Significance and prognostic value of Gli-1 and Snail/E-cadherin expression in progressive gastric cancer.

Abnormal activation of the hedgehog (Hh) signaling pathway has been found to be involved in the occurrence, invasion, and metastasis of cancers. Epithelial-mesenchymal transition (EMT) also plays an important role in the invasion and metastasis of cancers. However, the significance of the Hh signaling pathway and EMT in the invasion and metastasis of gastric cancer is still unclear. This study aimed to investigate the significance and prognostic value of the Hh signaling pathway and EMT in progressive gastric cancer. Immunohistochemistry was performed to detect the expression of the Hh-induced transcriptional factor Gli-1 and the EMT-related molecules Snail and E-cadherin in 121 patients with progressive gastric cancer. Histological type, depth of invasion, lymph node metastasis, and pTNM stage were also recorded. In progressive gastric cancer, Gli-1 expression increased markedly, and was closely associated with increased Snail expression and decreased E-cadherin expression. Diffuse type cancer, lymph node metastasis, and abnormal expression of E-cadherin were independent factors influencing the prognosis of patients with progressive gastric cancer. These findings suggest that abnormal activation of the Hh signaling pathway is closely related to the presence of EMT and is an important factor influencing the prognosis of patients with diffuse progressive gastric cancer.

Downregulation of metallothionein 1F, a putative oncosuppressor, by loss of heterozygosity in colon cancer tissue.

PURPOSE: Downregulation of metallothionein (MT) genes has been reported in several tumors with discrepant results. This study is to investigate molecular mechanism of MT gene regulation in colon cancer which is characterized by tumor suppressor gene alterations. EXPERIMENTAL DESIGN: Integral analysis of microarray data with loss of heterozygosity (LOH) information was employed. Quantitative real-time PCR and immunohistochemistry were used to validate MT isoform expression in colon cancer tissues and cell lines. The effects of MT1F expression on RKO cell survival and tumorigenesis was analyzed. Bisulphite sequencing PCR (BSP) and methylation-specific PCR were employed to detect the methylation status of the MT1F gene in colon cancer tissues and cell lines. DNA sequencing was used to examine the LOH at the MT1F locus. RESULTS: MT1F, MT1G, MT1X, and MT2A gene expression was significantly downregulated in colon cancer tissue (p<0.05). Exogenous MT1F expression increased RKO cell apoptosis and inhibited RKO cell migration, invasion and adhesion as well as in vivo tumorigenicity. Downregulation of MT1F gene in majority of human colon tumor tissues is mainly through mechanism by loss of heterozygosity (p=0.001) while CpG island methylation of MT1F gene promoter region was only observed in poorly differentiated, MSI-positive RKO and LoVo colon cancer cell lines. CONCLUSIONS: MT1F is a putative tumor suppressor gene in colon carcinogenesis that is downregulated mainly by LOH in colon cancer tissue. Further studies are required to elucidate a possible role for MT1F downregulation in colon cancer initiation and/or progression.

microRNA-148a suppresses human gastric cancer cell metastasis by reversing epithelial-to-mesenchymal transition.

MicroRNAs (miRNAs) are important regulators of gastric cancer development and progression. miR-148a is one of the most frequently and highly downregulated miRNAs in gastric cancer and is associated with advanced clinical stage and poor prognosis. In this study, we investigated the role of miR-148a in gastric cancer metastasis. Levels of miR-148a were determined by qRT-PCR in 60 gastric cancer samples. Cell migration and invasion assays were performed in a stably expressing miRNA-148a gastric cancer cell line established using a lentivirus expression system. Epithelial-mesenchymal transition (EMT) was evaluated using qRT-PCR and Western Blots to detect epithelial marker E-cadherin and mesenchymal marker, vimentin. Luciferase reporter assays were used to identify downstream targets and biological function of miR-148a. Gastric cancer tissue had significantly lower expression of miR-148a compared to non-tumor tissue. Low miR-148a levels were associated with lymph node metastasis, N stage, and blood vessel invasion. miR-148a overexpression inhibited metastasis of gastric cancer cells. miR-148a overexpression also downregulated vimentin expression and upregulated E-cadherin expression, suggesting that miR-148a inhibited EMT. Finally, the SMAD2 gene was identified as the direct and functional target of miR-148a. MiR-148a suppresses gastric cancer metastasis and EMT, likely via SMAD2. Restoration of miR-148a expression could have important implications in gastric cancer therapy.

Identification and validation of Kallikrein-ralated peptidase 11 as a novel prognostic marker of gastric cancer based on immunohistochemistry.

BACKGROUND AND OBJECTIVES: It is important to identify and validate the differentially expressed genes in gastric cancer to screen diagnostic and/or prognostic tumor markers. METHODS: cDNA expression microarray, gene set enrichment analysis, and bioinformatics approaches were used to screen the differentially expressed genes between gastric cancer tissues and adjacent non-cancerous mucosa. A novel candidate prognostic marker, Kallikrein-related peptidase 11 (KLK11), was validated in 400 Chinese gastric cancer patients. KLK11 expression in gastric cancer tissues was detected using real-time PCR and Western blot. KLK11 protein expression was further analyzed by immunostaining on tissue microarray, followed with clinicopathological significance and survival analysis. RESULTS: KLK11 expression was significantly decreased in gastric cancer compared with that in normal gastric mucosa (P<0.001). Furthermore, KLK11 expression was much lower in poorly differentiated cancer samples than that in well-differentiated group (P<0.01). Survival analysis showed that negative KLK11 expression was associated with nearly fivefold increased risk of distant metastasis after curative gastrectomy (HR 4.65, P<0.01). Multivariate Cox regression analysis showed that KLK11 expression emerged as a significant independent prognostic factor for disease-free survival and overall survival (P<0.05). CONCLUSIONS: The results indicated that KLK11 expression was decreased in gastric cancer and might serve as a novel independent prognostic marker.

Are there tumor suppressor genes on chromosome 4p in sporadic colorectal carcinoma?

AIM: To study the candidate tumor suppressor genes (TSG) on chromosome 4p by detecting the high frequency of loss of heterozygosity (LOH) in sporadic colorectal carcinoma in Chinese patients. METHODS: Seven fluorescent labeled polymorphic microsatellite markers were analyzed in 83 cases of colorectal carcinoma and matched normal tissue DNA by PCR. PCR products were electrophoresed on an ABI 377 DNA sequencer. Genescan 3.7 and Genotype 3.7 software were used for LOH scanning and analysis. The same procedure was performed by the other six microsatellite markers spanning D4S3013 locus to make further detailed deletion mapping. Comparison between LOH frequency and clinicopathological factors was performed by c2 test. RESULTS: Data were collected from all informative loci. The average LOH frequency on 4p was 24.25%, and 42.3% and 35.62% on D4S405 and D4S3013 locus, respectively. Adjacent markers of D4S3013 displayed a low LOH frequency (< 30%) by detailed deletion mapping. Significant opposite difference was observed between LOH frequency and tumor diameter on D4S412 and D4S1546 locus (0% vs 16.67%, P = 0.041; 54.55% vs 11.11%, P = 0.034, respectively). On D4S403 locus, LOH was significantly associated with tumor gross pattern (11.11%, 0, 33.33%, P = 0.030). No relationship was detected on other loci compared with clinicopathological features. CONCLUSION: By deletion mapping, two obvious high frequency LOH regions spanning D4S3013 (4p15.2) and D4S405 (4p14) locus are detected. Candidate TSG, which is involved in carcinogenesis and progression of sporadic colorectal carcinoma on chromosome 4p, may be located between D4S3017 and D4S2933 (about 1.7 cm).

Refined mapping of loss of heterozygosity on 1q31.1-32.1 in sporadic colorectal carcinoma.

AIM: To explore precise deleted regions and screen the candidate tumor suppressor genes related to sporadic colorectal carcinoma. METHODS: Six markers on 1q31.1-32.1 were chosen. These polymorphic microsatellite markers in 83 colorectal cancer patients tumor and normal DNA were analyzed via PCR. PCR products were electrophoresed on an ABI 377 DNA sequencer. Genescan 3.1 and Genotype 2.1 software were used for Loss of heterozygosity (LOH) scanning and analysis. Comparison between LOH frequency and clinicopathological factors was performed by c2 test. RESULTS: 1q31.1-32.1 exhibited higher LOH frequency in colorectal carcinoma. The average LOH frequency of 1q31.1-32.1 was 23.0%, with the highest frequency of 36.7% (18/49) at D1S2622, and the lowest of 16.4% (11/67) at D1S412, respectively. A minimal region of frequent deletion was located within a 2 cM genomic segment at D1S413-D1S2622 (1q31.3-32.1). There was no significant association between LOH of each marker on 1q31.1-32.1 and the clinicopathological data (patient sex, age, tumor size, growth pattern or Dukes stage), which indicated that on 1q31.1-32.1, LOH was a common phenomenon in all kinds of sporadic colorectal carcinoma. CONCLUSION: Through our refined deletion mapping, the critical and precise deleted region was located within 2 cM chromosomal segment encompassing 2 loci (D1S413, D1S2622). No significant association was found between LOH and clinicopathologic features in 1q31.1-32.1.

[Refined mapping of loss of heterozygosity of 1p36.33-36.31 in sporadic colorectal carcinoma].

OBJECTIVE: To screen the candidate tumor suppressor gene (TSG) related to colorectal cancer (CRC). METHODS: Seven fluorescent labeled polymorphic markers (1p36.33-36.31) were analyzed in the samples of tissues of CRC and adjacent normal tissues obtained during operation performed on 83 CRC patients, 40 males and 43 females, aged 31 - 84. The PCR products were electrophoresed. The softwares Genescan 3.1 and Genotype 2.1 were used for loss of heterozygosity (LOH) scanning and analysis. Comparison between the LOH frequency and the clinicopathological factors was performed by Chi-square test. RESULTS: The average LOH frequency in the 1p36.33-36.31 was 31.47%, with the highest LOH frequency of 47.22% at the D1S243 locus and the lowest LOH frequency of 7.35% at the locus D1S1347. Two obvious high LOH regions were detected: D1S1347 locus (1p36.33, about 1 cM) and a region between D1S468 and D1S2660 (1p36.32-36.31 about 3 cM). The LOH rats of the loci in 1p36.33-36.31 region were not correlated with the age and sex of the patient, and the size, growth pattern, and Dukes stage of the tumor. CONCLUSION: Two obvious high frequency LOH regions, 1p36.32-36.31 and 1p36.33 have been detected in sporadic CRC where tumor-suppressor-gene (s) may exist.

Detailed deletion mapping of loss of heterozygosity on 22q13 in sporadic colorectal cancer.

AIM: Both development and progression of malignancies occur as a multistep process, requiring the activation of oncogenes and the inactivation of several tumor suppressor genes. The loss of heterozygosity (LOH) of tumor suppressor genes is believed to play a key role in carcinogenesis of colorectal cancer (CRC). In this study, we analyzed the LOH of seven loci on chromosome 22q13 in an effort to identify candidate tumor suppressor genes involved in colorectal carcinogenesis. METHODS: Matched tumor and normal tissue DNA were analyzed by PCR using fluorescence-labeled polymorphic microsatellite markers in 83 CRC patients. PCR products were eletrophoresed and LOH was determined by calculating the peak height acquired through computer software. Comparisons between LOH frequency and clinicopathological features were performed by chi2 test. P<0.05 was considered as statistical significance. RESULTS: The average LOH frequency of chromosome 22q13 was 28.38%. The highest LOH frequency was 64.71% on D22S1160 locus, and the lowest was 21.43% on D22S1141 locus. We detected two obvious minimal deletion regions: one between markers D22S1171 and D22S274, the other flanked by markers D22S1160 and D22S1149, each about 2.7 and 1.8 cm, respectively. None had lost in all informative loci. LOH frequency on D22S1171 is 50% on distal colon, which was higher than that on proximal one (P = 0.020); on D22S114 locus, none LOH event occurred in patients with liver metastasis, whilst 46.94% occurred in patients without liver metastasis (P = 0.008); on D22S1160 locus, LOH frequency in lymph nodes metastasis patients was 83.33%, which was much higher than 43.75% without lymph nodes metastasis ones (P = 0.016). There was no statistical significance between clinicopathological features and other loci. CONCLUSION: This study provides evidence of two minimal deletion regions, which may harbor putative tumor suppressor genes related to progression and metastasis in sporadic colorectal carcinoma on chromosome 22q13.

[Refined mapping of loss of heterzygosity on 10q23-24 in sporadic colorectal carcinoma].

OBJECTIVE: Loss of heterozygosity (LOH) of tumor suppressor gene (TSG) is believed to play a key role in carcinogenesis of colorectal cancer (CRC). In this study, we performed refined mapping of LOH on 10q23-24 region and screened the candidate TSG related to sporadic CRC. METHODS: Seven fluorescent labeled polymorphic markers (encompassing D10S185 locus) were analyzed in 83 cases of colorectal carcinoma and normal tissues by PCR. PCR products were eletrophoresed on an ABI 377 DNA sequencer. GeneScan 3.1 and Genotyper 2.1 softwares were used for LOH scanning and analysis. Comparison between LOH frequency and clinicopathological factors were performed by Chi-square test. RESULTS: The average LOH frequency was 36.11%. The highest frequency of LOH (D10S583 locus) and the lowest (D10S205 locus) were 54.84% and 21.3%, respectively. Two obviously high LOH regions were detected: one between D10S583 locus and D10S185 locus (about 0.9 cm, 10q23.33); another flanked by D10S1709 and D10S1265 locus (about 1.5 cm, 10q24.2-24.31). Furthermore, significant difference was observed between LOH frequency and Dukes stages only on D10S1265 locus. CONCLUSION: Two obviously high frequency LOH regions, 10q23.33 and 10q24.2-24.31, were detected in sporadic CRC. Besides PTEN gene, the above two regions may harbor candidate TSG involved in development and progression of sporadic CRC.

Overexpression of HOXA10 promotes gastric cancer cells proliferation and HOXA10(+)/CD44(+) is potential prognostic biomarker for gastric cancer.

Gastric cancer (GC) is a malignant cancer with poor prognosis. This study aims to investigate the roles of homeobox A10 (HOXA10) in GC and the correlations between HOXA10/CD44 expression and GC prognosis. Based on qRT-PCR and Western Blot analyses in 50 pairs of fresh GC samples and adjacent normal samples, it is identified that HOXA10 was significantly up-regulated in GC tissues at mRNA and protein levels. Cell proliferation, migration, and invasion were enhanced in GC cells with overexpressed HOXA10, while inhibited in cells with silenced HOXA10. Through IPA software, HOXA10 was predicted to interact with CD44 via MSN, which was preliminarily confirmed by using Western Blot. Through immunohistochemistry and tissue microarray (N=264), it is found that HOXA10 expression was significantly correlated with tumor size (P=0.011) and CD44 expression (P<0.001), while CD44 expression was significantly correlated with tumor size (P<0.001), depth of tumor invasion (P<0.001), lymph node metastasis (P<0.001), distant metastasis (P=0.001), UICC stage (P<0.001), histological differentiation (P<0.001), and HOXA10 expression (P<0.001). Additionally, the over-all survival and disease-free survival of HOXA10(+)/CD44(+) patients were dramatically decreased in comparison with that of HOXA10(+)/CD44(-), HOXA10(-)/CD44(+), or HOXA10(-)/CD44(-) patients (P<0.001), suggesting that the combinatory expression of HOXA10 and CD44 was correlated with poor GC prognosis. In conclusion, HOXA10 and CD44 might play roles in GC tumorigenesis, metastasis, and invasion. HOXA10(+)/CD44(+) expression might serve as a prognostic biomarker for GC, which needs more studies to validate.

Loss of heterozygosity on chromosome 1 in sporadic colorectal carcinoma.

AIM: Loss of heterozygosity (LOH) on tumor suppressor genes is believed to play a key role in carcinogenesis of colorectal cancer. When it occurs at a tumor suppressor gene locus with abnormal allele, neoplastic transformation happens. In this study, we analyzed the LOH at 21 loci on chromosome 1 in sporadic colorectal cancer to identify additional loci involved in colorectal tumorigenesis. METHODS: Twenty-one polymorphic micro-satellite DNA markers were analyzed with PCR both in 83 cases of colorectal cancer and in normal tissues. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.1 and Genotype 2.1 software were used for LOH scanning and analysis. chi2 test was used to compare LOH frequency with clinicopathological data. P<0.05 was considered as statistically significant. RESULTS: The average LOH frequency of chromosome 1, short arm and long arm was 19.83%, 18.00% and 21.66%, respectively. The 2 highest LOH loci with a frequency of 36.54% and 32.50% were identified on D1S468 (1p36.33-p36.31) and D1S413 (1q31.3), respectively. On D1S2726 locus, LOH frequency of rectal cancer was 28.57% (6/21), which was higher than that of colon cancer (0.00%, 0/33) (P=0.002), suggesting that the mechanism of carcinogenesis was different in both groups. CONCLUSION: Putative tumor suppressor genes on chromosome 1 may relate to sporadic colorectal carcinomas. Tumor-suppressor-genes might locate on 1p36.33-36.31 and/or 1q31.3.

Expression of telomerase activity and oxidative stress in human hepatocellular carcinoma with cirrhosis.

AIM: To study the expression and significance of telomerase activity and oxidative stress in hepatocellular carcinoma (HCC) with cirrhosis. METHODS: In this study, TRAP-ELISA assay was used to determine telomerase activity in 21 cases of HCC as well as in 23 cases of hepatic cirrhosis. Malondialdehyde (MDA), glutathione S-transferase (GST) and total anti-oxidative capacity (T-AOC) were also examined in the same samples with human MDA, GST and T-AOC kits. RESULTS: Eighteen of 21 cases of HCC were found to have increased telomerase activity, whereas only three of the 23 non-cancerous cirrhotic samples were found to have weak telomerase activity, and the difference was significant (P<0.001). No significant difference in telomerase activity was detected according to different tumor size, tumor stage, histological grade, HBsAg, contents of albumin, bilirubin, ALT, AFP, r-GT and platelet. There were significant differences between HCC and cirrhosis in the expression of MDA, GST and T-AOC respectively. Telomerase activity correlated positively with the content of MDA (P<0.05). CONCLUSION: Telomerase activation is the early event of carcinogenesis, which is not correlated with clinicopathological factors of HCC. The dysfunction of the anti-oxidative system is closely correlated with the progression from cirrhosis to hepatocellular carcinoma. Oxidative stress may contribute partly to telomerase activation.

Loss of heterozygosity on long arm of chromosome 22 in sporadic colorectal carcinoma.

AIM: The loss of heterozygosity (LOH) on tumor suppressor genes is believed to play a key role in carcinogenesis of colorectal cancer. In this study, we analyzed the LOH at 5 loci on the long arm of chromosome 22 in sporadic colorectal cancer to identify additional loci involved in colorectal tumorigenesis. METHODS: Five polymorphic microsatellite markers were analyzed in 83 cases of colorectal and normal DNA by PCR. PCR products were eletrophoresed on an ABI 377 DNA sequencer; Genescan 3.1 and Genotype 2.1 software were used for LOH scanning and analysis. Comparison between LOH frequency and clinicopathological data were performed by chi(2) test. P<0.05 was considered as statistically significant. RESULTS: The average LOH frequency on chromosome 22q was 28.38 %. The region between markers D22S280 and D22S274 (22q12.2-q13.33) exhibited relatively high LOH frequency. The two highest LOH loci with frequencies of 35.09 % and 34.04 % was identified on D22S280 (22q12.2-12.3) and D22S274 (22q13.32-13.33). 8 cases showed LOH at all informative loci, suggesting that one chromosome 22q had been completely lost. On D22S274 locus, LOH frequency of rectal cancer was 50 % (9/18), which was higher than that of proximal colon cancer (12 %, 2/17) (P=0.018). The frequency of distal colon cancer was 42 % (5/12), also higher than that of proximal colon cancer. But there was no statistical significance. Putting both the tumors in distal colon and rectum together into consideration, the frequency, 47 % (14/30), was higher than that of proximal colon cancer (P=0.015),suggesting the mechanism of carcinogenisis was different in both groups. CONCLUSIONS: This study provided evidence for the involvement of putative tumor suppressor genes related to the sporadic colorectal carcinoma on chromosome 22q. The tumor-suppressor-gene(s) might locate on the 22q12.2-12.3 and/or 22q13.32-13.33.

Cullin 4B is a novel prognostic marker that correlates with colon cancer progression and pathogenesis.

Cullin 4B (CUL4B), a scaffold protein of the Cullin4B-RING E3 ligase complex, functions in proteolysis. The present study aims to investigate its expression pattern and evaluate whether CUL4B expression was associated with histopathological and prognosis in the patients with colon cancer. Real-time PCR and western blot were used to identify CUL4B expression in tumor tissue and the paired adjacent normal mucosa from patients with colon cancer. Immunohistochemistry on a tissue microarray containing 203 cases of colon cancer was performed to analyze the association between CUL4B expression and clinicopathological features. Results indicated that CUL4B mRNA and protein levels in tumor tissues were both higher than that in normal mucosae (P < 0.001). Immunohistochemical study displayed that high CUL4B expression was significantly associated with the depth of tumor invasion, lymph node metastasis, distant metastasis, histological differentiation, vascular invasion, and advanced tumor stage. Patients with CUL4B-positive tumors had a higher recurrence rate and poorer survival than patients with CUL4B-negative tumors. In multivariate analyses, CUL4B expression was an independent factor for determining colon cancer prognosis after surgery. In conclusion, CUL4B might promote the progression of colon cancer and can be served as a novel independent prognostic marker for the prediction of recurrence in colon cancer.

Identification and validation that up-expression of HOXA13 is a novel independent prognostic marker of a worse outcome in gastric cancer based on immunohistochemistry.

Homeobox (HOX) gene family is known to be classic examples of the intimate relationship between embryogenesis and tumorigenesis. However, less is known about the involvement of HOX gene family with gastric cancerogenesis. Here, we screened the expression of HOX gene family in gastric cancers and explored the relationships between them by cDNA microarray. We found several differentially expressed HOX genes in gastric cancers, especially HOXA10 (11/12) and HOXA13 (11/12) with significantly higher expression in the cancerous tissues. Furthermore, we validated HOXA13 as a novel prognostic marker in gastric cancer based on immunohistochemistry and statistical analysis. HOXA13 expression was significantly up-regulated in cancerous tissues compared with the corresponding non-cancerous mucosa (P < 0.001). Up-expression of HOXA13 was significantly correlated with T stage (P = 0.002), M stage (P = 0.024), advanced UICC stage (P < 0.001), histological differentiation (P = 0.005), and relapse (P = 0.001). Patients with positive HOXA13 expression had a obviously lower overall survival (OS) and disease-free survival (DFS) rate than patients with negative HOXA13 expression (HR 3.331, 95 % CI 1.722-6.442, P < 0.001; HR 3.289, 95 % CI 1.703-6.351, P < 0.001, respectively). Univariate and multivariate Cox analysis confirmed that HOXA13 could serve as a significant independent prognostic factor for DFS and OS. Therefore, our results indicated that several HOX genes might be closely involved in the process of the gastric tumorigenesis. Furthermore, up-expression of HOXA13 might be associated with highly aggressive phenotype of gastric cancer. HOXA13 was a significant independent prognostic factor and could serve as a putative biomarker for diagnosis and prognosis of gastric cancer.

Increased expression of TBX2 is a novel independent prognostic biomarker of a worse outcome in colorectal cancer patients after curative surgery and a potential therapeutic target.

T-box2 (TBX2) plays a critical role in embryonic development. Recently, deregulated expression of TBX2 has been implicated in several malignancies. However, the expression and the role of TBX2 in colorectal cancer (CRC) remain unclear. In this study, we found that TBX2 was obviously up-regulated in CRC in comparison with the corresponding normal mucosa at transcriptional and protein level. Up-expression of TBX2 was significantly associated with depth of tumor invasion (P = 0.006), distant metastasis (P = 0.038), advanced AJCC stage (P = 0.008), and relapse (P = 0.003). TBX2 was a significantly prognostic factor for decreased survival and increased disease recurrence independent of tumor stage(II, III stage) and functioned as a biomarker to identify prognosis of patients with CRC (OS: HR 2.154; 95% CI 1.019-4.551; P = 0.044, DFS: HR 2.253; 95% CI 1.109-4.575; P = 0.025). Furthermore, TBX2 could serve as a potential target of cancer drug therapy.

Upregulated INHBA expression is associated with poor survival in gastric cancer.

Expression microarrays are widely used for investigating the candidate molecular targets in human cancer. While genome-wide expression signatures screened by gene set enrichment analysis (GSEA) were not performed in Chinese gastric cancer (GC). To gain new molecular targets for GC, GSEA analysis was performed. In the present study, GSEA were used to pick out differentially expressed gene sets of our database. Total RNA of paired tissue samples (n = 48) and a tissue microarray containing 132 paired tissues were used to further validate expression levels of INHBA and its correction with clinicopathological factors. Upregulated INHBA expression in gastric cancer was screened and further confirmed by qPCR and immunostaining analysis. Increased INHBA expression was significantly correlated with the diameter of cancer and depth of tumor invasion. Patients with higher expression levels of INHBA had a shorter disease-free survival rate. It was effective to gain new molecular targets for GC by GSEA analysis. INHBA may be a poor survival indicator of GC.

Overexpression of Forkhead box M1 protein associates with aggressive tumor features and poor prognosis of hepatocellular carcinoma.

The aim of this study was to detect the expression of the Forkhead box M1 (FOXM1) protein in human hepatocellular carcinoma (HCC) and to associate FOXM1 expression with clinicopathological features of the patients, and predict the prognosis of patients with FOXM1 expression. Surgical tissue specimens from 151 HCC patients were subjected to a tissue microarray construction and immunohistochemistry analysis of FOXM1 and the proliferation marker proliferating cell nuclear antigen (PCNA). The data showed that the FOXM1 protein was expressed in 59.3% of the HCC tissues, which was significantly higher compared to that of the surrounding non-tumorous tissues (23.8%; P<0.001). Moreover, FOXM1 expression was positively correlated with the labeling index of PCNA (P<0.001) in HCC and with aggressive tumor phenotypes, such as larger tumor size, multiple tumors, bilobar involvement, poor tumor cell differentiation, advanced stage and macrovascular invasion (P<0.05). In addition, HCC patients with FOXM1-positive tumors had a poorer recurrence-free and overall survival after hepatectomy than those with FOXM1-negative tumors. Multivariate Cox regression analysis demonstrated that FOXM1 expression was an independent predictor of unfavorable outcome (P<0.05). The data from the current study suggest that FOXM1 may play an important role in HCC progression and could be further evaluated as a prognostic biomarker and potential therapeutic target.

Biglycan expression correlates with aggressiveness and poor prognosis of gastric cancer.

Biglycan, a member of the small leucine-rich proteoglycan family, has been implicated in the development and progression of human cancers. However, the clinical significance of biglycan expression in gastric cancer has not been determined. In the present study, biglycan mRNA and protein concentrations were analyzed using quantitative realtime reverse transcription polymerase chain reaction and Western blot in 69 gastric cancer and adjacent non-tumorous tissues, respectively. Biglycan expression was further assessed using immunohistochemistry in tissue microarrays that contained 264 cases of gastric cancer, and others containing normal or metastasized lymph node tumor tissues. Biglycan was upregulated at the transcriptional and translational levels and there was a correlation between the expression of biglycan mRNA and protein (P = 0.000, κ = 0.769). Over-expression of biglycan was strongly associated with lymph node metastasis, tumor (T) classification, metastasis (M) classification, vascular invasion and Union for International Cancer Control (UICC) stage. Patients with biglycan-positive tumors had a significantly higher disease recurrence rate and poorer survival than patients with biglycan-negative tumors after the radical surgery. Multivariate analysis revealed that biglycan expression is an independent prognostic indicator for survival of patients with gastric cancer. The data from the current study demonstrate that elevated expression of biglycan may play an important role in the development and progression of gastric cancer, and could be further evaluated as a biomarker for predication of a poor clinical outcome.

Mutation analysis of tumor suppressor gene PTEN in patients with gastric carcinomas and its impact on PI3K/AKT pathway.

The aim of this study was to clarify the participation of PTEN mutation in gastric carcinogenesis and its impact on PI3K/AKT pathway. All nine exons of PTEN were screened for mutations by direct sequencing in 144 patients with pathologically proven gastric carcinoma and their corresponding normal mucosae, followed by Western blotting to detect the changes in PI3K/AKT pathway. Direct sequencing indicated there were 27 cases with mutations among 144 patients consisting of 15 cases (55.6%) of missense mutation, nine nonsense mutations (33.3%), two 1-bp deletion (7.4%), and a mutation within intron 6 (3.7%). The mutation hot spots at codons 36, 75, 232 and 393 had not been observed previously, and the mutation sites in exons 3, 5, 6 and 8 were not found, suggesting that there might be some unique characteristic of PTEN inactivation mechanism in the Shanghai population. The PTEN mutation rate was significantly higher at pTMN stages III and IV than that at stages I and II (P<0.005), and it was higher in poorly differentiated gastric cancer than in well or moderately differentiated types (P<0.05). PTEN and E-cadherin protein expression in gastric cancer was significantly down-regulated comparing with that in paracancerous tissues, while the PI3K, AKT, MMP-2, MMP-9 and NF-kappaBp65 protein were overexpressed in cancer tissues. Our results implicated that the mutations of PTEN did not occur at a significant rate in gastric carcinoma in Shanghai, but might play a role in tumorigenesis. The mutation status of PTEN was significantly relevant to pTNM staging and degree of cell differentiation, hinting that PTEN might be a prognostic biomarker of gastric cancer. The decreased expression of PTEN and E-cadherin, together with the overexpression of PI3K, AKT, MMP-2, MMP-9 and NF-kappaBp65, contributed cooperatively to the accelerated progress of gastric cancer.