Rape Myth Acceptance: Implications for Counselor Education Programs

Abstract A sexually violent act or rape is committed every 1.9 minutes in the United States (USDJ, 2009, p.1). Blaming the rape victim for their perceived complicity is one component of the construct known as rape myth, a term identified by Burt (1980). This study explored and examined the perceptions, and understanding of sexual violence, rape, and rape myths by master’s level counselors-in-training (n=5). Phenomenology and naturalistic inquiry guided the qualitative design and implementation. Suggestions for implementing rape education and training into counseling curriculums and clinical supervision are provided. Keywords: rape myth, counselors-in-training, phenomenolog

Activation of Hsp90 Enzymatic Activity and Conformational Dynamics through Rationally Designed Allosteric Ligands

Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65 æ from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules’ effects on Hsp90 enzymatic, conformational, cochaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary

Asymmetric Hsp90 N domain SUMOylation recruits Aha1 and ATP-competitive inhibitors

The stability and activity of numerous signaling proteins in both normal and cancer cells depends on the dimeric molecular chaperone heat shock protein 90 (Hsp90). Hsp90's function is coupled to ATP binding and hydrolysis and requires a series of conformational changes that are regulated by cochaperones and numerous posttranslational modifications (PTMs). SUMOylation is one of the least-understood Hsp90 PTMs. Here, we show that asymmetric SUMOylation of a conserved lysine residue in the N domain of both yeast (K178) and human (K191) Hsp90 facilitates both recruitment of the adenosine triphosphatase (ATPase)-activating cochaperone Aha1 and, unexpectedly, the binding of Hsp90 inhibitors, suggesting that these drugs associate preferentially with Hsp90 proteins that are actively engaged in the chaperone cycle. Importantly, cellular transformation is accompanied by elevated steady-state N domain SUMOylation, and increased Hsp90 SUMOylation sensitizes yeast and mammalian cells to Hsp90 inhibitors, providing a mechanism to explain the sensitivity of cancer cells to these drugs. © 2014 Elsevier Inc

HSP90 inhibitors disrupt a transient HSP90-HSF1 interaction and identify a noncanonical model of HSP90-mediated HSF1 regulation

Heat shock factor 1 (HSF1) initiates a broad transcriptional response to proteotoxic stress while also mediating a cancer-specific transcriptional program. HSF1 is thought to be regulated by molecular chaperones, including Heat Shock Protein 90 (HSP90). HSP90 is proposed to sequester HSF1 in unstressed cells, but visualization of this interaction in vivo requires protein crosslinking. In this report, we show that HSP90 binding to HSF1 depends on HSP90 conformation and is only readily visualized for the ATP-dependent, N-domain dimerized chaperone, a conformation only rarely sampled by mammalian HSP90. We have used this mutationally fixed conformation to map HSP90 binding sites on HSF1. Further, we show that ATP-competitive, N-domain targeted HSP90 inhibitors disrupt this interaction, resulting in the increased duration of HSF1 occupancy of the hsp70 promoter and significant prolongation of both the constitutive and heat-induced HSF1 transcriptional activity. While our data do not support a role for HSP90 in sequestering HSF1 monomers to suppress HSF1 transcriptional activity, our findings do identify a noncanonical role for HSP90 in providing dynamic modulation of HSF1 activity by participating in removal of HSF1 trimers from heat shock elements in DNA, thus terminating the heat shock response

Integration of Gene Dosage and Gene Expression in Non-Small Cell Lung Cancer, Identification of HSP90 as Potential Target

BACKGROUND: Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC) represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC) is essential to improve early diagnosis and treatment for this disease. METHODOLOGY AND PRINCIPAL FINDINGS: In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%), which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008), survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04). Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines. CONCLUSIONS: We suggest that targeting HSP90 will have clinical impact for NSCLC patients

Rape Myth Acceptance: Implications for Counselor Education Programs

Abstract A sexually violent act or rape is committed every 1.9 minutes in the United States (USDJ, 2009, p.1). Blaming the rape victim for their perceived complicity is one component of the construct known as rape myth, a term identified by Burt (1980). This study explored and examined the perceptions, and understanding of sexual violence, rape, and rape myths by master’s level counselors-in-training (n=5). Phenomenology and naturalistic inquiry guided the qualitative design and implementation. Suggestions for implementing rape education and training into counseling curriculums and clinical supervision are provided. Keywords: rape myth, counselors-in-training, phenomenolog

Loss of Hsp90 Association Up-Regulates Src-Dependent ErbB2 Activity

The receptor tyrosine kinase ErbB2 plays a crucial role in tumorigenesis. We showed previously that the molecular chaperone Hsp90 protects ErbB2 from proteasome-mediated degradation by binding to a short loop structure in the N-lobe of the kinase domain. Here we show that loss of Hsp90 binding correlates with enhanced ErbB2 kinase activity and its transactivating potential, concomitant with constitutively increased phosphorylation of Tyr877, located in the activation loop of the kinase domain. We show further that Tyr877 phosphorylation is mediated by Src and that it is necessary for the enhanced kinase activity of ErbB2. Finally, computer modeling of the kinase domain suggests a phosphorylation-dependent reorientation of the activation loop, denoting the importance of Tyr877 phosphorylation for ErbB2 activity. These findings suggest that Hsp90 binding to ErbB2 participates in regulation of kinase activity as well as kinase stability

The HSP90 Inhibitor Ganetespib Synergizes with the MET Kinase Inhibitor Crizotinib in both Crizotinib-Sensitive and -Resistant MET-Driven Tumor Models

The proto-oncogene MET is aberrantly activated via overexpression or mutation in numerous cancers, making it a prime anticancer molecular target. However, the clinical success of MET-directed tyrosine kinase inhibitors (TKI) has been limited due, in part, to mutations in the MET kinase domain that confer therapeutic resistance. Circumventing this problem remains a key challenge to improving durable responses in patients receiving MET-targeted therapy. MET is an HSP90-dependent kinase, and in this report we show that HSP90 preferentially interacts with and stabilizes activated MET, regardless of whether the activation is ligand-dependent or is a consequence of kinase domain mutation. In contrast, many MET-TKI show a preference for the inactive form of the kinase, and activating mutations in MET can confer resistance. Combining the HSP90 inhibitor ganetespib with the MET-TKI crizotinib achieves synergistic inhibition of MET, its downstream signaling pathways, and tumor growth in both TKI-sensitive and -resistant MET-driven tumor models. These data suggest that inclusion of an HSP90 inhibitor can partially restore TKI sensitivity to previously resistant MET mutants, and they provide the foundation for clinical evaluation of this therapeutic combination in patients with MET-driven cancers. (C)2013 AACR

Computational polypharmacology analysis of the heat shock protein 90 interactome

The design of a single drug molecule that is able to simultaneously and specifically interact with multiple biological targets is gaining major consideration in drug discovery. However, the rational design of drugs with a desired polypharmacology profile is still a challenging task, especially when these targets are distantly related or unrelated. In this work, we present a computational approach aimed at the identification of suitable target combinations for multitarget drug design within an ensemble of biologically relevant proteins. The target selection relies on the analysis of activity annotations present in molecular databases and on ligand-based virtual screening. A few target combinations were also inspected with structure-based methods to demonstrate that the identified dual-activity compounds are able to bind target combinations characterized by remote binding site similarities. Our approach was applied to the heat shock protein 90 (Hsp90) interactome, which contains several targets of key importance in cancer. Promising target combinations were identified, providing a basis for the computational design of compounds with dual activity. The approach may be used on any ensemble of proteins of interest for which known inhibitors are available

Inhibition of HSP90 with Pochoximes: SAR and Structure-Based Insights

The pochoximes, based on the radicicol pharmacophore, are potent inhibitors of heat shock protein 90 (HSP90) that retain their activity in vivo. Herein we report an extended library that broadly explores the structure–activity relationship (SAR) of the pochoximes with four points of diversity. Several modifications were identified that afford improved cellular efficacy, new opportunities for conjugation, and further diversifications. Cocrystal structures of pochoximes A and B with HSP90 show that pochoximes bind to a different conformation of HSP90 than radicicol and provide a rationale for the enhanced affinity of the pochoximes relative to radicicol and the pochonins