Gene Body Methylation of the Lymphocyte-Specific Gene CARD11 Results in Its Overexpression and Regulates Cancer mTOR Signaling.

Investigations into the function of nonpromoter DNA methylation have yielded new insights into epigenetic regulation of gene expression. Previous studies have highlighted the importance of distinguishing between DNA methylation in discrete functional regions; however, integrated nonpromoter DNA methylation and gene expression analyses across a wide number of tumor types and corresponding normal tissues have not been performed. Through integrated analysis of gene expression and DNA methylation profiles, we examined 32 tumor types and identified 57 tumor suppressors and oncogenes out of 260 genes exhibiting a correlation of > 0.5 between gene body methylation and gene expression in at least one tumor type. The lymphocyte-specific gene CARD11 exhibits robust association between gene body methylation and expression across 19 of 32 tumor types examined. It is significantly overexpressed in kidney renal cell carcinoma (KIRC) and lung adenocarcinoma (LUAD) tumor tissues in comparison with respective control samples; and is significantly associated with lower overall survival in KIRC. Contrary to its canonical function in lymphocyte NFκB activation, CARD11 activates the mTOR pathway in KIRC and LUAD, resulting in suppressed autophagy. Furthermore, demethylation of a CpG island within the gene body of CARD11 decreases gene expression. Collectively, our study highlights how DNA methylation outside the promoter region can impact tumor progression. IMPLICATIONS: Our study describes a novel regulatory role of gene body DNA methylation-dependent CARD11 expression on mTOR signaling and its impact on tumor progression.

Gain-of-function p53 protein transferred via small extracellular vesicles promotes conversion of fibroblasts to a cancer-associated phenotype.

Tumor and stromal interactions consist of reciprocal signaling through cytokines, growth factors, direct cell-cell interactions, and extracellular vesicles (EVs). Small EVs (≤200 nm) have been considered critical messengers of cellular communication during tumor development. Here, we demonstrate that gain-of-function (GOF) p53 protein can be packaged into small EVs and transferred to fibroblasts. GOF p53 protein is selectively bound by heat shock protein 90 (HSP90), a chaperone protein, and packaged into small EVs. Inhibition of HSP90 activity blocks packaging of GOF, but not wild-type, p53 in small EVs. GOF p53-containing small EVs result in their conversion to cancer-associated fibroblasts. In vivo studies reveal that GOF p53-containing small EVs can enhance tumor growth and promote fibroblast transformation into a cancer-associated phenotype. These findings provide a better understanding of the complex interactions between cancer and stromal cells and may have therapeutic implications.

CD63-mediated cloaking of VEGF in small extracellular vesicles contributes to anti-VEGF therapy resistance.

Despite wide use of anti-vascular endothelial growth factor (VEGF) therapy for many solid cancers, most individuals become resistant to this therapy, leading to disease progression. Therefore, new biomarkers and strategies for blocking adaptive resistance of cancer to anti-VEGF therapy are needed. As described here, we demonstrate that cancer-derived small extracellular vesicles package increasing quantities of VEGF and other factors in response to anti-VEGF therapy. The packaging process of VEGF into small extracellular vesicles (EVs) is mediated by the tetraspanin CD63. Furthermore, small EV-VEGF (eVEGF) is not accessible to anti-VEGF antibodies and can trigger intracrine VEGF signaling in endothelial cells. eVEGF promotes angiogenesis and enhances tumor growth despite bevacizumab treatment. These data demonstrate a mechanism where VEGF is partitioned into small EVs and promotes tumor angiogenesis and progression. These findings have clinical implications for biomarkers and therapeutic strategies for ovarian cancer.

Pan-cancer genomic analysis links 3'UTR DNA methylation with increased gene expression in T cells.

BACKGROUND: Investigations into the function of non-promoter DNA methylation have yielded new insights into the epigenetic regulation of gene expression. However, integrated genome-wide non-promoter DNA methylation and gene expression analyses across a wide number of tumour types and corresponding normal tissues have not been performed. METHODS: To investigate the impact of non-promoter DNA methylation on cancer pathogenesis, we performed a large-scale analysis of gene expression and DNA methylation profiles, finding enrichment in the 3'UTR DNA methylation positively correlated with gene expression. Filtering for genes in which 3'UTR DNA methylation strongly correlated with gene expression yielded a list of genes enriched for functions involving T cell activation. FINDINGS: The important immune checkpoint gene Havcr2 showed a substantial increase in 3'UTR DNA methylation upon T cell activation and subsequent upregulation of gene expression in mice. Furthermore, this increase in Havcr2 gene expression was abrogated by treatment with decitabine. INTERPRETATION: These findings indicate that the 3'UTR is a functionally relevant DNA methylation site. Additionally, we show a potential novel mechanism of HAVCR2 regulation in T cells, providing new insights for modulating immune checkpoint blockade.

Role of YAP1 as a Marker of Sensitivity to Dual AKT and P70S6K Inhibition in Ovarian and Uterine Malignancies.

Background: The PI3K/AKT/P70S6K pathway is an attractive therapeutic target in ovarian and uterine malignancies because of its high rate of deregulation and key roles in tumor growth. Here, we examined the biological effects of MSC2363318A, which is a novel inhibitor of AKT1, AKT3, and P70S6K. Methods: Orthotopic murine models of ovarian and uterine cancer were utilized to study the effect of MSC2363318A on survival and regression. For each cell line, 10 mice were treated in each of the experimental arms tested. Moreover, in vitro experiments in 21 cell lines (MTT, immunoblot analysis, plasmid transfection, reverse phase protein array [RPPA]) were carried out to characterize underlying mechanisms and potential biomarkers of response. All statistical tests were two-sided. Results: MSC2363318A decreased tumor growth and metastases in multiple murine orthotopic models of ovarian (SKOV3ip1, HeyA8, and Igrov1) and uterine (Hec1a) cancer by reducing proliferation and angiogenesis and increasing cell death. Statistically significant prolonged overall survival was achieved with combination MSC2363318A and paclitaxel in the SKUT2 (endometrioid) uterine cancer mouse model ( P < .001). Mice treated with combination MSC2363318A and paclitaxel had the longest overall survival (mean = 104.2 days, 95% confidence interval [CI] = 97.0 to 111.4) compared with those treated with vehicle (mean = 61.9 days, 95% CI = 46.3 to 77.5), MSC2363318A alone (mean = 89.7 days, 95% CI = 83.0 to 96.4), and paclitaxel alone (mean = 73.6 days, 95% CI = 53.4 to 93.8). Regression and stabilization of established tumors in the Ishikawa (endometrioid) uterine cancer model was observed in mice treated with combination MSC2363318A and paclitaxel. Synergy between MSC2363318A and paclitaxel was observed in vitro in cell lines that had an IC50 of 5 µM or greater. RPPA results identified YAP1 as a candidate marker to predict cell lines that were most sensitive to MSC2363318A (R = 0.54, P = .02). After establishment of a murine ovarian cancer model of adaptive anti-angiogenic resistance (SKOV3ip1-luciferase), we demonstrate that resensitization to bevacizumab occurs with the addition of MSC2363318A, resulting in improved overall survival ( P = .01) using the Kaplan-Meier method. Mice treated with bevacizumab induction followed by MSC2363318A had the longest overall survival (mean = 66.0 days, 95% CI = 53.9 to 78.1) compared with mice treated with control (mean = 42.0 days, 95% CI = 31.4 to 52.6) and bevacizumab-sensitive mice (mean = 47.2 days; 95% CI = 37.5 to 56.9). Conclusions: MSC2363318A has therapeutic efficacy in multiple preclinical models of ovarian and uterine cancer. These findings support clinical development of a dual AKT/P70S6K inhibitor.

A miR-192-EGR1-HOXB9 regulatory network controls the angiogenic switch in cancer.

A deeper mechanistic understanding of tumour angiogenesis regulation is needed to improve current anti-angiogenic therapies. Here we present evidence from systems-based miRNA analyses of large-scale patient data sets along with in vitro and in vivo experiments that miR-192 is a key regulator of angiogenesis. The potent anti-angiogenic effect of miR-192 stems from its ability to globally downregulate angiogenic pathways in cancer cells through regulation of EGR1 and HOXB9. Low miR-192 expression in human tumours is predictive of poor clinical outcome in several cancer types. Using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) nanoliposomes, we show that miR-192 delivery leads to inhibition of tumour angiogenesis in multiple ovarian and renal tumour models, resulting in tumour regression and growth inhibition. This anti-angiogenic and anti-tumour effect is more robust than that observed with an anti-VEGF antibody. Collectively, these data identify miR-192 as a central node in tumour angiogenesis and support the use of miR-192 in an anti-angiogenesis therapy.

Improving vascular maturation using noncoding RNAs increases antitumor effect of chemotherapy.

Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessels and starving tumor cells. This strategy has shown transient and modest efficacy. Here, we report a better approach to target cancer-associated endothelial cells (ECs), reverse permeability and leakiness of tumor blood vessels, and improve delivery of chemotherapeutic agents to the tumor. First, we identified deregulated microRNAs (miRs) from patient-derived cancer-associated ECs. Silencing these miRs led to decreased vascular permeability and increased maturation of blood vessels. Next, we screened a thioaptamer (TA) library to identify TAs selective for tumor-associated ECs. An annexin A2-targeted TA was identified and used for delivery of miR106b-5p and miR30c-5p inhibitors, resulting in vascular maturation and antitumor effects without inducing hypoxia. These findings could have implications for improving vascular-targeted therapy.

Composite B-cell and T-cell non-Hodgkin lymphoma of the tibia.

We report a unique case of de novo composite lymphoma in the tibia of a 35-year-old man who presented with increasingly frequent and intense pain in the right upper leg. He was otherwise healthy without significant medical history. A plain radiograph of the right leg showed a permeative lesion with alternating areas of radiolucency and radiodensity in the upper third of the tibia. Magnetic resonance imaging showed a large, heterogeneous enhancing lesion involving the medullary and cortical bone of the proximal tibia with cortical disruption and extension into the adjacent soft tissue. A biopsy showed sheets and clusters of large cells, punctuated by clusters of small, irregular lymphocytes. Flow cytometry and immunohistochemical analysis showed composite lymphoma: diffuse large B-cell lymphoma (DLBCL) and peripheral T-cell non-Hodgkin lymphoma with predominantly small cell morphologic features. The DLBCL expressed CD19, CD20, CD79a, CD5, CD10, CD23, CD38, CD117, bcl-2, and bcl-6, with monotypic expression of immunoglobulin kappa light chain. The T cells expressed CD2, CD3, CD5, CD7, and CD8, with partial loss of CD4. Clonal rearrangement of T-cell receptor gamma chain gene was found. Neither the large B cells nor the small T cells expressed Epstein-Barr virus-encoded RNA. Physical examination and radiologic studies showed no evidence of lymphadenopathy, organomegaly, or other mass lesions in the body. No peripheral lymphocytosis or bone marrow involvement was present.

Inhibition of bone repair in a rat model for chronic and excessive alcohol consumption.

Alcohol abuse is associated with increases in both the incidence of fractures and complications in fracture healing. The purpose of this study was to determine the dose-dependent effects of ethanol on bone repair in a rat model. Three-month-old male Wistar rats were continuously fed liquid diets containing ethanol as either 36% or 26% of total calories or control diets for 6 weeks. Then, a bone repair model was created in all rats. Bone healing and liver metabolism were evaluated 7 weeks after bone injury. For each dose, there were three ethanol-feeding groups receiving (1) ethanol for 13 weeks, (2) control diet for 13 weeks (pair-fed), and (3) ethanol before bone injury and control diet (pair-fed) after injury. Another group was fed ethanol (36%) before injury and given control diet ad libitum after injury. There were also two nutritional controls consuming control diet and standard rat chow ad libitum for 13 weeks. Abnormal liver metabolism was evident at the higher ethanol dose - increases in cytochrome P4502E1 specific activity (5-fold; P < .01), triglyceride content (4-fold; P < .02), and liver weight (25%; P = .05) - compared with pair-fed controls. The higher dose of ethanol resulted in deficient bone repair when compared with rats receiving ethanol-free control diet by pair-feeding: 26% less (P = .02) rigidity of the repaired bone, 41% less (P = .02) intrinsic stiffness, 24% less intrinsic strength (P = .05), and 14% less (P = .001) ash density of the repair tissue. The reduced food consumption of ethanol-fed rats compared with that in the nutritional controls did not contribute to this deficiency. Furthermore, removal of ethanol (as 36% of calories) from the diet after bone injury completely restored normal bone healing and nearly normalized the liver metabolism. The lower ethanol dose (26% of calories) had a minimal effect on liver metabolism and bone repair. We conclude that ethanol (as 36% of calories) in the rat diet, especially during the postinjury period, was solely responsible for the observed inhibition of bone repair.