Suppression of FUT1/FUT4 expression by siRNA inhibits tumor growth

Lewis Y (LeY) antigen is highly expressed in a variety of human carcinomas of epithelial cell origin. Recent studies suggest functional blockade of LeY may provide a novel therapeutic approach for the treatment of cancers. However, suppressing LeY expression by genetic manipulation and its impact on neoplastic cell proliferation has not been investigated. We report here that different fucosyltransferases (FUTs) were expressed with the greatest expression of fucosyltransferase I or IV (FUT1/4), the two key enzymes for the synthesis of LeY in human epidermoid carcinoma A431 cells. Knocking down FUT1/4 expression by short interfering RNA technique dramatically reduced the expression of FUT1/4 and LeY and inhibited cell proliferation through decreasing epidermal growth factor receptor (EGFR) signaling pathway. Treatment of A431 cells that were inoculated into the nude mice with FUT1 siRNA or FUT4 siRNA greatly impeded tumor growth. Suppressing FUT1/4 expression also blocked EGF-induced tyrosine phosphorylation of EGFR and mitogen-activated protein kinases. In conclusion, suppressing the expression of FUT1/4 by RNAi technology reduces the synthesis of LeY and inhibits cancer growth. It may serve as a potential methodology for the treatment of cancers that express LeY glycoconjugates.     

Analysis of glycosyltransferase expression in metastatic prostate cancer cells capable of rolling activity on microvascular endothelial (E)-selectin

Prostate cancer (PCa) cell tethering and rolling on microvascular endothelium has been proposed to promote the extravasation of PCa cells. We have shown that these adhesive events are mediated through binding interactions between endothelial (E)-selectin and Lewis carbohydrates on PCa cells. Prior data indicate that E-selectin-mediated rolling of bone-metastatic PCa MDA PCa 2b (MDA) cells is dependent on sialyl Lewis X (sLe(X))-bearing glycoproteins. To explore the molecular basis of sLe(X) synthesis and E-selectin ligand (ESL) activity on PCa cells, we compared and contrasted the expression level of glycosyltransferases, characteristically involved in sLe(X) and ESL synthesis, in ESL(+) MDA cells among other ESL(-) metastatic PCa cell lines. We also created and examined ESL(hi) and ESL(lo) variants of MDA cells to provide a direct comparison of the glycosyltransferase expression level. We found that normal prostate tissue and all metastatic PCa cell lines expressed glycosyltransferases required for sialo-lactosamine synthesis, including N-acetylglucosaminyl-, galactosyl-, and sialyltransferases. However, compared with expression in normal prostate tissue, ESL(+) MDA cells expressed a 31- and 10-fold higher level of alpha1,3 fucosyltransferases (FT) 3 and 6, respectively. Moreover, FT3 and FT6 were expressed at 2- to 354-fold lower levels in ESL(-) PCa cell lines. Consistent with these findings, ESL(hi) MDA cells expressed a 131- and 51-fold higher level of FT3 and FT6, respectively, compared with expression in ESL(lo) MDA cells. We also noted that alpha1,3 FT7 was expressed at a 5-fold greater level in ESL(hi) MDA cells. Furthermore, ESL(lo) MDA cells did not display sLe(X) on glycoproteins capable of bearing sLe(X), notably P-selectin glycoprotein ligand-1. These results implicate the importance of alpha1,3 FT3, FT6, and/or FT7 in sLe(X) and ESL synthesis on metastatic PCa cells.     

FUT family mediates the multidrug resistance of human hepatocellular carcinoma via the PI3K/Akt signaling pathway

The fucosyltransferase (FUT) family is the key enzymes in cell-surface antigen synthesis during various biological processes such as tumor multidrug resistance (MDR). The aim of this work was to analyze the alteration of FUTs involved in MDR in human hepatocellular carcinoma (HCC) cell lines. Using mass spectrometry (MS) analysis, the composition profiling of fucosylated N-glycans differed between drug-resistant BEL7402/5-FU (BEL/FU) cells and the sensitive line BEL7402. Further analysis of the expressional profiles of the FUT family in three pairs of parental and chemoresistant human HCC cell lines showed that FUT4, FUT6 and FUT8 were predominant expressed in MDR cell lines. The altered levels of FUT4, FUT6 and FUT8 were responsible for changed drug-resistant phenotypes of BEL7402 and BEL/FU cells both in vitro and in vivo. In addition, regulating FUT4, FUT6 or FUT8 expression markedly modulated the activity of the phosphoinositide 3 kinase (PI3K)/Akt signaling pathway and MDR-related protein 1 (MRP1) expression. Inhibition of the PI3K/Akt pathway by its specific inhibitor wortmannin, or by Akt small interfering RNA (siRNA), resulted in decreased MDR of BEL/FU cells, partly through the downregulation of MRP1. Taken together, our results suggest that FUT4-, FUT6- or FUT8-mediated MDR in human HCC is associated with the activation of the PI3K/Akt pathway and the expression of MRP1, but not of P-gp, indicating a possible novel mechanism by which the FUT family regulates MDR in human HCC.     

鳞癌细胞增殖能力与岩藻糖基转移酶表达水平及糖蛋白岩藻糖基化修饰有关

岩藻糖基转移酶（fucosyltransferases,FUTs）是一类催化糖蛋白和糖脂发生岩藻糖基化（修饰）酶,主要包括FUT1~FUT9。已有研究证明,很多癌组织中都有不同FUT基因表达升高的现象。本研究证明,表皮鳞癌细胞的增殖能力与几种FUT基因表达水平有关。本文比较研究了人表皮鳞癌A431和SCC12细胞的增殖速度和几种FUT的表达状况,以揭示鳞癌细胞增殖能力与几种FUT基因表达水平的关系。细胞倍增时间结合MTT法揭示,鳞癌A431细胞的倍增时间约为26 h,而鳞癌SCC12细胞的倍增时间约为33 h（P < 0.05）,提示A431细胞增殖速度比SCC12细胞明显加快。与增殖速度一致的是,Western 印迹显示,A431细胞中与DNA合成相关的增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）蛋白表达水平比SCC12细胞高。实时定量PCR（qPCR）检测FUT1-9基因 mRNA转录本,揭示A431细胞中几种FUT基因的mRNA水平均显著高于SCC12细胞。凝集素免疫印迹法和Western 印迹法进一步证明,A431细胞中总蛋白的岩藻糖基化水平比SCC12细胞中的明显升高。敲低FUT4基因表达后,A431细胞中LeY寡糖的表达水平下调,细胞增殖被明显抑制。这些结果证明,较强的表皮鳞癌细胞增殖能力可能与几种FUT基因的高表达,以及糖蛋白的岩藻糖基化（修饰）相关。岩藻糖基转移酶表达水平与临床表皮鳞癌的恶性增生的相关性有待进一步证明。     

FUT7 antisense sequence inhibits the expression of FUT7/sLeX and adhesion between embryonic and uterine cells

Implantation is a complex developmental event that is initiated by recognition and adhesion of the embryo to the endometrial epithelium. sLeX is an oligosaccharide antigen acting as the ligand of L-selectin, and is stage-specifically expressed in the endometrial epithelium. The adhesion system mediated by L-selectin and sLeX oligosaccharide plays an important role in this process. FUT7 is a key enzyme for sLeX synthesis, and the regulation of sLeX through FUT7 may influence maternal-fetal recognition. In this study, we observed the effect of FUT7 antisense oligodeoxynucleotide on the expression of FUT7 and sLeX, as well as adhesion in an in vitro implantation model consisting of the human uterine epithelial cell line RL95-2 and the human embryonic cell line JAR. Results showed that the expression of FUT7 was significantly decreased, compared with controls, after FUT7 antisense oligodeoxynucleotide transfection into RL95-2 cells, as determined by RT-PCR, Western blotting, and indirect immunofluorescence. Synthesis of sLeX was also decreased, consistent with the FUT7 decrease, as shown by indirect immunofluorescence. The adhesion of embryonic cells to uterine epithelial cells was significantly reduced (P < 0.01) compared with the control. These data indicate that the use of a FUT7 antisense oligodeoxynucleotide can cause a significant reduction of both FUT7 and sLeX antigen, and thereby inhibit the adhesion of embryo cells to endometrium. This approach may provide a new way to regulate reproduction.     

长链非编码RNA LINC01006对前列腺癌细胞增殖和侵袭的影响

摘要 目的：探究长链非编码RNA LINC01006对前列腺癌（prostate cancer, PCa）细胞增殖和侵袭能力的影响。方法：体外培养人前列腺正常上皮细胞系RWPE-1,人PCa细胞系LNCaP、22Rv1、PC3、C4-2b,应用实时定量PCR（qRT-PCR）技术检测上述细胞LINC01006的表达;分别通过转染小干扰RNA（siRNA）或过表达LINC01006的慢病毒载体,在LNCaP和PC3细胞中敲减LINC01006或稳定过表达LINC01006;应用CCK8、克隆形成实验检测LINC01006对PCa细胞增殖能力的影响;应用Transwell侵袭实验检测LINC01006对PCa细胞侵袭能力的影响;通过网站预测LINC01006的转录调控因子及其结合位点。结果：相较于正常前列腺上皮细胞系RWPE-1,PCa细胞系LNCaP、22Rv1、C4-2b和PC3中LINC01006表达明显升高（P<0.05）。敲减LINC01006后的PCa细胞系LNCaP和PC3的增殖和侵袭能力被显著抑制（P<0.05）,过表达LINC1006则明显促进PCa细胞系LNCaP和PC3的增殖、侵袭能力（P<0.05）。通过PROMO网站预测可见AR是LINC01006的潜在转录调控因子,通过Cistrome DB数据库发现LINC01006上游启动子区域存在AR富集;敲减、抑制AR后LNCaP细胞中LINC01006表达明显升高（P<0.05）。结论：LINC01006在PCa细胞系中呈高表达,促进PCa细胞的增殖和侵袭,其受到AR负向调控,可能在PCa发生发展和去势抵抗形成过程中发挥作用。     

Functional analysis of α1,3/4-fucosyltransferase VI in human hepatocellular carcinoma cells

The α1,3/4-fucosyltransferases (FUT) subfamily are key enzymes in cell surface antigen synthesis during various biological processes. A novel role of FUTs in tumorigenesis has been discovered recently, however, the underlying mechanism remains largely unknown. Here, we characterized FUT6, a member of α1,3/4-FUT subfamily, in human hepatocellular carcinoma (HCC). In HCC tissues, the expression levels of FUT6 and its catalytic product SLe(x) were significantly up-regulated. Overexpression of FUT6 in HCC cells enhanced S-phase cell population, promoted cell growth and colony formation ability. Moreover, subcutaneously injection of FUT6-overexpressing cells in nude mice promoted cell growth in vivo. In addition, elevating FUT6 expression markedly induced intracellular Akt phosphorylation, and suppressed the expression of the cyclin-dependent kinases inhibitor p21. Bath application of the PI3K inhibitor blocked FUT6-induced Akt phosphorylation, p21 suppression and cell proliferation. Our results suggest that FUT6 plays an important role in HCC growth by regulating the PI3K/Akt signaling pathway.     

Partial silencing of fucosyltransferase 8 gene expression inhibits proliferation of Ishikawa cells,a cell line of endometrial cancer

Endometrial cancer is the most common gynecologic malignancy and is associated with increased morbidity each year, including young people. However, its mechanisms of proliferation and progression are not fully elucidated. It is well known that abnormal glycosylation is involved in oncogenesis, and fucosylation is one of the most important types of glycosylation. In particular, fucosyltransferase 8 (FUT8) is the only FUT responsible for α1, 6-linked fucosylation (core fucosylation), and it is involved in various physiological as well as pathophysiological processes, including cancer biology. Therefore, we aimed to identify the expression of FUT8 in endometrial endometrioid carcinoma and investigate the effect of the partial silencing of the FUT8 gene on the cell proliferation of Ishikawa cells, an epithelial-like endometrial cancer cell line. Quantitative real-time PCR analysis showed that FUT8 gene expression was significantly elevated in the endometrial endometrioid carcinoma, compared to the normal endometrium. The immunostaining of FUT8 and Ulex europaeus Agglutinin 1 (UEA-1), a kind of lectin family specifically binding to fucose, was detected endometrial endometrioid carcinoma. The proliferation assay showed FUT8 partial knockdown by transfection of siRNA significantly suppressed the proliferation of Ishikawa cells, concomitant with the upregulation in the gene expressions associated with the interesting pathways associated with de-ubiquitination, aspirin trigger, mesenchymal-epithelial transition (MET) et al. It was suggested that the core fucosylation brought about by FUT8 might be involved in the proliferation of endometrial endometrioid carcinoma cells.     

Upregulation of Fucosyltransferase 3, 8 and protein O-Fucosyltransferase 1, 2 genes in esophageal cancer stem-like cells (CSLCs)

Recently, studies have shown that Fucosylation plays an important role in the invasion and metastatic process of CSLCs. Understanding the expression pattern of fucosyltransferase (FUT) genes may help to suggest better-targeted therapy strategies for esophageal squamous cell carcinoma (ESCC). The study aimed to address the expression pattern of FUT gene variants in esophageal CSLCs and parental adherent cells. Sphere formation method was used to enrich CSLCs. Expression of FUT genes was examined in tumor sphere and parental adherent cells using the RT-PCR method and then relative expression of detected variants was performed by the Real-Time PCR method in both groups. The detected FUTs, also, were assessed in fresh ESCC tumors and the matched healthy controls. Analysis of The cell surface carbohydrate Lewis x (LeX, CD15) was performed by flow cytometry. Molecular analysis showed that the expression of FUT 3, 8 and POFUT1, 2 genes in tumorsphere were significantly higher than parental adherent cells. Analysis of fresh ESCC tumor tissues and the matched healthy controls showed that FUT8 and POFUT1, 2 genes in contrast to FUT 3 have higher expression in tumor tissues than controls. Flow cytometric analyses revealed that tumorsphere and their parent cells do not differ significantly in Lewis x surface marker. The present study showed that FUT 3, 8 and POFUT1, 2 genes upregulated in esophageal CSLCs in comparison to adherent cells. Understanding the expression pattern of FUT gene variants may help to suggest better-targeted therapy strategies for ESCC.     

Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC)

Background

Recent data show aberrant and altered expression of regulatory noncoding micro (mi) RNAs in prostate cancer (PCa). A large number of miRNAs are encoded in organized intronic clusters within many protein coding genes. While expression profiling studies of miRNAs are common place, little is known about the host gene and their resident miRNAs coordinated expression in PCa cells. Furthermore, whether expression of a subset of miRNAs is distinct in androgen-responsive and androgen-independent cells is not clear. Here we have examined the expression of mature miRNAs of miR 17–92, miR 106b-25 and miR 23b-24 clusters along with their host genes C13orf25, MCM7 and AMPO respectively in PCa cell lines.

Results

The expression profiling of miRNAs and host genes was performed in androgen-sensitive MDA PCa 2b and LNCaP as well as in androgen-refractory PC-3 and DU 145 cell culture models of PCa. No significant correlation between the miRNA expression and the intrinsic hormone-responsive property of PCa cells was observed. Androgen-sensitive MDA PCa 2b cells exhibited the highest level of expression of most miRNAs studied in this report. We found significant expression variations between host genes and their resident miRNAs. The expressions of C13orf25 and miR 17–92 cluster as well as MCM7 and miR 106b-25 cluster did not reveal statistically significant correlation, thus suggesting that host genes and resident miRNAs may be expressed independent of each other.

Conclusion

Our results suggest that miRNA expression profiles may not predict intrinsic hormone-sensitive environment of PCa cells. More importantly, our data indicate the possibility of additional novel mechanisms for intronic miRNA processing in PCa cells.     

Androgen-Regulated Expression of Arginase 1, Arginase 2 and Interleukin-8 in Human Prostate Cancer

Background

Prostate cancer (PCa) is the most frequently diagnosed cancer in North American men. Androgen-deprivation therapy (ADT) accentuates the infiltration of immune cells within the prostate. However, the immunosuppressive pathways regulated by androgens in PCa are not well characterized. Arginase 2 (ARG2) expression by PCa cells leads to a reduced activation of tumor-specific T cells. Our hypothesis was that androgens could regulate the expression of ARG2 by PCa cells.

Methodology/Principal Findings

In this report, we demonstrate that both ARG1 and ARG2 are expressed by hormone-sensitive (HS) and hormone-refractory (HR) PCa cell lines, with the LNCaP cells having the highest arginase activity. In prostate tissue samples, ARG2 was more expressed in normal and non-malignant prostatic tissues compared to tumor tissues. Following androgen stimulation of LNCaP cells with 10 nM R1881, both ARG1 and ARG2 were overexpressed. The regulation of arginase expression following androgen stimulation was dependent on the androgen receptor (AR), as a siRNA treatment targeting the AR inhibited both ARG1 and ARG2 overexpression. This observation was correlated in vivo in patients by immunohistochemistry. Patients treated by ADT prior to surgery had lower ARG2 expression in both non-malignant and malignant tissues. Furthermore, ARG1 and ARG2 were enzymatically active and their decreased expression by siRNA resulted in reduced overall arginase activity and l-arginine metabolism. The decreased ARG1 and ARG2 expression also translated with diminished LNCaP cells cell growth and increased PBMC activation following exposure to LNCaP cells conditioned media. Finally, we found that interleukin-8 (IL-8) was also upregulated following androgen stimulation and that it directly increased the expression of ARG1 and ARG2 in the absence of androgens.

Conclusion/Significance

Our data provides the first detailed in vitro and in vivo account of an androgen-regulated immunosuppressive pathway in human PCa through the expression of ARG1, ARG2 and IL-8.     

Metabolic Inhibition of Sialyl-Lewis X Biosynthesis by 5-Thiofucose Remodels the Cell Surface and Impairs Selectin-Mediated Cell Adhesion

Sialyl-Lewis X (sLe^X) is a tetrasaccharide that serves as a ligand for the set of cell adhesion proteins known as selectins. This interaction enables adhesion of leukocytes and cancer cells to endothelial cells within capillaries, resulting in their extravasation into tissues. The last step in sLe^X biosynthesis is the α1,3-fucosyltrasferase (FUT)-catalyzed transfer of an L-fucose residue to carbohydrate acceptors. Impairing FUT activity compromises leukocyte homing to sites of inflammation and renders cancer cells less malignant. Inhibition of FUTs is, consequently, of great interest, but efforts to generate glycosyltransferase inhibitors, including FUT inhibitors, has proven challenging. Here we describe a metabolic engineering strategy to inhibit the biosynthesis of sLe^X in cancer cells using peracetylated 5-thio-L-fucose (5T-Fuc). We show that 5T-Fuc is taken up by cancer cells and then converted into a sugar nucleotide analog, GDP-5T-Fuc, that blocks FUT activity and limits sLe^X presentation on HepG2 cells with an EC₅₀ in the low micromolar range. GDP-5T-Fuc itself does not get transferred by either FUT3 or FUT7 at a measurable rate. We further demonstrate that treatment of cells with 5T-Fuc impaired their adhesive properties to immobilized adhesion molecules and human endothelial cells. 5T-Fuc, therefore, is a useful probe that can be used to modulate sLe^X levels in cells to evaluate the consequences of inhibiting FUT-mediated sLe^X formation. These data also reveal the utility of using sugar analogues that lead to formation of donor substrate analogues within cells as a general approach to blocking glycosyltransferases in cells.     

CCR2 expression correlates with prostate cancer progression

Although the primary role of chemokines and their receptors is controlling the trafficking of leukocytes during inflammatory responses, they also play pleoitropic roles in cancer development. There is emerging evidence that cancer cells produce chemokines that induce tumor cell proliferation or chemotaxis in various cancer types. We have previously reported that MCP-1 acts as a paracrine and autocrine factor for prostate cancer (PCa) growth and invasion. As the cellular effects of MCP-1 are mediated by CC chemokine receptor 2 (CCR2), we hypothesized that CCR2 may contribute PCa progression. Accordingly, we first determined CCR2 mRNA and protein expression in various cancer cell lines, including PCa and other cancer types. All cells expressed CCR2 mRNA and protein, but in PCa, more aggressive cancer cells such as C4-2B, DU145, and PC3 expressed a higher amount of CCR2 compared with the less aggressive cancer cells such as LNCaP or non-neoplastic PrEC and RWPE-1 cells. Further, we found a positive correlation between CCR2 expression and PCa progression by analyzing an ONCOMINE gene array database. We confirmed that CCR2 mRNA was highly expressed in PCa metastatic tissues compared with the localized PCa or benign prostate tissues by real-time RT-PCR. Finally, CCR2 protein expression was examined by immunohistochemical staining on tissue microarray specimens from 96 PCa patients and 31 benign tissue controls. We found that CCR2 expression correlated with Gleason score and clinical pathologic stages, whereas lower levels of CCR2 were expressed in normal prostate tissues. These results suggest that CCR2 may contribute to PCa development.     

Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.     

Analysis of the expression and association of retinoblastoma binding protein 6 with the JNK signaling pathway in prostate cancers

This study aims to investigate the expression of retinoblastoma binding protein 6 (RBBP6) in prostate cancer (PCa) and its association with the c‐Jun N‐terminal kinase (JNK) pathway. Immunohistochemistry was used to detect RBBP6 and JNK1/2 expression in PCa and benign prostatic hyperplasia tissues. RBBP6 expression in PCa cells (LNCap, PC3, and DU145) and noncancerous prostate epithelial cells (RWPE‐1) was determined by quantitative real‐time polymerase chain reaction and western blot analysis. PC3 and DU145 cells were transfected with RBBP6 small interfering RNAs (siRNAs) to examine the biological characteristics. Anisomycin (a JNK activator) with/without RBBP6 siRNA was used to treat PC3 cells for further investigating the ramification of the RBBP6‐mediated JNK pathway in PCa. PCa tissues and cells showed higher RBBP6 and JNK1/2 expression. RBBP6 was positively correlated with JNK1/2 in PCa tissues. Besides, RBBP6 expression was correlated to clinical tumor stage, lymph node metastasis, Gleason grade, preoperative prostate‐specific antigen level, as well as prognosis of PCa. RBBP6 siRNA reduced cell proliferation, arrested cells at G2/M, and promoted cell apoptosis, and suppressed JNK pathway. In addition, migration and invasion decreased after the RBBP6 siRNA transfection with downregulated matrix metallopeptidase‐2 (MMP‐2) and MMP‐9. Anisomycin promoted the proliferation, invasion, and migration of PC3 cells and inhibited PC3 cell apoptosis, which could be reversed by RBBP6 siRNA. RBBP6 expression was upregulated in PCa tissues and positively correlated with expression level of JNK1/2. With inhibition of RBBP6 expression, the proliferation, invasion, and migration of PCa cells decreased dramatically, while PC3 cell apoptosis increased appreciably, accompanied by the suppression of the JNK pathway.     

HOTAIR/miR-326/FUT6 axis facilitates colorectal cancer progression through regulating fucosylation of CD44 via PI3K/AKT/mTOR pathway

Metastasis is the main cause of death in colorectal cancer (CRC) patients. Aberrant fucosylation, catalyzed by the specific fucosyltransferases (FUTs), is associated with malignant behaviors. Non-conding RNAs, including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), emerge as key molecules in cancer malignancy. The aim of this study was to investigate HOTAIR/miR-326/FUT6 axis modified fucosylation on sLe^X-CD44 (HCELL), which served as E-selectin ligand during CRC progression. Higher levels of HOTAIR and FUT6 were verified in CRC tissues and cell lines, with a positive correlation. HOTAIR was associated with poor clinical prognosis of CRC. Altered HOTAIR levels influenced proliferation, aggressiveness, apoptosis and tumorigenesis of CRC cells. HOTAIR directly harbored miR-326 binding sites and regulated FUT6 expression. Further results corroborated that HOTAIR/miR-326/FUT6 axis modified α1, 3-fucosylation of CD44, which mediated CRC malignancy. Co-modulation of HOTAIR, miR-326 and FUT6 impacted α1, 3-fucosylated CD44, which further triggered PI3K/AKT/mTOR pathway. HOTAIR also mediated CRC tumorigenesis and liver metastasis in vivo. Thus, our findings indicated that HOTAIR/miR-326/FUT6 axis mediated CRC procession through α1, 3-fucosylated CD44 via PI3K/AKT/mTOR pathway. This work rendered new therapeutic targets for CRC.     

Immunohistochemical characterisation of the monoclonal antibody BLCA-38 for the detection of prostate cancer

Background: Monoclonal antibodies (MAbs) can be used to detect, image and treat cancers. This study aimed to characterise the binding of BLCA-38 MAbs to human prostate cancer cell lines, human prostate cancer biopsy samples and normal tissues to enable future targeted studies. Methods: BLCA-38 antigen expression on cancer lines was determined by flow cytometry; that on patient specimens from normal tissues and cancers was tested by immunohistochemistry using fresh frozen tissues or paraffin-embedded tissues that had undergone antigen retrieval. Results: Cell surface BLCA-38 antigen expression was seen on DU-145, PC-3, PC-3 M and PC-3 M-MM2 prostate cancer lines, but LNCaP, MDA PCa 2a or MDA PCa 2b lines were negative. Other human lines, including 8/12 bladder cancer and A431 vulval epidermoid cells, but not breast cancer lines, expressed BLCA-38 antigen. Staining occurred in glandular epithelial cells in the majority of frozen, and paraffin-embedded prostate cancer tissues and was occasionally seen in prostatic intraepithelial neoplasia (PIN). No staining was observed in normal cadaver tissues or in benign areas from various other cancer tissues. Conclusions: The BLCA-38 antibody binds to the majority of human prostate cancers but not to normal cells, and has potential for targeting novel therapies in patients with this disease.     

Transfected Poly(I:C) Activates Different dsRNA Receptors,Leading to Apoptosis or Immunoadjuvant Response in Androgen-independent Prostate Cancer Cells

Despite the effectiveness of surgery or radiation therapy for the treatment of early-stage prostate cancer (PCa), there is currently no effective strategy for late-stage disease. New therapeutic targets are emerging; in particular, dsRNA receptors Toll-like receptor 3 (TLR3) and cytosolic helicases expressed by cancer cells, once activated, exert a pro-apoptotic effect in different tumors. We previously demonstrated that the synthetic analog of dsRNA poly(I:C) induces apoptosis in the androgen-dependent PCa cell line LNCaP in a TLR3-dependent fashion, whereas only a weak apoptotic effect is observed in the more aggressive and androgen-independent PCa cells PC3 and DU145. In this paper, we characterize the receptors and the signaling pathways involved in the remarkable apoptosis induced by poly(I:C) transfected by Lipofectamine (in-poly(I:C)) compared with the 12-fold higher free poly(I:C) concentration in PC3 and DU145 cells. By using genetic inhibition of different poly(I:C) receptors, we demonstrate the crucial role of TLR3 and Src in in-poly(I:C)-induced apoptosis. Therefore, we show that the increased in-poly(I:C) apoptotic efficacy is due to a higher binding of endosomal TLR3. On the other hand, we show that in-poly(I:C) binding to cytosolic receptors MDA5 and RIG-I triggers IRF3-mediated signaling, leading uniquely to the up-regulation of IFN-β, which likely in turn induces increased TLR3, MDA5, and RIG-I proteins. In summary, in-poly(I:C) activates two distinct antitumor pathways in PC3 and DU145 cells: one mediated by the TLR3/Src/STAT1 axis, leading to apoptosis, and the other one mediated by MDA5/RIG-I/IRF3, leading to immunoadjuvant IFN-β expression.     

Notch signaling and ERK activation are important for the osteomimetic properties of prostate cancer bone metastatic cell lines

Prostate cancer bone metastases are characterized by their ability to induce osteoblastic lesions and local bone formation. It has been suggested that bone metastatic prostate cancer cells are osteomimetic and capable of expressing genes and proteins typically expressed by osteoblasts. The ability of preosteoblasts to differentiate and express osteoblastic genes depends on several pathways, including Notch and MAPK. Here we show that notch1 expression is increased 4-5 times in C4-2B and MDA PCa 2b cells (osteoblastic skeletal prostate metastatic cancer cell lines) when compared with nonskeletal metastatic cell lines (LNCaP and DU145). Notch1 ligand, dll1, is expressed only in C4-2B cells. Immunohistochemical studies demonstrate that Notch1 is present in both human clinical samples from prostate cancer bone metastases and the C4-2B cell line. To determine whether prostate cancer bone metastases respond to osteogenic induction similar to osteoblasts, C4-2B cells were cultured in osteogenic medium that promotes mineralization. C4-2B cells mineralize and express HES-1 (a downstream target of Notch), an effect that is completely inhibited by L-685,458, a Notch activity inhibitor. Furthermore, osteogenic induction increases ERK activation, runx2 expression, and nuclear localization, independent of Notch signaling. Finally, we show that Notch and ERK activation are essential for Runx2 DNA binding activity and osteocalcin gene expression in C4-2B cells in response to osteogenic induction. These studies demonstrate that prostate cancer bone metastatic cell lines acquire osteoblastic properties through independent activation of ERK and Notch signaling; presumably, both pathways are activated in the bone microenvironment.