CDX2 Is a Prognostic Biomarker for Unresectable Metastatic Colorectal Cancer.

BACKGROUND/AIM: Colorectal cancer (CRC) with reduced expression of the homeobox transcription factor CDX2, a master gene essential for the development and maintenance of the intestinal tract, is known as a poor prognosis subtype of CRC. The recurrence rate is high in patients with CDX2low CRC. However, the prognostic significance of CDX2 in advanced CRC is unclear. This study aimed to elucidate the prognostic significance of CDX2 in unresectable metastatic CRC (mCRC). PATIENTS AND METHODS: Twenty-nine patients with unresectable mCRC who underwent primary site resection at the Kobe University Hospital during a 6-year period from January 2008 to January 2015 were included. The tissues from those patients were immunohistochemically stained with anti-CDX2 antibody (clone: CDX2-88). The patients were divided into CDX2high CRC group and CDX2low CRC group and their prognoses were analyzed. RESULTS: There were no clear differences in background between the two groups. A low CDX2 expression was associated with reduced overall survival (37.67 months vs. 25.32 months, p=0.03) and tended to associate with reduced progression-free survival (17.4 months vs. 12.9 months, p=0.37). Two patients received chemotherapy after resection of the primary lesion and obtained pathological complete response. CONCLUSION: CDX2 expression might be a possible prognostic biomarker for unresectable mCRC.

Upregulation of BMI1-suppressor miRNAs (miR-200c, miR-203) during terminal differentiation of colon epithelial cells.

BACKGROUND: MicroRNAs (miRNAs) are key regulators of stem cell functions, including self-renewal and differentiation. In this study, we aimed to identify miRNAs that are upregulated during terminal differentiation in the human colon epithelium, and elucidate their role in the mechanistic control of stem cell properties. METHODS: "Bottom-of-the-crypt" (EPCAM+/CD44+/CD66alow) and "top-of-the-crypt" (EPCAM+/CD44neg/CD66ahigh) epithelial cells from 8 primary colon specimens (6 human, 2 murine) were purified by flow cytometry and analyzed for differential expression of 335 miRNAs. The miRNAs displaying the highest upregulation in "top-of-the-crypt" (terminally differentiated) epithelial cells were tested for positive correlation and association with survival outcomes in a colon cancer RNA-seq database (n = 439 patients). The two miRNAs with the strongest "top-of-the-crypt" expression profile were evaluated for capacity to downregulate self-renewal effectors and inhibit in vitro proliferation of colon cancer cells, in vitro organoid formation by normal colon epithelial cells and in vivo tumorigenicity by patient-derived xenografts (PDX). RESULTS: Six miRNAs (miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-345) were upregulated in "top-of-the-crypt" cells and positively correlated in expression among colon carcinomas. Overexpression of the three miRNAs with the highest inter-correlation coefficients (miR-200a, miR-200b, miR-200c) associated with improved survival. The top two over-expressed miRNAs (miR-200c, miR-203) cooperated synergistically in suppressing expression of BMI1, a key regulator of self-renewal in stem cell populations, and in inhibiting proliferation, organoid-formation and tumorigenicity of colon epithelial cells. CONCLUSION: In the colon epithelium, terminal differentiation associates with the coordinated upregulation of miR-200c and miR-203, which cooperate to suppress BMI1 and disable the expansion capacity of epithelial cells.

Structural basis of cytokine-mediated activation of ALK family receptors.

Anaplastic lymphoma kinase (ALK)1 and the related leukocyte tyrosine kinase (LTK)2 are recently deorphanized receptor tyrosine kinases3. Together with their activating cytokines, ALKAL1 and ALKAL24-6 (also called FAM150A and FAM150B or AUGß and AUGα, respectively), they are involved in neural development7, cancer7-9 and autoimmune diseases10. Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain11, consistent with a metabolic role for Drosophila ALK12. Despite such functional pleiotropy and growing therapeutic relevance13,14, structural insights into ALK and LTK and their complexes with cognate cytokines have remained scarce. Here we show that the cytokine-binding segments of human ALK and LTK comprise a novel architectural chimera of a permuted TNF-like module that braces a glycine-rich subdomain featuring a hexagonal lattice of long polyglycine type II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicits similar dimeric assemblies with two-fold 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 these diverse structure-function findings, we propose a structural and mechanistic blueprint for complexes of ALK family receptors, and thereby extend the repertoire of ligand-mediated dimerization mechanisms adopted by receptor tyrosine kinases.

Simple Cancer Stem Cell Markers Predict Neoadjuvant Chemotherapy Resistance of Esophageal Squamous Cell Carcinoma.

BACKGROUND/AIM: Cancer stem cells (CSCs) contribute to resistance against neoadjuvant chemotherapy (NAC) in esophageal squamous cell carcinoma (ESCC). We conducted a retrospective observational study for the relationship between the expression levels of CSC markers in biopsy specimens prior to 5-fluorouracil plus cisplatin (FP)-NAC and the pathological responses. PATIENTS AND METHODS: We included 171 patients with ESCC who underwent the FP-NAC followed by radical resection. Biopsy specimens prior to the FP-NAC were obtained and immunochemically stained for CD44, CD133, and CD24. RESULTS: The biopsy specimens of the non-responders had the CD44high/CD24low expression at high levels, which was found as an independent predictor of not only FP-NAC resistance but also poor overall survival by multivariate analyses. CONCLUSION: CD44high/CD24low expression in the biopsy specimens prior to FP-NAC may be a predictor of FP-NAC resistance and poor prognosis of ESCC patients.

Treatment Strategy for Rectal Cancer Patients With Inguinal Lymph Node Metastasis.

BACKGROUND/AIM: To investigate the impact of inguinal lymph node dissection (ILND) following neoadjuvant chemoradiotherapy (NACRT) for rectal cancer patients with ILN metastasis. PATIENTS AND METHODS: Forty-three patients with rectal cancer underwent NACRT followed by curative surgery between January 2005 and December 2016. Seven patients underwent ILND after NACRT for clinically-positive ILN metastasis (ILND (+) group), while the remaining 36 did not receive ILND for clinically negative ILN metastasis (ILND (-) group). Their outcomes were retrospectively analyzed. RESULTS: Only one patient in the ILND (+) group had a local recurrence at six years after surgery. The 5-year recurrence-free survival was 100% and 65.4% in the ILND (+) and ILND (-) groups, respectively (p=0.09), and the 5-year overall survival was 100% and 83.2%, respectively (p=0.32). CONCLUSION: ILND following NACRT seems effective for rectal cancer patients with ILN metastasis.

miR-221 Targets QKI to Enhance the Tumorigenic Capacity of Human Colorectal Cancer Stem Cells.

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+/CD44+ cancer cells (enriched in CSCs) and EpCAM+/CD44neg 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+/CD44+ cancer cells. High levels of miR-221 expression were associated with Lgr5+ 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. SIGNIFICANCE: These findings uncover molecular mechanisms underlying the maintenance of cancer stem cell properties in colon cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5151/F1.large.jpg.

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.

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.

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

INTRODUCTION: Primary appendiceal cancer with fistula formation is extremely rare. We report a case of a patient with appendiceal cancer invading the ileum who underwent successful laparoscopic ileocecal resection. PRESENTATION OF CASE: A 76-year-old man who presented with fever and abdominal pain was diagnosed with acute appendicitis and received antibiotics at a local hospital. After a few days, he was referred to our hospital because of an abnormality found in the colonoscopy, which was an oozing ulcer in the terminal ileum. Laparoscopic ileocecal resection was performed with a preoperative diagnosis of ileal cancer. The tumor adhered to the right internal inguinal ring. We dissected the right spermatic cord involved in the tumor. The resected specimen revealed a fistula between the appendiceal orifice and ileac ulcer. Histopathological examination revealed a well differentiated tubular adenocarcinoma. We made the diagnosis of appendiceal cancer with an ileal fistula because the ileal ulcer was derived from the appendiceal site. DISCUSSION: Most cases of appendiceal cancer with a fistula undergo laparotomy, but in selected cases, laparoscopic resection should be considered a feasible, safe, and curative procedure. Our patient underwent laparoscopic ileocecal resection, whereby the tumor and other organs with invasion were resected successfully with a negative surgical margin. CONCLUSION: This is the first case report of appendiceal cancer with an ileal fistula successfully treated with laparoscopic resection. Laparoscopic ileocecal resection can be applied for appendiceal cancers with a fistula by experienced surgeons with careful consideration.

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.

Discordance of MCM7 mRNA and its Intronic MicroRNA Levels Under Hypoxia.

BACKGROUND: Intronic microRNAs (miRNAs) are considered to be transcribed using their host gene promoter. However, about one third of intronic miRNAs are predicted to have independent promoter elements. MATERIALS AND METHODS: Human breast cancer cells were cultured under normoxia or hypoxia, and expression levels of intronic miR-106b-25 cluster miRNAs and their host gene minichromosome maintenance complex component 7 (MCM7) transcripts were analyzed by semi-quantitative polymerase chain reaction. The putative promoter element of miR-106b-25 cluster was analyzed by chromatin immunoprecipitation and luciferase assays. RESULTS: Exposure to hypoxia reduced the expression of MCM7 mRNA and a primary transcript of miR-106b-25 cluster, but did not affect that of mature miRNAs. The putative promoter element of miR-106b-25 cluster was not bound by hypoxia-inducible factor 1-alpha (HIF1-α), and was not activated under hypoxia. CONCLUSION: Maintenance of miR-106b-25 cluster miRNA levels under hypoxia was not caused by the activation of an independent promoter element.

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.

Effect of Xenotransplantation Site on MicroRNA Expression of Human Colon Cancer Stem Cells.

BACKGROUND: Cancer stem cells (CSCs) have a high tumorigenic ability to form patient-derived tumor xenografts (PDXs). PDXs are an attractive pre-clinical model, but gene expression and biological behavior of cancer cells in the tumor will change during establishment and passage of PDXs. MATERIALS AND METHODS: Human colon cancer PDX was established and passaged either subcutaneously or orthotopically into the murine intestine. Histology and flow cytometric profile of the surgical specimen and the PDX were analyzed. CSCs were then isolated from the tumors and their microRNA (miRNA) expression was analyzed by semi-quantitative polymerase chain reaction. RESULTS: The surgical specimens and PDXs were histologically similar. The size of CSC population increased and expression of miRNAs in CSCs changed in the passaged PDXs. Expression of oncogenic miRNAs was highly up-regulated in the CSCs of the orthotopically passaged PDXs. CONCLUSION: The xenotransplantation site and the number of tumor passages affect the miRNA expression of human colon CSCs.

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.

[Effect of Natural Killer Cell Infiltration on the Growth of Breast Cancer Patient-Derived Tumor Xenografts].

Natural killer (NK) cells, a component of the innate immunity, play important roles in tumor suppression. In this study, three human breast cancer patient-derived tumor xenografts (PDXs), established by the transplantation of surgical specimens, were passaged in immunodeficient NOD/SCID mice or NSG mice, that further lacks NK cell activity. The intensity of the relative growth suppression between NOD/SCID and NSG mice was clearly different depending on the PDX lines, and it was associated with the intensities of the CD49b-positive NK cell infiltration in the PDX tumor tissues. However, no obvious association was observed between the mRNA expression levels of the NK cell ligands in the PDX tumor cells and the intensity of NK cell infiltration into the PDX tumors. These results suggest that the suppressive effect of NK cells on the growth of breast cancer PDX is highly variable depending on the PDX lines. Further studies are needed to elucidate the molecular mechanism of NK cell infiltration in PDX tumors.

[Breast cancer stem cell research utilizing clinical specimens].

The existence of cancer stem cells in solid tumors was first reported in 2003 based on the analyses of human breast cancers. Analyses of clinical specimens are especially important for the advancement of human cancer stem cell research. For example, the analyses of breast cancer stem cells directly isolated from human breast cancer specimens identified the microRNAs that are involved in the regulation of human breast cancer stem cells. In addition, human breast cancer xenograft is an attractive model to analyze cancer stem cells, as well as cancer metastases that have profound effect on the long-term survival of breast cancer patients. We will present the current status of human breast cancer stem cell research that utilizes clinical specimens.