Junko Mukohyama

MicroRNA Regulation of Human Breast Cancer Stem Cells

MicroRNAs (miRNAs) are involved in virtually all biological processes, including stem cell maintenance, differentiation, and development. The dysregulation of miRNAs is associated with many human diseases including cancer. We have identified a set of miRNAs differentially expressed between human breast cancer stem cells (CSCs) and non-tumorigenic cancer cells. In addition, these miRNAs are similarly upregulated or downregulated in normal mammary stem/progenitor cells. In this review, we mainly describe the miRNAs that are dysregulated in human breast CSCs directly isolated from clinical specimens. The miRNAs and their clusters, such as the miR-200 clusters, miR-183 cluster, miR-221-222 cluster, let-7, miR-142 and miR-214, target the genes and pathways important for stem cell maintenance, such as the self-renewal gene BMI1, apoptosis, Wnt signaling, Notch signaling, and epithelial-to-mesenchymal transition. In addition, the current evidence shows that metastatic breast CSCs acquire a phenotype that is different from the CSCs in a primary site. Thus, clarifying the miRNA regulation of the metastatic breast CSCs will further advance our understanding of the roles of human breast CSCs in tumor progression.

Roles of microRNAs and RNA-Binding Proteins in the Regulation of Colorectal Cancer Stem Cells

Colorectal cancer stem cells (CSCs) are responsible for the initiation, progression and metastasis of human colorectal cancers, and have been characterized by the expression of cell surface markers, such as CD44, CD133, CD166 and LGR5. MicroRNAs (miRNAs) are differentially expressed between CSCs and non-tumorigenic cancer cells, and play important roles in the maintenance and regulation of stem cell properties of CSCs. RNA binding proteins (RBPs) are emerging epigenetic regulators of various RNA processing events, such as splicing, localization, stabilization and translation, and can regulate various types of stem cells. In this review, we summarize current evidences on the roles of miRNA and RBPs in the regulation of colorectal CSCs. Understanding the epigenetic regulation of human colorectal CSCs will help to develop biomarkers for colorectal cancers and to identify targets for CSC-targeting therapies.

Evaluation of the risk of lymphomagenesis in xenografts by the PCR-based detection of EBV BamHI W region in patient cancer specimens

Establishment of patient-derived tumor xenografts (PDXs) is hampered by lymphomagenesis mostly caused by the latently-infected Epstein-Barr virus (EBV) contained in patient cancer tissues. However, the character of patient tissues that result in lymphomagenesis after xenotransplantation is not elucidated. In this study, we analyzed the patient colorectal cancer (CRC) tissues and the PDXs established by their xenotransplantation. We found that 2 of 9 (22%) PDX tumors were EBV-associated human diffuse large B cell lymphoma which was formed by clonal proliferation of human B-cell lymphocytes, were strongly positive for EBER-ISH, and were classified as type III latency. Expression of EBV genes and RNAs, such as EBNAs, LMP1, EBER and EBV-associated microRNAs in patient CRC tissues were unlikely to be associated with lymphomagenesis in PDXs. In contrast, the positive PCR-based amplification of BamHI W region, a major internal repeat in EBV genome, in the patient CRC tissues was correlated with lymphomagenesis in PDXs. These results suggest that the detection of the EBV BamHI W region in the patient surgical specimens will be an effective way to predict the risk of lymphomagenesis in PDXs before xenotransplantation.

Laparoscopic ileocecal resection can be applied for appendiceal cancer with an ileal fistula: A case report

Highlights • We experienced a case of appendiceal cancer invading the ileum with a fistula.• This is the first case report of appendiceal cancer with an ileal fistula that successfully treated with laparoscopic resection.• Laparoscopic resection can be a feasible, safe and curative procedure in selected cases of appendiceal cancer with a fistula.• Laparoscopic ileocecal resection can be applied for appendiceal cancers with a fistula by experienced surgeons with careful consideration.

Recent advances of neoadjuvant chemoradiotherapy in rectal cancer: Future treatment perspectives

Neoadjuvant chemoradiotherapy (nCRT) has been widely used as a multidisciplinary approach for stage II/III rectal cancer. However, its safety and efficacy are controversial because previous studies have shown conflicting outcomes. The present review aimed to elucidate the benefits and limitations of nCRT for patients with rectal cancer. Future perspectives of nCRT are also described. No recent randomized trials have been able to show a survival benefit, although many studies have demonstrated good local control with the use of fluoropyrimidine (e.g. 5‐fluorouracil [FU] or capecitabine)‐based nCRT. Addition of oxaliplatin (OX) to FU‐based nCRT might improve overall survival by preventing distant metastasis, as shown in recent meta‐analyses. However, control of adverse effects is an important concern with this treatment. New treatment strategies such as nonoperative management (watch and wait policy) and total neoadjuvant therapy (TNT) are promising, but the establishment of reliable diagnostic methods of metastasis is essential. Development of new biomarkers is also necessary to select patients who are more likely to benefit from nCRT.

Abstract 2890: MicroRNA-mediated upregulation of the WNT signaling activities in human breast cancer stem cells

The canonical WNT signaling plays a critical role in many adult stem cells, including those of the breast and intestine. The fact that the canonical WNT signaling is implicated in both stem cell self-renewal and cancer suggests that normal physiological regulator of stem cell functions might be “hijacked” in cancer. Adenomatous polyposis coli (APC) is a component of the destruction complex that destabilizes β-catenin and suppresses the activity of the canonical WNT signaling. MicroRNAs (miRNAs) are important regulators of stem cell functions. We have previously reported a set of 37 miRNAs that are upregulated or downregulated in human breast cancer stem cells (BCSCs, a CD44+CD24-/lowlineage- population of human breast cancer cells) as compared to non-tumorigenic breast cancer cells (NTCs). Among them, miR-200c targets BMI1 that is a critical regulator of the stem cell maintenance, and strongly impairs the functions of human BCSCs in vivo. In this study, we compared the expression profiles of miRNAs, mRNAs and proteins between BCSCs and NTCs isolated from the patient specimens of human breast cancers and patient-derived tumor xenografts (PDXs) established by their transplantation. Luciferase assays were performed using the plasmid in which the 3’UTR region of candidate mRNA was cloned downstream of a luciferase minigene. The effect of miRNAs on the activity of WNT signaling was evaluated using a TCF reporter plasmid. Finally, the abilities to form organoids and to form tumors in immunodeficient mice were evaluated using the human BCSCs infected with the miRNA inhibitor expressing lentivirus. We found that miR-142 was highly upregulated in BCSCs, but was hardly expressed in NTCs in the patient breast cancer specimens. We confirmed that miR-142 targeted the sequence within the 3’UTR of APC mRNA and suppressed APC protein expression. Accordingly, miR-142 activated the canonical WNT signaling pathway in an APC-suppression dependent manner. The results of mRNA and protein expression profiling of the BCSCs isolated from human breast cancer PDXs suggested that the canonical WNT signaling was activated in BCSCs. Finally, inhibition of miR-142 in the BCSCs suppressed the tumor growth in vivo. These results suggest that the miR-142, a miRNA frequently upregulated in human BCSCs, could provide at least a part of the molecular mechanism for aberrant activation of the canonical WNT signaling in breast cancer in which APC mutations are much less frequent than colon cancer. Citation Format: Yohei Shimono, Taichi Isobe, Andrei Turtoi, Junko Mukohyama, Toru Mukohara, Akira Suzuki, Vincent Castronovo, Hironobu Minami. MicroRNA-mediated upregulation of the WNT signaling activities in human breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2890. doi:10.1158/1538-7445.AM2017-2890

Organoid Culture of Human Cancer Stem Cells

Organoid culture is a three-dimensional culture method that enables ex vivo analysis of stem cell behavior and differentiation. This method is also applicable to the studies on stem cell characters of human cancer stem cells. The components of organoid culture include Matrigel® and a culture medium containing growth factor cocktails that mimic the microenvironments of organ stem cell niches. Here, we describe the basic methods for the organoid culture of dissociated or FACS-sorted human cancer stem cells. Then, we introduce a method to dissociate the organoids for serial passage and propagation.