David Fuller

Transcription Factors OVOL1 and OVOL2 Induce the Mesenchymal to Epithelial Transition in Human Cancer

Cell plasticity regulated by the balance between the mesenchymal to epithelial transition (MET) and the opposite program, EMT, is critical in the metastatic cascade. Several transcription factors (TFs) are known to regulate EMT, though the mechanisms of MET remain unclear. We demonstrate a novel function of two TFs, OVOL1 and OVOL2, as critical inducers of MET in human cancers. Our findings indicate that the OVOL-TFs control MET through a regulatory feedback loop with EMT-inducing TF ZEB1, and the regulation of mRNA splicing by inducing Epithelial Splicing Regulatory Protein 1 (ESRP1). Using mouse prostate tumor models we show that expression of OVOL-TFs in mesenchymal prostate cancer cells attenuates their metastatic potential. The role of OVOL-TFs as inducers of MET is further supported by expression analyses in 917 cancer cell lines, suggesting their role as crucial regulators of epithelial-mesenchymal cell plasticity in cancer.

IL-4 induces proliferation in prostate cancer PC3 cells under nutrient-depletion stress through the activation of the JNK-pathway and survivin up-regulation

Interleukin (IL)‐4 plays a critical role in the regulation of immune responses and has been detected at high levels in the tumor microenvironment of cancer patients where it correlates with the grade of malignancy. The direct effect of IL‐4 on cancer cells has been associated with increased cell survival; however, its role in cancer cell proliferation and related mechanisms is still unclear. Here it was shown that in a nutrient‐depleted environment, IL‐4 induces proliferation in prostate cancer PC3 cells. In these cells, under nutrient‐depletion stress, IL‐4 activates mitogen‐activated protein kinases (MAPKs), including Erk, p38, and JNK. Using MAP‐signaling‐specific inhibitors, it was shown that IL‐4‐induced proliferation is mediated by JNK activation. In fact, JNK‐inhibitor‐V (JNKi‐V) stunted IL‐4‐mediated cell proliferation. Furthermore, it was found that IL‐4 induces survivin up‐regulation in nutrient‐depleted cancer cells. Using survivin‐short‐hairpin‐RNAs (shRNAs), it was demonstrated that in this milieu survivin expression above a threshold limit is critical to the mechanism of IL‐4‐mediated proliferation. In addition, the significance of survivin up‐regulation in a stressed environment was assessed in prostate cancer mouse xenografts. It was found that survivin knockdown decreases tumor progression in correlation with cancer cell proliferation. Furthermore, under nutrient depletion stress, IL ‐4 could induce proliferation in cancer cells from multiple origins: MDA‐MB‐231 (breast), A253 (head and neck), and SKOV‐3 (ovarian). Overall, these findings suggest that in a tumor microenvironment under stress conditions, IL‐4 triggers a simultaneous activation of the JNK‐pathway and the up‐regulation of survivin turning on a cancer proliferation mechanism. J. Cell. Biochem. 113: 1569–1580, 2012.

Polyamines: A Dual Role in the Modulation of Cellular Sensitivity to Heat

The organic polycations known as polyamines occur at average intracellular concentrations (as determined by acid extraction) approaching the millimolar level. Positively charged at normal intracellular pHs, they have long been suspected of ionic interaction with cellular macromolecules of functional importance (1). The chain structure of the polyamines (see Table I for spermidine and spermine) distinguishes these polycations from the monoand divalent metal ions in a variety of biochemical interactions. The degree of specificity of polyamine interactions is, however, unclear and it may be that these polycations fulfill general stabilizing and charge-buffering functions within the cell [for general review, see Ref. (2)]. This report will discuss a small area within the vast polyamine literature, that of the relationship between these compounds and the responses of cell populations to elevated temperatures, or hyperthermia. The review will be divided into what have become two major areas of interest. The first involves the interaction of exogenous polyamines with what we believe to be membrane targets. In this mode, we believe the polyamines are acting directly, or immediately upon interaction with some membrane component, as heat sensitizers. The second and recently emerging body of work centers on the use of polyamine biosynthesis inhibitors to deplete critical intracellular target(s) of polyamines in such a way as to achieve delayed sensitivity to heat.

Chemical transfection of dye‐conjugated microRNA precursors for microRNA functional analysis of M2 macrophages

MicroRNAs (miRNAs) are short noncoding ribonucleic acids known to affect gene expression at the translational level and there is mounting evidence that miRNAs play a role in the function of tumor‐associated macrophages (TAMs). To aid the functional analyses of miRNAs in an in‐vitro model of TAMs known as M2 macrophages, a transfection method to introduce artificial miRNA constructs or miRNA molecules into primary human monocytes is needed. Unlike differentiated macrophages or dendritic cells, undifferentiated primary human monocytes have been known to show resistance to lentiviral transduction. To circumvent this challenge, other techniques such as electroporation and chemical transfection have been used in other applications to deliver small gene constructs into human monocytes. To date, no studies have compared these two methods objectively to evaluate their suitability in the miRNA functional analysis of M2 macrophages. Of the methods tested, the electroporation of miRNA‐construct containing plasmids and the chemical transfection of miRNA precursor molecules are the most efficient approaches. The use of a silencer siRNA labeling kit (Ambion) to conjugate Cy 3 fluorescence dyes to the precursor molecules allowed the isolation of successfully transfected cells with fluorescence‐activated cell sorting. The chemical transfection of these dye‐conjugated miRNA precursors yield an efficiency of 37.5 ± 0.6% and a cell viability of 74 ± 1%. RNA purified from the isolated cells demonstrated good quality, and was fit for subsequent mRNA expression qPCR analysis. While electroporation of plasmids containing miRNA constructs yield transfection efficiencies comparable to chemical transfection of miRNA precursors, these electroporated primary monocytes seemed to have lost their potential for differentiation. Among the most common methods of transfection, the chemical transfection of dye‐conjugated miRNA precursors was determined to be the best‐suited approach for the functional analysis of M2 macrophages. J. Cell. Biochem. 113: 1714–1723, 2012.

Sensitization to heat by the polyamines and their analogs

The polyamines putrescine, spermidine, and spermine have been shown to sensitize Chinese hamster ovary (CHO) cells to the cytotoxic effects of elevated temperatures. This sensitization occurs in the order spermine greater than spermidine greater than putrescine. A series of homologs of the diamine putrescine demonstrated little variance in the degree of potentiation until a chain length equivalent to that of spermidine was employed (1,8-diaminooctane). Two compounds bearing structural similarities to spermidine gave rather different results when combined with heat. Methylglyoxal bis(guanylhydrazone) (MGBG), an antiproliferative drug, sensitized cells to heat, while S-2-(3-aminopropylamino)-ethyl phosphoric acid (WR-2721), a radioprotector, had no measureable effect on 43 degrees C-induced cytotoxicity. Together, these results point out the primary importance of two terminal amino groups separated by either carbon, or carbon and nitrogen, atoms in an aliphatic chain in the observed sensitization of heat-induced cytotoxicity by polyamines. These data suggest specific dimensional characteristics of the presumed negatively charged structure(s) with which the polyamines are interacting to elicit this effect.

Abstract 1405: Macrophages induce a transcriptional program that triggers a stable epithelial to mesenchymal transition in prostate cancer cells

Epithelial-mesenchymal transition (EMT) is a process crucial for metastatic dissemination and the acquisition of therapeutic resistance in cancer cells. Previous studies demonstrated that the interaction between macrophages and prostate cancer cells generates cells that maintain stable-EMT characteristics in culture. These cells were isolated by direct interactions between M2-type macrophages and the prostate cancer epithelial PC3-lucE cells. The EMT lines were used in mouse experiments to elucidate tumorigenic potential of the stable-EMT cells compared to the parental epithelial cells. It was found that EMT cells develop and metastasize at a faster rate than PC3-Luc-E cells when injected via an intra-cardiac injection (ICI). However, the EMT-lines grew at a similar rate than PC3 Luc-E when the cells were injected subcutaneously in mice, suggesting that the ICI results cannot be explained by the difference in the proliferation potential of the cell lines but rather their capability to survive the circulation and invade and metastasize at distant sites. The transcriptomes of three stable-EMT cell lines were compared with the parental PC3-lucE by microarray expression profiling. A shared-signature of 983 genes, which are differentially expressed in these cells compared to the parental PC3-lucE cells, was identified. The biological processes associated with this signature were determined using ‘David Bioinformatics Resources,’ and the most significant were found to correlate with macrophage-associated functions including: 1)-response to wounding (p=4.6 E-14), 2)-inflammatory response (p=3 E-10), 3)-extracellular matrix organization (p=1.1 E-9), 4)-wound healing (p=8.8 E-8) and 5)-defense response (p=1.9 E-5), as well as functions also connected to EMT: 6)-cell migration (p=5.7 E-6) and 7)-cell motility (p=1.4 E-5). Since this signature was identified in our cancer cells, it suggests that these cells through their interactions with macrophages, acquired functions that enhance matrix remodeling, cell migration, invasion, tumor progression and metastasis. Furthermore, the EMT-signature revealed the induction of a transcriptional program characterized by the up-regulation of Zeb1 and the downregulation of other transcription factors that were also regulated by ZEB1: EHF, IRF6, OVOL1/L2 and ANKRD22. Two transcription factors were identified to play a critical role in the stable EMT: ZEB1 and OVOL1. It was shown that ZEB1 knockdown or the overexpression of OVOL1 induces the reverse MET (mesenchymal-epithelial transition) in these cells suggesting a critical regulatory circuit of EMT-MET transformation in prostate cancer cells. In conclusion, macrophages do play a role in initiating EMT by inducing a transcriptional program in prostate cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1405. doi:1538-7445.AM2012-1405

Abstract 3369: Macrophages and their induced cytokines drive the epithelial to mesenchymal transition in prostate cancer cells

Macrophages derived from circulating monocytic myeloid precursors are a major component of leukocyte infiltrate found in tumors and their role in prostate cancer progression is now emerging. Here we investigate the potential of macrophages to induce the epithelial-mesenchymal transition (EMT) in prostate cancer cells. EMT is a critical process for metastasis, and the elucidation of factors that initiate EMT would be beneficial in the development of treatments to halt the dissemination of cancer cells throughout the body. CD14+-peripheral blood monocytes were isolated from healthy donors and stimulated with either interferon gamma (INFγ) or interleukin (IL)-4, factors known to promote the differentiation of monocytes into M1 or M2-type macrophages, respectively. After 48 hours, cells from prostate cancer cell lines PC3 Luc, DU145 Luc, and ARCAP Luc were co-cultured with the macrophages for four days. The cells were passaged three times by trypsination and protein lysates were analyzed by Western Blot for the expression of EMT markers: E-cadherin and vimentin. Our results revealed that cells co-cultured with IL-4-stimulated macrophages expressed lower levels of E-cadherin and higher levels of vimentin compared to control epithelial cells. In addition, three mesenchymal-type subpopulations that were isolated from PC3 cells interacting with IL-4-treated CD14+-cells exhibited a complete loss of E-cadherin. After more than 20 passages, these cells maintained the mesenchymal characteristics in culture and showed a striking up-regulation of the transcription factor ZEB 1, whose expression has been previously correlated with Gleason grade in human prostate tumors. Concurrently, we set out to study the secreted factors by which macrophages may trigger EMT. We treated DU145 Luc and PC3 Luc cells with several cytokines found to be differentially expressed in media containing cancer cells co-cultured with IL-4-stimulated macrophages and evaluated whether any of these factors induced EMT. It was discovered that cells grown in media containing the cytokine, IL-1B, in combination with IL-4 showed a greater display of mesenchymal markers than control cells. In conclusion, these results establish that macrophages can act as potent EMT-inducers in prostate cancer cells, although the exact mediators of this function remain unknown. The data suggests that CD14+ cells stimulated with IL-4 are more effective at triggering EMT than non-stimulated or INFγ-stimulated macrophages, which may pinpoint M2-type macrophages as a mediator of EMT in vivo. Furthermore, the cytokine study revealed that macrophage-induced IL-1B in combination with IL-4 may be an important factor in the mechanism of macrophage-induced EMT. As this transition may represent a major mechanism of prostate cancer metastasis, future research will be focused on elucidating the molecules involved in order to develop novel therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3369. doi:10.1158/1538-7445.AM2011-3369