Quantification of Cinpanemab (BIIB054) Binding to α- Synuclein in Cerebrospinal Fluid of Phase 1 Single Ascending Dose Samples.

Through its pathological and genetic association to Parkinson's Disease (PD), α-synuclein (α-syn) remains a favorable therapeutic target that is being investigated using various modalities, including many passive immunotherapy approaches clinically targeting different forms of α-syn and epitopes. Whereas published studies from some immunotherapy trials have demonstrated engagement in plasma, none have shown direct drug-antigen interactions in the disease-relevant compartment, the central nervous system (CNS). Cinpanemab (BIIB054) selectively targets pathological aggregated α-syn with low affinity binding to monomeric forms. The avidity-driven binding, low drug concentration, and the very low α-syn levels plus its heterogeneous nature in cerebrospinal fluid (CSF) made it not possible to measure drug-target interactions by conventional assays. Here we overcame these challenges by using zero-length crosslinking to stabilize the BIIB054-α-syn complexes and then quantified the crosslinked complexes using a Meso Scale Discovery (MSD) electrochemiluminescence assay. CSF samples from healthy volunteers (HV, n=46) and individuals with PD (PD, n=18) from study 228HV101 (Phase I clinical trial of BIIB054), demonstrated dose- and time- dependent binding of cinpanemab to α-syn with measurable complexes detected at doses {greater than or equal to}15 mg/kg. Complex formation displayed a direct positive correlation to drug concentration (Spearman rank correlation = 0.8295 (HV), 0.8032 (PD) p < 0.0001 (HV, PD)). The observed binding of cinpanemab to α-syn in CSF is consistent with its low intrinsic affinity for α-syn monomer and provides evidence that the drug is behaving with expected binding dynamics in the central nervous system compartment. Significance Statement A zero-length cross-linking method with MSD detection was developed to enable quantification of cinpanemab-α-syn complexes in Phase 1 clinical CSF samples by preventing signal loss caused by their rapid dissociation. Observed dose- and time-dependent binding were consistent with cinpanemab's affinity for α-syn and provided confidence that the drug had engaged its target at the desired site of action. This is the first demonstration of α-syn binding by an antibody in clinical samples from the CNS.

Addendum: The antibody aducanumab reduces Aß plaques in Alzheimer's disease.

This corrects the article DOI: 10.1038/nature21361.

The antibody aducanumab reduces Aß plaques in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by deposition of amyloid-ß (Aß) plaques and neurofibrillary tangles in the brain, accompanied by synaptic dysfunction and neurodegeneration. Antibody-based immunotherapy against Aß to trigger its clearance or mitigate its neurotoxicity has so far been unsuccessful. Here we report the generation of aducanumab, a human monoclonal antibody that selectively targets aggregated Aß. In a transgenic mouse model of AD, aducanumab is shown to enter the brain, bind parenchymal Aß, and reduce soluble and insoluble Aß in a dose-dependent manner. In patients with prodromal or mild AD, one year of monthly intravenous infusions of aducanumab reduces brain Aß in a dose- and time-dependent manner. This is accompanied by a slowing of clinical decline measured by Clinical Dementia Rating-Sum of Boxes and Mini Mental State Examination scores. The main safety and tolerability findings are amyloid-related imaging abnormalities. These results justify further development of aducanumab for the treatment of AD. Should the slowing of clinical decline be confirmed in ongoing phase 3 clinical trials, it would provide compelling support for the amyloid hypothesis.

Characterization of tau binding by gosuranemab.

Deposition of tau aggregates in the brain is a pathological hallmark of several neurodegenerative diseases, termed tauopathies, such as Alzheimer's disease (AD), corticobasal degeneration, and progressive supranuclear palsy (PSP). As transcellular spread of pathological tau aggregates has been implicated in disease progression, immunotherapy is being considered as a treatment for tauopathies. Here we report a detailed biochemical and biophysical characterization of the tau-binding properties of gosuranemab, a humanized monoclonal antibody directed against N-terminal tau that is currently being investigated as a treatment for AD. Binding experiments showed that gosuranemab exhibited high affinity for tau monomer, tau fibrils, and insoluble tau from different tauopathies. Epitope mapping studies conducted using X-ray crystallography and mutagenesis showed that gosuranemab bound to human tau residues 15-22. Immunodepletion of pathological human brain homogenates and transgenic mouse interstitial fluid (ISF) with gosuranemab resulted in reduced tau aggregation in tau biosensor cells. Preincubation of seed-competent AD-tau with gosuranemab significantly inhibited tau aggregation in mouse primary cortical neurons. Gosuranemab also significantly reduced unbound N-terminal tau in cerebrospinal fluid (CSF) from individuals with PSP and AD, and in ISF and CSF of treated transgenic mice. These results are consistent with the >90% target engagement observed in the CSF of some clinical trial dosing cohorts and support the evaluation of gosuranemab as a potential treatment for AD.

Development of an aggregate-selective, human-derived α-synuclein antibody BIIB054 that ameliorates disease phenotypes in Parkinson's disease models.

Aggregation of α-synuclein (α-syn) is neuropathologically and genetically linked to Parkinson's disease (PD). Since stereotypic cell-to-cell spreading of α-syn pathology is believed to contribute to disease progression, immunotherapy with antibodies directed against α-syn is considered a promising therapeutic approach for slowing disease progression. Here we report the identification, binding characteristics, and efficacy in PD mouse models of the human-derived α-syn antibody BIIB054, which is currently under investigation in a Phase 2 clinical trial for PD. BIIB054 was generated by screening human memory B-cell libraries from healthy elderly individuals. Epitope mapping studies conducted using peptide scanning, X-ray crystallography, and mutagenesis show that BIIB054 binds to α-syn residues 1-10. BIIB054 is highly selective for aggregated forms of α-syn with at least an 800-fold higher apparent affinity for fibrillar versus monomeric recombinant α-syn and a strong preference for human PD brain tissue. BIIB054 discriminates between monomers and oligomeric/fibrillar forms of α-syn based on high avidity for aggregates, driven by weak monovalent affinity and fast binding kinetics. In efficacy studies in three different mouse models with intracerebrally inoculated preformed α-syn fibrils, BIIB054 treatment attenuated the spreading of α-syn pathology, rescued motor impairments, and reduced the loss of dopamine transporter density in dopaminergic terminals in striatum. The preclinical data reported here provide a compelling rationale for clinical development of BIIB054 for the treatment and prevention of PD.

Tau Antibody Targeting Pathological Species Blocks Neuronal Uptake and Interneuron Propagation of Tau in Vitro.

The clinical progression of Alzheimer disease (AD) is associated with the accumulation of tau neurofibrillary tangles, which may spread throughout the cortex by interneuronal tau transfer. If so, targeting extracellular tau species may slow the spreading of tau pathology and possibly cognitive decline. To identify suitable target epitopes, we tested the effects of a panel of tau antibodies on neuronal uptake and aggregation in vitro. Immunodepletion was performed on brain extract from tau-transgenic mice and postmortem AD brain and added to a sensitive fluorescence resonance energy transfer-based tau uptake assay to assess blocking efficacy. The antibodies reduced tau uptake in an epitope-dependent manner: N-terminal (Tau13) and middomain (6C5 and HT7) antibodies successfully prevented uptake of tau species, whereas the distal C-terminal-specific antibody (Tau46) had little effect. Phosphorylation-dependent (40E8 and p396) and C-terminal half (4E4) tau antibodies also reduced tau uptake despite removing less total tau by immunodepletion, suggesting specific interactions with species involved in uptake. Among the seven antibodies evaluated, 6C5 most efficiently blocked uptake and subsequent aggregation. More important, 6C5 also blocked neuron-to-neuron spreading of tau in a unique three-chamber microfluidic device. Furthermore, 6C5 slowed down the progression of tau aggregation even after uptake had begun. Our results imply that not all antibodies/epitopes are equally robust in terms of blocking tau uptake of human AD-derived tau species.

Inhibition of integrin αVß6 changes fibril thickness of stromal collagen in experimental carcinomas.

BACKGROUND: Chemotherapeutic efficacy can be improved by targeting the structure and function of the extracellular matrix (ECM) in the carcinomal stroma. This can be accomplished by e.g. inhibiting TGF-ß1 and -ß3 or treating with Imatinib, which results in scarcer collagen fibril structure in xenografted human KAT-4/HT29 (KAT-4) colon adenocarcinoma. METHODS: The potential role of αVß6 integrin-mediated activation of latent TGF-ß was studied in cultured KAT-4 and Capan-2 human ductal pancreatic carcinoma cells as well as in xenograft carcinoma generated by these cells. The monoclonal αVß6 integrin-specific monoclonal antibody 3G9 was used to inhibit the αVß6 integrin activity. RESULTS: Both KAT-4 and Capan-2 cells expressed the αVß6 integrin but only KAT-4 cells could utilize this integrin to activate latent TGF-ß in vitro. Only when Capan-2 cells were co-cultured with human F99 fibroblasts was the integrin activation mechanism triggered, suggesting a more complex, fibroblast-dependent, activation pathway. In nude mice, a 10-day treatment with 3G9 reduced collagen fibril thickness and interstitial fluid pressure in KAT-4 but not in the more desmoplastic Capan-2 tumors that, to achieve a similar effect, required a prolonged 3G9 treatment. In contrast, a 10-day direct inhibition of TGF-ß1 and -ß3 reduced collagen fibril thickness in both tumor models. CONCLUSION: Our data demonstrate that the αVß6-directed activation of latent TGF-ß plays a pivotal role in modulating the stromal collagen network in carcinoma, but that the sensitivity to αVß6 inhibition depends on the simultaneous presence of alternative paths for latent TGF-ß activation and the extent of desmoplasia.

Inhibition of αvß5 Integrin Attenuates Vascular Permeability and Protects against Renal Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury (IRI) is a leading cause of AKI. This common clinical complication lacks effective therapies and can lead to the development of CKD. The αvß5 integrin may have an important role in acute injury, including septic shock and acute lung injury. To examine its function in AKI, we utilized a specific function-blocking antibody to inhibit αvß5 in a rat model of renal IRI. Pretreatment with this anti-αvß5 antibody significantly reduced serum creatinine levels, diminished renal damage detected by histopathologic evaluation, and decreased levels of injury biomarkers. Notably, therapeutic treatment with the αvß5 antibody 8 hours after IRI also provided protection from injury. Global gene expression profiling of post-ischemic kidneys showed that αvß5 inhibition affected established injury markers and induced pathway alterations previously shown to be protective. Intravital imaging of post-ischemic kidneys revealed reduced vascular leak with αvß5 antibody treatment. Immunostaining for αvß5 in the kidney detected evident expression in perivascular cells, with negligible expression in the endothelium. Studies in a three-dimensional microfluidics system identified a pericyte-dependent role for αvß5 in modulating vascular leak. Additional studies showed αvß5 functions in the adhesion and migration of kidney pericytes in vitro Initial studies monitoring renal blood flow after IRI did not find significant effects with αvß5 inhibition; however, future studies should explore the contribution of vasomotor effects. These studies identify a role for αvß5 in modulating injury-induced renal vascular leak, possibly through effects on pericyte adhesion and migration, and reveal αvß5 inhibition as a promising therapeutic strategy for AKI.

Immunotherapy with Aducanumab Restores Calcium Homeostasis in Tg2576 Mice.

Calcium homeostasis plays a major role in maintaining neuronal function under physiological conditions. Amyloid-ß (Aß) initiates pathological processes that include disruption in intracellular calcium levels, so amelioration of the calcium alteration could serve as an indirect functional indicator of treatment efficacy. Therefore, calcium dynamics were used as a measure of functional outcome. We evaluated the effects of the anti-Aß antibody aducanumab on calcium homeostasis and plaque clearance in aged Tg2576 mice with in vivo multiphoton imaging. Acute topical application of aducanumab to the brain resulted in clearance of amyloid plaques. Although chronic systemic administration of aducanumab in 22-month-old mice did not clear existing plaques, calcium overload was ameliorated over time. Therefore, this antibody likely restores neuronal network function that possibly underlies cognitive deficits, indicating promise as a clinical treatment. In addition, functional readouts such as calcium overload may be a more useful outcome measure to monitor treatment efficacy in models of Alzheimer's disease compared with amyloid burden alone. SIGNIFICANCE STATEMENT: Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is currently without a cure. Aducanumab is an anti-amyloid-ß antibody being developed for the treatment of AD. Interim analyses of a phase 1b clinical trial have suggested potential beneficial effects on amyloid pathology and cognitive status in patients treated with aducanumab (Sevigny et al., 2016). Here, we show that a murine analog of aducanumab clears amyloid plaques in an acute setting and restores calcium homeostasis disrupted in a mouse model of AD upon chronic treatment. Therefore, we demonstrate that aducanumab reverses a functional outcome measure reflective of neural network activity.

Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvß6 Expression.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with high mortality. Active TGFß1 is considered central to the pathogenesis of IPF. A major mechanism of TGFß1 activation in the lung involves the epithelially restricted αvß6 integrin. Expression of the αvß6 integrin is dramatically increased in IPF. How αvß6 integrin expression is regulated in the pulmonary epithelium is unknown. Here we identify a region in the ß6 subunit gene (ITGB6) promoter acting to markedly repress basal gene transcription, which responds to both the Ets domain-containing protein Elk1 (Elk1) and the glucocorticoid receptor (GR). Both Elk1 and GR can regulate αvß6 integrin expression in vitro We demonstrate Elk1 binding to the ITGB6 promoter basally and that manipulation of Elk1 or Elk1 binding alters ITGB6 promoter activity, gene transcription, and αvß6 integrin expression. Crucially, we find that loss of Elk1 causes enhanced Itgb6 expression and exaggerated lung fibrosis in an in vivo model of fibrosis, whereas the GR agonist dexamethasone inhibits Itgb6 expression. Moreover, Elk1 dysregulation is present in epithelium from patients with IPF. These data reveal a novel role for Elk1 regulating ITGB6 expression and highlight how dysregulation of Elk1 can contribute to human disease.

Integrin αvß6 critically regulates hepatic progenitor cell function and promotes ductular reaction, fibrosis, and tumorigenesis.

UNLABELLED: Integrin αvß6 is rapidly up-regulated on cells of epithelial lineage during tissue injury, where one of its primary functions is activation of latent transforming growth factor beta 1 (TGFß1). In human liver cirrhosis, αvß6 is overexpressed by cells comprising the ductular reaction, and its inhibition suppresses experimental biliary fibrosis in rodents. Here, we show that αvß6 is expressed on the actively proliferating subset of hepatic progenitor cells and is required for their progenitor function in vivo and in vitro through integrin αvß6-dependent TGFß1 activation. Freshly isolated αvß6(+) liver cells demonstrate clonogenic potential and differentiate into cholangiocytes and functional hepatocytes in vitro, whereas colony formation by epithelial cell adhesion molecule-positive progenitor cells is blocked by αvß6-neutralizing antibody and in integrin beta 6-deficient cells. Inhibition of progenitors by anti-αvß6 antibody is recapitulated by TGFß1 neutralization and rescued by addition of bioactive TGFß1. Genetic disruption or selective targeting of αvß6 with 3G9 antibody potently inhibits progenitor cell responses in mouse models of chronic biliary injury and protects from liver fibrosis and tumorigenesis, two conditions clinically associated with exacerbated ductular reaction. CONCLUSION: These results suggest that αvß6 is a promising target for chronic fibrotic liver diseases and associated cancers.

Connective tissue growth factor and integrin αvß6: a new pair of regulators critical for ductular reaction and biliary fibrosis in mice.

UNLABELLED: Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell-matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvß6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvß6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter-driven green fluorescent protein reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvß6 during liver injury induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen-inducible Cre-loxP system down-regulated integrin αvß6 in DDC-damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvß6, by administrating the neutralizing antibody, 6.3G9 (10 mg/kg body weight), caused low levels of epithelial cell adhesion molecule and cytokeratin 19 gene messenger RNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within 2 weeks after DDC treatment. Associated fibrosis was attenuated, as indicated by reduced expression of fibrosis-related genes, smaller areas of alpha-smooth muscle actin staining, and low collagen production based on hydroxyproline content and Sirius Red staining. Finally, integrin αvß6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)-ß1 activation in vitro. CONCLUSIONS: CTGF and integrin αvß6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF-ß1. CTGF and integrin αvß6 are potential therapeutic targets to control DRs and fibrosis in related liver disease.

Influenza promotes collagen deposition via αvß6 integrin-mediated transforming growth factor ß activation.

Influenza infection exacerbates chronic pulmonary diseases, including idiopathic pulmonary fibrosis. A central pathway in the pathogenesis of idiopathic pulmonary fibrosis is epithelial injury leading to activation of transforming growth factor ß (TGFß). The mechanism and functional consequences of influenza-induced activation of epithelial TGFß are unclear. Influenza stimulates toll-like receptor 3 (TLR3), which can increase RhoA activity, a key event prior to activation of TGFß by the αvß6 integrin. We hypothesized that influenza would stimulate TLR3 leading to activation of latent TGFß via αvß6 integrin in epithelial cells. Using H1152 (IC50 6.1 µm) to inhibit Rho kinase and 6.3G9 to inhibit αvß6 integrins, we demonstrate their involvement in influenza (A/PR/8/34 H1N1) and poly(I:C)-induced TGFß activation. We confirm the involvement of TLR3 in this process using chloroquine (IC50 11.9 µm) and a dominant negative TLR3 construct (pZERO-hTLR3). Examination of lungs from influenza-infected mice revealed augmented levels of collagen deposition, phosphorylated Smad2/3, αvß6 integrin, and apoptotic cells. Finally, we demonstrate that αvß6 integrin-mediated TGFß activity following influenza infection promotes epithelial cell death in vitro and enhanced collagen deposition in vivo and that this response is diminished in Smad3 knock-out mice. These data show that H1N1 and poly(I:C) can induce αvß6 integrin-dependent TGFß activity in epithelial cells via stimulation of TLR3 and suggest a novel mechanism by which influenza infection may promote collagen deposition in fibrotic lung disease.

αvß6 integrin may be a potential prognostic biomarker in interstitial lung disease.

Idiopathic pulmonary fibrosis (IPF) and fibrotic nonspecific interstitial pneumonitis are progressive interstitial lung diseases (ILDs) with limited treatment options and poor survival. However, the rate of disease progression is variable, implying there may be different endotypes of disease. We hypothesised that immunophenotyping biopsies from ILD patients might reveal distinct endotypes of progressive fibrotic disease, which may facilitate stratification when undertaking clinical trials of novel therapies for IPF.43 paraffin-embedded, formalin-fixed lung tissue sections were immunostained for five molecules implicated in the pathogenesis of the fibrosis: α-smooth muscle actin (αSMA), αvß6 integrin, pro-surfactant protein C (SP-C), hepatocyte growth factor (HGF) and tenascin-C (TenC). Levels of immunostaining and numbers of fibroblastic foci were quantified using operator-dependent and -independent methods. The relationship of all these markers to overall survival was analysed.Staining revealed high levels of αSMA, αvß6 integrin, pro-SP-C, HGF and TenC, and fibroblastic foci. Immunostaining varied across samples for all molecules but only the extent of αvß6 integrin immunostaining was associated with increased mortality. There was no association with the other markers measured.Our data suggest high levels of αvß6 integrin may identify a specific endotype of progressive fibrotic lung disease.

Suppression of Hedgehog signalling promotes pro-tumourigenic integrin expression and function.

Aberrant Hedgehog (Hh) signalling has been reported in a number of malignancies, particularly basal cell carcinoma (BCC) of the skin. Clinical trials of Hh inhibitors are underway in many cancers, and these have produced significant clinical benefit in BCC patients, although regrowth of new, or clinically aggressive, variants, as well as development of secondary malignancies, has been reported. αvß6 integrin is expressed in many cancers, where it has been shown to correlate with an aggressive tumour phenotype and poor prognosis. We have previously reported αvß6 up-regulation in aggressive, morphoeic BCC variants, where it modulates the stromal response and induces invasion. To examine a possible link between Hh and αvß6 function, we generated BCC models, overexpressing Gli1 in immortalized keratinocytes (NTert1, HaCaT). Unexpectedly, we found that suppressing Gli1 significantly increased αvß6 expression. This promoted tumour cell motility and also stromal myofibroblast differentiation through integrin-dependent TGF-ß1 activation. Gli1 inhibited αvß6 expression by suppressing TGF-ß1-induced Smad2/3 activation, blocking a positive feedback loop maintaining high αvß6 levels. A similar mechanism was observed in AsPC1 pancreatic cancer cells expressing endogenous Gli1, suggesting a common mechanism across tumour types. In vitro findings were supported using human clinical samples, where we showed an inverse correlation between αvß6 and Gli1 expression in different BCC subtypes and pancreatic cancers. In summary, we show that expression of Gli1 and αvß6 inversely correlates in tumours in vivo, and Hh targeting up-regulates TGF-ß1/Smad2/3-dependent αvß6 expression, promoting pro-tumourigenic cell functions in vitro. These results have potential clinical significance, given the reported recurrence of BCC variants and secondary malignancies in patients treated by Hh targeting.

Inhibition of the αvß6 integrin leads to limited alteration of TGF-α-induced pulmonary fibrosis.

A number of growth factors and signaling pathways regulate matrix deposition and fibroblast proliferation in the lung. The epidermal growth factor receptor (EGFR) family of receptors and the transforming growth factor-ß (TGF-ß) family are active in diverse biological processes and are central mediators in the initiation and maintenance of fibrosis in many diseases. Transforming growth factor-α (TGF-α) is a ligand for the EGFR, and doxycycline (Dox)-inducible transgenic mice conditionally expressing TGF-α specifically in the lung epithelium develop progressive fibrosis accompanied with cachexia, changes in lung mechanics, and marked pleural thickening. Although recent studies demonstrate that EGFR activation modulates the fibroproliferative effects involved in the pathogenesis of TGF-ß induced pulmonary fibrosis, in converse, the direct role of EGFR induction of the TGF-ß pathway in the lung is unknown. The αvß6 integrin is an important in vivo activator of TGF-ß activation in the lung. Immunohistochemical analysis of αvß6 protein expression and bronchoalveolar analysis of TGF-ß pathway signaling indicates activation of the αvß6/TGF-ß pathway only at later time points after lung fibrosis was already established in the TGF-α model. To determine the contribution of the αvß6/TGF-ß pathway on the progression of established fibrotic disease, TGF-α transgenic mice were administered Dox for 4 wk, which leads to extensive fibrosis; these mice were then treated with a function-blocking anti-αvß6 antibody with continued administration of Dox for an additional 4 wk. Compared with TGF-α transgenic mice treated with control antibody, αvß6 inhibition significantly attenuated pleural thickening and altered the decline in lung mechanics. To test the effects of genetic loss of the ß6 integrin, TGF-α transgenic mice were mated with ß6-null mice and the degree of fibrosis was compared in adult mice following 8 wk of Dox administration. Genetic ablation of the ß6 integrin attenuated histological and physiological changes in the lungs of TGF-α transgenic mice although a significant degree of fibrosis still developed. In summary, inhibition of the ß6 integrin led to a modest, albeit significant, effect on pleural thickening and lung function decline observed with TGF-α-induced pulmonary fibrosis. These data support activation of the αvß6/TGF-ß pathway as a secondary effect contributing to TGF-α-induced pleural fibrosis and suggest a complex contribution of multiple mediators to the maintenance of progressive fibrosis in the lung.

Dynamic structural changes are observed upon collagen and metal ion binding to the integrin α1 I domain.

We have applied hydrogen-deuterium exchange mass spectrometry, in conjunction with differential scanning calorimetry and protein stability analysis, to examine solution dynamics of the integrin α1 I domain induced by the binding of divalent cations, full-length type IV collagen, or a function-blocking monoclonal antibody. These studies revealed features of integrin activation and α1I-ligand complexes that were not detected by static crystallographic data. Mg(2+) and Mn(2+) stabilized α1I but differed in their effects on exchange rates in the αC helix. Ca(2+) impacted α1I conformational dynamics without altering its gross thermal stability. Interaction with collagen affected the exchange rates in just one of three metal ion-dependent adhesion site (MIDAS) loops, suggesting that MIDAS loop 2 plays a primary role in mediating ligand binding. Collagen also induced changes consistent with increased unfolding in both the αC and allosteric C-terminal helices of α1I. The antibody AQC2, which binds to α1I in a ligand-mimetic manner, also reduced exchange in MIDAS loop 2 and increased exchange in αC, but it did not impact the C-terminal region. This is the first study to directly demonstrate the conformational changes induced upon binding of an integrin I domain to a full-length collagen ligand, and it demonstrates the utility of the deuterium exchange mass spectrometry method to study the solution dynamics of integrin/ligand and integrin/metal ion interactions. Based on the ligand and metal ion binding data, we propose a model for collagen-binding integrin activation that explains the differing abilities of Mg(2+), Mn(2+), and Ca(2+) to activate I domain-containing integrins.

Lysophosphatidic acid increases proximal tubule cell secretion of profibrotic cytokines PDGF-B and CTGF through LPA2- and Gαq-mediated Rho and αvß6 integrin-dependent activation of TGF-ß.

After ischemia-reperfusion injury (IRI), kidney tubules show activated transforming growth factor ß (TGF-ß) signaling and increased expression of profibrotic peptides, platelet-derived growth factor-B (PDGF-B) and connective tissue growth factor (CTGF). If tubule repair after IRI is incomplete, sustained paracrine activity of these peptides can activate interstitial fibroblast progenitors and cause fibrosis. We show that lysophosphatidic acid (LPA), a ubiquitous phospholipid that is increased at sites of injury and inflammation, signals through LPA2 receptors and Gαq proteins of cultured proximal tubule cells to transactivate latent TGF-ß in a Rho/Rho-kinase and αvß6 integrin-dependent manner. Active TGF-ß peptide then initiates signaling to increase the production and secretion of PDGF-B and CTGF. In a rat model of IRI, increased TGF-ß signaling that was initiated early during reperfusion did not subside during recovery, but progressively increased, causing tubulointerstitial fibrosis. This was accompanied by correspondingly increased LPA2 and ß6 integrin proteins and elevated tubule expression of TGF-ß1, together with PDGF-B and CTGF. Treatment with a pharmacological TGF-ß type I receptor antagonist suppressed TGF-ß signaling, decreased the expression of ß6 integrin, PDGF-B, and CTGF, and ameliorated fibrosis. We suggest that LPA-initiated autocrine signaling is a potentially important mechanism that gives rise to paracrine profibrotic signaling in injured kidney tubule cells.

Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a premalignant, fibrosing disorder of the mouth, pharynx, and oesophagus, with a malignant transformation rate of 7-13%. OSF is strongly associated with areca (betel) nut chewing and worldwide, over 5 million people are affected. As αvß6 integrin is capable of promoting both tissue fibrosis and carcinoma invasion, we examined its expression in fibroepithelial hyperplasia and OSF. αvß6 was markedly up-regulated in OSF, with high expression detected in 22 of 41 cases (p < 0.001). We investigated the functional role of αvß6 using oral keratinocyte-derived cells genetically modified to express high αvß6 (VB6), and also NTERT-immortalized oral keratinocytes, which express low αvß6 (OKF6/TERT-1). VB6 cells showed significant αvß6-dependent activation of TGF-ß1, which induced transdifferentiation of oral fibroblasts into myofibroblasts and resulted in up-regulation of genes associated with tissue fibrosis. These experimental in vitro findings were confirmed using human clinical samples, where we showed that the stroma of OSF contained myofibroblasts and that TGF-ß1-dependent Smad signalling was detectable both in keratinocytes and in myofibroblasts. We also found that arecoline, the major alkaloid of areca nuts, up-regulated keratinocyte αvß6 expression. This was modulated through the M(4) muscarinic acetylcholine receptor and was suppressed by the M(4) antagonist, tropicamide. Arecoline-dependent αvß6 up-regulation promoted keratinocyte migration and induced invasion, raising the possibility that this mechanism may support malignant transformation. Over 80% of OSF-related oral cancers examined had moderate/high αvß6 expression. These data suggest that the pathogenesis of OSF may be epithelial-driven and involve arecoline-dependent up-regulation of αvß6 integrin.

Stromal features are predictive of disease mortality in oral cancer patients.

Worldwide, approximately 405 000 cases of oral cancer (OSCC) are diagnosed each year, with a rising incidence in many countries. Despite advances in surgery and radiotherapy, which remain the standard treatment options, the mortality rate has remained largely unchanged for decades, with a 5-year survival rate of around 50%. OSCC is a heterogeneous disease, staged currently using the TNM classification, supplemented with pathological information from the primary tumour and loco-regional lymph nodes. Although patients with advanced disease show reduced survival, there is no single pathological or molecular feature that identifies aggressive, early-stage tumours. We retrospectively analysed 282 OSCC patients for disease mortality, related to clinical, pathological, and molecular features based on our previous functional studies [EGFR, αvß6 integrin, smooth muscle actin (SMA), p53, p16, EP4]. We found that the strongest independent risk factor of early OSCC death was a feature of stroma rather than tumour cells. After adjusting for all factors, high stromal SMA expression, indicating myofibroblast transdifferentiation, produced the highest hazard ratio (3.06, 95% CI 1.65-5.66) and likelihood ratio (3.6; detection rate: false positive rate) of any feature examined, and was strongly associated with mortality, regardless of disease stage. Functional assays showed that OSCC cells can modulate myofibroblast transdifferentiation through αvß6-dependent TGF-ß1 activation and that myofibroblasts promote OSCC invasion. Finally, we developed a prognostic model using Cox regression with backward elimination; only SMA expression, metastasis, cohesion, and age were significant. This model was independently validated on a patient subset (detection rate 70%; false positive rate 20%; ROC analysis 77%, p < 0.001). Our study highlights the limited prognostic value of TNM staging and suggests that an SMA-positive, myofibroblastic stroma is the strongest predictor of OSCC mortality. Whether used independently or as part of a prognostic model, SMA identifies a significant group of patients with aggressive tumours, regardless of disease stage.