Rangnath Mishra

Neuregulin-1-Human Epidermal Receptor-2 Signaling Is a Central Regulator of Pulmonary Epithelial Permeability and Acute Lung Injury

The mechanisms behind the loss of epithelial barrier function leading to alveolar flooding in acute lung injury (ALI) are incompletely understood. We hypothesized that the tyrosine kinase receptor human epidermal growth factor receptor-2 (HER2) would be activated in an inflammatory setting and participate in ALI. Interleukin-1β (IL-1β) exposure resulted in HER2 activation in human epithelial cells and markedly increased conductance across a monolayer of airway epithelial cells. Upon HER2 blockade, conductance changes were significantly decreased. Mechanistic studies revealed that HER2 trans-activation by IL-1β required a disintegrin and metalloprotease 17 (ADAM17)-dependent shedding of the ligand neuregulin-1 (NRG-1). In murine models of ALI, NRG-1-HER2 signaling was activated, and ADAM17 blockade resulted in decreased NRG-1 shedding, HER2 activation, and lung injury in vivo. Finally, NRG-1 was detectable and elevated in pulmonary edema fluid from patients with ALI. These results suggest that the ADAM17-NRG-1-HER2 axis modulates the alveolar epithelial barrier and contributes to the pathophysiology of ALI.

Contributions of KRAS and RAL in Non–Small-Cell Lung Cancer Growth and Progression

Introduction: KRAS mutations are poor prognostic markers for patients with non–small-cell lung cancer (NSCLC). RALA and RALB GTPases lie downstream of RAS and are implicated in RAS-mediated tumorigenesis. However, their biological or prognostic role in the context of KRAS mutation in NSCLC is unclear. Methods: Using expression analysis of human tumors and a panel of cell lines coupled with functional in vivo and in vitro experiments, we evaluated the prognostic and functional importance of RAL in NSCLC and their relationship to KRAS expression and mutation. Results: Immunohistochemical (N = 189) and transcriptomic (N = 337) analyses of NSCLC patients revealed high RALA and RALB expression was associated with poor survival. In a panel of 14 human NSCLC cell lines, RALA and RALB had higher expression in KRAS mutant cell lines whereas RALA but not RALB activity was higher in KRAS mutant cell lines. Depletion of RAL paralogs identified cell lines that are dependent on RAL expression for proliferation and anchorage independent growth. Overall, growth of NSCLC cell lines that carry a glycine to cystine KRAS mutation were more sensitive to RAL depletion than those with wild-type KRAS. The use of gene expression and outcome data from 337 human tumors in RAL-KRAS interaction analysis revealed that KRAS and RAL paralog expression jointly impact patient prognosis. Conclusion: RAL GTPase expression carries important additional prognostic information to KRAS status in NSCLC patients. Simultaneously targeting RAL may provide a novel therapeutic approach in NSCLC patients harboring glycine to cystine KRAS mutations.

HER2 activation results in β-catenin-dependent changes in pulmonary epithelial permeability.

The receptor tyrosine kinase human epidermal growth factor receptor-2 (HER2) is known to regulate pulmonary epithelial barrier function; however, the mechanisms behind this effect remain unidentified. We hypothesized that HER2 signaling alters the epithelial barrier through an interaction with the adherens junction (AJ) protein β-catenin, leading to dissolution of the AJ. In quiescent pulmonary epithelial cells, HER2 and β-catenin colocalized along the lateral intercellular junction. HER2 activation by the ligand neuregulin-1 was associated with tyrosine phosphorylation of β-catenin, dissociation of β-catenin from E-cadherin, and decreased E-cadherin-mediated cell adhesion. All effects were blocked with the HER2 inhibitor lapatinib. β-Catenin knockdown using shRNA significantly attenuated neuregulin-1-induced decreases in pulmonary epithelial resistance in vitro. Our data indicate that HER2 interacts with β-catenin, leading to dissolution of the AJ, decreased cell-cell adhesion, and disruption of the pulmonary epithelial barrier.

IL-13 induces periostin and eotaxin expression in human primary alveolar epithelial cells: Comparison with paired airway epithelial cells

Alveolar epithelial cells are critical to the pathogenesis of pulmonary inflammation and fibrosis, which are associated with overexpression of type 2 cytokine IL-13. IL-13 is known to induce the production of profibrotic (e.g., periostin) and pro-inflammatory (e.g., eotaxin-3) mediators in human airway epithelial cells, but it remains unclear if human primary alveolar epithelial cells increase periostin and eotaxin expression following IL-13 stimulation. The goals of this study are to determine if alveolar epithelial cells increase periostin and eotaxin expression upon IL-13 stimulation, and if alveolar and airway epithelial cells from the same subjects have similar responses to IL-13. Paired alveolar and airway epithelial cells were isolated from donors without any lung disease, and cultured under submerged or air-liquid interface conditions with or without IL-13. Up-regulation of periostin protein and mRNA was observed in IL-13-stimulated alveolar epithelial cells, which was comparable to that in IL-13-stimulated paired airway epithelial cells. IL-13 also increased eotaxin-3 expression in alveolar epithelial cells, but the level of eotaxin mRNA was lower in alveolar epithelial cells than in airway epithelial cells. Our findings demonstrate that human alveolar epithelial cells are able to produce periostin and eotaxin in responses to IL-13 stimulation. This study suggests the need to further determine the contribution of alveolar epithelial cell-derived mediators to pulmonary fibrosis.

Abstract 2138: Tumorigenic and migratory potential of Ral GTPases in non-small cell lung cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnThe Ral GTPase family, consisting of the Ral A and B isoforms contributes to tumor formation, invasion and metastasis, however their role in non-small cell lung cancer (NSCLC) is yet to be validated. Recently, we investigated the role of Ral GTPases in NSCLC by in vitro expression, colony formation and migration assay, in vivo tumor formation experiments, and analysis of microarray data from NSCLC patients. In vitro, Ral A and Ral B was expressed in all NSCLC cell lines studied (14) to varying levels. Increased expression of Ral A or Ral B was more frequent in Ras mutant cell lines (Ral A elevated in 2/8 cell lines with mutant KRas vs. 0/6 cell lines with WT KRas, Ral B elevated in 5/8 cell lines with mutant KRas vs. 2/6 WT KRas). Soft agar experiments with transient knockdown of Ral A, Ral B and Ral A+B in these cell lines demonstrated significant decrease (p<0.05) in colony formation. Dual knockdown of Ral A and B had a 2 to 3 fold decrease (p<0.05) in NSCLC cell motility in wound healing assays. In vivo, knockdown of Ral GTPases resulted in reduced subcutaneous tumor growth (p<0.05) in mice injected with the knockdown cells. Microarray data from NSCLC patients was analyzed to study the correlation between Ral A and B expression with overall patient survival. High Ral A expression was associated with poor patient survival in these microarray datasets (p<0.05). The above observations demonstrate that Ral GTPases play a role in lung cancer tumorigenesis and migration. In the Ras signaling cascade, Ral GTPases are downstream of Ras along with PI3K and MAPK signaling pathways. KRas mutations in NSCLC carry a poor patient prognosis. Based on our observations, we posit that Ral GTPases can be a valid target for drug development against KRas mutated lung cancers along with PI3K and MEK inhibitors.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2138. doi:1538-7445.AM2012-2138