Yoshimi Shoji

Combination effects of distinct cores in 11q13 amplification region on cervical lymph node metastasis of oral squamous cell carcinoma

Lymph node metastasis (LNM) in oral squamous cell carcinoma (OSCC) is known to associate with a significant decrease of 5-year survival. Genetic factors related to the difference of the LNM status in the OSCC have been not fully elucidated. Array-based comparative genomic hybridization (CGH) with individual gene-level resolution and real-time quantitative polymerase chain reaction (QPCR) were conducted using primary tumor materials resected from 54 OSCC patients with (n=22) or without (n=32) cervical LNM. Frequent gain was observed at the 11q13 region exclusively in patients with cervical LNM, which was confirmed by real-time QPCR experiments using 11 genes (TPCN2, MYEOV, CCND1, ORAOV1, FGF4, TMEM16A, FADD, PPFIA1, CTTN, SHANK2 and DHCR7) in this region. It was revealed that two distinct amplification cores existed, which were separated by a breakpoint between MYEOV and CCND1 in the 11q13 region. The combination of copy number amplification at CTTN (core 2) and/or TPCN2/MYEOV (core 1), selected from each core, was most significantly associated with cervical LNM (P=0.0035). Two amplification cores at the 11q13 region may have biological impacts on OSCC cells to spread from the primary site to local lymph nodes. Further study of a larger patient series should be conducted to validate these results.

Nitric oxide increases the invasion of pancreatic cancer cells via activation of the PI3K–AKT and RhoA pathways after carbon ion irradiation

Previous studies have shown that serine proteases and Rho‐associated kinase contribute to carbon ion radiation‐enhanced invasion of the human pancreatic cancer cell line PANC‐1. The results presented here show that nitric oxide synthase (NOS) also plays a critical role in this process. Irradiation of PANC‐1 cells promoted invasion and production of nitric oxide (NO), which activated the PI3K–AKT signaling pathway, while independently activating RhoA. Inhibition of PI3K, Rho‐associated kinase, and serine protease alone or in conjunction with NOS suppressed the radiation‐enhanced invasion of PANC‐1 cells, suggesting that they could serve as possible targets for the management of tumor metastasis.

Carbon-Ion Irradiation Suppresses Migration and Invasiveness of Human Pancreatic Carcinoma Cells MIAPaCa-2 via Rac1 and RhoA Degradation

PURPOSE To investigate the mechanisms underlying the inhibition of cancer cell migration and invasion by carbon (C)-ion irradiation. METHODS AND MATERIALS Human pancreatic cancer cells MIAPaCa-2, AsPC-1, and BxPC-3 were treated by x-ray (4 Gy) or C-ion (0.5, 1, 2, or 4 Gy) irradiation, and their migration and invasion were assessed 2 days later. The levels of guanosine triphosphate (GTP)-bound Rac1 and RhoA were determined by the active GTPase pull-down assay with or without a proteasome inhibitor, and the binding of E3 ubiquitin ligase to GTP-bound Rac1 was examined by immunoprecipitation. RESULTS Carbon-ion irradiation reduced the levels of GTP-bound Rac1 and RhoA, 2 major regulators of cell motility, in MIAPaCa-2 cells and GTP-bound Rac1 in AsPC-1 and BxPC-3 cells. Proteasome inhibition reversed the effect, indicating that C-ion irradiation induced Rac1 and RhoA degradation via the ubiquitin (Ub)-proteasome pathway. E3 Ub ligase X-linked inhibitor of apoptosis protein (XIAP), which directly targets Rac1, was selectively induced in C-ion--irradiated MIAPaCa-2 cells and coprecipitated with GTP-bound Rac1 in C-ion--irradiated cells, which was associated with Rac1 ubiquitination. Cell migration and invasion reduced by C-ion radiation were restored by short interfering RNA--mediated XIAP knockdown, indicating that XIAP is involved in C-ion--induced inhibition of cell motility. CONCLUSION In contrast to x-ray irradiation, C-ion treatment inhibited the activity of Rac1 and RhoA in MIAPaCa-2 cells and Rac1 in AsPC-1 and BxPC-3 cells via Ub-mediated proteasomal degradation, thereby blocking the motility of these pancreatic cancer cells.

A Genotoxic Stress-Responsive miRNA, miR-574-3p, Delays Cell Growth by Suppressing the Enhancer of Rudimentary Homolog Gene in Vitro

MicroRNA (miRNA) is a type of non-coding RNA that regulates the expression of its target genes by interacting with the complementary sequence of the target mRNA molecules. Recent evidence has shown that genotoxic stress induces miRNA expression, but the target genes involved and role in cellular responses remain unclear. We examined the role of miRNA in the cellular response to X-ray irradiation by studying the expression profiles of radio-responsive miRNAs and their target genes in cultured human cell lines. We found that expression of miR-574-3p was induced in the lung cancer cell line A549 by X-ray irradiation. Overexpression of miR-574-3p caused delayed growth in A549 cells. A predicted target site was detected in the 3′-untranslated region of the enhancer of the rudimentary homolog (ERH) gene, and transfected cells showed an interaction between the luciferase reporter containing the target sequences and miR-574-3p. Overexpression of miR-574-3p suppressed ERH protein production and delayed cell growth. This delay was confirmed by knockdown of ERH expression. Our study suggests that miR-574-3p may contribute to the regulation of the cell cycle in response to X-ray irradiation via suppression of ERH protein production.

Biological Effectiveness of Carbon-Ion Radiation on Various Human Breast Cancer Cell Lines

Introduction: Carbon-ion radiotherapy (C-ion RT) is known as a highly effective local treatment and its relative biological effectiveness (RBE) has been evaluated for various types of malignant tumors. There are only a few studies on C-ion radio sensitivity in breast cancer, and there has been no evaluation by subtypes. To estimate the impact of C-ion RT for breast cancer, RBE of C-ion beams of various types of human breast cancer cell lines was evaluated by comparison with X-rays. Methods: Six human breast cancer cell lines with different subtypes, Luminal-human epidermal growth factor receptor 2 (HER2)-negative (MCF-7), Luminal-HER2-positive (BT-474), Her2-enriched (SK-BR-3), Basal-like (MDAMB- 468, HCC1937) and ductal carcinoma in situ (MCF10DCIS.com) were used. Radio sensitivities were assessed with survival curves created from colony-forming assay (CFA) and high-density surviving assay (HDS). An X-ray generator was used with 200 kV, 20 mA. The Heavy Ion Medical Accelerator in Chiba (HIMAC) was used for C-ion irradiation, with 290 MeV/u, mono-peak, linear energy transfer (LET) of 80 KeV/μm. Results: CFA was not suitable for BT474, SK-BR-3, MDA-MB-468, and HCC1937 because of their low plating efficiency. The differences between the D10 values on HDS were large with X-ray, and the survival curve shoulders for MCF7, MDA-MB-468, and MCF10DCIS.com were wide. On the other hand, the differences between the D10 values were small with C-ion beams, and the survival curves were linear without shoulders for all cell lines except a small shoulder with MCF10DCIS.com. The RBE value of C-ion beams was 2.3 to 3.6, median 2.9 in all cell lines by CFA and HDS. Conclusion: RBE around 3 by C-ion beams was seen in many types of ductal cancer. The small survival curve shoulder on MCF10DCIS.com suggested that non invasive ductal carcinoma is relatively more resistant than invasive cancer.

Effects of carbon ion irradiation and X-ray irradiation on the ubiquitylated protein accumulation

C-ion radiotherapy is associated with improved local control and survival in several types of tumors. Although C-ion irradiation is widely reported to effectively induce DNA damage in tumor cells, the effects of irradiation on proteins, such as protein stability or degradation in response to radiation stress, remain unknown. We aimed to compare the effects of C-ion and X-ray irradiation focusing on the cellular accumulation of ubiquitylated proteins. Cells from two human colorectal cancer cell lines, SW620 and SW480, were subjected to C-ion or X-ray irradiation and determination of ubiquitylated protein levels. High levels of ubiquitylated protein accumulation were observed in the C-ion-irradiated SW620 with a peak at 3 Gy; the accumulation was significantly lower in the X-ray-irradiated SW620 at all doses. Enhanced levels of ubiquitylated proteins were also detected in C-ion or X-ray-irradiated SW480, however, those levels were significantly lower than the peak detected in the C-ion-irradiated SW620. The levels of irradiation-induced ubiquitylated proteins decreased in a time-dependent manner, suggesting that the proteins were eliminated after irradiation. The treatment of C-ion-irradiated SW620 with a proteasome inhibitor (epoxomicin) enhanced the cell killing activity. The accumulated ubiquitylated proteins were co-localized with γ-H2AX, and with TP53BP1, in C-ion-irradiated SW620, indicating C-ion-induced ubiquitylated proteins may have some functions in the DNA repair system. Overall, we showed C-ion irradiation strongly induces the accumulation of ubiquitylated proteins in SW620. These characteristics may play a role in improving the therapeutic ratio of C-ion beams; blocking the clearance of ubiquitylated proteins may enhance sensitivity to C-ion radiation.

Abstract 4140: Role of nitric oxide in invasiveness of tumor cells irradiated with carbon-ion beams

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA We previously reported that carbon-ion (C-ion) irradiation suppresses the invasiveness of several pancreatic cancer cell lines such as MIAPaCa-2, BxPC-3, and AsPC-1; however, we also observed that C-ion irradiation enhanced PANC-1 invasion. Most invading PANC-1 cells were nitric oxide (NO)-producing cells; the NOS-NO-PI3K-AKT pathway was activated in these cells. In addition, the actin-binding AKT substrate, Girdin, which modulates the actin organization and has important role in AKT-dependent cell motility, was activated and co-localized with p-AKT at the lamellipodia. C-ion irradiation increased the NO-producing cell population, thereby enhancing C-ion irradiation-enhanced PANC-1 invasion. In contrast to PANC-1 cells, NO levels as well as the number of NO-producing cells were decreased in C-ion irradiated MIAPaCa-2, indicated that the NO reduction might cause the suppression of MIAPaCa-2 invasion. The aim of this study is to clarify the role of NO in altered invasiveness of C-ion irradiated tumor cells. Treatment of PANC-1 with NO donor, DETA/NO, enhanced PANC-1 invasion. Interestingly, DETA/NO also increased MIAPaCa-2 invasion, indicated that NO contributes to the enhancement of MIAPaCa-2 invasiveness. The reduced invasiveness of C-ion irradiated MIAPaCa-2 was slightly but significantly recovered by the treatment with DETA/NO, but the recovered levels were still much less than the invasiveness of non-irradiated MIAPaCa-2. Thus, the reduction of NO levels involves in the decreased invasiveness of C-ion irradiated MIAPaCa-2, but there are additional factor regulating the invasiveness of cells irradiated with C-ion beams. Indeed, C-ion irradiation reduced GTP-Rac1 and GTP-RhoA expression, the active form of Rac1 and RhoA, which are known as two master regulators of cell motility. The reduction of GTP-Rac1 or GTP-RhoA was recovered by the treatment of proteasome inhibitor, indicated that those proteins were undergo degradation via the ubiquitin-proteasome pathway. So far, IAPs, Inhibitors of Apoptosis Proteins, and HACE1, HECT-domain containing E3 ubiquitin-ligase, were reported as direct E3 ubiquitin ligase of Rac1. Of those, XIAP was selectively induced and was co-precipitated with GTP-Rac1 in C-ion irradiated MIAPaCa-2. In conclusion, NO has a significant role in enhancing invasive potential of both PANC-1 and MIAPaCa-2 cells. The alteration of NO levels upon C-ion irradiation modulates the invasiveness of irradiated cells. Also, reductions of GTP-Rac1 and GTP-RhoA have the additional effects on the C-ion reduced MIAPaCa-2 invasion. Several studies have reported that NO modulates ubiquitin-proteasomal degradation of proteins. The effect of NO on the GTP-Rac1 and GTP-RhoA degradation remain unknown, and further researches are needed to clarify whether the NO reduction affects to the GTP-Rac1 or GTP-RhoA degradation observed in C-ion irradiated MIAPaCa-2. Citation Format: Mayumi Fujita, Kaori Imadome, Yoshimi Shoji, Robert Cheng, Aparna H. Kesarwala, David A. Wink, Takashi Imai. Role of nitric oxide in invasiveness of tumor cells irradiated with carbon-ion beams. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4140. doi:10.1158/1538-7445.AM2015-4140