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1.
Mol Ther ; 32(4): 1061-1079, 2024 Apr 03.
Article in English | MEDLINE | ID: mdl-38382529

ABSTRACT

Complement-mediated diseases can be treated using systemic inhibitors. However, complement components are abundant in circulation, affecting systemic inhibitors' exposure and efficacy. Furthermore, because of complement's essential role in immunity, systemic treatments raise infection risk in patients. To address these challenges, we developed antibody fusion proteins combining the alternative-pathway complement inhibitor factor H (fH1-5) with an anti-C3d monoclonal antibody (C3d-mAb-2fH). Because C3d is deposited at sites of complement activity, this molecule localizes to tissue complement while minimizing circulating complement engagement. These fusion proteins bind to deposited complement in diseased human skin sections and localize to activated complement in a primate skin injury model. We further explored the pharmacology of C3d-mAb-2fH proteins in rodent models with robust tissue complement activation. Doses of C3d-mAb-2fH >1 mg/kg achieved >75% tissue complement inhibition in mouse and rat injury models while avoiding circulating complement blockade. Glomerular-specific complement inhibition reduced proteinuria and preserved podocyte foot-process architecture in rat membranous nephropathy, indicating disease-modifying efficacy. These data indicate that targeting local tissue complement results in durable and efficacious complement blockade in skin and kidney while avoiding systemic inhibition, suggesting broad applicability of this approach in treating a range of complement-mediated diseases.


Subject(s)
Complement Factor H , Kidney Diseases , Humans , Mice , Rats , Animals , Complement Factor H/genetics , Complement C3d/metabolism , Kidney Diseases/etiology , Antibodies , Complement Activation
2.
Ann Am Thorac Soc ; 20(8): 1077-1087, 2023 08.
Article in English | MEDLINE | ID: mdl-37526479

ABSTRACT

Rationale: To identify barriers and opportunities for Ph.D., basic and translational scientists to be fully integrated into clinical units. Objectives: In 2022, an ad hoc committee of the American Thoracic Society developed a project proposal and workshop to identify opportunities and barriers for scientists who do not practice medicine to develop successful careers and achieve tenure-track faculty positions in clinical departments and divisions within academic medical centers (AMCs) in the United States. Methods: This document focuses on results from a survey of adult and pediatric pulmonary, critical care, and sleep medicine division chiefs as well as a survey of workshop participants, including faculty in departmental and school leadership roles in both basic science and clinical units within U.S. AMCs. Results: We conclude that full integration of non-clinically practicing basic and translational scientists into the clinical units, in addition to their traditional placements in basic science units, best serves the tripartite mission of AMCs to provide care, perform research, and educate the next generation. Evidence suggests clinical units do employ Ph.D. scientists in large numbers, but these faculty are often hired into non-tenure track positions, which do not provide the salary support, start-up funds, research independence, or space often associated with hiring in basic science units within the same institution. These barriers to success of Ph.D. faculty in clinical units are largely financial. Conclusions: Our recommendation is for AMCs to consider and explore some of our proposed strategies to accomplish the goal of integrating basic and translational scientists into clinical units in a meaningful way.


Subject(s)
Academic Medical Centers , Physicians , Adult , United States , Humans , Child , Personnel Selection , Leadership , Faculty, Medical
3.
Front Immunol ; 13: 869725, 2022.
Article in English | MEDLINE | ID: mdl-35784298

ABSTRACT

Sustained complement activation is an underlying pathologic driver in many inflammatory and autoimmune diseases. Currently approved anti-complement therapies are directed at the systemic blockade of complement. Consequently, these therapies provide widespread inhibition of complement pathway activity, beyond the site of ongoing activation and the intended pharmacodynamic (PD) effects. Given the essential role for complement in both innate and adaptive immunity, there is a need for therapies that inhibit complement in diseased tissue while limiting systemic blockade. One potential approach focuses on the development of novel fusion proteins that enable tissue-targeted delivery of complement negative regulatory proteins. These therapies are expected to provide increased potency and prolonged tissue PD, decreased dosing frequency, and the potential for improved safety profiles. We created a library of bifunctional fusion proteins that direct a fragment of the complement negative regulator, complement receptor type 1 (CR1) to sites of tissue injury. Tissue targeting is accomplished through the binding of the fusion protein to complement C3 fragments that contain a surface-exposed C3d domain and which are covalently deposited on tissues where complement is being activated. To that end, we generated a fusion protein that contains an anti-C3d monoclonal antibody recombinantly linked to the first 10 consensus repeats of CR1 (CR11-10) with the intention of delivering high local concentrations of this complement negative regulatory domain to tissue-bound complement C3 fragments iC3b, C3dg and C3d. Biochemical and in vitro characterization identified several fusion proteins that inhibit complement while maintaining the C3d domain binding properties of the parent monoclonal antibody. Preclinical in vivo studies further demonstrate that anti-C3d fusion proteins effectively distribute to injured tissue and reduce C3 fragment deposition for periods beyond 14 days. The in vitro and in vivo profiles support the further evaluation of C3d mAb-CR11-10 as a novel approach to restore proper complement activation in diseased tissue in the absence of continuous systemic complement blockade.


Subject(s)
Autoimmune Diseases , Complement C3 , Antibodies, Monoclonal , Complement Activation , Humans , Receptors, Complement/metabolism
5.
Am J Respir Crit Care Med ; 206(9): 1128-1139, 2022 11 01.
Article in English | MEDLINE | ID: mdl-35771569

ABSTRACT

Rationale: Treatment options for idiopathic pulmonary fibrosis (IPF) are limited. Objectives: To evaluate the efficacy and safety of BG00011, an anti-αvß6 IgG1 monoclonal antibody, in the treatment of patients with IPF. Methods: In a phase IIb randomized, double-blind, placebo-controlled trial, patients with IPF (FVC ⩾50% predicted, on or off background therapy) were randomized 1:1 to once-weekly subcutaneous BG00011 56 mg or placebo. The primary endpoint was FVC change from baseline at Week 52. Because of early trial termination (imbalance in adverse events and lack of clinical benefit), endpoints were evaluated at Week 26 as an exploratory analysis. Measurements and Main Results: One hundred six patients were randomized and received at least one dose of BG00011 (n = 54) or placebo (n = 52). At Week 26, there was no significant difference in FVC change from baseline between patients who received BG00011 (n = 20) or placebo (n = 23), least squares mean (SE) -0.097 L (0.0600) versus -0.056 L (0.0593), respectively (P = 0.268). However, after Week 26, patients in the BG00011 group showed a worsening trend. Eight (44.4%) of 18 who received BG00011 and 4 (18.2%) of 22 who received placebo showed worsening of fibrosis on high-resolution computed tomography at the end of treatment. IPF exacerbation/or progression was reported in 13 patients (all in the BG00011 group). Serious adverse events occurred more frequently in BG00011 patients, including four deaths. Conclusions: The results do not support the continued clinical development of BG00011. Further research is warranted to identify new treatment strategies that modify inflammatory and fibrotic pathways in IPF. Clinical trial registered with www.clinicaltrials.gov (NCT03573505).


Subject(s)
Idiopathic Pulmonary Fibrosis , Humans , Idiopathic Pulmonary Fibrosis/drug therapy , Antibodies, Monoclonal/therapeutic use , Treatment Outcome , Double-Blind Method , Immunoglobulin G
6.
J Extracell Vesicles ; 9(1): 1763594, 2020.
Article in English | MEDLINE | ID: mdl-32595914

ABSTRACT

Prostate cancer (PrCa) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sEVs). sEVs, as well as large extracellular vesicles (LEVs), isolated via iodixanol density gradients from PrCa cell culture media, express the epithelial-specific αvß6 integrin, which is known to be induced in cancer. In this study, we show sEV-mediated protein transfer of αvß6 integrin to microvascular endothelial cells (human microvascular endothelial cells 1 - HMEC1) and demonstrate that de novo αvß6 integrin expression is not caused by increased mRNA levels. Incubation of HMEC1 with sEVs isolated from PrCa PC3 cells that express the αvß6 integrin results in a highly significant increase in the number of nodes, junctions and tubules. In contrast, incubation of HMEC1 with sEVs isolated from ß6 negative PC3 cells, generated by shRNA against ß6, results in a reduction in the number of nodes, junctions and tubules, a decrease in survivin levels and an increase in a negative regulator of angiogenesis, pSTAT1. Furthermore, treatment of HMEC1 with sEVs generated by CRISPR/Cas9-mediated down-regulation of ß6, causes up-regulation of pSTAT1. Overall, our findings suggest that αvß6 integrin in cancer sEVs regulates angiogenesis during PrCa progression.

7.
Am J Respir Crit Care Med ; 199(5): 622-630, 2019 03 01.
Article in English | MEDLINE | ID: mdl-30141961

ABSTRACT

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal disease with a variable and unpredictable course. OBJECTIVES: To determine whether BAL cell gene expression is predictive of survival in IPF. METHODS: This retrospective study analyzed the BAL transcriptome of three independent IPF cohorts: Freiburg (Germany), Siena (Italy), and Leuven (Belgium) including 212 patients. BAL cells from 20 healthy volunteers, 26 patients with sarcoidosis stage III and IV, and 29 patients with chronic obstructive pulmonary disease were used as control subjects. Survival analysis was performed by Cox models and component-wise boosting. Presence of airway basal cells was tested by immunohistochemistry and flow cytometry. MEASUREMENTS AND MAIN RESULTS: A total of 1,582 genes were predictive of mortality in the IPF derivation cohort in univariate analyses adjusted for age and sex at false discovery rate less than 0.05. A nine-gene signature, derived from the discovery cohort (Freiburg), performed well in both replication cohorts, Siena (P < 0.0032) and Leuven (P = 0.0033). nCounter expression analysis confirmed the array results (P < 0.0001). The genes associated with mortality in BAL cells were significantly enriched for genes expressed in airway basal cells. Further analyses by gene expression, flow cytometry, and immunohistochemistry showed an increase in airway basal cells in BAL and tissues of IPF compared with control subjects, but not in chronic obstructive pulmonary disease or sarcoidosis. CONCLUSIONS: Our results identify and validate a BAL signature that predicts mortality in IPF and improves the accuracy of outcome prediction based on clinical parameters. The BAL signature associated with mortality unmasks a potential role for airway basal cells in IPF.


Subject(s)
Bronchoalveolar Lavage Fluid/cytology , Idiopathic Pulmonary Fibrosis/metabolism , Respiratory Mucosa/metabolism , Aged , Female , Gene Expression , Gene Expression Profiling , Humans , Idiopathic Pulmonary Fibrosis/mortality , Male , Oligonucleotide Array Sequence Analysis , Predictive Value of Tests , Proportional Hazards Models , Retrospective Studies , Survival Analysis
8.
Cell Commun Signal ; 16(1): 36, 2018 07 02.
Article in English | MEDLINE | ID: mdl-29966518

ABSTRACT

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.


Subject(s)
Antigens, Neoplasm/immunology , Collagen/chemistry , Integrins/immunology , Animals , Antibodies, Monoclonal/immunology , Antigens, Neoplasm/metabolism , Cell Line, Tumor , Cell Transformation, Neoplastic , Collagen/metabolism , Extracellular Fluid/metabolism , Female , Gene Expression Profiling , Humans , Integrins/metabolism , Mice , Pressure , Transforming Growth Factor beta/metabolism
10.
JCI Insight ; 2(9)2017 May 04.
Article in English | MEDLINE | ID: mdl-28469072

ABSTRACT

Fibrotic lung disease, most notably idiopathic pulmonary fibrosis (IPF), is thought to result from aberrant wound-healing responses to repetitive lung injury. Increased vascular permeability is a cardinal response to tissue injury, but whether it is mechanistically linked to lung fibrosis is unknown. We previously described a model in which exaggeration of vascular leak after lung injury shifts the outcome of wound-healing responses from normal repair to pathological fibrosis. Here we report that the fibrosis produced in this model is highly dependent on thrombin activity and its downstream signaling pathways. Direct thrombin inhibition with dabigatran significantly inhibited protease-activated receptor-1 (PAR1) activation, integrin αvß6 induction, TGF-ß activation, and the development of pulmonary fibrosis in this vascular leak-dependent model. We used a potentially novel imaging method - ultashort echo time (UTE) lung magnetic resonance imaging (MRI) with the gadolinium-based, fibrin-specific probe EP-2104R - to directly visualize fibrin accumulation in injured mouse lungs, and to correlate the antifibrotic effects of dabigatran with attenuation of fibrin deposition. We found that inhibition of the profibrotic effects of thrombin can be uncoupled from inhibition of hemostasis, as therapeutic anticoagulation with warfarin failed to downregulate the PAR1/αvß6/TGF-ß axis or significantly protect against fibrosis. These findings have direct and important clinical implications, given recent findings that warfarin treatment is not beneficial in IPF, and the clinical availability of direct thrombin inhibitors that our data suggest could benefit these patients.

11.
Sci Transl Med ; 9(384)2017 04 05.
Article in English | MEDLINE | ID: mdl-28381537

ABSTRACT

Pulmonary fibrosis is scarring of the lungs that can arise from radiation injury, drug toxicity, environmental or genetic causes, and for unknown reasons [idiopathic pulmonary fibrosis (IPF)]. Overexpression of collagen is a hallmark of organ fibrosis. We describe a peptide-based positron emission tomography (PET) probe (68Ga-CBP8) that targets collagen type I. We evaluated 68Ga-CBP8 in vivo in the bleomycin-induced mouse model of pulmonary fibrosis. 68Ga-CBP8 showed high specificity for pulmonary fibrosis and high target/background ratios in diseased animals. The lung PET signal and lung 68Ga-CBP8 uptake (quantified ex vivo) correlated linearly (r2 = 0.80) with the amount of lung collagen in mice with fibrosis. We further demonstrated that the 68Ga-CBP8 probe could be used to monitor response to treatment in a second mouse model of pulmonary fibrosis associated with vascular leak. Ex vivo analysis of lung tissue from patients with IPF supported the animal findings. These studies indicate that 68Ga-CBP8 is a promising candidate for noninvasive imaging of human pulmonary fibrosis.


Subject(s)
Collagen Type I/metabolism , Molecular Probes/chemistry , Positron-Emission Tomography , Pulmonary Fibrosis/diagnostic imaging , Pulmonary Fibrosis/diagnosis , Animals , Bleomycin , Capillary Permeability , Disease Models, Animal , Disease Progression , Gallium Radioisotopes , Humans , Idiopathic Pulmonary Fibrosis/pathology , Kidney/metabolism , Lung/pathology , Male , Mice, Inbred C57BL , Pulmonary Fibrosis/pathology
12.
Nat Commun ; 8: 14105, 2017 01 19.
Article in English | MEDLINE | ID: mdl-28102238

ABSTRACT

Functional interplay between tumour cells and their neoplastic extracellular matrix plays a decisive role in malignant progression of carcinomas. Here we provide a comprehensive data set of the human HNSCC-associated fibroblast matrisome. Although much attention has been paid to the deposit of collagen, we identify oncofetal fibronectin (FN) as a major and obligate component of the matrix assembled by stromal fibroblasts from head and neck squamous cell carcinomas (HNSCC). FN overexpression in tumours from 435 patients corresponds to an independent unfavourable prognostic indicator. We show that migration of carcinoma collectives on fibrillar FN-rich matrices is achieved through αvß6 and α9ß1 engagement, rather than α5ß1. Moreover, αvß6-driven migration occurs independently of latent TGF-ß activation and Smad-dependent signalling in tumour epithelial cells. These results provide insights into the adhesion-dependent events at the tumour-stroma interface that govern the collective mode of migration adopted by carcinoma cells to invade surrounding stroma in HNSCC.


Subject(s)
Carcinoma, Squamous Cell , Cell Movement/drug effects , Fibronectins/metabolism , Head and Neck Neoplasms , Adult , Aged , Aged, 80 and over , Cell Line, Tumor , Cell Movement/physiology , Extracellular Matrix , Female , Gene Expression Regulation, Neoplastic , Humans , Integrins/genetics , Integrins/metabolism , Male , Middle Aged , Squamous Cell Carcinoma of Head and Neck
13.
Mol Ther ; 25(1): 259-273, 2017 01 04.
Article in English | MEDLINE | ID: mdl-28129120

ABSTRACT

Expression of the αvß6 integrin is upregulated in several solid tumors. In contrast, physiologic expression of this epithelial-specific integrin is restricted to development and epithelial re-modeling. Here, we describe, for the first time, the development of a chimeric antigen receptor (CAR) that couples the recognition of this integrin to the delivery of potent therapeutic activity in a diverse repertoire of solid tumor models. Highly selective targeting αvß6 was achieved using a foot and mouth disease virus-derived A20 peptide, coupled to a fused CD28+CD3 endodomain. To achieve selective expansion of CAR T cells ex vivo, an IL-4-responsive fusion gene (4αß) was co-expressed, which delivers a selective mitogenic signal to engineered T cells only. In vivo efficacy was demonstrated in mice with established ovarian, breast, and pancreatic tumor xenografts, all of which express αvß6 at intermediate to high levels. SCID beige mice were used for these studies because they are susceptible to cytokine release syndrome, unlike more immune-compromised strains. Nonetheless, although the CAR also engages mouse αvß6, mild and reversible toxicity was only observed when supra-therapeutic doses of CAR T cells were administered parenterally. These data support the clinical evaluation of αvß6 re-targeted CAR T cell immunotherapy in solid tumors that express this integrin.


Subject(s)
Antigens, Neoplasm/immunology , Cell Engineering , Integrins/antagonists & inhibitors , Integrins/immunology , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Neoplasms/immunology , Neoplasms/metabolism , Receptors, Antigen, T-Cell/metabolism , Recombinant Fusion Proteins/metabolism , Animals , Antigens, Neoplasm/genetics , Cytokines/metabolism , Disease Models, Animal , Gene Expression , Gene Order , Genetic Vectors/genetics , Immunotherapy, Adoptive , Integrins/genetics , Mice , Mice, SCID , Neoplasms/therapy , Receptors, Antigen, T-Cell/genetics , Recombinant Fusion Proteins/genetics , Xenograft Model Antitumor Assays
14.
J Am Soc Nephrol ; 28(6): 1741-1752, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28062569

ABSTRACT

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.


Subject(s)
Capillary Permeability/drug effects , Kidney/blood supply , Receptors, Vitronectin/antagonists & inhibitors , Reperfusion Injury/prevention & control , Animals , Male , Rats , Rats, Sprague-Dawley
15.
PLoS One ; 11(8): e0158047, 2016.
Article in English | MEDLINE | ID: mdl-27494713

ABSTRACT

Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive disease with poor survival rates and limited treatment options. Upregulation of αvß6 integrins within the alveolar epithelial cells is a characteristic feature of IPF and correlates with poor patient survival. The pro-fibrotic cytokine TGFß1 can upregulate αvß6 integrin expression but the molecular mechanisms driving this effect have not previously been elucidated. We confirm that stimulation with exogenous TGFß1 increases expression of the integrin ß6 subunit gene (ITGB6) and αvß6 integrin cell surface expression in a time- and concentration-dependent manner. TGFß1-induced ITGB6 expression occurs via transcriptional activation of the ITGB6 gene, but does not result from effects on ITGB6 mRNA stability. Basal expression of ITGB6 in, and αvß6 integrins on, lung epithelial cells occurs via homeostatic αvß6-mediated TGFß1 activation in the absence of exogenous stimulation, and can be amplified by TGFß1 activation. Fundamentally, we show for the first time that TGFß1-induced ITGB6 expression occurs via canonical Smad signalling since dominant negative constructs directed against Smad3 and 4 inhibit ITGB6 transcriptional activity. Furthermore, disruption of a Smad binding site at -798 in the ITGB6 promoter abolishes TGFß1-induced ITGB6 transcriptional activity. Using chromatin immunoprecipitation we demonstrate that TGFß1 stimulation of lung epithelial cells results in direct binding of Smad3, and Smad4, to the ITGB6 gene promoter within this region. Finally, using an adenoviral TGFß1 over-expression model of pulmonary fibrosis we demonstrate that Smad3 is crucial for TGFß1-induced αvß6 integrin expression within the alveolar epithelium in vivo. Together, these data confirm that a homeostatic, autocrine loop of αvß6 integrin activated TGFß1-induced ITGB6 gene expression regulates epithelial basal αvß6 integrin expression, and demonstrates that this occurs via Smad-dependent transcriptional regulation at a single Smad binding site in the promoter of the ß6 subunit gene. Active TGFß1 amplifies this pathway both in vitro and in vivo, which may promote fibrosis.


Subject(s)
Idiopathic Pulmonary Fibrosis/pathology , Integrin beta Chains/metabolism , Transcription, Genetic/drug effects , Transforming Growth Factor beta/pharmacology , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Binding Sites , Cells, Cultured , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Idiopathic Pulmonary Fibrosis/metabolism , Integrin beta Chains/genetics , Integrins/genetics , Integrins/metabolism , Lung/drug effects , Lung/metabolism , Lung/pathology , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Promoter Regions, Genetic , RNA Stability/drug effects , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Smad3 Protein/genetics , Smad3 Protein/metabolism , Smad4 Protein/genetics , Smad4 Protein/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism
16.
Cancer Res ; 76(17): 5163-74, 2016 09 01.
Article in English | MEDLINE | ID: mdl-27450452

ABSTRACT

Androgen receptor signaling fuels prostate cancer and is a major therapeutic target. However, mechanisms of resistance to therapeutic androgen ablation are not well understood. Here, using a prostate cancer mouse model, Pten(pc-/-), carrying a prostate epithelial-specific Pten deletion, we show that the αvß6 integrin is required for tumor growth in vivo of castrated as well as of noncastrated mice. We describe a novel signaling pathway that couples the αvß6 integrin cell surface receptor to androgen receptor via activation of JNK1 and causes increased nuclear localization and activity of androgen receptor. This downstream kinase activation by αvß6 is specific for JNK1, with no involvement of p38 or ERK kinase. In addition, differential phosphorylation of Akt is not observed under these conditions, nor is cell morphology affected by αvß6 expression. This pathway, which is specific for αvß6, because it is not regulated by a different αv-containing integrin, αvß3, promotes upregulation of survivin, which in turn supports anchorage-independent growth of αvß6-expressing cells. Consistently, both αvß6 and survivin are significantly increased in prostatic adenocarcinoma, but are not detected in normal prostatic epithelium. Neither XIAP nor Bcl-2 is affected by αvß6 expression. In conclusion, we show that αvß6 expression is required for prostate cancer progression, including castrate-resistant prostate cancer; mechanistically, by promoting activation of JNK1, the αvß6 integrin causes androgen receptor-increased activity in the absence of androgen and consequent upregulation of survivin. These preclinical results pave the way for further clinical development of αvß6 antagonists for prostate cancer therapy. Cancer Res; 76(17); 5163-74. ©2016 AACR.


Subject(s)
Antigens, Neoplasm/metabolism , Integrins/metabolism , Mitogen-Activated Protein Kinase 8/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , Receptors, Androgen/metabolism , Animals , Cell Line, Tumor , Disease Models, Animal , Flow Cytometry , Fluorescent Antibody Technique , Gene Knockdown Techniques , Humans , Immunohistochemistry , Male , Mice , Mice, Knockout , Prostatic Neoplasms, Castration-Resistant/metabolism , Signal Transduction/physiology
17.
Cancer Med ; 5(8): 2000-11, 2016 08.
Article in English | MEDLINE | ID: mdl-27184932

ABSTRACT

Integrin αvß6 is involved in the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast. In addition, integrin ß6 (ITGB6) is of prognostic value in invasive breast cancers, particularly in HER2+ subtype. However, pathways mediating the activity of integrin αvß6 in clinical progression of invasive breast cancers need further elucidation. We have examined human breast cancer specimens (N = 460) for the expression of integrin ß6 (ITGB6) mRNA by qPCR. In addition, we have examined a subset (N = 147) for the expression of αvß6 integrin by immunohistochemistry (IHC). The expression levels of members of Rho-Rac pathway including downstream genes (ACTR2, ACTR3) and effector proteinases (MMP9, MMP15) were estimated by qPCR in the HER2+ subset (N = 59). There is a significant increase in the mean expression of ITGB6 in HER2+ tumors compared to HR+HER2- and triple negative (TNBC) subtypes (P = 0.00). HER2+ tumors with the highest levels (top quartile) of ITGB6 have significantly elevated levels of all the genes of the Rho-Rac pathway (P-values from 0.01 to 0.0001). Patients in this group have a significantly shorter disease-free survival compared to the group with lower ITGB6 levels (HR = 2.9 (0.9-8.9), P = 0.05). The mean level of ITGB6 expression is increased further in lymph node-positive tumors. The increased regional and distant metastasis observed in HER2+ tumors with high levels of ITGB6 might be mediated by the canonical Rho-Rac pathway through increased expression of MMP9 and MMP15.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Integrin beta Chains/genetics , Receptor, ErbB-2/metabolism , Signal Transduction , rac GTP-Binding Proteins/metabolism , Adult , Aged , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Female , Gene Amplification , Gene Expression , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Middle Aged , Neoplasm Metastasis , Prognosis , Proportional Hazards Models , ROC Curve , Receptor, ErbB-2/genetics
18.
J Biol Chem ; 291(18): 9540-53, 2016 Apr 29.
Article in English | MEDLINE | ID: mdl-26861876

ABSTRACT

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.


Subject(s)
Antigens, Neoplasm/biosynthesis , Gene Expression Regulation , Integrins/biosynthesis , Pulmonary Fibrosis/metabolism , Signal Transduction , Transcription, Genetic , ets-Domain Protein Elk-1/metabolism , Animals , Antigens, Neoplasm/genetics , Cell Line, Transformed , Humans , Integrins/genetics , Mice , Mice, Knockout , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/pathology , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolism , ets-Domain Protein Elk-1/genetics
19.
Hepatology ; 63(1): 217-32, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26448099

ABSTRACT

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.


Subject(s)
Antigens, Neoplasm/physiology , Carcinogenesis , Integrins/physiology , Liver Cirrhosis/etiology , Stem Cells/physiology , Animals , Cholangitis, Sclerosing/etiology , Fibrosis/etiology , Hepatocytes , Humans , Liver/pathology , Liver Neoplasms/etiology , Male , Mice , Mice, Inbred C57BL
20.
Eur Respir J ; 46(2): 486-94, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25745053

ABSTRACT

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.


Subject(s)
Antigens, Neoplasm/metabolism , Integrins/metabolism , Lung Diseases, Interstitial/pathology , Lung/pathology , Adult , Aged , Biomarkers/metabolism , Biopsy , Female , Humans , Lung Diseases, Interstitial/surgery , Male , Middle Aged , Prognosis
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