Yongjun Yin

FGF9 and SHH signaling coordinate lung growth and development through regulation of distinct mesenchymal domains

Morphogenesis of the lung is regulated by reciprocal signaling between epithelium and mesenchyme. In previous studies, we have shown that FGF9 signals are essential for lung mesenchyme development. Using Fgf9 loss-of-function and inducible gain-of-function mouse models, we show that lung mesenchyme can be divided into two distinct regions: the sub-mesothelial and sub-epithelial compartments, which proliferate in response to unique growth factor signals. Fibroblast growth factor (FGF) 9 signals from the mesothelium (the future pleura) to sub-mesothelial mesenchyme through both FGF receptor (FGFR) 1 and FGFR2 to induce proliferation. FGF9 also signals from the epithelium to the sub-epithelial mesenchyme to maintain SHH signaling, which regulates cell proliferation, survival and the expression of mesenchymal to epithelial signals. We further show that FGF9 represses peribronchiolar smooth muscle differentiation and stimulates vascular development in vivo. We propose a model in which FGF9 and SHH signals cooperate to regulate mesenchymal proliferation in distinct submesothelial and subepithelial regions. These data provide a molecular mechanism by which mesothelial and epithelial FGF9 directs lung development by regulating mesenchymal growth, and the pattern and expression levels of mesenchymal growth factors that signal back to the epithelium.

An FGF-WNT gene regulatory network controls lung mesenchyme development

Lung mesenchyme is a critical determinant of the shape and size of the lung, the extent and patterning of epithelial branching, and the formation of the pulmonary vasculature and interstitial mesenchymal components of the adult lung. Fibroblast growth factor 9 (FGF9) is a critical regulator of lung mesenchymal growth; however, upstream mechanisms that modulate the FGF mesenchymal signal and the downstream targets of mesenchymal FGF signaling are poorly understood. Here we have identified a robust regulatory network in which mesenchymal FGF signaling regulates beta-Catenin mediated WNT signaling in lung mesenchyme. By conditionally inactivating beta-Catenin in lung mesenchyme, we show that mesenchymal WNT-beta-Catenin signaling is essential for lung development and acts to regulate the cell cycle G1 to S transition and the FGF responsiveness of mesenchyme. Together, both FGF and WNT signaling pathways function to sustain mesenchymal growth and coordinate epithelial morphogenesis during the pseudoglandular stage of lung development.

Mesothelial- and epithelial-derived FGF9 have distinct functions in the regulation of lung development

Fibroblast growth factor (FGF) 9 is a secreted signaling molecule that is expressed in lung mesothelium and epithelium and is required for lung development. Embryos lacking FGF9 show mesenchymal hypoplasia, decreased epithelial branching and, by the end of gestation, hypoplastic lungs that cannot support life. Mesenchymal FGF signaling interacts with β-catenin-mediated WNT signaling in a feed-forward loop that functions to sustain mesenchymal FGF responsiveness and mesenchymal WNT/β-catenin signaling. During pseudoglandular stages of lung development, Wnt2a and Wnt7b are the canonical WNT ligands that activate mesenchymal WNT/β-catenin signaling, whereas FGF9 is the only known ligand that signals to mesenchymal FGF receptors (FGFRs). Here, we demonstrate that mesothelial- and epithelial-derived FGF9, mesenchymal Wnt2a and epithelial Wnt7b have unique functions in lung development in mouse. Mesothelial FGF9 and mesenchymal WNT2A are principally responsible for maintaining mesenchymal FGF-WNT/β-catenin signaling, whereas epithelial FGF9 primarily affects epithelial branching. We show that FGF signaling is primarily responsible for regulating mesenchymal proliferation, whereas β-catenin signaling is a required permissive factor for mesenchymal FGF signaling.

Signaling Networks Regulating Development of the Lower Respiratory Tract

The lungs serve the primary function of air-blood gas exchange in all mammals and in terrestrial vertebrates. Efficient gas exchange requires a large surface area that provides intimate contact between the atmosphere and the circulatory system. To achieve this, the lung contains a branched conducting system (the bronchial tree) and specialized air-blood gas exchange units (the alveoli). The conducting system brings air from the external environment to the alveoli and functions to protect the lung from debris that could obstruct airways, from entry of pathogens, and from excessive loss of fluids. The distal lung enables efficient exchange of gas between the alveoli and the conducting system and between the alveoli and the circulatory system. In this article, we highlight developmental and physiological mechanisms that specify, pattern, and regulate morphogenesis of this complex and essential organ. Recent advances have begun to define molecular mechanisms that control many of the important processes required for lung organogenesis; however, many questions remain. A deeper understanding of these molecular mechanisms will aid in the diagnosis and treatment of congenital lung disease and in the development of strategies to enhance the reparative response of the lung to injury and eventually permit regeneration of functional lung tissue.

Rapid Induction of Lung Adenocarcinoma by Fibroblast Growth Factor 9 Signaling through FGF Receptor 3

Fibroblast growth factors (FGF) are expressed in many non-small cell lung carcinoma (NSCLC) primary tumors and derived cell lines, and mutations in FGF receptor 3 (FGFR3) have been identified in human lung adenocarcinoma. FGF9 has been implicated in the pathogenesis of NSCLC by synergizing with EGFR pathways or by providing an escape pathway mediating resistance to EGFR inhibition. To model pathogenic mechanisms mediated by FGF signals, we have established a mouse model in which FGF9 expression can be induced in adult lung epithelium. Here, we show that induced expression of FGF9 in adult lung leads to the rapid proliferation of distal airway epithelial cells that express the stem cell marker, Sca-1, and the proximal and distal epithelial markers, Sftpc and CC10, the rapid formation of Sftpc-positive adenocarcinomas, and eventual metastasis in some mice. Furthermore, we have identified FGFR3 as the obligate receptor mediating the FGF9 oncogenic signal. These results identify an FGF9-FGFR3 signal as a primary oncogenic pathway for lung adenocarcinoma and suggest that this pathway could be exploited for customized therapeutic applications for both primary tumors and those that have acquired resistance to inhibition of other signaling pathways.

Characterization of the cell of origin and propagation potential of the fibroblast growth factor 9-induced mouse model of lung adenocarcinoma

Fibroblast growth factor 9 (FGF9) is essential for lung development and is highly expressed in a subset of human lung adenocarcinomas. We recently described a mouse model in which FGF9 expression in the lung epithelium caused proliferation of the airway epithelium at the terminal bronchioles and led to rapid development of adenocarcinoma. Here, we used this model to characterize the effects of prolonged FGF9 induction on the proximal and distal lung epithelia, and examined the propagation potential of FGF9‐induced lung tumours. We showed that prolonged FGF9 over‐expression in the lung resulted in the development of adenocarcinomas arising from both alveolar type II and airway secretory cells in the lung parenchyma and airways, respectively. We found that tumour cells harboured tumour‐propagating cells that were able to form secondary tumours in recipient mice, regardless of FGF9 expression. However, the highest degree of tumour propagation was observed when unfractionated tumour cells were co‐administered with autologous, tumour‐associated mesenchymal cells. Although the initiation of lung adenocarcinomas was dependent on activation of the FGF9–FGF receptor 3 (FGFR3) signalling axis, maintenance and propagation of the tumour was independent of this signalling. Activation of an alternative FGF–FGFR axis and the interaction with tumour stromal cells is likely to be responsible for the development of this independence. This study demonstrates the complex role of FGF–FGFR signalling in the initiation, growth and propagation of lung cancer. Our findings suggest that analysing the expressions of FGF–FGFRs in human lung cancer will be a useful tool for guiding customized therapy. Copyright

Fibroblast Growth Factor 9 Regulation by MicroRNAs Controls Lung Development and Links DICER1 Loss to the Pathogenesis of Pleuropulmonary Blastoma

Pleuropulmonary Blastoma (PPB) is the primary neoplastic manifestation of a pediatric cancer predisposition syndrome that is associated with several diseases including cystic nephroma, Wilms tumor, neuroblastoma, rhabdomyosarcoma, medulloblastoma, and ovarian Sertoli-Leydig cell tumor. The primary pathology of PPB, epithelial cysts with stromal hyperplasia and risk for progression to a complex primitive sarcoma, is associated with familial heterozygosity and lesion-associated epithelial loss-of-heterozygosity of DICER1. It has been hypothesized that loss of heterozygosity of DICER1 in lung epithelium is a non-cell autonomous etiology of PPB and a critical pathway that regulates lung development; however, there are no known direct targets of epithelial microRNAs (miRNAs) in the lung. Fibroblast Growth Factor 9 (FGF9) is expressed in the mesothelium and epithelium during lung development and primarily functions to regulate lung mesenchyme; however, there are no known mechanisms that regulate FGF9 expression during lung development. Using mouse genetics and molecular phenotyping of human PPB tissue, we show that FGF9 is overexpressed in lung epithelium in the initial multicystic stage of Type I PPB and that in mice lacking epithelial Dicer1, or induced to overexpress epithelial Fgf9, increased Fgf9 expression results in pulmonary mesenchymal hyperplasia and a multicystic architecture that is histologically and molecularly indistinguishable from Type I PPB. We further show that miR-140 is expressed in lung epithelium, regulates epithelial Fgf9 expression, and regulates pseudoglandular stages of lung development. These studies identify an essential miRNA-FGF9 pathway for lung development and a non-cell autonomous signaling mechanism that contributes to the mesenchymal hyperplasia that is characteristic of Type I PPB.

Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

ABSTRACT Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. Summary: This study validates the FGF9 lung adenocarcinoma mouse model as a tool to screen and evaluate potential therapeutics that are designed to inhibit FGF9 or its target receptor, FGFR3.

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer.

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

Tumor associated macrophages support the growth of FGF9-induced lung adenocarcinoma by multiple mechanisms

OBJECTIVES Tumor-associated macrophages (TAMs) are known to promote tumorigenesis but the mechanism(s) remain elusive. We have developed a mouse model of lung cancer that is initiated through an inducible overexpression of fibroblast growth factor 9 (FGF9) in type-2 pneumocytes. Expression of FGF9 in adult lungs resulted in a rapid development of multiple adenocarcinoma-like tumor nodules, and is associated with an intense immunological reaction. The purpose of this study is to characterize the immune response to the FGF9-induced lung adenocarcinoma and to determine the contribution of TAMs to growth and survival of these tumors. MATERIALS AND METHODS We used flow cytometry, immunostaining, RT-PCR and in vitro culture system on various cell populations isolated from the FGF9-induced adenocarcinoma mouse lungs. RESULTS Immunostaining demonstrated that the majority of the inflammatory cells recruited to FGF9-induced lung tumors were macrophages. These TAMs were enriched for the alternatively activated (M2) macrophage subtype. TAMs performed a significantly high immune suppressive function on T-cells and displayed high levels of arginase-1 expression and activity. The growth and colony forming potential of tumor cells was induced by co-culture with TAMs. Additionally, TAMs were shown to promote fibroblast proliferation and angiogenesis. TAMs had high expression of Tgf-β, Vegf, Fgf2, Fgf10, Fgfr2 and several matrix metalloproteinases; factors that play multiple roles in supporting tumor growth, immune protection, fibroblast activation and angiogenesis. CONCLUSION Our results provide evidence that the Fgf9-induced lung adenocarcinoma is associated with recruitment and activation of M2-biased TAMs, which provided multiple means of support to the tumor. This model represents an excellent means to further study the complex interactions between TAMs, their related chemokines, and progression of lung adenocarcinoma, and adds further evidence to support the importance of TAMs in tumorigenesis.