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Summary We here demonstrate that microsatellite (MS) alterations are elevated in both mouse and human induced pluripotent stem cells (iPSCs), but importantly we have now identified a type of human iPSC in which these alterations are considerably reduced. We aimed in our present analyses to profile the InDels in iPSC/ntESC genomes, especially in MS regions. To detect somatic de novo mutations in particular, we generated 13 independent reprogramed stem cell lines (11 iPSC and 2 ntESC lines) from an identical parent somatic cell fraction of a C57BL/6 mouse. By using this cell set with an identical genetic background, we could comprehensively detect clone-specific alterations and, importantly, experimentally validate them. The effectiveness of employing sister clones for detecting somatic de novo mutations was thereby demonstrated. We then successfully applied this approach to human iPSCs. Our results require further careful genomic analysis but make an important inroad into solving the issue of genome abnormalities in iPSCs., Graphical abstract, Highlights • InDels and microsatellite alterations are elevated in iPSCs • These alterations are reduced in human iPSCs derived from cord blood erythroblasts • Employing sister clones is an effective way to detect somatic de novo mutations, In this article, Abe and colleagues conclusively demonstrate that InDels and microsatellite alterations are elevated in reprogrammed pluripotent stem cells, both mouse and human, by employing sister clones and conducting large-scale validation experiments. Furthermore, they show that these alterations are considerably reduced in human iPSCs derived from cord blood erythroblasts.

We here demonstrate that microsatellite (MS) alterations are elevated in both mouse and human induced pluripotent stem cells (iPSCs), but importantly we have now identified a type of human iPSC in which these alterations are considerably reduced. We aimed in our present analyses to profile the InDels in iPSC/ntESC genomes, especially in MS regions. To detect somatic de novo mutations in particular, we generated 13 independent reprogramed stem cell lines (11 iPSC and 2 ntESC lines) from an identical parent somatic cell fraction of a C57BL/6 mouse. By using this cell set with an identical genetic background, we could comprehensively detect clone-specific alterations and, importantly, experimentally validate them. The effectiveness of employing sister clones for detecting somatic de novo mutations was thereby demonstrated. We then successfully applied this approach to human iPSCs. Our results require further careful genomic analysis but make an important inroad into solving the issue of genome abnormalities in iPSCs.

A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions., Point mutations have been found in induced pluripotent stem cells (iPSCs) but when they arise is unclear. Here, the authors show that a G1/S cell cycle checkpoint deficiency transiently occurs early in genome reprogramming, suggesting a common developmental pathway between iPSC and tumorigenesis, and generate genetic burden-free human iPSCs.

Highlights • Angiosarcoma is associated with a poor prognosis and is treated with radiotherapy. • Highly stable FGF1 mutants exhibit stronger mitogenic activity than wild-type FGF1 and are candidates for radioprotectors. • They were examined as strong signaling agonists to clarify the effects of FGF1 on the murine angiosarcoma cell line ISOS-1. • Strong FGF1 signaling reduced the proliferative, invasive, and migration capabilities of ISOS-1cells. • Their malignancy was reduced through the dual inhibition of EGFR and VEGFR pathways by strong FGF1 signaling., Background and purpose Angiosarcoma is associated with a poor prognosis and is treated with radiotherapy. Although FGF1 is a potential radioprotector, the influence of FGF1 on the malignancy of angiosarcoma remains unknown. Materials and methods Highly stable FGF1 mutants, which exhibit stronger mitogenic activity than wild-type FGF1, were examined as strong radioprotectors and signaling agonists to clarify the effects of FGF1 on the murine angiosarcoma cell line ISOS-1. Results FGF1 mutants reduced colony formation by and the in vitro invasion and migration of ISOS-1 cells, in addition to an increase in radiosensitivity to X-rays. In contrast, an FGFR inhibitor blocked the inhibitory effects of FGF1 mutants on colony formation, invasion, and migration. siRNA targeting the Fgfr1 gene showed that strong FGFR1 signaling reduced colony formation by ISOS-1 cells. However, the FGF1 mutant reduced the activation of VEGFRs and EGFRs in ISOS-1 cells more strongly than wild-type FGF1. Moreover, the inhibition of VEGFRs and EGFRs synergistically reduced colony formation by and invasion and migration of ISOS-1 cells. Conclusion These results suggest that strong FGF1 signaling exerts not only radioprotective effects, but also inhibitory effects on proliferative and metastatic capacities of angiosarcoma through the dual inhibition of EGFR and VEGFR pathways.

Pancreatic cancer is a highly metastatic tumor with an extremely low 5-year survival rate. Lack of efficient diagnostics and dearth of effective therapeutics that can target the cancer as well as the microenvironment niche are the reasons for limited success in treatment and management of this disease. Cell invasion through extracellular matrix (ECM) involves the complex regulation of adhesion to and detachment from ECM and its understanding is critical to metastatic potential of pancreatic cancer. To understand the characteristics of these cancer cells and their ability to metastasize, we compared human pancreatic cancer cell line, PANC-1 and its invading phenotype (INV) collected from transwell inserts. The invasive cell type, INV, exhibited higher resistance to Carbon-ion radiation compared to whole cultured (normally dish-cultured) PANC-1 (WCC), and had more efficient in vitro spheroid formation capability. Invasiveness of INV was hampered by nitric oxide synthase (NOS) inhibitors, suggesting that nitric oxide (NO) plays a cardinal role in PANC-1 invasion. In addition, in vitro studies indicated that a MEK-ERK-dependent, JAK independent mechanism through which NOS/NO modulate PANC-1 invasiveness. Suspended INV showed enhanced NO production as well as induction of several pro-metastatic, and stemness-related genes. NOS inhibitor, l-NAME, reduced the expression of these pro-metastatic or stemness-related genes, and dampened spheroid formation ability, suggesting that NO can potentially influence pancreatic cancer aggressiveness. Furthermore, xenograft studies with INV and WCC in NSG mouse model revealed a greater ability of INV compared to WCC, to metastasize to the liver and l-NAME diminished the metastatic lesions in mice injected with INV. Overall, data suggest that NO is a key player associated with resistance to radiation and metastasis of pancreatic cancer; and inhibition of NOS demonstrates therapeutic potential as observed in the animal model by specifically targeting the metastatic cells that harbor stem-like features and are potentially responsible for relapse., Highlights • Highly invasive pancreatic cancer cell line, collected from transwell inserts showed increased resistance to C-ion radiation. • NO is a key player in pancreatic cancer aggressiveness inducing pro-metastatic and stemness-related genes. • NOS/NO modulate invasiveness through a MEK-ERK dependent, JAK signaling independent mechanism. • NOS inhibition showed promising therapeutic potential in mouse model by reversing the pro-metastatic phenotype.

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.

Background: Metabolic reprogramming is being recognized as a fundamental hallmark of cancer, and efforts to identify drugs that can target cancer metabolism are underway. In this study, we used human breast cancer (BC) cell lines and established their invading phenotype (INV) collected from transwell inserts to compare metabolome differences and evaluate prognostic significance of the metabolome in aggressive BC invasiveness. Methods: The invasiveness of seven human BC cell lines were compared using the transwell invasion assay. Among these, INV was collected from SUM149, which exhibited the highest invasiveness. Levels of metabolites in INV were compared with those of whole cultured SUM149 cells (WCC) using CE-TOFMS. The impact of glycolysis in INV was determined by glucose uptake assay using fluorescent derivative of glucose (2-NBDG), and significance of glycolysis, or tricarboxylic acid cycle (TCA) and electron transport chain (ETC) in the invasive process were further determined in aggressive BC cell lines, SUM149, MDA-MB-231, HCC1937, using invasion assays in the presence or absence of inhibitors of glycolysis, TCA cycle or ETC. Results: SUM149 INV sub-population exhibited a persistent hyperinvasive phenotype. INV were hyper-glycolytic with increased glucose (2-NBDG) uptake; diminished glucose-6-phosphate (G6P) levels but elevated pyruvate and lactate, along with higher expression of phosphorylated-pyruvate dehydrogenase (pPDH) compared to WCC. Notably, inhibiting of glycolysis with lower doses of 2-DG (1 mM), non-cytotoxic to MDA-MB-231 and HCC1937, was effective in diminishing invasiveness of aggressive BC cell lines. In contrast, 3-Nitropropionic acid (3-NA), an inhibitor of succinate dehydrogenase, the enzyme that oxidizes succinate to fumarate in TCA cycle, and functions as complex II of ETC, had no significant effect on their invasiveness, although levels of TCA metabolites or detection of mitochondrial membrane potential with JC-1 staining, indicated that INV cells originally had functional TCA cycles and membrane potential. Conclusions: Hyper-glycolytic phenotype of invading cells caters to rapid energy production required for invasion while TCA cycle/ETC cater to cellular energy needs for sustenance in aggressive BC. Lower, non-cytotoxic doses of 2-DG can hamper invasion and can potentially be used as an adjuvant with other anti-cancer therapies without the usual side-effects associated with cytotoxic doses.

Pancreatic cancer is a metastatic tumor with an extremely low 5-year survival rate. Metastasis is a main cause of patient mortality as it is extremely difficult to treat. Thus, understanding the characteristics of cancer cell populations exhibiting the invasive phenotype is fundamental for developing novel strategies to counter metastasis and improve therapeutic efficacy. To characterize these invasive phenotypes, we compared the human PANC-1 parent cell line with PANC-1 cells (INV) that invaded through transwell inserts. INV cells exhibited increased invasiveness and higher resistance to Carbon-ion radiation compared to whole cultured, control PANC-1 cells (WCC). PANC-1 invasion was reduced by nitric oxide synthase (NOS) inhibitors, suggesting that nitric oxide (NO) plays a cardinal role in PANC-1 invasion. Cell invasion through extracellular membrane (ECM) involves complex regulation of cell adhesion and de-adhesion to ECM proteins. Suspended INV cells, as well as INV cells undergoing de-adhesion, showed enhanced NO production as well as induction of several pro-metastatic, and stemness-related genes. NOS inhibitor, L-NAME, reduced the expression of these pro-metastatic or stemness-related genes, and hampered spheroid formation ability, suggesting that NO can potentially influence PANC-1 aggressiveness. In addition, in vitro studies indicated that a MEK-ERK-dependent, JAK-independent mechanism promoted NOS/NO modulation of PANC-1 invasion. Furthermore, xenograft studies with INV and WCC in an NSG mouse model revealed a higher ability of INV in liver metastasis than WCC, and L-NAME diminished the metastasis observed in INV injected mice. In summary, these results implicate NO as a key mediator of therapeutic resistance to Carbon-ion radiation and metastasis of PANC-1 cells; inhibition of NOS demonstrates therapeutic potential as observed in the animal model by specifically targeting the metastatic cells. Citation Format: Mayumi Fujita, Veena Somasundaram, Debashree Basudhar Basudhar, Robert Y.s. Cheng, Lisa A. Ridnour, Kaori Imadome, Jae Hong No, Gaurav Bharadwaj, David A. Wink. Role of nitric oxide in the invasive pancreatic cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6071.

A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions.

We previously reported that about 0.4% of cells in the cultured human pancreatic cancer cell line, PANC-1, can invade matrigel during the transwell invasion assay, suggesting that these invaded PANC-1 cells may have specific characteristics to keep their invasive potential. To identify the metabolic characterization specific in the invaded PANC-1 cells, metabolome analysis of the invaded PANC-1 compared with the whole cultured PANC-1 was performed using CE-TOFMS, and concentrations of 110 metabolites were measured. In contrast to the whole cultured cells, the invaded PANC-1 was characterized as a population with reduced levels of amino acids and TCA cycle intermediates, and decreased and increased intermediates in glycolysis and nucleic acid metabolism. In particular, the ratio of both adenosine and guanosine energy charge was reduced in the invaded cells, revealing that the consumption of ATP and GTP was high in the invaded cells, and thus suggesting that ATP- or GTP-generating pathways are stimulated. In addition, the GSH/GSSG ratio was low in the invaded cells, but these cells had a higher surviving fraction after exposure to hydrogen peroxide. Thus, the invaded cells were the population resistant to oxidative stress. Furthermore, reduction in intracellular GSH content inhibited PANC-1 invasiveness, indicated that GSH has an important role in PANC-1 invasiveness. Overall, we propose the invaded cells have several unique metabolic profiles. This article is protected by copyright. All rights reserved.

PURPOSE: To examine whether inherent factors produce differences in lung morbidity in response to carbon ion (C-ion) irradiation, and to identify the molecules that have a key role in strain-dependent adverse effects in the lung. \nMETHODS AND MATERIALS: Three strains of female mice (C3H/He Slc, C57BL/6J Jms Slc, and A/J Jms Slc) were locally irradiated in the thorax with either C-ion beams (290 MeV/n, in 6 cm spread-out Bragg peak) or with (137)Cs gamma-rays as a reference beam. We performed survival assays and histologic examination of the lung with hematoxylin-eosin and Masson's trichrome staining. In addition, we performed immunohistochemical staining for hyaluronic acid (HA), CD44, and Mac3 and assayed for gene expression. \nRESULTS: The survival data in mice showed a between-strain variance after C-ion irradiation with 10 Gy. The median survival time of C3H/He was significantly shortened after C-ion irradiation at the higher dose of 12.5 Gy. Histologic examination revealed early-phase hemorrhagic pneumonitis in C3H/He and late-phase focal fibrotic lesions in C57BL/6J after C-ion irradiation with 10 Gy. Pleural effusion was apparent in C57BL/6J and A/J mice, 168 days after C-ion irradiation with 10 Gy. Microarray analysis of irradiated lung tissue in the three mouse strains identified differential expression changes in growth differentiation factor 15 (Gdf15), which regulates macrophage function, and hyaluronan synthase 1 (Has1), which plays a role in HA metabolism. Immunohistochemistry showed that the number of CD44-positive cells, a surrogate marker for HA accumulation, and Mac3-positive cells, a marker for macrophage infiltration in irradiated lung, varied significantly among the three mouse strains during the early phase. \nCONCLUSIONS: This study demonstrated a strain-dependent differential response in mice to C-ion thoracic irradiation. Our findings identified candidate molecules that could be implicated in the between-strain variance to early hemorrhagic pneumonitis after C-ion irradiation.

Purpose: To clarity how carbon-ion radiotherapy on primary tumors affects the characteristics of subsequently-arising metastatic tumor cells. Methods and Materials: Mouse squamous cell carcinomas, NR-S1, in synergic C3H/HeMsNrs mice were irradiated with a single dose of 5-50 Gy of carbon-ions (C-ion) (290 MeV/nucleon, 6 cm spread-out Bragg peak) or gamma-rays (GAMMA-rays) (137Cs source) as a reference beam. The volume of the primary tumors and the number of metastatic nodules in lung were studied, and histological analysis and microarray analysis of laser-microdissected tumor cells were also performed. Results: Including 5 Gy of C-ion and 8 Gy of GAMMA-rays, which did not inhibit the primary tumor growth, all doses used in this study inhibited lung metastasis significantly. Pathological findings showed no difference among the metastatic tumor nodules in the non-irradiated, carbon-ion-irradiated, and GAMMA- ray-irradiated groups. Clustering analysis of expression profiles among metastatic tumors and primary tumors revealed a single cluster consisting of metastatic tumors different from their original primary tumors, indicating that the expression profiles of the metastatic tumor cells were not affected by the local application of C-ion radiotherapy or GAMMA-ray radiotherapy. Conclusion: We found no difference in the incidence and histology, and only small differences in expression profile, of distant metastasis between local C-ion and GAMMA-ray radiotherapy. The application of local radiotherapy per se or the type of radiotherapy applied did not influence the transcriptional changes caused by metastasis in tumor cells.

Purpose: To elucidate the molecular changes in response to carbon beams (C-ions) in melanoma. Materials and methods: We examined expression profiles of 6 melanoma cell lines exposed to C-ions or X-rays with 2 Gy using single-color microarrays. Results: Twenty-two genes, including nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA), responded to C-ions in all six cell lines, based on analysis of variance (ANOVA) filtering (p < 0.001). We found 173 genes that responded in common to C-ions in four cell lines. We identified many down-regulated genes including the cell cycle - related genes that were more responsive to C-ions than X-rays. In contrast, most of the up-regulated genes including the tumor protein p53 (p53) target genes responded to both C-ions and X-rays. C-ions induced G2/M arrest significantly more than X-rays at 30 h (p < 0.05). Conclusion: Our findings suggest that down-regulation of gene expression plays a key role in the response to C-ions. Regulation of cell cycle - related genes and induction of prolonged G2/M arrest may be responsible for the extra sensitivity to C-ions, whereas p53-related genes may have similar roles in the sensitivities to both C-ions and X-rays.

Objective: To elucidate the in vivo biological effects induced by carbon-ion irradiation using comprehensive expression analysis. \nMaterials and Methods: We examined gene expression changes after carbon-ion (C-ion) irradiation (290 MeV/m, SOBP 6 cm middle, 50 kev/MUm) with a single dose of 30 Gy in four mouse tumors (NR-S1, SCCVII, NFSa, and #8520) transplanted into the hind legs of C3H/HeNrs mice, using 44K single-color oligo-microarrays at 6 hours (h), 1 day, and 3 days after irradiation. Gamma rays of 30 Gy and 50 Gy were used as a reference beam. Identification of C-ion-responsive genes was based on a false discovery rate of < 5% using the Wilcoxon test (P < 0.001) and the Benjamini-Hochberg correction. \nResults: In all tumors, the level of expression of several tens of genes, including Ccl3, Ccng1, Cd80, Cdkn1a, Cxcl2, IL7r, Lrdd, Mgmt, Mmp8, and Polk, was significantly altered 6 h and day 1 following C-ion irradiation. At day 3, several hundred genes, many of which are also classified as stress-response or cell-communication genes, including Tnfrsf5, Ikbke, and Icam1, were upregulated following C-ion irradiation. The expression level of the majority of these genes was similar following GAMMA-ray treatment, although the change was not as extensive and intertumor variance was apparent. Several genes, including Ikbke, Serpina3n, and Saa3, responded differentially following C-ion irradiation than after GAMMA-ray irradiation. Pathological investigation and immunohistochemical analysis of Cdkn1a revealed cell cycle arrest with mitotic catastrophe in tumors irradiated by C-ions. \nConclusions: This study revealed significant C-ion induced upregulation of stress-responsive and cell-communication genes common to different tumor types. These findings provide evidence for the efficacy of this modality for the treatment of local tumors.

Objective: To investigate the protein expression change of FGF2 in cervical cancers during chemoradiotherapy, as indicated in our previous study using microarray analysis. In addition, we sought to examine the predictive value of such changes in expression for disease failure after chemoradiotherapy. Patients and Methods: Biopsy specimens were obtained from 35 patients with cervical cancers before (pretreatment) and 1 week after initiation (midtreatment) of chemoradiotherapy (CRT) (9 Gy and 40 mg/m2 of cisplatin). Immunohistochemical studies (IHS) were performed to detect FGF2, laminin, and CD44 expression using an automated streptavidin-biotin immunoperoxidase staining system. Positive area proportion (%) of FGF2 and CD44 were analyzed using an image analysis system, and laminin staining pattern was scored by continuity of the basement membrane immunopositivity. Patients were defined as good (n =18) or poor responders (n =10) based on their two-year disease-free survival. Results: Protein expression of FGF2 in midtreatment samples (mid) was significantly higher than in pretreatment samples (pre). Discontinuity of laminin staining pattern in mid was significantly higher than in pre. Protein expression of CD44 was not significantly different between mid and pre. The ratio change (mid versus pre) of FGF2 expression in poor responders was significantly lower than that in good responders (P < 0.05). The number of cases with discontinuity of laminin staining pattern at pre was significantly increased in the poor responders (P < 0.05). Ratio changes of FGF2 or CD44 expression in mid correlated with laminin staining pattern in pre. Conclusions: Using biopsy specimens from pretreatment and midtreatment cervical cancers, we revealed significant changes in FGF2 protein expression during fractionated radiotherapy with cisplatin. We also found that FGF2 ratio change and laminin discontinuity staining pattern at pretreatment were significantly associated with prognosis. These molecular features might help us to identify patients at high risk of disease failure after CRT.

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.

Pancreatic cancer is an aggressive disease that responds poorly to conventional photon radiotherapy. Carbon-ion (C-ion) radiation has advantages compared with conventional radiotherapy, because it enables more accurate dose distribution and more efficient tumor cell killing. To elucidate the effects of local radiotherapy on the characteristics of metastatic tumors, it is necessary to understand the nature of motility in irradiated tumor cells; this will, in turn, facilitate the development of effective strategies to counter tumor cell motility, which can be used in combination with radiotherapy. The aim of the present study was to examine the invasiveness of pancreatic cancer cells exposed to C-ion irradiation. We found that C-ion irradiation suppressed the migration of MIAPaCa-2, BxPC-3 and AsPC-1; diminished the invasiveness of MIAPaCa-2; and tended to reduce the invasion of BxPC-3 and AsPC-1. However, C-ion irradiation increased the invasiveness of PANC-1 through the activation of plasmin and urokinase-type plasiminogen activator. Administration of serine protease inhibitor (SerPI) alone failed to reduce C-ion-induced PANC-1 invasiveness, whereas the combination of SerPI and Rho-associated coiled-coil forming protein kinase (ROCK) inhibitor suppressed it. Furthermore, PANC-1 showed mesenchymal–amoeboid transition when we treated with SerPI alone. In conclusion, C-ion irradiation is effective in suppressing the invasive potential of several pancreatic tumor cell lines, but not PANC-1; this is the first study showing that C-ion irradiation induces the invasive potential of a tumor cell line. Further in vivo studies are required to examine the therapeutic effectiveness of radiotherapy combined with inhibitors of both mesenchymal and amoeboid modes of tumor cell motility. (Cancer Sci 2012; 103: 677–683)

Radioprotection/Postirradiation administration/Mineral-yeast/Mice. Purpose: To elucidate the mechanism underlying the in vivo radioprotection activity by Zn-containing, heat-treat ed Saccharomyces cerevisiae yeast (Zn-yeast). Materials and Methods: Zn-yeast suspension was administered into C3H/He mice immediately after whole body irradiation (WBI) at 7.5 Gy. Bone marrow was extracted from the mice 6 hours after irradiation and analyzed on a microarray. Expression changes in the candidate responsive genes differentially expressed in treated mice were re-examined by qRT-PCR. The bone marrow was also examined pathologically at 6 h, 3, 7, and 14 days postirradiation. Results: Thirty-six genes, including Edn1 and Agpt2, were identified as candidate responsive genes in irradiated mouse bone marrow treated with Zn-yeast by showing a greater than three-fold change compared with control (no irradiation and no Zn-yeast) mice. The expressions of Cdkn1a, Bax, and Ccng, which are well known as radioresponsive genes, were upregulated in WBI mice and Zn-yeast treated WBI mice. Pathological examination showed the newly fo rmed microvessels lined with endothelial cells, and small round hematopoietic cells around vessels in bone marrow matrix of mice administered with Zn-yeast after WBI, while whole-body irradiated mice developed fatty bone marrow within 2 weeks after irradiation. Conclusion: This study identified a possible mechanism for the postirradiation protection conferred by Zn-yeast. The protective effect of Zn-yeast against WBI is related to maintaining the bone marrow microenvironment, including targeting endothelial cells and cytokine release.

Adenosquamous carcinoma (ASC) is a relatively uncommon histological subtype of cervical cancer (CC). A point of controversy is the relative prognosis of ASC compared to squamous cell carcinoma (SCC). We hypothesized that ASC could be classified into two intrinsic molecular subtypes with different outcomes. We examined 143 biopsy samples of CC patients to identify a molecule for classification using microarray expression analysis and immunohistochemical analysis (IHA). We found specific expression profiles of candidate genes that distinguished two clusters. All adenocarcinoma (AC) patients were classified into one cluster, and most SCC patients fell into the other cluster. ASC patients were classified across the two clusters, which showed significantly different prognoses. The SCC-like expression signature comprised ANXA8, CK5, IFI16, and nectin-1; and the AC-like signature comprised EpCAM, and TMEM98. These signature-specific genes hypothetically indicated specific pathways by ontological analysis. The AC-like signature suggested an epithelial-mesenchymal transition and activated beta-catenin pathway, while the SCC-like signature suggested keratinocyte differentiation, HPV infection, and p53-mediated apoptosis. IHA revealed that positive expression of the most promising protein, EpCAM, was significantly associated with poor prognosis. In addition, the inhibition of EpCAM expression using siRNA significantly increased radiation-induced cell death in the cervical cell line, ME-180. In conclusion, we identified two possible ASC subtypes associated with different expression profiles and different prognoses. This work provides a novel set of genes that could be used as independent prognostic markers and therapy targets.

While the pre-treatment status of cancer is generally correlated with outcome, little is known about microenvironmental change caused by anti-cancer treatment and how it may affect outcome. For example, treatment may lead to induction of gene expression that promotes resistance to therapy. In the present study, we attempted to find a gene that was both induced by irradiation and associated with radioresistance in tumors. Using single-color oligo-microarrays, we analyzed the gene expression profiles of two murine squamous cell carcinomas, NR-S1, which is highly radioresistant, and SCCVII, which is radiosensitive, after irradiation with 137-Cs gamma rays or carbon ions. Candidate genes were those differentially regulated between NR-S1 and SCCVII after any kind of irradiation. Four genes, Efna1 (Ephrin-A1), Sprr1a (small proline-rich protein 1A), Srgap3 (SLIT-ROBO Rho GTPase activating protein 3) and Xrra1 [RIKEN 2 days neonate thymus thymic cells (NOD) cDNA clone E430023D08 3′], were selected as candidate genes associated with radiotherapy-induced radioresistance. We focused on Efna1, which encodes a ligand for the Eph receptor tyrosine kinase known to be involved in the vascular endothelial growth factor (VEGF) pathway. We used immunohistochemical methods to detect expression of Ephrin-A1, VEGF, and the microvascular marker CD31 in radioresistant NR-S1 tumor cells. Ephrin-A1 was detected in the cytoplasm of NR-S1 tumor cells after irradiation, but not in SCCVII tumor cells. Irradiation of NR-S1 tumor cells also led to significant increases in microvascular density, and up-regulation of VEGF expression. Our results suggest that radiotherapy-induced changes in gene expression related with angiogenesis might also modulate microenvironment and influence responsiveness of tumors.

Evidence has accumulated that ionizing radiation induces biological effects in non-irradiated bystander cells having received signals from directly irradiated cells; however, energetic heavy ion-induced bystander response is incompletely characterized. Here we performed microarray analysis of irradiated and bystander fibroblasts in confluent cultures. To see the effects in bystander cells, each of 1, 5 and 25 sites was targeted with 10 particles of carbon ions (18.3 MeV/u, 103 keV/microm) using microbeams, where particles traversed 0.00026, 0.0013 and 0.0066% of cells, respectively. diated cells, cultures were exposed to 10% survival dose (D), 0.1D and 0.01D of corresponding broadbeams (108 keV/microm). Irrespective of the target numbers (1, 5 or 25 sites) and the time (2 or 6h postirradiation), similar expression changes were observed in bystander cells. Among 874 probes that showed more than 1.5-fold changes in bystander cells, 25% were upregulated and the remainder downregulated. These included genes related to cell communication (PIK3C2A, GNA13, FN1, ANXA1 and IL1RAP), stress response (RAD23B, ATF4 and EIF2AK4) and cell cycle (MYCN, RBBP4 and NEUROG1). Pathway analysis revealed serial bystander activation of G protein/PI-3 kinase pathways. Instead, genes related to cell cycle or death (CDKN1A, GADD45A, NOTCH1 and BCL2L1), and cell communication (IL1B, TCF7 and ID1) were upregulated in irradiated cells, but not in bystander cells. Our results indicate different expression profiles in irradiated and bystander cells, and imply that intercellular signaling between irradiated and bystander cells activate intracellular signaling, leading to the transcriptional stress response in bystander cells.

Objective: To identify changes in gene expression related to the concurrent use of platinum compounds with radiotherapy, in the treatment of cervical cancer. Patients and Methods: Biopsy specimens were obtained from 39 patients with squamous cell carcinoma of the uterine cervix, before and during fractionated radiotherapy. Twenty patients were treated with radiotherapy (RT) alone, while 19 received the same radiotherapy plus concomitant chemotherapy with cisplatin (CRT). Changes in gene expression induced by treatment were investigated using single-color oligo-microarrays consisting of 44K human sequences. Paraffin-embedded samples were used to examine apoptosis and the expression of protein by treatment-responsive genes. Changes in mRNA expression were assessed for these genes by real-time reverse transcriptase-polymerase chain reaction. Aberrant genomic change (detected using microarray-based comparative genomic hybridization), human papillomavirus infection, and p53 status were also evaluated. Results: The expression of CDKN1A, BAX, TNFSF8, and RRM2B was consistently upregulated by CRT (9 Gy with a single administration of cisplatin). Similar expression changes were induced by RT (9 Gy) alone, although the variability between tumors was greater. Apoptotic cells were significantly increased in both groups. CRT significantly increased the numbers of cases with diffusely distributed CDKN1A-positive cells. Genetic losses at 2q33-ter and gains of 3q26-ter were detected in the samples with high frequency; 60% were positive for human papillomavirus DNA; and three tumors had deletions/mutations of the p53 gene. There was no difference in the incidence of these genomic changes between the groups, and no association was found with the changes in expression of CDKN1A, BAX, TNFSF8 or RRM2B. Conclusions: Using biopsy samples from pretreatment and midtreatment cervical tumors, we identified therapy-induced genes related to the cell death signaling pathway. CRT produced a homogenous pattern of changes in expression of known radiation-responsive genes.

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