High Mobility Group A (HMGA) proteins: Molecular instigators of breast cancer onset and progression.

Cancer heterogeneity is one of the factors that constitute an obstacle towards an efficient targeting of this multifaceted disease. Molecular information can help in classifying cancer subtypes and in providing clinicians with novel targeted therapeutic opportunities. In this regard, classification of breast cancer into intrinsic subtypes based on molecular profiling represents a valuable prototype. The High Mobility Group A (HMGA) chromatin architectural factors (HMGA1a, HMGA1b, and HMGA2) have a relevant and causal role in breast cancer onset and development, by influencing virtually all cancer hallmarks. The regulation of HMGA expression is under the control of major pathways involved in cell proliferation and survival, as well as in other cancer-related processes, thereby suggesting, for the HMGA members, a high degree of homology and overlapping activities. Despite of this evidence, HMGA proteins display also specific functions. In this review, we provide an overview of (i) the pathways involved in HMGA transcriptional and post-transcriptional regulation, (ii) the utilization of HMGA as molecular markers, and (iii) the biological role of HMGA in the context of breast cancer. We focus on the potential significance of HMGA in governing the onset and development of this tumour, as well as on the potential of these factors as novel specific targets for preventing and treating strategies. The emerging picture is a highly interconnected triad of proteins that could mutually influence each other, either in a competitive or cooperative manner, and that, in our opinion, should be considered as a unified and integrated protein system.

High Mobility Group A (HMGA): Chromatin Nodes Controlled by a Knotty miRNA Network.

High mobility group A (HMGA) proteins are oncofoetal chromatin architectural factors that are widely involved in regulating gene expression. These proteins are unique, because they are highly expressed in embryonic and cancer cells, where they play a relevant role in cell proliferation, stemness, and the acquisition of aggressive tumour traits, i.e., motility, invasiveness, and metastatic properties. The HMGA protein expression levels and activities are controlled by a connected set of events at the transcriptional, post-transcriptional, and post-translational levels. In fact, microRNA (miRNA)-mediated RNA stability is the most-studied mechanism of HMGA protein expression modulation. In this review, we contribute to a comprehensive overview of HMGA-targeting miRNAs; we provide detailed information regarding HMGA gene structural organization and a comprehensive evaluation and description of HMGA-targeting miRNAs, while focusing on those that are widely involved in HMGA regulation; and, we aim to offer insights into HMGA-miRNA mutual cross-talk from a functional and cancer-related perspective, highlighting possible clinical implications.

SINEUP non-coding RNA activity depends on specific N6-methyladenosine nucleotides.

SINEUPs are natural and synthetic antisense long non-coding RNAs (lncRNAs) selectively enhancing target mRNAs translation by increasing their association with polysomes. This activity requires two RNA domains: an embedded inverted SINEB2 element acting as effector domain, and an antisense region, the binding domain, conferring target selectivity. SINEUP technology presents several advantages to treat genetic (haploinsufficiencies) and complex diseases restoring the physiological activity of diseased genes and of compensatory pathways. To streamline these applications to the clinic, a better understanding of the mechanism of action is needed. Here we show that natural mouse SINEUP AS Uchl1 and synthetic human miniSINEUP-DJ-1 are N6-methyladenosine (m6A) modified by METTL3 enzyme. Then, we map m6A-modified sites along SINEUP sequence with Nanopore direct RNA sequencing and a reverse transcription assay. We report that m6A removal from SINEUP RNA causes the depletion of endogenous target mRNA from actively translating polysomes, without altering SINEUP enrichment in ribosomal subunit-associated fractions. These results prove that SINEUP activity requires an m6A-dependent step to enhance translation of target mRNAs, providing a new mechanism for m6A translation regulation and strengthening our knowledge of SINEUP-specific mode of action. Altogether these new findings pave the way to a more effective therapeutic application of this well-defined class of lncRNAs.

HMGA1 positively regulates the microtubule-destabilizing protein stathmin promoting motility in TNBC cells and decreasing tumour sensitivity to paclitaxel.

High Mobility Group A1 (HMGA1) is an architectural chromatin factor involved in the regulation of gene expression and a master regulator in Triple Negative Breast Cancer (TNBC). In TNBC, HMGA1 is overexpressed and coordinates a gene network that controls cellular processes involved in tumour development, progression, and metastasis formation. Here, we find that the expression of HMGA1 and of the microtubule-destabilizing protein stathmin correlates in breast cancer (BC) patients. We demonstrate that HMGA1 depletion leads to a downregulation of stathmin expression and activity on microtubules resulting in decreased TNBC cell motility. We show that this pathway is mediated by the cyclin-dependent kinase inhibitor p27kip1 (p27). Indeed, the silencing of HMGA1 expression in TNBC cells results both in an increased p27 protein stability and p27-stathmin binding. When the expression of both HMGA1 and p27 is silenced, we observe a significant rescue in cell motility. These data, obtained in cellular models, were validated in BC patients. In fact, we find that patients with high levels of both HMGA1 and stathmin and low levels of p27 have a statistically significant lower survival probability in terms of relapse-free survival (RFS) and distant metastasis-free survival (DMFS) with respect to the patient group with low HMGA1, low stathmin, and high p27 expression levels. Finally, we show in an in vivo xenograft model that depletion of HMGA1 chemo-sensitizes tumour cells to paclitaxel, a drug that is commonly used in TNBC treatments. This study unveils a new interaction among HMGA1, p27, and stathmin that is critical in BC cell migration. Moreover, our data suggest that taxol-based treatments may be more effective in reducing the tumour burden when tumour cells express low levels of HMGA1.

Targeting the intrinsically disordered architectural High Mobility Group A (HMGA) oncoproteins in breast cancer: learning from the past to design future strategies.

INTRODUCTION: Triple-negative breast cancer (TNBC) is the most difficult breast cancer subtype to treat because of its heterogeneity and lack of specific therapeutic targets. High Mobility Group A (HMGA) proteins are chromatin architectural factors that have multiple oncogenic functions in breast cancer, and they represent promising molecular therapeutic targets for this disease. AREAS COVERED: We offer an overview of the strategies that have been exploited to counteract HMGA oncoprotein activities at the transcriptional and post-transcriptional levels. We also present the possibility of targeting cancer-associated factors that lie downstream of HMGA proteins and discuss the contribution of HMGA proteins to chemoresistance. EXPERT OPINION: Different strategies have been exploited to counteract HMGA protein activities; these involve interfering with their nucleic acid binding properties and the blocking of HMGA expression. Some approaches have provided promising results. However, some unique characteristics of the HMGA proteins have not been exploited; these include their extensive protein-protein interaction network and their intrinsically disordered status that present the possibility that HMGA proteins could be involved in the formation of proteinaceous membrane-less organelles (PMLO) by liquid-liquid phase separation. These unexplored characteristics could open new pharmacological avenues to counteract the oncogenic contributions of HMGA proteins.

HMGA1 Modulates Gene Transcription Sustaining a Tumor Signalling Pathway Acting on the Epigenetic Status of Triple-Negative Breast Cancer Cells.

Chromatin accessibility plays a critical factor in regulating gene expression in cancer cells. Several factors, including the High Mobility Group A (HMGA) family members, are known to participate directly in chromatin relaxation and transcriptional activation. The HMGA1 oncogene encodes an architectural chromatin transcription factor that alters DNA structure and interacts with transcription factors favouring their landing onto transcription regulatory sequences. Here, we provide evidence of an additional mechanism exploited by HMGA1 to modulate transcription. We demonstrate that, in a triple-negative breast cancer cellular model, HMGA1 sustains the action of epigenetic modifiers and in particular it positively influences both histone H3S10 phosphorylation by ribosomal protein S6 kinase alpha-3 (RSK2) and histone H2BK5 acetylation by CREB-binding protein (CBP). HMGA1, RSK2, and CBP control the expression of a set of genes involved in tumor progression and epithelial to mesenchymal transition. These results suggest that HMGA1 has an effect on the epigenetic status of cancer cells and that it could be exploited as a responsiveness predictor for epigenetic therapies in triple-negative breast cancers.

HMGA2 Antisense Long Non-coding RNAs as New Players in the Regulation of HMGA2 Expression and Pancreatic Cancer Promotion.

Background: Natural antisense long non-coding RNAs (lncRNAs) are regulatory RNAs transcribed from the opposite strand of either protein coding or non-coding genes, able to modulate their own sense gene expression. Hence, their dysregulation can lead to pathologic processes. Cancer is a complex class of diseases determined by the aberrant expression of a variety of factors, among them, the oncofetal chromatin architectural proteins High Mobility Group A (HMGA) modulate several cancer hallmarks. Thus, we decided to investigate the presence of natural antisense lncRNAs in HMGA1 and HMGA2 loci, and their possible involvement in gene expression regulation. Methods: We used FANTOM5 data resources, FANTOM-CAT genome browser and Zenbu visualization tool, which employ 1,829 human CAGE and RNA-sequencing libraries, to determine expression, ontology enrichment, and dynamic regulation of natural antisense lncRNAs in HMGA1 and HMGA2 loci. We then performed qRT-PCR in different cancer cell lines to validate the existence of HMGA2-AS1 transcripts. We depleted HMGA2-AS1 transcripts with siRNAs and investigated HMGA2 expression by qRT-PCR and western blot analyses. Moreover, we evaluated cell viability and migration by MTS and transwell assays, and EMT markers by qRT-PCR and immunofluorescence. Furthermore, we used bioinformatics approaches to evaluate HMGA2 and HMGA2-AS1 correlation and overall survival in tumor patients. Results: We found the presence of a promoter-associated lncRNA (CATG00000088127.1) in the HMGA1 gene and three antisense genes (RPSAP52, HMGA2-AS1, and RP11-366L20.3) in the HMGA2 gene. We studied the uncharacterized HMGA2-AS1 transcripts, validating their existence in cancer cell lines and observing a positive correlation between HMGA2 and HMGA2-AS1 expression in a cancer-derived patient dataset. We showed that HMGA2-AS1 transcripts positively modulate HMGA2 expression and migration properties of PANC1 cells through HMGA2. In addition, Kaplan-Meier analysis showed that high level of HMGA2-AS1 is a negative prognostic factor in pancreatic cancer patients. Conclusions: Our results describe novel antisense lncRNAs associated with HMGA1 and HMGA2 genes. In particular, we demonstrate that HMGA2-AS1 is involved in the regulation of its own sense gene expression, mediating tumorigenesis. Thus, we highlight a new layer of complexity in the regulation of HMGA2 expression, providing new potential targets for cancer therapy.

HMGA1 promotes breast cancer angiogenesis supporting the stability, nuclear localization and transcriptional activity of FOXM1.

BACKGROUND: Breast cancer is the most common malignancy in women worldwide. Among the breast cancer subtypes, triple-negative breast cancer (TNBC) is the most aggressive and the most difficult to treat. One of the master regulators in TNBC progression is the architectural transcription factor HMGA1. This study aimed to further explore the HMGA1 molecular network to identify molecular mechanisms involved in TNBC progression. METHODS: RNA from the MDA-MB-231 cell line, silenced for HMGA1 expression, was sequenced and, with a bioinformatic analysis, molecular partners HMGA1 could cooperate with in regulating common downstream gene networks were identified. Among the putative partners, the FOXM1 transcription factor was selected. The relationship occurring between HMGA1 and FOXM1 was explored by qRT-PCR, co-immunoprecipitation and protein stability assays. Subsequently, the transcriptional activity of HMGA1 and FOXM1 was analysed by luciferase assay on the VEGFA promoter. The impact on angiogenesis was assessed in vitro, evaluating the tube formation ability of endothelial cells exposed to the conditioned medium of MDA-MB-231 cells silenced for HMGA1 and FOXM1 and in vivo injecting MDA-MB-231 cells, silenced for the two factors, in zebrafish larvae. RESULTS: Here, we discover FOXM1 as a novel molecular partner of HMGA1 in regulating a gene network implicated in several breast cancer hallmarks. HMGA1 forms a complex with FOXM1 and stabilizes it in the nucleus, increasing its transcriptional activity on common target genes, among them, VEGFA, the main inducer of angiogenesis. Furthermore, we demonstrate that HMGA1 and FOXM1 synergistically drive breast cancer cells to promote tumor angiogenesis both in vitro in endothelial cells and in vivo in a zebrafish xenograft model. Moreover, using a dataset of breast cancer patients we show that the co-expression of HMGA1, FOXM1 and VEGFA is a negative prognostic factor of distant metastasis-free survival and relapse-free survival. CONCLUSIONS: This study reveals FOXM1 as a crucial interactor of HMGA1 and proves that their cooperative action supports breast cancer aggressiveness, by promoting tumor angiogenesis. Therefore, the possibility to target HMGA1/FOXM1 in combination should represent an attractive therapeutic option to counteract breast cancer angiogenesis.

The Architectural Chromatin Factor High Mobility Group A1 Enhances DNA Ligase IV Activity Influencing DNA Repair.

The HMGA1 architectural transcription factor is an oncogene overexpressed in the vast majority of human cancers. HMGA1 is a highly connected node in the nuclear molecular network and the key aspect of HMGA1 involvement in cancer development is that HMGA1 simultaneously confers cells multiple oncogenic hits, ranging from global chromatin structural and gene expression modifications up to the direct functional alterations of key cellular proteins. Interestingly, HMGA1 also modulates DNA damage repair pathways. In this work, we provide evidences linking HMGA1 with Non-Homologous End Joining DNA repair. We show that HMGA1 is in complex with and is a substrate for DNA-PK. HMGA1 enhances Ligase IV activity and it counteracts the repressive histone H1 activity towards DNA ends ligation. Moreover, breast cancer cells overexpressing HMGA1 show a faster recovery upon induction of DNA double-strand breaks, which is associated with a higher survival. These data suggest that resistance to DNA-damaging agents in cancer cells could be partially attributed to HMGA1 overexpression thus highlighting the relevance of considering HMGA1 expression levels in the selection of valuable and effective pharmacological regimens.

A novel HMGA1-CCNE2-YAP axis regulates breast cancer aggressiveness.

High Mobility Group A1 (HMGA1) is an architectural chromatin factor that promotes neoplastic transformation and progression. However, the mechanism by which HMGA1 exerts its oncogenic function is not fully understood. Here, we show that cyclin E2 (CCNE2) acts downstream of HMGA1 to regulate the motility and invasiveness of basal-like breast cancer cells by promoting the nuclear localization and activity of YAP, the downstream mediator of the Hippo pathway. Mechanistically, the activity of MST1/2 and LATS1/2, the core kinases of the Hippo pathway, are required for the HMGA1- and CCNE2-mediated regulation of YAP localization. In breast cancer patients, high levels of HMGA1 and CCNE2 expression are associated with the YAP/TAZ signature, supporting this connection. Moreover, we provide evidence that CDK inhibitors induce the translocation of YAP from the nucleus to the cytoplasm, resulting in a decrease in its activity. These findings reveal an association between HMGA1 and the Hippo pathway that is relevant to stem cell biology, tissue homeostasis, and cancer.

Expression and functional characterization of Xhmg-at-hook genes in Xenopus laevis.

High Mobility Group A proteins (HMGA1 and HMGA2) are architectural nuclear factors involved in development, cell differentiation, and cancer formation and progression. Here we report the cloning, developmental expression and functional analysis of a new multi-AT-hook factor in Xenopus laevis (XHMG-AT-hook) that exists in three different isoforms. Xhmg-at-hook1 and 3 isoforms, but not isoform 2, are expressed throughout the entire development of Xenopus, both in the maternal and zygotic phase. Localized transcripts are present in the animal pole in the early maternal phase; during the zygotic phase, mRNA can be detected in the developing central nervous system (CNS), including the eye, and in the neural crest. We show evidence that XHMG-AT-hook proteins differ from typical HMGA proteins in terms of their properties in DNA binding and in protein/protein interaction. Finally, we provide evidence that they are involved in early CNS development and in neural crest differentiation.

HMGA1 promotes metastatic processes in basal-like breast cancer regulating EMT and stemness.

Breast cancer is a heterogeneous disease that progresses to the critical hallmark of metastasis. In the present study, we show that the High Mobility Group A1 (HMGA1) protein plays a fundamental role in this process in basal-like breast cancer subtype. HMGA1 knockdown induces the mesenchymal to epithelial transition and dramatically decreases stemness and self-renewal. Notably, HMGA1 depletion in basal-like breast cancer cell lines reduced migration and invasion in vitro and the formation of metastases in vivo. Mechanistically, HMGA1 activated stemness and key migration-associated genes which were linked to the Wnt/beta-catenin, Notch and Pin1/mutant p53 signalling pathways. Moreover, we identified a specific HMGA1 gene expression signature that was activated in a large subset of human primary breast tumours and was associated with poor prognosis. Taken together, these data provide new insights into the role of HMGA1 in the acquisition of aggressive features in breast cancer.