Overexpression of p42.3 promotes cell growth and tumorigenicity in hepatocellular carcinoma

AIM:To investigate the association of p42.3 expression with clinicopathological characteristics and the biological function of p42.3 in human hepatocellular carcinoma(HCC).METHODS:We used reverse transcription-polymerase chain reaction(RT-PCR),quantitative real-time RT-PCR and western blotting to detect p42.3 mRNA and protein expression in hepatic cell lines.We examined primary HCC samples and matched adjacent normal tissue by immunohistochemistry to investigate the correlation between p42.3 expression and clinicopathological features.HepG2 cells were transfected with a pIRES2EGFP-p42.3 expression vector to examine the function of the p42.3 gene.Transfected cells were analyzed for their viability and malignant transformation abilities by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,colony formation assay,and tumorigenicity assay in nude mice.RESULTS:p42.3 is differentially expressed in primary HCC tumors and cell lines.Approximately 69.6%(96/138) of cells were p42.3-positive in hepatic tumor tissues,while 30.7%(35/114) were p42.3-positive in tumor-adjacent normal tissues.Clinicopathological characteristics of the HCC specimens revealed a significant correlation between p42.3 expression and tumor differentiation(P = 0.031).However,p42.3 positivity was not related to tumor tumor-node-metastasis classification,hepatitis B virus status,or hepatoma type.Regarding p42.3 overexpression in stably transfected HepG2 cells,we discovered significant enhancement of cancer cell growth and colony formation in vitro,and significantly enhanced tumorigenicity in nude mice.Western blot analysis of cell cycle proteins revealed that enhanced p42.3 levels promote upregulation of proliferating cell nuclear antigen,cyclin B1 and mitotic arrest deficient 2.CONCLUSION:p42.3 promotes tumorigenicity and tumor growth in HCC and may be a potential target for future clinical cancer therapeutics.

Prognostic significance of PTEN,Ki-67 and CD44s expression patterns in gastrointestinal stromal tumors

AIM:To develop a prognostic approach for gastrointestinal stromal tumors(GISTs) using a cluster of indicators and follow-up information.METHODS:One hundred and four GISTs that had not been subjected to targeted therapies were collected and classified by NIH risk assessment and anatomic location.By immunohistochemistry,the expressions of PTEN,Ki-67,CD44s matrix metalloproteinase(MMP)-9 and TIMP-1 were detected on tissue microarray.Univariate and multimarker survival analyses were performed and then a COX hazard proportion model was constructed to evaluate a cluster of predictors of GIST.RESULTS:Our data showed small intestinal GIST are more aggressive than gastric GIST.The NIH risk assessment correlated with disease-free survival foreither gastric GIST or small intestinal GIST.Immunohistochemical analysis revealed that Ki-67 labeling indexes(LIs) < 5% predicted higher disease-specific survival(DSS) in gastric and small intestinal GIST.CD44s positivity and PTEN LIs ≥ 50% correlated with higher DSS in gastric GIST.MMP-9 and TIMP-1 had no correlation with survival.Multimarker analysis revealed that the expression pattern of PTEN LIs ≥ 50% combined with Ki-67 LIs < 5% and CD44s positivity reliably predicted favorable outcomes for gastric GIST(P = 0.009),as did the combination of PTEN LIs ≥ 50% and Ki-67 LIs < 5% for small intestinal GIST(P = 0.011).Authors also found that high NIH risk grade was correlated with DSS in patients with gastric GIST and disease-free survival in patients with small intestinal GIST.CONCLUSION:PTEN LIs ≥ 50%,Ki-67 LIs < 5% and CD44s positivity provides an accurate,favorable prognosis for gastric GIST.PTEN LIs ≥ 50% and Ki-67 LIs < 5% does the same for small intestinal GIST.Ki-67 LIs enhances the NIH assessment.

Mutations in components of the Wnt signaling pathway in gastric cancer

AIM: To explore the contribution of AXIN1, AXIN2 and beta-catenin, components of Wnt signaling pathway, to the carcinogenesis of gastric cancer (GC), we examined AXIN1, AXIN2 exon7 and CTNNB1 (encoding beta- catenin) exon3 mutations in 70 GCs. METHODS: The presence of mutations was identified by polymerase chain reaction (PCR)-based denaturing high-performance liquid chromatography and direct DNA sequencing. Beta-catenin expression was detected by immunohistochemical analysis. RESULTS: Among the 70 GCs, 5 (7.1%) had mutations in one or two of these three components. A frameshift mutation (1 bp deletion) in exon7 of AXIN2 was found in one case. Four cases, including the case with a mutation in AXIN2, had frameshift mutations and missense mutations in AXIN1. Five single nucleotide polymorphisms (SNPs), 334 C>T, 874 C>T, 1396 G>A, 1690 C>T and 1942 T>G, were identified in AXIN1. A frameshift mutation (27 bp deletion) spanning exon3 of CTNNB1 was observed in one case. All four cases with mutations in AXIN1 and AXIN2 showed nuclear beta- catenin expression. CONCLUSION: These data indicate that the mutationsin AXIN1 and AXIN2 may contribute to gastric carcino- genesis.

MAWBP and MAWD inhibit proliferation and invasion in gastric cancer

AIM: To investigate role of putative mitogen-activated protein kinase activator with WD40 repeats (MAWD)/ MAWD binding protein (MAWBP) in gastric cancer (GC). METHODS: MAWBP and MAWD mRNA expression level was examined by real-time reverse transcriptasepolymerase chain reaction and semi-quantitative polymerase chain reaction in six GC cell lines. Western blotting was used to examine the protein expression levels. We developed GC cells that stably overexpressed MAWBP and MAWD, and downregulated expression by RNA interference assay. Proliferation and migration of these GC cells were analyzed by 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), soft agar, tumorigenicity, migration and transwell assays. The effect of expression of MAWBP and MAWD on transforming growth factor (TGF)-β1-induced epithelialmesenchymal transition (EMT) was examined by transfection of MAWBP and MAWD into GC cells. We detected the levels of EMT markers E-cadherin, N-cadherin and Snail in GC cells overexpressing MAWBP and MAWD by Western blotting. The effect of MAWBP and MAWD on TGF-β signal was detected by analysis of phosphorylation level and nuclear translocation of Smad3 using Western blotting and immunofluorescence. RESULTS: Among the GC cell lines, expression of endogenous MAWBP and MAWD was lowest in SGC7901 cells and highest in BGC823 cells. MAWBP and MAWD were stably overexpressed in SGC7901 cells and knocked down in BGC823 cells. MAWBP and MAWD inhibited GC cell proliferation in vitro and in vivo . MTT assay showed that overexpression of MAWBP and MAWD suppressed growth of SGC7901 cells (P < 0.001), while knockdown of these genes promoted growth of BGC823 cells (P < 0.001). Soft agar colony formation experiments showed that overexpression of MAWBP and MAWD alone or together reduced colony formation compared with vector group in SGC7901 (86.25±8.43, 12.75±4.49, 30±6.41 vs 336.75±22.55, P < 0.001), and knocked-down MAWBP and MAWD demonstrated opposite effects (131.25±16.54, 88.75±11.12, 341.75±22.23 vs 30.25±8.07, P < 0.001). Tumorigenicity experiments revealed that overexpressed MAWBP and MAWD inhibited GC cell proliferation in vivo (P < 0.001). MAWBP and MAWD also inhibited GC cell invasion. Transwell assay showed that the number of traverse cells of MAWBP, MAWD and coexpression group were more than that in vector group (84±16.57, 98.33±9.8, 29±16.39 vs 298±11.86, P < 0.001). Coexpression of MAWBP and MAWD significantly decreased the cells traversing the matrix membrane. Conversely, knocked-down MAWBP and MAWD correspondingly promoted invasion of GC cells (100.67±14.57, 72.66±8.51, 330.67±20.55 vs 27±11.53, P < 0.001). More importantly, coexpression of MAWBP and MAWD promoted EMT. Cells that coexpressed MAWBP and MAWD displayed a pebble-like shape and tight cell-cell adhesion, while vector cells showed a classical mesenchymal phenotype. Western blotting showed that expression of E-cadherin was increased, and expression of N-cadherin and Snail was decreased when cells coexpressed MAWBP and MAWD and were treated with TGF-β1. Nuclear translocation of p-Smad3 was reduced by attenuating its phosphorylation. CONCLUSION: Coexpression of MAWBP and MAWD inhibited EMT, and EMT-aided malignant cell progression was suppressed.