Tumorigenic Cell Lines Research Articles

New technologies and challenges of novel virus detection.

The use of new cell substrates for the development of biologicals, particularly tumorigenic and tumor-derived cell lines, can pose a major regulatory challenge due to safety concerns related to the presence of novel viruses, latent and occult viruses including oncogenic viruses, and endogenous retroviruses, since these may not be detected by the currently recommended conventional assays. This report is a summary of our laboratory's experiences using advanced nucleic acid-based technologies to evaluate a Madin-Darby canine kidney (MDCK) cell line and the insect Sf9 cell line derived from Spodoptera frugiperda, and presents some ongoing efforts to address the challenges of novel virus detection.

Mutation of β-catenin in a radiation and estrogen breast cancer model.

β-catenin plays a pivotal role in cell-to-cell adhesion as a transcriptional activator in signal transduction pathways. Potential role of this gene was studied in a radiation- and estrogen breast cancer model by analyzing differential expression of associated genes of β-catenin as E-cadherin and catenins. The aim was to identify whether β-catenin gene was mutated when associated with other genes as glycogen synthase kinase-3-β (GSK-3-β), T-cell factor (TCF) and other extracellular matrix genes related to cell adhesion. Results indicated that β-catenin gene had increased expression at mRNA and mutation at exon3 in irradiated and estrogen-treated cell lines when compared to MCF-10F. It was found that β-catenin and GSK-3-β had greater protein expression in the tumorigenic cell line, called Alpha5 and the tumor cell lines, called Tumor2 than control MCF-10F and non-malignant Alpha3 cell lines. The β-catenin/GSK-3-β complex was identified in non-malignant cell lines such as MCF-10F, Estrogen, Alpha1, Alpha3, and Alpha4 cell lines by immunoprecipitation assays. However, Alpha5 and Tumor2 did not form a complex in this assay. However, β-catenin/TCF-4 complex was found only in Alpha5 and Tumor2. Immunofluorescent studies confirmed these findings since co-localization in β-catenin and GSK-3-β was only found in MCF-10F and Alpha3 while β-catenin/TCF-4 was only observed in Alpha5 and Tumor2. It can be concluded that mutation of β-catenin and its interaction with other associated proteins may be an early event during radiation and estrogen induced progression of human breast carcinogenesis.

Abstract 3354: The level of aerobic glycolysis as an effective predictor of tumorigenicity

Abstract Introduction: Cancer cells have been known to harbor characteristic metabolic alterations such as enhanced glucose uptake and aerobic glycolysis. However, elucidation of molecular mechanisms underlying the association between aerobic glycolysis and tumorigenesis has remained elusive. Thus, this study aimed to investigate the role of altered metabolism in conferring advantage to tumorigenesis, using two different cell lines of immortalized human oral keratinocytes (IHOK) that differed in the level of aerobic glycolysis. Experimental procedures: Levels of glucose uptake and lactate production were measured. Reverse transcription polymerase chain reaction and western blot were employed to measure the expression of relevant genes. Proteomic analysis was performed to identify protein-level differences. Small interfering RNAs were used for transient knockdown of genes. The cell lines were injected into mouse tongue to assess subsequent tumor formation. Results: Although the two IHOK cell lines shared same origin, the cell line with enhanced glucose uptake and lactate production also had higher expression of genes involved in glycolysis, growth signal propagation, and matrix remodeling. In particular, matrix metalloproteinase (MMP)-1, 2, 9, and 14 expressions were significantly higher in the cell line with upregulated glycolysis. Upon injection into mice, the two cell lines differed in the rate of in vivo tumor formation and resulting tumor volume. Epidermal growth factor receptor (EGFR) was upregulated in the more tumorigenic IHOK cell line as well as in aggressive breast and oral cancer cell lines compared with their less aggressive counterparts. EGFR knockdown in the more tumorigenic cell lines led to reduction in lactate production and prevented high-level expression of glycolytic genes normally induced by EGF treatment. Proteomic analysis between the two IHOK cell lines revealed pyruvate kinase isozyme M2 (PKM2) as the protein whose level was significantly higher in the more tumorigenic cell line. To evaluate the relationship between upregulated PKM2 and high tumorigenic potentials, PKM2 was knocked down in the more tumorigenic cell line. MMP-1 level decreased upon PKM2 knockdown, indicating that upregulated PKM2 not only promotes aerobic glycolysis but also enhances MMP expression to facilitate tumorigenesis. Conclusions: Cells with upregulated PKM2 likely excel in tumorigenesis by enhancing MMP expression. In addition, upregulated EGFR amplifies the growth signals to enhance the expression of genes required for tumor progression. These results indicate that the degree of altered metabolic activity, aerobic glycolysis in particular, might be a significant predictor of tumorigenicity. Acknowledgements: This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(2009-0094027). Citation Format: Doo Young Lee, Young Jin Park, Jung Yoon Bae, Jin Kim. The level of aerobic glycolysis as an effective predictor of tumorigenicity. [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 3354. doi:10.1158/1538-7445.AM2014-3354

Abstract LB-90: Molecular mechanism of TAZ-induced lung tumorigenesis

Abstract The transcriptional co-activator with PDZ-binding domain (TAZ) is a transcriptional co-activator and major component of an emerging signaling pathway called the Hippo pathway that plays critical roles in tumorigenesis, organ size control, stem cell renewal, drug resistance, etc. Recently, we have provided the first biological evidence that TAZ is over-expressed in non-small cell lung cancer (NSCLC) cells and over-expression of TAZ can transform HBE135 immortalized non-tumorigenic human lung epithelial cells (Zhou et al., 2011). However, the cellular genes mediating TAZ-induced transformation are unknown. In addition, there is no in vivo mouse model established to study the roles of TAZ in lung tumorigenesis in vivo. To address these issues, we have carried out the following experiments: First, a Dox-inducible lentiviral expression system was used to overexpress constitutively active TAZ (TAZ-S89A) in a mouse immortalized lung epithelial cell line (E10). Overexpression of TAZ-S89A in E10 cells caused increased cell proliferation, transformation, and tumor formation in vivo in nude mice. Next, tumorigenic stable cell lines (TAZS89A-TM) were established after isolating TAZS89A-induced tumors from mice. Second, to identify the downstream genes mediating TAZ-induced tumor formation, the technology of Next-generation sequencing (RNA-seq) was performed on the new tumorigenic cell line E10-TAZ-TM to identify downstream genes transcriptionally regulated by TAZ after induction of TAZ by Dox. By analyzing the data and confirming them using qRT-PCR, multiple significant oncogenes were found activated by TAZ overexpression. Third, these genes were knocked down by shRNAs in the tumorigenic E10-TAZS89A-TM cell line and tested whether they are important in TAZ-induced cell proliferation, transformation and tumor formation in mice. Finally, since previous studies indicate that TAZ is involved in mammary cell growth by interacting with transcription factor TEAD or through its WW domain, we also tested whether these genes are activated through TEAD-binding domain or WW-domain of TAZ. E10 stable cell lines overexpressing inducible TAZ mutant with amino acid mutations in TEAD-binding domain (TAZ-F52A/F53A) or WW domain (TAZ-W152A/P155A) were established, followed by qRT-PCR analysis. The TEAD-binding domain of TAZ was found necessary for activation of the identified genes and maintaining TAZ-induced oncogenic effects in lung cells. In conclusion, this project established the first in vivo xenograft mouse model system using TAZ-overexpressing lung epithelial cells, and identified the downstream target genes mediating TAZ-induced lung tumorigenesis. Citation Format: Adel B. Alharbi, Hongchao Shan, Yawei Hao, Xiaolong Yang. Molecular mechanism of TAZ-induced lung tumorigenesis. [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 LB-90. doi:10.1158/1538-7445.AM2014-LB-90

Abstract 3297: PTEN loss enhances amphiregulin-specific signaling and gene expression in triple-negative breast cancer

Abstract The epidermal growth factor receptor (EGFR) is frequently overexpressed in triple-negative breast cancer, and approximately half of all basal breast cancers show autocrine activation of EGFR by amphiregulin (AREG). When AREG is the activating ligand, the dynamics of EGFR signaling are different than those of EGF-bound EGFR. AREG-ligated EGFR accumulates at the cell surface, resulting in altered receptor trafficking and downstream signaling. To further explore the role of amphiregulin in cell signaling, we performed reverse-phase protein array (RPPA) analysis on SUM149 cells, a highly tumorigenic and metastatic cell line that expresses high levels of AREG and EGFR, and MCF10A cells, a non-transformed mammary epithelial cell line cultured in the presence of either EGF or AREG. This analysis identified a number of signaling components mediated specifically by amphiregulin, including decreased caspase expression, increased Gab2 expression, elevated fibronectin levels and marked changes in integrin expression. In addition, AREG-mediated EGFR signaling resulted in increased expression and activation of c-src. Thus, a major effect of AREG signaling in both cell lines was enhanced activation of the canonical focal adhesion signaling pathway. In addition to increased expression of EGFR and AREG, SUM149 cells are known to have a genomic alteration at the PTEN locus resulting in complete abrogation of PTEN message and protein expression. RPPA analysis of signaling in SUM-149 cells confirmed this PTEN loss and, not surprisingly, revealed elevated AKT activity. In addition, several proteins in the WNT signaling pathway were altered in SUM-149 cells, including reduced expression of GSK3β, increased phosphorylation of GSK3β, and increased expression of Dvl. These results suggest enhanced Wnt-beta catenin activity in SUM-149 cells that is likely the result of enhanced AKT activity resulting from PTEN loss. Although we previously had difficulty measuring Wnt signaling in SUM-149 cells in monolayer culture, mammosphere assays performed with SUM149 cells expressing a Wnt reporter construct confirmed that Wnt signaling was highly active in mammospheres in 3D culture. We also analyzed the genes that are regulated in expression by EGFR in MCF-10A cells in the presence of EGF or AREG, and in SUM-149 cells. These results identified AREG-specific gene expression profiles in the isogenic system. In addition, SUM-149 cell-specific genes regulated by EGFR were also identified, some of which are likely to be the result of PTEN loss and enhanced AKT signaling in these cells. Finally, we found that AREG knockdown in SUM-149 cells significantly reduced the tumorigenicity of SUM149 cells in NOD-SCID mice. Analysis of signaling and gene expression in these AREG knock-down cells are consistent with the importance of AREG expression levels and of the modulation of the focal adhesion canonical pathway in tumor potential of the cells in vivo. Citation Format: Christiana Kappler, Robert Wilson, Bridget Varughese, Stephen P. Ethier. PTEN loss enhances amphiregulin-specific signaling and gene expression in triple-negative breast 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 3297. doi:10.1158/1538-7445.AM2014-3297

Abstract 4823: Circulating tumor cell miR-27a overexpression as a novel mechanism of hepatocellular carcinoma metastasis

Abstract Hepatocellular carcinoma is a very lethal disease, being the third most common cause of cancer-related deaths worldwide. The vast majority of these deaths are due to metastasis. Unfortunately, the mechanisms of metastasis are still largely unclear. Micro RNAs are non-coding RNAs that are typically about 22 nucleotides in length. They are now established as important regulators of gene expression. A number of different micro RNAs have been shown to contribute to hepatocellular carcinoma progression and metastasis. Although micro RNA 27a (miR-27a) has been implicated in the metastasis of other solid cancers, its role in hepatocellular carcinoma metastasis was previously unknown. We have recently established a novel circulating tumor cell line from a syngeneic metastatic hepatocellular carcinoma mouse model and we have demonstrated that it has enhanced tumorigenicity and metastatic capacity (1). In this study, our aim was to determine if miR-27a has a role in the development, progression and metastasis of hepatocellular carcinoma. To accomplish this aim, we used primary human hepatocytes (Hu 1476), a non-tumorigenic hepatocyte cell line established from the liver of wild-type immune competent Balb/c mouse (BNL CL.2), a highly tumorigenic hepatocellular carcinoma cell line derived from BNL CL.2 hepatocytes (BNL 1ME A. 7R.1), and a novel circulating tumor cell line derived from the syngeneic metastatic hepatocellular carcinoma Balb/c mouse model (OL0825). Total RNA was extracted from cultured cells. cDNA was made using a protocol suitable for assessment of miRNA expression. Real-time quantitative reverse transcription polymerase chain reaction (qPCR) was used to determine miR-27a expression using primers specific for human and mouse miR-27a. GAPDH was used as a house-keeping gene. The expression profile of a panel of four miRNAs (miR-27a, miR-27b, miR-103, and miR-107) in the BNL CL.2 hepatocytes was very similar to that of the Hu 1476 primary human hepatocytes. However, the BNL 1ME A. 7R.1 hepatocellular carcinoma cell line had about 42-fold greater expression of miR-27a than BNL CL.2 hepatocytes. Notably, the novel circulating tumor cell line, OL0825, had about 130-fold greater expression of miR-27a than the BNL CL.2 hepatocytes. Therefore, we conclude that circulating tumor cell miR-27a overexpression may be a novel mechanism of hepatocellular carcinoma metastasis. Furthermore, miR-27a may be a useful biomarker for hepatocellular carcinoma progression and metastasis. 1. Ogunwobi OO, Puszyk W, Dong H, Liu C. Epigenetic upregulation of c-Met and HGF drives metastasis in hepatocellular carcinoma. PLoS One, 2013, 8(5):e63765. Citation Format: Olorunseun O. Ogunwobi, Chen Liu. Circulating tumor cell miR-27a overexpression as a novel mechanism of hepatocellular carcinoma metastasis. [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 4823. doi:10.1158/1538-7445.AM2014-4823

Abstract 1458: The precursor miR-138/2, but not miR-138/1, targets p53 mRNA and contributes to the acquisition of melanoma metastatic phenotype: Are the miRNAs precursors important to direct mature miRNA to mRNA targets

Abstract MiRNAs regulate pathways associated with differentiation, proliferation, apoptosis, among others, miRNAs missregulation may contribute to malignant transformation. Although melanoma is one of the rarest dermatological cancers, it is responsible for the greatest number of skin cancer-related deaths. In that context, our aim is to identify miRNAs that could be involved with melanoma progression. Using a cellular murine model of melanoma development, we identified the miR-138 as an interesting target of studying. In this model, four cell lines (melan-a, 4C, 4C11- and 4C11+) mimics the distinct steps of human melanoma genesis. miR-138 is codified from two different regions on the genome and they were named according to their origin. miR-138/1 is coded from chromosome 9 in mouse (corresponding to 3, in humans) while miR138/2 is coded from chromosome 8 in mouse (equivalent to 16, in humans). Despite the mature sequence of these two miRNAs being exactly the same, the flanking regions that form the precursors are distinct. miR-138 coded from chromosome 8 but not from chromosome 9 promotes cells to acquire a more aggressive phenotype. When we transfected the tumorigenic but non-metastatic cell line 4C11- with the genomic DNA corresponding to miR-138/2, it promotes the increase of proliferation, migration, colony formation and resistance to anoikis by this cell line compared to its untransfected counterpart 4C11-. In vivo assays showed that 4C11- miR-138/2 is able to form fast-growing tumors and pulmonary metastasis. None of these phenotypic changes was observed in 4C11- cells overexpressing the miR-138 coded from chromosome 9. We also validated p53 (rarely mutated in melanomas) as a target of miR-138 coded from chromosome 8, but not from chromosome 9. Therefore, we hypothesized that the sequence of precursor miRNA (pre-miR) could be involved with the direction of mature miRNA to the mRNA target. Moreover, EZH2, validated as a target of miR-138, is downregulated only in 4C11- cells overexpressing the miR-138/1, emphasizing even more the importance of the precursor sequence in the regulation of the mRNA target. miR-138 expression in melanocytic lesion were evaluated as well. We observed increased expression of miR-138 in primary and metastatic human melanoma samples related to benign nevi. Therefore, in this work we showed that miR-138 may contribute to melanoma genesis and is capable to regulate the expression of p53. Moreover, we suggest for the first time that the precursor sequence of miRNA can direct the mature miRNA to mRNA targets. Supported by CNPq and FAPESP Citation Format: Adriana Taveira Da Cruz, Aline Hunger, Genevieve Paré, Dulcie Lai, Fabiana Henriques Machado Melo, Bryan Eric Strauss, Victor Tron, Miriam Galvonas Jasiulionis. The precursor miR-138/2, but not miR-138/1, targets p53 mRNA and contributes to the acquisition of melanoma metastatic phenotype: Are the miRNAs precursors important to direct mature miRNA to mRNA targets. [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 1458. doi:10.1158/1538-7445.AM2014-1458

Abstract 1566: Notch-1 regulation of the PTEN - mTOR axis in prostate

Abstract Prostate cancer affects one in three men over the age of 60. Loss of expression or function of PTEN is the most commonly observed molecular defect in human prostate cancer. Thirty to 70% of clinical cases exhibit loss of this critical tumor suppressor. PTEN is an essential negative regulator of PI3K/Akt signaling. Together with the mTOR pathway, the PTEN/PI3K/Akt cascade forms a network for cellular responses to growth factors and nutrients. Studies in mice have demonstrated that small changes in PTEN dose can influence cancer development. Thus, understanding the regulation of PTEN expression and function is key to understanding tumor progression. We have previously reported that signaling through the Notch-1 receptor pathway results in increased transcriptional expression of the tumor suppressor PTEN. We further reported that Notch-1 signaling was lost in the tumor foci of clinical prostate cancer cases as compared to the surrounding benign tissue. Herein, we report that Notch-1 signaling alters the pool of active, unphosphorylated PTEN in the human metastatic prostate tumor cell line DU145. Retroviral transduction was used to generate DU145 cells that express constitutively active Notch-1 (DU/ICN1). In cells with constitutively active Notch-1 (DU/ICN1), the pool of unphosphorylated PTEN was increased roughly 2.5 fold compared to the vector only control. Constitutive Notch-1 signaling also resulted in decreased mTOR signaling as determined by a decrease in phosphorlyated 4E-BP1 and phosphorylated S6 ribosomal protein. Decreased expression of Raptor and Rictor and decreased phosphorylation of mTOR were also observed in the presence of constitutively active Notch-1. To test if Notch-1 signaling influences tumor engraftment and growth in a syngeneic model, the tumorigenic C2 cell line, derived from TRAMP mice, was transduced with constitutively active Notch-1. C2/ICN1 and parental C2 cells were engrafted into C57/BL6 mice. All recipients of the parental C2 cell line developed tumors within 46 days, whereas none that received cells expressing constitutively active Notch-1 (C2/ICN1) had developed tumors. Only after 61 days did one of the four mice that received C2/ICN1 cells develop tumors. We next tested if loss of Notch-1 expression promotes characteristics associated with tumorigenicity by using lentiviral transduction to knock down endogenous Notch-1 expression in DU145 cells (DU/shN1). DU145 cells with loss of Notch-1 (DU/shN1) exhibited decreased expression of PTEN protein and a decreased ability to migrate in transwell experiments as compared with control cells expressing endogenous Notch-1. Collectively, these data indicate a role for Notch-1 receptor signaling in modulating the activity of PI3K/Akt and mTOR axis through regulation of the PTEN tumor suppressor, and suggest a mechanistic basis for Notch-1 tumor suppressive activity in prostate cells. Citation Format: Jennifer M. Nutter, C William Angus, Fred E. Bertrand. Notch-1 regulation of the PTEN - mTOR axis in prostate. [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 1566. doi:10.1158/1538-7445.AM2014-1566

Abstract 232: Extracellular matrix components influence prostate tumor cell sensitivity to cancer-preventive agents selenium and green tea polyphenols

Abstract Some cancer-preventive agents such as selenium and green tea polyphenols effectively prevent cancer in some cases, but fail in other cases. It could be possible that the cancer-preventive actions of these agents may be altered by the interaction of cancer cells with the extracellular matrix (ECM) components, thus making this an important issue to study. Others have previously cloned a family of human prostate cancer cell lines with a common lineage that show the progression of characteristics from low to high degrees of malignancy and mimic different stages of tumor progression seen in human prostate cancer. For this study, we used these prostate cancer cell lines with increasing tumorigenicity and invasive ability: WPE1-NA22 showing the lowest, WPE1-NB14 and WPE1-NB11 showing intermediate, and WPE1-NB26 showing the greatest. The low tumorigenic cell line, WPE1-NA22 resembles noninvasive prostatic intra-epithelial neoplasia and represents a good target for chemoprevention in humans. Initially, we grew these cells on the surface of ECM components, laminin, collagen I, and fibronectin and determined the tumor cell sensitivity to methylseleninic acid (MSA) and epigallocatechin-3-gallate (EGCG). Poly-L-lysine was used as a control substratum. Cell growth was determined by sulforhodamine B staining assay. Then we determined whether these ECM proteins could enhance tumor cell resistance to MSA and EGCG. ECM components, but not polylysine, decreased tumor cell sensitivity to these agents. The effect of ECM components was prevented by β1 integrin-blocking antibodies suggesting that β1 integrin plays a role in mediating intracellular signaling conferring resistance against MSA and EGCG. Previously, we have shown an increase in expression of protein kinase Cε (PKCε) in advanced prostate tumor cells WPE1-NB26. A decrease in expression of PKCε by siRNA approach decreased the tumor cell resistance caused by ECM components. Furthermore, an inhibition of cell survival enzymes phosphoinositide-3-kinase (PI3K) with its specific inhibitor LY294002 (50 μM) as well as Akt with its specific inhibitor MK-2206 (1 μM) decreased the effect of ECM components on tumor cell survival against MSA and EGCG. WPE1-NB14, a low tumorigenic cell line having low PKCε and Akt activities, responded less to ECM components and showed higher sensitivity to MSA and EGCG. Conceivably, ECM components, laminin, collagen, and fibronectin induced β1 integrin signaling to activate cell survival pathways involving PKCε and PI3K-Akt, thereby decreasing prostate tumor cell sensitivity to MSA and EGCG. Thus, ECM may be one of the limiting factors for the action of cancer-preventive agents. Citation Format: Rayudu Gopalakrishna, Tiffany Fan, Ronald Deng, David Rayudu, Zachary W. Chen, William S. Tzeng, Usha Gundimeda. Extracellular matrix components influence prostate tumor cell sensitivity to cancer-preventive agents selenium and green tea polyphenols. [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 232. doi:10.1158/1538-7445.AM2014-232

Abstract 4077: Impact of RET/PTC3 oncogene's inflammatory and transformative signaling on tumorigenicity of thyroid and non-thyroid tumorigenic cell lines

Abstract Rearranged in transformation/ papillary thyroid carcinoma 3 (RET/PTC3 or RP3), is a unique fusion oncogene, responsible for a benign version of PTC. RP3 has an active inflammatory component leading to the secretion of various pro-inflammatory cytokines, however the role of inflammation in development and progression of PTC has not been clearly defined. Facilitating investigation of this issue is our finding that the transforming and inflammatory signals of RP3 can be functionally separated allowing us to study each in isolation. We generated several RP3 mutants ablated in; 1) transformative signaling due to mutation of a tyrosine residue (RP3Y588Fmutant) which prevents the binding of downstream adaptor proteins 2) inflammatory signaling through mutation of binding sites for TRAF2 and TRAF6. Transformation only RP3 mutants grew tumors in immunodeficient and immunocompetent mice, strengthening our hypothesis that the inflammatory component of RP3 is responsible for the benign nature of PTC. To assess the impact of inflammatory RP3 on the progression of different tumors, various thyroid and non-thyroid tumorigenic cell lines were transduced with MSCV_IRES_GFP_RP3 mutants and sorted for GFP expression. With highly immunogenic MC57G cells (C57BL/6 mice), the transforming only mutants developed tumors whereas wild type RP3 along with inflammatory only RP3 were rejected. Inflammation only RP3 mutant also led to slower tumor growth in Renca cell line (Balb/c) demonstrating the effect to be independent of haplotype. We are currently extending the analysis to PTC lines with BRAF and KRAS mutations, which are usually associated with more aggressive tumor profiles without an inflammatory tumor microenvironment. Our data so far suggest that contrary to the contemporary view, inflammation can retard tumor progression. Thus, the RP3 oncogene might be exploited in this regard. Future experiments aim to further characterize the importance of RP3-driven inflammation in tumor growth through continued in vivo and in vitro studies. Citation Format: Suresh C. Kari, Laurence C. Eisenlohr. Impact of RET/PTC3 oncogene's inflammatory and transformative signaling on tumorigenicity of thyroid and non-thyroid tumorigenic cell lines. [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 4077. doi:10.1158/1538-7445.AM2014-4077

In vitro evaluation of novel N-acetylalaninate prodrugs that selectively induce apoptosis in prostate cancer cells.

BackgroundCancer cell esterases are often overexpressed and can have chiral specificities different from that of the corresponding normal cells and can, therefore, be useful targets for activating chemotherapeutic prodrug esters. Prodrug esters are inactive compounds that can be preferentially activated by esterase enzymes. Moreover, cancer cells often exhibit a high level of intrinsic oxidative stress due to an increased formation of reactive oxygen species (ROS) and a decreased expression of some enzymatic antioxidants. Prodrugs designed to induce additional oxidative stress can selectively induce apoptosis in cancer cells already exhibiting a high level of intrinsic oxidative stress. This study focused on the in vitro evaluation of four novel prodrug esters: the R- and S- chiral esters of 4-[(nitrooxy)methyl]phenyl N-acetylalaninate (R- and S-NPAA) and the R- and S- chiral esters of 4-[(nitrooxy)methyl]naphth-1-yl N-acetylalaninate (R- and S-NQM), which are activated, to varying extents, by oxidized protein hydrolase (OPH, EC 3.4.19.1) yielding a quinone methide (QM) intermediate capable of depleting glutathione (GSH), a key intracellular antioxidant. OPH is a serine esterase/protease that is overexpressed in some human tumors and cancer cell lines.MethodsTo evaluate the chiral ester prodrugs, we monitored cellular GSH depletion, cellular protein carbonyl levels (an oxidative stress biomarker) and cell viability in tumorigenic and nontumorigenic prostate cancer cell lines.ResultsWe found that the prodrugs were activated by OPH and subsequently depleted GSH. The S-chiral ester of NPAA (S-NPAA) was two-fold more effective than the R-chiral ester (R-NPAA) in depleting GSH, increasing oxidative stress, inducing apoptosis, and decreasing cell viability in tumorigenic prostate LNCaP cells but had little effect on non-tumorigenic RWPE-1 cells. In addition, we found that that S-NPAA induced apoptosis and decreased cell viability in tumorigenic DU145 and PC3 prostate cell lines. Similar results were found in a COS-7 model that overexpressed active human OPH (COS-7-OPH).ConclusionsOur results suggest that prostate tumors overexpressing OPH and/or exhibiting a high level of intrinsic oxidative stress may be susceptible to QM generating prodrug esters that are targeted to OPH with little effect on non-tumorigenic prostate cells.

Increased delivery of doxorubicin into tumor cells using extracellularly activated TAT functionalized liposomes: in vitro and in vivo study.

The development of highly efficient tumor-targeted delivery systems is crucial for successful tumor treatment. Previously, a novel cell-penetrating peptide TAT and cleavable polyethylene glycol (PEG) co-modified liposome delivery system (C-TAT-Lipo) showed enhanced accumulation in tumor regions. Under the control of cysteine (Cys), the liposomes were activated extracellularly and achieved increased delivery of their cargo into tumor cells efficiently. In this study, we developed an optimal formulation for the encapsulation of Doxorubicin (DOX) by this delivery system for tumor treatment. The in vitro study showed that the C-TAT-Lipo with Cys delivery system not only enhanced the amount of DOX delivered by at least 100% compared to other DOX-containing formulations, but also displayed high cytotoxicity against tumorigenic cell lines. Compared to other groups, the DOX-loaded C-TAT-Lipo formulation in the presence of cysteine enhanced treatment efficacy by lowering the IC50 (1.67 +/- 0.14 microM) and increasing the cancer cell apoptosis percentage (37.10%). Moreover, the in vivo antitumor activity also showed that DOX-loaded C-TAT-Lipo with injection of cysteine achieved the best tumor growth inhibition with a tumor growth rate of only 58.40 +/- 16.33% (% of initial volume/day), which was significant less than that achieved by other DOX formulations.

C-Jun promotes cell migration and drives expression of the motility factor ENPP2 in soft tissue sarcomas.

Genomic amplification of the c-Jun proto-oncogene has been identified in ∼30% of dedifferentiated liposarcomas (DDLPS), but the functional contribution of c-Jun to the progression of DDLPS remains poorly understood. In previous work we showed that knock-down of c-Jun by RNA interference impaired the in vitro proliferation and in vivo growth of a DDLPS cell line (LP6) with genomic amplification of the c-Jun locus. Here, we used gene expression analysis and functional studies in a broad panel of cell lines to further define the role of c-Jun in DDLPS and other soft tissue sarcomas. We show that c-Jun knock-down impairs transition through the G1 phase of the cell cycle in multiple DDLPS cell lines. We also found that high levels of c-Jun expression are both necessary and sufficient to promote DDLPS cell migration and invasion in vitro. Our data suggest that high levels of c-Jun enhance motility in part by driving the expression of ENPP2/Autotaxin. c-Jun over-expression has minimal effects on in vitro proliferation but substantially enhances the in vivo growth of weakly tumourigenic DDLPS cell lines. Finally, we provide evidence that c-Jun genomic amplification and over-expression may have similar functional consequences in other types of soft tissue sarcoma. Our data suggest a model in which relatively low levels of c-Jun are sufficient for in vitro proliferation, but high levels of c-Jun enhance invasiveness and capacity for in vivo tumour growth. These observations provide an explanation for the selective advantage provided by c-Jun genomic amplification in vivo and suggest that sarcomas with elevated c-Jun levels are likely to have a particularly high malignant potential. Data from exon array and RNA-Seq experiments have been deposited in the GEO database (Accession No. GSE57531).

Fibroblast growth factor receptor 4: a putative key driver for the aggressive phenotype of hepatocellular carcinoma.

Recently, we found upregulation of fibroblast growth factor receptor 4 (FGFR4) in a subset of hepatocellular carcinoma (HCC). Here, we provide mechanistic insight into the role of FGFR4-mediated signalling for the aggressive behaviour of HCC cells. To overexpress FGFR4, hepatoma/hepatocarcinoma cells were transfected with a construct coding for FGFR4. For downmodulation of endogenous FGFR4, we used small interfering RNA or adenoviral infection with dominant-negative FGFR4 constructs being either kinase dead (kdFGFR4) or coding for the autoinhibitory soluble domain (solFGFR4). FGFR4 overexpression in non-tumourigenic hepatocarcinoma cells significantly reduced cell-matrix adhesion, enabled cells to grow anchorage-independently in soft agar, to disintegrate the lymph-/blood-endothelial barrier for intra-/extravasation of tumour cells and to form tumours in SCID mice. Transcriptome analysis revealed altered expression of genes involved in cell-matrix interactions. Conversely, in highly tumourigenic cell lines, kdFGFR4 or solFGFR4 lowered the proportion of cells in S phase of the cell cycle, enhanced the G0/G1 and G2/M-phase proportions, reduced anchorage-independent growth in vitro and attenuated disintegration of the lymph-/blood-endothelium and tumour formation in vivo. These findings were confirmed by altered expression profiles of genes being important for late stages of cell division. Deregulated FGFR4 expression appears to be one of the key drivers of the malignant phenotype of HCC cells. Accordingly, blockade of FGFR4-mediated signalling by soluble dominant-negative constructs, like solFGFR4, may be a feasible and promising therapeutic approach to antagonize aggressive behaviour of hepatoma/hepatocarcinoma cells.

Establishment of highly tumorigenic human colorectal cancer cell line (CR4) with properties of putative cancer stem cells.

BackgroundColorectal cancer (CRC) has the third highest mortality rates among the US population. According to the most recent concept of carcinogenesis, human tumors are organized hierarchically, and the top of it is occupied by malignant stem cells (cancer stem cells, CSCs, or cancer-initiating cells, CICs), which possess unlimited self-renewal and tumor-initiating capacities and high resistance to conventional therapies. To reflect the complexity and diversity of human tumors and to provide clinically and physiologically relevant cancer models, large banks of characterized patient-derived low-passage cell lines, and especially CIC-enriched cell lines, are urgently needed.Principal FindingsHere we report the establishment of a novel CIC-enriched, highly tumorigenic and clonogenic colon cancer cell line, CR4, derived from liver metastasis. This stable cell line was established by combining 3D culturing and 2D culturing in stem cell media, subcloning of cells with particular morphology, co-culture with carcinoma associated fibroblasts (CAFs) and serial transplantation to NOD/SCID mice. Using RNA-Seq complete transcriptome profiling of the tumorigenic fraction of the CR4 cells in comparison to the bulk tumor cells, we have identified about 360 differentially expressed transcripts, many of which represent stemness, pluripotency and resistance to treatment. Majority of the established CR4 cells express common markers of stemness, including CD133, CD44, CD166, EpCAM, CD24 and Lgr5. Using immunocytochemical, FACS and western blot analyses, we have shown that a significant ratio of the CR4 cells express key markers of pluripotency markers, including Sox-2, Oct3/4 and c-Myc. Constitutive overactivation of ABC transporters and NF-kB and absence of tumor suppressors p53 and p21 may partially explain exceptional drug resistance of the CR4 cells.ConclusionsThe highly tumorigenic and clonogenic CIC-enriched CR4 cell line may provide an important new tool to support the discovery of novel diagnostic and/or prognostic biomarkers as well as the development of more effective therapeutic strategies.

The growth-inhibitory and apoptosis-inducing effect of deferoxamine combined with arsenic trioxide on HL-60 xenografts in nude mice

ObjectiveThe aim of this study is to investigate the growth-inhibitory and apoptosis-inducing effect of deferoxamine (DFO) combined with arsenic trioxide (ATO) on the human HL-60 xenografts in nude mice and its mechanism. MethodThe highly tumorigenic leukemia cell line HL-60 cells were inoculated subcutaneously into nude mice to establish a human leukemia xenograft model. The HL-60 xenograft nude mice models were randomly divided into four groups: control (Normal saline, NS), 50mg/kg DFO, 3mg/kg ATO, the combined treatment (50mg/kg DFO+1.5mg/kg ATO) once HL-60 cells were inoculated. Tumor sizes, growth curves, inhibitory rates, cell apoptosis, and the expression of apoptosis related markers were measured to evaluate the tumor growth. ResultsXenografted tumors were observed in all nude mice since the 5th day of inoculation. The inhibitory rates of tumor weight were 2.67%, 10.69%, and 25.57% in DFO, ATO and combination therapy groups, respectively. The combination of DFO with ATO induces significantly more tumor cell apoptosis than either agent alone (p<0.05). The expression of NF-κBp65 and survivin proteins decreased significantly while the expression of Caspase-3 and Bax increased in the combination therapy group (p<0.05). Double immunofluorescence for Caspase-3 and NFκBp65 demonstrated an inverse relationship between Caspase-3-positive areas and NFκBp65-positive areas, as well as the co-localization of Bax and survivin in xenografted tumor cells. ConclusionsCombination of DFO and ATO has synergistic effects on tumor growth inhibition and apoptosis-inducing in vivo with no significant side effects. The DFO and ATO can up-regulate the expression of Caspase-3 and Bax, and down-regulate the expression of NF-κBp65 and survivin, especially for their combination.

Thymidine phosphorylase is both a therapeutic and a suicide gene in a murine model of mitochondrial neurogastrointestinal encephalomyopathy.

Suicide gene therapy (SGT) is a promising strategy for treating cancer. In this work, we show that thymidine phosphorylase (TP) deficiency, the underlying genetic defect in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), presents an opportunity to apply SGT using capecitabine, a commonly used prodrug that is converted into 5-fluorouracil by TP. Using an immortalised B-lymphoblastoid cell line from a patient with MNGIE, the tumourigenic EL-4 cell line, lentiviral vectors encoding TP and a double knockout (Tymp(-/-)Upp1(-/-)) murine model, we found that EL-4 cell-derived TP(+) tumours were exquisitely sensitive to capecitabine and generated a significant local bystander effect. In addition, we detected a spontaneous cytolytic immune response in a significant fraction of the animals surviving more than 20 days after termination of the therapy. These data indicate that, in individuals lacking TP expression, TP is a highly specific suicide gene, which can be used to treat tumours that could hypothetically arise in MNGIE patients undergoing gene therapy, as these tumours will likely originate from the gene-modified cells and will be selectively targeted by capecitabine. These observations have important implications for gene therapy for MNGIE.

Anti-LRP/LR specific antibody IgG1-iS18 impedes adhesion and invasion of liver cancer cells.

Two key events, namely adhesion and invasion, are pivotal to the occurrence of metastasis. Importantly, the 37 kDa/67 kDa laminin receptor (LRP/LR) has been implicated in enhancing these two events thus facilitating cancer progression. In the current study, the role of LRP/LR in the adhesion and invasion of liver cancer (HUH-7) and leukaemia (K562) cells was investigated. Flow cytometry revealed that the HUH-7 cells displayed significantly higher cell surface LRP/LR levels compared to the poorly-invasive breast cancer (MCF-7) control cells, whilst the K562 cells displayed significantly lower cell surface LRP/LR levels in comparison to the MCF-7 control cells. However, Western blotting and densitometric analysis revealed that all three tumorigenic cell lines did not differ significantly with regards to total LRP/LR levels. Furthermore, treatment of liver cancer cells with anti-LRP/LR specific antibody IgG1-iS18 (0.2 mg/ml) significantly reduced the adhesive potential of cells to laminin-1 and the invasive potential of cells through the ECM-like Matrigel, whilst leukaemia cells showed no significant differences in both instances. Additionally, Pearson's correlation coefficients suggested direct proportionality between cell surface LRP/LR levels and the adhesive and invasive potential of liver cancer and leukaemia cells. These findings suggest the potential use of anti-LRP/LR specific antibody IgG1-iS18 as an alternative therapeutic tool for metastatic liver cancer through impediment of the LRP/LR- laminin-1 interaction.

Nuclear factor κB-dependent regulation of angiogenesis, and metastasis in an in vivo model of thyroid cancer is associated with secreted interleukin-8.

Development of novel strategies in the treatment of advanced thyroid cancer are needed. Our laboratory has previously identified a role for nuclear factor κB (NF-κB) signaling in human thyroid cancer cell growth, survival, and invasion. Our goal was to establish the role of NF-κB signaling on thyroid cancer growth and metastases in vivo and to begin to dissect mechanisms regulating this effect. We examined tumor formation of five thyroid cancer cell lines in an in vivo model of thyroid cancer and observed tumor establishment in two of the cell lines (8505C and BCPAP). Inhibition of NF-κB signaling by overexpression of a dominant-negative IκBα (mIκBα) significantly inhibited thyroid tumor growth in tumors derived from both cell lines. Further studies in an experimental metastasis model demonstrated that NF-κB inhibition impaired growth of tumor metastasis and prolonged mouse survival. Proliferation (mitotic index) was decreased in 8505C tumors, but not in BCPAP tumors, while in vitro angiogenesis and in vivo tumor vascularity were significantly inhibited by mIkBα only in the BCPAP cells. Cytokine antibody array analysis demonstrated that IL-8 secretion was blocked by mIκBα expression. Interestingly, basal NF-κB activity and IL-8 levels were significantly higher in the two tumorigenic cell lines compared with the nontumorigenic lines. Furthermore, IL-8 transcript levels were elevated in high-risk human tumors, suggesting that NF-κB and IL-8 are associated with more aggressive tumor behavior. These studies suggest that NF-κB signaling is a key regulator of angiogenesis and growth of primary and metastatic thyroid cancer, and that IL-8 may be an important downstream mediator of NF-κB signaling in advanced thyroid cancer growth and progression.

Possible Role of Arginase-1 in Concomitant Tumor Immunity

The expression of Adenovirus serotype 2 or serotype 5 (Ad2/5) E1A in tumor cells reduces their tumorigenicity in vivo by enhancing the NK cell mediated and T cell mediated anti-tumor immune response, an activity that correlates with the ability of E1A to bind p300. We determined if E1A could be used as a molecular adjuvant to enhance antigen-specific T cell responses to a model tumor antigen, ovalbumin (OVA). To achieve this goal, we stably expressed a fusion protein of E1A and OVA (MCA-205-E1A-OVA), OVA (MCA-205-OVA) or a mutant version of E1A unable to bind p300 and OVA (E1A-Δp300-OVA) in the B6-derived, highly tumorigenic MCA-205 tumor cell line. MCA-205-E1A-OVA tumor cells were over 10,000 fold less tumorigenic than MCA-205-OVA, MCA-205-E1A-Δp300-OVA, or MCA-205 in B6 mice. However, immunization of B6 mice with live MCA-205-OVA, MCA-205-E1A-Δp300-OVA and MCA-E1A-OVA tumor cells induced nearly equivalent OVA-specific CD4 T cells and CD8 CTL responses. Further studies revealed that mice with primary, enlarging MCA-205-OVA or MCA-205-E1A-Δp300-OVA tumors on one flank exhibited OVA-specific anti-tumor T cell responses that rejected a tumorigenic dose of MCA-205-OVA cells on the contralateral flank (concomitant tumor immunity). Next we found that tumor associated macrophages (TAMs) in progressive MCA-205-OVA tumors, but not MCA-205-E1A-OVA tumors that expressed high levels of arginase-1, which is known to have local immunosuppressive activities. In summary, immunization of mice with MCA-205 cells expressing OVA, E1A-Δp300-OVA or E1A-OVA induced equivalent OVA-specific CD4 and CD8 anti-tumor responses. TAMs found in MCA-205-OVA, but not MCA-205-E1A-OVA, tumors expressed high levels of arginase-1. We hypothesize that the production of arginase-1 by TAMs in MCA-205-OVA or MCA-205-E1A-Δp300-OVA tumor cells leads to an ineffective anti-tumor immune response in the tumor microenvironment, but does not result in inhibition of a systemic anti-tumor immunity.