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.

miRNAs are key players in the integrated regulation of cellular processes and shape many of the functional properties that define the "cancer stem cell" (CSC) phenotype. Little is known, however, about miRNAs that regulate such properties in human colorectal carcinoma. In this study, we compared the expression levels of 754 miRNAs between paired samples of EpCAM þ /CD44 þ cancer cells (enriched in CSCs) and EpCAM þ /CD44 neg cancer cells (with CSC depletion) sorted in parallel from human primary colorectal carcinomas and identified miR-221 as the miRNA that displayed the highest level of preferential expression in EpCAM þ /CD44 þ cancer cells. High levels of miR-221 expression were associated with Lgr5 þ cells in mouse colon crypts and reduced survival in patients with colorectal carcinoma. Constitutive overexpression of miR-221 enhanced organoid-forming capacity of both conventional colorectal carcinoma cell lines and patient-derived xenografts (PDX) in vitro. Importantly, constitutive downregulation of miR-221 suppressed organoid-forming capacity in vitro and substantially reduced the tumorigenic capacity of CSC populations from PDX lines in vivo. Finally, the most abundant splicing isoform of the human Quaking (QKI) gene, QKI-5, was identified as a functional target of miR-221; overexpression of miR-221-reduced QKI-5 protein levels in human colorectal carcinoma cells. As expected, overexpression of QKI-5 suppressed organoid-forming capacity in vitro and tumorigenic capacity of colorectal carcinoma PDX cells in vivo. Our study reveals a mechanistic link between miR-221 and QKI and highlights their key role in regulating CSC properties in human colorectal cancer.

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.

Anaplastic lymphoma kinase (ALK) 1 and the related leukocyte tyrosine kinase (LTK) 2 are recently deorphanized receptor tyrosine kinases (RTK) 3 . Together with their activating cytokines, ALKAL1 and ALKAL2 (also called FAM150A/FAM150B and AUGβ/AUGα) 4-6 , they are involved in neural development 7 , cancer [7][8][9] , and autoimmune diseases 10 . Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain 11 , consistent with a metabolic role in Drosphila 12 . Despite such functional pleiotropy and growing therapeutic relevance 13,14 , structural insights into ALK and LTK and their complexes with cognate cytokines had remained elusive. Here, we show that the cytokine-binding segments of human ALK and LTK comprise an unprecedented architectural chimera of a permuted TNF-like module that braces a Glycine-rich subdomain featuring a hexagonal lattice of long polyglycine-II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicit similar dimeric assemblies with twofold symmetry, that tent a single cytokine molecule proximal to the cell membrane. We show that the membrane-proximal EGF-like domain dictates the apparent cytokine preference of ALK. Assisted by diverse structure-function findings, we propose a structural and mechanistic blueprint for complexes of ALK family receptors, thereby extending the repertoire of ligand-mediated dimerization mechanisms adopted by RTK. MainALK is an evolutionarily ancient RTK with the vertebrate orthologues uniquely endowed with an Nterminal heparin binding domain (HBD) (Fig. 1a and Extended Data Fig. 1a,b). Gene duplication in vertebrates spawned LTK as a second ALK-like receptor 16 , which evolved divergently with loss of the HBD and additionally the MAM-LDLa-MAM module in mammals (Extended Data Fig. 1c). The common architectural hallmark in the ectodomains of ALK and LTK comprises their cytokine-binding segment, which is unique among cytokine receptors and features an intriguing array of a TNF-like (TNFL) module, a glycine-rich (GR) region, and a membrane-proximal EGF-like module (EGFL) (Fig. 1a). Whereas ALKAL1 and ALKAL2 are both strong activators of LTK 4 , only ALKAL2 potently activates ALK 5,6 coupled to additional regulation via glycosaminoglycan binding to its HBD 15 .

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.

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.

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.

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