Airway derived emphysema-specific alveolar type II cells exhibit impaired regenerative potential in COPD.

Emphysema, the progressive destruction of gas exchange surfaces in the lungs, is a hallmark of chronic obstructive pulmonary disease (COPD) that is presently incurable. This therapeutic gap is largely due to a poor understanding of potential drivers of impaired tissue regeneration, such as abnormal lung epithelial progenitor cells, including alveolar type II (ATII) and airway club cells. We discovered an emphysema-specific sub-population of ATII cells located in enlarged distal alveolar sacs, termed asATII cells. Single cell RNA-seq and in situ localisation revealed that asATII cells co-express the alveolar marker surfactant protein C (SPC) and the club cell marker secretaglobin-3A2 (SCGB3A2). A similar ATII sub-population derived from club cells was also identified in mouse COPD models using lineage labeling. Human and mouse ATII sub-populations formed 80-90% fewer alveolar organoids than healthy controls, indicating reduced progenitor function. Targeting asATII cells or their progenitor club cells could reveal novel COPD treatment strategies.

Cell-free DNA levels associate with COPD exacerbations and mortality.

THE QUESTION ADDRESSED BY THE STUDY: Good biological indicators capable of predicting chronic obstructive pulmonary disease (COPD) phenotypes and clinical trajectories are lacking. Because nuclear and mitochondrial genomes are damaged and released by cigarette smoke exposure, plasma cell-free mitochondrial and nuclear DNA (cf-mtDNA and cf-nDNA) levels could potentially integrate disease physiology and clinical phenotypes in COPD. This study aimed to determine whether plasma cf-mtDNA and cf-nDNA levels are associated with COPD disease severity, exacerbations, and mortality risk. MATERIALS AND METHODS: We quantified mtDNA and nDNA copy numbers in plasma from participants enrolled in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE, n = 2,702) study and determined associations with relevant clinical parameters. RESULTS: Of the 2,128 participants with COPD, 65% were male and the median age was 64 (interquartile range, 59-69) years. During the baseline visit, cf-mtDNA levels positively correlated with future exacerbation rates in subjects with mild/moderate and severe disease (Global Initiative for Obstructive Lung Disease [GOLD] I/II and III, respectively) or with high eosinophil count (≥ 300). cf-nDNA positively associated with an increased mortality risk (hazard ratio, 1.33 [95% confidence interval, 1.01-1.74] per each natural log of cf-nDNA copy number). Additional analysis revealed that individuals with low cf-mtDNA and high cf-nDNA abundance further increased the mortality risk (hazard ratio, 1.62 [95% confidence interval, 1.16-2.25] per each natural log of cf-nDNA copy number). ANSWER TO THE QUESTION: Plasma cf-mtDNA and cf-nDNA, when integrated into quantitative clinical measurements, may aid in improving COPD severity and progression assessment.

Loss of ANT1 Increases Fibrosis and Epithelial Cell Senescence in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by progressive lung scarring and remodeling. Although treatments exist that slow disease progression, IPF is irreversible, and there is no cure. Cellular senescence, a major hallmark of aging, has been implicated in IPF pathogenesis, and mitochondrial dysfunction is increasingly recognized as a driver of senescence. Adenine nucleotide translocases (ANTs) are abundant mitochondrial ATP-ADP transporters critical for regulating cell fate and maintaining mitochondrial function. We sought to determine how alterations in ANTs influence cellular senescence in pulmonary fibrosis. We found that SLC25A4 (solute carrier family 25 member 4) (ANT1) and SLC25A5 (ANT2) expression is reduced in the lungs of patients with IPF, particularly within alveolar type II (AT2) cells, by single-cell RNA sequencing and tissue staining. Loss of ANT1 by siRNA in lung epithelial cells resulted in increased senescence markers such as ß-galactosidase and p21, with a reduction in the ratio of nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide. Bleomycin-treated ANT1 knockdown cells also had increased senescence markers compared with bleomycin-treated control cells. Loss of ANT1 in AT2 cells resulted in a reduction in alveolar organoid growth, with an increase in p21 by staining. Global loss of ANT1 resulted in worse lung fibrosis and increased senescence in the bleomycin- and asbestos-induced mouse models of pulmonary fibrosis. In summary, loss of ANT1 contributes to IPF pathogenesis through mitochondrial dysfunction, increased senescence, and decreased regenerative capacity of AT2 cells, resulting in enhanced lung fibrosis. Modulation of ANTs presents a new therapeutic avenue that may alter cellular senescence pathways and limit pulmonary fibrosis.

Adenine nucleotide translocase regulates airway epithelial metabolism, surface hydration and ciliary function.

Airway hydration and ciliary function are critical to airway homeostasis and dysregulated in chronic obstructive pulmonary disease (COPD), which is impacted by cigarette smoking and has no therapeutic options. We utilized a high-copy cDNA library genetic selection approach in the amoeba Dictyostelium discoideum to identify genetic protectors to cigarette smoke. Members of the mitochondrial ADP/ATP transporter family adenine nucleotide translocase (ANT) are protective against cigarette smoke in Dictyostelium and human bronchial epithelial cells. Gene expression of ANT2 is reduced in lung tissue from COPD patients and in a mouse smoking model, and overexpression of ANT1 and ANT2 resulted in enhanced oxidative respiration and ATP flux. In addition to the presence of ANT proteins in the mitochondria, they reside at the plasma membrane in airway epithelial cells and regulate airway homeostasis. ANT2 overexpression stimulates airway surface hydration by ATP and maintains ciliary beating after exposure to cigarette smoke, both of which are key functions of the airway. Our study highlights a potential for upregulation of ANT proteins and/or of their agonists in the protection from dysfunctional mitochondrial metabolism, airway hydration and ciliary motility in COPD.This article has an associated First Person interview with the first author of the paper.

A comparison of visual and quantitative methods to identify interstitial lung abnormalities.

BACKGROUND: Evidence suggests that individuals with interstitial lung abnormalities (ILA) on a chest computed tomogram (CT) may have an increased risk to develop a clinically significant interstitial lung disease (ILD). Although methods used to identify individuals with ILA on chest CT have included both automated quantitative and qualitative visual inspection methods, there has been not direct comparison between these two methods. To investigate this relationship, we created lung density metrics and compared these to visual assessments of ILA. METHODS: To provide a comparison between ILA detection methods based on visual assessment we generated measures of high attenuation areas (HAAs, defined by attenuation values between -600 and -250 Hounsfield Units) in >4500 participants from both the COPDGene and Framingham Heart studies (FHS). Linear and logistic regressions were used for analyses. RESULTS: Increased measures of HAAs (in ≥ 10 % of the lung) were significantly associated with ILA defined by visual inspection in both cohorts (P < 0.0001); however, the positive predictive values were not very high (19 % in COPDGene and 13 % in the FHS). In COPDGene, the association between HAAs and ILA defined by visual assessment were modified by the percentage of emphysema and body mass index. Although increased HAAs were associated with reductions in total lung capacity in both cohorts, there was no evidence for an association between measurement of HAAs and MUC5B promoter genotype in the FHS. CONCLUSION: Our findings demonstrate that increased measures of lung density may be helpful in determining the severity of lung volume reduction, but alone, are not strongly predictive of ILA defined by visual assessment. Moreover, HAAs were not associated with MUC5B promoter genotype.

Interpretable machine learning uncovers epithelial transcriptional rewiring and a role for Gelsolin in COPD.

Transcriptomic analyses have advanced the understanding of complex disease pathophysiology including chronic obstructive pulmonary disease (COPD). However, identifying relevant biologic causative factors has been limited by the integration of high dimensionality data. COPD is characterized by lung destruction and inflammation with smoke exposure being a major risk factor. To define novel biological mechanisms in COPD, we utilized unsupervised and supervised interpretable machine learning analyses of single cell-RNA sequencing data from the gold standard mouse smoke exposure model to identify significant latent factors (context-specific co-expression modules) impacting pathophysiology. The machine learning transcriptomic signatures coupled to protein networks uncovered a reduction in network complexity and novel biological alterations in actin-associated gelsolin (GSN), which was transcriptionally linked to disease state. GSN was altered in airway epithelial cells in the mouse model and in human COPD. GSN was increased in plasma from COPD patients, and smoke exposure resulted in enhanced GSN release from airway cells from COPD patients. This method provides insights into rewiring of transcriptional networks that are associated with COPD pathogenesis and provide a novel analytical platform for other diseases.

Strategies for Achieving Gender Equity and Work-Life Integration in Physician-Scientist Training.

Substantial gender inequities persist across academic medicine. These issues are not new: Recent evidence still points to a chilly climate for women in academic medicine, including those in physician-scientist training. The discussion for how to address gender equity and issues of work-life integration typically centers around faculty and rarely includes trainees. The authors delineate specific strategies to address gender inequity in physician-scientist training by identifying key stakeholders for implementation and proposing areas to integrate these strategies with current training timelines. Strategies discussed include multiple-role mentoring, allyship training for trainees and faculty, early implementation of professional development sessions, incorporation of childcare and family-friendly policies, and additional policies for funding bodies to prioritize gender equity practices. The goal of this article is to equip trainees and the academic community with proactive strategies to create a more equitable environment for future generations of trainees in academic medicine.

Predoctoral MD-PhD grants as indicators of future NIH funding success.

MD-PhD trainees constitute an important source of physician-scientists. Persistence on this challenging path is facilitated by success in garnering independent (R grant) support from the NIH. Published research tracks academic appointments and global R01 success for MD-PhD trainees but has not included information on future funding success of individual MD-PhD predoctoral grant holders. Here, we used data from the NIH RePORTER database to identify and track the funding trajectory of physician-scientists who received predoctoral grant support through the F30 mechanism, which is specific for dual-degree candidates. Male and female F30 awardees did not differ in their success in garnering K (postdoctoral training) grants, but, among F30 grant awardees, men were 2.6 times more likely than women to receive R funding. These results underscore the need for analysis of factors that contribute to the disproportionate loss of NIH-supported female physician-scientists between the predoctoral F30 and the independent R grant-supported stages.

VEGF Receptor 1 Promotes Hypoxia-Induced Hematopoietic Progenitor Proliferation and Differentiation.

Although it is well known that hypoxia incites unleashed cellular inflammation, the mechanisms of exaggerated cellular inflammation in hypoxic conditions are not known. We observed augmented proliferation of hematopoietic stem and progenitor cells (HSPC), precursors of inflammatory leukocytes, in mice under hypoxia. Consistently, a transcriptomic analysis of human HSPC exposed to hypoxic conditions revealed elevated expression of genes involved in progenitor proliferation and differentiation. Additionally, bone marrow cells in mice expressed high amount of vascular endothelial growth factor (VEGF), and HSPC elevated VEGF receptor 1 (VEGFr1) and its target genes in hypoxic conditions. In line with this, VEGFr1 blockade in vivo and in vitro decreased HSPC proliferation and attenuated inflammation. In silico and ChIP experiments demonstrated that HIF-1α binds to the promoter region of VEGFR1. Correspondingly, HIF1a silencing decreased VEGFr1 expression in HSPC and diminished their proliferation. These results indicate that VEGF signaling in HSPC is an important mediator of their proliferation and differentiation in hypoxia-induced inflammation and represents a potential therapeutic target to prevent aberrant inflammation in hypoxia-associated diseases.

COVID-19 and the Early-Career Physician-Scientist. Fostering Resilience beyond the Pandemic.

The coronavirus disease (COVID-19) pandemic has created significant stressors for the academic and scientific community, with unique challenges for early-career physician-scientists. The pandemic-related disruptions have significantly affected research productivity, access to mentoring, professional development and networking opportunities, funding, and personal wellness. This is especially true for pulmonary and critical care medicine faculty because of the burden of specialized clinical care responsibilities that the COVID-19 pandemic has demanded. Departmental, institutional, and national leadership should foster open dialogue to identify and mitigate these challenges to promote ongoing career development of early-career physician-scientists. Implementation of thoughtful interventions to address these challenges will provide essential support for junior faculty and help retain a generation of physician-scientists.

Extracellular superoxide dismutase regulates cardiac function and fibrosis.

Extracellular superoxide dismutase (EC-SOD) is an antioxidant that protects the heart from ischemia and the lung from inflammation and fibrosis. The role of cardiac EC-SOD under normal conditions and injury remains unclear. Cardiac toxicity, a common side effect of doxorubicin, involves oxidative stress. We hypothesize that EC-SOD is critical for normal cardiac function and protects the heart from oxidant-induced fibrosis and loss of function. C57BL/6 and EC-SOD-null mice were treated with doxorubicin, 15 mg/kg (i.p.). After 15 days, echocardiography was used to assess cardiac function. Left ventricle (LV) tissue was used to assess fibrosis and inflammation by staining, Western blot, and hydroxyproline analysis. At baseline, EC-SOD-null mice have LV wall thinning and increases in LV end diastolic dimensions compared to wild-type mice but have normal cardiac function. After doxorubicin, EC-SOD-null mice have decreases in fractional shortening not apparent in WT mice. Lack of EC-SOD also leads to increases in myocardial apoptosis and significantly more LV fibrosis and inflammatory cell infiltration. Administration of the metalloporphyrin AEOL 10150 abrogates the loss of cardiac function, and potentially fibrosis, associated with doxorubicin treatment in both wild-type and EC-SOD KO mice. EC-SOD is critical for normal cardiac morphology and protects the heart from oxidant-induced fibrosis, apoptosis, and loss of function. The antioxidant metalloporphyrin AEOL 10150 effectively protects cardiac function from doxorubicin-induced oxidative stress in vivo. These findings identify targets for the use of antioxidant agents in oxidant-induced cardiac fibrosis.

Gremlin-mediated decrease in bone morphogenetic protein signaling promotes pulmonary fibrosis.

RATIONALE: Members of the transforming growth factor (TGF)-beta superfamily, including TGF-betas and bone morphogenetic proteins (BMPs), are essential for the maintenance of tissue homeostasis and regeneration after injury. We have observed that the BMP antagonist, gremlin, is highly up-regulated in idiopathic pulmonary fibrosis (IPF). OBJECTIVES: To investigate the role of gremlin in the regulation of BMP signaling in pulmonary fibrosis. METHODS: Progressive asbestos-induced fibrosis in the mouse was used as a model of human IPF. TGF-beta and BMP expression and signaling activities were measured from murine and human fibrotic lungs. The mechanism of gremlin induction was analyzed in cultured lung epithelial cells. In addition, the possible therapeutic role of gremlin inhibition was tested by administration of BMP-7 to mice after asbestos exposure. MEASUREMENTS AND MAIN RESULTS: Gremlin mRNA levels were up-regulated in the asbestos-exposed mouse lungs, which is in agreement with the human IPF biopsy data. Down-regulation of BMP signaling was demonstrated by reduced levels of Smad1/5/8 and enhanced Smad2 phosphorylation in asbestos-treated lungs. Accordingly, analyses of cultured human bronchial epithelial cells indicated that asbestos-induced gremlin expression could be prevented by inhibitors of the TGF-beta receptor and also by inhibitors of the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase pathways. BMP-7 treatment significantly reduced hydroxyproline contents in the asbestos-treated mice. CONCLUSIONS: The TGF-beta and BMP signaling balance is important for lung regenerative events and is significantly perturbed in pulmonary fibrosis. Rescue of BMP signaling activity may represent a potential beneficial strategy for treating human pulmonary fibrosis.

Extracellular superoxide dismutase protects against matrix degradation of heparan sulfate in the lung.

Asbestosis is a form of interstitial lung disease caused by the inhalation of asbestos fibers, leading to inflammation and pulmonary fibrosis. Inflammation and oxidant/antioxidant imbalances are known to contribute to the disease pathogenesis. Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that has been shown to protect the lung from oxidant-mediated damage, inflammation, and interstitial fibrosis. Extracellular matrix (ECM) components, such as collagen and glycosaminoglycans, are known to be sensitive to oxidative fragmentation. Heparan sulfate, a glycosaminoglycan, is highly abundant in the ECM and tightly binds EC-SOD. We investigated the protective role of EC-SOD by evaluating the interaction of EC-SOD with heparan sulfate in the presence of reactive oxygen species (ROS). We found that ROS-induced heparin and heparan sulfate fragments induced neutrophil chemotaxis across a modified Boyden chamber, which was inhibited by the presence of EC-SOD by scavenging oxygen radicals. Chemotaxis in response to oxidatively fragmented heparin was mediated by Toll-like receptor-4. In vivo, bronchoalveolar lavage fluid from EC-SOD knockout mice at 1, 14, and 28 days after asbestos exposure showed increased heparan sulfate shedding from the lung parenchyma. We demonstrate that one mechanism through which EC-SOD inhibits lung inflammation and fibrosis in asbestosis is by protecting heparin/heparan sulfate from oxidative fragmentation.

Neutrophil elastase promotes myofibroblast differentiation in lung fibrosis.

IPF is a progressive lung disorder characterized by fibroblast proliferation and myofibroblast differentiation. Although neutrophil accumulation within IPF lungs has been negatively correlated with outcomes, the role played by neutrophils in lung fibrosis remains poorly understood. We have demonstrated previously that NE promotes lung cancer cell proliferation and hypothesized that it may have a similar effect on fibroblasts. In the current study, we show that NE(-/-) mice are protected from asbestos-induced lung fibrosis. NE(-/-) mice displayed reduced fibroblast and myofibroblast content when compared with controls. NE directly both lung fibroblast proliferation and myofibroblast differentiation in vitro, as evidenced by proliferation assays, collagen gel contractility assays, and αSMA induction. Furthermore, αSMA induction occurs in a TGF-ß-independent fashion. Treatment of asbestos-recipient mice with ONO-5046, a synthetic NE antagonist, reduced hydroxyproline content. Thus, the current study points to a key role for neutrophils and NE in the progression of lung fibrosis. Lastly, the study lends rationale to use of NE-inhibitory approaches as a novel therapeutic strategy for patients with lung fibrosis.

Extracellular superoxide dismutase protects cardiovascular syndecan-1 from oxidative shedding.

The extracellular matrix is a complex system that regulates cell function within a tissue. The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is bound to the matrix, and previous studies show that a lack of EC-SOD results in increased cardiac injury, fibrosis, and loss of cardiac function. This study tests the hypothesis that EC-SOD protects against cardiac fibrosis mechanistically by limiting oxidative stress and oxidant-induced shedding of syndecan-1 in the extracellular matrix. Wild-type and EC-SOD null mice were treated with a single dose of doxorubicin, 15 mg/kg, and evaluated on day 15. Serum and left-ventricle tissue were collected for biochemical assays, including Western blot, mRNA expression, and immunohistochemical staining for syndecan-1. The loss of EC-SOD and doxorubicin-induced oxidative injury led to increases in shed syndecan-1 in the serum, which originates from the endothelium of the vasculature. The shed syndecan-1 ectodomain induces proliferation of primary mouse cardiac fibroblasts. This study suggests that one mechanism by which EC-SOD protects the heart against cardiac fibrosis is the prevention of oxidative shedding of cardiovascular syndecan-1 and its subsequent induction of fibroblast proliferation. This study provides potential new targets for understanding and altering fibrosis progression in the heart.

A novel method for accurate collagen and biochemical assessment of pulmonary tissue utilizing one animal.

AIM: The purpose of this study was to develop an improved method for collagen and protein assessment of fibrotic lungs while decreasing animal use. METHODS: 8-10 week old, male C57BL/6 mice were given a single intratracheal instillation of crocidolite asbestos or control titanium dioxide. Lungs were collected on day 14 and dried as whole lung, or homogenized in CHAPS buffer, for hydroxyproline analysis. Insoluble and salt-soluble collagen content was also determined in lung homogenates using a modified Sirius red colorimetric 96-well plate assay. RESULTS: The hydroxyproline assay showed significant increases in collagen content in the lungs of asbestos-treated mice. Identical results were present between collagen content determined on dried whole lung or whole lung homogenates. The Sirius red plate assay showed a significant increase in collagen content in lung homogenates however, this assay grossly over-estimated the total amount of collagen and underestimated changes between control and fibrotic lungs, conclusions: The proposed method provides accurate quantification of collagen content in whole lungs and additional homogenate samples for biochemical analysis from a single animal. The Sirius-red colorimetric plate assay provides a complementary method for determination of the relative changes in lung collagen but the values tend to overestimate absolute values obtained by the gold standard hydroxyproline assay and underestimate the overall fibrotic injury.

Paradoxical function for the receptor for advanced glycation end products in mouse models of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor survival. The identification of therapeutic targets is essential to improving outcomes. Previous studies found that expression of the receptor for advanced glycation end products (RAGE) in the lung is significantly decreased in human IPF lungs and in two animal models of pulmonary fibrosis. In addition, RAGE-null mice spontaneously develop pulmonary fibrosis with age and more severe fibrosis when challenged with asbestos. In contrast to the findings that the lack of RAGE enhanced pulmonary fibrosis, He et al. found that RAGE null mice were protected from bleomycin-induced fibrosis and suggested the effect was due to a lack of HMGB1 induced RAGE signaling. The current study further tests this hypothesis by blocking RAGE signaling via administration of soluble RAGE, a decoy receptor, to determine if this will also protect against pulmonary fibrosis. Wild-type, RAGE(+/-), and RAGE(-/-) mice were treated with bleomycin and assessed for fibrosis. Wild-type mice were also treated with exogenous soluble RAGE or vehicle control. In addition, in vitro studies with primary alveolar epithelial cells from wild-type and RAGE null mice were used to investigate the effect of RAGE on cell viability and migration in response to injury. A lack of RAGE was found to be protective against bleomycin injury in both in vivo and in vitro studies. However, soluble RAGE administration was unable to ameliorate fibrosis. This study confirms paradoxical responses to two different models of pulmonary fibrosis and suggests a further role for RAGE in cellular migration.

Oxidative stress, extracellular matrix targets, and idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by progressive fibrosis of the alveolar interstitium. The pathogenesis is thought to involve abnormal reepithelialization and dysregulated remodeling of the extracellular matrix after alveolar injury. There is growing evidence through human and animal studies that oxidative stress plays a role in this dysregulation. Markers of oxidative stress have been identified in the lungs of IPF patients and aberrant antioxidant activity exacerbates pulmonary fibrosis in animal models. In addition, the extracellular matrix is a critical component in regulating cellular homeostasis and appropriate wound healing. Recent investigations support that the matrix is a target of oxidative stress in the lung and IPF. Extracellular matrix degradation products, produced by reactive oxygen species, may promote fibrogenesis by influencing epithelial, mesenchymal, and inflammatory cell activity. The impact of the interactions of oxidative stress and the matrix of the lung remains unclear and may prove to be an important target for new therapies in IPF. Utilizing oxidative enzymes, antioxidants, or the matrix as therapeutic targets to control oxidative stress in IPF will continue be an area of active research and innovative discoveries in the coming years.

Neutrophil elastase-mediated degradation of IRS-1 accelerates lung tumor growth.

Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that tumor-associated inflammatory cells may modify lung tumor growth and invasiveness. To determine the role of neutrophil elastase (encoded by Elane) on tumor progression, we used the loxP-Stop-loxP K-ras(G12D) (LSL-K-ras) model of mouse lung adenocarcinoma to generate LSL-K-ras-Elane(-/-) mice. Tumor burden was markedly reduced in LSL-K-ras-Elane(-/-) mice at all time points after induction of mutant K-ras expression. Kaplan-Meier survival analysis showed that whereas all LSL-K-ras-Elane(+/+) mice died, none of the mice lacking neutrophil elastase died. Neutrophil elastase directly induced tumor cell proliferation in both human and mouse lung adenocarcinomas by gaining access to an endosomal compartment within tumor cells, where it degraded insulin receptor substrate-1 (IRS-1). Immunoprecipitation studies showed that, as neutrophil elastase degraded IRS-1, there was increased interaction between phosphatidylinositol 3-kinase (PI3K) and the potent mitogen platelet-derived growth factor receptor (PDGFR), thereby skewing the PI3K axis toward tumor cell proliferation. The inverse relationship identified between neutrophil elastase and IRS-1 in LSL-K-ras mice was also identified in human lung adenocarcinomas, thus translating these findings to human disease. This study identifies IRS-1 as a key regulator of PI3K within malignant cells. Additionally, to our knowledge, this is the first description of a secreted proteinase gaining access to the inside of a cell and altering intracellular signaling.