Longikaurin A - Mediated Regulation of ROS/JNK Signaling Counteracts Epithelial-Mesenchymal Transition in Glioblastoma.

Dichloroacetate prevents TGFβ-induced epithelial-mesenchymal transition of retinal pigment epithelial cells

Nitric oxide (NO) has been shown to mediate contrasting effects on tumorigenesis i.e. at low levels NO is pro-tumorigenic and at high levels NO is anti-tumorigenic. Recent studies revealed that treatment of tumor cell lines with NO donors at high levels significantly inhibited cell growth and survival pathways such as the constitutively activated NF-κB. Tumor cells with activated NF-κB were reported to have the capacity to initiate the metastatic cascade via induction of epithelial to mesenchymal transition (EMT). The EMT phenotype is characterized by decreased expression of epithelial gene products (e.g. E-cadherin, cytokeratin 18) and upregulation of mesenchymal gene products (e.g. vimentin, fibronectin). In addition, the tumor cells exhibit invasive and migratory properties. Based on these above findings, we hypothesized that treatment of metastatic prostate carcinoma cell lines, PC-3 and DU-145 with DETANONOate (500-1000μM) may reverse the EMT phenotype. This hypothesis was tested and treatment of the cells with DETANONOate resulted in inhibition of the EMT phenotype through inhibition of NF-κB activity. Inhibition of NF-κB was shown to be due, in part, to S-nitrosylation of p50. In addition to the inhibition of EMT phenotype, treatment with DETANONOate also inhibited invasive properties. The dual roles of DETANONOate-mediated inhibition of NF-κB and EMT were corroborated by the use of the NF-κB inhibitor DHMEQ. We examined the underlying mechanism by which NF-κB-induced inhibition by DETANONOate resulted in the inhibition of EMT. The EMT-induced transcription factor Snail was highly expressed in PC-3 and DU-145 cells. Treatment with DETANONOate inhibited Snail expression concomitant with inhibition of EMT. The direct role of Snail inhibition by DETANONOate and the inhibition of EMT was corroborated in cells transfected with Snail siRNA and such treatment reversed the EMT phenotype, mimicking DETANONOate. Inhibition of the repressor activity of Snail by DETANONOate resulted in the upregulation of the Raf-1 kinase inhibitory protein (RKIP). The role of RKIP induction in the reversal of EMT was corroborated in cells overexpressing RKIP. The in vitro findings with DETANONOate-induced inhibition of EMT were validated in mice bearing PC-3 xenografts. Treatment with DETANONOate and analysis by IHC of tumor biopsies revealed that the EMT phenotype was reversed. There was an inverse correlation between Snail inhibition and RKIP induction. The present findings demonstrate, for the first time, that NO donors (high concentrations) inhibit the EMT phenotype and suggest their potential therapeutic applications in vivo in patients. These studies also suggest that the NF-κB/Snail/RKIP circuitry is dysregulated in tumor cells and is responsible, in part, for the EMT phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1466.

Metastasis is the main cause of death in cancer patients. The underlying molecular mechanisms of the metastatic process and their regulation in different malignancies are unknown. Survival pathways, such as the constitutively activated NF-κB pathway, contribute to the initiation of metastasis, a process known as epithelial to mesenchymal transition (EMT). The EMT phenotype is characterized by several gene modifications such as inhibition of epithelial gene products (e.g. E-cadherin, cytokeratin 18) and upregulation of mesenchymal gene products (e.g. vimentin and fibronectin). In addition, the cells exhibit invasive properties. The metastasis-inducer transcription factor, Snail, is intimately involved in EMT and is transcriptionally regulated, in part, by NF-κB. A recent report demonstrated that Snail is transcriptionally regulated, in part, by the transcription factor Yin Yang 1 (YY1) and YY1 is downstream of NF-κB. We have reported that YY1 expression is a prognostic marker in prostate cancer and YY1 also regulates tumor cell resistance to apoptosis by cytotoxic therapeutics. Based on these findings, we hypothesized that the expression levels of YY1 in cancer may also regulate EMT directly and/or indirectly through its regulation of Snail transcription. This hypothesis was tested using the human metastatic prostate carcinoma cell lines, PC-3 and DU-145, that exhibit the EMT phenotype as models. The relationship between inhibition of YY1 and inhibition of EMT was established by several lines of evidence. Treatment of the tumor cells with the proteasome inhibitors, NPI-0052 or Bortezomib, inhibited NF-κB, Snail and YY1 activities and correlated with inhibition of EMT. The direct role of YY1 expression and activity in the regulation of EMT was shown by treating cells with YY1 siRNA and resulting in the inhibition of the EMT phenotype. In turn, inhibition of Snail resulted in upregulation of the expression of the metastasis-suppressor/immune surveillance cancer gene product, Raf-1 kinase inhibitor protein (RKIP). The upregulation of RKIP by YY1 siRNA was due to the inhibition of the RKIP transcription repressor Snail. These findings establish a novel role for YY1 in the regulation of EMT. The findings also establish the dysregulation of the circuitry NF-κB/Snail/YY1/RKIP in cancer and metastasis resulting in the initiation of EMT. Further, this dysregulation has also been recently reported by us to regulate tumor cell resistance to apoptotic-inducing stimuli. Therefore, this dysregulated circuitry and downstream regulated gene products may be considered as novel targets for the regulation of both metastasis and resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2290.

Background. Epithelial-mesenchymal transition (EMT), a process whereby tumorigenic epithelial cells acquire an invasive and migratory phenotype, is an important event in the invasion-metastasis cascade. As such, intracellular signaling pathways involved in the regulation of EMT represent potential therapeutic targets in the treatment and prevention of invasive cancer subtypes. The calcium ion, a highly versatile intracellular messenger, plays an important role in processes important in tumorigenesis including invasion and metastasis, and altered calcium signaling has been identified in various cancers. We recently identified that activation of signal transducer and activator of transcription 3 (STAT3) and expression of specific EMT markers in the MDA-MB-468 cell line model of epidermal growth factor (EGF) induced EMT display some calcium dependence. While the calcium permeable ion channel TRPM7 was shown to partially regulate this STAT3 activation and vimentin expression, the precise mechanisms of their regulation are not yet fully understood. The aim of this research was to investigate the upstream intracellular signaling pathway involved in EGF stimulated STAT3 activation and the subsequent induction of EMT in this model. Methods. MDA-MB-468 basal-like breast cancer cells were pre-treated for 1 hr with the Janus protein tyrosine kinase (JAK) inhibitor, JAK inhibitor I (1 and 10 μM), or the Src family tyrosine kinase inhibitor, PP2 (0.1, 1 and 10 μM), followed by stimulation with EGF (50 ng/mL) for 10 or 20 min, and 24 h to assess effects on STAT3 activation and/or EMT marker expression, respectively. Total cellular protein was isolated following inhibitor treatment ± EGF stimulation, and the level of phosphorylated STAT3 (10 or 20 min) or vimentin protein expression (24 h), was analyzed using Western blotting. Cellular RNA was isolated following inhibitor treatment ± EGF stimulation and levels of vimentin mRNA (24 h) were assessed using real time RT-PCR. Results. Treatment of MDA-MB-468 breast cancer cells with JAK inhibitor I resulted in a significant decrease in EGF stimulated STAT3 phosphorylation, while inhibition of Src family tyrosine kinases with PP2 also significantly decreased EGF stimulated STAT3 phosphorylation. In addition to its effects on STAT3 phosphorylation, pre-treatment of MDA-MB-468 cells with JAK inhibitor I also appeared to decrease EGF-induced vimentin protein and mRNA expression, indicating a potential role for Janus protein tyrosine kinases in the induction of EMT in this model. Conclusions. Janus protein tyrosine kinase signaling appears to play a role in the regulation of STAT3 activation, and the induction of the EMT marker vimentin in the MDA-MB-468 cell line model of EGF-induced EMT. Future studies will focus on investigating the specific JAK family member(s) involved in the EGF-STAT3 signaling pathway, as well as the nexus between calcium and identified regulators of EGF stimulated STAT3 activation, and EMT marker expression, in this model of breast cancer EMT. Citation Format: Teneale A Stewart, Iman Azimi, Felicity M Davis, Erik W Thompson, Andrew J Brooks, Sarah J Roberts-Thomson, Gregory R Monteith. A potential role for Janus protein tyrosine kinases in the regulation of epithelial-mesenchymal transition in a model of epidermal growth factor induced breast cancer epithelial-mesenchymal transition [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-07-05.

Black tea extract prevents inorganic arsenic induced uncontrolled proliferation, epithelial to mesenchymal transition and induction of metastatic properties in HaCaT keratinocytes - an in vitro study

Epithelial-mesenchymal transition (EMT) occurs during the progression of liver fibrosis in response to chronic liver injury. However, the molecular mechanism underlying the regulation of hepatocyte EMT remains unclear. The aim of this study was to determine whether advanced oxidation protein products (AOPP) had an effect on hepatocyte EMT. The human L02 hepatocyte cell line and hepatocytes from normal Sprague-Dawley rats were challenged with AOPP treatment in both in vitro and in vivo studies. The expression of cell and molecular markers of EMT in L02 hepatocytes were studied using Western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. Hepatocyte migratory potential was analyzed using a wound healing assay. Intracellular reactive oxygen species (ROS) were detected using the dichlorofluorescein (DCF) assay. In liver tissue sections, expression of EMT markers was evaluated using immunohistochemistry, and collagen was assessed using histochemical staining with Masson's trichrome. The findings were that AOPP treatment resulted in EMT in hepatocytes, which was associated with reduced expression of E-cadherin, increased expression of vimentin, increased deposition of collagen protein, and enhanced cell migration in vivo and in vitro. AOPP was also found to promote migration in L02 cells, and to promote the production of ROS and the activation of TGF-βR and Smad signaling. Inhibition of the generation of intracellular ROS and TGF-β receptor blocking could reverse AOPP-induced EMT in hepatocytes. This study has identified a novel mechanism in the regulation of hepatocyte EMT, and the findings may have implications for the control of liver fibrosis.

Background: Glioblastoma multiforme (GBM) represents one of the most aggressive and lethal primary brain malignancies, characterized by rapid proliferation, extensive invasiveness, and a dismal prognosis. Emerging evidence implicates nucleotide-binding oligomerization domain-containing protein 2 (NOD2), an intracellular pattern recognition receptor, as a potential driver of GBM progression. This study investigates NOD2’s role in promoting glioblastoma through its effects on the epithelial–mesenchymal transition (EMT) and cancer stem cell (CSC) markers. Methods: NOD2 expression levels and survival outcomes were assessed using TCGA data from GBM tumor samples (n = 153) and normal brain tissues (n = 5). NOD2 protein expression was validated in glioma cell lines using Western blot and immunofluorescence analyses. Functional studies employed siRNA-mediated NOD2 knockdown to evaluate effects on cellular proliferation, migration, invasion, and colony formation, while correlations between NOD2 and EMT/CSC markers were assessed. Results: The analysis of TCGA data revealed a significantly elevated NOD2 expression in GBM tumors compared to normal brain tissue, with a high NOD2 expression correlating with a reduced disease-free survival in GBM patients. All tested glioma cell lines demonstrated robust NOD2 expression. Functional analyses demonstrated that NOD2 depletion substantially impaired cellular proliferation, migration, invasion, and the colony-forming capacity. Mechanistically, siRNA-mediated NOD2 knockdown significantly decreased the expression of EMT (Snail, SLUG, Vimentin) and CSC markers (CD44, CD133) at both protein and mRNA levels. Conclusions: Our results indicate that NOD2 contributes to GBM progression by influencing EMT and CSC pathways. These findings suggest NOD2’s potential as a therapeutic target in glioblastoma, highlighting the need for further mechanistic studies and therapeutic exploration.

Introduction: Our laboratory has discovered a protein, BP1, which is a transcription factor of the homeobox gene family activated in 89% of the tumors of African American women (AAW) compared with 57% of the tumors of Caucasian American women (CW). High level BP1 expression has been shown to be associated with aggressive and larger tumors. Tumors of AAW are generally more aggressive – higher grade, larger, more frequently estrogen receptor (ER) negative, and more proliferative – than those of CW, resulting in an almost 50% higher mortality rate. Preliminary data suggest that BP1 is involved in the epithelial to mesenchymal transition (EMT) which is associated with the transdifferentiation of malignant epithelial cells to mesenchymal cells; BP1 up-regulates the Twist gene, an inducer of EMT. We hypothesize that EMT may be more frequent in tumors of AAW than CW, and that an important mechanism of EMT is via activation of BP1. Materials and Methods: Cell lines engineered to overexpress BP1 were compared with controls with respect to EMT characteristics using Western blot analysis, confocal microscopy, and migration assays. Breast cancer cell lines derived from the tumors of AAW and CW were characterized with respect to BP1 and EMT markers using Western blots. Selected AA or CW cell lines are being analyzed using microarrays to attempt to identify pathways that may differ between them. Several cell lines will be treated with siBP1 to reduce BP1 or used to overexpress BP1, followed by microarray analysis to identify EMT-related and other pathways dysregulated by BP1 in AAW or CW. Tumor tissues from six AAW and from six CW, matched for age and stage of breast cancer, were immunostained to determine differential expression of BP1 and/or EMT markers, including E-cadherin (ECA), vimentin (VIM), and smooth muscle actin (SMA). Results: (a) EMT. MCF7 cells overexpressing BP1 exhibit increased Twist and decreased E-cadherin (an epithelial marker), while Hs578T cells overexpressing BP1 show increased Twist, as well as vimentin and fibronectin (mesenchymal markers). Overexpression of BP1 also leads to increased migration of Hs578T cells. (b) Cell line analysis. We have analyzed five AAW cell lines and five CW cell lines; interestingly, in both types of cells, two were epithelial, one was mesenchymal, and two were not classifiable. Microarray analysis is underway. MDA-MB 157 and HCC70 cells, derived from tumors of AAW, and Hs578T, MCF7, and MDA-MB-231cells, derived from tumors of CW, were identified as candidates to perform siBP1 knock-down and microarray analysis. Upon siBP1 treatment of MDA-MB-231cells, vimentin expression decreased, further support for regulation of EMT by BP1. (c) Clinical Studies. The nuclear membrane area was stained most intensely for BP1, nucleus > cellular membrane > cytoplasm. Nuclear and cytoplasmic staining for BP1 tended to be less for CW than AAW and membrane ECD was less in CW than AAW. VIM and SMA were not informative. Additional cases are being studied. Conclusions: Our data support the hypothesis that BP1 overexpression upregulates EMT in breast cancer cells by directly stimulating Twist expression. If EMT is more prevalent in tumors of AAW than CW, genes expressed during the transition would make good targets for therapy, with the possibility of reducing or preventing metastases. If BP1 is a regulator of EMT in patients, there would be a strong rationale for targeting BP1 using small molecule inhibitors, siBP1 or other approaches. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B32.

Background: The pancreatic ductal adenocarcinoma tumor microenvironment plays an important role in promoting the epithelial to mesenchymal transition (EMT), an early event in pancreatic cancer, involved in cancer invasiveness and in tumor progression. Among the stromal components the cancer-associated fibroblasts (CAFs) are responsible for the peculiar pancreatic tumor microenvironment and are known to be linked to the induction of EMT. The EMT process requires a dynamic remodeling of the actin cytoskeleton and we have suggested that the splicing program of hMENA, an actin regulator, play a role in EMT. Two alternatively expressed isoforms, hMENA11a and hMENAΔv6, with opposite functions in invasiveness have been described in breast cancer (Di Modugno et al PNAS 2012). hMENA expression has not been detected in normal pancreatic ducts, whereas expressed in the human pancreatic ductal adenocarcinoma (PDAC) samples, but no data are available on hMENA alternative isoform expression in this neoplasia. The aim of this study is to investigate whether TGFβ1-mediated EMT in pancreatic cancer cells is affected by hMENA overexpression and splicing and how CAFs affect this process in cancer cell lines and in human tissues. Methods: hMENA isoform expression was evaluated in PDAC tissues by immunohistochemistry using isoform specific antibodies. hMENA isoforms and EMT markers expression were characterized in human PDAC cell lines, TGFβ1-treated or untreated, by qRT-PCR and WB analysis. The effects of either hMENA isoform specific knockdown or overexpression in the TGFβ1-induced EMT were also evaluated. Pancreatic CAFs were isolated from human tissues of resected PDAC patients. The effect of the conditioned medium of cultured CAFs was evaluated on hMENA expression. In parallel, the role of CAF-cancer cell interaction on the expression of the different hMENA isoforms was analysed using a co-culture system. Results: Freshly explanted CAFs expressed the “mesenchymal” hMENAΔv6, and not hMENA11a and secreted paracrine factors involved in the induction of hMENA isoforms in tumor cells. In a panel of pancreatic cancer cell lines, hMENA11a expression correlated with an epithelial phenotype, while hMENAΔv6 expression was correlated with a mesenchymal phenotype. Interestingly, the expression of the invasive hMENAΔv6 isoform is specifically up-regulated by TGFβ1 treatment. hMENA isoform expression levels influenced molecular changes induced by TGFβ1. Thus, the hMENA11a specific silencing led to E-cadherin down-regulation that is more evident in TGFβ1 treated cells. On the contrary, hMENA11a overexpression led to a reduction of vimentin expression and to E-cadherin up-regulation. Knockdown of the endogenous hMENA/hMENAΔv6 isoform expression prevented the activation of TGFβ1 signaling and up-regulation of mesenchymal markers. In addition, hMENA/hMENAΔv6 isoform depletion impaired the TGFβ1-induced invasiveness, migration and production of MMPs. IHC analysis of PDAC tissues revealed that the epithelial hMENA11a is rarely expressed in primary pancreatic tumour, while high levels of hMENA and hMENAΔv6 isoforms were found in 75% of primary tumours analysed. Conclusions: This data suggests that the lack of the epithelial hMENA11a isoform is an early event in pancreatic cancer, provides new insights into the role of hMENA splicing in TGFβ1-mediated EMT and highlights hMENA splicing program as an attractive pathway for the development of new therapies in PDAC. Citation Format: Roberta Melchionna, Pierluigi Iapicca, Francesca Di Modugno, Paola Trono, Novella Gualtieri, Maria Grazia Diodoro, Marcella Mottolese, Gian Luca Grazi, Matteo Fassan, Aldo Scarpa, Mina J. Bissell, Paola Nisticò. The hMENA Splicing Program: An important regulator of TGFβ1-driven EMT and invasiveness in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A60.

Paricalcitol attenuates cyclosporine-induced kidney injury in rats

The Role of EMT in Pancreatic Cancer Progression

Glioblastoma (GBM) is the most common and most aggressive central nervous system tumor in adults. Due to GBM cell invasiveness and resistance to chemotherapy, current medical interventions are not satisfactory, and the prognosis for GBM is poor. It is necessary to investigate the underlying mechanism of GBM metastasis and drug resistance so that more effective treatments can be developed for GBM patients. sushi repeat-containing protein, X-linked 2 (SRPX2) is a prognostic biomarker in many different cancer cell lines and is associated with poor prognosis in cancer patients. SRPX2 overexpression promotes interactions between tumor and endothelial cells, leading to tumor progression and metastasis. We hypothesize that SRPX2 also contributes to GBM chemotherapy resistance and metastasis. Our results revealed that GBM tumor samples from 42 patients expressed higher levels of SRPX2 than the control normal brain tissue samples. High-SRPX2 expression levels are correlated with poor prognosis in those patients, as well as resistance to temozolomide in cultured GBM cells. Up-regulating SRPX2 expression in cultured GBM cell lines facilitated invasiveness and migration of GBM cells, while down-regulating SRPX2 through RNA interference was inhibitory. These results suggest that SRPX2 plays an important role in GBM metastasis. Epithelial to mesenchymal transition (EMT) is one of the processes that facilitate GBM metastasis and resistance to chemotherapy. EMT marker expression was decreased in SRPX2 down-regulated GBM cells, and MAPK signaling pathway marker expression was also decreased when SRPX2 is knocked down in GBM-cultured cells. Blocking the MAPK signaling pathway inhibited GBM metastasis but did not inhibit cell invasion and migration in SRPX2 down-regulated cells. Our results indicate that SRPX2 facilitates GBM metastasis by enhancing the EMT process via the MAPK signaling pathway.

Extracellular Acidosis Modulates the Expression of Epithelial-Mesenchymal Transition (EMT) Markers and Adhesion of Epithelial and Tumor Cells

P4-04-02: Bromodomain and extra-terminal proteins regulate the epithelial-mesenchymal transition in breast cancer

Krüpple-like factor 5 (KLF5) is a basic transcriptional factor that has been well documented for its regulatory role in the proliferation and survival of epithelial cells. Recently, bioinformatic analysis suggests that KLF5 also regulates epithelial-to-mesenchymal transition (EMT), yet experimental evidence is still lacking regarding whether and how KLF5 regulates EMT. As a form of epithelial cell plasticity, EMT can be induced by many cytokines including TGF-β and EGF. Here we report a role of KLF5 in EMT. We found that KLF5 was significantly down-regulated during TGF-β- and EGF-induced EMT in epithelial cells including HaCaT cells, MCF-10A cells, and mouse primary keratinocytes; and that silencing KLF5 induced EMT as well, as indicated by altered cell morphology, enhanced migration, upregulation of CDH2, FN1 and ZEB1, dislocation of CDH1 from cell-cell junctions, and induction of actin fibers. To determine whether miRNAs mediate the role of KLF5 in EMT, a real-time qPCR assay was employed to search for miRNAs regulated by KLF5 during EMT. Five members of the miR-200 family, all of which have been implicated in EMT, were found to be down-regulated upon the downregulation of KLF5. KLF5 directly regulated the expression of miR-200s by binding to the GC boxes of their promoters, as demonstrated by functional and biochemical analyses including luciferase reporter assay, ChIP-PCR assay, and oligo pull-down assay. Functionally, overexpression of miR-200s inhibited EMT induced by KLF5 silencing or TGF-β and EGF treatment in HaCaT cells, and forced expression of KLF5 attenuated TGF-β- and EGF-induced EMT by rescuing miR-200s expression. To evaluate whether the KLF5-miR-200s axis modulates EMT in vivo, we determined the expression of Klf5, miR-200s and EMT markers in knockout mice and human breast cancer specimens. In mouse prostate neoplasms induced by Pten deletion, knockout of Klf5 repressed the expression of miR-200s and induced the downregulation of CDH1 and upregualtion of vimentin, which are indicative of EMT. Expression of KLF5 also correlated with miR-200s at the RNA level in human breast cancer specimens. These findings suggest that KLF5 directly regulates the transcription of miR-200s in epithelial cells, and that downregulation of the KLF5-miR-200 axis plays a causal role in TGF-β- and EGF-induced EMT, providing new insights into the regulation of EMT. Citation Format: Baotong Zhang, Jin-tang Dong. Downregulation of KLF5 contributes to TGF-β- and EGF-induced EMT by reducing miR-200s. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1491. doi:10.1158/1538-7445.AM2013-1491