Norikatsu Miyoshi

Reprogramming of Mouse and Human Cells to Pluripotency Using Mature MicroRNAs

Induced pluripotent stem cells (iPSCs) can be generated from differentiated human and mouse somatic cells using transcription factors such as Oct4, Sox2, Klf4, and c-Myc. It is possible to augment the reprogramming process with chemical compounds, but issues related to low reprogramming efficiencies and, with a number of protocols, residual vector sequences, remain to be resolved. We show here that it is possible to reprogram mouse and human cells to pluripotency by direct transfection of mature double-stranded microRNAs (miRNAs). Our approaches use a combination of mir-200c plus mir-302 s and mir-369 s family miRNAs. Because this reprogramming method does not require vector-based gene transfer, it holds significant potential for biomedical research and regenerative medicine.

Defined factors induce reprogramming of gastrointestinal cancer cells

Although cancer is a disease with genetic and epigenetic origins, the possible effects of reprogramming by defined factors remain to be fully understood. We studied the effects of the induction or inhibition of cancer-related genes and immature status-related genes whose alterations have been reported in gastrointestinal cancer cells. Retroviral-mediated introduction of induced pluripotent stem (iPS) cell genes was necessary for inducing the expression of immature status-related proteins, including Nanog, Ssea4, Tra-1-60, and Tra-1-80 in esophageal, stomach, colorectal, liver, pancreatic, and cholangiocellular cancer cells. Induced cells, but not parental cells, possessed the potential to express morphological patterns of ectoderm, mesoderm, and endoderm, which was supported by epigenetic studies, indicating methylation of DNA strands and the histone H3 protein at lysine 4 in promoter regions of pluripotency-associated genes such as NANOG. In in vitro analysis induced cells showed slow proliferation and were sensitized to differentiation-inducing treatment, and in vivo tumorigenesis was reduced in NOD/SCID mice. This study demonstrated that pluripotency was manifested in induced cells, and that the induced pluripotent cancer (iPC) cells were distinct from natural cancer cells with regard to their sensitivity to differentiation-inducing treatment. Retroviral-mediated introduction of iPC cells confers higher sensitivity to chemotherapeutic agents and differentiation-inducing treatment.

RB loss in resistant EGFR mutant lung adenocarcinomas that transform to small-cell lung cancer

Tyrosine kinase inhibitors are effective treatments for non-small-cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) mutations. However, relapse typically occurs after an average of 1 year of continuous treatment. A fundamental histological transformation from NSCLC to small-cell lung cancer (SCLC) is observed in a subset of the resistant cancers, but the molecular changes associated with this transformation remain unknown. Analysis of tumour samples and cell lines derived from resistant EGFR mutant patients revealed that Retinoblastoma (RB) is lost in 100% of these SCLC transformed cases, but rarely in those that remain NSCLC. Further, increased neuroendocrine marker and decreased EGFR expression as well as greater sensitivity to BCL2 family inhibition are observed in resistant SCLC transformed cancers compared with resistant NSCLCs. Together, these findings suggest that this subset of resistant cancers ultimately adopt many of the molecular and phenotypic characteristics of classical SCLC.

Detection of Sentinel Node in Gastric Cancer Surgery by Indocyanine Green Fluorescence Imaging: Comparison with Infrared Imaging

BackgroundSecure methods for clinical detection of the sentinel node (SN) are in great demand to avoid unnecessary resection. This was a clinical exploration/feasibility study of a novel detection system for SN biopsy using indocyanine green (ICG) fluorescence imaging in gastric cancer surgery.MethodsSN biopsy using ICG dye was performed in three patients who had gastric cancer. ICG fluorescence images were obtained using a detection system comprising a charge-coupled device (CCD) camera with a cut filter as the detector and light emitting diodes (LED) as the light source. The nodes were also examined simultaneously by an infrared (IR) imaging videoscope.ResultsImmediately after intraoperative ICG injection, the fluorescence imaging system allowed easy visualization of the lymphatic vessels draining from the primary gastric tumor toward the lymph nodes and tracing of the moving injected dye. Some lymph vessels and nodes were hardly recognized by ICG green color or IR imaging. The ICG fluorescence system also allowed visualization of the lymph node when ICG was injected the day before surgery, similar to the radio-guided method.ConclusionsDetection of SNs in gastric cancer surgery using the ICG fluorescence imaging system is a promising novel technique and may perhaps prove useful for laparoscopic surgery.

Epithelial-mesenchymal transition with expression of SNAI1-induced chemoresistance in colorectal cancer.

BACKGROUND Previous reports have demonstrated that SNAI1 plays a role in epithelial-mesenchymal transition (EMT) through the suppression of CDH1. Its role in the pathology and regulation of EMT expression to chemoresistance in colorectal cancer (CRC) has not yet been fully elucidated. METHODS Immunohistochemistry was performed to evaluate the expression of Snai1 protein in 30 primary CRC samples. The biological significance of Snai1 expression was studied by induction of the wild-type (WT) and mutant SNAI1 gene in CRC SW480 cells. RESULTS Examination of 20 surgical specimens of CRC indicated that Snai1 protein expression was localized outer regions of invasive tumors. Introduction of phosphorylation-defective active EMT forms, SNAI1-6SA and SNAI1-8SA, caused downregulation of CDH1 and upregulation of VIM compared with SNAI1-WT and the negative control (NC). Chemoresistance to 5-fluorouracil (IC50) was higher in SNAI1-6SA and SNAI1-8SA transfectants compared with SNAI1-WT and NC. All the above results were significantly different. CONCLUSION The present study demonstrated that Snai1 plays a role in CRC invasion through phosphorylation, suggesting a plausible mechanism for overcoming chemoresistance that will lead to the development of effective treatments for CRC.

Abnormal expression of TRIB3 in colorectal cancer: a novel marker for prognosis

Background:TRIB3 is a human homologue of Drosophila tribbles. Previous studies have shown that TRIB3 controls the cell growth through ubiquitination-dependent degradation of other proteins, whereas its significance in the prognosis of colorectal cancer (CRC) is not yet fully understood.Materials:This study comprised 202 patients who underwent surgery for CRC, as well as 22 cell lines derived from human gastrointestinal cancer. The correlation of gene expression with clinical parameters in patients was assessed. The biological significance was evaluated by knockdown experiments in seven colorectal cancer cell lines.Results:A total of 20 cancer cell lines (90.9%) expressed the TRIB3 gene. The assessment in surgical specimens indicated that the gene expression was significantly higher in the cancerous region than in the marginal non-cancerous region. Patients with high TRIB3 expression were statistically susceptible to a recurrence of the disease, and showed poorer overall survival than those with low expression. The assessment of TRIB3 knockdown in five cell lines showed that small interfering RNA (siRNA) inhibition resulted in a statistically significant reduction in cell growth.Conclusion:These data strongly suggest the usefulness of TRIB3 as a marker for predicting the prognosis of CRC patients, showing a basis for the development of effective treatments for CRC.

Emerging Methods for Preparing iPS Cells

In 1998, human embryonic stem cells were first generated and were expected to contribute greatly to regenerative medicine. However, when medical treatments were performed using human embryonic stem cells, there were problems, such as transplant rejection, as well as bioethical issues. Induced pluripotent stem cells were generated from mouse and human fibroblasts in 2006 and 2007 by introducing four transcription factors (Oct3/4, Sox2, c-Myc and Klf4). This process was defined as direct reprogramming, and induced pluripotent stem cells were better tolerated. Although induced pluripotent stem cells have contributed greatly to biomedical research and regenerative medicine, high tumorigenic potential is still a critical problem due to the introduction of the oncogene c-Myc and reprogramming with a virus vector. To address this, we reprogrammed somatic cells by transfection with microribonucleic acids to avoid using virus vectors for genomic integration into the host genome. We found that it was possible to reprogram mouse and human cells to pluripotency by direct transfection of three mature microribonucleic acids (mir-200c, -302s and -369s) with increased expression levels in embryonic stem cells and induced pluripotent stem cells. The microribonucleic acid-induced pluripotent stem cells have a reduced risk of mutations and tumorigenesis. Our laboratory also introduced four transcription factors (Oct3/4, Sox2, c-Myc and Klf4) into cancer cells, generating induced pluripotent cancer cells that exhibited strikingly less malignant features, suggesting the possibility of a novel type of cancer therapy. However, the gene transduction method is not yet safe for clinical applications, due to a genomic integration that may cause tumor formation. We are currently investigating the reprogramming method using microribonucleic acids in cancer cells to develop a very safe, highly efficient and highly complete reprogramming for clinical applications.

Expression of CLDN1 in colorectal cancer: A novel marker for prognosis

Claudin1 (CLDN1) plays an important role not only in the intercellular barrier function of tight junctions (TJs) but also in migration and invasiveness of cancer cells. Previous reports show that CLDN1 overexpression is significantly related to the malignant behavior in several cancer types whereas its significance in colorectal cancer (CRC) is not fully understood. The present study comprised 119 patients who underwent surgery for CRC, as well as 3 cell lines derived from human CRC. The correlation of gene expression with clinical parameters in patients was assessed by knockdown experiments using 3 cell lines. Patients with high CLDN1 expression were statistically shown to have a relatively better prognosis, and those with low CLDN1 expression showed poorer overall survival and disease-free survival than those with high expression. The assessment of CLDN1 knockdown in the 3 cell lines demonstrated that the siRNA inhibition resulted in a statistically significant increase in cell invasiveness. In conclusion, the present data strongly suggest that CLDN1 expression is a prognostic factor in CRC patients.

Long-term culture following ES-like gene-induced reprogramming elicits an aggressive phenotype in mutated cholangiocellular carcinoma cells

BACKGROUND We recently reported that gastrointestinal (GI) cancer cells can be reprogrammed to a pluripotent state by the ectopic expression of defined embryonic stem (ES)-like transcriptional factors. The induced pluripotent cancer (iPC) cells from GI cancer were sensitized to chemotherapeutic agents and differentiation-inducing treatment during a short-term culture, although a phenotype induced by long-term culture needs to be studied. METHODS A long-term cultured (Lc)-iPC cells were produced in GI cancer cell lines by virus-mediated introduction of four ES-like genes-c-MYC, SOX2, OCT3/4, and KLF4-followed by a culture more than three months after iPC cells induction. An acquired state was studied by expression of immature-related surface antigens, Tra-1-60, Tra-1-81, Tra-2-49, and Ssea-4; and epigenetic trimethyl modification at lysine 4 of histone H3. Sensitivity to chemotherapeutic agents and tumorigenicity were studied in Lc-iPC cells. RESULTS Whereas the introduction of defined factors of iPC cells once induced an immature state and sensitized cells to therapeutic reagents, the endogenous expression of the ES-like genes except for activated endogenous c-MYC was down-regulated in a long-term culture, suggesting a high magnitude of the reprogramming induction by defined factors and the requirement of therapeutic maintenance in Lc-iPC cells from cholangiocellular carcinoma HuCC-T1 cells, which harbor TP53(R175H) and KRAS(G12D). The Lc-iPC cells showed resistance to 5-fluorouracil in culture, and high tumorigenic ability with activated endogenous c-MYC in immunodeficient mice. CONCLUSION The Lc-iPC cells from HuCC-T1 might be prone to an undesirable therapeutic response because of an association with the activated endogenous c-MYC. To consider the possible therapeutic approach in GI cancer, it would be necessary to develop a predictive method for evaluating the improper reprogramming-associated aggressive phenotype of iPC cells.

SCRN1 is a novel marker for prognosis in colorectal cancer.

Secernin 1 (SCRN1) is a member of the secernin family and is reported to be a tumor‐associated antigen. Previous reports show that SCRN1 is upregulated in gastric cancer cell lines and may be a novel immunotherapy target, whereas its significance in colorectal cancer (CRC) is not fully understood.