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Summary StatementThe present study is the first report for reproducing human disease by utilizing blastocyst complementation method and would lead to the development of novel therapy for human disease.The application of pluripotent stem cell is expected to contribute to the elucidation of the unknown mechanism of human diseases. However, in vitro induction of cells in several organs, such as the pancreas and liver, remains difficult; therefore, reproduction of those diseases in a model has not been feasible. To reproduce human hereditary pancreatitis (HP), which is most frequently caused by the mutations in thePRSS1gene, we performed the blastocyst complementation (BC) method. In the BC method, mouse embryonic stem (ES) cells harboring CRISPR/CAS9-mediated mutations in thePrss1were injected into the blastocysts of deficientPdx1gene mice, which is a critical transcription factor in the pancreas. The results showed that the blastocysts injected into thePrss1-mutant ES cells induced trypsin activation. This implied that the mouse phenotype mimics that of human HP and that the BC method was useful for the reproduction and study of pancreatic disorders. The present study opens the possibility of investigating uncharacterized human diseases by utilizing the BC method.

Artificial intelligence (AI) trained with a convolutional neural network (CNN) is a recent technological advancement. Previously, several attempts have been made to train AI using medical images for clinical applications. However, whether AI can distinguish microscopic images of mammalian cells has remained debatable. This study assesses the accuracy of image recognition techniques using the CNN to identify microscopic images. We also attempted to distinguish between mouse and human cells and their radioresistant clones. We used phase-contrast microscopic images of radioresistant clones from two cell lines, mouse squamous cell carcinoma NR-S1, and human cervical carcinoma ME-180. We obtained 10,000 images of each of the parental NR-S1 and ME-180 controls as well as radioresistant clones. We trained the CNN called VGG16 using these images and obtained an accuracy of 96%. Features extracted by the trained CNN were plotted using t-distributed stochastic neighbor embedding, and images of each cell line were well clustered. Overall, these findings suggest the utility of image recognition using AI for predicting minute differences among phase-contrast microscopic images of cancer cells and their radioresistant clones. Significance: This study demonstrates rapid and accurate identification of radioresistant tumor cells in culture using artifical intelligence; this should have applications in future preclinical cancer research.

Cancer stem cells (CSCs) exhibit tumorigenic potential and can generate resistance to chemotherapy and radiotherapy. A labeled ornithine decarboxylase (ODC, a rate-limiting enzyme involved in polyamine [PA] biosynthesis) degradation motif (degron) system allows visualization of a fraction of CSC-like cells in heterogeneous tumor populations. A labeled ODC degradation motif system allowed visualization of a fraction of CSC-like cells in heterogeneous tumor populations. Using this system, analysis of polyamine flux indicated that polyamine metabolism is active in CSCs. The results showed that intracellular polyamines inhibited the activity of histone lysine 4 demethylase enzymes, including lysine-specific demethylase-1 (LSD1). Chromatin immunoprecipitation with Pol II antibody followed by massively parallel DNA sequencing, revealed the global enrichment of Pol II in transcription start sites in CSCs. Increase of polyamines within cells resulted in an enhancement of ID1 gene expression. The results of this study reveal details of metabolic pathways that drive epigenetic control of cancer cell stemness and determine effective therapeutic targets in CSCs.

The significance of mitochondrial metabolism in cancer cells has recently been gaining attention. Among other findings, One-carbon folate metabolism has been reported to be closely associated with cellular characteristics in cancer. To study molecular targets for efficient cancer therapy, we investigated the association between the expressions of genes that code enzymes involved in one-carbon metabolism and survival rate of patients with adenocarcinomas of the colorectum and lung. Patients with high expression of genes that control the metabolic cycle of tetrahydrofolate (THF) in mitochondria, SHMT2, MTHFD2, and ALDH1L2, have a shorter overall survival rate compared with patients with low expression of these genes. Our results revealed that these genes could be novel and more promising anticancer targets than dihydrofolate reductase (DHFR), the current target of drug therapy linked with folate metabolism, suggesting the rationale of drug discovery in cancer medicine.

The Golgi-associated PDZ- and coiled-coil motif-containing (GOPC) protein controls the intracellular trafficking of numerous integral membrane proteins. Knockdown of GOPC increases activation of the mitogen-activated protein kinase-extracellular signal-regulated kinase 1/2 pathway and cancer cell progression in colorectal cancer. The present study aimed to clarify the correlation between GOPC expression and prognosis in colorectal cancer. Total RNA was extracted from 153 clinical colorectal cancer specimens and GOPC expression was evaluated using reverse transcription-quantitative polymerase chain reaction. The correlation between GOPC expression and clinicopathological factors was analyzed, along with the association of GOPC expression with overall survival (OS) and with recurrence-free survival (RFS). Lower expression of GOPC was significantly associated with a high frequency of venous invasion (P=0.001) and to poorer OS and RFS based on Kaplan-Meier analysis. In addition, multivariate analyses using a Cox proportional hazards model identified lower expression of GOPC to be an independent prognostic factor for colorectal cancer (hazard ratio=2.800; 95% confidence interval; 1.121–7.648; P=0.027). Lower expression of GOPC revealed a high frequency of venous invasion and associated with poorer prognosis for patients with colorectal cancer.

The biological significance of micro (mi)RNAs has traditionally been evaluated according to their RNA expression levels based on the assumption that miRNAs recognize and regulate their targets in an unvarying fashion. Here we show that a fraction of mature miRNAs including miR-17-5p, -21-5p, and -200c-3p and let-7a-5p harbor methyl marks that potentially alter their stability and target recognition. Importantly, methylation of these miRNAs was significantly increased in cancer tissues as compared to paired normal tissues. Furthermore, miR-17-5p methylation level in serum samples distinguished early pancreatic cancer patients from healthy controls with extremely high sensitivity and specificity. These findings provide a basis for diagnostic strategies for early-stage cancer and add a dimension to our understanding of miRNA biology., In cancer it is assumed that microRNAs recognise and regulate their targets uniformly. Here, the authors show that in gastrointestinal cancers methylation of microRNAs may impact their stability, and that levels of microRNA methylation are distinct in pancreatic cancer patients compared to healthy controls with potential diagnostic implications.

Cancer stem cells (CSCs) are involved in metastatic colorectal cancer recurrence, but no effective therapy targeting these cells is currently available. Because trifluridine (FTD)/tipiracil therapy is used for refractory colorectal cancer, we sought to determine whether FTD is effective against CSC-like cells. CD44+CD133+ high-expressing and other populations of human DLD-1 colon cancer cells were separately isolated through fluorescence-activated cell sorting. The sphere-forming activity of each population and the anti-sphere-forming effects of FTD and fluorouracil (5-FU) on CD44+CD133+ cells were then measured. CD44+CD133+ DLD-1 cells formed substantially more spheres than other cells. Moreover, treating CD44+CD133+ DLD-1 cells with subtoxic concentrations of FTD (1 µM) inhibited sphere formation, and this was superior to the effect of subtoxic concentrations (1 µM) of 5-FU. The associated inhibition rates for FTD and 5-FU were 58.2% and 26.1%, respectively. Further, CD44+CD133+ DLD-1 cells expressed higher levels of thymidine kinase 1, which is responsible for FTD phosphorylation, than DLD-1 cells, and FTD was incorporated into the DNA of CD44+CD133+ DLD-1 cells. Thus, our data show that FTD treatment is effective against CSC-like cells and might be applied as CSC-targeting chemotherapy for tumor subtypes with high CD44 and CD133 expression.

Members of the ADAMTS family contain propeptide, metalloproteinase and disintegrin domains and serve key roles for cancer cell proliferation, progression and metastasis. Although overexpression of ADAMTS5 has been reported in glioblastoma, and head and neck cancer, little has been demonstrated in colorectal cancer types. The present study aimed to clarify the significance of ADAMTS5 for clinicopathological factors and prognosis in colorectal cancer. The mRNA expression of ADAMTS5 was measured in 143 colorectal cancer specimens. ADAMTS5 expression was increased as the pathological stage increased. The expression of ADAMTS5 in stage III-IV colorectal cancer was significantly greater compared with that of stage 0-II colorectal cancer (P=0.0003). The median expression of ADAMTS5 was used to divide the ADAMTS5 higher expressing group and the ADAMTS5 lower expressing group to assess the correlation of ADAMTS5 expression with clinicopathological factors and prognosis. The proportions of lymphatic invasion and lymph node metastasis were significantly greater in the ADAMTS5 higher expressing group (P=0.0214 and P=0.0289 respectively). Overall survival and disease-free survival were assessed by the Kaplan-Meier curve with calculation of significance by the log-rank test; however, no significant difference was observed between the ADAMTS5 higher expressing group and the ADAMTS5 lower expressing group (P=0.7490 and P=0.9455, respectively). The present study confirmed high expression of ADAMTS5 as a potent marker for lymphatic invasion and lymphnode metastasis in colorectal cancer. To clarify the function of ADAMTS5 in colorectal cancer, further molecular investigations are required.

There are several obstacles to overcome prior to achieving cellular reprogramming of pancreatic β cells in vitro and in vivo. The present study demonstrated that the transfer of epigenetic phenotypes was achieved in the cell-free conditioned medium (CM) of pancreatic insulinoma MIN6 cell cultures. The comparison of a subpopulation of MIN6, m14 and m9 cells indicated that MIN6-m14 cells were more prone to cellular reprogramming. Epigenetic profiling revealed that the transcription factor pancreas/duodenum homeobox protein 1 (Pdx1) was differentially associated among the clones. The culture of differentiated adipocytes in the CM of MIN6-m14 cells resulted in the induction of insulin mRNA expression, and was accompanied by epigenetic events of Pdx1 binding. The epigenetic profiling indicated that Pdx1 is preferentially associated with a previously uncharacterized region of the endoplasmic reticulum (ER) disulfide oxidase, ER oxidoreductin 1 gene. Therefore, the results of the present study indicated that the CM of MIN6 cells was able to induce a pancreatic β cell-like phenotype in differentiated adipocytes. These data provide additional support for the utility of cell-free CM for cellular reprogramming.

Hepcidin and ferroportin, which are known as key iron regulators, may be used in future treatments of pancreatic ductal adenocarcinoma. Iron is essential for life support; it helps oxygen molecules bind to hemoglobin and acts as an important catalytic enzyme center. However, iron overload is a risk factor for cancer, possibly through the generation of reactive oxygen species (ROS). Hepcidin, which is a peptide hormone mainly generated by the liver, inhibits iron absorption via enterocytes and iron release from macrophages. Notably, hepcidin regulates iron homeostasis in the body by regulating the iron transporter ferroportin. In the present study, it was assumed that high hepcidin expression and low ferroportin expression result in malignancy. Therefore, it was examined whether hepcidin and ferroportin expression levels were correlated with the prognosis of pancreatic cancer in patients. Results revealed that high hepcidin expression levels and low ferroportin expression levels in pancreatic cancer tissue were significantly associated with poor prognosis in the analyses of overall survival (P=0.0140 and 0.0478, respectively). Additionally, there was no significant difference in disease-free survival in the hepcidin- and ferroportin-staining groups. Hepcidin expression correlated with the pathological stage and vascular invasion (P=0.0493 and 0.0400, respectively), and ferroportin expression was correlated with age (P=0.0372). Multivariate analysis of overall survival in the hepcidin-staining group revealed that pathological N factor (pN), adjuvant chemotherapy, and hepcidin expression were independent prognostic factors (P=0.0450, 0.0002, and 0.0049, respectively). Similarly, multivariate analysis of overall survival in the ferroportin-staining group revealed that vascular invasion, and ferroportin expression were independent prognostic factors (P=0.0028, P

Fetal cells in the developmental stages function with a distinct mechanism in comparison to adult tissues, which may be a useful source for regenerative medicine in postnatal medicine; however, the precise molecular mechanism remains to be elucidated fully. The present study investigated murine fetal hepatocytes, which were cultured in vitro, and the supernatants were used for the culture with murine adipose tissue-derived cells. Notably, the results indicated that fetal hepatocyte-derived culture medium elicits the induction of differentiation of adipose tissue-derived cells to bile duct cell lineages, but not to hepatocyte lineages in mice. This indicates that fetal cells possess the multi potentials, which are already absent in adults, and may be useful for regenerative medicine in future.

Previous studies highlighted that aminopeptidase N (APN)/CD13 acts as a scavenger in the survival of hepatocellular carcinoma (HCC) stem cells by reducing reactive oxygen species (ROS) levels. Hence, it has been proposed that APN/CD13 inhibition can increase cellular ROS levels and sensitize cells to chemotherapeutic agents. Although ubenimex, also known as bestatin, competitively inhibits proteases such as APN/CD13 on the cellular membrane and it is clinically used for patients with acute myeloid leukemia and lymphedema, research has demonstrated that higher concentrations of the agent induce the death of APN/CD13+ HCC stem cells. In this study, we developed a poly(ethylene glycol)-poly(lysine) block copolymer-ubenimex conjugate (PEG-b-PLys(Ube)) to increase the efficacy of reagents in APN/CD13+ cancer stem cells. Exposure to PEG-b-PLys(Ube) increased the intracellular ROS concentration by inhibiting APN enzyme activity, permitting the induction of apoptosis and attenuation of HCC cell proliferation. In addition, PEG-b-PLys(Ube) exhibited a relatively stronger antitumor effect in mice than PEG-b-PLys alone or phosphate-buffered saline. Moreover, an isobologram analysis revealed that combinations of fluorouracil, cisplatin, or doxorubicin with PEG-b-PLys(Ube) exhibited synergistic effects. This study demonstrated that PEG-b-PLys(Ube) does not impair the properties of ubenimex and exerts a potent antitumor effect.

We investigated the relationship between methylomic [5-methylation on deoxycytosine to form 5-methylcytosine (5mC)] and transcriptomic information in response to chemotherapeutic 5-fluorouracil (5-FU) exposure and cisplatin (CDDP) administration using the ornithine decarboxylase (ODC) degron-positive cancer stem cell model of gastrointestinal tumour. The quantification of 5mC methylation revealed various alterations in the size distribution and intensity of genomic loci for each patient. To summarise these alterations, we transformed all large volume data into a smooth function and treated the area as a representative value of 5mC methylation. The present computational approach made the methylomic data more accessible to each transcriptional unit and allowed to identify candidate genes, including the tumour necrosis factor receptor-associated factor 4 (TRAF4), as novel therapeutic targets with a strong response to anti-tumour agents, such as 5-FU and CDDP, and whose significance has been confirmed in a mouse model in vivo. The present study showed that 5mC methylation levels are inversely correlated with gene expression in a chemotherapy-resistant stem cell model of gastrointestinal cancer. This mathematical method can be used to simultaneously quantify and identify chemoresistant potential targets in gastrointestinal cancer stem cells.

Pancreatic cancer has a poor prognosis because of its high invasiveness and recurrence, and these properties closely link to the phenomenon of epithelial-mesenchymal transition (EMT). Recently, it has been reported that Sox4 is indispensable for EMT in vitro and in vivo and regulates various master regulators of EMT including Zeb, Twist and Snail. Moreover, Sox4 induces the transcription of Ezh2 which is the histone methyltransferase, and reprograms the cancer epigenome to promote EMT and metastasis. Therefore, the present study evaluated the importance of Sox4, Ezh2 and miR-335, which regulate Sox4 expression epigenetically, in clinical samples with pancreatic cancer. This retrospective analysis included data from 36 consecutive patients who underwent complete surgical resection for pancreatic cancer and did not undergo any preoperative therapies. We assessed the clinical significance of Sox4/Ezh2 axis and miR-335 expression, using immunohistochemistry and qRT-PCR with laser captured microdissection (LCM). The Sox4 positive patients had significantly worse prognosis as for disease-free survival (DFS) (P=0.0154) and the Ezh2-positive patients had significantly worse prognosis as for overall survival (OS) (P=0.0347). The miR-335 expression was inversely correlated with Sox4 expression in the identical clinical specimens, but it was not related to the prognosis. Sox4/Ezh2 axis was closely associated with the prognosis in pancreatic cancer patients.

Cancer stem cells (CSCs) are a small population of cells in cancer with stem-like properties such as cell proliferation, multiple differentiation and tumor initiation capacities. CSCs are therapy-resistant and cause cancer metastasis and recurrence. One key issue in cancer therapy is how to target and eliminate CSCs, in order to cure cancer completely without relapse and metastasis. To target CSCs, many cell surface markers, DNAs and microRNAs are considered as CSC markers. To date, the majority of the reported markers are not very specific to CSCs and are also present in non-CSCs. However, the combination of several markers is quite valuable for identifying and targeting CSCs, although more specific identification methods are needed. While CSCs are considered as critical therapeutic targets, useful treatment methods remain to be established. Epigenetic gene regulators, microRNAs, are associated with tumor initiation and progression. MicroRNAs have been recently considered as promising therapeutic targets, which can alter the therapeutic resistance of CSCs through epigenetic modification. Moreover, carbon ion beam radiotherapy is a promising treatment for CSCs. Evidence indicates that the carbon ion beam is more effective against CSCs than the conventional X-ray beam. Combination therapies of radiosensitizing microRNAs and carbon ion beam radiotherapy may be a promising cancer strategy. This review focuses on the identification and treatment resistance of CSCs and the potential of microRNAs as new radiosensitizers and carbon ion beam radiotherapy as a promising therapeutic strategy against CSCs.

Previous reports have indicated that reprogramming technologies may be useful for altering the malignant phenotype of cancer cells. Although somatic stem cells in normal tissues are more sensitive to reprogramming induction than differentiated cells, it remains to be elucidated whether any specific subpopulations are sensitive to reprogramming in heterogeneous tumor tissues. Here we examined the susceptibility of pancreatic cancer stem cells (CSC) and non-CSC to reprogramming. To characterize CSC populations, we focused on c-Met signaling, which has been identified as a marker of CSC in mouse experiments in vivo. Cells that expressed high levels of c-Met showed higher CSC properties, such as tumor-initiating capacity, and resistance to gemcitabine. Real-time reverse transcription-polymerase chain reaction in cells expressing high levels of c-Met revealed endogenous expression of reprogramming factors, such as OCT3/4, SOX2, KLF4 and cMYC. Introduction of these four factors resulted in higher alkaline phosphatase staining in cells with high c-Met expression than in controls. Therefore, the study results demonstrate that cellular reprogramming may be useful for extensive epigenetic modification of malignant features of pancreatic CSC.

Background: SIRT4, which is localised in the mitochondria, is one of the least characterised members of the sirtuin family of nicotinamide adenine dinucleotide-dependent enzymes that play key roles in multiple cellular processes such as metabolism, stress response and longevity. There are only a few studies that have characterised its function and assessed its clinical significance in human cancers. Methods: We established colorectal cancer cell lines (SW480, HCT116, and HT29) overexpressing SIRT4 and investigated their effects on proliferation, migration and invasion, as well as E-cadherin expression, that negatively regulates tumour invasion and metastases. The associations between SIRT4 expression in colorectal cancer specimens and clinicopathological features including prognosis were assessed by immunohistochemistry. Results: SIRT4 upregulated E-cadherin expression and suppressed proliferation, migration and invasion through inhibition of glutamine metabolism in colorectal cancer cells. Moreover, SIRT4 expression in colorectal cancer decreased with the progression of invasion and metastasis, and a low expression level of SIRT4 was correlated with a worse prognosis. Conclusions: SIRT4 has a tumour-suppressive function and may serve as a novel therapeutic target in colorectal cancer.

Metabolism may determine the biologically malignant behavior of pancreatic cancer. To investigate the significance and prognostic value of cancer metabolism in cancer patients, we investigated the expression of two key enzymes in anaerobic glycolysis, hexokinase 2 (HK2) and pyruvate kinase isoenzyme type M2 (PKM2), in surgical specimens obtained from 36 patients who underwent curative resection of pancreatic ductal carcinoma. The hk2-glycolysis axis is a key system in the clinical imaging of tumors via positron emission tomography. Immunohistochemical staining for hk2 and pkm2 was performed and the data were statistically analyzed to evaluate their prognostic power. The expression of hk2 and pkm2 was associated with clinicopathological variables and patient prognosis, including overall survival, local recurrence-free survival and distant metastasis-free survival. Staining for hk2 was negative and positive in 42 and 58% of the patients, respectively, whereas staining for pkm2 was negative and positive in 56 and 44%, respectively; hk2-positive staining was correlated with progressive pathological tumor stage (pT3 vs. pT1 and pT2; P=0.017). In the univariate analysis, the positive expression of hk2 and pkm2, pathological stage (pT3 vs. pT1 and pT2) and nodal metastasis were significantly correlated with poor prognosis (P

Bioinformatics and computational modeling offer innovative approaches to investigate cancer metabolism and predict the secondary and tertiary cellular responses. Dysregulation of metabolism has also been implicated in the pathophysiology of cancer. A significant proportion of patients with glioblastoma and hematological malignancies harbor the mutated forms of the oxidative phosphorylation (OxPhos) enzymes, isocitrate dehydrogenase (IDH) 1 or 2. The mutated forms of IDH1 and IDH2 produce an oncogenic metabolite, D-2-hydroxyglutarate (D2HG). A recent study of breast cancer patients showed that D2HG can also be produced in the absence of mutated IDH, through an alternative route involving over-activated MYC signaling. We developed a novel methodology to computationally analyze gene expression in colorectal cancer (CRC), and identified novel sets of genes that are associated with patient survival. The study of OxPhos-related genes revealed that an imbalance between the expression of IDH1 and IDH2, defined as overexpression of one isoform in relation to the other, was associated with worse prognosis in CRC patients. This effect was further accentuated by reduced expression of the β-oxygenation enzyme, 3-D-hydroxyacyl-CoA dehydratase (HCDH) 4, which has been reported to contribute to metabolism of intracellular D2HG. The present computational analysis revealed a novel and potential mechanism of CRC development, through over-production of D2HG when there is an imbalance between IDH1 and IDH2 expression, resulting in decreased clearance of D2HG when the β-oxidization pathway is diminished. Additional validation analysis with another gene expression dataset resulted in IDH1/2 imbalanced expression with a shorter DFS compared with balanced expression. Altogether, these findings provide a strong rationale for studying this mechanism further in order to discover novel therapeutic targets for the treatment of CRC.

N6-methyladenosine (m6A) is the most abundant epitranscriptome modification in mammalian mRNA. Recent years have seen substantial progress in m6A epitranscriptomics, indicating its crucial roles in the initiation and progression of cancer through regulation of RNA stabilities, mRNA splicing, microRNA processing and mRNA translation. However, by what means m6A is dynamically regulated or written by enzymatic components represented by methyltransferase-like 3 (METTL3) and how m6A is significant for each of the numerous genes remain unclear. We focused on METTL3 in pancreatic cancer, the prognosis of which is not satisfactory despite the development of multidisciplinary therapies. We established METTL3-knockdown pancreatic cancer cell line using short hairpin RNA. Although morphologic and proliferative changes were unaffected, METTL3-depleted cells showed higher sensitivity to anticancer reagents such as gemcitabine, 5-fluorouracil, cisplatin and irradiation. Our data suggest that METTL3 is a potent target for enhancing therapeutic efficacy in patients with pancreatic cancer. In addition, we performed cDNA expression analysis followed by gene ontology and protein-protein interaction analysis using the Database for Annotation, Visualization, and Integrated Discovery and Search Tool for the Retrieval of Interacting Genes/Proteins databases, respectively. The results demonstrate that METTL3 was associated with mitogen-activated protein kinase cascades, ubiquitin-dependent process and RNA splicing and regulation of cellular process, suggesting functional roles and targets of METTL3.

Intrahepatic cholangiocarcinoma (ICC) is known to have a high malignant potential. Because of its high recurrence rate, ICC has a poor prognosis even after complete tumor resection. Compared with normal differentiated cells, cancer cells have an altered metabolism for supporting their survival in severe conditions. Cancer cells acquire additional malignant potential as a result of this metabolic alteration. Thus, the molecules known to be involved in cancer metabolism, could be novel therapeutic targets. The mitochondrial pyruvate carrier (MPC) is a recently discovered pyruvate transporter, which is located in the mitochondrial inner membrane. Although MPC is composed of two subunits, it has been reported that the MPC1 subunit is specifically associated with poor prognosis in several cancers, including colorectal and prostate cancer. However, only a few studies have assessed the clinical significance of MPC1 and the molecular mechanisms underlying its influence on cancer progression are not well understood. This study aimed to clarify the function of MPC1 that affects the malignant potential of ICC. The expression of MPC1 in ICC clinical specimens was determined by immunohistochemistry. In addition, the correlations between MPC1 expression and the survival rate, as well as various clinicopathological parameters were assessed. Low MPC1 expression correlated with poor ICC prognosis and was correlated with tumor invasion and distant metastasis. Both these phenomena are closely associated with the epithelial-mesenchymal transition (EMT). Therefore, we investigated the impact of altering the MPC1 gene expression on the malignant potential of cancer cells using biliary tract cancer cell lines in vitro. The expression of MPC1 was downregulated in the cells induced to undergo EMT following treatment with TGF-β. Furthermore, the inhibition of MPC1 expression induced EMT in cancer cells, and the overexpression of MPC1 suppressed the migration of tumor cells. These results indicated that MPC1 could be a novel therapeutic target in some cancers.

Osteosarcoma is the most common primary bone malignancy in pediatric and adolescent populations. Recurrence and metastatic potential can be due to a subpopulation of cells with stem cell-like characteristics, such as tumor-initiating cells (TICs), which maintain the capacity to regenerate entire tumors. Targeting the TICs in osteosarcoma is a promising avenue for the development of new therapies for this devastating disease. TICs are usually quiescent with a low protein turnover, decreased metabolism, and downregulation of proteasome activity. Recently, cancer cells with low proteasome activity have been identified as TICs in several types of cancer. We stably infected two osteosarcoma cell lines, MG-63 and U2-OS, with an expression vector for a fusion protein between the green fluorescent protein, ZsGreen, and the C-terminal degron of the murine ornithine decarboxylase to monitor the 26S proteasome activity in living cells. We separated the osteosarcoma cells with low proteasome activity using fluorescence-activated cell sorting (FACS) and verified whether these ZsGreen+ cells had TIC-like properties. The ZsGreen+ cells showed enhanced sphere formation capacity and underwent asymmetric divisions into ZsGreen+ and ZsGreen- cells, whereas ZsGreen- cells underwent only symmetric divisions into ZsGreen- cells. Moreover, the ZsGreen+ cells were more chemo- and radioresistant. Thus, the present study demonstrated that chemoradiation-resistant TICs can be visualized by this system and suggested the rationale for further study of osteosarcoma stem cells.

Cancer stem cells (CSCs), defined by their differentiation capacity, self-renewal capacity, and maintenance of proliferation, have been identified in many tumors, including cervical cancer. Current studies identify CSCs by several specific biomarkers; however, it is difficult to monitor cervical CSCs in real-time in vitro and in vivo. Recent research reported the visualization of CSCs in breast cancer and gliomas using green fluorescent protein, ZsGreen, fused to a degron motif ornithine decarboxylase (ODC), which is destroyed by proteasomes. Accordingly, CSCs have low 26S proteasome activity, whereas non-CSCs have high 26S proteasome activity. Therefore, it is possible to observe CSCs by their accumulation of the fluorescent ZsGreen protein. In this study, we investigated optical imaging parameters to evaluate CSCs using two human cervical cancer cell lines: CaSki and HeLa. We defined populations as cell types having high- and low ZsGreen-cODC (high- and low-Zs, respectively) expression levels. The results of a sphere-forming assay revealed that the self-renewal ability of the high-Zs population was significantly higher than that of the low-Zs population. A tumorigenicity assay confirmed that the high-Zs population exhibited higher tumorigenic potential than the low-Zs population. The radioresistance of the high-Zs population of both HeLa and CaSki cells and the chemoresistance of the high-Zs population of CaSki cells were confirmed by a clonogenic survival assay and the tetrazolium dye assay, respectively. These results indicate that high-Zs populations of both the HeLa and CaSki cell lines possess CSC-like properties and therapeutic resistance. In conclusion, we successfully visualized CSC-like cells using a fluorescent protein system.

Although numerous studies have shown the significance of cancer-specific aerobic glycolysis, how glycolysis contributes to tumor invasion, a critical phenomenon in metastasis, remains unclear. With regard to colorectal cancer (CRC), we studied two critical gate enzymes, hexokinase 2 (HK2), which is involved in glycolysis, and phosphorylated pyruvate dehydrogenase-E1α (p-PDH), which is involved in oxidative phosphorylation (OxPhos). Immunohistochemical analyses using anti-HK2 and p-PDH antibodies were performed on surgically resected CRC samples (n = 104), and the expression in invasive front lesions of tumors was assessed. Positive HK2 expression correlated with extensive tumor diameter (P = 0.0460), advanced tumor depth (P = 0.0395), and presence of lymph node metastasis (P = 0.0409). Expression of p-PDH tended to be higher in right-sided CRCs than in left-sided CRCs (P = 0.0883). In survival analysis, the combined evaluation of positive HK2 and negative p-PDH was associated with reduced recurrence-free survival (RFS) (P = 0.0169 in all stages and P = 0.0238 in Stage II and III patients, respectively). This evaluation could predict RFS more precisely than the independent evaluation. The present study indicated that high HK2 expression combined with low p-PDH expression in the invasive front lesions of CRC tumors is predictive of tumor aggressiveness and survival of CRC cases.

Background: Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance. Methods: We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining. Results: The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients. Conclusions: miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.

Background: Cancer stem cells (CSCs) are involved in disease recurrence, metastases, and therapeutic resistance. However, anticancer agents that target CSCs are not currently available. Trifluridine (FTD)/tipiracil (TPI), a novel oral antitumor drug, was approved for the treatment of patients with metastatic colorectal cancer who had been previously treated with anti-vascular endothelial growth factors; anti-epidermal growth factor receptors (in patients with ras wild-type genes); or fluoropyrimidine-, oxaliplatin-, irinotecan-based chemotherapies. FTD/TPI improves overall survival, and FTD is the key component of FTD/TPI. Here, we demonstrated the efficacy of FTD against CD44 and CD133 high-expressing (CD44+CD133+) colorectal cancer cells that possess CSC-like properties. Method: CD44+CD133+ and other populations of DLD-1, a colorectal cell line, were separated by fluorescence-activated cell sorting (FACS). The sphere-forming activity of each population, and anti- sphere-forming effects of FTD and fluorouracil (5-FU) on CD44+CD133+ cells, were measured. In addition, DLD-1 and HCT-116, two colorectal cell lines, were treated with FTD for 5 months. The CSC markers CD44 and CD133 were measured by FACS, and the cells' in vitro and in vivo tumor-initiating properties were evaluated. Results: CD44+CD133+ DLD-1 cells formed significantly more spheres than did CD44-CD133- cells (sphere ratio CD44+CD133+/CD44-CD133- = 4.0) and CD44+CD133- cells (sphere ratio CD44+CD133+/CD44+CD133- = 1.7). In the in vitro proliferation assay, CD44+CD133+ cells had greater 5-FU resistance, but not higher FTD resistance, than did unsorted cells. In addition, treatment of CD44+CD133+ DLD-1 cells with subtoxic concentrations of FTD (1 µM) disrupted sphere formation, which was superior to the effect of treatment with 1 µM 5-FU. The inhibition rates for FTD and 5-FU were 59.5% and 14.3%, respectively. DLD-1 and HCT-116 cell lines treated with FTD for 5 months had smaller CD44+CD133+ populations and lower sphere-forming activity than did untreated cell lines; population decline rates were 93.8% and 74.7%, respectively. Furthermore, 5 months of in vitro exposure to FTD reduced in vivo tumor formation potential in both cell lines. These results suggest that FTD is effective against CSC-like cells, and that treatment with FTD might reduce CSC-like populations. Conclusion: FTD had a direct effect on the sphere-forming activity of CSC-like CD44+CD133+ cells that was superior to that of treatment with 5-FU. Its effectiveness against CSC-like CD44+CD133+ cells suggests that FTD might be useful for CSC-targeted chemotherapy in tumors that highly express CD44 and CD133. Citation Format: Kenta Tsunekuni, Masamitsu Konno, Jun Koseki, Ayumu Asai, Kazuaki Matsuoka, Teiji Takechi, Yuichiro Doki, Masaki Mori, Hideshi Ishii. CD44+/CD133+ colorectal cancer stem cells are sensitive to trifluridine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2963.

Deranged metabolism is a hallmark of cancer, playing a significant role in driving the disease process. One such example is the induction of carcinogenesis by the oncometabolite D-2 hydroxyglutarate (D-2HG), which is produced by the mutated enzyme isocitrate dehydrogenase (IDH) occurring in subsets of leukaemias and brain tumours. The oncogenic property of D-2HG appears to stem from its ability to interfere with the activities of α-ketoglutarate-dependent dioxygenases, including the Jumonji family histone demethylases. Here, we find in colorectal cancer cells that even in the absence of IDH mutation, the levels of D-2HG and its enantiomer L-2HG were elevated through glutamine anaplerosis. D-2HG, but not L-2HG, increased the trimethylation of histone H3 lysine 4 of the promoter region of ZEB1, a master regulator of epithelial-mesenchymal transition (EMT), and increased the expression of the ZEB1 gene to directly induce EMT in colorectal cancer cells. EMT promotes the ability of cancer cells to invade the local tissue and enter into the bloodstream, leading to distant organ metastasis. D-2HG levels were elevated in colorectal cancer specimens, particularly in those associated with distant metastasis, supporting the observations in vitro and implicating the contribution of D-2HG in metastasis, the major cause of death in this disease.

Tumor cells respond to their microenvironment, which can include hypoxia and malnutrition, and adapt their metabolism to survive and grow. Some oncogenes are associated with cancer metabolism via regulation of the related enzymes or transporters. However, the importance of metabolism and precise metabolic effects of oncogenes in colorectal cancer remain unclear. We found that colorectal cancer cells survived under the condition of glucose depletion, and their resistance to such conditions depended on genomic alterations rather than on KRAS mutation alone. Metabolomic analysis demonstrated that those cells maintained tricarboxylic acid cycle activity and ATP production under such conditions. Furthermore, we identified pivotal roles of GLUD1 and SLC25A13 in nutritional stress. GLUD1 and SLC25A13 were associated with tumor aggressiveness and poorer prognosis of colorectal cancer. In conclusion, GLUD1 and SLC25A13 may serve as new targets in treating refractory colorectal cancer which survive in malnutritional microenvironments.

Folate plays a pivotal role in the one-carbon metabolism needed for methylation reactions, nucleotide synthesis, and DNA repair. Although folate metabolism was recently shown to be associated with carcinogenesis in some solid tumors, the importance of folate metabolism in colorectal cancer remains unclear. In the present investigation we found that expression of three mitochondrial folate metabolic enzymes, serine hydroxymethyl transferase (SHMT2), methylenetetrahydrofolate dehydrogenase (MTHFD2) and aldehyde dehydrogenase 1 family member L2 (ALDH1L2), were upregulated in human colorectal tumor tissues compared to normal tissues. Colorectal cancer tissue samples were obtained from 117 consecutive patients. We evaluated the expression of the enzymes with immunohistochemical analysis and determined their relevance to clinicopathological characteristics and prognosis. Rates of recurrence-free survival (RFS) and overall survival (OS) in patients with high expression of SHMT2, MTHFD2 and ALDH1L2 tended to be lower than in patients with low expression of SHMT2, MTHFD2 and ALDH1L2 (P=0.446 and P=0.337, P=0.099 and P=0.064, P=0.178 and P=0.257, respectively). Notably, the combined high expression of SHMT2, MTHFD2 and ALDH1L2 (triple high) was more highly associated with poor prognosis than the individual expression levels (RFS; P=0.004 and OS; P=0.037). A multivariate analysis showed that triple high expression was independently associated with RFS (P=0.017). These findings suggested that mitochondrial folate metabolic enzymes could provide a potential therapeutic strategy for treating colorectal cancer.

In this study, we compared the microRNA (miRNA) profiles of a control and X-ray- and carbon ion beam-resistant cells to identify miRNAs that can be used as radiosensitizers and biomarkers. Mouse squamous cell carcinoma line NR-S1, its X-ray-resistant derivative X60, and its carbon ion beam‑resistant derivative C30 were subjected to miRNA microarray analysis. Expression of miRNAs shown to be upregulated or downregulated in the microarray analysis was confirmed by qRT-PCR. Downregulated miRNAs were overexpressed in human pancreatic cancer cell lines PANC1 and MIA PaCa-2, and the resulting cells were tested for radiosensitivity using colony-forming and sphere-forming assays. Of 1,265 miRNAs analyzed, 4 were downregulated and 11 were upregulated in X-ray-resistant and carbon ion beam-resistant cells. Two of the downregulated miRNAs, miR-196 and miR-374, were selected for overexpression in PANC1 and MIA PaCa-2 cells. Overexpression of miR-374 sensitized PANC-1 and MIA PaCa-2 cells toward carbon ion beam radiation. miRNA miR-374 has the potential to be a new radiosensitizer for carbon ion beam radiotherapy and a new biomarker to determine the optimal treatment for cancer.

Although BRAFV600E mutation is associated with adverse clinical outcomes in patients with colorectal cancer (CRC), response and resistance mechanisms for therapeutic BRAFV600E inhibitors remains poorly understood. In the present study, we demonstrate that selective BRAFV600E inhibition activates AMP-activated protein kinase (AMPK), which induces autophagy as a mechanism of therapeutic resistance in human cancers. The present data show AMPK-dependent cytoprotective roles of autophagy under conditions of therapeutic BRAFV600E inhibition and AMPK was negatively correlated with BRAFV600E-dependent activation of MEK-ERK-RSK signaling and positively correlated with unc-51-like kinase 1 (ULK1), a key initiator of autophagy. Furthermore, selective BRAFV600E inhibition and concomitant suppression of autophagy led to the induction of apoptosis. Taken together, present experiments indicate that AMPK plays a role in the survival of BRAFV600E CRC cells by selective inhibition and suggest that the control of autophagy contributes to overcome the chemoresistance of BRAFV600E CRC cells.

Targeting epigenetic regulators is a promising therapeutic strategy against cancer. However, because of the broad spectrum of targets, selective inhibition of cancer-associated genes remains a major challenge. To address this issue, we focused on the oncogene-regulated histone demethylase, nucleolar protein 66 (NO66 [C14orf169/MAPJD]), which is known to work coordinately with the well-characterized oncogene, c-MYC. To investigate expression patterns and clinical significance of NO66 in colorectal cancer (CRC), we performed immunohistochemical staining in 114 CRC cases. We performed functional analysis of NO66 to evaluate its contribution to proliferation and migration ability in CRC cells in vitro. NO66 was selectively expressed in CRC tissues. Furthermore, high expression levels of NO66 were associated with cancer metastatic potential, including lymphatic duct invasion (p = 0.047), venous invasion (p = 0.033), and lymph node metastasis (p = 0.015). Multivariate analysis indicated that NO66 was an independent prognostic factor for overall survival. In vitro assays revealed that NO66 expression is closely associated with malignant potential, including proliferation, migration and anti-apoptotic activity. NO66 is an independent prognostic factor in CRC. The cancer-selective expression patterns and its involvement in metastatic phenotypes suggest that NO66 is not only a crucial biomarker but is also a promising therapeutic target in CRC.

Bioinformatics and computational modelling are expected to offer innovative approaches in human medical science. In the present study, we performed computational analyses and made predictions using transcriptome and metabolome datasets obtained from fluorescence-based visualisations of chemotherapy-resistant cancer stem cells (CSCs) in the human oesophagus. This approach revealed an uncharacterized role for the ornithine metabolic pathway in the survival of chemotherapy-resistant CSCs. The present study fastens this rationale for further characterisation that may lead to the discovery of innovative drugs against robust CSCs.

Noncoding microRNAs inhibit translation and lower the transcript stability of coding mRNA, however miR-369 s, in aberrant silencing genomic regions, stabilizes target proteins under cellular stress. We found that in vitro differentiation of embryonic stem cells led to chromatin methylation of histone H3K4 at the miR-369 region on chromosome 12qF in mice, which is expressed in embryonic cells and is critical for pluripotency. Proteomic analyses revealed that miR-369 stabilized translation of pyruvate kinase (Pkm2) splicing factors such as HNRNPA2B1. Overexpression of miR-369 stimulated Pkm2 splicing and enhanced induction of cellular reprogramming by induced pluripotent stem cell factors, whereas miR-369 knockdown resulted in suppression. Furthermore, immunoprecipitation analysis showed that the Argonaute complex contained the fragile X mental retardation-related protein 1 and HNRNPA2B1 in a miR-369-depedent manner. Our findings demonstrate a unique role of the embryonic miR-369-HNRNPA2B1 axis in controlling metabolic enzyme function, and suggest a novel pathway linking epigenetic, transcriptional, and metabolic control in cell reprogramming.

Background: Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which was approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. A comprehensive analysis of miRNA profiles was performed in cell lines resistant to FTD established by ourselves, in order to explore the underlying mechanisms of resistance to the drug. Method: We established subline resistance to FTD through continuous administration and increasing dose of the drug for 5 months using the colorectal cancer cell line DLD-1. Total RNA was extracted at intervals whilst establishing FTD resistant sublines, and miRNA expression was analyzed by microarray. The expression of miRNA that was significantly downregulated in the FTD resistant subline was knocked down to test its involvement in resistance to FTD. Cell viability was evaluated by crystal violet cytotoxicity test. Results: The established FTD-resistant sublines showed more than 22-fold higher resistance to FTD and no cross-resistance to 5-FU. miRNA and mRNA clustered in the genome locus located in chromosome 9, 9p22.32 were downregulated in the FTD-resistant cell line, one of which was the miRNA let-7d-5p, which is one of the let-7 family and is known to target oncogenes and several key components of the cell cycle and cell proliferation. Anti-let-7d-5p treated DLD-1 was less sensitive to FTD than compared to control. The IC50 values of FTD were 16.8 µM and 7.6 µM for the anti-let-7d-5p treated cells and control respectively. On the other hand, overexpression of let-7d-5p in DLD-1 using let-7d-5p mimic increased the sensitivity to FTD compared to control. The IC50 values of FTD were 3.7 µM and 13.9 µM in the let-7d-5p overexpression cells and control respectively. 5FU sensitivity was only altered slightly in anti- let-7d-5p treated or let-7d-5p mimic treated cells. These data suggest that let-7d-5p is more relevant to sensitivity for FTD than that of 5FU. Conclusion: Let-7d-5p expression level influenced FTD sensitivity more effectively than 5FU. It suggests that the let-7d-5p is a potential predictive marker for trifluridine/tipiracil treatment in the clinical setting. Citation Format: Kenta Tsunekuni, Jun Koseki, Masamitsu Konno, Ayumu Asai, Norihiro Nishida, Hugh Colvin, Koichi Kawamoto, Yuichiro Doki, Masaki Mori, Hideshi Ishii. Analysis of microRNA profiles involved in the resistance to trifluridine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3075. doi:10.1158/1538-7445.AM2017-3075

Pyruvate kinase M2 (PKM2) is an alternatively spliced variant of the pyruvate kinase gene that is preferentially expressed during embryonic development and in cancer cells. PKM2 alters the final rate-limiting step of glycolysis, resulting in the cancer-specific Warburg effect (also referred to as aerobic glycolysis). Although previous reports suggest that PKM2 functions in nonmetabolic transcriptional regulation, its significance in cancer biology remains elusive. Here we report that stimulation of epithelial–mesenchymal transition (EMT) results in the nuclear translocation of PKM2 in colon cancer cells, which is pivotal in promoting EMT. Immunoprecipitation and LC-electrospray ionized TOF MS analyses revealed that EMT stimulation causes direct interaction of PKM2 in the nucleus with TGF-β–induced factor homeobox 2 (TGIF2), a transcriptional cofactor repressor of TGF-β signaling. The binding of PKM2 with TGIF2 recruits histone deacetylase 3 to the E-cadherin promoter sequence, with subsequent deacetylation of histone H3 and suppression of E-cadherin transcription. This previously unidentified finding of the molecular interaction of PKM2 in the nucleus sheds light on the significance of PKM2 expression in cancer cells.

Background: Mesenchymal stem cells (MSCs), including adipose tissue-derived mesenchymal stem cells (ADSCs), are multipotent and can differentiate into various cell types, including pancreatic β cells. Therefore, ADSCs present a potential cell source for the treatment of type 1 diabetes mellitus (T1DM). However, current in vitro protocols are insufficient to induce fully matured insulin-producing β cells. In this study, we assessed the effectiveness of overexpression of ΒETA2 (NeuroD1), a member of the basic helix–loop–helix transcription factor family, with murine insulinoma cell line-derived conditioned medium (MIN6-CM) to improve the differentiation capacity of ADSCs into insulin-producing cells. Method: Murine ADSCs were isolated from C57BL/6 mice, transduced with several transcriptional factors (TFs), and stable transfectants were established. MIN6-CM was prepared. Syngeneic recipient mice were rendered diabetic by a single injection of streptozotocin, and differentiated cells were transplanted under the kidney capsule of recipient mice. Next, blood glucose levels were monitored. Results: CM alone was sufficient to induce insulin mRNA expression in vitro. However, other TFs were not detected. ADSCs cultured with MIN6-CM induced insulin expressions in vitro, but other β cell-related TFs were been detected. However, BETA2 transduction in MIN6-CM resulted in robust expression of multiple β cell phenotypic markers. Moreover, insulin content analysis revealed insulin protein expression in vitro. Furthermore, in vivo transplant studies revealed the effectiveness of the simultaneous use of BETA2 transduction with the CM. Conclusion: These results suggest that the balance of cytokines and growth factors in addition to gene manipulation would benefit the efficient differentiation of ADSCs into pancreatic β cells. Our technology could provide a path to β cell differentiation and novel cell replacement-based therapies for T1DM.

Trifluridine (FTD), known as main component of TAS-102, is beginning to be applied as anti-tumor agent due to the highly efficacious antitumor potency although there is little to distinguish FTD structure from thymidine structure. TAS-102 has first been used in Japan in clinical therapy as an oral agent. Some experimental results hypothesized that FTD pharmacological antitumor effect can be arisen by inhibition of thymidylate synthase and incorporation of FTD itself into DNA. One of them is the thermal denaturation experiments for DNA duplexes of containing some FTD or not, performed by J. C. Markley et al. Their experiments have shown that the DNA duplexes containing FTD are slightly less stable, with a melting temperature about three degrees lower than not containing duplexes. However, the change of thermodynamic structure and inter-nucleobase interaction changes are not yet known. In this presentation, we have performed molecular dynamics (MD) simulations and ab initio molecular orbital (MO) calculations to analyze the differences of thermodynamics structure and inter-nucleobase interaction between containing and not containing FTD. From these results, we sought to qualitatively understand the effects of FTD. At the first step, we have estimated the stabilization structures of same two DNA duplexes (containing FTD and not) used by J. C. Markley, with energy minimization. Then, a large differences were shown between these structures. The hydrogen bond breaking is observed between FTD and complementary Adenine base while the not containing DNA duplex keeps having a stabilized structure. The behavior means that the essential difference of intra-DNA interactions was arisen. At the second step, MD simulations were performed to analyze the distribution of distances between FTD and complementary base (i.e. thermally available conformations). With some conformations from MD sampling, the MO calculations were performed to discuss the difference of intra-base interactions. From our results, we found that thermodynamic conformational change of DNA duplex containing FTD occurs even if it is in internal body temperature, and the break hydrogen bonding between FTD and paired base was observed more frequently. In addition, strong interactions between FTD and nearby π-conjugate base were observed. These behaviors might be one of the factor of inducing unstabilization in DNA duplexes. Citation Format: Jun Koseki, Kenta Tsunekuni, Masamitsu Konno, Naohiro Nishida, Koichi Kawamoto, Yuichiro Doki, Masaki Mori, Hideshi Ishii. Thermodynamic and molecular orbital analysis of the effects caused by incorporation of novel anti-tumor agent Trifluridine to DNA. [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 2706.

Esophageal cancer is consists of highly heterogeneous populations, which include cancer initiating cells, so-called cancer stem cells. Although histone modification is shown to be an essential factor for normal stem cell maintenance, the involvement of histone demethylase in cancer progression has been poorly understood. We investigated the functional roles of the Histone H3 lysine 4 (H3K4) demethylase KDM5B, a crucial epigenetic regulator that is required for normal embryonic development and cell growth. KDM5B knockdown resulted in the suppression of esophageal cancer cell growth, sphere formation and invasion ability and was associated with loss of epithelial cell marker expression. These tumor inhibitory effects were reverted subsequent to subcutaneous inoculation of these cells into immune-deficient mice. These results indicated that KDM5B plays an important role in maintaining cancer stem cells and justifies the rationale for studying the effects of continuous inhibition of this epigenetic factor in esophageal cancer. Citation Format: Naohiro Nishida, Yoshihiro Kano, Jun Koseki, Masamitsu Konno, Koichi Kawamoto, Yuichiro Doki, Masaki Mori, Hideshi Ishii. KDM5B plays a central role in esophageal cancer progression. [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 102. doi:10.1158/1538-7445.AM2015-102

Bioinformatics and computational modeling offer innovative approaches to investigate cancer metabolism and predict the secondary and tertiary cellular responses. Dysregulation of metabolism has also been implicated in the pathophysiology of cancer. A significant proportion of patients with glioblastoma and hematological malignancies harbor the mutated forms of the oxidative phosphorylation (OxPhos) enzymes, isocitrate dehydrogenase (IDH) 1 or 2. The mutated forms of IDH1 and IDH2 produce an oncogenic metabolite, D-2-hydroxyglutarate (D2HG). A recent study of breast cancer patients showed that D2HG can also be produced in the absence of mutated IDH, through an alternative route involving over-activated MYC signaling. We developed a novel methodology to computationally analyze gene expression in colorectal cancer, and identified novel sets of genes that are associated with patient survival. The study of OxPhos-related genes revealed that an imbalance between the expression of IDH1 and IDH2, defined as overexpression of one isoform in relation to the other, was associated with worse prognosis in colorectal cancer patients. This effect was further accentuated by reduced expression of the beta-oxygenation enzyme, 3-D-hydroxyacyl-CoA dehydratase (HCDH) 4, which has been reported to contribute to metabolism of intracellular D2HG. The present computational analysis revealed a novel and potential mechanism of colorectal cancer development, through over-production of D2HG when there is an imbalance between IDH1 and IDH2 expression, resulting in decreased clearance of D2HG when the beta-oxidization pathway is diminished. Additional validation analysis with other gene expression dataset has resulted in that IDH1/2 imbalanced expression had a shorter DFS compared with balanced expression. Altogether, these findings provide a strong rationale for studying this mechanism further in order to discover novel therapeutic targets for the treatment of colorectal cancer. Citation Format: Jun Koseki, Hugh Colvin, Takahito Fukusumi, Naohiro Nishida, Masamitsu Konno, Koichi Kawamoto, Kenta Tsunekuni, Yuichiro Doki, Masaki Mori, Hideshi Ishii. Computational analysis predicts unbalanced IDH1/IDH2 expression associate with 2-HG-inactivating beta-oxygenation pathway in colorectal cancer. [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 4583. doi:10.1158/1538-7445.AM2015-4583

Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR) 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors.