The epithelial–mesenchymal transition (EMT) has been postulated as a mechanism by which cancer cells acquire the invasive and stem-like traits necessary for distant metastasis. However, direct in vivo evidence for the role of EMT in the formation of cancer stem–like cells (CSC) and the metastatic cascade remains lacking. Here we report the first isolation and characterization of mesenchymal-like and EMT tumor cells, which harbor both epithelial and mesenchymal characteristics, in an autochthonous murine model of prostate cancer. By crossing the established Pb-Cre+/−;PtenL/L;KrasG12D/+ prostate cancer model with a vimentin-GFP reporter strain, generating CPKV mice, we were able to isolate epithelial, EMT, and mesenchymal-like cancer cells based on expression of vimentin and EpCAM. CPKV mice (but not mice with Pten deletion alone) exhibited expansion of cells with EMT (EpCAM+/Vim-GFP+) and mesenchymal-like (EpCAM−/Vim-GFP+) characteristics at the primary tumor site and in circulation. These EMT and mesenchymal-like tumor cells displayed enhanced stemness and invasive character compared with epithelial tumor cells. Moreover, they displayed an enriched tumor-initiating capacity and could regenerate epithelial glandular structures in vivo, indicative of epithelia–mesenchyme plasticity. Interestingly, while mesenchymal-like tumor cells could persist in circulation and survive in the lung following intravenous injection, only epithelial and EMT tumor cells could form macrometastases. Our work extends the evidence that mesenchymal and epithelial states in cancer cells contribute differentially to their capacities for tumor initiation and metastatic seeding, respectively, and that EMT tumor cells exist with plasticity that can contribute to multiple stages of the metastatic cascade. Cancer Res; 75(13); 1–11. ©2015 AACR.

The epithelial-mesenchymal transition (EMT) has been postulated as a mechanism by which cancer cells acquire the invasive and stem-like traits necessary for distant metastasis. However, direct in vivo evidence for the role of EMT in the formation of cancer stem-like cells (CSC) and the metastatic cascade remains lacking. Here we report the first isolation and characterization of mesenchymal and EMT-like tumor cells, which harbor both epithelial and mesenchymal characteristics, in an autochthonous murine model of prostate cancer. By crossing the established Pb-Cre+/-;PtenL/L;KrasG12D/+ prostate cancer model with a vimentin-GFP reporter strain, generating CPKV mice, we were able to isolate epithelial, EMT-like and mesenchymal cancer cells based on expression of vimentin and EpCAM. CPKV mice (but not mice with Pten deletion alone) exhibited expansion of cells with EMT-like (EpCAM+/Vim-GFP+) and mesenchymal (EpCAM-/Vim-GFP+) characteristics at the primary tumor site and in circulation. These EMT-like and mesenchymal tumor cells displayed enhanced stemness and invasive character compared to epithelial tumor cells. Moreover, they displayed an enriched tumor-initiating capacity and could regenerate epithelial glandular structures in vivo, indicative of epithelia-mesenchyme plasticity. Interestingly, while mesenchymal tumor cells could persist in circulation and survive in the lung following intravenous injection, only epithelial and EMT-like tumor cells could form macrometastases. Our work extends the evidence that mesenchymal and epithelial states in cancer cells contribute differentially to their capacities for tumor initiation and metastatic seeding, respectively, and that EMT-like tumor cells exist with plasticity that can contribute to multiple stages of the metastatic cascade.

The Pim-1 kinase regulates cell survival, proliferation, and differentiation and is overexpressed frequently in many malignancies, including leukemia and skin cancer. In this study, we used kinase profiling analysis to demonstrate that 2′-hydroxycinnamicaldehyde (2′-HCA), a compound found in cinnamon, specifically inhibits Pim-1 activity. Cocrystallography studies determined the hydrogen bonding pattern between 2′-HCA and Pim-1. Notably, 2′-HCA binding altered the apo kinase structure in a manner that shielded the ligand from solvent, thereby acting as a gatekeeper loop. Biologically, 2′-HCA inhibited the growth of human erythroleukemia or squamous epidermoid carcinoma cells by inducing apoptosis. The compound was also effective as a chemopreventive agent against EGF-mediated neoplastic transformation. Finally, 2′-HCA potently suppressed the growth of mouse xenografts representing human leukemia or skin cancer. Overall, our results offered preclinical proof of concept for 2′-HCA as a potent anticancer principle arising from direct targeting of the Pim-1 kinase. Cancer Res; 75(13); 1–13. ©2015 AACR.

The Pim-1 kinase regulates cell survival, proliferation and differentiation and is overexpressed frequently in many malignancies including leukemia and skin cancer. In this study, we used kinase profiling analysis to demonstrate that 2′-hydroxycinnamicadelhyde (2′-HCA), a compound found in cinnamon, specifically inhibits Pim-1. Co-crystallography studies determined the hydrogen bonding pattern between 2′-HCA and Pim-1. Notably, 2′-HCA binding altered the apo kinase structure in a manner that shielded the ligand from solvent, thereby acting as a gatekeeper loop. Biologically, 2′-HCA inhibited the growth of human erythroleukemia or squamous epidermoid carcinoma cells by inducing apoptosis. The compound was also effective as a chemopreventive agent against EGF-mediated neoplastic transformation. Lastly, 2′-HCA potently suppressed the growth of mouse xenografts representing human leukemia or skin cancer. Overall, our results offered preclinical proof of concept for 2′-HCA as a potent anticancer principle arising from direct targeting of the Pim-1 kinase.

The redeployed drug combination of bezafibrate and medroxyprogesterone acetate (designated BaP) has potent in vivo anticancer activity in acute myelogenous leukemia (AML) and endemic Burkitt lymphoma (eBL) patients; however, its mechanism-of-action is unclear. Given that elevated fatty acid biosynthesis is a hallmark of many cancers and that these drugs can affect lipid metabolism, we hypothesized that BaP exerts anticancer effects by disrupting lipogenesis. We applied mass spectrometry–based lipidomics and gene and protein expression measurements of key lipogenic enzymes [acetyl CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and stearoyl CoA desaturase 1 (SCD1)] to AML and eBL cell lines treated with BaP. BaP treatment decreased fatty acid and phospholipid biosynthesis from 13C D-glucose. The proportion of phospholipid species with saturated and monounsaturated acyl chains was also decreased after treatment, whereas those with polyunsaturated chains increased. BaP decreased SCD1 protein levels in each cell line (0.46- to 0.62-fold; P < 0.023) and decreased FASN protein levels across all cell lines (0.87-fold decrease; P = 1.7 × 10−4). Changes to ACC1 protein levels were mostly insignificant. Supplementation with the SCD1 enzymatic product, oleate, rescued AML and e-BL cells from BaP cell killing and decreased levels of BaP-induced reactive oxygen species, whereas supplementation with the SCD1 substrate (and FASN product), palmitate, did not rescue cells. In conclusion, these data suggest that the critical anticancer actions of BaP are decreases in SCD1 levels and monounsaturated fatty acid synthesis. To our knowledge, this is the first time that clinically available antileukemic and antilymphoma drugs targeting SCD1 have been reported. Cancer Res; 75(12); 1–11. ©2015 AACR.