Octavio A. Quiñones

Chr 19A/J modifies tumor resistance in a sex and parent-of-origin specific manner

Neurofibromatosis type 1 (NF1) is one of the most common human genetic diseases affecting the nervous system and predisposes individuals to cancer, including peripheral nerve sheath tumors (PNSTs) and astrocytomas. Modifiers in the genetic background affect the severity of the disease and we have previously mapped two modifier loci, Nstr1 and Nstr2, that influence resistance to PNSTs in the Nf1−/+;Trp53−/+cis mouse model of NF1. We report here the analysis of Nstr1 in isolation from other epistatic loci using a chromosome substitution strain, and further show that a modifier locus (or loci) on chromosome 19 influences resistance to both PNSTs and astrocytomas. This modifier locus interacts with sex, resulting in sex-specific modification of tumors. Allele variability on chromosome 19 affects both the timing and the penetrance of the growth of different tumor types associated with NF1, specifically PNSTs and astrocytoma. These results indicate that modifiers of cancer susceptibility interact and affect tumorigenesis under different genetic conditions and demonstrate the power of chromosome substitution strains to study genetic modifiers.

Effect of suberoylanilide hydroxamic acid (SAHA) administration on the residual virus pool in a model of combination antiretroviral therapy-mediated suppression in SIVmac239-infected indian rhesus macaques.

ABSTRACT Nonhuman primate models are needed for evaluations of proposed strategies targeting residual virus that persists in HIV-1-infected individuals receiving suppressive combination antiretroviral therapy (cART). However, relevant nonhuman primate (NHP) models of cART-mediated suppression have proven challenging to develop. We used a novel three-class, six-drug cART regimen to achieve durable 4.0- to 5.5-log reductions in plasma viremia levels and declines in cell-associated viral RNA and DNA in blood and tissues of simian immunodeficiency virus SIVmac239-infected Indian-origin rhesus macaques, then evaluated the impact of treatment with the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA; Vorinostat) on the residual virus pool. Ex vivo SAHA treatment of CD4+ T cells obtained from cART-suppressed animals increased histone acetylation and viral RNA levels in culture supernatants. cART-suppressed animals each received 84 total doses of oral SAHA. We observed SAHA dose-dependent increases in acetylated histones with evidence for sustained modulation as well as refractoriness following prolonged administration. In vivo virologic activity was demonstrated based on the ratio of viral RNA to viral DNA in peripheral blood mononuclear cells, a presumptive measure of viral transcription, which significantly increased in SAHA-treated animals. However, residual virus was readily detected at the end of treatment, suggesting that SAHA alone may be insufficient for viral eradication in the setting of suppressive cART. The effects observed were similar to emerging data for repeat-dose SAHA treatment of HIV-infected individuals on cART, demonstrating the feasibility, utility, and relevance of NHP models of cART-mediated suppression for in vivo assessments of AIDS virus functional cure/eradication approaches.

C/EBPγ Suppresses Senescence and Inflammatory Gene Expression by Heterodimerizing with C/EBPβ

ABSTRACT C/EBPβ is an important regulator of oncogene-induced senescence (OIS). Here, we show that C/EBPγ, a heterodimeric partner of C/EBPβ whose biological functions are not well understood, inhibits cellular senescence. Cebpg−/− mouse embryonic fibroblasts (MEFs) proliferated poorly, entered senescence prematurely, and expressed a proinflammatory gene signature, including elevated levels of senescence-associated secretory phenotype (SASP) genes whose induction by oncogenic stress requires C/EBPβ. The senescence-suppressing activity of C/EBPγ required its ability to heterodimerize with C/EBPβ. Covalently linked C/EBPβ homodimers (β∼β) inhibited the proliferation and tumorigenicity of RasV12-transformed NIH 3T3 cells, activated SASP gene expression, and recruited the CBP coactivator in a Ras-dependent manner, whereas γ∼β heterodimers lacked these capabilities and efficiently rescued proliferation of Cebpg−/− MEFs. C/EBPβ depletion partially restored growth of C/EBPγ-deficient cells, indicating that the increased levels of C/EBPβ homodimers in Cebpg−/− MEFs inhibit proliferation. The proliferative functions of C/EBPγ are not restricted to fibroblasts, as hematopoietic progenitors from Cebpg−/− bone marrow also displayed impaired growth. Furthermore, high CEBPG expression correlated with poorer clinical prognoses in several human cancers, and C/EBPγ depletion decreased proliferation and induced senescence in lung tumor cells. Our findings demonstrate that C/EBPγ neutralizes the cytostatic activity of C/EBPβ through heterodimerization, which prevents senescence and suppresses basal transcription of SASP genes.

Serum S100A6 Concentration Predicts Peritoneal Tumor Burden in Mice with Epithelial Ovarian Cancer and Is Associated with Advanced Stage in Patients

Background Ovarian cancer is the 5th leading cause of cancer related deaths in women. Five-year survival rates for early stage disease are greater than 94%, however most women are diagnosed in advanced stage with 5 year survival less than 28%. Improved means for early detection and reliable patient monitoring are needed to increase survival. Methodology and Principal Findings Applying mass spectrometry-based proteomics, we sought to elucidate an unanswered biomarker research question regarding ability to determine tumor burden detectable by an ovarian cancer biomarker protein emanating directly from the tumor cells. Since aggressive serous epithelial ovarian cancers account for most mortality, a xenograft model using human SKOV-3 serous ovarian cancer cells was established to model progression to disseminated carcinomatosis. Using a method for low molecular weight protein enrichment, followed by liquid chromatography and mass spectrometry analysis, a human-specific peptide sequence of S100A6 was identified in sera from mice with advanced-stage experimental ovarian carcinoma. S100A6 expression was documented in cancer xenografts as well as from ovarian cancer patient tissues. Longitudinal study revealed that serum S100A6 concentration is directly related to tumor burden predictions from an inverse regression calibration analysis of data obtained from a detergent-supplemented antigen capture immunoassay and whole-animal bioluminescent optical imaging. The result from the animal model was confirmed in human clinical material as S100A6 was found to be significantly elevated in the sera from women with advanced stage ovarian cancer compared to those with early stage disease. Conclusions S100A6 is expressed in ovarian and other cancer tissues, but has not been documented previously in ovarian cancer disease sera. S100A6 is found in serum in concentrations that correlate with experimental tumor burden and with clinical disease stage. The data signify that S100A6 may prove useful in detecting and/or monitoring ovarian cancer, when used in concert with other biomarkers.

3′UTR elements inhibit Ras-induced C/EBPβ post-translational activation and senescence in tumour cells

C/EBPβ is an auto‐repressed protein that becomes post‐translationally activated by Ras‐MEK‐ERK signalling. C/EBPβ is required for oncogene‐induced senescence (OIS) of primary fibroblasts, but also displays pro‐oncogenic functions in many tumour cells. Here, we show that C/EBPβ activation by H‐RasV12 is suppressed in immortalized/transformed cells, but not in primary cells, by its 3′ untranslated region (3′UTR). 3′UTR sequences inhibited Ras‐induced cytostatic activity of C/EBPβ, DNA binding, transactivation, phosphorylation, and homodimerization, without significantly affecting protein expression. The 3′UTR suppressed induction of senescence‐associated C/EBPβ target genes, while promoting expression of genes linked to cancers and TGFβ signalling. An AU‐rich element (ARE) and its cognate RNA‐binding protein, HuR, were required for 3′UTR inhibition. These components also excluded the Cebpb mRNA from a perinuclear cytoplasmic region that contains activated ERK1/2, indicating that the site of C/EBPβ translation controls de‐repression by Ras signalling. Notably, 3′UTR inhibition and Cebpb mRNA compartmentalization were absent in primary fibroblasts, allowing Ras‐induced C/EBPβ activation and OIS to proceed. Our findings reveal a novel mechanism whereby non‐coding mRNA sequences selectively regulate C/EBPβ activity and suppress its anti‐oncogenic functions.

Molecularly tagged simian immunodeficiency virus SIVmac239 synthetic swarm for tracking independent infection events.

ABSTRACT Following mucosal human immunodeficiency virus type 1 transmission, systemic infection is established by one or only a few viral variants. Modeling single-variant, mucosal transmission in nonhuman primates using limiting-dose inoculations with a diverse simian immunodeficiency virus isolate stock may increase variability between animals since individual variants within the stock may have substantial functional differences. To decrease variability between animals while retaining the ability to enumerate transmitted/founder variants by sequence analysis, we modified the SIVmac239 clone to generate 10 unique clones that differ by two or three synonymous mutations (molecular tags). Transfection- and infection-derived virus stocks containing all 10 variants showed limited phenotypic differences in 9 of the 10 clones. Twenty-nine rhesus macaques were challenged intrarectally or intravenously with either a single dose or repeated, limiting doses of either stock. The proportion of each variant within each inoculum and in plasma from infected animals was determined by using a novel real-time single-genome amplification assay. Each animal was infected with one to five variants, the number correlating with the dose. Longitudinal sequence analysis revealed that the molecular tags are highly stable with no reversion to the parental sequence detected in >2 years of follow-up. Overall, the viral stocks are functional and mucosally transmissible and the number of variants is conveniently discernible by sequence analysis of a small amplicon. This approach should be useful for tracking individual infection events in preclinical vaccine evaluations, long-term viral reservoir establishment/clearance research, and transmission/early-event studies. IMPORTANCE Human immunodeficiency virus type 1 transmission is established by one or only a few viral variants. Modeling of limited variant transmission in nonhuman primates with a diverse simian immunodeficiency virus isolate stock may increase the variability between animals because of functional differences in the individual variants within the stock. To decrease such variability while retaining the ability to distinguish and enumerate transmitted/founder variants by sequence analysis, we generated a viral stock with 10 sequence-identifiable but otherwise genetically identical variants. This virus was characterized in vitro and in vivo and shown to allow discrimination of distinct transmission events. This approach provides a novel nonhuman primate challenge system for the study of viral transmission, evaluation of vaccines and other prevention approaches, and characterization of viral reservoirs and strategies to target them.

Mutations in HIV-1 Reverse Transcriptase Affect the Errors Made in a Single Cycle of Viral Replication

ABSTRACT The genetic variation in HIV-1 in patients is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Some of the mutations in reverse transcriptase (RT) that alter the deoxynucleoside triphosphate (dNTP)-binding pocket, including those that confer resistance to nucleoside/nucleotide analogs, affect dNTP selection during replication. The effects of mutations in RT on the spectrum (nature, position, and frequency) of errors made in vivo are poorly understood. We previously determined the mutation rate and the frequency of different types of mutations and identified hot spots for mutations in a lacZα (the α complementing region of lacZ) reporter gene carried by an HIV-1 vector that replicates using wild-type RT. We show here that four mutations (Y115F, M184V, M184I, and Q151M) in the dNTP-binding pocket of RT that had relatively small effects on the overall HIV-1 mutation rate (less than 3-fold compared to the wild type) significantly increased mutations at some specific positions in the lacZα reporter gene. We also show that changes in a sequence that flanks the reporter gene can affect the mutations that arise in the reporter. These data show that changes either in HIV-1 RT or in the sequence of the nucleic acid template can affect the spectrum of mutations made during viral replication. This could, by implication, affect the generation of drug-resistant mutants and immunological-escape mutants in patients. IMPORTANCE RT is the viral enzyme that converts the RNA genome of HIV into DNA. Errors made during replication allow the virus to escape from the hosts immune system and to develop resistance to the available anti-HIV drugs. We show that four different mutations in RT which are known to be associated with resistance to anti-RT drugs modestly increased the overall frequency of errors made during viral replication. However, the increased errors were not uniformly distributed; the additional errors occurred at a small number of positions (hot spots). Moreover, some of the RT mutations preferentially affected the nature of the errors that were made (some RT mutations caused an increase in insertion and deletion errors; others caused an increase in substitution errors). We also show that sequence changes in a region adjacent to a target gene can affect the errors made within the target gene.

C/EBPγ Is a Critical Regulator of Cellular Stress Response Networks through Heterodimerization with ATF4

ABSTRACT The integrated stress response (ISR) controls cellular adaptations to nutrient deprivation, redox imbalances, and endoplasmic reticulum (ER) stress. ISR genes are upregulated in stressed cells, primarily by the bZIP transcription factor ATF4 through its recruitment to cis-regulatory C/EBP:ATF response elements (CAREs) together with a dimeric partner of uncertain identity. Here, we show that C/EBPγ:ATF4 heterodimers, but not C/EBPβ:ATF4 dimers, are the predominant CARE-binding species in stressed cells. C/EBPγ and ATF4 associate with genomic CAREs in a mutually dependent manner and coregulate many ISR genes. In contrast, the C/EBP family members C/EBPβ and C/EBP homologous protein (CHOP) were largely dispensable for induction of stress genes. Cebpg−/− mouse embryonic fibroblasts (MEFs) proliferate poorly and exhibit oxidative stress due to reduced glutathione levels and impaired expression of several glutathione biosynthesis pathway genes. Cebpg−/− mice (C57BL/6 background) display reduced body size and microphthalmia, similar to ATF4-null animals. In addition, C/EBPγ-deficient newborns die from atelectasis and respiratory failure, which can be mitigated by in utero exposure to the antioxidant, N-acetyl-cysteine. Cebpg−/− mice on a mixed strain background showed improved viability but, upon aging, developed significantly fewer malignant solid tumors than WT animals. Our findings identify C/EBPγ as a novel antioxidant regulator and an obligatory ATF4 partner that controls redox homeostasis in normal and cancerous cells.

Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Single-Nucleotide Polymorphisms Identified in Clinical Samples Can Affect MicroRNA Processing, Level of Expression, and Silencing Activity

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs that can produce 25 KSHV mature microRNAs. We previously reported single-nucleotide polymorphisms (SNPs) in KSHV-encoded pre-microRNA and mature microRNA sequences from clinical samples (V. Marshall et al., J. Infect. Dis., 195:645–659, 2007). To determine whether microRNA SNPs affect pre-microRNA processing and, ultimately, mature microRNA expression levels, we performed a detailed comparative analysis of (i) mature microRNA expression levels, (ii) in vitro Drosha/Dicer processing, and (iii) RNA-induced silencing complex-dependent targeting of wild-type (wt) and variant microRNA genes. Expression of pairs of wt and variant pre-microRNAs from retroviral vectors and measurement of KSHV mature microRNA expression by real-time reverse transcription-PCR (RT-PCR) revealed differential expression levels that correlated with the presence of specific sequence polymorphisms. Measurement of KSHV mature microRNA expression in a panel of primary effusion lymphoma cell lines by real-time RT-PCR recapitulated some observed expression differences but suggested a more complex relationship between sequence differences and expression of mature microRNA. Furthermore, in vitro maturation assays demonstrated significant SNP-associated changes in Drosha/DGCR8 and/or Dicer processing. These data demonstrate that SNPs within KSHV-encoded pre-microRNAs are associated with differential microRNA expression levels. Given the multiple reports on the involvement of microRNAs in cancer, the biological significance of these phenotypic and genotypic variants merits further studies in patients with KSHV-associated malignancies.

Ontogeny-Driven rDNA Rearrangement, Methylation, and Transcription, and Paternal Influence

Gene rearrangement occurs during development in some cell types and this genome dynamics is modulated by intrinsic and extrinsic factors, including growth stimulants and nutrients. This raises a possibility that such structural change in the genome and its subsequent epigenetic modifications may also take place during mammalian ontogeny, a process undergoing finely orchestrated cell division and differentiation. We tested this hypothesis by comparing single nucleotide polymorphism-defined haplotype frequencies and DNA methylation of the rDNA multicopy gene between two mouse ontogenic stages and among three adult tissues of individual mice. Possible influences to the genetic and epigenetic dynamics by paternal exposures were also examined for Cr(III) and acid saline extrinsic factors. Variables derived from litters, individuals, and duplicate assays in large mouse populations were examined using linear mixed-effects model. We report here that active rDNA rearrangement, represented by changes of haplotype frequencies, arises during ontogenic progression from day 8 embryos to 6-week adult mice as well as in different tissue lineages and is modifiable by paternal exposures. The rDNA methylation levels were also altered in concordance with this ontogenic progression and were associated with rDNA haplotypes. Sperm showed highest level of methylation, followed by lungs and livers, and preferentially selected haplotypes that are positively associated with methylation. Livers, maintaining lower levels of rDNA methylation compared with lungs, expressed more rRNA transcript. In vitro transcription demonstrated haplotype-dependent rRNA expression. Thus, the genome is also dynamic during mammalian ontogeny and its rearrangement may trigger epigenetic changes and subsequent transcriptional controls, that are further influenced by paternal exposures.