Plasticity of melanoma and EMT-TF reprogramming.

Plasticity of melanoma and EMT-TF reprogramming

Combination treatment with ionising radiation and gefitinib ('Iressa', ZD1839), an epidermal growth factor receptor (EGFR) inhibitor, significantly inhibits bladder cancer cell growth in vitro and in vivo

Purpose: External beam radiotherapy (EBRT) is the principal bladder-preserving monotherapy for muscle-invasive bladder cancer. Seventy percent of muscle-invasive bladder cancers express epidermal growth factor receptor (EGFR), which is associated with poor prognosis. Ionising radiation (IR) stimulates EGFR causing activation of cytoprotective signalling cascades and thus may be an underlying cause of radioresistance in bladder tumours. Materials and methods: We assessed the ability of IR to activate EGFR in bladder cancer cells and the effect of the anti-EGFR therapy, gefitinib on potential radiation-induced activation. Subsequently we assessed the effect of IR on signalling pathways downstream of EGFR. Finally we assessed the activity of gefitinib as a monotherapy, and in combination with IR, using clonogenic assay in vitro, and a murine model in vivo. Results: IR activated EGFR and gefitinib partially inhibited this activation. Radiation-induced activation of EGFR activated the MAPK and Akt pathways. Gefitinib partially inhibited activation of the MAPK pathway but not the Akt pathway. Treatment with combined gefitinib and IR significantly inhibited bladder cancer cell colony formation more than treatment with gefitinib alone (p = 0.001-0.03). J82 xenograft tumours treated with combined gefitinib and IR showed significantly greater growth inhibition than tumours treated with IR alone (p = 0.04). Conclusions: Combining gefitinib and IR results in significantly greater inhibition of invasive bladder cancer cell colony formation in vitro and significantly greater tumour growth inhibition in vivo. Given the high frequency of EGFR expression by bladder tumours and the low toxicity of gefitinib there is justification to translate this work into a clinical trial

SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell-cell junctions.

SIP1/ZEB2 is a member of the deltaEF-1 family of two-handed zinc finger nuclear factors. The expression of these transcription factors is associated with epithelial mesenchymal transitions (EMT) during development. SIP1 is also expressed in some breast cancer cell lines and was detected in intestinal gastric carcinomas, where its expression is inversely correlated with that of E-cadherin. Here, we show that expression of SIP1 in human epithelial cells results in a clear morphological change from an epithelial to a mesenchymal phenotype. Induction of this epithelial dedifferentiation was accompanied by repression of several cell junctional proteins, with concomitant repression of their mRNA levels. Besides E-cadherin, other genes coding for crucial proteins of tight junctions, desmosomes and gap junctions were found to be transcriptionally regulated by the transcriptional repressor SIP1. Moreover, study of the promoter regions of selected genes by luciferase reporter assays and chromatin immunoprecipitation shows that repression is directly mediated by SIP1. These data indicate that, during epithelial dedifferentiation, SIP1 represses in a coordinated manner the transcription of genes coding for junctional proteins contributing to the dedifferentiated state; this repression occurs by a general mechanism mediated by Smad Interacting Protein 1 (SIP1)-binding sites

Behavior of the different proteins of the cadherin–catenin complex upon SIP1 induction

<p><b>Copyright information:</b></p><p>Taken from "SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell–cell junctions"</p><p>Nucleic Acids Research 2005;33(20):6566-6578.</p><p>Published online 24 Nov 2005</p><p>PMCID:PMC1298926.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Immunofluorescence microscopy of non-induced and induced DLD1Tr21/WTSIP1 cells using antibodies specific for adherens junction components. E-cadherin as well as αE-catenin, p120ctn and β-catenin became nearly undetectable at cell–cell contacts in the SIP1-induced cells. () Western blot analysis of the non-induced and induced DLD1Tr21/WTSIP1 cell line. E-cadherin and αE-catenin were downregulated at the protein level in the SIP1-expressing cells. β-catenin protein levels were unaltered in the SIP1-induced compared to non-induced cells. Protein expression of p120ctn isoform 1 was upregulated and that of isoform 3 downregulated after SIP1 induction (: addition of Dox every 2 days, : washing away Dox from the cell culture medium)

Reactive oxygen species and mitochondrial sensitivity to oxidative stress determine induction of cancer cell death by p21.

p21(Waf1/Cip1/Sdi1) is a cyclin-dependent kinase inhibitor that mediates cell cycle arrest. Prolonged p21 up-regulation induces a senescent phenotype in normal and cancer cells, accompanied by an increase in intracellular reactive oxygen species (ROS). However, it has been shown recently that p21 expression can also lead to cell death in certain models. The mechanisms involved in this process are not fully understood. Here, we describe an induction of apoptosis by p21 in sarcoma cell lines that is p53-independent and can be ameliorated with antioxidants. Similar levels of p21 and ROS caused senescence in the absence of significant death in other cancer cell lines, suggesting a cell-specific response. We also found that cells undergoing p21-dependent cell death had higher sensitivity to oxidants and a specific pattern of mitochondrial polarization changes. Consistent with this, apoptosis could be blocked with targeted expression of catalase in the mitochondria of these cells. We propose that the balance between cancer cell death and arrest after p21 up-regulation depends on the specific effects of p21-induced ROS on the mitochondria. This suggests that selective up-regulation of p21 in cancer cells could be a successful therapeutic intervention for sarcomas and tumors with lower resistance to mitochondrial oxidative damage, regardless of p53 status

Widespread FRA1-dependent control of mesenchymal transdifferentiation programs in colorectal cancer cells.

Tumor invasion and metastasis involves complex remodeling of gene expression programs governing epithelial homeostasis. Mutational activation of the RAS-ERK is a frequent occurrence in many cancers and has been shown to drive overexpression of the AP-1 family transcription factor FRA1, a potent regulator of migration and invasion in a variety of tumor cell types. However, the nature of FRA1 transcriptional targets and the molecular pathways through which they promote tumor progression remain poorly understood. We found that FRA1 was strongly expressed in tumor cells at the invasive front of human colorectal cancers (CRCs), and that its depletion suppressed mesenchymal-like features in CRC cells in vitro. Genome-wide analysis of FRA1 chromatin occupancy and transcriptional regulation identified epithelial-mesenchymal transition (EMT)-related genes as a major class of direct FRA1 targets in CRC cells. Expression of the pro-mesenchymal subset of these genes predicted adverse outcomes in CRC patients, and involved FRA-1-dependent regulation and cooperation with TGFβ signaling pathway. Our findings reveal an unexpectedly widespread and direct role for FRA1 in control of epithelial-mesenchymal plasticity in CRC cells, and suggest that FRA1 plays an important role in mediating cross talk between oncogenic RAS-ERK and TGFβ signaling networks during tumor progression

Enrichment of FRA1 in tumor cells at the invasive front of human CRCs.

<p>(A) Low power image of a representative colorectal carcinoma stained with an antibody detecting FRA1. The asterisk indicates the lumen, while the arrowheads indicate the deep invasive front. Scale bar represents 1 mm. (B and C) High power images of the tumor centre (TC) and invasive front (IF) shown in (A). Arrowheads indicate tumor buds. Scale bar represents 10 µM. (D) Relationship between the intensity of nuclear FRA1 expression and the tumor budding marker, cytokeratin AE1/AE3 in 25 CRC cases.</p

FRA1 knockdown suppresses mesenchymal-like features in CRC cells.

<p>(A) Immunoblot analysis of FRA1 and E-cadherin levels in BE cells stably transduced with a non-silencing control (shNS) or FRA1-targeting shRNAs (shFRA1-A and -B). (B) Effect of FRA1 knockdown on basal and c-Jun-induced AP-1 reporter gene activity in BE cells. (C) Phase-contrast images (top row) and immunofluorescence staining for DAPI with ZO-1 (middle row) or vimentin (bottom row) on the cells from (A). Scale bar represents 10 µM. (D–F) Analysis of <i>in vitro</i> migration, invasion and proliferation in cells from (A). Error bars represent S.E.M. for 3 independent experiments.</p

Clinical significance of FRA1 and FRA1^EMT genes in CRC.

<p>(A) Kaplan-Meier plots of recurrence-free survival in stage B and C CRC patients according to expression of the FRA1 gene (<i>FOSL1</i>). (B) Unsupervised clustering of stage B and stage C CRCs based on FRA1^EMT genes encompassing concordant probesets exhibiting significant expression differences between the two main groups. Clustering divides cancers into groups with mesenchymal and epithelial profiles. Samples are arranged along the X-axis and genes along the Y-axis. Genes are grouped into those downregulated (blue) or upregulated (orange) upon FRA1 knockdown in BE cells relative to the mean- and sample-centered scaled expression. (C) Kaplan-Meier plots of recurrence-free survival in stage B and C CRC patients based on expression of both <i>FOSL1</i> (low vs high) and mesenchymal (Mes, dark green) or epithelial (Epi, light green) subsets of FRA1^EMT genes. The log-rank test was used for comparisons.</p

A FRA1-dependent autocrine TGFβ2 loop promotes mesenchymal gene expression in BE CRC cells.

<p>(A and B) Expression of selected mesenchymal (<i>TGFBI</i>, <i>AXL</i>) and epithelial (<i>CDH1</i>, <i>CLDN7</i>) FRA1^EMT genes upon transient knockdown of the TGFβ pathway FRA1 targets <i>TGFB2</i> and <i>SMAD3</i> using siRNA pools in BE cells. Data are represented relative to levels of these genes in cells transfected with siRNAs targeting GFP. (C) Effects of the TGFβ receptor inhibitor SB43152 (10 µM for 72 h) on expression of a selected mesenchymal FRA1^EMT (<i>TGFBI</i>, <i>AXL</i>) genes in BE cells. Student's t-test was used for all comparisons (^*p&lt;0.05, ^**p&lt;0.01, ^***p&lt;0.001). Error bars represent S.E.M. for 3 independent experiments.</p