Mock Transfectants Research Articles

A Genetic Defect in Exportin-5 Traps Precursor MicroRNAs in the Nucleus of Cancer Cells

The global impairment of mature microRNAs (miRNAs) is emerging as a common feature of human tumors. One interesting scenario is that defects in the nuclear export of precursor miRNAs (pre-miRNAs) might occur in transformed cells. Exportin 5 (XPO5) mediates pre-miRNA nuclear export and herein we demonstrate the presence of XPO5-inactivating mutations in a subset of human tumors with microsatellite instability. The XPO5 genetic defect traps pre-miRNAs in the nucleus, reduces miRNA processing, and diminishes miRNA-target inhibition. The XPO5 mutant form lacks a C-terminal region that contributes to the formation of the pre-miRNA/XPO5/Ran-GTP ternary complex and pre-miRNAs accumulate in the nucleus. Most importantly, the restoration of XPO5 functions reverses the impaired export of pre-miRNAs and has tumor-suppressor features.

Fluid Flow-induced Soluble Vascular Endothelial Growth Factor Isoforms Regulate Actin Adaptation in Osteoblasts

Although load-induced mechanical signals play a key role in bone formation and maintenance of bone mass and structure, the cellular mechanisms involved in the translation of these signals are still not well understood. Recent identification of a novel flow-induced mechanosignaling pathway involving VEGF in osteoblasts and the known VEGF regulation of actin reorganization in various cell types has led us to hypothesize that fluid shear stress-induced Vegf up-regulation underlies the actin cytoskeleton adaptation observed in osteoblasts during mechanotransduction. Our results show that MC3T3-E1 cells secrete significant VEGF in response to 5 h of pulsatile fluid shear stress (PFSS; 5 dynes/cm(2) at 1 Hz), whereas expression of VEGF receptors (VEGFR-1, VEGFR-2, or NRP1) is unaffected. These receptors, in particular VEGFR-2, participate in PFSS-induced VEGF release. Exposure to flow-conditioned medium or exogenous VEGF significantly induces stress fiber formation in osteoblasts that is comparable with PFSS-induced stress fiber formation, whereas VEGF knockdown abrogates this response to PFSS, thereby providing evidence that flow-induced VEGF release plays a role in actin polymerization. Using neutralizing antibodies against the receptors and VEGF isoforms, we found that soluble VEGFs, in particular VEGF(164), play a crucial role in transient stress fiber formation during osteoblast mechanotransduction, most likely through VEGFR-2 and NRP1. Based on these data we conclude that flow-induced VEGF release from osteoblasts regulates osteoblast actin adaptation during mechanotransduction and that VEGF paracrine signaling may provide potent cross-talk among bone cells and endothelial cells that is essential for fracture healing, bone remodeling, and osteogenesis.

RAGE Signaling Significantly Impacts Tumorigenesis and Hepatic Tumor Growth in Murine Models of Colorectal Carcinoma

BackgroundThe receptor for advanced glycation end-products (RAGE) is a cell surface receptor implicated in tumor cell proliferation and migration. We hypothesized that RAGE signaling impacts tumorigenesis and metastatic tumor growth in murine models of colorectal carcinoma. Materials and MethodsTumorigenesis: Apc1638N/+ mice were crossed with Rage−/− mice in the C57BL/6 background to generate Apc1638N/+/Rage−/− mice. Metastasis: BALB/c mice underwent portal vein injection with CT26 cells (syngeneic) and received daily soluble (s)RAGE or vehicle. Rage−/− mice and Rage+/+ controls underwent portal vein injection with MC38 cells (syngeneic). Rage+/+ mice underwent portal vein injection with MC38 cells after stable transfection with full-length RAGE or mock transfection control. ResultsTumorigenesis: Apc1638N/+/Rage−/− mice had reduced tumor incidence, size, and histopathologic grade. Metastasis: Pharmacological blockade of RAGE with sRAGE or genetic deletion of Rage reduced hepatic tumor incidence, nodules, and burden. Gain of function by transfection with full-length RAGE increased hepatic tumor burden compared to vector control MC38 cells. ConclusionRAGE signaling plays an important role in tumorigenesis and hepatic tumor growth in murine models of colorectal carcinoma. Further work is needed to target the ligand–RAGE axis for possible prophylaxis and treatment of primary and metastatic colorectal carcinoma.

Down-regulation of fractalkine inhibits the in vitro and in vivo angiogenesis of the hepatocellular carcinoma HepG2 cells

There is increasing evidence that hepatocellular carcinoma (HCC) is inherently associated with the inflammatory process and the up-regulation of cytokines. Our study aimed at elucidating the role of the cytokine fractalkine in the process of HCC carcinogenesis. Expression of fractalkine in hepatocellular carcinoma cell line HepG2 was knocked-down by RNAi. Conditioned media (CMs) from HepG2 was used in angiogenesis assays both in vitro and in vivo. Compared with CMs from mock transfection and negative shRNA treated HepG2, CMs from fractalkine shRNA treated HepG2 highly suppressed the migration, proliferation, and differentiation of human umbilical vein endothelial cells. The results suggest that fractalkine may play a key role in the mechanism of angiogenesis and carcinogenesis of HCC.

Abstract B30: Altered response to the prodrug of gemcitabine LY2334737 by overexpression or knockdown of carboxylesterase 2 (CES2)

Abstract Gemcitabine (gem) is a nucleoside anticancer agent that inhibits cell cycle in S-phase and is registered clinically for the treatment of a number of solid tumors. A prodrug of gemcitabine, LY2334737, was developed for oral administration for metronomic dosing with the aim of growth inhibition of more tumor cells as they go through the cell cycle. LY is well absorbed and systemically cleaved in humans. The rate of hydrolysis is slow resulting in circulating levels of LY that are 10-fold higher than gemcitabine in plasma. Studies with cell lines and recombinant protein indicate that LY is cleaved to gemcitabine by carboxylesterase 2 (CES2). Because LY circulates in the plasma for an extended period of time, we wondered if expression of CES2 by the tumor could alter its response to the prodrug. The present study was undertaken to determine if the expression of CES2 alters cellular response to LY in vitro and in vivo. The constitutive expression of CES2 in SKOV-3 was knocked down by shRNA. A stable subclone was obtained that had CES2 expression reduced by 80%. Stable HCT-116 transfectants were prepared that overexpressed CES2. Cell lines, transfectants and knock down cells were evaluated for drug sensitivity in a cytotoxicity assay. They were also evaluated for CES2 expression by biochemical cellular assay, Western analysis, and qRT-PCR. A xenograft study was conducted employing transfected HCT-116 cells. Immunohistochemical (IHC) staining was used to evaluate cell lines and tumors for CES2 expression. LY was less cytotoxic to SKOV-3 cells when CES2 was knocked down while sensitivity to gem was unaltered. Transfection of CES2 into low CES2 endogenously expressing HCT-116 cells enhanced LY drug sensitivity with no change in gemcitabine sensitivity. Stably transfected CES2 and mock HCT-116 transfectants were grown as a xenograft. The transfectants had identical growth rates in vivo and were equally sensitive to gem treatment. IHC staining of the tumors indicated that CES2 expression was maintained throughout the growth period. CES2 expressing tumors demonstrated a statistically significant greater response to LY than mock transfectant tumors. Taken together, these studies indicate that CES2 cleaves the prodrug and tumor expression of CES2 can enhance response to treatment with LY2334737. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B30.

Cotransfection of Vascular Endothelial Growth Factor-A and Platelet-Derived Growth Factor-B Via Recombinant Adeno-Associated Virus Resolves Chronic Ischemic Malperfusion: Role of Vessel Maturation

We set out to investigate the ability of cardiotropic adeno-associated viral vector (AAV2.9 = recombinant adeno-associated virus [rAAV]) to induce prolonged expression of vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF)-B in a rabbit hindlimb ischemia model and a pig model of hibernating myocardium. Gene therapy to induce angiogenesis and arteriogenesis has produced mixed results. However, long-acting viruses, such as rAAV, as well as combined induction of angiogenesis and vessel maturation might extend the therapeutic potential. In rabbits, 0.5 x 10(11) particles rAAV.VEGF-A with or without 1 x 10(12) particles rAAV.PDGF-B were retroinfused at day 7 after femoral artery excision. At days 7 and 35, collateral counts and perfusion were determined, each value given as the day 35/day 7 ratio. Capillary-to-muscle fiber ratio was determined at day 35. In pigs, implantation of a reduction stent graft into the circumflex artery led to complete occlusion at day 28. At this time point, retroinfusion of rAAV.VEGF-A (1 x 10(13) particles), rAAV.VEGF-A/PDGF-B (2 x 10(12) and 4 x 10(12) particles, respectively) or mock transfection was performed. Ejection fraction and left ventricular end-diastolic pressure were assessed at days 28 and 56. In rabbits, rAAV.VEGF-A strongly induced angiogenesis (capillary-to-muscle fiber ratio; 1.67 +/- 0.09 vs. 1.32 +/- 0.11 in rAAV.LacZ-treated limbs, p < 0.05), but not collateral growth (125 +/- 7% vs. 106 +/- 7%, p = NS) or perfusion (136 +/- 12% vs. 107 +/- 9%, p = NS). With VEGF-A/PDGF-B cotransfection, collateral growth increased to 146 +/- 9%, perfusion to 163 +/- 8% of the respective day 7 value (p < 0.05). In the pig model, retroinfusion of rAAV.VEGF-A/PDGF-B increased regional myocardial blood flow reserve from 101 +/- 4% (rAAV.Mock) to 129 +/- 8% (p < 0.05), based on collateral growth (3.2 +/- 0.3 in rAAV.Mock vs. 9.0 +/- 0.4 in rAAV.VEGF-A/PDGF-B, p < 0.05), whereas rAAV.VEGF-A did not alter flow reserve (112 +/- 7%) or collateral count (5.2 +/- 0.7). rAAV.VEGF-A/PDGF-B improved ejection fraction (55 +/- 5% vs. 34 +/- 3% in rAAV.Mock, p < 0.05) unlike rAAV.VEGF-A (37 +/- 2%). Retroinfusion of rAAV.VEGF-A alone induces angiogenesis, but fails to enhance collateralization and perfusion, unless PDGF-B is cotransfected. In addition to neovascularization, rAAV.VEGF-A/PDGF-B improves regional and global myocardial function in hibernating myocardium.

Different angiogenesis effect of mini-TyrRS/mini-TrpRS by systemic administration of modified siRNAs in rats with acute myocardial infarction

We aimed to clarify the different angiogenesis effects of mini-tyrosyl-tRNA synthetase (TyrRS)/minitryptophanyl-tRNA synthetase (TrpRS) in rodent primates with acute myocardial infarction, by delivering small interfering RNAs (siRNAs) systemically in a liposomal formulation. Left coronary artery ligation was used to establish the model of acute myocardial infarction in rats; mini-TyrRS/mini-TrpRS-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP), and administered by intravenous injection to rats. Rats were divided into four experiment groups: sham operated group (no left anterior descending artery [LAD] occlusion); negative control group (LAD occlusion + saline injection); mock transfection group (LAD occlusion + mock transfected injection); experiment group (LAD occlusion + mini-TyrRS/mini-TrpRS-specific siRNAs injection). Silencing efficiency was assayed by Western blotting. To determine whether mini-TyrRS/mini-TrpRS affected the angiogenesis activity of rats with myocardial infarction, we measured the myocardial infarction size by TTC staining, and the capillary density using immunohistochemistry staining, to investigate the expression of factor VIII. The myocardial infarction size and the capillary density of mini-TyrRS-siRNA group were respectively 18.89% and 8.64/0.1 mm(2) 1 month after ligation, while in the mini-TrpRS-siRNA group these values were 7.33% and 17.32/0.1 mm(2), significantly different compared with the mock transfection group (14.19%; 13.56/0.1 mm(2)) and negative control group (14.28%; 13.89/0.1 mm(2)), P < 0.05. There were no significant changes between the mock transfection group and the negative control group, P > 0.05. These results indicated that angiogenesis is either stimulated by mini-TyrRS or inhibited by mini-TrpRS in rat models with acute myocardial infarction.

Core 2 N‐acetylglucosaminyltransferase‐1 expression induces aggressive potential of testicular germ cell tumor

We studied orchiectomy specimens from 130 patients immuhistochemically with testicular germ cell tumor (TGCT) using anti-core 2 N-acetylglucosaminyltransferase-1 (C2GnT-1) antibody. The incidence of C2GnT-1 positivity in stage I disease (29.5%, 21/71) was significantly lower than that in higher stages (84.7%, 50/59) (P < 0.001, chi(2) test). This significant difference was also found when the cases were divided into seminoma and NSGCT according to histopathological classification. Kaplan-Meier plots and the log rank test showed that in the patients with stage I seminoma, C2GnT-1-positive cases had a higher risk for recurrence (P < 0.001). This was also the case with the patients with stage I NSGCT (P < 0.001). To determine whether C2GnT-1 promotes aggressive behavior of cancer cells, a C2GnT-1-negative human TGCT cell line, JKT-1, was stably transfected with a mammalian expression vector containing C2GnT-1 cDNA. In vitro assays revealed that JKT-1-C2 cells are more invasive than mock transfectants, although there are no differences in proliferation activity. When orthotopically inoculated into athymic nude mice, JKT-1-C2 cells produced larger testicular tumors extending to the retroperitoneum with mesenteric metastasis, while mock transfectants produced small tumors without metastasis (P < 0.01, Mann-Whitney's U-test). When injected via the tail vein, JKT-1-C2 cells produced a number of metastatic lung foci. In contrast, mock transfectants produced a small number of nodules (p < 0.01, Mann-Whitney's U-test). These results strongly suggest that C2GnT-1 enhances the metastatic potential of TGCT and may be a reliable biomarker for aggressive potential of TGCT.

Abstract 219: Treatment with survivin siRNA augments cytotoxicity of paclitaxel in primary ovarian cancer cells

Abstract Introduction Chemoresistance is a major problem encountered during the treatment of ovarian cancer. Paclitaxel one of the first line drugs is less effective in patients whose cancer cells overexpress survivin. This study investigates whether reducing expression of survivin by siRNA treatment of primary cultures of ovarian cancer cells can increase sensitivity to paclitaxel. Methodology Five primary cultures were established from ascites obtained from untreated ovarian cancer patients. Ascites mixed with MCDB 105 and M199 medium (ratio 1:1) was plated in 75 mm2 flasks for establishment of primary cultures. Survivin siRNA was synthesized using in-vitro transcription technique and was quantified using pico green. A dose of 15 nM of Survivin siRNA was used for further experiments. Primary cultures were treated with survivin siRNA using olgofectamine as the transfecting agent. After 24 hours Survivin expression was quantified by RT-PCR. Cells plated in 96 well plates were treated with survivin siRNA (15nM). After 24 hours, paclitaxel treatment was given at four different doses (0.1, 1, 5 and 25 µg/ml) in separate wells. Untransfected cells, cells transfected with oligofectamine alone (mock transfection) and with universal control siRNA were also treated with same doses of paclitaxel after 24 hours. Cell survival was quantified using MTT assay. All experiments were done in triplicates. Results On treating the primary cultures with survivin siRNA there was a 6 fold decrease in Survivin expression. Survivin siRNA alone caused 23 ±3% cell deaths in the 5 primary cultures. When these cultures were treated with paclitaxel alone (untansfected cells) at different doses (0.1, 1, 5 and 25 µg/ml) the average cell death was 21.2%, 22.4%, 26% and 37.5% respectively. There was no significant difference in cell death in mock transfected and control siRNA transfected cells when compared to untransfected cells. However when the cells were pretreated with survivin siRNA and then followed by same doses of paclitaxel after 24 hours, cell death increased to 52.8%, 55.4%, 60% and 70% respectively. All the primary cultures were resistant to paclitaxel treatment as 62.5% of the cells survived the highest dose of paclitaxel. Survivin siRNA treatment could significantly increase the sensitivity of the primary ovarian cancer cell cultures to paclitaxel. Conclusion Survivin appears to play a major role in the response of ovarian cancer cells to paclitaxel treatment and knocking off survivin expression may prove beneficial in the treatment of ovarian cancer patients with paclitaxel whose tumors overexpress Survivin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 219.

Abstract 5284: The Oncogenic Role of MALL in NSCLC

Abstract MALL (T-cell differentiation protein-like) located on the human chromosome 2q13 locus encodes a 17.5KDa protein enriched in glycolipid and cholesterol enriched membrane raft (GEM). It is involved in the raft-mediated trafficking machinery in endothelial cells. Previous study showed that MALL can interact with Caveolin-1. However, Caveolin-1 can be phosphorylated by FAK and then promotes cell proliferation and colony formation in NSCLC (non-small cell lung cancer) cell lines. However, the role of MALL in cancer progression is totally unknown. In this study, we found that the expression of MALL is upregulated up to 250-fold in the high invasive lung cancer cells (CL1-5) than low invasive cells (CL1-0). To investigate the biological function of MALL, we first generated a constitutively MALL-expressed lung cancer cell line. We found that MALL can significantly enhance cancer invasiveness by the Boyden chamber matrigel assay (2.12 fold, p&amp;lt;0.01). MALL also promote cell migration by the transwell assay (1.53 fold, p&amp;lt;0.01) and wound healing assay. Moreover, MALL increased the colonogenesis of lung cancer cells by using an anchorage-dependent colony formation assay (1.73 fold, p=0.035) but did not affect the rate of cell proliferation by MTT assay. These findings indicated that MALL can promote tumor metastasis and tumorigenesis. To determine the underlying mechanisms by which MALL increases tumorigenesis and invasion Affymetrix oligonucleotide analysis was performed. The genes differentially expressed between MALL and mock transfectants with greater than two-fold change was identified by ANOVA analysis (FDR&amp;lt; 0.05). The most relevant networks involved were analyzed using MetaCore and IPA (ingenuity pathway analysis) software including cytoskeleton, ECM signaling, and other networks. Taken together, MALL may be a potential oncogene which might provide new insights into the future treatment of lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5284.

Abstract 2950: Silencing of STAT3 and Src gene by RNA interference enhances the cytotoxic effect of cisplatin in cervical cancer cell lines

Abstract Purpose: Signal transducer and activator of transcription-3 (STAT3) is well known to regulate oncogenesis, angiogenesis and immunosuppression in cancer cells. The Src family consists of nonreceptor tyrosine kinases and participates in cell cycle progression, adhesion, spreading, migration, and differentiation. Activation of STAT3 by constitutively activated Src pathway in cancer cells results in cancer progression. We investigated the cytotoxic effect of cisplatin in cervical cancer cell lines according to the presence of RNA interference-mediated STAT3 and Src gene silencing. Experimental procedures: We prepared two cervical cancer cell lines of C4I and HeLa. First of all, reverse transcription with an RT-PCR kit was performed in order to confirm the increased STAT3 expression in cervical cancer cells. We synthesized STAT3-specific siRNA, using six different sequences of oligonucleotides encoding STAT3 siRNA. After ligation and transformation into bacteria, the positive colonies were selected by antibiotic resistance. HeLa cells were transfected with the plasmids with luciferase reagent according to the established methods. As the same methods, we obtained the HeLa cells transfected with c-Src and v-Src-targeting siRNAs. We checked the reduction rate of each mRNAs by RT-PCR after 48 hours. HeLa and C4I cells transfected with indicated siRNAs, which showed the highest reduction rate at silencing STAT3, c-Src and v-Src, were cultured and mock transfection without any DNA was always included as experimental controls. At 36 hours after starting culture, cisplatin was administered as indicated concentrations (0, 100, 250, 500, 1000 ng/mL). At 96 hours after starting culture, the viability of HeLa and C4I cells was determined by the MTT assay. Results: We confirmed the increased expression of STAT3 in HeLa cell lines. STAT3 mRNAs were reduced very effectively in all cases of STAT3-targeting siRNAs with different 6 sequences to HeLa (80.1%∼93.4%) and the following sequence of oligonucleotides encoding STAT3 siRNA showed the highest reduction rate; 5′-GCGTCCAGTTCACTACTAAAGTCAG-3′ (upper strand) and 5′-CTGACTTTAGTAGTGAACTGGACGC-3′ (lower strand). The reduction rate at silencing c-Src with different 5 sequences siRNAs was high (73.8%∼88.9%). The reduction rate at silencing v-Src with different 5 siRNAs was lower than other two cases (40.5%∼66.6%). The enhancement of cytotoxicity of cisplatin by gene silencing of STAT3 and c-Src, but not of v-Src, was confirmed in HeLa and C4I cells. Conclusion: In this study, we confirmed that STAT3/c-Src targeting siRNAs down-regulated the expression of STAT3 and c-Src and they could enhance the cytotoxic effect of cisplatin in cervical cancer cell lines. We will continue the upgraded study to investigate methods for enhancement of drug susceptibility, including the effect of co-silencing STAT3 and c-Src and their in vivo responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2950.

Abstract 3908: Id1/Id3 Knockdown increases Chemosensitivity of Colorectal Cancer

Abstract Objective: The inhibitor of DNA binding/differentiation (Id) proteins belong to the helix-loop-helix transcriptional regulatory factors, and play important roles in tumor development. Recently, overexpression of Id-1 has been associated with more aggressive and metastatic cancers in various cancer types, as well as implicated in resistance to chemotherapy, and thus, its down-regulation has been shown to lead to increased chemotherapy-induced apoptosis. Previously, we have demonstrated that simultaneous targeting of Id1 and Id3 inhibited proliferation, migration, adhesion, and metastatic growth of colorectal cancers. In the present study, we aimed to evaluate the role of Id in chemosensitivity of colorectal cancer cells, and for this purpose, human colon cancer cells with downregulated expression of Id1/Id3 were treated with oxaliplatin, and the changes in the sensitivity to chemotherapy as well as the mechanisms underlying it were investigated. Methods: Id1 and Id3 were stably and simultaneously knockdown in human colorectal cancer cell line HT29 by means of RNA interference. Both Id1 and Id3 were double-knockdown since they are known to functionally compensate each other, and the downregulation of either is not enough for the inhibition of their effects. Parental HT29 cells and the empty vector (mock) transfectants were used as control. Oxaliplatin, one component of FOLFOX6, which is one of the standard treatment protocols for colorectal cancer, was chosen as the chemotherapeutic agent. The chemosensitivity was investigated as follows: the proliferative activity was analyzed by the MTS assay, the induction of apoptosis, by the flow-cytometry after double-staining of the cells with AnnexinV/PI. Induction of autophagy was investigated by flow-cytometry after staining of the cells with acridine orange for the detection of AVOs. The changes of the cell cycle were investigated by flow-cytometry after staining of the cells with propidium iodide (PI). Results: Id double-knockdown resulted in increased chemosensitivity to oxaliplatin, as observed by the reduced proliferative ability compared with parental and mock cells. And the reduced proliferative activity was associated with the arrest of cell to the G2-M phase of the cell cycle, and the increased cell apoptosis. The chemosensitivity of Id double-knockdown cells seemed to be associated with decreased ability to induce autophagy in response to the chemotherapeutic agent. Conclusions: Id1, 3 double-knockdown resulted in increased chemosensitivity of colorectal cancer cells to oxaliplatin, dependent on the cell cycle arrest and apoptosis induction. And the decreased ability to induce autophagy seemed to be a mechanism responsible for this effect. From these results, we concluded that Id proteins play an important role in the chemoresistance of colorectal cancer, and downregulation of Id should be a promising strategy to improve chemosensitivity of Id expressing cancers. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3908.

Abstract 3298: Elf3 as negative regulation for epithelial-mesenchymal transition in ovarian cancer

Abstract Objective: To delineate the role of Elf3, an epithelially restricted member of the ETS transcription factor family, in regulating epithelial-mesenchymal transition in malignant serous ovarian epithelial cells. Methods: Based on microarray study, Elf3 was indentified as one of the significantly upregulated transcription factor encoded genes in the malignant serous ovarian epithelia compared with that in normal ovarian surface epithelium. To further evaluate differential Elf3 expression in serous epithelia, immunolocalization of Elf3 was performed and clinical significance of nuclear Elf3 expression was evaluated by Cox and Kaplan-Meier analyses on patients with HGSC. In order to delineate the function of Elf3, expression levels of Elf3 in various ovarian cancer cell lines and normal human ovarian surface epithelial cells (HOSE) were evaluated by western blot analysis. Low Elf3 expresser, OVCA429 and SKOV3 ovarian cancer cells were stably transfected with full-length Elf3 to evaluate its effect on cell growth. To investigate the role of Elf3 in epithelial-mesenchymal transition, expression level, and subcellularlocalization of various transition markers were evaluated by western blot analysis and confocal microscopy. Results: Immunohistochemistry showed that nuclear Elf3 expression was significantly higher in benign, borderline and low-grade serous epithelia compared to that in high-grade serous epithelia, and down-regulation of Elf3 in advanced stage high-grade serous ovarian cancer was significantly associated with poor overall survival, suggesting that Elf3 is a favorable prognostic marker of this disease. Western blot and confocal microscopy confirmed that nuclear Elf3 expressed in ovarian cancer cells in vitro but not in normal HOSE. Cell proliferation assay demonstrated a significant decrease in growth rate in OVCA429 and SKOV3 cells transfected with Elf3 compared with that of the vector control (46.3%, p&amp;lt;0.001). Bright field microscopy showed that Elf3 overexpressing SKOV3 cells demonstrated mesechymal-epithelial transition. Cells were less elongated and gained cobble stone-like morphology. Forced expressing Elf3 in SKOV3 cells also induced translocation of Snail from the nucleus to the cytoplasm and β -catenin from the nucleus to the cell membrane. In addition, western blot analysis detected increased expression of cell-cell adhesion protein E-cadherin, and decreased expression of mesechymal elements N-cadherin, Slug and Vimentin in SKOV3 cells transfected with Elf3 compared with that of the mock transfectant. Conclusions: Our data suggest that Elf3 is a negative regulator of epithelial-mesechymal transition and demonstrates growth inhibitory effect in malignant ovarian epithelial cell lines. These observed supports our pervious clinical findings that nuclear Elf3 expression is associated with improved patient survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3298.

Upregulation of phagocytic clearance of apoptotic cells by autoimmune regulator

To investigate the effect of autoimmune regulator (AIRE) on phagocytic clearance of apoptotic cells, a recombinant expression vector containing full-length human AIRE cDNA was transfected into 16HBE cells. After incubation with transfected 16HBE cells, engulfment of apoptotic HL-60 cells induced by camptothecin was detected by myeloperoxidase (MPO) staining. The change in the expression of Rac 1 in transfected 16HBE cells was determined by RT-PCR and Western blotting. The results showed that the phagocytosis percentage of the experimental group, the mock transfection group and the negative control group (non-apoptotic cells) was (25.50 + or - 3.67)%, (6.25 + or - 1.58)% and (1.0 + or - 0.67)%, respectively. Moreover, the expressions of Rac 1 mRNA and protein were up-regulated in AIRE-transfected 16HBE cells, suggesting that AIRE may function as a regulator in the phagocytic clearance of apoptotic cells by promoting the expression of Rac 1.

Mutant p53 R248Q but not R248W enhances in vitro invasiveness of human lung cancer NCI-H1299 cells

More than half of all human cancers are associated with mutations of the TP53 gene. In regard to the functional interaction with the remaining wild-type (WT) p53 allele, p53 mutations are classified into two types, recessive and dominant-negative (DN) mutations. The latter mutant protein has a DN activity over the remaining WT allele. We previously showed that the DN p53 mutant was useful as a predictor of poor outcome or a risk factor for metastatic recurrence in patients with some types of cancers, regardless of the presence or absence of loss of heterozygosity (LOH) of WT p53, suggesting that the DN p53 had 'gain-of-function (GOF)' activity besides the transdominance function. In this study, we investigated GOF activity of two DN p53 mutants which had a point mutation at codon 248 (R248Q and R248W), one of the hot spots, by transfecting them respectively into H1299 cells which originally expressed no p53 protein. Growth activity of the transfectants with the two mutants was not different from that of parent or Mock transfectants. Meanwhile, in vitro invasions of Matrigel and type I collagen gel by R248Q-transfectants were significantly higher than those by R248W-transfectants or the control cells. However, there were no differences in cell motile activities, expressions of extracellular matrix-degradative enzymes such as matrix metalloproteinases, urokinase-type plasminogen activator and heparanase, and their inhibitors, between R248Q- and R248W-transfectants. These findings indicate that the p53 mutants have a different quality in GOF activities even if the mutations occurred at the same codon. And detailed information of the status of p53, including transdominancy and GOF activity, is expected to be useful for diagnosis and therapeutic strategy fitting the individual patients.

Identification of a bone marrow-derived mesenchymal progenitor cell subset that can contribute to the gastric epithelium

Recent studies with Helicobacter-infected mice have shown that bone marrow-derived cells can repopulate the gastric epithelium and progress to cancer. However, it has not been established which cellular subset can potentially contribute to the epithelium. The aim of this study was to investigate the ability of bone marrow-derived mesenchymal stem cells (MSCs) that express cytokeratin 19 (K19) to contribute to the gastric epithelium. MSCs cultures were established from whole bone marrow and expression of K19 was detected in a minority (1 of 13) of clones by real-time PCR and immunostaining. Transfection of a K19-green fluorescent protein (GFP) vector and isolation of GFP-expressing colonies generated high K19-expressing MSC clones (K19GFPMSC). Incubation of MSCs with gastric tissue extract markedly induced mRNA expression of gastric phenotypic markers and was observed to a greater extent in K19GFPMSCs compared with parental MSCs and mock transfectants. Both K19GFPMSCs and GFP-labeled control MSCs gave rise to gastric epithelial cells after injection into the murine stomach. In addition, after blastocyst injections, K19GFPMSCs gave rise to GFP-positive gastric epithelial cells in all 13 pups, whereas only 3 of 10 offspring showed GFP-positive gastric epithelial cells after injection of GFP-labeled control MSCs. Although K19 expression could not be detected in murine whole bone marrow, H. felis infection increased K19-expressing MSCs in the circulation. Taken together, our results show that bone marrow-derived MSCs can contribute to the gastric epithelium. The K19-positive MSC fraction that is induced by chronic H. felis infection appears to be the important subset in this process.

Influence of Mir-155 On B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia.

Abstract 2343Poster Board II-320B-cell receptor (BCR) signaling arguably plays an important role in the pathogenesis and/or progression of chronic lymphocytic leukemia. Ligation of the BCR by F(ab)2 anti-μ can induce phosphorylation of p72Syk, BLNK, phospholipase C-gamma (PLCγ) and other downstream adapter/signaling molecules, inducing intracellular calcium flux and cellular activation. Prior studies found that CLL cells that expressed unmutated Ig heavy-chain variable region genes (IGHV) and the zeta-associated protein of 70 kD (ZAP-70) generally experienced greater levels of activation following treatment with anti-μ than did CLL cells that lacked expression of ZAP-70. However, we found unusual cases that lacked expression of ZAP-70 that also responded vigorously to treatment with anti-μ, suggesting that other factors contribute to the noted differences in BCR-signaling. Analyses for expression of microRNAs by microarray revealed that CLL cells that used unmutated IGHV and that expressed ZAP-70 expressed higher levels of certain microRNAs than did cases that used mutated IGHV and that lacked expression of ZAP-70. One of such microRNA, miR-155, was found to target mRNA encoding SHIP-1, a phosphatase that plays a critical role in modulating the level of BCR signaling in normal B cells. Using quantitative assays for miR-155 we found high-level expression of this microRNA was associated with proficient BCR signaling in CLL. To examine whether miR-155 could modulate the levels of SHIP-1 and/or BCR signaling in CLL cells we transfected primary leukemia cells from each of multiple patients with control oligo-RNAs, miR-155, or a specific inhibitor of miR-155 (miR-155 inhibitor). Twenty-four hours later the cells were stimulated with anti-μ or control antibody and then examined 10 minutes later for expression of SHIP-1, induced calcium influx, or phosphorylation of kinases and adapter proteins that are involved in BCR signaling. CLL cells that had low expression levels of miR-155 and that were poorly responsive BCR had significantly higher levels of calcium influx and phosphorylated p72Syk, BLNK, and PLCγ in response to anti-μ following transfection with miR-155 than following mock transfection or transfection with control oligo-RNA. Conversely, CLL cells that had high expression levels of miR-155 and highly responsive BCR were made to have significantly higher amounts of SHIP-1 protein and to have significantly lower relative levels of phosphorylated protein and calcium influx in response to anti-μ following transfection with the miR-155 inhibitor than did mock transfected CLL cells. These results identify miR-155 as a factor that can modulate BCR signaling in CLL in part by regulating the relative expression level of SHIP-1. These results demonstrate that differential expression of microRNAs in CLL can influence physiologic features that potentially contribute to disease progression. Disclosures:No relevant conflicts of interest to declare.

Glucocorticoid-Induced TNFR-Related Protein (GITR) Ligand Mediates Tumor Immunoediting in Chronic Lymphocytic Leukemia and Impairs Direct and Rituximab-Induced NK Cell Anti-Leukemia Reactivity.

Abstract 4403Members of the TNF/TNF receptor (TNFR) family of proteins govern differentiation, proliferation, activation, and death of both tumor and immune effector cells and thus play an important role in tumor immunoediting, the reciprocal interaction of tumor cells and anti-tumor immunity. Activation of the TNFR family member GITR has recently been shown to stimulate T cell-mediated anti-tumor immunity in mice. However, available data suggest that GITR mediates different effects in mice and men, and may impair anti-tumor immunity of human NK cells. Here we studied the expression and function of GITR ligand (GITRL) in patients with chronic lymphocytic leukemia (CLL) and the consequences of GITR-GITRL interaction for NK cell reactivity against CLL cells. Substantial GITRL expression was detected on primary B-CLL cells in 38 of 48 (79%) investigated patients. Upon interaction with its cognate receptor, GITRL induced the release of immunoregulatory cytokines like TNF by the leukemia cells, which demonstrated that CLL-expressed GITRL is functional and capable to transduce bidirectional signals. Moreover, disruption of GITR-GITRL interaction in cultures of allogenic NK cells with patient CLL cells by addition of blocking antibody caused a significant increase in NK cell granule mobilization, cytotoxicity and IFN-γ production. The inhibitory effect of tumor-expressed GITRL on the reactivity of human NK cells was also confirmed in cocultures of C1R lymphoma cells transfected to express GITRL with mock transfectants serving as control. In addition, blocking GITR-GITRL interaction also considerably augmented both antibody-dependent cellular cytotoxicity (ADCC) and antibody-induced IFN-γ production of NK cells in cultures with allogenic CLL cells upon Rituximab exposure. Of note, GITR blockade also significantly enhanced anti-leukemia reactivity of autologous NK cells among PBMC of B-CLL patients, and this reinforcement of NK cell effector functions was observed both regarding the direct and, more pronounced, Rituximab-induced anti-leukemia reactivity (both n=10, p<0.01, Student's T test). Thus, expression of functional GITRL by CLL cells potently influences tumor immunoediting and impairs anti-tumor immunity by diminishing both direct and Rituximab-dependent anti-leukemia reactivity of NK cells. Modulation of the GITR-GITRL system might therefore serve to enhance the efficacy of therapeutic approaches in CLL which, like Rituximab-induced ADCC or stem cell transplantation, rely on a sufficient NK cell anti-tumor response. Disclosures:No relevant conflicts of interest to declare.

Age-Related Changes in Pericellular Hyaluronan Organization Leads to Impaired Dermal Fibroblast to Myofibroblast Differentiation

We have previously demonstrated that transforming growth factor-beta1 (TGF-beta1)-mediated fibroblast-myofibroblast differentiation is associated with accumulation of a hyaluronan (HA) pericellular coat. The current study demonstrates failure of fibroblast-myofibroblast differentiation associated with in vitro aging. This is associated with attenuation of numerous TGF-beta1-dependent responses, including HA synthesis and induction of the HA synthase enzyme HAS2 and the hyaladherin tumor necrosis factor-alpha-stimulated gene 6 (TSG-6), which led to an age-related defect in pericellular HA coat assembly. Inhibition of HAS2-dependent HA synthesis by gene silencing, removal of the HA coat by hyaluronidase digestion, or gene silencing of TSG-6 or cell surface receptor CD44 led to abrogation of TGF-beta1-dependent induction of alpha-smooth muscle actin in "young" cells. This result supports the importance of HAS2-dependent HA synthesis and the HA coat during phenotypic activation. Interleukin-1beta stimulation, however, failed to promote phenotypic conversion despite coat formation. A return to basal levels of HA synthesis in aged cells by HAS2 overexpression restored TGF-beta1-dependent induction of TSG-6 and pericellular HA coat assembly. However, this did not lead to the acquisition of a myofibroblast phenotype. Coordinated induction of HAS2 and TSG-6 facilitation of pericellular HA coat assembly is necessary for TGF-beta1-dependent activation of fibroblasts, and both components of this response are impaired with in vitro aging. In conclusion, the HA pericellular coat is integral but not sufficient to correct for the age-dependent defect in phenotypic conversion.

Role of Varp, a Rab21 exchange factor and TI‐VAMP/VAMP7 partner, in neurite growth

The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP/VAMP7) was previously shown to mediate an exocytic pathway involved in neurite growth, but its regulation is still largely unknown. Here we show that TI-VAMP interacts with the Vps9 domain and ankyrin-repeat-containing protein (Varp), a guanine nucleotide exchange factor (GEF) of the small GTPase Rab21, through a specific domain herein called the interacting domain (ID). Varp, TI-VAMP and Rab21 co-localize in the perinuclear region of differentiating hippocampal neurons and transiently in transport vesicles in the shaft of neurites. Silencing the expression of Varp by RNA interference or expressing ID or a form of Varp deprived of its Vps9 domain impairs neurite growth. Furthermore, the mutant form of Rab21, defective in GTP hydrolysis, enhances neurite growth. We conclude that Varp is a positive regulator of neurite growth through both its GEF activity and its interaction with TI-VAMP.