Naohiro Uraoka

MicroRNA‐148a is downregulated in gastric cancer, targets MMP7, and indicates tumor invasiveness and poor prognosis

Gastric cancer (GC) develops through deregulation of gene expression and accumulation of epigenetic abnormalities, leading to tumor cell acquisition of malignant features. MicroRNAs (miRNAs) play a critical role in cancer development where they can act as oncogenes or oncosuppressors. To identify miRNAs that are associated with some clinicopathologic features of GC and/or participate in tumor progression, miRNA expression in 20 GC tissues and five corresponding non‐neoplastic gastric mucosa was examined by miRNA microarray. Oligonucleotide array analysis was carried out for miRNA target prediction. The functions of candidate miRNAs and their target genes were also analyzed by quantitative RT‐PCR, Western blotting, reporter gene assay, and cell invasion assay. Comparison of miRNA expression profiles revealed that downregulation of miR‐148a was identified in most of the GC tissues. Downregulation of miR‐148a was significantly correlated with an advanced clinical stage, lymph node metastasis, and poor clinical outcome. Custom oligonucleotide array analysis revealed that MMP7 expression was markedly downregulated in miR‐148a‐overexpressing GC cells; MMP7 was found to be a direct and functional target of miR‐148a, participating in cell invasion. These results suggest that miR‐148a contributes to the maintenance of homeostasis in normal stomach tissue and plays an important role in GC invasion by regulating MMP7 expression.

Liver-intestine cadherin induction by epidermal growth factor receptor is associated with intestinal differentiation of gastric cancer.

Gastric cancer (GC) is one of the most common malignancies worldwide. The epidermal growth factor receptor (EGFR) molecule is very important in GC progression. To examine the correlation between EGFR and GC‐related genes, we analyzed gene expression profiles of HT‐29 cells treated with EGFR ligands and identified six genes upregulated by epidermal growth factor (EGF) and transforming growth factor (TGF)‐α treatment. Among these, we focused on cadherin 17 (CDH17) encoding liver–intestine cadherin (LI‐cadherin). Expression of LI‐cadherin was induced by both EGF and TGF‐α, as detected by quantitative RT‐PCR and Western blot analysis. A luciferase assay showed that LI‐cadherin promoter activity was enhanced by EGF or TGF‐α in both HT‐29 cells and MKN‐74 GC cells. Immunohistochemical analysis of 152 GC cases showed that out of 58 LI‐cadherin‐positive cases, 24 (41%) cases were also positive for EGFR, whereas out of 94 LI‐cadherin‐negative cases, only 9 (10%) cases were positive for EGFR (P < 0.0001). Double‐immunofluorescence staining revealed that EGFR and LI‐cadherin were coexpressed. Significant correlation was found between LI‐cadherin expression and advanced T grade and N grade. Both EGFR and LI‐cadherin expression were more frequently found in GC cases with an intestinal mucin phenotype than in cases with a gastric mucin phenotype. These results indicate that, in addition to the known intestinal transcription factor caudal type homeobox 2, EGFR activation induces LI‐cadherin expression and participates in intestinal differentiation of GC.

Overexpression of ZDHHC14 promotes migration and invasion of scirrhous type gastric cancer

Scirrhous type gastric cancer is highly aggressive and has a poorer prognosis than many other types of gastric carcinoma, due to its characteristic rapid cancer cell infiltration and proliferation, extensive stromal fibrosis, and frequent peritoneal dissemination. The aim of the present study was to identify novel prognostic markers or therapeutic targets for scirrhous type gastric cancer. We reviewed a list of genes with upregulated expression in scirrhous type gastric cancer and compared their expression with that in normal stomach from our previous Escherichia coli (E. coli) ampicillin secretion-trap (CAST) analysis. We focused on the ZDHHC14 gene, which encodes zinc finger, DHHC-type containing 14 protein. qRT-PCR analysis of ZDHHC14 in 41 gastric cancer cases revealed that compared to mRNA levels in normal non-neoplastic gastric mucosa, ZDHHC14 mRNA was overexpressed in 27% of gastric cancer tissue samples. The overexpression of ZDHHC14 was significantly associated with depth of tumor invasion, undifferentiated histology and scirrhous pattern. The invasiveness of ZDHHC14-knockdown HSC-44PE and 44As3 gastric cancer cells was decreased in comparison with that of the negative control siRNA-transfected cells, together with downregulation of MMP-17 mRNA. Integrins α5 and β1 were also downregulated in ZDHHC14-knockdown 44As3 cells. Forced expression of ZDHHC14 activated gastric cancer cell migration and invasion in vitro. These results indicate that ZDHHC14 is involved in tumor progression in patients with scirrhous type gastric cancer.

NRD1, which encodes nardilysin protein, promotes esophageal cancer cell invasion through induction of MMP2 and MMP3 expression

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. In the present study, to identify novel prognostic markers or therapeutic targets for ESCC, we reviewed a list of genes with upregulated expression in ESCC compared with normal esophagus, as identified by our serial analysis of gene expression (SAGE) analysis. We focused on the NRD1 gene, which encodes the nardilysin protein. Quantitative reverse transcription–polymerase chain reaction (qRT‐PCR) in 34 ESCC tissue samples revealed that mRNA expression of NRD1 was upregulated in 56% of ESCC tissue samples. Immunohistochemical analysis of nardilysin in 109 ESCC tissue samples demonstrated that 43 (39%) ESCC cases were positive for nardilysin. Nardilysin‐positive ESCC cases were more advanced in terms of T classification (P = 0.0007), N classification (P = 0.0164), and tumor stage (P < 0.0001) than nardilysin‐negative ESCC cases. Furthermore, nardilysin expression was significantly associated with poorer prognosis (P = 0.0258). Univariate and multivariate analyses revealed that nardilysin expression is an independent prognostic classifier of patients with ESCC. The invasiveness of NRD1‐knockdown TE1 and TE5 esophageal cancer cell lines was less than that of the negative control siRNA‐transfected cell lines. Expression of MMP2 and MMP3 mRNA was significantly lower in NRD1‐knockdown TE5 cells than in negative control siRNA‐transfected cells. These results suggest that nardilysin is involved in tumor progression, and is an independent prognostic classifier in patients with ESCC.

Signal peptidase complex 18, encoded by SEC11A, contributes to progression via TGF-α secretion in gastric cancer

We built an in-house oligonucleotide array on which 394 genes were selected based on our Serial Analysis of Gene Expression (SAGE) data and previously reported array data and listed several genes related to cancer progression. Among these, we focused on SEC11A, which encodes the SPC18 protein. SEC11A mRNA expression was measured by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) in gastric cancer (GC) tissue samples. Expression and distribution of SPC18 protein were investigated by immunohistochemical analysis in two independent GC cohorts (Hiroshima cohort, n=99 and Chiba cohort, n=989). To determine the effect of SPC18 on cell viability and invasiveness in vitro, MTT and Boyden chamber invasion assays were performed. To evaluate the influence of SPC18 on cell growth in vivo, GC cells were injected into severe combined immunodeficiency mice. Levels of TGF-α and EGF in media from the GC cells were measured by enzyme-linked immunosorbent assay (ELISA). Studies in human tissue revealed overexpression of SEC11A mRNA in 40% of 42 GC samples by qRT–PCR. Immunohistochemical analysis of SPC18 revealed that 26 and 20% of GC cases were SPC18-positive in the Hiroshima and Chiba cohorts, respectively. In both cohorts, the Kaplan–Meier analysis showed poorer survival in SPC18-positive GC cases than in SPC18-negative GC cases. Forced expression of SPC18 activates GC cell growth in vitro and in vivo. The levels of TGF-α in culture media from GC cells were reduced by knockdown of SPC18. These results indicate that SPC18 contributes to malignant progression through promotion of TGF-α secretion in GC.

Upregulation of HOXA10 in gastric cancer with the intestinal mucin phenotype: reduction during tumor progression and favorable prognosis

Gastric cancer (GC) is one of the most common malignancies worldwide. Better knowledge of the changes in gene expression that occur during gastric carcinogenesis may lead to improvements in diagnosis, treatment and prevention. In this study, we screened for genes upregulated in GC by comparing gene expression profiles from microarray and serial analysis of gene expression and identified the HOXA10 gene. The aim of the present study was to investigate the significance of HOXA10 in GC. Immunohistochemical analysis demonstrated that 221 (30%) of 749 GC cases were positive for HOXA10, whereas HOXA10 was scarcely expressed in non-neoplastic gastric mucosa except in the case of intestinal metaplasia. Next, we analyzed the relationship between HOXA10 expression and clinicopathological characteristics. HOXA10 expression showed a significant inverse correlation with the depth of invasion and was observed more frequently in the differentiated type of GC than in the undifferentiated type of GC. HOXA10 expression was associated with GC with the intestinal mucin phenotype and correlated with CDX2 expression. Furthermore, the prognosis of patients with positive HOXA10 expression was significantly better than in the negative expression cases. 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide and wound healing assay revealed that knockdown of HOXA10 in GC cells by short interfering RNA transfection significantly increased viability and motility relative to the negative control, indicating that HOXA10 expression inhibits cell growth and motility. These results suggest that expression of HOXA10 may be a key regulator for GC with the intestinal mucin phenotype.

Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues

Immune-profiling is becoming an important tool to identify predictive markers for the response to immunotherapy. Our goal was to validate multiplex immunofluorescence (mIF) panels to apply to formalin-fixed and paraffin-embedded tissues using a set of immune marker antibodies, with the Opal™ 7 color Kit (PerkinElmer) in the same tissue section. We validated and we described two panels aiming to characterize the expression of PD-L1, PD-1, and subsets of tumor associated immune cells. Panel 1 included pancytokeratin (AE1/AE3), PD-L1, CD4, CD8, CD3, CD68, and DAPI, and Panel 2 included pancytokeratin, PD-1, CD45RO, granzyme B, CD57, FOXP3, and DAPI. After all primary antibodies were tested in positive and negative controls by immunohistochemistry and uniplex IF, panels were developed and simultaneous marker expressions were quantified using the Vectra 3.0™ multispectral microscopy and image analysis InForm™ 2.2.1 software (PerkinElmer).These two mIF panels demonstrated specific co-localization in different cells that can identify the expression of PD-L1 in malignant cells and macrophages, and different T-cell subpopulations. This mIF methodology can be an invaluable tool for tumor tissue immune-profiling to allow multiple targets in the same tissue section and we provide that is accurate and reproducible method when is performed carefully under pathologist supervision.

The Potential Role of Inflammation Associated with Interaction between Osteopontin and CD44 in a Case of Pulmonary Tumor Thrombotic Microangiopathy Caused by Breast Cancer.

Pulmonary tumor thrombotic microangiopathy (PTTM) is a rare and fatal cancer-related complication. We herein present a case of PTTM that diagnosed antemortem by lung scintigraphy and pulmonary microvascular cytology. The patient was treated with steroid pulse therapy. Although her symptoms temporarily improved, she died of respiratory failure. An autopsy showed PTTM, and an immunohistochemical analysis revealed the expression of osteopontin and CD44 in macrophages that had migrated into the PTTM lesions. These findings suggest that inflammation associated with the interaction between osteopontin and CD44 may play an important role in PTTM.

Regression of Cecal MALT Lymphoma after Antibiotic Treatment in a Patient with Helicobacter pylori Infection.

A 63-year-old man with abdominal discomfort was referred to our hospital. Colonoscopy revealed a hemispherical-shaped protruding cecal mass of approximately 10 mm in size with a normal mucosal surface. Biopsy specimens showed nodules consisting of the proliferation of atypical lymphoid cells. Mucosa-associated lymphoid tissue (MALT) lymphoma was diagnosed based on the histological and immunohistochemical findings. Since upper gastrointestinal endoscopy demonstrated Helicobacter pylori-associated atrophic gastritis, eradication therapy was administered. The cecal mass disappeared completely within three months after triple therapy. Therefore, H. pylori eradication therapy may be a useful treatment option for cecal MALT lymphoma.

4-1BB Agonist Focuses CD8+ Tumor-Infiltrating T-Cell Growth into a Distinct Repertoire Capable of Tumor Recognition in Pancreatic Cancer

Purpose: Survival for pancreatic ductal adenocarcinoma (PDAC) patients is extremely poor and improved therapies are urgently needed. Tumor-infiltrating lymphocyte (TIL) adoptive cell therapy (ACT) has shown great promise in other tumor types, such as metastatic melanoma where overall response rates of 50% have been seen. Given this success and the evidence showing that T-cell presence positively correlates with overall survival in PDAC, we sought to enrich for CD8+ TILs capable of autologous tumor recognition. In addition, we explored the phenotype and T-cell receptor repertoire of the CD8+ TILs in the tumor microenvironment. Experimental Design: We used an agonistic 4-1BB mAb during the initial tumor fragment culture to provide 4-1BB costimulation and assessed changes in TIL growth, phenotype, repertoire, and antitumor function. Results: Increased CD8+ TIL growth from PDAC tumors was achieved with the aid of an agonistic 4-1BB mAb. Expanded TILs were characterized by an activated but not terminally differentiated phenotype. Moreover, 4-1BB stimulation expanded a more clonal and distinct CD8+ TIL repertoire than IL2 alone. TILs from both culture conditions displayed MHC class I-restricted recognition of autologous tumor targets. Conclusions: Costimulation with an anti-4-1BB mAb increases the feasibility of TIL therapy by producing greater numbers of these tumor-reactive T cells. These results suggest that TIL ACT for PDAC is a potential treatment avenue worth further investigation for a patient population in dire need of improved therapy. Clin Cancer Res; 23(23); 7263–75. ©2017 AACR.