Yoshihiro Kano

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.

Adipose-derived mesenchymal stem cells and regenerative medicine

Adipose tissue‐derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β‐cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow‐derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.

Aldehyde dehydrogenase high gastric cancer stem cells are resistant to chemotherapy

Cancer stem cells (CSCs) are known to influence chemoresistance, survival, relapse and metastasis. Aldehyde dehydrogenase (ALDH) functions as an epithelial CSC marker. In the present study, we investigated the involvement of ALDH in gastric CSC maintenance, chemoresistance and survival. Following screening for eight candidate markers (CD13, CD26, CD44, CD90, CD117, CD133, EpCAM and ALDH), five gastric cancer cell lines were found to contain small subpopulations of high ALDH activity (ALDH(high) cells). We also examined the involvement of ALDH(high) cell populations in human primary tumor samples. Immunodeficient NOD/SCID mice were inoculated with tumor tissues obtained from surgical specimens. ALDH(high) cells were found to persist in the xenotransplanted primary tumor samples. in the immunodeficient mice, ALDH(high) cells exhibited a greater sphere‑forming ability in vitro and tumorigenic potential in vivo, compared with subpopulations of low ALDH activity (ALDH(low) cells). Cell cultures treated with 5-fluoro-uracil and cisplatin exhibited higher numbers of ALDH(high) cells. Notch1 and Sonic hedgehog (Shh) expression was also found to increase in ALDH(high) cells compared with ALDH(low) cells. Therefore, it can be concluded that ALDH generates chemoresistance in gastric cancer cells through Notch1 and Shh signaling, suggesting novel treatment targets.

CD10 as a novel marker of therapeutic resistance and cancer stem cells in head and neck squamous cell carcinoma.

Background:Cancer stem cells (CSCs) are responsible for treatment failure. However, their identification and roles in resistance are not well established in head and neck squamous cell carcinoma (HNSCC).Methods:Three HNSCC cell lines (FaDu, Detroit562 and BICR6) were treated with cisplatin or radiation. Cell surface antigens were analysed by LyoPlate, a novel cell surface antigen array. The expression levels of antigens highly expressed after treatments were further compared between cisplatin-resistant Detroit562 cells and its parental line. Association of the candidate antigen with CSCs properties, namely sphere formation and in vivo tumourigenicity, was also examined.Results:CD10, CD15s, CD146 and CD282 were upregulated across the treated cell lines, while the increased expression of CD10 was prominent in the cisplatin-resistant cell line. Isolation mediated by FACS revealed that the CD10-positive subpopulation was more refractory to cisplatin, fluorouracil and radiation than the CD10-negative subpopulation. It also showed an increased ability to form spheres in vitro and tumours in vivo. Moreover, the CD10-positive subpopulation expressed the CSC marker OCT3/4 at a higher level than that in the CD10-negative subpopulation.Conclusions:CD10 is associated with therapeutic resistance and CSC-like properties of HNSCC. CD10 may serve as a target molecule in the treatment of refractory HNSCC.

Depletion of JARID1B induces cellular senescence in human colorectal cancer

The global incidence of colorectal cancer (CRC) is increasing. Although there are emerging epigenetic factors that contribute to the occurrence, development and metastasis of CRC, the biological significance of epigenetic molecular regulation in different subpopulations such as cancer stem cells remains to be elucidated. In this study, we investigated the functional roles of the H3K4 demethylase, jumonji, AT rich interactive domain 1B (JARID1B), an epigenetic factor required for the continuous cell growth of melanomas, in CRC. We found that CD44(+)/aldehyde dehydrogenase (ALDH)(+) slowly proliferating immature CRC stem cell populations expressed relatively low levels of JARID1B and the differentiation marker, CD20, as well as relatively high levels of the tumor suppressor, p16/INK4A. Of note, lentiviral‑mediated continuous JARID1B depletion resulted in the loss of epithelial differentiation and suppressed CRC cell growth, which was associated with the induction of phosphorylation by the c‑Jun N‑terminal kinase (Jnk/Sapk) and senescence‑associated β‑galactosidase activity. Moreover, green fluorescent‑labeled cell tracking indicated that JARID1B‑positive CRC cells had greater tumorigenicity than JARID1B‑negative CRC cells after their subcutaneous inoculation into immunodeficient mice, although JARID1B‑negative CRC cells resumed normal growth after a month, suggesting that continuous JARID1B inhibition is necessary for tumor eradication. Thus, JARID1B plays a role in CRC maintenance. JARID1B may be a novel molecular target for therapy‑resistant cancer cells by the induction of cellular senescence.

Cancer stem cell theory in gastrointestinal malignancies: recent progress and upcoming challenges

A growing body of evidence supports the notion that malignant tumors are heterogeneous and contain diverse subpopulations of cells with unique characteristics including the ability to initiate a tumor and metastasize. This phenomenon might be explained by the so-called cancer stem cell (CSC) theory. Recent technological developments have allowed a deeper understanding and characterization of CSCs. Even though the application of this theory to hematopoietic malignancies and solid tumors holds promise for new ways to treat cancer, it also brings some skepticism. Efficacious therapeutic approaches targeting the CSC population should be explored to overcome therapeutic failure and improve patient outcomes. This review will focus on the intrinsic and extrinsic regulation of CSCs, as well as the development of therapeutic approaches against CSCs, predominantly focusing on gastrointestinal malignancies.

Significance of INHBA expression in human colorectal cancer

Inhibin β A (INHBA) is a member of the transforming growth factor β (TGF-β) superfamily. INHBA expression is associated with several types of human cancers; however, its significance in colorectal cancer (CRC) is not fully understood. INHBA expression was studied in 126 primary CRC samples and 4 CRC cell lines. Cell growth was assessed after inhibition of INHBA expression or after exogenous overexpression of INHBA in CRC tissues. INHBA expression was significantly higher in CRC tissues when compared to that in the corresponding normal tissues (P<0.001). Patients in the high expression group showed a poorer overall survival rate when compared to those in the low expression group (P<0.001); the present study did not evaluate for an independent prognostic factor but showed the significance of lymph node metastasis as an independent prognostic factor. The present study suggests that INHBA is useful as a predictive marker for prognosis in CRC patients.

Significance of Polypyrimidine Tract–Binding Protein 1 Expression in Colorectal Cancer

Polypyrimidine tract–binding protein (PTBP1) is an RNA-binding protein with various molecular functions related to RNA metabolism and a major repressive regulator of alternative splicing, causing exon skipping in numerous alternatively spliced pre-mRNAs. Here, we have investigated the role of PTBP1 in colorectal cancer. PTBP1 expression levels were significantly overexpressed in cancerous tissues compared with corresponding normal mucosal tissues. We also observed that PTBP1 expression levels, c-MYC expression levels, and PKM2:PKM1 ratio were positively correlated in colorectal cancer specimens. Moreover, PTBP1 expression levels were positively correlated to poor prognosis and lymph node metastasis. In analyses of colorectal cancer cells using siRNA for PTBP1, we observed that PTBP1 affects cell invasion, which was partially correlated to CD44 splicing, and this correlation was also confirmed in clinical samples. PTBP1 expression also affected anchorage-independent growth in colorectal cancer cell lines. PTBP1 expression also affected cell proliferation. Using time-lapse imaging analysis, PTBP1 was implicated in prolonged G2–M phase in HCT116 cells. As for the mechanism of prolonged G2–M phase in HCT116 siPTBP1 cells, Western blotting revealed that PTBP1 expression level was correlated to CDK11p58 expression level, which was reported to play an important role on progression to complete mitosis. These findings indicated that PTBP1 is a potential therapeutic target for colorectal cancer. Mol Cancer Ther; 14(7); 1705–16. ©2015 AACR.

Cancer Stem-like Properties in Colorectal Cancer Cells with Low Proteasome Activity.

Purpose: One of the main reasons for cancer treatment resistance is the existence of cancer stem-like cells (CSCs). Here, we elucidated the relationship between low proteasome activity cells (LPACs) and CSCs. Experimental Design: The human colorectal cancer cell lines HCT116, SW480, DLD1, and KM12SM were engineered to stably express a green fluorescent molecule fused to the degron of ornithine decarboxylase, resulting in an accumulation of the fluorescence in LPACs. LPACs were isolated by flow cytometry. Treatment resistance (radio- and chemotherapy) and the capacity of LPACs to act as CSCs were analyzed. Microarray analysis was performed to reveal genes related to treatment resistance. The prognostic impact of potent genes was examined in 190 patients with colorectal cancer. Results: LPACs had a significantly increased capacity for radioresistance and chemoresistance (5-fluorouracil and oxaliplatin), significantly lower reactive oxygen species activity, and significantly increased sphere formation capacity compared with non-LPACs. The number of cells in the G0–G1 phase was significantly higher among LPACs. Subcutaneous injection of as few as 20 LPACs led to tumor formation in immunologically incompetent mice. Microarray analysis revealed that the expression of EP300-interacting inhibitor of differentiation 3 (EID3) was significantly increased in LPACs. In vitro assay revealed that EID3 positively controlled cell proliferation and treatment resistance. The high expression of EID3 was an adverse prognostic indicator in patients with colorectal cancer (P = 0.0400). Conclusions: LPACs have characteristic treatment resistance and act as CSCs in colorectal cancer. In addition, EID3 is one of the potential regulators of treatment resistance in colorectal cancer and may be a potential therapeutic target. Clin Cancer Res; 22(21); 5277–86. ©2016 AACR.

Jumonji/Arid1b (Jarid1b) protein modulates human esophageal cancer cell growth

Although esophageal cancer is highly heterogeneous and the involvement of epigenetic regulation of cancer stem cells is highly suspected, the biological significance of epigenetically modified molecules that regulate different subpopulations remains to be firmly established. Using esophageal cancer cells, we investigated the functional roles of the H3K4 demethylase Jumonji/Arid1b (Jarid1b) (Kdm5b/Plu-1/Rbp2-h1), an epigenetic factor that is required for continuous cell growth in melanoma. JARID1B knockdown resulted in the suppression of esophageal cancer cell growth, sphere formation and invasion ability and was associated with loss of epithelial marker expression. However, these inhibitory effects observed on tumor formation were reverted subsequent to subcutaneous inoculation of these cells into immune-deficient mice. These results indicated that JARID1B plays a role in maintaining cancer stem cells in the esophagus and justifies the rationale for studying the effects of continuous inhibition of this epigenetic factor in esophageal cancer.