Htoo Zarni Oo

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.

Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein.

Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification.

Targeting HER2 with T-DM1, an Antibody Cytotoxic Drug Conjugate, is Effective in HER2 Over Expressing Bladder Cancer

PURPOSE Systemic therapy for advanced bladder cancer has not changed substantially in more than 2 decades and mortality rates remain high. The recognition of HER2 over expression in bladder cancer has made HER2 a promising therapeutic target. T-DM1, a new drug consisting of the HER2 antibody trastuzumab conjugated with a cytotoxic agent, has been shown in breast cancer to be superior to trastuzumab. We tested T-DM1 in preclinical models of bladder cancer. MATERIALS AND METHODS We evaluated the effect of T-DM1 compared to trastuzumab in different in vitro and in vivo models of HER2 over expressing bladder cancer. RESULTS RT4V6 was the highest HER2 expressing bladder cancer cell line and it showed higher growth inhibition with T-DM1 compared to trastuzumab. T-DM1 but not trastuzumab induced apoptosis of RT4V6 cells after G2/M arrest on cell cycle analysis. HER2 expression was higher in cell lines with acquired cisplatin resistance compared to the corresponding parental cell lines. Resistant cells showed higher sensitivity to T-DM1 by the induction of apoptosis. In addition, cells cultured in anchorage independent conditions increased HER2 expression compared to cells cultured in adherent conditions and T-DM1 significantly inhibited colony formation in soft agar compared to trastuzumab. In an orthotopic bladder cancer xenograft model tumor growth of cisplatin resistant RT112 was significantly inhibited by T-DM1 via the induction of apoptosis compared to treatment with control IgG or trastuzumab. CONCLUSIONS T-DM1 has promising antitumor effects in preclinical models of HER2 over expressing bladder cancer.

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.

Not all NOTCH Is Created Equal: The Oncogenic Role of NOTCH2 in Bladder Cancer and Its Implications for Targeted Therapy

Purpose: Recent molecular analyses of bladder cancer open the door to significant advances in targeted therapies. NOTCH has been identified as a tumor suppressor in bladder cancer, but prior reports have focused on NOTCH1. Here we hypothesized that NOTCH2 is an oncogene suitable for therapeutic targeting in bladder cancer. Experimental design: We studied genomic aberrations of NOTCH, compared survival and tumor progression according to NOTCH2 expression levels, and studied NOTCH2 function in vitro and vivo. Results: We report a high rate of NOTCH2 copy number gain in bladder cancer. High NOTCH2 expression was identified especially in the basal subtype and in mesenchymal tumors. NOTCH2 activation correlated with adverse disease parameters and worse prognosis by immunohistochemistry. Forced overexpression of the intracellular domain of NOTCH2 (N2ICD) induced cell growth and invasion by cell-cycle progression, maintenance of stemness and epithelial-to-mesenchymal transition (EMT). These effects were abrogated by silencing of CSL, indicating that the effects were mediated through the canonical NOTCH signaling pathway. In an orthotopic xenograft model, forced overexpression of N2ICD increased growth, invasion, and metastasis. To explore the potential for therapeutic targeting of NOTCH2, we first silenced the receptor with shRNA and subsequently treated with a specific inhibitory antibody. Both interventions decreased cell growth, invasion, and metastasis in vitro and in the orthotopic xenograft model. Conclusions: We have demonstrated that NOTCH2 acts as an oncogene that promotes bladder cancer growth and metastasis through EMT, cell-cycle progression, and maintenance of stemness. Inhibition of NOTCH2 is a rational novel treatment strategy for invasive bladder cancer. Clin Cancer Res; 22(12); 2981–92. ©2016 AACR.

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.

MicroRNA-145 is a potential prognostic factor of scirrhous type gastric cancer.

Gastric cancer (GC) is one of the most common malignancies worldwide. In particular, scirrhous type GC is highly metastatic and is characterized clinically by rapid disease progression and poor prognosis. MicroRNAs (miRNAs) play crucial roles in cancer development and progression. We previously demonstrated by microarray analysis that microRNA-145 (miR-145) is one of the more highly expressed miRNAs in scirrhous type GC vs. non-scirrhous types of GC. In the present study, we investigated the role of miR-145 in scirrhous type GC. The expression levels of miR-145 assessed by quantitative RT-PCR were higher in scirrhous type GC tissue samples than in non-scirrhous type GC and corresponding normal tissues. GC patients with high miR-145 expression were at a more advanced tumor stage (P=0.0156) and had more scirrhous type histology (P=0.0054) than those with low miR-145 expression. Furthermore, miR-145 expression was significantly associated with poor prognosis in GC patients (P=0.0438). miR-145 expression was localized in stromal fibroblasts of scirrhous type GC but not in cancer cells. miR-145 was induced by treatment by transforming growth factor-β, and it enhanced the expression of α-smooth muscle actin, a marker of myofibroblasts, in both normal gastric fibroblasts and cancer-associated fibroblasts. These data suggest that miR-145 may contribute to the progression of scirrhous type GC by regulating activation of peri-tumoral fibroblasts.

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.

Identification of transmembrane protein in prostate cancer by the Escherichia coli ampicillin secretion trap: expression of CDON is involved in tumor cell growth and invasion.

Aims: Prostate cancer (PCa) is one of the most common malignancies worldwide. Genes expressed only in cancer tissue, and especially related to proteins located on the cell membrane, will be useful molecular markers for diagnosis and may also be good therapeutic targets. The aim of this study was to identify genes that encode transmembrane proteins present in PCa. Methods and Results: We generated Escherichia coli ampicillin secretion trap (CAST) libraries from 2 PCa cell lines and normal prostate tissues. By sequencing 3,264 colonies from CAST libraries, we identified 18 candidate genes that encode transmembrane proteins present in PCa. Quantitative RT-PCR analysis of these candidates revealed that STEAP1, ADAM9 and CDON were expressed much more highly in PCa than in 15 kinds of normal tissues. Among the candidates, CDON encodes the CDO protein, which is an orphan cell surface receptor of the immunoglobulin superfamily. Additional quantitative RT-PCR revealed that 83% of PCa tissues showed CDON overexpression. Knockdown of CDON in DU145 cells induced 5-fluorouracil-induced apoptosis and inhibited invasion ability. Conclusion: These results suggest that CDON has a high potential as a therapeutic target for PCa.

Identification of novel transmembrane proteins in scirrhous type gastric cancer by Escherichia coli ampicillin secretion trap (CAST) method : TM9SF3 participates in tumor invasion and serves as a prognostic factor

Objective: Scirrhous-type gastric cancer (GC) is highly aggressive and has a poor prognosis due to rapid cancer cell infiltration accompanied by extensive stromal fibrosis. The aim of this study is to identify genes that encode transmembrane proteins frequently expressed in scirrhous-type GC. Methods: We compared Escherichia coli ampicillin secretion trap (CAST) libraries from 2 human scirrhous-type GC tissues with a normal stomach CAST library. By sequencing 2,880 colonies from scirrhous CAST libraries, we identified a list of candidate genes. Results: We focused on the TM9SF3 gene because it has the highest clone count, and immunohistochemical analysis demonstrated that 46 (50%) of 91 GC cases were positive for TM9SF3, which was observed frequently in scirrhous-type GC. TM9SF3 expression showed a significant correlation with the depth of invasion, tumor stage and undifferentiated GC. There was a strong correlation between TM9SF3 expression and poor patient outcome, which was validated in two separate cohorts by immunostaining and quantitative RT-PCR, respectively. Transient knockdown of the TM9SF3 gene by siRNA showed decreased tumor cell-invasive capacity. Conclusion: Our results indicate that TM9SF3 might be a potential diagnostic and therapeutic target for scirrhous-type GC.