Higher mRNA Expression Levels Research Articles

Abstract P2-03-07: Exome sequencing of human breast cancer tissues resistant to taxanes

Abstract Background: Although taxanes are a mainstay of breast cancer treatment, some cases are resistant to these drugs. This is a crucial issue in breast cancer therapy. In the emerging era of next-generation sequencing, it is possible to obtain extensive genomic information on individual tumors in a very short time. Using this technology, it was reported that specific mutations might affect therapeutic efficacy and induce resistance to specific treatment. Objective: The aim of this study was to investigate the mechanisms of taxane resistance using whole exon sequencing and expression analyses in human breast cancer tissues. Materials and Methods: We selected six breast cancer patients whose tumors responded well to anthracycline treatment but suffered disease progression on taxane treatment. We then performed whole exon sequencing on these samples using HiSeq (Illumia). In this way, we identified somatic mutations of candidate genes considered to be instrumental for mediating resistance to taxanes. Next, we performed mRNA expression analyses of these candidate genes in a further 122 breast cancers treated with taxanes at our institute. Finally, we correlated mRNA expression levels of these genes with clinicopathological factors and prognosis. Results: We identified 9 mutations common to all 6 patients analyzed in this study, and a further 16 mutations shared by 5 of them. Kaplan-Meier analyses showed that high level mRNA expression of 3 of these 25 genes was significantly associated with poorer disease-free survival. Moreover, high level mRNA expression of one of these three genes was significantly associated with worse overall survival. However, there were no significant correlations between expression levels of these three genes and any clinicopathologeical features. Conclusion: Using next-generation sequencing, we have identified three candidate genes involved in resistance to taxane treatment in breast cancer. We are now analyzing the functional attributes of these three genes. Citation Format: Endo Y, Dong Y, Kondo N, Hato Y, Hisada T, Nishimoto M, Nishikawa S, Takahashi S, Toyama T. Exome sequencing of human breast cancer tissues resistant to taxanes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-03-07.

Selection of progesterone derivatives specific to membrane progesterone receptors

The search of selective agonists and antagonists of membrane progesterone receptors (mPRs) is a starting point for the study of progesterone signal transduction mechanisms mediated by mPRs, distinct from nuclear receptors. According to preliminary data, the ligand affinity for mPRs differs significantly from that for classical nuclear progesterone receptors (nPRs), which might indicate structural differences in the ligand-binding pocket of these proteins. In the present work, we analyzed the affinity of several progesterone derivatives for mPRs of human pancreatic adenocarcinoma BxPC3 cell line that is characterized by a high level of mPR mRNA expression and by the absence of expression of nPR mRNA. The values were compared with the affinity of these compounds for nPRs. All tested compounds showed almost no affinity for nPRs, whereas their selectivity towards mPRs was different. Derivatives with an additional 19-hydroxyl group and removed 3-keto group had the highest selectivity for mPRs. These results suggest these compounds as the most selective progesterone analogs for studying the mechanisms of progestin action via mPRs.

SIX1 maintains tumor basal cells via transforming growth factor-β pathway and associates with poor prognosis in esophageal cancer.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors. Although improvement in both surgical techniques and neoadjuvant chemotherapy has been achieved, the 5‐year survival rate of locally advanced tumors was, at best, still 55%. Therefore, elucidation of mechanisms of the malignancy is eagerly awaited. Epithelial‐mesenchymal transition (EMT) by transforming growth factor‐β (TGF‐β) has been reported to have critical biological roles for cancer cell stemness, whereas little is known about it in ESCC. In the current study, a transcriptional factor SIX1 was found to be aberrantly expressed in ESCCs. SIX1 cDNA transfection induced overexpression of transforming growth factors (TGFB1 and TGFB2) and its receptor (TGFBR2). Cell invasion was reduced by SIX1 knockdown and was increased in stable SIX1‐transfectants. Furthermore, the SIX1‐transfectants highly expressed tumor basal cell markers such as NGFR,SOX2,ALDH1A1, and PDPN. Although mock‐transfectants had only a 20% PDPN‐high population, SIX1‐transfectants had 60–70%. In two sets of 42 and 85 ESCC patients receiving surgery alone or neoadjuvant chemoradiotherapy followed by surgery, the cases with high SIX1 mRNA and protein expression level significantly showed a poor prognosis compared with those with low levels. These SIX1 high cases also expressed the above basal cell markers, but suppressed the differentiation markers. Finally, TGF‐β signaling blockade suppressed ESCC cell growth in association with the reduction of PDPN‐positive tumor basal cell population. The present results suggest that SIX1 accelerates self‐renewal of tumor basal cells, resulting in a poor prognosis for ESCC patients.

CDNA cloning, characterization and expression analysis of ATP-binding cassette transmembrane transporter in Exopalaemon carinicauda

ATP-binding cassette (ABC) transporters are integral membrane proteins which carry diverse substrates across biological membranes. In this study, we report the cloning of a full-length, 2468-bp cDNA sequence encoding a gene of the ABC transporter subfamily G (ABCG) from the shrimp Exopalaemon carinicauda (E. carinicauda). The deduced protein, EcABCG, contains 627 amino acid residues, with a theoretical molecular weight of 69.0 kDa. Phylogenetic analysis showed that EcABCG shares the highest identity with the homologs of the shrimp species Litopenaeus vannamei and Fenneropenaeus chinensis, and is also closely related to the insect ABC transporter/white proteins. High-level mRNA expression of EcABCG was detected in the tissue of the hepatopancreas and intestine, using a real-time quantitative reverse-transcriptase PCR. The ABC transporter mRNA transcript in the hepatopancreas of E. carinicauda was significantly upregulated after 6 h and after 1 h of exposure to acidic and alkaline conditions respectively. EcABCG was also induced in the intestine at 1–24 and 3–48 h after exposure to acidic and alkaline conditions respectively. Upon exposure to sulfamerazine diets (50, 100 and 200 mg kg−1) for 3 and 6 h, respectively, the mRNA expression of EcABCG was upregulated in the hepatopancreas. EcABCG was also induced in the intestine after exposure to 50, 100 and 200 mg kg−1 sulfamerazine diets for 6, 3 and 12 h respectively. These results suggest that the ABCG transporter of E. carinicauda might play a role in coping with pH stress and in exporting xenobiotics.

Expression Profiling of Ribosome Biogenesis Factors Reveals Nucleolin as a Novel Potential Marker to Predict Outcome in AML Patients.

Acute myeloid leukemia (AML) is a heterogeneous disease. Prognosis is mainly influenced by patient age at diagnosis and cytogenetic alterations, two of the main factors currently used in AML patient risk stratification. However, additional criteria are required to improve the current risk classification and better adapt patient care. In neoplastic cells, ribosome biogenesis is increased to sustain the high proliferation rate and ribosome composition is altered to modulate specific gene expression driving tumorigenesis. Here, we investigated the usage of ribosome biogenesis factors as clinical markers in adult patients with AML. We showed that nucleoli, the nucleus compartments where ribosome production takes place, are modified in AML by analyzing a panel of AML and healthy donor cells using immunofluorescence staining. Using four AML series, including the TCGA dataset, altogether representing a total of about 270 samples, we showed that not all factors involved in ribosome biogenesis have clinical values although ribosome biogenesis is increased in AML. Interestingly, we identified the regulator of ribosome production nucleolin (NCL) as over-expressed in AML blasts. Moreover, we found in two series that high NCL mRNA expression level was associated with a poor overall survival, particular in elderly patients. Multivariate analyses taking into account age and cytogenetic risk indicated that NCL expression in blast cells is an independent marker of reduced survival. Our study identifies NCL as a potential novel prognostic factor in AML. Altogether, our results suggest that the ribosome biogenesis pathway may be of interest as clinical markers in AML.

Upregulation of Far Upstream Element-Binding Protein 1 (FUBP1) Promotes Tumor Proliferation and Tumorigenesis of Clear Cell Renal Cell Carcinoma

ObjectiveThe far upstream element (FUSE)-binding protein 1 (FUBP1) is a transactivator of human c-myc proto-oncogene transcription, with important roles in carcinogenesis. However, the expression pattern and potential biological function of FUBP1 in clear cell renal cell carcinoma (ccRCC) is yet to be established.MethodsFUBP1 expression was detected in ccRCC tissues and cell lines by real-time RT-PCR, Western blot analysis, and immunohistochemistry. The correlations of FUBP1 mRNA expression levels with clinicopathological factors were evaluated. The biological function of FUBP1 during tumor cell proliferation was studied by MTS, colony formation, and soft-agar colony formation. The effects of FUBP1 on cell cycle distribution and apoptosis were analyzed by flow cytometry. Western blot analysis was used to identify the potential mechanism of FUBP1 regulating cell cycle and apoptosis.ResultsThe levels of FUBP1 mRNA and protein expression were upregulated in human ccRCC tissues compared with adjacent noncancerous tissues. High levels of FUBP1 mRNA expression were associated with higher tumor stage and tumor size. FUBP1 knockdown inhibited cell proliferation and induced cell cycle arrest and apoptosis. Meanwhile, the expression levels of c-myc and p21 mRNA were correlated with that of FUBP1 mRNA.ConclusionsFUBP1 acts as a potential oncogene in ccRCC and may be considered as a novel biomarker or an attractive treatment target of ccRCC.

Expression of Radioresistant Gene PEG10 in OSCC Patients and Its Prognostic Significance

Background:Oral squamous cell carcinoma (OSCC) is one of the most common forms of cancer occurring worldwide. PEG10 is well known as a paternally expressed gene from a newly recognized imprinted region at human chromosome 7q21. Previous study had demonstrated that the significant expression of PEG10 was found in radioresistant OSCC cell line and its expression was significantly associated with poor survival in several cancers. Therefore it has been evaluated as a potential marker in OSCC patients undergoing radiotherapy.Objective:This study was conducted to analyze the mRNA expression of PEG10 in OSCC and its expression in relation to clinicpathological features, radiotherapy treatment response and survival.Methods:This study included tissue specimens obtained via biopsy of 118 patients with OSCC who were recommended for radiotherapy treatment and 80 healthy control tissues analysis of mRNA expression of PEG10 was done by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Patients were treated with 70 Gy of radiation dose by shrinking field technique using Cobalt-60 teletherapy machine.Results:Significantly higher mRNA expression of PEG10 was found in OSCC patients when compared with matched controls. High level of PEG10 mRNA expression showed a significant correlation with lymph node metastasis (p = 0.0047) and tumor stage (p = 0.0499). Multivariate Cox regression analysis revealed that high level of mRNA expression of PEG10 was significantly associated with poor survival (p < 0.05). Our research demonstrated that the expression of PEG10 was higher in radioresistant tumor.Conclusion:We observed significantly increased expression of PEG10 in context of lymph node status, advanced stage and poor survival in our study. Thus PEG10 gene can be used as potential predictive and prognostic biomarker in OSCC patients undergoing radiotherapy.

MTE23.02 Biomarker Characterization: Challenges and Perspectives

Small molecule inhibitors are the current treatment for non-small-cell lung cancer (NSCLC), especially for tumors harboring an active mutation of epidermal growth factor receptor (EGFR). Approximately 90% of EGFR mutations are exon 19 deletions or exon 21 single-point L858R substitutions and are associated with sensitivity to EGFR tyrosine kinase inhibitors (EGFR TKIs), like gefitinib or erlotinib. However, less than 5% of EGFR-mutant NSCLC patients achieve a complete response to EGFR TKI and the overall median progression-free survival is no longer than 9-11 months. Accumulated studies report several mechanisms of early adaptive resistance that can occur as early as two hours after starting EGFR TKI therapy. The activation of signal transducer and activator of transcription 3 (STAT3) signaling is among these mechanisms of resistance. Furthermore, EGFR blockage enriches lung cancer stem cells through Notch3-dependent signaling pathway. We have previously demonstrated that NF-κB contributes to gefitinib resistance in EGFR-mutant NSCLC. The efficacy of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) is jeopardized by the activation of signaling pathways. We examined the relevance of co-targeting EGFR, signal transducer and activator of transcription 3 (STAT3) and Src-YES-associated protein 1 (YAP1) signaling. We conducted clinical and preclinical studies of key components of signaling pathways limiting EGFR TKI efficacy in EGFR-mutant NSCLC. High levels of STAT3 or YAP1 mRNA expression were associated with worse outcome to EGFR TKI in two independent cohorts of EGFR-mutant NSCLC patients. In the initial cohort of 64 patients, median progression-free survival was shorter among the patients with high STAT3 than among those with low STAT3 (hazard ratio [HR] for disease progression, 3·02; 95% confidence interval [CI], 1·54-5·93; P=0·0013). Median progression-free survival was shorter among the patients with high YAP1 than among those with low YAP1 (HR for disease progression, 2·57; 95%CI, 1·30-5·09; P=0·0067). The results were similar in the validation cohort of 55 patients. We demonstrated that gefitinib augments STAT3 signaling in EGFR-mutant NSCLC cells. Gefitinib with TPCA-1 (STAT3 inhibitor) blocked STAT3, but not the YAP1 phosphorylation on tyrosine residue 357 by Src family kinases (SFKs) that occurs downstream of IL-6. The triple combination of gefitinib, TPCA-1 and AZD0530 (SFK inhibitor) ablated both STAT3 and YAP1 phosphorylation and markedly and safely suppressed tumor growth. Added value of our research: We found that EGFR TKI therapy activates STAT3 and that EGFR blockage enriches lung cancer stem cells with up-regulation of the YAP1 and Notch downstream effectors connective tissue growth factor (CTGF) and hairy-enhancer of split-1 (HES1), respectively. We sought to demonstrate that EGFR TKI treatment cannot abrogate STAT3 and Src-YAP1-Notch activation in EGFR-mutant NSCLC cell lines, leading us to examine whether the combination of gefitinib with compounds targeting STAT3 and Src, suppresses the mechanisms of resistance. Nine days after gefitinib treatment, STAT3 mRNA level was significantly increased, as well as the fraction of ALDH positive cells. TPCA-1, a compound that targets STAT3, increases sensitivity to gefitinib in PC-9 and H1975 cells; however, neither gefitinib nor TPCA-1 inhibits Src or YAP1. The addition of the Src inhibitor, saracatinib, to the doublet of gefitinib and TPCA-1, was highly synergistic and abrogated STAT3, and Src-YAP1-Notch signaling. Implications: Treatment with single EGFR TKI can no longer be considered adequate for patients with EGFR mutant NSCLC. Our findings ultimately suggest that a clinical trial evaluating the co-targeted inhibition of STAT3 and Src is warranted. As a result, STAT3 and YAP1 mRNA levels could become important predictive biomarkers. We searched PubMed for English language reports published up to December, 2015 using the terms “non-small-cell lung cancer”, “STAT3”, “interleukin-6”, “NF-κB”, “aldehyde-dehydrogenase (ALDH)”, “integrin-linked kinase (ILK)”, “glycoprotein 130 (gp130)”, “Src-homology 2 domain-containing phosphatase 2 (SHP2)”, “the complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing protein-1 (CDCP1)”, “AXL”, “ephrin type-A receptor-2 (EphA2)”, “Src family kinases (SFK)”, “YES-associated protein 1 (YAP1)”, “Notch”, “cell migration, invasion and metastases” and “STAT3 inhibitors”. STAT3, EGFR TKI, YAP1, NSCLC

P3.02b-047 Co-Activation of STAT3 and YAP1 Signaling Pathways Limits EGFR Inhibitor Response in Lung Cancer: Topic: EGFR Clinical

EGFR tyrosine kinase inhibitors (TKIs) induce early activation of several signaling pathways. Interleukin-6 (IL-6) and signal transducer and activator of transcription 3 (STAT3) hyper-activation occur following EGFR TKI therapy in EGFR-mutant NSCLC cells. We explored the relevance of co-targeting EGFR, STAT3 and Src-YES-associated protein 1 (YAP1) signaling in EGFR-mutant NSCLC. We combined in vitro and in vivo approaches to explore whether concomitant activation of STAT3 and Src-YAP1 can limit the effectiveness of EGFR TKIs in EGFR-mutant NSCLC cells and xenograft models. In two cohorts of EGFR-mutant NSCLC patients, we examined messenger RNA (mRNA) gene expression within signaling pathways, leading to EGFR TKI resistance. Gefitinib suppressed EGFR, ERK1/2 and AKT phosphorylation but increased STAT3 phosphorylation (pSTAT3-Tyr705). In EGFR mutant cells, gefitinib plus TPCA-1 (STAT3 inhibitor) abolished pSTAT3-Tyr705 but not the YAP1 phosphorylation on tyrosine 357 by Src family kinases (SFKs). The triple combination of gefitinib, TPCA-1 and AZD0530 (SFK inhibitor) ablated both STAT3 and YAP1 phosphorylation and was highly synergistic, according to the combination index. In two EGFR mutant xenograft mouse models, the triple combination of gefitinib, TPCA-1 and AZD0530 markedly and safely suppressed tumor growth. High levels of STAT3 or YAP1 mRNA expression were associated with worse outcome to EGFR TKI in 64 EGFR-mutant NSCLC patients. Median progression-free survival (PFS) was 9.6 (95%CI, 5.9-14.1) and 18.4 months (95% CI, 8.8-30.2) for patients with high and low STAT3 mRNA, respectively (p<0.001), (HR for disease progression, 3.02; 95% CI, 1.54-5.93; p=0.0013). Median PFS was 9.6 (95% CI, 7.7-15.2) and 23.4 months (95%CI, 13.0-28.1) for patients with high and low YAP1 mRNA, respectively (p=0.005), (HR for disease progression, 2.57; 95% CI, 1.30-5.09; p=0.0067). The results were similar in the validation cohort of 55 EGFR-mutant NSCLC patients treated with first-line EGFR TKI in the Department of Oncology of Shanghai Pulmonary Hospital. Our study reveals that STAT3 and Src-YAP1 signaling activation occurs following single EGFR TKI in EGFR-mutant NSCLC. STAT3 and YAP1 mRNA levels were significantly predictive of progression-free survival in the original as well as in the validation cohort of EGFR-mutant NSCLC patients. Co-targeting STAT3 and Src in combination with EGFR TKI could substantially improve survival.

Wilms Tumor 1 (WT1) mRNA Expression Level at Diagnosis Is a Significant Prognostic Marker in Elderly Patients with Myelodysplastic Syndrome

Background/Aims: A high expression of Wilms tumor 1 (WT1) mRNA occurs in most cases of acute leukemia and myelodysplastic syndrome (MDS). Although there are many reports suggesting that acute myeloid leukemia patients with high expression levels of WT1 mRNA have a relatively poor long-term survival, there are few reports addressing the relationship between WT1 levels and prognosis in MDS. Methods: We retrospectively analyzed 42 elderly patients with MDS whose WT1 levels at diagnosis were available, and we assessed the relationships between WT1 levels in peripheral blood and preexisting prognostic factors such as World Health Organization prognostic scores and Revised International Prognostic Scoring System risk categories, bone marrow blast percentages, and chromosomal abnormalities linked to a poor prognosis. We also evaluated the relationship between WT1 levels and prognosis. Results:WT1 levels were significantly different between high- and low-risk MDS patients (p < 0.05). There was a trend towards a significant difference between those with and those without poor prognostic chromosomal rearrangements (p = 0.051). Moreover, the overall survival and progression-free survival were significantly worse in elderly patients with higher levels of WT1 (p = 0.00039 and p = 0.00077, respectively). Conclusions: The WT1 mRNA expression level at diagnosis may be a significant independent prognostic marker for elderly patients with MDS.

Molecular characterization and functional analysis of a piscidin gene in large yellow croaker (Larimichthys crocea).

The piscidin family, which includes potent antimicrobial peptides with broad-spectrum activity, plays an important role in the innate immune system of fish. In this study, we cloned piscidin-5-like type 3 (Lcpis5lt3) in large yellow croaker (Larimichthys crocea). Multiple alignments with other known piscidins revealed amino acid conservation throughout the fish, especially at the signal peptide (22 amino acids). The phylogenetic tree confirmed that Lcpis5lt3 and large yellow croaker piscidin-5-like proteins were grouped together to form a branch. Quantitative real-time PCR revealed that Lcpis5lt3 was expressed in a wide range of tissues, including the brain, muscle, gill, head kidney, intestine, kidney, liver, and spleen. The highest mRNA expression level of Lcpis5lt3 was found in the spleen. After Vibrio alginolyticus infection, mRNA expression was rapidly upregulated in the liver, head kidney, gill, kidney, and intestine at 4, 8, 12, and 24 h post infection (hpi), whereas there were no significant changes in the spleen. The antimicrobial spectrum showed that the synthetic mature peptide of Lcpis5lt3 exhibited different activity in vitro against various bacteria, such as Aeromonas hydrophila, V. anguillarum, V. alginolyticus, V. parahaemolyticus, Staphylococcus aureus, and Listeria monocytogenes. In addition, survival rates from the in vivo assay indicated that the synthetic peptide of Lcpis5lt3 increased the survival rate of large yellow croaker after V. alginolyticus challenge, resulting in a decline in bacterial burden and mRNA expression levels of interleukin-1β, interleukin-10, and tumor necrosis factor-α. These data suggest that Lcpis5lt3 plays an important role in innate immunity in large yellow croaker and might represent a potential therapeutic agent against pathogen invasion.

Adenosine receptors expression in cardiac fibroblasts of patients with left ventricular dysfunction due to valvular disease

Context: Adenosine restores tissue homeostasis through the interaction with its membrane receptors (AR) expressed on fibroblasts, endothelial cells, smooth muscle cells and leukocytes, but their modulation is still not fully understood.Objective: To evaluate whether changes in the transcriptomic profiling of adenosine receptors (AR) occur in cardiac fibroblasts (CF) of patients (pts) with LV dysfunction due to valvular disease (V). The secondary aim was to compare in the same pts the results obtained at cardiac level with those found in circulating leukocytes.Materials and methods: Auricle fragments were excised from 13 pts during prosthetic implantation while blood samples were collected from pts (n = 9) and from healthy subjects (C, n = 7). In 7 pts cardiac biopsy and blood samples were taken simultaneously. A human CF atrial cell line (cc) was used as control.Results: AR higher levels of mRNA expression were observed with real-time PCR in Vpts compared to C, both at cardiac (overexpression A1R:98%, A2AR:63%, A2BR:87%, A3R:85%, CD39:92%, CD73:93%) and at peripheral level (A1R vs C: p = .0056; A2AR vs C: p = .0173; A2BR vs C: p = .0272; A3R vs C: p = .855; CD39 vs C: p = .0001; CD73 vs C: p = .0091).Conclusion: All AR subtypes were overexpressed in CF of Vpts. The same trends in AR expression at cardiac level was assessed on circulating leukocytes, thus opening a new road to minimally invasive studies of the adenosinergic system in cardiac patients.

PELI1 expression is correlated with MYC and BCL6 expression and associated with poor prognosis in diffuse large B-cell lymphoma

AbstractPELI is a family of E3 ubiquitin ligases that regulate protein activity through a post-translational modification, ubiquitination. While PELI1 has been found to play a pivotal role in inflammatory processes through the activation of Toll-like receptor signaling and the NF-kB pathway, the role of PELI1 in oncogenesis has not been the subject of much investigation. We aimed to explore PELI1 expression in various malignant lymphomas and identify clinicopathologic significance. Immunohistochemistry for PELI1 was performed on a total of 502 cases, including 406 B-cell, 76 T or NK-cell, and 20 Hodgkin lymphomas. High expression of PELI1 was found in high-grade B-cell lymphoma cases such as diffuse large B-cell lymphoma, Burkitt lymphoma, and plasmablastic lymphoma, whereas low-grade B-cell lymphoma, T/NK-cell lymphoma, and Hodgkin lymphoma cases showed very low levels of expression. In vitro cell line studies, the results of western blot, and RT-PCR were concordant with those of the immunohistochemical results; RL7, Pfeiffer, SUDHL-2, DOHH2, and Ramos cell lines showed high levels of PELI1 protein and mRNA expression. In 182 diffuse large B-cell lymphoma, PELI1 expression was positively correlated with the expression of MYC, BCL6, BCL2, and MUM1 (Spearman's ρ=0.427, 0.507, 0.246, and 0.137, respectively; P<0.001, <0.001, 0.001, and 0.066, respectively). In diffuse large B-cell lymphoma, high expression of PELI1 was associated with frequent bone marrow involvement (P=0.013) and shorter relapse-free survival (P=0.002). Our results suggest that PELI1 might participate in B-cell maturation or oncogenic activation of aggressive B-cell lymphomas, both during and after germinal center stages.

Comparative characteristics of the transcriptional activity of CDH1, CTNNB1, VEGFA genes and expression of proteins E-cadherin, β-catenin and VEGFA, coded by these genes in metastatic and non-metastatic endometrioid endometrial carcinoma

The violation of Wnt-signaling pathway and cyclic neoangiogenesis is early events in endometrial carcinogenesis. In this process an important role is played by epigenetic modifications and mutations of CDH1, CTNNB1, VEGFA genes, followed by expression violations of E-cadherin, β-catenin, VEGFA encoded by them. The aim . To study the expression levels of mRNA of CDH1, CTNNB1, VEGFA genes and the expression levels of E-cadherin, β-catenin, VEGFA in non-metastatic endometrioid endometrial carcinoma (EEC) and in EEC with metastases to regional lymph nodes. Materials and methods. Immunohistochemical study of invasive рТ 1-4 N 0 M x endometrioid endometrial carcinoma (EEC) (n=56; age – 42–83 years), invasive рТ 1-4 N 1-2 M x EEC (n=30; age – 48–79 years), samples of normal proliferative endometrium (PE) (n=30; age – 41–62 years) was performed. Molecular-genetic study of invasive рТ 1-4 N 0 M x EEC (n=10; age – 45–67 years), invasive рТ 1-4 N 1-2 M x EEC (n=10; age – 44–63 years), samples of PE (n=10; age – 46–59 years) was performed. Results. EEC is characterized by the lower mRNA expression level of CDH1 gene and by the higher mRNA expression levels of CTNNB1, VEGFA genes in comparison with the PE. E-cadherin expression level is 32.6 % lower in non-metastatic EEC and also is 58.10 % lower in metastatic EEC in comparison with the PE. β-catenin expression level is 32.56 % lower in non-metastatic EEC and also is 58.11 % lower in metastatic EEC in comparison with the PE. VEGFA expression level is 27.72 % higher in non-metastatic EEC and also is 17.87 % higher in metastatic EEC in comparison with the PE. There is lower β-catenin expression level in metastatic EEC in comparison with non-metastatic EEC. Non-metastatic and metastatic EEC is characterized by the direct medium connections between the mRNA expression levels of CTNNB1 and VEGFA genes (γ=0.44), between the mRNA expression level of CTNNB1 gene and VEGFA expression level in the tumor (γ=0.37). Conclusions . These changes contribute to tumor growth, invasion and metastasis.

MRNA expression of delta-HER2 and its clinicopathological correlation in HER2-overexpressing breast cancer.

Delta-human epidermal growth factor receptor 2 (HER2) is a splice variant of HER2, which lacks 16 amino acids in the extracellular domain. The aim of the present study was to elucidate the expression of delta-HER2 and its clinicopathological correlation in Japanese patients with HER2-overexpressing breast cancer. A total of 40 cases of HER2-overexpressing breast cancer were investigated. The mRNA expression levels of wild type (wt)-HER2 and delta-HER2 were quantitated by quantitative polymerase chain reaction using RNA extracted from formalin-fixed paraffin-embedded specimens. In addition, the activated form of phosphorylated-SRC (pSRC) and Ki-67 were detected by immunostaining. The mRNA expression levels of wt-HER2 varied largely, and the mRNA levels of delta-HER2 were correlated with those of wt-HER2. Furthermore, the enhanced immunostaining of pSRC was detected in 15 cases. Unsupervised hierarchal clustering of wt-HER2, delta-HER2 and pSRC was used to separate the cases into three clusters. In the first cluster, the levels of wt-HER2 and delta-HER2 were low, and pSRC immunostaining was low. Two other clusters were characterized by moderately and highly increased levels of wt-HER2 and delta-HER2 mRNA expression and enhanced pSRC. The delta/wt ratio was higher in the first cluster. Positive lymph node status and recurrence were more frequent in the first cluster compared with those in the two other clusters. Furthermore, the delta/wt ratio was significantly increased in lymph node-positive and recurrent cases, compared with in lymph node-negative and non-recurrent cases. The present study demonstrated that delta-HER2 was expressed in Japanese patients with HER2-overexpressing breast cancer. High mRNA expression levels of delta-HER2 were associated with pSRC and increased proliferation of tumor cells. A poor prognosis may be predicted by the increase in delta/wt ratio in HER2-overexpressing breast cancer.

Reduced mRNA expression levels of NFE2L2 are associated with poor outcome in breast cancer patients.

BackgroundThe transcription factor nuclear factor erythroid 2-related factor 2 (NFE2L2; previously known as NRF2) is a crucial regulator of the intracellular antioxidant response. It controls the expression of genes involved in the detoxification and elimination of reactive oxidants and electrophilic agents. The role of NFE2L2 in cancer is subject of controversial discussion, as it has been reported to have both pro-and anti-tumourigenic functions. To shed some light on this paradox, we analysed the NFE2L2 mRNA expression levels in breast cancer and its association with clinicopathological features and survival.MethodsWe retrospectively evaluated the NFE2L2 mRNA expression levels in tumour tissue of two independent breast cancer patient cohorts. In the training set we analysed data from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). In the test set we measured the NFE2L2 mRNA expression levels in 176 breast tumour tissues by quantitative real-time reverse transcription PCR (qRT-PCR). Group differences were analysed using Mann–Whitney U-test, and associations between NFE2L2 mRNA expression levels and clinicopathological features were examined by means of univariate and multivariate survival analyses. Furthermore, we compared NFE2L2 mRNA expression levels between tumour and normal breast tissue samples by means of 108 paired samples from the The Cancer Genome Atlas (TCGA) dataset.ResultsIn the training set we identified an independent predictive value for high NFE2L2 mRNA expression levels [HRdisease specific death 0.8 (0.6–1.0), P = 0.041; HRdeath 0.8 (0.6–1.0), P = 0.023] especially in the subgroup of oestrogen receptor (ER) positive tumours [HRdisease specific death 0.6 (0.4–0.9), P = 0.008; HRdeath 0.6 (0.4–0.8), P = 0.001]. Similarly, we found this association also in the test set [HRrelapse 0.4 (0.2–0.9), P = 0.031] and again, more pronounced in patients with ER positive tumours [HRrelapse 0.2 (0.1–0.7), P = 0.012]. In addition, we observed generally lower NFE2L2 expression levels in tumour tissues than in normal breast tissues.ConclusionWe concluded that reduced NFE2L2 mRNA expression in tumour tissues is an independent predictor of shortened survival in breast cancer patients.

A Periodontal pathogen Porphyromonas gingivalis deteriorates Isoproterenol-Induced myocardial remodeling in mice.

Heart failure is a serious disease induced by several conditions, including hypertrophic cardiomyopathy. Although many reports suggest that there is an association between periodontal disease and cardiovascular disease, the mechanisms have yet to be elucidated. The purpose of this study was to clarify the relationship between periodontal disease and heart disease, especially in cardiac hypertrophy. We used C57BL/6J mice and implanted two types of subcutaneous chambers. First, we subcutaneously implanted a coil-shaped chamber into the back of a mouse. Porphyromonas gingivalis (P.g.), a major periodontal pathogen, was injected into the chamber. Then, an osmotic pump was implanted to infuse isoproterenol. Four weeks after the ISO infusion, we performed echocardiography and harvested the heart and blood. We measured the serum level of anti-P.g.-IgG using ELISA. The mRNA levels of several factors were measured using PCR. We found stronger cardiomyocyte hypertrophy in the ISO(+)/P.g.(+) mice compared with the ISO(+)/P.g.(-) mice. The total square of randomly selected cardiomyocytes was 23% larger in the ISO(+)/P.g.(+) mice than in the ISO(+)/P.g.(-) mice. We detected a higher level of mRNA expression in Toll-like receptor 2 and NADPH oxidase 4 in the ISO(+)/P.g.(-) mice compared with the control group. We revealed that a periodontal pathogen affected ISO-induced cardiac hypertrophy via oxidative stress.

Expression and significance of MET gene in non-small cell lung cancer

Objective To detect the mRNA expression of mesenchymal-epithelial transition factor (MET) gene in patients with non-small cell lung cancer (NSCLC), and to investigate the relationship and clinical significance between the mRNA expression of MET gene and clinical pathological characteristics. Methods From June 2011 to November 2013, 48 patients with pathologically confirmed NSCLC in Chinese People′s Liberation Army General Hospital were selected. All patients included in the study were not treated before surgery. The branched DNA liquid chip technology was used to detect mRNA expression of MET gene in tumor tissues. The relationship between mRNA expression of MET gene and clinical pathological characteristics was analyzed. Results The constituent ratios of low, moderate and high mRNA expression level of MET gene were 22.9%, 50.0% and 27.1% respectively, mainly for the moderate expression. The mRNA expression of MET gene was related to the pathologic type (χ2=7.183, P=0.020) and TNM stage (χ2=24.566, P=0.017) of the patients; but it was not related to the gender (χ2=0.566, P=0.754), age (χ2=1.857, P=0.395), smoking history (χ2=4.959, P=0.084), degrees of differentiation (χ2=5.749, P=0.067), lymph node metastasis (χ2=1.631, P=0.442) and distant metastasis (χ2=4.261, P=0.119). Conclusion mRNA expression of MET gene is more likely to present moderate and higher level in NSCLC patients. MET gene can also be used as a biomarker for judging tumor pathological type. Key words: Carcinoma, non-small-cell lung; RNA, messenger; Mesenchymal-epithelial transition factor

T-3256336, a novel and orally available small molecule IAP antagonist, induced tumor cell death via induction of systemic TNF alpha production

Inhibitors of apoptosis proteins (IAPs) are a family of antiapoptotic regulators that have attracted attention as potential targets for cancer therapeutics. Although recent studies have revealed that small-molecule IAP antagonists induce tumor selective cell death in an autocrine tumor necrosis factor (TNF)α-dependent manner, the single-agent efficacy of IAP antagonists is restricted to a small subset of cancer cells. In this study, we showed that the single-agent activity of T-3256336 was limited to a few cancer cell lines in vitro, and these cell lines were defined by relatively high levels of TNFα mRNA expression. However, some other cancer cells, including PANC-1 cells, become drastically sensitive to T-3256336 when costimulated with exogenous TNFα. In PANC-1 mouse xenograft models, the administration of T-3256336 increased levels of several cytokines including TNFα and lead to tumor regression as a single agent, which was attenuated by the neutralization of circulating mouse TNFα with an antibody. These results suggest dual roles of IAP antagonists, increase systemic cytokines including TNFα, and sensitization of tumors to IAP antagonist-induced death.

Molecular characterization and expression analysis of toll-like receptor 2 in response to bacteria in silvery pomfret intestinal epithelial cells

Toll-like receptor 2 (TLR2) has been shown to play a crucial role in the host defense of pathogenic microbes in innate immunity. In this study, the full-length cDNA of TLR2 in silvery pomfret (Pampus argenteus) was cloned by homology cloning and the rapid amplification of cDNA ends (RACE) technique. The complete cDNA sequence of TLR2 was 2932 bp, containing an open reading frame (ORF) of 2469 bp encoding 822 amino acids. A multiple alignment analysis of the silvery pomfret TLR2 protein-coding sequence with other known TLR2 sequences from Oplegnathus fasciatus, Epinephelus coioides, Larimichthys crocea, Miichthys miiuy, Oreochromis niloticus, Paralichthys olivaceus, Trematomus bernacchii, Sparus aurata, and Chionodraco hamatus exhibited a high degree of homology of 78.83%, 75.91%, 74.21%, 74.94%, 71.95%, 72.57%, 73.68%, 75%, and 72.52 respectively, between these fish. Analysis of the TLR2 domain structures indicated that TLR2 from the silvery pomfret has the typical structural features of proteins that belong to the TLR family, including one transmembrane domain, eleven leucine-rich repeats (LRRs), and one Toll/IL-1 receptor homology domain (TIR). In vitro immunostimulation experiments revealed that Lactobacillus plantarum and Clostridium butyricum induce high levels of TLR2 mRNA and protein expression, but they induce only moderate levels of IL-8 and TNF-α production compared to Vibrio anguillarum. This suggests that TLR2 might play a vital role in the L. plantarum and C. butyricum-mediated immune response. In contrast, V. anguillarum significantly increased the secretion of IL-8 and TNF-α and induced cell apoptosis and necrosis. Due to the lower expression of TLR2 and higher levels of IL-8 and TNF-α induced by V. anguillarum, we hypothesize that a V. anguillarum infection is independent of the TLR2-induced production of pro-inflammatory cytokines. These results indicate that TLR2 may be involved in molecular interactions between the host and commensal bacteria, that exist in the silvery pomfret intestinal tract.