A parasite DNA binding protein with potential to influence disease susceptibility acts as an analogue of mammalian HMGA transcription factors.

Intracellular pathogens construct their environmental niche, and influence disease susceptibility, by deploying factors that manipulate infected host cell gene expression. Theileria annulata is an important tick-borne parasite of cattle that causes tropical theileriosis. Excellent candidates for modulating host cell gene expression are DNA binding proteins bearing AT-hook motifs encoded within the TashAT gene cluster of the parasite genome. In this study, TashAT2 was transfected into bovine BoMac cells to generate three expressing and three non-expressing (opposite orientation) cell lines. RNA-Seq was conducted and differentially expressed (DE) genes identified. The resulting dataset was compared with genes differentially expressed between infected cells and non-infected cells, and DE genes between infected cell lines from susceptible Holstein vs tolerant Sahiwal cattle. Over 800 bovine genes displayed differential expression associated with TashAT2, 209 of which were also modulated by parasite infection. Network analysis showed enrichment of DE genes in pathways associated with cellular adhesion, oncogenesis and developmental regulation by mammalian AT-hook bearing high mobility group A (HMGA) proteins. Overlap of TashAT2 DE genes with Sahiwal vs Holstein DE genes revealed that a significant number of shared genes were associated with disease susceptibility. Altered protein levels encoded by one of these genes (GULP1) was strongly linked to expression of TashAT2 in BoMac cells and was demonstrated to be higher in infected Holstein leucocytes compared to Sahiwal. We conclude that TashAT2 operates as an HMGA analogue to differentially mould the epigenome of the infected cell and influence disease susceptibility.

Availability of bioactive flax lignan from foods and supplements.

Hyperlipidemia, high levels of blood lipids including cholesterol and triglycerides, is a major risk factor for cardiovascular disease. Traditional treatments of hyperlipidemia often include lifestyle changes and pharmacotherapy. Recently, flaxseed has been approved as a nutrient that lowers blood lipids. Several metabolites of flaxseed lignan secoisolariciresinol diglucoside (SDG), have been identified that reduce blood lipids. SDG is present in flaxseed hull as an ester-linked copolymer with 3-hydroxy-3-methylglutaric acid (HMGA). However, purification processes involved in hydrolysis of the copolymer and enriching SDG are often expensive. The natural copolymer of SDG with HMGA (SDG polymer) is a source of bioactive compounds useful in prophylaxis of hypercholesterolemia. After consumption of the lignan copolymer, SDG and HMGA are released in the stomach and small intestines. SDG is metabolized to secoisolariciresinol, enterolactone and enterodiol, the bioactive forms of mammalian lignans. These metabolites are then distributed throughout the body where they accumulate in the liver, kidney, skin, other tissues, and organs. Successively, these metabolites reduce blood lipids including cholesterol, triglycerides, low density lipoprotein cholesterol, and lipid peroxidation products. In this review, the metabolism and efficacies of flaxseed-derived enriched SDG and SDG polymer will be discussed.

Pleiotropic Effects of Hfq on the Cytochrome c Content and Pyomelanin Production in Shewanella oneidensis.

Shewanella oneidensis is the best understood model microorganism for the study of diverse cytochromes (cytos) c that support its unparallel respiratory versatility. Although RNA chaperone Hfq has been implicated in regulation of cyto c production, little is known about the biological pathways that it affects in this bacterium. In this study, from a spontaneous mutant that secretes pyomelanin and has a lowered cyto c content, we identified Hfq to be the regulator that critically associates with both phenotypes in S. oneidensis. We found that expression of the key genes in biosynthesis and degradation of heme is differentially affected by Hfq at under- and overproduced levels, and through modulating heme levels, Hfq influences the cyto c content. Although Hfq in excess results in overproduction of the enzymes responsible for both generation and removal of homogentisic acid (HGA), the precursor of pyomelanin, it is compromised activity of HmgA that leads to excretion and polymerization of HGA to form pyomelanin. We further show that Hfq mediates HmgA activity by lowering intracellular iron content because HmgA is an iron-dependent enzyme. Overall, our work highlights the significance of Hfq-mediated posttranscriptional regulation in the physiology of S. oneidensis, unraveling unexpected mechanisms by which Hfq affects cyto c biosynthesis and pyomelanin production. IMPORTANCE In bacteria, Hfq has been implicated in regulation of diverse biological processes posttranslationally. In S. oneidensis, Hfq affects the content of cytos c that serve as the basis of its respiratory versatility and potential application in bioenergy and bioremediation. In this study, we found that Hfq differentially regulates heme biosynthesis and degradation, leading to altered cyto c contents. Hfq in excess causes a synthetic effect on HmgA, an enzyme responsible for pyomelanin formation. Overall, the data presented manifest that the biological processes in a given bacterium regulated by Hfq are highly complex, amounting to required coordination among multiple physiological aspects to allow cells to respond to environmental changes promptly.

Fluctuating Bacteriophage-induced galU Deficiency Region is Involved in Trade-off Effects on the Phage and Fluoroquinolone Sensitivity in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, which causes chronic infections, has demonstrated rapid acquisition of antimicrobial resistance (AMR). Therefore, bacteriophages have received significant attention as promising antimicrobial agents; however, previous trials have reported the occurrence of phage-resistant variants. P. aeruginosa has lost large chromosomal fragments via evolutionary selection by MutL. Mutants lacking galU and hmgA, located in close proximity, exhibit phage resistance and brown color phenotype since hmgA encodes a homogentisic acid metabolic enzyme and deletion of galU results in a lack of O-antigen polysaccharide and absence of the phage receptor. In the present study, we evaluated this mechanism for controlling phage resistance in P. aeruginosa veterinary isolate Pa12. Phage-resistant Pa12 brown mutants (brmts) with galU and hmgA deletions were isolated. Whole-genome sequencing of the brmts revealed that regions 148-27 kbp upstream and 261-110 kbp downstream of galU were largely deleted from the Pa12 parental chromosome. Furthermore, all of these fluctuating deleted sequences in Pa12 brmts, tentatively designated bacteriophage-induced galU deficiency (BigD) regions, harbor multi-drug efflux system genes (mexXY). Minimum inhibitory concentration (MIC) assays demonstrated that brmts altered sensitivity to antibiotics and exhibited increased levofloxacin sensitivity compared with the Pa12 parent. Orbifloxacin and enrofloxacin also effectively suppressed growth of the Pa12 brmts, suggesting that MexXY, which mediates quinolone efflux and is located in the BigD region, might be associated with restoration of fluoroquinolone sensitivity. Our findings indicate that AMR-related genes in the BigD region could produce trade-off effects between phages and drug sensitivity and thereby contribute to a potential strategy to control and prevent phage-resistant variants in phage therapy.

Phenotypic Effects of Homeodomain-Interacting Protein Kinase 2 Deletion in Mice.

Homeodomain-interacting protein kinase 2 (HIPK2) is a serine-threonine kinase that phosphorylates various transcriptional and chromatin regulators, thus modulating numerous important cellular processes, such as proliferation, apoptosis, DNA damage response, and oxidative stress. The role of HIPK2 in the pathogenesis of cancer and fibrosis is well established, and evidence of its involvement in the homeostasis of multiple organs has been recently emerging. We have previously demonstrated that Hipk2-null (Hipk2-KO) mice present cerebellar alterations associated with psychomotor abnormalities and that the double ablation of HIPK2 and its interactor HMGA1 causes perinatal death due to respiratory failure. To identify other alterations caused by the loss of HIPK2, we performed a systematic morphological analysis of Hipk2-KO mice. Post-mortem examinations and histological analysis revealed that Hipk2 ablation causes neuronal loss, neuronal morphological alterations, and satellitosis throughout the whole central nervous system (CNS); a myopathic phenotype characterized by variable fiber size, mitochondrial proliferation, sarcoplasmic inclusions, morphological alterations at neuromuscular junctions; and a cardiac phenotype characterized by fibrosis and cardiomyocyte hypertrophy. These data demonstrate the importance of HIPK2 in the physiology of skeletal and cardiac muscles and of different parts of the CNS, thus suggesting its potential relevance for different new aspects of human pathology.

Interplay between HMGA and TP53 in cell cycle control along tumor progression.

The high mobility group A (HMGA) proteins are found to be aberrantly expressed in several tumors. Studies (in vitro and in vivo) have shown that HMGA protein overexpression has a causative role in carcinogenesis process. HMGA proteins regulate cell cycle progression through distinct mechanisms which strongly influence its normal dynamics along malignant transformation. Tumor protein p53 (TP53) is the most frequently altered gene in cancer. The loss of its activity is recognized as the fall of a barrier that enables neoplastic transformation. Among the different functions, TP53 signaling pathway is tightly involved in control of cell cycle, with cell cycle arrest being the main biological outcome observed upon p53 activation, which prevents accumulation of damaged DNA, as well as genomic instability. Therefore, the interaction and opposing effects of HMGA and p53 proteins on regulation of cell cycle in normal and tumor cells are discussed in this review. HMGA proteins and p53 may reciprocally regulate the expression and/or activity of each other, leading to the counteraction of their regulation mechanisms at different stages of the cell cycle. The existence of a functional crosstalk between these proteins in the control of cell cycle could open the possibility of targeting HMGA and p53 in combination with other therapeutic strategies, particularly those that target cell cycle regulation, to improve the management and prognosis of cancer patients.

Nonhost Disease Resistance in Pea: Chitosan's Suggested Role in DNA Minor Groove Actions Relative to Phytoalexin-Eliciting Anti-Cancer Compounds.

A stable intense resistance called "nonhost resistance" generates a complete multiple-gene resistance against plant pathogenic species that are not pathogens of pea such as the bean pathogen, Fusarium solani f. sp. phaseoli (Fsph). Chitosan is a natural nonhost resistance response gene activator of defense responses in peas. Chitosan may share with cancer-treatment compounds, netropsin and some anti-cancer drugs, a DNA minor groove target in plant host tissue. The chitosan heptamer and netropsin have the appropriate size and charge to reside in the DNA minor groove. The localization of a percentage of administered radio-labeled chitosan in the nucleus of plant tissue in vivo indicates its potential to transport to site(s) within the nuclear chromatin (1,2). Other minor groove-localizing compounds administered to pea tissue activate the same secondary plant pathway that terminates in the production of the anti-fungal isoflavonoid, pisatin an indicator of the generated resistance response. Some DNA minor groove compounds also induce defense genes designated as "pathogenesis-related" (PR) genes. Hypothetically, DNA targeting components alter host DNA in a manner enabling the transcription of defense genes previously silenced or minimally expressed. Defense-response-elicitors can directly (a) target host DNA at the site of transcription or (b) act by a series of cascading events beginning at the cell membrane and indirectly influence transcription. A single defense response, pisatin induction, induced by chitosan and compounds with known DNA minor groove attachment potential was followed herein. A hypothesis is formulated suggesting that this DNA target may be accountable for a portion of the defense response generated in nonhost resistance.

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.

Bimodal function of chromatin remodeler Hmga1 in neural crest induction and Wnt-dependent emigration.

During gastrulation, neural crest cells are specified at the neural plate border, as characterized by Pax7 expression. Using single-cell RNA sequencing coupled with high-resolution in situ hybridization to identify novel transcriptional regulators, we show that chromatin remodeler Hmga1 is highly expressed prior to specification and maintained in migrating chick neural crest cells. Temporally controlled CRISPR-Cas9-mediated knockouts uncovered two distinct functions of Hmga1 in neural crest development. At the neural plate border, Hmga1 regulates Pax7-dependent neural crest lineage specification. At premigratory stages, a second role manifests where Hmga1 loss reduces cranial crest emigration from the dorsal neural tube independent of Pax7. Interestingly, this is rescued by stabilized ß-catenin, thus implicating Hmga1 as a canonical Wnt activator. Together, our results show that Hmga1 functions in a bimodal manner during neural crest development to regulate specification at the neural plate border, and subsequent emigration from the neural tube via canonical Wnt signaling.

The neural plate is a structure that serves as the basis for the brain and central nervous system during the development of animals with a backbone. In particular, the tissues at the border of the neural plate become the neural crest, a group of highly mobile cells that can specialize to form nerves and parts of the face. The exact molecular mechanisms that allow the crest to emerge are still unknown. The protein Hmga1 alters how genes are packaged and organized inside cells, which in turn influences how genes are switched on and off. Here, Gandhi et al. studied how Hmga1 helps to shape the neural crest in developing chicken embryos. To do so, they harnessed a genetic tool called CRISPR-Cas9, and deleted the gene that encodes Hmga1 at specific developmental stages. This manipulation highlighted two periods where Hmga1 is active. First, Hmga1 helped to define neural crest cells at the neural plate border by activating a gene called pax7. Then, at a later stage, Hmga1 allowed these cells to move to other parts of the body by triggering the Wnt communication system. Failure for the neural crest to develop properly causes birth defects and cancers such as melanoma and childhood neuroblastoma, highlighting the need to better understand how this structure is formed. In addition, a better grasp of the roles of Hmga1 in healthy development could help to appreciate how it participates in a range of adult cancers.

HMGA Genes and Proteins in Development and Evolution.

HMGA (high mobility group A) (HMGA1 and HMGA2) are small non-histone proteins that can bind DNA and modify chromatin state, thus modulating the accessibility of regulatory factors to the DNA and contributing to the overall panorama of gene expression tuning. In general, they are abundantly expressed during embryogenesis, but are downregulated in the adult differentiated tissues. In the present review, we summarize some aspects of their role during development, also dealing with relevant studies that have shed light on their functioning in cell biology and with emerging possible involvement of HMGA1 and HMGA2 in evolutionary biology.

HMGA Proteins in Stemness and Differentiation of Embryonic and Adult Stem Cells.

HMGA1 and HMGA2 are chromatin architectural proteins that do not have transcriptional activity per se, but are able to modify chromatin structure by interacting with the transcriptional machinery and thus negatively or positively regulate the transcription of several genes. They have been extensively studied in cancer where they are often found to be overexpressed but their functions under physiologic conditions have still not been completely addressed. Hmga1 and Hmga2 are expressed during the early stages of mouse development, whereas they are not detectable in most adult tissues. Hmga overexpression or knockout studies in mouse have pointed to a key function in the development of the embryo and of various tissues. HMGA proteins are expressed in embryonic stem cells and in some adult stem cells and numerous experimental data have indicated that they play a fundamental role in the maintenance of stemness and in the regulation of differentiation. In this review, we discuss available experimental data on HMGA1 and HMGA2 functions in governing embryonic and adult stem cell fate. Moreover, based on the available evidence, we will aim to outline how HMGA expression is regulated in different contexts and how these two proteins contribute to the regulation of gene expression and chromatin architecture in stem cells.

LncRNA SNHG1 exerts a protective role in cardiomyocytes hypertrophy via targeting miR-15a-5p/HMGA1 axis.

Heart failure preceded by pathological cardiac hypertrophy is a leading cause of death. Long noncoding RNA small nucleolar RNA host gene 1 (SNHG1) was reported to inhibit cardiomyocytes apoptosis, but the role and underlying mechanism of SNHG1 in pathological cardiac hypertrophy have not yet been understood. This study was designed to investigate the role and molecular mechanism of SNHG1 in regulating cardiac hypertrophy. We found that SNHG1 was upregulated during cardiac hypertrophy both in vivo (transverse aortic constriction treatment) and in vitro (phenylephrine [PE] treatment). SNHG1 overexpression attenuated the cardiomyocytes hypertrophy induced by PE, while SNHG1 inhibition promoted hypertrophic response of cardiomyocytes. Furthermore, SNHG1 and high-mobility group AT-hook 1 (HMGA1) were confirmed to be targets of miR-15a-5p. SNHG1 promoted HMGA1 expression by sponging miR-15a-5p, eventually attenuating cardiomyocytes hypertrophy. There data revealed a novel protective mechanism of SNHG1 in cardiomyocytes hypertrophy. Thus, targeting of SNHG1-related pathway may be therapeutically harnessed to treat cardiac hypertrophy.

The Prominent Role of HMGA Proteins in the Early Management of Gastrointestinal Cancers.

GI tumors represent a heterogeneous group of neoplasms concerning their natural history and molecular alterations harbored. Nevertheless, these tumors share very high incidence and mortality rates worldwide and patients' poor prognosis. Therefore, the identification of specific biomarkers could increase the development of personalized medicine, in order to improve GI cancer management. In this sense, HMGA family members (HMGA1 and HMGA2) comprise an important group of genes involved in the genesis and progression of malignant tumors. Additionally, it has also been reported that HMGA1 and HMGA2 display an important role in the detection and progression of GI tumors. In this way, HMGA family members could be used as reliable biomarkers able to efficiently track not only the tumor per se but also the main risk conditions related with their development of GI cancers in the future. Finally, it shall be a promising option to revert the current scenario, once HMGA genes and proteins could represent a convergence point in the complex landscape of GI tumors.

HMGA and Cancer: A Review on Patent Literatures.

BACKGROUND: The high mobility group A proteins modulate the transcription of numerous genes by interacting with transcription factors and/or altering the structure of chromatin. These proteins are involved in both benign and malignant neoplasias as a result of several pathways. A large amount of benign human mesenchymal tumors has rearrangements of HMGA genes. On the contrary, malignant tumors show unarranged HMGA overexpression that is frequently and causally related to neoplastic cell transformation. Here, we review the function of the HMGA proteins in human neoplastic disorders, the pathways by which they contribute to carcinogenesis and the new patents focused on targeting HMGA proteins. OBJECTIVE: Current review was conducted to check the involvement of HMGA as a druggable target in cancer treatment. METHODS: We reviewed the most recent patents focused on targeting HMGA in cancer treatment analyzing patent literature published during the last years, including the World Intellectual Property Organization (WIPO®), United States Patent Trademark Office (USPTO®), Espacenet®, and Google Patents. RESULTS: HMGA proteins are intriguing targets for cancer therapy and are objects of different patents based on the use of DNA aptamers, inhibitors, oncolytic viruses, antisense molecules able to block their oncogenic functions. CONCLUSION: Powerful strategies able to selectively interfere with HMGA expression and function could represent a helpful approach in the development of new anti-cancer therapies.

Effect of Doxorubicin on Squamous Cell Carcinoma of Skin: Assessment by MRI Relaxometry at 4.7T.

Squamous cell carcinoma (SCC) of skin has no standard treatment regimen, resulting in recurrences/metastasis. Although, doxorubicin (Dox), an anthracycline antibiotic has demonstrated some degree of efficacy. Molecular imaging can help in assessment of treatment response and prognosis of SCCs. MRI data showed that spin-spin relaxation (T2) time was longer (138 ± 2 msec) in Dox treated Test-II and there is no significant difference in spin-lattice relaxation (T1) time with respective controls. These findings further corroborated with the histology, proliferation index, apoptotic index, and HMGA1 protein expression. Thus, MRI may be a useful tool for monitoring treatment response noninvasively for skin tumor prognosis.

The High Mobility Group A1 (HMGA1) Chromatin Architectural Factor Modulates Nuclear Stiffness in Breast Cancer Cells.

Plasticity is an essential condition for cancer cells to invade surrounding tissues. The nucleus is the most rigid cellular organelle and it undergoes substantial deformations to get through environmental constrictions. Nuclear stiffness mostly depends on the nuclear lamina and chromatin, which in turn might be affected by nuclear architectural proteins. Among these is the HMGA1 (High Mobility Group A1) protein, a factor that plays a causal role in neoplastic transformation and that is able to disentangle heterochromatic domains by H1 displacement. Here we made use of atomic force microscopy to analyze the stiffness of breast cancer cellular models in which we modulated HMGA1 expression to investigate its role in regulating nuclear plasticity. Since histone H1 is the main modulator of chromatin structure and HMGA1 is a well-established histone H1 competitor, we correlated HMGA1 expression and cellular stiffness with histone H1 expression level, post-translational modifications, and nuclear distribution. Our results showed that HMGA1 expression level correlates with nuclear stiffness, is associated to histone H1 phosphorylation status, and alters both histone H1 chromatin distribution and expression. These data suggest that HMGA1 might promote chromatin relaxation through a histone H1-mediated mechanism strongly impacting on the invasiveness of cancer cells.

RPSAP52 lncRNA is overexpressed in pituitary tumors and promotes cell proliferation by acting as miRNA sponge for HMGA proteins.

Long non-coding RNAs (lncRNAs) are emerging as fundamental players in cancer biology. Indeed, they are deregulated in several neoplasias and have been associated with cancer progression, tumor recurrence, and resistance to treatment, thus representing potential biomarkers for cancer diagnosis, prognosis, and therapy. In this study, we aimed to identify lncRNAs associated with pituitary tumorigenesis. We have analyzed the lncRNA expression profile of a panel of gonadotroph pituitary adenomas in comparison with normal pituitaries. Then, we focused on RPSAP52, a novel lncRNA antisense for the HMGA2 gene, whose overexpression plays a critical role in the development of pituitary adenomas. We report that RPSAP52 expression is highly upregulated in gonadotroph and prolactin-secreting pituitary adenomas, where it correlates with that of HMGA2, compared with normal pituitary tissues. Conversely, its expression showed a variable behavior in somatotroph adenomas. We also demonstrate that RPSAP52 enhances HMGA2 protein expression in a ceRNA-dependent way acting as sponge for miR-15a, miR-15b, and miR-16, which have been already described to be able to target HMGA2. Interestingly, RPSAP52 also positively modulates HMGA1, the other member of the High-Mobility Group A family. Moreover, functional studies indicate that RPSAP52 promotes cell growth by enhancing the G1-S transition of the cell cycle. The results reported here reveal a novel mechanism, based on the overexpression of the lncRNA RPSAP52, which contributes to pituitary tumorigenesis, and propose this lncRNA as a novel player in the development of these tumors. KEY MESSAGES: RPSAP52 is overexpressed in pituitary adenomas. RPSAP52 increases HMGA protein levels. A ceRNA mechanism is proposed for the increased HMGA1/2 expression.

Bortezomib inhibited the progression of diffuse large B-cell lymphoma via targeting miR-198.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, which is an aggressive malignancy with high variance of clinical features and response to the treatment. The proteasome inhibitor bortezomib (BTZ) has been demonstrated to suppress the progression of DLBCL, however, the underlying molecular mechanisms by which BTZ regulates the growth of DLBCL cells remain largely unknown. Increasing evidence has suggested that microRNAs (miRNAs) are novel targets of anti-cancer drugs to modulate the progression of cancers. Here, we showed BTZ treatment significantly inhibited the proliferation of DLBCL CRL-2630 cells. Mechanistically, exposure of BTZ up-regulated the expression of miR-198 in DLBCL cells. Depletion of miR-198 significantly reversed the inhibitory effect of BTZ on the proliferation of CRL-2630 cells. To further characterize the involvement of miR-198 in BTZ-induced growth defects of CRL-2630 cells, the downstream targets of miR-198 were predicted with the bioinformatics tools. The results showed that miR-198 bound the 3'-untranslated region (UTR) of the high mobility group AT-hook 1 (HMGA1) and suppressed the expression of HMGA1 in DLBCL cells. Consistently, BTZ treatment decreased the level of HMAG1 and inhibited the migration of DLBCL cells. Our results provided the possible mechanism by which BTZ suppressed the growth of DLBCL cells.

Extracellular HMGA1 Promotes Tumor Invasion and Metastasis in Triple-Negative Breast Cancer.

PURPOSE: The study of the cancer secretome suggests that a fraction of the intracellular proteome could play unanticipated roles in the extracellular space during tumorigenesis. A project aimed at investigating the invasive secretome led us to study the alternative extracellular function of the nuclear protein high mobility group A1 (HMGA1) in breast cancer invasion and metastasis. EXPERIMENTAL DESIGN: Antibodies against HMGA1 were tested in signaling, adhesion, migration, invasion, and metastasis assays using breast cancer cell lines and xenograft models. Fluorescence microscopy was used to determine the subcellular localization of HMGA1 in cell lines, xenograft, and patient-derived xenograft models. A cohort of triple-negative breast cancer (TNBC) patients was used to study the correlation between subcellular localization of HMGA1 and the incidence of metastasis. RESULTS: Our data show that treatment of invasive cells with HMGA1-blocking antibodies in the extracellular space impairs their migration and invasion abilities. We also prove that extracellular HMGA1 (eHMGA1) becomes a ligand for the Advanced glycosylation end product-specific receptor (RAGE), inducing pERK signaling and increasing migration and invasion. Using the cytoplasmic localization of HMGA1 as a surrogate marker of secretion, we showed that eHMGA1 correlates with the incidence of metastasis in a cohort of TNBC patients. Furthermore, we show that HMGA1 is enriched in the cytoplasm of tumor cells at the invasive front of primary tumors and in metastatic lesions in xenograft models. CONCLUSIONS: Our results strongly suggest that eHMGA1 could become a novel drug target in metastatic TNBC and a biomarker predicting the onset of distant metastasis.

Histone Citrullination Represses MicroRNA Expression, Resulting in Increased Oncogene mRNAs in Somatolactotrope Cells.

Peptidylarginine deiminase (PAD) enzymes convert histone arginine residues into citrulline to modulate chromatin organization and gene expression. Although PADs are expressed in anterior pituitary gland cells, their functional role and expression in pituitary adenomas are unknown. To begin to address these issues, we first examined normal human pituitaries and pituitary adenomas and found that PAD2, PAD4, and citrullinated histones are highest in prolactinomas and somatoprolactinomas. In the somatoprolactinoma-derived GH3 cell line, PADs citrullinate histone H3, which is attenuated by a pan-PAD inhibitor. RNA sequencing and chromatin immunoprecipitation (ChIP) studies show that the expression of microRNAs (miRNAs) let-7c-2, 23b, and 29c is suppressed by histone citrullination. Our studies demonstrate that these miRNAs directly target the mRNA of the oncogenes encoding HMGA, insulin-like growth factor 1 (IGF-1), and N-MYC, which are highly implicated in human prolactinoma/somatoprolactinoma pathogenesis. Our results are the first to define a direct role for PAD-catalyzed histone citrullination in miRNA expression, which may underlie the etiology of prolactinoma and somatoprolactinoma tumors through regulation of oncogene expression.