BMP7 Expression in Esophageal Squamous Cell Carcinoma and Its Potential Role in Modulating Metastasis

Background

Our previous study showed that BMP7 revealed significantly higher levels in esophageal squamous cell carcinoma (ESCC) tissues with lymph node metastasis compared with non-lymph node metastasis, using gene expression profiling assays. The roles of BMP7 in ESCC is not fully understood.

Aim

The aim of this study was to investigate the effect of BMP7 on lymph node metastasis of ESCC and to explore its potential mechanism.

Methods

Expression of BMP7 in ESCC tissues was evaluated by immunohistochemistry. BMP7 were down-regulated by RNA interference. The protein and mRNA levels of BMP7 were detected by western blot and RT-PCR, respectively. High content screening and transwell assay were used to identify the metastatic ability of tumor cells.

Results

Positivity of BMP7 staining was 57.5 % in the tissues of primary carcinoma with lymph node metastasis compared to tissues without lymph node metastasis, and expression of BMP7 was significantly higher in the cell lines with highly metastatic capacity than that in the cell lines without metastatic ability. Suppression of endogenous BMP7 expression by siRNA in the highly metastatic cell lines resulted in significant reduction in ability of cell migration and invasion in both in vitro and in vivo studies. In addition, inhibition of BMP7 by siRNA also leads to up-regulation of E-cadherin and down-regulation of MMP-9 in the highly metastatic cell lines.

Conclusions

These findings indicate that BMP7 modulates the expression of E-cadherin and MMP-9, and by which mechanism it may regulate cell migration and metastasis of ESCCs.     

Fusion between tumor cells enhances melanoma metastatic potential

Purpose

Malignant melanoma, characterized by early distant metastasis to the lungs and brain, is a leading cause of mortality related to skin cancer. Cell fusion and the subsequent aneuploidy, commonly observed in melanoma, are associated with poor prognosis. However, the pathological consequences of cell fusion in melanoma remain unknown. Therefore, the present study aims to investigate the pathological consequences of cell fusion in melanoma and the mechanism of melanoma metastasis.

Methods

Phytohemagglutinin–polyethylene glycol (PHA–PEG) fusion method was developed for the fusion of tumor cells. Melanoma cells were fused through the improved PHA–PEG fusion method and obtained by fluorescence-activated cell sorting. DNA content was analyzed through flow cytometry. Cell proliferation rate was detected by cell culture in vitro, and the cell number was counted daily. To detect the tumor growth rate in vivo, cells were injected subcutaneously and the tumor volumes were measured using a vernier caliper. To analyze the tumor metastatic potential, cells were injected intravenously, and the collected lung-metastasis samples were weighed by an electronic balance and the surface nodules were counted.

Results

We established an improved phytohemagglutinin–polyethylene glycol fusion method and successfully obtained stable melanoma tumor–tumor cell fusion hybrids. Cell size, DNA content, and chromosome numbers of the fusion hybrids were approximately twice those of the parents. The metastatic potential of the fusion hybrids was dramatically enhanced, in contrast to their proliferation rate. Their metastasis was specific to the lungs.

Conclusions

We developed a highly efficient cell fusion method that can be applied in many fields, particularly cancer research. Our study has proven that tumor–tumor cell fusion hybrids in melanoma can acquire enhanced and specific metastatic potential. Thus, blockage of cell fusion may be a new strategy for melanoma metastasis therapy.     

S100A6 as a Potential Serum Prognostic Biomarker and Therapeutic Target in Gastric Cancer

Background

Increased expression of S100A6 in many cancer tissues and its association with tumor behavior and patient prognosis were demonstrated, and there are no studies analyzing the serum levels of S100A6 in patients with gastric cancer (GC).

Aim

Serum S100A6 levels were investigated as a marker of tumor aggressiveness in patients with GC, and the S100A6 gene was examined as a potential therapeutic target in GC.

Methods

Serum S100A6 levels were detected in 103 GC patients and 72 healthy subjects by ELISA. Clinicopathological features of GC patients were analyzed in correlation to serum S100A6 levels. Two small interfering RNAs against S100A6 (siRNA1-S100A6 and siRNA2-S100A6) were generated and transfected into SGC7901 cells using pSUPER gfp-neo vector, and the effects of S100A6 knockdown on cell proliferation, invasion and apoptosis were evaluated in vitro. The effects of S100A6 silencing on tumor growth and metastasis were evaluated in vivo in a pseudo-metastatic GC nude mouse model.

Results

Serum S100A6 levels were significantly higher in GC patients than in healthy controls (P < 0.001). Serum S100A6 levels were significantly correlated with lymph node metastasis, TNM stage, perineural invasion and vascular invasion. Serum S100A6 level was an independent predictor of overall survival. SiRNA-mediated silencing of S100A6 significantly induced apoptosis and decreased proliferation, clone formation and the invasiveness of GC SGC7901 cells in vitro and significantly reduced tumor volume and number in vivo (P < 0.01).

Conclusion

Serum S100A6 level may serve as a potential prognostic biomarker in GC. Inhibition of S100A6 decreased the metastatic potential of GC cells.     

Epigenetic silencing of WIF-1 in hepatocellular carcinomas

Purpose

To examine the expression profile and promoter methylation status of WIF-1 in hepatocellular carcinoma (HCC) and identify the possible relationship between the WIF-1 expression pattern and promoter methylation status.

Methods

Quantitative real-time PCR was performed to detect mRNA level of WIF-1 in 4 HCC cell lines, 15 paired HCC clinical samples and 3 normal liver tissues. Methylation-specific PCR and bisulfite DNA sequencing were used in methylation analysis. In vitro assays for HCC cells, colony formation and cell proliferation assay were carried out to observe the effect of WIF-1 on cell growth; TOP-flash luciferase analysis was employed to determine its role in the Wnt pathway.

Results

Quantitative real-time PCR analysis showed the extensive low expression of WIF-1 mRNA in HCC, and this down-regulation was generally dependent on the degree of methylation at its promoter region. In vitro assays indicated WIF-1 can inhibit cell growth by blocking Wnt signaling in HCC cells.

Conclusions

WIF-1 silencing as a result of its promoter hypermethylation may be a frequent event in HCC.     

Epidemiology of Metastatic Hepatocellular Carcinoma,A Nationwide Perspective

Background

Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver.

Aims

The aim of this study was to describe the prevalence, trends, and predictors of metastatic HCC on a national scale.

Methods

We used two nationwide datasets for our study: the University Health Consortium (UHC) and the Nationwide Inpatient Sample (NIS) databases. We included adults with a primary diagnosis of HCC from 2000 to 2011. We collected information regarding demographics, insurance, HCC risk factors, liver decompensation, and the sites and frequencies of metastases. Multivariable regression analysis was used to examine predictors of metastatic HCC. Trend analysis was performed to examine the change in metastatic HCC prevalence over time.

Results

We included 25,671 and 26,054 HCC patients from UHC and NIS, respectively. Prevalence of metastatic HCC was 18 % with lung being the most frequent site (31 %). Compared with Caucasian, African American ethnicity was an independent predictor of metastasis in both the NIS [OR 1.13 (1.02–1.25)] and UHC [OR 1.4 (1.3–1.6)] databases. Lack of long-term insurance was associated with significantly higher prevalence of metastasis in both the NIS [OR 1.6 (1.4–1.9)] and UHC [OR 1.9 (1.6–2.2)] databases. There has been an increased prevalence of metastatic HCC over the last decade with an annual percentage change of +1.25 and +1.60 % (p = 0.03 and p = 0.08) for the NIS and UHC databases, respectively.

Conclusions

Metastasis is not rare among HCC patients and is rising in prevalence over the last decade. Lungs were the most common metastatic site. Ethnicity and insurance status are independent predictors of metastasis.     

Characteristics of aggressive variants in T1a renal cell carcinoma

Purpose

To explore factors associated with metastasis and prognosis in T1a renal cell carcinoma (RCC).

Methods

We retrospectively reviewed 451 cases of sporadic T1aRCC among 1,060 patients admitted to the Department of Urology at Hamamatsu University Hospital and affiliated hospitals between 1978 and 2007. Clinicopathological factors were analyzed for metastatic and prognostic risks.

Results

We identified 32 RCC patients with metastatic disease, 22 with synchronous and 10 with metachronous metastatic RCC. Patients with metastatic disease had a significantly higher incidence of symptomatic cancer, as well as greater tumor size, C-reactive protein (CRP) level, sarcomatoid component ratio, histological grade 3 and microvascular invasion than those without metastasis. Among the 32 patients with metastasis, there is no significant difference in clinicopathological factors. The most common site of metastasis was bone. Among patients with metastatic T1aRCC, findings at diagnosis of a symptomatic cancer, CRP level of 0.4?mg/dL or more, tumor size of 3.0?cm or greater, histological grade 3, a sarcomatoid component and microvascular invasion appeared to be significant and independent risk factors. Significant independent risk factors with metachronous metastatic RCC were a symptomatic cancer and a sarcomatoid component at diagnosis. A CRP level of 0.4?mg/dL or more was also an independent prognostic factor for overall survival.

Conclusion

RCC patients with findings at diagnosis of a symptomatic cancer, a sarcomatoid component and CRP level of 0.4?mg/dL or more require intensive follow-up.     

Fate of metastatic foci after chemotherapy and usefulness of contrast-enhanced intraoperative ultrasonography to detect minute hepatic lesions

Background/purpose

Along with advances in the chemotherapy for colorectal cancer, the strategy for hepatic metastasis has been changed. One of the most striking issues is that initially unresectable hepatic metastases can be resectable after chemotherapy with considerably high frequency. In addition, advanced chemotherapy leads to the downsizing of the metastatic foci in the liver, which is sometimes difficult to detect with conventional intraoperative ultrasonography (IOUS). To discover the undetectable hepatic lesions with IOUS, we have introduced contrast-enhanced intraoperative ultrasonography (CEIOUS). In the present study, we present evidence that viable cancer cells exist in even the shrunken tumors with high frequency and that CEIOUS contributes to detecting the minute foci.

Methods

This study was composed of eight patients; four of them had initially unresectable metastasis, and the remaining four had either H2 or H3 status of hepatic metastases. All of them underwent hepatic resection after chemotherapy.

Results

A total of 57 metastatic lesions were detected before chemotherapy. Thirty lesions were demonstrated by CEIOUS with perflubutane and resected. In the pathological examination, tumor cells were not found in 12 of the 30 resected lesions. The degree of pathological liver damage was grade 1 or less in all patients, and no serious complication occurred after surgery in any of the patients.

Conclusion

The present study showed that viable cancer cells remained with high frequency, even in the minute hepatic metastasis which was reduced in size after chemotherapy, and CEIOUS was a useful examination for detecting the minute hepatic foci.     

MYO5B Is Epigenetically Silenced and Associated with MET Signaling in Human Gastric Cancer

Background

Our previous study has shown that MYO5B is downregulated in gastric cancer. However, the mechanism by which the expression of MYO5B was inhibited remains unknown.

Methods

Inspection of the human MYO5B locus uncovered a large and dense CpG island within the 5′ region of this gene. Methylation-specific PCR (MSP) and bisulfite sequencing (BSP) were used for determination of MYO5B promoter methylation in gastric cancer cell lines and gastric cancer samples. Involvement of histone H3 methylation in those cell lines were examined by ChIP assay.

Results

The densely methylated MYO5B promoter region was confirmed by MSP and BSP. Enhanced gene expression was detected when the cells were treated with the DNA-demethylating agent 5-aza-2′-deoxycytidine (DAC) and trichostatin A (TSA), a histone deacetylase inhibitor. Knockdown of MYO5B expression in gastric cancer cells expressing endogenous MYO5B inhibits HGF-stimulated MET degradation, concomitant with sustained c-MET levels and signaling.

Conclusion

The results of our study showed for the first time that MYO5B is epigenetically silenced in gastric cancer cells by aberrant DNA methylation and histone modification. Inactivation of MYO5B expression in gastric cancer cells expressing endogenous MYO5B inhibits HGF-stimulated MET degradation, concomitant with sustained c-MET levels and signaling.     

Cytoplasmic hepatocyte growth factor immunoreactivity predicting lymph node metastasis in esophageal squamous cell carcinoma

Background

Esophageal squamous cell carcinoma (ESCC) is a highly malignant disease because of its aggressive biological behavior and metastatic potential. Although several molecular markers have been identified as prognostic factors and/or clinicopathological correlation for the patients of ESCC, it has not been established as the effective new treatment for ESCC patients yet. Hepatocyte growth factor (HGF) is a biomarker as known to promote cell proliferation, motility, and invasion in various human malignancies. But the correlation with cytoplasmic HGF and ESCC has been unclear in details.

Methods

We studied the correlation of HGF expression in ESCC tumor cells from 83 ESCC patients who were operated in our hospital using immunohistochemistry.

Results

High cytoplasmic HGF expression was detected in 50.6 % (42/83) of ESCC cases examined. HGF immunoreactivity was also focally detected in some cancer-associated fibroblasts. High HGF expression group was significantly correlated with lymph node metastasis (P = 0.029) and tumor differentiation (P = 0.018). Increased HGF immunoreactivity was also correlated with clinicopathological features associated with invasiveness of carcinoma cells (lymphatic invasion, venous invasion, distant metastasis, and pathological stage) but not with actual clinical outcome of the patients.

Conclusions

Results of our present study indicated that HGF cytoplasmic expression in ESCC was associated with increased potential for lymph node metastasis and carcinoma differentiation. These findings indicated that endogenous HGF did play an important role in progression and invasion of ESCC via c-Met/HGF autocrine loop together with HGF paracrine mechanisms.     

Clinical Impact of HAb18G/CD147 Expression in Esophageal Squamous Cell Carcinoma

Background

HAb18G/CD147 expression has been associated with many tumor invasion molecules, which play important roles in recurrence and poor differentiation of esophageal squamous cell carcinoma (ESCC). However, the clinical implications of HAb18G/CD147 in ESCC are still unclear.

Aims

In this study, we clarified the clinical significance of HAb18G/CD147 and characterized the association between HAb18G/CD147 and tumor invasion in ESCC cases.

Methods

Tumor tissues were obtained from 86 ESCC patients who underwent surgical resection between 2002 and 2005. All patients that had received previous therapy were excluded. ESCC tissues were analyzed by IHC using anti HAb18G/CD147 antibody. The expression of HAb18G/CD147 mRNA in esophageal cancer cell lines was analyzed by RT?CPCR.

Results

HAb18G/CD147 was uniformly expressed in EC109 and EC871214 cell lines, but negatively expressed in EPC2, esophageal normal squamous cell line. HAb18G/CD147 mainly localized to the membrane of tumor cells in 84.9% of ESCC patients (64 out of 86 cases). Furthermore, we also found that higher HAb18G/CD147 expression levels significantly correlated with lymph node metastasis, depth of tumor invasion and differentiation (P < 0.05). But the expression levels of HAb18G/CD147 in lymph node metastatic tissues were almost equal to that in the primary tumor tissues. Furthermore, lymph node metastasis and expression of HAB18G/CD147 were independent prognostic indicators in ESCC.

Conclusions

The expression of HAb18G/CD147 might be involved in the progression and survival of ESCC. Therefore, HAb18G/CD147 could be a clinical marker for the poor prognosis in ESCC patients and may also be a potentially therapeutic target to improve the progression of ESCC.     

Nanog: A Potential Biomarker for Liver Metastasis of Colorectal Cancer

Background

At present, the relationship between Nanog expression and the biological behavior and prognosis of colorectal cancer is still unclear.

Aim

The purpose of this study was to evaluate the expression and regulatory effects of Nanog in colorectal cancer and the correlation between Nanog protein expression and the prognosis of patients with colorectal cancer.

Materials and Methods

The differential expression of genes between CD133+ tumor cells and CD133? tumor cells were detected using RT² Profiler? PCR Array. The Nanog mRNA expression level was detected by RT-PCR and the protein level was detected using immunohistochemistry staining. The relationship between Nanog expression and clinicopathological parameters of colorectal cancer was determined.

Results

Nanog were expressed significantly higher in CD133+ tumor cells compared to CD133? tumor cells. It was observed that 72 (20.00?%) of the 360 cases positively expressed Nanog. Univariate analyses indicated that Nanog expression was related to histological grade, lymph node metastasis, TNM stage, and liver metastasis (P?=?0.005, 0.001, 0.001 and 0.012, respectively). Spearman correlation analysis showed that Nanog expression has a linear correlation to liver metastasis (P?=?0.001). After conducting multivariate analysis, histological grade, TNM stage, and Nanog were found to be related to liver metastasis (P?=?0.020, 0.01 and 0.001, respectively). In the Cox regression test, the histological grade, Lymph node metastasis, TNM stage, liver metastasis, and Nanog were detected as the independent prognostic factors (P?=?0.02, 0.045, 0.01, 0.001 and 0.001, respectively).

Conclusions

Nanog protein may be a potential biomarker for postoperative liver metastasis of colorectal cancer.     

Aggressive surgery for stage IV gallbladder carcinoma; what are the contraindications?

Background/Purpose

Advanced gallbladder carcinoma with paraaortic lymph node metastasis or distant metastasis is normally considered a contraindication for surgery. Our latest analyses suggest otherwise.

Methods

Records of 166 patients who underwent surgery for stage IV gallbladder carcinoma were reviewed retrospectively. Predictors of hospital mortality and long-term survival were analyzed. Long-term survival in patients with paraaortic lymph node metastasis and/or distant metastasis was also determined.

Results

Fifteen patients were 5-year survivors, with a 5-year survival rate of 12% among the 166 patients investigated. Overall hospital mortality was 14%. Male sex and portal vein resection were independent predictors of hospital mortality. Multivariate analysis of long-term survival failed to identify independent predictors. Patients with distant metastasis were divided into two groups based on whether or not the metastases were distant from the liver. Patients with paraaortic lymph node metastasis who underwent curative resection or who had isolated liver metastasis survived longer than those with other distant metastasis or those with unresectable advanced cancer.

Conclusions

Patients with advanced gallbladder carcinoma can benefit from surgical resection even when paraaortic lymph node metastasis and/or liver metastasis are present. However, surgical indications in advanced disease should be determined on an individual basis, based on clinical status.     

Identification of two new HLA-A*0201-restricted cytotoxic T lymphocyte epitopes from colorectal carcinoma-associated antigen PLAC1/CP1

Background

To explore the potential application of placenta-specific PLAC1/Cancer Placenta (CP) 1 antigen for immunotherapy in CRC patients, further identification of the cytotoxic T lymphocyte epitopes from this antigen is necessary.

Methods

We assessed the protein expression of PLAC1/CP1 using a tissue chip and immunochemistry staining in CRC samples. Simultaneously, we predicted four PLAC1/CP1-derived HLA-A*0201-restricted peptides by using reverse immunology methods. Peptide-specific CD8⁺ T cell responses were assessed by an IFN-γ release ELISPOT assay. Effector CD8⁺ T cells lyse HLA-A*0201 CRC cell line SW620 was detected in a granzyme-B release ELISPOT cytotoxicity assay.

Results

Our results indicated that PLAC1/CP1 was highly expressed in 56.7 % (55/97) of adenocarcinomas. PLAC1/CP1 protein expression was associated with CRC tumor differentiation, the tumor/node/metastasis stage, and lymph node metastasis. Two of four peptides showed high affinities in an HLA-A2 binding assay. In 66.7 % (6/9) of peripheral blood mononuclear cells of CRC samples with PLAC1/CP1 protein-positive expression, these two peptides, PLAC1/CP1 p41-50 (FMLNNDVCV) and PLAC1/CP1 p69-77 (HAYQFTYRV), were immunogenic in the induction of peptide-specific CD8⁺ T cell responses as assessed by an IFN-γ release ELISPOT assay. Furthermore, the generated effector CD8⁺ T cells could specifically lyse the PLAC1/CP1 HLA-A*0201 CRC cell line SW620 in a granzyme-B release ELISPOT cytotoxicity assay.

Conclusions

These results show that the PLAC1/CP1 antigen is a possible prognostic marker of CRC and that PLAC1/CP1 p41-50 and PLAC1/CP1 p69-77 are novel HLA-A*0201-restricted CD8⁺ T cell epitopes and potential targets for peptide-based immunotherapy in CRC patients.     

Should lymph nodes along the thoracic duct be dissected routinely in radical esophagectomy?

Background

Although dissection of mediastinal lymph nodes along the thoracic duct is included in standard radical esophagectomy, it is not routinely performed because of the undesirable hemodynamic effects. This study aims to investigate whether dissection of the nodes along the thoracic duct has prognostic benefits.

Methods

A total of 778 consecutive patients who underwent radical esophagectomy with three-field lymph node dissection for squamous cell carcinoma of the thoracic esophagus from 1984 to 2011 were included. The incidence of metastasis in thoracic duct nodes and that in nodes within #112 station excluding thoracic duct nodes were studied in relation to the depth of the main tumor. The survival curves of lymph node-positive patients were compared.

Results

The metastatic incidence was 2.2 % in T1b/T2, whereas it was 10.0 % in T3/T4. The survival curves in patients with metastasis in the thoracic duct nodes and in the #112 station were not statistically different.

Conclusion

The dissection of the nodes along the thoracic duct along with thoracic duct resection should be performed routinely; however, reliable indicator of the necessity of its dissection is awaited in T1b/T2 tumors because of the low metastatic rate and the potential risk associated with resection of the thoracic duct.     

DNA Methylation of NDRG2 in Gastric Cancer and Its Clinical Significance

Background

Gastric cancer is one of the most common digestive malignancies worldwide. N-myc downstream-regulated gene 2 (NDRG2) is a differentiation-related gene that is considered to be a metastasis suppressor gene. In this study, we examined the expression and DNA methylation of NDRG2 in gastric cancer cell lines and tissues, as well as its clinical significance.

Methods

Six gastric cancer cell lines and 42 paired normal and gastric cancer tissue samples were used to assess NDRG2 mRNA expression using RT-PCR. NDRG2 DNA methylation status was evaluated by methylation-specific PCR (MSP) in gastric cancer cell lines and tissues. The suppression of NDRG2 in BGC823 cells by siRNA transfection was utilized to detect the role of NDRG2 in gastric cancer progression.

Results

NDRG2 mRNA was down-regulated in gastric cancer cell lines and tissues, and its expression was just related to lymph node metastasis (p = 0.032). MSP showed methylation of NDRG2 in 54.0 % (47/87) of primary gastric cancer specimens and in 20.0 % (16/80) of corresponding nonmalignant gastric tissues. NDRG2 methylation was related to depth of tumor invasion, Borrmann classification and TNM stage (p < 0.05). Upon treatment with 5-aza-2′-deoxycytidine and trichostatin A, NDRG2 expression was upregulated in HGC27 cells, and demethylation of the highly metastatic cell line, MKN45, inhibited cell invasion. Furthermore, the suppression of NDRG2 by siRNA transfection enhanced BGC823 cells invasion.

Conclusions

Our results suggest that the aberrant methylation of NDRG2 may be mainly responsible for its downregulation in gastric cancer, and may play an important role in the metastasis of gastric cancer.     

Hepatic steatosis is associated with lower incidence of liver metastasis from colorectal cancer

Purpose

Both hepatic steatosis (HS) and colorectal cancer (CRC) are conditions associated with metabolic syndrome. The liver is the most frequent site of distant metastasis of CRC; however, the impact of HS on the incidence of liver metastasis of CRC is not clearly defined. Then, the correlation with the presence or absence of HS was analyzed.

Methods

A total of 604 CRC patients receiving curative surgical resection who had a preoperative non-enhanced computed tomography (CT) were enrolled. The mean attenuation values (in Hounsfield units) of the liver and spleen were obtained on a plain CT slice, and the patients with liver–spleen attenuation ratio lower than 1.1 were objectively defined as HS. The clinicopathological features of these patients were analyzed, and the association between HS and the clinical features of CRC was examined.

Results

Sixty-three (10.4 %) among the 604 patients were diagnosed as HS. Recurrence-free survival (RFS) and hepatic RFS, but not extrahepatic RFS, were significantly higher in the group with HS (p?=?0.04 and p?=?0.006). However, this effect was not evident in the group of patients with obesity, defined as body mass index?>?25.0. Among the stage I~III cases, HS was significantly associated with lower hepatic, but not extrahepatic, RFS. Moreover, absence of HS was an independent risk factor for hepatic RFS (p?=?0.003).

Conclusion

Metastases of CRC are less frequent in fatty liver. Steatosis may be an unfavorable microenvironment for metastatic formation in the liver.