Epithelial-mesenchymal transition (EMT) was shown to confer tumor cells with abilities essential for metastasis, including migratory phenotype, invasiveness, resistance to apoptosis, evading immune surveillance, and tumor stem cell traits. Therefore, inhibition of EMT can be an important therapeutic strategy to inhibit tumor metastasis. Here, we show that activation of peroxisome proliferator-activated receptor g (PPAR-g) inhibits transforming growth factor b (TGF-b)-induced EMT in lung cancer cells and prevents metastasis by antagonizing Smad3 function. Activation of PPAR-g by synthetic ligands (troglitazone and rosiglitazone) or by a constitutively active form of PPAR-g prevents TGF-b-induced loss of E-cadherin expression and inhibits the induction of mesenchymal markers (vimentin, N-cadherin, fibronectin) and matrix metalloproteases. Consistently, activation of PPAR-g also inhibited EMT-induced migration and invasion of lung cancer cells. Furthermore, effects of PPAR-g ligands were attenuated by siRNA-mediated knockdown of PPAR-g, indicating that the ligand-induced responses are PPAR-g dependent. Selective knockdown of Smad2 and Smad3 by siRNA showed that TGF-b-induced EMT is Smad3 dependent in lung cancer cells. Activation of PPAR-g inhibits TGF-b-induced Smad transcriptional activity but had no effect on the phosphorylation or nuclear translocation of Smads. Consistently, PPAR-g activation prevented TGFb-induced transcriptional repression of E-cadherin promoter and inhibited transcriptional activation of Ncadherin promoter. Finally, treatment of mice with troglitazone or knockdown of Smad3 in tumor cells significantly inhibited TGF-b-induced experimental metastasis in SCID-Beige mice. Together, with the low toxicity profile of PPAR-g ligands, our data show that these ligands may serve as potential therapeutic agents to inhibit metastasis. Mol Cancer Ther; 9(12); 3221-32. Ó2010 AACR.

Pancreatic cancer is the third leading cause of cancer-related death in the USA. Despite extensive research, minimal improvements in patient outcomes have been achieved. Early identification of treatment response and metastasis would be valuable to determine the appropriate therapeutic course for patients. In this work, we isolated exosomes from the serum of 10 patients with locally advanced pancreatic cancer at serial time points over a course of therapy, and quantitative analysis was performed using the iTRAQ method. We detected approximately 700–800 exosomal proteins per sample, several of which have been implicated in metastasis and treatment resistance. We compared the exosomal proteome of patients at different time points during treatment to healthy controls and identified eight proteins that show global treatment-specific changes. We then tested the effect of patient-derived exosomes on the migration of tumor cells and found that patient-derived exosomes, but not healthy controls, induce cell migration, supporting their role in metastasis. Our data show that exosomes can be reliably extracted from patient serum and analyzed for protein content. The differential loading of exosomes during a course of therapy suggests that exosomes may provide novel insights into the development of treatment resistance and metastasis.

Background Acquisition of mesenchymal phenotype by epithelial cells by means of epithelial mesenchymal transition (EMT) is considered as an early event in the multi-step process of tumor metastasis. Therefore, inhibition of EMT might be a rational strategy to prevent metastasis. Methods Utilizing the global gene expression profile from a cell culture model of TGF-β-induced EMT, we identified potential EMT inhibitors. We used a publicly available database (www.broad.mit.edu/cmap) comprising gene expression profiles obtained from multiple different cell lines in response to various drugs to derive negative correlations to EMT gene expression profile using Connectivity Map (C-Map), a pattern matching tool. Results Experimental validation of the identified compounds showed rapamycin as a novel inhibitor of TGF-β signaling along with 17-AAG, a known modulator of TGF-β pathway. Both of these compounds completely blocked EMT and the associated migratory and invasive phenotype. The other identified compound, LY294002, demonstrated a selective inhibition of mesenchymal markers, cell migration and invasion, without affecting the loss of E-cadherin expression or Smad phosphorylation. Conclusions Collectively, our data reveals that rapamycin is a novel modulator of TGF-β signaling, and along with 17-AAG and LY294002, could be used as therapeutic agent for inhibiting EMT. Also, this analysis demonstrates the potential of a systems approach in identifying novel modulators of a complex biological process.