Matrix Metalloproteinase 14 promotes lung cancer by cleavage of Heparin-Binding EGF-like Growth Factor.

Molecularly targeted therapies benefit approximately 15-20% of non-small cell lung cancer (NSCLC) patients carrying specific drug-sensitive mutations. Thus, there is a clinically unmet need for the identification of novel targets for drug development. Here, we performed RNA-deep sequencing to identify altered gene expression between malignant and non-malignant lung tissue. Matrix Metalloproteinase 14 (MMP14), a membrane-bound proteinase, was significantly up-regulated in the tumor epithelial cells and intratumoral myeloid compartments in both mouse and human NSCLC. Overexpression of a soluble dominant negative MMP14 (DN-MMP14) or pharmacological inhibition of MMP14 blocked invasion of lung cancer cells through a collagen I matrix in vitro and reduced tumor incidence in an orthotopic K-RasG12D/+p53-/- mouse model of lung cancer. Additionally, MMP14 activity mediated proteolytic processing and activation of Heparin-Binding EGF-like Growth Factor (HB-EGF), stimulating the EGFR signaling pathway to increase proliferation and tumor growth. This study highlights the potential for development of therapeutic strategies that target MMP14 in NSCLC with particular focus on MMP14-HB-EGF axis.

Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance.

The role of epithelial-to-mesenchymal transition (EMT) in metastasis is a longstanding source of debate, largely owing to an inability to monitor transient and reversible EMT phenotypes in vivo. Here we establish an EMT lineage-tracing system to monitor this process in mice, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We show that within a predominantly epithelial primary tumour, a small proportion of tumour cells undergo EMT. Notably, lung metastases mainly consist of non-EMT tumour cells that maintain their epithelial phenotype. Inhibiting EMT by overexpressing the microRNA miR-200 does not affect lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment owing to reduced proliferation, apoptotic tolerance and increased expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment.

Identification of Reprogrammed Myeloid Cell Transcriptomes in NSCLC.

Lung cancer is the leading cause of cancer related mortality worldwide, with non-small cell lung cancer (NSCLC) as the most prevalent form. Despite advances in treatment options including minimally invasive surgery, CT-guided radiation, novel chemotherapeutic regimens, and targeted therapeutics, prognosis remains dismal. Therefore, further molecular analysis of NSCLC is necessary to identify novel molecular targets that impact prognosis and the design of new-targeted therapies. In recent years, tumor "activated/reprogrammed" stromal cells that promote carcinogenesis have emerged as potential therapeutic targets. However, the contribution of stromal cells to NSCLC is poorly understood. Here, we show increased numbers of bone marrow (BM)-derived hematopoietic cells in the tumor parenchyma of NSCLC patients compared with matched adjacent non-neoplastic lung tissue. By sorting specific cellular fractions from lung cancer patients, we compared the transcriptomes of intratumoral myeloid compartments within the tumor bed with their counterparts within adjacent non-neoplastic tissue from NSCLC patients. The RNA sequencing of specific myeloid compartments (immature monocytic myeloid cells and polymorphonuclear neutrophils) identified differentially regulated genes and mRNA isoforms, which were inconspicuous in whole tumor analysis. Genes encoding secreted factors, including osteopontin (OPN), chemokine (C-C motif) ligand 7 (CCL7) and thrombospondin 1 (TSP1) were identified, which enhanced tumorigenic properties of lung cancer cells indicative of their potential as targets for therapy. This study demonstrates that analysis of homogeneous stromal populations isolated directly from fresh clinical specimens can detect important stromal genes of therapeutic value.

Transcriptome analysis of individual stromal cell populations identifies stroma-tumor crosstalk in mouse lung cancer model.

Emerging studies have begun to demonstrate that reprogrammed stromal cells play pivotal roles in tumor growth, metastasis, and resistance to therapy. However, the contribution of stromal cells to non-small-cell lung cancer (NSCLC) has remained underexplored. We used an orthotopic model of Kras-driven NSCLC to systematically dissect the contribution of specific hematopoietic stromal cells in lung cancer. RNA deep-sequencing analysis of individually sorted myeloid lineage and tumor epithelial cells revealed cell-type-specific differentially regulated genes, indicative of activated stroma. We developed a computational model for crosstalk signaling discovery based on ligand-receptor interactions and downstream signaling networks and identified known and novel tumor-stroma paracrine and tumor autocrine crosstalk-signaling pathways in NSCLC. We provide cellular and molecular insights into components of the lung cancer microenvironment that contribute to carcinogenesis. This study has the potential for development of therapeutic strategies that target tumor-stroma interactions and may complement conventional anti-cancer treatments.

A role for Egfl7 during endothelial organization in the embryoid body model system.

Epidermal growth factor-like domain 7, Egfl7, is a largely endothelial restricted gene which is thought to have a role during the differentiation of embryonic stem cells (ESCs) along the endothelial lineage. While it has been shown that Egfl7 knock-down in zebrafish impairs endothelial cord formation, the role of the gene in mammals has been unresolved. Interpretation of mouse knockout studies has been complicated by the fact that deletion of miR-126, an intronic microRNA located within Egfl7, results in vascular defects. Here we use an siRNA knock-down approach to target specific regions of Egfl7 without affecting miR-126 expression. Egfl7 was knocked down in mouse ESCs and the effect on vascular development was assessed using the in vitro embryoid body (EB) model after either 7 or 14 days of differentiation. Knock-down of Egfl7 resulted in the formation of abnormal sheet-like CD31+ structures that were abundant within EBs after 7 days of differentiation. Only up to 60% of these sheets co-expressed basement membrane and endothelial cell junction markers. Similar CD31+ sheets were also seen as outgrowths from 7 day EBs into collagen gels. A partial remodelling occurred by 14 days of differentiation when fewer CD31+ sheets were seen both within EBs, and as outgrowths from EBs. Formation of these sheets was due, at least in part, to increased proliferation specifically of CD31+ cells. Cell death within EBs was unaffected by Egfl7 knock-down. In conclusion, our work shows that knock-down of Egfl7 causes defects in early vascular cord formation, and results in the development of CD31+ sheet-like structures. This suggests that Egfl7 is vital for the formation of endothelial cell cords, and that the gene has an important role during both vasculogenesis and angiogenesis in mammalian cells.