The identification of Hoxc8 target

Haiyan Lei*, Hailong Wang†, Aster H. Juan*, and Frank H. Ruddle*‡

*Department of Molecular, Cellular, and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, CT 06511; and †PhytoCeutica, Inc., 5 Science Park, Suite 13, New Haven, CT 06511

Contributed by Frank H. Ruddle, December 23, 2004 Hox genes encode transcription factors that control spatial pat- Hox deregulation is implicated in human cancers includ- terning during embryogenesis. To date, downstream targets of ing leukemia and colorectal, breast, renal, and lung cancers Hox genes have proven difficult to identify. Here, we describe (16–21). Expression of Hoxc8 correlates with higher Gleason studies designed to identify target genes under the control of the scores in prostate cancers (22). Hoxc8 is selectively activated in murine Hoxc8. We used a mouse 16,463 gene cervical cancer cells (23). Hematopoietic progenitor cells show oligonucleotide microarray to identify mRNAs whose expression abnormalities in Hoxc8-null mutant mice (24). However, it is not was altered by the overexpression of Hoxc8 in C57BL͞6J mouse clear how deregulation specifically effects neoplastic embryo fibroblasts (MEF) in cell culture (in vitro). We identified a inception and progression. Few studies have established direct total of 34 genes whose expression was changed by 2-fold or functional roles for Hox genes in carcinogenesis. greater: 16 genes were up-regulated, and 18 genes were down- In the present study, we overexpressed mouse Hoxc8 gene in regulated. The majority of genes encoded involved in the C57BL͞6J mouse embryo fibroblasts (MEF) cells and then critical biological processes, such as cell adhesion, migration, me- applied microarray assay to identify possible Hoxc8 target genes. tabolism, apoptosis, and tumorigenesis. Two genes showed high Expression of candidate genes was also examined by semiquan- levels of regulation: (i) secreted phosphoprotein 1 (Spp1), also titative RT-PCR, and these data correlated well with the array known as osteopontin (OPN), was down-regulated 4.8-fold, and (ii) data. Chromatin immunoprecipitation (ChIP) assay confirmed frizzled homolog 2 (Drosophila)(Fzd2) was up-regulated 4.4-fold. that OPN is a direct transcriptional target of Hoxc8 in vivo. Most Chromatin immunoprecipitation (ChIP) analysis confirmed the di- of the 34 identified candidate target genes are involved in rect interaction between the OPN promoter and Hoxc8 in proliferation, adhesion, migration, metabolism, and related cel- vivo, supporting the view that OPN is a direct transcriptional target of Hoxc8. lular processes, and can be viewed as global regulators of growth and differentiation (25). In general, our results suggest that Hox chromatin immunoprecipitation ͉ microarray ͉ osteopontin genes may play important roles in cancer progression by serving as modulators in neoplastic pathways. ox genes regulate anterior͞posterior developmental pat- Materials and Methods Hterning in an extensive domain, extending from the mid- brain͞hindbrain junction to the tail. The individual genes are Cell Culture, Plasmid Construction, and Transfection. Cells were expressed in an overlapping array, each regulating differentia- cultured in DMEM (GIBCO͞BRL) supplemented with 10% tion and morphogenesis in their individual expression domains FCS (HyClone) and 100 ␮g͞ml penicillin–streptomycin– along the anterior͞posterior axis. The Hoxc8 gene has been glutamine (GIBCO͞BRL). MEF cell lines were cultured from studied in considerable detail by us and others (1–4). Expression 15.5-dpc C57BL͞6J embryos and were immortalized by trans- analysis shows that the gene is expressed initially at 8 days fecting an SV40 T-antigen plasmid (pPVU0neo) (26). Cells were postconception (dpc) in the tail bud and then extends to an plated in six-well plates and transfected with 1 ␮g of pPVU0neo anterior position at the level of the forelimbs. A posterior limit plasmid DNA by using FuGENE 6 Transfection Reagent (Roche of expression is defined later at the junction between the thoracic Molecular Biochemicals). Stable transfectants were selected in and lumbar regions. The gene is expressed in both the neural media containing 500 ␮g͞ml G-418 (GIBCO͞BRL). Hoxc8- tube and the somites in the prospective thorax (5–7). Null overexpressing cells were produced by transfecting immortalized mutants of Hoxc8 show neuromuscular defects in the forelimb MEFs with the pcDNA4͞Hoxc8 plasmid. The plasmid pcDNA4͞ and skeletal defects in the ribs and vertebrae of the thorax (8). Hoxc8 was constructed by RT-PCR amplification of Hoxc8 We have shown recently that a retardation of Hoxc8 expression cDNA and cloning the amplicon into the KpnI͞EcoRI site of the results in the phenocopy of Hoxc8-null mutations, demonstrating zeomycin-resistant expression vector pcDNA4 (Invitrogen). The the criticality of expression timing for the Hox transcription insert was sequenced to affirm that no enzymatic errors had been factors (9). introduced. Immortalized MEF cells were transfected with the The tissue-specific overexpression of Hoxc8 has been shown to pcDNA4͞Hoxc8 plasmid to produce Hoxc8-positive cell lines or inhibit chondrocyte maturation and stimulate chondrocyte pro- blank pcDNA4 vector to produce Hoxc8-negative control cell liferation (10). Bone morphogenetic protein (BMP) is a potent lines by using FuGENE 6 transfection reagent (Roche Molecular osteotropic protein that induces osteoblast differentiation and Biochemicals). Zeomycin (GIBCO͞BRL) at 500 ␮g͞ml was bone formation. Hox-binding elements (ATTA) are common in added to the cell culture medium to select for stable Hoxc8- promoters of osteoblast differentiation marker genes, especially overexpressing cell lines and control cell lines. Three Hoxc8- those that rapidly respond to BMP stimulation, such as osteo- positive and three Hoxc8-negative colonies were selected at protegerin, BMP-4, and osteonectin (11–14). Gel retardation random and paired into three independent positive and negative studies have shown that Hoxc8 binds to ATTA-rich sites in the sets. Each set was subjected to microarray analysis. osteopontin (OPN) promoter domain (15), but evidence for functional interaction in vivo is lacking. BMP stimulation acti-

vates gene transcription by depressing Hoxc8 protein through the Abbreviations: dpc, days postconception; BMP, bone morphogenetic protein; OPN, os- interaction of Smad1 and Hoxc8 proteins. These results suggest teopontin; MEF, mouse embryo fibroblast; ChIP, chromatin immunoprecipitation; Fzd2, that direct interaction between Smad1 and Hoxc8 proteins frizzled homolog 2. represents a major mechanism of osteoblast differentiation in ‡To whom correspondence should be addressed. E-mail: [email protected]. BMP-induced skeletal development (15). © 2005 by The National Academy of Sciences of the USA

2420–2424 ͉ PNAS ͉ February 15, 2005 ͉ vol. 102 ͉ no. 7 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409700102 Downloaded by guest on September 27, 2021 Table 1. Primers for RT-PCR and ChIP analysis Gene Forward primer Reverse primer Size, bp

GAPDH CATCACCATCTTCCAGGAGC ATGCCAGTGAGCTTCCCGTC 500 Cdh11 CGGGAACCATTTTTGTGATT TCCACCGAGAAATAGGGTTG 343 OPN TCTGATGAGACCGTCACTGC TCTCCTGGCTCTCTTTGGAA 349 PRDC CTGTCTCCAGCTCCTTCCAC GATCTGGTGATGCCACCTCT 394 Pmp22 GCCAGCTCTTCACTCTCACC AACAGCAATCCCCACTCAAC 394 Fzd2 ATCTTTCTGTCCGGCTGCTA GCCAAGATGGTGATGGTTTT 326 Gas1 GCGAATCGGTCAAAGAGAAC TACAAGTGTGACCCGAGCAG 349 Zac1 TGAAGAACCACCTCCAGACC CTCTGGGCACAGAACTGACA 346 Hoxc8 CTACCAGCAGAACCCATGCT GGCGCTTTCTGGTCAAATAA 323 OPN promoter for ChIP GAAAGTTCTGCCGAGACAGC TGAGGTTTTTGCCACTACCC 371

RNA Preparation, Real-Time PCR Quantification, and Semiquantitative transfectants were selected in media containing 500 ␮g͞ml RT-PCR. Total RNA was isolated with RNeasy miniprep columns G-418 (GIBCO͞BRL). Thirty colonies were typically obtained and an RNase-free DNaseI kit (Qiagen, Valencia, CA). The from transfection. Real-time PCR was used to quantitate the cDNA for real-time PCR and semiquantitative RT-PCR was Hoxc8 expression level in each cell line. The lowest-expressing made by using the OmniScript kit (Qiagen). Real-time PCR was Hoxc8 cell line was selected for further study (real-time PCR performed in triplicate by using TaqMan probes and the ABI data not shown). A Hoxc8 expression vector, pcDNA4͞Hoxc8, Prism 7900 sequence detection system (Applied Biosystems). was transfected into the above selected MEF cell line to generate The DNA Taq polymerase (Qiagen) was used to carry out PCR the stable Hoxc8-overexpressing cell line. As a control, the same amplification. Primers and the expected product sizes for the cell line was transfected with empty pcDNA4 vectors. Total semiquantitative PCR are detailed in Table 1. All primers were RNA was obtained from the parental cells, three sets of Hoxc8 synthesized by Invitrogen. The PCR products were resolved on transfectants, and control transfectants, and the expression a 1.5% agarose gel containing ethidium bromide. levels of Hoxc8 were compared by RT-PCR. In the Hoxc8 transfectants, higher levels of Hoxc8 expression were observed in Analysis. We used the RNeasy miniprep columns the Hoxϩ1, Hoxϩ2, and Hoxϩ3 cells than in the parent cells and (Qiagen) to isolate total RNA from three sets of Hoxc8-positive the control transfectants, Conϩ1, Conϩ2, and Conϩ3 cells (Fig. and -negative cells as above. Each set was subjected to microar- 1). The specificity of the RT-PCR products was confirmed by ray analysis. The mouse 16K 70-mer oligo-operon microarray sequencing the amplified PCR bands. Real-time PCR results (OMM16K) was obtained from the Keck facility at Yale Uni- Ͼ versity. The oligonucleotide array gene set is described at showed that the Hoxc8 expression level was increased 10,000 BIOLOGY http:͞͞keck.med.yale.edu͞dnaarrays͞genelists. The biotinylated times in the three Hoxc8 transfectants compared with the three cRNA preparation, hybridization, and scanning of the microar- control transfectants. DEVELOPMENTAL rays were performed according to the manufacturer’s protocols. In brief, a total of 50 ␮g of RNA was reverse-transcribed by using Expression Array Analysis. We used a mouse 16K oligonucleotide Superscript II reagents (Invitrogen) at 42°C for 2 h. After RNA microarray to analyze gene expression profiles between the removal and cDNA probe purification, Cy5 and Cy3 fluorescent overexpressing Hoxc8 cell lines and the control cell lines. Three dyes were coupled to aa-dUTP-labeled samples and controls, groups of total RNA samples were prepared from MEF cells respectively, and cohybridized to the microarray slides. Microar- transfected with plasmid pcDNA4͞Hoxc8 or control empty ray protocols, including probe-labeling, cleaning, hybridization, pcDNA4 vector (see Materials and Methods). Probe labeling, and posthybridization washing procedures, can be found at hybridization, and scanning were done by the Keck facility (Yale http:͞͞keck.med.yale.edu͞dnaarrays͞protocols.htm. Hybridized University). We identified 34 genes that showed a minimum slides were scanned with a GenePix 4000A scanner (Axon 2-fold change in expression (Table 2). Sixteen genes were Instruments, Union City, CA), and the acquired images were up-regulated 2-fold or more, and 18 genes were down-regulated analyzed with GENEPIX PRO 3.0. Only those genes that showed a 2-fold or more. The majority of the identified genes are known minimum 2-fold increase or decrease in all three sets were to play roles in cell proliferation, differentiation, integration, included in the results presented in Table 2. The final values of apoptosis, metabolism, and carcinogenesis, with the exception of increase or decrease were calculated as the average of the values four genes whose function is unknown. for the three independent sets. Semiquantitative RT-PCR of Selected Differentially Expressed Genes ChIP Assay. The ChIP protocol was modified from the ChIP Assay kit (Upstate Biotechnology, Lake Placid, NY). To improve Identified by cDNA Microarray. To corroborate the cDNA microar- specificity, we performed two sequential ChIPs using the same ray data, semiquantitative RT-PCR analysis was performed on antibody for both the first and second steps. Hoxc8 monoclonal seven selected genes identified as being differentially expressed anibody (MMS-266R) was purchased from Covance Research (Fig. 2). These included osteopontin (OPN), growth-arrest- Products (Princeton). Anti-mouse IgG antibody (A-4312) was specific 1 (Gas1), peripheral myelin protein 22 (Pmp22), zinc purchased from Sigma. finger protein regulator of apoptosis (Zac1), protein related to DAN and Cerberus (PRDC), cadherin 11 (Cdh11), and frizzled Results homolog 2 (Fzd2). All PCR products were sequenced to prove Overexpression of Hoxc8 in C57BL͞6J MEF Cell Lines. To identify the specificity. In general, the RT-PCR data were consistent with the Hoxc8 candidate target genes, we first generated primary cDNA microarray data, showing that expression of Fzd2 and C57BL͞6J MEF cell lines. The primary MEF cells prepared Pmp22 was significantly increased and expression of OPN and from 15.5-dpc mouse embryo bodies were immortalized by Gas1 was decreased in Hoxc8-overexpression cells when com- transfecting an SV-40 T antigen-containing plasmid. Stable pared with control cells.

Lei et al. PNAS ͉ February 15, 2005 ͉ vol. 102 ͉ no. 7 ͉ 2421 Downloaded by guest on September 27, 2021 Table 2. Table of genes deregulated by overexpression of Hoxc8 in C57BL͞6J MEF cell lines GenBank accession no. definition Fold change no.

mRNA that decreases in the overexpression of Hoxc8 NM࿝009263 Mus musculus-secreted phosphoprotein 1 (Spp1, OPN) 4.8 5 NM࿝013468 ankyrin repeat domain 1 (cardiac muscle) (CARP) 2.1 19 NM࿝008471 Mus musculus keratin complex 1, acidic, gene 19 (Krt1–19) 2.2 11 NM࿝080288 engulfment and cell motility 1, ced-12 homolog (Ced-12h) 2.7 13 NM࿝021274 Mus musculus chemokine (C-X-C motif) ligand 10 (Cxcl10) 3.3 5 AF147785 Mus musculus zinc finger protein regulator of apoptosis (Zacl) 2.1 10 NM࿝009922 Mus musculus calponin 1 (Cnn1) 2.4 9 X15052 Mus musculus neural cell adhesion molecule 1 (NCAM) 2.4 9 D50410 Mus musculus mRNA for meltrin beta (ADAM19) 2.2 11 NM࿝008086 Mus musculus growth-arrest-specific 1 (Gas1) 2.2 13 NM࿝011825 Mus musculus protein related to DAC and cerberus (PRDC) 2.4 1 NM࿝011526 Mus musculus transgelin (Tagln) 2.3 8 NM࿝011340 Serine (or cysteine) proteinase inhibitor (Serpinf1) 3.0 11 BC022623 Mus musculus cDNA sequence BC022623 2.4 16 AK002886 Mus musculus adult male kidney cDNA, clone:0610041G09 2.6 4 BC019134 Mus musculus, clone IMAGE:5037334, mRNA, partial cds 2.3 9 NM࿝008706 Mus musculus NAD(P)H dehydrogenase, quinone 1 (Nqo1) 2.1 8 NM࿝007494 Mus musculus argininosuccinate synthetase 1 (Ass1) 2.3 13 mRNA that increases in the overexpression of Hoxc8 NM࿝020510 Mus musculus frizzled homolog 2 (Drosophila)(Fzd2) 4.4 11 NM࿝030696 Mus musculus monocarboxylate transporter 4 (MCTs) 2.5 11 NM࿝010330 Mus musculus embigin (Emb), mRNA 3.0 13 NM࿝010555 Mus musculus interleukin 1 , type II (I11r2), mRNA 2.3 1 NM࿝009866 Mus musculus cadherin 11 (Cdh11), mRNA 3.0 8 NM࿝013654 Mus musculus chemokine (C™C motif) ligand 7 (Cc17), mRNA 2.4 11 NM࿝011333 Mus musculus chemokine (C™C motif) ligand 2 (Ccl2), mRNA 3.0 11 NM࿝018857 Mus musculus mesothelin (Msln), mRNA 2.5 17 NM࿝010738 Mus musculus lymphocyte antigen 6 complex, locus A (Ly6a) 2.9 15 NM࿝008530 Mus musculus lymphocyte antigen 6 complex, locus F (Ly6f) 3.2 15 NM࿝010741 Mus musculus lymphocyte antigen 6 complex, locus C (Ly6c) 2.8 15 NM࿝008236 Mus musculus hairy and enhancer of split 2 (Hes2) 2.2 4 NM࿝012011 eukaryotic translation initiation factor 2, subunit 3 (Eif2s3y) 3.8 Y NM࿝008885 Mus musculus peripheral myelin protein (Pmp22), mRNA 2.1 11 NM࿝053188 Mus musculus steroid 5 alpha-reductase 2 (Srd5a2), mRNA 2.3 17 AK014614 Mus musculus day 0 neonate skin cDNA, clone:4633401122 3.3 2

ChIP Assay Confirms OPN as a Direct Target of Hoxc8 in Vivo. To determine whether the Hoxc8 protein can bind directly to Mouse-secreted phosphoprotein 1, also known as ostepontin the OPN promoter in vivo, we performed a ChIP assay of the (OPN), was found to be down-regulated Ϸ5-fold in the microar- OPN promoter (Fig. 3). The ChIP assay relies on the ability of ray analysis. Semiquantitative RT-PCR further confirmed that specific antibodies to immunoprecipitate DNA-binding proteins OPN is down-regulated in the Hoxc8-overexpressing cell lines along with the associated genomic DNA. Immunoprecipitation (Fig. 2). Shi et al. (15) reported that, in Cos-1 cells, Hoxc8 of DNA–protein complex by using an antibody against Hoxc8 interacts with Smad1 and that this interaction specifically acti- was performed on formaldehyde-crosslinked extract from over- vates OPN gene transcription in response to BMP stimulation. expressed Hoxc8 MEF cell lines or control MEF cell lines. We Five putative Hox-binding sites were identified within the first then measured the abundance of genomic DNA containing the 382 bp of the 5Ј-flanking region in the OPN gene. A gel-shift OPN promoter sequence within the immunoprecipitate complex assay was performed to demonstrate that Hoxc8 binds to the by PCR amplification. PCR products after ChIP assay were OPN promoter in vitro (15). sequenced to verify the identity of the amplified DNA. ChIP

Fig. 1. Semiquantitative RT-PCR analysis of the Hoxc8 gene expression in parent C57BL͞6J MEF cells and zeomycin-selected transfectants. Total cellular RNA was isolated from parent cells and three sets of pcDNA4͞Hoxc8 (Hox) or pcDNA4 (Con) transfectants, and, after reverse transcription, PCR was performed with Hoxc8- and GAPDH-specific primers. The up-regulation of the 323-bp Hoxc8-specific band was detected in cells transfected with pcDNA4͞Hoxc8 plasmid. A 100-bp DNA ladder (Biolabs) was used for size markers.

2422 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409700102 Lei et al. Downloaded by guest on September 27, 2021 Fig. 2. Semiquantitative RT-PCR analysis of gene expressions of Pmp22, Fzd2, Cdh11, Gas1, PRDC, OPN, and Zac1 in pcDNA4͞Hoxc8 transfectant cells (Sample) and pcDNA4 empty-vector transfectant cells (Control). Total RNA was extracted from the cultured cells, and, after reverse transcription, PCR was performed with Pmp22-, Fzd2-, Cdh11-, Gas1-, PRDC-, OPN-, Zac1-, and GAPDH-specific primers. A 100-bp DNA ladder (Biolabs) was used for size markers.

assay showed that the OPN promoter sequence was present in a this report, we identify 34 candidate gene targets for Hoxc8.In complex immunoprecipitated by an antibody against Hoxc8 but the specific case of OPN, we present evidence based on ChIP that not by an anti-mouse IgG antibody (Fig. 3B, lanes 4–6). OPN supports its direct in vivo regulation by Hoxc8. promoter was detected in the input-positive control for PCR but Whereas cell proliferation, migration, adhesion, and differ- not in the immunoprecipitated sample from the control MEF entiation are mediators of normal development, they are also cells with an antibody against Hoxc8 (Fig. 3B, lanes 3 and 7). Our involved in abnormal growth when aberrantly regulated, as in results show that the Hoxc8 protein can interact with the OPN neoplasia. It should come as no surprise that master control promoter in vivo. genes such as the Hox genes should also be involved in atypical growth, given the hierarchical relationship between Hox genes Discussion and their mediators. In fact, Hoxc8 has been shown to play a Hox genes regulate pattern formation during early development. significant role in the progression of leukemias and lung and Pattern formation itself is mediated by the coordinated control prostate cancers, among others, and possibly in precancerous- of cell proliferation, migration, adhesion, and differentiation. like conditions such as polycystic kidney disease. Thus, Hox genes must directly and͞or indirectly exert control In the present study, the use of a gene-expression microarray BIOLOGY over such mediators. For this reason, it is necessary to identify permitted us to identify those genes that are positively or the gene targets of Hox genes to properly understand pattern negatively changed when Hoxc8 was overexpressed in MEF cells. DEVELOPMENTAL formation. We reason that a plethora of target genes serve as Overall, we identified 34 genes that were at least 2-fold up- or targets, but, to date, only a few have been properly identified. In down-regulated in comparisons between overexpressed Hoxc8

Fig. 3. The mouse OPN gene has a Hoxc8-responsive element in the promoter region. (A) Shown is a schematic of the approximate location of the primers (arrows) used in the ChIP experiments. The numbers are relative to ϩ1 being the 5Ј end of the mRNA (indicated by a bent arrow). (B) A ChIP experiment was performed from Hoxc8-transfectant MEF cells (Sample) or vector-transfectant MEF cells (Control). Crosslinked Hoxc8 protein–DNA complexes were immuno- precipitated by a Hoxc8 monoclonal antibody (lanes 4 and 7). PCR amplification of the immunopriecipitated samples was performed by using the primers that flank the OPN promoter (see A). Immnoprecipitates with an anti-mouse IgG (lanes 5 and 8) or in the absence of antibody (lanes 6 and 9) were used for controls. Input chromatin represents a portion of the sonicated chromatin before immunoprecipitation (lanes 2 and 3). DW indicates a no-template control (lane 1). A 100-bp DNA ladder (Biolabs) was used for size markers.

Lei et al. PNAS ͉ February 15, 2005 ͉ vol. 102 ͉ no. 7 ͉ 2423 Downloaded by guest on September 27, 2021 cells and control cells. Semiquantitative RT-PCR of selected skeletal abnormalities in ribs, sternum, and vertebra (8). More- genes performed on the same samples also confirmed the over, expression of Hoxc8 in skeletal tissue results in an accu- changed levels of expression detected on the microarray analysis. mulation of blastogenic cells in hypertrophic areas (10). Some genes involved in the induction of apoptosis, such as Shi et al. (15) have demonstrated that Hoxc8 functions as an growth arrest specific (Gas1), protein regulator of OPN transcription repressor and that the interaction of Smad1 apoptosis (Zac1), cardiac responsive adriamycin protein and Smad4 with Hoxc8 can stimulate OPN transcription in (CARP), serine (or cysteine) proteinase inhibitor (Serpinf1), response to BMP stimulation. They also performed gel-shift NAD(P)H, and quinone oxidoreductase1 (Nqo1), were found in assays that support the binding of Hoxc8 to the OPN promoter the present study to be down-regulated by Hoxc8 overexpression in vitro. In this report, we confirm and extend their results by (Table 2). The proliferation rate of a cell population reflects a showing, by means of ChIP analysis, that Hoxc8 interacts with balance between cell division, cell-cycle arrest, differentiation, the OPN promoter in vivo. This result strengthens our argument and apoptosis. The neural cell adhesion molecule (NCAM), that Hox genes may serve as modulators of neoplastic progres- cadherin 11 (Cdh11), and embigin (Emb) play important roles in sion. Studies in vitro and in animal models of cancer have clearly cell–cell adhesion. NCAM was down-regulated, whereas Cdh11 indicated that OPN can function to regulate tumor growth and and Emb were up-regulated (Table 2). Calponin h1 (Cnn1), a progression. Numerous reports of elevated OPN expression in protein related to DAN and Cerberus (PRDC), engulfment and human cancers support the idea that OPN should be considered cell motility 1 (Ced-12h), and transgelin (Tagln) have been as a potential prognostic marker for a variety of human categorized as genes related to cell migration, motility, and cancers (28). proliferation. These genes were down-regulated by Hoxc8 OPN is strongly regulated by Hoxc8 expression, showing a (Table 2). 4.8-fold reduction in the experiments reported here. We also Significantly, a majority of the 34 Hoxc8 target genes identified demonstrate a direct interaction between Hoxc8 and the OPN here are tumor-related genes. In general, our results support the promoter in vivo. All of the other candidate genes reported here, role of Hox genes as modulators of neoplastic progression. Hox with one exception, show significantly lower levels of change. genes are global regulators of growth and differentiation. They Possibly, Hoxc8 indirectly influences those genes showing lower can interact with different classes of genes at different develop- levels of modulation through intermediary genes. The excep- mental stages and in specific tissue types (25). It is particularly tional gene is Fzd2, which is a cell-surface receptor in the interesting to find that OPN, the major noncollagenous bone WNT–␤-catenin–TCF-signaling pathway. This pathway is im- matrix protein associated with osteoblastic cell adhesion and portant in dorsal patterning during early development and in the abundantly expressed during the early stages of osteoblast progression of intestinal cancers, among others (29, 30). Fzd2 is differentiation, is down-regulated in our microarray analysis. up-regulated 4.4-fold, and we might speculate that it is under Semiquantitative RT-PCR proved that OPN is dramatically direct control by Hoxc8. Our future aim is to determine the down-regulated by Hoxc8 overexpression (Fig. 2). OPN expres- intimate regulatory interactions involving Hoxc8, the candidate sion is rapidly induced by both BMP and transforming growth genes described here, and additional candidates genes as they are factor ␤ (TGF-␤). BMPs, members of the TGF-␤ superfamily, identified. play a pivotal role in signaling networks and are involved in nearly all processes associated with limb development (27). It is We thank Prof. Mary J. Tevethia (Pennsylvania State University, likely that Hoxc8 is involved in both osteo- and chondrogenic Hershey, PA) for providing pPVU0neo plasmid. This work was sup- processes in development, because Hoxc8 knockout mice display ported by National Institutes of Health Grant GM09966.

1. McGinnis, W. & Krumlauf, R. (1992) Cell 68, 283–302. 17. DeVita, G., Barba, P., Odartchenki, N., Givel, J.-C., Freschi, G., Bucciarelli, G., 2. Krumlauf, R. (1994) Cell 78, 191–201. Magli, M., Boncinelli, E. & Cillo, C. (1993) Eur. J. Cancer 29A, 887–893. 3. Belting, H. G., Shashikant, C. S. & Ruddle, F. H. (1998) Proc. Natl. Acad. Sci. 18. Chariot, A. & Castronovo, V. (1996) Biochem. Biophys. Res. Commun. 222, USA 95, 2355–2360. 292–297. 4. Shashikant, C. S., Bieberich, C. J., Belting, H. G., Wang, J. C. H., Borbe´ly, M. A. 19. Cillo, C., Barba, P., Freschi, G., Bucciarelli, G., Magli, M. C. & Boncinelli, E. & Ruddle, F. H. (1995) Development (Cambridge, U.K.) 121, 4339–4347. (1992) Int. J. Cancer 51, 892–897. 5. Bradshaw, M. S., Shashikant, C. S., Belting, H. G., Bollekens, J. A. & Ruddle, 20. Hamada, J.-I., Omatsu, T., Okada, F., Furuuchi, K., Okubo, Y., Takahashi, Y., F. H. (1996) Proc. Natl. Acad. Sci. USA 93, 2426–2430. Tada, M., Miyazaki, Y. J., Taniguchi, Y., Shirato, H., et al. (2001) Int. J. Cancer 6. Belting, H. G., Shashikant, C. S. & Ruddle, F. H. (1998) J. Exp. Zool. 282, 93, 516–525. 196–222. 21. Miller, G. J., Miller, H. L., Bokhoven, A. V., Lambert, J. R., Werahera, P. N., 7. Shashikant, C. S. & Ruddle, F. H. (1996) Proc. Natl. Acad. Sci. USA 93, Schirripa, O., Lucia, M. S. & Nordeen, S. K. (2003) Cancer Res. 63, 12364–12369. 5879–5888. 8. Mouellic, H. L., Lallemand, Y. & Brulet, P. (1992) Cell 69, 251–264. 22. Waltregny, D., Alami, Y., Clausse, N., de Leval, J. & Castronovo, V. (2002) 9. Juan, A. H. & Ruddle, F. H. (2003) Development (Cambridge, U.K.) 130, Prostate 50, 162–169. 4823–4834. 23. Alami, Y., Castronovo, V., Belotti, D., Flagiello, D. & Clausse, N. (1999) 10. Yueh, Y. G., Gardner, D. P. & Kappen, C. (1998) Proc. Natl. Acad. Sci. USA Biochem. Biophys. Res. Commun. 257, 738–745. 95, 9956–9961. 11. Hofbauer, L. C., Dunstan, C. R., Spelsberg, T. C., Riggs, B. L. & Khosla, S. 24. Shimamoto, T., Tang, Y., Naot, Y., Nardi, M., Brulet, P., Bieberich, C. J. & (1998) Biochem. Biophys. Res. Commun. 250, 776–781. Takeshita, K. (1999) J. Exp. Zool. 283, 186–193. 12. Feng. J., Chen, D., Cooney, A. J., Tsai, M., Harris, M. A., Tsai, S. Y., Feng, M., 25. Shen, C. A. (2002) Nat. Rev. Cancer 2, 777–785. Mundy, G. R. & Harris, S. E. (1995) J. Biol. Chem. 270, 28364–28373. 26. Kierstead, T. D. & Tevethia, M. J. (1993) J. Virol. 67, 1817–1829. 13. Zhou, H., Hammonds, R. G., Jr., Findlay, D. M., Martin, T. J. & Ng, K. W. 27. Cheifetz, S., Li, I. W., McCulloch, C. A., Sampath, K. & Sodek, J. (1996) (1993) J. Cell. Physiol. 155, 112–119. Connect. Tissue Res. 35, 71–78. 14. Wan, M., Shi, X., Feng, X. & Cao, X. (2001) J. Biol. Chem. 276, 10119–10125. 28. Rittiing, S. R. & Chambers, A. F. (2004) Br. J. Cancer 90, 1877–1881. 15. Shi, X., Yang, X., Chen, D., Chang, Z. & Cao, X. (1999) J. Biol. Chem. 274, 29. Brennan, K. R. & Brown, A. M. (2004) J. Mammary Gland Biol. Neoplasia 9, 13711–13717. 119–131. 16. Celetti, A., Barba, P., Cillo, C., Rotoli, B., Boncinelli, E. & Magli, M. C. (1993) 30. Sancho, E., Batle, E. & Clevers, H. (2004) Annu. Rev. Cell Dev. Biol. 20, Int. J. Cancer 53, 237–244. 695–723.

2424 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409700102 Lei et al. Downloaded by guest on September 27, 2021