Non-CpG sites preference in G:C > A:T transition of TP53 in gastric cancer of Eastern Europe (Poland, Romania and Hungary) compared to East Asian countries (China and Japan).

AIM: Mutation spectrum of TP53 in gastric cancer (GC) has been investigated world-widely, but a comparison of mutation spectrum among GCs from various regions in the world are still sparsely documented. In order to identify the difference of TP53 mutation spectrum in GCs in Eastern Europe and in East Asia, we sequenced TP53 in GCs from Eastern Europe, Lujiang (China), and Yokohama, Kanagawa (Japan) and identified the feature of TP53 mutations of GC in these regions. SUBJECTS AND METHOD: In total, 689 tissue samples of GC were analyzed: 288 samples from East European populations (25 from Hungary, 71 from Poland and 192 from Romania), 268 from Yokohama, Kanagawa, Japan and 133 from Lujiang, Anhui province, China. DNA was extracted from FFPE tissue of Chinese, East European cases; and from frozen tissue of Japanese GCs. PCR products were direct-sequenced by Sanger method, and in ambiguous cases, PCR product was cloned and up to 8 clones were sequenced. We used No. NC_000017.11(hg38) as the reference sequence of TP53. Mutation patterns were categorized into nine groups: six base substitutions, insertion, deletion and deletion-insertion. Within G:C > A:T mutations the mutations in CpG and non-CpG sites were divided. The Cancer Genome Atlas data (TCGA, ver.R20, July, 2019) having somatic mutation list of GCs from Whites, Asians, and other ethnicities were used as a reference for our data. RESULTS: The most frequent base substitutions were G:C > A:T transition in all the areas investigated. The G:C > A:T transition in non-CpG sites were prominent in East European GCs, compared with Asian ones. Mutation pattern from TCGA data revealed the same trend between GCs from White (TCGA category) vs Asian countries. Chinese and Japanese GCs showed higher ratio of G:C > A:T transition in CpG sites and A:T > G:C mutation was more prevalent in Asian countries. CONCLUSION: The divergence in mutation spectrum of GC in different areas in the world may reflect various pathogeneses and etiologies of GC, region to region. Diversified mutation spectrum in GC in Eastern Europe may suggest GC in Europe has different carcinogenic pathway of those from Asia.

Maspin subcellular expression in wild-type and mutant TP53 gastric cancers.

BACKGROUND: Although the role of p53 in the evolution and prognosis of gastric cancer (GC) has been extensively examined, the exact mechanism of action is incompletely understood. In the last years, p53-target genes were supposed to be involved in the p53 pathway. One of them is the tumor-suppressor gene Maspin, which codifies the protein with the same name. Maspin activity depends on its subcellular localization. To our knowledge, the possible role of TP53 gene in Maspin subcellular localization, in GC cells, has not yet been studied in a large number of human samples. AIM: To evaluate the possible role of wild-type and mutated p53 in Maspin subcellular localization. METHODS: The present study included 266 consecutive patients with GC in which TP53 gene status, and mutations in exons 2 to 11, respectively, were analyzed and correlated with immunohistochemical expression of p53 and Maspin. RESULTS: None of the 266 cases showed mutations in exon 9. The rate of TP53 mutations was 33.83%. The mutation rate was slightly higher in distally-located GCs, with a lower degree (≤ 5 buds/ high power fields) of dyscohesivity (P < 0.01). The wild-type cases had a longer survival, compared with mutant GCs, especially in patients without lymph node metastases, despite the high depth of tumor infiltration (P = 0.01). The Dukes-MAC-like staging system was proved to have the most significant independent prognostic value (P < 0.01). The statistical correlations proved that TP53 gene mutations in exon 7 might induce knockdown of Maspin, but wild-type p53 can partially restore nuclear Maspin expression and decrease the metastatic potential of gastric adenocarcinoma cells. CONCLUSION: Downregulated Maspin might be induced by mutations in exon 7 of the TP53 gene but wild-type p53 can partially restore nuclear Maspin expression. These findings should be proved in experimental studies.

PLK4 overexpression and its effect on centrosome regulation and chromosome stability in human gastric cancer.

Polo-like kinase 4 (PLK4) is a centrosomal protein that is involved in the regulation of centrosome duplication. This study aimed to determine whether the genetic abnormality of PLK4 is involved in human gastric cancer. First, we examined the status of PLK4 mRNA expression in 7 gastric cancer cell lines and 48 primary gastric cancers using an RT-PCR analysis. The upregulation of PLK4 mRNA expression was detected in 57.1 % (4/7) of the gastric cancer cell lines, and a novel PLK4 variant with exon 4, but without exon 5, was identified. In the primary gastric cancers, the upregulation of PLK4 mRNA expression in the cancerous cells was detected in 50.0 % (24/48) of the cases, and this upregulation was statistically significant (P value = 0.0139). Next, we established AGS gastric cancer cells capable of inducibly expressing PLK4 using the piggyBac transposon vector system and showed that PLK4 overexpression induced centrosome amplification and chromosome instability using immunofluorescence and FISH analyses, respectively. Furthermore, PLK4 overexpression suppressed primary cilia formation. Our current findings suggested that PLK4 is upregulated in a subset of primary gastric cancers and that PLK4 overexpression induces centrosome amplification and chromosome instability and causes the suppression of primary cilia formation.

The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer.

BACKGROUND: Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer. METHODS AND RESULTS: A genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide. CONCLUSION: These results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.

EPH-EPHRIN in human gastrointestinal cancers.

Ever since its discovery two decades ago, the erythropoietin-producing hepatoma (EPH)-EPHRIN system has been shown to play multifaceted roles in human gastroenterological cancer as well as neurodevelopment. Over-expression, amplification and point mutations have been found in many human cancers and many investigators have shown correlations between these up-regulations and tumor angiogenesis. Thus, the genes in this family are considered to be potential targets of cancer therapy. On the other hand, the down-regulation of some members as a result of epigenetic changes has also been reported in some cancers. Furthermore, the correlation between altered expressions and clinical prognosis seems to be inconclusive. A huge amount of protein-protein interaction studies on the EPH-EPHRIN system have provided a basic scheme for signal transductions, especially bi-directional signaling involving EPH-ERPHRIN molecules at the cell membrane. This information also provides a manipulative strategy for harnessing the actions of these molecules. In this review, we summarize the known alterations of EPH-EPHRIN genes in human tumors of the esophagus, stomach, colorectum, liver and pancreas and present the perspective that the EPH-EPHRIN system could be a potential target of cancer therapy.

The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to S phase.

Brd4 is a bromodomain protein that binds to acetylated chromatin. It regulates cell growth, although the underlying mechanism has remained elusive. Brd4 has also been shown to control transcription of viral genes, whereas its role in transcription of cellular genes has not been fully elucidated. Here we addressed the role of Brd4 in cell growth and transcription using a small hairpin (sh) RNA approach. The Brd4 shRNA vector stably knocked down Brd4 protein expression by approximately 90% in NIH3T3 cells and mouse embryonic fibroblasts. Brd4 knockdown cells were growth impaired and grew more slowly than control cells. When synchronized by serum starvation and released, Brd4 knockdown cells were arrested at G(1), whereas control cells progressed to S phase. In microarray analysis, although numerous genes were up-regulated during G(1) in control cells, many of these G(1) genes were not up-regulated in Brd4 knockdown cells. Reintroduction of Brd4 rescued expression of these G(1) genes in Brd4 knockdown cells, allowing cells to progress toward S phase. Chromatin immunoprecipitation analysis showed that Brd4 was recruited to the promoters of these G(1) genes during G(0)-G(1) progression. Furthermore, Brd4 recruitment coincided with increased binding of Cdk9, a component of P-TEFb and RNA polymerase II to these genes. Brd4 recruitment was low to absent at genes not affected by Brd4 shRNA. The results indicate that Brd4 stimulates G(1) gene expression by binding to multiple G(1) gene promoters in a cell cycle-dependent manner.

Embryonic lethal effect of expressing a dominant negative mutant human thyroid hormone receptor alpha1 in mice.

Resistance to thyroid hormone (RTH) is caused mainly by mutations of the thyroid hormone receptor (TR) beta gene. Although, in vitro, TRalpha1 and TRbeta1 mutants exhibit similar dominant negative effects against wild-type TR, no TRalpha mutants have ever been identified in RTH patients. It has been postulated that mutations in TRalpha gene may be lethal, compensated completely by intact TRbeta or associated with phenotypic manifestations different from RTH. To investigate the consequences of mutant TRalpha1 expression in vivo, we tried to generate two different lines of transgenic mice which express a strong or a weak dominant negative mutant TR alpha1, respectively. First, we expressed betaF451X identified in a patient with severe RTH and alphaF397X, which has an identical C-terminal truncation and a similarly strong dominant negative potency to betaF451X, under the control of human polypeptide chain elongation factor 1alpha promoter. Six betaF451X-transgenic mice were born from 223 transferred embryos, giving a transgenic frequency of 2.7%. By contrast, expression of alphaF397X resulted in quite a low transgenic frequency with 0.39% of the transferred embryos bearing the transgene. Only three transgenic mice were born with no apparently overt abnormalities, of which one male produced F1 offspring. The transgenic progeny expressed alphaF397X in the testis but we did not succeed in generating transgenic mice expressing alphaF397X in multiple organs. To avoid toxic effects mediated by a strong dominant negative activity of mutant TRalpha1, we exchanged alphaF397X for alphaK389E, which has an identical missense mutation and a relatively weak transdominant potency as betaK443E identified in a patient with mild RTH. When expressed by cytomegalovirus immediate early enhancer-chicken beta-actin promoter, we did not succeed in creating alphaK389E-transgenic mice despite three independent transgene-injections. These findings define crucial in vivo functions of mutant TRalpha1s during mouse fetal development and suggest the possibility that the expression of a dominant negative mutant TRalpha1 in extensive tissues from the early embryonal stages might be lethal.

Beta-catenin/Tcf-1-mediated transactivation of cyclin D1 promoter is negatively regulated by thyroid hormone.

Cyclin D1 is an oncogenic cyclin frequently over-expressed in cancer. To examine the effect of thyroid hormone (T3) and its receptor (TR) on the transcription of cyclin D1 gene, we co-transfected the chloramphenicol acetyl-transferase (CAT) reporter plasmid containing cyclin D1 promoter together with the expression plasmids for TRbeta1 and wild-type or mutant beta-catenin (SA) into 293T cells. In the presence of T3, beta-catenin-dependent transactivation of cyclin D1 promoter was suppressed by co-transfection of TRbeta1. The suppression by T3/TRbeta1 was in a dose-dependent manner. The CAT reporter gene in which Tcf/Lef-1 sites were fused to heterologous promoter was also suppressed by T3/TRbeta1. Furthermore, inhibition of endogenous wild-type beta-catenin by T3/TRbeta1 was observed in SW480 colon carcinoma cells with mutation of the adenomatous polyposis coli gene. These results indicate that the T3-bound TR inhibits the transcription of cyclin D1 through the Tcf/Lef-1 site, which is positively regulated by the Wnt-signaling pathway.