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PDF file - 133K, Table summarizing the data from Figure 6.

PDF file - 132K, TMEM16A expression influences cell size measured via flow cytometry.

Purpose:Despite the therapeutic success of existing HER2-targeted therapies, tumors invariably relapse. This study aimed at identifying new mechanisms responsible for HER2-targeted therapy resistance.Experimental Design:We have used a platform of HER2-targeted therapy–resistant cell lines and primary cultures of healthy and tumor-associated fibroblasts (TAF) to identify new potential targets related to tumor escape from anti-HER2 therapies.Results:We have shown that TAFs promote resistance to HER2-targeted therapies. TAFs produce and secrete high levels of FGF5, which induces FGFR2 activation in the surrounding breast cancer cells. FGFR2 transactivates HER2 via c-Src, leading to resistance to HER2-targeted therapies. In vivo, coinoculating nonresistant cell lines with TAFs results in more aggressive and resistant tumors. Resistant cells activate fibroblasts and secrete FGFR ligands, creating a positive feedback loop that fuels resistance. FGFR2 inhibition not only inhibits HER2 activation, but also induces apoptosis in cells resistant to HER2-targeted therapies. In vivo, inhibitors of FGFR2 reverse resistance and resensitize resistant cells to HER2-targeted therapies. In HER2 patients' samples, α-SMA, FGF5, and FGFR2 contribute to poor outcome and correlate with c-Src activation. Importantly, expression of FGF5 and phospho-HER2 correlated with a reduced pathologic complete response rate in patients with HER2-positive breast cancer treated with neoadjuvant trastuzumab, which highlights the significant role of TAFs/FGF5 in HER2 breast cancer progression and resistance.Conclusions:We have identified the TAF/FGF5/FGFR2/c-Src/HER2 axis as an escape pathway responsible for HER2-targeted therapy resistance in breast cancer, which can be reversed by FGFR inhibitors.

PDF file - 3718K, TMEM16A shRNA resistant DNA can rescue the expression from the stable shRNA knockdown.

PDF file - 472K, Over expression of TMEM16A in T24 cancer cells decreases motility and invasion.

PDF file - 78K, TMEM16A expression levels in primary SCCHN does not correlate with the presence of nodal metastases.

PDF file - 292K, Table of values for proteomics screen.

Supplementary Legend

Characterization of HER2 transactivation by FGFR2 in HER2 targeted therapies resistant cell lines

PDF file - 267K, Proposed model describing the role of TMEM16A in tumor progression.

Purpose: Tumor metastasis is the leading cause of death in patients with cancer. However, the mechanisms that underlie metastatic progression remain unclear. We examined TMEM16A (ANO1) expression as a key factor shifting tumors between growth and metastasis.Experimental Design: We evaluated 26 pairs of primary and metastatic lymph node (LN) tissue from patients with squamous cell carcinoma of the head and neck (SCCHN) for differential expression of TMEM16A. In addition, we identified mechanisms by which TMEM16A expression influences tumor cell motility via proteomic screens of cell lines and in vivo mouse studies of metastasis.Results: Compared with primary tumors, TMEM16A expression decreases in metastatic LNs of patients with SCCHN. Stable reduction of TMEM16A expression enhances cell motility and increases metastases while decreasing tumor proliferation in an orthotopic mouse model. Evaluation of human tumor tissues suggests an epigenetic mechanism for decreasing TMEM16A expression through promoter methylation that correlated with a transition between an epithelial and a mesenchymal phenotype. These effects of TMEM16A expression on tumor cell size and epithelial-to-mesenchymal transition (EMT) required the amino acid residue serine 970 (S970); however, mutation of S970 to alanine does not disrupt the proliferative advantages of TMEM16A overexpression. Furthermore, S970 mediates the association of TMEM16A with Radixin, an actin-scaffolding protein implicated in EMT.Conclusions: Together, our results identify TMEM16A, an eight transmembrane domain Ca2+-activated Cl− channel, as a primary driver of the “Grow” or “Go” model for cancer progression, in which TMEM16A expression acts to balance tumor proliferation and metastasis via its promoter methylation. Clin Cancer Res; 20(17); 4673–88. ©2014 AACR.

PDF file - 3396K, Overexpression of TMEM16A S970A increase anchorage dependent and independent tumor cell proliferation.

PDF file - 196K, Table summarizing the antibodies used for immunoblotting and IHC.

PDF file - 202K, Knockdown of TMEM16A in T24 cancer cells decreases motility and migration.

File describing the supplementary material and methods and the table for the antibodies and drugs used for treatment

PDF file - 87K

We present two high-throughput compatible methods to detect the interaction of ectopically expressed (RT-Bind) or endogenously tagged (EndoBind) proteins of interest. Both approaches provide temporal evaluation of dimer formation over an extended duration. Using examples of the Nrf2-KEAP1 and the CRAF-KRAS-G12V interaction, we demonstrate that our method allows for the detection of signal for more than 2 days after substrate addition, allowing for continuous monitoring of endogenous protein-protein interactions in real time., Bill et al describe two high-throughput methods to detect protein-protein interactions in cells in real-time using the split-NanoLuciferase-complementation system. They demonstrate the methods can detect exogenously (RT-bind) or endogenously (EndoBind) expressed proteins, respectively.

Purpose:Despite the therapeutic success of existing HER2-targeted therapies, tumors invariably relapse. This study aimed at identifying new mechanisms responsible for HER2-targeted therapy resistance.Experimental Design:We have used a platform of HER2-targeted therapy–resistant cell lines and primary cultures of healthy and tumor-associated fibroblasts (TAF) to identify new potential targets related to tumor escape from anti-HER2 therapies.Results:We have shown that TAFs promote resistance to HER2-targeted therapies. TAFs produce and secrete high levels of FGF5, which induces FGFR2 activation in the surrounding breast cancer cells. FGFR2 transactivates HER2 via c-Src, leading to resistance to HER2-targeted therapies. In vivo, coinoculating nonresistant cell lines with TAFs results in more aggressive and resistant tumors. Resistant cells activate fibroblasts and secrete FGFR ligands, creating a positive feedback loop that fuels resistance. FGFR2 inhibition not only inhibits HER2 activation, but also induces apoptosis in cells resistant to HER2-targeted therapies. In vivo, inhibitors of FGFR2 reverse resistance and resensitize resistant cells to HER2-targeted therapies. In HER2 patients' samples, α-SMA, FGF5, and FGFR2 contribute to poor outcome and correlate with c-Src activation. Importantly, expression of FGF5 and phospho-HER2 correlated with a reduced pathologic complete response rate in patients with HER2-positive breast cancer treated with neoadjuvant trastuzumab, which highlights the significant role of TAFs/FGF5 in HER2 breast cancer progression and resistance.Conclusions:We have identified the TAF/FGF5/FGFR2/c-Src/HER2 axis as an escape pathway responsible for HER2-targeted therapy resistance in breast cancer, which can be reversed by FGFR inhibitors.

α-Synuclein (αS) and tau have a lot in common. Dyshomeostasis and aggregation of both proteins are central in the pathogenesis of neurodegenerative diseases: Parkinson's disease, dementia with Lewy bodies, multi-system atrophy and other 'synucleinopathies' in the case of αS; Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy and other 'tauopathies' in the case of tau. The aggregated states of αS and tau are found to be (hyper)phosphorylated, but the relevance of the phosphorylation in health or disease is not well understood. Both tau and αS are typically characterized as 'intrinsically disordered' proteins, while both engage in transient interactions with cellular components, thereby undergoing structural changes and context-specific folding. αS transiently binds to (synaptic) vesicles forming a membrane-induced amphipathic helix; tau transiently interacts with microtubules forming an 'extended structure'. The regulation and exact nature of the interactions are not fully understood. Here we review recent and previous insights into the dynamic, transient nature of αS and tau with regard to the mode of interaction with their targets, the dwell-time while bound, and the cis and trans factors underlying the frequent switching between bound and unbound states. These aspects are intimately linked to hypotheses on how subtle changes in the transient behaviors may trigger the earliest steps in the pathogenesis of the respective brain diseases. Based on a deeper understanding of transient αS and tau conformations in the cellular context, new therapeutic strategies may emerge, and it may become clearer why existing approaches have failed or how they could be optimized.

TMEM16A, a Ca2+ -activated Cl- channel, contributes to tumor growth in breast cancer and head and neck squamous cell carcinoma (HNSCC). Here, we investigated whether TMEM16A influences the response to EGFR/HER family-targeting biological therapies. Inhibition of TMEM16A Cl- channel activity in breast cancer cells with HER2 amplification induced a loss of viability. Cells resistant to trastuzumab, a monoclonal antibody targeting HER2, showed an increase in TMEM16A expression and heightened sensitivity to Cl- channel inhibition. Treatment of HNSCC cells with cetuximab, a monoclonal antibody targeting EGFR, and simultaneous TMEM16A suppression led to a pronounced loss of viability. Biochemical analyses of cells subjected to TMEM16A inhibitors or expressing chloride-deficient forms of TMEM16A provide further evidence that TMEM16A channel function may play a role in regulating EGFR/HER2 signaling. These data demonstrate that TMEM16A regulates EGFR and HER2 in growth and survival pathways. Furthermore, in the absence of TMEM16A cotargeting, tumor cells may acquire resistance to EGFR/HER inhibitors. Finally, targeting TMEM16A improves response to biological therapies targeting EGFR/HER family members.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor's biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

// Anke Bill 1 , Abraham Gutierrez 1 , Sucheta Kulkarni 2 , Carolyn Kemp 2 , Debora Bonenfant 3 , Hans Voshol 3 , Umamaheswar Duvvuri 2, 4, * , L. Alex Gaither 1, * 1 Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA 2 University of Pittsburgh, Medical Center, Department of Otolaryngology, Pittsburgh, PA 15213, USA 3 Novartis Institutes for Biomedical Research, Basel, CH-4002, Switzerland 4 VA Pittsburgh HealthCare System, Pittsburgh, PA 15213, USA * These authors have contributed equally to this work Correspondence to: L. Alex Gaither, e-mail: alex.gaither@novartis.com Umamaheswar Duvvuri, e-mail: duvvuriu@upmc.edu Keywords: epidermal growth factor receptor (EGFR), EGFR-targeted therapy, biomarker, calcium-activated chloride channel, protein-protein interaction Received: December 01, 2014 Accepted: February 07, 2015 Published: March 16, 2015 ABSTRACT The epidermal growth factor receptor (EGFR) contributes to the pathogenesis of head&neck squamous cell carcinoma (HNSCC). However, only a subset of HNSCC patients benefit from anti-EGFR targeted therapy. By performing an unbiased proteomics screen, we found that the calcium-activated chloride channel ANO1 interacts with EGFR and facilitates EGFR-signaling in HNSCC. Using structural mutants of EGFR and ANO1 we identified the trans/juxtamembrane domain of EGFR to be critical for the interaction with ANO1. Our results show that ANO1 and EGFR form a functional complex that jointly regulates HNSCC cell proliferation. Expression of ANO1 affected EGFR stability, while EGFR-signaling elevated ANO1 protein levels, establishing a functional and regulatory link between ANO1 and EGFR. Co-inhibition of EGFR and ANO1 had an additive effect on HNSCC cell proliferation, suggesting that co-targeting of ANO1 and EGFR could enhance the clinical potential of EGFR-targeted therapy in HNSCC and might circumvent the development of resistance to single agent therapy. HNSCC cell lines with amplification and high expression of ANO1 showed enhanced sensitivity to Gefitinib, suggesting ANO1 overexpression as a predictive marker for the response to EGFR-targeting agents in HNSCC therapy. Taken together, our results introduce ANO1 as a promising target and/or biomarker for EGFR-directed therapy in HNSCC.

Multiple studies have described the high expression and amplification of Anoctamin 1 (ANO1) in various cancers, including, but not limited to breast cancer, head and neck cancer, gastrointestinal stromal tumors and glioblastoma. ANO1 has been demonstrated to be critical for tumor growth in breast and head and neck cancers through its regulation of EGFR signaling and pathway modulators like MAPK and protein kinase B. However, the discovery of ANO1 as a calcium activated chloride channel came as a surprise to the field and has given rise to many questions. How does a chloride channel promote oncogenesis? Is the chloride channel function of ANO1 important for its role in cancer? Does ANO1 exhibits chloride-independent functions in cancer cells? This review summarizes the current understanding of ANO1's function in cancer, provides a synopsis of the findings addressing the open questions in the field and gives an outlook on the promising future of ANO1 as a potential therapeutic target for the treatment of various cancers.

Multiple studies have described the high expression and amplification of Anoctamin 1 (ANO1) in various cancers, including, but not limited to breast cancer, head and neck cancer, gastrointestinal stromal tumors and glioblastoma. ANO1 has been demonstrated to be critical for tumor growth in breast and head and neck cancers through its regulation of EGFR signaling and pathway modulators like MAPK and protein kinase B. However, the discovery of ANO1 as a calcium activated chloride channel came as a surprise to the field and has given rise to many questions. How does a chloride channel promote oncogenesis? Is the chloride channel function of ANO1 important for its role in cancer? Does ANO1 exhibits chloride-independent functions in cancer cells? This review summarizes the current understanding of ANO1’s function in cancer, provides a synopsis of the findings addressing the open questions in the field and gives an outlook on the promising future of ANO1 as a potential therapeutic target for the treatment of various cancers.

Background: The calcium-activated chloride channel ANO1 is highly expressed in cancer. Results: Inhibition of ANO1 activity alone is not sufficient to inhibit cancer cell proliferation, suggesting a novel function of ANO1 protein in cancer. Conclusion: The ANO1 inhibitor CaCCinh-A01 inhibits cancer cell proliferation by facilitating degradation of ANO1. Significance: Our results may provide a new targeting approach for antitumor therapy in ANO1-amplified cancers., ANO1, a calcium-activated chloride channel, is highly expressed and amplified in human cancers and is a critical survival factor in these cancers. The ANO1 inhibitor CaCCinh-A01 decreases proliferation of ANO1-amplified cell lines; however, the mechanism of action remains elusive. We explored the mechanism behind the inhibitory effect of CaCCinh-A01 on cell proliferation using a combined experimental and in silico approach. We show that inhibition of ANO1 function is not sufficient to diminish proliferation of ANO1-dependent cancer cells. We report that CaCCinh-A01 reduces ANO1 protein levels by facilitating endoplasmic reticulum-associated, proteasomal turnover of ANO1. Washout of CaCCinh-A01 rescued ANO1 protein levels and resumed cell proliferation. Proliferation of newly derived CaCCinh-A01-resistant cell pools was not affected by CaCCinh-A01 as compared with the parental cells. Consistently, CaCCinh-A01 failed to reduce ANO1 protein levels in these cells, whereas ANO1 currents were still inhibited by CaCCinh-A01, indicating that CaCCinh-A01 inhibits cell proliferation by reducing ANO1 protein levels. Furthermore, we employed in silico methods to elucidate novel biological functions of ANO1 inhibitors. Specifically, we derived a pharmacophore model to describe inhibitors capable of promoting ANO1 degradation and report new inhibitors of ANO1-dependent cell proliferation. In summary, our data demonstrate that inhibition of the channel activity of ANO1 is not sufficient to inhibit ANO1-dependent cell proliferation, indicating that the role of ANO1 in cancer only partially depends on its function as a channel. Our results provide an impetus for gaining a deeper understanding of ANO1 modulation in cells and introduce a new targeting approach for antitumor therapy in ANO1-amplified cancers.

TMEM16A, a Ca2+-activated Cl− channel, contributes to tumor growth in breast cancer and head and neck squamous cell carcinoma (HNSCC). Here, we investigated whether TMEM16A influences the response to EGFR/HER family-targeting biological therapies. Inhibition of TMEM16A Cl− channel activity in breast cancer cells with HER2 amplification induced a loss of viability. Cells resistant to trastuzumab, a monoclonal antibody targeting HER2, showed an increase in TMEM16A expression and heightened sensitivity to Cl− channel inhibition. Treatment of HNSCC cells with cetuximab, a monoclonal antibody targeting EGFR, and simultaneous TMEM16A suppression led to a pronounced loss of viability. Biochemical analyses of cells subjected to TMEM16A inhibitors or expressing chloride-deficient forms of TMEM16A provide further evidence that TMEM16A channel function may play a role in regulating EGFR/HER2 signaling. These data demonstrate that TMEM16A regulates EGFR and HER2 in growth and survival pathways. Furthermore, in the absence of TMEM16A co-targeting, tumor cells may acquire resistance to EGFR/HER inhibitors. Finally, targeting TMEM16A improves response to biological therapies targeting EGFR/HER family members.

The beta-amyloid peptide (Abeta) is a major component of the Alzheimer's disease (AD)-associated senile plaques and is generated by sequential cleavage of the beta-amyloid precursor protein (APP) by beta-secretase and gamma-secretase. Since BACE1 initiates Abeta generation it represents a valuable target to interfere with Abeta production and treatment of AD. While the enzymatic activity of BACE1 resides in the extracellular domain, the protein also contains a short cytoplasmic tail (B1-CT). This domain serves as a binding site for at least two proteins, the copper chaperone for superoxide dismutase-1 (CCS), and the Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding (GGA1) protein, and contains a single phosphorylation site. However, the precise role of the B1-CT for the overall biological function of this protein is largely unknown. Functional studies focusing on the activity of this domain would strongly benefit from the availability of domain-specific inhibitors. Here we describe the isolation and characterization of RNA aptamers that selectively target the B1-CT. We show that these RNAs bind to authentic BACE1 and provide evidence that the binding site is restricted to the membrane-proximal half of the C terminus. Aptamer-binding specifically interferes with the recruitment of CCS, but still permits GGA1 association and casein kinase-dependent phosphorylation, consistent with selective binding site targeting within this short peptide. Because phosphorylation and GGA1 binding to B1-CT regulate BACE1 transport, these RNA inhibitors could be applied to investigate B1-CT activity without affecting the subcellular localization of BACE1.

The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases.

Purpose: Tumor metastasis is the leading cause of death in patients with cancer. However, the mechanisms that underlie metastatic progression remain unclear. We examined TMEM16A (ANO1) expression as a key factor shifting tumors between growth and metastasis. Experimental Design: We evaluated 26 pairs of primary and metastatic lymph node (LN) tissue from patients with squamous cell carcinoma of the head and neck (SCCHN) for differential expression of TMEM16A. In addition, we identified mechanisms by which TMEM16A expression influences tumor cell motility via proteomic screens of cell lines and in vivo mouse studies of metastasis. Results: Compared with primary tumors, TMEM16A expression decreases in metastatic LNs of patients with SCCHN. Stable reduction of TMEM16A expression enhances cell motility and increases metastases while decreasing tumor proliferation in an orthotopic mouse model. Evaluation of human tumor tissues suggests an epigenetic mechanism for decreasing TMEM16A expression through promoter methylation that correlated with a transition between an epithelial and a mesenchymal phenotype. These effects of TMEM16A expression on tumor cell size and epithelial-to-mesenchymal transition (EMT) required the amino acid residue serine 970 (S970); however, mutation of S970 to alanine does not disrupt the proliferative advantages of TMEM16A overexpression. Furthermore, S970 mediates the association of TMEM16A with Radixin, an actin-scaffolding protein implicated in EMT. Conclusions: Together, our results identify TMEM16A, an eight transmembrane domain Ca2+-activated Cl− channel, as a primary driver of the “Grow” or “Go” model for cancer progression, in which TMEM16A expression acts to balance tumor proliferation and metastasis via its promoter methylation. Clin Cancer Res; 20(17); 4673–88. ©2014 AACR.

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype β1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation.

(Cell 143, 201–211; October 15, 2010)In Bill et al., we identified the cytohesin ARNO as an ErbB receptor activator that enhances receptor autophosphorylation in H460 and SkBr3 cells. Cell-free reconstitution with purified proteins showed that ARNO enhances EGF receptor autophosphorylation by direct interaction with the intracellular domain of the receptor (EGFR-ICD). We were recently notified by Cell that the laboratory of Dr. Oreste Segatto was unable to replicate these effects in HeLa cells or under cell-free reconstituted conditions. Although we reproducibly observe 1.6-fold ARNO-dependent stimulation of cellular EGFR autophosphorylation and partial colocalization of ARNO with EGFR in plasma membrane sheets of HeLa cells, we cannot reproduce the data showing direct stimulation of EGFR-ICD autophosphorylation by ARNO-Sec7 in a cell-free reconstitution system (shown in Figures 5D and S5D). Thus, the conclusion that ARNO enhances receptor activation by direct interaction with EGFR-ICD is no longer supported by experimental evidence. We maintain the view that cellular EGFR autophosphorylation is enhanced by ARNO, that ARNO and EGFR colocalize at the plasma membrane, and that ARNO binds to the EGFR-ICD. Nevertheless, our inability to reproduce the data in Figures 5D and S5D compromises our confidence in the proposed mechanism of this regulation. Therefore, we wish to retract the paper. We apologize for any inconvenience that may have resulted from its publication. Franziska Thorwith could not be reached about this retraction.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib, have been proven to efficiently inhibit the proliferation of a subset of non small-cell lung cancers (NSCLC). Unfortunately, the majority of NSCLC expressing wild type EGFR is primarily resistant to EGFR-TKI treatment. Here, we show that the proliferation of the gefitinib-resistant NSCLC cell lines H460 and A549 is reduced by the small molecule SecinH3 which indirectly attenuates EGFR activation by inhibition of cytohesins, a class of recently discovered cytoplasmic EGFR activators. SecinH3 and gefitinib showed a synergistic antiproliferative effect, which correlated with a profound inhibition of Akt activation and survivin expression. Treating mice bearing H460 xenografts with SecinH3 showed the antiproliferative and pro-apoptotic effect of SecinH3 in vivo. Our data suggest that targeting the EGFR indirectly by inhibiting its cytoplasmic activators, the cytohesins, has the potential to improve the treatment of primarily EGFR-TKI resistant lung cancers.

A homogeneous fluorescence resonance energy transfer (FRET) system for the real-time monitoring of exchange factor-catalyzed activation of a ras-like small GTPase is described. The underlying design is based on supramolecular template effects exerted by protein-protein interactions between the GTPase adenosine diphosphate ribosylation factor (ARF) and its effector protein GGA3. The GTPase is activated when bound to guanosine triphosphate (GTP) and switched off in its guanosine diphosphate (GDP)-bound state. Both states are accompanied by severe conformational changes that are recognized by GGA3, which only binds the GTPase "on" state. GDP-to-GTP exchange, i.e., GTPase activation, is catalyzed by the guanine nucleotide exchange factor cytohesin-2. When GGA3 and the GTPase ARF1 are labeled with thoroughly selected FRET probes, with simultaneous recording of the fluorescence of an internal tryptophan residue in ARF1, the conformational changes during the activation of the GTPase can be monitored in real time. We applied the FRET system to a multiplex format that allows the simultaneous identification and distinction of small-molecule inhibitors that interfere with the cytohesin-catalyzed ARF1 activation and/or with the interaction between activated ARF1-GTP and GGA3. By screening a library of potential cytohesin inhibitors, predicted by in silico modeling, we identified new inhibitors for the cytohesin-catalyzed GDP/GTP exchange on ARF1 and verified their increased potency in a cell proliferation assay.

Virtual screening (VS) of chemical libraries formatted in silico pro- vides an alternative to experimental high-throughput screening (HTS) for the iden- tification of small molecule modulators of protein function. We have tailored a VS approach combining fingerprint similarity searching and support vector machine modeling toward the identification of small molecular probes for the study of cyto- hesins, a family of cytoplasmic regulator proteins with multiple cellular functions. A total of 40 new structurally diverse inhibitors were identified, and 26 of these compounds were more active than the primary VS template, a single known inhibi- tory chemotype, in at least one of three different assays (guanine nucleotide ex- change, Drosophila insulin signaling, and human leukocyte cell adhesion). More- over, these inhibitors displayed differential inhibitory profiles. Our findings demonstrate that, at least for the cytohesins, computational extrapolation from known active compounds was capable of identifying small molecular probes with highly diversified functional profiles.

Signaling by ErbB receptors requires the activation of their cytoplasmic kinase domains, which is initiated by ligand binding to the receptor ectodomains. Cytoplasmic factors contributing to the activation are unknown. Here we identify members of the cytohesin protein family as such factors. Cytohesin inhibition decreased ErbB receptor autophosphorylation and signaling, whereas cytohesin overexpression stimulated receptor activation. Monitoring epidermal growth factor receptor (EGFR) conformation by anisotropy microscopy together with cell-free reconstitution of cytohesin-dependent receptor autophosphorylation indicate that cytohesins facilitate conformational rearrangements in the intracellular domains of dimerized receptors. Consistent with cytohesins playing a prominent role in ErbB receptor signaling, we found that cytohesin overexpression correlated with EGF signaling pathway activation in human lung adenocarcinomas. Chemical inhibition of cytohesins resulted in reduced proliferation of EGFR-dependent lung cancer cells in vitro and in vivo. Our results establish cytohesins as cytoplasmic conformational activators of ErbB receptors that are of pathophysiological relevance.

Background: Acquired resistance to therapy is a main obstacle in the success of cancer treatment. In HER2+ breast cancer (BC) patient survival has raised considerably after the introduction of the anti-target therapies Trastuzumab and Lapatinib. However, a considerable number of patients develop resistance to anti-HER2 therapies by HER2-related and unrelated mechanisms. Cancer cells displaying HER2 overexpression initially rely on the oncogenic addiction for HER2-controlled survival signaling pathways. However, the fact that some tumors resistant to anti-HER2 therapies still present HER2 amplification suggest the activation of compensatory pathways for the control of cell survival. Objectives: We aimed to identify compensatory survival pathways activated after the acquisition of resistance to Trastuzumab and Lapatinib that could be therapeutically targeted to overcome resistance to anti-HER2 therapies. We focus our efforts in identifying activated RTKs given that they are easily targetable proteins. Experimental Procedures: To identify compensatory pathways related to tumor escape to anti-HER2 therapies we developed an in vitro model of drug resistance by continuous exposure of the MDA-MB-453, BT474, and SKBR3 cell lines to increased concentrations of Trastuzumab or Lapatinib for over 6 months. Activated RTKs were identified by a phosphoproteome array, and their role in cell survival in cell lines resistant to anti-HER2 therapies was validated by siRNA and chemical inhibition experiments. Results: The resistant phenotype of the cell lines was confirmed by determining cell proliferation and apoptosis upon drug treatment. Moreover, the resistant cell lines displayed the more aggressive biological behavior in vitro and in vivo, as evidenced by the increased clonogenic ability and their tumorigenicity in immmunosupressed mice. We did not observed changes in the copy number of 17q12 (HER2) or 7p12 (EGFR) by FISH or in the mRNA levels by qPCR in any of the resistant cell lines, although there was a significant increase in the protein levels as determined by immunohistochemistry. By phosphoproteome array we identified the activation of several RTKs. The INGENUITY pathway analysis of the receptors identified indicated a main relevance for the processes related to cell signaling and cell cycle, cellular growth and proliferation, and more interestingly with axonal guidance signaling and human embryonic stem cell pluripotency. To confirm the involvement of those receptors in the activation of compensatory pathways in Trastuzumab and Lapatinib resistant cell lines, we performed a chemical screening. We further validated the involvement of several receptors in cell survival by siRNA. Conclusion: Several compensatory pathways for cell survival are activated after the acquisition of resistance to Trastuzumab and Lapatinib. The inhibition of such pathways could be used a new therapeutic approach for the treatment of resistant tumors, or to avoid the development of resistant cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A60. Citation Format: Patricia Fernandez Nogueira, Anke Bill, Inga Rye, Domiziana Costamagna, Hege Russnes, Pedro Gascón, Alex Gaither, Vanessa Almendro. Activation of compensatory receptor tyrosine kinases after the acquisition of resistance to anti-Her2 therapies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A60.

Genomic alterations are the underlining cause of many human cancers: Amplified and overexpressed genes can drive neoplastic transformation and become essential survival factors for cancer cells. Thus, they represent promising targets for anti-cancer therapies and their identification and validation is of paramount importance. In a search for novel survival factors contributing to breast cancer oncogenesis, we performed genomic fine mapping of the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human neoplasia, in a large dataset of breast cancer patients. Remarkably, we found ANO1, a calcium-activated chloride channel, to be located within the summit of the most frequently and highly amplified genomic region. Furthermore, amplification of ANO1 correlates with its overexpression and elevated protein levels and is a negative predictor for overall survival in breast cancer patients. Next, we examined how ANO1 contributes to cancer cell survival and proliferation. We found that inhibition of ANO1 expression or function reduced cancer cell viability and colony formation, and triggered apoptosis in 11q13 amplified breast cancer, HNSCC and ESCC. Furthermore, expression of ANO1 in non-transformed mammary cells increased cell viability and established a novel addiction to ANO1 biochemical activity. We next applied inducible shRNAs against ANO1 in vivo to assess the effect of ANO1 inhibition on the maintenance of established tumors. We found in four different 11q13-amplified tumor models that knockdown of ANO1 reduced tumor growth, indicating an important role for ANO1 not only in oncogenesis, but also in tumor maintenance of 11q13 amplified cancers. To explore the mechanism by which inhibition of ANO1 contributes to cancer cell viability and tumor growth, we performed antibody arrays to measure the activity of major oncogenic signaling pathways after knockdown of ANO1. Upon depletion of ANO1, activation of EGFR and several survival signaling pathways (AKT-, SRC- and ERK1/2 pathways) were reduced in different models of human cancer. Subsequent experiments showed that ANO1 modulates both EGFR- and Ca2+/calmodulin-dependent protein kinase (CAMK) signaling in breast cancer and HNSCC cells. Consistently, activation of EGFR- and CAMK correlated with the expression of ANO1 in human primary breast tumor samples. Lastly, we found that only the simultaneous stimulation of EGFR- and CAMK-signaling pathways rescued the effect of the ANO1-inhibition. In summary, our study establishes ANO1 as a key tumor-promoting factor in 11q13-amplified breast and other malignancies. Our results highlight the importance of chloride channels in cancer and provide the first detailed mechanistic insight into the activity of ANO1 in tumorigenesis. Most importantly, our findings open up new opportunities for therapeutic intervention in several prevalent cancers. Citation Format: Adrian Britschgi, Anke Bill, Heike Brinkhaus, Christopher Rothwell, Ieuan Clay, Stephan Duss, Michael Rebhan, Pichai Raman, Chantale Guy, Kristie Wetzel, Elizabeth George, M. Oana Popa, Sarah Lilley, Hedaythul Choudhury, Martin Gosling, Louis Wang, Stephanie Fitzgerald, Jason Borawski, Jonathan Baffoe, Mark Labow, L. Alex Gaither, Mohamed Bentires-Alj. The calcium activated chloride channel ANO1 promotes breast cancer progression by activating EGFR- and CAMK-signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-205. doi:10.1158/1538-7445.AM2013-LB-205

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).This article has been retracted at the request of the authors.In Bill et al., we identified the cytohesin ARNO as an ErbB receptor activator that enhances receptor autophosphorylation in H460 and SkBr3 cells. Cell-free reconstitution with purified proteins showed that ARNO enhances EGF receptor autophosphorylation by direct interaction with the intracellular domain of the receptor (EGFR-ICD). We were recently notified by Cell that the laboratory of Dr. Oreste Segatto was unable to replicate these effects in HeLa cells or under cell-free reconstituted conditions. Although we reproducibly observe 1.6-fold ARNO-dependent stimulation of cellular EGFR autophosphorylation and partial colocalization of ARNO with EGFR in plasma membrane sheets of HeLa cells, we cannot reproduce the data showing direct stimulation of EGFR-ICD autophosphorylation by ARNO-Sec7 in a cell-free reconstitution system (shown in Figures 5D and S5D). Thus, the conclusion that ARNO enhances receptor activation by direct interaction with EGFR-ICD is no longer supported by experimental evidence. We maintain the view that cellular EGFR autophosphorylation is enhanced by ARNO, that ARNO and EGFR colocalize at the plasma membrane, and that ARNO binds to the EGFR-ICD. Nevertheless, our inability to reproduce the data in Figures 5D and S5D compromises our confidence in the proposed mechanism of this regulation. Therefore, we wish to retract the paper. We apologize for any inconvenience that may have resulted from its publication. Franziska Thorwith could not be reached about this retraction.