viral vectors (2, 38). The potential “early” toxicity associated with oncolytic vaccinia viruses has not been completely elucidated heretofore (36, 46).Given the high vascularization of renal cell cancers and the pressing need to generate new antitumor agents with increased safety and efficacy, we hypothesized that an oncolytic vaccinia virus targeted by TK and VGF deletions and armed with VEGFR-1-Ig would exhibit enhanced antitumor efficacy due to its antiangiogenic properties in renal cell cancer models compared to a nonarmed control virus, allowing reduction of the treatment dose.     

Clathrin- and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae

Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)-deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1-independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.     

Inducible ablation of melanopsin-expressing retinal ganglion cells reveals their central role in non-image forming visual responses

Rod/cone photoreceptors of the outer retina and the melanopsin-expressing retinal ganglion cells (mRGCs) of the inner retina mediate non-image forming visual responses including entrainment of the circadian clock to the ambient light, the pupillary light reflex (PLR), and light modulation of activity. Targeted deletion of the melanopsin gene attenuates these adaptive responses with no apparent change in the development and morphology of the mRGCs. Comprehensive identification of mRGCs and knowledge of their specific roles in image-forming and non-image forming photoresponses are currently lacking. We used a Cre-dependent GFP expression strategy in mice to genetically label the mRGCs. This revealed that only a subset of mRGCs express enough immunocytochemically detectable levels of melanopsin. We also used a Cre-inducible diphtheria toxin receptor (iDTR) expression approach to express the DTR in mRGCs. mRGCs develop normally, but can be acutely ablated upon diphtheria toxin administration. The mRGC-ablated mice exhibited normal outer retinal function. However, they completely lacked non-image forming visual responses such as circadian photoentrainment, light modulation of activity, and PLR. These results point to the mRGCs as the site of functional integration of the rod/cone and melanopsin phototransduction pathways and as the primary anatomical site for the divergence of image-forming and non-image forming photoresponses in mammals.     

Biological diversity and cultural heritage

In this paper some examples of the development of communities of microorganisms and plants on historic buildings and montiments are shown. When the building stones differ from the surrounding natural substrata, an increase in the biological diversity of the area is produced. In some cases, monuments can come to constitute a true refuge for a few species when the natural habitat is threatened. It is suggested that biological diversity, when it does not represent a threat for the cultural heritage, should be considered worthy of preservation.     

Phosphorylation-dependent regulation of Kv2.1 Channel activity at tyrosine 124 by Src and by protein-tyrosine phosphatase epsilon

Voltage-gated potassium (Kv) channels are a complex and heterogeneous family of proteins that play major roles in brain and cardiac excitability. Although Kv channels are activated by changes in cell membrane potential, tyrosine phosphorylation of channel subunits can modulate the extent of channel activation by depolarization. We have previously shown that dephosphorylation of Kv2.1 by the nonreceptor-type tyrosine phosphatase PTPepsilon (cyt-PTPepsilon) down-regulates channel activity and counters its phosphorylation and up-regulation by Src or Fyn. In the present study, we identify tyrosine 124 within the T1 cytosolic domain of Kv2.1 as a target site for the activities of Src and cyt-PTPepsilon. Tyr(124) is phosphorylated by Src in vitro; in whole cells, Y124F Kv2.1 is significantly less phosphorylated by Src and loses most of its ability to bind the D245A substrate-trapping mutant of cyt-PTPepsilon. Phosphorylation of Tyr(124) is critical for Src-mediated up-regulation of Kv2.1 channel activity, since Y124F Kv2.1-mediated K(+) currents are only marginally up-regulated by Src, in contrast with a 3-fold up-regulation of wild-type Kv2.1 channels by the kinase. Other properties of Kv2.1, such as expression levels, subcellular localization, and voltage dependence of channel activation, are unchanged in Y124F Kv2.1, indicating that the effects of the Y124F mutation are specific. Together, these results indicate that Tyr(124) is a significant site at which the mutually antagonistic activities of Src and cyt-PTPepsilon affect Kv2.1 phosphorylation and activity.     

Epithelial-specific histone modification of the <Emphasis Type="Italic">miR-96/182</Emphasis> locus targeting <Emphasis Type="Italic">AMAP1</Emphasis> mRNA predisposes p53 to suppress cell invasion in epithelial cells

Background

TP53 mutations in cancer cells often evoke cell invasiveness, whereas fibroblasts show invasiveness in the presence of intact TP53. AMAP1 (also called DDEF1 or ASAP1) is a downstream effector of ARF6 and is essential for the ARF6-driven cell-invasive phenotype. We found that AMAP1 levels are under the control of p53 (TP53 gene product) in epithelial cells but not in fibroblasts, and here addressed that molecular basis of the epithelial-specific function of p53 in suppressing invasiveness via targeting AMAP1.

Methods

Using MDA-MB-231 cells expressing wild-type and p53 mutants, we identified miRNAs in which their expression is controlled by normal-p53. Among them, we identified miRNAs that target AMAP1 mRNA, and analyzed their expression levels and epigenetic statuses in epithelial cells and nonepithelial cells.

Results

We found that normal-p53 suppresses AMAP1 mRNA in cancer cells and normal epithelial cells, and that more than 30 miRNAs are induced by normal-p53. Among them, miR-96 and miR-182 were found to target the 3′-untranslated region of AMAP1 mRNA. Fibroblasts did not express these miRNAs at detectable levels. The ENCODE dataset demonstrated that the promoter region of the miR-183-96-182 cistron is enriched with H3K27 acetylation in epithelial cells, whereas this locus is enriched with H3K27 trimethylation in fibroblasts and other non-epithelial cells. miRNAs, such as miR-423, which are under the control of p53 but not associated with AMAP1 mRNA, demonstrated similar histone modifications at their gene loci in epithelial cells and fibroblasts, and were expressed in these cells.

Conclusion

Histone modifications of certain miRNA loci, such as the miR-183-96-182 cistron, are different between epithelial cells and non-epithelial cells. Such epithelial-specific miRNA regulation appears to provide the molecular basis for the epithelial-specific function of p53 in suppressing ARF6-driven invasiveness.

     

Heritable Custom Genomic Modifications in Caenorhabditis elegans via a CRISPR–Cas9 System

We adapted the CRISPR–Cas9 system for template-mediated repair of targeted double-strand breaks via homologous recombination in Caenorhabditis elegans, enabling customized and efficient genome editing. This system can be used to create specific insertions, deletions, and base pair changes in the germline of C. elegans.