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The loss of the p53 activator HIPK2 is responsible for galectin-3 overexpression in well differentiated thyroid carcinomas.

Lavra L, Rinaldo C, Ulivieri A, Luciani E, Fidanza P, Giacomelli L, Bellotti C, Ricci A, Trovato M, Soddu S, Bartolazzi A, Sciacchitano S - PLoS ONE (2011)

Bottom Line: We found LOH at HIPK-2 gene locus in 37.5% of PTCs, 14.3% of FTCs and 18.2% of follicular adenomas.To causally link these data with Gal-3 upregulation, we performed in vitro experiments, using the PTC-derived K1 cells, in which HIPK2 expression was manipulated by RNA interference (RNAi) or plasmid-mediated overexpression.HIPK2 RNAi was associated with Gal-3 upregulation, while HIPK2 overexpression with Gal-3 downregulation.

View Article: PubMed Central - PubMed

Affiliation: Research Center, St. Pietro Fatebenefratelli Hospital, Rome, Italy.

ABSTRACT

Background: Galectin-3 (Gal-3) is an anti-apoptotic molecule involved in thyroid cells transformation. It is specifically overexpressed in thyroid tumour cells and is currently used as a preoperative diagnostic marker of thyroid malignancy. Gal-3 expression is downregulated by wt-p53 at the transcriptional level. In well-differentiated thyroid carcinomas (WDTCs) there is an unexplained paradoxical concomitant expression of Gal-3 and wt-p53. HIPK2 is a co-regulator of different transcription factors, and modulates basic cellular processes mainly through the activation of wt-p53. Since we demonstrated that HIPK2 is involved in p53-mediated Gal-3 downregulation, we asked whether HIPK2 deficiency might be responsible for such paradoxical Gal-3 overexpression in WDTC.

Methodology/principal findings: We analyzed HIPK2 protein and mRNA levels, as well as loss of heterozygosity (LOH) at the HIPK2 locus (7q32-34), in thyroid tissue samples. HIPK2 protein levels were high in all follicular hyperplasias (FHs) analyzed. Conversely, HIPK2 was undetectable in 91.7% of papillary thyroid carcinomas (PTCs) and in 60.0% of follicular thyroid carcinomas (FTCs). HIPK2 mRNA levels were upregulated in FH compared to normal thyroid tissue (NTT), while PTC showed mean HIPK2 mRNA levels lower than FH and, in 61.5% of cases, also lower than NTT. We found LOH at HIPK-2 gene locus in 37.5% of PTCs, 14.3% of FTCs and 18.2% of follicular adenomas. To causally link these data with Gal-3 upregulation, we performed in vitro experiments, using the PTC-derived K1 cells, in which HIPK2 expression was manipulated by RNA interference (RNAi) or plasmid-mediated overexpression. HIPK2 RNAi was associated with Gal-3 upregulation, while HIPK2 overexpression with Gal-3 downregulation.

Conclusions/significance: Our results indicate that HIPK2 expression and function are impaired in WDTCs, in particular in PTCs, and that this event explains Gal-3 overexpression typically observed in these types of tumours. Therefore, HIPK2 can be considered as a new tumour suppressor gene for thyroid cancers.

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Effects of modulation of HIPK2 protein levels on Gal-3 expression.(A) Western blot analysis, using antibodies to HIPK2 and Gal-3, performed on TCE obtained from PTC-derived K1 cell line stably transfected with pSUPER.retro control vectors (ctr) and pSUPER-HIPK2 interfering construct (HIPK2i). (B) Inverted images of agarose gels showing semiquantitative RT-PCR analyses of HIPK2 and Gal-3 gene expression performed on total RNA extracted from K1 cells described in (A). (C) Western blot analysis of HIPK2 and Gal-3 in K1 cell transiently transfected with control vector pCMV-FLAG (Flag) and with pCMV-FLAG-HIPK2 (Flag-HIPK2) expression construct. (D) Inverted images of agarose gels showing semiquantitative RT-PCR analyses of HIPK2 and Gal-3 gene expression performed on total RNA extracted from K1 cells described in (C). (E) Western blot analysis of HIPK2, p53Ser46, total p53 and Gal-3 in K1 cell transiently transfected with the kinase-dead pEGFP-HIPK2K221R (GFP-HIPK2K221R) and the wild type pEGFP-HIPK2 (GFP-HIPK2) HIPK2 expression constructs. Western blot experiments were normalized using β-actin protein expression while the 18S gene was amplified as control for semiquantitative RT-PCR analyses.
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pone-0020665-g005: Effects of modulation of HIPK2 protein levels on Gal-3 expression.(A) Western blot analysis, using antibodies to HIPK2 and Gal-3, performed on TCE obtained from PTC-derived K1 cell line stably transfected with pSUPER.retro control vectors (ctr) and pSUPER-HIPK2 interfering construct (HIPK2i). (B) Inverted images of agarose gels showing semiquantitative RT-PCR analyses of HIPK2 and Gal-3 gene expression performed on total RNA extracted from K1 cells described in (A). (C) Western blot analysis of HIPK2 and Gal-3 in K1 cell transiently transfected with control vector pCMV-FLAG (Flag) and with pCMV-FLAG-HIPK2 (Flag-HIPK2) expression construct. (D) Inverted images of agarose gels showing semiquantitative RT-PCR analyses of HIPK2 and Gal-3 gene expression performed on total RNA extracted from K1 cells described in (C). (E) Western blot analysis of HIPK2, p53Ser46, total p53 and Gal-3 in K1 cell transiently transfected with the kinase-dead pEGFP-HIPK2K221R (GFP-HIPK2K221R) and the wild type pEGFP-HIPK2 (GFP-HIPK2) HIPK2 expression constructs. Western blot experiments were normalized using β-actin protein expression while the 18S gene was amplified as control for semiquantitative RT-PCR analyses.

Mentions: To demonstrate that Gal-3 overexpression in WDTCs is a consequence of HIPK2 protein loss, we modulated HIPK2 expression levels in vitro by RNAi or by vector-mediated overexpression in a wt-p53-carring PTC-derived cell line, namely the K1 cells (ECACC, Salisbury, United Kingdom). Stable RNAi of HIPK2 expression in K1 cells induced upregulation of Gal-3 both at the protein (Figure 5, panel A) and mRNA (Figure 5, panel B) levels. Conversely, overexpression of HIPK2 in the same cells caused a concomitant downregulation of Gal-3 protein (Figure 5, panel C) and mRNA (Figure 5, panel D) levels. Moreover, overexpression of the HIPK2 kinase-dead mutant K221R, in K1 cells was unable to phosphorylate p53 on Ser46 and to downregulate Gal-3 expression levels compared to wt-HIPK2 (Figure 5, panel E). These results demonstrate that Gal-3 protein levels are regulated by HIPK2 expression and kinase activity probably through the specific phosphorylation of p53 protein at its residue Ser46. These data reinforce our hypothesis that the loss of HIPK2 expression observed in WDTC could be responsible for the concomitant overexpression of Gal-3.


The loss of the p53 activator HIPK2 is responsible for galectin-3 overexpression in well differentiated thyroid carcinomas.

Lavra L, Rinaldo C, Ulivieri A, Luciani E, Fidanza P, Giacomelli L, Bellotti C, Ricci A, Trovato M, Soddu S, Bartolazzi A, Sciacchitano S - PLoS ONE (2011)

Effects of modulation of HIPK2 protein levels on Gal-3 expression.(A) Western blot analysis, using antibodies to HIPK2 and Gal-3, performed on TCE obtained from PTC-derived K1 cell line stably transfected with pSUPER.retro control vectors (ctr) and pSUPER-HIPK2 interfering construct (HIPK2i). (B) Inverted images of agarose gels showing semiquantitative RT-PCR analyses of HIPK2 and Gal-3 gene expression performed on total RNA extracted from K1 cells described in (A). (C) Western blot analysis of HIPK2 and Gal-3 in K1 cell transiently transfected with control vector pCMV-FLAG (Flag) and with pCMV-FLAG-HIPK2 (Flag-HIPK2) expression construct. (D) Inverted images of agarose gels showing semiquantitative RT-PCR analyses of HIPK2 and Gal-3 gene expression performed on total RNA extracted from K1 cells described in (C). (E) Western blot analysis of HIPK2, p53Ser46, total p53 and Gal-3 in K1 cell transiently transfected with the kinase-dead pEGFP-HIPK2K221R (GFP-HIPK2K221R) and the wild type pEGFP-HIPK2 (GFP-HIPK2) HIPK2 expression constructs. Western blot experiments were normalized using β-actin protein expression while the 18S gene was amplified as control for semiquantitative RT-PCR analyses.
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Related In: Results  -  Collection

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pone-0020665-g005: Effects of modulation of HIPK2 protein levels on Gal-3 expression.(A) Western blot analysis, using antibodies to HIPK2 and Gal-3, performed on TCE obtained from PTC-derived K1 cell line stably transfected with pSUPER.retro control vectors (ctr) and pSUPER-HIPK2 interfering construct (HIPK2i). (B) Inverted images of agarose gels showing semiquantitative RT-PCR analyses of HIPK2 and Gal-3 gene expression performed on total RNA extracted from K1 cells described in (A). (C) Western blot analysis of HIPK2 and Gal-3 in K1 cell transiently transfected with control vector pCMV-FLAG (Flag) and with pCMV-FLAG-HIPK2 (Flag-HIPK2) expression construct. (D) Inverted images of agarose gels showing semiquantitative RT-PCR analyses of HIPK2 and Gal-3 gene expression performed on total RNA extracted from K1 cells described in (C). (E) Western blot analysis of HIPK2, p53Ser46, total p53 and Gal-3 in K1 cell transiently transfected with the kinase-dead pEGFP-HIPK2K221R (GFP-HIPK2K221R) and the wild type pEGFP-HIPK2 (GFP-HIPK2) HIPK2 expression constructs. Western blot experiments were normalized using β-actin protein expression while the 18S gene was amplified as control for semiquantitative RT-PCR analyses.
Mentions: To demonstrate that Gal-3 overexpression in WDTCs is a consequence of HIPK2 protein loss, we modulated HIPK2 expression levels in vitro by RNAi or by vector-mediated overexpression in a wt-p53-carring PTC-derived cell line, namely the K1 cells (ECACC, Salisbury, United Kingdom). Stable RNAi of HIPK2 expression in K1 cells induced upregulation of Gal-3 both at the protein (Figure 5, panel A) and mRNA (Figure 5, panel B) levels. Conversely, overexpression of HIPK2 in the same cells caused a concomitant downregulation of Gal-3 protein (Figure 5, panel C) and mRNA (Figure 5, panel D) levels. Moreover, overexpression of the HIPK2 kinase-dead mutant K221R, in K1 cells was unable to phosphorylate p53 on Ser46 and to downregulate Gal-3 expression levels compared to wt-HIPK2 (Figure 5, panel E). These results demonstrate that Gal-3 protein levels are regulated by HIPK2 expression and kinase activity probably through the specific phosphorylation of p53 protein at its residue Ser46. These data reinforce our hypothesis that the loss of HIPK2 expression observed in WDTC could be responsible for the concomitant overexpression of Gal-3.

Bottom Line: We found LOH at HIPK-2 gene locus in 37.5% of PTCs, 14.3% of FTCs and 18.2% of follicular adenomas.To causally link these data with Gal-3 upregulation, we performed in vitro experiments, using the PTC-derived K1 cells, in which HIPK2 expression was manipulated by RNA interference (RNAi) or plasmid-mediated overexpression.HIPK2 RNAi was associated with Gal-3 upregulation, while HIPK2 overexpression with Gal-3 downregulation.

View Article: PubMed Central - PubMed

Affiliation: Research Center, St. Pietro Fatebenefratelli Hospital, Rome, Italy.

ABSTRACT

Background: Galectin-3 (Gal-3) is an anti-apoptotic molecule involved in thyroid cells transformation. It is specifically overexpressed in thyroid tumour cells and is currently used as a preoperative diagnostic marker of thyroid malignancy. Gal-3 expression is downregulated by wt-p53 at the transcriptional level. In well-differentiated thyroid carcinomas (WDTCs) there is an unexplained paradoxical concomitant expression of Gal-3 and wt-p53. HIPK2 is a co-regulator of different transcription factors, and modulates basic cellular processes mainly through the activation of wt-p53. Since we demonstrated that HIPK2 is involved in p53-mediated Gal-3 downregulation, we asked whether HIPK2 deficiency might be responsible for such paradoxical Gal-3 overexpression in WDTC.

Methodology/principal findings: We analyzed HIPK2 protein and mRNA levels, as well as loss of heterozygosity (LOH) at the HIPK2 locus (7q32-34), in thyroid tissue samples. HIPK2 protein levels were high in all follicular hyperplasias (FHs) analyzed. Conversely, HIPK2 was undetectable in 91.7% of papillary thyroid carcinomas (PTCs) and in 60.0% of follicular thyroid carcinomas (FTCs). HIPK2 mRNA levels were upregulated in FH compared to normal thyroid tissue (NTT), while PTC showed mean HIPK2 mRNA levels lower than FH and, in 61.5% of cases, also lower than NTT. We found LOH at HIPK-2 gene locus in 37.5% of PTCs, 14.3% of FTCs and 18.2% of follicular adenomas. To causally link these data with Gal-3 upregulation, we performed in vitro experiments, using the PTC-derived K1 cells, in which HIPK2 expression was manipulated by RNA interference (RNAi) or plasmid-mediated overexpression. HIPK2 RNAi was associated with Gal-3 upregulation, while HIPK2 overexpression with Gal-3 downregulation.

Conclusions/significance: Our results indicate that HIPK2 expression and function are impaired in WDTCs, in particular in PTCs, and that this event explains Gal-3 overexpression typically observed in these types of tumours. Therefore, HIPK2 can be considered as a new tumour suppressor gene for thyroid cancers.

Show MeSH
Related in: MedlinePlus