Deregulation of HMGA1 expression induces chromosome instability through regulation of spindle assembly checkpoint genes

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

HIPK2 deficiency causes chromosomal instability by cytokinesis failure and increases tumorigenicity

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

HIGH-MOBILITY GROUP A1 (HMGA1) PROTEIN INHIBITS P53-MEDIATED INTRINSIC APOPTOSIS INTERACTING WITH Bcl-2 AT MITOCHONDRIA

ABSTRACT 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 rearrangement are frequent events in human cancer. Their importance in cancer progression and their role in apoptosis has been defined by a new physical and functional interaction between HMGA1 and p53 proteins. HMGA1 inhibits p53-mediated apoptosis modulating the transcription of p53 target genes as Mdm2, p21Waf1, Bax, Bcl-2 and promoting Hipk2 relocalization from the nucleus to the cytoplasm. Even though HMGA1 proteins have been identified as nuclear proteins, abundant HMGA1 expression has been frequently detected in the cytoplasm of cancer cells but it has been often considered as an artefact likely do a very abundant HMGA1 expression, and no deep investigation has been undertaken to clearly demonstrate the presence of these proteins in the cytoplasm, and to unveil the functional role of this localization. Preliminary studies obtained by a screening of an Antibody ArrayTM strongly suggest possible cytoplasmic interactors of HMGA1 proteins in tumoral cell lines. My thesis project is focalized on the interaction between HMGA1 and anti-apoptotic factor Bcl-2. I have identified and characterized a new subcellular localization of HMGA1 proteins by analysis of total, nuclear and cytoplasmic cell lysates from several normal, and tumor-derived cell lines. Then, I confirmed the interaction HMGA1-Bcl-2 in cytoplasm in vivo and in vitro. Moreover, since p53 interacts with Bcl-2 blocking its anti-apoptotic function, I identified a new mechanism that allows HMGA1 proteins to inhibit the p53-mediated intrinsic-apoptotic pathway. Indeed, HMGA1 localizes at mitochondria and displaces it from the binding to Bcl-2 enhancing its anti-apoptotic function. Finally, I reported the correlation between the HMGA1 cytoplasmic localization and a more aggressive phenotype in thyroid, breast and colon cancer

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

Down-regulation of oestrogen receptor-?? 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-?? (ER??) and ER?? subtypes, but only ER?? 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 ER?? interacts with PATZ1 in normal germ cells, while down-regulation of ER?? 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 ER??, while this effect is counteracted by using the anti-oestrogen ICI 182-780

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

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

HMGA1 pseudogenes as candidate proto-oncogenic competitive endogenous RNAs

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