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Claus Vogelmeier,1 Nanshan Zhong,2 Michael J Humphries,3 Karen Mezzi,4 Robert Fogel,5 Giovanni Bader,4 Francesco Patalano,4 Donald Banerji5 1Department of Medicine, Pulmonary and Critical Care Medicine, University Medical Center Giessen and Marburg, Philipps-University Marburg, Member of the German Center for Lung Research (DZL), Marburg, Germany; 2State Key Laboratory, Guangzhou Institute of Respiratory Diseases, First Affiliated Hospital, Guangzhou Medical University, Guangzhou, 3Beijing Novartis Pharma Co. Ltd., Shanghai, People’s Republic of China; 4Novartis Pharma AG, Basel, Switzerland; 5Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA Background: Indacaterol/glycopyrronium (IND/GLY) is approved for maintenance treatment of adult patients with COPD. This post hoc analysis explored the efficacy and safety of IND/GLY versus salmeterol/fluticasone (SFC) in symptomatic (Global Initiative for Chronic Obstructive Lung Disease [GOLD] B and GOLD D) patients with moderate-to-severe COPD.Patients and methods: Data from LANTERN and ILLUMINATE studies were pooled and analyzed. In both studies, symptomatic COPD patients were randomized to once-daily IND/GLY 110 µg/50 µg or twice-daily SFC 50 µg/500 µg. End points were pre-dose trough forced expiratory volume in one second (FEV1), standardized area under the curve for FEV1 from 0 to 12 hours (FEV1 AUC0–12 hours), peak FEV1, peak forced vital capacity (FVC), pre-dose trough FVC, Transition Dyspnea Index (TDI) total score, St George’s Respiratory Questionnaire total score, rescue medication use and safety.Results: A total of 1,263 patients were classified as either GOLD B (n=809) or GOLD D (n=454). At week 26, IND/GLY demonstrated statistically significant improvement in all lung function parameters versus SFC in patients in both the GOLD B and GOLD D subgroups. TDI total score and rescue medication use were significantly improved with IND/GLY versus SFC in the overall population and in the GOLD B (TDI total score only) and GOLD D (rescue medication only) subgroups. IND/GLY also reduced the rate of exacerbations in the pooled population. Overall safety profile was comparable with a higher incidence of pneumonia in the SFC-treated group.Conclusion: In this pooled analysis, IND/GLY demonstrated superior efficacy compared with SFC in patients in the GOLD B and GOLD D subgroups and supported its use in symptomatic COPD patients. Keywords: COPD, exacerbation, LABA/LAMA, LABA/ICS, lung function

Rabbit synovial fibroblasts (RSF) express basal levels of the metalloproteinases (MMP) collagenase, stromelysin-1 and 92-kD gelatinase when plated on intact fibronectin (FN), but elevated levels when plated on either the central RGD-containing cell-binding region of FN (120FN) or antibody against the alpha 5 beta 1 integrin, suggesting that domains outside 120FN may suppress the induction of MMP (Werb, Z., P. M. Tremble, O. Behrendtsen, E. Crowley, and C.H. Damsky. 1989. J. Cell Biol. 109:877-889). We therefore attempted to reconstitute the basal signaling of intact FN by plating RSF on 120FN together with domains of FN outside this region. Large COOH-terminal fragments containing both the heparin-binding and HICS domains suppressed MMP when combined with 120FN. To map the active sequences, peptides from this region and larger fragments that did, or did not, include the CS-1 portion of IIICS were tested. Only CS-1 peptide, or larger fragments containing CS-1, suppressed MMP expression induced by 120FN. In contrast, peptide V from the heparin-binding region, shown previously to stimulate focal contact formation, further enhanced MMP expression by RSF when present on the substrate with 120FN. RSF expressed alpha 4 beta 1 integrin, the receptor for CS-1, and the anti-alpha 4 mAb blocked the ability of CS-1 to suppress MMP induction by 120FN. These results show that signals modulating MMP expression and focal contact assembly are regulated independently, and that cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins plays a dominant role in regulating expression of these extracellular matrix-remodeling genes in response to FN. This work demonstrates directly the modular way in which information in the extracellular matrix is detected and processed by cell surface receptors.

Radiotherapy of prostate cancer (PC) can lead to the acquisition of radioresistance through molecular mechanisms that involve, in part, cell adhesion-mediated signaling. To define these mechanisms, we employed a DU145 PC model to conduct a comparative mass spectrometry-based proteomic analysis of the purified integrin nexus, i.e., the cell-matrix junction where integrins bridge assembled extracellular matrix (matrisome components) to adhesion signaling complexes (adhesome components). When parental and radioresistant cells were compared, the expression of integrins was not changed, but cell radioresistance was associated with extensive matrix remodeling and changes in the complement of adhesion signaling proteins. Out of 72 proteins differentially expressed in the parental and radioresistant cells, four proteins were selected for functional validation based on their correlation with biochemical recurrence-free survival. Perlecan/heparan sulfate proteoglycan 2 (HSPG2) and lysyl-like oxidase-like 2 (LOXL2) were upregulated, while sushi repeat-containing protein X-linked (SRPX) and laminin subunit beta 3 (LAMB3) were downregulated in radioresistant DU145 cells. Knockdown of perlecan/HSPG2 sensitized radioresistant DU145 RR cells to irradiation while the sensitivity of DU145 parental cells did not change, indicating a potential role for perlecan/HSPG2 and its associated proteins in suppressing tumor radioresistance. Validation in androgen-sensitive parental and radioresistant LNCaP cells further supported perlecan/HSPG2 as a regulator of cell radiosensitivity. These findings extend our understanding of the interplay between extracellular matrix remodeling and PC radioresistance and signpost perlecan/HSPG2 as a potential therapeutic target and biomarker for PC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Samaržija, Lukiyanchuk, Lončarić, Rac-Justament, Stojanović, Gorodetska, Kahya, Humphries, Fatima, Humphries, Fröbe, Dubrovska and Ambriović-Ristov.)

CAR (CARSKNKDC) is a wound-homing peptide that recognises angiogenic neovessels. Here we discover that systemically administered CAR peptide has inherent ability to promote wound healing: wounds close and re-epithelialise faster in CAR-treated male mice. CAR promotes keratinocyte migration in vitro. The heparan sulfate proteoglycan syndecan-4 regulates cell migration and is crucial for wound healing. We report that syndecan-4 expression is restricted to epidermis and blood vessels in mice skin wounds. Syndecan-4 regulates binding and internalisation of CAR peptide and CAR-mediated cytoskeletal remodelling. CAR induces syndecan-4-dependent activation of the small GTPase ARF6, via the guanine nucleotide exchange factor cytohesin-2, and promotes syndecan-4-, ARF6- and Cytohesin-2-mediated keratinocyte migration. Finally, we show that genetic ablation of syndecan-4 in male mice eliminates CAR-induced wound re-epithelialisation following systemic administration. We propose that CAR peptide activates syndecan-4 functions to selectively promote re-epithelialisation. Thus, CAR peptide provides a therapeutic approach to enhance wound healing in mice; systemic, yet target organ- and cell-specific., (© 2023. The Author(s).)

Introduction: Vasculogenic mimicry (VM), the process of tumor cell transdifferentiation to endow endothelial-like characteristics supporting de novo vessel formation, is associated with poor prognosis in several tumor types, including SCLC. In genetically engineered mouse models (GEMMs) of SCLC, NOTCH, and MYC co-operate to drive a neuroendocrine (NE) to non-NE phenotypic switch, and co-operation between NE and non-NE cells is required for metastasis. Here, we define the phenotype of VM-competent cells and molecular mechanisms underpinning SCLC VM using circulating tumor cell-derived explant (CDX) models and GEMMs., Methods: We analyzed perfusion within VM vessels and their association with NE and non-NE phenotypes using multiplex immunohistochemistry in CDX, GEMMs, and patient biopsies. We evaluated their three-dimensional structure and defined collagen-integrin interactions., Results: We found that VM vessels are present in 23/25 CDX models, 2 GEMMs, and in 20 patient biopsies of SCLC. Perfused VM vessels support tumor growth and only NOTCH-active non-NE cells are VM-competent in vivo and ex vivo, expressing pseudohypoxia, blood vessel development, and extracellular matrix organization signatures. On Matrigel, VM-primed non-NE cells remodel extracellular matrix into hollow tubules in an integrin β1-dependent process., Conclusions: We identified VM as an exemplar of functional heterogeneity and plasticity in SCLC and these findings take considerable steps toward understanding the molecular events that enable VM. These results support therapeutic co-targeting of both NE and non-NE cells to curtail SCLC progression and to improve the outcomes of patients with SCLC in the future., (Copyright © 2023 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Aims: Endothelial erosion of plaques is responsible for ∼30% of acute coronary syndromes (ACS). Smoking is a risk factor for plaque erosion, which most frequently occurs on the upstream surface of plaques where the endothelium experiences elevated shear stress. We sought to recreate these conditions in vitro to identify potential pathological mechanisms that might be of relevance to plaque erosion., Methods and Results: Culturing human coronary artery endothelial cells (HCAECs) under elevated flow (shear stress of 7.5 Pa) and chronically exposing them to cigarette smoke extract (CSE) and tumour necrosis factor-alpha (TNFα) recapitulated a defect in HCAEC adhesion, which corresponded with augmented Nrf2-regulated gene expression. Pharmacological activation or adenoviral overexpression of Nrf2 triggered endothelial detachment, identifying Nrf2 as a mediator of endothelial detachment. Growth/Differentiation Factor-15 (GDF15) expression was elevated in this model, with protein expression elevated in the plasma of patients experiencing plaque erosion compared with plaque rupture. The expression of two Nrf2-regulated genes, OSGIN1 and OSGIN2, was increased by CSE and TNFα under elevated flow and was also elevated in the aortas of mice exposed to cigarette smoke in vivo. Knockdown of OSGIN1&2 inhibited Nrf2-induced cell detachment. Overexpression of OSGIN1&2 induced endothelial detachment and resulted in cell cycle arrest, induction of senescence, loss of focal adhesions and actin stress fibres, and disturbed proteostasis mediated in part by HSP70, restoration of which reduced HCAEC detachment. In ACS patients who smoked, blood concentrations of HSP70 were elevated in plaque erosion compared with plaque rupture., Conclusion: We identified a novel Nrf2-OSGIN1&2-HSP70 axis that regulates endothelial adhesion, elevated GDF15 and HSP70 as biomarkers for plaque erosion in patients who smoke, and two therapeutic targets that offer the potential for reducing the risk of plaque erosion., Competing Interests: Conflict of interest: None of the authors have received any financial, personal, or professional support, other than the funding disclosed above that has a bearing on the data presented here. Dr Libby is an unpaid consultant to or involved in clinical trials for Amgen, AstraZeneca, Baim Institute, Beren Therapeutics, Esperion Therapeutics, Genentech, Kancera, Kowa Pharmaceuticals, Medimmune, Merck, Norvo Nordisk, Novartis, Pfizer, and Sanofi-Regeneron. Dr Libby is a member of the scientific advisory board for Amgen, Caristo Diagnostics, Cartesian Therapeutics, CSL Behring, DalCor Pharmaceuticals, Dewpoint Therapeutics, Kancera, Kowa Pharmaceuticals, Olatec Therapeutics, Medimmune, Novartis, PlaqueTec, TenSixteen Bio, and XBiotech, Inc. Dr Libby’s laboratory has received research funding in the last 2 years from Novartis. Dr Libby is on the Board of Directors of XBiotech, Inc. Dr Libby has a financial interest in XBiotech, a company developing therapeutic human antibodies. Dr Libby has a financial interest in TenSixteen Bio, a company targeting somatic mosaicism and clonal haematopoiesis of indeterminate potential (CHIP) to discover and develop novel therapeutics to treat age-related diseases. Dr Libby’s interests were reviewed and managed by Brigham and Women’s Hospital and Mass General Brigham in accordance with their conflict-of-interest policies., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: Focal adhesions (FAs) are integrin-containing, multi-protein structures that link intracellular actin to the extracellular matrix and trigger multiple signaling pathways that control cell proliferation, differentiation, survival and motility. Microtubules (MTs) are stabilized in the vicinity of FAs through interaction with the components of the cortical microtubule stabilizing complex (CMSC). KANK (KN motif and ankyrin repeat domains) family proteins within the CMSC, KANK1 or KANK2, bind talin within FAs and thus mediate actin-MT crosstalk. We previously identified in MDA-MB-435S cells, which preferentially use integrin αVβ5 for adhesion, KANK2 as a key molecule enabling the actin-MT crosstalk. KANK2 knockdown also resulted in increased sensitivity to MT poisons, paclitaxel (PTX) and vincristine and reduced migration. Here, we aimed to analyze whether KANK1 has a similar role and to distinguish which talin isoform binds KANK2., Methods: The cell model consisted of human melanoma cell line MDA-MB-435S and stably transfected clone with decreased expression of integrin αV (3αV). For transient knockdown of talin1, talin2, KANK1 or KANK2 we used gene-specific siRNAs transfection. Using previously standardized protocol we isolated integrin adhesion complexes. SDS-PAGE and Western blot was used for protein expression analysis. The immunofluorescence analysis and live cell imaging was done using confocal microscopy. Cell migration was analyzed with Transwell Cell Culture Inserts. Statistical analysis using GraphPad Software consisted of either one-way analysis of variance (ANOVA), unpaired Student's t-test or two-way ANOVA analysis., Results: We show that KANK1 is not a part of the CMSC associated with integrin αVβ5 FAs and its knockdown did not affect the velocity of MT growth or cell sensitivity to PTX. The talin2 knockdown mimicked KANK2 knockdown i.e. led to the perturbation of actin-MT crosstalk, which is indicated by the increased velocity of MT growth and increased sensitivity to PTX and also reduced migration., Conclusion: We conclude that KANK2 functionally interacts with talin2 and that the mechanism of increased sensitivity to PTX involves changes in microtubule dynamics. These data elucidate a cell-type-specific role of talin2 and KANK2 isoforms and we propose that talin2 and KANK2 are therefore potential therapeutic targets for improved cancer therapy., (© 2023. The Author(s).)

Connexin37-mediated regulation of cell cycle modulators and, consequently, growth arrest lack mechanistic understanding. We previously showed that arterial shear stress up-regulates Cx37 in endothelial cells and activates a Notch/Cx37/p27 signaling axis to promote G1 cell cycle arrest, and this is required to enable arterial gene expression. However, how induced expression of a gap junction protein, Cx37, up-regulates cyclin-dependent kinase inhibitor p27 to enable endothelial growth suppression and arterial specification is unclear. Herein, we fill this knowledge gap by expressing wild-type and regulatory domain mutants of Cx37 in cultured endothelial cells expressing the Fucci cell cycle reporter. We determined that both the channel-forming and cytoplasmic tail domains of Cx37 are required for p27 up-regulation and late G1 arrest. Mechanistically, the cytoplasmic tail domain of Cx37 interacts with, and sequesters, activated ERK in the cytoplasm. This then stabilizes pERK nuclear target Foxo3a, which up-regulates p27 transcription. Consistent with previous studies, we found this Cx37/pERK/Foxo3a/p27 signaling axis functions downstream of arterial shear stress to promote endothelial late G1 state and enable up-regulation of arterial genes., (© 2023 Acharya et al.)

Introduction: Patients with non-small-cell lung cancer (NSCLC) whose tumors harbor anaplastic lymphoma kinase (ALK) rearrangements can be treated with ALK tyrosine kinase inhibitors. We assessed real-world ALK biomarker testing and treatment patterns of patients with NSCLC in the United States., Patients and Methods: Data were extracted from the Flatiron Health electronic health record-derived deidentified database for patients aged ≥18 years with stage IIIB or IV NSCLC and ≥2 clinic visits between January 2011 and December 2019., Results: Among 60,025 eligible patients, tumors from 36,691 (61.1%) patients were tested for ALK rearrangements, and 1042 (2.8%) tested positive (ALK+). From 2011 to 2019, ALK testing rates increased from 33.1% to 73.0%; testing via fluorescence in situ hybridization declined from 68.3% to 32.1% while next-generation sequencing increased from <1% to 52.2%. Although tissue samples were more commonly used than blood (85.1% vs. 13.5% of tests), blood sample testing increased from 0.1% in 2011 to 28.2% in 2019. Median (interquartile range) time from diagnosis of advanced NSCLC to first ALK+ test result was 23 (13-43) days, including laboratory processing time of 9 (6-14) days. For the 24.7% of patients with an ALK+ test result who began treatment before receiving the positive result, chemotherapy was initiated most often overall until 2018 when immuno-oncology agents became most common., Conclusion: Although ALK testing in NSCLC increased over time, testing rates among eligible patients did not reach 100% during the study period. Treatment decisions for some patients with NSCLC may have been made without important, guideline-recommended biomarker data., (Copyright © 2022. Published by Elsevier Inc.)

Modulation of integrin function is required in many physiological and pathological settings, such as angiogenesis and cancer. Integrin allosteric changes, clustering, and trafficking cooperate to regulate cell adhesion and motility on extracellular matrix proteins via mechanisms that are partly defined. By exploiting four monoclonal antibodies recognizing distinct conformational epitopes, we show that in endothelial cells (ECs), the extracellular βI domain, but not the hybrid or I-EGF2 domain of active β1 integrins, promotes their FAK-regulated clustering into tensin 1-containing fibrillar adhesions and impairs their endocytosis. In this regard, the βI domain-dependent clustering of active β1 integrins is necessary to favor fibronectin-elicited directional EC motility, which cannot be effectively promoted by β1 integrin conformational activation alone., (© 2022 Mana et al.)

BACKGROUND: The discovery of specific oncogenic drivers in non-small cell lung cancer (NSCLC) has led to the development of highly targeted anaplastic lymphoma kinase tyrosine kinase inhibitors (ALKis). Brigatinib is a next-generation ALKi associated with prolonged progression-free survival in patients with ALKi-naive ALK + NSCLC. OBJECTIVE: To estimate the cost-effectiveness of brigatinib compared with crizotinib and alectinib in patients with ALKi-naive ALK + NSCLC, from a US payer perspective. METHODS: A lifetime area under the curve-partitioned survival model with 4 health states was used to evaluate the relative cost-effectiveness of brigatinib in the ALKi-naive ALK + NSCLC setting. Brigatinib was compared with crizotinib within a cost-effectiveness framework and compared with alectinib in a cost-comparison framework, where all efficacy outcomes were assumed equal. The efficacy of brigatinib and crizotinib was informed by the ALTA-1L trial, and an indirect treatment comparison was performed to inform the efficacy of brigatinib vs alectinib owing to a lack of head-to-head data. Costs were derived from public sources. The main outcomes of the model were total costs, quality-adjusted life-years (QALYs), life-years, and incremental cost-effectiveness ratios. Univariate and probabilistic sensitivity analyses, in addition to multiple scenario analyses, were conducted to assess the robustness of the model outcomes. RESULTS: The improved outcomes observed in ALTA-1L translated into QALY gains (+0.97) in the comparison of brigatinib vs crizotinib. The superior efficacy profile was associated with increased time on treatment with brigatinib, which drove the increase in costs vs crizotinib (+$210,519). The resulting base-case incremental cost-effectiveness ratio was $217,607/QALY gained. Compared with alectinib, brigatinib was associated with a cost difference of -$8,546. Sensitivity analysis suggested that extrapolation of overall survival, the assumptions relating to time on treatment, and subsequent therapy costs were the most influential determinants of results. Probabilistic sensitivity analysis suggested brigatinib had the highest probability of being cost-effective beyond willingness-to-pay thresholds of $236,000 per QALY vs crizotinib and alectinib. CONCLUSIONS: At list prices and under base-case assumptions in the current analysis, brigatinib was associated with cost-savings vs alectinib, and QALY gains but at higher costs vs crizotinib. Additional research into the real-world efficacy of ALKis is warranted to further understand the comparative cost-effectiveness of these therapies. DISCLOSURES: Ms Cranmer and Ms Kearns are employees of Takeda UK Ltd. Dr Young is a former employee of Takeda Pharmaceuticals America, Inc. Dr Humphries is an employee of Takeda Pharmaceuticals U.S.A., Inc. Mr Trueman is an employee of Source Health Economics, the consultancy company that provided health economic and writing services. This work was funded by ARIAD Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited. Work by Source Health Economics was funded by ARIAD Pharmaceuticals, Inc. Professional medical writing assistance was provided by Phillipa White, of Source Health Economics, and funded by ARIAD Pharmaceuticals, Inc.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6β4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin β4 reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, suggesting a requirement for α6β4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC. Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Experimental in vitro models that capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel extracellular matrix designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from genetically engineered murine models and human patients, essential adhesive cues were empirically defined and replicated in the hydrogel scaffold, revealing a functional role of laminin-integrin α 3 /α 6 signalling in establishment and survival of pancreatic organoids. Altered tissue stiffness-a hallmark of pancreatic cancer-was recapitulated in culture by adjusting the hydrogel properties to engage mechano-sensing pathways and alter organoid growth. Pancreatic stromal cells were readily incorporated into the hydrogels and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Integrins are heterodimeric cell surface glycoproteins used by cells to bind to the extracellular matrix (ECM) and regulate tumor cell proliferation, migration and survival. A causative relationship between integrin expression and resistance to anticancer drugs has been demonstrated in different tumors, including head and neck squamous cell carcinoma. Using a Cal27 tongue squamous cell carcinoma model, we have previously demonstrated that de novo expression of integrin αVβ3 confers resistance to several anticancer drugs (cisplatin, mitomycin C and doxorubicin) through a mechanism involving downregulation of active Src, increased cell migration and invasion. In the integrin αVβ3 expressing Cal27-derived cell clone 2B1, αVβ5 expression was also increased, but unrelated to drug resistance. To identify the integrin adhesion complex (IAC) components that contribute to the changes in Cal27 and 2B1 cell adhesion and anticancer drug resistance, we isolated IACs from both cell lines. Mass spectrometry (MS)-based proteomics analysis indicated that both cell lines preferentially, but not exclusively, use integrin α6β4, which is classically found in hemidesmosomes. The anticancer drug resistant cell clone 2B1 demonstrated an increased level of α6β4 accompanied with increased deposition of a laminin-332-containing ECM. Immunofluorescence and electron microscopy demonstrated the formation of type II hemidesmosomes by both cell types. Furthermore, suppression of α6β4 expression in both lines conferred resistance to anticancer drugs through a mechanism independent of αVβ3, which implies that the cell clone 2B1 would have been even more resistant had the upregulation of α6β4 not occurred. Taken together, our results identify a key role for α6β4-containing type II hemidesmosomes in regulating anticancer drug sensitivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tadijan, Humphries, Samaržija, Stojanović, Zha, Čuljak, Tomić, Paradžik, Nestić, Kang, Humphries and Ambriović-Ristov.)

Intercellular mechanisms by which the stromal microenvironment contributes to solid tumor progression and targeted therapy resistance remain poorly understood, presenting significant clinical hurdles. PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is an actin cytoskeleton- and focal adhesion-associated pseudokinase that promotes cell state plasticity and cancer metastasis by mediating growth factor-integrin signaling crosstalk. Here, we determined that stromal PEAK1 expression predicts poor outcomes in HER2-positive breast cancers high in SNAI2 expression and enriched for MSC content. Specifically, we identified that the fibroblastic stroma in HER2-positive breast cancer patient tissue stains positive for both nuclear SNAI2 and cytoplasmic PEAK1. Furthermore, mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) express high PEAK1 protein levels and potentiate tumorigenesis, lapatinib resistance and metastasis of HER2-positive breast cancer cells in a PEAK1-dependent manner. Analysis of PEAK1-dependent secreted factors from MSCs revealed INHBA/activin-A as a necessary factor in the conditioned media of PEAK1-expressing MSCs that promotes lapatinib resistance. Single-cell CycIF analysis of MSC-breast cancer cell co-cultures identified enrichment of p-Akt high /p-gH2AX low , MCL1 high /p-gH2AX low and GRP78 high /VIM high breast cancer cell subpopulations by the presence of PEAK1-expressing MSCs and lapatinib treatment. Bioinformatic analyses on a PEAK1-centric stroma-tumor cell gene set and follow-up immunostaining of co-cultures predict targeting antiapoptotic and stress pathways as a means to improve targeted therapy responses and patient outcomes in HER2-positive breast cancer and other stroma-rich malignancies. These data provide the first evidence that PEAK1 promotes tumorigenic phenotypes through a previously unrecognized SNAI2-PEAK1-INHBA stromal cell axis., (© 2021. The Author(s).)

Retinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch's membrane, are essential for RPE cell health and function, but the signals induced by Bruch's membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch's membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and β1 integrin subunits. These short-term experiments reveal an immediate protein-integrin interaction that were obtained from primary RPE cells and replicated using the hTERT-RPE1 cell line. Separate, longer term experiments utilising RNAseq analysis of hTERT-RPE1 cells bound to FHL-1, showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H 2 O 2 to induce oxidative stress-induced cell death suggest hTERT-RPE1 cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.

Talin (TLN1) is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behavior of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronized phosphorylation of a large number of protein targets. CDK1 activity maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesion that are required for successful mitosis. Using a combination of biochemical, structural, and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8. Talin also contains a consensus CDK1 phosphorylation motif centered on S1589, a site shown to be phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of the cytoskeletal adaptor KANK and weakened the response of this region to force as measured by single molecule stretching, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator CDK1 to the primary integrin effector talin represents a coupling of cell proliferation and cell adhesion machineries and thereby indicates a mechanism by which the microenvironment can control cell division in multicellular organisms., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.)

Background: Accumulation of extracellular matrix in organs and tissues is a feature of both aging and disease. In the kidney, glomerulosclerosis and tubulointerstitial fibrosis accompany the decline in function, which current therapies cannot address, leading to organ failure. Although histologic and ultrastructural patterns of excess matrix form the basis of human disease classifications, a comprehensive molecular resolution of abnormal matrix is lacking., Methods: Using mass spectrometry-based proteomics, we resolved matrix composition over age in mouse models of kidney disease. We compared the changes in mice with a global characterization of human kidneymatrix during aging and to existing kidney disease datasets to identify common molecular features., Results: Ultrastructural changes in basement membranes are associated with altered cell adhesion and metabolic processes and with distinct matrix proteomes during aging and kidney disease progression in mice. Within the altered matrix, basement membrane components (laminins, type IV collagen, type XVIII collagen) were reduced and interstitial matrix proteins (collagens I, III, VI, and XV; fibrinogens; and nephronectin) were increased, a pattern also seen in human kidney aging. Indeed, this signature of matrix proteins was consistently modulated across all age and disease comparisons, and the increase in interstitial matrix was also observed in human kidney disease datasets., Conclusions: This study provides deep molecular resolution of matrix accumulation in kidney aging and disease, and identifies a common signature of proteins that provides insight into mechanisms of response to kidney injury and repair., (Copyright © 2021 by the American Society of Nephrology.)

The Kank (kidney or KN motif and ankyrin repeat domain-containing) family of proteins has been described as essential for crosstalk between actin and microtubules. Kank1, 2, 3 and 4 arose by gene duplication and diversification and share conserved structural domains. KANK proteins are localised mainly to the plasma membrane in focal adhesions, indirectly affecting RhoA and Rac1 thus regulating actin cytoskeleton. In addition, Kank proteins are part of the cortical microtubule stabilisation complex regulating microtubules. Most of the data have been collected for Kank1 protein whose expression promotes apoptosis and cell-cycle arrest while Kank3 was identified as hypoxia-inducible proapoptotic target of p53. A discrepancy in Kanks role in regulation of cell migration and sensitivity to antitumour drugs has been observed in different cell models. Since expression of Kank1 and 3 correlate positively with tumour progression and patient outcome, at least in some tumour types, they are candidates for tumour suppressors., (Copyright © 2020. Published by Elsevier Ltd.)

Tumour growth and invasiveness require extracellular matrix (ECM) degradation and are stimulated by the GALA pathway, which induces protein O-glycosylation in the endoplasmic reticulum (ER). ECM degradation requires metalloproteases, but whether other enzymes are required is unclear. Here, we show that GALA induces the glycosylation of the ER-resident calnexin (Cnx) in breast and liver cancer. Glycosylated Cnx and its partner ERp57 are trafficked to invadosomes, which are sites of ECM degradation. We find that disulfide bridges are abundant in connective and liver ECM. Cell surface Cnx-ERp57 complexes reduce these extracellular disulfide bonds and are essential for ECM degradation. In vivo, liver cancer cells but not hepatocytes display cell surface Cnx. Liver tumour growth and lung metastasis of breast and liver cancer cells are inhibited by anti-Cnx antibodies. These findings uncover a moonlighting function of Cnx-ERp57 at the cell surface that is essential for ECM breakdown and tumour development.

BioID, a proximity biotinylation technique, offers a valuable approach to examine the interactions occurring within protein complexes that complements traditional protein biochemical methods. BioID has various advantages that are beneficial to the study of complexes, including an ability to detect insoluble and transient proteins. We have applied BioID to the study of integrin adhesion complexes (IACs), which are located at the junction between the plasma membrane and actin cytoskeleton. The use of multiple BioID baits enables a complex-wide, spatial annotation of IACs, which in turn facilitates the detection of novel proximal interactors and provides insights into IAC architecture. This article describes the labeling and affinity purification of IAC-proximal proteins and their analysis by label-free quantitative mass spectrometry. The article also outlines steps to identify high-confidence proximity interactors, and to interrogate the topology and functional relevance of proximity interaction networks through bioinformatic analyses. © 2020 The Authors. Basic Protocol 1: Proximity biotinylation of integrin adhesion complex components Basic Protocol 2: Mass spectrometry data processing by MaxQuant and detection of high-confidence proximal interactors Basic Protocol 3: Bioinformatic analysis and data visualization., (© 2020 The Authors.)

Background: Insulin signalling contributes to diverse cellular activities including protein synthesis, proliferation and cell survival. Insulin resistance describes the inability of cells to activate the insulin signalling pathway effectively; leading to pathological effects in multiple organ systems including the kidney. In diabetic kidney disease, there is progressive glomerular dysfunction and recent studies have demonstrated that the kidney podocyte is a direct target for insulin action. In this study we defined the literature-based insulin receptor (INSR) interactome and utilised an unbiased proteomic approach to examine INSR interactors in podocytes. Methods: Human podocytes expressing the INSR were characterised under basal and insulin resistant conditions. The INSR was isolated by whole cell immunoprecipitation following a time course stimulation of 2, 7, and 15 minutes with of 100nM insulin. The resulting INSR complexes were analysed by label-free mass spectrometry (MS) to detect protein interactors. Results: We identified 27 known, direct INSR interactors in addition to novel interactors including doublecortin domain-containing protein 2 (DCDC2). The interaction of DCDC2 with the INSR was confirmed by immunoprecipitation and immunofluorescence, and under insulin resistant conditions, DCDC2 had increased association with the INSR. siRNA knockdown of DCDC2 in podocytes resulted in cell morphological change and altered INSR localisation. Conclusion: This study provides insight into the complexity of INSR interactors in podocytes and highlights DCDC2 as a novel INSR binding protein. Involvement of this novel interactor in insulin signalling and podocyte biology may explain how insulin resistance alters morphology and integrity of the glomerular filtration barrier., Competing Interests: No competing interests were disclosed., (Copyright: © 2020 Hosawi SB et al.)

Integrin adhesion complexes (IACs) bridge the extracellular matrix to the actin cytoskeleton and transduce signals in response to both chemical and mechanical cues. The composition, interactions, stoichiometry, and topological organization of proteins within IACs are not fully understood. To address this gap, we used multiplexed proximity biotinylation (BioID) to generate an in situ, proximity-dependent adhesome in mouse pancreatic fibroblasts. Integration of the interactomes of 16 IAC-associated baits revealed a network of 147 proteins with 361 proximity interactions. Candidates with underappreciated roles in adhesion were identified, in addition to established IAC components. Bioinformatic analysis revealed five clusters of IAC baits that link to common groups of prey, and which therefore may represent functional modules. The five clusters, and their spatial associations, are consistent with current models of IAC interaction networks and stratification. This study provides a resource to examine proximal relationships within IACs at a global level., (© 2020 Chastney et al.)

Cells have evolved mechanisms to sense the composition of their adhesive microenvironment. Although much is known about general mechanisms employed by adhesion receptors to relay signals between the extracellular environment and the cytoskeleton, the nuances of ligand-specific signalling remain undefined. Here, we investigated how glomerular podocytes, and four other basement membrane-associated cell types, respond morphologically to different basement membrane ligands. We defined the composition of the respective adhesion complexes using mass spectrometry-based proteomics. On type IV collagen, all epithelial cell types adopted a round morphology, with a single lamellipodium and large adhesion complexes rich in actin-binding proteins. On laminin (511 or 521), all cell types attached to a similar degree but were polygonal in shape with small adhesion complexes enriched in endocytic and microtubule-binding proteins. Consistent with their distinctive morphologies, cells on type IV collagen exhibited high Rac1 activity, while those on laminin had elevated PKCα. Perturbation of PKCα was able to interchange morphology consistent with a key role for this pathway in matrix ligand-specific signalling. Therefore, this study defines the switchable basement membrane adhesome and highlights two key signalling pathways within the systems that determine distinct cell morphologies. Proteomic data are availableviaProteomeXchange with identifier PXD017913., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Integrins are heterodimeric cell surface receptors composed of α and β subunits that control adhesion, proliferation and gene expression. The integrin heterodimer binding to ligand reorganises the cytoskeletal networks and triggers multiple signalling pathways that can cause changes in cell cycle, proliferation, differentiation, survival and motility. In addition, integrins have been identified as targets for many different diseases, including cancer. Integrin crosstalk is a mechanism by which a change in the expression of a certain integrin subunit or the activation of an integrin heterodimer may interfere with the expression and/or activation of other integrin subunit(s) in the very same cell. Here, we review the evidence for integrin crosstalk in a range of cellular systems, with a particular emphasis on cancer. We describe the molecular mechanisms of integrin crosstalk, the effects of cell fate determination, and the contribution of crosstalk to therapeutic outcomes. Our intention is to raise awareness of integrin crosstalk events such that the contribution of the phenomenon can be taken into account when researching the biological or pathophysiological roles of integrins.

Integrins are heterodimeric glycoproteins that bind cells to extracellular matrix. Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) are formed, creating links to the cell cytoskeleton. We have previously observed decreased cell migration and increased sensitivity to microtubule (MT) poisons, paclitaxel and vincristine, in the melanoma cell line MDA-MB-435S upon transfection with integrin αV-specific siRNA, suggesting a link between adhesion and drug sensitivity. To elucidate the underlying mechanism, we determined αV-dependent changes in IAC composition. Using mass spectrometry (MS)-based proteomics, we analyzed the components of isolated IACs of MDA-MB-435S cells and two MDA-MB-435S-derived integrin αV-specific shRNA-expressing cell clones with decreased expression of integrin αV. MS analysis showed that cells preferentially use integrin αVβ5 for the formation of IACs. The differential analysis between MDA-MB-435S cells and clones with decreased expression of integrin αV identified key components of integrin αVβ5 adhesion complexes as talins 1 and 2, α-actinins 1 and 4, filamins A and B, plectin and vinculin. The data also revealed decreased levels of several components of the cortical microtubule stabilization complex, which recruits MTs to adhesion sites (notably liprins α and β, ELKS, LL5β, MACF1, KANK1, and KANK2), following αV knockdown. KANK2 knockdown in MDA-MB-435S cells mimicked the effect of integrin αV knockdown and resulted in increased sensitivity to MT poisons and decreased migration. Taken together, we conclude that KANK2 is a key molecule linking integrin αVβ5 IACs to MTs, and enabling the actin-MT crosstalk that is important for both sensitivity to MT poisons and cell migration., (Copyright © 2020 Paradžik, Humphries, Stojanović, Nestić, Majhen, Dekanić, Samaržija, Sedda, Weber, Humphries and Ambriović-Ristov.)

Electrodes with low work functions are required to efficiently inject electrons into semiconductor devices. However, when the work function drops below about 4 electronvolts, the electrode suffers oxidation in air, which prevents its fabrication in ambient conditions. Here we show that multivalent anions such as oxalate, carbonate and sulfite can act as powerful latent electron donors when dispersed as small ion clusters in a matrix, while retaining their ability to be processed in solution in ambient conditions. The anions in these clusters can even n-dope the semiconductor core of π-conjugated polyelectrolytes that have low electron affinities, through a ground-state doping mechanism that is further amplified by a hole-sensitized or photosensitized mechanism in the device. A theoretical analysis of donor levels of these anions reveals that they are favourably upshifted from ionic lattices by a decrease in the Coulomb stabilization of small ion clusters, and by irreversibility effects. We attain an ultralow effective work function of 2.4 electronvolts with the polyfluorene core. We realize high-performance, solution-processed, white-light-emitting diodes and organic solar cells using polymer electron injection layers with these universal anion donors, demonstrating a general approach to chemically designed and ambient-processed Ohmic electron contacts for semiconductor devices.

Cell division, the purpose of which is to enable cell replication, and in particular to distribute complete, accurate copies of genetic material to daughter cells, is essential for the propagation of life. At a morphological level, division not only necessitates duplication of cellular structures, but it also relies on polar segregation of this material followed by physical scission of the parent cell. For these fundamental changes in cell shape and positioning to be achieved, mechanisms are required to link the cell cycle to the modulation of cytoarchitecture. Outside of mitosis, the three main cytoskeletal networks not only endow cells with a physical cytoplasmic skeleton, but they also provide a mechanism for spatio-temporal sensing via integrin-associated adhesion complexes and site-directed delivery of cargoes. During mitosis, some interphase functions are retained, but the architecture of the cytoskeleton changes dramatically, and there is a need to generate a mitotic spindle for chromosome segregation. An economical solution is to re-use existing cytoskeletal molecules: transcellular actin stress fibres remodel to create a rigid cortex and a cytokinetic furrow, while unipolar radial microtubules become the primary components of the bipolar spindle. This remodelling implies the existence of specific mechanisms that link the cell-cycle machinery to the control of adhesion and the cytoskeleton. In this article, we review the intimate three-way connection between microenvironmental sensing, adhesion signalling and cell proliferation, particularly in the contexts of normal growth control and aberrant tumour progression. As the morphological changes that occur during mitosis are ancient, the mechanisms linking the cell cycle to the cytoskeleton/adhesion signalling network are likely to be primordial in nature and we discuss recent advances that have elucidated elements of this link. A particular focus is the connection between CDK1 and cell adhesion. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

An understanding of the mechanisms whereby cell adhesion complexes (ACs) relay signals bidirectionally across the plasma membrane is necessary to interpret the role of adhesion in regulating migration, differentiation, and growth. A range of AC types has been defined, but to date all have similar compositions and are dependent on a connection to the actin cytoskeleton. Recently, a new class of AC has been reported that normally lacks association with both the cytoskeleton and integrin-associated adhesome components, but is rich in components of the clathrin-mediated endocytosis machinery. The characterization of this new type of adhesion structure, which is emphasized by mitotic cells and cells in long-term culture, identifies a hitherto underappreciated link between the adhesion machinery and clathrin structures at the plasma membrane. While this discovery has implications for how ACs are assembled and disassembled, it raises many other issues. Consequently, to increase awareness within the field, and stimulate research, we explore a number of the most significant questions below., (© 2019 Lock et al.)

The sinus node is a collection of highly specialised cells constituting the heart's pacemaker. The molecular underpinnings of its pacemaking abilities are debated. Using high-resolution mass spectrometry, we here quantify >7,000 proteins from sinus node and neighbouring atrial muscle. Abundances of 575 proteins differ between the two tissues. By performing single-nucleus RNA sequencing of sinus node biopsies, we attribute measured protein abundances to specific cell types. The data reveal significant differences in ion channels responsible for the membrane clock, but not in Ca 2+ clock proteins, suggesting that the membrane clock underpins pacemaking. Consistently, incorporation of ion channel expression differences into a biophysically-detailed atrial action potential model result in pacemaking and a sinus node-like action potential. Combining our quantitative proteomics data with computational modeling, we estimate ion channel copy numbers for sinus node myocytes. Our findings provide detailed insights into the unique molecular make-up of the cardiac pacemaker.

Endothelial cell (EC) sensing of fluid shear stress direction is a critical determinant of vascular health and disease. Unidirectional flow induces EC alignment and vascular homeostasis, whereas bidirectional flow has pathophysiological effects. ECs express several mechanoreceptors that respond to flow, but the mechanism for sensing shear stress direction is poorly understood. We determined, by using in vitro flow systems and magnetic tweezers, that β1 integrin is a key sensor of force direction because it is activated by unidirectional, but not bidirectional, shearing forces. β1 integrin activation by unidirectional force was amplified in ECs that were pre-sheared in the same direction, indicating that alignment and β1 integrin activity has a feedforward interaction, which is a hallmark of system stability. En face staining and EC-specific genetic deletion studies in the murine aorta revealed that β1 integrin is activated and is essential for EC alignment at sites of unidirectional flow but is not activated at sites of bidirectional flow. In summary, β1 integrin sensing of unidirectional force is a key mechanism for decoding blood flow mechanics to promote vascular homeostasis.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)