Atsushi Hamabe

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.

Role of pyruvate kinase M2 in transcriptional regulation leading to epithelial–mesenchymal transition

Significance Our study shows that pyruvate kinase M2 (PKM2), an alternatively spliced variant of the pyruvate kinase gene, mediates epithelial–mesenchymal transition (EMT), which is critical for cancer cells to acquire invasive potential. Our study demonstrates that EMT stimulates nuclear translocation of PKM2 and decreases epithelial cadherin transcription (a requirement for EMT induction). Our results also demonstrate that PKM2 interacts with the transcriptional factor TGF-β–induced factor homeobox 2, which induces the deacetylation of histone H3, resulting in repressed E-cadherin expression. The precise understanding of nuclear PKM2 function suggests the potential for a model preventing cancer metastasis. 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.

Tumour-suppressive function of SIRT4 in human colorectal cancer.

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.

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.

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.

Combined evaluation of hexokinase 2 and phosphorylated pyruvate dehydrogenase-E1α in invasive front lesions of colorectal tumors predicts cancer metabolism and patient prognosis.

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.

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.

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.