The tamoxifen-regulated, long non-coding RNA LINC00992 affects proliferation, migration, and expression of tamoxifen resistance-associated genes in MCF-7 breast cancer cells.

Introduction: Tamoxifen-adapted MCF-7 breast cancer cells (MCF-7-TAM-R) are a model for acquired tamoxifen resistance in oestrogen receptor-positive breast cancer. In this system, the expression of long-non-coding RNA LINC00992 is decreased. LINC00992 might therefore contribute to tamoxifen adaption and associated gene expres-sion changes. Here, we investigated whether a modulation of LINC00992 modifies gene expression, proliferation, and migration. Material and methods: Up- and down-- regulation of LINC00992 was performed using plasmid vectors and siRNA. Gene expression was measured via nCounter® and quantitative real-time polymerase chain reaction. Database analysis was performed using GEPIA2 and cBioportal. Furthermore, we performed scratch assays, colony-forming assays, and proliferation assays with MCF-7 and MCF-7-TAM-R after up-regulation of LINC00992. Results: Up- and down-regulation of LINC00992 caused gene expression changes in 4 of the 42 tamoxifen-regulated genes tested. Especially ubiquitin D, single-minded homologue 1 (SIM1) carcinoembryonic antigen-related cell adhesion molecule 5 and the G-protein coupled oestrogen receptor 1 were affected. In tamoxifen-adapted MCF-7-TAM-R cells, LINC00992 overexpression resulted in augmented viability and proliferation and enhanced migration. Database analyses revealed that luminal breast cancers have increased expression of LINC00992 compared to Her2-type/neu- or basal type. Furthermore, higher expression of LINC00992 was associated with poor prognosis in luminal-A carcinomas. Conclusions: Changes in the expression of tamoxifen-regulated genes could be induced by manipulating LINC00992's abundance, suggesting that it is at least partially involved in the establishment of the tamoxifen-induced gene expression pattern. LINC00992 may also serve as a prognostic biomarker and may indicate the development of tamoxifen resistance.

GPER-1 expression is associated with a decreased response rate to primary tamoxifen therapy of breast cancer patients.

PURPOSE: Endocrine therapies using tamoxifen and/or aromatase inhibitors are important therapeutic options for the targeted treatment of hormone-responsive breast cancer. In addition to nuclear estrogen receptors ERα and ß, G-protein-coupled estrogen receptor GPER-1 is a third receptor-mediating estrogen effects in breast cancer cells. The aim of this study was to examine to what extent GPER-1 expression might affect the efficacy of primary endocrine treatment of breast cancer. METHODS: GPER-1 expression was determined in tissue samples from patients with early breast cancer by means of immunohistochemistry and a GPER-1 score of ≥ 3 was considered to be positive. In a total of 165 patients, the response to a primary therapy with tamoxifen (TAM) or aromatase inhibitors (AI) was assessed by ultrasound imaging for up to 6 months. The primary endpoint of this study was the response to treatment evaluated by RECIST 1.1 criteria. RESULTS: GPER-1 expression was observed in 127 (77.0%) out of 165 cases. Based on GPER-1 expression and the type of endocrine treatment, the patients were divided into 4 groups: GPER-1 negative/TAM (12.1%), GPER-1 negative/AI (10.9%), GPER-1 positive/TAM (44.8%), and GPER-1 positive/AI (32.1%). The groups were well balanced regarding different clinical and pathological factors. After 4 and 6 months of treatment, a high level of stable disease or progressive disease was observed in the GPER-1 positive/TAM group only (p < 0.0001), whereas in the other three groups of patients, the most common objective response was classified as partial response. We observed a continuous reduction of mean tumor size in patients treated with aromatase inhibitors irrespective of the GPER-1 status and in GPER-1 negative patients treated with TAM. In contrast, in GPER-1 positive patients treated with TAM, a reduction of mean tumor size was observed only in the first 2 months after beginning of treatment. Four and six months after start of treatment, no reduction, but even a slight increase of tumor size was observed in this patients group. CONCLUSIONS: GPER-1 expression is significantly associated with a reduced effect of primary treatment with tamoxifen in breast cancer patients.

The IL-17A/IL-17RA Axis Is Not Related to Overall Survival and Cancer Stem Cell Modulation in Pancreatic Cancer.

(1) Background: IL-17A accelerates pancreatic intraepithelial neoplasia (PanIN) progression. In this study, we examined whether IL-17A/IL-17RA promotes pancreatic ductal adenocarcinoma (PDAC) aggressiveness in terms of survival and cancer stem cell modulation. (2) Methods: In vitro, the wound-healing assay, the sphere formation assay, and flow cytometry were applied to assess cancer stem cell features. In vivo, pancreatic tumors were induced in C57BL/6 mice using electroporation with oncogenic plasmids (P53-/- R172H; KrasG12V). Anti-IL-17 antibodies were administered as immunotherapy. We analyzed IL-17A/IL-17RA related survival using publicly available transcriptomic data (n = 903). (3) Results: IL-17A/IL-17RA expression was not related to survival in PDAC patients. IL-17A neither induces stem cell markers nor increases sphere formation and cell motility in vitro. Blocking the IL-17A/IL-17RA axis in a murine pancreatic cancer model did not improve the survival of mice, but reduced the tumor burden slightly. (4) Conclusions: IL-17A does not promote stem cell expansion in PDAC cell lines. Blocking IL-17A/IL-17RA signaling does not interfere with pancreatic cancer development and progression and may not be considered as a promising monotherapy for PDAC.

G-protein-coupled estrogen receptor GPER-1 expression in hormone receptor-positive breast cancer is associated with poor benefit of tamoxifen.

BACKGROUND: The role of G-protein-coupled estrogen receptor 1 (GPER-1) in the development of tamoxifen resistance in breast cancer is a highly controversial issue. The aim of this study was to determine the expression of GPER-1 in the clinical routine under conditions of endocrine treatment. PATIENTS AND METHODS: GPER-1 expression was analyzed in 442 patients with primary invasive breast cancer. GPER-1 score of > 3 was determined as positive. Expression data were correlated with clinical and pathological characteristics and patient survival. RESULTS: GPER-1 expression was observed in 352 (80.9%) cases, and positively correlated with estrogen and progesterone receptor status (p = 0.0001). GPER-1 positivity was associated with an increased grade of differentiation (p = 0.0001) and with a low level of Ki-67 expression (p = 0.0001). High GPER-1 expression was associated with a decreased level upon systemic treatment (p = 0.011). In the whole cohort, GPER-1 expression was associated with prolonged disease-free survival (DFS). DFS between tamoxifen- and aromatase inhibitor-treated GPER-1-positive patients was similar (p = 0.090). Notably, after matching the analysis for the most important prognostic factors, DFS for tamoxifen-treated GPER-1-positive patients was 69.1%, which is a percentage that is significantly lower compared to DFS for GPER-1-positive patients treated with aromatase inhibitors (92.7%) (p = 0.005). CONCLUSION: GPER-1 expression is a favorable prognostic factor in breast cancer patients. Its predictive role for poor benefit form tamoxifen treatment should be investigated in further studies.

Time resolved gene expression analysis during tamoxifen adaption of MCF-7 cells identifies long non-coding RNAs with prognostic impact.

Acquired tamoxifen resistance is a persistent problem for the treatment of estrogen receptor positive, premenopausal breast cancer patients and predictive biomarkers are still elusive. We here analyzed gene expression changes in a cellular model to identify early and late changes upon tamoxifen exposure and thereby novel prognostic biomarkers. Estrogen receptor positive MCF-7 cells were incubated with 4OH-tamoxifen (10 nM) and gene expression analyzed by array hybridization during 12 weeks. Array results were confirmed by nCounter- and qRT-PCR technique. Pathway enrichment analysis revealed that early responses concerned mainly amine synthesis and NRF2-related signaling and evolved into a stable gene expression pattern within 4 weeks characterized by changes in glucuronidation-, estrogen metabolism-, nuclear receptor- and interferon signaling pathways. As a large number of long non coding RNAs was subject to regulation, we investigated 5 of these (linc01213, linc00632 linc0992, LOC101929547 and XR_133213) in more detail. From these, only linc01213 was upregulated but all were less abundant in estrogen-receptor negative cell lines (MDA-MB 231, SKBR-3 and UACC3199). In a web-based survival analysis linc01213 and linc00632 turned out to have prognostic impact. Linc01213 was investigated further by plasmid-mediated over-expression as well as siRNA down-regulation in MCF-7 cells. Nevertheless, this had no effect on proliferation or expression of tamoxifen regulated genes, but migration was increased. In conclusion, the cellular model identified a set of lincRNAs with prognostic relevance for breast cancer. One of these, linc01213 although regulated by 4OH-tamoxifen, is not a central regulator of tamoxifen adaption, but interferes with the regulation of migration.

Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies.

Trefoil factor family 2 (TFF2) and the mucin MUC6 are co-secreted from human gastric and duodenal glands. TFF2 binds MUC6 as a lectin and is a constituent of the gastric mucus. Herein, we investigated human gastric extracts by FPLC and identified mainly high- but also low-molecular-mass forms of TFF2. From the high-molecular-mass forms, TFF2 can be completely released by boiling in SDS or by harsh denaturing extraction. The low-molecular-mass form representing monomeric TFF2 can be washed out in part from gastric mucosa specimens with buffer. Overlay assays with radioactively labeled TFF2 revealed binding to the mucin MUC6 and not MUC5AC. This binding is modulated by Ca2+ and can be blocked by the lectin GSA-II and the monoclonal antibody HIK1083. TFF2 binding was also inhibited by Me-ß-Gal, but not the α anomer. Thus, both the α1,4GlcNAc as well as the juxtaperipheral ß-galactoside residues of the characteristic GlcNAcα1→4Galß1→R moiety of human MUC6 are essential for TFF2 binding. Furthermore, there are major differences in the TFF2 binding characteristics when human is compared with the porcine system. Taken together, TFF2 appears to fulfill an important role in stabilizing the inner insoluble gastric mucus barrier layer, particularly by its binding to the mucin MUC6.

Inactivation of JNK2 as carcinogenic factor in colitis-associated and sporadic colorectal carcinogenesis.

We recently reported that dysregulated c-Jun N-terminal kinases (JNK) activity causes defective cell cycle checkpoint control, inducing neoplastic transformation in a cellular ulcerative colitis (UC) model. In the quiescent chronic phase of UC, p-p54 JNK was down-regulated and p-p46 JNK was up-regulated. Both were up-regulated in the acute phase. Consequently, increased p21WAF1 and γ-H2AX, two JNK-regulated proteins, induced cell cycle arrest. Their down-regulation led to checkpoint override, causing increased proliferation and undetected DNA damage in quiescent chronic phase, all characteristics of tumorigenesis. We investigated expression of p-JNK2, p-JNK1-3, p21WAF1, γ-H2AX and Ki67 by immunohistochemistry in cases of quiescent UC (QUC), active UC (AUC), UC-dysplasia and UC-related colorectal carcinoma (UC-CRC). Comparison was made to normal healthy colorectal mucosa, sporadic adenoma and colorectal carcinoma (CRC), diverticulitis and Crohns disease (CD). We found p-JNK2 up-regulation in AUC and its early down-regulation in UC-CRC and CRC carcinogenesis. With down-regulated p-JNK2, p21WAF1 was also decreased. Ki67 was inversely expressed, showing increased proliferation early in UC-CRC and CRC carcinogenesis. p-JNK1-3 was increased in AUC and QUC. Less increased γ-H2AX in UC-CRC compared to CRC gave evidence that colitis-triggered inflammation masks DNA damage, thus contributing to neoplastic transformation. We hypothesize that JNK-dependent cell cycle arrest is important in AUC, while chronic inflammation causes dysregulated JNK activity in quiescent phase that may contribute to checkpoint override, promoting UC carcinogenesis. We suggest restoring p-JNK2 expression as a novel therapeutic strategy to early prevent the development of UC-related cancer.

Oxidative stress and glyoxalase I activity mediate dicarbonyl toxicity in MCF-7 mamma carcinoma cells and a tamoxifen resistant derivative.

BACKGROUND: Acquired tamoxifen resistance is a significant problem in estrogen receptor positive breast cancer. In a cellular model, tamoxifen resistance was associated with increased sensitivity towards toxic dicarbonyls and reduced free sulfhydryl group content. We here analyzed the role of oxidative stress and glyoxalase I activity on dicarbonyl resistance and the significance of glyoxalase I expression for survival. METHODS: Reactive oxygen species were determined by 2,7-dihydrochlorofluorescein diacetate. Inhibitors for NADPH-oxidase (diphenyleneiodonium), p38 MAPK (SB203580) and ERK1/2 (UO126) were applied to investigate interactions of these signaling molecules. N-acetyl cysteine was used to evaluate the effect of oxidative stress on cell viability, which was assessed by the resazurin assay. Gene expression was analyzed by real time qRT-PCR. Glyoxalase activity was inhibited by the specific inhibitor CS-0683 and siRNA. The relevance of glyoxalase 1 mRNA abundance on survival of breast cancer patients was evaluated by the KM-plotter web interface. RESULTS: α-Oxo-aldehydes caused an immediate increase in reactive oxygen species where the tamoxifen resistant cell line (TamR) responded at lower concentrations than the MCF-7 parental cell line. Inhibitor studies placed ROS production by NADPH-oxidase downstream of p38 MAPK. The antioxidant N-acetyl cysteine (NAC) increased survival, whereas glyoxalase (GLO1) inhibition increased dicarbonyl toxicity. GLO1 mRNA abundance was correlated with unfavorable prognosis of breast cancer patients. CONCLUSIONS: Dicarbonyl toxicity was mediated by oxidative stress and GLO1 activity determines aldehyde toxicity in tamoxifen resistant cells. GENERAL SIGNIFICANCE: Glyoxalases might be predictive biomarkers for tamoxifen resistance and a putative target for the treatment of tamoxifen resistant breast cancer patients.

Accumulation of the advanced glycation end product carboxymethyl lysine in breast cancer is positively associated with estrogen receptor expression and unfavorable prognosis in estrogen receptor-negative cases.

Advanced glycation end products (AGEs) accumulate as a result of high concentrations of reactive aldehydes, oxidative stress, and insufficient degradation of glycated proteins. AGEs are therefore accepted biomarkers for aging, diabetes, and several degenerative diseases. Due to the Warburg effect and increased oxidative stress, cancer cells frequently accumulate significant amounts of AGEs. As the accumulation of AGEs may reflect the metabolic state and receptor signaling, we evaluated the potential prognostic and predictive value of this biomarker. We used immunohistochemistry to determine the AGE Nε-carboxymethyl lysine (CML) in 213 mammary carcinoma samples and Western blotting to detect AGEs in cell cultures. Whereas no significant correlation between hormone receptor status and CML was observed in cell lines, CML accumulation in tumors was positively correlated with the presence of estrogen receptor alpha, the postmenopausal state, and age. A negative correlation was found for grade III carcinomas and triple-negative cases. In a retrospective Kaplan-Meier survival analysis, there was a statistical trend that high CML accumulation correlated with a more favorable prognosis (relapse-free survival, RFS) under tamoxifen treatment (p = 0.1). In estrogen receptor-negative cases, the high CML content was significantly correlated with an unfavorable outcome (RFS) of chemotherapy (p = 0.046). CML is a therefore a potentially predictive marker for the treatment of breast cancer patients with tamoxifen or chemotherapy.

GPER Promoter Methylation Controls GPER Expression in Breast Cancer Patients.

Recently, we found that G-protein-coupled estrogen receptor (GPER) protein expression decreased during breast carcinogenesis, and that GPER promoter is methylated. Here we analyzed GPER promoter methylation in 260 primary breast cancer specimens by methylation-specific polymerized chain reaction. The results demonstrated that GPER protein down-regulation significantly correlated with GPER promoter hypermethylation (p < .001). Comparison of 108 tumors and matched normal breast tissues indicated a significant GPER down-regulation in cancer tissues correlating with GPER promoter hypermethylation (p < .001). The latter was an unfavorable factor for overall survival of patients with triple-negative breast cancer (p = .025). Thus GPER promoter hypermethylation might be used as a prognostic factor.

Protocol of the PANCALYZE trial: a multicenter, prospective study investigating the tumor biomarkers CXCR4, SMAD4, SOX9 and IFIT3 in patients with resected pancreatic adenocarcinoma to predict the pattern of recurrence of the disease.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies today with an urgent need for novel therapeutic strategies. Biomarker analysis helps to better understand tumor biology and might emerge as a tool to develop personalized therapies. The aim of the study is to investigate four promising biomarkers to predict the clinical course and particularly the pattern of tumor recurrence after surgical resection. DESIGN: Patients undergoing surgery for PDAC can be enrolled into the PANCALYZE trial. Biomarker expression of CXCR4, SMAD4, SOX9 and IFIT3 will be prospectively assessed by immunohistochemistry and verified by rt.-PCR from tumor and adjacent healthy pancreatic tissue of surgical specimen. Immunohistochemistry expression pattern of all four biomarkers will be combined into a single score. Beginning with the hospital stay clinical data from enrolled patients will be collected and followed. Different adjuvant chemotherapy protocols will be used to create subgroups. The combined biomarker expression score will be correlated with the further clinical course of the patients to test the hypothesis if CXCR4 positive, SMAD4 negative, SOX9 positive, IFIT3 positive tumors will predominantly develop metastatic spread. DISCUSSION: Pancreatic cancer is associated with different patterns of progression requiring personalized therapeutic strategies. Biomarker expression analysis might be a tool to predict the pattern of tumor recurrence and discriminate patients that develop systemic metastatic disease from those with tumors that rather develop local recurrence over time. This data might lead to personalized adjuvant treatment decisions as patients with tumors that stay localized might benefit from adjuvant local therapies like radiochemotherapy as compared to those with systemic recurrence who would benefit exclusively from chemotherapy. Moreover, the pattern of propagation might be a predefined characteristic of pancreatic cancer determined by the genetic signature of the tumor. In the future, biomarker expression analysis could be performed on tumor biopsies to develop personalized therapeutic pathways right after diagnosis of cancer. TRIAL REGISTRATION: German Clinical Trials Register, DRKS00006179 .

miR-221 Mediates Chemoresistance of Esophageal Adenocarcinoma by Direct Targeting of DKK2 Expression.

BACKGROUND: Chemoresistance is a main obstacle to effective esophageal cancer (EC) therapy. We hypothesize that altered expression of microRNAs (miRNAs) play a role in EC cancer progression and resistance to 5-fluorouracil (5-FU) based chemotherapeutic strategies. METHODS: Four pairs of esophageal adenocarcinoma (EAC) cell lines and corresponding 5-FU resistant variants were established. The expression levels of miRNAs previously shown to be involved in the general regulation of stem cell pathways were analyzed by qRT-PCR. The effects of selected miRNAs on proliferation, apoptosis, and chemosensitivity were evaluated both in vitro and in vivo. We identified a particular miRNA and analyzed its putative target genes in 14 pairs of human EC tumor specimens with surrounding normal tissue by qRT-PCR as well as Wnt pathway associated genes by immunohistochemistry in another 45 EAC tumor samples. RESULTS: MiR-221 was overexpressed in 5-FU resistant EC cell lines as well as in human EAC tissue. DKK2 was identified as a target gene for miR-221. Knockdown of miR-221 in 5-FU resistant cells resulted in reduced cell proliferation, increased apoptosis, restored chemosensitivity, and led to inactivation of the Wnt/ß-catenin pathway mediated by alteration in DKK2 expression. Moreover, miR-221 reduction resulted in alteration of EMT-associated genes such as E-cadherin and vimentin as well as significantly slower xenograft tumor growth in nude mice. RT profiler analysis identified a substantial dysregulation of 4 Wnt/ß-catenin signaling and chemoresistance target genes as a result of miR-221 modulation: CDH1, CD44, MYC, and ABCG2. CONCLUSION: MiR-221 controls 5-FU resistance of EC partly via modulation of Wnt/ß-catenin-EMT pathways by direct targeting of DKK2 expression. MiR-221 may serve as a prognostic marker and therapeutic target for patients with 5-FU resistant EAC.

The asphericity of the metabolic tumour volume in NSCLC: correlation with histopathology and molecular markers.

PURPOSE: Asphericity (ASP) is a tumour shape descriptor based on the PET image. It quantitates the deviation from spherical of the shape of the metabolic tumour volume (MTV). In order to identify its biological correlates, we investigated the relationship between ASP and clinically relevant histopathological and molecular signatures in non-small-cell lung cancer (NSCLC). METHODS: The study included 83 consecutive patients (18 women, aged 66.4 ± 8.9 years) with newly diagnosed NSCLC in whom PET/CT with 18F-FDG had been performed prior to therapy. Primary tumour resection specimens and core biopsies were used for basic histopathology and determination of the Ki-67 proliferation index. EGFR status, VEGF, p53 and ALK expression were obtained in a subgroup of 44 patients. The FDG PET images of the primary tumours were delineated using an automatic algorithm based on adaptive thresholding taking into account local background. In addition to ASP, SUVmax, MTV and some further descriptors of shape and intratumour heterogeneity were assessed as semiquantitative PET measures. RESULTS: SUVmax, MTV and ASP were associated with pathological T stage (Kruskal-Wallis, p = 0.001, p < 0.0005 and p < 0.0005, respectively) and N stage (p = 0.017, p = 0.003 and p = 0.002, respectively). Only ASP was associated with M stage (p = 0.026). SUVmax, MTV and ASP were correlated with Ki-67 index (Spearman's rho = 0.326/p = 0.003, rho = 0.302/p = 0.006 and rho = 0.271/p = 0.015, respectively). The latter correlations were considerably stronger in adenocarcinomas than in squamous cell carcinomas. ASP, but not SUVmax or MTV, showed a tendency for a significant association with the extent of VEGF expression (p = 0.058). In multivariate Cox regression analysis, ASP (p < 0.0005) and the presence of distant metastases (p = 0.023) were significantly associated with progression-free survival. ASP (p = 0.006), the presence of distant metastases (p = 0.010), and Ki-67 index (p = 0.062) were significantly associated with overall survival. CONCLUSION: The ASP of primary NSCLCs on FDG PET images is associated with tumour dimensions and molecular markers of proliferation and angiogenesis.

Dual time point imaging for F18-FDG-PET/CT does not improve the accuracy of nodal staging in non-small cell lung cancer patients.

OBJECTIVES: To analyze the diagnostic performance of dual time point imaging (DTPI) for pre-therapeutic lymph node (LN) staging in non-small cell lung cancer (NSCLC). METHODS: This was a retrospective analysis of 47 patients with NSCLC who had undergone DTPI by PET (early + delayed) using F18-fluorodeoxyglucose (FDG). PET raw data were reconstructed iteratively (point spread function + time-of-flight). LN uptake in PET was assessed visually (four-step score) and semi-quantitatively (SUVmax, SUVmean, ratios LN/primary, LN/liver, and LN/mediastinal blood pool). DTPI analyses included retention indices (RIs), Δ-ratios and changes in visual score. Histology or cytology served as standards of reference. Accuracy was determined based on ROC analyses. RESULTS: Thirty-six of 155 LNs were malignant. DTPI accuracy was low for all measures (visual assessment, 24.5%; RI SUVmax, 68.4%; RI SUVmean, 65.8%; Δ-ratios, 63.9-76.1%) and significantly inferior to early PET. Accuracies of early (range, 86.5-92.9%) and delayed PET (range, 85.2-92.9%) were comparable. At early PET, accuracy of the visual score (92.9%) was similar or superior to semi-quantitative analyses (range, 86.5-92.3%). CONCLUSIONS: Using a modern PET/CT device and novel image reconstruction, neither additional delayed PET nor DTPI analyses improved the accuracy of PET-based LN staging. Dedicated visual assessment criteria performed very well. KEY POINTS: • DTPI did not improve accuracy of PET-based LN staging in NSCLC. • Analyzed SUV ratios were not superior to LN SUVmax or SUVmean. • A four-step visual score may allow highly accurate, standardized LN assessment.

1,25-dihydroxyvitamin D3 inhibits corneal wound healing in an ex-vivo mouse model.

PURPOSE: Impaired healing of corneal injuries can result in ulceration and complete loss of vision, especially in the elderly. Such patients frequently also exhibit vitamin D insufficiency. 1,25-dihydroxyvitamin D3 is the active vitamin D metabolite. As it affects cell proliferation and inflammation, we herein aimed at elucidating its influence on corneal wound healing after alkali burn by using in vitro and ex vivo techniques. METHODS: mRNA abundance in human corneal epithelial cells in response to vitamin D3 was determined by RT-PCR. Corneal re-epithelialization after alkaline burn was analyzed using enucleated mouse eyes and fluorescein staining. RESULTS: Human corneal epithelial cells (HCEC) expressed the vitamin D receptor (VDR) and retinoid x receptor (RXR) and were responsive to 1,25- dihydroxyvitamin D3, as shown by induction of the 1,25- dihydroxyvitamin D3 responsive gene cyp-24A1 and slightly reduced abundance of IL-6 mRNA. However, no effect on cell vitality and migration was observed. In contrast, re-epithelialization of mouse corneas ex vivo was dose dependently inhibited by 1,25- dihydroxyvitamin D3. CONCLUSIONS: These data indicate that topically applied 1,25- dihydroxyvitamin D3 does not seem to be suitable for therapy of corneal lesions.

Sustained remission of blastic plasmacytoid dendritic cell neoplasm after unrelated allogeneic stem cell transplantation--a single center experience.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematodermic neoplasm which typically presents with skin infiltrates with or without lymphadenopathy and bone marrow involvement. No standard of care exists for this aggressive disease and prognosis is particularly poor. Here, we present our experience with nine BPDCN patients diagnosed at our institution between 2005 and 2012. BPDCN patients were identified in the databases at the Department of Hematology and Oncology, the Department of Dermatology, and the Institute of Pathology at the Otto-von-Guericke-University Magdeburg. There were six male and three female patients with a median age at diagnosis of 66 years. Sites involved were skin (five cases), lymph nodes (five cases), and bone marrow (five cases). Treatments varied from single agent chemotherapy to polychemotherapy and allogeneic stem cell transplantation for consolidation. The three patients that were treated with acute leukemia-type induction therapy followed by allogeneic stem cell transplantation (one after standard conditioning and two after reduced intensity conditioning using fludarabine in combination with thiotepa) achieved sustained remissions and are alive with a follow-up of 8, 35, and 41 months. In contrast, median survival in the less intensively treated patients was only 9.5 (range 1 to 29) months.

Human gastric TFF2 peptide contains an N-linked fucosylated N,N'-diacetyllactosediamine (LacdiNAc) oligosaccharide.

In the human stomach, the peptide trefoil factor family 2 (TFF2) is secreted together with the mucin MUC6 by mucous neck cells (MNCs) and antral gland cells. TFF2 is strongly associated with the gastric mucus and promotes gastric restitution. Here, TFF2 was purified from the human corpus and antrum, respectively, by size-exclusion chromatography, and the N-linked glycan structure at N-15 of the mature peptide was determined. As a hallmark, the unusual monofucosylated N,N'-diacetylhexosediamine (tentatively assigned as GalNAcß1 â†’ 4GlcNAc, LacdiNAc) modification was detected as the terminal structure of a bi-antennary complex type N-glycan exhibiting also core fucosylation. Replicate analyses did not show microheterogeneities in the fraction of peptide-N-glycosidase F cleaved and permethylated N-glycans when analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). On the glycopeptide level, a minor glycan microheterogeneity was evident in liquid chromatography-electrospray ionization (ESI)-MS, demonstrating the presence of underfucosylated species. The tryptic TFF2 N-glycopeptide p34-39 (LSPHNR N-glycosylated with Fuc3Hex3HexNAc6) was identified by both ESI-tandem mass spectrometry and MALDI-post-source decay analysis. Lectin analyses with the Wisteria floribunda agglutinin indicated the potential presence of LacdiNAc terminating glycans and revealed minor differences between TFF2 from fundic units, i.e. MNCs, and antral units, i.e. antral gland cells. Strikingly, on the level of the primary structure, there was no indication that the formation of the proposed LacdiNAc structure is cis-controlled by a peptidic determinant related to the published sequences.

GPER-1 expression decreases during breast cancer tumorigenesis.

GPER-1 protein expression was immunohistochemically examined in 164 primary breast cancer specimens and their matched normal breast epithelium. GPER-1 down-regulation correlated significantly with increased histological grading (p = .015), lymph node metastases (p = .032), and negative estrogen receptor status (p = .018). The decrease of GPER-1 expression in breast cancer tissue, relative to normal tissue, was associated with poor overall survival (p = .043) and disease-free survival (p = .037) and remained a significant unfavorable factor in multivariate analysis for DFS (HR = 1.569; 95% CI, 1.024-2.797; p = .041) and OS (HR = 2.082; 95% CI, 1.248-4.773; p = .039). Thus aberrant GPER-1 expression seems to be an important factor in breast cancer progression.

Expression of Eukaryotic Translation Initiation Factors in the Urothelial Carcinoma of the Bladder.

BACKGROUND/AIM: Urothelial carcinoma (UC) of the urinary bladder is the second most common tumor in the field of urology and is characterized by a relatively aggressive growth behavior. New therapeutic approaches are required to improve the prognosis of affected patients. We hypothesized a link between dysregulation of eIFs and the development of UC. Therefore, in the present work, we investigated the expression behavior of eIF1, eIF1AY, eIF1AX, eIF2α, eIF3a, eIF3b, eIF4B, eIF4E, eIF4G, eIF5A, eIF5B, and eIF6 in UC compared with that in urothelial tissue. MATERIALS AND METHODS: Paraffin-embedded tumor tissue samples from 107 patients suffering from UC were examined. Seventy-six patients contained adjacent urothelial tissue. Three tumor tissue cylinders (tumor collective) and two urothelial tissue cylinders (control collective) were collected per patient and embedded in tissue microarray (TMA) blocks. Immunohistochemical staining of the TMA sections was then performed. The staining results were assessed semi-quantitatively. Staining intensities and immunoreactive scores (IRS) of both collectives were compared. In each case, a distinction was made between cytoplasmic and nuclear staining. RESULTS: Significant up-regulation of eIF1AY, eIF2α, eIF3a, eIF3b, eIF4B, eIF4G, eIF5B, and eIF6 was found in the cytoplasm of UC. In contrast, eIF1 and eIF5A were significantly down-regulated in the cytoplasm of UC. eIF5A and eIF6 were significantly down-regulated in the nuclei of UC. CONCLUSION: Dysregulation of eIFs in the urothelium of the urinary bladder is linked to carcinogenesis at this site.