Rashika Joshi

Localization and stretch-dependence of lung elastase activity in development and compensatory growth

Synthesis and remodeling of the lung matrix is necessary for primary and compensatory lung growth. Because cyclic negative force is applied to developing lung tissue during the respiratory cycle, we hypothesized that stretch is a critical regulator of lung matrix remodeling. By using quantitative image analysis of whole-lung and whole-lobe elastin in situ zymography images, we demonstrated that elastase activity increased twofold during the alveolar stage of postnatal lung morphogenesis in the mouse. Remodeling was restricted to alveolar walls and ducts and was nearly absent in dense elastin band structures. In the mouse pneumonectomy model of compensatory lung growth, elastase activity increased threefold, peaking at 14 days postpneumonectomy and was higher in the accessory lobe compared with other lobes. Remodeling during normal development and during compensatory lung growth was different with increased major airway and pulmonary arterial remodeling during development but not regeneration, and with homogenous remodeling throughout the parenchyma during development, but increased remodeling only in subpleural regions during compensatory lung growth. Left lung wax plombage prevented increased lung elastin during compensatory lung growth. To test whether the adult lung retains an innate capacity to remodel elastin, we developed a confocal microscope-compatible stretching device. In ex vivo adult mouse lung sections, lung elastase activity increased exponentially with strain and in peripheral regions of lung more than in central regions. Our study demonstrates that lung elastase activity is stretch-dependent and supports a model in which externally applied forces influence the composition, structure, and function of the matrix during periods of alveolar septation.

HER2-mTOR signaling–driven breast cancer cells require ER-associated degradation to survive

Exploiting the dependence of some cancer cells on an ER stress response pathway may overcome resistance to some chemotherapeutics. Putting more stress on HER2-driven cancer Cancer cells have increased gene expression and hence protein synthesis, which can induce endoplasmic reticulum (ER) stress. This stress can trigger apoptosis if not alleviated. Using patient and cancer cell line genomic databases, Singh et al. found that HER2-driven breast cancers have increased ER stress–induced signaling. Pharmacologically blocking this pathway caused cell death in cultured HER2-positive cells, including those that were resistant to clinically used HER2 inhibitors. Targeting non-oncogenic vulnerabilities may provide additional therapeutic approaches in tumors that are resistant to oncogene-targeted therapy. Using a computational pathway-based approach, we interrogated clinical breast cancer genomic data sets for candidate non-oncogenic vulnerabilities in breast cancers that have genomic amplification of ERBB2, which encodes human epidermal growth factor 2 (HER2). HER2-positive (HER2+) breast cancers showed increased expression of genes encoding proteins in the endoplasmic reticulum (ER)–associated degradation (ERAD) pathway. Genetic ablation or pharmacological inhibition of ERAD led to irrecoverable ER stress and selectively killed HER2+ breast cancer cells. Cell death caused by ERAD inhibition partially depended on increased HER2-mTOR signaling, which imposed an increased proteotoxic burden on the ER. Cell death in response to ER stress required the IRE1α-JNK pathway, which was selectively suppressed in HER2+ breast cancers by phosphatases that inactivate JNK. Accordingly, the cytotoxicity of inhibiting ERAD as well as JNK phosphatases was synergistic in HER2+ but not in HER2-negative breast cancer cells. Therefore, our study suggests that reactivation of oncogene-induced stress by targeting stress-adaptive pathways may be a beneficial approach for therapy-resistant breast cancers.

Stretch regulates expression and binding of chymotrypsin-like elastase 1 in the postnatal lung

Lung stretch is critical for normal lung development and for compensatory lung growth after pneumonectomy (PNX), but the mechanisms by which strain induces matrix remodeling are unclear. Our prior work demonstrated an association of chymotrypsin‐like elastase 1 (Cela1) with lung elastin remodeling, and that strain triggered a near‐instantaneous elastin‐remodeling response. We sought to determine whether stretch regulates Cela1 expression and Cela1 binding to lung elastin. In C57BL/6J mice, Cela1 protein increased 176‐fold during lung morphogenesis. Cela1 was covalently bound to serpin peptidase inhibitor, clade A, member 1, resulting in a higher molecular mass in lung homogenate compared to pancreas homogenate. Post‐PNX, Cela1 mRNA increased 6‐fold, protein 3‐fold, and Cela1‐positive cells 2‐fold. Cela1 was expressed predominantly in alveolar type II cells in the embryonic lung and predominantly in CD90‐positive lung fibroblasts postnatally. During compensatory lung growth, Cela1 expression was induced in nonproliferative mesenchymal cells. In ex vivo mouse lung sections, stretch increased Cela1 binding to lung tissue by 46%. Competitive inhibition with soluble elastin completely abrogated this increase. Areas of stretch‐induced elastase activity and Cela1 binding colocalized. The stretch‐dependent expression and binding kinetics of Cela1 indicate an important role in stretch‐dependent remodeling of the peripheral lung during development and regeneration.—Joshi, R., Liu, S., Brown, M. D., Young, S. M., Batie, M., Kofron, J. M., Xu, Y., Weaver, T. E., Apsley, K., Varisco, B. M. Stretch regulates expression and binding of chymotrypsin‐like elastase 1 in the postnatal lung. FASEB J. 30, 590‐600 (2016). www.fasebj.org

Excessive Reversal of Epidermal Growth Factor Receptor and Ephrin Signaling Following Tracheal Occlusion in Rabbit Model of Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) causes severe pulmonary hypoplasia from herniation of abdominal contents into the thorax. Tracheal occlusion (TO) for human CDH improves survival, but morbidity and mortality remain high, and we do not fully understand the cellular pathways and processes most severely impacted by CDH and TO. We created a left diaphragmatic hernia (DH) in rabbit fetuses with subsequent TO and collected left lung sections for NextGen mRNA sequencing. DH, TO and DHTO fetuses had comparable body and organ growth to control except for lower lung weights in DH (p < 0.05). Of 13,687 expressed genes, DHTO had 687 differentially expressed genes compared with DH, but no other group-group comparison had more than 10. Considering genes in combination, many of the genes reduced in DH were more highly expressed in DHTO than in control. Benchmarking fetal rabbit lung gene expression to published lung development data, both DH and DHTO lungs were more highly correlated with the gene expression of immature lung. DNA synthesis was upregulated in DHTO compared with DH and ribosome and protein synthesis pathways were downregulated. DH reduced total and epithelial cell proliferation by half and two-thirds respectively, and DHTO increased proliferation by 2.5 and 3.4-fold respectively. Signaling pathways downregulated by DH and upregulated in DHTO were epidermal growth factor receptor signaling, ephrin signaling and cell migration; however, levels of ephrin and EGFR signaling in DHTO exceeded that of control. Identification and inhibition of the ligands responsible for this dysregulated signaling could improve lung development in CDH.

Role for Cela1 in Postnatal Lung Remodeling and Alpha-1 Antitrypsin–Deficient Emphysema

&NA; Alpha‐1 antitrypsin (AAT) deficiency‐related emphysema is the fourth leading indication for lung transplant. Chymotrypsin‐like elastase 1 (Cela1) is a digestive protease that is expressed during lung development in association with regions of elastin remodeling, exhibits stretch‐dependent expression during lung regeneration, and binds lung elastin in a stretch‐dependent manner. AAT covalently neutralizes Cela1 in vitro. We sought to determine the role of Cela1 in postnatal lung physiology, whether it interacted with AAT in vivo, and to detect any effects it may have in the context of AAT deficiency. The lungs of Cela1−/− mice had aberrant lung elastin structure and higher elastance as assessed with the flexiVent system. On the basis of in situ zymography with ex vivo lung stretch, Cela1 was solely responsible for stretch‐inducible lung elastase activity. By mass spectrometry, Cela1 degraded mature elastin similarly to pancreatic elastase. Cela1 promoter and protein sequences were phylogenetically distinct in the placental mammal lineage, suggesting an adaptive role for lung‐expressed Cela1 in this clade. A 6‐week antisense oligonucleotide mouse model of AAT deficiency resulted in emphysema with increased Cela1 mRNA and reduction of approximately 70 kD Cela1, consistent with covalent binding of Cela1 by AAT. Cela1−/− mice were completely protected against emphysema in this model. Cela1 was increased in human AAT‐deficient emphysema. Cela1 is important in physiologic and pathologic stretch‐dependent remodeling processes in the postnatal lung. AAT is an important regulator of this process. Our findings provide proof of concept for the development of anti‐Cela1 therapies to prevent and/or treat AAT‐deficient emphysema.

Proteomic Profiling of Tracheal Fluid in an Ovine Model of Congenital Diaphragmatic Hernia and Fetal Tracheal Occlusion

Congenital diaphragmatic hernia (CDH) occurs in ~1:2,000 pregnancies and is associated with substantial morbidity and mortality. Fetal tracheal occlusion (TO) is an emerging therapy that improves lung growth and reduces mortality, although substantial respiratory compromise persists in survivors. In this study, we used tracheal fluid in a fetal sheep model of CDH with TO for proteomic analysis with subsequent validation of findings in sheep lung tissue. We found that the proteomic profiles of CDH tracheal fluid was most similar to control lung and CDH/TO lung most similar to TO lung. Among 118 proteins altered in CDH, only 11 were reciprocally regulated in CDH/TO. The most significantly altered pathways and processes were cell proliferation, phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling, inflammation, and microtubule dynamics. CDH suppressed and TO promoted cell proliferation and AKT-related signaling cascades. By Western blot analysis and immunohistochemistry, epithelial PCNA and phosphorylated AKT were decreased in CDH and increased in TO and CDH/TO lungs. The Wnt target Axin2 was decreased threefold in CDH lung compared with control without a significant increase in CDH/TO lung. Cilia-related pathways were among the most dysregulated with CDH lung having a nearly twofold increase in acetylated α-tubulin and a relative increase in the number of ciliated cells. While TO improves lung growth and patient survival in CDH, the procedure substantially alters many processes important in lung development and cell differentiation. Further elucidation of these changes will be critical to improving lung health in infants with CDH treated with TO.

Role Of Chymotrypsin-Like Elastase 1 In Lung Physiology And in α1-Antitrypsin Deficiency

α1-antitrypsin (AAT) deficiency-related emphysema is the fourth leading indication for lung transplantation. We previously demonstrated that AAT covalently neutralizes chymotrypsin-like elastase 1 (Cela1) in vitro, that Cela1 is expressed during the alveolar stage of lung development in association with regions of lung elastin remodeling, and that lung stretch increases Cela1 expression and binding to lung elastin. Here we show that Cela1 is exclusively responsible for stretch-inducible lung elastase activity, reduces postnatal lung elastance, and is required for emphysema in an antisense oligo model of AAT deficiency. Cela1 mRNA is present in the human lung, and in the placental mammal lineage, Cela1 is more conserved than Cela2 or Cela3 with unique promoter and protein elements indicating a unique role for Cela1 in this lineage. These data demonstrate an adaptive role for Cela1 in placental mammal lung biology with physiologic relevance to AAT-deficient lung disease in humans. α1-antitrypsin related lung disease (AAT-RLD) is the fourth leading indication for lung transplantation and is characterized by protease-mediated progressive emphysema that manifests in the 4th or 5th decade of life. Chymotrypsin-like elastase 1 (Cela1) is a digestive enzyme that binds to elastin in a stretch-dependent manner and is covalently neutralized by AAT. We hypothesized a role for Cela1 in AAT-RLD. Cela1-/- mice where phenotypically similar to wild type but had higher lung elastance and lacked stretch-inducible elastase activity. Wild-type mice administered anti-AAT oligo had reduced amounts of lung Cela1-AAT fusion protein in lung homogenate and spontaneously developed emphysema after 6 weeks. Cela1-/- mice administered anti-AAT oligo were completely protected from these emphysematous changes. Cela1 recombinant protein did not require propeptide cleavage for elastolysis, and its elastolytic profile was similar to that of other pancreatic elastases. Phylogenetic analysis of vertebrate Cela promoter and protein sequences showed that placental mammal Cela1 was distinct from other Cela’s, and that the placental mammal the Cela1 gene was invariantly conserved despite variable loss of other Cela genes in non-carnivores. These data demonstrate that the pancreatic enzyme Cela1 has been evolutionarily co-opted for a role in reducing lung elastance in the placental mammal lineage and that its stretch-regulated expression and elastolytic activity is responsible for emphysema in the absence of its anti-protease: AAT.

Abstract A2-53: Integrative systems analyses reveal addiction of HER2+ breast cancers to ERAD for survival

ERBB2/HER2-positive breast cancers are associated with poor prognosis; and resistance to HER2-targeted therapy is one of the significant clinical challenges. Trastuzumab and lapatinib are two agents that target the HER2 protein in the clinic and constitute the standard of care for HER2+ breast cancers (BCs). However, the clinical benefits of HER2-targeted therapies are limited due to significant de novo (>60%) and acquired resistance (~100%) to these agents during prolonged phases of treatment. No treatment options exist for metastatic HER2+ BCs that have progressed on HER2-targeted therapy. Therefore, targeting of non-oncogenic vulnerabilities is a promising therapeutic approach in tumors that are resistant to oncogene-targeted therapy. Using an evolutionary selection criterion, we computationally interrogated targetable non-oncogenic vulnerabilities in ERBB2/HER2+ breast cancers, which suffer from a high rate of de novo and acquired resistance to HER2-targeted therapy. We found that the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway is induced in HER2+ breast cancers, partly due to adaptive genomic rearrangements, to counter ER stress caused by increased proteotoxic burden due to heightened HER2/mTOR activity. Genetic and pharmacologic targeting of ERAD lead to irrecoverable ER stress and selectively killed HER2+ breast cancer cells, including those that are resistant to conventional therapy. Cell death due to ERAD inhibition was executed by the IRE1α/JNK pathway, which was suppressed in HER2+ breast cancers due to the activation of JNK phosphatases. Accordingly, inhibiting ERAD in combination with JNK phosphatases synergized in killing HER2+, but not HER2-, breast cancer cells. Our study supports exploiting oncogenic proteotoxicity as a novel therapeutic approach for therapy-refractory breast cancers, and presents an integrated strategy to identify non-oncogenic vulnerabilities in cancers. Citation Format: Navneet Singh, Rashika Joshi, Kakajan Komurov. Integrative systems analyses reveal addiction of HER2+ breast cancers to ERAD for survival. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-53.

Abstract C17: Revealing targetable cancer cell vulnerabilities by in silico pathway profiling

Successful targeted therapy of cancers relies on comprehensive understanding of vulnerabilities of the tumorigenic platform. Recent cancer genomics efforts have provided with an unprecedented opportunity to gain an unbiased insight into the functional make-up of human cancers. We have devised a computational strategy to leverage cancer genomic datasets to identify candidate pathway vulnerabilities in cancers. The core of our approach is in the delineation of molecular pathways that are activated in tumor cells as a result of secondary selection events during tumor evolution. These secondary pathways are likely to be required for tumorigenesis in the given tumor context. We applied this strategy on HER2+ breast cancers using breast cancer genomic data in the TCGA, the recently published METABRIC and the Cancer Cell Line Encyclopedia (CCLE) datasets. Although HER2+ breast cancers are treated with HER2-targeting agents in the clinic, the response is usually short-lived due to de novo and acquired resistance. We have found that HER2+ breast cancers are characterized by extensive and robust secondary upregulation of the endoplasmic reticulum (ER) quality control (ERQC) pathways, including the ER-associated degradation (ERAD) and PERK/eIF2 pathways. Our follow-up combined in silico and in vitro analyses on a panel of HER2+ and HER2- breast cancer cell lines revealed a model of HER2-driven tumorigenesis where yin-yang interplay between hyperactive HER2/mTOR signaling and the ERQC machinery regulate the ER protein homeostasis to control survival of HER2+ cells. Furthermore, we report that this delicate regulatory architecture can be exploited for therapeutic purposes, such that, combinatorial targeting of ERQC pathways to disturb the proteostatic balance synergized in selectively inducing proteotoxic cell death in therapy-refractory HER2+ cells. This study presents a computational method for identification of cancer pathway vulnerabilities, and suggests that exploiting proteotoxic ER stress may be an effective therapeutic strategy in therapy-refractory HER2+ breast cancers. Note: This abstract was not presented at the conference. Citation Format: Rashika Joshi, Kakajan Komurov. Revealing targetable cancer cell vulnerabilities by in silico pathway profiling. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C17.