Decoding high mobility group A2 protein expression regulation and implications in human cancers

High Mobility Group A2 (HMGA2) oncofetal proteins are a distinct category of Transcription Factors (TFs) known as “architectural factors” due to their lack of direct transcriptional activity. Instead, they modulate the three-dimensional structure of chromatin by binding to AT-rich regions in the minor grooves of DNA through their AT-hooks. This binding allows HMGA2 to interact with other proteins and different regions of DNA, thereby regulating the expression of numerous genes involved in carcinogenesis. Consequently, multiple mechanisms exist to finely control HMGA2 protein expression at various transcriptional levels, ensuring precise concentration adjustments to maintain cellular homeostasis. During embryonic development, HMGA2 protein is highly expressed but becomes absent in adult tissues. However, recent studies have revealed its re-elevation in various cancer types. Extensive research has demonstrated the involvement of HMGA2 protein in carcinogenesis at multiple levels. It intervenes in crucial processes such as cell cycle regulation, apoptosis, angiogenesis, epithelial-to-mesenchymal transition, cancer cell stemness, and DNA damage repair mechanisms, ultimately promoting cancer cell survival. This comprehensive review provides insights into the HMGA2 protein, spanning from the genetic regulation to functional protein behavior. It highlights the significant mechanisms governing HMGA2 gene expression and elucidates the molecular roles of HMGA2 in the carcinogenesis process. Graphical Abstract


Introduction
High Mobility Group (HMG) proteins are abundant and heterogeneous chromatin components, that represent a unique class of nonhistone chromatin structure.They were identified in 1973 by isolating them from calf's thymus [1,2].
These proteins are characterized by their abundance, heterogeneity, and rapid electrophoretic mobility, with molecular weights ranging from 10 to 15 kDa [2].Approximately 3% of the histone total content by weight is attributed to HMG proteins, with an estimated 10 6 molecules per nucleus [3].They are readily released from nuclei upon mild digestion with DNase I, suggesting an association of these proteins with structurally active genomic regions [4].
Studies have demonstrated the involvement of HMGA subfamily proteins (HMGA1a, HMGA1b, HMGA1c, and HMGA2) in a diverse range of cellular processes, including proliferation, differentiation, senescence, apoptosis, inflammation, metabolism, and autophagy.Their involvement in these critical cellular pathways underscores the importance of understanding their abundance within the nucleus where the amount of HMGB in the cell is approximately 10 times less than the amount of histones, the amount of HMGC is 10 times less than the amount of HMGB, and the amount of HMGA is 10 times less than the amount of HMGC, highlighting the relative scarcity of HMGA subfamily [8].

HMGA2 gene
The HMGA2 gene exhibits greater structural complexity compared to the HMGA1 gene, primarily due to its extended length and the presence of a large third intron.This intron plays a crucial role in HMGA2 gene rearrangements, particularly in benign mesenchymal tumors [11,13].These rearrangements can lead to the production of truncated or chimeric HMGA2 proteins.
Analysis of the HMGA2 promoter region revealed the absence of TATAA and CAAAT boxes, commonly recognized promoter elements.Instead, the major transcription start site of HMGA2 is positioned adjacent to the CGC GTG sequence, which closely resembles the consensus E-box (CAC GTG ).This similarity suggests that the HMGA2 promoter is susceptible to regulation by a wide range of transcription factors during carcinogenesis, including Sp1 and Sp3 [14].Additional transcription start sites are located within a CpG-rich island, reflecting the high frequency of CpG dinucleotides and multiple GC boxes in the promoter region [11,15].These elements can bind to the transcriptional activator Sp1, a critical factor for transcription from TATAA-less promoters [16][17][18].The HMGA2 gene spans approximately 140 kb of chromosomal DNA and comprises at least five exons [19].The first three exons encode the AT-binding domain sites, while exon 4 encodes a protein linker, a short peptide of 11 amino acids separating the last DNA-binding domain from the acidic tail.Exon 5 encodes the acidic domain; therefor, the HMGA2 protein harbors three DNA-binding regions and an acidic terminus (Fig. 1) [11,15,20,21].
Unlike HMGA1, which possesses a single 3′ UTR, HMGA2 exhibits multiple splicing variants with distinct 3′ UTRs compared to the canonical 3′ UTR.This diversity in 3′ UTRs may influence miRNA-mediated HMGA2 regulation, further contributing to the complexity of the HMGA2 gene.

HMGA2 protein structure and its functional domains
The HMGA2 protein comprises of 108 amino acid residues [24,25], with AT-hook motifs consisting of a positively charged 9-amino-acid extension [26] containing the Arg-Gly-Arg-Pro (R-G-R-P) constant repeat [27].This structural feature enables HMGA2 to bind to B-form DNA, inducing a conformational transition from a disordered to an ordered state, which influences gene transcription regulation [28].The number and spacing of AT-rich binding sites within the target DNA region modulate HMGA2's ability to interact with the minor groove of AT-rich regions on DNA and nucleosomes through its AT-hooks [26,29].Consequently, HMGA2 can enhance or repress the transcriptional activity of numerous human genes involved in diverse biological processes [30].This regulatory versatility has earned HMGA2 proteins the designation of "architectural factors" [31].
HMGA2 also possesses a negatively charged acidic tail composed of 15 amino acid residues, including glutamic acid, aspartic acid, serine, and threonine.This acidic tail is a substrate for phosphorylation by Casein Kinase 2 (CK2) [32].In the fully phosphorylated state, the acidic tail could carry up to 19 negative charges.Electrostatic interactions play a crucial role in HMGA2's binding to AT-rich DNA [33], suggesting that the acidic C-terminus may regulate HMGA2's DNA-binding affinity in addition to potentially mediating protein-protein interactions [5,34].Notably, HMGA2 isoforms retaining the three AT-hooks but lacking the acidic C-terminus exhibit DNA-binding specificity comparable to the wild-type protein.However, this modification can alter transcription by influencing protein-protein interactions at gene promoters or enhancers [35].

The role of HMGA2 protein in modifying chromatin structure
The HMGA2 protein exhibits a high degree of plasticity attributed to its intrinsically disordered structure, which is a distinct feature of HMGA proteins [36].This structural flexibility is hypothesized to enable HMGA proteins to interact with DNA, modify its conformational state, and engage with a diverse array of proteins, including numerous transcription factors [20,37,38].HMGA2's ability to utilize only one or two AT-hooks while leaving the remaining hooks available for trans-interactions with other DNA regions facilitates the formation of regulatory complexes termed "enhanceosomes" and contributes to the establishment of higher-order chromatin structures [39].
The HMGA2 protein also appears to play a role in alleviating nucleosomal constraints that impede the formation of Transcription Factor-DNA (TF-DNA) complexes.This is supported by the observation that HMGA2 binding sites within chromatin resemble those of histone H1, suggesting that HMGA2 competes with H1 for binding to linker DNA and thereby catalyzes chromatin decondensation, ultimately promoting target gene expression [8,35,[40][41][42], as histone H1 is known to act as a transcriptional repressor [43], and the ability of HMGA2 to interact with both nucleosomes and chromatin remodelers suggests a potential role in facilitating histone clearance and/or packaging during transcriptional regulation [39] (Fig. 2).
Due to their ability to interact with numerous molecular players across diverse regulatory pathways, HMGA proteins have been dubbed "molecular glue" and are implicated in various aspects of gene regulation and cellular biological processes [37].

HMGA2 expression levels
HMGA2 expression is generally low or absent in adult tissues, with peak expression observed in undifferentiated cells during early development and embryogenesis [44].As fetal development progresses, HMGA2 expression becomes more restricted, with the protein being primarily expressed during embryonic development [24,29,45], HMGA2 is also found in Embryonic Stem Cells (ESCs) and in adult stem cell populations, where it plays a critical role in selfrenewal and differentiation [46], spermatids, and spermatocytes [47,48].
Careful regulation of HMGA2 expression is essential for proper development and maintenance of cellular homeostasis in adults.Dysregulation of HMGA2 expression has been implicated in various pathological conditions, including: • Benign tumors [49,50]: HMGA2 overexpression is frequently observed in benign tumors such as lipomas [15,31,46,51,52], fibroadenomas [53], salivary gland adenomas [54], hamartomas [55], and pituitary adenomas [56,57].In these tumors, chromosomal rearrangements involving the HMGA2 gene lead to the expression of truncated forms of the protein or the fusion of the HMGA2 N-terminus with the C-terminus of other proteins, and these alterations often result in the deletion of the natural 3′ UTR of HMGA2 mRNAs [45].• Preneoplastic lesions: HMGA2 expression has consistently shown an increase in various preneoplastic lesions compared to adjacent normal tissue.For endometrial cancers and their primary lesions, HMGA2 showed an important role in their evaluation, as HMGA2 expression gradually increased from precancerous lesion endometrial glandular dysplasia to intraepithelial serous endometrial carcinoma and eventually to fully developed endometrial serous carcinoma [58].Also in ovarian tissue, overexpression of HMGA2 in normal ovarian epithelial cells has been associated with malignant transformation, as HMGA2 exhibited increased overexpression in Serous Tubal Intraepithelial Carcinoma (STIC) lesions, suggesting an early event in the formation of high-grade serous carcinomas [59].Similarly, in the prostate, HMGA2 overexpression in the stroma contributes to the development of multifocal precancerous prostate lesions.This process is dependent on the Wnt/β-catenin pathway and occurs in conjunction with stromal Androgen Receptor (AR) activity.Intriguingly, this suggests that cancer initiation can occur solely through epigenetic changes involving HMGA2 in the stromal environment, preceding any mutations in neighboring epithelial cells via paracrine signaling [60].Furthermore, studies have demonstrated that HMGA2 acts as an oncoprotein by enhancing the Wnt/β-catenin signaling pathway in sporadic colorectal tubular adenomas [61].Additionally, HMGA2 expression increases in pancreatic cancer and high-grade Pancreatic Intraepithelial Neoplasia (PanIN), but not in low-grade PanIN or benign lesions.The progressive elevation of HMGA2 expression from PanIN lesions to Pancreatic Ductal Adenocarcinoma (PDAC) suggests its involvement in pancreatic carcinogenesis and the transition to a more aggressive phenotype [62].Consequently, HMGA2 has been proposed as a valuable molecular marker for the differential diagnosis preneoplastic lesions and malignant tumors.

Regulating the expression of the HMGA2 protein
Aberrant expression of the HMGA2 protein can transform the normal cell phenotype to a more motile and invasive state [88]; stringent control mechanisms at multiple levels are essential to regulate the biological activity of HMGA2 protein within cells.These regulatory mechanisms operate at various levels:

Posttranscriptional regulation
Posttranscriptional regulation is the most critical level for controlling HMGA2 expression.This regulation is mediated by noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs).ncRNAs are classified based on their length, with less than 200 nucleotides (nt) for miRNAs, while lncRNAs are longer than 200 nt [88].

HMGA2 regulation by miRNAs
miRNAs are often dysregulated in cancer cells [99], and over 100 miRNAs are implicated in HMGA mRNA regulation [12].The 3′ UTR located within the C-terminal tail is a primary target for miRNAs.Deletion or replacement of the 3′ UTR with other transcripts can lead to the repression of miRNA function in reducing HMGA expression through either mRNA degradation or translational repression [100].
The discrepancy between HMGA protein and mRNA levels, particularly for HMGA2, suggests that regulatory elements within the 3′ UTR could mediate posttranscriptional control of HMGA protein expression.This is particularly relevant in cases of aberrant HMGA2 transcripts, which contribute to a more aggressive tumor phenotype [101,102], Interestingly, miRNA seed sequences can imperfectly bind to miRNA Response Elements (MREs) on the 3′ UTR of HMGA mRNA [100].
Lineage-28 (LIN28) is another key player in this regulatory mechanism.LIN28A and LIN28B are highly conserved RNA-binding proteins that restrict the biogenesis of a subset of the mammalian Let-7 family [104].The primary mechanisms for this restriction include: Long noncoding RNAs (lncRNAs) are another class of ncRNAs that play a crucial role in posttranscriptional regulation by interacting with mRNAs, proteins, and other ncRNAs, including miRNAs [100].These interactions can either enhance or suppress miRNA-mediated regulation [108] through the process of miRNA sponging [88].
Evidence suggests that lncRNAs influence HMGA2 protein expression.The abundance of similar MREs in both HMGA1 and HMGA2 sequences enables them to bind to the same miRNAs, thereby affecting other transcripts, ncRNAs, circular RNAs (circRNAs), and pseudogenes that share the same MREs [100] (Fig. 5).This interaction leads to mutual regulation of HMGA1 and HMGA2 mRNAs, functioning as competing endogenous RNAs (ceRNAs) within a ceRNA network (ceRNET).Two types of connections exist between ceRNET components: (1) direct linkages between two ceRNAs sharing the same MREs and (2) indirect linkages between two ceRNAs that do not share the same MREs but are linked to a common ceRNA [109].
In addition to the HMGA1 and HMGA2 genes, two HMGA1-processed noncoding pseudogenes (generated by mRNA retrotransposition [110]), HMGA1P6 and HMGA1P7, exhibit high sequence homology with HMGA1 in both the 5′ and 3′ UTRs and the coding region.Consequently, they share the same MREs and interact with miRNAs targeting HMGA1 and HMGA2.Overexpression of these pseudogenes can promote cancer cell proliferation and migration.Moreover, elevated expression of the HMGA1 gene or its pseudogenes can increase HMGA2 protein levels, contributing to cancer progression [110,111].This highlights the significant role of these pseudogenes in gene expression regulation through their involvement in the ceRNA hypothesis and the formation of a complex regulatory network at the transcriptome level.3 The regulatory mechanism of LIN-28A&B on the biogenesis of Let-7 miRNA.Let-7 miRNA is transcribed by RNA pol.II.The initial transcript called primary microRNA (pri-miRNA) that contains an imperfectly double-stranded region within a hairpin loop, in addition to 5′ and 3′ ends, then it is cleaved by DROSHA, which removes the 5′ and 3′ ends and produces a short hairpin called the pre-miRNA in the nucleus.After that, pre-miRNA is transferred through Exportin-5 to the cytoplasm where it is bound by the RISC that contains DICER, which cleaves the pre-miRNA and produces Let-7 dsRNA, which will be bound by the RISC that contains DICER and cleaved to two separate stands, one of them, the passenger strand will be removed while the guide strand will be retained.LIN28B inhibits the formation of mature Let-7 by inhibiting DROSHA, while LIN28A inhibits DICER in the cytoplasm and promotes the uridynylation of pre-Let-7 thus preventing the formation of mature Let-7
Fig. 4 The LIN28-Let7-HMGA2 axis, which controls HMGA2 levels.LIN28A&B inhibit the formation of mature Let-7 miRNAs and thus increase the HMGA2 levels, as mature Let-7 miRNAs leads either to the cleavage and degradation of HMGA2 mRNA, or to block the HMGA2 mRNA translation Fig. 5 Competing endogenous RNA (ceRNA) hypothesis.lncRNAs, circRNAs, and mRNAs form complex interaction networks, where the type and abundance of molecules and the number of MREs that interact with miRNA can influence the way these molecules interact with each other through the ceRNA machinery.These miRNAs bind to the 3′ UTR of the target mRNA, which leads either to the inhibition of the translation process or mRNA degradation.However, other RNA molecules can interact with miRNAs and control their abundance, thus creating a crosstalk interaction with other target molecules [114] (2024) 15:322 | https://doi.org/10.1007/s12672-024-01202-xReview 6.2 HMGA2 R-loop-mediated transcriptional regulation R-loops are three-stranded nucleic acid structures formed when an RNA strand invades a double-stranded DNA helix [115].These structures typically arise during transcription and referred to as Watson-Crick RNA-DNA hybrid [116,117].R-loop formation often occurs co-transcriptionally near gene promoters enriched in C/G content, such as the HMGA2 promoter.The presence of R-loops can induce an open chromatin conformation, facilitating access of transcription factors and regulatory proteins to HMGA2 transcriptional cis-regulatory sequences [118].
In cancer cells, the lncRNA RPSAP52 plays a crucial role in this network.The RPSAP52 pseudogene overlaps with the HMGA2 gene, and the presence of a C/G skew in the HMGA2 gene promoter favors R-loop formation between RPSAP52 ncRNA and genomic DNA (Fig. 6).This R-loop structure stimulates chromatin decompaction and transcription of the HMGA2 gene, leading to increased HMGA2 protein levels [108,118].
RPSAP52 also exerts regulatory effects in the cytoplasm by interacting with Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), an RNA-binding protein that regulates the translation of numerous mRNAs, including HMGA2 mRNA and LIN28 mRNA, with a preference for these transcripts.The interaction of RPSAP52 with IGF2BP2 enhances the binding of IGF2BP2 to HMGA2 mRNA, thereby promoting its translation [108].Interestingly, IGF2BP2 is also considered a downstream target of HMGA2.HMGA2 binds to and recruits NF-κB to an AT-rich region in the IGF2BP2 promoter, leading to their mutual upregulation within a positive feedback loop [120].

HMGA2 posttranslational regulation
Posttranslational modifications (PTMs) represent a critical level of regulation that controls HMGA2 protein function.These modifications influence HMGA2's ability to interact with DNA and other factors, contributing significantly to the regulation of its activity.The occurrence of these modifications is dependent on both intracellular and extracellular signals, reflecting the strong link between HMGA2 protein activity and internal and external cues [121].
Phosphorylation is one of the most important PTMs affecting HMGA2 function.The protein is rich in proline, serine, and threonine residues, and each AT-hook is flanked by two phosphorylation sites.These phosphorylation events significantly impact HMGA2's DNA-binding affinity.It has been proposed that phosphorylation of the acidic tail can enhance protein compaction, while truncated forms exhibit a more relaxed structure.These structural differences likely influence the accessibility of modifying enzymes [21,33,121].
Fig. 6 The overlap of the pseudogene encoding RPSAP52 with HMGA2 gene in the promoter region.During transcription, RPSAP52 lncRNA forms an R-loop structure in HMGA2 gene promoter, which induces the expression of the HMGA2 protein by facilitating the access of transcription factors to the HMGA2 promoter [119]

The role of HMGA2 protein in cell cycle
Tight regulation of the cell cycle is essential for maintaining a balance in cell proliferation.Disruption of this balance can lead to neoplastic transformation.Several studies have demonstrated a direct role for HMGA2 protein in regulating cell cycle progression (Fig. 7) [30,128].
• HMGA2-mediated Cyclin A2 expression: HMGA2 binds to the Cyclic AMP (cAMP)-Responsive Element (CRE) in the Cyclin A2 gene promoter, displacing p120 E4F , a cell cycle inhibitor.This displacement facilitates the binding of ATF/ CREB family TFs, leading to the induction of Cyclin A2 expression and subsequent cell cycle progression in ovarian serous carcinoma [127].• HMGA2-mediated activation of the AP-1 transcriptional complex: HMGA2 enhances Cyclin A2 expression through the activation of the Activator Protein-1 (AP-1) transcriptional complex, which comprises Jun proteins (JUN, JUNB, JUND) and FOS proteins (FOS, FOSB, FRA1, FRA2) [129].JUNB and FRA1 play the most crucial roles in activating Cyclin A2 gene expression.FRA1 is recruited to the Cyclin A2 gene promoter and increases JUNB expression, which in turn binds to the Cyclin A2 gene promoter and promotes its expression, which has been reported in breast and thyroid cancers [130].• HMGA2-mediated E2F1 activation: HMGA2 displaces Histone DeACetylase 1 (HDAC1) from the Retinoblastoma protein-E2F1 (pRB-E2F1) complex located at the promoters of transcription factor genes.This displacement leads to increased acetylation of both E2F1 and histones at E2F1 target gene sites, ultimately promoting cell cycle progression in pituitary tumors [28,130].pRB, a tumor suppressor protein, strictly controls cell cycle entry into the S phase.It acts as the master regulator of the cell cycle by maintaining E2F1 in its inactive form through its interaction with HDAC1 [129].• HMGA2-mediated regulation of the Cyclin D1/CDK4/CDK6/pRB-E2F1 axis: HMGA2 modulates the cyclin D1/CDK4/ CDK6/pRB-E2F1 axis by increasing cyclin D1 and CDK6 levels and stimulating their complex formation.Cyclin D1/ CDK4/CDK6 activation phosphorylates RB, abrogating its cell cycle inhibitory activity in metastatic renal carcinoma cell line ACHN [131].• HMGA2-mediated activation of the PI3K/AKT/mTOR/p70 S6K signaling pathway: In Acute Myeloid Leukemia (AML), HMGA2 overexpression directly activates the Phosphatidylinositide 3-Kinase (PI3K)/AKT/mTOR/p70 S6K signaling pathway, resulting in Cyclin E activation and suppression of p16 INK4A as well as p21 CIP1/WAF1 activity.These cyclin-dependent kinase inhibitors play a critical role in restricting cell cycle progression by inhibiting E2F1 release [132,133].• HMGA2-mediated Cyclin B2 expression: HMGA2 binds to the ccnb2 promoter and promotes Cyclin B2 expression to enhance cell growth.Cyclin B2, encoded by the ccnb2 gene, is a cell cycle-dependent protein that regulates the G2-M transition [64].

Apoptosis
Apoptosis, a genetically programmed cell death mechanism, is crucial for maintaining a balance between cell proliferation and cell death in multicellular organisms, eliminating abnormal cells and ensuring tissue homeostasis.Two main pathways contribute to programmed cell death: the extrinsic (receptor-mediated) and the intrinsic (mitochondria-mediated) pathways [134].
Studies have demonstrated that HMGA2 plays a dual role in regulating apoptosis in cancer cells, contributing to cancer cell survival:

HMGA2 catalytic role in apoptosis
Paradoxically, HMGA2 can also play a pro-apoptotic role as a defense mechanism to eliminate cancer cells harboring fatal genetic defects.Elevated HMGA2 expression can induce caspase-2 cleavage, triggering apoptosis.Caspase-2, an initiator caspase, promotes the release of cytochrome c from mitochondria, a critical step in apoptosis induction [134].

The role of HMGA2 protein in Angiogenesis
Angiogenesis, the formation of new blood vessels from pre-existing ones, is a crucial process in tumor development.It provides the growing tumor cell mass with the necessary oxygen and nutrients and facilitates the removal of waste products from the tumor site.This process is not merely a consequence of tumor growth; rather, it is an active and essential feature of tumor development [138].
Several studies have implicated HMGA2 in the signaling pathways for both Vascular Endothelial Growth Factor (VEGF) and Transforming Growth Factor-β (TGF-β), key regulators of angiogenesis.HMGA2 promotes the upregulation of VEGF-A, Fig. 9 The role of the HMGA2 protein and its effect on angiogenesis.A The HMGA2 protein recruits NF-kB to the IGF2PB2 gene and upregulates its expression, which in turns induces angiogenesis, as IGF2PB2 is a growth factor.B The HMGA2 protein upregulates the expression of TGF-β and VEGF growth factors resulting in the induction of their signaling pathways, which also leads to angiogenesis https://doi.org/10.1007/s12672-024-01202-xReview VEGF-C, FGF-2, and TGF-β, contributing to angiogenesis [139].Additionally, HMGA2 and Nuclear Factor-κB (NF-κB) bind to the AT-rich regulatory region of the IGF2BP2 gene, leading to increased IGF2BP2 expression, as mentioned before, and further promoting angiogenesis [63,140] (Fig. 9).

The role of HMGA2 protein in EMT
Epithelial-Mesenchymal Transition (EMT) is a process whereby epithelial cells undergo transdifferentiation into motile mesenchymal cells [30].This process plays a critical role in embryonic development, wound healing, stem cell behavior, and cancer development, as it enables cancer cells to invade and metastasize to distant organs [65].
EMT involves significant phenotypic changes, including loss of adhesion to neighboring cells, loss of cell polarity, and acquisition of migratory and invasive properties [141].It is characterized by the downregulation of epithelial markers, such as E-cadherin and zonula-1, and the upregulation of mesenchymal markers, including vimentin, fibronectin, Snail1/2, ZEB1/2, and Twist [30,65].Additionally, detachment of cells from the surrounding tissue occurs due to increased expression of MMP2 and MMP9, proteins belonging to the MMP family that are responsible for extracellular matrix degradation, leading to cell migration, invasion, and angiogenesis [142].
Numerous studies have demonstrated the involvement of HMGA2 in stimulating EMT by activating various signaling pathways, resulting in increased expression of mesenchymal markers, decreased expression of epithelial markers, and elevated levels of MMP2 and MMP9 proteins, which are essential for metastasis.The mechanisms underlying HMGA2induced EMT are as follows (Fig. 10): • TGF-β signaling pathway: Extracellular signals, particularly TGF-β, bind to TGFβRII on the cell surface, stimulating the Smad pathway, which in turn enhances HMGA2 expression [143].This considers the main driver of tumor development and metastasis where TGFβRII is expressed exclusively at the invasive front of human tumors [144].HMGA2, in cooperation with Smad proteins, binds to the Snail1 promoter, increasing its expression [123], leading to the suppression of occludin and E-cadherin [129,137,145].• DNA methylation: Prolonged activation of the TGF-β signaling pathway causes HMGA2 to recruit DNMT3A to the E-cadherin gene promoter, silencing its transcription via DNA methylation [145].• Has2-CD44-AKT/ERK1/2 signaling axis: Within the TGF-β pathway, Smads cooperate with HMGA2 to increase Has2 expression, which then binds to CD44, activating the AKT/ERK1/2 signaling pathway [146,147].• NF-κB activation: HMGA2 promotes the binding of NF-κB to the Positive Regulatory Domain II (PRDII) TF, which is a characteristic feature of the β-interferon gene promoter [132].
• MAPK and PI3K signaling pathways: Activation of the MAPK and PI3K pathways leads to the induction of growth factors such as FGF-1 and platelet-derived growth factor-BB (PDGF-BB), potent stimulators of HMGA2 expression [132,148].
HMGA2 increases the expression of Twist1, which suppresses E-cadherin expression, leading to β-catenin translocation from the cell membrane to the cytoplasm and nucleus, the initial step in EMT.In addition, HMGA2 reduces AXIN1 expression, which phosphorylates β-catenin and reduces its levels, preventing nuclear entry and activation of the Wnt/β-catenin pathway [151].
In summary, HMGA2 plays a pivotal role in driving EMT by modulating various signaling pathways and promoting the expression of mesenchymal markers while suppressing epithelial markers.This ability to induce EMT contributes significantly to the metastatic potential of cancer cells.

The role of HMGA2 protein in cancer stemness
Cancer Stem Cells (CSCs) are a subpopulation of cancer cells with the capacity for self-renewal and differentiation into various cell types, including wild-type stem cells.CSCs reside within tumors as a distinct population and possess the ability to initiate tumor formation due to their self-renewal and differentiation properties.Moreover, CSCs exhibit a high degree of drug resistance, rendering them a major challenge in cancer therapy [129,152].HMGA2 protein plays a crucial role in maintaining the undifferentiated state of cancer cells and their self-renewal properties.Studies have demonstrated the involvement of HMGA2 in cancer stemness across various cancer types.HMGA2 directly binds to the SOX2 promoter, a TF critical for stem cell maintenance, and enhances its expression.Additionally, HMGA2 upregulates the expression of other cancer stem cell markers, such as CD44, Oct4, c-Myc, ALDH1, and Twist1, in addition to the activation of the Wnt/β-catenin pathway, which is known to be responsible for the ability of the self-renewal property, further promoting cancer cell aggressiveness, metastasis, and resistance to cancer therapies [63,67,151,153] (Fig. 11).
The ability of HMGA2 to regulate cancer stemness highlights its importance in tumor development and progression.Understanding the mechanisms by which HMGA2 modulates CSC properties may provide novel therapeutic strategies for targeting CSCs and improving cancer treatment outcomes.

The role of the HMGA2 protein in DNA repair mechanisms
Upon encountering DNA damage, cells activate a complex multistep process known as the DNA Damage Response (DDR) to repair the damaged DNA.This process involves the activation of various DNA repair pathways, including NonHomologous End-Joining (NHEJ), Base Excision Repair (BER), and Nucleotide Excision Repair (NER).
HMGA2, a non-histone chromosomal protein, is widely recognized for its ability to interact with other proteins and DNA, making it a key regulator of DNA repair processes.Through these interactions, HMGA2 influences the function of numerous DNA repair-related proteins, thereby modulating the overall efficiency of DNA repair mechanisms.
Specifically, HMGA2 has been shown to interact with and regulate the activity of proteins involved in NHEJ, BER, and NER.For instance, HMGA2 interacts with Ku70/80, a heterodimeric protein complex essential for NHEJ, influencing its ability to stabilize double-strand breaks and promote repair.Similarly, HMGA2 interacts with AP Endonuclease 1 (APE1), a key enzyme in BER, modulating its activity in base excision repair.Additionally, HMGA2 influences NER by interacting with Excision Repair Cross-Complementation group 1 (ERCC1) protein, a protein involved in NER initiation, affecting its ability to recognize and repair nucleotide excision sites.
These interactions between HMGA2 and DNA repair proteins highlight the multifaceted role of HMGA2 in maintaining genomic integrity.By influencing the function of these proteins, HMGA2 plays a critical role in regulating DNA repair processes and ensuring the stability of the genome.

Base excision repair (BER) mechanism
Mammalian cells encounter approximately 70,000 base lesions daily, necessitating a robust DNA repair mechanism.This mechanism is particularly crucial for highly proliferating tumor cells, where high-fidelity DNA replication is essential for their rapid growth.Unrepaired base lesions can lead to replication fork stalling and an increased risk of Double-Strand Breaks (DSBs) upon replication fork collapse.The BER-supporting function of HMGA2 enhances the ability of cancer cells to efficiently repair underlying lesions at the appropriate time [72].
HMGA2 possesses intrinsic Apurinic/Apyrimidinic (AP) site cleavage activity, enabling it to recognize and cleave AP sites, facilitating BER initiation.Furthermore, HMGA2 physically interacts with human AP Endonuclease 1 (APE1) in cancer cells, stimulating its activity and promoting the removal of AP sites [154] (Fig. 12).
Additionally, HMGA2 binds with high affinity to DNA replication forks through interactions with the replication fork proteins PCNA and RPA, contributing to the stabilization of stalled replication forks and protecting them from endonucleolytic attack.All three AT hooks of HMGA2 participate in this process, allowing it to act as a scaffold protein that stabilizes DNA branching from stalled replication forks [72,155].
These dual roles of HMGA2 in BER and replication fork stabilization highlight its critical contribution to maintaining genomic integrity in highly proliferating tumor cells.By promoting efficient base lesion repair and stabilizing stalled replication forks, HMGA2 helps to prevent DNA damage accumulation and DSB formation, thereby contributing to tumor cell survival and progression.

Nucleotide excision repair (NER) mechanism
HMGA2 promotes Nucleotide Excision Repair (NER) by upregulating the transcription of Excision Repair Cross-Complementation group 1 (ERCC1) protein [156].NER is a crucial pathway for repairing bulky helix-distorting adducts that arise from exposure to ultraviolet radiation or chemical mutagens.
The NER process initiates when the damage encompasses a significant portion of the nitrogenous bases.The XPC-RAD23b complex binds to the damage site, recruiting the TFIIH complex, which contains two helicase subunits (XPB and XPD).These helicases unwind the DNA duplex, creating a bubble around the damaged region.Subsequently, endonucleases, including ERCC1/XPF and XPG, excise the damaged DNA segment.Finally, DNA polymerases fill the resulting gap with newly synthesized DNA, completing the repair process [30,156] (Fig. 12).

NonHomologous end-joining (NHEJ) mechanism
The role of HMGA2 in the NHEJ process remains controversial, but some studies have indicated that HMGA2 has both positive and negative effects on NHEJ.
The precise role of HMGA2 in NHEJ remains a subject of debate, with evidence suggesting both positive and negative effects.HMGA2 can repress NHEJ by disrupting DNA-PK dynamics, altering the binding of Ku70 and Ku80 to DNA ends, and leading to the persistence of γ-H2AX, a DDR recognition signal that facilitates chromatin opening and allows DNA repair proteins to access the break site [157][158][159][160] (Fig. 12).The failure to remove γ-H2AX at the appropriate time indicates impaired DNA repair, increasing the risk of DNA deformities and potentially contributing to carcinogenesis [157].
Conversely, HMGA2 can also enhance the NHEJ mechanism by activating the Ataxia Telangiectasia Mutated (ATM) protein.HMGA2 serves as a substrate for ATM and its downstream tumor suppressor CHeckpoint Kinase 2 (CHK2), both of which are crucial for DNA damage signal transduction [159,160] (Fig. 12).Fig. 12 The role of the HMGA2 protein in DNA repair mechanisms.The HMGA2 protein has intrinsic apurinic/apyrimidinic (AP) site cleavage activity and it interacts with PCNA and RPA proteins, leading to the stabilization of stalled replication forks and thus inducing BER mechanism, it also increases the transcription of ERCC1 protein that acts as an endonuclease and is involved in cutting the area surrounding the site of damage to be repaired by DNA polymerase, while the HMGA2 protein has a dual role in NHEJ mechanism, as it impairs DNA-PK dynamics and causes persistence of γ-H2AX leading to the repression of NHEJ and starting tumor formation, and it activates the ATM protein, which is essential for DNA damage signal transduction, which maintains cancer cell survival (2024) 15:322 | https://doi.org/10.1007/s12672-024-01202-xReview Furthermore, HMGA2 has been implicated in maintaining the phosphorylation of ATR and CHK1, potentially switching the cell state from apoptosis to DNA repair, thereby promoting cancer cell survival and resistance to chemotherapy [161,162].
Given these conflicting findings, further investigations are warranted to elucidate the exact role of HMGA2 in NHEJ and its implications for cancer cell survival.

Conclusion
HMGA2, an architectural TF, plays a critical role in embryonic development but is typically absent in adult tissues.Its reexpression in adult tissues disrupts cellular homeostasis by dysregulating the expression of numerous genes involved in cell cycle regulation, apoptosis, angiogenesis, EMT, cancer stem cell maintenance, and DNA repair mechanisms.HMGA2 exerts its effects through several key signaling pathways, including TGF-β, AKT/ERK1/2, MAPK, and Wnt/β-catenin.These pathways contribute to the upregulation of cancer stem cell markers, enabling cancer cells to detach from the primary tumor site and migrate to distant organs, a hallmark of metastasis.
Due to its multifaceted role in cancer development and progression, HMGA2 has emerged as a potential diagnostic and prognostic cancer marker.High HMGA2 expression levels may correlate with tumor aggressiveness and treatment response, guiding clinicians in selecting appropriate treatment strategies.The development of targeted therapies against HMGA2 holds promise for reducing cancer incidence and improving prognosis across various cancer types.However, further research is required to establish the correlation between HMGA2 protein levels in blood and cancer tissues.This could validate HMGA2 as an accurate, easily measurable cancer marker that reflects the protein status within the tumor tissue.

Fig. 1
Fig. 1 Schematic diagram of the HMGA2 gene, mRNA, and protein.The HMGA2 gene is located on chromosome 12, and it encodes a small protein that can bind to AT-rich sites in the minor groove of DNA in conjunction with other parts of DNA and other proteins through its AT-hooks, thus contributing to transcription regulation by inducing conformational changes in the DNA

Fig. 2
Fig. 2 Schematic diagram of the role of the HMGA2 protein in modifying chromatin structure.HMGA2 can facilitate TF access to chromatin through histone H1 translocation, thus inducing gene expression.TF transcriptional factor

Fig.
Fig.3The regulatory mechanism of LIN-28A&B on the biogenesis of Let-7 miRNA.Let-7 miRNA is transcribed by RNA pol.II.The initial transcript called primary microRNA (pri-miRNA) that contains an imperfectly double-stranded region within a hairpin loop, in addition to 5′ and 3′ ends, then it is cleaved by DROSHA, which removes the 5′ and 3′ ends and produces a short hairpin called the pre-miRNA in the nucleus.After that, pre-miRNA is transferred through Exportin-5 to the cytoplasm where it is bound by the RISC that contains DICER, which cleaves the pre-miRNA and produces Let-7 dsRNA, which will be bound by the RISC that contains DICER and cleaved to two separate stands, one of them, the passenger strand will be removed while the guide strand will be retained.LIN28B inhibits the formation of mature Let-7 by inhibiting DROSHA, while LIN28A inhibits DICER in the cytoplasm and promotes the uridynylation of pre-Let-7 thus preventing the formation of mature Let-7

Fig. 7
Fig. 7 The role of the HMGA2 protein and its effect on cell cycle proteins.The HMGA2 protein has both direct and indirect effects on cell cycle progression.It activates Cyclin A, Cyclin E, Cyclin D1, Cyclin B2, and E2F1 to promote cell cycle progression

Fig. 10
Fig.10 The role of the HMGA2 protein in EMT.This figure shows the main pathways affected by the HMGA2 protein in the context of EMT process.The HMGA2 protein expression is stimulated by TGFβRII/TGF-β, MAPK/PI3K, and RAF/MEK/ERK pathways resulting in the upregulation of mesenchymal proteins such as Vimentin, Snail, and Twist, and the downregulation of epithelial proteins such as E-cadherin, MMP2, MMP9, and Occludin and the stimulation of Wnt/ β-catenin and AKT/ERK1/2 pathways, which together leads to the induction of EMT process

Fig. 11
Fig.11The role of the HMGA2 protein in cancer stemness.The HMGA2 protein upregulates the expression of SOX2, Oct4, CD44, Twist1, ALDH1, and c-Myc proteins by its binding to the AT-rich regions in each gene promoter, which results in the formation of complexes of transcription factors within these promoters, and that leads to upregulation of gene expression.As a result, cancer cells acquires stem-like properties