Ganetespib limits ciliation and cystogenesis in autosomal-dominant polycystic kidney disease (ADPKD)

© 2018 FASEB. Autosomal-dominant polycystic kidney disease (ADPKD) is associated with progressive formation of renal cysts, kidney enlargement, hypertension, and typically end-stage renal disease. In ADPKD, inherited mutations disrupt functionofthe polycystins (encoded byPKD1 and PKD2), thus causing loss of a cyst-repressive signal emanating from the renal cilium. Genetic studies have suggested ciliary maintenance is essential for ADPKD pathogenesis. Heat shock protein 90 (HSP90) clients include multiple proteins linked to ciliary maintenance. We determined that ganetespib, a clinical HSP90 inhibitor, inhibited proteasomal repression of NEK8 and the Aurora-A activator trichoplein, rapidly activating Aurora-A kinase and causing ciliary loss in vitro. Using conditional mouse models for ADPKD, we performed long-term (10 or 50 wk) dosing experiments that demonstrated HSP90 inhibition caused durable in vivo loss of cilia, controlled cystic growth, and ameliorated symptoms induced by loss of Pkd1 or Pkd2.Ganetespib efficacy was not increased by combination with 2-deoxy-D-glucose, aglycolysis inhibitor showing some promise for ADPKD. These studies identify a new biologic activity for HSP90 and support a cilia-based mechanism for cyst repression

NEDD9 promotes oncogenic signaling, a stem/mesenchymal gene signature, and aggressive ovarian cancer growth in mice

© 2018, Macmillan Publishers Limited, part of Springer Nature. Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) supports oncogenic signaling in a number of solid and hematologic tumors. Little is known about the role of NEDD9 in ovarian carcinoma (OC), but available data suggest elevated mRNA and protein expression in advanced stage high-grade cancers. We used a transgenic MISIIR-TAg mouse OC model combined with genetic ablation of Nedd9 to investigate its action in the development and progression of OC. A Nedd9−/− genotype delayed tumor growth rate, reduced incidence of ascites, and reduced expression and activation of signaling proteins including SRC, STAT3, E-cadherin, and AURKA. Cell lines established from MISIIR-TAg;Nedd9−/− and MISIIR-TAg;Nedd9+/+ mice exhibited altered migration and invasion. Growth of these cells in a syngeneic allograft model indicated that systemic Nedd9 loss in the microenvironment had little impact on tumor allograft growth, but in a Nedd9 wild-type background Nedd9−/− allografts exhibited significantly reduced growth, dissemination, and oncogenic signaling compared to Nedd9+/+ allografts. Gene expression analysis revealed that Nedd9+/+ tumors exhibited more mesenchymal “stem-like” transcriptional program, including increased expression of Aldh1a1 and Aldh1a2. Conversely, loss of Nedd9 resulted in increased expression of differentiation genes, including fallopian tube markers Foxj1, Ovgp1, and Pax8. Collectively, these data suggest that tumor cell-intrinsic Nedd9 expression promotes OC development and progression by broad induction of oncogenic protein signaling and stem/mesenchymal gene expression

Embryonal Fyn-associated substrate (EFS) and CASS4: The lesser-known CAS protein family members

© 2015 Elsevier B.V.. The CAS (Crk-associated substrate) adaptor protein family consists of four members: CASS1/BCAR1/p130Cas, CASS2/NEDD9/HEF1/Cas-L, CASS3/EFS/Sin and CASS4/HEPL. While CAS proteins lack enzymatic activity, they contain specific recognition and binding sites for assembly of larger signaling complexes that are essential for cell proliferation, survival, migration, and other processes. All family members are intermediates in integrin-dependent signaling pathways mediated at focal adhesions, and associate with FAK and SRC family kinases to activate downstream effectors regulating the actin cytoskeleton. Most studies of CAS proteins to date have been focused on the first two members, BCAR1 and NEDD9, with altered expression of these proteins now appreciated as influencing disease development and prognosis for cancer and other serious pathological conditions. For these family members, additional mechanisms of action have been defined in receptor tyrosine kinase (RTK) signaling, estrogen receptor signaling or cell cycle progression, involving discrete partner proteins such as SHC, NSP proteins, or AURKA. By contrast, EFS and CASS4 have been less studied, although structure-function analyses indicate they conserve many elements with the better-known family members. Intriguingly, a number of recent studies have implicated these proteins in immune system function, and the pathogenesis of developmental disorders, autoimmune disorders including Crohn's disease, Alzheimer's disease, cancer and other diseases. In this review, we summarize the current understanding of EFS and CASS4 protein function in the context of the larger CAS family group

Mechanisms for nonmitotic activation of Aurora-A at cilia

© 2017 The Author(s).Overexpression of the Aurora kinase A (AURKA) is oncogenic in many tumors. Many studies of AURKA have focused on activities of this kinase in mitosis, and elucidated the mechanisms by which AURKA activity is induced at the G2/M boundary through interactions with proteins such as TPX2 and NEDD9. These studies have informed the development of small molecule inhibitors of AURKA, of which a number are currently under preclinical and clinical assessment. While the first activities defined for AURKA were its control of centrosomal maturation and organization of the mitotic spindle, an increasing number of studies over the past decade have recognized a separate biological function of AURKA, in controlling disassembly of the primary cilium, a small organelle protruding from the cell surface that serves as a signaling platform. Importantly, these activities require activation of AURKA in early G1, and the mechanisms of activation are much less well defined than those in mitosis. A better understanding of the control of AURKA activity and the role of AURKA at cilia are both important in optimizing the efficacy and interpreting potential downstream consequences of AURKA inhibitors in the clinic. We here provide a current overview of proteins and mechanisms that have been defined as activating AURKA in G1, based on the study of ciliary disassembly

The WW-HECT protein Smurf2 interacts with the Docking Protein NEDD9/HEF1 for Aurora A activation

The multi-functional adaptor protein NEDD9/HEF1/Cas-L regulates cell motility, invasion and cell cycle progression, and plays key roles in cancer progression and metastasis. NEDD9 is localized to the centrosome and is required for activation of Aurora A kinase in mitosis. Here we demonstrate that the HECT-WW protein Smurf2 physically associates with NEDD9 and is required for the stability of NEDD9 protein. Smurf2 depletion results in a marked decrease in NEDD9 protein levels, by facilitating polyubiquitination and proteasomal degradation of NEDD9. Conversely, forced overexpression of Smurf2 results in upregulation of endogenous NEDD9 protein, confirming the role for Smurf2 in NEDD9 stability. Cells with Smurf2 depletion fail to activate Aurora A at the G2/M boundary, leading to a marked delay in mitotic entry. These observations suggest that the stable complex of Smurf2 and NEDD9 is required for timely entry into mitosis via Aurora A activation

Tumor-targeted SN38 inhibits growth of early stage non-small cell lung cancer (NSCLC) in a KRas/p53 transgenic mouse model

© 2017 Deneka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide, with a 5-year survival of only 16%. Potential strategies to address NSCLC mortality include improvements in early detection and prevention, and development of new therapies suitable for use in patients with early and late stage diagnoses. Controlling the growth of early stage tumors could yield significant clinical benefits for patients with comorbidities that make them poor candidates for surgery: however, many drugs that limit cancer growth are not useful in the setting of long-term use or in comorbid patients, because of associated toxicities. In this study, we explored the use of a recently described small molecule agent, STA-8666, as a potential agent for controlling early stage tumor growth. STA-8666 uses a cleavable linker to merge a tumor-targeting moiety that binds heat shock protein 90 (HSP90) with the cytotoxic chemical SN38, and has been shown to have high efficacy and low toxicity, associated with efficient tumor targeting, in preclinical studies using patient-derived and other xenograft models for pancreatic, bladder, and small cell lung cancer. Using a genetically engineered model of NSCLC arising from induced mutation of KRas and knockout of Trp53, we continuously dosed mice with STA-8666 from immediately after tumor induction for 15 weeks. STA-8666 significantly slowed the rate of tumor growth, and was well tolerated over this extended dosing period. STA-8666 induced DNA damage and apoptosis, and reduced proliferation and phosphorylation of the proliferation-associated protein ERK1/2, selectively in tumor tissue. In contrast, STA-8666 did not affect tumor features, such as degree of vimentin staining, associated with epithelial-mesenchymal transition (EMT), or downregulate tumor expression of HSP90. These data suggest STA-8666 and other similar targeted compounds may be useful additions to control the growth of early stage NSCLC in patient populations

Comprehensive characterization of PTEN mutational profile in a series of 34,129 colorectal cancers

Loss of expression or activity of the tumor suppressor PTEN acts similarly to an activating mutation in the oncogene PIK3CA in elevating intracellular levels of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), inducing signaling by AKT and other pro-tumorigenic signaling proteins. Here, we analyze sequence data for 34,129 colorectal cancer (CRC) patients, capturing 3,434 PTEN mutations. We identify specific patterns of PTEN mutation associated with microsatellite stability/instability (MSS/MSI), tumor mutational burden (TMB), patient age, and tumor location. Within groups separated by MSS/MSI status, this identifies distinct profiles of nucleotide hotspots, and suggests differing profiles of protein-damaging effects of mutations. Moreover, discrete categories of PTEN mutations display non-identical patterns of co-occurrence with mutations in other genes important in CRC pathogenesis, including KRAS, APC, TP53, and PIK3CA. These data provide context for clinical targeting of proteins upstream and downstream of PTEN in distinct CRC cohorts.Loss of the tumour suppressor gene PTEN leads to the activation of pro-tumourigenic signalling pathways. Here, the authors analyse sequencing data from a large cohort of colorectal cancer patients harbouring PTEN mutations and identify distinct patterns of associations with genomic and clinical features

Opposing effects of inhibitors of Aurora-A and EGFR in autosomal-dominant polycystic kidney disease

© 2015 Nikonova, Deneka, Eckman, Kopp, Hensley, Egleston and Golemis. Aurora-A kinase (AURKA) overexpression in numerous tumors induces aneuploidy, in part because of cytokinetic defects. Alisertib and other small-molecule inhibitors targeting AURKA are effective in some patients as monotherapies or combination therapies. Epidermal growth factor receptor (EGFR) pro-proliferative signaling activity is commonly elevated in cancer, and the EGFR inhibitor erlotinib is commonly used as a standard of care agent for cancer. An erlotinib/alisertib combination therapy is currently under assessment in clinical trials, following pre-clinical studies that indicated synergy of these drugs in cancer. We were interested in further exploring the activity of this drug combination. Beyond well-established functions for AURKA in mitotic progression, additional non-mitotic AURKA functions include control of ciliary stability and calcium signaling. Interestingly, alisertib exacerbates the disease phenotype in mouse models for autosomal-dominant polycystic kidney disease (ADPKD), a common inherited syndrome induced by aberrant signaling from PKD1 and PKD2, cilia-localized proteins that have calcium channel activity. EGFR is also more active in ADPKD, making erlotinib also of potential interest in this disease setting. In this study, we have explored the interaction of alisertib and erlotinib in an ADPKD model. These experiments indicated erlotinib-restrained cystogenesis, opposing alisertib action. Erlotinib also interacted with alisertib to regulate proliferative signaling proteins, albeit in a complicated manner. Results suggest a nuanced role of AURKA signaling in different pathogenic conditions and inform the clinical use of AURKA inhibitors in cancer patients with comorbidities

Identification of evolutionarily conserved DNA damage response genes that alter sensitivity to cisplatin

Ovarian, head and neck, and other cancers are commonly treated with cisplatin and other DNA damaging cytotoxic agents. Altered DNA damage response (DDR) contributes to resistance of these tumors to chemotherapies, some targeted therapies, and radiation. DDR involves multiple protein complexes and signaling pathways, some of which are evolutionarily ancient and involve protein orthologs conserved from yeast to humans. To identify new regulators of cisplatin-resistance in human tumors, we integrated high throughput and curated datasets describing yeast genes that regulate sensitivity to cisplatin and/or ionizing radiation. Next, we clustered highly validated genes based on chemogenomic profiling, and then mapped orthologs of these genes in expanded genomic networks for multiple metazoans, including humans. This approach identified an enriched candidate set of genes involved in the regulation of resistance to radiation and/or cisplatin in humans. Direct functional assessment of selected candidate genes using RNA interference confirmed their activity in influencing cisplatin resistance, degree of γH2AX focus formation and ATR phosphorylation, in ovarian and head and neck cancer cell lines, suggesting impaired DDR signaling as the driving mechanism. This work enlarges the set of genes that may contribute to chemotherapy resistance and provides a new contextual resource for interpreting next generation sequencing (NGS) genomic profiling of tumors

Compounds identified by virtual docking to a tetrameric EGFR extracellular domain can modulate Grb2 internalization

© Ramirez et al. 2015. Background: Overexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the growth of many solid tumors. Tumor cells frequently display resistance to mechanistically-distinct EGFR-directed therapeutic agents, making it valuable to develop therapeutics that work by additional mechanisms. Current EGFR-targeting therapeutics include antibodies targeting the extracellular domains, and small molecules inhibiting the intracellular kinase domain. Recent studies have identified a novel prone extracellular tetrameric EGFR configuration, which we identify as a potential target for drug discovery. Methods: Our focus is on the prone EGFR tetramer, which contains a novel protein-protein interface involving extracellular domain III. This EGFR tetramer is computationally targeted for stabilization by small molecule ligand binding. This study performed virtual screening of a Life Chemicals, Inc. small molecule library of 345,232 drug-like compounds against a molecular dynamics simulation of protein-protein interfaces distinct to the novel tetramer. One hundred nine chemically diverse candidate molecules were selected and evaluated using a cell-based high-content imaging screen that directly assessed induced internalization of the EGFR effector protein Grb2. Positive hits were further evaluated for influence on phosphorylation of EGFR and its effector ERK1/2. Results: Fourteen hit compounds affected internalization of Grb2, an adaptor responsive to EGFR activation. Most hits had limited effect on cell viability, and minimally influenced EGFR and ERK1/2 phosphorylation. Docked hit compound poses generally include Arg270 or neighboring residues, which are also involved in binding the effective therapeutic cetuximab, guiding further chemical optimization. Conclusions: These data suggest that the EGFR tetrameric configuration offers a novel cancer drug target