The Effect of HMGB1 and HMGB2 on Transcriptional Regulation Differs in Neuroendocrine and Adenocarcinoma Models of Prostate Cancer.

Human high-mobility group-B (HMGB) proteins regulate gene expression in prostate cancer (PCa), a leading cause of oncological death in men. Their role in aggressive PCa cancers, which do not respond to hormonal treatment, was analyzed. The effects of HMGB1 and HMGB2 silencing upon the expression of genes previously related to PCa were studied in the PCa cell line PC-3 (selected as a small cell neuroendocrine carcinoma, SCNC, PCa model not responding to hormonal treatment). A total of 72% of genes analyzed, using pre-designed primer panels, were affected. HMGB1 behaved mostly as a repressor, but HMGB2 as an activator. Changes in SERPINE1, CDK1, ZWINT, and FN1 expression were validated using qRT-PCR after HMGB1 silencing or overexpression in PC-3 and LNCaP (selected as an adenocarcinoma model of PCa responding to hormonal treatment) cell lines. Similarly, the regulatory role of HMGB2 upon SERPINE1, ZWINT, FN1, IGFPB3, and TYMS expression was validated, finding differences between cell lines. The correlation between the expression of HMGB1, HMGB2, and their targets was analyzed in PCa patient samples and also in PCa subgroups, classified as neuroendocrine positive or negative, in public databases. These results allow a better understanding of the role of HMGB proteins in PCa and contribute to find specific biomarkers for aggressive PCa.

Identification of lncRNAs Deregulated in Epithelial Ovarian Cancer Based on a Gene Expression Profiling Meta-Analysis.

Epithelial ovarian cancer (EOC) is one of the deadliest gynecological cancers worldwide, mainly because of its initially asymptomatic nature and consequently late diagnosis. Long non-coding RNAs (lncRNA) are non-coding transcripts of more than 200 nucleotides, whose deregulation is involved in pathologies such as EOC, and are therefore envisaged as future biomarkers. We present a meta-analysis of available gene expression profiling (microarray and RNA sequencing) studies from EOC patients to identify lncRNA genes with diagnostic and prognostic value. In this meta-analysis, we include 46 independent cohorts, along with available expression profiling data from EOC cell lines. Differential expression analyses were conducted to identify those lncRNAs that are deregulated in (i) EOC versus healthy ovary tissue, (ii) unfavorable versus more favorable prognosis, (iii) metastatic versus primary tumors, (iv) chemoresistant versus chemosensitive EOC, and (v) correlation to specific histological subtypes of EOC. From the results of this meta-analysis, we established a panel of lncRNAs that are highly correlated with EOC. The panel includes several lncRNAs that are already known and even functionally characterized in EOC, but also lncRNAs that have not been previously correlated with this cancer, and which are discussed in relation to their putative role in EOC and their potential use as clinically relevant tools.

The HMGB protein Ixr1 interacts with Ssn8 and Tdh3 involved in transcriptional regulation.

Ixr1 is a Saccharomyces cerevisiae transcriptional factor that extensively regulates the response to hypoxia and controls other important cellular functions and DNA repair. During aerobic growth, the Ixr1 repressor function is predominant on regulated promoters of hypoxic genes, although activator effects are also observed on other genes. During hypoxia, Ixr1 expression increases and the number of genes activated by Ixr1 also increase. In this work we demonstrate that the NH2-terminal region of Ixr1 is involved in transcriptional activation. We also present the first analysis about Ixr1 interactions with three factors that have been previously identified as important players in the yeast hypoxic response, Cyc8, Tup1 and Ssn8; results demonstrate that only Ssn8 binds to Ixr1. We have also looked for other Ixr1-binding proteins associated with transcriptional regulation, by co-purification and mass spectrometry identification. Tdh3, a protein involved in transcriptional silencing, is among the new identified Ixr1-binding proteins. Differential phosphorylation of Ixr1 is found when comparing aerobic and hypoxic yeast growth. Implication of these results in transcriptional regulation mediated by Ixr1 is discussed.

Promoter-Terminator Gene Loops Affect Alternative 3'-End Processing in Yeast.

Many eukaryotic genes undergo alternative 3'-end poly(A)-site selection producing transcript isoforms with 3'-UTRs of different lengths and post-transcriptional fates. Gene loops are dynamic structures that juxtapose the 3'-ends of genes with their promoters. Several functions have been attributed to looping, including memory of recent transcriptional activity and polarity of transcription initiation. In this study, we investigated the relationship between gene loops and alternative poly(A)-site. Using the KlCYC1 gene of the yeast Kluyveromyces lactis, which includes a single promoter and two poly(A) sites separated by 394 nucleotides, we demonstrate in two yeast species the formation of alternative gene loops (L1 and L2) that juxtapose the KlCYC1 promoter with either proximal or distal 3'-end processing sites, resulting in the synthesis of short and long forms of KlCYC1 mRNA. Furthermore, synthesis of short and long mRNAs and formation of the L1 and L2 loops are growth phase-dependent. Chromatin immunoprecipitation experiments revealed that the Ssu72 RNA polymerase II carboxyl-terminal domain phosphatase, a critical determinant of looping, peaks in early log phase at the proximal poly(A) site, but as growth phase advances, it extends to the distal site. These results define a cause-and-effect relationship between gene loops and alternative poly(A) site selection that responds to different physiological signals manifested by RNA polymerase II carboxyl-terminal domain phosphorylation status.

Transcriptome analysis of the thermotolerant yeast Kluyveromyces marxianus CCT 7735 under ethanol stress.

The thermotolerant yeast Kluyveromyces marxianus displays a potential to be used for ethanol production from both whey and lignocellulosic biomass at elevated temperatures, which is highly alluring to reduce the cost of the bioprocess. Nevertheless, contrary to Saccharomyces cerevisiae, K. marxianus cannot tolerate high ethanol concentrations. We report the transcriptional profile alterations in K. marxianus under ethanol stress in order to gain insights about mechanisms involved with ethanol response. Time-dependent changes have been characterized under the exposure of 6% ethanol and compared with the unstressed cells prior to the ethanol addition. Our results reveal that the metabolic flow through the central metabolic pathways is impaired under the applied ethanol stress. Consistent with these results, we also observe that genes involved with ribosome biogenesis are downregulated and gene-encoding heat shock proteins are upregulated. Remarkably, the expression of some gene-encoding enzymes related to unsaturated fatty acid and ergosterol biosynthesis decreases upon ethanol exposure, and free fatty acid and ergosterol measurements demonstrate that their content in K. marxianus does not change under this stress. These results are in contrast to the increase previously reported with S. cerevisiae subjected to ethanol stress and suggest that the restructuration of K. marxianus membrane composition differs in the two yeasts which gives important clues to understand the low ethanol tolerance of K. marxianus compared to S. cerevisiae.

KlGcr1 controls glucose-6-phosphate dehydrogenase activity and responses to H2O2, cadmium and arsenate in Kluyveromyces lactis.

It has been previously reported that Gcr1 differentially controls growth and sugar utilization in Saccharomyces cerevisiae and Kluyveromyces lactis, although the regulatory mechanisms causing activation of glycolytic genes are conserved (Neil et al., 2004). We have found that KlGCR1 deletion diminishes glucose consumption and ethanol production, but increases resistance to oxidative stress caused by H2O2, cadmium and arsenate, glucose 6P dehydrogenase activity, and the NADPH/NADP(+) and GSH/GSSG ratios in K. lactis. The gene KlZWF1 that encodes for glucose 6P dehydrogenase, the first enzyme in the pentose phosphate pathway, is transcriptionally regulated by KlGcr1. The high resistance to oxidative stress observed in the ΔKlgcr1 mutant strain, could be explained as a consequence of an increased flux of glucose through the pentose phosphate pathway. Since mitochondrial respiration decreases in the ΔKlgcr1 mutant (García-Leiro et al., 2010), the reoxidation of the NADPH, produced through the pentose phosphate pathway, has to be achieved by the reduction of other molecules implied in the defense against oxidative stress, like GSSG. The higher GSH/GSSG ratio in the mutant would explain its phenotype of increased resistance to oxidative stress.

Thanksgiving to Yeast, the HMGB Proteins History from Yeast to Cancer.

Yeasts have been a part of human life since ancient times in the fermentation of many natural products used for food. In addition, in the 20th century, they became powerful tools to elucidate the functions of eukaryotic cells as soon as the techniques of molecular biology developed. Our molecular understandings of metabolism, cellular transport, DNA repair, gene expression and regulation, and the cell division cycle have all been obtained through biochemistry and genetic analysis using different yeasts. In this review, we summarize the role that yeasts have had in biological discoveries, the use of yeasts as biological tools, as well as past and on-going research projects on HMGB proteins along the way from yeast to cancer.

Ixr1p and the control of the Saccharomyces cerevisiae hypoxic response.

In Saccharomyces cerevisiae, adaptation to hypoxia/anaerobiosis requires the transcriptional induction or derepression of multiple genes organized in regulons controlled by specific transcriptional regulators. Ixr1p is a transcriptional regulatory factor that causes aerobic repression of several hypoxic genes (COX5B, TIR1, and HEM13) and also the activation of HEM13 during hypoxic growth. Analysis of the transcriptome of the wild-type strain BY4741 and its isogenic derivative Δixr1, grown in aerobic and hypoxic conditions, reveals differential regulation of genes related not only to the hypoxic and oxidative stress responses but also to the re-adaptation of catabolic and anabolic fluxes in response to oxygen limitation. The function of Ixr1p in the transcriptional regulation of genes from the sulfate assimilation pathway and other pathways producing α-keto acids is of biotechnological importance for industries based on yeast-derived fermentation products.

The yeast hypoxic responses, resources for new biotechnological opportunities.

Recent advances in the knowledge of molecular mechanisms that control the adaptation to low oxygen levels in yeast and their biotechnological applications, including bioproduct synthesis, such as ethanol, glutathione or recombinant proteins, as well as pathogenic virulence, are reviewed. Possible pathways and target genes, which might be of particular interest for the improvement of biotechnological applications, are evaluated.

HMG Proteins from Molecules to Disease.

High Mobility Group (HMG) proteins are today the focus of interest due to their participation in human degenerative diseases and inflammatory responses [...].

Transcriptional repression by Kluyveromyces lactis Tup1 in Saccharomyces cerevisiae.

The general repression complex, constituted by the yeast Tup1 and Ssn6 factors, is a conserved global regulator of transcription in eukaryotes. In the yeast Saccharomyces cerevisiae, it is an important repressor of hypoxic genes, such as ANB1, under aerobic conditions and deletion of the TUP1 gene causes a flocculation phenotype. The KlTUP1 gene from the yeast Kluyveromyces lactis encodes for a protein with 83% similarity to Tup1 in S. cerevisiae. Despite the general domain conservation, the database searches showed the absence of a characteristic Tup1 glutamine-rich domain (Q1 at positions 96-116). Instead, there was a non-conserved sequence lacking the α-helix structure in this region. The ability to act as a transcriptional repressor was tested by expressing the KlTUP1 gene, in both high- and low-copy vectors, in an S. cerevisiae tup1 mutant strain. Repression effects were studied using the aerobic repressible reporter ANB1-lacZ and the effect on flocculation. In both regulatory systems, low levels of KlTup1 caused moderate (~30%) repression, but when the number of KlTup1 copies was increased, only the ANB1 reporter raised the repression levels of S. cerevisiae Tup1. These results show the capability of KlTup1 to act as a repressor in S. cerevisiae. The lower repression reached in S. cerevisiae is discussed in terms of structural differences.

A stress response related to the carbon source and the absence of KlHAP2 in Kluyveromyces lactis.

The Kluyveromyces lactis HIS4 gene (KlHIS4) is transcriptionally regulated by the carbon source. The promoter region encompassing positions -238 to -139 is responsible for this regulation according to lacZ reporter assays. Electrophoretic Mobility Shift Assay (EMSA) experiments on KlHIS4 promoter (positions -218 to -213, Fragment 6, F6) show a specific gel-shift band, CS1, whose intensity is carbon-source dependent in K. lactis hap2 (klhap2) knock-out strains. The klhap3 mutation is not able to cause this effect by itself, but the combination of klhap2 and klhap3 mutations has an enhanced effect on CS1 band formation. Introducing a heat shock element (HSE) at the sequence in the F6 fragment (mutated F6, F6*) increases the binding activity in the klhap2 mutant. KlHIS4 mRNA levels in the klhap2 or the double Klhap2/3p mutant do not correlate with the increase in CS1 binding activity, indicating that the factor causing CS1 is acting and only detectable in vitro. EMSA experiments with K. lactis wild-type cells under temperature stress conditions show a band enhancement (Ts1), similar in size to CS1. Cross-competition experiments between F6 and F6* show that F6* competes more efficiently than F6 for both CS1 and Ts1 formation, indicating the involvement of the HSE in the formation of the specific gel-shift bands. Moreover, the similar gel-shift patterns suggest that both bands are caused by the same heat shock-like factor under different stress conditions. Therefore, the enhancement of the CS1 band signal in the klhap2 (and klhap2/3) mutants is due to the increase in heat shock-like factors in the protein extracts from these mutant cells grown in a non-fermentable carbon source. This Klhap2-dependent stress effect was not previously described in K. lactis.

The HMGB Protein KlIxr1, a DNA Binding Regulator of Kluyveromyces lactis Gene Expression Involved in Oxidative Metabolism, Growth, and dNTP Synthesis.

In the traditional fermentative model yeast Saccharomyces cerevisiae, ScIxr1 is an HMGB (High Mobility Group box B) protein that has been considered as an important regulator of gene transcription in response to external changes like oxygen, carbon source, or nutrient availability. Kluyveromyces lactis is also a useful eukaryotic model, more similar to many human cells due to its respiratory metabolism. We cloned and functionally characterized by different methodologies KlIXR1, which encodes a protein with only 34.4% amino acid sequence similarity to ScIxr1. Our data indicate that both proteins share common functions, including their involvement in the response to hypoxia or oxidative stress induced by hydrogen peroxide or metal treatments, as well as in the control of key regulators for maintenance of the dNTP (deoxyribonucleotide triphosphate) pool and ribosome synthesis. KlIxr1 is able to bind specific regulatory DNA sequences in the promoter of its target genes, which are well conserved between S. cerevisiae and K. lactis. Oppositely, we found important differences between ScIrx1 and KlIxr1 affecting cellular responses to cisplatin or cycloheximide in these yeasts, which could be dependent on specific and non-conserved domains present in these two proteins.

HMGB1 Protein Interactions in Prostate and Ovary Cancer Models Reveal Links to RNA Processing and Ribosome Biogenesis through NuRD, THOC and Septin Complexes.

This study reports the HMGB1 interactomes in prostate and ovary cancer cells lines. Affinity purification coupled to mass spectrometry confirmed that the HMGB1 nuclear interactome is involved in HMGB1 known functions such as maintenance of chromatin stability and regulation of transcription, and also in not as yet reported processes such as mRNA and rRNA processing. We have identified an interaction between HMGB1 and the NuRD complex and validated this by yeast-two-hybrid, confirming that the RBBP7 subunit directly interacts with HMGB1. In addition, we describe for the first time an interaction between two HMGB1 interacting complexes, the septin and THOC complexes, as well as an interaction of these two complexes with Rab11. Analysis of Pan-Cancer Atlas public data indicated that several genes encoding HMGB1-interacting proteins identified in this study are dysregulated in tumours from patients diagnosed with ovary and prostate carcinomas. In PC-3 cells, silencing of HMGB1 leads to downregulation of the expression of key regulators of ribosome biogenesis and RNA processing, namely BOP1, RSS1, UBF1, KRR1 and LYAR. Upregulation of these genes in prostate adenocarcinomas is correlated with worse prognosis, reinforcing their functional significance in cancer progression.

Ixr1p regulates oxygen-dependent HEM13 transcription.

In Saccharomyces cerevisiae, HEM13 encodes the enzyme coproporphyrinogen III oxidase, which catalyzes the rate-limiting step in heme biosynthesis. HEM13 is a regulated hypoxic gene repressed by Rox1p and Mot3p under aerobic conditions. In this study, we further investigate the hypoxic expression of HEM13, focusing on the promoter regions that are functionally important during hypoxia and on the effect of deleting the transcriptional regulators Sut1p, Sut2p, Upc2p, Ecm22p and Ixr1p. Ixr1p is necessary for the high expression of HEM13 under hypoxic conditions and its function is exerted in vivo through the HEM13 promoter region extending from -577 to -419. Ixr1p binds in vivo to the HEM13 promoter both under aerobic and under hypoxic conditions. Purified Ixr1p binds in vitro to two sequences extending from -534 to -509 and from -497 to -450, respectively. These DNA regions compete for Ixr1p binding and the consensus KTTSAAYKGTTYASA is important for the regulatory protein to interact. These results suggest that the regulation of HEM13 expression is dependent on two proteins with high mobility group (HMG) domains: Rox1p and Ixr1p. Their interactions with the HEM13 promoter might change in the transition from aerobiosis to hypoxia.

Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 by oxygen levels: a model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1 and IXR1 cross-regulation.

Ixr1p from Saccharomyces cerevisiae has been previously studied because it binds to DNA containing intrastrand cross-links formed by the anticancer drug cisplatin. Ixr1p is also a transcriptional regulator of anaerobic/hypoxic genes, such as SRP1/TIR1, which encodes a stress-response cell wall manoprotein, and COX5B, which encodes the Vb subunit of the mitochondrial complex cytochrome c oxidase. However, factors controlling IXR1 expression remained unexplored. In the present study we show that IXR1 mRNA levels are controlled by oxygen availability and increase during hypoxia. In aerobiosis, low levels of IXR1 expression are maintained by Rox1p repression through the general co-repressor complex Tup1-Ssn6. Ixr1p itself is necessary for full IXR1 expression under hypoxic conditions. Deletion analyses have identified the region in the IXR1 promoter responsible for this positive auto-control (nucleotides -557 to -376). EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays show that Ixr1p binds to the IXR1 promoter both in vitro and in vivo. Ixr1p is also required for hypoxic repression of ROX1 and binds to its promoter. UPC2 deletion has opposite effects on IXR1 and ROX1 transcription during hypoxia. Ixr1p is also necessary for resistance to oxidative stress generated by H2O2. IXR1 expression is moderately activated by H2O2 and this induction is Yap1p-dependent. A model of IXR1 regulation as a relay for sensing different signals related to change in oxygen availability is proposed. In this model, transcriptional adaptation from aerobiosis to hypoxia depends on ROX1 and IXR1 cross-regulation.

The HMGB1-2 Ovarian Cancer Interactome. The Role of HMGB Proteins and Their Interacting Partners MIEN1 and NOP53 in Ovary Cancer and Drug-Response.

High mobility group box B (HMGB) proteins are overexpressed in different types of cancers such as epithelial ovarian cancers (EOC). We have determined the first interactome of HMGB1 and HMGB2 in epithelial ovarian cancer (the EOC-HMGB interactome). Libraries from the SKOV-3 cell line and a primary transitional cell carcinoma (TCC) ovarian tumor were tested by the Yeast Two Hybrid (Y2H) approach. The interactome reveals proteins that are related to cancer hallmarks and their expression is altered in EOC. Moreover, some of these proteins have been associated to survival and prognosis of patients. The interaction of MIEN1 and NOP53 with HMGB2 has been validated by co-immunoprecipitation in SKOV-3 and PEO1 cell lines. SKOV-3 cells were treated with different anti-tumoral drugs to evaluate changes in HMGB1, HMGB2, MIEN1 and NOP53 gene expression. Results show that combined treatment of paclitaxel and carboplatin induces a stronger down-regulation of these genes in comparison to individual treatments. Individual treatment with paclitaxel or olaparib up-regulates NOP53, which is expressed at lower levels in EOC than in non-cancerous cells. On the other hand, bevacizumab diminishes the expression of HMGB2 and NOP53. This study also shows that silencing of these genes affects cell-viability after drug exposure. HMGB1 silencing causes loss of response to paclitaxel, whereas silencing of HMGB2 slightly increases sensitivity to olaparib. Silencing of either HMGB1 or HMGB2 increases sensitivity to carboplatin. Lastly, a moderate loss of response to bevacizumab is observed when NOP53 is silenced.

The Challenges and Opportunities of LncRNAs in Ovarian Cancer Research and Clinical Use.

Ovarian cancer is one of the most lethal gynecological malignancies worldwide because it tends to be detected late, when the disease has already spread, and prognosis is poor. In this review we aim to highlight the importance of long non-coding RNAs (lncRNAs) in diagnosis, prognosis and treatment choice, to make progress towards increasingly personalized medicine in this malignancy. We review the effects of lncRNAs associated with ovarian cancer in the context of cancer hallmarks. We also discuss the molecular mechanisms by which lncRNAs become involved in cellular physiology; the onset, development and progression of ovarian cancer; and lncRNAs' regulatory mechanisms at the transcriptional, post-transcriptional and post-translational stages of gene expression. Finally, we compile a series of online resources useful for the study of lncRNAs, especially in the context of ovarian cancer. Future work required in the field is also discussed along with some concluding remarks.

Differential Characteristics of HMGB2 Versus HMGB1 and their Perspectives in Ovary and Prostate Cancer.

We have summarized common and differential functions of HMGB1 and HMGB2 proteins with reference to pathological processes, with a special focus on cancer. Currently, several "omic" approaches help us compare the relative expression of these 2 proteins in healthy and cancerous human specimens, as well as in a wide range of cancer-derived cell lines, or in fetal versus adult cells. Molecules that interfere with HMGB1 functions, though through different mechanisms, have been extensively tested as therapeutic agents in animal models in recent years, and their effects are summarized. The review concludes with a discussion on the perspectives of HMGB molecules as targets in prostate and ovarian cancers.