Mara Tornincasa

Down-Regulation of the miR-25 and miR-30d Contributes to the Development of Anaplastic Thyroid Carcinoma Targeting the Polycomb Protein EZH2

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.

High-Mobility Group A1 Proteins Regulate p53-Mediated Transcription of Bcl-2 Gene

We have previously described a mechanism through which the high-mobility group A1 (HMGA1) proteins inhibit p53-mediated apoptosis by delocalizing the p53 proapoptotic activator homeodomain-interacting protein kinase 2 from the nucleus to the cytoplasm. By this mechanism, HMGA1 modulates the transcription of p53 target genes such as Mdm2, p21(waf1), and Bax, inhibiting apoptosis. Here, we report that HMGA1 antagonizes the p53-mediated transcriptional repression of another apoptosis-related gene, Bcl-2, suggesting a novel mechanism by which HMGA1 counteracts apoptosis. Moreover, HMGA1 overexpression promotes the reduction of Brn-3a binding to the Bcl-2 promoter, thereby blocking the Brn-3a corepressor function on Bcl-2 expression following p53 activation. Consistently, a significant direct correlation between HMGA1 and Bcl-2 overexpression has been observed in human breast carcinomas harboring wild-type p53. Therefore, this study suggests a novel mechanism, based on Bcl-2 induction, by which HMGA1 overexpression contributes to the escape from apoptosis leading to neoplastic transformation.

Down‐regulation of oestrogen receptor‐β associates with transcriptional co‐regulator PATZ1 delocalization in human testicular seminomas

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. Copyright

The high-mobility group A1-estrogen receptor β nuclear interaction is impaired in human testicular seminomas.

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. J. Cell. Physiol. 227: 3749–3755, 2012.

Homeodomain-interacting protein kinase-2 stabilizes p27(kip1) by its phosphorylation at serine 10 and contributes to cell motility.

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 p27kip1. HIPK2 phosphorylates p27kip1 in vitro and in vivo at serine 10, an event that accounts for 80% of the total p27kip1 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 p27kip1 phosphorylation at serine 10 and of p27kip1 stability. An initial evaluation of the functional relevance of this HIPK2-mediated regulation of p27kip1 revealed a contribution to cell motility, rather than to cell proliferation, but only in cells that do not express wild-type p53.

Genetic ablation of homeodomain-interacting protein kinase 2 selectively induces apoptosis of cerebellar Purkinje cells during adulthood and generates an ataxic-like phenotype

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.

Hmga1 null mouse embryonic fibroblasts display downregulation of spindle assembly checkpoint gene expression associated to nuclear and karyotypic abnormalities

ABSTRACT 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.

High mobility group A1 protein modulates autophagy in cancer cells

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.

Retraction to: High-mobility group A1 protein inhibits p53-mediated intrinsic apoptosis by interacting with Bcl-2 at mitochondria

Retraction to: High-mobility group A1 protein inhibits p53-mediated intrinsic apoptosis by interacting with Bcl-2 at mitochondria

Homeodomain-interacting protein kinase-2 stabilizes p27kip1 by its phosphorylation at serine 10 and contributes to cell motility (Journal of Biological Chemistry (2011) 286, (29005-29013) DOI: 10.1074/jbc.A111.230854)

Homeodomain-interacting protein kinase-2 stabilizes p27 by its phosphorylation at serine 10 and contributes to cell motility Giovanna Maria Pierantoni, Francesco Esposito, Mara Tornincasa, Cinzia Rinaldo, Giuseppe Viglietto, Silvia Soddu, and Alfredo Fusco This article has been retracted by the publisher. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 25, p. 17426, June 20, 2014