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High-mobility group AT-hook 2 (HMGA2) is an architectural transcription factor that plays essential roles in embryonic development and cancer progression. However, the mechanism of HMGA2 regulation remains largely uncharacterized. Here, we demonstrate that HMGA2 can be modulated by hepatitis B X-interacting protein (HBXIP), an oncogenic transcriptional coactivator, in esophageal squamous cell carcinoma (ESCC). HMGA2 expression was positively associated with HBXIP expression in clinical ESCC tissues, and their high levels were associated with advanced tumor stage and reduced overall and disease-free survival. We found that oncogenic HBXIP could posttranslationally upregulate HMGA2 protein level in ESCC cells. HBXIP induced HMGA2 acetylation at the lysine 26 (K26), resulting in HMGA2 protein accumulation. In this process, HBXIP increased the acetyltransferase p300/CBP-associated factor (PCAF) phosphorylation and activation via the Akt pathway, then PCAF directly interacted with HMGA2, leading to HMGA2 acetylation in the cells. HMGA2 K26 acetylation enhanced its DNA binding capacity and blocked its ubiquitination and then inhibited proteasome-dependent degradation. Functionally, HBXIP-stabilized HMGA2 could promote ESCC cell growth in vitro and in vivo. Strikingly, aspirin suppressed ESCC growth by inhibiting HBXIP and HMGA2. Collectively, our findings disclose a new mechanism for the posttranslational regulation of HMGA2 mediated by HBXIP in ESCC.

Although oxidative stress has been shown to induce senescence and replication stress independently, no study has implicated unresolved replication stress as the driver for cellular senescence in response to oxidative stress. Using cells exposed to increasing concentrations of hydrogen peroxide, we show that sub-lethal amount of exogenous hydrogen peroxide induces two waves of DNA damage. The first wave is rapid and transient while the second wave coincides with the cells transition from the S to the G2/M phases of cell cycle. Subsequently, cells enter growth arrest accompanied by the acquisition of senescence-associated characteristics. Furthermore, a p53-dependent decrease in Rad51, which is associated with the formation of DNA segments with chromatin alterations reinforcing senescence, and Lamin B1 that is involved in chromatin remodeling, is observed during the establishment of the senescent phenotype. On the other hand, increase in senescence associated-β-Gal activity, a classical marker of senescence and HMGA2, a marker of the senescence-associated heterochromatin foci, is shown to be independent of p53. Together, our findings implicate replication stress-induced endogenous DNA damage as the driver for the establishment of cellular senescence upon sub-lethal oxidative stress, and implicate the role of p53 in some but not all hallmarks of the senescent phenotype.

The tumor-suppressive let-7 microRNA family targets various oncogene-encoding mRNAs. We identify the let-7 targets HMGA2, LIN28B and IGF2BP1 to form a let-7 antagonizing self-promoting oncogenic triangle. Surprisingly, 3'-end processing of IGF2BP1 mRNAs is unaltered in aggressive cancers and tumor-derived cells although IGF2BP1 synthesis was proposed to escape let-7 attack by APA-dependent (alternative polyadenylation) 3' UTR shortening. However, the expression of the triangle factors is inversely correlated with let-7 levels and promoted by LIN28B impairing let-7 biogenesis. Moreover, IGF2BP1 enhances the expression of all triangle factors by recruiting the respective mRNAs in mRNPs lacking AGO proteins and let-7 miRNAs. This indicates that the downregulation of let-7, largely facilitated by LIN28B upregulation, and the protection of let-7 target mRNAs by IGF2BP1-directed shielding in mRNPs synergize in enhancing the expression of triangle factors. The oncogenic potential of this triangle was confirmed in ovarian cancer (OC)-derived ES-2 cells transduced with let-7 targeting decoys. In these, the depletion of HMGA2 only diminishes tumor cell growth under permissive conditions. The depletion of LIN28B and more prominently IGF2BP1 severely impairs tumor cell viability, self-renewal and 2D as well as 3D migration. In conclusion, this suggests the targeting of the HMGA2-LIN28B-IGF2BP1 triangle as a promising strategy in cancer treatment.

HMGA proteins are not translated in normal human somatic cells, but are present in high copy numbers in pluripotent embryonic stem cells and most neoplasias. Correlations between the degree of malignancy, patient prognostic index and HMGA levels have been firmly established. Intriguingly, HMGA2 is also found in rare tumor-inducing cells which are resistant to chemotherapy. Here, we demonstrate that HMGA1a/b and HMGA2 possess intrinsic dRP and AP site cleavage activities, and that lysines and arginines in the AT-hook DNA-binding domains function as nucleophiles. We also show that HMGA2 can be covalently trapped at genomic abasic sites in cancer cells. By employing a variety of cell-based assays, we provide evidence that the associated lyase activities promote cellular resistance against DNA damage that is targeted by base excision repair (BER) pathways, and that this protection directly correlates with the level of HMGA2 expression. In addition, we demonstrate an interaction between human AP endonuclease 1 and HMGA2 in cancer cells, which supports our conclusion that HMGA2 can be incorporated into the cellular BER machinery. Our study thus identifies an unexpected role for HMGA2 in DNA repair in cancer cells which has important clinical implications for disease diagnosis and therapy.

High Mobility Group A (HMGA) is a family of architectural nuclear factors which play an important role in neoplastic transformation. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes including transcription. HMGA localization is exclusively nuclear but, to date, the mechanism of nuclear import for these proteins remains unknown. Here, we report the identification and characterization of a nuclear localization signal (NLS) for HMGA2, a member of the HMGA family. The NLS overlaps with the second of the three AT-hooks, the DNA-binding domains characteristic for this group of proteins. The functionality of this NLS was demonstrated by its ability to target a heterologous beta-galactosidase/green fluorescent protein fusion protein to the nucleus. Mutations to alanine of basic residues within the second AT-hook resulted in inhibition of HMGA2 nuclear localization and impairment of its function in activating the cyclin A promoter. In addition, HMGA2 was shown to directly interact with the nuclear import receptor importin-alpha2 via the second AT-hook. HMGA proteins are overexpressed and rearranged in a variety of tumors; our findings can thus help elucidating their role in neoplastic transformation.

The high-mobility-group A2 protein (HMGA2) plays important functional roles in transcriptional regulation, DNA replication and chromatin structure. In this study, the effect of histone deacetylase inhibition on the transcriptional activity of the Hmga2 gene was investigated in vivo both at the endogenous gene level and in a variety of cell lines using transiently transfected promoter constructs. Trichostatin A (TSA) repressed both transfected murine and human Hmga2 promoter constructs 3-8-fold in NIH3T3, F9 and HeLa cells. Steady-state Hmga2 mRNA levels in NIH3T3 cells decreased 4-5-fold following TSA treatment, while pre- treatment of NIH3T3 cells with the transcriptional inhibitor, actinomycin D, completely blocked TSA mediated repression of the Hmga2 gene. Cross-linked chromatin immunoprecipitation (X-ChIP) analysis revealed a 5-6-fold decrease in endogenous Hmga2 promoter bound Sp1 and Sp3 proteins following TSA treatment in parallel with observed loss of acetylated histone H3 and H4. In addition, the poly-pyrimidine-tract-binding protein (PTB) was observed to bind to the Hmga2 promoter in both TSA treated and untreated NIH3T3 cells. Together, these results suggest TSA treatment leads to a decrease in Hmga2 gene transcription, and a significant decrease in promoter bound Sp1, Sp3 and acetylated histones H3 and H4.

The murine Hmgi-c gene, a member of the Hmgi gene family, contains five exons encompassing >110 kb of genomic DNA at the pygmy locus on mouse chromosome 10. Northern analysis identified a 4.1 kb transcript which contains a 324 bp open reading frame encoding a 12 kDa HMGI-C protein. Further analysis defined both the 5' and 3' untranslated regions of the Hmgi-c mRNA species as 658 and 2967 bp respectively. The HMGI-C protein has three consecutive AT hook DNA binding domains and an acidic domain, each of which are encoded by individual exons; such an organization is conserved among the HMGI gene family members from insects to mammals. Similar to the HMGI/Y proteins, the HMGI-C protein does not function as a typical transcriptional activator. Developmental studies revealed that the Hmgi-c gene is expressed predominantly during mouse embryogenesis. Since the human homolog is disrupted in a number of tumors, HMGI-C could play an important role in cell proliferation and differentiation during mammalian development.

The HMGI-C protein is a nuclear phosphoprotein expressed at high levels in transformed cells. The cDNA encoding the mouse protein has been isolated and the sequence of the encoded protein shows that it is related to the HMGY and I proteins, proteins which bind in the minor groove of DNA containing stretches of A and T. The HMGI-C protein has three short highly basic domains, an acidic C-terminal domain, and potential CDC2/p34 and casein kinase II phosphorylation sites. Analysis of mRNA levels demonstrate that the HMGI-C gene is not expressed in a variety of mouse tissues but is expressed in Lewis lung carcinoma cells.

DNA-binding and RNA-binding proteins are usually considered 'undruggable' partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein-nucleic acids interactions based on protein-DNA or protein-RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian high-mobility-group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2-DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2-DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2-DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.

High mobility group I proteins (HMGI, HMGY and HMGI-C) are a family of low molecular mass non-histone nuclear proteins which constitute an important component of the active chromatin structure. Two members of this family, HMGI and HMGY, have been demonstrated to contribute to the transcriptional regulation of several promoters by interacting with the DNA and with different transcription factors. On the contrary, very little is known about the third member, HMGI-C, which plays an important role during embryonic growth and in the process of cell transformation, its gene being rearranged in a large number of mesenchimal tumors. In this paper we show for the first time that HMGI-C is also able to function as architectural factor, enhancing the activity of a transcription factor, NF-kappaB, through the PRDII element of the beta-interferon enhancer. Moreover we show that this enhancement is absolutely dependent on the binding of HMGI-C to its target sequence. The demonstration that HMGI-C is able to modulate transcription is thus an important initial step in the identification of genes regulated by this factor.