Development, validation, and comparison of gene analysis methods for detecting EGFR mutation from non-small cell lung cancer patients-derived circulating free DNA.

The feasibility and required sensitivity of circulating free DNA (cfDNA)-based detection methods in second-line epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) treatment are not well elucidated. We examined T790M and other activating mutations of EGFR by cfDNA to assess the clinical usability. In 45 non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations, cfDNAs were prepared from the plasma samples. EGFR mutations in cfDNA were detected using highly sensitive methods and originally developed assays and these results were compared to tissue-based definitive diagnoses. The specificity of each cfDNA-based method ranged 96-100% whereas the sensitivity ranged 56-67%, indicating its low pseudo-positive rate. In EGFR-TKI failure cohort, 41-46% samples were positive for T790M by each cfDNA-based method, which was comparable to re-biopsy tissue-based T790M positive rates in literature. The concordance of the results for each EGFR mutation ranged from 83-95%. In eight patients, the results of the cfDNA-based assays and re-biopsy-derived tissue-based test were compared. The observed overall agreement ranged in 50-63% in T790M, and in 63-100% in activating EGFR mutations. In this study, we have newly developed three types of assay which have enough sensitivity to detect cfDNA. We also detected T790M in 44% of patients who failed prior EGFR-TKI treatment, indicating that cfDNA-based assay has clinical relevance for detecting acquired mutations of EGFR.

TAS-116, a highly selective inhibitor of heat shock protein 90α and ß, demonstrates potent antitumor activity and minimal ocular toxicity in preclinical models.

The molecular chaperone HSP90 plays a crucial role in cancer cell growth and survival by stabilizing cancer-related proteins. A number of HSP90 inhibitors have been developed clinically for cancer therapy; however, potential off-target and/or HSP90-related toxicities have proved problematic. The 4-(1H-pyrazolo[3,4-b]pyridine-1-yl)benzamide TAS-116 is a selective inhibitor of cytosolic HSP90α and ß that does not inhibit HSP90 paralogs such as endoplasmic reticulum GRP94 or mitochondrial TRAP1. Oral administration of TAS-116 led to tumor shrinkage in human tumor xenograft mouse models accompanied by depletion of multiple HSP90 clients, demonstrating that the inhibition of HSP90α and ß alone was sufficient to exert antitumor activity in certain tumor models. One of the most notable HSP90-related adverse events universally observed to differing degrees in the clinical setting is visual disturbance. A two-week administration of the isoxazole resorcinol NVP-AUY922, an HSP90 inhibitor, caused marked degeneration and disarrangement of the outer nuclear layer of the retina and induced photoreceptor cell death in rats. In contrast, TAS-116 did not produce detectable photoreceptor injury in rats, probably due to its lower distribution in retinal tissue. Importantly, in a rat model, the antitumor activity of TAS-116 was accompanied by a higher distribution of the compound in subcutaneously xenografted NCI-H1975 non-small cell lung carcinoma tumors than in retina. Moreover, TAS-116 showed activity against orthotopically transplanted NCI-H1975 lung tumors. Together, these data suggest that TAS-116 has a potential to maximize antitumor activity while minimizing adverse effects such as visual disturbances that are observed with other compounds of this class.

Nuclear localization of bradykinin B(2) receptors reflects binding to the nuclear envelope protein lamin C.

The mechanism of action of bradykinin (BK), a pro-inflammatory mediator, is thought to be mediated by specific cell surface membrane bradykinin B2 receptors. Some evidence suggests that there are both intracellular and nuclear bradykinin B2 receptors. This study identified proteins that interact with the C-terminus of the bradykinin B2 receptor (in particular, the nuclear membrane protein lamin C), using the yeast two-hybrid system. The motif of the C-terminal domain (CT) mutant 303-320 in bradykinin B2 receptor was identified as a lamin C protein binding motif. Immunohistochemistry revealed colocalization of FLAG- bradykinin B2 receptor with HA-lamin C in the nucleus of HEK 293T cells. In situ proximity ligation assay (PLA) showed that FLAG-bradykinin B2 receptor formed heterodimers with HA-lamin C in the nucleus. In addition, live cell fluorescence imaging showed that bradykinin B2 receptor-EGFP was located in the nucleus and co-localized with HcRed-lamin C. Interestingly, neither BK addition nor bradykinin B2 receptor CT mutation reduced the binding to lamin C or changed the distribution of bradykinin B2 receptor. Taken together, these findings demonstrate that bradykinin B2 receptor-lamin C heterodimers form in the nucleus independent of BK stimulation and CT mutation. We propose that heterodimerization of bradykinin B2 receptor with lamin C is essential to nuclear localization of bradykinin B2 receptor and plays an important role in cell signaling and function.

Loss of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 and reduced O-glycosylation in colon carcinoma cells selected for hepatic metastasis.

O-glycosylation of mucin is initiated by the attachment of N-acetyl-D-galactosamine (GalNAc) to serine or threonine residues in mucin core polypeptides by UDPGalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). It is not well understood how GalNAc attachment is regulated by multiple ppGalNAc-Ts in each cell. In the present study, the expression levels of murine ppGalNAc-Ts (mGalNAc-Ts), T1, T2, T3, T4, T6, and T7 were compared between mouse colon carcinoma colon 38 cells and variant SL4 cells, selected for their metastatic potentials, by using the competitive RT-PCR method. The expression levels of mGalNAc-T1, T2, and T7 were slightly higher in the SL4 cells than in the colon 38 cells, whereas the expression level of mGalNAc-T3 in the SL4 cells was 1.5% of that in the colon 38 cells. Products of enzymatic incorporations of GalNAc residues into FITCPTTTPITTTTK peptide by the use of microsome fractions of these cells as the enzyme source were separated and characterized for the number of attached GalNAc residues and their positions. The maximum number of attached GalNAc residues was 6 and 4 when the microsome fractions of the colon 38 cells and SL4 cells were used, respectively. When the microsome fractions of the colon 38 cells were treated with a polyclonal antibody raised against mGalNAc-T3, the maximum number of incorporated GalNAc residues was 4. These results strongly suggest that mGalNAc-T3 in colon 38 cells is involved in additional transfer of GalNAc residues to this peptide.

Interleukin-4 induces specific pp-GalNAc-T expression and alterations in mucin O-glycosylation in colonic epithelial cells.

Mucus hypersecretion occurs as a consequence of the Th2 immune response in epithelia, yet it was not previously known whether the degree of O-glycosylation was modulated under such conditions. A colonic carcinoma cell line LS174T was used to assess the effect of interleukin (IL)-4 on the mRNA levels of eight pp-GalNAc-Ts. A three- to four-fold increase in pp-GalNAc-T1, T4, and T7 levels was observed. Lysates of untreated or IL-4-treated cells were examined for their ability to transfer GalNAc residues onto a peptide corresponding to the tandem repeat portion of human MUC2. The number of incorporated GalNAc residues was greater after incubation with lysates of IL-4-treated cells than with lysates of untreated cells. Mucin-like large glycoproteins secreted by IL-4-treated cells had higher binding capacity to PNA and VVA-B(4) than those secreted by untreated cells. The results indicated that IL-4-treated LS174T cells are able to produce mucins with a higher degree of O-glycosylation than untreated counterparts.

Ectopic expression of N-acetylglucosamine 6-O-sulfotransferase 2 in chemotherapy-resistant ovarian adenocarcinomas.

Mucinous and clear cell adenocarcinomas are the major histological types of ovarian epithelial cancer and are associated with a poor prognosis due to their resistance to chemotherapy. A novel tumor marker specific for ovarian mucinous and clear cell adenocarcinomas would be helpful for overcoming these serious diseases. We showed previously by enzymological characterization and RT-PCR that colonic mucinous adenocarcinoma tissues ectopically express GlcNAc6ST-2, a member of the carbohydrate 6-O-sulfotransferase family (Seko, A. et al. (2002) Glycobiology 12, 379-388). Here, we prepared a GlcNAc6ST-2-specific polyclonal antibody for immunohistochemical analysis and found that GlcNAc6ST-2 is ectopically expressed by not only colonic mucinous adenocarcinomas but also ovarian mucinous, clear cell and papillary serous adenocarcinomas. In contrast, solid serous adenocarcinomas, endometrioid adenocarcinomas, and mucinous adenomas expressed GlcNAc6ST-2 much less frequently or not at all. RT-PCR analysis confirmed that GlcNAc6ST-2 transcripts are expressed in ovarian mucinous adenocarcinomas but not in mucinous adenomas. In addition, immunohistochemical analysis using sulfated glycan-specific monoclonal antibodies showed that ovarian adenocarcinoma cells express GlcNAc 6-O-sulfated glycans, including an L-selectin ligand and its related glycans. These results indicate that GlcNAc6ST-2 would be a novel tumor antigen that is specifically expressed in ovarian mucinous, clear cell and papillary serous adenocarcinomas.

Structures of the carbohydrate recognition domain of Ca2+-independent cargo receptors Emp46p and Emp47p.

Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions. Both proteins fold as a beta-sandwich, and resemble that of the mammalian ortholog, p58/ERGIC-53. However, the nature of metal binding is distinct from that of Ca(2+)-dependent p58/ERGIC-53. Interestingly, the CRD of Emp46p does not bind Ca(2+) ion but instead binds K(+) ion at the edge of a concave beta-sheet whose position is distinct from the corresponding site of the Ca(2+) ion in p58/ERGIC-53. Binding of K(+) ion to Emp46p appears essential for transport of a subset of glycoproteins because the Y131F mutant of Emp46p, which cannot bind K(+) ion fails to rescue the transport in disruptants of EMP46 and EMP47 genes. In contrast the CRD of Emp47p binds no metal ions at all. Furthermore, the CRD of Emp46p binds to glycoproteins carrying high mannosetype glycans and the is promoted by binding not the addition of Ca(2+) or K(+) ion in These results suggest that Emp46p can be regarded as a Ca(2+)-independent intracellular lectin at the ER exit sites.

Expression levels of FUT6 gene transfected into human colon carcinoma cells switch two sialyl-Lewis X-related carbohydrate antigens with distinct properties in cell adhesion.

A human colon carcinoma cell line KM12-LX, expressing low levels of monoclonal antibody (mAb) FH6 epitope, was transfected with alpha 1,3-fucosyltransferase VI cDNA. Clonal populations with high or intermediate expression levels of the mRNA, shown by RT-PCR (FT6hi and FT6in cells, respectively) were obtained. FT6hi cells were found to express both mAb FH6 and KM93 epitopes by flow-cytometric analysis, whereas FT6in cells expressed mAb FH6 epitopes but not mAb KM93 epitopes. The mAb FH6-binding was abrogated by endo-beta-galactosidase treatment of FT6in, but not FT6hi, cells. FT6hi but not FT6in cells adhered to Chinese-hamster-ovary cells expressing human E-selectin. FT6in cells adhered to sections of mouse liver and the adhesion was blocked by treatment of the cells with endo-beta-galactosidase. The results indicate that endo-beta-galactosidase-sensitive and mAb FH6-reactive carbohydrate chains are generated under the control of expression levels of FUT6 and involved in the adhesion of colon carcinoma cells to liver sections.