HMGA1 exacerbates tumor progression by activating miR-222 through PI3K/Akt/MMP-9 signaling pathway in uveal melanoma.

High-mobility group A1 (HMGA1), an architectural transcription factor, participates in different human tumors' biological progression. HMGA1 overexpression is associated with malignant cellular behavior in a wide range of cancers but the underlying mechanism remains poorly illuminated. In this study, we showed PI3K/Akt/MMP9 pathway activity could be positively regulated by HMGA1 using western blotting, real-time polymerase chain reaction (RT-PCR) and immunochemistry both in vitro (C918 and MUM-2B cell lines) and in vivo (xenograft mouse model). Later, MiRTarBase was used to identify the relationship between HMGA1 and miR-222-3p, we found miR-222 is positively regulated by HMGA1. Moreover, the proliferation and migration of UM cells significantly increased in the miR-222 mimics group and decreased in the miR-222 inhibitor group detected by the Annexin V-FITC apoptosis detection kit, CCK-8 and scratch wound-healing. The p-PI3K, p-Akt and MMP9 expressions were elevated in UM cells transfected with miR-222 mimics, and suppressed in the miR-222 inhibitor group. Together, our study highlights that HMGA1 acts as a pivotal regulator in UM tumor growth, proposing a critical viewpoint that HMGA1 expedites progression through the PI3K/Akt/MMP9 pathway and oncogenic miR-222 in UM.

IL-24 modulates the high mobility group (HMG) A1/miR222 /AKT signaling in lung cancer cells.

Interleukin (IL)-24, a novel tumor suppressor/cytokine exhibits antitumor activity against a broad-spectrum of human cancer cells. In a recent study, we showed that IL-24 inhibited AKT in lung cancer cells. However, the molecular mechanism of AKT inhibition by IL-24 remains elusive.The high mobility group (HMG) A1 a member of the non-histone chromosomal proteins and commonly referred to as architectural transcription factor, regulates transcription of various genes involved in cell growth and survival. Overexpression of HMGA1 has been shown to be associated with tumor progression and metastasis in several cancers, including human lung cancer. A recent study demonstrated that HMGA1 activates AKT function by reducing the activity of the protein phosphatase, phosphatase 2A subunit B (PPP2R2A) via the oncogenic micro (mi) RNA-222. Based on this report we hypothesized that IL-24-mediated AKT inhibition involved the HMGA1/miR-222 axis.To test our hypothesis, in the present study we used a H1299 lung cancer cell line that expressed exogenous human IL-24 when induced with doxycycline (DOX). Induction of IL-24 expression in the tumor cells markedly reduced HMGA1 mRNA and protein levels. Using a mechanistic approach, we found that IL-24 reduced miR-222-3p and -5p levels, as determined by qRT-PCR. Associated with HMGA1 and miR-222 inhibition was a marked increase in PPP2R2A, with a concomitant decrease in phosphorylated AKTT308/S473 expression. SiRNA-mediated knockdown of HMGA1 in combination with IL-24 significantly reduced AKT T308/S473 protein expression and greatly reduced cell migration and invasion compared with individual treatments. Further combination of IL-24 and a miR-222-3p inhibitor significantly increased PPP2R2A expression.Our results demonstrate for the first time that IL-24 inhibits AKT via regulating the HMGA1/miR-222 signaling node in human lung cancer cells and acts as an effective tumor suppressor. Thus, a therapy combining IL-24 with HMGA1 siRNA or miR-222-3p inhibitor should present effective treatment of lung cancer.

MicroRNA-214 suppresses growth, migration and invasion through a novel target, high mobility group AT-hook 1, in human cervical and colorectal cancer cells.

BACKGROUND: MicroRNA-214 (miR-214) has been shown to act as a tumour suppressor in human cervical and colorectal cancer cells. The aim of this study was to experimentally validate high mobility group AT-hook 1 as a novel target for miR-214-mediated suppression of growth and motility. METHODS: HMGA1 and miR-214 expression levels were estimated in cervical and colorectal clinical specimens using qPCR. HMGA1 3' untranslated region luciferase assays were performed to validate HMGA1 as a target of miR-214. Effect of altering the expression of miR-214 or HMGA1 on proliferation, migration and invasion of human cervical and colorectal cancer cells was investigated. RESULTS: miR-214 expression was poor while that of HMGA1 was high in cervical and colorectal cancer tissues. miR-214-re-expression or HMGA1 downregulation inhibited proliferation, migration and invasion of cancer cells while miR-214 inhibition had opposite effects. miR-214 was demonstrated to bind to the wild-type 3' untranslated region of HMGA1 but not with its mutant. CONCLUSIONS: Low expression of miR-214 concurrent with elevated levels of HMGA1 may contribute to cervical and colorectal cancer progression. miR-214-mediated regulation of HMGA1 is a novel mechanism for its tumour-suppressive actions in human cervical and colorectal cancer cells and opens up avenues for novel therapeutic strategies for these two cancers.

HMGA1 drives metabolic reprogramming of intestinal epithelium during hyperproliferation, polyposis, and colorectal carcinogenesis.

Although significant progress has been made in the diagnosis and treatment of colorectal cancer (CRC), it remains a leading cause of cancer death worldwide. Early identification and removal of polyps that may progress to overt CRC is the cornerstone of CRC prevention. Expression of the High Mobility Group A1 (HMGA1) gene is significantly elevated in CRCs as compared with adjacent, nonmalignant tissues. We investigated metabolic aberrations induced by HMGA1 overexpression in small intestinal and colonic epithelium using traveling wave ion mobility mass spectrometry (TWIMMS) in a transgenic model in which murine Hmga1 was misexpressed in colonic epithelium. To determine if these Hmga1-induced metabolic alterations in mice were relevant to human colorectal carcinogenesis, we also investigated tumors from patients with CRC and matched, adjacent, nonmalignant tissues. Multivariate statistical methods and manual comparisons were used to identify metabolites specific to Hmga1 and CRC. Statistical modeling of data revealed distinct metabolic patterns in Hmga1 transgenics and human CRC samples as compared with the control tissues. We discovered that 13 metabolites were specific for Hmga1 in murine intestinal epithelium and also found in human CRC. Several of these metabolites function in fatty acid metabolism and membrane composition. Although further validation is needed, our results suggest that high levels of HMGA1 protein drive metabolic alterations that contribute to CRC pathogenesis through fatty acid synthesis. These metabolites could serve as potential biomarkers or therapeutic targets.

The high mobility group A1 molecular switch: turning on cancer - can we turn it off?

INTRODUCTION: Emerging evidence demonstrates that the high mobility group A1 (HMGA1) chromatin remodeling protein is a key molecular switch required by cancer cells for tumor progression and a poorly differentiated, stem-like state. Because the HMGA1 gene and proteins are expressed at high levels in all aggressive tumors studied to date, research is needed to determine how to 'turn off' this master regulatory switch in cancer. AREAS COVERED: In this review, we describe prior studies that underscore the central role of HMGA1 in refractory cancers and we discuss approaches to target HMGA1 in cancer therapy. EXPERT OPINION: Given the widespread overexpression of HMGA1 in diverse, aggressive tumors, further research to develop technology to target HMGA1 holds immense promise as potent anticancer therapy. Previous work in preclinical models indicates that delivery of short hairpin RNA or interfering RNA molecules to 'switch off' HMGA1 expression dramatically impairs cancer cell growth and tumor progression. The advent of nanoparticle technology to systemically deliver DNA or RNA molecules to tumors brings this approach even closer to clinical applications, although further efforts are needed to translate these advances into therapies for cancer patients.

The HMGA1-COX-2 axis: a key molecular pathway and potential target in pancreatic adenocarcinoma.

CONTEXT: Although pancreatic cancer is a common, highly lethal malignancy, the molecular events that enable precursor lesions to become invasive carcinoma remain unclear. We previously reported that the high-mobility group A1 (HMGA1) protein is overexpressed in >90% of primary pancreatic cancers, with absent or low levels in early precursor lesions. METHODS: Here, we investigate the role of HMGA1 in reprogramming pancreatic epithelium into invasive cancer cells. We assessed oncogenic properties induced by HMGA1 in non-transformed pancreatic epithelial cells expressing activated K-RAS. We also explored the HMGA1-cyclooxygenase (COX-2) pathway in human pancreatic cancer cells and the therapeutic effects of COX-2 inhibitors in xenograft tumorigenesis. RESULTS: HMGA1 cooperates with activated K-RAS to induce migration, invasion, and anchorage-independent cell growth in a cell line derived from normal human pancreatic epithelium. Moreover, HMGA1 and COX-2 expression are positively correlated in pancreatic cancer cell lines (r(2) = 0.93; p < 0.001). HMGA1 binds directly to the COX-2 promoter at an AT-rich region in vivo in three pancreatic cancer cell lines. In addition, HMGA1 induces COX-2 expression in pancreatic epithelial cells, while knock-down of HMGA1 results in repression of COX-2 in pancreatic cancer cells. Strikingly, we also discovered that Sulindac (a COX-1/COX-2 inhibitor) or Celecoxib (a more specific COX-2 inhibitor) block xenograft tumorigenesis from pancreatic cancer cells expressing high levels of HMGA1. CONCLUSIONS: Our studies identify for the first time an important role for the HMGA1-COX-2 pathway in pancreatic cancer and suggest that targeting this pathway could be effective to treat, or even prevent, pancreatic cancer.

Cis-regulation of microRNA expression by scaffold/matrix-attachment regions.

microRNAs (miRNAs) spatio-temporally modulate gene expression; however, very little is known about the regulation of their expression. Here, we hypothesized that the well-known cis-regulatory elements of gene expression, scaffold/matrix-attachment regions (MARs) could modulate miRNA expression. Accordingly, we found MARs to be enriched in the upstream regions of miRNA genes. To determine their role in cell type-specific expression of miRNAs, we examined four individual miRNAs (let-7b, miR-17, miR-93 and miR-221) and the miR-17-92 cluster, known to be overexpressed in neuroblastoma. Our results show that MARs indeed define the cell-specific expression of these miRNAs by tethering the chromatin to nuclear matrix. This is brought about by cell type-specific binding of HMG I/Y protein to MARs that then promotes the local acetylation of histones, serving as boundary elements for gene activation. The binding, chromatin tethering and gene activation by HMG I/Y was not observed in fibroblast control cells but were restricted to neuroblastoma cells. This study implies that the association of MAR binding proteins to MARs could dictate the tissue/context specific regulation of miRNA genes by serving as a boundary element signaling the transcriptional activation.

HMGA1a is involved in specific splice site regulation of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) utilizes a highly complex splice site regulation system, taking advantage of host proteins, to express its own viral protein in an orderly way. We show here that one of the host proteins, high mobility group A protein 1a (HMGA1a), is involved in splice site regulation of 3' splice site 2 (A2) and 5'splice site 3 (D3) of HIV-1 genomic RNA. shRNA knockdown of HMGA1 in HeLa cells resulting in a decrease of HMGA1 showed a significant decrease of Vpr mRNA. RNA electrophoretic mobility shift assays showed HMGA1a specifically binds to a sequence adjacently upstream D3. In vitro splicing using heterologous pre-mRNA with A2 and D3, showed HMGA1a induced a splicing intermediate which decreased when an RNA decoy of the HMGA1a binding site was added. RT-PCR of in vitro splicing products revealed that HMGA1a induced an incomplete splicing product resulting from usage of A2 but inhibition of D3, which is reminiscent of the splicing pattern necessary for Vpr mRNA formation. HMGA1a interacted with hnRNPA1 shown by coimmunoprecipitation and supershifted U1 snRNP in an RNA electrophoretic mobility shift assay. We conclude that HMGA1a anchors U1 snRNP to inhibit D3 function, and that HMGA1a inhibits hnRNPA1 function on exon splicing silencer of Vpr (ESSV) to activate A2 function. We show here for the first time that HMGA1a is involved in specific splice site regulation of HIV-1.

H5N1 virus activates signaling pathways in human endothelial cells resulting in a specific imbalanced inflammatory response.

H5N1 influenza virus infections in humans cause a characteristic systemic inflammatory response syndrome; however, the molecular mechanisms are largely unknown. Endothelial cells (ECs) play a pivotal role in hyperdynamic septic diseases. To unravel specific signaling networks activated by H5N1 we used a genome-wide comparative systems biology approach analyzing gene expression in human ECs infected with three different human and avian influenza strains of high and low pathogenicity. Blocking of specific signaling pathways revealed that H5N1 induces an exceptionally NF-κB-dependent gene response in human endothelia. Additionally, the IFN-driven antiviral program in ECs is shown to be dependent on IFN regulatory factor 3 but significantly impaired upon H5N1 infection compared with low pathogenic influenza virus. As additional modulators of this H5N1-specific imbalanced gene response pattern, we identified HMGA1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not antiviral response, whereas NFATC4 was found to regulate transcription of specifically H5N1-induced genes. We describe for the first time, to our knowledge, defined signaling patterns specifically activated by H5N1, which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of an overwhelming proinflammatory and impaired antiviral gene program.

HMGA1 protein is a positive regulator of the insulin-like growth factor-I receptor gene.

The IGF-I receptor (IGF-IR) is often overexpressed in cancer and is believed to play a crucial role in cancer progression. High Mobility Group A1 (HMGA1) is a non-histone chromatin protein that has the ability to regulate gene expression through DNA binding and involvement in enhanceosome complexes. HMGA1 is expressed at low level in adult differentiated cells, whereas it is expressed at high level in embryonic and malignant cells. We evaluated whether the HMGA1 aberrant expression has a role in IGF-IR overexpression in cancer. We found that HMGA1 silencing induces a marked decrease in IGF-IR expression in various human cancer cell lines. Conversely, forced HMGA1 overexpression in cells with low endogenous HMGA1 levels was associated with IGF-IR upregulation. HMGA1 silencing reduced igf-ir promoter activity whereas forced HMGA1 expression increased it. Using the chromatin immunoprecipitation assay, HMGA1 protein was found to bind to the igf-ir promoter. Moreover, HMGA1 was found to associate with both p53 and Sp1, two major regulators of igf-ir gene transcription and to antagonise the p53 inhibitory activity while enhancing the Sp1 stimulatory activity. Our data indicate, therefore, that HMGA1 protein is a positive regulator of IGF-IR expression and that HMGA1 overexpression may contribute to IGF-IR dysregulation in cancer cells.

High-mobility group A1 (HMGA1) protein expression correlates with cisplatin-induced cell death in squamous cell carcinoma of skin.

It is well known that HMGA1 group of non-histone chromosomal proteins are up-regulated in several human cancers. We studied the HMGA1 expression in squamous cell carcinoma of skin in mice followed by the treatment with Cisplatin, which is often used in combination therapies of cancers. A short course of Cisplatin treatment led to apoptotic cell death and downregulation (by 40%) of HMGA1. However, extended treatment of Cisplatin caused necrotic cell death; concomitantly HMGA1 expression decreased by 90%. Present results indicate a strong association of HMGA1 with Cisplatin-linked tumor regression. Therefore, HMGA1 could be a potential target in designing therapeutic strategies against cancer.

HMGA proteins up-regulate CCNB2 gene in mouse and human pituitary adenomas.

The high mobility group As (HMGAs) belong to a family of nonhistone nuclear proteins that orchestrate the assembly of nucleoprotein complexes. Through a complex network of protein-DNA and protein-protein interaction, they play important roles in gene transcription, recombination, and chromatin structure. This protein family is involved, through different mechanisms, in both benign and malignant neoplasias. We have recently reported that transgenic mice carrying the Hmga1 or Hmga2 genes under transcriptional control of the cytomegalovirus promoter develop pituitary adenomas secreting prolactin and growth hormone. We have shown that the mechanism of the HMGA2-induced pituitary adenoma is based on the increased E2F1 activity. The expression profile of mouse normal pituitary glands and adenomas induced in HMGA transgenic mice revealed an increased expression of the ccnb2 gene, coding for the cyclin B2 protein, in the neoplastic tissues compared with the normal pituitary gland. Here, we show, by electrophoretic mobility shift assay and chromatin immunoprecipitation, a direct binding of HMGA proteins to the promoter of ccnb2 gene, whereas luciferase assays showed that HMGAs are able to up-regulate ccnb2 promoter activity. Finally, we report an increased CCNB2 expression in human pituitary adenomas of different histotypes that is directly correlated with HMGA1 and HMGA2 expression. Because cyclin B2 is involved in the regulation of the cell cycle, these results taken together indicate that HMGA-induced cyclin B2 overexpression gives an important contribution to experimental and human pituitary tumorigenesis.

Regulation of microRNA expression by HMGA1 proteins.

The High Mobility Group proteins HMGA1 are nuclear architectural factors that play a critical role in a wide range of biological processes. Since recent studies have identified the microRNAs (miRNAs) as important regulators of gene expression, modulating critical cellular functions such as proliferation, apoptosis and differentiation, the aim of our work was to identify the miRNAs that are physiologically regulated by HMGA1 proteins. To this purpose, we have analysed the miRNA expression profile of mouse embryonic fibroblasts (MEFs) carrying two, one or no Hmga1 functional alleles using a microarray (miRNA microarray). By this approach, we found a miRNA expression profile that differentiates Hmga1-null MEFs from the wild-type ones. In particular, a significant decrease in miR-196a-2, miR-101b, miR-331 and miR-29a was detected in homozygous Hmga1-knockout MEFs in comparison with wild-type cells. Consistently, these miRNAs are downregulated in most of the analysed tissues of Hmga1-null mice in comparison with the wild-type mice. ChIP assay shows that HMGA1 is able to bind regions upstream of these miRNAs. Moreover, we identified the HMGA2 gene product as a putative target of miR-196a-2, suggesting that HMGA1 proteins are able to downregulate the expression of the other member of the HMGA family through the regulation of the miR-196a-2 expression. Finally, ATXN1 and STC1 gene products have been identified as targets of miR-101b. Therefore, it is reasonable to hypothesize that HMGA1 proteins are involved in several functions by regulating miRNA expression.

The HMGA proteins: a myriad of functions (Review).

The 'high mobility group' HMGA protein family consists of four members: HMGA1a, HMGA1b and HMGA1c, which result from translation of alternative spliced forms of one gene and HMGA2, which is encoded for by another gene. HMGA proteins are characterized by three DNA-binding domains, called AT-hooks, and an acidic carboxy-terminal tail. HMGA proteins are architectural transcription factors that both positively and negatively regulate the transcription of a variety of genes. They do not display direct transcriptional activation capacity, but regulate gene expression by changing the DNA conformation by binding to AT-rich regions in the DNA and/or direct interaction with several transcription factors. In this way, they influence a diverse array of normal biological processes including cell growth, proliferation, differentiation and death. Both HMGA1 and HMGA2 are hardly detectable in normal adult tissue but are abundantly and ubiquitously expressed during embryonic development. In malignant epithelial tumors as well as in leukemia, however, expression of HMGA1 is again strongly elevated to embryonic levels thus leading to ectopic expression of (fetal) target genes. HMGA2 overexpression also has a causal role in inducing neoplasia. Besides overexpression of full length HMGA proteins in different tumors, the HMGA genes are often involved in chromosomal rearrangements. Such translocations are mostly detected in benign tumors of mesenchymal origin and are believed to be one of the most common chromosomal rearrangements in human neoplasia. To provide clarity in the abundance of articles on this topic, this review gives a general overview of the nuclear functions and regulation of the HMGA genes and corresponding proteins.

HMGA1a: sequence-specific RNA-binding factor causing sporadic Alzheimer's disease-linked exon skipping of presenilin-2 pre-mRNA.

Aberrant exon 5 skipping of presenilin-2 (PS2) pre-mRNA produces a deleterious protein isoform PS2V, which is almost exclusively observed in the brains of sporadic Alzheimer's disease patients. PS2V over-expression in vivo enhances susceptibility to various endoplasmic reticulum (ER) stresses and increases production of amyloid-beta peptides. We previously purified and identified high mobility group A protein 1a (HMGA1a) as a trans-acting factor responsible for aberrant exon 5 skipping. Using heterologous pre-mRNAs, here we demonstrate that a specific HMGA1a-binding sequence in exon 5 adjacent to the 5' splice site is necessary for HMGA1a to inactivate the 5' splice site. An aberrant HMGA1a-U1 snRNP complex was detected on the HMGA1a-binding site adjacent to the 5' splice site during the early splicing reaction. A competitor 2'-O-methyl RNA (2'-O-Me RNA) consisting of the HMGA1a-binding sequence markedly repressed exon 5 skipping of PS2 pre-mRNA in vitro and in vivo. Finally, HMGA1a-induced cell death under ER stress was prevented by transfection of the competitor 2'-O-Me RNA. These results provide insights into the molecular basis for PS2V-associated neurodegenerative diseases that are initiated by specific RNA binding of HMGA1a.

High mobility group protein HMGA1 inhibits retinoblastoma protein-mediated cellular G0 arrest.

Retinoblastoma protein (RB) acts as a tumor suppressor in many tissue types, by promoting cell arrest via E2F-mediated transcriptional repression. In addition to the aberrant forms of the RB gene found in different types of cancers, many viral oncoproteins including the simian virus 40 large T antigen target RB. However, cellular factors that inhibit RB function remain to be elucidated. Here, we report that RB interacts with the high mobility group protein A1 (HMGA1), a-non-histone architectural chromatin factor that is frequently overexpressed in cancer cells. HMGA1 binds the small pocket domain of RB, and competes with HDAC1. Subsequently, overexpression of HMGA1 abolishes the inhibitory effect of RB on E2F-activated transcription from the cyclin E promoter. Under serum starvation, T98G cells had been previously shown to be arrested in the G0 phase in an RB-mediated manner. The G0 phase was characterized by growth arrest and low levels of transcription, together with the hypophosphorylation of RB and the downregulation of HMGA1. In contrast, such serum-depleted G0 arrest was abrogated in T98G cells overexpressing HMGA1. The overexpressed HMGA1 was found to form complexes with cellular RB, suggesting that downregulation of HMGA1 is required for G0 arrest. There were no phenotypic changes in HMGA1-expressing T98G cells in the presence of serum, but the persistent expression of HMGA1 under serum starvation caused various nuclear abnormalities, which were similarly induced in T antigen-expressing T98G cells. Our present findings indicate that overexpression of HMGA1 disturbs RB-mediated cell arrest, suggesting a negative control of RB by HMGA1.

HMGA1 mediates the activation of the CRYAB promoter by BRG1.

alphaB-Crystallin (CRYAB) is a small heat-shock protein that is implicated in many cellular processes, such as transcription and differentiation, as well as pathologic process. It is expressed at high levels in vertebrate eye lens and at low levels in a variety of other cell types. We previously identified CRYAB as a target gene of the chromatin-remodeling SWI/SNF-like Brg or hBrm-associated factors (BAF) complexes. In this report, we identify a 30 bp DNA element required for mediating the activation of CRYAB by brahma-related gene 1 (BRG1). This BRG1-response element is located at the edge of a positioned nucleosome immediately upstream of the transcription initiation site. An AT-rich sequence within this region is bound by the high-mobility group AT-hook 1 (HMGA1) proteins in vitro and in vivo. We demonstrate that the HMGA1 target sequences and HMGA1 proteins are required for the maximal activation of the CRYAB promoter by BRG1. Our data indicate that HMGA1 nonhistone chromatin proteins, the SWI/SNF chromatin remodeling complexes, and sequence-specific transcription factors act together to regulate the expression of the CRYAB gene.

High-mobility group A1 proteins inhibit expression of nucleotide excision repair factor xeroderma pigmentosum group A.

Cells that overexpress high-mobility group A1 (HMGA1) proteins exhibit deficient nucleotide excision repair (NER) after exposure to DNA-damaging agents, a condition ameliorated by artificially lowering intracellular levels of these nonhistone proteins. One possible mechanism for this NER inhibition is down-regulation of proteins involved in NER, such as xeroderma pigmentosum complimentation group A (XPA). Microarray and reverse transcription-PCR data indicate a 2.6-fold decrease in intracellular XPA mRNA in transgenic MCF-7 cells overexpressing HMGA1 proteins compared with non-HMGA1-expressing cells. XPA protein levels are also approximately 3-fold lower in HMGA1-expressing MCF-7 cells. Moreover, whereas a >2-fold induction of XPA proteins is observed in normal MCF-7 cells 30 min after UV exposure, no apparent induction of XPA protein is observed in MCF-7 cells expressing HMGA1. Mechanistically, we present both chromatin immunoprecipitation and promoter site-specific mutagenesis evidence linking HMGA1 to repression of XPA transcription via binding to a negative regulatory element in the endogenous XPA gene promoter. Phenotypically, HMGA1-expressing cells exhibit compromised removal of cyclobutane pyrimidine dimer lesions, a characteristic of cells that express low levels of XPA. Importantly, we show that restoring expression of wild-type XPA in HMGA1-expressing cells rescues UV resistance comparable with that of normal MCF-7 cells. Together, these data provide strong experimental evidence that HMGA1 proteins are involved in inhibiting XPA expression, resulting in increased UV sensitivity in cells that overexpress these proteins. Because HMGA1 proteins are overexpressed in most naturally occurring cancers, with increasing cellular concentrations correlating with increasing metastatic potential and poor patient prognosis, the current findings provide new insights into previously unsuspected mechanisms contributing to tumor progression.

Gene-specific nucleotide excision repair is impaired in human cells expressing elevated levels of high mobility group A1 nonhistone proteins.

Previous work has established that stably transfected human MCF7 cells over-expressing high mobility group A1 proteins (HMGA1) are deficient in global genomic repair (GGR) following exposure to either UV light or cisplatin. To investigate whether HMGA1 over-expression also interferes with gene-specific repair, we employed a rapid and convenient quantitative polymerase chain reaction assay for measuring repair in unique DNA sequences. Efficiency of UV-induced lesion removal was assessed for two genes in MCF7 cells either induced, or not, to over-express transgenic HMGA1 proteins: the constitutively active HPRT gene and the transcriptionally silent beta-globin gene. As controls, similar experiments were also performed in non-transgenic MCF7 cells that do not express detectable levels of HMGA1 and in normal human embryonic fibroblasts that naturally over-express HMGA1 proteins. Our results indicate that exposure of cells to a UV dose of 20 J/m2 produced an average of 0.21+/-0.03 and 0.19+/-0.02 lesions/kb in the HPRT and beta-globin genes, respectively, with no significant difference between HMGA1 over-expressing cells and non-expressing cells. On the other hand, analysis of repair following UV exposure revealed that, compared to controls, HMGA1 over-expressing cells take considerably longer to repair photo-lesions in both the active HPRT and the silent beta-globin loci, with non-expressing cells repairing 50% of lesions in HPRT 3-4 h faster than HMGA1 over-expressing cells. Interestingly, the delay in repair is even more prolonged in the silent beta-globin locus in HMGA1 over-expressing cells compared to control cells. To our knowledge, this is the first report of HMGA1 proteins inhibiting nucleotide excision repair (NER) within specific genes located in either transcriptionally active "open", or inactive "closed", chromatin domains. Furthermore, taken together with previous findings, these results suggest that HMGA1 over-expression interferes with repair processes common to both the GGR and transcription-coupled repair pathways.