Open Access

All trans-retinoic acid (ATRA) induces re-differentiation of early transformed breast epithelial cells

  • Authors:
    • Maria F. Arisi
    • Rebecca A. Starker
    • Sankar Addya
    • Yong Huang
    • Sandra V. Fernandez
  • View Affiliations

  • Published online on: Friday, March 21, 2014
  • Pages: 1831-1842
  • DOI: 10.3892/ijo.2014.2354

Abstract

Retinoids have been used as potential chemotherapeutic or chemopreventive agents because of their differentiative, anti-proliferative, pro-apoptotic and antioxidant properties. We investigated the effect of all trans-retinoic acid (ATRA) at different stages of the neoplastic transformation using an in vitro model of breast cancer progression. This model was previously developed by treating the MCF-10F human normal breast epithelial cells with high dose of estradiol and consists of four cell lines which show a progressive neoplastic transformation: MCF-10F, normal stage; trMCF, transformed MCF-10F; bsMCF, invasive stage; and caMCF, tumorigenic stage. In 3D cultures, MCF-10F cells form tubules resembling the structures in the normal mammary gland. After treatment with estradiol, these cells formed tubules and spherical masses which are indicative of transformation. Cells that only formed spherical masses in collagen were isolated (trMCF clone 11) and treated with ATRA. After treatment with 10 or 1 µM ATRA, the trMCF clone 11 cells showed tubules in collagen; 10 and 43% of the structures were tubules in cells treated with 10 and 1 µM ATRA, respectively. Gene expression studies showed that 207 genes upregulated in transformed trMCF clone 11 cells were downregulated after 1 µM ATRA treatment to levels comparable to those found in the normal breast epithelial cells MCF-10F. Furthermore, 236 genes that were downregulated in trMCF clone 11 were upregulated after 1 µM ATRA treatment to similar levels shown in normal epithelial cells. These 443 genes defined a signature of the ATRA re-programming effect. Our results showed that 1 µM ATRA was able to re-differentiate transformed cells at early stages of the neoplastic process and antagonistically regulate breast cancer associated genes. The invasive and tumorigenic cells did not show any changes in morphology after ATRA treatment. These results suggest that ATRA could be used as a chemopreventive agent to inhibit the progression of premalignant lesions of the breast.

Introduction

Vitamin A is obtained through the diet in the form of retinol, retinyl ester or β-carotene (1). Retinoic acid (RA) is one of the principal active metabolites of vitamin A which plays a critical role in cell proliferation, differentiation and apoptosis in normal tissues during embryonic development (2). RA induces differentiation in many cell types and is the most widely used differentiating therapeutic agent (3,4). Retinol has 6 biologically active isoforms that among others includes all-trans (ATRA, tretinoin) and 9-cis RA (alitretinoin); ATRA is the predominant physiological form (5). RA mediates the transcriptional regulation of several genes by binding to the nuclear retinoic acid receptors (RARs), namely RARα, RARβ and RARγ (6,7). Like other nuclear receptors, RARs contain a domain that mediates interaction with ATRA, a zinc finger-containing DNA binding domain that binds to RA response elements (RAREs) in target genes, and a dimerization domain that engages members of the retinoid X receptor (RXR) subfamily in RXR/RAR heterodimers (8). Different isomers activate different receptors and thus lead to different biological effects. RARs can be activated by both all-trans (ATRA) and 9-cis-RA, while RXR are exclusively activated by 9-cis RA; however, due to the conversion of ATRA to 9-cis RA, high concentrations (10−5 M) of ATRA can also activate gene transcription in cells transfected with RXRs (9). It has also been shown that retinoids exert their effects via the nuclear receptor independent pathway (5).

RA and its derivatives are promising anti-neoplastic agents endowed with both therapeutic and chemopreventive potential because they are able to regulate cell growth, differentiation and apoptosis (10,11). It is believed that the anti-neoplastic pathways induced by RA are regulated predominantly by RAR-β, which is known to induce apoptosis; thus it has been suggested that RAR-β plays a critical role in mediating the growth arrest and differentiation in several breast cancer cell types (1214).

We have developed an in vitro-in vivo model of breast cancer progression by treating the human normal-like breast epithelial cells MCF-10F with a high dose of estradiol (70 nM) (Fig. 1) (15,16). This model consists of four cell lines: i) the spontaneously immortalized cell line MCF-10F, which is considered to be a normal-like breast epithelial cell line; ii) the transformed trMCF cells; iii) the invasive bsMCF cells; and iv) cells isolated from xenografts, caMCFs, which show all characteristics of fully malignant breast cancer cells (Fig. 1). Gene expression studies showed the highest number of deregulated genes in caMCF, being slightly lower in bsMCF, and lowest in trMCF and, this order was consistent with the extent of chromosome aberrations (caMCF>bsMCF>>trMCF) (16). This model of breast cancer progression resembles the different steps of neoplastic transformation of the mammary gland; it is widely held that breast cancer initiates as the premalignant stage of atypical ductal hyperplasia (ADH), progresses into the pre-invasive stage of ductal carcinoma in situ (DCIS), and culminates in the potentially lethal stage of invasive ductal carcinoma (IDC) (17). In collagen, the normal-like MCF-10F cells form tubules resembling the structures observed in the normal mammary gland although after treatment with estradiol, the transformed trMCF cells form tubules and spherical masses, which are indicative of cell transformation (8,19). The spherical masses showed a partial filling of the lumen that would result from decreased central apoptosis, enhanced cellular proliferation or both (18). The filling of the lumen of the tubular structures of the breast is the earliest morphologic alteration and is common in atypical ductal hyperplasia and ductal carcinoma in situ (DCIS) (18). In the presented study, we studied the effect of all trans-RA (ATRA) using this model of breast cancer progression. Our results showed that ATRA was able to re-program early transformed cells to a normal stage.

Materials and methods

Cells and media

The human normal-like breast epithelial cells MCF-10F are estrogen receptor (ER) negative, progesterone receptor (PR) negative and HER2 negative. Cells were cultured in Dulbecco’s modified Eagle’s medium [DMEM/F-12, Gibco, Carlsbad, CA; formula 90–5212 EF: containing DMEM/F12 (1:1) with L-glutamine and phenol red, D-glucose 315 mg/l, sodium pyruvate 55 mg/l] with 5% horse serum, 2.43 g/l sodium bicarbonate, 20 mg/l epidermal growth factor (EGF), 100 mg/l Vibrio cholerae toxin, 10 mg/l insulin, 0.5 mg/l hydrocortisone, 1.05 mM calcium, antibiotics and antimicotic (100 U/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin). A 10-mM solution of all trans-retinoic acid (ATRA, Cat# R2625, Sigma, St. Louis, MO) was prepared as a stock solution by dissolving ATRA in dimethylsulphoxide (DMSO).

The trMCF clone 11 was isolated by seeding 100–1,000 trMCF cells in a 100-mm cell culture plate and after 1 day in culture, several colonies were isolated by ring cloning. The trMCF clone 11 cells were generated by expanding the cells from one of these colonies; trMCF clone 11 cells only formed spherical masses on collagen. To study the effect of ATRA, trMCF clone 11 cells were treated continuously for 26 days with 10−5 M (10 μM) to 10−8 M (0.01 μM) ATRA (media was replaced daily). As control, the cells were treated with 0.1% DMSO (vehicle). The bsMCF and caMCF cells were treated with 10−5 to 10−8 M ATRA alone or in combination with 2.5 μM 5-aza-dC.

Collagen assays

The cells were resuspended at a final density of 1.5×104 cells/ml in collagen matrix consisting of 2.68 mg/ml (89.3%) type I collagen (PureCol, Inamed Biomaterials Co., Fremont, CA), 8% 12.5X DMEM-F12 with antibiotics, 0.1 mg/ml insulin, 14 mM NaHCO3 and 0.01 N NaOH. A total of 400 μl (3,000 cells) were plated on the top four 24-well chambers pre-coated with 400 μl of 89.3% collagen mix. Per each treatment, cells were plated in 4 wells and fed daily with the medium described before. The structures in collagen matrix were observed daily under an inverted microscope and at the end of the observation period (8 days), the structures (spherical masses, tubules and intermediate structures) were counted, photographed and fixed in 10% neutral buffered formalin and processed for histological examination. Results were expressed as the total number of structures per well (spherical masses, tubules and intermediate structures) and percentage of the different structures per treatment. The t-test was used to determine if the differences were significant.

Invasion assays

Cell invasion in real-time were performed using xCELLigence RTCA DP device from Roche Diagnostics (Mannheim, Germany). For this purpose, each well of the upper chamber of the CIM-Plate 16 was covered with Matrigel (BD Biosciences, Franklin Lakes, NJ) basement membrane matrix (1:20 in cell culture media) and 10% fetal bovine serum (chemo-attractant) was added in the lower chamber. A total of 40,000 cells suspended in 100 μl serum free media were seeded per well in CIM-Plates 16 (Roche Diagnostics). Data acquisition and analysis was performed with the RTCA software (version 1.2, Roche Diagnostics). Changes in impedance from cells that invade and migrate to the underside of wells were recorded and monitored for a total of 24 h.

Gene expression profiling

RNA was isolated from the cells using RiboPure™ kit (Life Technologies, Frederick, MD) and RNA quality was controlled using the Agilent 2100 Bioanalyzer. Gene expression studies were performed using Affymetrix U133 Plus 2.0 (Affymetrix, Santa Clara, CA) human oligonucleotide microarrays containing over 47,000 transcripts and variants, including 38,500 well characterized human genes. After hybridization, the chips were scanned using GeneChip Scanner 3000. The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. Background correction and normalization was done using Iterative Plier 16 with GeneSpring V11.5 software (Agilent, Palo Alto, CA). The criteria for differentially expressed genes was set at ≥2-fold changes (p-value <0.05). The differentially expressed gene list was loaded into Ingenuity Pathway Analysis (IPA) 8.0 software (Ingenuity Systems, Redwood City, CA) to perform biological network and functional analyses.

Results

Treatment with ATRA induced branching morphogenesis in early transformed breast epithelial cells

MCF-10F cells are normal-like breast epithelial cells that form tubules in collagen matrix (3D culture); when these cells were treated with high dose of estradiol (70 nM), the cells (trMCF) formed tubules and spherical masses. To isolate transformed cells that only form spherical masses, trMCF cells were seeded at low density in cell culture dishes and several clones were isolated by ring cloning. One of these clones, trMCF clone 11, did not form tubules in collagen; instead these cells formed spherical masses and intermediate structures (Fig. 2A and B). The trMCF clone 11 cells were treated continuously for 26 days with 10−5 to 10−8 M all trans-retinoic acid (ATRA) and, we found that cells treated with 10−5 and 10−6 M ATRA were able to form tubules in collagen (Fig. 2C and D). Furthermore, the spherical masses formed by trMCF clone 11 treated with 10−5 and 10−6 M ATRA (Fig. 2C and D) were smaller compared to the ones formed by the controls (Fig. 2A and B) or cells treated with 10−7 and 10−8 M ATRA (Fig. 2E and F). The trMCF clone 11 cells treated with 10−7 and 10−8 M ATRA (Fig. 2E and F) did not show any difference in morphology when compared to the controls (Fig. 2A and B). The number of spherical masses, intermediate structures and tubules for trMCF clone 11 cells treated with different concentrations of ATRA was counted (Fig. 3). The total number of structures in collagen was significantly lower in cells treated with ATRA compared with the controls suggesting that ATRA treatment decrease the proliferation rate of the cells (p<0.01) (Fig. 3A). The control trMCF clone 11 showed spherical masses and intermediate structures but no tubules in collagen while cells treated with 10−6 and 10−5 M ATRA formed tubules and less spherical masses (Fig. 3A). The cells treated with 10−5 or 10−6 M ATRA formed significantly less spherical masses than the cells treated with 10−7 or 10−8 M ATRA (p<0.01) (Fig. 3A). A total of 43% of the structures were tubules in the wells containing cells treated with 10−6 M ATRA and 10% tubules in wells with 10−5 M ATRA-treated cells (Fig. 3B).

The invasion capacity of trMCF clone 11 was studied before and after ATRA treatment but, no differences were observed (Fig. 4). The bsMCF and caMCF cells did not show any changes in their morphology or invasion capacity after treatment with ATRA alone or in combination with the demethylating agent 5-aza-cytidine (data not shown).

Treatment with ATRA re-programmed gene expression of early transformed cells

As trMCF clone 11 cells that only formed spherical masses on collagen were able to form tubules after treatment with 10−5 or 10−6 M ATRA, gene expression studies were performed on these cells. The microarray data have been deposited into the NCBI gene expression omnibus (GEO) datasets (GSE51549). The unsupervised sample classification by PCoA (principle coordinate analysis) revealed that trMCF clone 11 cells treated with 10−5 or 10−6 M ATRA demonstrated a major difference with trMCF clone 11 cells, and a minor difference with MCF-10F; also sample differences between 10−5 M ATRA and 10−6 M ATRA were weak (Fig. 5). Although, trMCF clone 11 cells treated with 10−5 M ATRA and 10−6 M ATRA showed minor differences at the expression level, we considered trMCF clone 11 treated with 10−6 M ATRA for the expression analysis since the number of tubules in collagen matrix was higher for this concentration (43% tubules with 10−6 M ATRA vs. 10% tubules with 10−5 M ATRA). For gene expression studies, three experimental groups were compared using empirical Bayesian-moderated t-test implemented in R package ‘limma’: the normal breast epithelial cells MCF-10F, the cells transformed by treatment with estradiol trMCF clone 11 (that only formed spherical masses on collagen) and the trMCF clone 11 after treatment with 10−6 M ATRA. We generated three gene lists at criteria of fold change ≥2 and p≤0.05: gene list-1 (trMCF clone11 vs. MCF-10F) with 1,409 probes (613 probes upregulated; 796 probes downregulated), gene list-2 (ATRA trMCF clone 11 vs. trMCF clone 11) with 1,859 probe sets (1,053 probes upregulated; 806 probes downregulated) and gene list-3 (ATRA trMCF clone 11 vs. MCF-10F) with 870 probe sets (308 probes upregulated; 562 probes downregulated) (Fig. 6). Most importantly, 207 genes (271 probes) upregulated in the transformed trMCF clone 11 (compared to the normal MCF-10F) were downregulated after treatment with 10−6 M ATRA (Fig. 6 and Table IA) and 236 genes (316 probes) that were downregulated in trMCF clone 11 (compared to MCF-10F) were upregulated by 10−6 M ATRA treatment (Fig. 6 and Table IB). These 443 genes defined a gene signature programming the reverse-transformation effect by ATRA (Table I). The relatively smaller number of significant probe sets in gene list-3 compared with other gene lists (Fig. 6) further supported the findings that ATRA-treatment reprograms the gene expression status of trMCF clone 11 cells to MCF-10F.

A, ATRA-downregulated genes (207 genes).

A, ATRA-downregulated genes (207 genes).

ACSS3DNAJB9KLF11PL-5283/SLC13A4TIMP3a
ALDH3A2DSC3KLHDC8BPLAG1TMEM167B
ALDOCDUSP5PLCA5PLD1TMEM27
ALPK1EFHC1LOC100288092PLD6TMEM40
ANKRD37EFNB3LOC100289187PLK1S1TMEM59
AQPEPEPB41L4BLOC100505894POFUT2TNFRSF25
ARG2ERCC1a LOC100506057/STK32CPOLR1DTNFSF11
ARHGAP19ETS2aLOC100507303PPIL6TNKS
ARHGEF10FABP6LOC100507547PPOXTP63
ATF2FAM117ALOC100507644PPP1R13LTPD52L1a
ATG14FAM168ALOC439938PPP1R3CTPRG1
ATP2C2FAM46CLOC642587PRKAB2TRAF3IP2
ATP5C1FAT2LOXPRMT2TRAPPC6A
BCAS4FBXO2LRIG1PROCRTSC22D3
BFSP1FEM1BLYSTPTENTTBK2
BLNKFLCNMAP2K5PTEN/PTENP1TTC39B
BTBD3FLJ37644MAPTPTPN14TXNIP
C11orf80FLJ45244MGEA5RAB11FIP4UFM1
C16orf46FNBP1LMGPaRAB38 UGT1A1/1A4/1A6/1A8/1A9/1A10
C17orf39FNTAMLF1RAB40CUSP3
C17orf48FNTBMRAP2RAB4AUSP32
C1orf133FSIP1MXD1RAB7L1VPS8
C1orf161FXYD2MYLIPRASSF6WAC
C20orf111GBASN4BP2L1RMND1WDR59
C21orf7GGNBP2NDE1RNF169WDR91
C5orf41GGTA1NDUFB4SCARA3WWOXa
C7orf68GIT2NEK2SCRG1YOD1
C9orf9GJA3NEURL1BSEMA6AZBTB34
CCDC28AGKAP1NFKBIL1SFT2D1ZFAND5
CD44aGNA13NGLY1SHOX2ZNF836
CELSR2GNAI1NMNAT3SLC25A37ZNRF1
CLCA2GOSR2NPLSLC2A12
CMBLGPM6AOGFRL1SLC2A9
COBLGPNMBPALMDSLC5A3
CRIP2H19PDCD4aSOCS3
CSNK2A2HAS3PDCD5SORL1
CYP1B1HBP1PDE7ASPATA17
CYP39A1HERPUD1PDZD2STAU2
DBPHMGCLPER1STMN3
DCDIFNAR1PER3STX6
DDAH2IRF6PHF21ASUSD4
DDCaIRX2PHLDB3TESK2
DDIT3KCMF1PHTF2THBS2
DHX40KDM5BPIK3CD THSD1///THSD1P1

{ label (or @symbol) needed for fn[@id='tfn1-ijo-44-06-1831'] } Genes upregulated in trMCF clone 11 that were downregulated after 10−6 M ATRA-treatment to similar levels found in MCF-10F are indicated.

a Genes with binding sites for RARα or RARβ described by Hua et al (29).

B, ATRA-upregulated genes (236 genes).

B, ATRA-upregulated genes (236 genes).

ABHD13COX7BHIATL1OSTM1SLC43A3TSPAN2
ACP2CRELD2HIGD1AP2RY2SMPDL3AUBE2Na
ADAM12CST6aHOXA11PAPPASNRNP25UBE2Q1
ALDH1A3CSTF2HPGDPARVASNX19UBP1
ANO1CYB561D2HS3ST1PCSK5SOAT1UNK
AOX1DCBLD2HS6ST2PDE12SPAG1VARS2
APOL6DHRS9IFI44PHACTR3SPATA13VGLL3
ARGLU1DHX9IFIT3PHLDA1SRPX2VSIG10
ARHGAP26DNAJA1IFIT5PHLDA2SRSF10ZADH2
ARHGAP42DOLKIFNAR1PITPNC1SRSF2IPZBED4
ARHGDIBDPH3KHNYNPKIBSSPNZDHHC2
ARIH2EFCAB2KLHL18PLCXD2STK39ZMPSTE24
ASPHD2EFNB2KLHL23 PLGLA/PLGLB1/PLGLB2STSZNF252
ATP6V0A2EHD4KRT80PNO1STYK1ZNF271
B3GALNT1EIF2AK1LOC100131993PNPLA3SUPT7LZNF326
BRI3BPEIF5BLOC100505759PODXLSUSD5ZNF35
BTG1ELOVL6LOC100507192POLR3KSYNCRIP
C12orf26ENC1aLOC283278PPP2R1BSYNJ2BP
C12orf5ENY2LOC728903PRPS1SYTL2
C1GALT1C1ERLIN2MACC1PRR15SYTL5
C1orf116EXOGMARCKSPSCATBC1D30
C1orf135FADS1aMAT2APSME3TFDP1
C1orf212FAIMMCFD2PTGR1TFRC
C1orf226FAM118BMEIS3P1PTP4A2TGFB2
C6orf223FAM119AMFAP3LPTPRBTGFBR2a
CALM1aFAM83AMFI2PTPRJTGM2
CCDC68FBXW2MFSD1RABIFTHSD4
CCDC88AFDX1MICALL1RBM25TIMM23
CCND1aFN1aMMACHCRBM45TIMM8A
CDAFNIP2MRPL35RGS17TIMM8B
CDC42EP2FRMD3MST1RRHOBTB1TLCD1
CDH2FUCA1MTERFD3RHOFTLR3
CEP78FXNMYEOVRPL27ATLR4
CFH/CFHR1FZD8MYO5CRPS6KA2TMC5
CFIGALNT7NAA40S1PR3TMEM133
CHAC2GATAD2ANAV3SAMHD1TMEM177
CHMLGBP1NECAP1SCELTMEM9B
CHRNA5GDANIPAL1SGK223TP53I3
CLDN23GGCXNMISH3TC2TPCN2
CMAHGPATCH2NRP2SLC16A5TRAK2
CNPY2GPX8NSD1SLC1A1TRIM45
COL4A3GXYLT1OLAHSLC35B4TRIOBP
COL4A4HAS2OR7E14PSLC35C1TRNT1
COX7A1HERC6OR7E47PSLC37A1TSPAN12

{ label (or @symbol) needed for fn[@id='tfn3-ijo-44-06-1831'] } Genes downregulated in trMCF clone 11 that were upregulated after 10−6 M ATRA-treatment to similar levels found in the normal breast epithelial cells MCF-10F.

a Genes with binding sites for RARα or RARβ described by Hua et al (29).

Ingenuity pathway analysis (IPA) revealed 4 canonical pathways significantly dysregulated in the transformed cells trMCF clone 11: aryl hydrocarbon receptor signaling, retinoic acid activation, xenobiotic metabolism signaling and molecular mechanism of cancer (Table II). Several genes of these pathways that were up- or downregulated in trMCF clone 11 show similar levels of expression to MCF-10F after trMCF clone 11 was treated with 10−6 M ATRA (Table II and Fig. 7). Genes from the aryl hydrocarbon receptor signaling ALDH1A3, CCND1, TGFBR2, TGM2 and TFDP1 were downregulated in the transformed cells trMCF clone 11 when compared to their expression in the normal breast epithelial cells MCF-10F and, the expression of these genes was upregulated after these cells were treated with 10−6 M ATRA reaching similar levels to the expression in MCF-10F (Table II and Fig. 7). One of the functions that show enrichment of dysregulated genes in the transformed trMCF clone 11 cells is cell morphology and the expression of most of these genes reached similar levels to MCF-10F after trMCF cells were treated with 10−6 M ATRA. The expression of some genes related to cell morphology such as PLD1, CD44, STX6, STMN3, ATF2, ETS2, NEK2, HAS3, MGP, GNA13 were upregulated in the transformed trMCF clone 11 and their expression reached normal levels after 10−6 M ATRA treatment (Fig. 7); other genes related to cell morphology such as PHLDA1, GBP1, HS6ST2 and TLR3 were downregulated in trMCF clone 11 and their expressions increased after 10−6 M ATRA treatment reaching similar levels to those found in the normal MCF-10F breast epithelial cells (Fig. 7). Also, the expression of several genes that encode enzymes involved in chromatin modifications such as MGEA5, ATF-2, KDM5B, PRMT2 (PRM2), PHF21A and NSD1, were dysregulated in trMCF clone 11, reaching normal levels after 10−6 M ATRA treatment (Fig. 7).

Table II.

Canonical pathways enriched with differentially expressed genes.

Table II.

Canonical pathways enriched with differentially expressed genes.

trMCF clone 11 vs. MCF-10FATRA trMCF clone 11 vs. trMCF clone 11
Aryl hydrocarbon receptor signalingALDH1A3↓, CCND1↓, TGFBR2↓, TGM2 ↓, TFDP1↓, ALDH3A2↑, CYP1B1↑ALDH1A3↑, CCND1↑, TGFB2↑, TGM2↑,TFDP1↑, ALDH3A2↓, CYP1B1↓
Other genes: CDKN1A↓, JUN↓ ALDH7A1↑, CSNK2A1↑, TGFB1↑, MAPK1↑, NFE2L2↑Other genes: CCNE1↑, CCNE2↑, CDK6↑,DHFR↑, IL1B↑, IL6↑, NR2F1↑, NRIP1↑, POLA1↑ ALDH3B2↓, ARNT↓, NCOA3↓, HSPB2↓, ALDH6A1↓
RAR activationALDH1A3↓, DHRS9↓, NSD1↓, TGFB2↓, CSNK2A2↑, PIK3CD↑, PRMT2↑, PTEN↑ALDH1A3↑, DHRS9↑, NSD1↑, TGFB2↑, CSNK2A2↓, PIK3CD↓, PRMT2↓, PTEN↓
Other genes: JUN↓, NR2F2↓, RBP1↓, CSNK2A1↑, CSNK2A2↑, MAPK1↑, MAPK14↑, TGFB1↑, GNAS↑Other genes: GTF2H2↑, IGFBP3↑, MAP2K1↑, MAPK13↑, NR2F1↑, NRIP1↑, RKAR2B↑, DH10↑, CITED2↓, PNRC1↓, PRKAR1A↓, SMARCD2↓
Xenobiotic metabolism signalingALDH1A3↓, HS3ST1↓, HS6ST2↓, PPP2R1B↓, ALDH3A2↑, CYP1B1↑, MAP2K5↑, PIK3CD↑, UGT1A1 (and others UGT)↑ALDH1A3↑, HS3ST1↑, HS6ST2↑, PPP2R1B↑, ALDH3A2↓, CYP1B1↓, MAP2K5↓, PIK3CD↓, UGT1A1 (and others UGT)↓
Other genes: CHST15↓, ALDH7A1↑, CAMK1D↑, HDAC4↑, MAPK1↑, MAPK14↑, MGMT↑, UGT8↑, NFE2L2↑Other genes: ILIB↑, IL6↑, NRIP1↑, MAP2K1↑, MAPK13↑, ALDH3B2↓, ARNT↓, CAMK2D↓, CITED2↓, MAP3K8↓, PPP2R3A↓, ALDH6A1↓, MAP3K2↓
Molecular mechanisms of cancerTGFB2↓, TGFBR2↓, CCND1↓, FZD8↓, PLCB4↓, RABIF↓, RHOF↓, GNA13↑, GNAI1↑, CD44↑TGFB2↑, TGFBR2↑, CCND1↑, FZD8↑, PLCB4↑, RABIF↑, RHOF↑, GNA13↓, GNAI1↓, CD44↓
Other genes: CDKN1A↓, CTNNB1↓, FYN↓, IRS1↓, JUN↓, SMAD4↓, TCF4↓, XIAP↓, PIK3CD↑, TCF3↑, TGFB1↑, GNAL↑, MAPK1↑, MAPK14↑Other genes: APC↑, CCNE1↑, CCNE2↑, CDC25A↑, CDK6↑, CYCS↑, E2F2↑, MAP2K1↑, MAPK13↑, PRKAR2B↑, RAPGEF3↑, RBL1↑, TFDP1↑, ARHGEF10↓, FOXO1↓, HHAT↓, IRS1↓, NF1↓, PAK3↓, PIK3CD↓, PRKAR1A↓, RALGDS↓, RHOV↓

[i] Genes downregulated (↓) or upregulated (↑) are shown. ATRA trMCF clone 11 refers to trMCF clone 11 treated with 10−6 M ATRA.

Discussion

In this study we showed that all trans-retinoic acid (ATRA) induced branching of early transformed human breast epithelial cells. The transformed trMCF clone 11 cells form spherical masses in collagen (3D culture) and treatment with 10−6 M ATRA produced a significant decrease in spherical masses and an increased number of tubules. Cells at an advanced stage of transformation (bsMCF and caMCF) did not show any change in morphology after being treated with ATRA. Our previous results showed that RARβ (retinoic acid receptor β) was unmethylated in MCF-10F and trMCF cells and became hypermethylated at the invasive (bsMCF) and tumorigenic (caMCF) stages (19); although bsMCF and caMCF were treated with 5-aza-dC to reactivate the expression of RARβ in combination with ATRA, no changes in the phenotype of these cells in collagen were observed. Our results indicate that ATRA is able to re-differentiate early transformed cells to a normal stage but, not tumor cells at later stages of the neoplastic process. We previously showed that bsMCF and caMCF had important chromosomal gains and losses and the earlier transformed cells trMCF showed small genomic changes (16); this could explain why ATRA was only effective as a re-differentiation agent in the early transformed breast epithelial cells. Different studies indicate that epigenetic modifications play important roles in RA transcriptional regulation (2024). Histones have a long N-terminal tail extending outside the nucleosome that is subject to acetylation, phosphorylation, and methylation (25). In the absence of RA, co-repressive elements (SMRT, NCoR and SIN3A) inhibit transcription; the presence of RA releases co-repressors and histone deacetylases allowing chromatin remodeling and access to specific RAREs (20,24). RA treatment leads to acetylation of histones H3 and H4 that lead to a more open stage of the chromatin allowing the transcription of ATRA regulated genes. However, only a limited number of information is currently available on the epigenetic dynamics of RA response.

Recently, analysis of gene expression array datasets of different FDA approved drugs revealed that ATRA (tretinoin) is a drug that is negatively associated with cancer stem cell (CSC) enriched gene expression signature (26). We found that ATRA treatment reduced the expression of the stem cell marker CD44 in early transformed cells. ATRA exerts effects on stem cell differentiation in part via the modulation of the epigenome. Numerous enzymes that alter the modifications on histones are involved in transcriptional activation of specific genes in stem cells, and many of these enzymes are modulated by RA treatment of stem cells (27). The expression of several genes encoding enzymes involved in chromatin modifications such as MGEA5, ATF-2, KDM5B, PRMT2, PHF21A and NSD1 were dysregulated in trMCF clone 11, reaching normal levels after ATRA treatment. Others have shown that in breast cancer, retinoids are effective inhibitors of breast cancer cells at early stages of tumor progression, but their effectiveness diminishes as the tumors become more aggressive (28). Our results support these findings.

Our results show that the RA concentration is important to induced re-differentiation of early transformed breast epithelial cells. The treatment of transformed cells with either 10−7 or 10−8 M ATRA did not induced any change in morphology although, cells were able to form tubules after treatment with 10−5 and 10−6 M ATRA, more tubules being developed after treatment with 10−6 M (1 μM) ATRA.

Little is known about the genomic targets and effects of the different isoforms of the RARs and mechanism or extent of crosstalk between RA signaling and other signaling pathways. It has been recently shown that RAR binding through the genome is highly coincident with estrogen receptor α binding, resulting in widespread crosstalk of RA and estrogen signaling to antagonistically regulate breast-cancer associated genes (29). Our gene expression studies determined 443 genes which defined a signature of ATRA re-programming effect in early transformed breast epithelial cells; these genes were dysregulated in the early transformed cells and they reached normal levels after the cells were treated with 10−6 M ATRA. Genes from the aryl hydrocarbon receptor (AhR), retinoic acid receptor (RAR) and the xenobiotic pathways were dysregulated in the early transformed breast epithelial cells and their expression reached normal levels after ATRA treatment. It has been shown that there is an interaction between AhR and RAR activation and that AhR not only binds to polycyclic aromatic hydrocarbon family of environmental contaminants but also to some synthetic retinoids (30,31).

N-(4-hydoxyphenyl) retinamide (fenretinide or 4HPR) is a synthetic retinoid that is currently one of the most promising clinically tested retinoids. The modification of the carboxyl end of all-trans RA with N-4-hydroxyphenyl group resulted in increased efficacy as a chemoprevention agent as well as reduced toxicity when compared with other retinoids (32). Animal models have demonstrated that treatment with fenretinide prevents chemically induced cancers of the breast, prostate, bladder and skin (3336).

In conclusion, our results showed that 1 μM ATRA was able to re-differentiate transformed cells at early stages of the neoplastic process and antagonistically regulated breast cancer associated genes. Our results support previous findings that 1 μM ATRA could be used as a chemo-preventive agent to inhibit the progression of premalignant lesions of the breast.

Acknowledgements

We thank Karen Trush for helping in the preparation of the final figures. This study was supported by The Pennsylvania Breast Cancer Coalition and Friends for an Earlier Breast Cancer Test.

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Journal Cover

June 2014
Volume 44 Issue 6

Print ISSN: 1019-6439
Online ISSN:1791-2423

2013 Impact Factor: 2.773
Ranked #30/202 Oncology
(total number of cites)

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