Skip to main content
NCBI home page
Translational Oncology logoLink to Translational Oncology
. 2010 Aug 1;3(4):218–229. doi: 10.1593/tlo.09325

Artemin Reduces Sensitivity to Doxorubicin and Paclitaxel in Endometrial Carcinoma Cells through Specific Regulation of CD241,2

Vijay Pandey *, Yewon Jung *, Jian Kang *, Michael Steiner *, Peng-Xu Qian †, Arindam Banerjee *, Murray D Mitchell *,§, Zheng-Sheng Wu ‡, Tao Zhu †, Dong-Xu Liu *, Peter E Lobie *,
PMCID: PMC2915413  PMID: 20689763

Abstract

We have previously reported that artemin (ARTN) stimulates the oncogenicity and invasiveness of endometrial carcinoma cells. Herein, we demonstrate that ARTN modulates the sensitivity of endometrial carcinoma cells to agents used to treat late-stage endometrial carcinoma. Forced expression of ARTN in endometrial carcinoma cells decreased sensitivity to doxorubicin and paclitaxel. Accordingly, depletion of ARTN by small interfering RNA or functional inhibition of ARTN with antibodies significantly increased sensitivity of endometrial carcinoma cells to doxorubicin and paclitaxel. Forced expression of ARTN in endometrial carcinoma cells abrogated doxorubicin-induced G2-M arrest and paclitaxel-induced apoptosis. ARTN increased CD24 expression in endometrial carcinoma cells by transcriptional up-regulation, and CD24 was partially correlated to ARTN expression in endometrial carcinoma. Forced expression of CD24 in endometrial carcinoma cells stimulated cell proliferation and oncogenicity, enhanced cell invasion, and decreased sensitivity to doxorubicin and paclitaxel. Depletion of CD24 in endometrial carcinoma cells abrogated ARTN-stimulated resistance to doxorubicin and paclitaxel. ARTN-stimulated resistance to doxorubicin and paclitaxel in endometrial carcinoma cells is therefore mediated by the specific regulation of CD24. Functional inhibition of ARTN may therefore be considered as an adjuvant therapeutic approach to improve the response of endometrial carcinoma to specific chemotherapeutic agents.

Introduction

Endometrial carcinoma (EC) is the most common malignancy of the female reproductive tract. Most cases diagnosed at an early stage (I/II) of the disease are treated with hysterectomy followed by radiation and exhibit a good prognosis [1]. Chemotherapy followed by hysterectomy is the only option for the treatment of late-stage and recurrent EC [1]. However, chemotherapy is not sufficient to produce long-lasting tumor regression in patients with late-stage (III/IV) and recurrent EC [1]. Patients with late-stage EC invariably exhibit a multidrug-resistant phenotype and experience a recurrence after therapy, with a median survival time less than 12 months [1]. Poor survival of late-stage and recurrent EC patients, particularly with an aggressive histological subtype, necessitates the development of new therapeutic modalities for advanced-stage and recurrent EC.

Artemin (ARTN) is a neurotrophic factor belonging to the glial cell-derived neurotrophic factor family of ligands. An elevated expression of ARTN has been observed in pancreatic, mammary, and ECs [2–4]. In mammary carcinoma, an elevated expression of ARTN predicted residual disease after chemotherapy, metastases, relapse, and death [4]. An elevated expression of ARTN in EC is associated with high tumor grade and myometrial invasion [2]. Functionally, the expression of ARTN promoted oncogenicity, tumor growth, and invasion of both mammary and EC cells [2,4].

CD24 is a small, heavily glycosylated protein with frequently increased expression in a wide range of human carcinomas including EC [5,6]. Elevated CD24 expression is a prognostic indicator of poor survival in non-small cell lung [7], prostate [6], mammary [8], and ovarian carcinomas [9]. In addition, CD24 has been repeatedly identified in gene expression profiling screens used to identify genes whose expression correlates with oncogenesis and tumor development [10–12]. CD24 has been reported to support the acquisition of multiple cellular properties associated with tumor development and metastasis [13]. Concordantly, transient down-regulation of CD24 expression in human carcinoma cell lines (mammary, urothelial, and prostate) resulted in growth inhibition and reduced clonogenicity and cell migration [14]. Similarly, functional inhibition of CD24 using small interfering RNA (siRNA) or a monoclonal antibody (mAb) abrogated cell growth of colorectal and pancreatic carcinoma cells in vitro and in vivo [15].

We therefore speculated that ARTN expression may modulate sensitivity to chemotherapeutics used in EC. In this article, we determined the effects of ARTN expression on the sensitivity of EC cells toward doxorubicin and paclitaxel, the therapeutic agents used to treat late stage EC [16]. Antibodies to ARTN increased the sensitivity of EC cells to doxorubicin and paclitaxel, indicating a potential therapeutic strategy to increase the efficacy of chemotherapeutic agents in EC.

Materials and Methods

Cell Culture and Reagents

The human EC cell lines RL95-2 and AN3 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and were cultured as per ATCC propagation instructions. Stable cell lines were generated as previously described [17]. Doxorubicin and paclitaxel were purchased from Sigma-Aldrich (Auckland, New Zealand). Bioassays with ARTN polyclonal chicken immunoglobulin (IgY) were performed as previously described [4].

Plasmids and Luciferase Assay

ARTN expression vector and siRNA plasmid constructs were previously described [4]. The CD24 expression vector was as a generous gift from Drs H. Kataoka and T. Fukushima (University of Miyazaki, Japan) [18]. Short-hairpin RNA (shRNA) for CD24 was obtained from Clontech Laboratories, Inc (Mountain View, CA). The CD24-luciferase reporter plasmid was a generous gift from Dr C. Sakanaka (Genentech, Inc, San Francisco, CA) [19]. EC cells were transfected in a 12-well plate at 5 x 105 cells per well using FuGENE6 (Roche Molecular Biochemicals, Indianapolis, IN) transfection reagent. Transfections were carried out in triplicate using 1 µg of the appropriate CD24 promoter luciferase reporter plasmid and empty vector per transfection along with 0.2 µg of pSV-β-galactosidase expression plasmid as control for transfection efficiency. Luciferase activities were assayed 48 hours after transfection using the dual Luciferase Assay System (Promega Corp, Madison, WI) as previously described [19].

Reverse Transcription-Polymerase Chain Reaction and Quantitative Polymerase Chain Reaction

Total RNA was isolated from cells (cultured in 10% fetal bovine serum) using TRIzol Plus RNA Purification system at 1 ml/10 cm2 according to the manufacturer's instructions (Invitrogen). RNA samples were further treated with DNase I for 30 minutes at 37°C. Total RNA was converted to complementary DNA (cDNA) by using Super-Script III First-Strand Synthesis SuperMix for quantitative polymerase chain reaction (qPCR) according to the manufacturer's instructions (Invitrogen). Platinum PCR SuperMix High-Fidelity Kit (Invitrogen) was used for amplification of cDNA according to manufacturer's instructions (Invitrogen). The ABI 7700 Real-Time PCR System (Applied Biosystems, Foster City, CA) was used for analysis. QPCR was performed as previously described [2]. Gene analysis and the sequences of the primers used are as previously described [2,17].

Immunoblot Analysis and Immunofluorescence

Western blot analysis was performed as previously described [20] using the following antibodies: goat anti-ARTN polyclonal antibody (R&D Systems, Minneapolis, MN), mouse anti-CD24 monoclonal IgG1 (Santa Cruz Biotechnology, Santa Cruz, CA), and a mAb against β-actin (Santa Cruz Biotechnology). Confocal laser scanning microscopy was performed as previously described [21] using tetramethyl-rhodamine B isothiocyanate-phalloidin (Sigma, St Louis, MO).

Apoptosis, Proliferation, and Oncogenicity Assays

A total of 5 x 105 cells per well were seeded in a six-well plate for the evaluation of apoptosis and nuclear ring formation. Apoptosis was determined by using Annexin V-FLUOS staining kit (Roche Diagnostics GmbH, Roche Applied Science Nonnenwald, Penzberg, Germany) following the manufacturer's instructions. For nuclear ring formation, cells were fixed for 20 minutes in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4) at roomtemperature and were stained with the tetramethyl-rhodamine B isothiocyanate-phalloidin (Sigma) for 45 minutes at room temperature. Ten photos from each well were obtained, and cells with nuclear ring formation were analyzed using ImageJ software (version 2.02; National Institutes of Health, Bethesda, MD). For the determination of total cell number, cells were seeded in six-well plates at a density of 5 x 104 cells per well in 10% FBS medium. Cells counts were conducted every 2 days for 14 days. Entry to the S phase was assayed by measuring the incorporation of 5-bromo-2-deoxyuridine as previously described [2,17]. In vitro cell invasion assays were performed in BD BioCoat Matrigel invasion chambers (BD Biosciences, Bedford, MA) according to the manufacturer's instructions as described previously [2,17]. A total of 2.5 x 104 cells were seeded in serum-free medium in the upper chamber. The lower chamber contained 10% FBS medium. After a 48-hour incubation, filters were washed with PBS, fixed with paraformaldehyde, and stained with crystal violet, and cells were counted. Other biological assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, anchorage-independent growth (soft agar colony formation and foci formation), and three-dimensional morphogenesis were performed as previously described [2,17]. The MTT assay was performed, and half maximal inhibitory concentration (IC50) values were calculated on the basis of the percentage of treatment over control as described previously [22].

Flow Cytometry

Cell cycle analysis was performed as previously described [23]. Fluorescence emitted from the propidium iodide-DNA complex was quantified using BD FACSAria Cell Sorter (BD Biosciences, San Jose, CA). For CD24 expression, cells were harvested and resuspended in PBS/1% FBS. The antihuman CD24 antibody (BD Pharmingen, San Diego, CA) was applied according to the manufacturer's manual. After antibody binding for 25 minutes at room temperature, cells were rinsed three times with PBS, and flow cytometry analyses were performed in triplicate. As negative controls, cells were stained either with isotype-matched control antibodies or with no primary antibody.

Xenograft Analyses

RL95-2-vector or RL95-2-ARTN cells (1.5 x 108) were suspended in 100 µl of PBS and were injected subcutaneously into 3- to 4-week-old BALBc nu/nu mice (Shanghai Slaccas Co, Shanghai, China). Histological analyses were performed as previously described [2]. Experiments were performed according to Institutional Animal Care and Use Committee guidelines (available at http://www.iacuc.org) with local institutional approval.

EC Tissue Microarray and Immunohistochemistry

EC tissue samples were collected from 45 female patients from the First Affiliated Hospital of Anhui Medical University (Hefei, Anhui, China) between 2004 and 2006. All 45 EC cases are endometrioid adenocarcinoma. The negative internal controls were the stromal cells of endometrium or smooth muscle cells of the uterus as background. Positive internal controls were EC samples with an intensity score of 3. Immunohistochemical analysis was performed as previously described [2] with polyclonal antibody to human ARTN (Abcam, Ltd, Cambridge, UK) and mAb against human CD24 (Thermo Fisher Scientific, Rockford, IL). Method for localization and histoscore of stained sections were performed as previously described [2].

Statistics

All experiments were performed at least three times. All numerical data are expressed as mean ± SD from a representative experiment performed in triplicate. Data were analyzed using an unpaired two-tailed t test or analysis of variance by GraphPad Prism 5 (GraphPad Software, Inc, La Jolla, CA).

Results

Forced Expression of ARTN in EC Cells Decreased Sensitivity to Doxorubicin and Paclitaxel

Appreciable differences in cell proliferation were evident by the forced expression of ARTN in RL95-2 and AN3 cell lines as previously reported [2]. We therefore determined the IC50 of doxorubicin and paclitaxel in RL95-2 and AN3 cell lines with forced expression of ARTN after exposure to doxorubicin or paclitaxel using the MTT viability assay (Figure W1). The IC50 values of doxorubicin and paclitaxel in RL95-2-ARTN cells were nearly 2-fold and 1.5-fold higher than those of the RL95-2-vector cells, respectively. The IC50 values of doxorubicin and paclitaxel in AN3-ARTN cells were nearly twofold and threefold higher than those of the AN3-vector cells, respectively. Concordantly, the IC50 values of doxorubicin and paclitaxel for RL95-2- siARTN and AN3-siARTN cells were reduced when compared with RL95-2-sivector and AN3-sivector cells, respectively. The IC50 values of doxorubicin or paclitaxel in RL95-2 and AN3 cell lines with forced or depleted expression of ARTN are tabulated (Table 1). IC50 values of doxorubicin and paclitaxel in control cells (transfected with empty vector or negative control sequence for siRNA) were not significantly different when compared with the parental cell lines, RL95-2 and AN3 (Table 1).

Table 1.

IC50 of Doxorubicin (DOX) or Paclitaxel (PTX) on the Viability of RL95-2 and AN3 Cell Lines with Forced or Depleted Expression of ARTN.

Cell Line IC50
DOX (µM) PTX (nM)
RL95-2 Wild-type 6.19 ± 0.17 119.21 ± 7.24
Vector 6.17 ± 0.26 129.00 ± 8.34
ARTN 12.33 ± 0.60 199.90 ± 4.80
sivector 6.16 ± 0.26 125.20 ± 9.03
siARTN 3.27 ± 0.42 72.34 ± 6.23
AN-3 Wild-type 6.23 ± 0.11 96.84 ± 8.27
Vector 6.24 ± 0.22 82.12 ± 6.90
ARTN 12.56 ± 0.36 279.19 ± 15.41
sivector 6.17 ± 0.29 100.10 ± 6.05
siARTN 3.04 ± 0.47 50.05 ± 2.99
Open in a new tab

Cell viability was measured using the MIT assay after a 72-hour drug exposure period with DOX or PTX followed by a 24-hour drug-free period as described in Materials and Methods. IC50 values are presented as mean ± SD of three independent experiments.

To determine the functional consequences of ARTN expression on the response of EC cells to doxorubicin and paclitaxel, we used the IC50 relevant for each cell line for further bioassays. Forced expression of ARTN in RL95-2 cells significantly enhanced their capacity for anchorage-independent growth when compared with vector control cells, and on exposure to either doxorubicin or paclitaxel, both RL95-2-vector and RL95-2-ARTN cells exhibited decreased anchorage-independent growth. However, the anchorage-independent growth of RL95-2-ARTN cells decreased significantly less on exposure to doxorubicin and paclitaxel compared with RL95-2-vector cells (Figure 1A). Depletion of ARTN in RL95-2 cells drastically decreased anchorage-independent growth on exposure to these drugs when compared with negative control siRNA control cells (Figure 1A). Doxorubicin and paclitaxel not only inhibit cell viability but also independently inhibit invasion of human carcinoma cells [24,25]. RL95-2-ARTN cells also exhibited increased invasion compared with RL95-2-vector cells [2], and on exposure to either doxorubicin or paclitaxel, both RL95-2-vector and RL95-2-ARTN cells exhibited decreased invasion through the Matrigel. However, invasion of RL95-2-ARTN cells decreased significantly less compared with RL95-2-vector cells on exposure to doxorubicin or paclitaxel (Figure 1B). siRNA-mediated depletion of ARTN in RL95-2 cells further significantly decreased invasion on exposure to doxorubicin and paclitaxel when compared with respective control cells treated with doxorubicin and paclitaxel (Figure 1B).

Figure 1.

Figure 1

Open in a new tab

ARTN expression in EC cells decreased sensitivity to doxorubicin and paclitaxel. IC50 doses of doxorubicin or paclitaxel against RL95-2 and AN3 cell lines were used, respectively. (A) Anchorage-independent growth of RL95-2 (left side) and AN3 (right side) cells with forced or depleted expression of ARTN on exposure to doxorubicin or paclitaxel was evaluated using soft agar colony formation. (B) Cellular invasion with RL95-2 (left side) and AN3 (right side) cells with forced or depleted expression of ARTN on exposure to doxorubicin or paclitaxel was evaluated using transwell assay. (C) Cell viability of RL95-2 and AN3 cells on exposure to doxorubicin or paclitaxel in combination with anti-ARTN IgY or control IgY was evaluated using MTT assay. (D) Anchorage-independent growth of RL95-2 and AN3 cells on exposure to doxorubicin or paclitaxel in combination with anti-ARTN IgY or control IgY was evaluated using soft agar colony formation. (E) Cellular invasion of RL95-2 and AN3 cells on exposure to doxorubicin or paclitaxel in combination with anti-ARTN IgY or control IgY was evaluated using transwell assay. Columns are mean of triplicate experiments; bars, ±SD. **P < .001, *P < .05.

In addition, forced or depleted expression of ARTN in a second EC cell line, AN3, exhibited similar effects on anchorage-independent capacity and cell invasion on exposure to doxorubicin or paclitaxel (Figure 1).

Functional Inhibition of ARTN in EC Cells Enhanced the Efficacy of Doxorubicin and Paclitaxel

Functional inhibition of ARTN by antibodies has been reported to reduce oncogenicity and invasiveness of EC cells [2]. We therefore evaluated the effect of ARTN IgY in combination with doxorubicin or paclitaxel on EC cells. Functional inhibition of ARTN in RL95-2 cells resulted in increased sensitivity to doxorubicin and paclitaxel up to 40% and 61% in monolayer culture (Figure 1C), up to 60% and 66% in soft agar colony formation (Figure 1D), and up to 36% and 68% during Matrigel invasion (Figure 1E), respectively, when compared with cells exposed to drug with control IgY alone. Similarly, in AN3 cells, IgY inhibition of ARTN resulted in increased sensitivity to doxorubicin and paclitaxel, up to 58% and 39% in monolayer culture (Figure 1C), up to 65% and 50% in soft agar colony formation (Figure 1D), and up to 25% and 60% during Matrigel invasion (Figure 1E), respectively, when compared with cells exposed to drug with control IgY alone.

Forced Expression of ARTN in RL95-2 Cells Abrogated Doxorubicin-Induced G2-M Phase Arrest and Paclitaxel-Induced Apoptosis

In response to DNA damage, cells arrest at G1, intra-S, or G2-M cell cycle checkpoints to allow for repair or to induce apoptosis [26]. We therefore used FACS analysis to determine the cell cycle profiles of RL95-2-vector and RL95-2-ARTN cells treated with various doses of doxorubicin. Vehicle-treated RL95-2-vector cells were proportionally distributed in the G0–G1 (55%), S (17%), and G2-M (27%) phases. Treatment of RL95-2-vector cells with doxorubicin induced a major shift in the cell cycle from the G0–G1 phase to the G2-M phase; the G2-M phase peak was elevated at 2 µM doxorubicin and increased with increasing concentration of doxorubicin to 12.5 µM, with 92% of the cells arrested in the G2-M phase. Accordingly, the percentage of RL95-2-vector cells at the G0-G1 and S phases decreased (Figure 2A). Vehicle-treated RL95-2-ARTN cells exhibited an altered cell cycle profile compared with RL95-2-vector cells and were distributed in the G0–G1 (49%), S (35%), and G2-M (15%) phases. Treatment of RL95-2-ARTN cells with doxorubicin induced a minor shift of the cell cycle from the G0–G1 phase to the G2-M phase when compared with RL95-2-vector cells; only 42% of the RL95-2-ARTN cells were arrested in the G2-M-phase at 12.5 µM doxorubicin, significantly less than RL95-2-vector cells treated with a similar dose of doxorubicin. Accordingly, the percentage of RL95-2-ARTN at the G0–G1 and S phases were higher when compared with RL95-2-vector cells treated with doxorubicin (Figure 2A).

Figure 2.

Figure 2

Open in a new tab

Forced expression of ARTN in RL95-2 cells abrogated doxorubicin-induced G2-M phase arrest and paclitaxel-induced apoptosis. (A) RL95-2-vector and RL95-2-ARTN cells were treated for 72 hours with doxorubicin (DOX) of the indicated dose and determination of cell cycle profile by FACS analysis. The cell counts versus PI staining are shown. (B) RL95-2-vector and RL95-2-ARTN cells were treated for 72 hours with paclitaxel (PTX) of the indicated dose and determination of cell cycle profile by FACS analysis. The cell counts versus PI staining are shown. (C) Confocal microscopic visualization of F-actin network after treatment with the indicated dose of paclitaxel (PTX). Red arrows indicate the positive nuclear F-actin ring formation. (D) Effect of forced expression of ARTN in RL95-2 cells on paclitaxel-induced nuclear ring formation. Cells were treated with the indicated dose of PTX and visualized for nuclear ring formation. The figure represents the average percentage of cells with positive nuclear ring versus total number of cells counted. Columns are mean of triplicate experiments; bars, ±SD. (E) Effect of forced expression of ARTN in RL95-2 cells on paclitaxel-induced apoptosis. Cells were treated with the indicated dose of paclitaxel for 72 hours and then assayed using Annexin V-FLUOS staining kit. Points are mean of triplicate experiments; bars, ±SD. **P < .001, *P < .05.

We also performed FACS analysis to determine the cell cycle profiles of RL95-2-vector and RL95-2-ARTN cells treated with paclitaxel. Paclitaxel induced a major shift from the G0–G1 phase to the S phase; the S-phase peak was elevated at 25 nM paclitaxel and increased with increasing concentration of paclitaxel to 200 nM, with 65% of the cells arrested in the S phase. Paclitaxel treatment of RL95-2-vector cells induced apoptosis at 200 nM, with a 26% apoptotic rate. Paclitaxel treatment of RL95-2-ARTN cells also produced a shift from the G0-G1 phase to the S phase and induced apoptosis (Figure 2B). However, paclitaxel treatment of RL95-2-ARTN cells produced a less pronounced shift to the S phase (57% vs 65%) and apoptosis (18% vs 26%) compared with that of RL95-2-vector cells, indicative of a protective effect of ARTN in paclitaxel-induced apoptosis (Figure 2B).

Forced Expression of ARTN in EC Cells Diminished the Paclitaxel-Induced Nuclear F-actin Ring Formation and Apoptosis

Paclitaxel interferes with microtubule depolymerization and results in stabilized microtubule structures, which disrupt the organization of F-actin and lead to apoptosis [27,28]. Development of circumferential actin bundles (also known as nuclear actin ring formation) has been observed in mammary carcinoma cells after treatment with paclitaxel [29]. We therefore visualized the F-actin network in RL95-2-vector and RL95-2-ARTN cells after exposure to paclitaxel (Figure 2C). In RL95-2-vector control (vehicle-treated) cells, F-actin was observed as fine, diffuse, and evenly distributed cytoplasmic filaments. RL95-2-vector cells treated with paclitaxel exhibited relatively thicker circumferential F-actin bundles organized around nuclei; the number of cells with nuclear ring was increased at 25 nM paclitaxel and continued to increase with increasing concentration of paclitaxel to 200 nM (Figure 2D). Accordingly, the number of apoptotic cells also increased with paclitaxel treatment of RL95-2-vector cells (Figure 2E). RL95-2-ARTN control (vehicle-treated) cells exhibited actin extensions at the rim of the cell and diffuse F-actin stress fibers throughout the cell cytoplasm (Figure 2C). RL95-2-ARTN cells treated with various doses of paclitaxel exhibited a significantly lower number of cells with nuclear ring compared with RL95-2-vector cells (Figure 2D). RL95-2-ARTN cells treated with paclitaxel also exhibited significantly less apoptosis than RL95-2-vector cells (Figure 2E). We also observed similar results with forced expression of ARTN in AN3 cells after exposure to paclitaxel (Figure W2).

ARTN Specifically Regulated Expression of CD24

We performed microarray analyses of RL95-2 cells with forced expression of ARTN compared with vector control cells to identify ARTN-regulated genes in EC and therefore potential mediators of ARTN-stimulated chemoresistance. Therein, we observed that the expression of CD24 messenger RNA (mRNA) was increased by approximately twofold by forced expression of ARTN in RL95-2 cells (data not shown). To verify this result, we performed luciferase assays using the 5′ 1.2-kb region of the CD24 gene promoter [19]. The promoter activity of CD24 was significantly increased by forced expression of ARTN in RL95-2 cells (Figure 3A). Furthermore, CD24 promoter activity was suppressed by siRNA-mediated depletion of ARTN expression in RL95-2 cells (Figure 3A). Forced expression of ARTN in RL95-2 cells increased levels of CD24 mRNA, as demonstrated using qPCR analyses (Figure 3B). Furthermore, we confirmed by FACS analysis a higher expression of CD24 surface protein consequent to the forced expression of ARTN in RL95-2 cells (Figure 3C). Depletion of ARTN in RL95-2 cells also decreased CD24 mRNA (Figure 3B). Moreover, a lower expression of CD24 surface protein was observed consequent to the depletion of ARTN in RL95-2 cells (Figure 3C). Similar results for the regulation of CD24 by ARTN were observed in AN3 cells (Figure 3). We also determined CD24 expression by immunohistochemistry (IHC) in tumors generated in vivo from RL95-2-vector and RL95-2-ARTN cells [2]. RL95-2-ARTN cell-generated tumors exhibited increased CD24 expression compared with RL95-2-vector cell-generated tumors (Figure 3D).

Figure 3.

Figure 3

Open in a new tab

ARTN regulates CD24 expression in EC cells. (A) Effect of ARTN on the activity of the CD24 promoter in RL95-2 and AN3 cell lines. The luciferase assay was peformed as described in Materials and Methods. (B) Effect of ARTN on the mRNA levels of CD24 in RL95-2 and AN3 cell lines. QPCR analysis of CD24 in RL95-2-vector/RL95-2-ARTN, RL95-2-sivector/RL95-2-siARTN, AN3-vector/AN3-ARTN, and AN3-sivector/AN3-siARTN cells. (C) Cells were cultured in serum replete medium for 72 hours. Flow cytometric analysis was performed with phycoerythrin (PE) antihuman CD24 antibody using the BD FACSAria Cell Sorter. Values in the figure represent the geometric mean fluorescence of CD24 detected. Black line represents CD24 expression in control cells, and red line represents CD24 expression with forced or depleted expression of ARTN in RL95-2 and AN3 cell lines, respectively. (D) Representative images of IHC staining for CD24 expression in RL95-2-vector and RL95-2-ARTN cell-derived xenograft tumors. (E) Representative images of IHC staining for ARTN and CD24 expression in EC patient tissues. (F) Graphical representation of the Spearman correlation of CD24 expression with ARTN expression in EC patients. Columns are mean of triplicate experiments; bars, ±SD. **P < .001, *P < .05.

We next determined whether ARTN expression correlated with CD24 expression in EC. Hence, we examined ARTN and CD24 expression by IHC staining in serial histological sections of EC from 45 patients (Figure 3E). In tumors with high ARTN expression (scores 2 and 3) [2], 72% also highly expressed CD24, whereas in tumors with low ARTN expression (scores 0 and 1), only 38% expressed either low or high levels of CD24 (Spearman coefficient, ρ = 0.36, P < .016; Figure 3F). Hence, CD24 expression in EC is partially correlated with ARTN expression.

Forced Expression of CD24 in EC Cells Stimulated Cellular Proliferation, Oncogenicity, and Invasion

To determine the functional consequences of CD24 expression in EC cells, we stably transfected RL95-2 and AN3 cells with an expression plasmid encoding CD24 cDNA (designated as RL95-2-CD24 and AN3-CD24) or with the respective empty plasmids as control (designated as RL95-2-vector and AN3-vector). We also depleted the expression of CD24 in RL95-2 and AN3 cells using shRNA (designated as RL95-2-shCD24 and AN3-shCD24). Control cells were transfected with respective scrambled controls (designated as RL95-2-vector and AN3-vector). The expression of CD24 mRNA and surface protein was demonstrated by qPCR and FACS analysis, respectively (Figure 4, A and B).

Figure 4.

Figure 4

Open in a new tab

Forced expression of CD24 stimulates cellular proliferation, oncogenicity, and invasion of EC cells. RL95-2 and AN3 cells were stably transfected with an expression vector containing CD24 cDNA (designated as RL95-2-CD24 and AN3-CD24). Control cells lines were transfected with the vector (designated as RL95-2-vector and AN3-vector). CD24 expression was depleted in RL95-2 and AN3 cell line using shRNA (designated as RL95-2-shCD24 and AN3-shCD24). Control cells lines were transfected with the scrambled shRNA control vector (designated as RL95-2-shvector and AN3-shvector). (A) QPCR analysis of CD24 expression in RL95-2-vector/RL95-2-CD24, RL95-2-shvector/RL95-2-shCD24, AN3-vector/AN3-CD24, and AN3-shvector/AN3-shCD24 cells. (B) Cells were cultured in serum replete medium for 72 hours. To determine CD24 cell surface expression, flow cytometry analysis was performed with PE antihuman CD24 antibody by use of BD FACSAria Cell Sorter. Values in the figure represent the geometric mean fluorescence of CD24 detected. (C) Total cell number of RL95-2-vector and RL95-2-CD24 cell lines were assessed in serum replete medium. (D) Anchorage-independent growth of RL95-2-vector and RL95-2-CD24 cells was evaluated by soft agar colony formation. (E) Cell invasion of RL95-2-vector and RL95-2-CD24 cells was determined by transwell chamber assay. (F) Three-dimensional growth of RL95-2-vector and RL95-2-CD24 cells cultured in growth factor-reduced Matrigel with serum replete medium. Graph represents the number of colonies produced by RL95-2-vector and RL95-2-CD24 cells at the indicated period. (G) Foci formation of RL95-2-vector and RL95-2-CD24 cells in serum replete medium. Columns are mean of triplicate experiments; bars, ±SD. **P < .001, *P < .05.

In monolayer culture, total cell number of RL95-2-CD24 increased significantly more than the cell number of RL95-2-vector during a period of 14 days in serum replete medium (Figure 4C). In addition, RL95-2-CD24 cells exhibited significantly enhanced cell viability (RL95-2-vector 100% ± 2.1% vs RL95-2-CD24 172.7% ± 1.6%, P < .001) compared with RL95-2-vector cells. RL95-2-CD24 cells exhibited significantly increased cell cycle S-phase entry as determined by 5-bromo-2-deoxyuridine incorporation (43% ± 5.1% vs 30% ± 4.7%, P < .001) compared with RL95-2-vector cells. RL95-2-CD24 cells also exhibited diminished apoptotic cell death compared with RL95-2-vector cells (12.8% ± 0.8% vs 19.8% ± 3%, P < .05) as a consequence of serum deprivation. In addition, we used qPCR analysis to examine the effect of forced expression of CD24 on the relative expression levels of genes involved in cell cycle progression and cell survival (Table 2). Forced expression of CD24 increased the mRNA levels of CCND1, Cyclin E1, CDC25A, and CDK2 required for cell cycle progression and of BCL-2 and BCL2L1, antiapoptotic genes required for cell survival. In addition, decreased mRNA levels of proapoptotic genes including TP53 and BAD were observed in RL95-2 cells consequent to the forced expression of CD24. Forced expression of CD24 in RL95-2 cells also increased levels of TERT mRNA indicative of decreased cellular senescence and enhanced cell survival [30].

Table 2.

qPCR Analysis of the Effect of Forced Expression of CD24 on mRNA Levels of Several Key Genes Functionally Involved in Oncogenicity and Neoplastic Progression of EC Cells*.

Functional Gene Grouping Gene Fold Change† P
Cell cycle control and DNA damage repair CCND1 3.92 1.36e-06
ATM 2.14 2.71e-02
BRCA1 2.26 6.49e-06
Cyclin E1 1.90 2.19e-05
CDC25A 3.09 2.10e-03
CDK2 3.13 2.94e-02
CDKN1A 1.75 1.86e-02
CDKN2A 1.53 2.81e-02
CHEK2 1.72 6.10e-04
E2F1 2.48 4.36e-04
CDKN1B -1.31 9.19e-04
RB1 2.03 2.81e-05
Apoptosis and cell senescence S100A4 1.79 4.46e-05
TP53 -1.84 1.03e-04
APAF1 2.20 3.16e-04
BAD -3.20 5.91e-03
BAX 2.19 1.28e-04
BCL2 3.64 2.35e-05
BCL2L1 1.86 6.11e-03
CASP7 2.11 1.44e-06
HTATIP2 -7.28 1.43e-02
TERT 2.89 3.39e-02
TNFRSF1A 2.91 1.98e-03
TNFRSF10B 2.06 2.57e-04
Signal transduction molecules and transcription factors AKT1 2.27 6.11e-06
ERBB2 3.61 4.86e-03
ETS2 2.16 2.80e-05
FOS 1.86 2.54e-02
JUN 2.03 4.43e-04
MAP2K1 2.05 1.19e-02
MYC 2.19 8.43e-03
NFKB1 2.34 1.16e-04
NFKBIA 2.04 1.60e-04
PIK3R1 2.77 6.79e-03
RAF1 2.75 5.59e-03
SNCG 1.81 1.24e-05
TWIST1 2.46 2.31e-02
Invasion and metastasis MET 2.06 7.36e-06
MTA1 2.38 2.50e-02
MTA2 2.28 4.26e-03
NME1 1.69 4.56e-02
PLAU -2.83 8.21e-04
PLAUR 1.85 2.41e-03
SERPINB5 2.08 1.59e-02
Cell remodeling VIM 35.40 1.57e-02
OCLN -2.85 4.23e-04
FN1 1.93 2.17e-02
Open in a new tab

Positive and negative fold changes indicate an increase or a decrease in mRNA levels respectively. A list of the genes analyzed by real-time PCR and the primer sequences used are previously described [2].

*

Results are presented as a fold change in mRNA levels in RL95-2-CD24 cells relative to RL95-2-vector cells.

†

Fold change values are representative of three independent experiments of mRNA samples.

Forced expression of CD24 in RL95-2 cells significantly enhanced anchorage-independent growth as indicated by colony formation in soft agar (Figure 4D) and foci formation (Figure 4G). Furthermore, qPCR analysis comparing RL95-2-vector and RL95-2-CD24 cell lines demonstrated increased mRNA levels of various signal transduction molecules and transcription factors (Table 2). Of note, mRNA levels for AKT1, JUN, ETS2, ERBB2, TWIST1, and MYC, previously demonstrated to be associated with increased oncogenicity of EC cells [2,31–33], were increased by forced expression of CD24 in RL95-2 cells. Three-dimensional structures were also generated by plating RL95-2-vector and RL95-2-CD24 cells as single cells in Matrigel. RL95-2-vector colonies appeared well defined with regular spheroid morphology. In contrast, RL95-2-CD24 colonies exhibited markedly increased sizes of the individual colony, and colonies were of irregular morphology (Figure 4F). Moreover, RL95-2-CD24 cells produced a significantly increased total number of colonies compared with RL95-2-vector cells (Figure 4F).

RL95-2 cells with forced expression of CD24 exhibited significantly increased invasion through Matrigel (Figure 4E). In addition, the mRNA levels of invasion- and metastasis-promoting genes, MET, PLAUR, and PI5/SERPINB5, were significantly increased in RL95-2-CD24 cells relative to RL95-2-vector cells (Table 2). Also, in accord with themorphological changes observed with forced expression of CD24, qPCR analysis demonstrated markedly increased mRNA levels of the mesenchymal marker, vimentin, in RL95-2-CD24 cells relative to RL95-2-vector cells (Table 2). Thus, forced expression of CD24 in EC cells produced an aggressive cellular phenotype with increased invasion.

In addition, we examined the effect of forced expression of CD24 on a second EC cell line, AN3. Forced expression of CD24 in AN3 cells was verified by qPCR and FACS analysis (Figure 4, A and B). Forced expression of CD24 in AN3 cells resulted in increased cell viability, anchorage-independent growth, and cell invasion (Figure W3). In addition, similar directional changes in the expression of genes involved in increased oncogenicity were observed after forced expression of CD24 in AN3 cells (Table W1).

Forced Expression of CD24 in EC Cells Decreased Sensitivity to Doxorubicin and Paclitaxel

EC cells with forced expression of CD24 exhibited increased viability compared with vector control cells, and on exposure to either doxorubicin or paclitaxel, both RL95-2-vector and RL95-2-CD24 cells exhibited decreased viability. However, RL95-2-CD24 cells exhibited enhanced cell viability when compared with RL95-2-vector cells on exposure to doxorubicin and paclitaxel (Figure 5A). Accordingly, shRNA-mediated depletion of CD24 in RL95-2 cells drastically decreased cell viability when compared with respective control cells on exposure to doxorubicin and paclitaxel (Figure 5B). Furthermore, forced expression of CD24 in RL95-2 cells significantly enhanced their capacity for anchorage-independent growth when compared with vector control cells, and on exposure to either doxorubicin or paclitaxel, both RL95-2-vector and RL95-2-CD24 cells decreased their capacity for anchorage-independent growth (Figure 5C). However, anchorage-independent growth of RL95-2-CD24 cells was inhibited significantly less compared with RL95-2-vector cells on exposure to doxorubicin and paclitaxel (Figure 5C). Depletion of CD24 in RL95-2 cells drastically decreased their capacity for anchorage-independent growth on exposure to doxorubicin and paclitaxel when compared with respective control cells (Figure 5D). RL95-2-CD24 cells exhibited increased invasion compared with RL95-2-vector cells, and on exposure to either doxorubicin or paclitaxel, both RL95-2-vector and RL95-2-CD24 cells exhibited decreased invasion. However, RL95-2-CD24 cells exhibited no change in invasive capacity on exposure to doxorubicin, whereas RL95-2-CD24 cell invasion decreased significantly less on exposure to paclitaxel when compared with RL95-2-vector cells (Figure 5E). Depleted expression of CD24 in RL95-2 cells resulted in further significant decrease in invasion on exposure to these drugs individually when compared with respective control cells (Figure 5F).

Figure 5.

Figure 5

Open in a new tab

Forced expression of CD24 in EC cells decreased sensitivity to doxorubicin and paclitaxel. (A and B) Cell viability of RL95-2 cells with forced or depleted expression of CD24 on exposure to doxorubicin and paclitaxel was evaluated using the MTT assay. (C and D) Anchorage-independent growth of RL95-2 cells with forced or depleted expression of CD24 on exposure to doxorubicin and paclitaxel was evaluated using soft agar colony formation. (E and F) Cellular invasion of RL95-2 cells with forced or depleted expression of CD24 on exposure to doxorubicin and paclitaxel was evaluated using transwell assay. Columns are mean of triplicate experiments; bars, ±SD. **P < .001, *P < .05.

In addition, forced or depleted expression of CD24 in AN3 cells exhibited similar directional effects on cell viability, anchorage-independent growth, and invasion on exposure to doxorubicin and paclitaxel when compared with vector-transfected control cells (Figure W3).

CD24 Expression Is Essential for ARTN-Stimulated Resistance to Doxorubicin and Paclitaxel in EC Cells

We next evaluated the effect of doxorubicin and paclitaxel on the anchorage-independent capacity and invasion of RL95-2-vector/RL95-2-ARTN and RL95-2-sivector/RL95-2-siARTN cells with depleted expression of CD24. As previously described, RL95-2-ARTN cells exhibited significantly increased capacity for anchorage-independent growth and invasion compared with RL95-2-vector cells. Depletion of CD24 reduced both basal and ARTN-stimulated anchorage-independent growth and invasion (Figure 6, A and C). Depletion of CD24 also eliminated or largely abrogated the stimulatory effect of ARTN on anchorage-independent growth and invasion in the presence of doxorubicin and paclitaxel. Combined depletion of ARTN and CD24 expression in RL95-2 cells drastically decreased the capacity for anchorage-independent growth and invasion on exposure to doxorubicin and paclitaxel when compared with their respective control cells or individual depletion of either ARTN or CD24 alone (Figure 6, B and D). In addition, AN3-vector/AN3-ARTN cells with depleted expression of CD24 exhibited similar directional effects on anchorage-independent growth and invasion on exposure to doxorubicin and paclitaxel when compared with vector-transfected control cells (Figure W4). Thus, depleted expression of CD24 in EC cells abolished ARTN-stimulated resistance to doxorubicin and paclitaxel.

Figure 6.

Figure 6

Open in a new tab

CD24 expression mediates ARTN-induced resistance to doxorubicin and paclitaxel in EC cells. (A and B) Anchorage-independent growth of forced or depleted expression of ARTN with RL95-2 cells with depleted expression of CD24 on exposure to doxorubicin and paclitaxel was evaluated using soft agar colony formation. (C and D) Cellular invasion of forced or depleted expression of ARTN with RL95-2 cells with depleted expression of CD24 on exposure to doxorubicin and paclitaxel was evaluated using transwell assay. Columns are mean of triplicate experiments; bars, ±SD. **P < .001, *P < .05.

Discussion

Advanced and recurrent EC present a difficult therapeutic challenge for clinicians. The mainstay treatment of recurrent and metastatic EC remains systemic therapy in the form of hormonal therapy (progestins, tamoxifen combined with progestins, and aromatase inhibitors) and/or cytotoxic chemotherapy [16]. Unfortunately, new approaches for the treatment of patients with advanced and recurrent EC have been slow to materialize. Treatment with single-agent doxorubicin or paclitaxel results in median overall survival intervals of less than 10 months [1,16]. Even when given as combined therapy, median overall survival after administration of doxorubicin and paclitaxel is marginally enhanced to 13.6 months [34]. In this report, we observed that functional inhibition of ARTN with antibody, when combined with doxorubicin or paclitaxel, resulted in enhanced reductions in oncogenicity and invasiveness of EC cell lines. Therefore, an inhibitor such as a mAb targeting ARTN may be a viable adjuvant therapy in EC.

Herein, we demonstrated that CD24 enhanced the oncogenicity and invasion of EC cells. Elevated levels of CD24 have also been reported to be associated with the progression of EC and indicate a worse prognosis [5,35]. Several studies have also reported the association of CD24 expression with a more aggressive phenotype and poor prognosis in other types of carcinomas, such as mammary, prostate, ovarian, and pancreatic carcinomas [6–9,36]. Transient depletion of CD24 expression or mAb inhibition of CD24 resulted in reduced oncogenicity and xenograft growth of various human carcinoma cell lines [14,15]. CD24 therefore exerts similar functional effects in EC and other carcinoma cells as ARTN [2,4]. It should be noted, however, that ARTNis amore potent promoter of oncogenicity and invasion than CD24, at least in the cell lines used herein. ARTN, therefore, will presumably use other CD24-independent pathways to exert its oncogenic and invasive effects in EC cells.

Herein, we demonstrated that CD24 expression mediated ARTN-induced resistance to doxorubicin and paclitaxel in EC cells. The role of CD24 in chemosensitivity was first suggested when high expression of CD24 was reported to be associated with tamoxifen-resistant ductal mammary carcinoma patients [37]. Also, higher levels of CD24 surface protein were observed in a Hoechst 33342 side population of stem cell-like carcinoma cells from NXS2 murine neuroblastoma cells, which exhibit increased resistance to doxorubicin [38]. In human pancreatic carcinoma stem cells, gemcitabine-resistant cells express increased CD24 surface protein along with CD44 and epithelial-specific antigen, the increased expression of which is associated with the carcinoma stem cell population [39]. Furthermore, primary pancreatic carcinoma xenografts treated with ionizing radiation and the chemotherapeutic agent gemcitabine resulted in the enrichment of the CD44-, CD24-, and epithelial-specific antigen-positive cell population [39]. In this regard, it is interesting that ARTN has recently been identified as an embryonic stem cell factor [40]. Gene expression profiles of aggressive carcinoma correlate with gene expression profiles of embryonic stem cells [41], and ARTN may therefore influence chemosensitivity of EC cells by modulating cancer stem cell activity. In contrast, mammary carcinoma stem cells with low or negative expression of CD24 are associated with increased resistance of cells to gemcitabine, paclitaxel, and 5-fluorouracil [42], suggestive of CD24 tissue specificity. In any case, we have demonstrated that CD24mediates ARTN-stimulated resistance to doxorubicin and paclitaxel in EC cells. It is possible that CD24 is an intermediate in ARTN-stimulated gene expression required for the effects of ARTN in EC described herein. Indeed, both ARTN [2] and CD24 stimulated the expression of genes such as CCND1, BCL2, AKT1, TWIST1, and VIM and decreased BAD and PLAU mRNA in RL95-2 cells. It remains to be determined whether ARTN stimulates other oncogenic pathways in EC independent of CD24.

In summary, functional inhibition of ARTN in EC cells increased the sensitivity to doxorubicin and paclitaxel. Therefore, development of function-inhibiting antibodies to target ARTN is a viable adjuvant therapeutic approach to improve the response of EC to specific chemotherapeutic agents.

Supplementary Material

Supplementary Figures and Tables
tlo0304_0218SD1.pdf (1.1MB, pdf)

Footnotes

1

This work was supported by the Breast Cancer Research Trust and by the Foundation of Research Science and Technology, the Hundred-Talent Scheme of Chinese Academy of Sciences, the National Natural Science Foundation of China (2007CB914801 and 2007CB914503), and the National Basic Research Program of China (30571030). Y.J., M.S., P.-X.Q., A.B., M.D.M., and Z.-S.W. have nothing to disclose. V.P., J.K., D.-X.L., and P.E.L. have equity interests in Saratan Therapeutics Ltd. D.-X.L. and P.E.L. are inventors on PCT application PCT/NZ2008/000152 and US provisional applications 61/234902. P.E.L. is an inventor on US provisional application 61/252513.T.Z. and P.E.L. are consultants of Saratan Therapeutics Ltd.

2

This article refers to supplementary materials, which are designated by Figures W1 to W4 and Table W1 and are available online at www.transonc.com.

References

  • 1.Ray M, Fleming G. Management of advanced-stage and recurrent endometrial cancer. Semin Oncol. 2009;36:45–154. doi: 10.1053/j.seminoncol.2008.12.006. [DOI] [PubMed] [Google Scholar]
  • 2.Pandey V, Qian PX, Kang J, Perry JK, Mitchell MD, Yin Z, Wu ZS, Liu DX, Zhu T, Lobie PE. Artemin stimulates oncogenicity and invasiveness of endometrial carcinoma. Endocrinology. 2010;151:909–920. doi: 10.1210/en.2009-0979. [DOI] [PubMed] [Google Scholar]
  • 3.Ceyhan GO, Giese NA, Erkan M, Kerscher AG, Wente MN, Giese T, Buchler MW, Friess H. The neurotrophic factor artemin promotes pancreatic cancer invasion. Ann Surg. 2006;244:274–281. doi: 10.1097/01.sla.0000217642.68697.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Kang J, Perry JK, Pandey V, Fielder GC, Mei B, Qian PX, Wu ZS, Zhu T, Liu DX, Lobie PE. Artemin is oncogenic for human mammary carcinoma cells. Oncogene. 2009;28:2034–2045. doi: 10.1038/onc.2009.66. [DOI] [PubMed] [Google Scholar]
  • 5.Kim KH, Choi JS, Kim JM, Choi YL, Shin YK, Lee HC, Seong IO, Kim BK, Chae SW, Kim SH. Enhanced CD24 expression in endometrial carcinoma and its expression pattern in normal and hyperplastic endometrium. Histol Histopathol. 2009;24:309–316. doi: 10.14670/HH-24.309. [DOI] [PubMed] [Google Scholar]
  • 6.Kristiansen G, Sammar M, Altevogt P. Tumour biological aspects of CD24, a mucin-like adhesion molecule. J Mol Histol. 2004;35:255–262. doi: 10.1023/b:hijo.0000032357.16261.c5. [DOI] [PubMed] [Google Scholar]
  • 7.Kristiansen G, Schluns K, Yongwei Y, Denkert C, Dietel M, Petersen I. CD24 is an independent prognostic marker of survival in nonsmall cell lung cancer patients. Br J Cancer. 2003;88:231–236. doi: 10.1038/sj.bjc.6600702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Kristiansen G, Winzer KJ, Mayordomo E, Bellach J, Schluns K, Denkert C, Dahl E, Pilarsky C, Altevogt P, Guski H, et al. CD24 expression is a new prognostic marker in breast cancer. Clin Cancer Res. 2003;9:4906–4913. [PubMed] [Google Scholar]
  • 9.Kristiansen G, Denkert C, Schluns K, Dahl E, Pilarsky C, Hauptmann S. CD24 is expressed in ovarian cancer and is a new independent prognostic marker of patient survival. Am J Pathol. 2002;161:1215–1221. doi: 10.1016/S0002-9440(10)64398-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Boon K, Edwards JB, Siu IM, Olschner D, Eberhart CG, Marra MA, Strausberg RL, Riggins GJ. Comparison of medulloblastoma and normal neural transcriptomes identifies a restricted set of activated genes. Oncogene. 2003;22:7687–7694. doi: 10.1038/sj.onc.1207043. [DOI] [PubMed] [Google Scholar]
  • 11.Kohlgraf KG, Gawron AJ, Higashi M, Meza JL, Burdick MD, Kitajima S, Kelly DL, Caffrey TC, Hollingsworth MA. Contribution of the MUC1 tandem repeat and cytoplasmic tail to invasive and metastatic properties of a pancreatic cancer cell line. Cancer Res. 2003;63:5011–5020. [PubMed] [Google Scholar]
  • 12.Nestl A, Von Stein OD, Zatloukal K, Thies WG, Herrlich P, Hofmann M, Sleeman JP. Gene expression patterns associated with the metastatic phenotype in rodent and human tumors. Cancer Res. 2001;61:1569–1577. [PubMed] [Google Scholar]
  • 13.Baumann P, Cremers N, Kroese F, Orend G, Chiquet-Ehrismann R, Uede T, Yagita H, Sleeman JP. CD24 expression causes the acquisition of multiple cellular properties associated with tumor growth and metastasis. Cancer Res. 2005;65:10783–10793. doi: 10.1158/0008-5472.CAN-05-0619. [DOI] [PubMed] [Google Scholar]
  • 14.Smith SC, Oxford G, Wu Z, Nitz MD, Conaway M, Frierson HF, Hampton G, Theodorescu D. The metastasis-associated gene CD24 is regulated by Ral GTPase and is a mediator of cell proliferation and survival in human cancer. Cancer Res. 2006;66:1917–1922. doi: 10.1158/0008-5472.CAN-05-3855. [DOI] [PubMed] [Google Scholar]
  • 15.Sagiv E, Starr A, Rozovski U, Khosravi R, Altevogt P, Wang T, Arber N. Targeting CD24 for treatment of colorectal and pancreatic cancer by monoclonal antibodies or small interfering RNA. Cancer Res. 2008;68:2803–2812. doi: 10.1158/0008-5472.CAN-07-6463. [DOI] [PubMed] [Google Scholar]
  • 16.Temkin SM, Fleming G. Current treatment of metastatic endometrial cancer. Cancer Control. 2009;16:38–45. doi: 10.1177/107327480901600106. [DOI] [PubMed] [Google Scholar]
  • 17.Pandey V, Perry JK, Mohankumar KM, Kong XJ, Liu SM, Wu ZS, Mitchell MD, Zhu T, Lobie PE. Autocrine human growth hormone stimulates oncogenicity of endometrial carcinoma cells. Endocrinology. 2008;149:3909–3919. doi: 10.1210/en.2008-0286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Fukushima T, Tezuka T, Shimomura T, Nakano S, Kataoka H. Silencing of insulin-like growth factor-binding protein-2 in human glioblastoma cells reduces both invasiveness and expression of progression-associated gene CD24. J Biol Chem. 2007;282:18634–18644. doi: 10.1074/jbc.M609567200. [DOI] [PubMed] [Google Scholar]
  • 19.Shulewitz M, Soloviev I, Wu T, Koeppen H, Polakis P, Sakanaka C. Repressor roles for TCF-4 and Sfrp1 in Wnt signaling in breast cancer. Oncogene. 2006;25:4361–4369. doi: 10.1038/sj.onc.1209470. [DOI] [PubMed] [Google Scholar]
  • 20.Liu DX, Lobie PE. Transcriptional activation of p53 by Pitx1. Cell Death Differ. 2007;14:1893–1907. doi: 10.1038/sj.cdd.4402209. [DOI] [PubMed] [Google Scholar]
  • 21.Rahnama F, Shafiei F, Gluckman PD, Mitchell MD, Lobie PE. Epigenetic regulation of human trophoblastic cell migration and invasion. Endocrinology. 2006;147:5275–5283. doi: 10.1210/en.2006-0288. [DOI] [PubMed] [Google Scholar]
  • 22.Hao J, Song X, Song B, Liu Y, Wei L, Wang X, Yu J. Effects of lentivirus-mediated HIF-1α knockdown on hypoxia-related cisplatin resistance and their dependence on p53 status in fibrosarcoma cells. Cancer Gene Ther. 2008;15:449–455. doi: 10.1038/cgt.2008.4. [DOI] [PubMed] [Google Scholar]
  • 23.Li S, Szymborski A, Miron MJ, Marcellus R, Binda O, Lavoie JN, Branton PE. The adenovirus E4orf4 protein induces growth arrest and mitotic catastrophe in H1299 human lung carcinoma cells. Oncogene. 2009;28:390–400. doi: 10.1038/onc.2008.393. [DOI] [PubMed] [Google Scholar]
  • 24.Kajiyama H, Shibata K, Terauchi M, Yamashita M, Ino K, Nawa A, Kikkawa F. Chemoresistance to paclitaxel induces epithelial-mesenchymal transition and enhances metastatic potential for epithelial ovarian carcinoma cells. Int J Oncol. 2007;31:277–283. [PubMed] [Google Scholar]
  • 25.Pichot CS, Hartig SM, Xia L, Arvanitis C, Monisvais D, Lee FY, Frost JA, Corey SJ. Dasatinib synergizes with doxorubicin to block growth, migration, and invasion of breast cancer cells. Br J Cancer. 2009;101:38–47. doi: 10.1038/sj.bjc.6605101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Engel JB, Schonhals T, Weidler C, Hausler S, Krockenberger M, Rieger L, Dietl J, Wischhusen J, Honig A. Tubulin inhibitor AEZS 112 inhibits the growth of experimental human ovarian and endometrial cancers irrespective of caspase inhibition. Oncol Rep. 2009;22:361–367. [PubMed] [Google Scholar]
  • 27.Etienne-Manneville S, Manneville JB, Adamson P, Wilbourn B, Greenwood J, Couraud PO. ICAM-1-coupled cytoskeletal rearrangements and transendothelial lymphocyte migration involve intracellular calcium signaling in brain endothelial cell lines. J Immunol. 2000;165:3375–3383. doi: 10.4049/jimmunol.165.6.3375. [DOI] [PubMed] [Google Scholar]
  • 28.Bijman MN, van Nieuw Amerongen GP, Laurens N, van Hinsbergh VW, Boven E. Microtubule-targeting agents inhibit angiogenesis at subtoxic concentrations, a process associated with inhibition of Rac1 and Cdc42 activity and changes in the endothelial cytoskeleton. Mol Cancer Ther. 2006;5:2348–2357. doi: 10.1158/1535-7163.MCT-06-0242. [DOI] [PubMed] [Google Scholar]
  • 29.Rosenblum MD, Shivers RR. “Rings“ of F-actin form around the nucleus in cultured human MCF7 adenocarcinoma cells upon exposure to both taxol and taxotere. Comp Biochem Physiol C Toxicol Pharmacol. 2000;125:121–131. doi: 10.1016/s0742-8413(99)00101-2. [DOI] [PubMed] [Google Scholar]
  • 30.Lee J, Sung YH, Cheong C, Choi YS, Jeon HK, Sun W, Hahn WC, Ishikawa F, Lee HW. TERT promotes cellular and organismal survival independently of telomerase activity. Oncogene. 2008;27:3754–3760. doi: 10.1038/sj.onc.1211037. [DOI] [PubMed] [Google Scholar]
  • 31.Chen ML, Xu PZ, Peng XD, Chen WS, Guzman G, Yang X, Di Cristofano A, Pandolfi PP, Hay N. The deficiency of Akt1 is sufficient to suppress tumor development in Pten+/- mice. Genes Dev. 2006;20:1569–1574. doi: 10.1101/gad.1395006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Dai D, Litman ES, Schonteich E, Leslie KK. Progesterone regulation of activating protein-1 transcriptional activity: a possible mechanism of progesterone inhibition of endometrial cancer cell growth. J Steroid Biochem Mol Biol. 2003;87:123–131. doi: 10.1016/j.jsbmb.2003.08.004. [DOI] [PubMed] [Google Scholar]
  • 33.Santin AD, Bellone S, Van Stedum S, Bushen W, Palmieri M, Siegel ER, De Las Casas LE, Roman JJ, Burnett A, Pecorelli S. Amplification of c-erbB2 oncogene: a major prognostic indicator in uterine serous papillary carcinoma. Cancer. 2005;104:1391–1397. doi: 10.1002/cncr.21308. [DOI] [PubMed] [Google Scholar]
  • 34.Fleming GF, Filiaci VL, Bentley RC, Herzog T, Sorosky J, Vaccarello L, Gallion H. Phase III randomized trial of doxorubicin + cisplatin versus doxorubicin + 24-h paclitaxel + filgrastim in endometrial carcinoma: a Gynecologic Oncology Group study. Ann Oncol. 2004;15:1173–1178. doi: 10.1093/annonc/mdh316. [DOI] [PubMed] [Google Scholar]
  • 35.Karahan N, Guney M, Oral B, Kapucuoglu N, Mungan T. CD24 expression is a poor prognostic marker in endometrial carcinoma. Eur J Gynaecol Oncol. 2006;27:500–504. [PubMed] [Google Scholar]
  • 36.Kristiansen G, Pilarsky C, Pervan J, Sturzebecher B, Stephan C, Jung K, Loening S, Rosenthal A, Dietel M. CD24 expression is a significant predictor of PSA relapse and poor prognosis in low grade or organ confined prostate cancer. Prostate. 2004;58:183–192. doi: 10.1002/pros.10324. [DOI] [PubMed] [Google Scholar]
  • 37.Surowiak P, Materna V, Paluchowski P, Matkowski R, Wojnar A, Maciejczyk A, Pudelko M, Kornafel J, Dietel M, Kristiansen G, et al. CD24 expression is specific for tamoxifen-resistant ductal breast cancer cases. Anticancer Res. 2006;26:629–634. [PubMed] [Google Scholar]
  • 38.Kruger JA, Kaplan CD, Luo Y, Zhou H, Markowitz D, Xiang R, Reisfeld RA. Characterization of stem cell-like cancer cells in immune-competent mice. Blood. 2006;108:3906–3912. doi: 10.1182/blood-2006-05-024687. [DOI] [PubMed] [Google Scholar]
  • 39.Lee CJ, Dosch J, Simeone DM. Pancreatic cancer stem cells. J Clin Oncol. 2008;26:2806–2812. doi: 10.1200/JCO.2008.16.6702. [DOI] [PubMed] [Google Scholar]
  • 40.Li J, Klein C, Liang C, Rauch R, Kawamura K, Hsueh AJ. Autocrine regulation of early embryonic development by the artemin-GFRA3 (GDNF family receptor-alpha 3) signaling system in mice. FEBS Lett. 2009;583:2479–2485. doi: 10.1016/j.febslet.2009.06.050. [DOI] [PubMed] [Google Scholar]
  • 41.Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A, Weinberg RA. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet. 2008;40:499–507. doi: 10.1038/ng.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Mine T, Matsueda S, Li Y, Tokumitsu H, Gao H, Danes C, Wong KK, Wang X, Ferrone S, Ioannides CG. Breast cancer cells expressing stem cell markers CD44+CD24lo are eliminated by Numb-1 peptide-activated T cells. Cancer Immunol Immunother. 2009;58:1185–1194. doi: 10.1007/s00262-008-0623-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figures and Tables
tlo0304_0218SD1.pdf (1.1MB, pdf)

Articles from Translational Oncology are provided here courtesy of Neoplasia Press

RESOURCES