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The serine–threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) modulates important cellular functions during development, acting as a signal integrator of a wide variety of stress signals, and as a regulator of transcription factors and cofactors. We have previously demonstrated that HIPK2 binds and phosphorylates High-Mobility Group A1 (HMGA1), an architectural chromatinic protein ubiquitously expressed in embryonic tissues, decreasing its binding affinity to DNA. To better define the functional role of HIPK2 and HMGA1 interaction in vivo, we generated mice in which both genes are disrupted. About 50% of these Hmga1/Hipk2 double knock-out (DKO) mice die within 12 h of life (P1) for respiratory failure. The DKO mice present an altered lung morphology, likely owing to a drastic reduction in the expression of surfactant proteins, that are required for lung development. Consistently, we report that both HMGA1 and HIPK2 proteins positively regulate the transcriptional activity of the genes encoding the surfactant proteins. Moreover, these mice display an altered expression of thyroid differentiation markers, reasonably because of a drastic reduction in the expression of the thyroid-specific transcription factors PAX8 and FOXE1, which we demonstrate here to be positively regulated by HMGA1 and HIPK2. Therefore, these data indicate a critical role of HIPK2/HMGA1 cooperation in lung and thyroid development and function, suggesting the potential involvement of their impairment in the pathogenesis of human lung and thyroid diseases.

The High Mobility Group A (HMGA) are nuclear proteins that participate in the organization of nucleoprotein complexes involved in chromatin structure, replication and gene transcription. HMGA overexpression is a feature of human cancer and plays a causal role in cell transformation. Since non-coding RNAs and pseudogenes are now recognized to be important in physiology and disease, we investigated HMGA1 pseudogenes in cancer settings using bioinformatics analysis. Here we report the identification and characterization of two HMGA1 non-coding pseudogenes, HMGA1P6 and HMGA1P7. We show that their overexpression increases the levels of HMGA1 and other cancer-related proteins by inhibiting the suppression of their synthesis mediated by microRNAs. Consistently, embryonic fibroblasts from HMGA1P7-overexpressing transgenic mice displayed a higher growth rate and reduced susceptibility to senescence. Moreover, HMGA1P6 and HMGA1P7 were overexpressed in human anaplastic thyroid carcinomas, which are highly aggressive, but not in differentiated papillary carcinomas, which are less aggressive. Lastly, the expression of the HMGA1 pseudogenes was significantly correlated with HMGA1 protein levels thereby implicating HMGA1P overexpression in cancer progression. In conclusion, HMGA1P6 and HMGA1P7 are potential proto-oncogenic competitive endogenous RNAs.

High Mobility Group A1 (HMGA1) is an architectural chromatin protein whose overexpression is a feature of malignant neoplasias with a causal role in cancer initiation and progression. HMGA1 promotes tumor growth by several mechanisms, including increase of cell proliferation and survival, impairment of DNA repair and induction of chromosome instability. Autophagy is a self-degradative process that, by providing energy sources and removing damaged organelles and misfolded proteins, allows cell survival under stress conditions. On the other hand, hyper-activated autophagy can lead to non-apoptotic programmed cell death. Autophagy deregulation is a common feature of cancer cells in which has a complex role, showing either an oncogenic or tumor suppressor activity, depending on cellular context and tumor stage. Here, we report that depletion of HMGA1 perturbs autophagy by different mechanisms. HMGA1-knockdown increases autophagosome formation by constraining the activity of the mTOR pathway, a major regulator of autophagy, and transcriptionally upregulating the autophagy-initiating kinase Unc-51-like kinase 1 (ULK1). Consistently, functional experiments demonstrate that HMGA1 binds ULK1 promoter region and negatively regulates its transcription. On the other hand, the increase in autophagosomes is not associated to a proportionate increase in their maturation. Overall, the effects of HMGA1 depletion on autophagy are associated to a decrease in cell proliferation and ultimately impact on cancer cells viability. Importantly, silencing of ULK1 prevents the effects of HMGA1-knockdown on cellular proliferation, viability and autophagic activity, highlighting how these effects are, at least in part, mediated by ULK1. Interestingly, this phenomenon is not restricted to skin cancer cells, as similar results have been observed also in HeLa cells silenced for HMGA1. Taken together, these results clearly indicate HMGA1 as a key regulator of the autophagic pathway in cancer cells, thus suggesting a novel mechanism through which HMGA1 can contribute to cancer progression.

HIPK2 is a serine/threonine kinase that acts as a coregulator of an increasing number of factors involved in cell survival and proliferation during development and in response to different types of stress. Here we report on a novel target of HIPK2, the cyclin-dependent kinase inhibitor p27(kip1). HIPK2 phosphorylates p27(kip1) in vitro and in vivo at serine 10, an event that accounts for 80% of the total p27(kip1) phosphorylation and plays a crucial role in the stability of the protein. Indeed, HIPK2 depletion by transient or stable RNA interference in tumor cells of different origin was consistently associated with strong reduction of p27(kip1) phosphorylation at serine 10 and of p27(kip1) stability. An initial evaluation of the functional relevance of this HIPK2-mediated regulation of p27(kip1) revealed a contribution to cell motility, rather than to cell proliferation, but only in cells that do not express wild-type p53.

Oestrogen exposure has been linked to a risk for the development of testicular germ cell cancers. The effects of oestrogen are now known to be mediated by oestrogen receptor-α (ERα) and ERβ subtypes, but only ERβ has been found in human germ cells of normal testis. However, its expression was markedly diminished in seminomas, embryonal cell carcinomas and mixed germ cell tumours, but remains high in teratomas. PATZ1 is a recently discovered zinc finger protein that, due to the presence of the POZ domain, acts as a transcriptional repressor affecting the basal activity of different promoters. We have previously described that PATZ1 plays a crucial role in normal male gametogenesis and that its up-regulation and mislocalization could be associated with the development of testicular germ cell tumours. Here we show that ERβ interacts with PATZ1 in normal germ cells, while down-regulation of ERβ associates with transcriptional co-regulator PATZ1 delocalization in human testicular seminomas. In addition, we show that the translocation of PATZ1 from the cytoplasm into the nucleus is regulated by cAMP, which also induces increased expression and nuclear localization of ERβ, while this effect is counteracted by using the anti-oestrogen ICI 182-780.

[This article][1] ([1][2]) has been retracted at the request of the editors. The editors were made aware of concerns regarding potential manipulation of data in the article. An internal review by the editors determined that multiple γ-tubulin Western blot bands appear to be duplicated in Figs. 1A

The High Mobility Group A1 proteins (HMGA1) are nonhistone chromatinic proteins with a critical role in development and cancer. We have recently reported that HMGA1 proteins are able to increase the expression of spindle assembly checkpoint (SAC) genes, thus impairing SAC function and causing chromosomal instability in cancer cells. Moreover, we found a significant correlation between HMGA1 and SAC genes expression in human colon carcinomas. Here, we report that mouse embryonic fibroblasts null for the Hmga1 gene show downregulation of Bub1, Bub1b, Mad2l1 and Ttk SAC genes, and present several features of chromosomal instability, such as nuclear abnormalities, binucleation, micronuclei and karyotypic alterations. Interestingky, also MEFs carrying only one impaired Hmga1 allele present karyotypic alterations. These results indicate that HMGA1 proteins regulate SAC genes expression and, thereby, genomic stability also in embryonic cells.

Homeodomain-interacting protein kinase 2 (HIPK2) is a multitalented coregulator of an increasing number of transcription factors and cofactors involved in cell death and proliferation in several organs and systems. As Hipk2−/− mice show behavioral abnormalities consistent with cerebellar dysfunction, we investigated whether Hipk2 is involved in these neurological symptoms. To this aim, we characterized the postnatal developmental expression profile of Hipk2 in the brain cortex, hippocampus, striatum, and cerebellum of mice by real-time PCR, western blot analysis, and immunohistochemistry. Notably, we found that whereas in the brain cortex, hippocampus, and striatum, HIPK2 expression progressively decreased with age, that is, from postnatal day 1 to adulthood, it increased in the cerebellum. Interestingly, mice lacking Hipk2 displayed atrophic lobules and a visibly smaller cerebellum than did wild-type mice. More important, the cerebellum of Hipk2−/− mice showed a strong reduction in cerebellar Purkinje neurons during adulthood. Such reduction is due to the activation of an apoptotic process associated with a compromised proteasomal function followed by an unpredicted accumulation of ubiquitinated proteins. In particular, Purkinje cell dysfunction was characterized by a strong accumulation of ubiquitinated β-catenin. Moreover, our behavioral tests showed that Hipk2−/− mice displayed muscle and balance impairment, indicative of Hipk2 involvement in cerebellar function. Taken together, these results indicate that Hipk2 exerts a relevant role in the survival of cerebellar Purkinje cells and that Hipk2 genetic ablation generates cerebellar dysfunction compatible with an ataxic-like phenotype.

// Giovanna Maria Pierantoni 1, * , Andrea Conte 1, * , Cinzia Rinaldo 2 , Mara Tornincasa 1 , Raffaele Gerlini 1 , Antonella Federico 1 , Davide Valente 2 , Enzo Medico 3 , Alfredo Fusco 1 1 Istituto di Endocrinologia ed Oncologia Sperimentale del CNR and Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita di Napoli “Federico II”, 80131, Naples, Italy 2 Istituto di Biologia e Patologie Molecolari del CNR c/o Universita “Sapienza” di Roma, 00185, Rome, Italy 3 Laboratorio di Oncogenomica, Istituto per la ricerca sul cancro, 10060 Candiolo, Turin, Italy * These authors have contributed equally to this work Correspondence to: Alfredo Fusco, e-mail: alfusco@unina.it Giovanna Maria Pierantoni, e-mail: gmpieran@unina.it Keywords: HMGA1, CIN, SAC, transcriptional regulation Received: February 27, 2015 Accepted: May 05, 2015 Published: May 15, 2015 ABSTRACT The mitotic spindle assembly checkpoint (SAC) is an essential control system of the cell cycle that contributes to mantain the genomic stability of eukaryotic cells. SAC genes expression is often deregulated in cancer cells, leading to checkpoint impairment and chromosome instability. The mechanisms responsible for the transcriptional regulation and deregulation of these genes are still largely unknown. Herein we identify the nonhistone architectural nuclear proteins High Mobility Group A1 (HMGA1), whose overexpression is a feature of several human malignancies and has a key role in cancer progression, as transcriptional regulators of SAC genes expression. In particular, we show that HMGA1 proteins are able to increase the expression of the SAC genes Ttk, Mad2l1, Bub1 and Bub1b , binding to their promoter regions. Consistently, HMGA1-depletion induces SAC genes downregulation associated to several mitotic defects. In particular, we observed a high number of unaligned chromosomes in metaphase, a reduction of prometaphase time, a delay of anaphase, a higher cytokinesis time and a higher percentage of cytokinesis failure by using live-cell microscopy. Finally, a significant direct correlation between HMGA1 and SAC genes expression was detected in human colon carcinomas indicating a novel mechanism by which HMGA1 contributes to cancer progression.

It is well established that estrogens participate in the control of normal spermatogenesis and endogenous or environmental estrogens are involved in pathological germ cell proliferation including testicular germ cell tumors. The effects of estrogen are now known to be mediated by estrogen receptor-α (ERα) and ERβ subtypes, but only ERβ has been found in human germ cells of normal testis. However, its expression was markedly diminished in human testicular seminomas. The expression and the possible interaction of ERβ and HMGA1 were studied in normal germ cells and in human testicular seminomas. GC1 and TCam-2 germ cell lines, were used; in addition, a tissue micro-array (TMA) was built using the most representative areas from 35 cases of human testicular seminomas. The expression and the interaction of ERβ and HMGA1 were observed by using immunoprecipitation and Western blot analyses in combination with immunocytochemistry and immunofluorescence analyses. Here, we show that ERβ interacts with HMGA1 in normal germ cells, while down regulation of ERβ associates with transcriptional co-regulator HMGA1 over-expression and cytoplasmic localization both in human testicular seminomas and in TCam-2 cell line. In addition, we show that 17β-oestradiol induces an HMGA1 increased cytoplasmic expression associated to an ERβ down-regulation in TCam-2 cell line. Taken together, our results suggest that exposure to estrogens or estrogen-mimics, in some as of yet undefined manner, diminishes the ERβ-mediated growth restraint in human testicular seminoma, probably due to the HMGA1 cytoplasmic delocalization associated with ERβ down-regulation.

Context:We have previously demonstrated that a set of micro-RNA (miRNA) is significantly down-regulated in anaplastic thyroid carcinomas with respect to normal thyroid tissues and to differentiated thyroid carcinomas.Objective:The objective was to evaluate the role of two of these down-regulated miRNA, miR-25 and miR-30d, in thyroid carcinogenesis.Design:miR-25 and miR-30d expression was restored in the ACT-1, 8505c, and FRO anaplastic thyroid cell lines, and their effects on cell proliferation, migration, and target expression were evaluated.Results:We report that miR-25 and miR-30d target the polycomb protein enhancer of zeste 2 (EZH2) that has oncogenic activity and is drastically up-regulated in anaplastic thyroid carcinomas but not in the differentiated ones. Ectopic expression of miR-25 and miR-30d inhibited proliferation and colony formation of anaplastic thyroid carcinoma cells by inducing G2/M-phase cell-cycle arrest. Finally, we found an inverse correlation between the expression of these miRNA and the EZH2 protein levels in anaplastic thyroid carcinomas, suggesting a critical role of these miRNA in regulating EZH2 expression also in vivo.Conclusion:The down-regulation of miR-25 and miR-30d could contribute to the process of thyroid cancer progression, leading to the development of anaplastic carcinomas targeting EZH2 mRNA.

Oestrogen exposure has been linked to a risk for the development of testicular germ cell cancers. The effects of oestrogen are now known to be mediated by oestrogen receptor-α (ERα) and ERβ subtypes, but only ERβ has been found in human germ cells of normal testis. However, its expression was markedly diminished in seminomas, embryonal cell carcinomas and mixed germ cell tumours, but remains high in teratomas. PATZ1 is a recently discovered zinc finger protein that, due to the presence of the POZ domain, acts as a transcriptional repressor affecting the basal activity of different promoters. We have previously described that PATZ1 plays a crucial role in normal male gametogenesis and that its up-regulation and mislocalization could be associated with the development of testicular germ cell tumours. Here we show that ERβ interacts with PATZ1 in normal germ cells, while down-regulation of ERβ associates with transcriptional co-regulator PATZ1 delocalization in human testicular seminomas. In addition, we show that the translocation of PATZ1 from the cytoplasm into the nucleus is regulated by cAMP, which also induces increased expression and nuclear localization of ERβ, while this effect is counteracted by using the anti-oestrogen ICI 182-780.

The high mobility group A (HMGA) non-histone chromosomal proteins constitute a subgroup of HMG accessory factors that play key roles in chromatin architecture and orchestrate the assembly of nucleoprotein complexes involved in gene transcription, replication, and chromatin structure through a complex network of protein-DNA and protein-protein interactions. HMGA overexpression and gene rearrangements are frequent events in human cancer, but the molecular basis of HMGA oncogenic activity remains partially unclear. Recently, we have defined a new physical and functional interaction between HMGA1 and p53. This interaction modulates the transcription of p53 target genes such as Mdm2, p21waf1, Bax, and inhibits p53-mediated apoptosis. Moreover, we have described a new mechanism through which HMGA1 inhibits p53-mediated apoptosis by counteracting the p53 proapoptotic activator homeodomain-interacting protein kinase 2 (HIPK2), which binds to and activates p53 by phosphorylating it at Ser46. We found that HMGA1 overexpression promoted HIPK2 relocalization from the nucleus to the cytoplasm, with consequent inhibition of p53 apoptotic function. During my doctorate course, I looked for other target genes that could be regulated by the HMGA1-p53 complex. So, I focused my attention on the apoptosis inhibitor gene Bcl-2, because this gene belongs to Bax family, is regulated by p53, and its promoter has several possible target regions for the HMGA1 proteins binding. I demonstrated that HMGA1 binds Bcl-2 promoter in vitro and in vivo, and that this binding exerts regulatory effects on Bcl-2 transcription. In fact, HMGA1 is able to abolish the repression promoted by p53 on Bcl-2 expression. This effect, as for the Bax promoter regulation, is due to HIPK2 delocalization, from the nucleus to the cytoplasm. Therefore, also Bcl-2 is a gene regulated by HMGA1-p53-HIPK2 complex. Subsequently, my studies were focalized in finding other possible components of this multiprotein complex. Using the neuronal cell line ND7, I found that the transcription factor Brn-3a belongs to this complex. Brn-3a is one of the most important activators of Bcl-2 transcription but, when p53 is overexpressed, it acts as a corepressor for this gene. Moreover, the binding of Brn-3a to its consensus sequences is positively regulated by HIPK2. I demonstrated that HMGA1 overexpression, in this cellular context, promotes not only HIPK2 delocalization from nucleus to the cytoplasm, but also the reduction of Brn-3a binding to the Bcl-2 promoter, removing Brn-3a from its role of corepressor following p53 overexpression. These data support the causal role suggested for the HMGA1 proteins in the neoplastic transformation and provide another mechanism of inactivation of p53 apoptotic function

RET/papillary thyroid carcinoma 1 (PTC1) oncogene is frequently activated in human PTCs. It is characterized by the fusion of the intracellular kinase-encoding domain of RET to the first 101 amino acids of CCDC6. The aim of our work is to characterize the function of the CCDC6 protein to better understand the function of its truncation, that results in the loss of the expression of one allele, in the process of thyroid carcinogenesis. Here, we report that CCDC6 interacts with CREB1 and represses its transcriptional activity by recruiting histone deacetylase 1 and protein phosphatase 1 proteins at the CRE site of the CREB1 target genes. Finally, we show an increased CREB1 phosphorylation and activity in PTCs carrying the RET/PTC1 oncogene. Consistently, an increased expression of two known CREB1 target genes, AREG and cyclin A, was observed in this subgroup of thyroid papillary carcinomas. Therefore, the repression of CREB1 activity by CCDC6 has a critical function in the development of human thyroid papillary carcinomas carrying RET/PTC1 activation. © 2010 Macmillan Publishers Limited All rights reserved.

The high-mobility group A (HMGA) proteins are a family of non-histone chromatin factors, encoded by the HMGA1 and HMGA2 genes. Several studies demonstrate that HMGA proteins have a critical role in neoplastic transformation, and their overexpression is mainly associated with a highly malignant phenotype, also representing a poor prognostic index. Even though a cytoplasmic localization of these proteins has been previously reported in some highly malignant neoplasias, a clear role for this localization has not been defined. Here, we first confirm the localization of the HMGA1 proteins in the cytoplasm of cancer cells, and then we report a novel mechanism through which HMGA1 inhibits p53-mitochondrial apoptosis by counteracting the binding of p53 to the anti-apoptotic factor Bcl-2. Indeed, we demonstrate a physical and functional interaction between HMGA1 and Bcl-2 proteins. This interaction occurs at mitochondria interfering with the ability of p53 protein to bind Bcl-2, thus counteracting p53-mediated mitochondrial apoptosis. This effect is associated with the inhibition of cytochrome c release and activation of caspases. Consistent with this mechanism, a strong correlation between HMGA1 cytoplasmic localization and a more aggressive histotype of thyroid, breast and colon carcinomas has been observed. Therefore, cytoplasmic localization of HMGA1 proteins in malignant tissues is a novel mechanism of inactivation of p53 apoptotic function.

The high-mobility group A (HMGA) proteins are a family of non-histone chromatin factors, encoded by the HMGA1 and HMGA2 genes. Several studies demonstrate that HMGA proteins have a critical role in neoplastic transformation, and their overexpression is mainly associated with a highly malignant phenotype, also representing a poor prognostic index. Even though a cytoplasmic localization of these proteins has been previously reported in some highly malignant neoplasias, a clear role for this localization has not been defined. Here, we first confirm the localization of the HMGA1 proteins in the cytoplasm of cancer cells, and then we report a novel mechanism through which HMGA1 inhibits p53-mitochondrial apoptosis by counteracting the binding of p53 to the anti-apoptotic factor Bcl-2. Indeed, we demonstrate a physical and functional interaction between HMGA1 and Bcl-2 proteins. This interaction occurs at mitochondria interfering with the ability of p53 protein to bind Bcl-2, thus counteracting p53-mediated mitochondrial apoptosis. This effect is associated with the inhibition of cytochrome c release and activation of caspases. Consistent with this mechanism, a strong correlation between HMGA1 cytoplasmic localization and a more aggressive histotype of thyroid, breast and colon carcinomas has been observed. Therefore, cytoplasmic localization of HMGA1 proteins in malignant tissues is a novel mechanism of inactivation of p53 apoptotic function.

The high mobility group A (HMGA) non-histone chromosomal proteins play key roles in chromatin architecture and orchestrate the assembly of nucleoprotein complexes involved in gene transcription, replication, and chromatin structure. HMGA overexpression and gene rearrangements are frequent events in human cancer, but the molecular basis of HMGA oncogenic activity has not been completely elucidated. Recently, we have defined a new physical and functional interaction between HMGA1 and p53. This interaction modulates the transcription of p53 target genes such as Mdm2, p21waf1, Bax, Bcl-2, and inhibits p53-mediated apoptosis. Here we report that HMGA1 does not act only at transcriptional level but it has also a direct, anti-apoptogenic role at the mitochondria. In fact, we found that in mouse embryonic fibroblast (MEF) and several tumour cell lines overexpressing HMGA1, this protein is present at cytoplasmic level other than in the nucleus. Moreover, we demonstrate that HMGA1 stably localizes at the mitochondria, in which it binds Bcl-2, displacing it from the binding to p53, thus counteracting the release of cytochrome C. In agreement with this observation, the HMGA1-overexpressing cells show a significant reduction of cytochrome C release, after lethal dose of Ultraviolet (UV) light, whereas HMGA1-depleted cells are more susceptibile to UV treatment. These data define a new localization of HMGA1 at the mitochondria level that allows HMGA1 to inhibit by a new mechanism the p53-mediated intrinsic-apoptotic pathway. This mechanism may also have a role in the resistance of malignant cells to chemo- and radio-therapy, in particularly for the tumor cells harboring wild-type form of p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 206. doi:10.1158/1538-7445.AM2011-206