Facilitates Chromatin Transcription Complex Is an “Accelerator” of Tumor Transformation and Potential Marker and Target of Aggressive Cancers

SummaryThe facilitates chromatin transcription (FACT) complex is involved in chromatin remodeling during transcription, replication, and DNA repair. FACT was previously considered to be ubiquitously expressed and not associated with any disease. However, we discovered that FACT is the target of a class of anticancer compounds and is not expressed in normal cells of adult mammalian tissues, except for undifferentiated and stem-like cells. Here, we show that FACT expression is strongly associated with poorly differentiated aggressive cancers with low overall survival. In addition, FACT was found to be upregulated during in vitro transformation and to be necessary, but not sufficient, for driving transformation. FACT also promoted survival and growth of established tumor cells. Genome-wide mapping of chromatin-bound FACT indicated that FACT’s role in cancer most likely involves selective chromatin remodeling of genes that stimulate proliferation, inhibit cell death and differentiation, and regulate cellular stress responses

Uncovering the fine print of the CreER^T2<i>-LoxP</i> system while generating a conditional knockout mouse model of <i>Ssrp1</i> gene

<div><p><b>FA</b>cilitates <b>C</b>hromatin <b>T</b>ranscription (<b>FACT</b>) is a complex of SSRP1 and SPT16 that is involved in chromatin remodeling during transcription, replication, and DNA repair. FACT has been mostly studied in cell-free or single cell model systems because general FACT knockout (KO) is embryonically lethal (E3.5). FACT levels are limited to the early stages of development and stem cell niches of adult tissues. FACT is upregulated in poorly differentiated aggressive tumors. Importantly, FACT inhibition (RNAi) is lethal for tumors but not normal cells, making FACT a lucrative target for anticancer therapy. To develop a better understanding of FACT function in the context of the mammalian organism under normal physiological conditions and in disease, we aimed to generate a conditional FACT KO mouse model. Because SPT16 stability is dependent on the SSRP1-SPT16 association and the presence of <i>SSRP1</i> mRNA, we targeted the <i>Ssrp1</i> gene using a CreER^T2- LoxP approach to generate the FACT KO model. Here, we highlight the limitations of the CreER^T2-LoxP (Rosa26) system that we encountered during the generation of this model. <i>In vitro</i> studies showed an inefficient excision rate of ectopically expressed CreER^T2 (retroviral CreER^T2) in fibroblasts with homozygous floxed <i>Ssrp1</i>. <i>In vitro</i> and <i>in vivo</i> studies showed that the excision efficiency could only be increased with germline expression of two alleles of Rosa26CreER^T2. The expression of one germline Rosa26CreER^T2 allele led to the incomplete excision of <i>Ssrp1</i>. The limited efficiency of the CreER^T2-LoxP system may be sufficient for studies involving the deletion of genes that interfere with cell growth or viability due to the positive selection of the phenotype. However, it may not be sufficient for studies that involve the deletion of genes supporting growth, or those crucial for development. Although CreER^T2-LoxP is broadly used, it has limitations that have not been widely discussed. This paper aims to encourage such discussions.</p></div

Histone chaperone FACT and curaxins: effects on genome structure and function

The histone chaperone facilitates chromatin transcription (FACT) plays important roles in essentially every chromatin-associated process and is an important indirect target of the curaxin class of anti-cancer drugs. Curaxins are aromatic compounds that intercalate into DNA and can trap FACT in bulk chromatin, thus interfering with its distribution and its functions in cancer cells. Recent studies have provided mechanistic insight into how FACT and curaxins cooperate to promote unfolding of nucleosomes and chromatin fibers, resulting in genome-wide disruption of contact chromatin domain boundaries, perturbation of higher-order chromatin organization, and global dysregulation of gene expression. Here, we discuss the implications of these insights for cancer biology

Targeted Modulation of Interferon Response-Related Genes with IFN-Alpha/Lambda Inhibition

Interferon (IFN) signaling resulting from external or internal inflammatory processes initiates the rapid release of cytokines and chemokines to target viral or bacterial invasion, as well as cancer and other diseases. Prolonged exposure to IFNs, or the overexpression of other cytokines, leads to immune exhaustion, enhancing inflammation and leading to the persistence of infection and promotion of disease. Hence, to control and stabilize an excessive immune response, approaches for the management of inflammation are required. The potential use of peptides as anti-inflammatory agents has been previously demonstrated. Our team discovered, and previously published, a 9-amino-acid cyclic peptide named ALOS4 which exhibits anti-cancer properties in vivo and in vitro. We suggested that the anti-cancer effect of ALOS4 arises from interaction with the immune system, possibly through the modulation of inflammatory processes. Here, we show that treatment with ALOS4 decreases basal cytokine levels in mice with chronic inflammation and prolongs the lifespan of mice with acute systemic inflammation induced by irradiation. We also show that pretreatment with ALOS4 reduces the expression of IFN alpha, IFN lambda, and selected interferon-response genes triggered by polyinosinic-polycytidylic acid (Poly I:C), a synthetic analog of viral double-stranded RNA, while upregulating the expression of other genes with antiviral activity. Hence, we conclude that ALOS4 does not prevent IFN signaling, but rather supports the antiviral response by upregulating the expression of interferon-response genes in an interferon-independent manner

Generation of conditional <i>Ssrp1</i> KO mice.

<p>A) Schematic representation of the wild-type <i>Ssrp1</i> allele (<i>Ssrp1</i>⁺) and the targeting vector with 5’ and 3’ homology arms (pink dashed lines). The black dashed lines indicate intron regions in <i>Ssrp1</i>⁺ where LoxP sites, FRT sites, and the synthetic cassette are inserted. B) Schematic representation of the mutant allele (<i>Ssrp1</i> ^{<i>Neo LacZ fl</i>}) after homologous recombination, representation of mutant allele without the synthetic cassette (<i>Ssrp1</i>^<i>fl</i>) after Flp-FRT recombination and representation of <i>Ssrp1</i>^<i>Δ</i> allele with deletion of the critical exons after CreER^T2-LoxP recombination. C) Southern blot hybridization of DNA from ESC cells to determine clones with correct (red) and incorrect (black) recombination of 3’ and 5’ arm. Wild-type DNA used as control for 5’ probe is C. D) PCR of genomic DNA from F1 progeny of wild-type (<i>Ssrp1</i>^<i>+/+</i>) crossed with chimera (<i>Ssrp1</i> ^{<i>Neo LacZ fl /</i>+}). Two bands are present in heterozygous pups (<i>Ssrp1</i> ^{<i>Neo LacZ fl</i> /+}) (red). Numbers indicate individual pup IDs in one litter. E) β-Galactosidase assay to determine LacZ activity (Blue-Green stain for positive activity) on SSRP1 positive (colon, pancreas and testis) and negative (lung) tissues isolated from <i>Ssrp1</i>^+/+ and <i>Ssrp1</i> ^{<i>Neo LacZ fl</i> /+} mice.</p

Excision efficiency of two alleles of germline <i>CreER</i>^<i>T2</i> in vivo.

<p>A) PCR of genomic DNA to determine <i>Ssrp1</i> excision in <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/- and <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/+ mice treated with 1 mg tamoxifen or <i>Ssrp1</i>^<i>fl/fl</i><i>CreER</i>^<i>T2</i>+/- mice treated with vehicle control i.p. for 5 days. T = tamoxifen-treated mice and C = vehicle-treated control mice; T2 = <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/ mouse treated twice with tamoxifen. For T2, the image shows the PCR results following the second round of treatment.^-B) qPCR to determine the relative expression of the excised gene in <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/- and <i>Ssrp1</i>^<i>fl/fl</i> <i>CreER</i>^<i>T2</i>+/+ mice treated with 1 mg tamoxifen i.p. for 5 days. p-value = 0.0022.</p

Small-Molecule Inhibitor Which Reactivates p53 in Human T-Cell Leukemia Virus Type 1-Transformed Cells▿

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of the aggressive and fatal disease adult T-cell leukemia. Previous studies have demonstrated that the HTLV-1-encoded Tax protein inhibits the function of tumor suppressor p53 through a Tax-induced NF-κB pathway. Given these attributes, we were interested in the activity of small-molecule inhibitor 9-aminoacridine (9AA), an anticancer drug that targets two important stress response pathways, NF-κB and p53. In the present study, we have examined the effects of 9AA on HTLV-1-transformed cells. Treatment of HTLV-1-transformed cells with 9AA resulted in a dramatic decrease in cell viability. Consistent with these results, we observed an increase in the percentage of cells in sub-G1 and an increase in the number of cells positive by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay following treatment of HTLV-1-transformed cells with 9AA. In each assay, HTLV-1-transformed cells C8166, Hut102, and MT2 were more sensitive to treatment with 9AA than control CEM and peripheral blood mononuclear cells. Analyzing p53 function, we demonstrate that treatment of HTLV-1-transformed cells with 9AA resulted in an increase in p53 protein and activation of p53 transcription activity. Of significance, 9AA-induced cell death could be blocked by introduction of a p53 small interfering RNA, linking p53 activity and cell death. These results suggest that Tax-repressed p53 function in HTLV-1-transformed cells is “druggable” and can be restored by treatment with 9AA. The fact that 9AA induces p53 and inhibits NF-κB suggests a promising strategy for the treatment of HTLV-1-transformed cells