Chae Hwa Kwon

Overexpression of Snail is associated with lymph node metastasis and poor prognosis in patients with gastric cancer

BackgroundEpithelial–mesenchymal transition (EMT) plays a significant role in tumor progression and invasion. Snail is a known regulator of EMT in various malignant tumors. This study investigated the role of Snail in gastric cancer.MethodsWe examined the effects of silenced or overexpressed Snail using lenti-viral constructs in gastric cancer cells. Immunohistochemical analysis of tissue microarrays from 314 patients with gastric adenocarcinoma (GC) was used to determine Snail’s clinicopathological and prognostic significance. Differential gene expression in 45 GC specimens with Snail overexpression was investigated using cDNA microarray analysis.ResultsSilencing of Snail by shRNA decreased invasion and migration in GC cell lines. Conversely, Snail overexpression increased invasion and migration of gastric cancer cells, in line with increased VEGF and MMP11. Snail overexpression (≥75% positive nuclear staining) was also significantly associated with tumor progression (P < 0.001), lymph node metastases (P = 0.002), lymphovascular invasion (P = 0.002), and perineural invasion (P = 0.002) in the 314 GC patients, and with shorter survival (P = 0.023). cDNA microarray analysis revealed 213 differentially expressed genes in GC tissues with Snail overexpression, including genes related to metastasis and invasion.ConclusionSnail significantly affects invasiveness/migratory ability of GCs, and may also be used as a predictive biomarker for prognosis or aggressiveness of GCs.

S100A8 and S100A9 promotes invasion and migration through p38 mitogen-activated protein kinase-dependent NF-κB activation in gastric cancer cells

S100A8 and S100A9 (S100A8/A9) are low-molecular weight members of the S100 family of calcium-binding proteins. Recent studies have reported S100A8/A9 promote tumorigenesis. We have previously reported that S100A8/A9 is mostly expressed in stromal cells and inflammatory cells between gastric tumor cells. However, the role of environmental S100A8/A9 in gastric cancer has not been defined. We observed in the present study the effect of S100A8/A9 on migration and invasion of gastric cancer cells. S100A8/ A9 treatment increased migration and invasionat lower concentrations that did not affect cell proliferation and cell viability. S100A8/A9 caused activation of p38 mitogenactivated protein kinase (MAPK) and nuclear factor-κB (NF-κB). The phosphorylation of p38 MAPK was not affected by the NF-κB inhibitor Bay whereas activation of NF-κB was blocked by p38 MAPK inhibitor SB203580, indicating that S100A8/A9-induced NF-κB activation is mediated by phosphorylation of p38 MAPK. S100A8/A9-induced cell migration and invasion was inhibited by SB203580 and Bay, suggesting that activation of p38 MAPK and NF-κB is involved in the S100A8/A9 induced cell migration and invasion. S100A8/A9 caused an increase in matrix metalloproteinase 2 (MMP2) and MMP12 expression, which were inhibited by SB203580 and Bay. S100A8/A9-induced cell migration and invasion was inhibited by MMP2 siRNA and MMP12 siRNA, indicating that MMP2 and MMP12 is related to the S100A8/A9 induced cell migration and invasion. Taken together, these results suggest that S100A8/A9 promotes cell migration and invasion through p38 MAPKdependent NF-κB activation leading to an increase of MMP2 and MMP12 in gastric cancer.

PSMB8 and PBK as potential gastric cancer subtype-specific biomarkers associated with prognosis.

Gastric adenocarcinoma is a common form of cancer associated with a poor prognosis. We analyzed microarray profiling data from 48 patients with gastric adenocarcinoma to characterize gastric cancer subtypes and identify biomarkers associated with prognosis. We identified two major subtypes of gastric adenocarcinoma differentially associated with overall survival (P = 0.025). Genes that were differentially expressed were identified using specific criteria (P < 0.001 and >1.5-fold); expression of 294 and 116 genes was enriched in good and poor prognosis subtypes, respectively. Genes related to translational elongation and cell cycle were upregulated in the poor prognosis group. Of these genes, upregulation of proteasome subunit beta type 8 PSMB8 and PDZ binding kinase PBK was confirmed by real-time reverse transcription-PCR analysis. PSMB8 or PBK knockdown had no effect on gastric cancer cell proliferation but suppressed cell migration and invasion, respectively. Furthermore, immunohistochemistry analysis of 385 gastric cancer patients revealed that increased nuclear expression of PSMB8 and PBK was correlated with depth of invasion, lymph node metastasis, and lower survival rates. Taken together, two gastric adenocarcinoma subtypes were predictive of prognosis. PSMB8 and PBK were predictive of gastric cancer prognosis and could be potential gastric cancer subtype-specific biomarkers.

Decreased Muc5AC expression is associated with poor prognosis in gastric cancer

Mucins reportedly play numerous key roles in carcinogenesis, including in tumor invasion, regulation of differentiation and tumor cell proliferation. We investigated the effect of Muc5AC, a secreted mucin, on the invasiveness/migratory capability of gastric cancer cells and the prognostic significance of Muc5AC in gastric cancer patients. The clinicopathological and prognostic significance of Muc5AC expression was validated using immunohistochemical analysis in 412 gastric cancer patients. Differential gene expression was investigated using complementary DNA microarray analysis of 48 fresh tumor tissue samples. Silencing of Muc5AC by using a small hairpin RNA‐containing lentivirus increased the invasion and migration of SNU216 and AGS cells as well as Akt phosphorylation and the expression of vascular endothelial growth factor and matrix metalloproteinase‐7, which were blocked by inhibitors of the phosphatidylinositol 3‐kinase/Akt pathway. Loss of Muc5AC expression was significantly associated with tumor progression (advanced T stage; p = 0.004), lymph node metastases (p = 0.001), lymphovascular invasion (p < 0.0001), and increased tumor size (p = 0.027). Lower MUC5AC expression was identified as an independent poor prognostic factor in diffuse‐type gastric cancer by using the Cox regression proportional hazard model (hazard ratio, 2.39; p = 0.043). Complementary DNA microarray analysis revealed 86 differentially expressed genes, including genes related to metastasis and invasion, in gastric cancer tissues with high (≥25%) and low (<25%) Muc5AC expression levels. Low Muc5AC expression increased the invasion and migration of gastric cancer cells and could be a useful biomarker of poor prognosis in gastric cancer.

NTRK1 fusions for the therapeutic intervention of Korean patients with colon cancer

The identification and clinical validation of cancer driver genes are essential to accelerate the translational transition of cancer genomics, as well as to find clinically confident targets for the therapeutic intervention of cancers. Here we identified recurrent LMNA-NTRK1 and TPM3-NTRK1 fusions in Korean patients with colon cancer (3 out of 147, 2%) through next-generation RNA sequencing (RNA-seq). NTRK1 fusions were mutually exclusive oncogenic drivers of colon cancer that were accompanied with in vitro potential of colony formation and in vivo tumorigenicity comparable to KM12, a human colon cancer cell line harboring TPM3-NTRK1 fusion. NTRK1-encoded TrkA protein was prevalent in 11 out of 216 Korean (5.1%) and 28 out of 472 Chinese patients (5.9%) from independent cohorts, respectively. The expression level of TrkA was significantly correlated with NTRK1 fusion (p = 0.0192), which was verified by a fluorescence in situ hybridization (FISH). Korean patients with TrkA-positive colon cancer had a marginal but significant shorter overall survival time than TrkA-negative colon cancer [hazard ratio (HR) = 0.5346, 95% confidential interval (CI) = 0.2548-0.9722, p = 0.0411]. In addition, KM12 cell line was sensitive to selective TrkA inhibitors. These results demonstrate that NTRK1 fusion is granted as a clinically relevant target for therapeutic intervention of colon cancer.

Sporadic Early-Onset Diffuse Gastric Cancers Have High Frequency of Somatic CDH1 Alterations, but Low Frequency of Somatic RHOA Mutations Compared With Late-Onset Cancers

BACKGROUND & AIMS: Early-onset gastric cancer, which develops in patients younger than most gastric cancers, is usually detected at advanced stages, has diffuse histologic features, and occurs more frequently in women. We investigated somatic genomic alterations associated with the unique characteristics of sporadic diffuse gastric cancers (DGCs) from younger patients. METHODS: We conducted whole exome and RNA sequence analyses of 80 resected DGC samples from patients 45 years old or younger in Korea. Patients with pathogenic germline mutations in CDH1, TP53, and ATM were excluded from the onset of this analysis, given our focus on somatic alterations. We used MutSig2CV to evaluate the significance of mutated genes. We recruited 29 additional early-onset Korean DGC samples and performed SNP6.0 array and targeted sequencing analyses of these 109 early-onset DGC samples (54.1% female, median age, 38 years). We compared the SNP6.0 array and targeted sequencing data of the 109 early-onset DGC samples with those from diffuse-type stomach tumor samples collected from 115 patients in Korea who were 46 years or older (late onset) at the time of diagnosis (controls; 29.6% female, median age, 67 years). We compared patient survival times among tumors from different subgroups and with different somatic mutations. We performed gene silencing of RHOA or CDH1 in DGC cells with small interfering RNAs for cell-based assays. RESULTS: We identified somatic mutations in the following genes in a significant number of early-onset DGCs: the cadherin 1 gene (CDH1), TP53, ARID1A, KRAS, PIK3CA, ERBB3, TGFBR1, FBXW7, RHOA, and MAP2K1. None of 109 early-onset DGC cases had pathogenic germline CDH1 mutations. A higher proportion of early-onset DGCs had mutations in CDH1 (42.2%) or TGFBR1 (7.3%) compared with control DGCs (17.4% and 0.9%, respectively) (P < .001 and P = .014 for CDH1 and TGFBR1, respectively). In contrast, a smaller proportion of early-onset DGCs contained mutations in RHOA (9.2%) than control DGCs (19.1%) (P = .033). Late-onset DGCs in The Cancer Genome Atlas also contained less frequent mutations in CDH1 and TGFBR1 and more frequent RHOA mutations, compared with early-onset DGCs. Early-onset DGCs from women contained significantly more mutations in CDH1 or TGFBR1 than early-onset DGCs from men. CDH1 alterations, but not RHOA mutations, were associated with shorter survival times in patients with early-onset DGCs (hazard ratio, 3.4; 95% confidence interval, 1.5–7.7). RHOA activity was reduced by an R5W substitution—the RHOA mutation most frequently detected in early-onset DGCs. Silencing of CDH1, but not RHOA, increased migratory activity of DGC cells. CONCLUSIONS: In an integrative genomic analysis, we found higher proportions of early-onset DGCs to contain somatic mutations in CDH1 or TGFBR1 compared with late-onset DGCs. However, a smaller proportion of early-onset DGCs contained somatic mutations in RHOA than late-onset DGCs. CDH1 alterations, but not RHOA mutations, were associated with shorter survival times of patients, which might account for the aggressive clinical course of early-onset gastric cancer. Female predominance in early-onset gastric cancer may be related to relatively high rates of somatic CDH1 and TGFBR1 mutations in this population.

Transcriptome analysis of paired primary colorectal carcinoma and liver metastases reveals fusion transcripts and similar gene expression profiles in primary carcinoma and liver metastases

BackgroundDespite the clinical significance of liver metastases, the difference between molecular and cellular changes in primary colorectal cancers (CRC) and matched liver metastases is poorly understood.MethodsIn order to compare gene expression patterns and identify fusion genes in these two types of tumors, we performed high-throughput transcriptome sequencing of five sets of quadruple-matched tissues (primary CRC, liver metastases, normal colon, and liver).ResultsThe gene expression patterns in normal colon and liver were successfully distinguished from those in CRCs; however, RNA sequencing revealed that the gene expression between primary CRCs and their matched liver metastases is highly similar. We identified 1895 genes that were differentially expressed in the primary carcinoma and liver metastases, than that in the normal colon tissues. A major proportion of the transcripts, identified by gene expression profiling as significantly enriched in the primary carcinoma and metastases, belonged to gene ontology categories involved in the cell cycle, mitosis, and cell division. Furthermore, we identified gene fusion events in primary carcinoma and metastases, and the fusion transcripts were experimentally confirmed. Among these, a chimeric transcript resulting from the fusion of RNF43 and SUPT4H1 was found to occur frequently in primary colorectal carcinoma. In addition, knockdown of the expression of this RNF43-SUPT4H1 chimeric transcript was found to have a growth-inhibitory effect in colorectal cancer cells.ConclusionsThe present study reports a high concordance of gene expression in the primary carcinoma and liver metastases, and reveals potential new targets, such as fusion genes, against primary and metastatic colorectal carcinoma.

Gastric poorly cohesive carcinoma: a correlative study of mutational signatures and prognostic significance based on histopathological subtypes

Genome‐wide next‐generation sequencing has revealed several driver mutations and has allowed the establishment of a molecular taxonomy of gastric cancer. However, there are few detailed studies on the mutational spectrum of poorly cohesive gastric carcinoma. Thus, this study aim to investigate its mutation profile based on clinicopathological characteristics.

TWIST mediates resistance to paclitaxel by regulating Akt and Bcl-2 expression in gastric cancer cells.

The transcription factor TWIST has been reported to play an important role in tumor progression as well as resistance to anti-cancer drugs. However, the role of TWIST in gastric cancer and the molecular mechanisms by which this protein elicits drug resistance remain poorly understood. We transfected gastric cancer cell lines with lentiviral vector to generate TWIST-overexpressing stable cell lines. Our study showed that overexpression of TWIST not only increased cell migration and invasion but also induced resistance to the anti-cancer drug paclitaxel in gastric cancer. Paclitaxel increased gastric cancer cell death in dose-dependent manner; this was decreased following TWIST overexpression. Furthermore, treatment with paclitaxel decreased Akt phosphorylation and Bcl-2 expression, whereas these effects were suppressed by TWIST overexpression. Treatment of cells with Akt inhibitor or small interfering RNA targeting for Bcl-2 led to increased paclitaxel-induced cell death, indicating that TWIST elicits resistance to paclitaxel via the regulation of the Akt and Bcl-2 pathway. Our results suggest an underlying mechanism for TWIST-mediated paclitaxel resistance and indicate that TWIST represents a potential target for overcoming paclitaxel resistance in gastric cancer cells.

Abstract 3417: Snail and serpinA1 promote tumor progression and predict prognosis in colorectal cancer

Purpose: Snail and serpin peptidase inhibitor clade A member 1 (serpinA1) are involved in tumor progression. In this study, we investigated the role of Snail and serpinA1 in colorectal cancer (CRC) and examined the mechanisms through which these proteins mediate CRC progression. Experimental Design: Tumors from 528 patients with CRC were examined to determine the clinicopathological significance of Snail and serpinA1 expression. Cell migration and invasion were assessed to determine the functional roles of Snail and serpinA1. Snail binding to the serpinA1 promoter was analyzed by chromatin immunoprecipitation (ChIP) assays. Results: Immunohistochemical analysis of 528 samples from patients with CRC showed that elevated expression of Snail or serpinA1 was correlated with advanced stage, lymph node metastasis, and poor prognosis. Moreover, we detected a correlation between Snail and serpinA1 expression. Functional studies performed using the CRC cell lines DLD-1 and SW-480 showed that overexpression of Snail or serpinA1 significantly increased CRC cell invasion and migration. Conversely, knockdown of Snail or serpinA1 expression suppressed CRC cell invasion and migration. ChIP analysis revealed that Snail regulated serpinA1 by binding to its promoter. In addition, fibronectin mediated Snail and serpinA1 signaling and was involved in CRC cell invasion and migration. Conclusion: Taken together, our data showed that Snail and serpinA1 promoted CRC progression through fibronectin. These findings suggested that Snail and serpinA1 were novel prognostic biomarkers and candidate therapeutic targets in CRC. Note: This abstract was not presented at the meeting. Citation Format: Chae Hwa Kwon, Hye Ji Park, Ja Rang Lee, Hye Kyung Kim, Do Youn Park. Snail and serpinA1 promote tumor progression and predict prognosis in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3417. doi:10.1158/1538-7445.AM2015-3417