HPVE6-USP46 Mediated Cdt2 Stabilization Reduces Set8 Mediated H4K20-Methylation to Induce Gene Expression Changes

E6 from high-risk strains of HPV is well known to transform cells by deregulating p53. We reported that in HPV transformed cell-lines E6 from high-risk HPV can recruit the USP46 deubiquitinase to substrates such as Cdt2 and stabilize the latter, and that USP46 is important for growth of HPV induced tumors in xenografts. Here we show that in cervical cancer biopsies the stabilization of Cdt2 in the HPV-induced cancers leads to the decrease of a CRL4-Cdt2 substrate, the histone H4K20 mono-methyltransferase Set8, and decrease in H4K20me1 or H4K20me3 that can be detected by immunohistochemistry. In HPV-transformed cancer cell lines in vitro, knockdown of E6 decreases Cdt2 and increases Set8. Co-knockdown of Set8 shows that some of the gene expression changes produced by E6 knockdown is due to the increase of Set8. EGFR and EGFR regulated genes were identified in this set of genes. Turning to the mechanism by which E6 stabilizes Cdt2, we find that a purified E6:USP46 complex has significantly more de-ubiquitinase activity in vitro than USP46 alone, demonstrating that E6 can directly interact with USP46 in the absence of other proteins and that it can substitute for the known activators of USP46, UAF1 and WDR20. Deletion mapping of Cdt2 shows that there are three discrete, but redundant, parts of the substrate that are essential for stabilization by E6: USP46. The helix–loop–helix region or the WD40 repeat driven beta-propeller structure of Cdt2 are dispensable for the stabilization implying that interaction with DDB1 (and the rest of the CRL4 complex) or with the substrate of the CRL4-Cdt2 E3 ligase is not necessary for E6:USP46 to interact with and stabilize Cdt2. The identification of 50 amino acid stretches in the 731 amino acid Cdt2 protein as being important for the stabilization by E6 underlines the specificity of the process. In summary, E6 activates the deubiquitinase activity of USP46, stabilizes Cdt2 utilizing multiple sites on Cdt2, and leads to degradation of Set8 and changes in gene-expression in HPV-transformed cells.

Cutaneous Malignancies in Tattoos, a Case Series of Six Patients

Background: A variety of side effects following the tattooing of the skin were reported over the years. Analytical studies showed that some tattoo inks contain harmful compounds. Methods: We presented six patient cases with cutaneous malignancies in tattooed skin and performed an extensive literature research. Results: Two patients with black ink tattoos that were diagnosed with malignant melanoma raises the number of described cases to 36 patients. One of the patients developed an immunologic reaction limited to the tattoo area after treatment with a targeted immune therapy. In the other patient, the malignancy (malignant melanoma) was fatal. Basal cell carcinoma was seen in four patients with tattoos containing varying ink colors (black, green, red). This increased the number of described patient cases to 18. Although some ink components and their cleavage products have carcinogenic properties, epidemiological evidence for a causative correlation fails. Further epidemiologic studies on tattoos and malignancies, as well as on the appearance of naevi in tattoos, are necessary. Determining the type of mutation might be helpful to separate sun-induced tumors from skin cancers due to other pathogenic mechanisms.

Targeting MICA/B with cytotoxic therapeutic antibodies leads to tumor control

Background: MICA and MICB are tightly regulated stress-induced proteins that trigger the immune system by binding to the activating receptor NKG2D on cytotoxic lymphocytes. MICA and MICB are highly polymorphic molecules with prevalent expression on several types of solid tumors and limited expression in normal/healthy tissues, making them attractive targets for therapeutic intervention. Methods: We have generated a series of anti-MICA and MICB cross-reactive antibodies with the unique feature of binding to the most prevalent isoforms of both these molecules. Results: The anti-MICA and MICB antibody MICAB1, a human IgG1 Fc-engineered monoclonal antibody (mAb), displayed potent antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of MICA/B-expressing tumor cells in vitro. However, it showed insufficient efficiency against solid tumors in vivo, which prompted the development of antibody-drug conjugates (ADC). Indeed, optimal tumor control was achieved with MICAB1-ADC format in several solid tumor models, including patient-derived xenografts (PDX) and carcinogen-induced tumors in immunocompetent MICAgen transgenic mice. Conclusions: These data indicate that MICA and MICB are promising targets for cytotoxic immunotherapy.

Targeting MICA/B with cytotoxic therapeutic antibodies leads to tumor control

Background: MICA and MICB are tightly regulated stress-induced proteins that trigger the immune system by binding to the activating receptor NKG2D on cytotoxic lymphocytes. MICA and MICB are highly polymorphic molecules with prevalent expression on several types of solid tumors and limited expression in normal/healthy tissues, making them attractive targets for therapeutic intervention. Methods: We have generated a series of anti-MICA and MICB cross-reactive antibodies with the unique feature of binding to the most prevalent isoforms of both these molecules. Results: The anti-MICA and MICB antibody MICAB1, a human IgG1 Fc-engineered monoclonal antibody (mAb), displayed potent antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of MICA/B-expressing tumor cells in vitro. However, it showed insufficient efficiency against solid tumors in vivo, which prompted the development of antibody-drug conjugates (ADC). Indeed, optimal tumor control was achieved with MICAB1-ADC format in several solid tumor models, including patient-derived xenografts (PDX) and carcinogen-induced tumors in immunocompetent MICAgen transgenic mice. Conclusions: These data indicate that MICA and MICB are promising targets for cytotoxic immunotherapy.

OS02.4.A Molecular characterization of adult cerebellar glioblastomas identifies distinct prognosis subgroups

BACKGROUND Adult cerebellar glioblastomas (cGBM) are very rare and recent studies have shown that they constitute a heterogeneous group of gliomas. The aim of the present study was to characterize the prevalence and prognostic significance of major driver molecular alterations in a large series of cGBM. MATERIAL AND METHODS Adults with histologically proven cGBM diagnosed between 2003 and 2017 were identified from the French brain tumor database and the Club de Neuro-Oncologie of the Société Française de Neurochirurgie. Tumors were reviewed and reclassified according to WHO 2016. Targeted sequencing was performed, including determination of H3F3A, TERTp, IDH1/2, FGFR1, BRAF and EGFR status. RESULTS A total of 83 adult patients (median age 57 years) with cGBM (hemispheric n= 47, vermian n=14 or both n=22) were identified. Median overall survival was 10 months. Main molecular alterations observed were TERT promoter, H3F3A K27M, hotspot FGFR1 (N546 and K656), BRAF mutations, EGFR amplification and ATRX loss of expression in 19.2%, 18.8%, 10.9%, 2.6%, 19.5% and 22.7% of patients, respectively. cGBM could be classified into 6 mutually exclusive subgroups associated with age at diagnosis and prognosis: pTERT and/or EGFR amplified tumors (n=22, 26.5%, median age = 62 years, median OS = 4 months), H3K27M-mutant tumors (n=15, 18.1%, median age = 48 years, median OS =8 months), tumors with MAPK pathway activating mutations (FGFR1, BRAF) or occurring in NF1 patients (n=15, 18.1%, median age = 41 years, median OS = 57 months), radiation-induced tumors (n=5, 6%, median age = 47 years, median OS = 5 months), IDH-mutant tumors (n=1) and unclassified tumors (n=25, 30.1%, median age = 63 years, median OS = 17 months). In multivariate analysis, MAPK activating mutations and ATRX loss of expression were independently associated with a better outcome and pTERT/EGFR alterations with a worse outcome. CONCLUSION About 18% of tumors diagnosed as cGBM harbor actionable MAPK activating genetic alterations. Targeted sequencing enables to classify these tumors into clinically relevant subgroups.

Prodigiosin purification from Serratia marcescens M10 and its antitumor activities

Prodigiosin (Pg) is a bright red pigment, which is produced by gram negative and gram positive bacteria including Serratia marcescens, Hahella chejuensis, Vibrio psychroerythrus, Streptomyces coelicolor and many other marine bacteria such as Pseudomonas sp., Vibrio sp. Pg induces apoptosis in different kinds of cancer cells with low toxicity on normal cells. In this study, we purified Pg from solid fermentation of S. marcescens M10 strain and assessed its anticancer activity in tumorized mice. The results showed that Pg was purified by running through a silicagel 60 column with a suitable solvent system of n-hexane:toluene at the rate of 1:1 (v/v) combined with toluene:ethyl acetate at the rate of 9:1 (v/v). TLC plate was used to test the presence of active substances with separation solvent n-hexane:ethyl acetate ratio of 1:1 (v/v). The purity of fractions tested by high performance liquid chromatography method showed as 98%. Purified fractions also showed promising anti-tumor activity in Lewis lung carcinoma induced tumors in BALB/c mice. The tumor volumes in Pg treated groups decreased by 34.18% after 28 days of administration.

Epidemiological features of meningiomas: a single Brazilian center’s experience with 993 cases

ABSTRACT Background: Meningiomas are the most frequent primary central nervous system (CNS) tumors. Their geographical and ethnic characteristics need to be known, in order to enable rational treatment. Objective: To investigate clinical and epidemiological aspects in a series of patients with meningiomas. Methods: Retrospective analysis on the demographic profile, location and histopathology of 993 patients with meningiomas (768 operated and 225 not operated). Results: Meningiomas represented 43.8% of the primary CNS tumors; 6.8% were multiple tumors (14.7% with neurofibromatosis 2) and 0.6% were radiation-induced tumors. The mean ages were 53.0 and 63.9 years for operated and non-operated patients and the female/male ratios were 3.2:1 and 6.3:1. Diagnosis was made later among females. The peak incidences were in the 6th and 7th decades respectively for operated and non-operated patients. The incidence was low at early ages and higher among patients aged 70+ years. The meningiomas were intracranial in 96.5% and most were WHO grade I (88.9%) and transitional. In the spinal canal (3.5%), they occurred mainly in the dorsal region (all grade I; mostly transitional). The racial distribution was 1.0% in Asian-Brazilians, 87% in Caucasians and 12% in African-Brazilians. 83.4% and 51.6% of the patients were estimated to be recurrence-free at 10 and 20 years, and the mortality rate was 3%. Conclusions: Most of the demographic data were similar to what has been observed in other western centers. Differences were higher incidence of meningiomas, female and older predominance in non-operated patients, predominance in Caucasian, and higher association with neurofibromatosis 2.

Fermented Brown Rice as a Functional Food

Brown rice, especially in a part of rice bran, contains many kinds of nutrients and biologically active components such as plant polyphenols and phytic acid, but is hard to eat. “Brown rice and rice bran fermented with Aspergillus oryzae (FBRA)” is a processed food that is easier for daily intake, commercially available, and rich in eating experience. During the fermentation process, dietary fibers is partially digested, and free vitamins and phenolic compounds have increased. These fermentation products are utilized for quality control to manage FBRA production. Recently, plant-derived polyphenols have shown anti-oxidative activity and biological function in various disease models. We and other research groups used raw powder FBRA to examine its biological activity through pathological and/or molecular biological analysis. Dietary administration of FBRA showed anti-tumorigenic effects in chemically induced tumors in rodents. Anti-inflammatory effects have been observed in DSS-induced colitis in rat and inflammation-mediated rodent tumor models. I will give an outline of the characteristic of FBRA, and then introduce our recently published work about “Preventive effect of FBRA on spontaneous type 1 diabetes in NOD female mice”, including how to estimate the in vivo effect of dietary FBRA, its possible mechanisms and the limit of this study.

Less Carcinogenic Chlorinated Estrogens Applicable to Hormone Replacement Therapy

Human estrogens prescribed for hormone replacement therapy (HRT) are known to be potent carcinogens. To find safer estrogens, several chlorinated estrogens were synthesized and their carcinogenic potential were determined. A pellet containing either 2-chloro-17β-estradiol (2-ClE2) or 4-chloro-17β-estradiol (4-ClE2) was implanted subcutaneously for 52 weeks into August Copenhagen Irish (ACI) rats, a preferred animal model for human breast cancer. 17β-Estradiol (E2) frequently induced mammary tumors while both 2-ClE2 and 4-ClE2 did not. Their 17α-ethinyl forms, thought to be orally active estrogens, were also synthesized. Neither 2-chloro-17α-ethinylestradiol (2-ClEE2) nor 4-chloro-17α-ethinylestradiol (4-ClEE2) induced tumors. The less carcinogenic effects were supported by histological examination of mammary glands of ACI rats treated with the chlorinated estrogens. A chlorine atom positioned at the 2- or 4-position of E2 may prevent the metabolic activation, resulting in reducing the carcinogenicity. 2-ClE2 and 4-ClE2 administered subcutaneously and 2-ClEE2 and 4-ClEE2 given orally to ovariectomized rats all showed uterotrophic potency, albeit slightly weaker than that of E2. Our results indicate that less carcinogenic chlorinated estrogens retaining estrogenic potential could be safer alternatives to the carcinogenic estrogens now in use for HRT.