Epigenetic Contribution of High-Mobility Group A Proteins to Stem Cell Properties.

High-mobility group A (HMGA) proteins have been examined to understand their participation as structural epigenetic chromatin factors that confer stem-like properties to embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and cancer stem cells (CSCs). The function of HMGA was evaluated in conjunction with that of other epigenetic factors such as histones and microRNAs (miRs), taking into consideration the posttranscriptional modifications (PTMs) of histones (acetylation and methylation) and DNA methylation. HMGA proteins were coordinated or associated with histone and DNA modification and the expression of the factors related to pluripotency. CSCs showed remarkable differences compared with ESCs and iPSCs.

Translating Proteomic Into Functional Data: An High Mobility Group A1 (HMGA1) Proteomic Signature Has Prognostic Value in Breast Cancer.

Cancer is a very heterogeneous disease, and biological variability adds a further level of complexity, thus limiting the ability to identify new genes involved in cancer development. Oncogenes whose expression levels control cell aggressiveness are very useful for developing cellular models that permit differential expression screenings in isogenic contexts. HMGA1 protein has this unique property because it is a master regulator in breast cancer cells that control the transition from a nontumorigenic epithelial-like phenotype toward a highly aggressive mesenchymal-like one. The proteins extracted from HMGA1-silenced and control MDA-MB-231 cells were analyzed using label-free shotgun mass spectrometry. The differentially expressed proteins were cross-referenced with DNA microarray data obtained using the same cellular model and the overlapping genes were filtered for factors linked to poor prognosis in breast cancer gene expression meta-data sets, resulting in an HMGA1 protein signature composed of 21 members (HRS, HMGA1 reduced signature). This signature had a prognostic value (overall survival, relapse-free survival, and distant metastasis-free survival) in breast cancer. qRT-PCR, Western blot, and immunohistochemistry analyses validated the link of three members of this signature (KIFC1, LRRC59, and TRIP13) with HMGA1 expression levels both in vitro and in vivo and wound healing assays demonstrated that these three proteins are involved in modulating tumor cell motility. Combining proteomic and genomic data with the aid of bioinformatic tools, our results highlight the potential involvement in neoplastic transformation of a restricted list of factors with an as-yet-unexplored role in cancer. These factors are druggable targets that could be exploited for the development of new, targeted therapeutic approaches in triple-negative breast cancer.

The expression of the high-mobility group A2 protein in colorectal cancer and surrounding fibroblasts is linked to tumor invasiveness.

Tumor staging of colorectal cancer is typically based on conventional TNM and Dukes classifications. However, additional information could be useful, and there is a significant interest in identifying molecular markers that are related to genetic or epigenetic processes. Using immunohistochemistry, we analyzed the expression of the high-mobility group A2 (previously high-mobility group 1-C [HMGI-C]) protein in 103 colorectal cancer cases to determine its use as a biomarker in colorectal cancer to integrate morphological staging. We found a progressive increase of the high-mobility group A2 protein expression in colorectal cancer tumor samples from cases in which all of the tumor cells were negative up to cases in which all of the tumor cells stained positive. Increased high-mobility group A2 expression is strongly associated with an increase in tumor invasiveness, which was measured through both budding and vascular invasion (P < .0001). Kaplan-Meier estimates showed a decrease in overall survival when vascular invasion is present (P = .023). Moreover, a fraction of the analyzed samples showed high-mobility group A2-positive stromal fibroblasts. Although high-mobility group A2-positive tumors were associated with cell invasiveness, high-mobility group A2-positive stromal fibroblasts were correlated with less invasive tumors. High-mobility group A2 protein expression could be used as a prognostic marker to provide prospective information on patient outcome, complementing the data obtained using conventional pathologic staging systems.

Conformational role for the C-terminal tail of the intrinsically disordered high mobility group A (HMGA) chromatin factors.

The architectural factors HMGA are highly connected hubs in the chromatin network and affect key cellular functions. HMGA have a causal involvement in cancer development; in fact, truncated or chimeric HMGA forms, resulting from chromosomal rearrangements, lack the constitutively phosphorylated acidic C-terminal tail and display increased oncogenic potential, suggesting a functional role for this domain. HMGA belong to the intrinsically disordered protein category, and this prevents the use of classical approaches to obtain structural data. Therefore, we combined limited proteolysis, ion mobility separation-mass spectrometry (IMS-MS), and electrospray ionization-mass spectrometry (ESI-MS) to obtain structural information regarding full length and C-terminal truncated HMGA forms. Limited proteolysis indicates that HMGA acidic tail shields the inner portions of the protein. IMS-MS and ESI-MS show that HMGA proteins can assume a compact form and that the degree of compactness is dependent upon the presence of the acidic tail and its constitutive phosphorylations. Moreover, we demonstrate that C-terminal truncated forms and wild type proteins are post-translationally modified in a different manner. Therefore, we propose that the acidic tail and its phosphorylation could affect HMGA post-translational modification status and likely their activity. Finally, the mass spectrometry-based approach adopted here proves to be a valuable new tool to obtain structural data regarding intrinsically disordered proteins.

HMGA molecular network: From transcriptional regulation to chromatin remodeling.

Nuclear functions rely on the activity of a plethora of factors which mostly work in highly coordinated molecular networks. The HMGA proteins are chromatin architectural factors which constitute critical hubs in these networks. HMGA are referred to as oncofetal proteins since they are highly expressed and play essential functions both during embryonic development and neoplastic transformation. A particular feature of HMGA is their intrinsically disordered status, which confers on them an unusual plasticity in contacting molecular partners. Indeed these proteins are able to bind to DNA at the level of AT-rich DNA stretches and to interact with several nuclear factors. In the post-genomic era, and with the advent of proteomic tools for the identification of protein-protein interactions, the number of HMGA molecular partners has increased rapidly. This has led to the extension of our knowledge of the functional involvement of HMGA from the transcriptional regulation field to RNA processing, DNA repair, and chromatin remodeling and dynamics. This review focuses mainly on the protein-protein interaction network of HMGA and its functional outcome. HMGA molecular partners have been functionally classified and all the information collected in a freely available database (http://www.bbcm.units.it/ approximately manfiol/INDEX.HTM).

Macroscopic differences in HMGA oncoproteins post-translational modifications: C-terminal phosphorylation of HMGA2 affects its DNA binding properties.

HMGA is a family of nuclear proteins involved in a huge number of functions at the chromatin level. It consists of three members, HMGA1a, HMGA1b, and HMGA2, having high sequence homology and sharing the same structural organization (three highly conserved DNA-binding domains, an acidic C-terminal tail, and a protein-protein interaction domain). They are considered important nodes in the chromatin context, establishing a complex network of interactions with both promoter/enhancer sequences and nuclear factors. They are involved in a plethora of biological processes and their activities are finely tuned by several different post-translational modifications. We have performed an LC/MS screening on several different cell lines to investigate HMGA proteins expression and their post-translational modifications in order to detect distinctive modification patterns for each. Our analyses evidenced relevant macroscopic differences in the phosphorylation and methylation patterns of these proteins. These differences occur both within the HMGA family members and in the different cell types. Focusing on HMGA2, we have mapped its in vivo phosphorylation sites demonstrating that, similarly to the HMGA1 proteins, it is highly phosphorylated on the acidic C-terminal tail and that these modifications affect its DNA binding properties.

Interaction proteomics of the HMGA chromatin architectural factors.

The high mobility group A (HMGA) chromatin architectural transcription factors are a group of proteins involved in development and neoplastic transformation. They take part in an articulated interaction network, both with DNA and other nuclear proteins, organizing multimolecular complexes at chromatin level. Here, we report the development of a novel in vitro strategy for the identification of HMGA molecular partners based on the combination of an RP-HPLC prefractionation procedure, 2-DE gels, blot-overlay and MS. To demonstrate that our approach could be a reliable screening method we confirmed a representative number of interactions in vitro by GST pull-down and far-Western and in vivo by co-affinity purification. This approach allowed us to enlarge the HMGA molecular network confirming their involvement also in non-transcriptional-related processes such as RNA processing and DNA repair.

The second AT-hook of the architectural transcription factor HMGA2 is determinant for nuclear localization and function.

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.

HMGA1 inhibits the function of p53 family members in thyroid cancer cells.

HMGA1 is an architectural transcription factor expressed at high levels in transformed cells and tumors. Several lines of evidence indicate that HMGA1 up-regulation is involved in the malignant transformation of thyroid epithelial cells. However, the mechanisms underlying the effect of HMGA1 on thyroid cancer cell phenotype are not fully understood. We now show that in thyroid cancer cells, HMGA1 down-regulation by small interfering RNA and antisense techniques results in enhanced transcriptional activity of p53, TAp63alpha, TAp73alpha, and, consequently, increased apoptosis. Coimmunoprecipitation and pull-down experiments with deletion mutants showed that the COOH-terminal oligomerization domain of p53 family members is required for direct interaction with HMGA1. Moreover, inhibition of HMGA1 expression in thyroid cancer cells resulted in increased p53 oligomerization in response to the DNA-damaging agent doxorubicin. Finally, electrophoretic mobility shift assay experiments showed that the p53-HMGA1 interaction results in reduced DNA-binding activity. These results indicate a new function of HMGA1 in the regulation of p53 family members, thus providing new mechanistic insights in tumor progression.

Breast cancer markers.

This review illustrates the relationships linking the ER and the ErbB family of receptor tyrosine kinases and their kinase pathways in breast cancer. The central role of the ER in activating tumour growth linked gene transcription as well as the cooperating nuclear co-factors very likely implicated in breast cancer tumourigenesis is discussed. The action of ErbB family members has been located upstream of the kinase pathways that begin at plasma membrane and end at the nucleus after complex interconnections with many factors, such as AP-1. The important role of MAPKs and PKB/Akt in cell survival and tumour proliferation is highlighted. Also other factors are discussed such as Fra-1 (a member of the AP-1 complex), E-cadherin (a tumour suppressor), and BRCA1 (another factor involved in tumour growth inhibition). Lactoferrin protein (characteristic of healthy tissues) and resistance proteins have also been briefly discussed.

The AT-hook of the chromatin architectural transcription factor high mobility group A1a is arginine-methylated by protein arginine methyltransferase 6.

The HMGA1a protein belongs to the high mobility group A (HMGA) family of architectural nuclear factors, a group of proteins that plays an important role in chromatin dynamics. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes, such as transcriptional regulation, viral integration, DNA repair, RNA processing, and chromatin remodeling. The activity of HMGA proteins is finely modulated by a variety of post-translational modifications. Arginine methylation was recently demonstrated to occur on HMGA1a protein, and it correlates with the apoptotic process and neoplastic progression. Methyltransferases responsible for these modifications are unknown. Here we show that the protein arginine methyltransferase PRMT6 specifically methylates HMGA1a protein both in vitro and in vivo. By mass spectrometry, the sites of methylation were unambiguously mapped to Arg(57) and Arg(59), two residues which are embedded in the second AT-hook, a region critical for both protein-DNA and protein-protein interactions and whose modification may cause profound alterations in the HMGA network. The in vivo association of HMGA and PRMT6 place this yet functionally uncharacterized methyltransferase in the well established functional context of the chromatin structure organization.

Discovering high mobility group A molecular partners in tumour cells.

DNA-based activities rely on an extremely coordinated sequence of events performed by several chromatin-associated proteins which act in concert. High Mobility Group A (HMGA) proteins are non-histone architectural nuclear factors that participate in the regulation of specific genes but they are also believed to have a more general role in chromatin dynamics. The peculiarity of these proteins is their flexibility, both in terms of DNA-binding and in protein-protein interactions. Since these proteins act as core elements in the assembly of multiprotein complexes called enhanceosomes, and have already displayed the ability to interact with several different proteins, we started a proteomic approach for the systematic identification of their molecular partners. By a combination of affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry we have identified about twenty putative HMGA interactors which could be roughly assigned to three different classes: mRNA processing proteins, chromatin remodelling related factors and structural proteins. Direct HMGA interaction with some of these proteins was confirmed by glutathione-S-transferase pull-down assays and the HMGA domain involved was mapped. Blot-overlay experiments reveal that members of the HMGA family share most of their molecular partners but, interestingly, it seems that there are some cell-type specific partners. Taken together, these experimental data indicate that HMGA proteins are highly connected nodes in the chromatin protein network. Since these proteins are strongly implicated with cancer development, the identification of molecules able to perturb the HMGA molecular network could be a possible tool to interfere with their oncogenic activity.

Differential HMGA expression and post-translational modifications in prostatic tumor cells.

The HMGA architectural nuclear factors are involved in chromatin dynamics and their overexpression has been strongly linked to the neoplastic transformation process. Here we investigate the expression and post-translational modifications (PTMs) of HMGA proteins (HMGA1a, HMGA1b and HMGA2) in the rat prostatic cancer Dunning model (G, AT-1, and MAT-Ly-Lu cell lines). We demonstrate the expression of HMGA2, in addition to HMGA1a and HMGA1b, in both the anaplastic cell lines AT-1 and MAT-Ly-Lu and an extremely specific HMGA1a mono-methylation only in the most metastatic cell line MAT-Ly-Lu. The HMGA ectopic expression in HMGA-negative Dunning G cells does not significantly alter their growth ability, suggesting that, although HMGA expression is necessary for the progression of neoplastic transformation in several cellular models, in these cells it is not sufficient. These data suggest exploring HMGA2 as a potential marker in human prostate tumor and moreover indicate PTMs as an additional tool in the staging of tumor progression.

HMGA1 protein overexpression in human breast carcinomas: correlation with ErbB2 expression.

We measured, by immunohistochemistry, HMGA1 protein expression in 212 breast tissue specimens: 6 normal samples, 28 hyperplastic lesions (13 with cellular atypia), 11 fibroadenomas, 10 in situ ductal carcinomas, 144 ductal carcinomas, and 13 lobular carcinomas. HMGA1 was not expressed in normal breast tissue; HMGA1 staining was intense in 40% of hyperplastic lesions with cellular atypia and in 60% of ductal carcinomas and weak in fibroadenomas and in hyperplastic lesions without cellular atypia. Because HMGA1 expression was similar among ductal breast carcinomas with different histologic grading, we evaluated the association between HMGA1 expression and that of other markers of breast carcinoma invasion (estrogen and progesterone receptors, Ki-67 antigen, and ErbB2) in 21 cases of grade 3 breast ductal carcinomas and 7 cases of breast lobular carcinomas. We found that HMGA1 expression tended to be associated only with c-erbB-2 expression (Spearman rho: 0.36; P=0.065). Taken together, these results suggest that HMGA1 expression might be a novel indicator for the diagnosis and prognosis of human breast cancer.

Nuclear phosphoproteins HMGA and their relationship with chromatin structure and cancer.

The structural characteristics of the three nuclear phosphoproteins of the high mobility group A family are outlined and related to their participation in chromatin structure alteration in many biological processes such as gene expression, neoplastic transformation, differentiation, and apoptosis. The elevated expression of these proteins in tumor cells and their post-translational modifications, such as phosphorylation, acetylation and methylation, are discussed and suggested as suitable targets for cancer chemotherapy.

Transcriptional activation of the cyclin A gene by the architectural transcription factor HMGA2.

The HMGA2 protein belongs to the HMGA family of architectural transcription factors, which play an important role in chromatin organization. HMGA proteins are overexpressed in several experimental and human tumors and have been implicated in the process of neoplastic transformation. Hmga2 knockout results in the pygmy phenotype in mice and in a decreased growth rate of embryonic fibroblasts, thus indicating a role for HMGA2 in cell proliferation. Here we show that HMGA2 associates with the E1A-regulated transcriptional repressor p120(E4F), interfering with p120(E4F) binding to the cyclin A promoter. Ectopic expression of HMGA2 results in the activation of the cyclin A promoter and induction of the endogenous cyclin A gene. In addition, chromatin immunoprecipitation experiments show that HMGA2 associates with the cyclin A promoter only when the gene is transcriptionally activated. These data identify the cyclin A gene as a cellular target for HMGA2 and, for the first time, suggest a mechanism for HMGA2-dependent cell cycle regulation.

Hmga2 promoter analysis in transgenic mice.

HMGA2(2) belongs to the high mobility group A (HMGA) family of architectural transcription factors which participate in a wide variety of nuclear processes ranging from transcription to recombination, playing an important role in chromatin remodelling. HMGA2 is expressed during embryogenesis but not by adult somatic tissues, yet it becomes re-expressed following neoplastic transformation. A role in development is underscored by the finding that the inactivation of the Hmga2 gene is responsible for the murine pygmy phenotype. To elucidate mechanisms that control HMGA2 expression, we have previously cloned the gene and identified functional elements involved in its regulation. In this paper, transgenic mice were generated to define genomic regions involved in Hmga2 developmental and tissue-specific transcriptional regulation. A genomic region from -8.1 to -3.7kb upstream from the initiation site has been found to recapitulate most of the spatial and temporal endogenous Hmga2 gene expression.

HMGA1 protein over-expression is a frequent feature of epithelial ovarian carcinomas.

High mobility group A 1 (HMGA1) proteins are chromatinic factors, which are absent or expressed at very low levels in normal adult tissues, while they are over-expressed in several human malignant tumors. In this study, HMGA1 protein expression was investigated by immunohistochemistry in a series of 44 epithelial ovarian specimens, which included four normal ovarian tissues, 29 primary invasive carcinomas, one metastatic ovarian tumor and 10 low malignant potential (LMP) tumors. HMGA1 staining was not detected in normal ovarian surface epithelium, which is the area from which ovarian adenocarcinoma frequently arises. HMGA1 proteins were expressed at low levels in some LMP tumors, whereas they were present in abundance in most of the primary ovarian adenocarcinomas. RT-PCR and western blot analysis correlated with immunohistochemical data. We demonstrated that the suppression of HMGA1 protein synthesis by an adenovirus carrying the HMGA1 gene in antisense orientation (Ad-Yas-GFP) inhibited the growth of two human ovarian carcinoma cell lines (OVCAR-5 and OVCAR-8). These results confirm HMGA1 over-expression as a general feature of human malignant neoplasias, including ovarian cancer and suggest that suppression of HMGA1 protein synthesis by an antisense adenoviral vector may represent a new and promising gene therapy for the treatment of ovarian cancer.

Molecular dissection of the architectural transcription factor HMGA2.

HMGA2 protein belongs to the High Mobility Group A (HMGA) family of architectural transcription factors. These proteins establish a network of protein-protein and protein-DNA interactions resulting in the formation of enhanceosomes at promoters and enhancers regulating the expression of several genes. HMGA2 dysregulation, as a result of specific chromosomal rearrangements, has been identified in a variety of common benign mesenchymal tumors, and transgenic mice expressing a truncated form of HMGA2 protein demonstrated a causal relationship between the expression of the HMGA2 protein and tumorigenesis. In this paper, using several recombinant mutant proteins, we have investigated the role played by the different domains of HMGA2 in protein-protein and protein-DNA interaction. Using the IFN-beta gene as a model, we have shown that a short region of HMGA2, comprising the second DNA-binding domain, is critical for enhancing the NF-kappaB complex formation, for binding to the PRDII element, and also for protein-protein interaction with the NF-kappaB p50 subunit. Moreover, we have analyzed the interaction of HMGA2 mutant proteins with different DNA targets demonstrating that the absence of the C-terminal tail alters HMGA2/DNA complexes in a subset of DNA sequences. Our results suggest possible implications for the role of HMGA2 in tumorigenesis.

During apoptosis of tumor cells HMGA1a protein undergoes methylation: identification of the modification site by mass spectrometry.

Programmed cell death is characterized by posttranslational modifications of a limited and specific set of nuclear proteins. We demonstrate that during apoptosis of different types of tumor cells there is a monomethylation of the nuclear protein HMGA1a that is associated to its previously described hyperphosphorylation/dephosphorylation process. HMGA1a methylation is strictly related to the execution of programmed cell death and is a massive event that involves large amounts of the protein. In some tumor cells, HMGA1a protein is already methylated to an extent that depends on cell type. The degree of methylation in any case definitely increases during apoptosis. In the studied cell systems (human leukaemia, human prostate tumor, and rat thyroid transformed cells) among the low-molecular-mass HMG proteins, only HMGA1a was found to be methylated. A tryptic digestion map of HPLC-purified HMGA1a protein showed that methylation occurs at arginine 25 in the consensus G(24)R(25)G(26) that belongs to one of the DNA-binding AT-hooks of the protein. An increase of HMGA1a methylation could be related to heterochromatin and chromatin remodeling of apoptotic cells.