Seok-Hyung Kim

Inactivation of SMAD4 Tumor Suppressor Gene During Gastric Carcinoma Progression

Purpose: Mothers against decapentaplegic homologue 4 (SMAD4) is a tumor suppressor gene associated with gastrointestinal carcinogenesis. The aim of the present study is to more precisely characterize its role in the development and progression of human gastric carcinoma. Experimental Design: The expression of SMAD4 was investigated in 283 gastric adenocarcinomas and related lesions, as well as in 9 gastric carcinoma cell lines. We also analyzed the methylation status of SMAD4 gene by using methylation-specific PCR, examined loss of heterozygosity (LOH) of this gene locus by using a vicinal marker, and detected exon mutation of SMAD4 through exon-by-exon amplification. Moreover, we assessed whether MG132, a proteasome inhibitor, affected the SMAD4 protein level. Results: We found loss of SMAD4 protein expression in the cytoplasm (36 of 114, 32%) and in the nucleus (46 of 114, 40%) of gastric cancer cells. The loss of nuclear SMAD4 expression in primary tumors correlated significantly with poor survival, and was an independent prognostic marker in multivariate analysis. We also found a substantial decrease in SMAD4 expression at both the RNA and protein level in several human gastric carcinoma cell lines. In addition, we found that LOH (20 of 70, 29%) and promoter hypermethylation (4 of 73, 5%) were associated with the loss of SMAD4 expression. SMAD4 protein levels were also affected in certain gastric carcinoma cell lines following incubation with MG132. Conclusion: Taken together, our results indicate that the loss of SMAD4, especially loss of nuclear SMAD4 expression, is involved in gastric cancer progression. The loss of SMAD4 in gastric carcinomas was due to several mechanisms, including LOH, hypermethylation, and proteasome degradation.

Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients

Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy.

Overexpression of CD24: association with invasiveness in urothelial carcinoma of the bladder.

CONTEXT CD24, originally described as a B-cell marker, has gained considerable attention in tumor research. High rates of CD24 expression have been found in several types of carcinomas that are significantly associated with a more aggressive course of the disease. To our knowledge, the expression of CD24 in urothelial carcinoma (UC) of the bladder has not been previously reported. OBJECTIVE To determine the expression of CD24 in UCs and the association between CD24 levels and tumor grade and stage. DESIGN Urothelial carcinomas (48 cystectomy, 87 transurethral biopsy), including 56 pTa, 29 pT1, 19 pT2, and 31 pT3, were analyzed immunohistochemically using an anti-CD24 monoclonal antibody. The intensity of CD24 staining was semiquantitatively scored as high-level or low-level expression. RESULTS In normal urothelium, CD24 was localized to the cytoplasm of the luminal cell layer with very low intensity. CD24 expression was upregulated in noninvasive UCs, and a high level of expression was correlated with the tumor grade (P = .003). Invasive UCs demonstrated strong diffuse cytoplasmic overexpression of CD24 and the difference in CD24 expression between invasive and noninvasive UC was statistically significant (P < .001). CONCLUSIONS CD24 protein is overexpressed in a significant number of bladder UCs. The high level of CD24 expression with loss of apical localization is a marker for stromal invasion and high tumor grade in UC. This study provides the basis for future investigations of CD24 as a potential serum marker or target of antibody-based therapeutics in bladder UC.

Clinical significance of CD99 down-regulation in gastric adenocarcinoma.

Purpose: CD99 is a cell adhesion molecule associated with human tumors. The aim of the present study was to characterize its role in the development and progression of human gastric adenocarcinoma. Experimental Design: The expression of CD99 was investigated in 283 gastric adenocarcinomas and related lesions and 9 gastric carcinoma cell lines. We also analyzed the methylation status of CD99 gene by using methylation-specific PCR and examined loss of heterozygosity (LOH) of this gene locus by using an intragenic marker. Moreover, we assessed whether SP1, a positive transcription factor for CD99, is expressed in these samples. Results: We found that the decreased expression of CD99 was strongly associated with poor survival and unfavorable clinicopathologic variables. Promoter region methylation (15 of 89, 16.9%) and LOH (21 of 74, 28.4%) were observed and significantly associated with CD99 down-regulation (P < 0.05). In addition, most of the gastric adenocarcinoma cases with CD99 down-regulation had reduced expression of SP1 (47 of 103, 45.6%; P < 0.01). This relationship between CD99 and SP1 was consolidated by using SP1 small interfering RNA transfection experiment and CD99 promoter luciferase assay. Furthermore, we showed that CD99 down-regulation was associated with proliferation and migration in gastric carcinoma cell line. Conclusion: These observations suggest that CD99 down-regulation is a critical event in the progression of gastric adenocarcinoma, and CD99 promoter methylation, CD99 LOH, and SP1 down-regulation were responsible for the down-regulation of CD99.

Gα12 gep oncogene deregulation of p53-responsive microRNAs promotes epithelial-mesenchymal transition of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a poor prognosis owing to aggressive phenotype. Gα12 gep oncogene product couples to G-protein-coupled receptors, whose ligand levels are frequently increased in tumor microenvironments. Here, we report Gα12 overexpression in human HCC and the resultant induction of zinc-finger E-box-binding homeobox 1 (ZEB1) as mediated by microRNA deregulation. Gα12 expression was higher in HCC than surrounding non-tumorous tissue. Transfection of Huh7 cell with an activated mutant of Gα12 (Gα12QL) deregulated microRNA (miRNA or miR)-200b/a/429, -194-2/192 and -194-1/215 clusters in the miRNome. cDNA microarray analyses disclosed the targets affected by Gα12 gene knockout. An integrative network of miRNAs and mRNA changes enabled us to predict ZEB1 as a key molecule governed by Gα12. Decreases of miR-200a/b, -192 and -215 by Gα12 caused ZEB1 induction. The ability of Gα12 to decrease p53 levels, as a result of activating protein-1 (AP-1)/c-Jun-mediated mouse double minute 2 homolog induction, contributed to transcriptional deregulation of the miRNAs. Gα12QL induced ZEB1 and other epithelial–mesenchymal transition markers with fibroblastoid phenotype change. Consistently, transfection with miR-200b, -192 or -215 mimic prevented the ability of Gα12QL to increase tumor cell migration/invasion. In xenograft studies, sustained knockdown of Gα12 decreased the overall growth rate and average volume of tumors derived from SK-Hep1 cell (mesenchymal-typed). In HCC patients, miR-192, -215 and/or -200a were deregulated with microvascular invasion or growth advantage. In the HCC samples with higher Gα12 level, a correlation existed in the comparison of relative changes of Gα12 and ZEB1. In conclusion, Gα12 overexpressed in HCC causes ZEB1 induction by deregulating p53-responsive miRNAs, which may facilitate epithelial–mesenchymal transition and growth of liver tumor. These findings highlight the significance of Gα12 upregulation in liver tumor progression, implicating Gα12 as an attractive therapeutic target.

MHC class II engagement inhibits CD99-induced apoptosis and up-regulation of T cell receptor and MHC molecules in human thymocytes and T cell line.

Major histocompatibility complex (MHC) class II surface levels on thymocytes increase after CD99 ligation. The functional implication of the up‐regulated MHC class II was assessed by engaging MHC class II on CD99‐ligated cells. MHC class II engagement down‐modulated surface levels of T cell receptor and MHC molecules, and inhibited apoptosis of CD99‐ligated thymocytes and CEM tumor cells, antagonistic effects on the previously reported CD99 functions. The results were reproducible regardless of the order of ligation of MHC class II and CD99. We suggest that signaling via MHC class II on CD99‐engaged cells might be involved in the thymic maturation process by damping CD99 ligation effects.

SUMOylation of ZFP282 potentiates its positive effect on estrogen signaling in breast tumorigenesis.

Estrogen receptor α (ERα) has critical roles in the development and progression of breast cancer, and the coiled-coil co-activator (CoCoA) is an important ERα co-activator for estrogen-induced gene expression. The small ubiquitin-like modifier (SUMO) pathway is hyperactivated in breast cancer, but the mechanism by which SUMOylation regulates ERα-mediated transcription remains poorly understood. Here, we identified ZFP282 as a CoCoA-binding protein. ZFP282 associates directly with ERα and cooperates synergistically with CoCoA to enhance ERα function. ZFP282 is required for estrogen-induced expression of ERα target genes and estrogen-dependent breast cancer cell growth and tumorigenesis. In addition, we found that ZFP282 is SUMOylated and that SUMOylation positively regulates the co-activator activity of ZFP282 by increasing its binding affinity to ERα and CoCoA, and consequently increasing recruitment of ZFP282–CoCoA complex to the promoter of ERα target genes. These findings reveal essential roles for ZFP282 and its SUMOylation in estrogen signaling and breast tumorigenesis.

Low expression of transforming growth factor beta-1 in cancer tissue predicts a poor prognosis for patients with stage III rectal cancers.

Objective: Transforming growth factor beta (TGF-β) plays an important role in tumorigenesis and metastasis. It works as a tumor suppressor in the normal colon, but acts as a cancer promoter during the late stages of colorectal carcinogenesis. High expression of TGF-β is known to be associated with advanced stages, tumor recurrence and decreased survival of patients. We investigated the expression of TGF-β and its signaling axis molecules and evaluated their prognostic significance in patients with stage III rectal cancers. Methods: Tissues from 201 cases of stage III rectal cancer were subjected to immunohistochemistry for TGF-β1, type II TGF-β receptor, Smad3, Smad4 and Smad7 proteins. The immunoactivities of these molecules were evaluated and the results were compared with clinicopathological variables including patient survival. Results: Low expression of TGF-β1 protein was correlated with a decreased disease-free survival in univariate Kaplan-Meier (p = 0.003) and multivariate Cox regression (HR 9.188 and 95% CI 1.256-67.198, p = 0.029) analyses. The loss of Smad4 protein expression was associated with a reduction in disease-free survival in the univariate analysis, but this finding was not significant after the multivariate analysis. Conclusion: Low expression of TGF-β1 protein is associated with a poor prognosis for patients with stage III rectal cancers.

Differential expression of Yes-associated protein and phosphorylated Yes-associated protein is correlated with expression of Ki-67 and phospho-ERK in colorectal adenocarcinoma.

Yes-associated protein (YAP) is a transcriptional co-activator and functions as a nuclear downstream effector of the Hippo pathway. Differential expression of YAP and phosphorylated Yes-associated protein (pYAP), which are involved in the expression of Ki-67 and phosphorylated extracellular signal-regulated kinase (pERK) in colorectal adenocarcinoma (CRAC), is not clear. Herein, we hypothesized that nuclear expression of YAP could predict cell proliferation and poor prognosis, while cytoplasmic expression of pYAP would show a reverse correlation with cell proliferation. Paraffin-embedded samples from 144 CRAC patients were studied using immunohistochemistry for YAP, pYAP, Ki-67 and pERK. Frozen samples from 20 CRAC patients were examined for YAP mRNA in tumor and non-tumor tissues, using quantitative real-time PCR. High nuclear YAP expression coincided with high Ki-67 expression (P=0.002). The high nuclear YAP expression group tended to display a poor overall and disease-free survival (P=0.089 and P=0.089, respectively), but YAP mRNA levels in the 20 CRAC tissues were not significantly different in comparison with the 20 non-tumor tissues (P=0.929). We observed an inverse correlation between high cytoplasmic pYAP expression and high Ki-67 expression (P=0.001). Nuclear pERK expression was positively correlated with nuclear YAP expression, but negatively correlated with cytoplasmic pYAP expression (P=0.017 and P=0.020, respectively). Activated nuclear YAP and inactivated cytoplasmic pYAP in CRAC showed a positive correlation with Ki-67 and nuclear pERK expression, suggesting that the expression of YAP and pYAP is a possible predictor of tumor cell proliferation and prognosis in CRAC.

Comparative study of non‐functional islet cell tumors and pancreatic solid and papillary neoplasms: Biological behavior and immunohistochemistry

Although non‐functional islet cell tumor (NFICT) and solid and papillary neoplasm (SPN) share similar clinical and pathological features, the outcome of each is different. Because NFICT often follow a malignant course and SPN are usually benign, the correct differential diagnosis is very important. We investigated the clinical and pathological findings in 10 cases of NFICT and 12 cases of SPN, including immunohistochemical analysis for chromogranin, vimentin, neuron‐specific enolase, somatostatin, alpha‐1‐antitrypsin, estrogen receptor, progesterone receptor, CD99, p21 and Ki‐67. The current study shows that chromogranin is the most valuable marker in differentiating between the tumors (P < 0.01). In contrast to previous reports stating that SPN express the progesterone and/or estrogen receptors, which are absent in other pancreatic tumors, our results show that one‐third of SPN were positive for the progesterone receptor. Downregulation of p21 was found more frequently in NFICT (40%) than SPN (17%). The mean value of the Ki‐67 proliferation index for NFICT (2.77% ± 2.53%) was significantly higher than that for SPN (0.94% ± 0.89%; P = 0.043). These results are consistent with NFICT having more malignant behavior than SPN.