In the U.S., lung cancer is the leading cause of cancer death, with a median survival of eight months following diagnosis and only 16% of patients surviving more than five years. The low survival rate is attributable, in part, to a lack of early detection, limiting the benefit of surgical resection as metastatic progression has already occurred. The early events of lung cancer pathogenesis have not yet been well defined, contributing to the paucity of early detection techniques clinically. For tumors to progress to metastatic disease, they must acquire characteristics that allow them to become migratory, degrade and invade their local basement membrane, and migrate to a new site before forming a micrometastasis. As part of this process, tumors undergo a series of events known as epithelial-to-mesenchymal transition (EMT), where they lose epithelial characteristics and become mesenchymal in phenotype and molecular profile.
Snail is a zinc-finger transcription factor that exerts global effects on epithelial cell gene expression profiles, resulting in regulation of EMT. Recent studies by our lab have shown that Snail is upregulated in human non-small cell lung cancer (NSCLC) tissues, is associated with poor prognosis, and promotes NSCLC tumor progression in vivo. Furthermore, Snail overexpression in NSCLC is associated with differential gene expression related to diverse aspects of lung cancer progression, including angiogenesis. Herein we demonstrate that overexpression of Snail leads to upregulation of Secreted Protein, Acidic and Rich in Cysteine (SPARC). Immunoblot and qRT-PCR analysis of multiple NSCLC cell lines with and without Snail overexpression validated the relationship between Snail and SPARC in established cancers. Similar results were found in Snail-overexpressing Human Bronchial Epithelial Cells (HBECs), a model of early pathogenesis, as well as in human lung adenocarcinomas and squamous cell carcinomas immunostained for Snail and SPARC. Taken together, these data indicate that SPARC is upregulated by Snail at early and late points during lung carcinogenesis and may play a role in lung cancer initiation and progression.
In all cell lines evaluated, Snail overexpression leads to increased SPARC-dependent invasion in vitro. The promoter region of SPARC does not contain a binding site for Snail, indicating that Snail upregulates SPARC by an indirect mechanism. Bioinformatic analysis of array data revealed potential intermediaries in Snail-mediated upregulation of SPARC, including miR-29b and the TGF-β and MEK/ERK pathways. Both the TGF-β1 ligand and TGF-βR2 are upregulated following Snail overexpression. In addition, treatment of HBEC cell lines with TGF-β1 resulted in phosphorylation of ERK1/2 as well as upregulation of SPARC and Snail. Inhibition of TGF-β1 mRNA decreased the Snail-dependent activation of ERK1/2 and protein expression of SPARC. Inhibition of MEK phosphorylation by the chemical inhibitor U0126 in Snail-overexpressing cell lines leads to a loss of SPARC upregulation, indicating that the TGF-β-driven MEK/ERK pathway is necessary for Snail-dependent upregulation of SPARC. The microRNA miR-29b is downregulated in Snail-overexpressing cell lines. As the 3'UTR of SPARC mRNA contains multiple consensus sequences for miR-29b, we have hypothesized that downregulation of miR-29b by Snail, downstream of ERK, allows for upregulation of SPARC. Transient overexpression of a miR-29b mimic in both the vector control and Snail-overexpressing cell lines inhibited SPARC expression, confirming the ability of miR-29b to regulate SPARC. In addition, miR-29b was upregulated following ERK inhibition, indicating a pathway by which Snail overexpression leads to activation of TGF-β and ERK signaling, resulting in downregulation of miR-29b and upregulation of SPARC.
Upregulation of SPARC is associated with metastatic potential of melanomas and gliomas as well as an invasive phenotype in breast, prostate, and colorectal carcinomas. Expression of SPARC in the tumor stroma of NSCLC is associated with poor patient prognosis, though its role in tumor progression, especially in relation to Snail expression, has not been evaluated. Our novel discovery of SPARC overexpression as an early event during lung carcinogenesis has potentially important implications, especially as related to the parallel model of cancer development and metastatic progression. Delineating pathways involved in Snail-dependent and SPARC-mediated parallel progression may yield new targets for lung cancer prevention and treatment.