MODL-32. IMPACT OF PRDM6 ON CHROMATIN ACCESSIBILITY, GENE EXPRESSION, AND MEDULLOBLASTOMA FORMATION

Rivaroxaban does not affect growth of human pancreatic tumors in mice

Combination of clotam and vincristine enhances anti-proliferative effect in medulloblastoma cells

We administered serum fractions obtained from cancer patients by double-filtration plasmapheresis (DFPP) to cancer-bearing mice to examine the effects on tumor growth and metastasis. Fraction 1 (whole plasma), fraction 2 (a plasma fraction containing substances with higher particle size), fraction 3 (a plasma fraction containing substances with smaller particle size) and saline were administered intravenously to cancer-bearing mice for 10 days following the inoculation of tumor cells. The tumor growth and metastasis in mice administered fraction 2 was far more rapid than that in the control mice. On the other hand, tumor growth in mice administered fraction 3 was significantly delayed compared with that in mice injected with fraction 2. These results suggest that factors in the higher particle-size fraction of cancer patients' sera promote the growth and the metastasis of tumors in mice, and that DFPP, which remove these factors, is an effective therapy against cancer.

Animal studies have shown that virus infection causes changes in host chromatin accessibility, but little is known about changes in chromatin accessibility of plants infected by viruses and its potential impact. Here, rice infected by rice stripe virus (RSV) was used to investigate virus-induced changes in chromatin accessibility. Our analysis identified a total of 6462 open- and 3587 closed-differentially accessible chromatin regions (DACRs) in rice under RSV infection by ATAC-seq. Additionally, by integrating ATAC-seq and RNA-seq, 349 up-regulated genes in open-DACRs and 126 down-regulated genes in closed-DACRs were identified, of which 34 transcription factors (TFs) were further identified by search of upstream motifs. Transcription levels of eight of these TFs were validated by reverse transcription-PCR. Importantly, four of these TFs (OsWRKY77, OsWRKY28, OsZFP12 and OsERF91) interacted with RSV proteins and are therefore predicted to play important roles in RSV infection. This is the first application of ATAC-seq and RNA-seq techniques to analyse changes in rice chromatin accessibility caused by RSV infection. Integrating ATAC-seq and RNA-seq provides a new approach to select candidate TFs in response to virus infection.

Comprehensive sequencing of human cancers has identified recurrent mutations in genes encoding chromatin regulatory proteins. For clear cell renal cell carcinoma (ccRCC), three of the five commonly mutated genes encode the chromatin regulators PBRM1, SETD2, and BAP1. How these mutations alter the chromatin landscape and transcriptional program in ccRCC or other cancers is not understood. Here, we identified alterations in chromatin organization and transcript profiles associated with mutations in chromatin regulators in a large cohort of primary human kidney tumors. By associating variation in chromatin organization with mutations in SETD2, which encodes the enzyme responsible for H3K36 trimethylation, we found that changes in chromatin accessibility occurred primarily within actively transcribed genes. This increase in chromatin accessibility was linked with widespread alterations in RNA processing, including intron retention and aberrant splicing, affecting ∼25% of all expressed genes. Furthermore, decreased nucleosome occupancy proximal to misspliced exons was observed in tumors lacking H3K36me3. These results directly link mutations in SETD2 to chromatin accessibility changes and RNA processing defects in cancer. Detecting the functional consequences of specific mutations in chromatin regulatory proteins in primary human samples could ultimately inform the therapeutic application of an emerging class of chromatin-targeted compounds.

Inhibitory CpG sequences reduced ischemia/reperfusion-induced hepatic metastases of liver tumor in a murine model

Sulfate plays an important role in maintaining normal structure and function of tissues, and its content is decreased in certain cancers including lung carcinoma. In this study, we investigated tumor growth in a mouse model of hyposulfatemia (Nas1(-/-)) and compared it to wild-type (Nas1(+/+)) mice. Lung epithelial tumor cells (TC-1 cell line) were injected subcutaneously into male Nas1(-/-) and Nas1(+/+) mice on a mixed 129Sv and C57BL/6 genetic background. Tumor sections were stained with anti-glycosaminoglycan antibodies to assess the distribution of proteoglycans and Gomori's trichrome to detect collagen. After 14 days, tumor weights were markedly increased (by approximately 12-fold) in Nas1(-/-) mice when compared with Nas1(+/+) mice. Histological analyses of tumors revealed increased (by approximately 2.4-fold) vessel content, as well as markedly reduced collagen and immunoreactivity against glycosaminoglycan structural epitopes in the tumors from Nas1(-/-) mice. No significant differences were found for the growth of cultured TC-1 cells supplemented with Nas1(-/-) or Nas1(+/+) serum, as determined by (3)H-thymidine incorporation, implying that the cell culture conditions may not reflect the in vivo situation of enhanced tumor growth. This study has revealed increased tumor growth and an altered extracellular tumor matrix in hyposulfatemic Nas1(-/-) mice. These findings highlight the importance of blood sulfate levels as a possible modulator of tumor growth, and could lead to future cancer studies in humans with altered sulfate homeostasis.

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1), which generates active glucocorticoids from inactive precursors, is expressed in glucocorticoid target tissues, including the arterial wall. Since active glucocorticoids are anti-angiogenic 11β-HSD1 inhibitors enhance angiogenesis and may have therapeutic potential in ischaemia. However increased angiogenesis may be detrimental in tumours. This investigation tested the hypothesis that 11β-HSD1 inhibition promotes angiogenesis and tumour growth in mouse models of squamous cell carcinoma (SCC) and pancreatic ductal adenocarcinoma (PDA). Murine tumour cells (1 × 106) were injected subcutaneously into mice (female, 10–12 weeks, FVB/C57Bl6/J) receiving RM-1 diet with, or without (Control), the 11β-HSD1 inhibitor UE2316 (175 mg/kg). Tumour size was measured every 2–3 days for 2–3 weeks. Sections of tumours were stained for vascular markers (CD31, alpha-smooth muscle actin) for quantification of vessel density. Steroid/drug levels were measured in plasma/tissues using liquid chromatography tandem mass spectrometry whilst 11β-HSD1 activity was assayed in tissue homogenates by high performance liquid chromatography. The effects of 11β-HSD1 inhibition on angiogenesis were examined ex vivo using an aortic ring assay. Data are mean±SEM. 11β-HSD1 inhibition increased SCC tumour growth in FVB mice (p 11β-HSD1 inhibition does not promote angiogenesis in SCC or PDA tumours, but may increase SCC growth through a mechanism involving reduced fibrosis. Whether 11β-HSD1 inhibition increases angiogenesis in glucocorticoid-sensitive tumours remains to be established.

Myeloid-derived suppressor cells (MDSCs) are "polarized" myeloid cells that effectively promote tumorigenesis by inhibiting antitumor immunity. How myeloid cells acquire the protumoral properties during tumorigenesis is poorly understood. We report here that the polarity protein TIPE2 (tumor necrosis factor-α-induced protein 8-like 2) mediates the functional polarization of murine and human MDSCs by specifying their pro- and antitumoral properties. Tumor cells induced the expression of TIPE2 in Gr1+CD11b+ cells through reactive oxygen species (ROS). TIPE2 in turn increased the expression of protumoral mediators such as CCAAT/enhancer-binding protein-β while inhibiting the expression of antitumoral mediators. Consequently, tumor growth in TIPE2-deficient mice was significantly diminished, and TIPE2-deficient MDSCs markedly inhibited tumor growth upon adoptive transfer. Pharmaceutical blockade of ROS inhibited TIPE2 expression in MDSCs and reduced tumor growth in mice. These findings indicate that TIPE2 plays a key role in the functional polarization of MDSCs and represents a new therapeutic target for cancer immunotherapy.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a promising and novel anticancer cytokine, specifically kills numerous tumor cells by apoptosis. However, some malignancies are resistant to TRAIL treatment in clinical trials, thus limiting its therapeutic potential. In the present study, the TRAIL-resistant murine hepatocellular carcinoma cell line Hepa1-6 was used to elucidate the physiological significance of TRAIL resistance, especially with respect to the immune regulatory function of TRAIL. Hepa1-6 cells were resistant to TRAIL-induced apoptosis in vitro; however, intratumoral injection of recombinant soluble TRAIL inhibited tumor growth and prolonged survival time in tumor-bearing mice. Local TRAIL treatment decreased the number of intratumoral CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) but did not affect CD4(+)CD25(+)Foxp3(+) Tregs in the draining lymph nodes and spleen. Further investigation showed that TRAIL induced apoptosis of tumor-activated CD4(+)CD25(+)Foxp3(+) Tregs, but not of CD4(+)CD25(-) T cells. Moreover, mouse TRAIL receptor DR5 expression was detected on the surface of the tumor-infiltrating CD4(+)CD25(+)Foxp3(+) Tregs, but not on naïve CD4(+)CD25(+)Foxp3(+) Tregs. Interestingly, intratumoral injection of TRAIL not only decreased the number of CD4(+)CD25(+)Foxp3(+) Tregs but also increased the number of tumor-specific CD8(+) CTL and augmented their cytotoxicity to the tumor cells. These data provide the novel evidence for an immune regulatory function of TRAIL and may shed light on the clinical application of TRAIL.

Backround and aimsComplex host-virus interactions account for adaptive and innate immunity dysfunctions and viral cccDNA mini-chromosome persistence, key features of HBV chronicity and challenges for HBV cure. The extent of HBV direct impact on liver transcriptome remains controversial. Transcriptional activation in eukaryotic cells is tightly linked with disruption of nucleosome organization at accessible genomic sites of remodeled chromatin. We sought to investigate the impact of HBV on chromatin accessibility and transcription.MethodsWe used ATAC-seq (Assay for Transposase Accessible Chromatin followed by high throughput sequencing) to detect early changes in chromatin accessibility coupled with RNA-seq in HBV-infected Primary Human Hepatocytes (PHHs).ResultsAn increasing number of genomic sites change their nucleosome organization over time after HBV infection, with a prevalent, but not exclusive, reduction of chromatin accessibility at specific sites that is partially prevented by inhibiting HBV transcription and replication. ATAC-seq and RNA-seq integration showed that HBV infection impacts on liver fatty acids, bile acids, iron metabolism and liver cancer pathways. The upregulation of iron uptake genes leads to a significant increase of iron content in HBV-infected PHHs whereas iron chelation inhibits cccDNA transcription and viral replication. The chromatin accessibility and transcriptional changes imposed by HBV early after infection persist, as an epigenetic scar, in chronic HBV (CHB) patients and in HBV-related HCCs. These changes are to a large extent independent from viral replication levels and disease activity.ConclusionsAltogether our results show that HBV infection impacts on host cell chromatin landscape and specific transcriptional programs including liver metabolism and liver cancer pathways. Re-wiring of iron metabolism boosts viral replication early after infection. The modulation of genes involved in cancer-related pathways may favor the development or the selection of a pro-neoplastic phenotype and persists in HBV-related HCCs.

Significance of host heparanase in promoting tumor growth and metastasis

BackgroundDecoding the genetic mechanisms of late‐onset Alzheimer’s disease (LOAD) is a major challenge in the post‐GWAS era, since the majority of the LOAD associated SNPs are in noncoding regions. Noncoding disease‐associated loci have been shown to be enriched for regulatory elements in tissues and cells relevant to the disease. Thus, post‐GWAS research requires an in‐depth characterization of cell‐type‐specific DNA regulatory elements and gene expression patterns.MethodsWe conducted ATAC‐seq profiling using NeuN sorted‐nuclei from 40 frozen brain tissues to determine LOAD‐specific changes in chromatin accessibility landscape in a cell‐type specific manner. We complemented these experiments by generating single‐nuclei(sn)RNA‐seq data from a subset group of the same brain samples.ResultsThe study has five major findings for the field of LOAD epigenetics. First, we generated a map of LOAD‐associated cell‐type specific chromatin accessibility sites. Second, we identified 211 LOAD‐specific differential chromatin accessibility sites in neuronal‐nuclei. Third, we provided a catalogue of 842 female‐specific disease‐associated chromatin accessibility sites in non‐neuronal cells. Forth, we showed that the LOAD‐associated cell‐type‐specific changes in chromatin accessibility overlapped with ∼25% of the LOAD‐GWAS regions. Thus, suggesting a noncoding regulatory mechanism, namely chromatin accessibility, by which several LOAD‐GWAS loci may exert their pathogenic effect. Fifth, we functionally validated the ATAC‐seq findings using snRNA‐seq datasets. Data integration identified overlaps between the LOAD‐specific ATAC‐seq signals with the transcriptome profiles of known and new LOAD loci and demonstrated that LOAD‐associated changes in chromatin accessibility can result in gene dysregulation.ConclusionTo our knowledge, this study represents the first and most comprehensive systematic interrogation of the chromatin accessibility landscape in LOAD and the functional effect on gene dysregulation. Moreover, the outcomes unveiled how cell‐type‐specific and sex‐dependent alterations in chromatin structure and transcription programs are associated with LOAD. These findings enhance the interpretation of LOAD‐GWAS discoveries towards translating association to causation, provide potential pathomechanisms, and suggest novel LOAD‐loci. Furthermore, our results convey mechanistic insights into sex differences in LOAD risk and clinicopathology.

Abnormal increases in nucleolar size and number caused by dysregulation of ribosome biogenesis has emerged as a hallmark in the majority of spontaneous cancers. The observed ribosome hyperactivity can be directly induced by the MYC transcription factors controlling the expression of RNA and protein components of the ribosome. Neuroblastoma, a highly malignant childhood tumor of the sympathetic nervous system, is frequently characterized by MYCN gene amplification and high expression of MYCN and c-MYC signature genes. Here, we show a strong correlation between high-risk disease, MYCN expression, poor survival, and ribosome biogenesis in neuroblastoma patients. Treatment of neuroblastoma cells with quarfloxin or CX-5461, two small molecule inhibitors of RNA polymerase I, suppressed MycN expression, induced DNA damage, and activated p53 followed by cell cycle arrest or apoptosis. CX-5461 repressed the growth of established MYCN-amplified neuroblastoma xenograft tumors in nude mice. These findings suggest that inhibition of ribosome biogenesis represent new therapeutic opportunities for children with high-risk neuroblastomas expressing high levels of Myc.

BackgroundWe have previously shown that 5-fluorouracil (5-FU) selectively kills myeloid-derived suppressor cells (MDSCs) and activates NLRP3 (NOD-leucine rich repeat and pyrin containing protein 3) inflammasome. NLRP3 activation leads to caspase-1...