Agonist and Antagonist of Retinoic Acid Receptors Cause Similar Changes in Gene Expression and Induce Senescence-Like Growth Arrest in MCF-7 Breast Carcinoma Cells

Research Article

Agonist and Antagonist of Retinoic Acid Receptors Cause Similar Changes in Gene Expression and Induce Senescence-like Growth Arrest in MCF-7 Breast Carcinoma Cells

Yuhong Chen,1 Milos Dokmanovic,1 Wilfred D. Stein,1,2 Robert J. Ardecky,3 and Igor B. Roninson1

1Cancer Center, Ordway Research Institute, Albany, New York; 2Institute of Life Sciences, Hebrew University, Jerusalem, Israel; and 3Ligand Pharmaceuticals, Inc., San Diego, California

Abstract retinoids is most often attributed to the induction of differentia- Biological effects of retinoids are mediated via retinoic acid tion, but these compounds were also shown to stop the growth of (RA) receptors (RAR) and retinoid X receptors (RXR). The tumor cells by inducing apoptosis or accelerated senescence (1, 2). best-characterized mechanism of retinoid action is stimula- In particular, treatment of two human breast carcinoma cell lines tion of transcription from promoters containing RA response with all-trans retinoic acid (RA) or fenretinide, in vitro or in vivo, elements (RARE). Retinoids induce senescence-like growth induces a senescence-like phenotype characterized by increased h arrest in MCF-7 breast carcinoma cells; this effect is cell size and expression of senescence-associated -galactosidase h associated with the induction of several growth-inhibitory (SA- -gal; refs. 3, 4). This phenotype, as investigated in MCF-7 cells, genes. We have nowfound that these genes are induced by is associated with irreversible growth arrest and up-regulation of RAR-specific but not by RXR-specific ligands. Genome-scale several intracellular and secreted proteins with known growth- microarray analysis of gene expression was used to compare inhibitory activities. These include intracellular growth-inhibitory the effects of two pan-RAR ligands, one of which is a strong proteins, such as UBD (also known as FAT10) and putative tumor agonist of RARE-dependent transcription, whereas the other suppressor EPLIN, as well as secreted growth-inhibitory factors, induces such transcription only weakly and antagonizes the including insulin-like growth factor-binding protein 3 (IGFBP3) inducing effect of RAR agonists. Both RAR ligands, however, and an extracellular matrix component, TGFBI also known as h produced very similar effects on gene expression in MCF-7 IG-h3 (ref. 4). cells, suggesting that RARE-dependent transcription is only a Induction of gene expression by retinoids is mediated at the level minor component of retinoid-induced changes in gene of transcription through binding to dimeric transcription factors expression. The effects of RAR ligands on gene expression formed by RA receptors (RAR) and retinoid X receptors (RXR). The parallel changes associated with damage-induced senescence, best-known mechanism of action of these receptors involves their binding to RA response elements (RARE) in the promoters of andbothligandsinducedG1 arrest and the senescent phenotype in MCF-7 cells. The RAR ligands up-regulated retinoid-responsive genes. Nevertheless, retinoid receptors also many tumor-suppressive genes and down-regulated multiple affect transcription through RARE-independent mechanisms, such genes with oncogenic activities. Genes that are strongly as repression of transcription factor activator protein (AP-1; Jun/ induced by RAR ligands encode secreted bioactive proteins, Fos; ref. 5), or by modulating the interaction of Sp1 and GC-rich including several tumor-suppressing factors. In agreement DNA via ternary complex formation (6). Remarkably, a survey of with these observations, retinoid-treated MCF-7 cells inhibited Balmer and Blomhoff (7) concluded that only a minority of all the the growth of retinoid-insensitive MDA-MB-231 breast published retinoid-inducible genes are induced through the RARE- carcinoma cells in coculture. These results indicate that dependent mechanism. In the case of retinoid-treated MCF-7 cells, RARE-independent transcriptional effects of RAR ligands lead only 1 of 13 genes found to be strongly up-regulated at the onset of to senescence-like growth arrest and paracrine growth- senescence-like growth arrest contained a putative RARE sequence inhibitory activity in MCF-7 breast carcinoma cells. (Cancer in its promoter, whereas the other genes had no identifiable RARE Res 2006; 66(17): 8749-61) sites and showed a slow kinetics of retinoid response, requiring up to 3 days for maximal induction (4). Such genes may be induced Introduction either by an entirely RARE-independent mechanism or as a secondary consequence of some early RARE-dependent changes Retinoids, natural and synthetic derivatives of vitamin A, in gene expression. regulate growth, differentiation, and survival of different types of In the present study, we have used pan-RAR– and pan-RXR– normal and tumor cells. Retinoids are used in the treatment of specific agonists and antagonists to investigate the roles of promyelocytic leukemia and in chemoprevention of several retinoid receptors in the induction of growth-inhibitory genes in cancers, including breast carcinoma. The antitumor effect of MCF-7 cells. We have found that these genes are induced by the agonist of RAR (but nor RXR) and, surprisingly, by an RAR ligand that was developed as an antagonist of RARE-dependent Note: Current address for Milos Dokmanovic: Memorial Sloan-Kettering Cancer transcriptional activation. Microarray analysis of gene expression Center, Cell Biology Program, Sloan-Kettering Institute for Cancer Research, New York, showed that the agonist and the antagonist of RARE-dependent NY 10021. Requests for reprints: Igor B. Roninson, Cancer Center, Ordway Research transcription produced very similar effects on global gene Institute, 150 New Scotland Avenue, Albany, NY 12208. Phone: 518-641-6471; Fax: 518- expression in MCF-7 cells. In agreement with these effects, both 641-6305; E-mail: [email protected]. I2006 American Association for Cancer Research. RAR agonist and RAR antagonist induced senescence-like growth doi:10.1158/0008-5472.CAN-06-0581 arrest in MCF-7 cells. We have also identified numerous genes with www.aacrjournals.org 8749 Cancer Res 2006; 66: (17). September 1, 2006

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research oncogenic or tumor-suppressive properties that are affected by retinoid-treated MCF-7 cells, we have analyzed the effects of pan- RAR ligands and shown that the up-regulation of secreted tumor- RAR– and pan-RXR–specific agonists and antagonists on these cells. suppressive factors in retinoid-treated MCF-7 cells is associated The tested compounds include LGD1550, a pan-RAR agonist that with paracrine growth-inhibitory effect on retinoid-insensitive shows greater selectivity for RAR than natural retinoids (10); breast carcinoma cells. These results indicate that retinoids inhibit LGD1268 (a pan-RXR agonist); LG100815 (a pan-RAR antagonist); MCF-7 cell growth primarily through RARE-independent effects on and LG101208 (a pan-RXR antagonist). We have also used all-trans- cellular gene expression. RA, a natural RAR agonist that was originally used to define retinoid- induced senescence and changes in gene expression in MCF-7 cells Materials and Methods (3, 4). The structures of these compounds are shown in Fig. 1. In the first set of experiments, we asked how these compounds Cellular assays. MCF-7 cells were obtained from American Type Culture affect the expression of several growth-inhibitory genes previously Collection (Manassas, VA) and cultured in DMEM with 10% fetal bovine found to be strongly up-regulated under the conditions of retinoid- serum. Cells were plated at 105 per P100 (for assays requiring up to 6 days of 4 induced senescence of MCF-7 cells. The tested genes included culture) or at 10 per P60 (for longer assays), in the presence of different b concentrations of all-trans-RA (from Sigma, St. Louis, MO), pan-RAR EPLIN- , UBD (also known as FAT10), IGFBP3, and TGFBI (also b agonist LGD1550, pan-RXR agonist LGD1268, pan-RAR antagonist known as IG-h3), as well as TRIM31, a gene strongly induced by LG100815 or pan-RXR antagonist LG101208 (Ligand Pharmaceuticals, RA that has a putative RARE element in its promoter (4). RNA was Inc., San Diego, CA), or DMSO carrier. Following treatment, the number extracted from MCF-7 cells that were either untreated or treated of attached cells was measured using Coulter counter, and staining for with individual compounds or their combinations for 3 days, the SA-h-gal was carried out as described (8). For cell cycle analysis, cells were period previously shown to be required for the maximal effect of stained with propidium iodide by standard procedures. Cellular DNA RA or fenretinide (4). Gene expression was analyzed both by content was determined by flow cytometry using BD LSRII fluorescence- semiquantitative RT-PCR (not shown) and by QPCR; both assays activated cell sorting, and the percentages of cells in G1,S,orG2-M were produced essentially the same results. The outcome of a determined using ModFit software. representative set of QPCR assays is shown in Table 1. For coculture assays, MDA-MB-231 cells that were modified to express green fluorescent protein (GFP) from lentiviral vector pLL3.7 (9) were plated In agreement with the previous study, all five of the tested genes either alone (at 2 Â 105 per P100) or mixed with MCF-7 (at 105 cells from were induced by 100 nmol/L RA, the concentration found to be each cell line). In some assays, MCF-7 cells pretreated with 100 nmol/L RA sufficient for maximal induction of gene expression (4), with for 8 days were collected by trypsinization before mixing with MDA-MB- TRIM31 showing greater fold induction than the other four genes. 231. After culture in the presence or in the absence of 100 nmol/L RA, total The pan-RAR agonist LGD1550 induced all five genes as strongly as cell number was counted and the percentage of MDA-MB-231 GFP cells was RA; a 100 nmol/L concentration of this agonist was also sufficient determined by flow cytometry. for the maximal effect. In contrast, the pan-RXR agonist LGD1268 Â 5 Gene expression analysis. MCF-7 cells were plated at 5 10 per P100; showed detectable induction of only two of the five genes (TGFBI exposed to different drugs or carrier on the next day; and cells were and TRIM31), and the extent of induction was much lower than collected after 24, 48, or 72 hours treatment. Total cellular RNA was isolated observed for RA or the pan-RAR agonist. The pan-RXR antagonist using the RNeasy kit (Qiagen, Valencia, CA). Gene expression levels of IGFBP3, EPLIN, UBD, TGFBI, and TRIM31 were quantitated by reverse LG101208 showed no effect on the expression of any of the five A transcription-PCR (RT-PCR) as described (4) and by quantitative real-time genes (Table 1). When combined with 100 nmol/L RA, 10 mol/L of RT-PCR (QPCR), using an ABI 7900HT real-time PCR instrument. In QPCR, the pan-RXR antagonist inhibited the induction of only one of serial cDNA dilutions were used for primer validation and the comparative these genes (IGBP3) by RA (Table 1). These findings suggest that CT method for relative quantitation of gene expression (Applied Biosystems, retinoid-inducible expression of growth-inhibitory genes is acti- Foster City, CA) was used to determine expression levels for target genes. vated primarily through RAR rather than RXR. h-Actin was used as a normalization standard. Primer sequences will be Surprising results were obtained, however, with the pan-RAR provided upon request. For gene expression profiling, RNA samples were antagonist LG100815. This compound acts as a competitive provided to the Microarray Core Facility at the Genomics Institute of the inhibitor of RAR agonists, which binds to RAR but does not NYSDOHWadsworth Center, which carried out biotinylated target efficiently activate RARE-dependent transcription (11).4 To verify preparation (using 2 Ag RNA per assay) and hybridization with Affymetrix U133 Plus 2.0 microarrays. Data analysis was carried out using GeneSpring the antagonistic effect of LG100815 on RARE-dependent transcrip- software (Agilent, Palo Alto, CA). Gene function analysis was carried out tion, we analyzed the effects of this compound and of RAR agonists using Pathway Assist (Ariadne Genomics, Rockville, MD) and PubMed. RA and LGD1550 on the expression of firefly luciferase reporter RARE-dependent transcription was analyzed using a plasmid construct transcribed from a RARE-containing artificial promoter, DR5. that expresses firefly luciferase from a RARE-containing artificial promoter Figure 2 shows the results of DR5-luciferase transient transfection DR5 (Stratagene, La Jolla, CA). Cells were plated at 3 Â 105 per P60 24 hours assays, carried out in the presence of LGD1550, RA, and LG100815, before transient transfection. DR5 reporter plasmid (4 Ag) was mixed with alone or in pairwise combinations. One hundred nanomoles per A the SV40-driven Renilla luciferase control plasmid (0.04 g) and transfected liter concentrations of RA or LGD1550 agonists activated the using LipofectAMINE Plus (Invitrogen, Carlsbad, CA) as described by the RARE-containing promoter f50-fold, whereas 10 Amol/L LG100815 manufacturer. Three hours after transfection, cells were rinsed thrice with 4 antagonist (the concentration used in the literature for maximal PBS, trypsinized, and replated in a 12-well plate to the density of 5 Â 10 per well. Retinoid agonists or antagonists were added 48 hours later, and the effect) produced an order of magnitude weaker (4.2-fold) induction. luciferase assay was done after another 24 hours. On the other hand, the addition of LG100815 to RAR agonists RA or LGD1550 diminished the induction of transcription by the latter compounds 2.5 to 3 times (Fig. 2). These results confirm that Results LG100815 is relatively inefficient in stimulating RARE-dependent RAR agonists and an antagonist induce the expression of growth-inhibitory genes. To determine which classes of retinoid receptors mediate the induction of growth-inhibitory genes in 4 W. Lamph, personal communication.

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Figure 1. Structures of retinoid receptor ligands used in the present study. Structure representations in two-dimensional and three-dimensional formats were generated using ChemDraw Pro version 10.0 and Chem3D Ultra version 10.0, respectively.

transcription and that it antagonizes the effect of RAR agonists on agonists (Table 1). The fold induction of these genes by 1 or such transcription. 10 Amol/L LG100815 was only slightly lower than the highest In contrast to the results of the promoter assays, QPCR analysis induction by either RA or LGD1550 agonist, with the only large showed that LG100815 antagonist induced RNA expression of all difference (3- to 4-fold) observed for TRIM31. When 10 Amol/L the tested endogenous genes to a level comparable with RAR LG100815 was combined with RA, it decreased the induction of

Table 1. QPCR analysis of the effects of retinoid receptor ligands on gene expression

Compounds Fold induction

IGFBP3 EPLIN UBD TGFBI TRIM31

RA (100 nmol/L) 2.5 F 1.36 3.1 F 1.38 8.5 F 1.62 6.5 F 1.27 34.4 F 1.12 LG1550 (100 nmol/L) 4.2 F 1.34 4.2 F 1.21 9.5 F 1.02 5.1 F 1.13 42.7 F 1.04 LG1550 (1 Amol/L) 3.7 F 1.17 3.2 F 1.1 7.7 F 1.15 3.5 F 1.11 30.3 F 1.04 LG1550 (10 Amol/L) 2.8 F 1.29 4.9 F 1.55 7.3 F 1.16 5.6 F 1.02 45.0 F 1.04 LGD100815 (1 Amol/L) 2.1 F 1.3 3.8 F 1.5 4.6 F 1.14 4.3 F 1.2 7.7 F 1.62 LGD100815 (10 Amol/L) 2.0 F 1.28 3.2 F 1.61 2.8 F 1.24 4.4 F 1.09 11.7 F 1.4 RA (100 nmol/L) + LGD100815 (1 Amol/L) 2.4 F 1.35 1.7 F 1.3 8.0 F 1.22 6.4 F 1.38 30.3 F 1.12 RA (100 nmol/L) + LGD100815 (10 Amol/L) 1.9 F 1.31 0.9 F 1.63 2.2 F 1.06 3.7 F 1.28 10.0 F 1.56 LG1268 (100 nmol/L) 1.2 F 1.26 1.2 F 1.13 1.0 F 1.16 1.9 F 1.72 1.3 F 1.38 LG1268 (1 Amol/L) 0.9 F 1.23 1.0 F 1.14 0.8 F 1.31 2.2 F 1.57 1.4 F 1.56 LG1268 (10 Amol/L) 1.2 F 1.21 1.4 F 1.4 1.0 F 1.25 2.4 F 1.12 3.8 F 1.21 LGD101208 (10 Amol/L) 1.0 F 1.24 0.8 F 1.53 1.0 F 1.33 0.9 F 1.47 1.0 F 1.3 RA (100 nmol/L) + LGD101208 (10 Amol/L) 1.1 F 1.46 2.9 F 1.25 7.8 F 1.13 5.7 F 1.23 28.6 F 1.73

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the phenotype of MCF-7 cells. In most of the subsequent studies, we used 100 nmol/L LGD1550 agonist and 10 Amol/L LG100815 antagonist, the concentrations that produced the maximal effects in the above-described assays. RAR agonist and antagonist produce similar effects on genome-scale gene expression. To analyze the effects of RAR ligands on the expression of essentially all the cellular genes, we treated MCF-7 cells with RAR agonist LGD1550 (100 nmol/L) or RAR antagonist LG100815 (10 Amol/L) for 24, 48, or 72 hours, and analyzed RNA from the untreated or treated cells by hybridization with Affymetrix U133 Plus 2.0 oligonucleotide arrays, containing 56,000 probe sets representing 48,500 human transcripts. The time course of changes in gene expression observed in the microarrays (Fig. 3A) was in agreement with the results of RT- PCR assays (not shown) and with previous studies on RA- or Figure 2. Effects of RAR agonists (RA and LGD1550, 100 nmol/L each) and RAR antagonist LG100815 (10 Amol/L) on luciferase expression from DR5 fenretinide-treated MCF-7 cells (4), with most of the responsive RARE-containing promoter in MCF-7 cells. The assays were carried out as genes showing the response on day 1 with subsequent increases described in Materials and Methods, in triplicate. up to day 3. All 13 genes shown by RT-PCR to be induced by RA or fenretinide (4) also showed induction by the pan-RAR agonist gene expression by RA to the levels that were similar to those and antagonist (Fig. 3B). observed with the antagonist alone, as expected for a competitive Strikingly, the effects of the RAR agonist and the RAR antagonist effect (Table 1). Hence, LG100815, although acting as a weak were exceedingly similar. Seventy-four percent of the genes inducer and a competitive antagonist of RARE-dependent tran- showing z1.5-fold induction and 77% of the genes showing z1.5- scription, mimics the effects of RAR agonists in stimulating the fold inhibition by the antagonist were also induced or inhibited, expression of the tested growth-inhibitory genes. This surprising respectively, at least 1.3-fold by the agonist, and vice versa (77% and observation prompted us to compare the effects of the RAR agonist 69%, respectively). Figure 4A plots (on the log scale) the maximal and the RAR antagonist on the expression of other genes and on changes in gene expression (at any time point) produced by the

Figure 3. Microarray analysis of changes in gene expression in MCF-7 cells treated with RAR agonist LGD1550 or RAR antagonist LG100815, plotted using GeneSpring software. X axis, different time points of treatment with RAR ligands (0 point correspond to cells cultured for 3 days with DMSO carrier). Y axis, changes in gene expression on log scale. The groups of genes shown in (A), (G), and (H) represent GO categories, with the exclusion of genes showing raw signal intensity <10 in MCF-7 cells. The selection of the other gene groups is described in the text.

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sequences in its promoter (12). Another cytochrome P450 involved in RA metabolism, CYP26B1, showed the most selective response to the RAR agonist among all the genes (180-fold induction by the agonist, compared with only 2.4-fold induction by the antagonist). We have also looked at the effects of the agonist and the antagonist on the expression of 40 human genes, identified by Balmer and Blomhoff (13) as containing canonical and evolutionarily conserved RARE sequences in their promoters. Only 7 of these 40 genes were induced z1.5-fold in MCF-7 cells by the RAR agonist and just three genes were induced by the antagonist. RARE-containing genes induced by both ligands showed 3- to 5-fold stronger response to the agonist than to the antagonist (Fig. 3C). The most responsive gene in this group is HOXA1, induced 95-fold by the agonist but only 18-fold by the antagonist. However, when we examined promoter sequences of 10 randomly chosen genes that show preferential induction by the agonist for the presence of putative RARE sequences (this analysis was carried out using MatInspector program as previously described; ref. 4), only 1 of 10 promoters was found to contain putative RARE sequences. We also examined 28 randomly selected genes from the group identified by Balmer and Blomhoff (7) as indirectly inducible by retinoids (‘‘category 0’’) and found that these genes showed a similar response to the agonist and the antagonist (Fig. 3D and data not shown). Altogether, these results indicate that the majority of genes induced by the RAR ligands are induced through indirect, RARE-independent mechanisms. RAR agonist and antagonist induce senescence-associated changes in gene expression and cellular phenotype. MCF-7 cells treated with 100 nmol/L RA eventually (after z4 days) undergo cytostatic growth arrest, which is accompanied by the loss Figure 4. Comparison of changes in gene expression produced by RAR agonist of clonogenic potential upon removal of the drug and the and antagonist. The maximal changes in gene expression for 11,729 probe sets representing genes that show >1.3-fold (A) or >5-fold (B) effect by either the development of the senescent phenotype (3, 4). We compared agonist or the antagonist (dots) are plotted on a log scale. Trend lines represent changes in gene expression induced in MCF-7 cells by LGD1550 power regression. Gene names for selected probe sets strongly affected by and LG100815 with the results obtained in a well-characterized either ligand are shown in (B). system of drug-induced senescence of tumor cells. In that system, HCT116 colon carcinoma cells were transiently exposed to agonist versus those produced by the antagonist for 11,729 probe doxorubicin, the surviving cells were separated into proliferating sets that showed >1.3-fold changes in gene expression after and senescent populations after drug treatment, and differentially treatment with either the agonist or the antagonist. The effects expressed genes were identified using a cDNA microarray (14). We of the agonist and antagonist on gene expression show highly have recently repeated this analysis using Affymetrix U133 Plus 2.0 significant correlation. The regression through all the data points array.5 We now asked how treatment of MCF-7 cells with RAR has an R2 value of 0.6955 with n = 11,729 (Student’s t test yields ligands affects the expression of genes found in the latter analysis P < 0.0001). The regression line has a slope of 1.027 F 0.006, to be strongly (>5-fold) induced or inhibited in senescent HCT116 indicating that the RAR agonist and the RAR antagonist have the cells. As shown in Fig. 3E and F, most of these genes changed their same effect on the majority of the responsive genes. The similarity expression in the same direction in MCF-7 cells treated with RAR of the overall effects of the agonist and the antagonist on gene agonist or antagonist. Specifically, 88% of 231 genes down- expression agrees with the results of QPCR analysis of selected regulated in senescent HCT116 cells and expressed in MCF-7 cells genes (Table 1), but contrasts with an order of magnitude weaker were down-regulated by the RAR agonist, and only 3% were up- effect of the antagonist on RARE-dependent transcription (Fig. 1). regulated. Among 353 genes up-regulated in senescent HCT116 On the other hand, 316 probe sets showing the strongest cells and expressed in MCF-7, 53% were up-regulated by the (>5-fold) induction or inhibition by RAR ligands were significantly agonist and 10% were down-regulated. Similar results were more responsive to the agonist than to the antagonist (at P < obtained with the RAR antagonist. This analysis indicates 0.0001), with the regression slope increasing to 1.297 F 0.038 (this profound similarities between the effects of retinoids and translates to f2-fold stronger average effect of the agonist relative doxorubicin-induced senescence on gene expression. On the other to the antagonist; Fig. 4B). We considered whether preferential hand, there were also notable differences in gene expression induction of the most responsive genes by the agonist could between RAR ligand-treated MCF-7 cells and HCT116 cells indicate the presence of RARE sequences in the corresponding undergoing doxorubicin-induced senescence. These differences promoters. Indeed, the gene showing the strongest induction by may be attributed to a large extent to the fact that only the latter the agonist (Fig. 4B) encodes RA-metabolizing enzyme CYP26A1 (induced 220-fold by the agonist and 23.5-fold by the antagonist), which was reported to contain two synergistically acting RARE 5 Y. Chen and I.B. Roninson, in preparation. www.aacrjournals.org 8753 Cancer Res 2006; 66: (17). September 1, 2006

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research but not the former up-regulated cyclin-dependent kinase (CDK) SA-h-gal marker of senescence (Fig. 5C, D; ref. 8). The RXR agonist inhibitor p21Waf1 (CDKN1A), a key regulator of gene expression in LGD1268 did not inhibit MCF-7 cell growth (Fig. 5A) and did not senescent cells (see Discussion). induce the senescent phenotype (Fig. 5D); in fact, LGD1268 The results of gene expression studies suggested that RAR treatment produced a moderate but reproducible increase in cell agonist and antagonist (but not RXR ligands) are likely to induce growth (Fig. 5A). The RXR antagonist LG101208 had no effect senescence-like growth arrest in MCF-7 cells. To test this, we on either the cell growth (Fig. 5A) or the senescent phenotype treated MCF-7 cells with RAR and RXR agonists and antagonists for (Fig. 5D). In contrast, the RAR agonist LGD1550 and the RAR 7 days and analyzed the effects of the treatment on the cell number antagonist LG100815 inhibited cell growth to the extent similar to (Fig. 5A), cell cycle distribution (Fig. 5B), and the expression of the that of RA (Fig. 5A). Cell cycle analysis showed that RA, LGD1550, and LG100815 all increased the G1 and decreased the S fraction, indicating cell cycle arrest in G1 (Fig. 5B), in agreement with previous observations on RA-treated MCF-7 cells (15). LGD1550 and LG100815 increased SA-h-gal activity (Fig. 5C), as has also been shown for RA (3), and this effect of all three RAR ligands was quantitatively similar (Fig. 5D). Hence, RAR agonists and the antagonist induce senescence-like growth arrest in MCF-7 cells with a similar efficiency. Effects of RAR ligands on tumor suppressor and oncogene expression and paracrine growth-inhibitory activity of retinoid-treated MCF-7 cells. The most prominent Gene Ontology categories of genes that were largely inhibited by RAR ligands are genes involved in DNA replication (Fig. 3G) or mitosis (Fig. 3H). Strong down-regulation of such genes has been associated with cell cycle arrest induced by chemotherapeutic drugs. In contrast, inhibition of these genes by RAR ligands was moderate (f2-fold), with none of the genes showing z5-fold inhibition. On the other hand, we noticed that many of the genes that show the greatest fold inhibition by RAR ligands are known or putative oncogenes. (Here, we define genes as putative oncogenes or tumor suppressors if such genes have been reported in the literature to play a functional role in tumor cell growth, survival, tumorigenesis, or metastasis, as determined by targeted inhibition, gene over- expression or protein addition studies.) A total of 26 oncogenes were found to be inhibited by RAR ligands, with the strongest inhibition found for VAV3, SPDEF (also known as PDEF), AMIGO2, MYB, RET, C4.4A, and MAFB (Table 2). On the other hand, we also identified a smaller number (10) of genes with reported tumor- suppressive activities that were inhibited by RAR ligands (Table 2), most notably caveolin proteins CAV1 and CAV2, as well as cellular RA-binding protein 2 (CRABP2), a retinoid-binding protein reported to sensitize MCF-7 cells to growth inhibition by RA (16). We have also identified 22 known or putative oncogenes and 34 tumor suppressors as up-regulated by RAR ligands (Table 3). The products of these genes include both secreted factors (see below) and cell-associated proteins. The most highly induced cell- associated growth inhibitors were the previously identified retinoid-inducible genes UBD, EPLIN (4), and SOX9 (17), followed

Figure 5. Effects of retinoid agonists and antagonists on MCF-7 cell growth and the senescent phenotype. A, cell number after 7 days of culture with the addition of DMSO (control), 100 nmol/L RA, 100 nmol/L RAR agonist LGD1550, 100 nmol/L RXR agonist LGD1268, 10 Amol/L RAR antagonist LG100815, and 10 Amol/L RXR antagonist LG101208. Experiments were done in triplicate, and the results are expressed relative to the average of the control. B, changes in cell cycle distribution in untreated cells or in cells treated with 100 nmol/L RA, 100 nmol/L LGD1550 RAR agonist, 10 Amol/L LG100815 RAR antagonist, or DMSO carrier (untreated). For cells treated with RAR ligands, 0 point represents cells cultured for 2 days with DMSO carrier. The analysis was carried out as described in Materials and Methods. C, staining of cells that were treated for 8 days with DMSO carrier (control), 100 nmol/L LGD1550, or 10 Amol/L LG100815 for senescence marker SA-h-gal. Photographed at Â100 magnification. D, percentages of SA-h-gal+ cells after 8 days of treatment with the indicated compounds (in triplicate), at the same concentrations as in (A).

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Table 2. Oncogenes and tumor suppressors inhibited by RAR ligands

Gene name Affymetrix Genbank Description Basal raw Max fold inhibition Max fold inhibition probe no. signal by agonist by antagonist

Oncogenes Cell -associated VAV3 218807_at NM_006113 Vav 3 oncogene 1,181 14.5 2.7 SPDEF 220192_x_at NM_012391 SAM pointed domain containing 522 14.2 3.0 ets transcription factor AMIGO2 222108_at AC004010 Adhesion molecule with 1,051 11.7 4.1 immunoglobulin-like domain 2 MYB 204798_at NM_005375 V-myb myeloblastosis viral 716 9.9 2.2 oncogene homologue RET 211421_s_at M31213 Ret proto-oncogene 350 8.5 1.8 C4.4A 204952_at NM_014400 GPI-anchored metastasis- 190 8.1 3.2 associated protein homologue MAFB 218559_s_at NM_005461 V-maf musculoaponeurotic 774 7.5 2.1 fibrosarcoma oncogene homologue B FGFR3 204379_s_at NM_000142 Fibroblast growth factor 173 5.0 2.4 receptor 3 GREB1 205862_at NM_014668 GREB1 protein 285 4.8 2.8 MSX2 210319_x_at D89377 Msh homeo box homologue 2 395 4.0 3.2 PDLIM2 219165_at NM_021630 PDZ and LIM domain 2 (mystique) 136 4.0 2.2 ENTPD5 205757_at NM_001249 Ectonucleoside triphosphate 51 3.4 3.0 diphosphohydrolase 5 MALAT1 224559_at AF001540 Metastasis-associated lung 200 2.5 3.4 adenocarcinoma transcript 1 PBX3 204082_at NM_006195 Pre-B-cell leukemia 311 3.4 2.5 transcription factor 3 CCNA2 203418_at NM_001237 Cyclin A2 415 3.3 2.3 G6PD 202275_at NM_000402 Glucose-6-phosphate 1,380 2.2 3.2 dehydrogenase HMMR 207165_at NM_012485 Hyaluronan-mediated 540 2.9 2.1 motility receptor (RHAMM) CCND1 208711_s_at BC000076 Cyclin D1 695 2.1 2.9 ECT2 219787_s_at NM_018098 Epithelial cell transforming 545 2.8 1.6 sequence 2 oncogene PTTG1 203554_x_at NM_004219 Pituitary tumor-transforming 1 1,236 2.7 2.2 MYBL1 213906_at AW592266 V-myb myeloblastosis viral 387 2.6 2.0 oncogene homologue-like 1 IRS1 204686_at NM_005544 Insulin receptor substrate 1 2,117 2.6 1.6 PIK3R1 212240_s_at AI679268 Phosphoinositide-3-kinase, 1,206 2.0 2.1 regulatory subunit 1 (p85a) Secreted CXCL12 209687_at U19495 Chemokine (C-X-C motif) ligand 12 596 5.7 1.6 (stromal cell-derived factor 1) TFF3 204623_at NM_003226 Trefoil factor 3 (intestinal) 387 4.0 3.0 BMP7 209591_s_at M60316 Bone morphogenetic protein 7 1,008 1.8 2.0 (osteogenic protein 1) Tumor suppressors (cell associated) CAV1 212097_at AU147399 Caveolin 1, caveolae protein, 22 kDa 516 21.8 4.3 GPR30 210640_s_at U63917 G protein-coupled receptor 30 163 7.0 2.6 CRABP2 202575_at NM_001878 Cellular RA binding protein 2 4354 6.1 3.5 CAV2 203324_s_at NM_001233 Caveolin 2 643 5.0 2.8 PHLDA2 209803_s_at AF001294 Pleckstrin homology-like domain, 351 4.4 2.7 family A, member 2 BARD1 205345_at NM_000465 BRCA1-associated RING domain 1 543 4.1 2.3 NAT1 214440_at NM_000662 N-acetyltransferase 1 1484 4.1 2.2 (arylamine N-acetyltransferase) BLM 205733_at NM_000057 Bloom syndrome 122 3.3 2.0 SFN 33322_i_at X57348 Stratifin 2,066 3.1 1.9 TOB1 228834_at BF240286 Transducer of ERBB2, 1 3,287 2.9 2.1

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Table 3. Oncogenes and tumor suppressors induced by RAR ligands

Gene name Affymetrix Genbank Description Basal raw Max fold induction Max fold induction probe no. signal by agonist by antagonist

Oncogenes Cell-associated HOXA1 214639_s_at S79910 Homeobox A1 8 95.3 18.3 RGC32 218723_s_at NM_014059 Response gene to complement 32 9 49.6 34.0 ATF3 202672_s_at NM_001674 Activating transcription 24 6.9 3.3 factor 3 CXCR4 217028_at AJ224869 Homo sapiens CXCR4 gene 213 5.5 2.5 encoding receptor CXCR4. ELF3 210827_s_at U73844 E74-like factor 3 (ets domain 318 4.9 2.9 transcription factor, epithelial-specific) SIX1 228347_at N79004 Sine oculis homeobox 179 4.6 2.3 homologue 1 MITF 226066_at AL117653 Microphthalmia-associated 27 4.1 3.2 transcription factor FGFR2 203638_s_at NM_022969 Fibroblast growth factor 57 3.3 4.1 receptor 2 BHLHB2 201170_s_at NM_003670 Basic helix-loop-helix domain 95 3.5 2.1 containing, class B, 2 MDM2 217373_x_at AJ276888 Mdm2, transformed 3T3 cell 39 3.1 1.9 double minute 2, p53 binding protein LYN 202625_at AI356412 V-yes-1 Yamaguchi sarcoma viral 77 2.8 3.0 related oncogene homologue JUN 201464_x_at BG491844 V-jun sarcoma virus 17 190 2.6 2.6 oncogene homologue S100P 204351_at NM_005980 S100 calcium binding protein P 523 2.3 2.1 JAG1 209099_x_at U73936 Jagged 1 111 2.2 1.9 Secreted IL-8 202859_x_at NM_000584 Interleukin-8 10 14.4 8.2 PLA2G2A 203649_s_at NM_000300 Phospholipase A2, group IIA 30 7.4 1.9 EDN1 222802_at J05008 Endothelin-1 22 4.1 1.7 CLU 208792_s_at M25915 Clusterin (complement lysis inhibitor, 723 3.4 2.7 SP-40,40, sulfated glycoprotein 2, testosterone-repressed prostate message 2, apolipoprotein J) PDGFC 218718_at NM_016205 Platelet-derived growth factor C 11 3.4 2.6 FGF13 205110_s_at NM_004114 Fibroblast growth factor 13 186 2.9 2.2 TGFA 205016_at NM_003236 Transforming growth factor, a 61 2.7 1.5 PSAP 200866_s_at M32221 Prosaposin 674 1.9 1.8 Tumor suppressors Cell-associated UBD 205890_s_at NM_006398 Ubiquitin D 36 35.9 17.5 SOX9 202935_s_at AI382146 SRY (sex-determining region Y)-box 9 50 26.0 10.8 EPLIN 217892_s_at NM_016357 Epithelial protein lost in neoplasm h 128 18.8 7.4 CEACAM1 206576_s_at NM_001712 Carcinoembryonic antigen-related 15 16.2 2.8 cell adhesion molecule 1 PPARG 208510_s_at NM_015869 Peroxisome proliferative 64 15.9 4.0 activated receptor, g MARCKS 201669_s_at NM_002356 Myristoylated alanine-rich protein 64 9.3 4.6 kinase C substrate BTG2 201236_s_at NM_006763 BTG family, member 2 87 8.3 6.5 NKX3-1 209706_at AF247704 NK3 transcription factor-related, locus 1 35 7.5 2.9 IRF1 202531_at NM_002198 IFN regulatory factor 1 43 5.3 2.4 SOD2 215223_s_at W46388 Superoxide dismutase 2, mitochondrial 327 4.9 4.8 NBL1 37005_at D28124 Neuroblastoma, suppression of 61 4.0 3.0 tumorigenicity 1

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Table 3. Oncogenes and tumor suppressors induced by RAR ligands (Cont’d)

Gene name Affymetrix Genbank Description Basal raw Max fold induction Max fold induction probe no. signal by agonist by antagonist

GADD45G 204121_at NM_006705 Growth arrest and DNA-damage- 37 3.8 2.7 inducible, g PHLDA1 217996_at AA576961 Pleckstrin homology-like domain, 359 3.7 2.7 family A, member 1 HIPK2 225368_at BF218115 Homeodomain interacting 114 3.7 2.8 protein kinase 2 CDKN2B 236313_at AW444761 Cyclin-dependent kinase inhibitor 2B 14 3.2 3.1 (p15, inhibits CDK4) BATF 205965_at NM_006399 Basic leucine zipper transcription 213 2.7 2.9 factor, ATF-like VHL 1559227_s_at BF972755 Von Hippel-Lindau tumor 25 2.0 2.7 suppressor EI24 216396_s_at AF131850 Etoposide induced 2.4 mRNA 147 2.2 2.6 KLF6 208961_s_at AB017493 Kruppel-like factor 6 25 2.2 2.5 BTG1 200921_s_at NM_001731 B-cell translocation gene 1, 509 2.5 1.8 antiproliferative DDIT3 209383_at BC003637 DNA-damage-inducible transcript 3 131 2.5 2.2 FOXO3A 224891_at AV725666 Forkhead box O3A 1172 2.3 1.8 GSN 200696_s_at NM_000177 Gelsolin 435 1.7 1.9 PDCD4 212593_s_at N92498 Programmed cell death 4 1433 1.7 1.6 (neoplastic transformation inhibitor) Secreted TGFBI 201506_at NM_000358 Transforming growth factor, 127 53.0 12.8 h-induced, 68 kDa IGFBP3 210095_s_at M31159 Insulin-like growth factor 15 32.2 12.3 binding protein 3 FBLN5 203088_at NM_006329 Fibulin 5 7 10.1 19.0 GDF15 221577_x_at AF003934 Growth differentiation factor 15 378 7.2 3.4 TGM2 211003_x_at BC003551 Transglutaminase 2 (C polypeptide, 17 5.8 2.9 protein-glutamine-g- glutamyltransferase) SULF1 212354_at BE500977 Sulfatase 1 126 2.9 2.9 TGFB2 228121_at AU145950 Transforming growth factor, h2 20 2.0 2.7 IGFBP6 203851_at NM_002178 Insulin-like growth factor 81 2.6 2.1 binding protein 6 PRSS8 202525_at NM_002773 Protease, serine, 8 (prostasin) 186 2.4 1.9 PRSS11 201185_at NM_002775 Protease, serine, 11 (IGF binding) 50 1.9 1.5

by CEACAM1, PPARG, MARCKS, BTG2, and NKX3-1. The most functional test to determine whether such cells produce primarily strongly induced oncogenes were the RARE-regulated transcription promitogenic or antimitogenic paracrine effects. In this assay, we factor HOXA1 and RGC32, a positive regulator of the cell cycle. The mixed MCF-7 cells 1:1 with MDA-MB-231 breast carcinoma cells time course of the induction or inhibition of cell-associated (insensitive to retinoids). The latter cells had been transduced oncogenes and tumor suppressors listed in Table 2 is shown in Fig. with GFP, allowing us to distinguish and quantitate the two cell 3I and J, respectively. lines by flow cytometry. The cocultures were treated for 5 days Many genes up-regulated by RAR ligands encode secreted with 100 nmol/L RA (used because of limited availability of proteins. Some of these proteins have cancer-relevant activities, LGD1550 and LG100815). RA or MCF-7 cells alone had no effect stimulating or inhibiting cell growth, survival, invasion, or on the growth of MDA-MB-231 cells. In contrast, retinoid angiogenesis. The induced secreted proteins with tumor-promoting treatment in coculture with MCF-7 decreased the number of (oncogenic) or tumor-suppressing activities are listed in Table 3, MDA-MB-231 cells by f25% (Fig. 6A). In another type of whereas the inhibited secreted proteins are listed in Table 2. Figure experiment, shown in Fig. 6B, MCF-7 cells were pretreated for 3K and L show the effects of RAR ligands on the expression of the 8 days with RA to allow for complete growth arrest and corresponding genes. The genes for secreted proteins showing the development of the senescent phenotype. The treated MCF-7 strongest induction include four tumor-suppressing factors cells were collected by trypsinization and cocultured for 3 days (TGFBI, IGFBP3, FBLN5, and GDF15) and two tumor-promoting with MDA-MB-231, in the presence or in the absence of RA. proteins [interleukin-8 (IL-8) and PLAG2A]. Coculture with RA-pretreated MCF-7 cells was sufficient to Because retinoid-treated MCF-7 cells up-regulate genes for inhibit MDA-MB-231 cell growth by f30%, compared with secreted factors with different activities, we carried out a coculture with untreated MCF-7 cells (Fig. 6B). The addition of www.aacrjournals.org 8757 Cancer Res 2006; 66: (17). September 1, 2006

Figure 6. Effects of coculture with RA-treated MCF-7 cells on MDA-MB-231 cell growth. A, MDA-MB-231 cells (GFP-expressing) were plated either alone or in 1:1 mixture with MCF-7 cells, in the presence of 100 nmol/L RA or DMSO carrier. Columns, mean MDA-MB-231 cell number after 5 days of culture relative to cell number in the absence of MCF-7 or RA, calculated from three independent experiments; bars, SD. B, MDA-MB-231 cells (GFP- expressing) were plated as 1:1 mixtures with MCF-7 cells that were either untreated or treated for 8 days with 100 nmol/L, and grown in the presence of DMSO carrier or 100 nmol/L RA. Columns, mean MDA-MB-231 cell number relative to MDA-MB-231 cell number in coculture with untreated MCF-7 without RA, calculated from three independent experiments; bars, SD.

RA to the coculture had no significant effect on this growth contain RARE elements. Indeed, we have identified a set of genes, inhibition (Fig. 6B), indicating that MDA-MB-231 cell growth was primarily among the strongest responders, which are preferentially inhibited not by the retinoid but by factors secreted by retinoid- induced or inhibited by the agonist relative to the antagonist. Such treated MCF-7 cells. genes, however, were a small minority (e.g., 1,616 genes were up- regulated >2-fold by either ligand, but only 261 of these genes showed z2-fold stronger response to the agonist than to the anta- Discussion gonist), and the average effects of the agonist and the antagonist on In the present study, we have used pan-RAR– or pan-RXR– the bulk of the responsive genes were essentially the same (Fig. 4A). specific agonists and antagonists to investigate the mechanism of (c) Nevertheless, one could still argue that RARE-dependent trans- changes in gene expression associated with senescence-like growth cription, which is preferentially induced by the agonist, could be the arrest, which is induced by retinoids in MCF-7 breast carcinoma key determinant of the effect of retinoids on the growth of MCF-7 cells. Biological effects of retinoids are commonly attributed to the cells, whereas RARE-independent changes in global gene expression effects of ligand-bound retinoid receptors on transcription from could be epiphenomena irrelevant to the biological response. RARE-containing promoters. However, the majority of genes However, the RAR antagonist LG100815 was just as efficient as the induced by retinoids in different systems (7), including MCF-7 RAR agonists (RA and LGD1550) in inducing cell growth inhibition, cells treated with RA or fenretinide (4), do not contain RARE G1 arrest, and the senescent phenotype (Fig. 5). elements in their promoters. Nevertheless, RARE-mediated induc- A number of RARE-independent mechanisms of regulation of tion of transcription could still be the primary response to gene expression by retinoids have been described in the literature. retinoids, triggering a chain of events leading to indirect changes in Not all of these mechanisms are transcriptional; for example, the expression of other genes. The results of the present study retinoids were suggested to affect RNA stability (18). We have suggest, however, that RARE-dependent transcription plays only a analyzed mRNA stability of IGFBP3, TGFBI, UBD, and EPLIN minor role in the effects of retinoids on gene expression in MCF-7 during 24-hour treatment with actinomycin D and found these cells, and that changes in gene expression responsible for messages to be as stable as h-actin mRNA, with no detectable senescence-like growth arrest are due primarily to RARE-indepen- effect of RA on their stability (19). Hence, the effect of retinoids dent transcriptional effects. on these genes is more likely to be exerted at the level of These conclusions are based on the following arguments. (a) The transcription. In the case of IGFBP3, the induction of transcrip- limited role of RARE promoter sequences in determining transcription by RA in MCF-7 cells has been shown by nuclear run-on tional effects of retinoids is indicated by the findings that only a assays (20). Some examples of RARE-independent transcription small fraction of the genes induced by RAR ligands contain RARE regulatory mechanisms include the binding of RAR/RXR–based elements in their promoter, and that the majority of RARE- transcription factor complexes to cis-acting sequences distinct containing genes examined are not affected by the RAR ligands. (b) from RARE (21) or interactions between retinoid receptors and RARE-dependent transcription is also unlikely to be the initial effect other transcription factors, such as Sp1 (6, 22) or AP-1 (5, 23). responsible for subsequent global changes in gene expression, as Furthermore, retinoids can regulate the activity of protein kinase indicated by the comparison of the effects of RAR agonist LGD1550 C (PKC; ref. 24), and RA-activated PKC was shown to bind to RAR and RAR antagonist LG100815. The RAR agonist was an order of and stimulate its transcriptional activity (25). The effects of RAR magnitude more efficient than the antagonist in stimulating a ligands on gene expression, observed in the present study, most RARE-containing promoter (at concentrations producing the likely reflect many different RARE-independent transcriptional maximal effects), and it induced the responsive genes known to effects of retinoids rather than any single mechanism. contain functional RARE sequences 3 to 10 times stronger than the In light of our findings, it would seem more appropriate to antagonist. One would expect therefore that genes that are induced describe compounds such as LG100815 not as RAR antagonists as a consequence of RARE-dependent early events should also show but rather as a novel type of RAR modulators that are poor preferential response to the agonist, even if such genes do not inducers of RARE-dependent transcription and that act primarily

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. RAR Ligand Effects on Gene Expression and Cell Growth by stimulating RARE-independent transcriptional effects. Not all could potentially represent a negative regulatory mechanism that the RAR antagonists seem to belong to this class. For example, a limits retinoid-induced apoptosis. Another surprising observation RAR-a–specific antagonist LG100629 was shown to block essen- is the inhibition of caveolin proteins CAV1 and CAV2. Caveolins tially completely the induction of IGFBP3 and TGFB2 by RA in were shown to act as tumor suppressors in the mammary gland human bronchial epithelial cells (26), in contrast to our findings but as oncogenes in other tissue contexts, such as the prostate (40). with pan-RAR modulator LG100815. On the other hand, several CAV1 expression in MCF-7 cells was found to inhibit anchorage- RAR antagonists, similarly to LG100815, were found to inhibit independent growth but at the same time protect cells from tumor cell growth (27–30). It would be of interest to determine if apoptosis (41). Altogether, despite some changes to the contrary, these compounds act as LG100815-type RAR modulators. the overall effect of RAR modulators on the expression of Genome-scale microarray analysis of the effects of RAR ligands oncogenes and tumor suppressors in MCF-7 cells seems to be has confirmed and expanded the results of our previous analysis of indicative of the reversal of the neoplastic phenotype. the effects of RA in MCF-7 cells, carried out using a smaller Several retinoid-induced tumor suppressors (BTG2, BTG1, microarray (4). The principal conclusion of the prior study was that GDF15, EPLIN, and CEACAM1) are also induced by DNA-damaging retinoid-induced arrest of MCF-7 cells was associated with drugs (such as doxorubicin) and remain constitutively up-regulated concerted induction of several growth-inhibitory genes encoding in HCT116 colon carcinoma cells that become permanently growth both cell-associated (UBD and EPLIN) and secreted proteins arrested and develop the senescent phenotype after exposure to (TGFBI and IGFBP3). The present analysis showed that RAR doxorubicin (14). Aside from these genes, we have found a striking ligands LGD1550 and LG100815 also strongly induced these genes, overlap between the large groups of genes that are induced or and it revealed many additional tumor-suppressive genes induced inhibited in doxorubicin-induced senescence of HCT116 cells and by retinoids (e.g., SOX9, CEACAM1, MARCKS, GDF15, FBLN5, in retinoid-induced senescence of MCF-7 cells (Fig. 3K and L). On BTG2, NKX3-1, NBL1, and IRF1). Importantly, we discovered in the the other hand, an important difference between retinoid-treated present study that RAR ligands not only induce tumor suppressors MCF-7 cells and most of the characterized systems of DNA but also inhibit multiple oncogenes or proto-oncogenes expressed damage–induced senescence is the lack of induction of the in MCF-7 cells, with the strongest effects observed for VAV3, damage-responsive CDK inhibitor p21Waf1 (CDKN1A), which was SPDEF, AMIGO2, MYB, RET, and C4.4A. We have investigated found here and in previous studies (15) to be moderately down- the effects of overexpressing several retinoid-inducible growth- regulated in MCF-7 cells after retinoid treatment. In fact, the only inhibitory genes, including UBD (4), EPLIN, and TGFBI, as well as CDK inhibitor that we found to be up-regulated in retinoid-treated IGFBP3 protein product (19), on MCF-7 cells, and found that each MCF-7 cells was p15Ink4b (CDKN2B), which was recently identified of these genes produced only weak growth inhibition and did not as a marker of oncogene-induced senescence (42). In contrast, p21 induce the senescent phenotype. In light of these findings, we is drastically induced in MCF-7 cells after doxorubicin treatment, hypothesize that retinoids induce senescence-like growth arrest in concordantly with the development of the senescent phenotype MCF-7 cells through a cumulative effect of multiple changes in the (43). p21 induction leads to rapid inhibition of genes involved in expression of different growth-regulatory genes. mitosis or DNA replication (44), and p21 knockout prevents the Many of the genes found in the present study to be affected to a shutdown of such genes in doxorubicin-treated cells (14). Although similar extent by RAR agonist and antagonist (i.e., RARE- genes involved in mitosis and DNA replication are clearly inhibited independent genes) have been previously reported to be retinoid in RAR ligand–treated MCF-7 cells (Fig. 2F and G), their inhibition responsive in MCF-7 and other breast carcinoma cell lines. Of is not as drastic as in DNA-damaged cells (14), possibly due to the special interest, these include tumor suppressor genes proposed to low levels of p21. The lack of p21 induction is also likely to account mediate the antiproliferative effects of retinoids, such as IGFBP3, for our observations that a number of genes that are induced in induced by RA in MCF-7 and Hs578T lines (31), as well as SOX9 and response to p21 and implicated in tumor promotion or the PDCD4, induced by RAR-specific but not by RXR-specific agonists development of age-related diseases, such as LGALS3, APP, or SHC1 in MCF-7 and T-47D cells (17, 32). Other genes identified by our (44), were not induced in RAR modulator–treated MCF-7 cells. microarray analysis have been implicated specifically in the Induction of senescence-like permanent growth arrest could be phenotype of breast carcinoma cells, including MCF-7. Among one of the most desirable treatment responses in tumor cells, the genes that we found to be inhibited by retinoids, ets trans- because it occurs at low drug concentrations that produce little cription factor SPDEF (PDEF) was shown to be a key mediator of systemic toxicity, and because senescent cells not only fail to grow motility and invasion in MCF-7 and other breast carcinoma cell but also secrete proteins with paracrine growth-inhibitory effects. lines (33), PDLIM2 (Mystique) is a negative regulator of anchorage- On the other hand, senescence induced by treatment with p21- independent growth and migration in MCF-7 cells (34), and GREB1 inducing DNA-damaging agents is also associated with overpro- was reported to mediate the proliferative response of MCF-7 cells duction of proteins with the opposite, tumor-promoting activities, to estrogen (35). Among the genes found here to be induced by which stimulate the growth or survival of the neighboring RAR modulators, an isoform of adhesion molecule CEACAM1 nonsenescent cells (45). In our previous study on retinoid-induced inhibits cell growth and restores normal morphology of MCF-7 senescence of MCF-7 cells (4), we observed overproduction of cells (36), and IRF1 was shown to mediate p53-independent tumor secreted growth-inhibitory proteins (TGFBI and IGFBP3). We have suppression and apoptosis in MCF-7 and other breast carcinoma now found that MCF-7 cells treated with RAR ligands up-regulate cell lines (37). Growth inhibition and apoptosis in retinoid-treated not only additional tumor-suppressing proteins (GDF15 and MCF-7 cells have been previously associated with the drastically FBLN5) but also tumor-promoting factors (IL-8 and PLAG2A), overexpressed IGFBP3 (38) and with the retinoid-binding protein although tumor-suppressing proteins show overall greater induc- CRABP2 (39), which, paradoxically, we find here to be strongly tion, at least at the RNA level (Fig. 3K and L). The results of our down-regulated rather than induced by RAR ligands. Down- coculture studies with retinoid-sensitive and retinoid-insensitive regulation of CRABP2 gene expression upon retinoid treatment breast carcinoma cell lines (Fig. 6) show that retinoid-treated www.aacrjournals.org 8759 Cancer Res 2006; 66: (17). September 1, 2006

MCF-7 cells indeed secrete tumor-suppressing factors that produce LG100815-type RAR modulators are inefficient in inducing the a moderate but reproducible decrease in the growth of neighboring major effect of conventional retinoids (RARE-dependent trans- retinoid-insensitive MDA-MB-231 cells. Exploitation of this para- activation), which provides the likeliest explanation for their crine tumor-suppressive effect of retinoid-treated tumor cells offers nontoxicity. On the other hand, LG100815 mimics the effects of a new insight into the clinical applications of retinoids. conventional RAR agonists in inducing senescence-like growth Despite the shown efficacy of retinoids in PML treatment arrest of tumor cells and up-regulating the expression of genes andincancerchemopreventiontrials,retinoidtreatmentis for tumor-suppressing secreted factors. This novel type of RAR complicated by systemic toxic responses, such as intracranial modulators may be viewed therefore as a prototype of a potentially hypertension and headaches, dyspnea, hypertriglyceridemia, hyper- interesting new class of drugs that should provide a high calcemia, and hyperleukocytosis (46). An experimental approach therapeutic ratio for cancer treatment or chemoprevention. proposed to avoid these side effects is the use of RXR-selective agonists that show lower toxicity than the conventional RAR Acknowledgments agonists (47). However, RXR agonists may not be effective against all the types of transformed mammary epithelial cells, as indicated Received 2/14/2006; revised 5/22/2006; accepted 6/28/2006. Grant support: NIHgrants R01 CA62099 and R01 AG17921 (I.B. Roninson). in the present study by the observation that LGD1268 did not inhibit The costs of publication of this article were defrayed in part by the payment of page but rather stimulated MCF-7 cell growth (Fig. 5A). As an alternative charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. approach, RAR antagonists have been developed to prevent or treat We thank Dr. William Lamph (Ligand Pharmaceuticals, Inc., San Diego, CA), for retinoid toxicity (48). RAR antagonists were reported to be very well providing RAR and RXR ligands used in this study and for helpful advice; Dr. Chang tolerated even at very high doses (49) and in some cases produced Lim for assistance with flow cytometry; Drs. Eugenia Broude, Errin Lagow, and Charitha Madiraju for helpful discussions; and the late Dr. Robin Pietropaolo and the in vivo tumor-suppressive effects on their own (49, 50). The results Microarray Core Facility at the Genomics Institute of the New York State Department of the present study suggest an explanation for these observations. of Health Wadsworth Center, who carried out microarray hybridization.

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