Kinome-Wide Functional Genomics Screen Reveals a Novel Mechanism of TNFα-Induced Nuclear Accumulation of the HIF-1α Transcription Factor in Cancer Cells

Hypoxia-inducible factor-1 (HIF-1) and its most important subunit, HIF-1α, plays a central role in tumor progression by regulating genes involved in cancer cell survival, proliferation and metastasis. HIF-1α activity is associated with nuclear accumulation of the transcription factor and regulated by several mechanisms including modulation of protein stability and degradation. Among recent advances are the discoveries that inflammation-induced cytokines and growth factors affect protein accumulation of HIF-1α under normoxia conditions. TNFα, a major pro-inflammatory cytokine that promotes tumorigenesis is known as a stimulator of HIF-1α activity. To improve our understanding of TNFα-mediated regulation of HIF-1α nuclear accumulation we screened a kinase-specific siRNA library using a cell imaging–based HIF-1α-eGFP chimera reporter assay. Interestingly, this systematic analysis determined that depletion of kinases involved in conventional TNFα signaling (IKK/NFκB and JNK pathways) has no detrimental effect on HIF-1α accumulation. On the other hand, depletion of PRKAR2B, ADCK2, TRPM7, and TRIB2 significantly decreases the effect of TNFα on HIF-1α stability in osteosarcoma and prostate cancer cell lines. These newly discovered regulators conveyed their activity through a non-conventional RELB-depended NFκB signaling pathway and regulation of superoxide activity. Taken together our data allow us to conclude that TNFα uses a distinct and complex signaling mechanism to induce accumulation of HIF-1α in cancer cells. In summary, our results illuminate a novel mechanism through which cancer initiation and progression may be promoted by inflammatory cytokines, highlighting new potential avenues for fighting this disease

Enhanced progression of human prostate cancer PC3 cells induced by the microenvironment of the seminal vesicle

The objective of this study was to characterise the mechanism mediating the prostate cancer progression induced by the microenvironment of seminal vesicle (SV). The invasive potential of PC3 cells significantly increased after treatment with extract from SV of NOD/SCID mouse. Among several growth factors and cytokines that were present in the SV extract, transforming growth factor-β1 (TGF-β1) significantly enhanced the invasive potential of PC3 cells; however, the additional treatment with neutralising antibody against TGF-β1 suppressed the enhanced invasive potential induced by the SV extract. Changes in the invasive potential in PC3 cells after treatment with the SV extract and/or TGF-β1 were in proportion to those in the production of urokinase-type plasminogen activator (uPA) by PC3 cells. Tumour growth as well as the incidence of lymph node metastasis in NOD/SCID mice after the injection of PC3 cells into the SV were significantly greater than those after the injection into the prostate. These findings suggest that the microenvironment of SV enhances the progression of prostate cancer through a stimulated invasive potential, and that enhanced uPA production in prostate cancer cells induced by TGF-β1 could therefore be one of the most important mechanisms involved in the progression of prostate cancer after SV invasion

Multifunctional Properties of Chicken Embryonic Prenatal Mesenchymal Stem Cells- Pluripotency, Plasticity, and Tumor Suppression

The chick embryo represents an accessible and economical in vivo model, which has long been used in developmental biology, gene expression analysis, and loss/gain of function experiments. In the present study, we assessed and characterized bone marrow derived mesenchymal stem cells from prenatal day 13 chicken embryos (chBMMSCs) and determined some novel properties. After assessing the mesenchymal stem cell (MSC) properties of these cells by the presence of their signature markers (CD 44, CD 73, CD 90, CD 105, and vimentin), we ascertained a very broad spectrum of multipotentiality as these MSCs not only differentiated into the classic tri-lineages of MSCs but also into ectodermal, endodermal, and mesodermal lineages such as neuron, hepatocyte, islet cell, and cardiac. In addition to wide plasticity, we detected the presence of several pluripotent markers such as Oct4, Sox2, and Nanog. This is the first study characterizing prenatal chBMMSCs and their ability to not only differentiate into mesenchymal lineages but also into all the germ cell layer lineages. Furthermore, our studies indicate that prenatal chBMMSCs derived from the chick provide an excellent model for multi-lineage development studies because of their broad plasticity and faithful reproduction of MSC traits as seen in the human. Here, we also present evidence for the first time that media derived from prenatal chBMMSC cultures have an anti-tumorigenic, anti-migratory, and pro-apoptotic effect on human tumors cells acting through the Wnt-ß-catenin pathway. These data confirm that chBMMSCs are enriched with factors in their secretome that are able to destroy tumor cells. This suggests a commonality of properties of MSCs across species between human and chicken

Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Mice

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor which regulates the expression of many cytoprotective genes. In the present study, we found that the expression of Nrf2 was suppressed in prostate tumor of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Similarly, the expression of Nrf2 and the induction of NQO1 were also substantially suppressed in tumorigenic TRAMP C1 cells but not in non-tumorigenic TRAMP C3 cells. Examination of the promoter region of the mouse Nrf2 gene identified a CpG island, which was methylated at specific CpG sites in prostate TRAMP tumor and in TRAMP C1 cells but not in normal prostate or TRAMP C3 cells, as shown by bisulfite genomic sequencing. Reporter assays indicated that methylation of these CpG sites dramatically inhibited the transcriptional activity of the Nrf2 promoter. Chromatin immunopreceipitation (ChIP) assays revealed increased binding of the methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in the TRAMP C1 cells as compared to TRAMP C3 cells. In contrast, the binding of RNA Pol II and acetylated histone H3 to the Nrf2 promoter was decreased. Furthermore, treatment of TRAMP C1 cells with DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-aza) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) restored the expression of Nrf2 as well as the induction of NQO1 in TRAMP C1 cells. Taken together, these results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice. Our present findings reveal a novel mechanism by which Nrf2 expression is suppressed in TRAMP prostate tumor, shed new light on the role of Nrf2 in carcinogenesis and provide potential new directions for the detection and prevention of prostate cancer

Inhibition of Histone Deacetylase Activity in Human Endometrial Stromal Cells Promotes Extracellular Matrix Remodelling and Limits Embryo Invasion

Invasion of the trophoblast into the maternal decidua is regulated by both the trophoectoderm and the endometrial stroma, and entails the action of tissue remodeling enzymes. Trophoblast invasion requires the action of metalloproteinases (MMPs) to degrade extracellular matrix (ECM) proteins and in turn, decidual cells express tissue inhibitors of MMPs (TIMPs). The balance between these promoting and restraining factors is a key event for the successful outcome of pregnancy. Gene expression is post-transcriptionally regulated by histone deacetylases (HDACs) that unpacks condensed chromatin activating gene expression. In this study we analyze the effect of histone acetylation on the expression of tissue remodeling enzymes and activity of human endometrial stromal cells (hESCs) related to trophoblast invasion control. Treatment of hESCs with the HDAC inhibitor trichostatin A (TSA) increased the expression of TIMP-1 and TIMP-3 while decreased MMP-2, MMP-9 and uPA and have an inhibitory effect on trophoblast invasion. Moreover, histone acetylation is detected at the promoters of TIMP-1 and TIMP-3 genes in TSA-treated. In addition, in an in vitro decidualized hESCs model, the increase of TIMP-1 and TIMP-3 expression is associated with histone acetylation at the promoters of these genes. Our results demonstrate that histone acetylation disrupt the balance of ECM modulators provoking a restrain of trophoblast invasion. These findings are important as an epigenetic mechanism that can be used to control trophoblast invasion

The Role of EZH2 in the Regulation of the Activity of Matrix Metalloproteinases in Prostate Cancer Cells

Degradation of the extracellular matrix (ECM), a critical step in cancer metastasis, is determined by the balance between MMPs (matrix metalloproteinases) and their inhibitors TIMPs (tissue inhibitors of metalloproteinases). In cancer cells, this balance is shifted towards MMPs, promoting ECM degradation. Here, we show that EZH2 plays an active role in this process by repressing the expression of TIMP2 and TIMP3 in prostate cancer cells. The TIMP genes are derepressed by knockdown of EZH2 expression in human prostate cancer cells but repressed by overexpression of EZH2 in benign human prostate epithelial cells. EZH2 catalyzes H3K27 trimethylation and subsequent DNA methylation of the TIMP gene promoters. Overexpression of EZH2 confers an invasive phenotype on benign prostate epithelial cells; however, this phenotype is suppressed by cooverexpression of TIMP3. EZH2 knockdown markedly reduces the proteolytic activity of MMP-9, thereby decreasing the invasive activity of prostate cancer cells. These results suggest that the transcriptional repression of the TIMP genes by EZH2 may be a major mechanism to shift the MMPs/TIMPs balance in favor of MMP activity and thus to promote ECM degradation and subsequent invasion of prostate cancer cells

DNA Methylation Profiles of Primary Colorectal Carcinoma and Matched Liver Metastasis

BACKGROUND: The contribution of DNA methylation to the metastatic process in colorectal cancers (CRCs) is unclear. METHODS: We evaluated the methylation status of 13 genes (MINT1, MINT2, MINT31, MLH1, p16, p14, TIMP3, CDH1, CDH13, THBS1, MGMT, HPP1 and ERα) by bisulfite-pyrosequencing in 79 CRCs comprising 36 CRCs without liver metastasis and 43 CRCs with liver metastasis, including 16 paired primary CRCs and liver metastasis. We also performed methylated CpG island amplification microarrays (MCAM) in three paired primary and metastatic cancers. RESULTS: Methylation of p14, TIMP3 and HPP1 in primary CRCs progressively decreased from absence to presence of liver metastasis (13.1% vs. 4.3%; 14.8% vs. 3.7%; 43.9% vs. 35.8%, respectively) (P<.05). When paired primary and metastatic tumors were compared, only MGMT methylation was significantly higher in metastatic cancers (27.4% vs. 13.4%, P = .013), and this difference was due to an increase in methylation density rather than frequency in the majority of cases. MCAM showed an average 7.4% increase in DNA methylated genes in the metastatic samples. The numbers of differentially hypermethylated genes in the liver metastases increased with increasing time between resection of the primary and resection of the liver metastasis. Bisulfite-pyrosequencing validation in 12 paired samples showed that most of these increases were not conserved, and could be explained by differences in methylation density rather than frequency. CONCLUSIONS: Most DNA methylation differences between primary CRCs and matched liver metastasis are due to random variation and an increase in DNA methylation density rather than de-novo inactivation and silencing. Thus, DNA methylation changes occur for the most part before progression to liver metastasis

Epigenetic Drugs Can Stimulate Metastasis through Enhanced Expression of the Pro-Metastatic Ezrin Gene

Ezrin has been reported to be upregulated in many tumors and to participate in metastatic progression. No study has addressed epigenetic modification in the regulation of Ezrin gene expression, the importance of which is unknown. Here, we report that highly metastatic rhabdomyosarcoma (RMS) cells with high levels of Ezrin have elevated acetyl-H3-K9 and tri-methyl-H3-K4 as well as reduced DNA methylation at the Ezrin gene promoter. Conversely, poorly metastatic RMS cells with low levels of Ezrin have reduced acetyl-H3-K9 and elevated methylation. Thus epigenetic covalent modifications to histones within nucleosomes of the Ezrin gene promoter are linked to Ezrin expression, which in fact can be regulated by epigenetic mechanisms. Notably, treatment with histone deacetylase (HDAC) inhibitors or DNA demethylating agents could restore Ezrin expression and stimulate the metastatic potential of poorly metastatic RMS cells characterized by low Ezrin levels. However, the ability of epigenetic drugs to stimulate metastasis in RMS cells was inhibited by expression of an Ezrin-specific shRNA. Our data demonstrate the potential risk associated with clinical application of broadly acting covalent epigenetic modifiers, and highlight the value of combination therapies that include agents specifically targeting potent pro-metastatic genes