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1.
Cell Death Dis ; 13(7): 595, 2022 07 11.
Article in English | MEDLINE | ID: mdl-35817766

ABSTRACT

Age-related hearing loss (ARHL) is the most common sensory impairment mainly caused by degeneration of sensory hair cells in the cochlea with no causal medical treatment available. Auditory function and sensory hair cell survival critically depend on the Kv7.4 (KCNQ4) channel, a voltage-gated potassium channel expressed in outer hair cells (OHCs), with its impaired function or reduced activity previously associated with ARHL. Here, we investigated the effect of a potent small-molecule Kv7.4 agonist on ARHL in the senescence-accelerated mouse prone 8 (SAMP8) model. For the first time in vivo, we show that Kv7.4 activation can significantly reduce age-related threshold shifts of auditory brainstem responses as well as OHC loss in the SAMP8 model. Pharmacological activation of Kv7.4 thus holds great potential as a therapeutic approach for ARHL as well as other hearing impairments related to Kv7.4 function.


Subject(s)
Hair Cells, Auditory, Outer , Hearing Loss , Animals , Cochlea , Disease Models, Animal , Hearing , Hearing Loss/drug therapy , Mice , Potassium Channels
2.
Cell Death Dis ; 10(10): 747, 2019 10 03.
Article in English | MEDLINE | ID: mdl-31582725

ABSTRACT

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.


Subject(s)
HMGA1a Protein/genetics , Protein Serine-Threonine Kinases/genetics , Respiratory Tract Diseases/genetics , Thyroid Gland/abnormalities , Animals , Animals, Newborn , Embryonic Development , Gene Deletion , Gene Expression Regulation , HMGA1a Protein/metabolism , HeLa Cells , Humans , Lung/metabolism , Lung/pathology , Mice, Inbred C57BL , Mice, Knockout , Protein Serine-Threonine Kinases/metabolism , Pulmonary Surfactant-Associated Proteins , Respiratory Tract Diseases/pathology , Thyroid Gland/embryology , Thyroid Gland/pathology
4.
Cell Death Differ ; 24(11): 1948-1962, 2017 11.
Article in English | MEDLINE | ID: mdl-28777374

ABSTRACT

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.


Subject(s)
Autophagy , HMGA1a Protein/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Animals , Autophagy-Related Protein 5/metabolism , Autophagy-Related Protein-1 Homolog/genetics , Autophagy-Related Protein-1 Homolog/metabolism , Cell Proliferation , Cell Survival , Gene Knockdown Techniques , Gene Silencing , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Microtubule-Associated Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Transcription, Genetic
5.
Cell Cycle ; 15(6): 812-8, 2016.
Article in English | MEDLINE | ID: mdl-26889953

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.


Subject(s)
Chromosomal Instability , Fibroblasts/metabolism , G2 Phase Cell Cycle Checkpoints , HMGA1a Protein/genetics , M Phase Cell Cycle Checkpoints/genetics , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Nucleus/genetics , Cell Nucleus/pathology , Embryo, Mammalian , Fibroblasts/pathology , Gene Expression Regulation , Genetic Complementation Test , HMGA1a Protein/deficiency , Karyotype , Mice , Mice, Knockout , Micronuclei, Chromosome-Defective , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Signal Transduction
6.
Oncotarget ; 6(19): 17342-53, 2015 Jul 10.
Article in English | MEDLINE | ID: mdl-26009897

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.


Subject(s)
Chromosomal Instability/genetics , Colonic Neoplasms/genetics , Gene Expression Regulation, Neoplastic/genetics , HMGB Proteins/genetics , M Phase Cell Cycle Checkpoints/genetics , Animals , Blotting, Western , Chromatin Immunoprecipitation , Fluorescent Antibody Technique , HeLa Cells , Humans , Immunohistochemistry , Mice , NIH 3T3 Cells , Polymerase Chain Reaction , Transfection
7.
Oncotarget ; 6(12): 10320-34, 2015 Apr 30.
Article in English | MEDLINE | ID: mdl-25868975

ABSTRACT

HIPK2, a cell fate decision kinase inactivated in several human cancers, is thought to exert its oncosuppressing activity through its p53-dependent and -independent apoptotic function. However, a HIPK2 role in cell proliferation has also been described. In particular, HIPK2 is required to complete cytokinesis and impaired HIPK2 expression results in cytokinesis failure and tetraploidization. Since tetraploidy may yield to aneuploidy and chromosomal instability (CIN), we asked whether unscheduled tetraploidy caused by loss of HIPK2 might contribute to tumorigenicity. Here, we show that, compared to Hipk2+/+ mouse embryo fibroblasts (MEFs), hipk2-null MEFs accumulate subtetraploid karyotypes and develop CIN. Accumulation of these defects inhibits proliferation and spontaneous immortalization of primary MEFs whereas increases tumorigenicity when MEFs are transformed by E1A and Harvey-Ras oncogenes. Upon mouse injection, E1A/Ras-transformed hipk2-null MEFs generate tumors with genetic alterations resembling those of human cancers derived by initial tetraploidization events, such as pancreatic adenocarcinoma. Thus, we evaluated HIPK2 expression in different stages of pancreatic transformation. Importantly, we found a significant correlation among reduced HIPK2 expression, high grade of malignancy, and high nuclear size, a marker of increased ploidy. Overall, these results indicate that HIPK2 acts as a caretaker gene, whose inactivation increases tumorigenicity and causes CIN by cytokinesis failure.


Subject(s)
Carcinogenesis/pathology , Chromosomal Instability , Cytokinesis/physiology , Protein Serine-Threonine Kinases/deficiency , Animals , Carcinogenesis/genetics , Carcinogenesis/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Differentiation/physiology , Cell Proliferation/physiology , Female , HeLa Cells , Humans , Mice , Mice, Nude , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Transfection
8.
Oncotarget ; 5(18): 8341-54, 2014 Sep 30.
Article in English | MEDLINE | ID: mdl-25268743

ABSTRACT

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.


Subject(s)
HMGA Proteins/genetics , MicroRNAs/genetics , Ovarian Neoplasms/genetics , Pseudogenes/genetics , Thyroid Carcinoma, Anaplastic/genetics , Thyroid Neoplasms/genetics , Animals , Apoptosis , Cell Proliferation , Cellular Senescence , Computational Biology , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , HEK293 Cells , HMGA Proteins/metabolism , Humans , MCF-7 Cells , Mice, Inbred C57BL , Mice, Transgenic , MicroRNAs/metabolism , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , RNA Interference , Thyroid Carcinoma, Anaplastic/metabolism , Thyroid Carcinoma, Anaplastic/pathology , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/pathology , Time Factors , Transfection
10.
J Clin Endocrinol Metab ; 97(5): E710-8, 2012 May.
Article in English | MEDLINE | ID: mdl-22399519

ABSTRACT

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.


Subject(s)
Carcinoma/genetics , DNA-Binding Proteins/genetics , Down-Regulation , MicroRNAs/genetics , Thyroid Gland/metabolism , Thyroid Neoplasms/genetics , Transcription Factors/genetics , Carcinoma/metabolism , Carcinoma/pathology , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation , DNA-Binding Proteins/metabolism , Enhancer of Zeste Homolog 2 Protein , Gene Expression/genetics , Humans , MicroRNAs/metabolism , Polycomb Repressive Complex 2 , Thyroid Gland/pathology , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/pathology , Transcription Factors/metabolism
11.
J Cell Physiol ; 227(12): 3749-55, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22392906

ABSTRACT

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 ERß subtypes, but only ERß 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 ERß 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 ERß and HMGA1 were observed by using immunoprecipitation and Western blot analyses in combination with immunocytochemistry and immunofluorescence analyses. Here, we show that ERß interacts with HMGA1 in normal germ cells, while down regulation of ERß 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 17ß-oestradiol induces an HMGA1 increased cytoplasmic expression associated to an ERß 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 ERß-mediated growth restraint in human testicular seminoma, probably due to the HMGA1 cytoplasmic delocalization associated with ERß down-regulation.


Subject(s)
Estrogen Receptor beta/metabolism , Gene Expression Regulation, Neoplastic/physiology , HMGA1a Protein/metabolism , Seminoma/metabolism , Animals , Cell Line, Tumor , Estradiol/pharmacology , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/genetics , HMGA1a Protein/genetics , HMGA2 Protein/genetics , HMGA2 Protein/metabolism , Humans , Male , Mice , Protein Array Analysis , Protein Binding , Testicular Neoplasms/metabolism
12.
J Biol Chem ; 286(33): 29005-29013, 2011 08 19.
Article in English | MEDLINE | ID: mdl-21715331

ABSTRACT

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.


Subject(s)
Carrier Proteins/metabolism , Cell Movement/physiology , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Protein Serine-Threonine Kinases/metabolism , Carrier Proteins/genetics , Cell Line , Cyclin-Dependent Kinase Inhibitor p27/genetics , Humans , Phosphorylation/physiology , Protein Serine-Threonine Kinases/genetics , RNA Interference , Serine/genetics , Serine/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
13.
J Pathol ; 224(1): 110-20, 2011 May.
Article in English | MEDLINE | ID: mdl-21381029

ABSTRACT

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 ERß subtypes, but only ERß 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 ERß interacts with PATZ1 in normal germ cells, while down-regulation of ERß 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 ERß, while this effect is counteracted by using the anti-oestrogen ICI 182-780.


Subject(s)
Estrogen Receptor beta/metabolism , Kruppel-Like Transcription Factors/metabolism , Repressor Proteins/metabolism , Seminoma/metabolism , Testicular Neoplasms/metabolism , Antineoplastic Agents, Hormonal/pharmacology , Cell Nucleus/metabolism , Cyclic AMP/pharmacology , Cytoplasm/metabolism , Down-Regulation , Estradiol/analogs & derivatives , Estradiol/pharmacology , Estrogen Antagonists/pharmacology , Fulvestrant , Gene Expression Regulation, Neoplastic , Humans , Kruppel-Like Transcription Factors/genetics , Male , Neoplasm Proteins/metabolism , Repressor Proteins/genetics , Seminoma/pathology , Translocation, Genetic/drug effects , Tumor Cells, Cultured
14.
Cancer Res ; 70(13): 5379-88, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20530667

ABSTRACT

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.


Subject(s)
Breast Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Genes, bcl-2 , HMGA1a Protein/genetics , Tumor Suppressor Protein p53/genetics , Animals , Apoptosis/physiology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Carrier Proteins/genetics , Carrier Proteins/metabolism , Down-Regulation , Female , HMGA1a Protein/biosynthesis , Humans , Mice , Promoter Regions, Genetic , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics , Transcription Factor Brn-3A/genetics , Transcription Factor Brn-3A/metabolism , Transcription, Genetic , Tumor Suppressor Protein p53/metabolism
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