Fumiaki Tanaka

Long Noncoding RNA HOTAIR Regulates Polycomb-Dependent Chromatin Modification and Is Associated with Poor Prognosis in Colorectal Cancers

The functional impact of recently discovered long noncoding RNAs (ncRNAs) in human cancer remains to be clarified. One long ncRNA which has attracted attention is the Hox transcript antisense intergenic RNA termed HOTAIR, a long ncRNA expressed from the developmental HOXC locus located on chromosome 12q13.13. In cooperation with Polycomb complex PRC2, the HOTAIR long ncRNA is reported to reprogram chromatin organization and promote breast cancer metastasis. In this study, we examined the status and function of HOTAIR in patients with stage IV colorectal cancer (CRC) who have liver metastases and a poor prognosis. HOTAIR expression levels were higher in cancerous tissues than in corresponding noncancerous tissues and high HOTAIR expression correlated tightly with the presence of liver metastasis. Moreover, patients with high HOTAIR expression had a relatively poorer prognosis. In a subset of 32 CRC specimens, gene set enrichment analysis using cDNA array data revealed a close correlation between expression of HOTAIR and members of the PRC2 complex (SUZ12, EZH2, and H3K27me3). Our findings suggest that HOTAIR expression is associated with a genome-wide reprogramming of PRC2 function not only in breast cancer but also in CRC, where upregulation of this long ncRNA may be a critical element in metastatic progression.

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.

CD13 is a therapeutic target in human liver cancer stem cells

Cancer stem cells (CSCs) are generally dormant or slowly cycling tumor cells that have the ability to reconstitute tumors. They are thought to be involved in tumor resistance to chemo/radiation therapy and tumor relapse and progression. However, neither their existence nor their identity within many cancers has been well defined. Here, we have demonstrated that CD13 is a marker for semiquiescent CSCs in human liver cancer cell lines and clinical samples and that targeting these cells might provide a way to treat this disease. CD13+ cells predominated in the G0 phase of the cell cycle and typically formed cellular clusters in cancer foci. Following treatment, these cells survived and were enriched along the fibrous capsule where liver cancers usually relapse. Mechanistically, CD13 reduced ROS-induced DNA damage after genotoxic chemo/radiation stress and protected cells from apoptosis. In mouse xenograft models, combination of a CD13 inhibitor and the genotoxic chemotherapeutic fluorouracil (5-FU) drastically reduced tumor volume compared with either agent alone. 5-FU inhibited CD90+ proliferating CSCs, some of which produce CD13+ semiquiescent CSCs, while CD13 inhibition suppressed the self-renewing and tumor-initiating ability of dormant CSCs. Therefore, combining a CD13 inhibitor with a ROS-inducing chemo/radiation therapy may improve the treatment of liver cancer.

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.

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.

MicroRNA-125a-5p Is an Independent Prognostic Factor in Gastric Cancer and Inhibits the Proliferation of Human Gastric Cancer Cells in Combination with Trastuzumab

Purpose: MicroRNA 125a-5p (miR-125a-5p) has been reported to be a tumor suppressor in malignancies of the breast, ovary, lung, and central nervous system. However, the clinical significance of miR-125a-5p in human gastrointestinal cancer has not been explored. We investigated a tumor inhibitory effect of miR-125a-5p in gastric cancer, focusing in particular on the miR-125a-ERBB2 (HER2, HER-2/neu) pathway. Experimental Design: Quantitative RT-PCR was used to evaluate miR-125a-5p expression in 87 gastric cancer cases to determine the clinicopathologic significance of miR-125a-5p expression. The regulation of ERBB2 by miR-125a-5p was examined with precursor miR-125a–transfected cells. Furthermore, we investigated whether miR-125a-5p suppresses proliferation of gastric cancer cells in combination with trastuzumab, a monoclonal antibody against ERBB2. Results: Low expression levels of miR-125a-5p were associated with enhanced malignant potential such as tumor size (P = 0.0068), tumor invasion (P = 0.031), liver metastasis (P = 0.029), and poor prognosis (P = 0.0069). Multivariate analysis indicated that low miR-125a-5p expression was an independent prognostic factor for survival. In vitro assays showed that ERBB2 is a direct target of miR-125a-5p, which potently suppressed the proliferation of gastric cancer cells, and, interestingly, the growth inhibitory effect was enhanced in combination with trastuzumab. Conclusions:miR-125a-5p is a meaningful prognostic marker. Furthermore, miR-125a-5p mimic alone or in combination with trastuzumab could be a novel therapeutic approach against gastric cancer. Clin Cancer Res; 17(9); 2725–33. ©2011 AACR.

Clinical Significance of miR-146a in Gastric Cancer Cases

Purpose: The profiles of microRNAs change significantly in gastric cancer. MiR-146a is reported to be a tumor suppressor in pancreatic cancer, breast cancer, and prostate cancer. We investigated the clinical significance of miR-146a in gastric cancer, in particular focusing on hypothetical miR-146a target genes, such as epidermal growth factor receptor (EGFR) and interleukin-1 receptor-associated kinase (IRAK1). Experimental Design: We examined miR-146a levels in 90 gastric cancer samples by q-real-time (qRT)–PCR and analyzed the association between miR-146a levels and clinicopathologic factors and prognosis. The regulation of EGFR and IRAK1 by miR-146a was examined with miR-146a–transfected gastric cancer cells. Moreover, we analyzed the association between miR-146a levels and the G/C single nucleotide polymorphism (SNP) within pre-miR-146a seed sequences in 76 gastric cancer samples, using direct sequencing of genomic DNA. Results: In 90 clinical samples of gastric cancer, miR-146a levels in cancer tissues were significantly lower than those in the corresponding noncancerous tissue (P < 0.001). Lower levels of miR-146a were associated with lymph node metastasis and venous invasion (P < 0.05). Moreover, a lower level of miR-146a was an independent prognostic factor for overall survival (P = 0.003). Ectopic expression of miR-146a inhibited migration and invasion and downregulated EGFR and IRAK1 expression in gastric cancer cells. In addition, G/C SNP within the pre-miR-146a seed sequence significantly reduced miR-146a levels in the GG genotype compared with the CC genotype. Conclusions:MiR-146a contains an SNP, which is associated with mature miR-146a expression. MiR-146a targeting of EGFR and IRAK1 is an independent prognostic factor in gastric cancer cases. Clin Cancer Res; 17(13); 4277–84. ©2011 AACR.

Plastin3 Is a Novel Marker for Circulating Tumor Cells Undergoing the Epithelial–Mesenchymal Transition and Is Associated with Colorectal Cancer Prognosis

Circulating tumor cells (CTC) in blood have attracted attention both as potential seeds for metastasis and as biomarkers. However, most CTC detection systems might miss epithelial-mesenchymal transition (EMT)-induced metastatic cells because detection is based on epithelial markers. First, to discover novel markers capable of detecting CTCs in which EMT has not been repressed, microarray analysis of 132 colorectal cancers (CRC) from Japanese patients was conducted, and 2,969 genes were detected that were overexpressed relative to normal colon mucosa. From the detected genes, we selected those that were overexpressed CRC with distant metastasis. Then, we analyzed the CRC metastasis-specific genes (n = 22) to determine whether they were expressed in normal circulation. As a result, PLS3 was discovered as a CTC marker that was expressed in metastatic CRC cells but not in normal circulation. Using fluorescent immunocytochemistry, we validated that PLS3 was expressed in EMT-induced CTC in peripheral blood from patients with CRC with distant metastasis. PLS3-expressing cells were detected in the peripheral blood of approximately one-third of an independent set of 711 Japanese patients with CRC. Multivariate analysis showed that PLS3-positive CTC was independently associated with prognosis in the training set (n = 381) and the validation set [n = 330; HR = 2.17; 95% confidence interval (CI) = 1.38-3.40 and HR = 3.92; 95% CI = 2.27-6.85]. The association between PLS3-positive CTC and prognosis was particularly strong in patients with Dukes B (HR = 4.07; 95% CI = 1.50-11.57) and Dukes C (HR = 2.57; 95% CI = 1.42-4.63). PLS3 is a novel marker for metastatic CRC cells, and it possesses significant prognostic value.

Biological and Genetic Characteristics of Tumor-Initiating Cells in Colon Cancer

BackgroundHuman prominin-1 (PROM1, CD133) was used as a marker to detect stem cells (progenitor cells) and cancer stem cells (tumor-initiating cells) in various tissues. The purpose of this study was to investigate the biological and genetic characteristics of tumor-initiating cells in colon cancer with both in vitro and in vivo analyses.MethodsThe CD133 expression of 12 colon cancer cell lines was evaluated. CD133+ cells were isolated by flow cytometry and examined for in vivo tumor formation, in vitro proliferation, colony formation, and invasion ability. Additionally, we used microarray analysis to compare gene expression profiles between CD133+ and CD133– isolated cells.ResultsCD133+ cells were found in 5 of 12 colon cancer cell lines. Isolated CD133+ cells from the HT29 colon cancer cell line exhibited a higher tumorigenic potential than CD133– cells in the in vivo tumor formation assay. Furthermore, it was shown that CD133+ cells are more proliferative and have higher colony-forming and invasive abilities than CD133– cells in vitro. Microarray analysis found differential gene expression correlating with CD133 expression.ConclusionsIt was confirmed that CD133+ cells in colon cancer are useful markers for the detection of tumor-initiating cells. Intimate biological and genetic features of CD133+ cells in colon cancer cell lines were also revealed. The biological characteristics of CD133+ cells and differentially expressed genes in these cells will help elucidate more details of tumor-initiating cells in colon cancer.

Expression of multiple cancer-testis antigen genes in gastrointestinal and breast carcinomas

Cancer-testis antigens (CTAs) such as MAGE are selectively expressed in various types of human neoplasms but not in normal tissues other than testis. This characteristic feature of CTAs makes them promising antigens for cancer-specific immunotherapy. A critical requirement for this therapy is identification of promising antigens. In this study, we investigated the expression of 6 genes recently identified by serological analysis of antigens by recombinant expression (SEREX) libraries: NY-ESO-1, LAGE-1, SCP-1, SSX-1, SSX-2, and SSX-4, in many surgical samples of gastrointestinal and breast carcinomas using reverse transcription-polymerase chain reaction. We found relatively high expression of SCP-1 (23.5%) and SSX-4 (20.6%) in gastric carcinoma, LAGE-1 (39.1%) and NY-ESO-1 (23.9%) in oesophageal carcinoma, and SCP-1 (34.1%) in breast carcinoma. We also found frequent synchronous expression with MAGE, including LAGE-1 (46.2%) in oesophageal carcinoma, SSX-4 (46.7%) in gastric carcinoma, and SCP-1 (38.3%) in breast carcinoma. Immunohistochemical analysis of the tumour samples expressing both MAGE-4 and NY-ESO-1 genes demonstrated differences in distribution between MAGE-4 and NY-ESO-1 in serial sections. We concluded that NY-ESO-1, LAGE-1, SCP-1 and SSX-4 genes may be promising candidates for cancer-specific immunotherapy in addition to MAGE, and that polyvalent cancer vaccines may be useful in cases of heterogeneous expressions of CTA genes in gastrointestinal and breast carcinomas.