CD44 enhances tumor aggressiveness by promoting tumor cell plasticity

Aggressive tumor cells can obtain the ability to transdifferentiate into cells with endothelial features and thus form vasculogenic networks. This phenomenon, called vasculogenic mimicry (VM), is associated with increased tumor malignancy and poor clinical outcome. To identify novel key molecules implicated in the process of vasculogenic mimicry, microarray analysis was performed to compare gene expression profiles of aggressive (VM+) and non-aggressive (VM-) cells derived from Ewing sarcoma and breast carcinoma. We identified the CD44/c-Met signaling cascade as heavily relevant for vasculogenic mimicry. CD44 was at the center of this cascade, and highly overexpressed in aggressive tumors. Both CD44 standard isoform and its splice variant CD44v6 were linked to increased aggressiveness in VM. Since VM is most abundant in Ewing sarcoma tumors functional analyses were performed in EW7 cells. Overexpression of CD44 allowed enhanced adhesion to its extracellular matrix ligand hyaluronic acid. CD44 expression also facilitated the formation of vasculogenic structures in vitro, as CD44 knockdown experiments repressed migration and vascular network formation. From these results and the observation that CD44 expression is associated with vasculogenic structures and blood lakes in human Ewing sarcoma tissues, we conclude that CD44 increases aggressiveness in tumors through the process of vasculogenic mimicry

Targeting PDGF-mediated recruitment of pericytes blocks vascular mimicry and tumor growth

Aggressive tumor cells can adopt an endothelial cell-like phenotype and contribute to the formation of a tumor vasculature, independent of tumor angiogenesis. This adoptive mechanism is referred to as vascular mimicry and it is associated with poor survival in cancer patients. To what extent tumor cells capable of vascular mimicry phenocopy the angiogenic cascade is still poorly explored. Here, we identify pericytes as important players in vascular mimicry. We found that pericytes are recruited by vascular mimicry-positive tumor cells in order to facilitate sprouting and to provide structural support of the vascular-like networks. The pericyte recruitment is mediated through platelet-derived growth factor (PDGF)-B. Consequently, preventing PDGF-B signaling by blocking the PDGF receptors with either the small tyrosine kinase inhibitor imatinib or blocking antibodies inhibits vascular mimicry and tumor growth. Collectively, the current study identifies an important role for pericytes in the formation of vascular-like structures by tumor cells. Moreover, the mechanism that controls the pericyte recruitment provides therapeutic opportunities for patients with aggressive vascular mimicry-positive cancer types. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.status: publishe

The Immediate Early Gene Product EGR1 and Polycomb Group Proteins Interact in Epigenetic Programming during Chondrogenesis

<div><p>Initiation of and progression through chondrogenesis is driven by changes in the cellular microenvironment. At the onset of chondrogenesis, resting mesenchymal stem cells are mobilized <i>in vivo</i> and a complex, step-wise chondrogenic differentiation program is initiated. Differentiation requires coordinated transcriptomic reprogramming and increased progenitor proliferation; both processes require chromatin remodeling. The nature of early molecular responses that relay differentiation signals to chromatin is poorly understood. We here show that immediate early genes are rapidly and transiently induced in response to differentiation stimuli <i>in vitro</i>. Functional ablation of the immediate early factor EGR1 severely deregulates expression of key chondrogenic control genes at the onset of differentiation. In addition, differentiating cells accumulate DNA damage, activate a DNA damage response and undergo a cell cycle arrest and prevent differentiation associated hyper-proliferation. Failed differentiation in the absence of EGR1 affects global acetylation and terminates in overall histone hypermethylation. We report novel molecular connections between EGR1 and Polycomb Group function: Polycomb associated histone H3 lysine27 trimethylation (H3K27me3) blocks chromatin access of EGR1. In addition, EGR1 ablation results in abnormal <i>Ezh2</i> and <i>Bmi1</i> expression. Consistent with this functional interaction, we identify a number of co-regulated targets genes in a chondrogenic gene network. We here describe an important role for EGR1 in early chondrogenic epigenetic programming to accommodate early gene-environment interactions in chondrogenesis.</p> </div

Loss of EGR1 affects chondrogenic histone modification and epigenetic modifier expression.

<p>(A, B) IB analysis of histone modifications ATDC5 <i>shcon</i> and <i>shEgr1</i> cultures as a function of chondrogenic differentiation time (as indicated): reduced histone acetylation (A) and abnormal histone trimethylation (B); bActin: loading control (* as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058083#pone-0058083-g005" target="_blank">Figure 5B</a>). (C) IC Detection of enhanced H3K9me3 staining (upper panels) and H3K27me3 staining in ATDC5 large flat <i>shEgr1</i> cells at 3 days <i>pid</i>; DAPI counterstaining by DAPI (lower panels). (D) IC detection of abnormal epigenetic regulator protein expression (BMI1, EZH2, KAP1) as a function of differentiation time; Tubulin loading control. Samples corresponding to control and experiment (Figures A, B, D; <i>shcon, shEgr1</i>) were loaded on the same gel to enable direct quantitative comparison (corresponding sections are shown separately; representative experiments shown). (E) EGR1 enrichment on BMI1 and EZH2 promoters at 0, 2 and 8 hours <i>pid</i>. *: P values (EGR1/chromatin enrichment at t = 2 vs t = 0): 0.045 and 0.05.</p

EGR1 depletion reduces chondrogenic differentiation.

<p>(A) EGR1-protein expression (protein) in ATDC5 cells stably expressing control short hairpin sequences (<i>shcon</i>) (upper panel); absent EGR1 in cells expressing <i>shEgr1</i> vectors (lower panel) at 0, 1, 2 and 4 hours pid. GAPDH is used as loading control. Samples corresponding to control and experiment (<i>shcon, shEgr1</i>) were loaded on the same gel to enable direct quantitative comparison (corresponding sections are shown separately; representative experiment shown. Selection pressure on <i>shRNA</i> expression was maintained for the duration of the experiments. (B–D) Reduced <i>Egr1</i> (B), <i>Sox9</i> (C) and <i>Col2a1</i> (D) expression (mRNA) in ATDC5 <i>shEgr1</i> compared to <i>shcon cultures</i>; standard error is based on three independent, parallel experiments; expression was normalized to <i>cyclophilin A</i>. (E) Reduced chondrogenic marker protein expression in ATDC5 cells stably expressing <i>shEgr1</i>. Samples corresponding to control and experiment (<i>shcon, shEgr1</i>) were loaded on the same gel to enable direct quantitative comparison (corresponding sections are shown separately; representative experiment shown).</p

EGR1-LOF elicits DNA damage in hyper-proliferating chondrocytes.

<p>(A) Immunoblot (IB) detection of enhanced phospho-CHK2 (pCHK2) in <i>shEgr1</i> cultures early in chondrogenesis; FC <i>shcon</i>/<i>shEgr1</i>: 1,1 (t = 0), 1.0 (t = 8), 0.5 (t = 24), 4.5 (t = 72 days <i>pid</i>). (B) Detection of early DNA damage by IC analysis of γH2A.X (upper panels) in ATDC5 <i>shEgr1</i> cultures at 2 days <i>pid</i>; DAPI counterstaining by DAPI (lower panels). (C) IB detection of proteins related to DNA damage responses and cell-cycle arrest in ATDC5 cells stably expressing <i>shEgr1</i>; bActin loading control (* as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058083#pone-0058083-g006" target="_blank">Figure 6B</a>). Samples corresponding to control and experiment (<i>shcon, shEgr1</i>) were loaded on the same gel to enable direct quantitative comparison (corresponding sections are shown separately; representative experiment shown). (D) Comparative mRNA expression analysis of genes involved in cellular senescence, DNA damage response and stress signalling in <i>shcon</i> ATDC5 cells and cells stably expressing <i>shEgr1</i> throughout chondrogenesis (arbitrary expression units).</p