Vorinostat ameliorates IL-1α-induced reduction of type II collagen by inhibiting the expression of ELF3 in chondrocytes.

Osteoarthritis (OA) is a common joint disease that ultimately causes physical disability and imposes an economic burden on society. Cartilage destruction plays a key role in the development of OA. Vorinostat is an oral histone deacetylase (HDAC) inhibitor and has been used for the treatment of T-cell lymphoma. Previous studies have reported the anti-inflammatory effect of HDAC inhibitors in both in vivo and in vitro models. However, it is unknown whether vorinostat exerts a protective effect in OA. In this study, our results demonstrate that treatment with vorinostat prevents interleukin 1α (IL-1α)-induced reduction of type II collagen at both gene and protein levels. Treatment with vorinostat reduced the IL-1α-induced production of mitochondrial reactive oxygen species (ROS) in T/C-28a2 cells. Additionally, vorinostat rescued the IL-1α-induced decrease in the expression of the collagen type II a1 (Col2a1) gene and the expression of Sry-related HMG box 9 (SOX-9). Importantly, we found that vorinostat inhibited the expression of matrix metalloproteinase-13 (MMP-13), which is responsible for the degradation of type II collagen. Furthermore, vorinostat suppressed the expression of E74-like factor 3 (ELF3), which is a key transcription factor that plays a pivotal role in the IL-1α-induced reduction of type II collagen. Also, the overexpression of ELF3 abolished the protective effects of vorinostat against IL-1α-induced loss of type 2 collagen by inhibiting the expression of SOX-9 whilst increasing the expression of MMP-13. In conclusion, our findings suggest that vorinostat might prevent cartilage destruction by rescuing the reduction of type II collagen, mediated by the suppression of ELF3.

Assessment of mucin-related gene alterations following treatment with rebamipide ophthalmic suspension in Sjögren's syndrome-associated dry eyes.

Ocular surface mucins are thought to play vital roles in maintaining the homeostasis of the pre-ocular surface tear film. We performed ocular surface tests with impression cytology to assess the expression levels of mucin-related genes on the ocular surface in healthy eyes. In addition, we investigated alterations in mucin-related gene expression secondary to treatment with rebamipide ophthalmic suspension in patients with Sjögren's syndrome-associated dry eyes (SS-DE). Thirty-three healthy individuals (control group) and 13 patients from our hospital with SS-DE were enrolled. Impression cytology was performed using Schirmer's test paper for RNA sampling. The mRNA levels of SAM-pointed domain-containing ETS-like factor (SPDEF), mucin 5AC (MUC5AC), and mucin 16 (MUC16) were determined using a real-time reverse transcription-polymerase chain reaction. The ocular surface test was performed once for the control group, and at baseline as well as 2, 4, 8, and 12 weeks after treatment in the Sjögren's syndrome-associated dry eyes group. mRNA levels of SPDEF, MUC5AC, and MUC16 were not significantly different between the control and SS-DE groups before rebamipide ophthalmic suspension treatment. SPDEF mRNA levels in control subjects were significantly correlated with levels of MUC5AC. Among SS-DE patients, SPDEF mRNA levels were significantly increased at 2, 4, and 8 weeks after treatment compared with baseline levels. MUC16 mRNA levels were significantly decreased from baseline levels at 4 and 8 weeks post-treatment. Ocular surface test using impression cytology is a clinically useful tool for assessing mucous conditions on the ocular surface and can be used to determine the effects of instillation treatment with eye drops that affect mucin production at the ocular surface.

A novel ETV6-miR-429-CRKL regulatory circuitry contributes to aggressiveness of hepatocellular carcinoma.

BACKGROUND: Tumor metastasis is one of the main causes of the high mortality of hepatocellular carcinoma (HCC). E-Twenty Six variant gene 6 (ETV6) is a strong transcriptional repressor, associated with the development and progression of tumors. However, the exact role and underlying mechanism of ETV6 in HCC remain unclear. METHODS: Western blotting, quantitative real-time PCR and immunohistochemistry were used to detect the expression levels of ETV6, CRKL (v-crk sarcoma virus CT10 oncogene homologue (avian)-like) and miR-429 in HCC tissues and cells; Transwell chamber and F-actin cytoskeleton staining assay to examine the effects of ETV6 and CRKL deregulation on the migration, invasion and cytoskeleton of HCC cells; Co-immunoprecipitation assay to determine the interaction between CRKL and ETV6; Chromatin immunoprecipitation assay to investigate the interaction between ETV6 and miR-429. RESULTS: We established a novel ETV6-miR-429-CRKL regulatory circuitry contributes to HCC metastasis. ETV6 and CRKL were frequently increased, while miR-429 was downregulated in both hepatocarcinoma tissues and hepatocarcinoma cells. Moreover, ETV6 upregulation was positively correlated with CRKL upregulation, and two negative correlations were also established for ETV6 and CRKL upregulation with miR-429 downregulation in both hepatocarcinoma patients' tumorous tissues and hepatocarcinoma cells. Functional investigations revealed that overexpression and knockdown of ETV6 was remarkably effective in promoting and suppressing HCC cell migration, invasion, cytoskeleton F-actin expression and arrangement, whereas, CRKL overexpression exhibited similar effects to the overexpression of ETV6. Mechanistically, ETV6 negatively regulates miR-429 expression by directly binding to the promoter region of miR-429; miR-429 negatively regulates CRKL expression by selectively targeting CRKL-3'-UTR; ETV6 directly binds to CRKL and positively regulates its expression, which in turn CRKL positively regulates ETV6 expression. CONCLUSIONS: Our data demonstrated that ETV6 promotes migration and invasion of HCC cells by directly binding to promoter region of miR-429 via modulating CRKL expression. The newly identified ETV6-miR-429-CRKL regulatory circuitry contributes to the aggressiveness of HCC, which provides new clues for fundamental research on diagnosis and treatment parameters for HCC.

Silencing of ELK3 Induces S-M Phase Arrest and Apoptosis and Upregulates SERPINE1 Expression Reducing Migration in Prostate Cancer Cells.

ELK3, an ETS domain-containing transcription factor, participates in various physiological and pathological processes including cell proliferation, migration, angiogenesis, and malignant progression. However, the role of ELK3 in prostate cancer cells and its mechanism are not fully understood. The contribution of ELK3 to prostate cancer progression was investigated in the present study. We showed that silencing of ELK3 by siRNA in prostate cancer cell DU145 induced S-M phase arrest, promoted apoptosis, inhibited cell proliferation and migration in vitro, and suppressed xenograft growth in mice in vivo. In accordance with its ability to arrest cells in S-M phase, the expression of cyclin A and cyclin B was downregulated. In addition, the expression of p53 was upregulated following ELK3 knockdown, while that of antiapoptotic Bcl-2 was decreased. The migration inhibition may partly due to upregulation of SERPINE1 (a serine protease inhibitor) followed ELK3 knockdown. Consistently, downregulation of SERPINE1 resulted in a modest elimination of migration inhibition resulted from ELK3 knockdown. Furthermore, we found that the AKT signaling was activated in ELK3 knockdown cells, and treatment these cells with AKT inhibitor attenuated SERPINE1 expression induced by ELK3 silencing, suggesting that activation of AKT pathway may be one of the reasons for upregulation of SERPINE1 after ELK3 knockdown. In conclusion, modulation of ELK3 expression may control the progression of prostate cancer partly by regulating cell growth, apoptosis, and migration.

Clonal evaluation of early onset prostate cancer by expression profiling of ERG, SPINK1, ETV1, and ETV4 on whole-mount radical prostatectomy tissue.

BACKGROUND: Expression profiles of erythroblast transformation-specific (ETS)-related gene fusions and serine protease inhibitor Kazal-type 1 (SPINK1) in early onset prostate cancer have not been thoroughly explored. METHODS: We retrieved 151 radical prostatectomy specimens from young men with prostate cancer (<55 years) and characterized the expression of ETS-related gene (ERG), SPINK1, ETS Variant 1 (ETV1), and ETV4 by dual immunohistochemistry and dual RNA in situ hybridization. Age, race, family history, preoperative prostate-specific antigen, biochemical recurrence, and pathological variables using whole-mount radical prostatectomy tissue were collected. RESULTS: A total of 313 tumor nodules from 151 men including 68 (45%) Caucasians and 61 (40%) African Americans were included in the analysis. Positive family history of prostate cancer was seen in 65 (43%) patients. Preoperative prostate-specific antigen ranged from 0.3 to 52.7 ng/mL (mean = 7.04). The follow-up period ranged from 1 to 123.7 months (mean = 30.3). Biochemical recurrence was encountered in 8 of 151 (5%). ERG overexpression was observed in 85 of 151 (56%) cases, followed by SPINK1 in 61 of 151 (40%), ETV1 in 9 of 149 (6%), and ETV4 in 4 of 141 (3%). There were 25 of 151 (17%) cases showing both ERG and SPINK1 overexpression within different regions of either the same tumor focus or different foci. Higher frequency of ERG overexpression was seen in younger patients (≤45 years old; 76% vs 49%, P = .002), Caucasian men (71% vs 41% P = .0007), organ-confined tumors (64% vs 33%, P = .0008), and tumors of Gleason Grade groups 1 and 2 (62% vs 26%, P = .009). SPINK1 overexpression was more in African American men (68% vs 26%, P = .00008), in tumors with high tumor volume (>20%) and with anterior located tumors. ETV1 and ETV4 demonstrated rare overexpression in these tumors, particularly in the higher-grade tumors. CONCLUSION: This study expands the knowledge of the clonal evolution of multifocal cancer in young patients and support differences in relation to racial background and genetics of prostate cancer.

Overexpression of E74-Like Factor 5 (ELF5) Inhibits Migration and Invasion of Ovarian Cancer Cells.

BACKGROUND E74-like factor 5 (ELF5) plays a key role in the processes of cell differentiation, apoptosis, and occurrence of tumors. However, the effect of ELF5 on metastasis and invasion in human ovarian cancer remains poorly understood. MATERIAL AND METHODS Quantitative real-time polymerase chain reaction (qPCR) was performed to measure the expression of ELF5. The viability of cells was detected by cell counting kit (CCK-8). Cell apoptosis was tested by flow cytometry. Matrigel plug angiogenesis assay was employed to determine angiogenesis rate. The protein expression levels of vascular endothelial growth factor (VEGF), cleaved caspase-3, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), E-cadherin, N-cadherin, Snail, phosphoinositide 3 kinase (PI3K), phosphorylated (p)-PI3K, tyrosine kinase B (AKT), and phosphorylated (p)-AKT were determined by Western blot. Wound-healing assay and Transwell were used to determine invasion and migration. RESULTS We found that expression of ELF5 was obviously decreased in ovarian cancer cell lines. The cells viability, invasion and metastasis were inhibited by overexpression ELF5. ELF5 suppressed angiogenesis rate and the expression of VEGF. Changes of the expressions of Bcl-2, cleaved caspase-3 and Bax showed that anti-apoptosis ability was improved by ELF5. ELF5 also repressed N-cadherin and Snail and increased E-cadherin. The expressions of p-PI3K and p-AKT were decreased by ELF5. Further study showed that IGF-I reversed the inhibitory effect of ELF5 on growth and metastasis of SKOV3 cells. CONCLUSIONS Overexpression of ELF5 promoted the apoptosis and reduced the migration and invasion of ovarian cancer cells; therefore, it could provide a new approach to gene treatment of ovarian carcinoma.

Clinical significance of PDEF factor expression and its relation to androgen receptor in ER- breast cancer.

AIMS: The mechanism of androgen receptor (AR) promoting tumour growth in oestrogen receptor-negative (ER- ) breast cancer (BC) is undetermined. Prostate-derived ETS factor (PDEF) is highly restricted to the hormone-regulated tissues of epithelial cells, such as those in the prostate, breast and other tissues. It has been demonstrated that PDEF expression is associated with AR in prostate cancer. In this research, we aimed to investigate the relationship between PDEF and AR in ER- BC. METHODS AND RESULTS: We immunohistochemically evaluated the correlation between PDEF and AR expression in 246 cases of ER- invasive BC, and investigated their relationship in ER- BC cell lines. The expression of PDEF was associated with the positive expression of AR (P < 0.001) and a worse survival rate (P = 0.006). PDEF+ tumours were significantly more often AR+ (P < 0.001). AR and PDEF were more often co-expressed and the series of AR+ PDEF+ (126 of 246, 51.2%) had a poor survival rate (P = 0.046). In Cox models, PDEF expression (P = 0.028) was an independent predictor for overall survival (OS). At the cellular protein and mRNA levels, our experiments also showed a statistically significant positive correlation between PDEF and AR, and that PDEF may be regulated by AR. CONCLUSIONS: PDEF is associated with markers of bad prognosis, supporting its role as a growth promoter in ER- BC. Our findings also provide evidence that PDEF is strongly correlated with AR expression in ER- breast cancer; it may be a downstream target gene of AR and a potential prognostic factor in ER- BC.

Altered goblet cell function in Hirschsprung's disease.

AIMS AND OBJECTIVES: Hirschsprung's disease-associated enterocolitis (HAEC) is the most serious complication of Hirschsprung's disease (HSCR). HAEC occurs in 17-50% of patients with HSCR and may occur before or after a properly performed pull-through operation. The pathogenesis of HAEC is poorly understood. It is well recognized that a complex mucosal barrier protects, as the first line of defense, the surface of healthy intestinal tract from adhesion and invasion by luminal micro-organisms. Within the intestinal epithelium, goblet cells secrete gel-forming mucins, the major component of mucus, which block the direct attachment of commensal bacteria to the epithelial layer. Mucin 2 (MUC2) is the predominant mucin expressed in humans. Trefoil factor 3 (TFF3) synergizes with mucin and enhances the protective barrier properties of the mucus layer. SAM pointed domain-containing ETS transcription factor (SPDEF) drives terminal differentiation and maturation of secretory progenitors into goblet cells. Krueppel-like factor 4 (KLF4) is a goblet cell-specific differentiation factor in the colon and controls goblet cell differentiation and activates mucin synthesis. We hypothesized that the goblet cell function in the ganglionic pulled-through bowel in HSCR is abnormal and, therefore, we investigated the changes in goblet cell differentiation and functional expression of mucin in the bowel specimens from patients with HSCR. MATERIAL AND METHODS: We investigated MUC2, TFF3, SPDEF and KLF4 expression, and the goblet cell population in the ganglionic and aganglionic bowel of HSCR patients (n = 10) and controls (n = 10) by qPCR, Western blotting, confocal immunofluorescence, and alcian blue staining. RESULTS: The qPCR and Western blotting analysis revealed that TFF3, SPDEF and KLF4 expressions were significantly downregulated in the aganglionic and ganglionic colon of patients with HSCR as compared to controls (p < 0.05). Alcian blue staining revealed that the goblet cell population was significantly decreased in aganglionic and ganglionic colon as compared to controls (p < 0.05). Confocal microscopy revealed a markedly decreased expression of TFF3, SPDEF and KLF4 in colonic epithelium of patients with HSCR as compared to controls. CONCLUSION: This is, to our knowledge, the first report of decreased expression of TFF3, SPDEF, KLF4, and goblet cell population in the colon of patients with HSCR. Altered goblet cell function may result in intestinal barrier dysfunction contributing to the development of HAEC.

Androgen deprivation therapy-induced epithelial-mesenchymal transition of prostate cancer through downregulating SPDEF and activating CCL2.

The chemokine CC motif ligand 2 (CCL2) is important in recruiting tumor-associated macrophages and is involved in the development of castration-resistance prostate cancer (CRPC) after androgen-deprivation therapy (ADT); however, the underlying mechanism remains unclear. We found that inactivation of the androgen receptor (AR) reduces a transcriptional repressor (SAM pointed domain-containing ETS transcription factor, SPDEF) of CCL2, which mediates epithelial-to-mesenchymal transition (EMT) of prostate tumor cells. Cell lines derived from a prostate-specific Pten/Trp53-null mouse and capable of a spontaneous EMT were utilized for identification of CCL2, and showed that reduced SPDEF expression was associated with an elevated CCL2-activated EMT. AR signaling inhibits CCL2 through a SPDEF-mediated mechanism in that the SPDEF recognizes the CCL2 promoter and transcriptionally represses its activity. Ectopically expressed SPDEF reduced the EMT and rescued expression of CCL2 in SPDEF-expressing cells, which induced the EMT and promotes malignant functions of prostate cancer cells. In tissues from prostate cancer patients with ADT, low SPDEF levels were correlated with high CCL2 expression compared to patients without ADT. We present a novel mechanism that contributes to the EMT and metastatic phenotype observed in a subset of ADT-resistant prostate cancer, where the CCL2 is stimulated through the inactivated of AR-mediated SPDEF.

Transcriptional Analysis of Endothelial Cell Alternation Induced by Atrial Natriuretic Polypeptide in Human Umbilical Vein Endothelial Cells.

The aim of this study was to explore how atrial natriuretic polypeptide (ANP) affects the properties and function of endothelial cells. Gene expression data GSE56976 generated at 0, 1, and 6 hours after ANP incubation in human umbilical vein endothelial cells (HUVEC) was used. Microarray data were preprocessed for differentially expressed genes (DEGs) in each time-dependent group. Next, gene ontology (GO), pathway analysis, and transcriptional regulation were performed. Co-expression clustering analysis of DEGs and functional enrichment analysis of co-expression modules were processed. RT-PCR analysis was performed to validate gene expression. DEGs were obtained and their counts were increased from 0 hours to 6 hours. No overlapping DEGs were obtained among the 3 groups. The DEGs of ANP_6hours, including TGFB2 (transforming growth factor, beta 2), LTF (lactotransferrin/lactoferrin), and ETV7 (Ets variant 7) were mainly related with cell apoptosis and immune responses. The DEGs in the network of ANP_0hour were mainly associated with epithelial ion transport processes. In addition, 3 co-expressed modules were detected. CSF2 (colony stimulating factor 2) and PF4 (platelet factor 4) of the blue module were related with cytolysis, while FXYD1 (FXYD domain containing ion transport regulator 1) and TGFB2 of the yellow module were mainly enriched in ion transport and the ovulation cycle. The expression of TGFB2 obtained by microarray analysis was consistent with that of RT-PCR. Ion transport could be affected promptly after ANP treatment, and subsequently, the cytolysis of vein endothelial cells may be promoted and endothelial permeability would be enhanced, followed by activated immune responses.

Prostate-derived Ets factor, an oncogenic driver in breast cancer.

Prostate-derived Ets factor (PDEF), a member of the Ets family of transcription factors, differs from other family members in its restricted expression in normal tissues and its unique DNA-binding motif. These interesting attributes coupled with its aberrant expression in cancer have rendered PDEF a focus of increasing interest by tumor biologists. This review provides a current understanding of the characteristics of PDEF expression and its role in breast cancer. The bulk of the evidence is consistent with PDEF overexpression in most breast tumors and an oncogenic role for this transcription factor in breast cancer. In addition, high PDEF expression in estrogen receptor-positive breast tumors showed significant correlation with poor overall survival in several independent cohorts of breast cancer patients. Together, these findings demonstrate PDEF to be an oncogenic driver of breast cancer and a biomarker of poor prognosis in this cancer. Based on this understanding and the limited expression of PDEF in normal human tissues, the development of PDEF-based therapeutics for prevention and treatment of breast cancer is also discussed.

Combined DOG1 and Mammaglobin Immunohistochemistry Is Comparable to ETV6-breakapart Analysis for Differentiating Between Papillary Cystic Variants of Acinic Cell Carcinoma and Mammary Analogue Secretory Carcinoma.

BACKGROUND: We investigated the reliability of combined DOG1 and mammaglobin immunohistochemistry compared with ETV6 fluorescence in situ hybridization (FISH) in the assessment of salivary tumors previously diagnosed as acinic cell carcinoma (ACC). Ultrastructural features of cases reclassified as mammary analogue secretory carcinoma (MASC) were assessed by transmission electron microscopy (TEM). METHODS: Immunohistochemical (IHC) reactivity to DOG1 and mammaglobin was validated against FISH targeting the ETV6 gene in all 14 cases. RESULTS: Three cases with papillary cystic histomorphology previously diagnosed as ACC were revised to MASC. TEM features of the ETV6 rearrangement-positive MASC cases showed large numbers of secretory granules with extrusion into the intercellular spaces, well-developed endoplasmic reticulum, lipid-laden vacuoles, well-formed microvilli, and large lining cystic spaces. CONCLUSIONS: Combined DOG1 and mammaglobin immunohistochemistry is comparable to ETV6 -breakapart analysis for differentiating between papillary cystic variants of ACC and MASC.

ELF5 isoform expression is tissue-specific and significantly altered in cancer.

BACKGROUND: E74-like factor 5 (ELF5) is an epithelial-specific member of the E26 transforming sequence (ETS) transcription factor family and a critical regulator of cell fate in the placenta, pulmonary bronchi, and milk-producing alveoli of the mammary gland. ELF5 also plays key roles in malignancy, particularly in basal-like and endocrine-resistant forms of breast cancer. Almost all genes undergo alternative transcription or splicing, which increases the diversity of protein structure and function. Although ELF5 has multiple isoforms, this has not been considered in previous studies of ELF5 function. METHODS: RNA-sequencing data for 6757 samples from The Cancer Genome Atlas were analyzed to characterize ELF5 isoform expression in multiple normal tissues and cancers. Extensive in vitro analysis of ELF5 isoforms, including a 116-gene quantitative polymerase chain reaction panel, was performed in breast cancer cell lines. RESULTS: ELF5 isoform expression was found to be tissue-specific due to alternative promoter use but altered in multiple cancer types. The normal breast expressed one main isoform, while in breast cancer there were subtype-specific alterations in expression. Expression of other ETS factors was also significantly altered in breast cancer, with the basal-like subtype demonstrating a distinct ETS expression profile. In vitro inducible expression of the full-length isoforms 1 and 2, as well as isoform 3 (lacking the Pointed domain) had similar phenotypic and transcriptional effects. CONCLUSIONS: Alternative promoter use, conferring differential regulatory responses, is the main mechanism governing ELF5 action rather than differential transcriptional activity of the isoforms. This understanding of expression and function at the isoform level is a vital first step in realizing the potential of transcription factors such as ELF5 as prognostic markers or therapeutic targets in cancer.

A Double Negative Loop Comprising ETV6/RUNX1 and MIR181A1 Contributes to Differentiation Block in t(12;21)-Positive Acute Lymphoblastic Leukemia.

Childhood acute lymphoblastic leukemia (ALL) with t(12;21), which results in expression of the ETV6/RUNX1 fusion gene, is the most common chromosomal lesion in precursor-B (pre-B) ALL. We identified 17 microRNAs that were downregulated in ETV6/RUNX1+ compared with ETV6/RUNX1- clinical samples. Among these microRNAs, miR-181a-1 was the most significantly reduced (by ~75%; P < 0.001). Using chromatin immunoprecipitation, we demonstrated that ETV6/RUNX1 directly binds the regulatory region of MIR181A1, and knockdown of ETV6/RUNX1 increased miR-181a-1 level. We further showed that miR-181a (functional counterpart of miR-181a-1) could target ETV6/RUNX1 and cause a reduction in the level of the oncoprotein ETV6/RUNX1, cell growth arrest, an increase in apoptosis, and induction of cell differentiation in ETV6/RUNX1+ cell line. Moreover, ectopic expression of miR-181a also resulted in decreased CD10 hyperexpression in ETV6/RUNX1+ primary patient samples. Taken together, our results demonstrate that MIR181A1 and ETV6/RUNX1 regulate each other, and we propose that a double negative loop involving MIR181A1 and ETV6/RUNX1 may contribute to ETV6/RUNX1-driven arrest of differentiation in pre-B ALL.

Expression of ETV6/TEL is associated with prognosis in non-small cell lung cancer.

The ETV6/TEL gene is a member of the ETS family of transcription factors that has been mainly studied in hematological diseases. This study provides the first investigation of ETV6 expression in non-small cell lung cancer (NSCLC). In this study, ETV6 expression was immunohistochemically studied in 170 consecutive patients with NSCLC. The association between ETV6 expression and clinicopathological parameters was evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to estimate the effect of ETV6 expression on survival. ETV6 expression was observed in 135 of the 170 (79.4%) patients. ETV6 expression was positive for nuclear staining. From the clinicopathological standpoint, the expression of ETV6 was significantly correlated with age (P=0.014). The overall survival was significantly enhanced in the group with a low expression of ETV6 compared with the group with a high expression of ETV6 (five-year survival rates, 56.53% versus 29.88%; P=0.002), and the same finding was obtained for disease-free survival (five-year survival rates, 52.24% versus 30.47%; P=0.001). Multivariable analysis confirmed that ETV6 expression increased the hazard of death after adjusting for other clinicopathological factors (hazard ratio, 2.002; 95% confidence interval, 1.303-3.074; P=0.002). Our study demonstrated that ETV6 was markedly involved in the development of NSCLC and could serve as a potential prognostic marker for this deadly disease.

Expression of the ETS transcription factor GABPα is positively correlated to the BCR-ABL1/ABL1 ratio in CML patients and affects imatinib sensitivity in vitro.

In Philadelphia-positive chronic myeloid leukemia (CML), imatinib resistance frequently emerges because of point mutations in the ABL1 kinase domain, but may also be the consequence of uncontrolled upstream signaling. Recently, the heteromeric transcription factor GA-binding protein (GABP) was found to promote CML-like myeloproliferative disease in mice. In a cohort of 70 CML patients, we found that expression of the GABP α subunit (GABPα) is positively correlated to the BCR-ABL1/ABL1 ratio. Moreover, significantly higher GABPα expression was detected in blast crisis than in chronic phase CML after performing data mining on 91 CML patients. In functional studies, imatinib sensitivity is enhanced after GABPα knockdown in tyrosine kinase inhibitors (TKI)-sensitive K-562, as well as by overexpression of a deletion mutant in TKI-resistant NALM-1 cells. Moreover, in K-562 cells, GABP-dependent expression variations of PRKD2 and RAC2, relevant signaling mediators in CML, were observed. Notably, protein kinase D2 (Prkd2) was reported to be a GABP target gene in mice. In line with this, we detected a positive correlation between GABPA and PRKD2 expression in primary human CML, indicating that the effects of GABP are mediated by PRKD2. These findings illustrate an important role for GABP in disease development and imatinib sensitivity in human CML.

Elf5 inhibits TGF-ß-driven epithelial-mesenchymal transition in prostate cancer by repressing SMAD3 activation.

BACKGROUND: The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of migration, invasiveness, and metastasis traits. During tumor progression, EMT can be induced by transforming growth factor-ß (TGF-ß) signal that epithelial cells receive from their microenvironment. However, the master regulatory controls on TGF-ß-EMT axis are not understood. METHODS: The protein expression in human specimens was measured by immunohistochemical staining. E74-like factor 5 (Elf5) was silenced by short interfering RNAs in LNCaP cells and stably overexpressed by HA-tagged Elf5 cDNAs in 22Rv1 cells. These cells were used to study migration and anchorage-independent growth. RESULTS: Our data reveal that Elf5 results in the failure of mesenchymal morphogenesis, upregulation of EMT markers, spheres formation, and migration in the presence of TGF-ß. Furthermore, Elf5 blocks TGF-ß signaling, through decreasing drosophila mothers against decapentaplegic protein (SMAD3) activation by binding to it, one of the major effector of TGF-ß-induced EMT. Moreover, Elf5 can serve as a prognostic marker of metastasis-free survival in patients with TGF-ß-positive prostate cancer. CONCLUSIONS: Elf5 expression is inversely correlated with EMT. Elf5 inhibits TGF-ß-driven EMT via repressing SMAD3 phosphorylation in prostate cancer cells. In addition, Elf5 can be used as a biomarker of metastasis-free survival in patients with TGF-ß-positive prostate cancer.

Distal airways are protected from goblet cell metaplasia by diminished expression of IL-13 signalling components.

BACKGROUND: Increased mucus production is a critical factor impairing lung function in patients suffering from bronchial asthma, the most common chronic inflammatory lung disease worldwide. OBJECTIVE: This study aimed at investigating whether goblet cell (GC) metaplasia and mucus production are differentially regulated in proximal and distal airways. METHODS: Female Balb/c mice were sensitized to ovalbumin (OVA) and challenged with an OVA-aerosol on two consecutive days for 1 week (acute) or 12 weeks (chronic). Real-time RT-PCR analysis was applied on microdissected airways. RESULTS: In acutely and chronically OVA-challenged mice, GC metaplasia and mucus production were observed in proximal but not in distal airways. In contrast, inflammation reflected by the infiltration of eosinophils and expression of the TH2-type cytokines IL-4 and IL-13 was increased in both proximal and distal airways. Abundance of IL-13Rα1 was lower in distal airways of healthy control mice. Under acute and chronic OVA-exposure, activation of IL-13Rα1-dependent signalling cascade, reflected by Spdef and Foxo3A transcription factors, was attenuated in distal compared to proximal airways. CONCLUSION AND CLINICAL RELEVANCE: These data indicate that distal airways might be less sensitive to IL-13-induced GC metaplasia and mucus production through lower expression of IL-13Rα1 and attenuated activation of downstream signalling. This might represent a protective strategy to prevent mucus plugging of distal airways and thus impaired ventilation of attached alveoli.

LCK over-expression drives STAT5 oncogenic signaling in PAX5 translocated BCP-ALL patients.

The PAX5 gene is altered in 30% of BCP-ALL patients and PAX5 chromosomal translocations account for 2-3% of cases. Although PAX5 fusion genes significantly affect the transcription of PAX5 target genes, their role in sustaining leukemia cell survival is poorly understood. In an in vitro model of PAX5/ETV6 leukemia, we demonstrated that Lck hyper-activation, and down-regulation of its negative regulator Csk, lead to STAT5 hyper-activation and consequently to the up-regulation of the downstream effectors, cMyc and Ccnd2. More important, cells from PAX5 translocated patients show LCK up-regulation and over-activation, as well as STAT5 hyper-phosphorylation, compared to PAX5 wt and PAX5 deleted cases. As in BCR/ABL1 positive ALL, the hyper-activation of STAT5 pathway can represent a survival signal in PAX5 translocated cells, alternative to the pre-BCR, which is down-regulated. The LCK inhibitor BIBF1120 selectively reverts this phenomenon both in the murine model and in leukemic primary cells. LCK inhibitor could therefore represent a suitable candidate drug to target this subgroup of ALL patients.