Scirrhous-type Gastric Cancer Research Articles

Treatment development targeting Hippo pathway for scirrhous type gastric cancer.

398 Background: Treatment outcome of gastric cancer (GC) has been improved by minimally invasive surgery, and development of new drugs including immune checkpoint inhibitors. However, survival for patients with scirrhous type (SGC, Type4) gastric cancer is dismal. Methods: We collected miRNAs form exosomes released from cancer associated fibroblasts which is developed from resected gastric cancer tissue. Pathway enrichment analysis (PEA) of top miRNAs showed that they are significantly associated with Hippo signaling pathway (Hippo pathway). The molecules relating Hippo pathway was evaluated by Western blotting in cell line, and IHC of tissue array. Results: PEA using top miRNAs showed the significant relationship to Hippo pathway. Yap is a final effector in this pathway, and we found that YAP is not expressed in MKN45 cells (diffuse type), which is supposed to be degraded in cytoplasm. On the contrary, MKN74 cells express both YAP and pYAP. IHC showed that YAP translocation to nucleus is significantly associated with CTGF expression. CTGF low expression is significantly associated with worse prognosis especially in diffuse type GC (Log rank p = 0.022). This trend is similar in the analysis of patients with SGC. Conclusions: The role of Hippo pathway is different between intestinal type and diffuse type of GC. YAP activator could be effective for the treatment of SGC, and we are focusing on the treatment development.

Candidate Oncogenes,ARHGAP4, NOS3, andOR51B5, for the Development of Scirrhous-type Gastric Cancer

Scirrhous-type gastric cancer (SGC), one of the most intractable cancer subtypes, is characterized by rapid cancer cell proliferation and infiltration accompanied by extensive stromal fibrosis. One of the reasons for its poor prognosis may be the lack of molecular target drugs for SGC, because of the unknown driver genes. Exploration of somatic mutations in the human samples of SGC using next-generation sequencing (NGS) has been hampered by abundant fibrous tissues in these samples. Therefore, this study aimed to determine a novel oncogene by RNA-sequencing using SGC cell lines, avoiding contamination with fibrosis. In silico analysis of RNA-sequencing public data of the gastric cancer cell line, and RNA- sequencing using five of our unique SGC cell lines, OCUM1, OCUM2MLN, OCUM8, OCUM12, and OCUM14 were performed. We found three differentially expressed genes, ARHGAP4, NOS3, and OR51B5 that are significantly over-expressed in SGC cells. Immunohistochemical analysis indicated that the protein expression levels of these three genes were significantly higher in SGC than in other types of gastric cancer. The prognosis of patients with positive expression of these three genes was significantly poorer than those with negative expression. In particular, ARHGAP4 expression was an independent predictor of poor prognosis and recurrence. ARHGAP4, NOS3, and OR51B5 may be candidate driver genes for SGC. ARHGAP4 may be a promising molecular target for SGC.

Search for driver gene of scirrhous-type gastric cancer by RNA-seq using Next-Generation Sequencer

Search for driver gene of scirrhous-type gastric cancer by RNA-seq using Next-Generation Sequencer

Dipeptidyl Peptidase-4 from Cancer-associated Fibroblasts Stimulates the Proliferation of Scirrhous-type Gastric Cancer Cells.

Cancer-associated fibroblasts (CAFs) may promote the malignancy of human scirrhous gastric cancer (SGC) cells. We conducted the present study to identify novel growth factors from CAFs. OCUM-12 and 2 CAF cell lines were used. The proliferation of cancer cells was determined by the number of cancer cells or the MTT assay. The growth factor(s) were purified and characterized by the gel filtration chromatography and protein array. The molecular weight of the growth-stimulating factor was estimated to be approximately 66-669 kDa. Protein array of conditioned medium (CM) from CAFs indicated that dipeptidyl peptidase-4 (DPP-4) was one of the growth factors. The addition of CM increased the phosphorylation of C-X-C chemokine receptor 4 (CXCR4). The DPP-4 inhibitor significantly inhibited the growth-stimulating activity of CM. DPP-4 from CAFs might be one of the growth-stimulating factors for SGC through CXCR4.

P3-8 Lipocalin-2 expression might be a novel biomarker for patients with scirrhous-type gastric cancer

P3-8 Lipocalin-2 expression might be a novel biomarker for patients with scirrhous-type gastric cancer

Abstract 258: Identification of characteristic genes of scirrhous-type gastric cancer cells by RNAseq

Abstract Introduction: Scirrhous gastric cancer shows an extremely poor prognosis with rapid growth and infiltration activity. One of the reasons for poor prognosis is a lack of the identification of driver gene for scirrhous gastric cancer. FGFR2 (fibroblast growth factor receptor 2), E-cadherin, TGFβR (transforming growth factor β receptor), CD44/IGFIR (insulin-like growth factor I receptor), have been reported as candidates of driver for scirrhous-type of gastric cancer, however useful molecular-targeted therapeutic agents against these driver molecules have not yet been clinically approved. In this study, we identified the characteristic genes of scirrhous-type gastric cancer by RNA-seq of gastric cancer cell lines. Methods: RNA-seq was performed using 6 scirrhous gastric cancer cell lines (OCUM1, OCUM2MLN, OCUM8, OCUM12, OCUM13, OCUM14) using a next-generation sequencer (ION S5 system, Thermo Fisher Scientific). Furthermore, RNA-seq public data of 4 strains of scirrhous gastric cancer cell lines (KATO3, NUGC4, SNU601, SNU668) and 10 strains of non-scirrhous gastric cancer cell lines (MKN7, MKN45, MKN74, HS746T, SNU1, SNU16, SNU638, AZ521, HGC27, TMK1) on the Sequence Read Archive were added, and the genes expressed in scirrhous gastric cancer were compared and examined. The analysis tools used were kallisto and sleuth. We set p-value < 0.05 and log2 (fold change) > 2 as the cut-offs to screen out differentially expressed genes (DEGs). Pathway enrichment and protein-protein interaction (PPI) network analysis were performed based on metascape databases. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was also performed for the genes. Results: A total of 234 DEGs were identified from this analysis. A gene co-expression network was constructed containing 8 DEGs. The genes were mainly associated with response to chemokine, and cytokine-mediated signaling pathway. Several critical genes were disclosed, such as C-C motif chemokine ligand 20 (CCL20) and C-X-C subfamiliy. Genes highly expressed in scirrhous gastric cancer cell lines were CCL20 (10.6-fold change, p=9.89×10-4) and C-X-C Motif Chemokine Ligand 3 (CXCL3, 19.5-fold change, p=1.67×10-5). The mean of abundances across samples (=log(counts+0.5)), which represent the average expression levels of all samples, were CCL20 and CXCL3, 4.04 and 4.53, respectively. KEGG pathway enrichment analysis showed that viral protein interaction with cytokine and cytokine receptor, Cytokine-cytokine receptor interaction and IL-17 signaling pathway were significantly over-represented. Conclusion: CCL20 and CXCL3 might be characteristic genes for scirrhous-type of gastric cancer. Citation Format: Tomohiro Sera, Masakazu Yashiro, Gen Tsujio, Yurie Yamamoto, Atsushi Sugimoto, Shuhei Kushiyama, Sadaaki Nishimura, Masaichi Ohira. Identification of characteristic genes of scirrhous-type gastric cancer cells by RNAseq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 258.

Crosstalk Between Cancer Associated Fibroblasts and Cancer Cells in Scirrhous Type Gastric Cancer.

Gastric cancer (GC) is the third leading cause among all cancer deaths globally. Although the treatment outcome of GC has improved, the survival of patients with GC at stages III and IV remains unsatisfactory. Among several types of GC, scirrhous type GC (SGC) shows highly aggressive growth and invasive activity, leading to frequent peritoneal metastasis. SGC is well known to accompany abundant stromal cells that compose the tumor microenvironment (TME) along with the produced extracellular matrix (ECM) and secreted factors. One of the main stromal components is cancer associated fibroblast (CAF). In the SGC microenvironment, CAFs are a source of various secreted factors, including fibroblast growth factors (FGFs), which mediate prominent tumor-stimulating activity. In turn, cancer cells also secrete numerous factors, which can activate and educate CAFs. Current findings suggest that cancer cells and stromal cells communicate interactively via the soluble factors, the ECM, and likely also by exosomes. In this review, we focus on the soluble factors mediating communication between cancer cells and CAFs in SGC, and consider how they are related to the modulation of TME and the high rate of peritoneal metastasis. At last, we discuss the perspectives on targeting these communication pathways for improved future treatment.

Abstract 1217A: Serine threonine kinase 11/liver kinase B1 mutation in sporadic scirrhous-type gastric cancer cells

Abstract Background: Scirrhous-type gastric carcinoma (SGC), which is characterized by the rapid proliferation of cancer cells accompanied by extensive fibrosis, shows extremely poor survival. No driver gene responsible for the malignant potential of SGC has not been identified. Next-generation sequencing (NGS) technology has defined as the genomic landscape of various types of cancers, and has elucidated a number of novel driver mutations of gastric cancer. RHOA and CDH1 are characteristic mutations for SGC, but these mutations are not druggable. Novel molecular target therapy has been necessary for SGC based on its biological behavior. Objective: We sought to identify the characteristic driver gene which is responsible for SGC. Methods: DNA was isolated from six human SGC cell lines: OUCM-1, OCUM-2M, OCUM-8, OCUM-9, OCUM-12, and OCUM-14. The mutational status of 90 cancer-associated genes was examined by next-generation sequencing. The expression level of serine threonine kinase 11/liver kinase B1 (STK11/LKB1) in 708 gastric cancers was examined immunochemically, and the correlations between STK11/LKB1 expression and clinicopathologic features of gastric cancer were analyzed. Results: The nonsynonymous mutations of STK11/LKB1 gene were found in three of the six SGC cell lines. Nonsense mutation of STK11/LKB1, which was associated with STK11/LKB1 loss (Y253*), was found in OCUM-12 cells, and missense mutation of STK11/LKB1 (F354L) was found in OCUM-2M cells and OCUM-14 cells. Rapamycin, an inhibitor of STK11/ AMP-activated protein kinase (AMPK)/mammalian target of rapamycin signaling (mTOR), showed inhibitory effects of SGC cells harboring STK11/LKB1 mutation. The low STK11/LKB1 expression group was significantly associated with SGC, high invasion depth, and nodal involvement, in compared to the high STK11/LKB1 expression group. The 5-year survival rate of patients with low STK11/LKB1 expression was significantly poorer compared to those with high STK11/LKB1 expression (p<0.001). A conditional multivariate analysis showed that STK11/LKB1 expression status was an independent prognostic factor. Conclusion: SGC cells frequently showed the nonsynonymous mutations of STK11/LKB1. STK11/LKB1 mutation might be responsible for the malignant potential of SGC. STK11/AMPK/mTOR signaling might be one of the therapeutic targets for patients with SGC.1 Citation Format: Sadaaki Nishimura, Masakazu Yashiro, Tomohiro Sera, Yurie Yamamoto, Atsushi Sugimoto, Shuhei Kushiyama, Shingo Togano, Kenji Kuroda, Tomohisa Okuno, Masaichi Ohira. Serine threonine kinase 11/liver kinase B1 mutation in sporadic scirrhous-type gastric cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1217A.

Identification of candidates for driver oncogenes in scirrhous-type gastric cancer cell lines.

Scirrhous‐type gastric cancer (SGC) is one of the most intractable cancer subtypes in humans, and its therapeutic targets have been rarely identified to date. Exploration of somatic mutations in the SGC genome with the next‐generation sequencers has been hampered by markedly increased fibrous tissues. Thus, SGC cell lines may be useful resources for searching for novel oncogenes. Here we have conducted whole exome sequencing and RNA sequencing on 2 SGC cell lines, OCUM‐8 and OCUM‐9. Interestingly, most of the mutations thus identified have not been reported. In OCUM‐8 cells, a novel CD44‐IGF1R fusion gene is discovered, the protein product of which ligates the amino‐terminus of CD44 to the transmembrane and tyrosine‐kinase domains of IGF1R. Furthermore, both CD44 and IGF1R are markedly amplified in the OCUM‐8 genome and abundantly expressed. CD44‐IGF1R has a transforming ability, and the suppression of its kinase activity leads to rapid cell death of OCUM‐8. To the best of our knowledge, this is the first report describing the transforming activity of IGF1R fusion genes. However, OCUM‐9 seems to possess multiple oncogenic events in its genome. In particular, a novel BORCS5‐ETV6 fusion gene is identified in the OCUM‐9 genome. BORCS5‐ETV6 possesses oncogenic activity, and suppression of its message partially inhibits cell growth. Prevalence of these novel fusion genes among SGC awaits further investigation, but we validate the significance of cell lines as appropriate reagents for detailed genomic analyses of SGC.

Clinico-pathological significance of exosome marker CD63 expression on cancer cells and stromal cells in gastric cancer

BackgroundIt has been reported that CD63, an exosome marker, is expressed in solid cancer tissues. However, its significance in patients with gastric cancer has not been clarified. Exosomes derived from cancer cells and stromal cells might play an important role in the intracellular communications involved in the development of carcinoma. This study aimed to clarify the relationship between CD63 expression in cancer cells and stromal cells and clinical-pathologic factors.MethodsA total of 595 gastric cancer patients were enrolled in this study. CD63 expression in cancer cells and stromal cells was analyzed by immunohistochemistry. The correlations between CD63 expression and several clinicopathological factors were investigated.ResultsCD63 expression was mainly observed on the cell membranes of cancer cells, and in the cytoplasm of stromal cells. Of 595 patients, 247 cases had CD63-positive cancer cells, and 107 cases had CD63-positive stromal cells. Cases with CD63-positive cancer cells were significantly correlated with scirrhous-type gastric cancer, tumor depth, lymph node metastasis, lymphatic invasion, and tumor size. Cases with CD63-positive stromal cells were significantly correlated with age (≥65), tumor depth (T3-4), lymphatic invasion, and tumor size (≥ 5 cm). The 5-year survival rate was significantly lower (p<0.001) in patients with CD63-positive than CD63-negative tumors. Multivariate analysis showed that CD63 expression in cancer cells was a significant independent prognostic factor in patients with gastric cancer.ConclusionCD63 might be a prognostic marker for patients with gastric cancer. CD63-positive exosomes might be associated with the interaction between stromal cells and cancer cells.

Recent updates in perioperative chemotherapy and recurrence pattern of gastric cancer.

Gastrectomy with D2 lymph node dissection has become the global standard procedure for locally advanced gastric cancer to maximally reduce locoregional recurrence. In East Asia, based on the evidence of the ACTS‐GC and the CLASSIC trials, postadjuvant chemotherapy with S‐1 monotherapy or capecitabine and oxaliplatin after curative D2 gastrectomy is the current standard strategy. However, approximately 20% to 30% of patients still develop distant recurrence even after these postadjuvant chemotherapies, especially in those with pathological stage III disease. This review summarizes recent (2008‐2018) evidence on the benefits of adjuvant therapy for locally advanced gastric cancer. JACRO GC‐07, a Phase III trial, recently showed a superior 3‐year recurrence‐free survival of the S‐1 plus docetaxel regimen in comparison to S‐1 monotherapy for patients with pathological stage III gastric cancer after curative D2 gastrectomy. With regard to recent new evidence on neoadjuvant strategy, JCOG0501, a Phase III trial, did not show any superiority in 3‐year overall survival (OS) of additional neoadjuvant chemotherapy with S‐1/cisplatin over postadjuvant S‐1 monotherapy in scirrhous type gastric cancer. Further clinical trials of neoadjuvant chemotherapy are ongoing to improve the poor prognosis for gastric cancer with extensive lymph node metastases. These trials could lead to new evidence for improved treatment of gastric cancer in the near future.

CD9-positive exosomes from cancer-associated fibroblasts stimulate the migration ability of scirrhous-type gastric cancer cells

Background:Crosstalk between cancer cells and fibroblasts is crucial for tumour progression. It has been reported that exosomes derived from cancer cells play an important role in the intracellular communications involved in the development of carcinoma. However, the role of exosomes from fibroblasts remains unclear. This study aimed to clarify the effect of exosomes from fibroblasts on the motility of gastric cancer cells.Methods:5 gastric cancer cell lines were used: OCUM-12, NUGC-3, MKN45, FU97 and MKN74. 2 cancer-associated fibroblasts (CAFs) were used. CD9 expression of exosomes from fibroblasts was examined by western blot. The effect of exosomes on the motility of cancer cells was analysed by migration assays. MMP2 was examined by RT-PCR or gelatin zymography. Then, CD9 and MMP2 expressions of 619 gastric cancers were analysed by immunohistochemistry.Results:Exosomes from CAFs were taken into scirrhous-type gastric cancer cells, namely OCUM-12 cells and NUGC-3 cells, but not into other types of gastric cancer cells. Exosomes from CAFs were positive for CD9. Exosomes from CAFs significantly stimulated the migration and invasion of OCUM-12 and NUGC-3 cells, which was inhibited by anti-CD9 antibody or CD9-siRNA. MMP2 expression of OCUM-12 and NUGC-3 cells was significantly decreased by CD9-siRNA. 116 CD9-positive cases were significantly correlated with scirrhous-type gastric cancer, lymph node metastasis and venous invasion. The 5-year survival rate of patients with CD9-positive tumours was significantly lower (P<0.001) than in those with CD9-negative tumours.Conclusions:CD9-positive exosomes from CAFs might stimulate the migration ability of scirrhous-type gastric cancer cells.

Expression level of microRNA-200c is associated with cell morphology in vitro and histological differentiation through regulation of ZEB1/2 and E-cadherin in gastric carcinoma.

Scirrhous type gastric cancer is characterized by diffuse infiltration of poorly differentiated adenocarcinoma cells and poor prognosis. Although association of poorly differentiated histology with reduction in E-cadherin expression, as well as association of microRNA (miR)-200c with E-cadherin through regulation of ZEB1/2, has been reported, participation of miR-200c in gastric carcinogenesis is not fully understood. We used 6 cell lines originating from gastric cancers, and investigated levels of miR-200c along with its target mRNAs ZEB1/2 and E-cadherin by qRT-PCR. ZEB1 and E-cadherin protein expression was also assessed via western blotting. Furthermore, we investigated the expression levels of miR-200c by in situ hybridization, along with the expression of ZEB1 and E-cadherin by immunohistochemistry, in 97 gastric adenocarcinoma tissues. Inverse correlation between miR-200c and ZEB1 levels were obtained by qRT-PCR in cell lines (P<0.05). Cell lines with low miR-200c and high ZEB1 exhibited low E-cadherin expression in both qRT-PCR and western blotting, and exhibited spindle-shaped morphology, in contrast to round cell morphology in those cell lines with high miR-200c levels. Inverse correlations were also obtained between miR-200c and ZEB1 as well as between ZEB1 and E-cadherin levels in tissue samples (P<0.001). Cancer tissues with low miR-200c, high ZEB1, and low E-cadherin expression were associated with poorly differentiated histology, in contrast to tubular form in cancers with high miR-200c expression levels (P<0.001). Our data revealed that downregulation of miR-200c primarily regulated cell morphology by downregulation of E-cadherin through upregulation of ZEB1, leading to poorly differentiated histology in 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.

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.

Identification of Novel Transmembrane Proteins in Scirrhous-Type Gastric Cancer by the 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.

MicroRNA‐143 regulates collagen type III expression in stromal fibroblasts 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. In the present study, we identified several miRNAs that are expressed at higher levels in scirrhous type GC than in non-scirrhous type GC by miRNA microarray analysis. Among these, microRNA-143 (miR-143) expression was higher in scirrhous type GC than in non-scirrhous types of GC. In situ hybridization and quantitative RT-PCR analysis showed that miR-143 is expressed by stromal fibroblasts but not by cancer cells. In stromal cells, miR-143 enhanced collagen type III expression in normal gastric fibroblasts and cancer-associated fibroblasts through activation of transforming growth factor-β)/SMAD signaling. Furthermore, high miR-143 expression in GC was associated with worse cancer-specific mortality (P = 0.0141). Multivariate analysis revealed that miR-143 was an independent prognostic factor. Treatment of GC cell lines with 5-aza-2′-deoxycytidine restored the expression of miR-143, and precursor miR-143 caused the inhibition of cancer cell invasion. These data suggest that miR-143 regulates fibrosis of scirrhous type GC through induction of collagen expression in stromal fibroblasts and that miR-143 expression serves as a prognostic marker of GC.

Abstract 4197: Function of microRNA-143 and its potential of prognostic factor in scirrhous type gastric cancer.

Abstract Gastric cancer (GC) is one of the most common malignancies worldwide. Scirrhous type GC is highly metastatic and characterized clinically by rapid disease progression and poor prognosis. MicroRNAs (miRNAs), small non-coding RNA of 19-25 nucleotides, play a part in crucial biological processes and also have an important role in cancer development and progression. In the present study, we show that microRNA-143 (miR-143) is expressed at higher level in diffuse type GC than in intestinal type GC by microRNA microarray analysis. We investigated whether miR-143 is useful as a diagnostic marker of scirrhous type GC. Quantitative RT-PCR analysis revealed that miR-143 expression was low in GC in comparison with adjacent non-tumorous mucosa. Expression levels of miR-143 were higher in scirrhous type GC than those in other types of GC. Treatment of 5-aza-2′-deoxycytidine retrieved miR-143 expression, suggesting that miR-143 might be repressed by DNA methylation. Quantitative RT-PCR and in situ hybridization analysis showed that miR-143 is expressed at higher level in normal fibroblast and cancer associated fibroblast cell lines than in GC cell lines, indicating that major source of miR-143 in cancer tissue is stromal fibroblasts rather than cancer cells. miR-143 knock down by RNAi repressed collagen type III expression in normal fibroblast and cancer associated fibroblast cell lines. Furthermore, TGF-β treatment induced expression of miR-143 and collagen type III mRNA, and this induction of collagen type III mRNA was repressed by miR-143 knock down. We also studied the relation of miR-143 expression with GC patient prognosis (n=68). High miR-143 expression was associated with worse prognosis (P=0.0141). These data suggest that miR-143 regulates fibrosis of scirrhous type GC through induction of collagen expression in stromal fibroblasts and miR-143 expression is a prognostic marker of GC. Citation Format: Yutaka Naito, Naoya Sakamoto, Naohiro Uraoka, Kazuhiro Sentani, Naohide Oue, Masakazu Yashiro, Kosei Hirakawa, Wataru Yasui. Function of microRNA-143 and its potential of prognostic factor in scirrhous type gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4197. doi:10.1158/1538-7445.AM2013-4197

Abstract 1925: ZDHHC14, a potential regulator in peritoneal dissemination of scirrhous type gastric cancer, identified by CAST method.

Abstract Gastric cancer (GC) is one of the most common malignancies worldwide and is one of the leading causes of cancer related mortality. Among gastric cancer subtypes, scirrhous type GC is highly aggressive and has a worse prognosis because of its diffuse infiltration pattern. Because of such pathological features, early clinical diagnosis of scirrhous gastric carcinoma remains difficult. Moreover, peritoneal dissemination occurs frequently even after radical surgery and is the cause of death in many patients. A potential diagnostic or therapeutic target to identify or to inhibit the mechanism of peritoneal dissemination would be an advantageous in this field and need to be elucidated. Escherichia coli ampicillin secretion trap (CAST) method is a powerful technique to identify novel genes that encode secreted or transmembrane proteins. We previously reported DSC2 (desmocollin 2), identified by this method, as a key regulator of GC with intestinal phenotype, using MKN-1 and MKN-28 GC cell lines. In this study, mRNAs were extracted from HSC-44PE and 44As3 scirrhous GC cell lines to analyze the gene expression profile. HSC-44PE cell line was established from pleural fluid of a patient with scirrhous GC. 44As3, derived from HSC-44PE cells, by isolating a highly disseminated clones with a strong capability of inducing ascites in mouse model. Randomly primed cDNAs ligated with pCAST vector were picked up and sequenced thereafter. Among the candidate genes, ZDHHC14 (zinc finger, DHHC-type containing 14) showed higher expression in 44As3 CAST library than in HSC-44PE library. ZDHHC14 is one of the palmitoyl transferase family members and is supposed to be associated with substrates linked to dissemination cascade in scirrhous type GC. Indirect immunofluorescence microscopic analysis also revealed that ZDHHC14 expression was found mostly in large cells of both cell lines, however, ZDHHC14 staining was more frequently observed in 44As3 than in HSC-44PE. High level of ZDHHC14 expression was observed in 27% of GC samples by qRT-PCR. Cell invasion assay and MTT assay revealed that ZDHHC14 influenced the invasion and growth capacity of GC cells. Identification of the palmitoylated proteins, the substrates of ZDHHC14, remain under investigation. ZDHHC14 identified by CAST method, might contribute in the process of peritoneal dissemination of the gastric cancer cells and it might be a potential therapeutic target for scirrhous type GC. Citation Format: Htoo Zarni Oo, Naoya Sakamoto, Naohiro Uraoka, Katsuhiro Anami, Kazuhiro Sentani, Naohide Oue, Kazuyoshi Yanagihara, Wataru Yasui. ZDHHC14, a potential regulator in peritoneal dissemination of scirrhous type gastric cancer, identified by CAST method. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1925. doi:10.1158/1538-7445.AM2013-1925

Abstract 4468: In vitro evaluation of multi-targeted kinase inhibitors in scirrhous gastric cancer cell lines by tyrosine kinase activity profiling

Abstract Scirrhous-type gastric cancer, which accounts for about 10% of all gastric cancers, interacts with stromal cells with involvement of multiple growth factors. Although a fluoropyrimidine and platinum-containing regimen has been established as the standard chemotherapy, its efficacy is not satisfactory. Multi-targeted kinase inhibitors (MTKIs) such as sunitinib, sorafenib and pazopanib are active in renal cell carcinoma and are under clinical evaluation for other types of solid tumors. Here we examined the efficacy of the MTKIs in scirrhous gastric cancer cell lines and conducted an exploratory predictive biomarker study by tyrosine kinase activity profiling with PamChip® peptide micro-arrays. For 11 scirrhous and 8 non-scirrhous gastric cancer cell lines, the growth-inhibitory effect of the MTKIs sunitinib, sorafenib and pazopanib was evaluated by the MTT assay. For assessment of protein tyrosine kinase activity, cells were grown until semi-confluent, lysed, aliquoted and stored at -80°C until use. The tyrosine kinase activity of the lysates was measured in the absence and presence of the MTKIs on PamChip® peptide micro-arrays, containing 144 peptides derived from known human phosphorylation sites. Data analysis was carried out on inhibition profiles calculated for each MTKI by taking the log-ratio of peptide phosphorylation measured in the absence and presence of the MTKIs. Peptides with a low basal signal were excluded from the analysis. In the MTT assay, cell lines HSC-39, HSC-40A, KATO-III, HSC-43 (scirrhous) and SNU-16 (non-scirrhous) were sensitive to all three MTKIs with IC50 values &amp;lt; 1 μmol/L. The other 14 cell lines were classified as insensitive, except that three cell lines showed intermediate sensitivity to sunitinib with IC50 values &amp;lt; 3 μmol/L. Per peptide analysis showed a clear correlation between sensitivity to the MTKIs and in vitro inhibition of tyrosine kinase activity, with stronger response associated with more inhibition for a larger number of peptides (out of 100 peptides 48, 46, and 30 with p &amp;lt; 0.05 for sunitinib, sorafenib and pazopanib, respectively). Principal component analysis of the inhibition profiles revealed a clustering of sensitive vs. insensitive cell lines. Partial least squares-discriminant analysis was used to correlate the in vitro inhibition on the PamChip® micro-arrays to the sensitivity to the MTKIs. Good predictive performance with a misclassification rate &amp;lt; 20% was obtained with leave-one-out-cross-validation for all 3 inhibitors. MTKIs seem to be efficacious in a subset of scirrhous-type gastric cancer. Sunitinib showed the most potent growth-inhibitory effect, suggesting that it should be considered for further evaluation in scirrhous-type gastric cancer. Sensitivity to the MTKIs in the MTT assay could be correlated to the in vitro inhibition of kinase activity on the PamChip® peptide micro-array. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4468. doi:10.1158/1538-7445.AM2011-4468