Yukio Kamohara

CD133 + CD44 + Population Efficiently Enriches Colon Cancer Initiating Cells

BackgroundPrevious reports have demonstrated that CD133+ cells or CD44+ cells might be cancer initiating cells (CIC) of colon cancer. However, the association between the two cell types is unclear. In this study, we evaluated the tumorigenicity of each population of human colon cancer divided by CD133 and CD44 using non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice.MethodsUsing the colon cancer cell lines HT29 and Caco2 we evaluated the change of expression status of CD133 or CD44 by a treatment with sodium butyrate (NaBT) that can induce cellular differentiation. Next, we prepared ten clinical samples of colon cancer and analyzed the expression and tumorigenicity of CD133 and CD44.ResultsWith NaBT treatment, CD44 expression was greatly downregulated in both HT29 and Caco2 (HT29: nontreatment versus treatment; 77.8% versus 0.6%, Caco2: 14.0% versus 0.4%, respectively), more than CD133 expression (HT29: nontreatment versus treatment; 90.1% versus 67.7%, Caco2: 98.9% versus 76.3%, respectively). In clinical samples, the percentages of CD133+ cells and CD44+ cells varied from 0.3% to 82.0% (mean 35.5%), and from 11.5% to 58.4% (mean 30.0%), respectively. Subcutaneous injection of CD133+ or CD44+ cells made a tumor in all mice (3/3 and 4/4, respectively). The combined analysis of CD133 and CD44 revealed that only the CD133+CD44+ population had the ability to produce a tumor (3/3).ConclusionThe findings demonstrate that, at present, the CD133+CD44+ population may be the best to identify tumor initiating cells of human colon cancer.

The search for cancer stem cells in hepatocellular carcinoma

BACKGROUNDnRecent evidence suggests that some solid cancers originate from cancer stem cells. We have identified a subset of candidate stem cells, which are termed side population (SP) cells, in the hepatocellular carcinoma cell line Huh7. Because most stem cells reside in the G0 phase of the cell cycle, G0 cells were isolated, and the relationship between SP cells and G0 cells was investigated to clarify the biological characteristics of G0 cells.nnnMETHODSnHuh7 cells were sorted using Hoechst 33342 and Pyronin Y. The cells were then divided into G0, G1, and G2/M fractions and cultured under low-attachment conditions to obtain cellular spheres. Tumorigenetic ability was investigated using subcutaneous transplantation to NOD/SCID mice. G0 and G1 cells were analyzed for markers indicative of hepatocytic (albumin expression) and cholangiocytic (keratin 19 expression) differentiation and DNA synthesis (Ki67).nnnRESULTSnThe cell-cycle distribution of cultured Huh7 cells was 0.7% (G0), 63.8% (G1), and 34.5% (G2/M, S). The G0 cells were located within the neck of the SP fraction. The G0 cells showed spheroid formation and 3-dimensional growth. Those cells showed marked tumorigenesis in NOD/SCID transplantation. G0 cells, which did not express Ki67, were weakly positive for expression of albumin and were clearly positive for the expression of keratin 19. In contrast, G1 cells were positive for Ki67 and albumin expression but negative for keratin 19.nnnCONCLUSIONnG0 cells are present in the SP fraction of Huh7. They show self-renewal, tumorigenesis, and bidirectional lineage. These findings suggest that the G0 cells within the Huh7 cell line are promising candidates as cancer stem cells for future studies of hepatocellular carcinoma.

Mitotic centromere-associated kinesin is a novel marker for prognosis and lymph node metastasis in colorectal cancer.

Mitotic centromere-associated kinesin (MCAK) is a microtubule depolymerase that is essential for proper kinetochore–microtubule attachment during spindle formation. Overexpression of MCAK has been correlated with aggressive forms of carcinoma, resulting in poor prognosis of colorectal cancer. The purpose of this study was to quantify MCAK expression in malignant and benign colorectal tissues and to determine if MCAK expression levels correlate with clinicopathologic factors and prognosis in colorectal cancer patients. Paired colorectal tissue samples from tumours and the corresponding normal tissues were obtained from 120 patients with colorectal cancer who underwent surgical resection. The real-time reverse transcriptase-PCR and immunohistochemistry were used to analyse mRNA and protein expression status with respect to various clinicopathological factors. MCAK expression was higher in colorectal cancer tissue (P<0.01) than in corresponding normal tissue, and this elevated expression level was markedly associated with factors such as lymph node metastasis (P=0.0023), venous invasion (P=0.019), peritoneal dissemination (P=0.021) and Dukes classification (P=0.0023). Patients with high MCAK mRNA expression also showed a far poorer survival rate than those with low MCAK mRNA expression (P<0.01). Elevated MCAK expression was an independent predictor of overall survival and lymph node metastasis. These data suggest that MCAK expression may serve as a good marker of prognosis and lymph node metastasis in colorectal cancer.

Clinical Significance of Stanniocalcin 2 as a Prognostic Marker in Gastric Cancer

BackgroundStanniocalcins are glycoprotein hormones that were originally found in the endocrine gland of bony fish. Microarray expression data from 32 paired samples of gastric cancer and normal mucosa in a public microarray database showed that the expression level of Stanniocalcin 2 was higher in gastric cancer than in normal gastric mucosa. The clinical significance of Stanniocalcin 2 expression has been observed for several cancers. However, the relationship between Stanniocalcin 2 and clinicopathological factors in gastric cancer has not yet been investigated.Materials and MethodsWe examined the clinical significance of Stanniocalcin 2 in gastric cancer in 108 gastric cancer samples using real-time reverse transcription-polymerase chain reaction (RT–PCR). Immunohistochemical studies were conducted with paraffin sections. The suppression analysis of Stanniocalcin 2 using siRNA was done to determine Stanniocalcin 2’s biological roles.ResultsThe level of Stanniocalcin 2 in cancer tissues was higher than in normal tissues (Pxa0=xa0.0002). The high Stanniocalcin 2 expression group (nxa0=xa054) had more progressive lymph node metastasis (Pxa0=xa0.07) and venous invasion (Pxa0=xa0.028) than the low-expression group (nxa0=xa054). High Stanniocalcin 2 expression was an independent prognostic factor in gastric cancer patients (Pxa0=xa0.02). Moreover, siRNA suppression of Stanniocalcin 2 in a gastric cancer cell line inhibited cellular proliferation (Pxa0<xa0.05).ConclusionsThe high expression level of Stanniocalcin 2 in gastric cancer tissues could be a very powerful marker of poor prognosis. Therefore, Stanniocalcin 2 is a promising candidate for a molecular target for the treatment of gastric cancer.

Clinical Significance of Loss of Fhl1 Expression in Human Gastric Cancer

BackgroundHuman four-and-a-half LIM domains 1 (Fhl1) gene has been reported to achieve cancer suppressive effects. The purpose of this study is to clarify clinical significance of Fhl1 expression in gastric cancer.MethodsFhl1 mRNA expression was quantified by real-time reverse transcription (RT)-polymerase chain reaction (PCR) using paired tumor and normal clinical samples from a total of 110 gastric cancer patients. Fhl1 protein expression was investigated by immunohistochemistry. The relationship between Fhl1 mRNA expression and clinicopathological factors was statistically analyzed.ResultsFhl1 mRNA expression in tumor tissue was significantly downregulated compared with that in normal mucosa (Pxa0<xa00.0001). Fhl1 protein expression was also diminished in cancer cells by immunohistochemistry. Kaplan–Meier survival curves demonstrated that the patients with low Fhl1 expression tumor showed significantly shorter survival (Pxa0<xa00.05) than those with high Fhl1 expression tumor. Furthermore, the tumors with low Fhl1 expression showed deeper invasion into the serosal layer (Pxa0<xa00.05) or more frequent distant metastasis (Pxa0<xa00.01).ConclusionFhl1 gene was underexpressed in clinical gastric cancer. Loss of Fhl1 expression would be a novel biomarker to determine biological aggressiveness of gastric cancer.

Prognostic Relevance of Tensin4 Expression in Human Gastric Cancer

BackgroundThe Tensin4 gene is involved in various biological events by mediating signal transduction. This study was designed to clarify its clinical significance in gastric cancer.MethodsA total of 114 gastric cancer patients were enrolled in this study, and we obtained paired samples from tumor tissue and matched normal mucosa. Total RNA was extracted, and TNS4 mRNA expression was quantified using real-time reverse-transcription polymerase chain reaction. TNS4 protein expression was investigated by immunohistochemistry. Additionally, the relationship between TNS4 expression and clinicopathologic factors was statistically analyzed.ResultsTNS4 mRNA expression was significantly higher in tumor tissue than in normal mucosa (pxa0<xa00.0001). Additional immunohistochemical analysis demonstrated that TNS4 protein was predominantly expressed in gastric cancer. Tumors with higher TNS4 mRNA expression showed histologically poorer grade (pxa0<xa00.02), deeper invasion into the serosa (pxa0<xa00.01), more positive lymph node metastasis (pxa0<xa00.02) or peritoneal dissemination (pxa0<xa00.05). Patients with high TNS4 mRNA expression tumor were more likely to have cancer-related death (pxa0<xa00.05). A Kaplan–Meier curve also demonstrated that patients with high TNS4 expression tumor had significantly poorer prognosis (pxa0<xa00.01) than those with low TNS4 expression tumor. In particular, the 2-year overall survival rates were 48.2% and 83.1%, respectively, a very significant difference. Multivariate analysis revealed that high TNS4 mRNA expression was an independent prognostic predictor (relative risk 1.41, 95% confidence interval 1.03–1.96, pxa0=xa00.03).ConclusionTNS4 was frequently overexpressed in gastric cancer, and tumors with high TNS4 mRNA expression showed biologically aggressive behavior. High TNS4 mRNA expression may be a novel prognostic predictor for those patients.

Identification of HLA-A*0201/-A*2402-restricted CTL epitope-peptides derived from a novel cancer/testis antigen, MCAK, and induction of a specific antitumor immune response.

Cancer immunotherapy is a potential therapeutic strategy, in addition to surgical treatment, radiotherapy, and chemotherapy. Cancer-specific immunotherapy, such as the MAGE peptide vaccine, has been utilized clinically. How-ever, there are inherent limits to the effectiveness of vaccinotherapy using a single antigen because of the expression frequency of cancer-specific antigens on tumor cells. Thus, identification of a new cancer-specific antigen is needed. In this study, we examined the possibility of using cancer-specific immunotherapy based upon mitotic centromere-associated kinesin (MCAK) which was previously identified as a novel cancer/testis antigen. To evaluate the feasibility of developing cancer immunotherapy using MCAK peptides, we studied HLA-A*0201 and *2402 as targets for CTLs in the context of HLA class I molecules. By using a peptide with a sequence of AINPELLQL (amino acid positions 63-71 in MCAK, HLA-A*0201) and FFEIYNGKL (amino acid positions 401-409 in MCAK, HLA-A*2402), CTL responses could be induced from unseparated PBMCs by stimulation of freshly isolated, peptide-pulsed PBMCs as antigen-presenting cells (APCs) and also by using interleukin-7 and keyhole limpet hemocyanin in primary culture. The induced CTLs could lyse HLA-A-*0201/*2402 colon and gastric cancer cells expressing MCAK, as well as the peptide-pulsed target cells, in an HLA class l, and CD8 restricted manner. The identification of the MCAK/HLA-A*0201 and *2402 peptides suggests the possibility of designing peptide-based immunotherapeutic approaches that might prove effective in treating patients with MCAK-positive cancer.

Long-term outcome of adipose-derived regenerative cell-enriched autologous fat transplantation for reconstruction after breast-conserving surgery for Japanese women with breast cancer

PurposeMore effective methods are needed for breast reconstruction after breast-conserving surgery for breast cancer. The aim of this clinical study was to assess the perioperative and long-term outcomes of adipose-derived regenerative cell (ADRC)-enriched autologous fat grafting.MethodsTen female patients who had undergone breast-conserving surgery and adjuvant radiotherapy for breast cancer were enrolled. An ADRC-enriched fat graft prepared from the patient’s adipose tissue was implanted at the time of adipose tissue harvest. The perioperative and long-term outcomes of the grafts, which included safety, efficacy, and questionnaire-based patient satisfaction, were investigated.ResultsThe mean operation time was 188xa0±xa030xa0min, and the mean duration of postoperative hospitalization was 1.2xa0±xa00.4xa0days. No serious postoperative complications were associated with the procedure. Neither recurrence nor metastatic disease was observed during the follow-up period (7.8xa0±xa01.5xa0years) after transplantation. Of 9 available patients, “more than or equal to average” satisfaction with breast appearance and overall satisfaction were reported by 6 (66.7%) and 5 (55.6%) patients, respectively.ConclusionsADRC-enriched autologous fat transplantation is thus considered to be safe perioperatively, with no long-term recurrence, for patients with breast cancer treated by breast-conserving surgery, and it may be an option for breast reconstruction, even after adjuvant radiotherapy.