Kozo Noguchi

Metabolic Adaptation to Nutritional Stress in Human Colorectal Cancer

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.

Identification of chemoradiation-resistant osteosarcoma stem cells using an imaging system for proteasome activity

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: The potential of carbon ion beam radiation and new radiosensitizers (Review)

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.

Susceptibility of pancreatic cancer stem cells to reprogramming

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.

Visualization and characterization of cancer stem-like cells in cervical cancer.

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.

Pancreatic intraductal tubulopapillary neoplasm with associated invasive cancer successfully treated by total pancreatectomy: A case report

A 74-year-old male was admitted to Departments of Surgery, Toyonaka Municipal Hospital (Osaka, Japan) for treatment of a pancreatic tumor. Contrast enhanced computed tomography (CT) revealed a mass with small cystic lesions in the pancreatic head and body. Fluorodeoxyglucose-positron emission tomography/CT revealed an abnormal uptake of fluorodeoxyglucose, corresponding to the mass lesions. Upper gastrointestinal endoscopy revealed rough mucosa near the opening of the accessory pancreatic duct, and the mucosa biopsy exhibited adenocarcinoma with no mucin observed. The preoperative diagnosis was pancreatic intraductal tubulopapillary neoplasm (ITPN) with cancerous lesions, and a total pancreatectomy with splenectomy was performed. The resected tissue specimen revealed a solid tumor occupying the entire pancreas with intraductal growth into the main pancreatic duct. Histological examination revealed high-grade dysplastic cells in a tubulopapillary growth pattern without overt mucin production beyond the pancreatic duct. Immunohistochemical staining analysis of the tumor was positive for cytokeratin (CK)7, CK19 and mucin (MUC)1, and negative for MUC2, MUC5AC, MUC6 and caudal type homeobox 2. The tumor was finally diagnosed as pancreatic ITPN with associated invasive cancer. The patient remains well without evident recurrence nine months post-surgery. ITPN is a rare type of epithelial neoplasm of the pancreas, and is characterized by intraductal tubulo-papillary growth, ductal differentiation, limited intracellular mucin production, and cellular dysplasia. The present case report may contribute to improved understanding of how to effectively treat patients with ITPN.

Feasibility of laparoscopic cholecystectomy in patients with cerebrospinal fluid shunt

Previous reports of laparoscopic surgery in patients with cerebrospinal fluid (CSF) shunts for intracranial hypertension described shunt‐related complications. Thus, the shunts have been considered a contraindication for laparoscopic procedures. However, with the implementation of recent improvements in surgical techniques, perioperative management, and shunt technology, laparoscopic surgery may now be safe in cases with shunts. The aim of the present study was to examine the safety of such procedures based on our own experiences with laparoscopic surgery in patients with CSF shunts.

The mitochondrial one‑carbon metabolic pathway is associated with patient survival in pancreatic cancer

The expression levels of one-carbon metabolic enzymes were investigated and observed to be correlated with clinicopathological parameters in patients with pancreatic cancer. Mitochondrial one-carbon metabolism comprises a network of biological reactions that integrate nutrient status with nucleotide synthesis, amino acid metabolism, antioxidant reduced nicotinamide adenine dinucleotide phosphate production and epigenetic methylation processes. Previous studies have reported that the hyper-activation of mitochondrial one-carbon metabolism serves a significant role in malignant cancer phenotypes. A total of 103 patients underwent surgical resection of pancreatic ductal adenocarcinomas (PDAC) at Osaka University Hospital between April 2007 and December 2013 and were enrolled in this study. Subsequently, the expression of the one-carbon metabolic enzymes methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), aldehyde dehydrogenase 1 family member L2 (ALDH1L2), and serine hydroxymethyltransferase (SHMT2) was examined using immunohistochemical analysis. The immunohistochemical analyses demonstrated that patients with high expression levels of MTHFD2, ALDH1L2 or SHMT2 had significantly poor overall survival (OS) and disease-free survival (DFS) rates, as compared with patients with low expression levels. Furthermore, multivariate Cox proportional hazards analysis indicated that MTHFD2 and ALDH1L2 were independent prognostic factors for OS and DFS, whereas SHMT2 was not predictive of DFS. However, high and low expression levels of all three folate metabolic enzymes were significantly associated with improved OS and DFS, compared with the high expression of one or two folate metabolic enzymes. The expression levels of mitochondrial one-carbon metabolic enzymes are independent prognostic factors and potential therapeutic targets for future pancreatic cancer treatments.

C-met affects gemcitabine resistance during carcinogenesis in a mouse model of pancreatic cancer

Pancreatic adenocarcinoma is thought to develop from histologically identifiable intraductal lesions known as pancreatic intraepithelial neoplasias (PanINs), which exhibit similar morphological and genetic features to pancreatic ductal adenocarcinoma (PDAC). Therefore, a better understanding of the biological features underlying the progression of PanIN is essential to development more effective therapeutic interventions for PDAC. In recent years, numerous studies have reported that MET proto-oncogene receptor tyrosine kinase (c-MET) is a potential marker of pancreatic cancer stem cells (CSCs). CSCs have been revealed to initiate and propagate tumors in vitro and in vivo, and are associated with a chemoresistant phenotype. However, in vivo models using a xenograft approach are limited. In the present study, the morphological phenotype, molecular alteration and biological behavior of neoplasia in Pdx-1Cre/+, KrasLSL-G12D/+ and Metflox/flox and wild-type mice was analyzed. The results demonstrated that while oncogenic KrasLSL-G12D/+ increased PanIN initiation and significantly decreased survival rate compared with wild-type mice, no additive effect of c-Met receptor signaling on PanIN progression or prognosis was observed. Following gemcitabine administration, c-Met inhibition in Kras LSL-G12D/+ mice significantly decreased the total surface area of PanIN lesions and the number of anti-proliferation marker protein Ki-67 positive cells occupying PanIN lesions compared with Met+/+ mice. In conclusion, complete inhibition of the c-Met signaling pathway with chemotherapy may be useful for the treatment of pancreatic cancer.

Surgical outcomes of laparoscopic cholecystectomy for acute cholecystitis in elderly patients: Lap cholecystectomy in the elderly

The clinical significance of laparoscopic cholecystectomy (LC) for acute cholecystitis in elderly patients aged 80 years or older has not been determined. This study aimed to investigate surgical outcomes of LC for acute cholecystitis in elderly patients compared to non‐elderly patients.