Keiichiro Sakuma

Transcription factors c-Myc and CDX2 mediate E-selectin ligand expression in colon cancer cells undergoing EGF/bFGF-induced epithelial–mesenchymal transition

Sialyl Lewis x (sLex) and sialyl Lewis a (sLea) glycans are expressed on highly metastatic colon cancer cells. They promote extravasation of cancer cells and tumor angiogenesis via interacting with E-selectin on endothelial cells. Recently, epithelial–mesenchymal transition (EMT) has been noted as a critical phenotypic alteration in metastatic cancer cells. To address the association between sLex/a expression and EMT, we assessed whether sLex/a are highly expressed on colon cancer cells undergoing EMT. Treatment of HT29 and DLD-1 cells with EGF and/or basic FGF (bFGF) induced EMT and significantly increased sLex/a expression resulting in enhanced E-selectin binding activity. The transcript levels of the glycosyltransferase genes ST3GAL1/3/4 and FUT3 were significantly elevated and that of FUT2 was significantly suppressed by the treatment. We provide evidence that ST3GAL1/3/4 and FUT3 are transcriptionally up-regulated by c-Myc with probable involvement of Ser62 phosphorylation, and that FUT2 is transcriptionally down-regulated through the attenuation of CDX2. The contribution of c-Myc and CDX2 to the sLex/a induction was proved to be significant by knockdown or forced expression experiments. Interestingly, the cells undergoing EMT exhibited significantly increased VEGF secretion, which can promote tumor angiogenesis in cooperation with sLex/a. Finally, immunohistological study indicated high E-selectin ligand expression on cancer cells undergoing EMT in vivo, supporting their coexistence observed in vitro. These results suggest a significant link between sLex/a expression and EMT in colon cancer cells and a pivotal role of c-Myc and CDX2 in regulating sLex/a expression during EMT.

Altered expression of glycan genes in cancers induced by epigenetic silencing and tumor hypoxia: Clues in the ongoing search for new tumor markers

(Cancer Sci 2010; 101: 586–593)

Colonic Epithelial Cells Express Specific Ligands for Mucosal Macrophage Immunosuppressive Receptors Siglec-7 and -9

Immune cells are known to express specific recognition molecules for cell surface glycans. However, mechanisms involved in glycan-mediated cell–cell interactions in mucosal immunity have largely been left unaccounted for. We found that several glycans preferentially expressed in nonmalignant colonic epithelial cells serve as ligands for sialic acid-binding Ig-like lectins (siglecs), the immunosuppressive carbohydrate-recognition receptors carried by immune cells. The siglec ligand glycans in normal colonic epithelial cells included disialyl Lewisa, which was found to have binding activity to both siglec-7 and -9, and sialyl 6-sulfo Lewisx, which exhibited significant binding to siglec-7. Expression of these siglec-7/-9 ligands was impaired upon carcinogenesis, and they were replaced by cancer-associated glycans sialyl Lewisa and sialyl Lewisx, which have no siglec ligand activity. When we characterized immune cells expressing siglecs in colonic lamina propriae by flow cytometry and confocal microscopy, the majority of colonic stromal immune cells expressing siglec-7/-9 turned out to be resident macrophages characterized by low expression of CD14/CD89 and high expression of CD68/CD163. A minor subpopulation of CD8+ T lymphocytes also expressed siglec-7/-9. Siglec-7/-9 ligation suppressed LPS-induced cyclooxygenase-2 expression and PGE2 production by macrophages. These results suggest that normal glycans of epithelial cells exert a suppressive effect on cyclooxygenase-2 expression by resident macrophages, thus maintaining immunological homeostasis in colonic mucosal membranes. Our results also imply that loss of immunosuppressive glycans by impaired glycosylation during colonic carcinogenesis enhances inflammatory mediator production.

Clinical significance of pretreatment serum amphiregulin and transforming growth factor‐α, and an epidermal growth factor receptor somatic mutation in patients with advanced non‐squamous, non‐small cell lung cancer

Circulating amphiregulin and transforming growth factor‐α (TGF‐α) have been found to be correlated with an unfavorable response to gefitinib based on the identification of patients with a higher probability of resistance to the drug. However, the association between an epidermal growth factor receptor (EGFR) somatic mutation and the overexpression of its ligands has not been determined. To verify the clinical significance of the two serum markers and EGFR mutation status, we determined serum amphiregulin and TGF‐α levels by enzyme‐linked immunosorbent assay in 93 patients with advanced non‐squamous, non‐small cell lung cancer and EGFR somatic mutation status using the peptic nucleic acid‐locked nucleic acid clamp method in 46 cases. The relationship between each independent clinicopathological variable and the response to gefitinib therapy was examined. We also evaluated the risk factors associated with prognosis. Fourteen (41.0%) of 34 progressive disease cases were positive for amphiregulin (P = 0.007). Eleven (32.4%) of 34 progressive disease cases were positive for TGF‐α (P = 0.005). The median survival time of patients with the EGFR somatic mutation was significantly longer (P = 0.01). The same was true of amphiregulin‐ (P = 0.046) and TGF‐α‐negative patients (P < 0.01). In multivariate analysis, serum TGF‐α positivity (hazard ratio, 2.558; P = 0.005) and the wild type EGFR gene (hazard ratio, 1.894; P = 0.003) were significant independent prognostic factors. Our study demonstrates that the status of the serum EGFR ligand, in addition to EGFR activating mutation, is a predictive factor for response to gefitinib therapy. (Cancer Sci 2008; 99: 2295–2301)

Significance of NF-κB/GATA axis in TNF-α-induced expression of 6-sulfated cell-recognition glycans in human T-lymphocytes

Sulfated glycans play critical roles in various cell recognition events among leukocytes. The 6-sulfated lactosamine glycans in particular have been widely noted for their importance because they are involved in cell recognition events mediated by cell-adhesion molecules such as selectins and sialic acid-recognizing molecules such as siglecs and also in the activation of CD44 in binding to extracellular matrix hyaluronate. A pro-inflammatory cytokine, tumor necrosis factor-α, induces expression of 6-sulfated glycans on human leukocytes. Here we report that the transcription of the GlcNAc6ST-1 gene, the gene encoding a sulfotransferase for 6-sulfated glycan synthesis, is induced in human T-lymphoid cells through tandem NF-κB and GATA motifs in its 5′-regulatory region. Results of our reporter assays, immunoprecipitation, and chromatin immunoprecipitation analyses indicated that GATA-3 and/or GATA-2, but not GATA-1, associates with NF-κB in a transcription factor complex on the 5′-regulatory region of the gene and acts synergistically with NF-κB in triggering GlcNAc6ST-1 transcription. Recently, a skin-homing subset of helper memory T cells exhibiting the Th2 marker CCR4 was shown to specifically express 6-sulfated glycans. The transactivation mechanism described here suggested that GlcNAc6ST-1 transcription is coordinated with the NF-κB/GATA-3 axis, which is known to figure heavily in Th2 cell differentiation. In line with this, in vitro differentiation of human T cells to Th2 cells was found to significantly induce GlcNAc6ST-1 transcription and 6-sulfated glycan expression.

Significance of NF-κB/GATA Axis in Tumor Necrosis Factor-α-induced Expression of 6-Sulfated Cell Recognition Glycans in Human T-lymphocytes

Sulfated glycans play critical roles in various cell recognition events among leukocytes. The 6-sulfated lactosamine glycans in particular have been widely noted for their importance because they are involved in cell recognition events mediated by cell-adhesion molecules such as selectins and sialic acid-recognizing molecules such as siglecs and also in the activation of CD44 in binding to extracellular matrix hyaluronate. A pro-inflammatory cytokine, tumor necrosis factor-α, induces expression of 6-sulfated glycans on human leukocytes. Here we report that the transcription of the GlcNAc6ST-1 gene, the gene encoding a sulfotransferase for 6-sulfated glycan synthesis, is induced in human T-lymphoid cells through tandem NF-κB and GATA motifs in its 5′-regulatory region. Results of our reporter assays, immunoprecipitation, and chromatin immunoprecipitation analyses indicated that GATA-3 and/or GATA-2, but not GATA-1, associates with NF-κB in a transcription factor complex on the 5′-regulatory region of the gene and acts synergistically with NF-κB in triggering GlcNAc6ST-1 transcription. Recently, a skin-homing subset of helper memory T cells exhibiting the Th2 marker CCR4 was shown to specifically express 6-sulfated glycans. The transactivation mechanism described here suggested that GlcNAc6ST-1 transcription is coordinated with the NF-κB/GATA-3 axis, which is known to figure heavily in Th2 cell differentiation. In line with this, in vitro differentiation of human T cells to Th2 cells was found to significantly induce GlcNAc6ST-1 transcription and 6-sulfated glycan expression.

Accuracy of epidermal growth factor receptor gene mutation analysis by direct sequencing method based on small biopsy specimens from patients with non-small cell lung cancer : analysis of results in 19 patients

BackgroundThe importance of an epidermal growth factor receptor (EGFR) gene mutation has been recognized in patients with non-small cell lung cancer (NSCLC), and many reports have indicated that the presence of somatic mutations in the EGFR gene is a strong predictor of both clinical and in vitro sensitivity to EGFR tyrosine kinase inhibitors; thus necessitating the standardization of a mutation screening system based on the sources of tissue samples.MethodsIn this study, we compared the results of EGFR mutation analyses in 19 small biopsy specimens with results obtained in surgical materials from the same patients with NSCLC. We used a laser microdissection method and a direct sequencing method, and we confirmed the accuracy of EGFR mutation analysis with small biopsy specimens.ResultsThe results obtained from the biopsy specimens were identical to those obtained from the surgical materials in 18 of the 19 patients analyzed. For the 1 patient in whom the results obtained from the two sets of materials were not identical, the number of cancer cells in one bronchoscopic specimen was insufficient to perform analyses of all three exons of interest (i.e., exons 18, 19, and 21), and so only exon 19 was sequenced, and no mutation was demonstrated.ConclusionWe conclude that satisfactory accuracy can be achieved by the genomic analysis of a small biopsy specimen from a patient with NSCLC and we note that it is possible to conduct prospective clinical trials that include patient assignment for treatment based on the results obtained.

Sialylated and Sulfated Carbohydrate Ligands for Selectins and Siglecs: Involvement in Traffic and Homing of Human Memory T and B Lymphocytes

Selectin-mediated adhesion to endothelial cells is the first step of extravasation of leukocytes. Neutrophils constitutively express sialyl Lewis x, the selectin ligand, and are ready to adhere to endothelial cells once the latter cells express E- or P-selectin. This leads to rapid and massive extravasation of neutrophils into acute inflammatory lesions.

HNRNPLL, a newly identified colorectal cancer metastasis suppressor, modulates alternative splicing of CD44 during epithelial-mesenchymal transition

Objective Despite the recent advances in treatment of colon cancer, the prognosis is unfavourable for patients with distant metastases. The aim of this study was to identify targets for prevention and/or therapy of colon cancer metastasis. Design CMT93 cells, a murine rectal cancer cell line with poor metastasising activity, were transduced with lentiviral shRNA library and transplanted into the rectum of syngeneic C57BL/6 mice. Genomic DNA was collected from metastatic lesions, and the integrated shRNA were retrieved by PCR for sequencing, followed by identification of the candidate genes targeted by the shRNA. Results The genome-wide shRNA library screen identified Hnrnpll (heterogeneous nuclear ribonucleoprotein L-like) encoding a pre-mRNA splicing factor as a candidate metastasis suppressor gene. Knockdown of Hnrnpll enhanced matrigel invasion activity of colon cancer cells in vitro, as well as their metastatic ability in vivo. An RNA-immunoprecipitation analysis showed Hnrnpll-binding to Cd44 pre-mRNAs, and the level of Cd44 variable exon 6 (Cd44v6), a poor prognosis marker of colorectal cancer, was increased by knocking down Hnrnpll. A neutralising Cd44v6 antibody suppressed the matrigel invasion ability induced by Hnrnpll knockdown. HNRNPLL expression was downregulated when colon cancer cells were induced to undergo epithelial-mesenchymal transition (EMT). Immunohistochemistry of clinical samples indicated that colorectal cancer cells with low E-cadherin expression at the invasion front exhibited decreased HNRNPLL expression. Conclusions HNRNPLL is a novel metastasis suppressor of colorectal cancer, and modulates alternative splicing of CD44 during EMT.

Tumor-Associated Glycans and Their Functional Roles in the Multistep Process of Human Cancer Progression

Cancer develops through a multistep process of carcinogenesis. This process accompanies incremental alterations of expression of biologically functional glycans on the surface of cancer cells. A variety of glycans are expressed in nonmalignant epithelial cells, including several normal glycans serving as ligands for siglecs, the immunosuppressive molecules carried by interstitial immune cells. These normal glycans decrease or disappear and are replaced by cancer-associated glycans at the early stages of carcinogenesis. This glycan transition facilitates production by mucosal immune cells of inflammatory mediators that are known to promote cancer progression. Expression of glycans that regulate growth factor receptor functions is also affected at the early stages of cancers. The major mechanism involved in glycan alteration at the early stages is epigenetic silencing through DNA methylation and/or histone deacetylation/methylation of genes responsible for synthesis of normal glycans, leading to their incomplete synthesis. In the locally advanced stages, multiple glycan-related genes are induced transcriptionally and posttranscriptionally by tumor hypoxia and epithelio-mesenchymal transition, thus further culminating in abnormal expression of cancer-associated glycans. Some such glycans serve as specific ligands for selectins, the cell adhesion molecules carried by vascular endothelial cells, and facilitate tumor vascularization and ultimately hematogenous metastasis. Advanced cancer cells which have undergone epithelio-mesenchymal transition share biological characteristics with so-called cancer stem cells, and glycans associated with such cells are currently known to be frequently expressed in human embryonic stem cells as well.