Control Transfectants Research Articles

Abstract 912A: Cadherins control Src kinase induced fibroblast cell motility

Abstract Cancer associated fibroblasts (CAFs) are a part of the tumor microenvironment, and help remodel the extracellular matrix to pave the way for tumor cells to forge invasive fronts. Src tyrosine kinase activity is often increased in the tumor microenvironment, and has been shown to induce cell motility and disrupt adherens junctions. Adherens junctions are formed by cadherin proteins that mediate intercellular adhesion and control cell migration. Previous studies have found correlations between cadherin expression and cancer cell motility. However, effects of cadherin expression on CAF motility have not been thoroughly elucidated. We are investigating how junctions formed by E-cadherin (ECAD) and N-cadherin (NCAD) affect individual and collective migration of fibroblasts expressing different levels of Src kinase activity. We are utilizing fluorescently tagged ECAD or NCAD to study the localization and effects of cadherin proteins in real time by live cell imaging along with wound healing and Transwell migration assays. Results from these studies indicate that ECAD and NCAD have different effects on migration of cells depending on Src kinase activity. Src kinase activity did not affect collective cell migration, but increased individual cell migration by over 4 fold (p<0.0001 by t-test) in the absence of forced cadherin expression. In addition, our data indicate that ECAD increases collective cell motility, while NCAD decreases collective cell migration in the absence of strong Src kinase activity. Collective migration by ECAD expressing cells was over 4 fold higher than cells expressing NCAD or control transfectants (p<0.0001 by t-test) in the absence of strong Src activity. Conversely, collective migration of cells expressing NCAD was over 5 fold lower than control transfectants, and over 30 fold lower than cells expressing ECAD (p<0.005 by t-test) without strong Src kinase activity. In contrast, both cadherins decrease individual cell motility promoted by Src kinase activity. ECAD and NCAD transfected cells migrated over 4 fold less than control transfectants with high levels of Src kinase expression (p<0.0001 by t-test). Interestingly, human oral squamous cell carcinoma (OSCC) cells that express high levels of both ECAD and NCAD exhibit collective migration equal to cells with low Src kinase activity, but individual migration over 5 fold higher than nontransformed cells (p<0.0001 by t-test). This system should prove useful to elucidate how cadherins and Wnt signaling pathways affect motility of CAFs and tumor cells. For example, western blotting analysis suggest that both ECAD and NCAD may protect β-catenin from destruction initiated by Src kinase activity. Taken together this work should elucidate how cadherins affect the ability of the Src kinase to increase CAF motility, and identify mechanisms that can be exploited to identify potential chemotherapeutic targets. Citation Format: Stephanie A. Sheehan, Edward P. Retzbach, Yongquan Shen, Gary S. Goldberg. Cadherins control Src kinase induced fibroblast cell motility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 912A. doi:10.1158/1538-7445.AM2017-912A

Abstract 4855: Loss of USP18 represses invasion and metastasis of lung cancer

Abstract Metastasis is a major cause of human lung cancer mortality. Uncovering novel targets and mechanisms involved in regulating metastasis is critical for developing effective ways to improve lung cancer survival. It is reported that ubiquitin and ubiquitin-like pathways can regulate invasion and metastasis. We previously found that USP18 (Ubiquitin Specific Peptidase 18) is substantially upregulated in several cancers, including lung cancer. Engineered repression of the deubiquitinase USP18 decreased growth, increased apoptosis and augmented chemotherapeutic agent response of lung cancer cells. Our prior work showed that loss of USP18 can destabilize specific oncogenic proteins and this results in the marked reduction of lung cancer cell growth and in vivo tumorigenicity. This study sought to explore the precise role of USP18 expression in invasion, migration and metastasis of lung cancer. In murine (KC2 and 344SQ) and human (A549 and H1299) lung cancer cells with varying degrees of metastatic potential, knock-down of USP18 by different short hairpin RNAs (shRNAs) decreased cell growth and increased cell death as compared to vector control transfectants. The findings from wound-healing migration and Transwell invasion assays established that USP18 knock-down in all studied lung cancer cell lines conferred substantial reduction of migration and invasion versus vector control transfected cell lines (p < 0.001). In mouse models, USP18 knock-down of lung cancer cell lines displayed a lower number of lung cancer metastasis in vivo. To discern engaged mechanisms, Reverse Phase Protein Arrays (RPPAs) were performed to interrogate over 300 growth-regulatory proteins in murine (344SQ and KC2) and human (A549 and H1299) lung cancer cells following USP18 knock-down. Using RPPAs, potential targets identified were differentially expressed between USP18 knock-down and vector control transfected lung cancer cells. Highlighted species included Myc, eEF2K (Eukaryotic Elongation Factor 2 Kinase), Programmed Cell Death 4 (PDCD4), Hes Family BHLH Transcription Factor 1 (Hes) and Polo Like Kinase 1 (PLK1), among other species. In addition, phosphorylated Acetyl-CoA Carboxylase (ACC), a key component of the 5'-adenosine monophosphate-activated protein kinase (AMPK) pathway, was significantly affected in lung cancer cell lines following USP18 knock-down (p < 0.05). Ingenuity Pathway Analysis and functional validation are now underway to uncover specific pathways directly involved in the observed reduction of lung cancer metastases. Taken together, this study implicates the deubiquitinase USP18 as a molecular target to combat lung cancer metastases. Citation Format: Lin Zheng, Lisa Mustachio, Yulong Chen, Xi Liu, Jason Roszik, Jonathan Kurie, Ethan Dmitrovsky. Loss of USP18 represses invasion and metastasis of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4855. doi:10.1158/1538-7445.AM2017-4855

Syntaphilin Regulates Mitochondrial Dynamics and Tumor Cell Invasion

Recent studies support the concept of spatiotemporal regulation of energy production as a driver of tumor cell motility and invasiveness. However, the molecular mechanisms governing mitochondria repositioning to the leading edge of the tumor cells have remained elusive. In this study, we identify the molecular motors and adaptors responsible for trafficking of mitochondria in tumor cells by a genome‐wide shRNA screen. One of the top hits in the screen was Syntaphilin (SNPH), a molecule that arrests mitochondrial trafficking at sites of high energy demands in neurons. Previously thought to be neuronal specific, SNPH was expressed in brain and non‐neuronal tissues, and in non‐transformed and cancer cell lines. To test the relevance of this pathway, we next silenced SNPH using siRNA or shRNA sequences. Depletion of SNPH constitutively accelerated focal adhesion complex dynamics, stimulated 2D cell motility and increased tumor cell invasion. Furthermore, depletion of SNPH stimulated the repositioning of mitochondria from their perinuclear localization to the cortical cytoskeleton of tumor cells. Reconstitution of SNPH‐depleted cells with siRNA‐insensitive SNPH cDNA reversed the increase in tumor chemotaxis and prevented the increased accumulation of mitochondria to the cortical cytoskeleton. Time lapse imaging revealed that mitochondria in SNPH‐depleted tumor cells exhibited faster speed of movements, higher processivity (time that mitochondria spend in motion/time of analysis), and greater distance traveled, compared to control transfectants. In neurons, SNPH interfaces with microtubule‐mediated trafficking by interacting with kinesin motors and atypical Rho GTPases (Miro) adapters. siRNA silencing of the anterograde kinesin KIF5B or Miro 1 suppressed the increased tumor cell invasion induced by loss of SNPH. In contrast, silencing of Miro2 did not reduce tumor cell invasion in SNPH‐knockdown cells. Next, we expressed various deletion mutants of SNPH that lack the microtubule binding domain (ΔMBD), kinesin‐binding domain (ΔKBD), or LC8‐binding domain (ΔLBD), and looked at their effects on tumor cell invasion. While expression of full length SNPH suppressed tumor cell invasion, ΔMBD, ΔKB, or ΔLBD mutants had no effect. In previous studies, mitochondrial trafficking required energetically active organelles. Here, loss of SNPH was sufficient to deregulate this process, and resulted in robust mitochondrial recruitment to the cortical cytoskeleton in nutrient‐starved tumor cells or oxidative phosphorylation‐deficient ρ0 cells. However, mitochondria from ρ0 cells depleted of SNPH failed to support tumor chemotaxis. In conclusion, we provided compelling evidence that SNPH‐directed mitochondrial dynamics regulates the bioenergetics requirements of tumor cell invasion.Support or Funding InformationThis work was supported by National Institutes of Health (NIH) grants P01 CA140043 (D.C.A.), R01 CA78810 and CA190027 (D.C.A.), F32 CA177018 (M.C.C.), the Office of the Assistant Secretary of Defense for Health Affairs through the Prostate Cancer Research Program under Award No. W81XWH‐13‐1‐0193 (D.C.A.), and a Challenge Award from the Prostate Cancer Foundation (PCF) to M.C.C. and D.C.A.

The Effects of Antisense miRNA-20a Alone or in Combination with Imatinib on K562 Cell Proliferation.

Objective: The effects of microRNA-20a (miR-20a) antisense oligonucleotides (ASODNs) on the proliferation and apoptosis of K562 cells were investigated, and the effects of these ASODNs in combination with imatinib on K562 cells were preliminarily observed.Methods: miR-20a ASODNs and scrambled oligonucleotides (SODNs) were chemically synthesized, and the later was used as the control. miR-20a ASODNs were transfected into K562 cells using Lipofectamine 2000 transfection reagent, and the expression of miR-20a was detected using real-time quantitative RT-PCR (qRT-PCR). The CCK8 assay was performed to detect the inhibition of the cell growth rate. The cells were stained by Hoechst 33258 to detect apoptotic cell morphology. Annexin V/PI double staining was used to detect the cell apoptosis rate using flow cytometry. The protein expression levels of E2F1, P21, and Bim in the K562 cell line were detected using western blotting.Results: The qRT-PCR results showed that the expression level of miR-20a in K562 cells transfected with miR-20a ASODNs was lower than those in the normal control, SODN and blank transfection groups (p < 0.05). miR-20a ASODNs significantly inhibited the growth of K562 cells as compared to the controls (p < 0.05). The Hoechst staining results showed morphological changes, suggesting apoptosis. The cell apoptosis rates in the ASODN group was (13.9 ± 1.5)%, which was significantly higher than that in the normal control group (1.84 ± 0.21)%, blank transfection group (3.21 ± 0.32)%, and SODN group (3.72 ± 0.44)% (p < 0.05). The protein expression of E2F1 and P21 in K562 cells transfected with miR-20a ASODNs were higher, while the level of Bim protein was significantly lower than that in the control groups. When miR-20a ASODNs were combined with imatinib, the growth of K562 cells was significantly inhibited as compared to the ASODN treatment alone, imatinib alone, and SODN+imatinib groups (p < 0.05).Conclusions: miR-20a ASODNs could induce apoptosis and inhibit the proliferation of K562 cells. In addition, imatinib combined with miR-20a ASODNs can increase the inhibitory effect on K562 cell proliferation.

The hematopoietic cell-specific transcription factor PU.1 is critical for expression of CD11c.

PU.1 is a hematopoietic cell-specific transcription factor belonging to the Ets family, which plays an important role in the development of dendritic cells (DCs). CD11c (encoded by Itgax) is well established as a characteristic marker of hematopoietic lineages including DCs. In the present study, we analyzed the role of PU.1 (encoded by Spi-1) in the expression of CD11c. When small interfering RNA (siRNA) for Spi-1 was introduced into bone marrow-derived DCs (BMDCs), the mRNA level and cell surface expression of CD11c were dramatically reduced. Using reporter assays, the TTCC sequence at -56/-53 was identified to be critical for PU.1-mediated activation of the promoter. An EMSA showed that PU.1 directly bound to this region. ChIP assays demonstrated that a significant amount of PU.1 bound to this region on chromosomal DNA in BMDCs, which was decreased in LPS-stimulated BMDCs in accordance with the reduced levels of mRNAs of Itgax and Spi-1, and the histone acetylation degree. Enforced expression of exogenous PU.1 induced the expression of the CD11c protein on the cell surface of mast cells, whereas control transfectants rarely expressed CD11c. Quantitative RT-PCR also showed that the expression of a transcription factor Irf4, which is a partner molecule of PU.1, was reduced in PU.1-knocked down BMDCs. IRF4 transactivated the Itgax gene in a synergistic manner with PU.1. Taken together, these results indicate that PU.1 functions as a positive regulator of CD11c gene expression by directly binding to the Itgax promoter and through transactivation of the Irf4 gene.

Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Activates Type I Interferon Signals in Lupus Nephritis.

Type I interferon (IFN) plays a central role in pathogenesis of systemic lupus erythematosus (SLE); tumor necrosis factor-like weak inducer of apoptosis (TWEAK) has been associated with a pathogenic role in lupus nephritis (LN). Thus we investigated whether TWEAK could induce the activation of type I IFN pathway in LN. We examined this in patient-derived peripheral blood mononuclear cells (PBMCs) as well as MRL/lpr mice, a murine LN model. Relative to the control cohorts, MRL/lpr mice showed severe histological changes, high index levels of renal damage, and elevated expression of type I IFN-inducible genes. After shRNA suppression of TWEAK, we observed that renal damage was significantly attenuated and expression of type I IFN-inducible genes was reduced in MRL/lpr mice. In parallel, siRNA of TWEAK also significantly reduced the expression of type I IFN-inducible genes in PBMCs relative to control transfections. In PBMCs, TWEAK stimulation also led to expression of type I IFN-inducible genes. Our results illustrate a novel regulatory role of TWEAK, in which its activity positively regulates type I IFN pathway in LN based on preclinical models. Our findings suggest TWEAK could act as a critical target in preventing renal damage in patients with LN.

Circulating exosomal microRNA-96 promotes cell proliferation, migration and drug resistance by targeting LMO7.

Detection and treatment of lung cancer still remain a clinical challenge. This study aims to validate exosomal microRNA‐96 (miR‐96) as a serum biomarker for lung cancer and understand the underlying mechanism in lung cancer progression. MiR‐96 expressions in normal and lung cancer patients were characterized by qPCR analysis. Changes in cell viability, migration and cisplatin resistance were monitored after incubation with isolated miR‐96‐containing exosomes, anti‐miR‐96 and anti‐miR negative control (anti‐miR‐NC) transfections. Dual‐luciferase reporter assay was used to study interaction between miR‐96 and LIM‐domain only protein 7 (LMO7). Changes induced by miR‐96 transfection and LMO7 overexpression were also evaluated. MiR‐96 expression was positively correlated with high‐grade and metastatic lung cancers. While anti‐miR‐96 transfection exhibited a tumour‐suppressing function, exosomes isolated from H1299 enhanced cell viability, migration and cisplatin resistance. Potential miR‐96 binding sites were found within the 3′‐UTR of wild‐type LMO7 gene, but not of mutant LMO7 gene. LMO7 expression was inversely correlated with lung cancer grades, and LMO7 overexpression reversed promoting effect of miR‐96. We have identified exosomal miR‐96 as a serum biomarker of malignant lung cancer. MiR‐96 promotes lung cancer progression by targeting LMO7. The miR‐96‐LMO7 axis may be a therapeutic target for lung cancer patients, and new diagnostic or therapeutic strategies could be developed by targeting the miR‐96‐LMO7 axis.

Expression of SET8 in renal cell carcinoma and the effects of SET8 silencing on proliferation and migration of 786-O cells

Objective To investigate the expression of lysine methyltransferase SET8 protein in the clear cell carcinoma of kidney and the effects of silencing SET8 gene by shRNA on proliferation and migration of human renal cell carcinoma cell line 786-O in vitro. Methods Immunohistochemistry(IHC) was employed to detect the expression of SET8 in 156 tissue samples of clear cell renal cell carcinoma in the study between December, 2006 and December, 2011.Four pairs of SET8-shRNA were designed and synthesized. Transfection was performed with cationic lipid vectors(LipofectamineTM2000). The transfection efficiency was observed by fluorescence confocal microscopy. The effecive shRNA sequences were screened by Western blot. After transfection with effective shRNA sequences, 786-O cells were divided into 3 groups: (1)SET8-shRNA transfection; (2)negative control transfection; (3)blank control.The assays of methyl thiazolyl tetrazolium(MTT), colony formation and scratch wound were applied to examine variations in cell proliferation, colony formation and migration. Results The positive rate of SET8 expression was 98.7%(154/156), and its expression was closely associated with TNM staging, the size of tumor and lymph node metastasis(all P<0.05). The transfection efficiency was around 70% with a concentration of SET8-shRNA 2μg/L and lipofectamine 2000 4μl/well. SET8-shRNA2 was chosen as the effective sequences with a suppression ratio up to 64%. At 48h post-transfection, the expression of SET8 protein was significantly lower in SET8-shRNA2 group than that in negative control transfection group and blank control group(t=-38.174, P<0.01; t=-58.766, P<0.01). While SET8 gene was silenced by shRNA in SET8-shRNA transfection group, the proliferation, colony formation and migration ability were decreased markedly. Conclusion SET8 protein plays a significant role in the carcinogenesis and progression of renal cell carcinoma. SET8-shRNA can effectively inhibit the expression of SET8 protein in 786-O cells and thus suppress malignant phenotypes of RCC. Key words: Lysine methyltransferaseSET8; Clear cell renal cell carcinoma; 786-O cell; Cell proliferation; Colony formation; Migration

MicroRNA-124 enhances response to radiotherapy in human epidermal growth factor receptor 2-positive breast cancer cells by targeting signal transducer and activator of transcription 3.

AimTo determine whether microRNA (miR)-124 enhances the response to radiotherapy in human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells by targeting signal transducer and activator of transcription 3 (Stat3).MethodsmiR-29b expression was measured in 80 pairs of breast tumor samples and adjacent normal tissues collected between January 2013 and July 2014. Activity changes of 50 canonical signaling pathways upon miR-124 overexpression were determined using Cignal Signal Transduction Reporter Array. Target gene of miR-124 was determined using Targetscan and validated by Western blotting and dual-luciferase assay. Cell death rate was assessed by propidium iodide (PI)/ Annexin V staining followed by flow cytometry analysis. Stat3 and miR-124 expression was further measured in 10 relapsed (non-responder) and 10 recurrence-free HER2-positive breast cancer patients.ResultsMiR-124 expression was down-regulated in HER2 positive breast cancers compared with normal tissues, and was negatively associated with tumor size. MiR-124 overexpression in HER2 positive breast cancer cell line SKBR3 significantly reduced the activity of Stat3 signaling pathway compared with control transfection (P < 0.001). Bioinformatic prediction and function assay suggested that miR-124 directly targeted Stat3, which is a key regulator of HER2 expression. MiR-124 overexpression down-regulated Stat3 and potently enhanced cell death upon irradiation. Consistently, chemical inhibitor of Stat3 also sensitized HER2-positive breast cancer cells to irradiation. Moreover, increased Stat3 expression and reduced miR-124 expression were associated with a poor response to radiotherapy in HER2-positive breast cancers.ConclusionsWeak miR-124 expression might enhance Stat3 expression and radiotherapy resistance in HER2-positive breast cancer cells.

Abstract C41: Altered control of intracellular androgen availability by glucuronidation contributes to loss of androgen dependence in prostate tumor cells

Abstract Locally advanced and metastatic cancers are frequently treated by androgen deprivation therapy. Tumors that recur despite this treatment, termed castration resistant prostate cancer, are highly aggressive and have acquired the capacity to survive and expand in conditions of low circulating androgens. Prostate epithelial cells control the potency and overall availability of androgen hormones in part by inactivation and excretion of excess quantities. The enzymes that catalyze inactivation, specifically by covalently modifying the hormone with a glucuronate adduct, are called UDP-glucuronosyltransferases (UGT). Genetic mutations that reduce levels of the UGT isozymes needed to eliminate excess androgens from the prostate have been correlated with prostate cancer incidence. The ability to alter androgen elimination at the level of individual cells within a tumor is one mechanism by which tumors become heterogeneous with respect to molecular features and therapeutic resistance. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD+-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. Our laboratory has found that non-metastatic prostate tumor cell lines express UGDH in an androgen sensitive manner, that its expression increases the level of UDP-glucuronate, and that these precursors are then channeled to the UGT-catalyzed glucuronidation pathway. The level of UGDH is androgen controlled and serves to regulate tumor cell growth, inversely to its expression. Loss of androgen stimulated UGDH, or pharmacological antagonism of its activity, translated to increased proliferation rate, specifically of androgen dependent tumor cells. We knocked down expression of UGDH in androgen dependent cells to test the role of UGDH in driving cellular glucuronate levels, intratumoral steroid availability, and response to androgen. Flux of UDP sugar metabolites and levels of androgen glucuronide accumulation were compared in control and UGDH knockdown lines by liquid chromatography and mass spectrometry analysis. Effects on androgen sensitive gene expression were monitored by western analysis. Selection of manipulated cells in decremental DHT levels that simulated androgen deprivation was found to reduce steady state UGDH expression, thereby activating the UGT-driven steroid clearance mechanism, as reflected in increased PSA levels and de-repression of UGT2B17. Whereas restimulation of control transfectants with physiological levels of DHT could only increase UGDH expression and maintain full androgen sensitivity in cells experiencing complete androgen loss, the knockdown of UGDH permitted cells to accumulate UDP-glucuronate pools, and decreased UGT-driven glucuronidation of DHT and androsterone. These results support a model in which maintenance of partial UGDH activity through gradual androgen deprivation facilitates loss of sensitivity in androgen elimination pathways and suggests adjuvant UGDH inhibition could prevent or delay onset of castration resistant recurrence. Citation Format: Michelle Palmer, Linlin Ma, Trevor Romsdahl, Qin Wei, Eileen Loughman, Jonathan Markham, Joseph Barycki, Melanie A. Simpson. Altered control of intracellular androgen availability by glucuronidation contributes to loss of androgen dependence in prostate tumor cells. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C41.

Characterization of MUDENG, a novel anti-apoptotic protein.

MUDENG (Mu-2-related death-inducing gene, MuD) is revealed to be involved in cell death signaling. Astrocytes, the major glial cell type in the central nervous system, are a source of brain tumors. In this study, we examined MuD expression and function in human astroglioma cells. Stimulation of U251-MG cells with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resulted in a 40% decrease in cell viability and a 33% decrease in MuD protein levels, although not in MuD mRNA levels. To study the functional relevance of MuD expression, stable transfectants expressing high levels of MuD were generated. Stimulation of these transfectants with TRAIL resulted in enhanced cell survival (77% for stable and 46% for control transfectants). Depletion of MuD led to a marked reduction upon TRAIL stimulation in cell viability (22% in MuD-depleted cells and 54% in control cells). In addition, we observed that MuD depletion increased the susceptibility of the cells to TRAIL by enhancing the cleavage of caspase-3/-9 and BH3-interacting domain death agonist (Bid). A unique 25-kDa fragment of B-cell lymphoma 2 (Bcl-2) lacking BH4 was observed 60–180 min post TRAIL treatment in MuD-depleted cells, suggesting that Bcl-2 is converted from its anti-apoptotic form to the truncated pro-apoptotic form. Importantly, the TRAIL-mediated decrease in cell viability in MuD-depleted cells was abrogated upon Bid depletion, indicating that the role of MuD in apoptotic signaling takes place at the Bid and Bcl-2 junction. MuD localizes predominantly in the endoplasmic reticulum and partly in the mitochondria and its amounts are reduced 6 h post TRAIL stimulation, presumably via caspase-3-mediated MuD cleavage. Collectively, these results suggest that MuD, a novel signaling protein, not only possesses an anti-apoptotic function but may also constitute an important target for the design of ideal candidates for combinatorial treatment strategies for glioma cells.

Construction of SET overexpression vector and its effects on the proliferation and apoptosis of 293T cells.

The expression of SET nuclear proto‑oncogene (SET) is commonly associated with cell proliferation and tumorigenesis. In the present study, a eukaryotic SET expression plasmid (pEGFP‑N1‑SET) was constructed and transiently transfected into 293T human embryonic kidney cells. Transfection led to expression of the SET oncoprotein at high levels, as indicated by polymerase chain reaction and western blot analysis. In addition, the relative mRNA and protein expression of protein phosphatase2A in pEGFP‑N1‑SET‑transfected 293T cells was downregulated compared with that in empty vector‑transfected cells. Furthermore, overexpression of SET increased the percentage of 293T cells in S and G2/M phases compared with the control transfectants. An increase in B‑cell lymphoma2 (Bcl‑2) and a decrease in Bcl‑2‑associatedX (Bax) protein expression was observed in the pEGFP‑N1‑SET‑transfected cells compared with that in the controls, and their susceptibility to As4S4‑induced apoptosis was decreased. The protein SET is involved in a number of cellular processes, including DNA replication, chromatin remodeling, gene transcription, differentiation, migration and cell cycle regulation. SET is overexpressed in several neoplasms, particularly in acute myeloid leukemia. The findings of the present study suggested that the SET gene may contribute to tumorigenesis and may be a potential novel effective therapeutic target for leukemia and other cancer types.

Enhanced plasmid DNA utilization in transiently transfected CHO-DG44 cells in the presence of polar solvents.

Although the protein yields from transient gene expression (TGE) with Chinese hamster ovary (CHO) cells have recently improved, the amount of plasmid DNA (pDNA) needed for transfection remains relatively high. We describe a strategy to reduce the pDNA amount by transfecting CHO-DG44 cells with 0.06 μg pDNA/10(6) cells (10% of the optimal amount) in the presence of nonspecific (filler) DNA and various polar solvents including dimethylsufoxide, dimethyl formamide, acetonitrile, dimethyl acetamide (DMA), and hexamethyl phosphoramide (HMP). All of the polar solvents with the exception of HMP increased the production of a recombinant antibody in comparison to the untreated control transfection. In the presence of 0.25% DMA, the antibody yield in a 7-day batch culture was 500 mg/L. This was fourfold higher than the yield from the untreated control transfection. Mechanistic studies revealed that the polar solvents did not affect polyethylenimine-mediated pDNA delivery into cells or nuclei. The steady-state transgene mRNA level was elevated in the presence of each of the polar solvents tested, while the transgene mRNA half-life remained the same. These results indicated that the polar solvents enhanced transgene transcription. When screening a panel of recombinant antibodies and Fc-fusion proteins for production in the presence of the polar solvents, the highest increase in yield was observed following DMA addition for 11 of the 12 proteins. These results are expected to enhance the applicability of high-yielding TGE processes with CHO-DG44 cells by decreasing the amount of pDNA required for transfection.

Abstract 450: Suppression of innate and adaptive immune responses in the human lung tumor microenvironment via microRNA-183

Abstract Innate and adaptive immunity plays a critical role in defense against cancer but can be undermined by the tumor microenvironment. To understand the underlying mechanism, we set out to analyze the molecular and cellular basis for natural killer (NK) and CD8 T cell suppression by human lung tumor cells. We found that transforming growth factor-beta (TGFb), abundantly produced by tumor cells, effectively reduced tumoricidal function in activated NK cells and CD8 T cells by specifically targeting a key adaptor protein, DAP12, which is required for surface anchoring of multiple NK receptors, including NKp44. For proof of the TGFb-miR-183-DAP12 circuit, flow cytometric analysis demonstrated that TGFb reduced surface levels of NKp44 on activated primary human NK and CD8 T cells, but western blot analysis demonstrated their intact presence in the cytoplasm. This was due to loss of DAP12, which is critical for surface expression of NKp44 and for downstream signaling to mobilize lytic granules to kill tumor cells. Mechanistically, TGFb readily induced miR-183 expression in both primary human NK cells and T cells which could bind specifically to the 3′UTR of human DAP12 to inhibit transcription and translation. Only miR-183 but not miR-185 or control (CT) transfection repressed luciferase activity of DAP12 3′UTR luciferase reporter constructs transferred into HELA cells. Moreover, we overexpressed lentiviral miR-183 in either primary NK and T cells and found that such expression effectively reduced DAP12 levels in both cell types. Flow cytometric analysis of these same cells indicated that loss of DAP12 was accompanied by loss of NKp44 surface expression and by loss of cytotoxic function against tumor cells. As further proof, we showed that lentiviral anti-sense miR-183 transfection into NK cells and T cells prevented TGFb from suppressing DAP12 expression. Thus, we have uncovered a seminal TGFb-miR-183-DAP12 pathway utilized by tumor cells to silence infiltrating NK and T cells, constituting the first report of miR control of an adaptor protein to control both NK and T cell immune function. Citation Format: Melba Marie Tejera, Melba Marie Tejera, Sarah S. Donatelli, Jun-Min Zhou, Danielle L. Gilvary, Xianghong Chen, Erika A.. Suppression of innate and adaptive immune responses in the human lung tumor microenvironment via microRNA-183. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 450. doi:10.1158/1538-7445.AM2015-450

Cyclic stretch and compression forces alter microRNA-29 expression of human periodontal ligament cells

MicroRNAs (miRs) play an important role in the development and remodeling of tissues through the regulation of large cohorts of extracellular matrix (ECM) genes. The purpose of the present study was to determine the response of miR-29 family expression to loading forces and their effects on ECM gene expression in periodontal ligament cells, the key effector cell population during orthodontic tooth movement. In a comparison between miRs from human periodontal ligament cells (PDLCs) and alveolar bone cells (ABCs) from healthy human subjects, the ABC cohort of miRs was substantially greater than the corresponding PDLC cohort. Cyclic mechanical stretch forces at 12% deformation at 0.1Hz for 24h decreased expression of miR-29 family member miRs about 0.5 fold while 2g/cm2 compression force for 24h increased miR-29 family member expression in PDLCs 1.8–4 folds. Cyclic stretch up-regulated major ECM genes in PDLCs, such as COL1A1, COL3A1 and COL5A1, while the compression force resulted in a down-regulation of these ECM genes. Direct interactions of miR-29 and Col1a1, Col3a1 and Col5a1 were confirmed using a dual luciferase reporter gene assay. In addition, transient transfection of a miR-29b mimic in mouse PDLCs down-regulated Col1a1, Col3a1 and Col5a1 while the transfection of miR-29b inhibitor up-regulated these genes compared to control transfection indicating that these target ECM genes directly responded to the altered level of miR-29b. These results provided a possible explanation for the effects of the miR-29 family on loaded PDLCS and their roles in extracellular matrix gene expression.

Partial inhibition of differentiation associated with elevated protein levels of pluripotency factors in mouse embryonic stem cells expressing exogenous EGAM1N homeoprotein

We previously reported that transcripts encoding the homeoprotein EGAM1N are expressed in preimplantation mouse embryos and embryonic stem (ES) cells, and the exogenous expression of EGAM1N inhibits the differentiation of ES cells. In order to clarify the relationship between the inhibition of differentiation and EGAM1N, we generated mouse MG1.19 ES cells stably expressing EGAM1N. Control transfectants with an empty vector formed relatively flattened cell colonies similar to those observed in parental MG1.19 cells. In contrast, Egam1n transfectants formed tightly aggregated cell colonies with increased localization of CDH1 at cell-to-cell interfaces. The protein levels of pluripotency factors, including TBX3 and SOX2, were also increased. The expression of Tbx3 transcripts was induced, although the level of Sox2 transcripts was almost unchanged. The expression of EGAM1N resulted in no obvious changes in the expression of genes encoding receptors, protein kinases, transcription factors, and their encoded proteins involved in the LIF-STAT3 signaling pathway. Alkaline phosphatase activity, a marker for the undifferentiated state, in Egam1n transfectants was exhibited in a clonal proliferation assay. When differentiation of Egam1n transfectants was induced, progression was prevented with increases in transcript levels of Pou5f1, Sox2, Nanog, Klf4, Tbx3, and their encoded proteins. However, Egam1n transfectants formed relatively flattened-cell layers as observed in the control, indicating that the expression of EGAM1N could not maintain LIF-independent self-renewal of ES cells. Overall, we suggest that expression of EGAM1N could inhibit differentiation, at least in part, by elevating the protein levels of pluripotency factors in MG1.19 ES cells.

Internal Tandem Duplication Mutation of FLT3 (FLT3/ITD) Significantly Increases Inter Chromosomal Homologous Recombination Providing a Direct Link Between Constitutive FLT3-ITD Activity and Propagation of Copy Neutral Loss of Heterozygosity (CN-LOH) in Acute Myeloid Leukaemia

The majority of patients with adult acute myeloid leukaemia (AML) that present with an apparently normal karyotype (NK-AML) are grouped together in the “intermediate” risk category and constitute 40-45% of all adult AML patients. Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor whether it is internal tandem duplication (ITD) of its juxtamembrane domain or point mutations in its kinase domain are one of the most common mutations in NK-AML. The presence of FLT3-ITD mutation in NK-AML results in a more aggressive disease, resistance to therapy and poor survival. Acquired copy neutral loss of heterozygosity (CN-LOH) also referred to as uniparental disomy (UPD) is a common phenomenon of myeloid malignancies where an oncogenic allele is duplicated on the other chromosome. The use of single nucleotide phenotype analysis (SNP-A) karyotyping detects CN-LOH in 20% and 40% of newly diagnosed and relapsed AML respectively. CN-LOH at 6p or at the FLT3 locus 13q associated with a FLT3-ITD mutation in NK-AML results in an even more aggressive disease compared to NK-AML + FLT3-ITD without CN-LOH. It has been proposed that CN-LOH is the result of a homologous recombination (HR) DNA repair event. However, the underlying mechanisms that confer CN-LOH have yet to be determined.To elucidate the mechanisms that produce CN-LOH in NK-AML we determined whether oncogenes such as FLT3-ITD have the propensity to generate CN-LOH through up-regulation of inter chromosomal (between maternal and paternal chromosomes) HR DNA repair. We have showed previously that constitutional FLT3-ITD kinase activity increased reactive oxygen species resulting in elevated double strand breaks. Moreover, FLT3-ITD mutation increases homologous recombination activity through transcriptional augmentation of HR factor, RAD51 expression.Firstly, to evaluate FLT3-ITD induced genomic instability we measured sister chromatid exchanges (SCE) that result from HR mediated chromosome cross-over. It has been shown previously in the pre-leukaemic chromosomal instability disorder, Blooms syndrome is characterised by increased SCE and an increased propensity for CN-LOH. FLT3/ITD primary AML (n=17), MOLM-13 and FLT3-ITD transfected U937 cells show a significant increase in SCE compared to WT FLT3 primary AML cells, U937 cells transfected with empty vector, (10 vs. 6 SCE per metaphase, p<0.001) (Figure 1A-B). Importantly, primary AML with CN-LOH at 13q + FLT3-ITD mutation gives significantly higher HR activity and SCE than primary AML + FLT3-ITD mutation without CN-LOH (p<0.05). Moreover, treatment of FLT3/ITD primary AML, MOLM-13 and FLT3-ITD transfected U937 cells with the FLT3 inhibitor, CEP701 and the anti-oxidant agent, N-acetyl cysteine restored SCE to levels observed for WT FLT3. Next, we investigated whether inappropriate HR activity at G0 rather than at S phase of the cell cycle could result in illegitimate HR resulting in inter chromosomal HR. Western blot studies showed that G0 primary AML with FLT3/ITD, synchronised G0 MOLM 13 and FLT3-ITD transfected U937 cells showed augmented RAD51 expression compared to non-mutated FLT3 primary AML cells, cell lines and KG-1 transfected with empty vector (Figure 1C). Lastly, we used a thymidine kinase (TK) gene mutation assay system to screen for FLT3-ITD derived CN-LOH. The human lymphoblastoid cell line, TK6 is heterozygous for TK and becomes resistant to trifluorothymidine (TFT) upon loss of the wild type TK allele on 17q. FLT3-ITD transfected TK6 cells (using 2 different expression constructs) demonstrated significantly increased mutation frequency (grey bars) and mutation ate (Black bars) and TFT resistance compared to WT FLT3 and emptor vector control transfection (p<0.001) (Figure 1D). Colony PCR of TFT resistant colonies revealed that resistance was due primarily (80%) to inter chromosomal HR at 17q in contrast to empty vector (20%) and cisplatin treated (45%) TK6 cells (Figure 1E). The findings that FLT3-ITD mutation induces increased ROS production inducing DNA double strand breaks and the concomitant increase in the rad51 dependent inter homologous recombination provide a strong proof of principle that constitutive FLT3-ITD kinase activity directly confers CN-LOH that furthers our understanding of leukaemic progression and relapse in NK-AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Influence of recombinant lentiviral vector encoding miR-15a/16-1 in biological features of human nasopharyngeal carcinoma CNE-2Z cells.

MicroRNAs can function as tumor suppressor miRNAs. Bcl-2 is an antiapoptotic gene overexpressed in many tumors, including nasopharyngeal carcinoma (NPC). It is reported that microRNA-15a (miR-15a) and microRNA-16-1 (miR-16-1) could act as bcl-2 inhibitors. To investigate their effects on NPC, the authors used recombinant lentiviral vector to upregulate the expression of miR-15a/16-1 in NPC CNE-2Z cells. The authors divided cells into the control group, transfection group, radiotherapy group, and transfection-radiotherapy group. In this experiment, reverse transcription-quantitative polymerase chain reaction was used to detect the expression of miR-15a/16-1 and bcl-2 mRNA. Cell proliferation was analyzed by MTT assay. Flow cytometry was used to measure cell apoptosis. Radiosensitivity was measured using colony-forming experiment. The protein expression of bcl-2 was measured by western blot, the activation levels of caspase were detected by a spectrophotometric method. After transfection, cell proliferation was inhibited, while the apoptosis rate and radiosensitivity were increased. In addition, the activation of caspase-2 and caspase-3 was aggrandized correspondingly. Although the expression levels of bcl-2 mRNA in each group had no difference, the protein expression of bcl-2 was downregulated. These results suggested that miR-15a/16-1 could inhibit cell proliferation and increase the apoptosis and radiosensitivity of CNE-2 cells, by regulating the bcl-2 gene at post-transcriptional level and by increasing the activation of caspase-2 and caspase-3.

Abstract 2096: HIF-1α is a crucial factor in the development of peritoneal dissemination via natural metastatic routes in scirrhous gastric cancer

Abstract Background The molecular mechanisms of peritoneal dissemination in gastric cancer still remain unclear. This study was designed to clarify the role of HIF-1α in the development of peritoneal dissemination of gastric cancer. Methods HIF-1α knockdown (KD) cells were established in the scirrhous gastric cancer cell line 58As9 by siRNA transfection. In western blot analysis, HIF-1α expression was assessed using KD and the control transfectant (SC) cells under normoxia (20%O2) as well as hypoxia (1%O2). A series of in vitro analysis were conducted and compared between the 2 transfectants. Using nude mice, peritoneal dissemination was evaluated by orthotopic implantation (o.i.) and intraperitoneal injection (i.p.) models. Finally, tumor angiogenesis of venous and lymph vessels was immunohistochemically analyzed. Results Deficient HIF-1α expression was observed in KD, but not SC cells under normoxia and hypoxia. In vitro proliferation and invasion was significantly reduced in KD compared with SC cells. On contrary, HIF-1α exerts unfavorable effects on anoikis resistance and adhesion ability. In o.i. model, stomach tumor was highly developed in both of KD (86.7%) and SC mice(100%). On the other hand, peritoneal dissemination was more frequently observed in the SC mice (93%) than in the KD mice (13%) (p &amp;lt; 0.001). In the i.p. model, the dissemination highly occurred in both mice (each 100%), while greater number of nodules was observed in the KD mice (p = 0.017). Angiogenesis with vascular invasion were more highly developed in the SC stomach tumors (venous vessels: p=0.001, lymph vessels: p=0.043). In the disseminated nodules, the vascular invasion was developed at intratumoral regions in the SC o.i. mice, however such appearance was not found in i.p. mice. Conclusions This study clearly showed that HIF-1α is crucial for the development of peritoneal dissemination in the spontaneous metastatic (o.i.) model. Moreover, in i.p. model, larger number of dissemination was found in KD than SC mice, indicating that the dissemination by o.i. would not be developed via seeding mechanism. Analysis of tumor angiogenesis in o.i. model demonstrated that HIF-1α activated venous as well as lymph angiogenesis in the stomach tumor. In peritoneal disseminated nodules, vascular invasion was observed at intratumoral area in o.i. SC mice, but not in the i.p. mice. This unique feature further suggests that the dissemination in o.i. mice might be developed by extravasation of cancer cells, which are micro-connected to the stomach tumor via trans-vessel route. Citation Format: Shuusuke Miyake, Yoshihiko Kitajima, Jun Nakamura, Keita Kai, Kazuyoshi Yanagihara, Tomokazu Tanaka, Masatsugu Hiraki, Kohji Miyazaki, Hirokazu Noshiro. HIF-1α is a crucial factor in the development of peritoneal dissemination via natural metastatic routes in scirrhous gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2096. doi:10.1158/1538-7445.AM2014-2096

Analgesic effect of lentivirus-mediated RNA interference of the expression of glial cell line-derived neurotrophic factor gene on bone cancer pain

Objective To observe the analgesic effects of lentivirus-mediated RNA interference of the expression of glial cell line-derived neurotrophic factor (GDNF) on bone cancer pain.Methods (1)Lvs-siGDNF was constructed and bone cancer pain models were established by intra-tibial injection of MRMT-1 cells.(2) The rats were randomly divided into 4 groups according to different drugs which were infused into subarachnoid space through the intrathecal tubes:psiHIV-U6,psiHIV-GDNF-mU6,mor-phine,and normal saline.(3) The pain threshold at different time points (normal rats,7 days and 14 days after dosing) was measured.(4) The changes of pain threshold were observed from preoperative day to 7 days and 14 days after administration.The levels of GDNF,glial fibrillary acidic protein (GFAP),and Fos were detected by immunofluorescent staining technique and Western blotting.Results (1) As compared with control group (0.117 ±0.060) and transfection group (0.113 ±0.072),GDNF siRNA sequence 3 (0.045 ± 0.034) was the most effective interference segment confirmed by Q-PCR (P ＜ 0.05).The percentage of fluorescent cells to the brightfield was 0.87.TLvs-siGDNF was 3.48 × 108 pfu/ml.(2)Thermal pain threshold at 7th day (3.80 ±0.69) and 14th day (3.71 ± 1.10),and mechanical pain thresh-old at 14th day (2.58 ± 1.93) were all increased in siGDNF group than normal saline group (1.38 ± 1.01,1.11 ± 1.02,and 1.53 ±0.69) and psiHIV-U6 group (1.39 ± 1.18,0.67 ±0.27,and 1.21 ± 1.06)(P ＜ 0.05),while there was no significant difference in mechanical pain threshold at 7 day after administration (P ＞ 0.05).(3) RNA interference effectively decreased the expression level of GDNF in siGDNF group (P ＜0.05).(4) As compared with other groups,the expression levels of Fos and GFAP in the spinal dorsal horn were decreased in morphine group and siGDNF group (P ＜ 0.05).Conclusion The analgesic effect of siGDNF is similar to that of morphine in bone cancer rats,and siGDNF worked effectively more than 2 weeks.SiGDNF realized analgesic effect probably by inhibiting the astrocyte activation. Key words: Bone cancer pain; Glial cell line-derived neurotrophic factor; RNA interference; Lentiviral