Faculty Bibliography

Field of cancerization in the airway epithelium has been increasingly examined to understand early pathogenesis of non-small cell lung cancer. However, the extent of field of cancerization throughout the lung airways is unclear. Here we sought to determine the differential gene and microRNA expressions associated with field of cancerization in the peripheral airway epithelial cells of patients with lung adenocarcinoma. We obtained peripheral airway brushings from smoker controls (n=13) and from the lung contralateral to the tumor in cancer patients (n=17). We performed gene and microRNA expression profiling on these peripheral airway epithelial cells using Affymetrix GeneChip and TaqMan Array. Integrated gene and microRNA analysis was performed to identify significant molecular pathways. We identified 26 mRNAs and 5 miRNAs that were significantly (FDR <0.1) up-regulated and 38 mRNAs and 12 miRNAs that were significantly down-regulated in the cancer patients when compared to smoker controls. Functional analysis identified differential transcriptomic expressions related to tumorigenesis. Integration of miRNA-mRNA data into interaction network analysis showed modulation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in the contralateral lung field of cancerization. In conclusion, patients with lung adenocarcinoma have tumor related molecules and pathways in histologically normal appearing peripheral airway epithelial cells, a substantial distance from the tumor itself. This finding can potentially provide new biomarkers for early detection of lung cancer and novel therapeutic targets.

Over the past two decades, the secreted protein sonic hedgehog (SHH) has emerged as a critical morphogen during embryonic lung development, regulating the interaction between epithelial and mesenchymal cell populations in both the airway and alveolar compartments. There is increasing evidence that the SHH pathway is active in adult lung diseases such as pulmonary fibrosis, asthma, chronic obstructive pulmonary disease (COPD) and lung cancer, which raises two questions: (1) what role does SHH signaling play in these diseases? (2) Is it a primary driver of the disease, or a response (perhaps beneficial) to the primary disturbance? In this review we aim to fill the gap between the well-studied period of embryonic lung development and the adult diseased lung by reviewing the HH pathway during the postnatal period, and in adult uninjured and injured lungs. We elucidate the similarities and differences in the epithelial-mesenchymal interplay during the fibrosis response to injury in lung compared to other organs, and present a critical appraisal of tools and agents available to evaluate HH signaling.

Sonic Hedgehog (Shh) signals from epithelium to mesenchyme during embryonic lung development, but the roles of Hedgehog (Hh) signaling in postnatal lung development and adult lung are not known. Using Gli1nlacZ reporter mice to identify cells with active Hh signaling, we found that Gli1nlacZ-positive mesenchymal cells are densely and diffusely present up to 2 weeks after birth and decline in number thereafter. In adult mice, Gli1nlacZ-positive cells are present around large airways and vessels and are sparse in alveolar septa. Hh-stimulated cells are mostly fibroblasts; only 10% of Gli1nlacZ-positive cells are smooth muscle cells, and most smooth muscle cells do not have activation of Hh signaling. After bleomycin injury there are abundant Gli1nlacZ-positive mesenchymal cells in fibrotic lesions and increased numbers of Gli1nlacZ-positive cells in preserved alveolar septa. Inhibition of Hh signaling with an antibody against all Hedgehog isoforms does not reduce bleomycin-induced fibrosis, but adenovirus-mediated over-expression of Shh increases collagen production in this model. Inhibition of Hh signaling during early postnatal lung development causes airspace enlargement without diminished alveolar septation. Reduction of Hh signaling in the later stages of postnatal lung development may be required for normal thinning and maturation of alveolar septa.

The growth factor TGF-beta is secreted in a latent complex consisting of three proteins: TGF-beta, an inhibitor (latency-associated protein, LAP, which is derived from the TGF-beta propeptide) and an ECM-binding protein (one of the latent TGF-beta binding proteins, or LTBPs). LTBPs interact with fibrillins and other ECM components and thus function to localize latent TGF-beta in the ECM. LAP contains an integrin-binding site (RGD), and several RGD-binding integrins are able to activate latent TGF-beta through binding this site. Mutant mice defective in integrin-mediated activators, and humans and mice with fibrillin gene mutations, show the critical role of ECM and integrins in regulating TGF-beta signaling

The arginine-glycine-aspartate (RGD)-binding integrins alphavbeta6 and alphavbeta8 activate latent TGFbeta1 and TGFbeta3 in vivo, but it is uncertain whether other RGD-binding integrins such as integrins alphavbeta5 and alphavbeta3 activate these TGFbeta isoforms. To define the combined role of alphavbeta6- and alphavbeta8-integrin in TGFbeta activation, we analyzed mice lacking function of both integrins by means of gene deletion and/or pharmacologic inhibition. Most Itgb6-/-;Itgb8-/- embryos die at mid-gestation; those that survive develop cleft palate-as observed in Tgfb3-/- mice. Itgb8-/- mice treated with an anti-alphavbeta6-integrin antibody develop severe autoimmunity and lack Langerhans cells-similar to Tgfb1-null mice. These results support a model in which TGFbeta3-mediated palate fusion and TGFbeta1-mediated suppression of autoimmunity and generation of Langerhans cells require integrins alphavbeta6 and alphavbeta8 but not other RGD-binding integrins as TGFbeta activators

Gene deletion experiments have shown that the three TGFbeta isoforms regulate distinct developmental processes. Recent work by our group and others showed that the integrins alphavbeta6 and alphavbeta8 activate latent forms of TGFbeta1 and TGFbeta3. This raises the possibility that TGFbeta1 and TGFbeta3 act redundantly in developmental processes where both isoforms are expressed and activation is by integrins. To investigate this issue, we generated mice with defective integrin-mediated TGFbeta1 activation (Tgfb1(RGE/RGE)) that were also homozygous for a null mutation in the TGFbeta3 gene. Tgfb1(RGE/RGE); Tgfb3(-/-) mice have severely perturbed development of the brain vasculature that is highly similar to that in mice lacking alphavbeta8. Some Tgfb1(RGE/RGE); Tgfb3(+/-) and Tgfb1(RGE/RGE); Tgfb3(+/+) mice have milder, background-dependent versions of the phenotype. In addition, we found that Tgfb3 gene status influences embryonic lethality due to TGFbeta1 deficiency after limited backcrossing to the BALB/c background. Conversely, Tgfb1 gene status modifies the extent of palate fusion in Tgfb3(-/-) mice after limited backcrossing to the ICR background. Our results are consistent with a functional connection between TGFbeta1 and TGFbeta3 during development based on a shared mechanism of activation

RATIONALE: In experimental models, lung fibrosis is dependent on transforming growth factor (TGF)-beta signaling. TGF-beta is secreted in a latent complex with its propeptide, and TGF-beta activators release TGF-beta from this complex. Because the integrin alpha(v)beta6 is a major TGF-beta activator in the lung, inhibition of alpha(v)beta6-mediated TGF-beta activation is a logical strategy to treat lung fibrosis. OBJECTIVES: To determine, by genetic and pharmacologic approaches, whether murine radiation-induced lung fibrosis is dependent on alpha(v)beta6. METHODS: Wild-type mice, alpha(v)beta6-deficient (Itgb6-/-) mice, and mice heterozygous for a Tgfb1 mutation that eliminates integrin-mediated activation (Tgfb1(+/RGE)) were exposed to 14 Gy thoracic radiation. Some mice were treated with an anti-alpha(v)beta6 monoclonal antibody or a soluble TGF-beta receptor fusion protein. Alpha(v)beta6 expression was determined by immunohistochemistry. Fibrosis, inflammation, and gene expression patterns were assessed 20-32 weeks postirradiation. MEASUREMENTS AND MAIN RESULTS: Beta6 integrin expression increased within the alveolar epithelium 18 weeks postirradiation, just before onset of fibrosis. Itgb6-/- mice were completely protected from fibrosis, but not from late radiation-induced mortality. Anti-alpha(v)beta6 therapy (1-10 mg/kg/wk) prevented fibrosis, but only higher doses (6-10 mg/kg/wk) caused lung inflammation similar to that in Itgb6-/- mice. Tgfb1-haploinsufficient mice were also protected from fibrosis. CONCLUSIONS: Alpha(v)beta6-mediated TGF-beta activation is required for radiation-induced lung fibrosis. Together with previous data, our results demonstrate a robust requirement for alpha(v)beta6 in distinct fibrosis models. Inhibition of alphavbeta6-mediated TGF-beta activation is a promising new approach for antifibrosis therapy