Fer Expression Research Articles

SKOR1 mediates FER kinase-dependent invasive growth of breast cancer cells.

High expression of the non-receptor tyrosine kinase FER is an independent prognostic factor that correlates with poor survival in breast cancer patients. To investigate whether the kinase activity of FER is essential for its oncogenic properties, we developed an ATP analogue-sensitive knock-in allele (FERASKI). Specific FER kinase inhibition in MDA-MB-231 cells reduces migration and invasion, as well as metastasis when xenografted into a mouse model of breast cancer. Using the FERASKI system, we identified Ski family transcriptional corepressor 1 (SKOR1) as a direct FER kinase substrate. SKOR1 loss phenocopies FER inhibition, leading to impaired proliferation, migration and invasion, and inhibition of breast cancer growth and metastasis formation in mice. We show that SKOR1 Y234, a candidate FER phosphorylation site, is essential for FER-dependent tumor progression. Finally, our work suggests that the SKOR1 Y234 residue promotes Smad2/3 signaling through SKOR1 binding to Smad3. Our study thus identifies SKOR1 as a mediator of FER-dependent progression of high-risk breast cancers.

FER regulates endosomal recycling and is a predictor for adjuvant taxane benefit in breast cancer.

Elevated expression of non-receptor tyrosine kinase FER is an independent prognosticator that correlates with poor survival of high-grade and basal/triple-negative breast cancer (TNBC) patients. Here, we show that high FER levels are also associated with improved outcomes after adjuvant taxane-based combination chemotherapy in high-risk, HER2-negative patients. In TNBC cells, we observe a causal relation between high FER levels and sensitivity to taxanes. Proteomics and mechanistic studies demonstrate that FER regulates endosomal recycling, a microtubule-dependent process that underpins breast cancer cell invasion. Using chemical genetics, we identify DCTN2 as a FER substrate. Our work indicates that the DCTN2 tyrosine 6 is essential for the development of tubular recycling domains in early endosomes and subsequent propagation of TNBC cell invasion in 3D. In conclusion, we show that high FER expression promotes endosomal recycling and represents a candidate predictive marker for the benefit of adjuvant taxane-containing chemotherapy in high-risk patients, including TNBC patients.

Fer exacerbates renal fibrosis and can be targeted by miR-29c-3p.

AimRenal fibrosis (RF) is a common clinical condition leading to irreversible renal function loss. Tyrosine kinase proteins and microRNAs (miRs) are associated with pathogenesis and we aim to investigate the role of Fer and its partner miR(s) in RF.MethodIn silico reproduction of Mouse Kidney FibrOmics browser was performed to identify potential miR(s) and target gene(s). In vivo validation was performed in C57BL/6 mice with unilateral ureteral obstruction (UUO). In vitro validation was performed in rat kidney fibroblast NRK-49F cells. Mimics and inhibitors of miR-29c-3p were constructed. The target gene Fer was monitored by RT-PCR and western blotting. The levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α in serum and media were measured by ELISA.ResultsThe Fer expression and protein level were gradually increased during 14 days of UUO modeling. miR-29c-3p expression was strongly correlated with that of Fer. In vivo validation showed increased expressions of fibrosis-associated genes and increased phospoho-Smad3 level in the UUO model. Fer-knockdown (KD) significantly decreased expressions of fibrosis-associated genes. Pharmaceutical inhibition of Fer showed similar effects to miR-29c-3p, and miR inhibition showed a significant decrease of excretion of inflammatory factors.ConclusionDysregulation of miR-29c-3p and Fer plays a role in RF. Pharmaceutical or genetic inhibition of Fer may serve as the potential treatment for RF.

Nitrogen stress inhibits root growth by regulating cell wall and hormone changes in cotton (Gossypium hirsutum L.)

AbstractRoots play an important role in the response to nitrogen stress in cotton. To better understand the molecular and physiological mechanisms underlying the adaptation of cotton roots to nitrogen stress, the responses of genes in roots to nitrogen changes were analysed by RNA sequencing (RNA‐seq), and morphological and physiological indicators were measured for verification. This study revealed that lack of nitrogen stress (LN, 0 mm) and excess nitrogen stress (HN, 8 mm) inhibited root growth. Moderate nitrogen (MN, 4 mm) resulted in the largest values for root length, projected area and volume. Nitrogen stress regulated root growth via two mechanisms. Nitrogen stress decreased root growth by regulating cell wall growth via alterations in the cell wall composition and the expression of cell wall‐related genes. Under LN and HN, the contents of cellular polysaccharide and glucan reached their lowest levels, whereas the contents of lignin and cellulose reached their highest levels. Lower expression of EXPA17, EXLB1 and PME3 as well as higher expression of EXLA1, WAT1, CESA1, CESA3, CAD6, COMT1, SAMS, LAC4 and NAC081 were obtained under nitrogen stress compared to MN, indicating that nitrogen stress increases lignin and cellulose synthesis and promotes root ageing. Nitrogen stress also decreased root growth by affecting the contents and regulation of salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA). Nitrogen significantly upregulated the expression of PAD4, ALD1, WRKY70, WAKL10, MYB44, FER, LHY and BHLH112 as well as significantly downregulated that of TGA6, BT1, BT2 and PYL4. The changes in the expression of these genes under nitrogen stress resulted in significant increases in the SA, JA and ABA contents in roots. Our results suggested that nitrogen stress inhibits the growth of the cotton root system by affecting cell wall growth and hormone regulation. These results contribute to our understanding of how nitrogen regulates cotton root growth at the genetic and physiological levels, and they provide a theoretical basis for the efficient utilization of nitrogen resources.

FER Regulated by miR-206 Promotes Hepatocellular Carcinoma Progression via NF-κB Signaling.

ObjectivesFeline sarcoma-related protein (FER) is known to play a critical regulatory role in several carcinomas. However, the exact biological function of FER in hepatocellular carcinoma (HCC) still needs to be investigated. The primary objective of this research was to investigate the unknown function and molecular mechanisms of FER in HCC.Materials and MethodsThe expression level of FER in HCC tissue samples and cells was examined by RT-qPCR, immunohistochemistry and western blot. Cellular and animal experiments were used to explore the effect of FER on the proliferative and metastatic capacities of HCC cells. The crosstalk between FER and NF-κB signaling was explored by western blot. The upstream factors that regulate FER were evaluated through dual-luciferase experiments and western blot assays.ResultsFER was overexpressed in HCC specimens and HCC cell lines. FER expression levels were positively associated with unfavorable clinicopathological characteristics. The higher the expression of FER was, the worse the overall survival of HCC patients was. The results of loss-of-function and gain-of-function experiments indicated that knockdown of FER decreased, while overexpression of FER increased, the proliferation, invasion and metastasis of HCC cells in vitro and in vivo. Mechanistically, we found that FER activated the NF-κB signaling pathway and stimulated epithelial-to-mesenchymal transition (EMT). We also found that FER was directly regulated by miR-206, and the downregulation of miR-206 was associated with proliferation and metastatic progression in HCC.ConclusionsThe present research was the first to reveal that a decrease in miR-206 levels results in an increase in FER expression in HCC, leading to enhanced cell growth and metastatic abilities via activation of the NF-κB signaling pathway.

Abstract LB203: FER regulates endosomal recycling and is a candidate predictor for taxane benefit in breast cancer

Abstract To this day, targeted treatment options for patients with triple negative metastatic breast cancer (TNBC) are still virtually absent. Previously, we showed that elevated expression of the tyrosine kinase FER is an independent prognosticator that correlates with poor survival of high-grade and basal/TNBC patients. Here, we show that high FER levels are also associated with improved outcome after adjuvant taxane-based combination chemotherapy, in high-risk, HER2-negative patients. In TNBC cells, we observed a causal relation between high FER levels and sensitivity to taxanes. Our proteomics and mechanistic studies demonstrated that FER regulates endosomal recycling, a microtubule-dependent process that underpins breast cancer cell invasion. Using chemical genetics, we identified DCTN2 as a novel FER substrate. Our work indicates that the DCTN2 tyrosine 6 is essential for tubular recycling domains development in early endosomes and subsequent propagation of TNBC cell invasion in 3D. In conclusion, we show that high FER expression promotes endosomal recycling and represents a candidate predictive marker for benefit of adjuvant taxane-containing chemotherapy in high-risk patients, including TNBC patients. Citation Format: Sandra Tavares, Nalan Liv, Milena Pasolli, Mark Opdam, Max Ratze, Manuel Saornil, Lilian Sluimer, Rutger Hengeveld, Robert van Es, Erik van Werkhoven, Harmjan Vos, Holger Rehmann, Boudewijn Burgering, Hendrika Oosterkamp, Susanne Lens, Judith Klumperman, Sabine Linn, Patrick Derksen. FER regulates endosomal recycling and is a candidate predictor for taxane benefit in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB203.

The phyB-dependent induction of HY5 promotes iron uptake by systemically activating FER expression.

Iron (Fe) deficiency affects global crop productivity and human health. However, the role of light signaling in plant Fe uptake remains uncharacterized. Here, we find that light-induced Fe uptake in tomato (Solanum lycopersicum L.) is largely dependent on phytochrome B (phyB). Light induces the phyB-dependent accumulation of ELONGATED HYPOCOTYL 5 (HY5) protein both in the leaves and roots. HY5 movement from shoots to roots activates the expression of FER transcription factor, leading to the accumulation of transcripts involved in Fe uptake. Mutation in FER abolishes the light quality-induced changes in Fe uptake. The low Fe uptake observed in phyB, hy5, and fer mutants is accompanied by lower photosynthetic electron transport rates. Exposure to red light at night increases Fe accumulation in wild-type fruit but has little effects on fruit of phyB mutants. Taken together, these results demonstrate that Fe uptake is systemically regulated by light in a phyB-HY5-FER-dependent manner. These findings provide new insights how the manipulation of light quality could be used to improve Fe uptake and hence the nutritional quality of crops.

CD20-Modified Exosomes Delivered E260 Inhibits Progression of Diffuse Large B-Cell Lymphoma By Restoring Hippo Activity

Introduction: Fer tyrosine kinase (Fer) is ubiquitously expressed in various cancer types and has been proved to play a pivotal role in tumorigenesis and metastasis. E260, as an effective inhibitor of Fer, is selective lethal to cancer cells. However, the effect of Fer and E260 in diffuse large B-cell lymphoma (DLBCL) remain elusive. Thus, we set out to investigate the function and mechanisms of E260 targeting Fer in DLBCL for a treatment purpose. Methods: The impact of Fer expression to predict clinical outcome was evaluated in 50 newly diagnosed DLBCL and 30 healthy donors between 2014 and 2019 by immunohistochemistry. This study was approved by the ethics board of Shandong Provincial Hospital, and informed consent was obtained. Next, we assessed the oncogenic effect of Fer by using gain-and loss-of-function assays. Fer inhibitor E260 was used in the treatment of DLBCL cells. CD20 targeted exosomes were isolated from CD20 transfected HEK293T cells and then incubated with E260 in order to generate CD20-EXO-E260. The anti-tumor efficacy of E260 and CD20-EXO-E260 were assessed by cell proliferation, apoptosis, migration and drug sensitivity assays. To elucidate the regulatory mechanism, we test the expression of Hippo-YAP pathway and YAP location after E260 treated by western blotting and immunofluorescent staining. Interaction of the Fer and AJUBA was further validated by coimmunoprecipitation (Co-IP) assay. To further explore the clinical value of Fer, we isolated and test Fer expression in exosomes from the plasma of DLBCL patients. ROC curve was performed to test the diagnostic value of exosomal Fer in DLBCL. Results: To explore the association of Fer expression with DLBCL tumorigenesis, we examined Fer expression using immunohistochemical staining. Fer was upregulated in DLBCL patients (Fig.1A) and correlated with adverse clinical parameters (Fig.1B) and shorter overall survival time (Fig.1C). Since Fer was also increased in the DLBCL cell lines, we then set out to explore the biological function of Fer in DLBCL cells (Fig.1D). Transfected efficacy was verified by western blot (Fig2.A). In vitro gain-and loss-of-function assays demonstrated the stimulatory role of Fer on cell viability, migration and chemoresistance (Fig2.B-D). Recently, exosomes have been emerged as promising drug delivery carriers. Thus, we generated CD20-modified exosomes as an alternative to targeting deliver E260 to DLBCL cells. This event not only facilitated the targeting efficacy of E260 (Fig.3A) but also showed higher efficacy than E260 in eliminating DLBCL progression through inhibiting cell proliferation and chemotaxis, enhancing cell apoptosis and drug sensitivity both in vitro and in vivo (Fig.3B-F). In the view of GO and KEGG analysis, Fer mainly enrichment in regulating cell proliferation, adhesion, response to drug and Hippo pathway in cancer (Fig.4A). Consistent with this, we demonstrated that E260 activated Hippo pathway and leading to YAP cytoplasmic sequestration (Fig.4B-C). Co-IP assay confirmed the physical interaction between Fer and AJUBA. Furthermore, upregulated AJUBA reversed E260 mediated cell proliferation inhibition (Fig.4D). Thus, we suggest that E260 inhibits DLBCL progression by downregulating AJUBA which restores Hippo tumor-suppressor activity. In the mice model, the levels of plasma exosomal Fer could reflex the tumor burden (Fig.5A). We next detected that exosomal Fer and AJUBA expression increased in the plasma of DLBCL patients (Fig.5B-C) and released after R-CHOP therapy (Fig.5D). Besides, Fer expression was positively related with AJUBA (Fig.5E). As shown in the Figure 5F-G, exosomal Fer could act as a promising indicator in early diagnosis and disease progression for DLBCL patients. Conclusion: We demonstrated for the first time the oncogenic role of Fer in DLBCL progression and therefore provided an appealing therapeutic target. CD20-targeted exosomes delivery E260 are more effective in eliminating DLBCL progression through diminishing AJUBA and activating Hippo pathway. Furthermore, we provided circulating exosomal Fer as a novel non-invasive biomarker for DLBCL diagnosis. Disclosures No relevant conflicts of interest to declare.

P0717EPIGENETIC REGULATION OF CHRONIC KIDNEY DISEASE ACCELERATED-ATHEROSCLEROSIS

Abstract Background and Aims Atherosclerosis and its related clinical complications are the leading causes of death in western countries. Multiple processes are involved in atherosclerosis like endothelial dysfunction, inflammation, vascular proliferation, neovascularization, oxidative stress, apoptosis, extracellular matrix degradation and thrombosis. Moreover atherosclerosis could be accelerated in some diseases like chronic kidney disease (CKD). In this regard, miRNAs have been elucidated as important factors in the regulation of different cellular and molecular processes involved in atherosclerosis development. Our aim is to identify specific miRNAs that could be involved in chronic kidney disease (CKD) accelerated-atherosclerosis. Method A CKD accelerated-atherosclerosis mouse model was developed by subtotal nephrectomy (5/6 nephrectomy) in APOE-/- mice fed on a high fat diet (HFD) during 10 weeks and compared to mice with normal renal function. Urine and blood samples were collected every week. Western-blot, RT-PCR, immunohistochemistry and confocal microscopy analysis were performed. In previous studies mir23a-3p and miR652-3p have been suggested as possible regulators of the atherosclerotic process. Therefore the expression levels of these miRNAs and its possible target genes (FER and BTLA) were analyzed in mice total blood. Results APOE-/-+ERC+HFD mouse showed a progressive decrease in renal function and an increase of renal damage markers such as KIM-1. These animals showed structural glomerular damage characterized by de-cellularization, mesangial cell expansion and podocyte (WT1+,podocalixin+), endothelial (CD31+) and mesangial (GATA3+) loss. Lipid profile modifications were also observed in APOE-/-+ERC+HFD mice with an increase in plasma total cholesterol levels. Moreover atherosclerotic plaque size was also increased in APOE-/-+ERC+HFD animals in comparison with atherosclerotic plaque size of APOE-/-+HFD mice. CKD mice showed lower fibrosis (sirius red staining) and higher inflammatory markers in its atherosclerotic plaques. The presence of macrophages (CD68+) and T lymphocytes (CD3+) was increased in APOE-/-+ERC+HFD group in contrast with APOE-/-+HFD animals. Moreover, in samples from the aortic arch of APOE-/-+ERC+HFD mice, ICAM1 protein levels were increased. Finally we found decreased levels of mir23a-3p and mir652-3p gene in total blood samples of APOE-/-+ ERC+ HFD mice. On the other hand, in aortic arc and descendent aorta tissues, mir23a-3p and mir652-3p expression levels were similar between groups. The expression levels of mRNAs of potential targets of this miRNAs (FER and BTLA) were increased in total blood and aortic arch samples. In addition, we observed that BTLA and FER expression were localized in vascular smooth muscle cells and endothelial cells respectively. Conclusion Levels of mir23a-3p, mir652-3p and its related mRNA (FER and BTLA) are altered in CKD and may be a potential therapeutic target for CKD-accelerated atherosclerosis treatment.

Mask, a component of the Hippo pathway, is required for Drosophila eye morphogenesis

Hippo signaling is an important regulator of tissue size, but it also has a lesser-known role in tissue morphogenesis. Here we use the Drosophila pupal eye to explore the role of the Hippo effector Yki and its cofactor Mask in morphogenesis. We found that Mask is required for the correct distribution and accumulation of adherens junctions and appropriate organization of the cytoskeleton. Accordingly, disrupting mask expression led to severe mis-patterning and similar defects were observed when yki was reduced or in response to ectopic wts. Further, the patterning defects generated by reducing mask expression were modified by Hippo pathway activity. RNA-sequencing revealed a requirement for Mask for appropriate expression of numerous genes during eye morphogenesis. These included genes implicated in cell adhesion and cytoskeletal organization, a comprehensive set of genes that promote cell survival, and numerous signal transduction genes. To validate our transcriptome analyses, we then considered two loci that were modified by Mask activity: FER and Vinc, which have established roles in regulating adhesion. Modulating the expression of either locus modified mask mis-patterning and adhesion phenotypes. Further, expression of FER and Vinc was modified by Yki. It is well-established that the Hippo pathway is responsive to changes in cell adhesion and the cytoskeleton, but our data indicate that Hippo signaling also regulates these structures.

The immune defense response of Pacific white shrimp (Litopenaeus vannamei) to temperature fluctuation

Temperature is a significant environmental factor contributing to the success of aquaculture. To investigate the immune defense response during temperature fluctuation, Litopenaeus vannamei (L. vannamei) was treated under conditions of gradual cooling from an acclimation temperature of 28 °C (C group) to 13 °C (T group) in 2 days with a cooling rate of 7.5 °C/d and then rewarmed to 28 °C (R group) using the same rate. Relative expression of immune defense system-related genes and intestinal microbial composition in L. vannamei were investigated. The results showed that with a decrease in temperature, the expression of TLR, IMD, proPO and Casp3 in intestine was significantly decreased, while the expression of Muc-3A, Muc-5AC, Muc-17, IAP, p53, HSP70, MT and Fer was significantly increased after cooling. After temperature recovery, gene expression generally showed a trend of recovering to the normal level (C). Intestinal microbial analysis showed that, compared with the C group, the Chao and Ace indexes, the relative abundance of microflora from the Phylum Bacteroidetes, Class Alphaproteobacteria, and Class Bacteroidia, significantly decreased in the R group. The results revealed that cold-stress may decrease microbial community richness, alter the bacterial community in general and reduce shrimp immunity to pathogens and antibacterial activity. As a result, during temperature fluctuation shrimp may mobilize the immune defense system through upregulating the expression of Muc genes, anti-apoptosis related genes, and antioxidant related genes in order to maintain organism homeostasis as well as to repair damaged intestinal tissue.

Transcriptome profiling of high oleic peanut under low temperatureduring germination

High oleic (HO) peanut (Arachishypogaea L.) oils benefit human health and industrial production due to its superior nutritional composition and thermo-oxidative stability. However, HO peanut is sensitive to cold stress especially during germination, which limits its distribution in low temperature areas. To understand the molecular mechanism of cold responses in HO peanuts at germination stage, four HO peanut varieties with different cold tolerance were selected in field experiments to analyze their genome-wide gene regulation under low temperatures. High-throughput sequencing and transcriptome analysis revealed a total of 139 429 unigenes. Among these, 3520 common differentially expressed genes (DEG) were detected between two groups of cold-tolerant and cold-sensitive peanuts, and the number of up-regulated genes was greater than that of down-regulated genes in the cold-tolerant peanuts. Gene ontology analysis indicates that the number of DEGs involved in cell membrane metabolism and integrity as well as proteins located in the cell periphery were significantly higher in the cold-tolerant peanuts. KEGG pathway analysis suggests that plant-pathogen interaction and plant hormone signal transduction pathway play important roles in cold tolerance. Four cold-induced genes, TIC(TIME FOR COFFEE), ATX3(histone-lysine N-methyltransferase ATX3-like), AGO4(argonaute 4-like), FER(FERONIA-like receptor protein kinase), and three transcription factor genes, bHLH(bHLH49-like transcription factor), MYB(MYB-related protein 3R-1-like)and EREB(Ethylene-responsive element binding factor 6)were selected to verify the expression profile via real-time quantitative PCR detection. The expression of TIC, ATX3, AGO4, bHLH, MYB and EREB significantly increased within 3 hours after low temperature stress, while the expression of FER significantlyincreased after 12 hours, suggesting that these genes responded to low temperature stress during peanut germination. This work not only sheds light on the transcriptional regulation of HO peanut under low-temperature stress during germination but also provides data resources for screening candidate genes in improving peanuts stress resistance.

YY1 inhibits the migration and invasion of pancreatic ductal adenocarcinoma by downregulating the FER/STAT3/MMP2 signaling pathway

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and a high mortality rate. The transcription factor YY1 acts as an inhibitor of many types of tumors. We found that YY1 knockdown promoted the invasion and migration of PANC-1 and BxPC-3 cells; FER knockdown partially restored the promotion of pancreatic cancer caused by YY1 knockdown. In vivo experiments yielded the same results. According to luciferase reporter gene, electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays, YY1 directly binds to the FER promoter region. Moreover, higher level FER expression results in a worse TNM stage and prognosis for patients with PDAC. Furthermore, by downregulating FER, YY1 inhibits the formation of the STAT3-MMP2 complex, thereby suppressing expression of MMP2 and ultimately inhibiting the migration and invasion of pancreatic cancer. Our study demonstrates that the YY1/FER/STAT3/MMP2 axis is associated with the progression of pancreatic cancer and may provide a new therapeutic target for the treatment of pancreatic cancer.

Role of feline sarcoma‑related protein in the viability and apoptosis of bladder cancer cells.

Feline sarcoma‑related protein (Fer) is a type of nuclear and cytoplasmic non‑receptor protein tyrosine kinase, which is associated with the progression of numerous types of cancer. Previously, we identified that Fer is associated with the migration and invasion of bladder cancer. The present study aimed to investigate the role of Fer in bladder cancer cell viability and apoptosis. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis were performed to detect the expression levels of Fer; short interference RNA (siRNA) and overexpression vectors were used to downregulate or upregulate Fer expression, respectively. The effects on cell proliferation ability and cell apoptosis were then tested by MTT assay and flow cytometry. The results revealed that Fer expression was upregulated in bladder cancer cell lines. Downregulation of Fer expression by siRNA significantly suppressed T24 cell viability and induced apoptosis, as well as inducing cell cycle arrest. Conversely, Fer overexpression in 5637cells significantly promoted cell viability and cell cycle progression, but inhibited cell apoptosis. Furthermore, the suppression and overexpression of Fer significantly altered the expression of cleaved caspase‑3 and Bcl‑2, and dysregulated the P38 mitogen‑activated protein kinase signaling pathway. The findings of the present study indicate a possible molecular mechanism of Fer in bladder cancer and may be considered as a potential target in the treatment of this disease.

Stromal expression of Fer suppresses tumor progression in renal cell carcinoma and is a predictor of survival.

Fps/Fes related (Fer) is a non-receptor tyrosine kinase that is expressed in fibroblasts, immune cells and endothelial cells. Fer serves an important pathological role in cell survival, angiogenesis and the immune system. However, the pathological role of Fer expression in the stromal cells surrounding renal cell carcinoma (RCC) has not been previously investigated. In the present study, immunohistochemical analysis of Fer was performed using the formalin-fixed tissue samples of 152 patients with RCC. The proliferative and apoptotic indices were used to represent the percentage of proliferation marker protein Ki-67- and cleaved caspase-3-positive cells, respectively. The microvessel density was defined as the number of cluster of differentiation (CD) 31-positively stained vessels/mm2. In addition, CD57+ and CD68+ cells were counted using semi-quantification of natural killer (NK) cells and macrophages. Fer expression in stromal cells was negatively associated with Fuhrman grade, pathological tumor stage and metastasis (P<0.001). Fer expression in stromal cells was negatively associated with CD68+ macrophage density, whereas it was positively associated with CD57+ NK cell density. Kaplan-Meier estimators indicated that decreased stromal Fer expression was a predictive marker of decreased cause-specific survival rate (P<0.001). Furthermore, low expression of Fer was identified as being an independent marker of decreased cause-specific survival using multivariate analysis (hazard ratio, 7.4; 95% confidence interval, 1.7-33.0; P<0.001). The results of the present study suggested that low Fer expression in stromal cells is associated with increased malignant aggressiveness and decreased survival in patients with RCC. CD57+ NK cell and CD68+ macrophage regulation in cancer-stromal tissue is considered to affect RCC pathology.

Downregulation of feline sarcoma-related protein inhibits cell migration, invasion and epithelial-mesenchymal transition via the ERK/AP-1 pathway in bladder urothelial cell carcinoma.

Feline sarcoma-related protein (Fer) is a nuclear and cytoplasmic non-receptor protein tyrosine kinase and Fer overexpression is associated with various biological processes. However, the clinicopathological characteristics and molecular mechanisms of Fer expression in bladder urothelial cell carcinoma (UCC) have yet to be elucidated. The present study demonstrated that Fer was significantly upregulated in bladder UCC tissues and cell lines. A clinicopathological analysis suggested that Fer expression was significantly associated with tumor stage, histological grade and lymph node status, and Fer expression was a prognostic factor for overall survival in a multivariate analysis. Furthermore, small interfering RNA (siRNA) was used to silence the expression of the Fer gene in human bladder UCC T24 cells, and was shown to significantly reduce the migration and invasion of the cells. It was also observed that Fer-siRNA caused the T24 cells to acquire an epithelial cobblestone phenotype, and was able to reverse the epithelial-mesenchymal transition of the cells. Subsequently, Fer-knockdown was shown to deactivate the extracellular signal-regulated kinase/activator protein-1 signaling pathway in T24 cells. These results indicated, for the first time, that Fer has a critical role in bladder UCC progression and may be a potential therapeutic target for bladder UCC metastasis.

Electroporation-mediated delivery of the FER gene in the resolution of trauma-related fatal pneumonia.

Injured patients with lung contusion (LC) are at risk of developing bacterial pneumonia (PNA) followed by sepsis and death. A recent Genome-wide Association Study, showed FER gene expression positively correlating with survival rates among individuals with above conditions. We sought to determine if electroporation-mediated (EP) delivery of FER gene could indeed improve survival, in a lethal model of combined LC and PNA. C57BL/6 mice sustained unilateral LC, which preceded a 500 Klebsiella CFU inoculation by 6 hrs. In-between these insults, human FER plasmid (pFER) was introduced into the lungs followed by eight EP pulses applied externally (10ms at 200V/cm). Control groups included EP of empty vector (pcDNA3) or Na+/K+-ATPase genes (pPump) and no treatment (LC + PNA). We recorded survival, histology, lung mechanics, bronchial alveolar fluid (BAL), FER and inflammatory gene expression and bacteriology. The data shows that 7-day survival was significantly improved by pFER compared to control groups. pFER increased BAL monocytes and activated antibacterial response genes (NOS, Fizz). pFER treatment showed decreased lung and blood Klebsiella counts reaching, in some cases, complete sterilization. In conclusion, FER gene delivery promoted survival in LC+PNA mice via recruitment of activated immune cells, improving efficiency of bacterial clearance within contused lung

173. Electroporation-Mediated FER Gene Delivery in the Resolution of Severe Gram Negative Pneumonia

Introduction: The emergence of multi drug resistant bacterial pneumonia is a major concern among Intensive Care Units across the nation. Severely injured patients including those with lung contusion (LC) and requiring mechanical ventilation are particularly vulnerable in developing above forms of virulent pneumonia (PNA). The continued deterioration of these patients evolves into highly mortal Acute Respiratory Distress Syndrome. A recent GWAS study showed that FER expression, a non-receptor cytosolic tyrosine-kinase, was associated with survival from pneumonia and sepsis. We elected to study the role of FER gene in a murine model of bacterial PNA. We hypothesized that electroporation-mediated (EP) delivery of the FER gene would decrease mortality in severe PNA complicated by trauma. Methods: Two separate PNA models were created in C57Bl/6 mice by airway inoculation of 500 CFU of Klebsiella sp., in naive (Primary PNA) or after blunt trauma-induced lung contusion (Secondary PNA). Treatment group(s) consisted on delivering naked plasmid DNA encoding FER to the lung (pFER) followed by square wave EP at 200 V/cm, eight 10 ms pulses, 1 s apart using external forelimb flat electrodes, either before or after insult. These plasmids were delivered with appropriate controls (saline EP {EP-sham} and plasmids encoding the Na+/K+-ATPase- which has shown favorable response in sterile inflammatory conditions {pPump}). 7-day survival curves were recorded. Specific readouts included parameters of lung inflammation (Histology, Bronchial Alveolar Lavage [BAL], Taq-man PCR) and infection (blood-lung bacterial quantification) at 24 and 72 hrs. Results: Survival was markedly improved by pFER treatment, both in primary and secondary bacterial PNA as compared to EP-sham or pPump controls. (Figure 1 leftFigure 1 left). Assessed parameters of inflammation showed also favorable changes being more prominent in secondary bacterial PNA, as visualized by H-E staining (Figure 1 rightFigure 1 right). With pFER treatment there was a robust recruitment of monocytes in BAL (~20 fold increase over LC+PNA control, p<0.0001), with cells showing activation of bactericidal Fizz (~110 fold change over LC + PNA control, p<0.0001) and NOS (~160 fold change over LC + PNA, p<0.0001) genes. Blood and lung, bacterial counts were significantly decreased in the pFER group with some animals able to completely clear the lung of infection (Figure 2Figure 2). Conclusion: EP-mediated delivery of the FER gene significantly improved mouse survival in severe primary and secondary models of bacterial PNA. Mechanisms include recruitment of monocytes, activation of lung immune cells and improved bacterial clearance.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide

Influence of feline sarcoma-related protein on cell cycle and apoptisis of human prostate cancer cells and the mechanism

Objective To investigate the influence of small interference RNA(siRNA) silencing feline sarcoma-related protein(Fer)on cell cycle and apoptosis of prostate cancer cells. Methods PC-3 cells were transfected with three siRNAs targeting human Fer by liposome transfection reagent.Real -time reverse transcriptase-polymerase chain reaction(RT-qPCR) and Western blotting were used to detect the expression of Fer mRNA and protein respectively, and the most efficient siRNA was screened out for the following experiments.The cell cycle and apoptosis rate were analyzed by flow cytometry.Furthermore, we observed the expression levels of the cell cycle-and apoptosisrelated genes in the transfected cells with down-regulation of the Fer expression. Results RT-qPCR showed that Fer mRNA relative expression levels of three siRNAs was 0. 288,0. 369 and 0. 623, respectively, which were significantly lower than those in the negative control group(l,P< 0. 05).Western blotting also demonstrated that the expression of Fer protein in PC-3 cells transfected with three siRNAs could significantly inhibited,and siRNA-1 was selected as the most efficient siRNA(P<0. 01).Flow cytometry revealed the percentage of G0/G1 phase cells in RNAi(Si) group was(52. 340± 2. 049)%, significantly higher than that in negative control group[(40. 230±2. 065)%](P< 0. 05), and that of S phase cells was(35. 040±3. 810)%, significantly lower than that in the negative control group[(50. 650±3. 693)%](P< 0. 05). The apoptosis rate in Si group was(33. 6± 3. 3)%, significanfly higher than that in negative control group[(7. 8±1. 0)%](P< 0. 05).Meanwhile, with the inhibition of Fer, the expression levels of Cyclin D1 and B cell lymphoma/leukemia-2(bcl-2) were reduced obviously, and p21 and cleaved Caspase-3 upregulated obviously as compared with negative control group(P< 0. 05). Conclusion Fer-siRNA could significantly inhibit the expression of Fer and the progression of G1/S in human prostate cancer cells, and then induced apoptosis of PC-3 cells in vitro.The mechanism probably correlates with regulation of the cell cycle -and apoptosis-related genes expression. Key words: Prostate cancer; Feline sarcoma-related protein; Small interfering RNA; Cell cycle; Apoptosis

195 Stromal expression of Fer plays as suppressor for tumour progression and it was a useful predictor for survival in patients with renal cell carcinoma

195 Stromal expression of Fer plays as suppressor for tumour progression and it was a useful predictor for survival in patients with renal cell carcinoma