Novel integrin endocytosis motif

Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-μ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions.We gratefully acknowledge the following funding sources: N.d.F. FinPharma Doctoral Program, Instrumentarium Foundation, Orion Research Foundation, Liv och Halsa foundation, Finsk-Norska Medicinska Stiftelsen and the Magnus Ehrnrooth Foundation; J.I. Academy of Finland CoE, European Research Council Consolidator Grant, the Sigrid Juselius Foundation, The Finnish Heart Foundation and Finnish Cancer Organizations. DJO, AGW and TW are funded by Wellcome Trust fellowship 090909 (DJO).This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nsmb.3161

Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells

Estrogen receptor α (ERα) is expressed in tissues as diverse as brains and mammary glands. In breast cancer, ERα is a key regulator of tumor progression. Therefore, understanding what activates ERα is critical for cancer treatment in particular and cell biology in general. Using biochemical approaches and superresolution microscopy, we show that estrogen drives membrane ERα into endosomes in breast cancer cells and that its fate is determined by the presence of fibronectin (FN) in the extracellular matrix; it is trafficked to lysosomes in the absence of FN and avoids the lysosomal compartment in its presence. In this context, FN prolongs ERα half-life and strengthens its transcriptional activity. We show that ERα is associated with β1-integrin at the membrane, and this integrin follows the same endocytosis and subcellular trafficking pathway triggered by estrogen. Moreover, ERα+ vesicles are present within human breast tissues, and colocalization with β1-integrin is detected primarily in tumors. Our work unravels a key, clinically relevant mechanism of microenvironmental regulation of ERα signaling.Fil: Sampayo, Rocío Guadalupe. Universidad Nacional de San Martin. Instituto de Nanosistemas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Toscani, Andrés Martin. Universidad Nacional de Luján; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Rubashkin, Matthew G.. University of California; Estados UnidosFil: Thi, Kate. Lawrence Berkeley National Laboratory; Estados UnidosFil: Masullo, Luciano Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Violi, Ianina Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Lakins, Jonathon N.. University of California; Estados UnidosFil: Caceres, Alfredo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Hines, William C.. Lawrence Berkeley National Laboratory; Estados UnidosFil: Coluccio Leskow, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad Nacional de Luján; ArgentinaFil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Chialvo, Dante Renato. Universidad de Buenos Aires; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro Internacional de Estudios Avanzados; ArgentinaFil: Bissell, Mina J.. Lawrence Berkeley National Laboratory; Estados UnidosFil: Weaver, Valerie M.. University of California; Estados UnidosFil: Simian, Marina. Universidad Nacional de San Martin. Instituto de Nanosistemas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentin

Ex vivo modelling of drug efficacy in a rare metastatic urachal carcinoma

Background Ex vivo drug screening refers to the out-of-body assessment of drug efficacy in patient derived vital tumor cells. The purpose of these methods is to enable functional testing of patient specific efficacy of anti-cancer therapeutics and personalized treatment strategies. Such approaches could prove powerful especially in context of rare cancers for which demonstration of novel therapies is difficult due to the low numbers of patients. Here, we report comparison of different ex vivo drug screening methods in a metastatic urachal adenocarcinoma, a rare and aggressive non-urothelial bladder malignancy that arises from the remnant embryologic urachus in adults. Methods To compare the feasibility and results obtained with alternative ex vivo drug screening techniques, we used three different approaches; enzymatic cell viability assay of 2D cell cultures and image-based cytometry of 2D and 3D cell cultures in parallel. Vital tumor cells isolated from a biopsy obtained in context of a surgical debulking procedure were used for screening of 1160 drugs with the aim to evaluate patterns of efficacy in the urachal cancer cells. Results Dose response data from the enzymatic cell viability assay and the image-based assay of 2D cell cultures showed the best consistency. With 3D cell culture conditions, the proliferation rate of the tumor cells was slower and potency of several drugs was reduced even following growth rate normalization of the responses. MEK, mTOR, and MET inhibitors were identified as the most cytotoxic targeted drugs. Secondary validation analyses confirmed the efficacy of these drugs also with the new human urachal adenocarcinoma cell line (MISB18) established from the patient’s tumor. Conclusions All the tested ex vivo drug screening methods captured the patient’s tumor cells’ sensitivity to drugs that could be associated with the oncogenic KRASG12V mutation found in the patient’s tumor cells. Specific drug classes however resulted in differential dose response profiles dependent on the used cell culture method indicating that the choice of assay could bias results from ex vivo drug screening assays for selected drug classes

Beta 1-integrin-c-Met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/R.B.M. was a recipient of a UK Medical Research Council (MRC) studentship, MRC Centenary Award, Barts and The London Charity (472/1711) and Rosetrees Trust (M314), N.K. was a recipient of an MRC studentship (MR/J500409/1), C.J. was a recipient of the Barts and The London Charitable Foundation Scholarship (RAB 05/PJ/07), L.M. was supported by CR-UK, Breast Cancer Now (2008NovPR10) and Rosetrees Trust (M346), A.H. was a recipient of a CR-UK studentship (C236/A11795). P.J.P. was supported by CR-UK. J.I. was supported by grants from the Academy of Finland, ERC Starting grant, Finnish Cancer Organisations and Sigrid Juselius Foundation. S.K. was supported by the MRC (G0501003) and The British Lung Foundation (CAN09-4)

Image-based ex vivo drug screen to assess targeted therapies in recurrent thymoma

Objectives Thymoma is a rare malignancy derived from the thymic epithelial cells. No standard salvage treatments are available for recurrent thymoma and due to the low number of cases, alternative treatment regimens have been assessed only in small case series with varying success. The aim of this study was to use an image-based ex vivo drug screening strategy to assess efficacy of a large panel of anti-cancer agents for thymoma using patient derived tumor cells. Materials and Methods Vital tumor and tumor associated cells were used to assess the efficacy of 147 anti-cancer drugs including approved and experimental agents. Drug efficacy was analyzed at single cell resolution using image-based high content drug screening to assess tumor cell specific responses. Molecular profiling and histopathology was used to confirm the drug targets identified by the screen. Results The ex vivo drug screen identified selective sensitivity of the cancerous epithelial thymoma cells to EGFR-, HDAC- and mTOR-inhibition. Histopathology confirmed high protein level expression of EGFR in the patient’s tumor. Patient was initiated treatment with Cetuximab resulting in stable disease after relapse on five different chemotherapy regimens. Conclusion The results show that the image-based ex vivo therapy efficacy screening strategy can be used to identify patient and tumor relevant drug sensitivity patterns in thymoma. The results also warrant continued research on EGFR as a biomarker and therapy target in recurrent thymomas

Formin-like 2 promotes beta 1-integrin trafficking and invasive motility downstream of PKC alpha.

Regulated turnover of integrin receptors is essential for cell adhesion and migration. Pathways selectively regulating beta 1-integrin recycling are implicated in cancer invasion and metastasis, yet proteins required for the internalization of this pro-invasive integrin remain to be identified. Here, we uncover formin-like 2 (FMNL2) as a critical regulator of beta 1-integrin internalization downstream of protein kinase C (PKC). PKC alpha associates with and phosphorylates FMNL2 at S1072 within its Diaphanous autoregulatory region, leading to the release of formin autoinhibition. Phosphorylation of FMNL2 triggers its rapid relocation and promotes its interaction with the cytoplasmic tails of the a-integrin subunits for beta 1-integrin endocytosis. FMNL2 drives beta 1-integrin internalization and invasive motility in a phosphorylationdependent manner, while a FMNL2 mutant defective in actin assembly interferes with beta 1-integrin endocytosis and cancer cell invasion. Our data establish a role for FMNL2 in the regulation of beta 1-integrin and provide a mechanistic understanding of the function of FMNL2 in cancer invasiveness

Lamellipodin promotes invasive 3D cancer cell migration via regulated interactions with Ena/VASP and SCAR/WAVE

Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlate with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement, we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation and matrix degradation was impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not with Ena/VASP is required for random 2D cell migration. We identified a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, whereas Src-dependent phosphorylation enhances binding to Scar/WAVE but not to Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of epidermal growth factor (EGF) gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis.Virginia and D.K. Ludwig Fund for Cancer Research (Postdoctoral fellowship)King's College London (Overseas Research PhD Studentship (KORS))National Cancer Institute (U.S.) (U54-CA112967)National Cancer Institute (U.S.) (U54-CA163109)Ludwig Center for Molecular Oncology at MITDavid H. Koch Institute for Integrative Cancer Research at MIT (Support Grant P30-CA14051)National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051)Biotechnology and Biological Sciences Research Council (Great Britain) (BB/F011431/1)Biotechnology and Biological Sciences Research Council (Great Britain) (BB/J000590/1)Biotechnology and Biological Sciences Research Council (Great Britain) (BB/N000226/1)Wellcome Trust (London, England) (082907/Z/07/Z