Nuclear Expression Levels Research Articles

Abstract B083: Dysregulation of the AR-Nrdp1-ErbB3 axis occurs in African American prostate cancer patients and is associated with worse outcomes

Abstract Background: We previously showed that Nrdp1, an E3 ubiquitin ligase, is transcriptionally regulated by the androgen receptor (AR) in prostate cancer (CaP) cells and that Nrdp1 can post-translationally regulate ErbB3 (an EGFR family member) levels via ubiquitination and subsequent proteasomal degradation. Increased levels of ErbB3 are associated with worse CaP patient outcome. The goal of the current study was to determine whether dysregulation of the AR-Nrdp1-ErbB3 axis contributes to prostate cancer health disparities for African American (AA) men, and to identify the underlying mechanisms involved. Methods: Expression levels and localization of AR, Nrdp1, and ErbB3 were assessed in a total of 208 CaP patient samples (50 African American (AA) and 158 Caucasian (CA) CaP patients) and two cell lines (LNCaP (CA) and MDAPCa2b (AA)). Expression levels and localization of these molecules were determined by immunohistochemistry (IHC) and subcellular fractionation and western blot. A combination of knockdown (siRNA), forced overexpression (Nrdp1-FLAG construct), and treatment with enzalutamide or synthetic androgen (R1881) experiments were employed to investigate the relationship between AR, Nrdp1, and ErbB3 expression levels and localization and to identify differences in AA and CA cell lines. Immunoprecipitation allowed for investigation of the mechanisms which facilitate nuclear transport of Nrdp1. Results: A statistically significant negative association between cytoplasmic levels of AR (AR C) and nuclear Nrdp1 (Nrdp1 N) exists in both Caucasian (CA) and African American (AA) prostate cancer (CaP) patients, but this association was stronger in AA patients compared to CA patients (Spearman correlation coefficient of -0.62 for AA patients, and -0.36 for CA patients). Increased nuclear Nrdp1 expression levels were associated with increased 5-year survival rates, and reduced nuclear Nrdp1 expression levels predicted biochemical recurrence (AUC 0.63). Dysregulation of the AR-Nrdp1-ErbB3 axis in AA CaP cells was also observed in cell line studies; AA CaP cells expressed significantly lower levels of both total and nuclear Nrdp1 compared to their CA CaP counterparts. Manipulation of AR expression levels and localization via knockdown and inhibition with enzalutamide had a lesser impact on Nrdp1 expression levels and localization in AA versus CA CaP cells. Immunoprecipitation studies demonstrated that Nrdp1 can bind to AR in CA CaP cells but not AA CaP cells. Conclusions: Our combined data indicate that dysregulation of the AR-Nrdp1-ErbB3 axis occurs to a larger extent in AA than CA CaP cells and that this can contribute to worse patient outcomes. Our data also indicate that lack of binding between AR and Nrdp1 in AA CaP cells may account for the lower levels of nuclear Nrdp1 observed in AA CaP cells. Further understanding of the mechanisms which regulate Nrdp1 levels and localization may allow for the development of treatments which help abrogate cancer health disparities. Citation Format: Anhao Sam, Shawna Evans, Elizabeth Browning, Sheryl Krig, Neelu Batra, Thomas Steele, Salma Siddqui, Paramita Ghosh, Ruth Vinall. Dysregulation of the AR-Nrdp1-ErbB3 axis occurs in African American prostate cancer patients and is associated with worse outcomes [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B083.

Cathepsin L promotes angiogenesis by regulating the CDP/Cux/VEGF-D pathway in human gastric cancer

BackgroundIncreasing evidence indicates that angiogenesis plays an important role in tumor progression. The function of cathepsin L (CTSL), an endosomal proteolytic enzyme, in promoting tumor metastasis is well recognized. The mechanisms by which CTSL has promoted the angiogenesis of gastric cancer (GC), however, remains unclear.MethodsThe nuclear expression levels of CTSL were assessed in GC samples. The effects of CTSL on GC angiogenesis were determined by endothelial tube formation analysis, HUVEC migration assay, and chick embryo chorioallantoic membrane (CAM) assay. The involvement of the CDP/Cux/VEGF-D pathway was analyzed by angiogenesis antibody array, Western blot, co-immunoprecipitation (Co-IP) and dual-luciferase reporter assay.ResultsIn this study, we found that the nuclear CTSL expression level in GC was significantly higher than that in adjacent nontumor gastric tissues and was a potential important clinical prognostic factor. Loss- and gain-of-function assays indicated that CTSL promotes the tubular formation and migration of HUVEC cells in vitro. The CAM assay also showed that CTSL promotes angiogenesis of GC in vivo. Mechanistic analysis demonstrated that CTSL can proteolytically process CDP/Cux and produce the physiologically relevant p110 isoform, which stably binds to VEGF-D and promotes the transcription of VEGF-D, thus contributing to the angiogenesis of GC.ConclusionThe findings of the present study suggested that CTSL plays a constructive role in the regulation of angiogenesis in human GC and could be a potential therapeutic target for GC.

Liraglutide Ameliorates Lipotoxicity-Induced Oxidative Stress by Activating the NRF2 Pathway in HepG2 Cells.

Although glucagon-like peptide-1 (GLP-1) analogue has been reported to suppress oxidative stress in non-alcoholic fatty liver disease (NAFLD), an effective therapeutic agent for NAFLD is currently unavailable. Therefore, in this study, we aimed to investigate the protective effects of the GLP-1 analogue liraglutide against lipotoxicity-induced oxidative stress in HepG2 cells and to elucidate the underlying mechanisms. HepG2 cells were cultured for 48 hours and treated with a free fatty acid (FFA) mixture: FFA mixture and liraglutide or FFA mixture, liraglutide, and exendin (9-39). Lipid accumulation was examined by oil red O staining. Oxidative stress was assessed by measuring the levels of intracellular reactive oxygen species using 2',7'-dichlorofluorescein diacetate and thiobarbituric acid-reactive substances, whereas antioxidant capacity was assessed by measuring the activity of superoxide dismutase and catalase. Expression of the nuclear factor erythroid-2-related factor 2 (NRF2) gene and the genes encoding antioxidant enzymes was analyzed using quantitative RT-PCR. Cellular and nuclear NRF2 expression levels were assessed using immunofluorescence cell staining and western blotting. Liraglutide treatment reduced high fat-induced lipid formation and the levels of oxidative stress markers and increased antioxidant enzyme activity in HepG2 cells. Liraglutide treatment increased the mRNA expression of NRF2 target genes, induced NRF2 nuclear translocation, and increased nuclear NRF2 levels without altering NRF2 mRNA expression. Collectively, these results indicate that liraglutide exhibits a protective effect against lipotoxicity-induced oxidative stress, possibly via modulation of NRF2 and expression of antioxidant enzymes in liver cells.

Correlation between p53 expression and
 hepatocellular carcinoma differentiation grade

Relevance: Hepatocellular carcinoma (HCC) is one of the world’s most common liver cancers and fatal malignant neoplasms. According to GLOBOCAN 2018, liver cancer ranks sixth in incidence (842,000 new cases) and fourth in mortality
 (782,000 deaths) globally. In Kazakhstan, there is an increase in HCC incidence from 879 cases in 2016 up to 984 cases in 2017,
 which amounted to 5.5 cases per 100 000 population. In 2017, the five-year survival rate was 23.7%.
 The mutations in the p53 gene that encodes the p53 protein are most frequent in HCC (35.2%). A high level of nuclear expression of p53 and a decrease in apoptosis of tumor cells with a simultaneous increase in their proliferative activity, noted in
 HCC cells, are important for the prognosis of the disease. There is a correlation between poor prognosis, a decreased survival of
 patients with HCC, and high expression of the p53 oncoprotein in HCC.
 U. Nzeako et al. have revealed a strong correlation of HCC histological differentiation with the patient survival rate. Therefore, a study of the correlation between p53 expression and HCC differentiation grade is very relevant.
 The purpose of this study was to analyze the correlation between p53 expression and HCC differentiation grade.
 Results: p53 was overexpressed in 18% of HCC cases; the expression was high in 62% and low in 20% of HCC cases. In
 well-differentiated HCC (G1), the p53-immunopositive nuclei expression was low in 10 cases and high in 2 cases. In moderately
 differentiated HCC (G2), p53 was highly expressed in 21 cases and overexpressed in 2 cases. In poorly-differentiated HCC (G3),
 p53 was overexpressed in 7 cases and highly expressed in 8 cases of HCC. The analysis of the correlation between p53 expression and HCC differentiation grade by Spearman showed a high correlation (r=0.79, p<0.01), evidencing a direct dependence
 of p53 expression level on HCC gradation.
 Conclusion: HCC differentiation grade highly correlates with p53 expression level in immunopositive nuclei of HCC cells.
 The obtained data evidences a dependence between p53 expression level and HCC gradation.

Cytoplasmic expression of B7-H3 and membranous EpCAM expression are associated with higher grade and survival outcomes in patients with clear cell renal cell carcinoma

B7-H3 and EpCAM are overexpressed in cancer and play a role in tumorigenesis and metastasis. In this study, the membranous, cytoplasmic and nuclear expression levels of B7-H3 and EpCAM biomarkers were mapped in three major subtypes of renal cell carcinoma (RCC). Expression of B7-H3 and EpCAM were evaluated using immunohistochemistry in RCC samples on tissue microarrays (TMAs), including clear cell RCCs (ccRCCs), type I and II papillary RCCs (pRCCs), and chromophobe RCCs (chRCCs). The association between B7-H3 and EpCAM expression and clinicopathological features as well as survival outcomes was determined. There was a statistically significant difference between B7-H3 and EpCAM expression among the different RCC subtypes. In ccRCC, higher cytoplasmic expression of B7-H3 was significantly associated with increase in nucleolar grade, lymph node invasion (LNI), invasion of the Gerota's fascia, and tumor necrosis, while no association was found with the membranous and nuclear expression. Moreover tumors with cytoplasmic expression of B7-H3 tended to have a worse prognosis for disease-specific survival (DSS) than those with membranous expression. In case of EpCAM, increased membranous expression of EpCAM was associated with nucleolar grade and tumor necrosis in ccRCC. Additionally, membranous EpCAM expression added prognostic value in patients with ccRCC who had high nucleolar grade versus low nucleolar grade. Moreover, membranous EpCAM expression was found to be an independent favorable prognostic marker for progression-free survival (PFS) in ccRCC. Our results demonstrated that higher cytoplasmic B7-H3 and membranous EpCAM expression are clinically significant in ccRCC and are associated with more aggressiveness tumor behavior.

Inflammatory breast cancer cells are characterized by abrogated TGFβ1-dependent cell motility and SMAD3 activity.

Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with elevated metastatic potential, characterized by tumor emboli in dermal and parenchymal lymph vessels. This study has investigated the hypothesis that TGFβ signaling is implicated in themolecular biology of IBC. TGFβ1-induced cell motility and gene expression patterns were investigated in three IBC and three non-IBC (nIBC) cell lines. Tissue samples from IBC and nIBC patients were investigated for the expression of nuclear SMAD2, SMAD3, and SMAD4. SMAD protein levels were related to gene expression data. TGFβ1-induced cell motility was strongly abrogated in IBC cells (P = 0.003). Genes differentially expressed between IBC and nIBC cells post TGFβ1 exposure revealed attenuated expression of SMAD3 transcriptional regulators, but overexpression of MYC target genes in IBC. IBC patient samples demonstrated a near absence of SMAD3 and -4 expression in the primary tumor compared to nIBC patient samples (P < 0.001) and a further reduction of staining intensity in tumor emboli. Integration of gene and protein expression data revealed that a substantial fraction of the IBC signature genes correlated with SMAD3 and these genes are indicative of attenuated SMAD3 signaling in IBC. We demonstrate attenuated SMAD3 transcriptional activity and SMAD protein expression in IBC, together with obliterated TGFβ1-induced IBC cell motility. The further reduction of nuclear SMAD expression levels in tumor emboli suggests that the activity of these transcription factors is involved in the metastatic dissemination of IBC cells, possibly by enabling collective invasion after partial EMT.

Expression level of androgen receptors in tumor tissue and its prognostic significance in primary operable luminal breast cancer without overexpression of Her2/neu in postmenopausal women

Aim. To evaluate the expression level and prognostic significance of androgen in primary operable luminal breast cancer without overexpression of Her2/neu in postmenopausal women.&#x0D; Methods. We analyzed treatment outcomes of 60 cases of primary operable (T1-2N0M0) luminal breast cancer without overexpression of Her2/neu in postmenopausal women. The follow-up period was 5 years. All cases were divided by immunohistochemical method into luminal A (20 females) and luminal B (40 patients) subtypes. Along with the standard panel of immunohistochemical markers, expression of nuclear androgen receptors was measured in tumor tissues of all patients. Depending on the expression levels, patients with luminal A and B subtypes were divided into three groups: (1) with high, (2) moderate and (3) low or negative expression.&#x0D; Results. Mean levels of androgen receptor expression in the nuclei of tumor cells in patients with luminal A subtype (57.3±5.9%) were higher than those of luminal B subtype (21.4±4.04%) by 62.7% (Mann-Whitney test, р=0.0026). In patients with luminal A subtype, the maximal accumulation of androgen receptors in the nuclei of tumor cells was 2.7 times higher (р=0.0023) than in patients with luminal B subtype. All cases diagnosed with the disease progression were characterized by low or negative level of nuclear androgen receptor expression.&#x0D; Conclusion. Negative and low levels of androgen receptor expression in tumor tissues of postmenopausal patients with luminal primary operable breast cancer without overexpression of Her2/neu might be an independent factor associated with poor prognosis.

The regulative effects of levetiracetam on adult hippocampal neurogenesis in mice via Wnt/β-catenin signaling

Adult hippocampal neurogenesis plays the pivotal roles in central nervous system diseases. Recently, it has been reported that levetiracetam (LEV), a new antiepileptic drug with novel chemical construction and unique pharmacological properties, suppressed aberrant adult subventricular zone (SGZ) neurogenesis in kainite-induced epileptic mice, while promoted adult SGZ neuroblast differentiation in normal mice. These studies indicate LEV can modulate adult hippocampal neurogenesis, but the exact mechanism remained unknown. Thus, the present study aimed to investigate the effects of subchronic and chronic LEV treatments on neural stem cell by lineage tracing in adult hippocampal dentate gyrus of mice, as well as the potential mechanism related to Wnt/β-catenin signaling pathway. The data showed that both subchronic and chronic LEV treatments had no effects on body weight, locomotor activity and anxiety-like behavior in mice. Notably, subchronic LEV treatment significantly suppressed the proliferation of intermediate progenitor cell and neuroblast, decreased the number of intermediate progenitor cell and neuroblast, but increased the number of quiescent neural stem cell. On the contrary, chronic LEV treatment promoted the proliferation of neural stem cell, intermediate progenitor cell and neuroblast, increased the number of neural stem cell, intermediate progenitor cell and neuroblast, and promoted differentiation of newborn immature neuron and mature neuron. Furthermore, subchronic LEV treatment decreased the level of Wnt 3a and nuclear β-Catenin expression, which led to the inhibition on Wnt/β-catenin signaling pathway. Chronic LEV treatment increased the level of Wnt 3a, cytosolic β-catenin and nuclear β-Catenin, decreased the expression of GSK-3β, p-Tyr216-GSK-3β and Axin2, resulting in the enhancement of Wnt/β-catenin signaling pathway. These results demonstrated that LEV significantly suppressed or promoted adult hippocampal neurogenesis in mice by subchronic or chronic treatment possibly through the regulation of Wnt/β-catenin signaling pathway. Our findings provided the new perspectives of LEV on adult hippocampal neurogenesis underlying its clinical application.

High nuclear NADPH oxidase 4 expression levels are correlated with cancer development and poor prognosis in hepatocellular carcinoma

NADPH oxidase (NOX) is a key source of reactive oxygen species (ROS). This study aimed to verify NOX2 and NOX4 expression levels in hepatocellular carcinoma (HCC). A total of 134 matched pairs of HCC cells and non-tumour hepatocytes from 134 patients were examined by immunohistochemical staining, and the association of NOX2 and NOX4 expression with clinicopathological parameters was analysed. Western blotting in four HCC cell lines and reverse transcription digital droplet polymerase chain reaction (RT-ddPCR) in 20 pairs of HCC and non-tumour tissue samples were also performed to detect NOX4. Cytoplasmic NOX2 and nuclear NOX4 expression levels were shown by immunohistochemistry to be higher in HCC cells than in non-tumour hepatocytes (p<0.001 each). The western blotting results for NOX4 in four HCC cell lines were consistent with the immunohistochemical results. Increased cytoplasmic expression of NOX2 and NOX4 in HCC cells was significantly correlated with liver cirrhosis (p<0.001 and p<0.031, respectively). However, decreased cytoplasmic expression of NOX2 and NOX4 was significantly correlated with advanced pathological TNM stage (p<0.029 and p<0.007, respectively). Multivariate analysis with clinicopathological parameters showed that high nuclear and low cytoplasmic NOX4 expression levels are correlated with short overall survival (p=0 .021). Our findings imply that cytoplasmic NOX2 and nuclear NOX4 expression is upregulated during HCC development. In particular, NOX4 translocation into the nucleus may affect the development and progression of HCC. NOX2 and NOX4 could be diagnostic markers and have therapeutic implications in HCC.

Effect of estradiol on high glucose‑induced osteoblast injury.

Estradiol (E2) serves an important role in the changes of postmenopausal bone turnover rate and the development of osteoporosis. The present study aimed to investigate the effects of E2 on high glucose (HG)-induced osteoblast injury. Cell Counting Kit-8 was used to determine cell viability. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting was used to analyze the mRNA and protein expression levels of osteocalcin, Runt-related transcription factor 2 (Runx2), nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO1). Flow cytometry was performed to analyze apoptosis. The results revealed that cell viability was lower in cells treated with HG (100, 200 or 300 mg/dl) compared with the control group. Cell viability was decreased in cells treated with 200 mg/dl HG on days 3, 5 and 7. In addition, cell viability was increased by 0.1 µM E2. E2 with HG co-treatment increased cell viability, osteocalcin and Runx2 mRNA expression levels and nuclear Nrf2 and HO1 protein expression levels compared with the HG-only group. All these changes, with the exception of Runx2, were reversed by silencing Nrf2 expression using small interfering (si)RNA (siNrf2). Additionally, apoptosis was reduced by E2 in HG-treated cells, which was reversed by siNrf2 transfection. These results demonstrated that E2 may prevent HG-induced osteoblast injury by activating Nrf2/HO1 signaling pathways.

Increased glucocorticoid receptor activity and proliferation in metastatic colon cancer

Metastasis is regarded as the fatal hallmark for colon cancer, but molecular mechanisms responsible for it have remained poorly defined. Glucocorticoid receptor (GR) within the tumor microenvironment mediates the effects of stress hormones which are used in clinics for their inflammation-modulatory and immunosuppressive properties. Further, epigenetic activation of GR promotes tumor heterogeneity and metastasis. Here, we sought to investigate the correlation between GR activation and proliferation and invasion in metastatic colon cancer microenvironment. We used proliferation/invasion assays, western blot, RT-qPCR, immunofluorescence staining and quantitative methylation to study glucocorticoid-GR signaling, including the involvement of CDK1, in human colon adenocarcinoma cell lines HT29 and T84 (a representative metastatic cell line). Nuclear expression levels of GR were significantly upregulated in metastatic T84 cells, and glucocorticoid derivative, dexamethasone (DEX) treatment caused increased proliferation and invasion in T84 cell, compared to HT29 cell. DEX treatment induced CDK1 expression which was accompanied by reduced CDK1 methylation, indicating epigenetic regulation. Depletion of GR suppressed proliferation of metastatic colon carcinoma cells and depletion of CDK1 had similar suppressing effects on proliferation as well as invasion of metastatic cells. Our study suggests that glucocorticoid-GR-CDK1 signaling induces proliferation and invasion of colon cancer cells and therapies involving the use of glucocorticoids need to exercise caution and re-evaluation.

Temporal expression patterns of the melatoninergic system in the human thymus of children

ObjectivesTo obtain greater knowledge of the extra-pineal sources of melatonin during development, the amount of indolamine and the expression levels of the last two enzymes involved in its biosynthesis, Arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT), were analyzed in the human thymus from children from three different age groups (from days to years). The melatonin membrane and nuclear receptor expression levels also were studied. MethodsQuantitative reverse transcriptase PCR and western blot were performed to investigate the receptor and enzyme expression levels. The results were examined and correlated with the ages of the thymuses. ResultsWe found high levels of indolamine in the thymuses of newborns (younger than 1 month), which decreased during development; thymuses from the months (from 2 to 11 months) and years (from 1 to 12 years) groups showed lower levels. A similar decline was also observed in the mRNA of the AANAT enzyme and the expression levels of melatonin receptors. However, ASMT expression was exactly the opposite, with low levels in the newborn group and higher levels in the years group. Our results show that the thymic synthesis of melatonin occurs very early in childhood. Additionally, this is the first report that is focused on melatonin receptors expression in the human thymus. ConclusionConsidering the limited melatonin synthesis performed by the newborn pineal gland, we suggest that the high levels of melatonin found in human thymus in this experimental group arise from synthesis in the tissue itself, which could be contributing to the immune efficiency at the thymic level.

Expressions of Sry-related High Mobility Group Box 9 and Gastrokine-1 in Gastric Cancer and Their Relationships with Prognosis

Objective To explore the expressions of Sry-related high mobility group box 9(SOX9)and gastrokine-1(GKN1) in gastric cancer tissues and their relationships with clinicopathologic features and prognosis of patients.Methods Immunohistochemistry was used to detect the expressions of SOX9 and GKN1 in 70 cases of gastric cancer tissues and corresponding paracancerous tissues including 27 cases of intestinal metaplasia and 43 cases of normal gastric mucosa. The relationships of SOX9 and GKN1 expressions with clinicopathological features and prognosis were analyzed in gastric cancer tissues.Results The high expression rates of SOX9 in gastric cancer tissues,intestinal metaplasia,and normal gastric mucosa were 92.9%(65/70),77.8%(21/27),and 55.8%(24/43),respectively(χ 2=21.722,P<0.001). Positive nuclear and cytoplasmic staining was observed. The high nuclear expression rate of SOX9 in gastric cancer tissues was 67.1%,which was significantly higher than those of intestinal metaplasia(37.0%,P=0.007)and normal gastric mucosa(23.3%,P<0.001). The high cytoplasmic expression rate of GKN1 in normal gastric mucosa was 76.7%,which was significantly higher than those of intestinal metaplasia(44.4%,P=0.006)and gastric cancer tissues(37.1%,P<0.001). Univariate analysis demonstrated that the nuclear expression of SOX9 in gastric cancer was associated with the degree of tissue differentiation(P=0.007),while the cytoplasmic expression of GKN1 was associated with both the degree of tissue differentiation(P=0.002)and whether the pathological type was a signet-ring cell carcinoma(P=0.009). Furthermore,the nuclear expression of SOX9 was negatively correlated with the expression of GKN1 in gastric cancer(χ 2=15.424,P<0.001). The 5-year survival rates of patients with high or low nuclear expression of SOX9 were 33.8% and 67.5%,respectively(P=0.016).The 5-year survival rates of patients with high or low expression of GKN1 were 60.0% and 35.6%,respectively(P=0.044). Further research indicated that 5-year survival rate of patients with high nuclear expression of SOX9 and low expression of GKN1 was 28.8%. Cox multivariate regression analysis showed that TNM stage(stage Ⅱ:HR=7.435,95%CI:1.313-42.096,P=0.023;stage Ⅲ:HR=12.214,95%CI:2.677-55.721,P=0.001)and nuclear expression level of SOX9(HR=3.297,95%CI:1.199-9.065,P=0.021)were independent risk factors for the prognosis of gastric cancer patients.Conclusions Changes in the expressions of SOX9 and GKN1 may be associated with the malignant biological behavior of gastric cancer. SOX9 may be a potential prognostic factor. The combined detection of SOX9 and GKN1 expression and the further study of their molecular mechanism may provide new clues for early diagnosis,targeted therapy,and prognostic prediction of gastric cancer.

Loss of nuclear localization of thyroid transcription factor 1 and adverse outcomes in papillary thyroid cancer

Function of the thyroid follicular cell depends on nuclear expression of thyroid transcription factor 1 (TTF1). Regulation of this key protein regulating iodide transport is not well known, but its loss is linked to the most lethal of thyroid malignancies. We examined TTF1 nuclear expression in the context of adverse pathological features, disease recurrence, and BRAF status in papillary thyroid carcinomas with (n = 182) and without (n = 303) nodal metastases. Overall nuclear expression level of TTF1 was strong and diffuse in approximately 73%, whereas 27% exhibited lower levels or a paucity of nuclear staining. In the same cohort, approximately 59% exhibited the BRAF mutation. On univariate analysis, low levels of TTF1 nuclear expression was linked to vascular invasion, extrathyroidal extension, and nodal metastases. Multivariate analysis indicated that low levels of TTF1 were most strongly linked to nodal metastases and vascular invasion. Interestingly, TTF1 levels were not linked to the BRAF mutation. TTF1 staining alone predicted disease recurrence, but when combined with BRAF status, the 2 markers exhibited a more marked influence. Patients lacking the BRAF mutation and exhibiting normal levels of TTF1 exhibited very low levels of disease recurrence (11% at 10 years). Conversely, patient tumors with low levels of TTF1 and the BRAF mutation recurred in 31% of cases in the same time frame. The mixed expression of BRAF under varying levels of differentiation may explain, in part, the contradictory studies regarding the impact of BRAF mutations on patient prognosis and also indicates a complex genomic signature for dedifferentiated thyroid cancer.

EGR1 IS NEGATIVELY ASSOCIATED WITH HNSCC CELL INVASION VIA INHIBITION OF MMP9 AND MDM2

Objectives The effect of early growth response-1 (EGR1) on cancer invasion remains controversial depending on the cancer type. EGR1 is known to slow the progression of cancer by inhibiting the expression of MMP2. However, the effect of EGR1 on MMP9, which is important for HNSCC invasion, is disputed. Our aim is to clarify the tumor suppressor role of EGR1 in downregulating MMP9. We also consider MDM2, an enhancer of MMP9 expression. Findings EGR1 mRNA and protein expression were compared in normal and HNSCC tissues using The Cancer Genome Atlas (TCGA) dataset analysis as well as immunohistochemistry (IHC). In vitro cell invasion was performed by two-dimension (2-D) and three-dimension (3-D) spheroids Matrigel invasion assay. TCGA data showed significantly higher EGR1 mRNA levels in nonmetastatic HNSCC tissues than in metastatic tissues. IHC analysis showed significantly higher levels of nuclear EGR1 expression in primary tumor tissues than in paired metastatic lymph node tissues. Transient EGR1 overexpression inhibited the Matrigel invasion of HNSCC cells, as well as decreasing mRNA of MMP9 and MDM2. Consistent with these observations, TCGA data analysis found significantly fewer metastatic patients among a subgroup of a large population presenting higher EGR1 expressions with lower MMP9 and/or MDM2. Conclusions Our data suggests that EGR1 might be a potential candidate to attenuate HNSCC metastasis.

Chitosan oligosaccharide-mediated attenuation of LPS-induced inflammation in IPEC-J2 cells is related to the TLR4/NF-κB signaling pathway

The protective mechanism of chitosan oligosaccharide (COS) against lipopolysaccharides (LPS) -induced inflammatory responses in IPEC-J2 and in mice with DSS dextran sulfate sodium (DSS) -induced colitis is reported. Upon exposure to LPS, the proliferation rate of IPEC-J2 cells markedly decreased, and epithelial cell integrity was compromised. However, COS pretreatment significantly reduced these changes. Low-concentration (200 μg/mL) COS up-regulated Toll-like receptor 4 (TLR4) and nuclear p65 expression, but inhibited LPS-induced expression of nuclear p65, IL-6, and IL-8. Addition of the TLR4 inhibitor reduced nuclear p65, IL-6, and IL-8 expression in IPEC-J2 cells exposed to COS or LPS alone, and a slight up-regulation in nuclear p65 was observed in COS and LPS co-treated cells. Medium-dose COS (600 mg/kg/d) protected against DSS-induced colitis, in which TLR4 and nuclear p65 expression levels were decreased. We postulate that the prevention of both LPS- and DSS -induced inflammatory responses in IPEC-J2 cells and mice by COS are related to the inhibition of the TLR4/NF-κB signaling pathway.

Circular RNA CDR1as acts as a sponge of miR-135b-5p to suppress ovarian cancer progression.

Background and objectives: Ovarian cancer is the most fatal primary malignancy among gynecological cancers. Circular RNAs (circRNAs) play an important role in the development of various cancers, but the functions of circRNAs in ovarian cancer development have not been studied. We aim to explore the function and mechanism of CDR1as in the progression of ovarian cancer and to provide a new target for the diagnosis and treatment of ovarian cancer.Methods: Ovarian cancer cell proliferation was assessed according to proliferating cell nuclear antigen (PCNA) and Ki67 protein expression levels and MTT and CCK8 assays. The migration ability of cells was detected by scratch-wound assays, and the invasion ability of the cells was determined by Transwell® assays. qRT-PCR and Western blotting were used to verify miRNA and protein expression.Results: CDR1as expression in ovarian tissues was significantly lower in ovarian cancer patients than in patients without ovarian cancer. CDR1as overexpression inhibited the proliferation, invasion and migration of ovarian cancer cells. Silencing CDR1as increased the expression of miR-135b-5p and decreased the expression of hypoxia-inducible factor 1-alpha inhibitor (HIF1AN), thus increasing the proliferation capacity of ovarian cancer cells.Conclusions: CDR1as, acting as a sponge of miR-135b-5p, promotes the expression of HIF1AN and therefore plays a role in tumor inhibition.

Differential Expression of HMGA1 and HMGA2 in pituitary neuroendocrine tumors

Defining biomarkers for invasive pituitary neuroendocrine tumors (PitNETs) is highly desirable. The high mobility group A (HMGA) proteins are among the most widely expressed cancer-associated proteins. Indeed, their overexpression is a frequent feature of human malignancies, including PitNETs. We show that nonfunctioning PitNETs (NF-PitNETs) express significantly higher levels of HMGA1 than somatotropinomas (GHs) and corticotropinomas (ACTHs). Furthermore, HMGA2 expression was detected only in NF-PitNETs and was significantly higher in larger tumors than in smaller tumors. HMGA expression analysis generally focuses on nuclear staining. Here, cytoplasmic HMGA staining was also found. PitNETs displayed strong nuclear HMGA1 and strong cytoplasmic HMGA2 immunoreactivity. Interestingly, the HMGA1 and HMGA2 nuclear expression levels were significantly higher in invasive adenomas than in noninvasive adenomas. The highest levels of nuclear HMGA2 were found in GHs. In conclusion, we show that overexpression of nuclear HMGA proteins could be a potential biomarker of invasive PitNETs, particularly HMGA2 for GHs. HMGA2 might be a reliable biomarker for NF-PitNETs.

Expression of tSTAT3, pSTAT3727 , and pSTAT3 705 in the epithelial cells of hormone-naïve prostate cancer.

BackgroundThe signal transducer and activator of transcription 3 (STAT3) pathway is observed to be constitutively activated in several malignancies including prostate cancer (PCa). In the present study, we investigated the expression of total STAT3 (tSTAT3) and two forms of activated phosphorylated STAT3 (pSTAT3727 and pSTAT3705) in tissue microarrays (TMA) of two cohorts of localized hormone‐naïve PCa patients and analyzed associations between the expression and disease outcome.MethodsThe expression of tSTAT3, pSTAT3727, and pSTAT3705 was scored in the nuclei and cytoplasm of prostatic gland epithelial cells in two TMAs of paraffin‐embedded prostatic tissue. The TMAs consisted of tissue originated from hormone‐naïve radical prostatectomy patients from two different sites: Malmö, Sweden (n = 300) and Dublin, Ireland (n = 99).ResultsThe nuclear expression levels of tSTAT3, pSTAT3727, and pSTAT3705 in the epithelial cells of benign glands were significantly higher than in the cancerous glands. Cytoplasmic tSTAT3 levels were also higher in benign glands. Patients with low pSTAT3727 and pSTAT3705 levels in the cancerous glands showed reduced times to biochemical recurrence, compared with those with higher levels. No significant trends in nuclear nor in cytoplasmic tSTAT3 were observed in relation to biochemical recurrence in the Malmö cohort. Higher cytoplasmic tSTAT3 was associated with reduced time to biochemical recurrence in the Dublin cohort. Adding the tSTAT3 and pSTAT3 expression data to Gleason score or pathological T stage did not improve their prognostic values.ConclusionsLow pSTAT3727 and pSTAT3705 expression in epithelial cells of cancerous prostatic glands in hormone‐naïve PCa was associated with faster disease progression. However, pSTAT3 and tSTAT3 expression did not improve the prognostic value of Gleason score or pathological T stage and may not be a good biomarker in the early hormone naïve stages of PCa.

Nuclear lncRNA HOXD-AS1 suppresses colorectal carcinoma growth and metastasis via inhibiting HOXD3-induced integrin \u03b23 transcriptional activating and MAPK/AKT signalling

BackgroundLong noncoding RNAs (lncRNAs) have been indicated to play critical roles in cancer development and progression. LncRNA HOXD cluster antisense RNA1 (HOXD-AS1) has recently been found to be dysregulated in several cancers. However, the expression levels, cellular localization, precise function and mechanism of HOXD-AS1 in colorectal carcinoma (CRC) are largely unknown.MethodsReal-time PCR and in situ hybridization were used to detect the expression of HOXD-AS1 in CRC tissue samples and cell lines. Gain- and loss-of-function experiments were performed to investigate the biological roles of HOXD-AS1 in CRC cell line. RNA pull down, RNA immunoprecipitation and chromatin immunoprecipitation assays were conducted to investigate the mechanisms underlying the functions of HOXD-AS1 in CRC.ResultsWe observed that HOXD-AS1 was located in the nucleus of CRC cells and that nuclear HOXD-AS1 was downregulated in most CRC specimens and cell lines. Lower levels of nuclear HOXD-AS1 expression were associated with poor outcomes of CRC patients. HOXD-AS1 downregulation enhanced proliferation and migration of CRC cells in vitro and facilitated CRC tumourigenesis and metastasis in vivo. Mechanistic investigations revealed that HOXD-AS1 could suppress HOXD3 transcription by recruiting PRC2 to induce the accumulation of the repressive marker H3K27me3 at the HOXD3 promoter. Subsequently, HOXD3, as a transcriptional activator, promoted Integrin β3 transcription, thereby activating the MAPK/AKT signalling pathways.ConclusionOur results reveal a previously unrecognized HOXD-AS1-HOXD3-Integrin β3 regulatory axis involving in epigenetic and transcriptional regulation constitutes to CRC carcinogenesis and progression.