A Novel Molecular Pathway for Snail-Dependent, SPARC-Mediated Invasion in Non–Small Cell Lung Cancer Pathogenesis

Abstract

Definition of the molecular pathogenesis of lung cancer allows investigators an enhanced understanding of the natural history of the disease, thus fostering development of new prevention strategies. In addition to regulating epithelial-to-mesenchymal transition (EMT), the transcription factor Snail exerts global effects on gene expression. Our recent studies reveal that Snail is upregulated in non-small cell lung cancer (NSCLC), is associated with poor prognosis, and promotes tumor progression in vivo. Herein, we demonstrate that overexpression of Snail leads to upregulation of Secreted Protein, Acidic and Rich in Cysteine (SPARC) in models of premalignancy and established disease, as well as in lung carcinoma tissues in situ. Snail overexpression leads to increased SPARC-dependent invasion in vitro, indicating that SPARC may play a role in lung cancer progression. Bioinformatic analysis implicates TGF-β, ERK1/2, and miR-29b as potential intermediaries in Snail-mediated upregulation of SPARC. Both the TGF-β1 ligand and TGF-βR2 are upregulated following Snail overexpression. Treatment of human bronchial epithelial cell (HBEC) lines with TGF-β1 and inhibition of TGF-β1 mRNA expression modulated SPARC expression. Inhibition of MEK phosphorylation downregulated SPARC. MiR-29b is downregulated in Snail overexpressing cell lines, while overexpression of miR-29b inhibited SPARC expression. In addition, miR-29b was upregulated following ERK inhibition, suggesting a Snail-dependent pathway by which Snail activation of TGF-β and ERK signaling results in downregulation of miR-29b and subsequent upregulation of SPARC. Our discovery of pathways responsible for Snail-induced SPARC expression contributes to the definition of NSCLC pathogenesis.

The role of miR-29 in pulmonary fibrosis

“…Interestingly, all these are predicted targets of miR-29. SPARC is a direct target of miR-29 in different cell types, including osteoblasts, human trabecular meshwork cells, breast carcinoma cells, hepatocellular carcinoma, and non-small cell lung cancer cells (Gerson et al 2012;Grant et al 2014;James et al 2014;Kapinas et al 2009;Luna et al 2009;Wang et al 2012b;Zhu et al 2012). miR-29 is known to directly target COL4A1/Col4a1, COL4A2/Col4a2, and LAMC1/Lamc1 in different cell types, including rat renal medullary epithelial cells, osteoblast, MCF-7 human breast cancer cells, and prostate cancer cells (Du et al 2010;Liu et al 2010;Luna et al 2009;Nishikawa et al 2014;Steele et al 2010).…”

Secreted protein acidic and rich in cysteine promotes epithelial–mesenchymal transition of hepatocellular carcinoma cells and acquisition of cancerstem cell phenotypes

“…Secreted protein acidic and rich in cysteine regulates EMT in endometrial cancer and non-small-cell lung cancer. 22,23 The process of EMT includes downregulating expression of epithelial markers, such as E-cadherin, and upregulating mesenchymal markers, such as N-cadherin and vimentin. We hypothesized that SPARC regulates EMT in liver cancer cells.…”

Identification of a Human Airway Epithelial Cell Subpopulation with Altered Biophysical, Molecular, and Metastatic Properties

“…However, the isolated subpopulation of cells did not exhibit morphological characteristics associated with EMT (FIG S1b). To evaluate EMT-related phenomena in our highly migratory model, we performed selection in HBECs that have been genetically engineered to over-express SNAIL (23). Similarly to isogenic cells, enhanced migration was observed in these SNAIL over-expressing HM cells (FIG S2a, Table S2).…”

“…They comprise at least some of the progenitors for human lung cancer, most accurately recapitulating central airway differentiation and squamous cell carcinoma development under the defined culture conditions we utilize. Detailed information about the generation of HBEC-vector and -Snail lines was previously published, along with detailed cell culture maintenance methods (23). All cells were genotyped by STR profiling (DNA IQ System and Powerplex 1.2 system, Promega) before and after migration selection at the UCLA Genotyping and Sequencing Core.…”

“…For IHC analysis, the mouse organs were fixed in 10% neutral buffered formalin for 48hr before being rinsed thoroughly and transferred to 70% ethanol. The tissues were then processed routinely, sectioned, H&E stained, and immunostained using standard methods previously described (23). Detection of single cells or small aggregates of HBECs within the mouse lungs was confirmed by CD44 (Cell Signaling #3570) immunostaining at 1:400 in EDTA with antigen retrieval.…”