Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation

RESEARCH ARTICLE Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation Kishore Polireddy1, Ruochen Dong1, Peter R. McDonald2, Tao Wang1, Brendan Luke1, Ping Chen1, Melinda Broward2, Anuradha Roy2, Qi Chen1* a11111 1 Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, Kansas, United States of America, 2 High-Throughput Screening Core Facility, Structural Biology Center, University of Kansas, Lawrence, Kansas, United States of America * OPEN ACCESS Citation: Polireddy K, Dong R, McDonald PR, Wang T, Luke B, Chen P, et al. (2016) Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation. PLoS ONE 11(10): e0164811. doi:10.1371/journal.pone.0164811 Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute, UNITED STATES Received: April 14, 2016 Accepted: September 30, 2016 Published: October 20, 2016 Copyright: © 2016 Polireddy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number 8P30GM103495 and the State of Kansas (https://projectreporter.nih.gov/project_info_ description.cfm?projectnumber=8P30GM10349503). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract Background Pancreatic cancer has an enrichment of stem-like cancer cells (CSCs) that contribute to chemoresistant tumors prone to metastasis and recurrence. Drug screening assays based on cytotoxicity cannot identify specific CSC inhibitors, because CSCs comprise only a small portion of cancer cell population, and it is difficult to propagate stable CSC populations in vitro for high-throughput screening (HTS) assays. Based on the important role of cancer cell epithelial-to-mesenchymal transition (EMT) in promoting CSCs, we hypothesized that inhibition of EMT can be a useful strategy for inhibiting CSCs, and therefore a feasible approach for HTS can be built for identification of CSC inhibitors, based on assays detecting EMT inhibition. Methods An immunofluorescent assay was established and optimized for HTS to identify compounds that enhance E-cadherin expression, as a hallmark of inhibition of EMT. Four chemical libraries containing 41,472 compounds were screened in PANC-1 pancreatic cancer cell line. Positive hits were validated for EMT and CSC inhibition in vitro using sphere formation assay, western blotting, immune fluorescence, and scratch assay. Results Initial hits were refined to 73 compounds with a secondary screening, among which 17 exhibited concentration dependent induction of E-cadherin expression. Six compounds were selected for further study which belonged to 2 different chemical structural clusters. A novel compound 1-(benzylsulfonyl) indoline (BSI, Compound #38) significantly inhibited pancreatic cancer cell migration and invasion. BSI inhibited histone deacetylase, increased PLOS ONE | DOI:10.1371/journal.pone.0164811 October 20, 2016 1 / 23 Inhibitors of Pancreatic Cancer EMT Inhibiting CSC Competing Interests: The authors have declared that no competing interests exist. histone 4 acetylation preferably, resulting in E-cadherin up-regulation. BSI effectively inhibited tumor spheres formation. Six more analogues of BSI were tested for anti-migration and anti-CSC activities. Conclusion This study demonstrated a feasible approach for discovery of agents targeting EMT and CSCs using HTS, and identified a class of novel chemicals that could be developed as antiEMT and anti-CSC drug leads. Introduction The American Cancer Society estimated 48,960 new cases and 40,560 deaths from pancreatic cancer in the US in 2015 (>111 deaths per day) [1]. With 6% of 5-year survival, pancreatic cancer has the highest fatality rate among cancers [2, 3]. In addition to late detection and fulminant disease course, the high mortality rate is a consequence of disappointing treatment efficacy [4, 5]. The current standard therapy using the nucleoside analogue gemcitabine produces little impact on median overall survival for patients with locally advanced or metastatic disease, who comprise the majority of cases [6–8]. Recent clinical trials achieved statistical significance adding agents (e.g. erlotinib, or nab-paclitaxel) to gemcitabine, or attempting to develop gemcitabine-free combination regimen (e.g. FOLFIRINOX), but these regimen also added significant toxic side effects [9–14]. Recent studies suggested that within a heterogeneous tumor, a small subpopulation of cancer cells have enhanced capacity to form a tumor, are responsible for propagation, relapse, metastasis, and treatment resistance [15–22]. These cells are referred to as tumor-initiating cells (TICs) or circulating tumor cells (CTCs). Because these cells also possess stem cell-like properties such as quiescence, self-renew, asymmetric division, and multidrug resistance, they are also called cancer stem cells (CSCs) [15]. In the past a few years, CSCs have been isolated from almost every type of solid tumor. In pancreatic cancer, CSCs has been identified that may be the root of the tumor’s high metastasis rate, and the extremely poor prognosis and treatment outcomes in patients [22–24]. Therefore, eliminating CSCs has emerged to be an important step for ultimate elimination of the entire cancer cell population. Because CSCs are generally resistant to current chemo and radiation therapies [18, 25–30], many cancer therapies, while effective in killing the bulk of tumor cells, may eventually fail because they do not eliminate CSCs, which survive to regenerate new tumors. However, developing drugs preferentially kill CSCs has been a challenge. Few approaches have been described to directly screen for agents that are specifically cytotoxic to CSCs. One reported high throughput screening (HTS) approach is by utilizing a genetically modified breast cancer cell line that has low expression of a cell adherent protein E-cadherin and is therefore forced into a mesenchymal status [31]. Other approaches use suspension culture of tumor spheres as indication of CSCs [32]. The challenge is likely due to two reasons: First, although CSCs can be identified and isolated by cell surface marker profiles, and tumor spheres are cultured in vitro, it remains difficult to propagate a stable, undifferentiated CSC population in cell culture suitable for high throughput screening for many solid (...truncated)