Oncogene (2004) 23, 777–785 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc

High-mobility group A1a regulates Ras/ERK signaling in MCF-7 human breast cells

Nathan R Treff1, Derek Pouchnik1, Gregory A Dement1, Rachel L Britt1 and Raymond Reeves*,1

1School of Molecular Biosciences, Washington State University, PO Box 644660, Pullman, WA 99164-4660, USA

High-mobility group (HMG)A1 are (formerly called HMG-1/2) and HMGN (formerly regulatory factors whose overexpression is frequently called HMG-14/17 (Bustin, 2001)), which have structu- observed in naturally occurring human . The rally distinct DNA-binding domains (Thomas and overexpression of transgenic HMGA1 proteins in cells Travers, 2001). The distinguishing feature of the results in neoplastic transformation and promotes pro- HMGA proteins is the presence in each protein of three gression to malignant cellular phenotypes. To understand independent DNA-binding peptide motifs, called ‘AT the underlying molecular and biological events involved in hooks’, which preferentially bind to the minor groove of these phenomena, we used oligonucleotide microarray AT-rich regions of B-form DNA (Reeves and Nissen, analyses to generate an HMGA1a-induced expression 1990). Members of the HMGA are coded profile for approximately 22 000 . This gene expres- for by two different genes, HMGA1 and HMGA2, which sion profile was generated using a well-characterized are located on different in humans transgenic human MCF-7 mammary adenocarcinoma cell (reviewed in Reeves, 2000). The human HMGA1 gene line in which overexpression of transgenic HMGA1 codes for two major protein isoforms, HMGA1a (a.k.a, promotes a transition to a more malignant and metastatic HMG-I) and HMGA1b (a.k.a, HMG-Y), which differ phenotype. Microarray expression analyses, together with by 11 internal amino acids as a result of alternative independent quantitative real-time reverse transcriptase messenger RNA splicing (Johnson et al., 1989; Friedmann polymerase chain reaction results, indicate that HMGA1a et al., 1993). HMGA1 proteins are architectural regulates genes involved in the Ras-extracellular signal- transcription factors that regulate the transcriptional related kinase (Ras/ERK)mitogenic signaling pathway, activity of a large number of mammalian genes by including KIT ligand and caveolins 1 and 2. We also found inducing changes in chromatin structure and by that many cholesterol biosynthesis genes were decreased controlling the formation of stereo-specific, multiprotein in cells overexpressing HMGA1a. Cholesterol depletion, complexes called ‘enhancesomes’ on the promoter/ decreased caveolin, and increased KIT ligand expression, enhancer regions of genes (reviewed in Reeves and are all independently associated with the activation of Beckerbauer, 2001; Reeves, 2001). HMGA1 proteins are Ras/ERK signaling. Upon further analysis, we found that also among the most highly modified of all the sensitivity to epidermal growth factor activation of ERK mammalian nuclear proteins, and their complex pat- phosphorylation was significantly higher, and that choles- terns of secondary biochemical modifications have been terol was significantly depleted, in cells overexpressing demonstrated to modulate their in vivo biological HMGA1a. The cumulative evidence indicates that one activity and function (Banks et al., 2000; review in likely mechanism by which the HMGA1a protein Reeves, 2001). Both the post-transcriptional biochem- promotes malignant changes in cells is through increased ical modifications found on HMGA1 proteins in vivo sensitivity to the activation of the Ras/ERK signaling and the transcriptional activity of the HMGA1 gene pathway. itself (Friedmann et al., 1993; Ogram and Reeves, 1995) Oncogene (2004) 23, 777–785. doi:10.1038/sj.onc.1207167 are direct downstream targets of numerous signal transduction pathways making them highly responsive Keywords: HMGA1; MCF-7 cells; Ras/ERK; caveolin; to various external environmental stimuli (review in KIT ligand; cholesterol Reeves, 2001). HMGA1 is a bona fide oncogene whose artificially induced overexpression causes both neoplastic transfor- mation of nonmalignant cells (Wood et al., 2000a) and Introduction promotes the progression of tumor cells from a low grade of malignancy to a highly metastatic and The mammalian high-mobility group (HMG) nonhis- aggressive phenotype (Reeves et al., 2001). Importantly, tone chromatin proteins are composed of three protein constitutively high levels of HMGA1 proteins are also families, HMGA (formerly called HMG-I/Y), HMG-B found in many different types of naturally occurring cancers, including those of the colon, prostate, thyroid, *Correspondence: R Reeves; E-mail: [email protected] breast, pancreas, uterus, skin, intestine, brain, lung, and Received 8 July 2003; revised 26 August 2003; accepted 27 August 2003 blood (Giancotti et al., 1989; Ram et al., 1993; Tamimi HMGA1-induced profile NR Treff et al 778 et al., 1993; Chiappetta et al., 1995; Fedele et al., 1996; expression of HMGA1-regulated genes, with functions Bandiera et al., 1998; Giannini et al., 1999; Abe et al., related to malignant progression of breast cancer, has 2000; Rajeswari et al., 2001; Lee et al., 2002; Pierantoni been suggested to promote these pathological pheno- et al., 2003, respectively), while the expression levels in typic changes. For example, prior work from our normal differentiated somatic tissues are usually ex- laboratory revealed that genes coding for certain cell tremely low and often nearly undetectable (Johnson surface integrin proteins and intracellular proteins in et al., 1988, 1989; Lundberg et al., 1989). Indeed, integrin signaling pathways are upregulated in response HMGA1 overexpression is such a consistent and wide- to overexpression of the HMGA1b isoform protein in spread feature of cancers that it has been suggested to be transgenic MCF-7 cells (Reeves et al., 2001). Here, we a diagnostic marker of neoplastic transformation with report results from expression analyses using high- increasing cellular protein concentrations being asso- density oligonucleotide microarrays, indicating that the ciated with increasing degrees of malignancy and HMGA1a isoform protein, like the HMGA1b protein, metastatic potential (reviewed in Tallini and Dal Cin, also regulates genes in MCF-7 cells associated with 1999; Reeves, 2000; Reeves and Beckerbauer, 2001). increased tumorigenic potential. In addition, we have Intriguingly, transcription of the HMGA1 gene is identified the Ras-extracellular signal-related kinase regulated by both c- (Wood et al., 2000b) and (Ras/ERK) signaling pathway as one of the likely AP-1 (Ogram and Reeves, 1995) oncogenic transcription molecular mechanisms by which overexpression of factors, which are aberrantly expressed in a variety of HMGA1a proteins promotes malignant metastatic tumors. progression of mammary cell tumors. Importantly, the Together, the above results imply that the HMGA1 present results provide additional support to the notion gene and its protein products are attractive targets for that the HMGA1 gene and its encoded proteins are potential anticancer chemotherapy. Consistent with this potential new chemotherapeutic targets for inhibiting suggestion is the observation that antisense inhibition of the activation of the Ras/ERK pathway in cancer cells. HMGA1 gene expression prevents neoplastic transfor- mation of rat thyroid cells (Berlingieri et al., 1995) and partially reverses the malignant phenotype of both Burkitt’s lymphoma (Wood et al., 2000b) and other Results highly malignant cancer cells (Reeves et al., 2001). Antisense inhibition of HMGA1 expression also induces RNA was extracted from well-characterized parental apoptosis in anaplastic human thyroid cancer cells but (control) MCF-7 cells and from ‘HMGA1a-ON’ trans- not in normal thyroid cells (Scala et al., 2000). genic MCF-7 cells that constitutively overexpress Furthermore, a mitosense-based antitumor drug transgenic HMGA1a protein in the absence of tetra- (FK317) that chemically crosslinks HMGA1 proteins cycline (Reeves et al., 2001) and used to synthesize to the minor groove of DNA in vivo (Beckerbauer et al., biotinylated ‘target’ cRNAs. These ‘target’ cRNAs were 2000) is currently being evaluated in clinical trials as a then used as hybridization probes for screening high- potential new anticancer drug. Since increased HMGA1 density oligonucleotide microarrays. The resulting protein expression is widespread in human cancers and hybridization signals on the microarrays allowed for a has shown promise as a chemotherapeutic target, quantitative determination of the relative amount of understanding the molecular mechanisms that underlie transcriptional expression of individual genes in both its oncogenic activity should have important clinical the control and the ‘ON’ MCF-7 cells. The hybridiza- implications. tion data also provided an unbiased assessment of In this study, we investigated the effects that whether or not the relative expression of a particular physiologically relevant overexpression of the HMGA1a endogenous gene was decreased or increased in response isoform protein has on the transcription of endogenous to HMGA1a protein overexpression in cells. Target genes in genetically engineered MCF-7 cells. In order to cRNAs were prepared in three independent isolations identify HMGA1a-regulated genes, we used a stable from both control and HMGA1a-overexpressing cell MCF-7 cell line (M/tet/HA-I) that exhibited constitutive lines. For statistical purposes, three independent micro- expression of a transgenic HA-tagged HMGA1a pro- array analyses were performed in order to reduce the tein, in the absence of tetracycline (Reeves et al., 2001), occurrence of both false-positive and false-negative gene and characterized the expression profile of over 22 000 classification (Lee et al., 2000) significantly. Generating endogenous human genes for a response to HMGA1a three replicates also allowed for reliable analysis of overexpression. Previous studies have demonstrated that genes whose expression exhibited a change of 71.5-fold when HMGA1a protein is overexpressed in these or greater (Daoud et al., 2003). The Affymetrix U133A transgenic MCF-7 cells they acquire a much more oligonucleotide microarray chips used for these hybri- malignant phenotype. These HMGA1a-overexpressing dization analyses contained probe sets for approxi- cells acquire the ability to grow in an anchorage- mately 22 000 well-characterized human genes. independent manner in soft agarose, form primary and The analysis of the oligonucleotide microarrays using metastatic tumors when injected into nude mice, and the Microarray Suite program (MAS, vers. 5.0; Affyme- undergo morphological and biochemical changes char- trix) provided a statistical means to determine the acteristic of a transition from an epithelial to a changes in the levels of expression of a given gene in mesenchymal phenotype (Reeves et al., 2001). Aberrant an experimental sample. The outcome was based on

Oncogene HMGA1-induced gene expression profile NR Treff et al 779 comparison of the hybridization efficiency, or signal, of microarray data sets analysed, the substantial upregula- the target cRNA to its complementary sequence. This tion of HMGA1a seen in HMGA1a-ON transgenic comparison took into account crosshybridization of the MCF-7 cells represented the most significant fold target cRNA of that gene to a mismatch sequence that is change in gene expression levels observed among all of exactly the same as the complementary sequence with the 22 215 human genes represented on the arrays. In the exception of one base. For each gene on the array, each experiment, this marked increase in HMGA1a there are 11 exact match complementary probes, each expression levels in the HMGA1a-ON, versus control, with a corresponding noncomplementary probe with cells was independently confirmed by reverse transcrip- one mismatch. The sequences of the complementary tase polymerase chain reaction (PCR), Northern, and probes for any given gene were selected from distinct Western analyses prior to microarray analysis (Figure 1). regions of that gene. Of particular importance is the fact that the 36.8713.9- Comparative analyses were carried out with the MAS fold change in HMGA1 gene expression, observed here, program using the levels of endogenous gene expression is within the physiological range of overexpression found in the parental MCF-7 cell line (control), which observed previously in naturally occurring cancer tissues was not overexpressing HMGA1a protein, as the (from 5- to 200-fold) (Giancotti et al., 1987; Johnson baseline. The genes whose expression in HMGA1a- et al., 1988; Ram et al., 1993; Giannini et al., 2000; Nam overexpressing cells, relative to the control, was et al., 2003; reviewed in Bustin and Reeves, 1996; Tallini consistently regulated either upward or downward by and Dal Cin, 1999). more than twofold in each of the three independent To further validate the microarray data, quantitative replicate microarray hybridization experiments, are real-time reverse transcriptase PCR (Q-PCR) was used listed in Table 1. A 36.8713.9-fold increase in HMGA1 to characterize the differential expression of several of gene expression was observed by microarray analysis in the genes in Table 1, including the caveolin 1, caveolin 2, the HMGA1a-ON transgenic MCF-7 cells compared to and Kit ligand (KITLG) genes (Figure 2), which play a the nontransgene-expressing parental (control) MCF-7 role in Ras/ERK signaling. The overall trend in altered cells. As expected, in each of the three independent gene expression levels for caveolin 1 (À2.970.3),

Table 1 Genes regulated by HMGA1 with greater than twofold change in the expression in three replicate data sets Gene title GenBank Accession # Fold changea

Upregulated High-mobility group AT-hook 1 NM_145899 36.8713.9 Dehydrogenase/reductase (SDR family) member 2 NM_005794 14.372.9 H. sapiens cDNA FLJ20338 fis, clone HEP12179, mRNA sequence AK000345 10.373.2 KIT ligand NM_000899 4.271.4 Ribosomal protein, large P2 NM_001004 3.670.6 Carcinoembryonic antigen-related cell adhesion molecule 7 NM_006890 3.570.7 Hypothetical protein, estradiol-induced NM_015516 3.271.0 Sperm-specific antigen 2 NM_006751 3.271.2 H. sapiens clone 24566 mRNA sequence AF070536 3.070.9 Solute carrier family 7 (cationic amino-acid transporter, y+ system), member 5 NM_003486 2.970.6 Argininosuccinate synthetase NM_000050 2.870.2 S100 calcium-binding protein A9 (calgranulin B) NM_002965 2.670.4 Monocytic leukemia zinc-finger protein-related factor NM_012330 2.571.1 Sarcoglycan, epsilon NM_003919 2.370.3 Tyrosinase-related protein 1 NM_000550 2.270.2 Stomatin NM_004099 2.270.5 B (flavin reductase (NADPH)) NM_000713 2.170.0

Downregulated MADS box transcription enhancer factor 2, polypeptide A (myocyte enhancer factor 2A) NM_005587 À7.670.3 Caveolin 1, caveolae protein, 22 kDa NM_001753 À4.172.2 Transforming growth factor, beta 2 NM_003238 À3.971.9 Transmembrane, prostate androgen-induced RNA NM_020182 À3.671.3 Sarcoglycan, gamma (35 kDa dystrophin-associated glycoprotein) NM_003238 À3.671.4 Carboxypeptidase E NM_001873 À3.671.2 ESTs, weakly similar to ubiquitously transcribed tetratricopeptide repeat gene BF511231 À3.570.6 Phosphodiesterase 8A NM_002605 À3.170.8 G-protein-coupled 51 NM_005458 À2.971.1 Hypothetical protein FLJ11619 NM_144710 À2.670.5 ESTs, moderately similar to hypothetical protein MGC5297 AW139448 À2.670.4 Insulin-induced gene 1 NM_005542 À2.570.4 Caveolin 2 NM_001233 À2.571.7 Homo sapiens mRNA; cDNA DKFZp434E033 (from clone DKFZp434E033) AL080130 À2.370.3 Dipeptidylpeptidase 7 NM_013379 À2.170.1 aMean71 s.d.

Oncogene HMGA1-induced gene expression profile NR Treff et al 780

Figure 2 Q- PCR analysis. Panel a shows linear amplification profiles for caveolin 1, caveolin 2, and KITLG in cells overexpressing transgenic HMGA1a (ON) and cells not expressing transgenic HMGA1a (OFF) and include no template controls. Panel b shows a comparison of fold-change values determined by Affymetrix micro- Figure 1 Characterization of differential expression of HMGA1a in array analysis and by Q-PCR. Error bars correspond to 71 s.d. MCF-7 cells, with (ON) or without (OFF) stable transfection of the pTRE-HA7C expression vector, using (a) multiplex quantitative RT– PCR (b) Northern and (c) Western blot analysis. For RT–PCR, the addition of primers for the housekeeping gene hypoxanthine phos- Table 2 Pathway-associated genes regulated by HMGA1 phoribosyl (HPRT1) was used as a normalization control, Gene title GenBank Accession # Fold changea the positive control reaction ( þ ) was carried out with pTRE-HA7C expression vector DNA as template, and the negative control (À) was Cholesterol biosynthesis carried out with no template. For Northern analysis, the 18S Insulin-induced gene 1 NM_005542 À2.570.4 ribosomal RNA band from the ethidium-stained formaldehyde gel HMG-CoA reductase NM_000859 À1.571.1 was used as a loading control. For Western analysis, the minor Mevalonate kinase NM_000431 À1.671.7 crossreactivity of crude anti-HMGA1 serum for H1 (H1) on Mevalonate pyrophosphate NM_002461 À1.771.3 the blots was used, along with Coomassie Blue staining of the proteins decarboxylase on SDS–polyacrylamide gels, used as a protein loading control 7-Dehydrocholesterol NM_001360 À1.671.1 reference standard reductase Isopentenyl pyrophosphate NM_004508 À1.571.3 caveolin 2 (À2.270.1), and KITLG (4.270.3) obtained by Q-PCR were similar to those obtained by microarray Ras/Erk signaling analysis. The fold-change averages of three replicates KIT ligand NM_000899 4.271.4 determined by Q-PCR are within the standard deviation ELK1 NM_005229 1.871.2 of and have a significantly smaller standard deviation Stat-3 NM_003150 1.871.5 Shc NM_003029 1.771.3 than averages obtained by microarray analysis. NM_000125 1.671.2 Microarray data were also characterized using Gen- c-Kit NM_000222 1.571.6 MAPP (Gene Microarray Pathway Profiler; Gladstone Caveolin 1 NM_001753 À4.172.2 Institutes), a program designed to view and analyse gene Caveolin 2 NM_001233 À2.271.7 7 expression data in the context of biological pathways PPAR-g NM_005037 À1.7 1.4 (Dahlquist, 2002; Doniger et al., 2003). The criteria for aMean71 s.d. increased and decreased gene expression levels using this program were set at X1.5 and pÀ1.5-fold change, respectively. Gene expression data were entered into we also experimentally demonstrated (Figure 3) that the GenMAPP as the average of three replicate data sets. total cholesterol level was significantly lower (o45%) in This provided statistical confidence for using the 71.5- HMGA1a-ON transgenic cells overexpressing fold difference in expression levels as valid criteria HMGA1a (6.671.2 ng cholesterol/mg cellular protein) (Daoud et al., 2003). Analysis using the subdirectory compared to untreated control cells not overexpressing ‘Metabolic MAPPs’ of the GenMAPP program revealed HMGA1a (14.971.4 ng cholesterol/mg cellular protein). subtle decreases in the expression of a number of genes Additional GenMAPP analyses using the same criteria involved in cholesterol biosynthesis, including (71.5-fold change) revealed both subtle and significant HMG-CoA reductase, mevalonate kinase, mevalonate changes in the expression of genes involved in Ras- pyrophosphate decarboxylase, 7-dehydrocholesterol re- extracellular signal-regulated kinase (Ras/ERK) signal ductase, and isopentenyl pyrophosphate isomerase transduction (Figure 4). These included decreased (Table 2). As an independent confirmation of the expression of caveolin 1, caveolin 2, and PPAR-g, and prediction of these microarray and GenMAPP analyses, increased expression of KITLG, its receptor, c-kit, and

Oncogene HMGA1-induced gene expression profile NR Treff et al 781

Figure 5 Western blot analysis of phosphorylated ERK-1/2 and total ERK-1/2 protein in parental control (OFF), and HMGA1a-over- expressing (ON) MCF-7 cells, with 0, 15, 30, and 60 min exposure to 30 ng/ml EGF. The intensity for phospho-ERK was determined by Figure 3 Cholesterol content of MCF-7 cells with (ON) or without densitometry, normalized to total ERK, and reported in arbitrary (OFF) expression of transgenic HMGA1a units. A significant change in phospho-ERK in ON versus OFF cells is indicated with an asterisk and a corresponding P-value above and within each bar, respectively

gating the properties of the ERK protein, the last common downstream member of this signaling cascade (Figure 4). Since microarray analyses did not reveal a significant difference in the levels of ERK mRNA expression in control versus HMGA1a-overexpressing cells (Figure 5; unpublished data), we assessed whether there might be detectable differences in the levels of phosphorylated ERK protein in these cells. For these experiments, we used Western blot analysis with a specific anti-phospho-ERK antibody to determine the sensitivity of control and HMGA1a-overexpressing cells to epidermal growth factor (EGF) exposure. As shown in Figure 5, and consistent with the microarray and GenMAPP predictions, results from these Western blot experiments demonstrated that transgenic MCF-7 cells overexpressing HMGA1a protein were significantly (2.170.3-fold) more sensitive to 15, 30, and 60 min of EGF activation of the Ras/ERK signaling pathway than were the control parental MCF-7 cells (P ¼ 0.006, 0.013, and 0.009 for 15, 30, and 60 min of EGF treatment, respectively).

Discussion

Induced overexpression of HMGA1 proteins promotes the progression of MCF-7 human breast cancer cells from a state of very low malignancy to a highly aggressive and metastatic phenotype (Reeves et al., 2001). In this study, oligonucleotide microarray analyses Figure 4 GenMAPP-generated Ras/ERK signaling pathway shaded were used to identify genes that were regulated, either to correspond with gene expression data. CAV1 ¼ caveolin 1; directly or indirectly, by the HMGA1a protein in MCF- CAV2 ¼ caveolin 2; ER ¼ estrogen receptor 7 cells overexpressing this protein. These expression array analyses led to the identification, among other Shc, as well as downstream target genes, Elk-1, Stat-3, things, that the HMGA1a protein regulates sensitivity to and estrogen receptor (ER). the activation of the Ras/ERK signaling pathway To further validate these GenMAPP predictions, we (Figure 5). Activated Ras/Raf/MEK/ERK signaling performed experiments to determine whether there were is a major pathway involved in the control of cell detectable differences in the Ras/ERK pathway in the growth, survival, and invasion in numerous types of untreated ‘control’ and HMGA1a-ON cells by investi- cancer cells and is, therefore, an attractive target for

Oncogene HMGA1-induced gene expression profile NR Treff et al 782 chemotherapeutic interventions (Weinstein-Oppenhei- by the demonstration that MCF-7 cells overexpressing mer et al., 2000; Peyssonnaux and Eychene, 2001; HMGA1a protein are significantly more sensitive to Hilger et al., 2002; Kolch, 2002; Lee and McCubrey, exogenous agents that stimulate the Ras/ERK pathway 2002; Smalley, 2003). (such as EGF) leading to the phosphorylation and The Ras/ERK pathway is initiated by extracellular activation of the ERK protein (Figure 5). Quantitative signals that lead to the activation of cytoplasmic immunocytochemical experiments employing the signaling molecules containing Src homology 2 (SH2) anti-phospho-ERK antibody to probe control and domains such as Shc. Shc is an SH2-phosphotyrosine- HMGA1-overexpressing cells also show a similar binding domain adaptor protein that links various HMGA1a-dependent increased EGF sensitivity of the tyrosine kinases to Ras by recruiting the Grb2/SOS Ras/ERK pathway (data not shown). complex to the plasma membrane (Pawson and Scott, The observed HMGA1a-induced changes in the 1997). SOS activation of Ras then initiates a kinase expression of endogenous genes in MCF-7 cells are in cascade that culminates in the activation of the mitogen- agreement with a number of other previously reported activated protein kinase ERK (Marshall, 1995). Acti- findings relating to cancer. For example, it has been vated ERK phosphorylates transcription factors such as demonstrated that artificially induced expression of Elk-1 and promotes transcription of various target genes caveolin 1 in highly malignant MCF-7 cells inhibits leading to the proliferation of the cell. their anchorage-independent growth (Fiucci et al., In this study, we show that overexpression of the 2002). Consistent with this finding, here we demonstrate HMGA1a isoform upregulates the expression of genes that the overexpression of HMGA1a downregulates the coding for both KITLG and its receptor c-kit. The expression of caveolin 1 in MCF-7 cells, while previous KITLG/c-kit complex is known to activate the Ras/ studies from this laboratory have shown that such ERK cascade through the phosphorylation of Shc overexpression also induces anchorage-independent (Lennartsson et al., 1999). We, likewise, demonstrate growth of MCF-7 cells (Reeves et al., 2001). Similarly, that caveolins 1 and 2 are downregulated by the other researchers have reported that expression of overexpression of HMGA1a protein. Caveolin 1 is TGFb2, which is downregulated by HMGA1a over- known to regulate negatively the Ras/ERK pathway expression (Table 1), is correlated with phenotypically (Engelman et al., 1998) and it has been suggested that less malignant human breast cancers that respond better caveolin 1 may inhibit laminin-induced activation of to tamoxifen chemotherapy (Kopp et al., 1995; Brandt ERK by sequestering Fyn and Shc in lipid rafts, thereby et al., 2003). Likewise, it has been shown that expression preventing them from interacting with a-integrins of the insulin-induced protein 1 gene is significantly (Fiucci et al., 2002). Genes encoding involved decreased in human gastric cancers (Kaneda et al., in the synthesis of cholesterol, which is an integral part 2002), and here we demonstrate that the expression of of lipid rafts, were found to be downregulated by this gene is downregulated approximately 2.5-fold in HMGA1a (Table 2). Likewise, insulin-induced gene 1, cells overexpressing the HMGA1a protein (Table 1). known to play an important role in regulating the levels Recently, Charpentier et al. (2000) reported that the of cholesterol within the cell (Janowski, 2002), was expression of the estradiol-induced hypothetical protein, downregulated in HMGA1a-overexpressing cells E2IG4, is increased in MCF-7 cells following exposure (Table 1). Cholesterol depletion of caveolae has been to mitogenic concentrations of estrogen (Charpentier shown to result in the activation of Ras/ERK signaling et al., 2000). Finally, there are reports that increased (Furuchi and Anderson, 1998). An enzymatic assay for transcriptional expression of the KITLG gene, shown total cholesterol demonstrated that cholesterol was, as here to be upregulated by overexpression of HMGA1a predicted, significantly depleted in HMGA1a-overex- (Table 1), is associated with both density-independent pressing cells (Figure 3). Together, these results indicate cell growth (Caceres-Cortes et al., 2001) and increased that HMGA1 regulates a number of factors that can angiogenesis of cancer cells (Zhang et al., 2000). lead to the activation of Ras/ERK signaling at or near In combination with the previous findings (Reeves the cell surface. et al., 2001), the results reported here strongly suggest In addition to regulating expression of genes involved that one of the key (but certainly not only) ways that in cell surface initiated events, evidence presented here overexpression of HMGA1 proteins promotes tumor also indicates that HMGA1 causes subtle changes in the progression and increased metastatic potential of human expression of genes coding for proteins that participate MCF-7 mammary breast cancer cells is through in downstream parts of the Ras/ERK signaling path- significantly increasing sensitivity to the activation of way. For example, we demonstrate that the expression the Ras/ERK signaling pathway. The HMGA1 genes of Shc is increased by 1.7-fold in cells overexpressing and proteins may, therefore, represent new chemother- HMGA1a. Other downstream targets of the Ras/ERK apeutic targets for breast cancer treatment. Most of the pathway are likewise regulated by HMGA1a over- present approaches to inhibiting the Ras/ERK pathway expression including increased expression of Stat-3 and in cancerous cells focus on drugs that interfere with the ER, and decreased expression of PPAR-g (Figure 4). enzymatic activity of various kinases in the signaling These results, while subtle, correspond to the expected cascade. In combination with these existing drug changes in cells with activated Ras/ERK signaling strategies, targeting the activity of the HMGA1 genes (Peyssonnaux and Eychene, 2001; Lee and McCubrey, or proteins could prove to be a more effective means of 2002; Smalley, 2003) and are independently supported cancer treatment than is currently available.

Oncogene HMGA1-induced gene expression profile NR Treff et al 783 Materials and methods used along with anti-p44/42 MAPK (ERK-1/2) antibody (antibody #9102), as recommended by the suppliers, to Cell culture characterize Ras/ERK signaling activation. Molecular Dy- namics ImageQuaNT version 4.1b was used to quantify the The MCF-7 cell line (catalog no. HTB-22, ATCC; Manassas, intensity of phospho-ERK and total ERK bands for each VA, USA) and the hemagglutinin (HA)-tagged HMGA1a- treatment (n ¼ 3). An unpaired Student’s t-test was used to expressing derivative (M/tet/HA-I-Cs) (HMGA1a-ON), stably calculate the probability that the differences are due to chance transfected with the pTRE-HA7C (HMGA1a) expression (P-value). A P-value less than 0.05 was considered statis- vector, were maintained as described previously in Reeves tically significant. et al. (2001). Briefly, cells were grown in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with 2 mML-gutamine, 10 mM HEPES, RT–PCR analysis 10% fetal bovine serum, penicillin G sodium at 100 U/ml, Total RNA (1 mg) from TRIzol (Invitrogen) extraction streptomycin sulfate at 100 mg/ml, and hygromycin B at was reverse-transcribed using murine Moloney leukemia 100 mg/ml, and maintained as recommended by the supplier. virus reverse transcriptase (MMLV-RT (Invitrogen) and For the activation of the Ras/ERK pathway, three replicates oligonucleotide (dT) of cells were treated for 0, 15, 30, and 60 min with 30 ng/ml 12À18 primer (Invitrogen). One-twentieth murine natural EGF (Invitrogen, Carlsbad, CA, USA). of the first-strand synthesis reaction was used as template in a PCR with both HMGA1-specific primers (upper, 50-AG ATCTATGAGTGAGT CGAGCTCGAAG-30; lower-50 GG Northern analysis ATCCTGCTGCTCCTCCTCCG-30) and HPRT1-specific pri- Total RNA was isolated from the HMGA1a-expressing, and mers (Clontech). Reaction conditions included 1.5 mM MgCl2, parental, MCF-7 cells by using TRIzol reagent (Life Technol- 200 nM dNTPs, and 0.75 mM of each primer. PCR was set up ogies, Inc., Gaithersburg, MD, USA) and analysed for for 30 cycles of 961C for 30 s, 521C for 45 s, and 721C for 1 min. quantity and quality using ultraviolet spectrophotometry and Amplification was visualized on an ethidium-stained 1% agarose-gel electrophoresis, respectively. Total RNA (15 mg) agarose gel. for each sample was electrophoresed on a 1% agarose gel with 5% formaldehyde for 3 h at 100 V. Separated RNA was Microarray analysis transferred to a nylon membrane (Micron Separations Inc., Westboro, MA, USA) and incubated with PerfectHyb Plus TRIzol (Invitrogen) extracted and RNeasy column (QIAGEN, hybridization buffer (Sigma) for 1 h at 651C. An HMGA1- Valencia, CA, USA) purified total RNA (10 mg) was used to specific probe was generated by using an HMGA1a restriction synthesize the microarray target. Target synthesized from fragment from the pTRE-derived (BD Biosciences Clontech, RNA from three independent parental and HMGA1a-expres- Mountain View, CA, USA) HMGA1a expression vector DNA sing MCF-7 cell preparations was hybridized to the microarrays. (pTRE-HA7C; Reeves et al., 2001) as template for incorpor- RNA was reverse transcribed to generate double-stranded ating [a-32P]dATP with the RadPrime labeling kit (Invitrogen). cDNA with a T7 promoter-containing primer using Super- Approximately, 3 Â 108 c.p.m. of probe was incubated with the script II, RNase H, and DNA polymerase (Invitrogen). After prehybridized membrane overnight at 651C. The membrane extraction with phenol–chloroform and ethanol precipitation was then washed once with 2 Â SSC and 0.1% sodium dodecyl with ammonium acetate, the cDNA was used as template in an sulfate (SDS) at room temperature for 10 min, and twice with in vitro transcription reaction with biotin-labeled RNA 0.2 Â SSC and 0.1% SDS at 651C for 10 min. Following (MEGAscript T7, High Yield Translation kit, Ambion, overnight exposure to a phosphorimager screen, the hybridiza- Austin, TX, USA). Resulting target cRNA was collected on tion signal was recorded with a Molecular Dynamics RNeasy columns (QIAGEN) and then fragmented for PhosphorImager (Amersham Biosciences, Piscataway, NJ, hybridization to the microarrays. Affymetrix USA). U133A gene chip arrays were used to analyse gene expression (Affymetrix Inc., Santa Clara, CA, USA). This array consists of approximately 22 000 genes from Homo sapiens. Probes Western blot analysis consist of 11 pairs of 25-mer oligonucleotides for each gene. Total protein was prepared from TRIzol extractions by One member of each pair contains a single base point following the manufacturer’s protocol. Equal amounts of mutation, and the signals of the pairs are compared to assess protein were separated by electrophoresis on an SDS–15% specificity of hybridization. Biotinylated target cRNA (15 mg) polyacrylamide minigel and transferred to a nitrocellulose was hybridized to the array and then processed using the membrane (Immobilon-P, Millipore, Bedford, MA, USA) Affymetrix GeneChip Fluidics Workstation 400, using the using a semidry electrophoretic transfer cell (Trans-Blot SD; Mini Euk 2v3 protocol. After binding with phycoerythrin- BioRad, Hercules, CA, USA). Immunodetection of the coupled avidin, microarrays were scanned on an Agilent HMGA1 proteins on the membrane was performed using a Technologies Gene Array Scanner (Agilent Technologies, Palo rabbit polyclonal anti-HMGA1 antibody (MR19), as de- Alto, CA, USA). Results were analysed using Affymetrix scribed in Reeves and Nissen (1999) and a goat anti-rabbit Microarray Software (MAS) 5.0. Individual microarrays were horseradish peroxidase (HRP)-conjugated secondary antibody scaled to produce a mean signal intensity of 125. Iterative (BioRad Laboratories, Hercules, CA, USA). Enhanced comparisons of different microarray data sets were performed chemiluminescence detection (Pierce Biotechnology Inc., with MAS 5.0 comparison analysis as described previously Rockford, IL, USA) and hyperfilm MP (Amersham) were with the modifications in Chen et al. (2000). Three replicate used for visualization. Anti-HMGA1 histone H1 cross- HMGA1a-expressing MCF-7 cell microarray data sets were reactive band of the membrane, along with Coomassie Blue compared with each of the three replicate parental MCF-7 cell staining of the proteins on SDS–polyacrylamide gels, was used (control) microarray data sets to determine the expression to confirm equal protein loading. Anti-phospho ERK-1/2 difference between HMGA1a and control. This comparison (phospho-p44/42 MAPK Thr202/Tyr204) antibody (antibody strategy allowed statistical comparison of fold-change values #9101; Cell Signaling Technology, Beverly, MA, USA) was (converted from signal log ratio obtained from MAS 5.0) for

Oncogene HMGA1-induced gene expression profile NR Treff et al 784 genes that survived the three pairwise comparisons with a fold signal occurs only if the target sequence is complementary to change X2. For analysis with the Gene MicroArray Pathway the probe and is amplified during PCR. Fluorescence is Profiler (GenMAPP, Version 1.0; Gladstone Institutes, Uni- detected at each cycle using an ABI PRISMs 7000 Sequence versity of California, SF, USA; www.GenMAPP.org), the Detection System (Applied Biosystems). Relative quantitation average fold-change values for each gene on the microarray, was carried out using the comparative cycle threshold (CT) along with the Swiss-Prot database ID supplied by Affymetrix, method as recommended by the supplier. were uploaded as a comma separated value-formatted file. An average fold change of greater than or equal to 1.5 was used as the criteria for whether a specific gene was up- or down- Cholesterol assay regulated by HMGA1. Isolation of total cholesterol was carried out following Heider and Boyett (1978). Approximately, 1 Â 106 parental and Quantitative real-time RT–PCR analysis HMGA1a-overexpressing MCF-7 cells were washed in phos- phate-buffered saline and spun at 1000 r.p.m. for 10 min. The Q-PCR was performed using Assays-on-Demand and an ABI s pellet was resuspended in 1 ml isopropanol and sonicated for PRISM 7000 Sequence Detection System on human target 15 s at 35% output (Fisher sonic dismembrator model 300) to genes, caveolin 1, caveolin 2, and c-kit ligand, and an extract cholesterol and cholesterol esters. The sonicated endogenous control, HPRT1 (assay IDs Hs00184697_m1, suspension was spun at 12 000 g for 15 min. Supernatant Hs00184597_m1, Hs00241497_m1, and Hs99999909_m1, re- (100 ml) was added to 700 ml Infinity Cholesterol Reagent spectively; Applied Biosystems, Foster City, CA, USA). (Sigma Diagnostics, St Louis, MO, USA) for 15 min at B241C TRIzol (1 mg) extracted total RNA was reverse transcribed before recording the absorbance at 510 nm. A standard curve using 20 mM oligonucleotide (dT)12À18, and 1 U MMLV-RT was generated by diluting 50 ml of 100, 200, and 400 mg/dl (Invitrogen). One-twentieth of the RT reaction (50 ng of RNA cholesterol standards into 950 ml of isopropanol. A measure of converted to cDNA) was used as template for the PCR step 100 ml of each solution, along with isopropanol alone (used for using TaqMan Universal PCR Master Mix, No AmpErase background correction), were added, in parallel, to 700 ml UNG, as recommended by the supplier (Applied Biosystems). Infinity Cholesterol Reagent for 15 min at B241C before Q-PCR was performed using Assays-on Demand and TaqMan 0 recording the absorbance at 510 nm. For each experimental 5 fluorogenic nuclease chemistry (Applied Biosystems). Each sample, total cholesterol was normalized to total protein from assay consists of two unlabeled PCR primers, and an FAM the isopropanol pellet as determined by absorbance at 280 nm dye-labeled TaqMan minor groove binder (MGB) probe. and a standard curve with bovine serum albumin. During PCR, the TaqMan MGB probe anneals specifically to cDNA between the forward and reverse primer sites. When the probe is intact, the proximity of the reporter dye to the Acknowledgements quencher dye results in the suppression of the reporter This work was supported, in part, by NIH Grant GM-46352 fluorescence. AmpliTaq Gold DNA polymerase cleaves only and WSU Cancer Research Development Fund grant 17A- probes that are hybridized to the target. Cleavage separates the 0165 (both to RR), by NIH Grant T-32 GM008336 for reporter dye from the quencher dye, which results in increased predoctoral training in biotechnology (to NRT), and by the fluorescence by the reporter. The increase in fluorescence WSU Laboratory for Biotechnology and Bioanalysis I.

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