沉默ST3Gal III抑制MDA-MB-231细胞黏附和侵袭能力

我们前期研究表明α2,3-唾液酸水平与乳腺癌侵袭转移密切相关。人α2,3-唾液酸转移酶（ST3Gal Ⅲ）可催化合成细胞表面的α2,3-唾液酸,并在乳腺癌组织中高表达,此酶活性与肿瘤转移潜能密切相关,但其机制尚未阐明。本研究中我们将继续探讨ST3Gal Ⅲ在对乳腺癌转移关键步骤粘附和侵袭中的作用。构建特异靶向ST3Gal Ⅲ的短发夹RNA（shRNA）序列的慢病毒载体,采用细胞转染沉默乳腺癌MDA-MB-231细胞的ST3Gal Ⅲ,经实时定量PCR及Western印迹检测转染后细胞ST3Gal Ⅲ mRNA及蛋白表达,验证构建了稳定下调ST3Gal Ⅲ表达的两个细胞克隆,分别记作shRNA-2、shRNA-4。细胞表面α2,3-唾液酸是ST3Gal Ⅲ下游产物,可代表酶活性。流式细胞术分析结果证实,shRNA-2、shRNA-4细胞表面α2,3-唾液酸的含量显著降低（P<0.05）。细胞黏附、细胞迁移及侵袭能力等功能学检测结果表明,shRNA细胞黏附能力及侵袭能力明显降低（P<0.05）。β1整合素表达与肿瘤侵袭能力获取密切相关。本研究中,沉默ST3Gal Ⅲ可抑制β1整合素表达（P<0.05）。这些结果提示,ST3Gal Ⅲ在乳腺癌转移关键步骤黏附和侵袭中具有重要作用,沉默ST3Gal Ⅲ抑制MDA MB-231细胞黏附和侵袭能力,其作用机制可能是通过下调β1整合素表达。此研究从新的视角认识了乳腺癌转移的机制,并可能提供乳腺癌转移治疗的新靶点。     

过表达外源ST3Gal I增加MCF-7细胞与胞外基质粘附和侵袭能力

为探讨过表达外源α2,3-唾液酸转移酶（ST3Gal Ⅰ）对乳腺癌MCF-7细胞粘 附和侵袭能力的影响,构建pEGFP-N1-ST3Gal I真核表达载体.采用GenEscortTM Ⅱ包裹后转染MCF-7细胞. MCF-7细胞为3组：未转染组 (M)、转染空质粒组 (P) 和转染ST3Gal I组 (ST3); 荧光显微镜观察融合蛋白EGFP ST3Gal I的表达.采用 半定量RT-PCR、Western印迹法分析转染后MCF-7细胞ST3Gal Ⅰ基因mRNA水平和 蛋白表达水平;流式细胞术分析ST3Gal Ⅰ下游产物细胞表面α2,3-唾液酸含量;采用细胞粘附实验及transwell小室检测转染前后细胞与基质胶Matrigel粘附、迁移和侵袭运动能力的变化.结果表明, 荧光显微镜下P组细胞内绿色荧光呈弥散分 布,而ST3组绿色荧光主要集中在细胞质中,RT-PCR与Western印迹也证实了外源 ST3Gal Ⅰ基因mRNA和蛋白表达均明显增加（P<0.05）,其下游产物细胞表面 α2,3-唾液酸含量明显增加（P<0.05）;与M、P组相比,ST3组表现为粘附、迁移和侵袭能力明显增强（P<0.05）.利用转染技术可明显提高外源ST3Gal Ⅰ在MCF -7细胞表达,明显增加MCF-7细胞与胞外基质（ECM）粘附、迁移和侵袭能力,可形成肿瘤入侵表型,将有望成为治疗乳腺癌转移的新靶点.     

Enhancement of anti-murine colon cancer immunity by fusion of a SARS fragment to a low-immunogenic carcinoembryonic antigen

Background  

It is widely understood that tumor cells express tumor-associated antigens (TAAs), of which many are usually in low immunogenicity; for example, carcinoembryonic antigen (CEA) is specifically expressed on human colon cancer cells and is viewed as a low-immunogenic TAA. How to activate host immunity against specific TAAs and to suppress tumor growth therefore becomes important in cancer therapy development.     

Guggulsterone targets smokeless tobacco induced PI3K/Akt pathway in head and neck cancer cells

Background

Epidemiological association of head and neck cancer with smokeless tobacco (ST) emphasizes the need to unravel the molecular mechanisms implicated in cancer development, and identify pharmacologically safe agents for early intervention and prevention of disease recurrence. Guggulsterone (GS), a biosafe nutraceutical, inhibits the PI3K/Akt pathway that plays a critical role in HNSCC development. However, the potential of GS to suppress ST and nicotine (major component of ST) induced HNSCC remains unexplored. We hypothesized GS can abrogate the effects of ST and nicotine on apoptosis in HNSCC cells, in part by activation of PI3K/Akt pathway and its downstream targets, Bax and Bad.

Methods and Results

Our results showed ST and nicotine treatment resulted in activation of PI3K, PDK1, Akt, and its downstream proteins - Raf, GSK3β and pS6 while GS induced a time dependent decrease in activation of PI3K/Akt pathway. ST and nicotine treatment also resulted in induction of Bad and Bax phosphorylation, increased the association of Bad with 14-3-3ζresulting in its sequestration in the cytoplasm of head and neck cancer cells, thus blocking its pro-apoptotic function. Notably, GS pre-treatment inhibited ST/nicotine induced activation of PI3K/Akt pathway, and inhibited the Akt mediated phosphorylation of Bax and Bad.

Conclusions

In conclusion, GS treatment not only inhibited proliferation, but also induced apoptosis by abrogating the effects of ST / nicotine on PI3K/Akt pathway in head and neck cancer cells. These findings provide a rationale for designing future studies to evaluate the chemopreventive potential of GS in ST / nicotine associated head and neck cancer.     

Tumor cell-mediated induction of the stromal factor stromelysin-3 requires heterotypic cell contact-dependent activation of specific protein kinase C isoforms

Stromelysin-3 (ST3, MMP-11) has been shown to be strongly overexpressed in stromal fibroblasts of most invasive human carcinomas. However, the molecular mechanisms leading to ST3 expression in nonmalignant fibroblasts remain unknown. The aim of the present study was to analyze the signaling pathways activated in normal pulmonary fibroblasts after their interaction with non-small cell lung cancer (NSCLC) cells and leading to ST3 expression. The use of selective signaling pathway inhibitors showed that conventional and novel protein kinase Cs (PKC) were required for ST3 induction, whereas Src kinases exerted a negative control. We observed by both conventional and real time confocal microscopy that green fluorescent protein-tagged PKCalpha and PKCepsilon, but not PKCdelta, transfected in fibroblasts, accumulate selectively at the cell-cell contacts between fibroblasts and tumor cells. In agreement, RNAi-mediated depletion of PKCalpha and PKCepsilon, but not PKCdelta significantly decreased co-culture-dependent ST3 production. Finally, a tetracycline-inducible expression model allowed us to confirm the central role of these PKC isoforms and the negative regulatory function of c-Src in the control of ST3 expression. Altogether, our data emphasize signaling changes occurring in the tumor microenvironment that may define new stromal targets for therapeutic intervention.     

GD₃ synthase expression enhances proliferation and tumor growth of MDA-MB-231 breast cancer cells through c-Met activation

The disialoganglioside G(D3) is overexpressed in ～50% of invasive ductal breast carcinoma, and the G(D3) synthase gene (ST8SIA1) displays higher expression among estrogen receptor-negative breast cancer tumors, associated with a decreased overall survival of breast cancer patients. However, no relationship between ganglioside expression and breast cancer development and aggressiveness has been reported. We have previously shown that overexpression of G(D3) synthase induces the accumulation of b- and c-series gangliosides (G(D3), G(D2), and G(T3)) at the cell surface of MDA-MB-231 breast cancer cells together with the acquisition of a proliferative phenotype in the absence of serum. Here, we show that phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways are constitutively activated in G(D3) synthase-expressing cells. Analysis of phosphorylation of tyrosine kinase receptors shows a specific c-Met constitutive activation in G(D3) synthase-expressing cells, in the absence of its ligand, hepatocyte growth factor/scatter factor. In addition, inhibition of c-Met or downstream signaling pathways reverses the proliferative phenotype. We also show that G(D3) synthase expression enhances tumor growth in severe combined immunodeficient mice. Finally, a higher expression of ST8SIA1 and MET in the basal subtype of human breast tumors are observed. Altogether, our results show that G(D3) synthase expression is sufficient to enhance the tumorigenicity of MDA-MB-231 breast cancer cells through a ganglioside-dependent activation of the c-Met receptor.     

Cyclooxygenase-2 Enzyme Induces the Expression of the α-2,3-Sialyltransferase-3 (ST3Gal-I) in Breast Cancer

Aberrant glycosylation is a common feature of malignant change. Changes in mucin-type O-linked glycosylation in breast cancer can result in the expression of truncated core 1-based sialylated glycans rather than the core 2-based glycans observed in normal mammary epithelium cells. This has been shown, in part, to be due to changes in the expression of glycosyltransferases, including the up-regulation of some sialyltransferases. Using the breast cancer cell line T47D, we have shown that PGE2, one of the final products of the cyclooxygenase-2 (COX-2) pathway, can induce the mRNA expression of the sialyltransferase α-2,3-sialyltransferase-3 (ST3Gal-I), resulting in increased sialyltransferase activity, demonstrated by a reduction in PNA lectin staining. Induction of COX-2 in the MDA-MB-231 breast cancer cell line also results in the increased expression of ST3Gal-I, leading to increased sialylation of the substrate of ST3Gal-I, core 1 Galβ1,3GalNAc. This effect on sialylation could be reversed by the selective COX-2 inhibitor celecoxib. The use of siRNA to knock down COX-2 and overexpression of COX-2 in MDA-MD-231 cells confirmed the involvement of COX-2 in the up-regulation of ST3Gal-I. Moreover, analysis of the expression of ST3Gal-I and COX-2 by 74 primary breast cancers showed a significant correlation between the two enzymes. COX-2 expression has been associated with a number of tumors, including breast cancer, where its expression is associated with poor prognoses. Thus, these results suggest the intriguing possibility that some of the malignant characteristics associated with COX-2 expression may be via the influence that COX-2 exerts on the glycosylation of tumor cells.     

Conversion of human fibroblasts to tissue macrophages by the Snyder-Theilen feline sarcoma virus (ST:FeSV): tumoricidal potential in monolayers and in agar suspensions.

In earlier studies [1-3], we have demonstrated the conversion of human fibroblasts (HF) to tissue macrophages (TM) by the Snyder-Theilen feline sarcoma virus (ST:(FeSV)). The purpose of the present study is to determine the cytolytic potential of ST:FeSV(FeLV)-induced TM against tumorigenic target cells under defined conditions in vitro. The results show that ST:FeSV-induced TM, but not mock-infected HF, produced significant lysis of human colon adenocarcinoma cells (LS-180) after a 3-day preincubation period, followed by a 4-day coincubation period at an effector to target cell ratio of 5:1. The presence of IFN-gamma, or lipopolysaccharides (LPS), and especially of M-CSF, during the coincubation period generally yielded optimal lysis of the tumor cells. Addition of LS-180 specific antibody (NRCO-4) substantially increased the cytolytic potential of TM. Significantly, coincubation of TM with LS-180 tumor cells in an agar medium, where no direct contact between cells occurs, resulted in the inhibition of tumor cell proliferation. Addition of LPS has further accentuated this inhibition. The results indicate that ST:FeSV-induced macrophages are potent oncocytolytic agents of LS-180 tumor cells in the absence and in the presence of direct contact between effector and target cells.     

Human Natural Killer-1 Sulfotransferase (HNK-1ST)-induced Sulfate Transfer Regulates Laminin-binding Glycans on α-Dystroglycan

Retinoic acid (RA) is a well established anti-tumor agent inducing differentiation in various cancer cells. Recently, a robust up-regulation of human natural killer-1 sulfotransferase (HNK-1ST) was found in several subsets of melanoma cells during RA-mediated differentiation. However, the molecular mechanism underlying the tumor suppression mediated by HNK-1ST remains unclear. Here, we show that HNK-1ST changed the glycosylation state and reduced the ligand binding activity of α-dystroglycan (α-DG) in RA-treated S91 melanoma cells, which contributed to an attenuation of cell migration. Knockdown of HNK-1ST restored the glycosylation of α-DG and the migration of RA-treated S91 cells, indicating that HNK-1ST functions through glycans on α-DG. Using CHO-K1 cells, we provide direct evidence that HNK-1ST but not other homologous sulfotransferases (C4ST1 and GalNAc4ST1) suppresses the glycosylation of α-DG. The activity-abolished mutant of HNK-1ST did not show the α-DG-modulating function, indicating that the sulfotransferase activity of HNK-1ST is essential. Finally, the HNK-1ST-dependent incorporation of [(35)S]sulfate groups was detected on α-DG. These findings suggest a novel role for HNK-1ST as a tumor suppressor controlling the functional glycans on α-DG and the importance of sulfate transfer in the glycosylation of α-DG.     

The effect of ST2 gene product on anchorage-independent growth of a glioblastoma cell line, T98G.

The ST2 gene, which is specifically induced by growth stimulation in fibroblasts, encodes interleukin-1 receptor-related proteins and is widely expressed in hematopoietic, helper T, and various cancer cells. However, the physiological as well as pathological functions of the ST2 gene products are not yet fully understood. In this study, we analyzed the expression of the ST2 gene in human glioma cell lines and human brain tumor samples with real-time polymerase chain reaction method, the results of which revealed that the expression level of the ST2 gene in glioma cell lines and glioblastoma samples is significantly lower than that in a fibroblastic cell line, TM12, and benign brain tumors, suggesting the reverse relationship between malignancy and ST2 expression. As we could not detect the soluble ST2 protein in the culture fluid of the T98G glioblastic cell line by ELISA, we established stable transformants of T98G that continuously produce and secrete the ST2 protein, in order to study the effect of the ST2 protein on malignancy. Although we could not detect a remarkable difference in proliferation between transformants and control cells in conventional tissue culture dishes, the efficiency of colony formation in soft agar was significantly decreased in the case of cells that continuously produce the ST2 protein. Furthermore, inhibition of colony formation in soft agar was observed in wild-type T98G cells when purified soluble ST2 protein was added to the culture, in a dose-dependent manner. Taken together, the results suggest that the expression of ST2 suppressed the anchorage-independent growth and malignancy.     

Hedgehog signaling: networking to nurture a promalignant tumor microenvironment

In addition to its role in embryonic development, the Hedgehog pathway has been shown to be an active participant in cancer development, progression, and metastasis. Although this pathway is activated by autocrine signaling by Hedgehog ligands, it can also initiate paracrine signaling with cells in the microenvironment. This creates a network of Hedgehog signaling that determines the malignant behavior of the tumor cells. As a result of paracrine signal transmission, the effects of Hedgehog signaling most profoundly influence the stromal cells that constitute the tumor microenvironment. The stromal cells in turn produce factors that nurture the tumor. Thus, such a resonating cross-talk can amplify Hedgehog signaling, resulting in molecular chatter that overall promotes tumor progression. Inhibitors of Hedgehog signaling have been the subject of intense research. Several of these inhibitors are currently being evaluated in clinical trials. Here, we review the role of the Hedgehog pathway in the signature characteristics of cancer cells that determine tumor development, progression, and metastasis. This review condenses the latest findings on the signaling pathways that are activated and/or regulated by molecules generated from Hedgehog signaling in cancer and cites promising clinical interventions. Finally, we discuss future directions for identifying the appropriate patients for therapy, developing reliable markers of efficacy of treatment, and combating resistance to Hedgehog pathway inhibitors.     

Soyasaponin I, a potent and specific sialyltransferase inhibitor

A growing number of reports demonstrate that hypersialylation, which is observed in certain pathological processes, such as oncogenic transformation, tumor metastasis, and invasion, is associated with enhanced sialyltransferase (ST) activity. There is therefore a need for the development of ST inhibitors to modulate ST activity and thus alleviate the disease processes caused by STs. In the present study, soyasaponin I had been discovered to be a potent and specific ST inhibitor by screening strategy from 7500 samples including micribial extracts and natural products. Kinetic analysis shows that it is a CMP-Neu5Ac competitive inhibitor with for ST3Gal I with an inhibition constant (K(i)) of 2.1 microM. In addition, it is only active against ST, but not against the other tested glycosyltransferases and glycosidases. Our study is the first report to discover ST inhibitor by screening method and also to provide the new chemical structure information that should be useful in the development of other novel ST inhibitors.     

Characterization of cancer associated mucin type O-glycans using the exchange sialylation properties of mammalian sialyltransferase ST3Gal-II

Our previous studies suggest that the α2,3sialylated T-antigen (NeuAcα2,3Galβ1,3GalNac-) and associated glycan structures are likely to be elevated during cancer. An easy and reliable strategy to label mucinous glycans that contain such carbohydrates can enable the identification of novel glycoproteins that are cancer associated. To this end, the present study demonstrates that the exchange sialylation property of mammalian ST3Gal-II can facilitate the labeling of mucin glycoproteins in cancer cells, tumor specimens, and glycoproteins in cancer sera. Results show that (i) the radiolabeled mucin glycoproteins of each of the cancer cell lines studied (T47D, MCF7, LS180, LNCaP, SKOV3, HL60, DU4475, and HepG2) is distinct either in terms of the specific glycans presented or their relative distribution. While some cell lines like T47D had only one single sialylated O-glycan, others like LS180 and DU4475 contained a complex mixture of mucinous carbohydrates. (ii) [14C]sialyl labeling of primary tumor cells identified a 25-35 kDa mucin glycoprotein unique to pancreatic tumor. Labeled glycoproteins for other cancers had higher molecular weight. (iii) Studies of [14C] sialylated human sera showed larger mucin glycopeptides and >2-fold larger mucin-type chains in human serum compared to [14C]sialyl labeled glycans of fetuin. Overall, the exchange sialylation property of ST3Gal-II provides an efficient avenue to identify mucinous proteins for applications in glycoproteomics and cancer research.     

ST6Gal-I–mediated sialylation of the epidermal growth factor receptor modulates cell mechanics and enhances invasion

Heterogeneity within the glycocalyx influences cell adhesion mechanics and signaling. However, the role of specific glycosylation subtypes in influencing cell mechanics via alterations of receptor function remains unexplored. It has been shown that the addition of sialic acid to terminal glycans impacts growth, development, and cancer progression. In addition, the sialyltransferase ST6Gal-I promotes epidermal growth factor receptor (EGFR) activity, and we have shown EGFR is an ‘allosteric mechano-organizer’ of integrin tension. Here, we investigated the impact of ST6Gal-I on cell mechanics. Using DNA-based tension gauge tether probes of variable thresholds, we found that high ST6Gal-I activity promotes increased integrin forces and spreading in Cos-7 and OVCAR3, OVCAR5, and OV4 cancer cells. Further, employing inhibitors and function-blocking antibodies against β1, β3, and β5 integrins and ST6Gal-I targets EGFR, tumor necrosis factor receptor, and Fas cell surface death receptor, we validated that the observed phenotypes are EGFR-specific. We found that while tension, contractility, and adhesion are extracellular-signal-regulated kinase pathway-dependent, spreading, proliferation, and invasion are phosphoinositide 3-kinase-Akt serine/threonine kinase dependent. Using total internal reflection fluorescence microscopy and flow cytometry, we also show that high ST6Gal-I activity leads to sustained EGFR membrane retention, making it a key regulator of cell mechanics. Our findings suggest a novel sialylation-dependent mechanism orchestrating cellular mechanics and enhancing cell motility via EGFR signaling.     

Pharmacological Inhibition of polysialyltransferase ST8SiaII Modulates Tumour Cell Migration

Polysialic acid (polySia), an α-2,8-glycosidically linked polymer of sialic acid, is a developmentally regulated post-translational modification predominantly found on NCAM (neuronal cell adhesion molecule). Whilst high levels are expressed during development, peripheral adult organs do not express polySia-NCAM. However, tumours of neural crest-origin re-express polySia-NCAM: its occurrence correlates with aggressive and invasive disease and poor clinical prognosis in different cancer types, notably including small cell lung cancer (SCLC), pancreatic cancer and neuroblastoma. In neuronal development, polySia-NCAM biosynthesis is catalysed by two polysialyltransferases, ST8SiaII and ST8SiaIV, but it is ST8SiaII that is the prominent enzyme in tumours. The aim of this study was to determine the effect of ST8SiaII inhibition by a small molecule on tumour cell migration, utilising cytidine monophosphate (CMP) as a tool compound. Using immunoblotting we showed that CMP reduced ST8iaII-mediated polysialylation of NCAM. Utilizing a novel HPLC-based assay to quantify polysialylation of a fluorescent acceptor (DMB-DP3), we demonstrated that CMP is a competitive inhibitor of ST8SiaII (K _i = 10 µM). Importantly, we have shown that CMP causes a concentration-dependent reduction in tumour cell-surface polySia expression, with an absence of toxicity. When ST8SiaII-expressing tumour cells (SH-SY5Y and C6-STX) were evaluated in 2D cell migration assays, ST8SiaII inhibition led to significant reductions in migration, while CMP had no effect on cells not expressing ST8SiaII (DLD-1 and C6-WT). The study demonstrates for the first time that a polysialyltransferase inhibitor can modulate migration in ST8SiaII-expressing tumour cells. We conclude that ST8SiaII can be considered a druggable target with the potential for interfering with a critical mechanism in tumour cell dissemination in metastatic cancers.     

Hypoxic colorectal cancer-secreted exosomes deliver miR-210-3p to normoxic tumor cells to elicit a protumoral effect

Hypoxia, the most common feature in the tumor microenvironment, is closely related to tumor malignant progression and poor patient’s prognosis. Exosomes, initially recognized as cellular “garbage dumpsters”, are now known to be important mediums for mediating cellular communication in tumor microenvironment. However, the mechanisms of hypoxic tumor cell-derived exosomes facilitate colorectal cancer progression still need further exploration. In the present study, we found that exosomes from hypoxic colorectal cancer cells (H-Exos) promoted G1-S cycle transition and proliferation while preventing the apoptosis of colorectal cancer cells by transmitting miR-210-3p to normoxic tumor cells. Mechanistic investigation indicated that miR-210-3p from H-Exos elicited its protumoral effect via suppressing CELF2 expression. A preclinical study further confirmed that H-Exos could promote tumorigenesis in vivo. Clinically, the expression of miR-210-3p in circulating plasma exosomes was markedly upregulated in colorectal cancer patients, which were closely associated with multiple unfavorable clinicopathological features. Taken together, these results suggest that hypoxia may stimulate colorectal cancer cells to secrete miR-210-3p-enriched exosomes in tumor microenvironment, which elicit protumoral effects by inhibiting CELF2 expression. These findings provide new insights on the mechanism of colorectal cancer progression and potential therapeutic targets for colorectal cancer.     

Enhanced Active Targeting via Cooperative Binding of Ligands on Liposomes to Target Receptors

To achieve effective active targeting in a drug delivery system, we previously developed dual-targeting (DT) liposomes decorated with both vascular endothelial growth factor receptor-1 (VEGFR-1)-targeted APRPG and CD13-targeted GNGRG peptide ligands for tumor neovessels, and observed the enhanced suppression of tumor growth in Colon26 NL-17 tumor-bearing mice by the treatment with the DT liposomes encapsulating doxorubicin. In this present study, we examined the binding characteristics of DT liposomes having a different couple of ligands, namely, APRPG and integrin α_vβ₃-targeted GRGDS peptides. These DT liposomes synergistically associated to stimulated human umbilical vein endothelial cells compared with single-targeting (ST) liposomes decorated with APRPG or GRGDS. The results of a surface plasmon resonance assay showed that ST liposomes modified with APRPG or GRGDS peptide selectively bound to immobilized VEGFR-1 or integrin α_vβ₃, respectively. DT liposomes showed a higher affinity for a mixture of VEGFR-1 and integrin α_vβ₃ compared with ST liposomes, suggesting the cooperative binding of these 2 kinds of ligand on the liposomal surface. In a biodistribution assay, the DT liposomes accumulated to a significantly greater extent in the tumors of Colon26 NL-17 tumor-bearing mice compared with other liposomes. Moreover, the intratumoral distribution of the liposomes examined by confocal microscopy suggested that the DT liposomes targeted not only angiogenic endothelial cells but also tumor cells due to GRGDS-decoration. These findings suggest that "dual-targeting" augmented the affinity of the liposomes for the target cells and would thus be useful for active-targeting drug delivery for cancer treatment.