HIV-1 Tat induces DNMT over-expression through microRNA dysregulation in HIV-related non Hodgkin lymphomas
© Luzzi et al.; licensee BioMed Central. 2014
Received: 25 August 2014
Accepted: 14 November 2014
Published: 9 December 2014
A close association between HIV infection and the development of cancer exists. Although the advent of highly active antiretroviral therapy has changed the epidemiology of AIDS-associated malignancies, a better understanding on how HIV can induce malignant transformation will help the development of novel therapeutic agents.
HIV has been reported to induce the expression of DNMT1 in vitro, but still no information is available about the mechanisms regulating DNMT expression in HIV-related B-cell lymphomas.
In this paper, we investigated the expression of DNMT family members (DNMT1, DNMT3a/b) in primary cases of aggressive B-cell lymphomas of HIV-positive subjects.
Our results confirmed the activation of DNMT1 by HIV in vivo, and reported for the first time a marked up-regulation of DNMT3a and DNMT3b in HIV-positive aggressive B-cell lymphomas. DNMT up-regulation in HIV-positive tumors correlated with down-regulation of specific microRNAs, as the miR29 family, the miR148-152 cluster, known to regulate their expression. Literature reports the activation of DNMTs by the human polyomavirus BKV large T-antigen and adenovirus E1a, through the pRb/E2F pathway. We have previously demonstrated that the HIV Tat protein is able to bind to the pocket proteins and to inactivate their oncosuppressive properties, resulting in uncontrolled cell proliferation. Therefore, we focused on the role of Tat, due to its capability to be released from infected cells and to dysregulate uninfected ones, using an in vitro model in which Tat was ectopically expressed in B-cells.
Our findings demonstrated that the ectopic expression of Tat was per se sufficient to determine DNMT up-regulation, based on microRNA down-regulation, and that this results in aberrant hypermethylation of target genes and microRNAs.
These results point at a direct role for Tat in participating in uninfected B-cell lymphomagenesis, through dysregulation of the epigenetical control of gene expression.
A close association between Human Immunodeficiency Virus (HIV) infection and the development of a number of cancers, NHL being the second most common, has been described. Interestingly, AIDS-associated lymphomas are of B-lymphoid origin in at least 95% of all cases described despite the fact that HIV infects T-lymphocytes , raising the question whether HIV may have a direct role in B-cell lymphomagenesis. To date, there are no clear answers to explain how HIV leads to transformation, even though several events have been proposed as co-factors in HIV-related tumorigenesis. The frequencies of different subpopulations of B-cells have been reported altered in the presence of HIV Reviewed in . These changes include increased frequency of activated and terminally differentiated B-cells expressing low levels of CD21 that have been associated with ongoing viral replication [3, 4], a decreased frequency of memory B-cells that is not reversed by antiretroviral therapy , and an increased frequency of immature/transitional B-cells that has been associated with CD4+ T-cell lymphopenia [6–8]. Although there is no indication of a direct role for the virus in the B-cell transformation, lymphadenopathy, polyclonal B-cell proliferation, and even lymphoma may precede overt compromise of T-cell immunity [9, 10]. In addition, changes in the microenvironment of the host cells have been recorded following HIV infection , as well as chronic immune activation and dysfunctional cytokine production that have been described throughout all stages of HIV-1 infection .
In addition to these indirect effects, HIV may directly contribute to B-cell transformation through its encoded proteins and/or viral microRNAs (miRNAs), using which it can disturb gene and miRNA expression in host cells . It is noteworthy that most transformed B-cells do not contain the virus, therefore some other mechanisms and/or viral factors may contribute to transformation. In particular, several findings support an oncogenic role of the HIV-1 Tat protein, which is essential for viral gene expression and virus production [14–16]. A soluble, biologically active form of Tat is released by HIV-infected cells, taken up and translocated to the nucleus by neighbouring uninfected ones [17–19], and may directly contribute to B-cell abnormalities in HIV-positive patients . We have previously demonstrated that B-cell lymphomas of HIV-infected individuals may be positively stained by an anti-Tat antibody . Therefore it is reasonable to hypothesize that the endocytosed Tat, released from infected cells, may then exert its pleiotropic activities in uninfected B-cells. Tat has been reported to modulate the expression of several cellular genes, including cytokines and their receptors [22–24]. In particular, the ability of Tat to increase the expression of interleukins-6 (IL-6) and 10 (IL-10) [25, 26], which in turn promote B-cell stimulation, and the evidence that about 30% of Tat-transgenic mice develop B-cell lymphomas , suggest that Tat might play a role in the pathogenesis of HIV-related B-lymphomas. In particular, IL-6 has been reported to induce the over-expression of the DNA Methyltransferase 1 (DNMT1), which has a key role in the maintenance of DNA methylation, and epigenetically regulate the expression of several genes, in liver cancer through miRNA dysregulation, which correlates with increased genomic methylation . Interestingly, HIV has been reported to induce the expression of DNMT1 [29, 30]in vitro, though there is no evidence that this can be exerted through IL-6 in HIV-positive individuals, although serum IL-6 is significantly elevated in HIV+ subjects who develop aggressive B-cell lymphomas . In addition, induction of DNMT aberrant activity has been reported by several human viruses through the pRb/E2F pathway . In particular, this occurs through the interaction of viral products with the RB proteins and their consequent inactivation [33–37]. Noteworthy, we have previously reported the physical interaction of Tat with the pocket proteins, which results in their inactivation and inhibition of their growth regulatory properties [21, 38]. This suggests that Tat may contribute to DNMT aberrant expression in HIV-positive subjects.
In this paper we have investigated the possible mechanisms used by HIV to induce DNMT over-expression. In particular, we have analyzed whether DNMT induction by HIV could depend on specific miRNA dysregulation, as reported in liver cancer [28, 39]. Our results show that DNMT1, DNMT3a/b are up-regulated in B-cell lymphomas, and that this relies on down-regulation of specific miRNAs. To assess the possible contribution of Tat, we used an in vitro model, in which Tat was ectopically expressed in uninfected B-cells. The ectopic expression of Tat resulted in the up-regulation of DNMT1, DNMT3a/b based on down-regulation of specific miRNAs, in accordance to what we observed in HIV-positive primary tumors.
DNMT over-expression may result in altered methylation pattern of genes and/or microRNAs, therefore we investigated whether it may affect the expression of genes frequently reported to be inactivated by hypermethylation, as INK4/p16, TP53 and RB1. In addition, we tested whether down-regulation of DNMT-regulating miRNAs detected in our cell model was possibly dependent on hypermethylation as well, in a feedback-loop mechanism fashion. Here we show that the ectopic expression of Tat determines an altered methylation pattern of INK4/p16 and of specific miRNAs, this finding being also confirmed in HIV-positive tumors.
These results point out at the possible role for Tat in participating in B-cell lymphomagenesis in uninfected cells, through dysregulation of the host cell miRNA machinery and of the epigenetic control of gene expression, and provide novel information to the molecular mechanisms of B-cell lymphomagenesis in HIV-infected individuals.
The Institutional Review Board of the University of Siena (Italy) and the Ethics and Research Committee of the University of Nairobi (Kenya) gave ethics approval for this study. Informed written consent was obtained in all cases.
Case selection and immunophenotype
List of the antibodies used for immunohistochemistry
PCR for detection of HIV infection
All of the HIV-positive lymphomas were tested for HIV genome presence. A fragment of the HIV-1 DNA was amplified by nested PCR using the lentivirus universal primer pair UNIPOL1/2 as outer primers (25 cycles) and the degenerate primers UNIPOL3 (50-GAAACAGGAMRRGAGACAGC-30) and UNIPOL4 (50-TTCATDGMTTCCACTACTCCTTG-30) as inner primers (30 cycles) . This nested primer set, when used at low-stringency annealing, specifically amplifies all HIV-1 and HIV-2 pol sequences known to date. PCR products were visualized on agarose gels and the specificity of the products was confirmed by direct sequencing.
miRNAs predicted to regulate the expression of DNMT1 (hsa-miR-130a, hsa-miR-130b, hsa-miR-148a, hsa-miR-148b, hsa-miR-152, hsa-miR-301) and DNMT3a/b (hsa-miR-29a, hsa-miR-29b and hsa-miR-29c, hsa-miR-148a, hsa-miR-148b) were identified by computational analysis, using web-available resources (Mirnaviewer, PicTar, Tarbase  and miRBase ; mirnaviewer is available at http://cbio.mskcc.org/mirnaviewer; PicTar is a project of the Rajewsky lab at NYU's Center for Comparative Functional Genomics and the Max Delbruck Centrum, Berlin). Among the many available by bioinformatics predictions, these specific miRNAs were selected for this study as regulation of DNMTs by these miRNAs through direct mRNA binding has been previously proved [45, 46].
Extraction of miRNAs from FFPE sections of primary tumors and reactive lymph nodes was performed using the miRNA easy FFPE kit (Qiagen, Carlsbad, CA), following manufacturer’s instructions. Quality and purity of RNA were assessed by spectrophotometric read using Nanodrop (Thermo Scientific, Wilmington, DE) and by Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Analysis of miRNA expression
MiRNA expression was analyzed by RT-qPCR as previously described . For each sample, 10 ng of total RNA were reverse transcribed. Real-time PCR was performed using Taqman probes specific for each miRNA (hsa-miR-130a, hsa-miR-130b, hsa-miR-148a, hsa-miR-148b, hsa-miR-152, hsa-miR-301, hsa-miR-29a, hsa-miR-29b and hsa-miR-29c), and for RNU43, used as an endogenous control (Applied Biosystems, Applera, Italy). Amount and quality of RNA were evaluated measuring the OD at 260 nm, the 260/230 and the 260/280 ratios by Nanodrop (Celbio, Italy).
Gene expression analysis
Primers used for qPCR
CYCLIN A- FORWARD
5’-AGG CTT CAA AGT ACC TGT GTG-3’
CYCLIN A- REVERSE
5’-TTG ATC CCA CGT GCA GAA G-3’
5’-TAT TGA TGA GCG CAC AAG AGA GC-3'
5’-GGG TGT TCC AGG GTA ACA TTG AG-3'
5’-GGC AAG TTC TCC GAG GTC TCTG-3'
5’-TGG TAC ATG GCT TTT CGA TAG GA-3'
5’-CAC CAA TAC CTC ACA TTC CTC-3'
5’-TTC TCA GAA GTC CCG AAT G-3’
5’-CCA TCC TCA CCA TCA TCA C-3'
5’-GGC AGT GCT CGC TTA GTG G-3'
5'-GGA AGG TCC CTC AGA CAT C-3'
5’-GCA GTT GTG GCC CTG TAG-3'
The recombinant Tat HIV-1 IIIB (aa 1–86) from Dr J Raina was obtained through the EU Programme EVA/MRC Centralised Facility for AIDS Reagents, NIBSC, UK (Grant numbers QLK2-CT-1999-00609 and GP828102). The stock solution was diluted in saline citrate buffer as recommended, and aliquots were stored at -80°C until use. The concentration of endotoxin was below 0.01 endotoxin unit (EU)/mg of protein. Extracellular Tat (50 ng/ml) was added to the medium culture of cells for 48 h. Cells grown in the absence of Tat were used as a control.
Ectopic expression of Tat in vitro
Tat ectopic expression was either obtained through exposure to recombinant Tat or through transient and stable transfections, by nucleofection. A Burkitt lymphoma-derived EBV-negative cell line (Ramos) was used to perform the in vitro experiments. Briefly, cells were cultured in RPMI supplemented with 10% FBS, 1% L-glutamine, penicillin/streptomycin, with 5% CO2, at 37°C. The recombinant Tat was used as previously described . Cells grown in the absence of Tat were used as a negative control. Transient and stable transfections were performed by nucleofection, using an Amaxa apparatus, program T16 and solution T (Amaxa, Cologne, Germany). A transfection efficiency of 45% was obtained, as assessed by FACS analysis for a GFP reporter. Cells (2x106) were transfected with 10 μg of pCDNA3-Tat , using the empty vector as negative control. In addition, a stable Tat-transfected Ramos cell line was obtained by antibiotic selection with G-418, at the concentration of 2 mg/ml.
The effect of Tat on cell proliferation was assessed as previously described . Briefly, cell counts were established by Tripan blue staining and the possible effect of Tat on the cell cycle was monitored by analysis of Cyclin A expression, used as a specific S-phase marker, by RT-qPCR, as described above. In addition, proliferation was also monitored following transfections with miRNA mimics and inhibitors (see below). Statistical significance was assessed by the analysis of variance (ANOVA) test.
To assess the regulation of the target genes by the predicted miRNAs in our cell model, modulation of the endogenous miRNAs was obtained by synthetic miRNAs and inhibitors (Dharmacon, Celbio, Milan, Italy), through nucleofection, followed by detection of the expression of the genes of interest. Briefly, cells were split the day before nucleofection and 5x106 cells were transfected with different concentrations of either the miRNA mimic or inhibitor (10 nM, 50 nM or 100 nM), to assess the best dose–response concentration. Negative control of mimics and inhibitors (NC, NCI, respectively) were used at the 10 nM concentration (Dharmacon, Euroclone, Milan, Italy). As the selected miRNAs regulating DNMT1 map in clusters, we used one mimic/inhibitor for each cluster. In particular, mimics and inhibitors of hsa-miR130a, hsa-miR152 and hsa-miR29 were used to modulate the endogenous expression of DNMT1 and DNMT3a/b, respectively (all from Dharmacon, Euroclone, Milan, Italy). To analyze the downstream DNMT modulation, mimics and inhibitors were used at the concentration which gave the best effect. RNA was extracted 24 hours after nucleofection and both gene expression for DNMTs and miRNA expression were checked by Real-Time RT-PCR, as previously described.
Cells pellets were lysed on ice for in EBC buffer (50 nM Tris–HCl pH 8.0, 130 mM NaCl, 1% Triton X-100, 0.1% SDS) supplemented with protease inhibitor cocktail (Sigma, Milan-Italy). Cell lysates were separated by 10% SDS-PAGE gel followed by transfer to Hybond ECL nitrocellulose membrane (GE Healthcare, Milan, Italy). Western blotting was made using anti-DNMT1 (1:400, BD, NJ USA), anti-DNMT3a (1:250, Abcam, UK) and anti-actin (1:1000, BD, NJ USA). Secondary antibodies conjugated with HRP were used at a dilution of 1:5000 and the reaction was revealed using the ECL Western Blotting Kit (Promega, Milan-Italy) according to the manufacturer’s instructions.
DNA extraction and methylation assay
Primer sequences for MSP
INK4/p16 U FORWARD
5’-TTA TTA GAG GGT GGG GTG GAT TGT-3’
INK4/p16 U REVERSE
5’-CAA CCC CAA ACC ACA ACC ATA A-3’
INK4/p16 M FORWARD
5’-TTA TTA GAG GGT GGG GCG GAT CGC-3’
INK4/p16 M REVERSE
5’-GAC CCC GAA CCG CGA CCG TAA-3’
TP53 U FORWARD
5’-TTT TTT AGG TAG TTT TTG GTT TTG T-3’
TP53 U REVERSE
5’-ACC AAA CCT CTC AAA TTA CAA CAA T-3’
TP53 M FORWARD
5’-ATT TTT TTA GGT AGT TTT CGG TTT C-3’
TP53 M REVERSE
5’-GAA CCT CTC AAA TTA CGA CGA T-3’
Primer sequences for microRNA methylation analysis
hsa-miR148a FORWARD (BSP)
hsa-miR148a REVERSE (BSP)
hsa-miR152 FORWARD (BSP)
hsa-miR152 REVERSE (BSP)
hsa-miR148a U-FORWARD (MSP)
hsa-miR148a U-REVERSE (MSP)
hsa-miR148a M-FORWARD (MSP)
hsa-miR148a M-REVERSE (MSP)
Treatment with 5-aza-2-deoxycitidine
To test whether miRNA down-regulation in HIV-positive tumors was possibly due to hypermethylation following DNMT over-expression, Ramos cells (either transfected or not with a vector coding for Tat) were treated with 1 μM 5-aza-2-deoxycitidine as reported  and relative quantification of miRNAs was made by RT-qPCR two days after treatment, as previously described.
HIV induces the aberrant expression of DNMTs
DNMT expression is increased in Tat-transfected cells
DNMT-regulating miRNAs are dysregulated in Tat-transfected cell lines
Over-expression of DNMTs determines an increase of gene and microRNA methylation
Analysis of methylated CpG sites for hsa-miR-148a and hsa-miR-152 in HIV-positive vs. negative primary tumors
Total CpG islands
Total CpG islands
As DNMT-mediated silencing of INK4/p16 through methylation may affect cell proliferation, due to the inactivation of its growth arrest properties, we monitored whether modulation of DNMT-regulating miRNAs through mimics/inhibitors may eventually affect cell growth. Our results show that inhibition of endogenous DNMT-regulating miRNAs through antagomirs results in an increased cell proliferation. Inhibition of endogenous miRNAs may affect cell proliferation through the up-regulation of DNMTs and consequent silencing of INK4/p16, which results in the abolishment of cell growth arrest. Conversely, DNMT down-regulation through miRNA mimics determines an opposite effect on cell growth (Figure 8e-f).
Tumor viruses has been implicated in the etiology of many cancers including malignant mesotheliomas, non-Hodgkin's lymphoma and tumors of the bone, brain and urinary tract [51–53]. The most commonly explored role for viruses in cancer involves the expression of viral oncogenes, such as polyomavirus large T-antigens (TAg), adenovirus E1a and E1b, and papillomavirus E6 and E7 . These proteins share the ability to interact with and inactivate the pocket protein family (pRb, p107, p130) and/or the p53 tumor suppressor [55–58]. This results in the activation of the cellular DNA replication machinery needed to replicate the viral genome and promotes increased cellular proliferation, delayed differentiation, and often malignant transformation .
HIV infection is often associated with the onset of malignant lymphomas, 95% of which are of B-cell origin. Some of them, as Burkitt lymphoma, may arise in immunocompetent patients, even before the AIDS manifestation. Due to the high CD4+ cell number of these patients, it is reasonable to hypothesize that malignant transformation in these cases may not be a consequence of the immunodepression of infected individuals, suggesting that HIV itself may be involved in driving the transformation process. The virus encodes for many proteins and viral microRNAs, using which it may compete with cellular proteins/RNAs, thus disturbing the physiological regulation of the host cell. As most transformed B-cells do not contain the virus, some other mechanisms and/or viral factors may be involved. Among these, the most reliable candidate is the Tat protein, as it may function as a soluble effector, being released from infected cells and taken up by uninfected B-cells, in a biologically active form. Our previous studies demonstrated that HIV-positive B-cell lymphomas may be positively stained by an anti-Tat antibody  and again here we show positivity for Tat in our set of B-cell tumors. Therefore the endocytosed Tat may directly exert its biological functions in uninfected B-cells.
The ability of Tat to act directly on B-cells and differentially modulate the B-cell response of naïve/memory and germinal center (GC) B-cells has been previously reported . Tat-mediated induction of GC B-cell proliferation might therefore contribute to promote HIV-associated follicular hyperplasia, autoimmune disorders and B-cell malignancies. The effects of Tat are likely to impact on the early stages of the cell cycle, before the G1 to S phase transition, and on B-cell differentiation, rather than affecting isotype switching . In addition, Tat has been shown to bind to the pocket proteins, thus interfering with control of cell growth [21, 38, 50], which may eventually result in transformation.
However, while genetic perturbations have been shown to play key roles in viral transformation, epigenetic modifications such as DNA methylation may also play important roles during viral infection and transformation, as viral infection affects de novo methylation and transcription of cellular genes as well [60–62].
In this paper we have analyzed the expression of a particular class of proteins, the DNA Methyl Transferases, which epigenetically regulate gene expression, as HIV-1 has been reported to induce the expression of DNMT1 in vitro, which has a key role in the maintenance of DNA methylation, and epigenetically regulate the expression of several genes . Over-expression of DNMT1 correlates with increased genomic methylation  and has been associated with miRNA dysregulation in liver cancer, in which DNMT1 over-expression is induced by IL-6 over-production . Here, we show that HIV enhances the expression of DNMTs involved in both the basic and the de novo methylation. Such up-regulation relies on the down-regulation of specific miRNAs predicted to regulate DNMTs, in primary tumors of aggressive B-cell lymphomas, compared to HIV-negative tumors and normal tissues. The increased expression of DNMTs could result in an altered pattern of methylation of target genes/miRNAs in HIV-positive subjects. Noteworthy, reduction of the global methylation has been recently reported in HIV-positive subjects following HAART treatment , thus supporting the finding that HIV is able to increase global DNA methylation. As the HIV genome has not been detected in the tumor tissues of the aggressive B-cell lymphomas we analyzed, we hypothesized that Tat, released in a soluble form from infected cells, could contribute to malignant transformation of these cases. To test this hypothesis, we used an in vitro model obtained by ectopic expression of Tat in B-cells, either by cell transfections or exposure to recombinant Tat. In line with our in vivo results, we observed that the ectopic expression of Tat was able to induce overexpression of DNMTs based on down-regulation of DNMT-regulating miRNAs, pointing at a direct role for Tat in regulating DNMT expression.
Based on the growth capability acquired by Tat-transfected cells, which may depend on silencing of cell cycle progression inhibitory genes, we have then analyzed whether key cell cycle regulatory genes, as INK4/p16, TP53 and RB1, may be possibly silenced through methylation, thus leading to the loss of the G1/S control , in Tat-transfected cells. Our results demonstrate a down-regulated expression of INK4/p16 and TP53, in Tat-transfected cells, whereas no difference is observed for RB1. In addition, we show that the reduced expression of INK4/p16, following ectopic expression of Tat, was specifically due to hypermethylation, whereas no methylation was detected for TP53. 5-aza-2-deoxycitidine treatment restored the expression of down-regulated miRNAs, for which CpG islands have been described, thus suggesting that miRNA down-regulation Tat-transfected cell lines may depend on hypermethylation resulting from aberrant DNMT expression. The in vitro results were then confirmed in HIV-positive primary tumors. Interestingly, cell growth was affected by transfections of mimics/inhibitors, as inhibition of the endogenous miRNAs resulted in a higher proliferation rate. This may be due to the up-regulation of DNMTs and consequent INK4/p16 silencing through methylation, which removes the control on cell growth and speeds up cell proliferation.
Transcriptional inactivation of DNMTs has been recently reported to occur by Rb proteins and this effect was shown to be reversible by Rb-inactivating viral oncoproteins such as the T-antigen [32–36]. In particular, decreased DNMT expression has been linked to the activity of the RBL2 gene product . Notably, we have previously demonstrated that Tat is able to inactivate RBL2/p130 through a physical binding [21, 38]. This may represent an intriguing mechanism through which Tat up-regulates DNMT expression in B-cells of infected patients by inactivating RBL2 activity.
The Tat-dependent modulation of DNA Methyl Transferases provides an attractive mechanism through which it can restore and maintain methylation of critical genes in HIV-infected individuals.
This work has been supported by the “Progetto Salute Regione Toscana” grant.
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