Norihiko Kawamura

Regulation of alternative polyadenylation by Nkx2-5 and Xrn2 during mouse heart development

Transcription factors organize gene expression profiles by regulating promoter activity. However, the role of transcription factors after transcription initiation is poorly understood. Here, we show that the homeoprotein Nkx2-5 and the 5’-3’ exonuclease Xrn2 are involved in the regulation of alternative polyadenylation (APA) during mouse heart development. Nkx2-5 occupied not only the transcription start sites (TSSs) but also the downstream regions of genes, serving to connect these regions in primary embryonic cardiomyocytes (eCMs). Nkx2-5 deficiency affected Xrn2 binding to target loci and resulted in increases in RNA polymerase II (RNAPII) occupancy and in the expression of mRNAs with long 3’untranslated regions (3’ UTRs) from genes related to heart development. siRNA-mediated suppression of Nkx2-5 and Xrn2 led to heart looping anomaly. Moreover, Nkx2-5 genetically interacts with Xrn2 because Nkx2-5+/-Xrn2+/-, but neither Nkx2-5+/-nor Xrn2+/-, newborns exhibited a defect in ventricular septum formation, suggesting that the association between Nkx2-5 and Xrn2 is essential for heart development. Our results indicate that Nkx2-5 regulates not only the initiation but also the usage of poly(A) sites during heart development. Our findings suggest that tissue-specific transcription factors is involved in the regulation of APA. DOI: http://dx.doi.org/10.7554/eLife.16030.001

Peripheral blood monocyte count reflecting tumor-infiltrating macrophages is a predictive factor of adverse pathology in radical prostatectomy specimens.

Tumor‐infiltrating macrophages, which are thought to be derived from blood monocytes, interact with tumor cells to promote cancer progression. The aim of this study was to assess the association of peripheral blood monocyte count with pathological findings and local tumor‐infiltrating macrophages in prostatectomy specimens.

MP99-06 HIGH FAT DIET-INDUCED INFLAMMATION ACCELERATES TUMOR PROGRESSION IN MICE MODEL FOR PROSTATE CANCER

retention at DSB sites in prostate cancer cells, and also impaired recruitment of Ku70/Ku80 to DSB sites. The impaired recruitment of Ku70 and Ku80 proteins to DNA damage sites upon ELL2 knockdown was rescued by re-expression of an ELL2 transgene insensitive to siELL2. CONCLUSIONS: This study suggests that ELL2 is an important factor mediating androgen protection of DNA damage via Ku70/Ku80 in prostate cancer cells.

MP73-02 IMMUNOLOGICAL CLASSIFICATION IN RENAL CELL CARCINOMA BASED ON IMMUNOCHECKPOINT MOLECULES: THE RELATIONSHIP WITH TUMOR AGGRESSIVENESS AND THE PRESENCE OF INTRA-TUMOR DIVERSITY

INTRODUCTION AND OBJECTIVES: Immune checkpoint inhibitors-based therapy is rapidly developing into an effective treatment option for a surprising range of cancers. Despite the autoimmune or inflammatory immune-mediated adverse effects which have been seen, the responses and overall survival benefits exhibited thus far warrant further clinical development. Lycorine, an alkaloid extracted from plants in the Amaryllidaceae family, which showed a strong anticancer effect by inducing cell lysis of various carcinomas. This study investigated the effectiveness of a combination therapy of lycorine and anti-CTLA-4 in a mouse model of renal cell carcinoma (RCC). METHODS: We investigated the anti-tumor efficacy of lycorine in various RCC cell lines including Caki-1, ACHN, KPK-1 and Renca through XTT proliferation, scratch motility, migration and invasion assays. We also discuss that mechanism underlying this anticancer potential with flow cytometry and western blot. Furthermore, luciferase-expressing Renca cells were implanted in the left kidney and the lung of BALB/c mice to develop a RCC metastatic mouse model for investigated the combination therapy effect of lycorine and antiCTLA-4. RESULTS: We confirmed the anticancer potential of lycorine in vitro as observed time-dependent inhibition in several RCC cell lines. Moreover, lycorine suppressed the migratory and invasive abilities of Renca cells. The possible mechanism underlying this anticancer potential was cell cycle profile arrest. Lycorine and anti-CTLA-4 additively decreased tumor weight, lung metastasis, and luciferin-stained tumor images. Importantly, these effects were dependent on significantly suppressing regulatory T-cells while upregulating effector T-cells. CONCLUSIONS: Herein, as we know, it’s the first report that lycorine in combination with anti-CTLA-4 inhibited orthotopic and metastatic tumors by downregulating Treg, which was accompanied with upregulation of effector T-cells. Our findings suggest and indicate lycorine as a potent candidate for treating RCC and will serve as an excellent aid for developing a better treatment strategy for the use of immune checkpoint inhibitors in RCC.

Abstract 2911: Gene knockout of NANOG and NANOGP8 mediated by CRISPR/Cas9 system decreases the malignant potential of prostate cancer cells

Introduction & objectives : NANOG is an essential transcription factor for self-renewal and pluripotency of embryonal stem cells. Recently, it has been reported that NANOG is expressed in various somatic cancers, including prostate cancer, and drive tumor development, and that increased NANOG expression in human prostate cancer tissues is correlated with an increased Gleason score. NANOG (hereinafter NANOG1) has many pseudogenes, and only the NANOGP8 pseudogene encodes the full-length NANOG1 protein with high sequence similarity. NANOGP8 is reported to be expressed in most types of cancer as a primary contributor of NANOG mRNA expression and to increase the malignant potential. However, the proportion of NANOG protein expression that comes from NANOG1 and NANOG8 in cancer cells is not known because of the high similarity between them. Therefore, a causal role of NANOG1 and NANOGP8 in prostate cancer cells is not clear. Materials & methods : We established NANOG1-/- and NANOGP8-/- prostate cancer cell lines from DU145 cells using CRISPR/Cas9, and also we established NANOG1-rescued cells and NANOGP8-rescued cells from each NANOG knockout cells for rescue experiments. We examined cancer properties associated with malignant potential in these cells and DU145 cells, including self-renewal, sphere-formation, migration, drug resistance and tumorigenic potential. Results : Colony formation assays were performed to examine the role of NANOG1 and NANOGP8 in self-renewal. The colony-forming capacity of NANOG1-/- and NANOGP8-/- cells was decreased compared to parental cells. Similary, the sphere-forming capacity of NANOG1-/- and NANOGP8-/- cells decreased to approximately 50% compared to the parental cells. Wound-healing assays were performed to examine the migration capacity, and migration was decreased in NANOG1-/- and NANOGP8-/- cells by 40-60%. MTS assays 48 hours after docetaxel administration were performed to evaluate the effect of NANOG1 and NANOGP8 on drug sensitivity. NANOG1-/- and NANOP8-/- cells showed increased sensitivity to docetaxel. NANOG1 and NANOGP8 knockout did not inhibit in vitro cell proliferation, but in vivo tumorigenic potential decreased significantly. These phenotypes were recovered in NANOG1- and NANOGP8-rescued cell lines. Conclusion : These results indicate that both NANOG1 and NANOGP8 proteins are expressed in prostate cancer cell lines, and both genes equally contribute to the high malignant potential of prostate cancer cells. Citation Format: Norihiko Kawamura, Keisuke Nimura, Atsunari Kawashima, Takeshi Ujike, Akira Nagahara, Kazutoshi Fujita, Motohide Uemura, Yasufumi Kaneda, Norio Nonomura. Gene knockout of NANOG and NANOGP8 mediated by CRISPR/Cas9 system decreases the malignant potential of prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2911.