Azusa Yoneshige

Increased ectodomain shedding of lung epithelial cell adhesion molecule 1 as a cause of increased alveolar cell apoptosis in emphysema

Rationale Alveolar epithelial cell apoptosis and protease/antiprotease imbalance based proteolysis play central roles in the pathogenesis of pulmonary emphysema but molecular mechanisms underlying these two events are not yet clearly understood. Cell adhesion molecule 1 (CADM1) is a lung epithelial cell adhesion molecule in the immunoglobulin superfamily. It generates two membrane associated C terminal fragments (CTFs), αCTF and βCTF, through protease mediated ectodomain shedding. Objective To explore the hypothesis that more CADM1-CTFs are generated in emphysematous lungs through enhanced ectodomain shedding, and cause increased apoptosis of alveolar epithelial cells. Methods and results Western blot analyses revealed that CADM1-CTFs increased in human emphysematous lungs in association with increased ectodomain shedding. Increased apoptosis of alveolar epithelial cells in emphysematous lungs was confirmed by terminal nucleotide nick end labelling (TUNEL) assays. NCI-H441 lung epithelial cells expressing mature CADM1 but not CTFs were induced to express αCTF both endogenously (by shedding inducers phorbol ester and trypsin) and exogenously (by transfection). Cell fractionation, immunofluorescence, mitochondrial membrane potentiometric JC-1 dye labelling and TUNEL assays revealed that CADM1-αCTF was localised to mitochondria where it decreased mitochondrial membrane potential and increased cell apoptosis. A mutation in the intracytoplasmic domain abrogated all three abilities of αCTF. Conclusions CADM1 ectodomain shedding appeared to cause alveolar cell apoptosis in emphysematous lungs by producing αCTF that accumulated in mitochondria. These data link proteolysis to apoptosis, which are two landmark events in emphysema.

Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing

Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10-5). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.

Increased Ectodomain Shedding of Cell Adhesion Molecule 1 from Pancreatic Islets in Type 2 Diabetic Pancreata: Correlation with Hemoglobin A1c Levels

Pulmonary emphysema and type 2 diabetes mellitus (T2DM), both caused by lifestyle factors, frequently concur. Respectively, the diseases affect lung alveolar and pancreatic islet cells, which express cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member. Protease-mediated ectodomain shedding of full-length CADM1 produces C-terminal fragments (CTFs) with proapoptotic activity. In emphysematous lungs, the CADM1 shedding rate and thus the level of CTFs in alveolar cells increase. In this study, CADM1 expression in islet cells was examined by western blotting. Protein was extracted from formalin-fixed, paraffin-embedded sections of pancreata isolated from patients with T2DM (n = 12) or from patients without pancreatic disease (n = 8) at autopsy. After adjusting for the number of islet cells present in the adjacent section, we found that full-length CADM1 decreased in T2DM islets, while ectodomain shedding increased. Hemoglobin A1c levels, measured when patients were alive, correlated inversely with full-length CADM1 levels (P = 0.041) and positively with ectodomain shedding rates (P = 0.001). In immunofluorescence images of T2DM islet cells, CADM1 was detected in the cytoplasm, but not on the cell membrane. Consistently, when MIN6-m9 mouse beta cells were treated with phorbol ester and trypsin to induce shedding, CADM1 immunostaining was diffuse in the cytoplasm. When a form of CTFs was exogenously expressed in MIN6-m9 cells, it localized diffusely in the cytoplasm and increased the number of apoptotic cells. These results suggest that increased CADM1 ectodomain shedding contributes to blood glucose dysregulation in T2DM by decreasing full-length CADM1 and producing CTFs that accumulate in the cytoplasm and promote apoptosis of beta cells. Thus, this study has identified a molecular alteration shared by pulmonary emphysema and T2DM.

The intracellular domain of cell adhesion molecule 1 is present in emphysematous lungs and induces lung epithelial cell apoptosis.

BackgroundPulmonary emphysema is characterized histologically by destruction of alveolar walls and enlargement of air spaces due to lung epithelial cell apoptosis. Cell adhesion molecule 1 (CADM1) is an immunoglobulin superfamily member expressed in lung epithelial cells. CADM1 generates a membrane-associated C-terminal fragment, αCTF, through A disintegrin- and metalloprotease-10-mediated ectodomain shedding, subsequently releasing the intracellular domain (ICD) through γ-secretase-mediated intramembrane shedding of αCTF. αCTF localizes to mitochondria and induces apoptosis in lung epithelial cells. αCTF contributes to the development and progression of emphysema as a consequence of increased CADM1 ectodomain shedding. The purpose of this study was to examine whether the ICD makes a similar contribution.ResultsThe ICD was synthesized as a 51-amino acid peptide, and its mutant was synthesized by substituting seven amino acids and deleting two amino acids. These peptides were labeled with fluorescein isothiocyanate and were introduced into various cell lines. ICD peptide-derived fluorescence was well visualized in lung epithelial cells at the site of Mitotracker mitochondrial labeling, but was detected in locations other than mitochondria in other cell types. Mutant peptide-derived fluorescence was detected in locations other than mitochondria, even in lung epithelial cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays revealed that transduction of the ICD peptide increased the proportion of apoptotic cells 2- to 5-fold in the lung epithelial cell lines, whereas the mutant peptide did not. Abundance of the ICD was below the Western blot detection limit in emphysematous (n = 4) and control (n = 4) human lungs. However, the ICD was detected only in emphysematous lungs when it was immunoprecipitated with anti-CADM1 antibody (4/4 vs. 0/4, P = 0.029).ConclusionsAs the abundance of ICD molecules was sparse but present, increased CADM1 shedding appeared to contribute to the development of emphysema by generating αCTF and the ICD in lung epithelial cells.

Significance of FGF9 gene in resistance to anti-EGFR therapies targeting colorectal cancer: A subset of colorectal cancer patients with FGF9 upregulation may be resistant to anti-EGFR therapies

Although fibroblast growth factor (FGF) signals are strongly associated with malignancy, limited information is available regarding the role of the FGF9 signal in colorectal cancer (CRC). In this study, we investigated the frequency of FGF9 amplification in CRC clinical specimens and the association between the FGF9 gene and resistance to anti‐EGFR therapies. In clinical samples, an FGF9 copy number gain of >5 copies was observed at a frequency of 8/145 (5.5%) and tended to be related to wild‐type KRAS (7/96, 7.3%). Furthermore, FGF9 amplification was not observed in any of the samples from the 15 responders to anti‐EGFR therapies but was observed in one sample from the seven non‐responders with wild‐type KRAS, and two samples from non‐responders also had high FGF9 mRNA expression levels. FGF9 amplification was validated using a fluorescence in situ hybridization (FISH) analysis, and FGF9‐amplified sections showed readily detectable signals originating from FGF9 protein when examined using immunohistochemistry. In both the in vitro and in vivo experiments using FGF9‐overexpressing CRC cell lines, FGF9 overexpression induced strong resistance to anti‐EGFR therapies via the enforced FGFR signal, and this resistance was cancelled by the application of an FGFR inhibitor. Considering these results, the FGF9 gene may play an important role in resistance to anti‐EGFR therapies in patients with CRC, and such resistance might be overcome by combined treatment with an anti‐FGFR inhibitor. These findings strongly encourage the development of FGFR‐targeted therapy for CRC patients with FGF9 gene upregulation.

Performance of a novel KRAS mutation assay for formalin-fixed paraffin embedded tissues of colorectal cancer

We compared the performance of the 3D-Gene® mutation assay (3D-Gene® KRAS mutation assay kit) with the Scorpion-ARMS (therascreen® KRAS RGQ PCR Kit) and Luminex (MEBGEN™ KRAS kit) assays for the detection of KRAS mutations in formalin-fixed, paraffin-embedded tissue samples from 150 patients diagnosed with colorectal cancer. DNA was extracted from the paraffin-embedded tissue samples with or without macrodissection under hematoxylin and eosin staining and the KRAS mutation status was independently determined using these assays. Discordant results were re-analyzed by Sanger sequencing. Mutation detection analysis was successfully performed in all 150 specimens using the 3D-Gene® mutation assay without an invalid case. The concordance rate between the 3D-Gene® mutation assay and Scorpion-ARMS or Luminex was 98.7% (148/150). KRAS mutations were detected at a frequency of 35.3% (53/150) in colorectal cancer specimens. Three discrepant cases were found between the three assays. Overall, our results demonstrate a high concordance rate of between the 3D-Gene® mutation assay and the two existing in-vitro diagnostics kits. All three assays proved to be validated methods for detecting clinically significant KRAS mutations in paraffin-embedded tissue samples.

Pathogenic Actions of Cell Adhesion Molecule 1 in Pulmonary Emphysema and Atopic Dermatitis

Cell adhesion mediated by adhesion molecules is of central importance in the maintenance of tissue homeostasis. Therefore, altered expression of adhesion molecules leads to the development of various tissue disorders involving cell activation, degeneration, and apoptosis. Nevertheless, it still remains unclear what initiates the altered expression of adhesion molecules and how the subsequent pathological cascades proceed. In this regard, cell adhesion molecule 1 (CADM1) is one of the candidates that is involved in the development of pathological lesions; it is an intercellular adhesion molecule that is expressed in various types of cells such as pulmonary cells, neurons, and mast cells. Recent studies have revealed that alterations in the transcriptional or post-transcriptional expressions of CADM1 correlate with the pathogenesis of pulmonary diseases and allergic diseases. In this review, we specifically focus on how CADM1 is involved in the development of pathological lesions in pulmonary emphysema and atopic dermatitis.

Manifestation of osteoblastic phenotypes in the sarcomatous component of epithelial carcinoma and sarcomatoid carcinoma

Epithelial carcinomas occasionally have sarcomatous components that consist primarily of spindle and cuboidal cells, which often resemble osteoblasts. Sarcomatoid carcinomas consist of similar cells. Recent studies have characterized these phenomena as a manifestation of epithelial–mesenchymal transition in carcinoma cells, but the mesenchymal phenotypes that manifest in sarcomatous cells of epithelial carcinomas are not well understood. Here, we examined the expression profiles of four osteoblastic differentiation biomarkers in the sarcomatous components of multiple carcinoma types, including five renal clear cell, four breast invasive ductal, two esophageal, one maxillary squamous cell, three larynx, three lung, one liver, and one skin sarcomatoid carcinoma. Expression was analyzed by immunohistochemistry using antibodies against cell adhesion molecule 1, a member of the IgCAM superfamily, osterix transcription factor (Osterix), cluster of differentiation 151, a transmembrane 4 superfamily member, and alkaline phosphatase. Immunostaining intensity was rated in scale 0 (negative), 0.5 (weak), and 1 (strong) for each marker, and the four scale values were summed to calculate osteoblastic scores. In all, 10 cases had a osteoblastic score ≥3, and all of these 10 cases were cell adhesion molecule 1- and Osterix-positive. Eight and five of the nine samples with a osteoblastic score <3 were negative for cell adhesion molecule 1 (p < 0.0001) and Osterix (p = 0.006), respectively. The other markers showed no statistical significance. These results indicate that osteoblastic differentiation can occur in carcinoma cells and that cell adhesion molecule 1 could be a useful marker for identifying this phenomenon in carcinoma tissues.

Expression of cell adhesion molecule 1 in gastric neck and base glandular cells: Possible involvement in peritoneal dissemination of signet ring cells

Aims: To determine cellular distribution of cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, in the human oxyntic gastric mucosa, and to explore possible involvement in the development and peritoneal dissemination of signet ring cell (SRC) gastric carcinoma, which often develops in the oxyntic mucosa. Main methods: Immunohistochemistry and double immunofluorescence were conducted on surgical specimens of normal and SRC‐bearing stomachs and peritoneal metastatic foci of SRCs. KATO‐III (lacking CADM1) and HSC‐43 (expressing CADM1) SRC cell lines were cocultured on a Met‐5A mesothelial or TIG‐1 fibroblastic cell monolayer. Key findings: In the oxyntic gland, some neck and nearly all base glandular cells were CADM1‐positive, and mucin 5AC‐positive cells were CADM1‐negative, while some mucin 6‐positive neck cells were CADM1‐positive. Foveolar‐epithelial, parietal, and endocrine cells were CADM1‐negative. CADM1 was negative in all SRC carcinomas that were confined within the submucosa (n = 11) and all but one of those invading deeper (n = 15). In contrast, peritoneal metastatic foci of SRCs were CADM1‐positive in five out of eleven cases (P < 0.01). In the cocultures, exogenous CADM1 made KATO‐III cells adhere more and grow faster on a Met‐5A monolayer, not on TIG‐1 monolayers. HSC‐43 cells adhered more and grew faster on Met‐5A than on TIG‐1 monolayers, which were partly counteracted by a function‐neutralizing anti‐CADM1 antibody. Significance: Nearly all chief cells and a part of mucous neck cells express CADM1. SRC gastric carcinoma appears to emerge as a CADM1‐negative tumor, but CADM1 may help SRCs develop peritoneal dissemination through promoting their adhesion and growth in the serosal tissue.

Early Gene Expression Profile in Retinal Ganglion Cell Layer After Optic Nerve Crush in Mice

Purpose Optic nerve crush (ONC) induces retinal ganglion cell (RGC) death, which causes vision loss in glaucoma. To investigate early events leading to apoptosis of RGCs, we performed gene expression analysis of injured retinas in the period before RGC loss. Methods The temporal changes of gene profiles at 0, 1, and 4 days after ONC were determined by DNA microarray. To verify the gene expression changes in RGCs, we enriched RGCs by laser-captured microdissection and performed real-time RT-PCR of 14 selected genes. In situ localization study was performed by immunohistochemistry. Results At 1 day and 4 days after ONC, 1423 and 2010 retinal genes were changed compared with 0 day, respectively; these genes were mainly related to apoptotic process, immune process, regulation of cell cycle, and ion transport. RT-PCR analysis revealed that expression levels of Activating transcription factor 3 (Atf3), Lipocalin 2 (Lcn2), and tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a) were remarkably changed in RGC-enriched fraction within 4 days postcrush. Immunohistochemical analysis confirmed that all of these genes expressed highly in the ganglion cell layer of crushed retinas. Conclusions In response to ONC, the expression of apoptotic genes was stimulated soon after crush. Atf3, Lcn2, and Tnfrsf12a might be key molecules responsible for RGC loss in glaucoma.