HAND1 gene expression is negatively regulated by the High ... - Nature

Oncogene (2009) 28, 876–885

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ORIGINAL ARTICLE

HAND1 gene expression is negatively regulated by the High Mobility Group A1 proteins and is drastically reduced in human thyroid carcinomas J Martinez Hoyos1,2, A Ferraro2, S Sacchetti2, S Keller1,2, I De Martino1,2, E Borbone2, P Pallante2, M Fedele1, D Montanaro2, F Esposito1, P Cserjesi3, L Chiariotti1,2, G Troncone4 and A Fusco1,2 1

Dipartimento di Biologia e Patologia Cellulare e Molecolare e/o Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, Facolta` di Medicina e Chirurgia di Napoli, Universita` degli Studi di Napoli ‘Federico II’, Naples, Italy; 2NOGEC (Naples Oncogenomic Center), CEINGE Biotecnologie Avanzate, Naples, Italy; 3Department of Cell and Molecular Biology, Tulane University, New Orleans, LA, USA and 4Dipartimento di Anatomia Patologica e Citopatologia, Facolta` di Medicina e Chirurgia, Universita` di Napoli ‘Federico II’, Naples, Italy

HMGA1 proteins exert their major physiological function during embryonic development and play a critical role in neoplastic transformation. Here, we show that Hand1 gene, which codes for a transcription factor crucial for differentiation of trophoblast giant cells and heart development, is upregulated in hmga1 minus embryonic stem cells. We demonstrate that HMGA1 proteins bind directly to Hand1 promoter both in vitro and in vivo and inhibit Hand1 promoter activity. We have also investigated HAND1 expression in human thyroid carcinoma cell lines and tissues, in which HMGA proteins are overexpressed, with respect to normal thyroid; an inverse correlation between HMGA1 and HAND1 expression was found in all thyroid tumor histotypes. A correlation between HAND1 gene repression and promoter hypermethylation was found in anaplastic carcinomas but not in other thyroid tumor histotypes. Therefore, we can hypothesize that HMGA1 overexpression plays a key role on HAND1 silencing in differentiated thyroid carcinomas and that promoter hypermethylation occurs in later stages of thyroid tumor progression. Finally, the restoration of the HAND1 gene expression reduces the clonogenic ability of two human thyroid carcinomaderived cell lines, suggesting that HAND1 downregulation may have a role in the process of thyroid carcinogenesis. Oncogene (2009) 28, 876–885; doi:10.1038/onc.2008.438; published online 8 December 2008 Keywords: HMGA1; HAND1; knockout mice; ES cells; thyroid carcinomas

Correspondence: Dr A Fusco, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facolta` di Medicina e Chirurgia di Napoli, via Pansini 5, 80131 Naples, Italy. E-mail: [email protected] Received 21 May 2008; revised 16 October 2008; accepted 1 November 2008; published online 8 December 2008

Introduction The high mobility group A (HMGA) protein family includes HMGA1a and HMGA1b, which are encoded by the same gene through alternative splicing, and the closely related HMGA2 protein (Fedele et al., 2001; Fusco and Fedele, 2007). These proteins are non-histone architectural nuclear factors that bind the minor groove of AT-rich DNA sequences through three short basic repeats, called ‘AT-hooks’, located at the NH2-terminal region of the proteins (Thanos and Maniatis, 1995; Reeves, 2001). The involvement of HMGA proteins in embryogenesis, cell proliferation, differentiation, apoptosis and above all, cancer development has been extensively demonstrated (Fedele et al., 2001; Reeves, 2001; Fusco and Fedele, 2007). In particular, HMGA proteins seem to play their major physiological role during embryonic development; in fact, their expression is very high during embryogenesis, whereas it is very low or negligible in normal adult tissues (Chiappetta et al., 1996). HMGA1 proteins have been found abundant in several malignant neoplasias, including colorectal, prostate, cervical, lung and thyroid carcinoma (Fedele et al., 2001; Fusco and Fedele, 2007) and, recently, also in glioblastoma (Donato et al., 2004). We identified HMGA1-regulated genes analysing by microarrays the expression profile of murine embryonic stem (ES) cells carrying two, one and no hmga1 functional alleles (Martinez Hoyos et al., 2004; Fedele et al., 2006). In this study, we focused our attention on the Hand1 gene, which showed by microarrays a 14-fold change in the hmga1-null ES in comparison to the wild-type cells (Martinez Hoyos et al., 2004). Hand1 (also named eHand/Hxt/Thing1) belongs to the Twist subfamily of Class B bHLH transcription factors. Murine Hand1 is expressed in developing heart tissue and derivatives of neural crest cells (Cserjesi et al., 1995). Extra-embryonic mRNA production has been detected in the ectoplacental cone, in giant cells as well as in distinct regions of the spongiotrophoblast cell layer (Cross et al., 1995). Embryos carrying a homozygous mutation of Hand1, arrested at E7.5 with defects in trophoblast giant cells differentiation. Upon rescue of

HAND1 regulation by HMGA1 proteins J Martinez Hoyos et al

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Results Loss of HMGA1 correlates with an increased Hand1 expression in murine ES cells, heart and thyroid tissues Expression profile, as assessed by microarray analysis of ES cells bearing one or two disrupted hmga1 alleles, identified Hand1 as a candidate gene negatively regulated by HMGA1. In fact, microarray data showed an increase of 3.8- and a 14.0-fold for the heterozygous and homozygous ES cells, respectively, when compared with wild-type cells (Martinez Hoyos et al., 2004). Our first aim was to validate the results obtained by microarray analysis by semiquantitative and quantitative RT–PCR (Figures 1a and b). These analyses confirmed that Hand1 was strongly overexpressed in hmga1-knockout ES compared with wild-type cells. Data clearly showed that regulation of Hand1 expression was HMGA1-dosedependent as an intermediate level of Hand1 expression was observed in the hmga1-heterozygous ES cells. The analysis of Hand1 expression in heart and thyroid tissues derived from hmga1-knockout mice also revealed a negative regulation by HMGA1 proteins (Figure 1c). Conversely, no changes in Hand1 expression were observed when embryonic fibroblasts, brain, spleen, pancreas, liver, kidney and thymus from hmga1-null mice were analysed (data not shown). These results are consistent with the general concept that HMGA1mediated gene regulation depends on the cellular context (Martinez Hoyos et al., 2004). Interestingly,

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the placental defect by aggregating wild-type tetraploid embryos, the fetuses died at E10.5 due to cardiac failure, demonstrating that Hand1 has essential roles in both trophoblast giant cell differentiation and cardiac morphogenesis (Riley et al., 1998). In humans, HAND1 expression has been detected in trophoblast-like cells, the amniotic epithelium and in adult heart tissue, suggesting that the protein may fulfill similar functions (Knofler et al., 2002). The mechanisms, however, which initiate and maintain HAND1 expression in extraembryonic cell types are not well known. A role of HAND1 in cell proliferation and neoplastic transformation has been recently envisaged because HAND1 gene has been found silenced and hypermethylated in human gastric (Kaneda et al., 2002), pancreatic (Hagihara et al., 2004) and ovarian carcinomas (Takada et al., 2004). Here, we demonstrate that Hand1 is upregulated in hmga1-null ES cells, and that HMGA1 proteins bind directly to the Hand1 promoter in vitro and in vivo resulting in the inhibition of its activity. We also found that HAND1 was downregulated in human thyroid tumors. Such downregulation was associated with HMGA1 overexpression and, limited to anaplastic carcinomas, with promoter hypermethylation. The restoration of HAND1 expression led to a reduced cell growth of two human papillary thyroid carcinoma cell lines, indicating a critical role of the HAND1 downregulation, likely mediated by HMGA1, in thyroid carcinogenesis.

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Figure 1 Hand1 expression in hmga1-knockout cells and tissues. Semiquantitative (a and c) and quantitative (b) RT–PCR analyses for Hand1 and Hand2 expression were performed on RNA extracted from wild-type ( þ / þ ), hmga1-single knockout ( þ /) and hmga1-double knockout (/) ES cells and tissues. b-actin expression was evaluated as internal control.

when we analysed the same tissues from the hmga2-null mice, no changes in Hand1 expression were observed (data not shown), indicating that Hand1 regulation was HMGA1-specific. It is noteworthy that no changes in the expression of Hand2, another gene that belongs to the Twist subfamily of class B bHLH transcription factors, were observed neither in ES cells, MEFs, nor in adult tissues from hmga1- and hmga2-knockout mice (Figures 1a and c). HMGA1 proteins bind to murine and human Hand1 promoter in vitro and in vivo To evaluate whether the differential Hand1 gene expression was a direct effect of HMGA1 binding to Hand1 gene regulatory regions, we performed an electrophoretic mobility shift assay. In particular, we analysed a region spanning nucleotides from 2326 to 2296 related to the transcription start site (TSS) of the murine Hand1 gene which contains AT-rich putative HMGA1 binding sites. As shown in Figure 2a, a recombinant HMGA1 protein was able to bind directly to this region. Binding specificity was demonstrated by competition experiments showing loss of binding with the addition of 200-fold molar excess of specific, unlabeled oligonucleotides. Subsequently, we performed binding assays with total extracts from wild-type and hmga1-knockout ES cells. A specific complex, with a mobility corresponding to the HMGA1 proteins, was present in extracts from wild-type mice whereas it was absent in extracts from homozygous hmga1-knockout ES cells. This complex was specifically displaced by incubation with HMGA1-specific antibodies (Figure 2b), demonstrating the specificity of the HMGA1 binding to the Hand1 promoter. To verify whether HMGA1 proteins bind to murine and human Hand1 promoter in vivo we performed chromatin immunoprecipitation (ChIP) experiments. Anti-HMGA1 antibodies precipitated murine Hand1 promoter from wild-type and heterozygous, but not Oncogene


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Figure 2 HMGA1 binding to Hand1 upstream regulating region. (a) Electrophoretic mobility shift assay performed incubating radiolabeled oligonucleotides spanning from 2326 to 2296 of the 50 untranslated region of the murine Hand1 gene with 5 or 20 ng of the recombinant HMGA1 as indicated. To assess the specificity of the binding, a 200 molar excess of unlabeled oligonucleotides were incubated as specific competitor. (b) Electrophoretic mobility shift assay performed with the same oligonucleotides as in (a), incubated with total extracts from wild-type ( þ / þ ) and hmga1- knockout (/) ES cells. Where indicated, the extracts were preincubated with HMGA1-specific antibodies. (c) Chromatin immunoprecipitation (ChIP) assay was performed on wild-type, hmga1single knockout ( þ /) and hmga1-double knockout (/) ES cells. The presence of 2367 to 2246 sequence of the murine Hand1 promoter was detected by PCR. Anti-HA was used as a negative immunoprecipitation control. (d) ChIP assay was performed on wild-type and hmga1-double knockout (/) thyroid tissue. Murine Hand1 promoter region 2367 to 2246 was detected by PCR. Anti-HA was used as a negative immunoprecipitation control. (e) Soluble chromatin from human HEK293 cells untransfected or transfected with HMGA1 was immunoprecipitated with anti-HMGA1 antibodies. The DNAs were then amplified by semiquantitative PCR using primers that cover two regions of human HAND1 promoter region (1589/1294 and 1311/1020). Coimmunoprecipitations with isotype-matched IgG were also performed as control of the specificity of the interactions. To assess the specificity of the binding, the panel shows PCR amplification of the immunoprecipitated DNA using primers for the human GAPDH gene promoter.

from homozygous hmga1-knockout ES cells (Figure 2c). No amplification was observed in samples immunoprecipitated with an unrelated antibody. Analogous results were obtained when the ChIP experiments were performed on thyroid tissues originating from wild-type and hmga1-null mice (Figure 2d). Occupancy of human HAND1 promoter region by HMGA1 was detected in anti-HMGA1-precipitated chromatin from HEK293 (Figure 2e). As HMGA1 proteins are lowly expressed in HEK293, a weak signal was detected in untransfected cells whereas a stronger signal was detected in HEK293 transfected with HMGA1. No amplification was observed in samples immunoprecipitated with an unrelated antibody.

luciferase gene under the control of the mouse Hand1 promoter region from nucleotides 2367 to 2084 related to TSS. As shown in Figure 3a, when the HMGA1 expression vector was co-transfected, a reduction of the luciferase activity was observed in a dosedependent manner. No decrease in Hand1 promoter activity was obtained when the cells were co-transfected with a construct expressing HMGA2. Then, we generated two point mutations in the putative binding site for the HMGA1 protein, replacing adenine 2320 and thymidine 2319 with two guanines. Overexpression of HMGA1 was able to reduce the activity of the wild-type construct, but it completely failed in its inhibitory effect if the HMGA1-binding site was mutated (Figure 3b).

HMGA1 proteins repress murine Hand1 promoter To investigate the functional effect of HMGA1 binding to Hand1 promoter, we transiently transfected the rat thyroid cells FRTL-5, that do not express detectable levels of HMGA1, with a construct expressing the

Hand1 expression is drastically decreased in thyroid cell transformation The HMGA1 proteins are overexpressed in several malignant neoplasias. To evaluate a possible role of Hand1 regulation by HMGA1 in cell transformation, we

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Figure 3 HMGA1 proteins repress murine Hand1 promoter. (a) Effect of HMGA1 expression on the activity of murine Hand1 promoter transfected in FRTL-5 cells. (b) Effect of HMGA1 expression on the activity of murine Hand1 promoter (black boxes) and same promoter in which A 2320 and T 2319 were replaced with G (gray boxes). The results are reported as mean expression values of three independent experiments, with bars indicating standard deviation (mean±s.d.).

analysed, by RT–PCR, the expression of Hand1 in FRTL-5 normal rat thyroid cells, which do not express significant levels of HMGA1, in the same cells transformed by the Kirsten murine sarcoma virus (KiMSV), which express high levels of HMGA1 (FRTL-5 –KiMSV) and in KiMSV-infected FRTL-5 cells in which the expression of HMGA1 was downregulated by an antisense construct (FRTL-5HMGA1as-KiMSV cells). A significant Hand1 expression was observed in the cells, which do not express the HMGA1 proteins, such as the FRTL-5 and FRTL-5HMGA1as-KiMSV, whereas no expression was observed in the FRTL-KiMSV cells expressing high HMGA1 levels (Figure 4a). These results confirm an inverse correlation between HMGA1 and Hand1 expression also in transformed cells. Subsequently, we analysed the expression of HMGA1 and HAND1 in human thyroid carcinoma cell lines and tissues in comparison with normal controls. To this aim a panel of 12 human thyroid carcinoma-derived cell lines and 20 surgically removed human thyroid carcinomas were analysed by semi-quantitative and quantitative RT–PCR, respectively (Figures 4b and c). Again, an inverse correlation between HMGA1 and HAND1 expression levels was observed. In fact, HMGA1 expression was increased in all the tumor-derived cell lines and tumors tested when compared with normal primary cultured cells and normal tissues, respectively. Conversely, HAND1 expression was much lower in all the thyroid carcinoma cell lines and tumor samples than in normal cells and tissues, respectively (Figures 4b and c). Immunohistochemical analysis of HAND1 expression in human thyroid carcinomas Subsequently, we carried out an immunohistochemical analysis of paraffin-embedded thyroid tissues. Ten cases of follicular thyroid adenoma (FTA), 15 cases of classical papillary thyroid carcinoma (PTC), 15 cases of follicular thyroid carcinoma (FTC), 10 cases of anaplastic thyroid carcinoma (ATC) and 10 normal

thyroid areas were scored for the expression of HAND1 by quantitative analysis performed with a computerized analyzer system (Ibas 2000, Kontron, Zeiss) and relative data were reported in Table 1. As shown in Figure 5 (Panels a and b), most of the normal thyroid follicular cells expressed the HAND1 protein at a very high level (median percentage of stained cells 66%) with the staining evenly distributed in all follicles. A high percentage of HAND1 nuclear labeling was also displayed by FTA (67% median value), whereas HAND1 expression was reduced in both differentiated and undifferentiated malignant histotypes. In the former group, HAND1 median expression values were 22% in PTC and 25% in FTC, whereas in ATC HAND1 loss of expression was even more evident (15% median value). In Figure 5, (at low and high magnification, respectively) the sporadic HAND1 staining of scattered neoplastic cells typical of PTC (Panels c and d), FTC (Panels e and f) and ATC (g and h) is shown. No staining was observed when normal thyroid gland samples were stained with antibodies pre-incubated with the peptide against which the antibodies were raised (Figure 5, Panels i and l) or in the absence of the primary antibodies (data not shown). Therefore, immunohistochemical analysis confirmed that HAND1 is expressed in normal thyroid and in benign neoplastic lesions and that its expression is significantly decreased in differentiated and undifferentiated carcinomas (Po0.001) (Figure 6). LOH and DNA methylation analyses of HAND1 gene To find out the mechanisms, other than HMGA1 overexpression, underlying the block of HAND1 expression in thyroid carcinomas, we analysed 37 informative carcinoma samples including 30 PTCs and seven ATCs for loss of heterozygosity. The analysis of eight single nucleotide polymorphisms showed that all the carcinoma samples were negative for loss of heterozygosity. Then, we investigated whether epigenetic mechanisms may contribute to silence HAND1 Oncogene

HAND1 regulation by HMGA1 proteins J Martinez Hoyos et al FRTL-5-HMGA1asKiMSV

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Figure 4 Hand1 expression is decreased in thyroid tumorigenesis. (a) RT–PCR analysis of Hand1 and HMGA1 expression in rat thyroid cells. Sources of RNA are FRTL-5, rat thyroid epithelial cell line; FRTL-5-KiMSV, FRTL-5 infected with the Kirsten murine sarcoma virus; FRTL-5-HMGA1as-KiMSV, FRTL-5 transfected with a construct carrying HMGA1 mRNA in an antisense orientation, and then infected with the Kirsten murine sarcoma virus. (b) RT–PCR analysis of HAND1 and HMGA1 expression in human thyroid carcinoma-derived cell lines. (c) Quantitative RT–PCR analysis was performed on human thyroid samples of different histotype. Sources of RNA are: 1, normal thyroid (NT); 2, follicular thyroid carcinoma (FTC); 3, medullary thyroid carcinoma (MTC); 4–14, papillary thyroid carcinoma (PTC); 15–21, anaplastic thyroid carcinoma (ATC).

Table 1 HAND1 protein immunohistochemical expression in different diagnostic categories Diagnostic Category

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Non-neoplastic thyroid Thyroid adenoma Papillary thyroid carcinoma Follicular thyroid carcinoma Anaplastic thyroid carcinoma

10 10 15 15 10

66 67 22 25 15

21 33 27 31 15

57 45 11 14 11

78 78 38 45 26

gene in thyroid tumors. We performed a quantitative DNA methylation analysis of HAND1 promoter using pyrosequencing technology. We chose to analyse four CpG sites (nucleotide positions 85, 94, 96 and Oncogene

Figure 5 Immunohistochemical analysis of HAND1 protein expression in benign and malignant thyroid tissues. Paraffin sections from hyperlastic and neoplasic thyroid tissues were analysed by immunohistochemistry using antibodies raised against a specific HAND1 peptide. (a and b) Immunostaining of a normal thyroid ( 10 and 40 magnification, respectively). A strong positive reactivity was observed. (c and d) Immunostaining of a thyroid papillary carcinoma ( 10 and 40 magnification, respectively). The signal is weak or absent in the neoplastic cells. (e and f) Immunostaining of a thyroid follicular carcinoma ( 10 and 40 magnification, respectively). The signal is weak or absent in the neoplastic cells. (g and h) Immunostaining of an anaplastic thyroid carcinoma ( 10 and 40 magnification, respectively). No positivity was observed. (i and l) Immunostaining of a normal thyroid with antibodies pre-incubated with the peptide against which the antibodies were raised ( 10 and 40 magnification, respectively). No immunoreactivity was detected.

109) that have been previously shown to be hypermethylated in gastric, pancreatic and ovarian tumors (Kaneda et al., 2002; Hagihara et al., 2004 and Takada et al., 2004). A representative pyrogram showing a bisulfite methylation profile of these CpG sites is shown in Figure 7. Comparison of HAND1 methylation state (Figure 7c) with HAND1 expression levels (Figure 4c) in thyroid tumor samples, indicated that a significant correlation between HAND1 hypermethylation and


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lower number of colonies compared with the cells transfected with the empty vector. This result suggests that HAND1 downregulation could have an important role in thyroid carcinoma cell proliferation, and then in the process of carcinogenesis.

Discussion

Figure 6 Boxplot and results of Kruskal–Wallis test for HAND1 protein expression in different diagnostic categories. Data are displayed as median, highest and lowest values and an interquartile range containing 50% of values.

silencing is present in anaplastic carcinomas. By contrast, no such correlation was observed in the other thyroid carcinoma histotypes. These data suggest that silencing of HAND1 in papillary carcinomas is primarily because of HMGA1 overexpression whereas, in the most aggressive and undifferentiated ATC, additional mechanisms of epigenetic nature may contribute to keep HAND1 repressed. Restoration of HAND1 gene expression inhibits the growth of thyroid carcinoma cell lines The reduced expression of HAND1 in thyroid carcinomas prompted us to investigate if its downregulation could be a causative event of the thyroid cell proliferation. To this purpose we performed a colony-forming assay on the NPA and FB2 human thyroid carcinoma cells transfecting them with 5 and 10 mg of pCDNA3Hand1 or the corresponding empty vector. As shown in Table 2, the cells transfected with the HAND1 expression vector gave rise to a significant dose-dependent

In our previous work, we identified HMGA1-regulated genes analysing by microarrays the expression profile of embryonic stem (ES) cells bearing one or two disrupted hmga1 alleles (Martinez Hoyos et al., 2004). The generation of hmga1 null mice unveiled a critical role of HMGA1 proteins on cardiomyocytic cell growth: in fact, both heterozygous and homozygous mice for the hmga1-null allele showed cardiac hypertrophy. Moreover, these mice also developed hematologic malignancies, including B-cell lymphoma and myeloid granuloerythroblastic leukemia (Fedele et al., 2006). Here, we focused our attention on Hand1, a gene that encodes a transcription factor crucial for differentiation of trophoblast giant cells and heart development (Riley et al., 1998). We validated the microarray results by semiquantitative and quantitative RT–PCR in ES cells. As in microarrays, we found that Hand1 expression displayed HMGA1 dose-dependency: the phenotype of heterozygous cells was intermediate between those of wild-type and homozygous knockout cells. We found an increased Hand1 expression also in heart and thyroid but not other tissues from hmga1-knockout mice with respect to wild-types. These results indicated that HMGA1-mediated Hand1 regulation depends on the cellular context, as occurs for other HMGA1-regulated genes (Martinez Hoyos et al., 2004). In this report, we show that HMGA1 proteins bind directly to Hand1 promoter in vitro and in vivo, by electrophoretic mobility shift assay and chromatin immunoprecipitation experiments. Moreover, this binding has a functional effect as we have seen by luciferase assay that hmga1 expression reduces the activity of Hand1 promoter. When we analysed by RT–PCR the expression of Hand1 in MEF and adult tissues taken from hmga2-knockout mice, we did not find changes between wild-type and knockout cells and tissues. This result indicated that Hand1 was probably not regulated by HMGA2. In fact, when we co-transfected hmga2 expression construct together with Hand1 promoter we did not find any alteration in Hand1-promoter activity by luciferase assay. This specific responsiveness to HMGA1 and not HMGA2 confirms that even though HMGA1 and HMGA2 have a similar structure and expression profile (high during embryogenesis and in neoplastic tissue), they exert different functions. This is consistent with our previous findings showing that the phenotype of the hmga1- and hmga2-knockout mice is divergent: cardiac hypertrophy and B-cell lymphomas in hmga1-null mice and a reduction in size and fat tissue in hmga2-null mice (Fedele et al., 2006). Oncogene


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Figure 7 DNA methylation analysis on HAND1 gene. (a) Sequence of the analysed HAND1 promoter region. The transcriptional start site is underlined. The analysed CpGs are boxed. (b) Representative pyrogram. The four targeted cytosines are enclosed in unshaded squares (as reverse strand was read, G peaks—arrowed—indicate methylated cytosine whereas A indicates unmethylated cytosine). The control, non-CpG cytosine residue showing complete conversion of cytosine to uracil by bisulfite treatment, is shown. (c) Hystogram of pyrosequencing results. For each sample the percentage of methylation represents the average of the methylation degree of the four CpG sites analysed. MTC, FTC, PTC and ATC indicate medullary, follicular, papillary and anaplatic thyroid carcinomas, respectively. NT, Normal thyroid samples.

Table 2 Restoration of HAND1 gene expression reduces clonogenic capacities of thyroid cancer cells Cell lines FB-2 FB-2 FB-2 FB-2 N-PA N-PA N-PA N-PA

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95±11.7 75±10* 99±9.5 58±12.2* 258±13.3 167±16.7* 263±11.4 122±14.6*

The results are reported as mean expression values with bars indicating standard deviation (mean±s.d.). *Po0.05 with respect to the negative control (empty vector).

As Hand1 shares 87% homology in bHLH region with Hand2, another gene that belongs to the Twist subfamily of class B bHLH transcription factors, we were intrigued if HMGA proteins could regulate even Oncogene

Hand2. This gene was on the microarray but was not changed in the hmga1-knockout cells compared with wild-types. Anyway, we performed semiquantitative PCR, but we did not find altered expression of Hand2 neither in ES cells, MEFs, nor in adult tissues from hmga1- and hmga2-knockout mice. HAND-1 and -2 gene expression has been studied in normal and hypertrophied human ventricle (Ritter et al., 1999) and found increased in both the hypertrophied left and right ventricle of patients with hypertrophic obstructive cardiomyopathy and tetralogy of Fallot. Consistently, we found an increased Hand1 mRNA expression in the hypertrophied heart of hmga1-knockout mice. Given this parallelism, it would be interesting to see the status of HMGA1 proteins in those patients. Therefore, it is reasonable to hypothesize that the HMGA1-mediated regulation of Hand1 may have a critical role in the cardiac hypertrophy that develops in the absence of an appropriate HMGA1 expression.


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The other tissue in the hmga1-knockout mice where we found Hand1 upregulation was the thyroid. In our laboratory, we have previously demonstrated that HMGA1 proteins have a major role in thyroid transformation (Berlingieri et al., 2002). As, in the present paper, we demonstrate that HMGA1 proteins regulate Hand1, we suspected that Hand1 could have a role in thyroid cell transformation. First of all, we studied Hand1 expression in a cell system constituted by normal rat thyroid cells (FRTL-5) that do not express HMGA1 proteins, the same cells malignantly transformed by the KiMSV (FRTL-5-KiMSV) that express high HMGA1 levels and, FRTL-5-KiMSV cells in which the synthesis of the HMGA1 protein was blocked by an antisense construct (FRTL-5-HMGA1asKiMSV). We found Hand1 expression in cells that do not express HMGA1 proteins but not in cells expressing HMGA1 proteins, confirming an inverse correlation between HMGA1 and Hand1 expression. When we analysed by RT–PCR 12 human thyroid carcinomaderived cell lines and by real-time–PCR 20 surgically removed human thyroid carcinomas, we found again an inverse correlation between HMGA1 and HAND1 expression. As it has been described that HAND1 is silenced and hypermethylated in gastric, pancreatic and ovarian carcinomas (Kaneda et al., 2002; Hagihara et al., 2004; Takada et al., 2004), we analysed the methylation status of HAND1 promoter in thyroid carcinomas of different histotypes and compared with normal thyroid. A significant correlation between HAND1 hypermethylation and gene repression was found only in anaplastic carcinomas whereas only in few cases of papillary carcinomas HAND1 silencing was accompanied by a high methylation degree of the promoter region. Based on the knowledge that ATCs derive from PTCs by neoplastic progression (Kondo et al., 2006), we suggest that HMGA1 may have a critical role in HAND1 silencing in PTCs, whereas in later stages of thyroid tumor progression, promoter hypermethylation could stabilize HAND1 repression. In conclusion, our data clearly demonstrate that HMGA1 proteins are able to directly downregulate HAND1 expression in embryonic and adult tissues, and also in cell transformation. Moreover, the reduction of the clonogenic ability after the restoration of the HAND1 gene expression in two human thyroid carcinoma-derived cell lines suggests that HAND1 downregulation by HMGA1 proteins may have a role in the process of thyroid carcinogenesis.

Materials and methods Plasmids For the Hand1 promoter construct, the region from 2367 to 2084, related to transcription start sites (TSS), of the mouse Hand1 gene was amplified using as primers 50 -ctgagatcccagat cactca-30 (forward) and 50 -gggatacacgaaggtcagtttt -30 (reverse), cloned in TA Cloning Vector (Invitrogen, Singapore, Singapore) and subcloned in pGL3 (Promega, Madison, WI,

USA) KpnI–XhoI cloning site. The point mutations in the HMGA binding site of the Hand1 promoter were generated using the QuikChange Mutagenesis Kit (Stratagene, La Jolla, CA, USA) in accordance with the manufacturer’s protocols. The primers used were: 50 -ggcaggaggagactctgttattaCCtaattagt taaaata-30 (forward) and 50 -tattttaactaattaGGtaataacagagtctc ctcctgcc-30 (reverse), where point mutations are shown in uppercase type. Hand1, HMGA1 and HMGA2 expression plasmids were constructed by cloning the murine full-length cDNAs of Hand1, Hmga1b or Hmga2 into the mammalian expression vector pcDNA3.1 (Invitrogen). Cell culture and transfections The generation and culture of hmga1 þ / and hmga1/ ES cells are described elsewhere (Battista et al., 2003). FRTL-5, FRTL-5-KiMSV and FRTL-5–HMGA1as-KiMSV cells and their culture conditions, are reported elsewhere (Berlingieri et al., 2002). Cells were harvested 24 h post-transfection and lysates were analysed for luciferase activity. Transfection efficiency was normalized using the b-galactosidase activity and fold of activation were calculated by dividing for pGL3 luciferase activity. All the assays were performed in triplicate and repeated in three independent experiments. Human thyroid primary cultures and carcinoma cell lines (TPC-1, WRO, NPA, ARO, FRO, NIM 1, B-CPAP, FB-1, FB-2, Kat-4 and Kat-18) are described elsewhere (Pallante et al., 2005). For cloning efficiency assays, FB-2 and NPA cells were either transfected with 5 and 10 mg of pCDNA3-Hand1 or the corresponding empty vector. Then, 48 h post-transfection of the plasmids 5 105 cells were plated on 10-cm dishes and cultured with a medium supplemented with G418 (Invitrogen) for selection of transfected cells. Colonies were stained with 1.5% glutaraldehyde and 0.06% methylene blue in Hanks’ balanced salt solution and counted. All the transfections were performed by Lipofectamine 2000 (Invitrogen), as suggested by the manufacturer. Tissue samples Neoplastic human thyroid tissues and normal adjacent tissue or the controlateral normal thyroid lobe were obtained from surgical specimens and immediately frozen in liquid nitrogen. Thyroid tumors were collected at the Service d’AnatomoPathologie, Centre Hospitalier Lyon Sud, Pierre Benite, France. RNA extraction from tissues and cells Total RNAs were extracted from frozen tissues and cell culture using TRI REAGENT (Molecular Research Center Inc., Cincinnati, OH, USA) solution, according to the manufacturer’s instructions. Semiquantitative and quantitative RT–PCR RNAs were treated with DnaseI (Invitrogen) and reversetranscribed using random exonucleotides as primers and MuLV reverse transcriptase (Applied Biosystems, Foster City, CA, USA). For semiquantitative PCR, reactions were optimized for the number of cycles to ensure product intensity within the linear phase of amplification. Digitized data were analyzed using Imagequant (Molecular Dynamics, Sunnyvale, CA, USA). Quantitative PCR was performed in triplicate with SYBR Green PCR Master Mix (Applied Biosystems). Primers for amplification of Hand1 gene were: 50 -gatgccttctcgagttaaaa30 (forward) and 50 -aagtgtagcgacaagaagga-30 (reverse), for murine; 50 -gttcaggacccaaaaagg-30 (forward) and 50 -gcagagtcttgatcttggag-30 (reverse), for rat; 50 -ctggctctttctctcttgtc-30 (forward) and 50 -cgtctggttctctttctcag-30 (reverse), for human. Oncogene


884 Primers for amplification of Hand2 gene were: 50 -ccaaactctccaagatcaag-30 (forward) and 50 -ctcttctcctctttcacgtc-30 (reverse).

provides an internal control of the completeness of bisulphite treatment.

Electrophoretic mobility shift assay Five or 20 ng of recombinant protein (Baldassarre et al., 2001) were incubated with radiolabeled double-strand oligonucleotides, corresponding to the region spanning bases from -2326 to -2296 with respect of TSS of the murine Hand1 gene (50 -ttattaattaattagttaaaataaaataaat-30 ). A 200-fold excess of specific unlabeled competitor oligonucleotide was added. The same oligonucleotides were also used in binding assays with total extract from wild-type and hmga1-knockout ES cells. Experimental conditions and anti-HMGA1 antibodies used were as previously described (Martinez Hoyos et al., 2004).

SNP-based Loss of Heterozygosity analysis The primers, SNP reference (NCBI database) and alleles were: 50 -cgaaataggcaaacaggctc-30 (forward) and 50 -aaagctcatccagg gacga-30 (reverse) for rs924581 (A/G); 50 -gaagacccgatctgttt tacct-30 (forward) and 50 -cttcaaggctgaactcaagaa-30 (reverse) for rs4370323 (A/G), rs1846966 (C/G), rs11748765 (A/T); 50 -cgctgttaatgctctcagt-30 (forward) and 50 -gtaaaacctgggatagcca-30 (reverse) for rs6880185 (A/G), rs13171812 (C/T), rs993098 (A/C) and rs3822714 (A/G). PCR conditions were: 94 1C for 2 min, 35 cycles at 94 1C for 20 s, 58 1C for 10 s, 70 1C for 40 s followed by 5 min at 70 1C.

Chromatin immunoprecipitation Chromatin immunoprecipitation was performed as previously described (Martinez Hoyos et al., 2004). For mouse Hand1 promoter, the primers used were: 50 -ctgagatcccagatcactca-30 (forward) and 50 -cttggtgacaagcacctt-30 (reverse). For human HAND1 promoter were analyzed two sequences spanning nucleotides from -1589 to -1294 and from -1311 to -1020 related to TSS. The primers used were: 50 -TCTGGGCTT CACGTTCCATA-30 (forward-prA) and 50 -GTTCCTTTCT TGGCCGATGT-30 (reverse-prA) and 50 -ATCGGCCAAGA AAGGAACCA-30 (forward-prB) and 50 -AGCATTCGGCGT TAGACACA-30 (reverse-prB). As specificity control of the binding the primers used were GAPDH 50 -GTATTCCCC CAGGTTTACATG-30 (forward) and GAPDH 50 -TTCTCCA TGGTGGTGAAGAC-30 (reverse).

Immunohistochemical analysis Immunostaining was performed with indirect-peroxidase assay, as previously described (Pallante et al. 2005). Archivial tumor blocks were from ‘Dipartimento di Scienze Biomorfologiche e Funzionali’ (University of Naples, Naples, Italy). A polyclonal antibody specific for the carboxy-terminal region of human HAND1 (1:200; Sigma, St Louis, MO, USA) was used. Results were scored by quantitative analysis performed with a computerized analyzer system (Ibas, 2000, Kontron, Zeiss) by using as cut-off the staining of X10% of cells displaying evident nuclear signal.

Methylation analysis Sodium bisulphite conversion of genomic DNA (about 2 mg for each conversion) was obtained using Epitect Bisulphite kit (Qiagen, Valencia, CA, USA) following the purchaser’s instructions. Amplicons used for methylation analysis were obtained from approximately 50 ng of bisulfite treated genomic DNA. Quantitative DNA methylation analysis was performed using the PSQ 96MA instrument from Pyrosequencing (Biotage AB, Uppsala, Sweden) following the protocol suggested by the manufacturer. The reactions were assayed on the PSQ 96MA using the SNP analysis software provided by the manufacturer. Primers used for PCR reactions were: BHand1FW (-175/-151) 50 -aagttygtagagtagggagttgag-30 (50 -Biotinylated); Hand1RV (19/ þ 4) 50 -accrcractttaatatcaacctc-30 . Amplification were carried out on 10 ng of bisulfite treated DNA using HotStarTaq DNA polymerase (Qiagen) under the following conditions: 15 min at 95 1C, followed by 50 cycles of 30 s at 95 1C, 40 s at 57,5 1C and 1 min at 72 1C, then a final elongation of 10 min at 72 1C before holding at 4 1C, in a final reaction volume of 50 ml. Sequencing primer (Hand1S1) was 50 -ataactatcccaaattttac-30 . The analysis of a non-CpG cytosine

Statistical analysis For the comparison of statistical significance between two groups of experiments, Student’s t test was used. A P-value of o0.05 was considered statistically significant. For immunohistochemical studies, differences between the different diagnostic groups were analysed by the nonparametric Kruskal–Wallis one-way ANOVA test. A P-value o0.001 was considered statistically significant. Databases The Hand1 gene sequences were retrieved by the Ensembl database. Accession numbers were: Human ENSG000001 13196; Mouse ENSMUS00000037335 (transcript ENSMU ST00000036917); Rat ENSRNOG00000002582. Acknowledgements This work was supported by grants from the Associazione Italiana Ricerca sul Cancro (AIRC), and the Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MIUR). This work was supported from NOGEC-Naples Oncogenomic Center. We thank the Associazione Partenopea per le Ricerche Oncologiche (APRO) for its support. We thank Gabriella Sole, INN-CNR of Cagliari, for statistical analysis.

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