Tumor-specific Manner Research Articles

MicroRNA-16 suppresses metastasis in an orthotopic, but not autochthonous, mouse model of soft tissue sarcoma.

ABSTRACTMicroRNAs (miRNAs) can regulate tumor cell invasion and metastasis in a tumor-specific manner. We recently demonstrated that global downregulation of miRNAs after deleting dicer can promote development of distant metastases in a mouse model of primary soft tissue sarcoma (STS). In this study, we identified miRNAs that are differentially downregulated in metastatic STS in both human and mouse, and investigated the role of these miRNAs in metastasis. miRNA- TaqMan PCR arrays showed a global downregulation of miRNAs in metastatic human sarcomas. Similar analysis in mouse metastatic sarcomas revealed overlap for several downregulated miRNAs including miR-16, miR-103, miR-146a, miR-223, miR-342 and miR-511. Restoration of these downregulated miRNAs in mouse primary sarcoma cell lines showed that miR-16, but not other downregulated miRNAs, was able to significantly suppress both migration and invasion in vitro, without altering cell proliferation. In addition, orthotopic transplantation of a sarcoma cell line stably expressing miR-16 into the muscle of immunocompromised mice revealed that restoration of miR-16 can significantly decrease lung metastasis in vivo. However, no change in the rate of lung metastasis was observed when miR-16 was deleted in mouse primary sarcomas at sarcoma initiation. Taken together, these results indicate that miR-16 can have metastasis-suppressing properties both in vitro and in vivo. However, the loss-of-function experiments in autochthonous tumors indicate that loss of miR-16 is not sufficient to promote metastasis in vivo.

Palmitic Acid, As Verified Using Lipid Profiling By Secondary Ion Mass Spectrometry, Demonstrates Anti-Tumor Activity Against Multiple Myeloma

Introduction Many recent studies have examined lipid metabolic changes in multiple myeloma (MM). Changes in lipid metabolism affect the survival of MM cells. Developments in imaging mass spectrometry (IMS) have facilitated research on the lipid profiles of tumors. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an IMS technique that uses a focused ion beam as the primary source for ionization. TOF-SIMS imaging is used to analyze the surface of specimens at sub-micrometer resolution, enabling analyses of the subcellular distribution of molecules in individual cells. TOF-SIMS analysis has enabled the detection of multiple fatty acid groups from single cells. Therefore, we applied this method to human clinical specimens to analyze the membrane fatty acid composition and determine candidate molecules for MM therapies. Using the different lipid profiles for MM cells and normal plasma cells (PCs), we conducted a cytocidal assay with MM cell lines supplemented with the fatty acids screened out by the profiles to assess lipotoxicity against MM. The molecules demonstrating distinct differences among cell types (i.e., MM and PC) were considered candidates for which supplementation leads to imbalanced lipid metabolism and cell death in a tumor-specific manner. We further evaluated the induction of apoptosis.Methods Primary patient MM cells and normal PCs were isolated from the bone marrow aspirates of two patients and two healthy volunteers using fluorescence-activated cell sorting. These separated cells were analyzed with PHI TRIFT V (ULVAC-PHI, Inc.). Analyses were performed in negative ion mode, and signals in the mass range of m/z 0 to 1850 were monitored. We performed pairwise comparisons of mean signal intensities for five types of fatty acids between MM cells and PCs.MM cell lines (U266 and RPMI-8226) were treated with 0–1000 µM of palmitic acid, palmitoleic acid, linoleic acid, oleic acid, and stearic acid. The number of viable cells in suspension at 72 hours after treatment was determined by the trypan blue exclusion test. HS-5, a human bone marrow stromal cell line, was used in the co-culture experiment. Healthy volunteers’ normal peripheral blood mononuclear cells (PBMCs) were purified by Ficoll-Hypaque density-gradient centrifugation. The distribution of apoptotic and necrotic cells were analyzed by measuring AnnexinV binding and propidium iodide uptake.Results The amounts of MM cells and PCs relative to the total nucleated cells were 3.38%, 35.9% for MM cells, 0.0368% and 0.246% for PCs. Multiple ions, including phosphoric acid, and five species of fatty acids (palmitoleic acid, palmitic acid, linoleic acid, oleic acid, and stearic acid) were detected. The mean signal intensities of palmitoleic acid and palmitic acid of MM cells were significantly lower than those of normal PCs (P = .00081 and .0018, respectively). These results were replicated in a second pairwise comparison. We did not observe statistically significant differences in intensities for linoleic acid, oleic acid, or stearic acid.In the cytocidal assay, palmitic acid reduced U266 cell viability dose-dependently for doses of 50–1000 μM. High concentrations of the other fatty acids also reduced cell viability; however, the effect on cell death was not observed at the low dose of 50–100 µM, as it was for palmitic acid. Even in co-culture experiments, palmitic acid decreased the viability of MM cells. Moreover, the proportions of both apoptotic and necrotic cells increased and the proportion of viable cells decreased 24 hours after palmitic acid treatment in MM cells. Palmitic acid also reduced the viability of RPMI-8226 cell lines. Meanwhile, cell viabilities of normal PBMCs were not affected by palmitic acid, even at 100–500 µM.Conclusion We applied the single-cell TOF-SIMS lipid analysis effectively to a very small population of cells. Significantly smaller intensities of palmitoleic acid and palmitic acid were observed in MM cells compared to normal cells. We also demonstrated an inhibitory effect of palmitic acid on the survival of MM cells. Palmitic acid is a potential candidate for novel therapeutic agents that specifically attack MM and should be considered in future studies of MM in a lipid biology framework. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3003: Targeting nucleolin: A potential strategy to overcome stroma-mediated bevacizumab resistance in lung cancer

Abstract Background Noticing the limitations of chemotherapy and the possibility of interrupting the tumor vascular network, there has been an increasing interest in targeting the tumor vasculature and developing agents to disrupt angiogenesis. Anti-angiogenic drugs, such as bevacizumab, have become a standard treatment option in lung cancer. In spite some clinical success with these inhibitors, disappointing results have been reported with patients being intrinsically refractory to anti-VEGF therapies or, following treatment response, ultimately becoming refractory and showing relapse. Moreover, it has been described that not only the tumor cells, but several host factors and stromal components also play an important role in resistance to bevacizumab (Cascone et al., JCI 2011). Aims 1) Validate nucleolin as a therapeutic target for a previous developed F3 peptide-targeted pH sensitive liposome containing doxorubicin (Moura et al., BCRT 2012) in human lung cancer models resistant to bevacizumab and assess the potential to overcome anti-angiogenic resistance. 2) Assess nucleolin expression in patients-derived lung cancer. Experimental design/Results Aiming at confirming the potential application of a previously developed nanoparticle on lung cancer, cellular association studies were carried with flow cytometry. Cell lines with intrinsic (A549 cells) and acquired (H1975, H441 cells) resistance to bevacizumab were incubated with fluorescently-labeled liposomes, either non-targeted (SLpH) or targeted by a non-specific (SLpHNS) or F3 (SLpHF3) peptide, for 1 h at 37 or 4ºC (temperature not permissive to endocytosis). Results demonstrated a 30 to 170-fold higher extent of association of F3-targeted liposomes by lung cancer cells than the the controls, suggesting a ligand-specific interaction. Improved association and intracellular delivery of encapsulated DXR enabled by F3-targeted liposomes, justified a 9.6-fold increase of DXR cytotoxicity relative to the non-targeted counterparts. In order to assess the clinical potential of the developed nucleolin-targeted strategy, immunohistochemistry of human specimens derived from lung cancer patients with surgical staged disease was performed aiming at investigating the expression of nucleolin. Results generated so far revealed that nucleolin was highly expressed in different types of cells in the tumor microenvironment of patient-derived lung tumors, in a tumor-specific manner. Main Conclusion The generated results render an important indication of the therapeutic potential of the tested nucleolin-targeted strategy against Bevazcizumab-resistant lung cancer. Acknowledgements Grants: QREN/FEDER/COMPETE (Ref 23240); PEst-C/SAU/LA0001/2011 Â. Valério-Fernandes is a graduate student from PhD program on Biomedicine and Experimental Biology, Center for Neuroscience and Cell Biology, University of Coimbra (FCT fellowship SFRH/BD/51191/2010) Citation Format: Ângela Valério-Fernandes, Nuno Fonseca, Vera Moura, Ana Ladeirinha, Teresa Ferreira, Ana Alarcão, Lina Carvalho, Sérgio Simões, João Nuno Moreira. Targeting nucleolin: A potential strategy to overcome stroma-mediated bevacizumab resistance in lung 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 3003. doi:10.1158/1538-7445.AM2014-3003

Abstract 1760: Tumor-specific targeting of the NAD metabolome with β-lapachone and NamPT inhibition

Abstract Cancer cells require increased NAD+ synthesis to support anabolic metabolism, to sustain signaling processes such as sirtuin activity and ADP-ribosylation, and to maintain redox balance. Inhibitors of Nicotinamide phosphoribosyltransferase (NamPT), the rate-limiting step in NAD+ biosynthesis, have modest anti-tumor activity in monotherapy in vitro and in vivo, but failed clinical trials due to a lack of tumor specificity attributed to the necessity of NAD+ synthesis in normal proliferating cells. We found that the tumor specificity and efficacy of NamPT inhibition can be greatly increased when small molecule NamPT inhibitors, such as FK866, are used in combination with the NQO1 bioactivated therapeutic ß-lapachone (ß-lap). Most solid tumor cells overexpress NQO1, which catalyzes a futile redox cycle with ß-lap, resulting in the formation of a burst of reactive oxygen species (ROS). This results in substantial DNA single strand breaks and base damage in a tumor-specific manner, which hyperactivates poly(ADP-ribose) polymerase-1 (PARP-1), a DNA repair enzyme that utilizes NAD+ to generate poly(ADP-ribose) (PAR)-protein moieties. Rapid NAD+ and ATP loss occurs and tumor cells die through programmed necrosis. PARP-1-mediated NAD+ depletion caused by ß-lap treatment synergizes with reduced NAD+ synthesis as a result of FK866-mediated NamPT inhibition. Enhanced cancer cell death in terms of loss of viability and clonogenicity at lower ß-lap doses was noted, while tumor specificity for NQO1 overexpressing cells was maintained. With combination treatment, cells are primed for ROS-mediated damage due to reduced glutathione and NAD(P)+ levels, they are unable to recover ATP, NAD+, and NADP+ synthesis even after only a 2 hour treatment, and they rapidly die through a caspase-independent mechanism. This therapeutic approach is highly effective against a variety of tumor cell lines from pancreatic, breast, and non-small cell lung, and is expected to improve the efficacy and tumor specificity of both of these drugs by allowing their use at lower doses in vivo. These studies were supported by an AACR/PanCan Innovator Award and NIH R01 CA102972 to DAB. Citation Format: Zachary Moore, David A. Boothman. Tumor-specific targeting of the NAD metabolome with β-lapachone and NamPT inhibition. [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 1760. doi:10.1158/1538-7445.AM2014-1760

Drug-induced amplification of nanoparticle targeting to tumors

Nanomedicines have the potential to significantly impact cancer therapy by improving drug efficacy and decreasing off-target effects, yet our ability to efficiently home nanoparticles to disease sites remains limited. One frequently overlooked constraint of current active targeting schemes is the relative dearth of targetable antigens within tumors, which restricts the amount of cargo that can be delivered in a tumor-specific manner. To address this limitation, we exploit tumor-specific responses to drugs to construct a cooperative targeting system where a small molecule therapeutic modulates the disease microenvironment to amplify nanoparticle recruitment in vivo. We first administer a vascular disrupting agent, ombrabulin, which selectively affects tumors and leads to locally elevated presentation of the stress-related protein, p32. This increase in p32 levels provides more binding sites for circulating p32-targeted nanoparticles, enhancing their delivery of diagnostic or therapeutic cargos to tumors. We show that this cooperative targeting system recruits over five times higher doses of nanoparticles to tumors and decreases tumor burden when compared with non-cooperative controls. These results suggest that using nanomedicine in conjunction with drugs that enhance the presentation of target antigens in the tumor environment may be an effective strategy for improving the diagnosis and treatment of cancer.

Esophageal cancer-selective expression of TRAIL mediated by MREs of miR-143 and miR-122

Esophageal cancer is one of the most common digestive system neoplasms and has a quite poor prognosis. TNF-related apoptosis-inducing ligand (TRAIL) induces the apoptosis in a wide range of cancer cells including esophageal cancers. However, TRAIL also activates apoptotic pathway in normal cells. To improve the specificity of TRAIL action, we employed the microRNA (miRNA) response elements (MREs) of miR-143 and miR-122 to restrict TRAIL expression mediated by an adenoviral vector (Ad-TRAIL-143-122) in esophageal cancer cells. The experiments showed that Ad-TRAIL-143-122 was able to highly express TRAIL in esophageal cancer cells, but not normal cells. The selective TRAIL expression also led to selective apoptosis in esophageal cancer cells. Ad-TRAIL-143-122 greatly reduced the viability of esophageal cancer cells without cytotoxicity to normal cells. In mice, Ad-TRAIL-143-122 suppressed the growth of esophageal cancer xenografts and protected liver from TRAIL-induced toxicity. In this study, we constructed a biologic vector that can express exogenous genes in a tumor-specific manner. This strategy can simultaneously treat cancer and prevent hepatoxicity and thus may be a promising way for esophageal cancer treatment.

Association between leukocyte telomere length and glioma risk: a case-control study

Compelling epidemiological evidence indicates that alterations of telomere length are associated with risks of many malignancies in a tumor-specific manner, such as lung cancer, breast cancer, and non-Hodgkin's lymphoma. However, the association between leukocyte telomere length and glioma risk has not been investigated. Relative telomere length (RTL) of peripheral blood leukocytes from 467 glioma patients and 467 healthy controls, matched by age and sex, was measured using the real-time PCR-based method in a case-control study. An unconditional multivariate logistic regression model was applied to estimate the association between RTL and glioma risk. Glioma patients showed notably longer RTL than controls (median, 0.555 vs 0.444; P > .04). RTL was negatively correlated with age in both cases (ρ = -0.430; P < .001) and controls (ρ = -0.388; P < .001). After adjusting for age, sex, smoking status and family history of cancer, multivariate logistic regression analysis showed that there was a U-shaped association between RTL and glioma risk (P for nonlinearity <.001). Compared with individuals in the second tertile of RTL, the odds ratios (95% CI) for participants in the first and third tertiles were 2.16 (range, 1.52-3.09) and 3.51 (range, 2.45-5.00), respectively. Stratified analysis showed that the association between RTL and glioma risk was not modulated by major host characteristics. Our study demonstrates for the first time that either shorter or longer RTL in peripheral blood leukocytes is associated with increased glioma risk, which warrants further investigation in the future.

Enhancing The Efficacy and Specificity Of Antibody-Based T Cell Retargeting Strategies Against Hematological Malignancies

New promising candidates for cancer immunotherapy are bispecific antibodies (bsAbs) which have already shown a convincing anti-tumor effect in first clinical trials. BsAbs are composed of two single-chain fragments variable (scFvs), derived from the variable heavy and light chain of a monoclonal Ab (mAb), which bind to the activating CD3-complex on T cells and a tumor-associated antigen (TAA) on cancer cells. Consequently, the cross-linkage of effector and target cells by bsAbs results in an efficient T cell-mediated cancer cell killing. However, several serious adverse effects were observed in patients after application of Ab constructs. Most critical is an overall activation of T cells which bears the risk of a systemic release of pro-inflammatory cytokines.With respect to the idea that the CD3 binding of an Ab construct is critical for T cell activation, the CD3 domain has to be optimized in order to prevent or even reduce unspecific T cell responses. For this reason, we cloned several anti-CD3 scFvs derived from different anti-CD3 mAb clones and compared their functionality and safety. The anti-CD3 scFv with lowest risk of side effects was introduced in different anti-TAA bsAbs. Functional and safety studies in vitro revealed that development of each bsAb requires extensive and time-consuming optimization steps to gain high efficacy but low risk of side effects. To improve the process of Ab engineering, we recently introduced a novel modular Ab platform that can be rapidly and cost-effectively adapted for redirection of T cells to any TAA (Arndt and Feldmann et al. accepted for publication in Leukemia 2013). In this novel modular system the dual specificity of a conventional bsAb is distributed to two separate Ab modules, (i) the effector module and (ii) the target module. Only the complex of both Ab modules mediates the cross-linkage of effector and target cells resulting in T cell activation and redirected cancer cell lysis similar to conventional T cell engaging bsAbs. The universal effector module is a well optimized bsAb balancing efficacy and safety. It binds CD3 on T cells and the E5B9 tag of the target module. The individual target module comprises an anti-TAA scFv and the peptide epitope E5B9. For treatment of lymphoid or myeloid malignancies a series of different target modules were designed. In vitro and in vivo data clearly underline that the combination of the established effector module with different target modules efficiently activated T cells against hematological malignancies. Both CD4+ and CD8+ T cells from healthy donors were efficiently activated to kill TAA-positive tumor cell lines at low effector to target cell ratios and Ab concentrations in a tumor-specific manner. Most importantly, we could demonstrate that patient-derived T cells were able to kill autologous malignant cells upon Ab-mediated cross-linkage. Another unique feature of the modular system is that multispecific or multifunctional target modules could be easily included to further improve the therapeutic effect. In order to increase anti-tumor specificity and reduce the risk of tumor escape variants, bispecific target modules that recognize different TAAs at the same time, were constructed. Cytotoxicity assays investigating dual targeting of double-positive tumor cells via the modular system demonstrated that target cell lysis can be considerably improved in comparison to single targeting modules. Moreover, proliferation and cytokine release of redirected T cells could be enhanced by supplying costimulatory immunoligands (e.g. 4-1BBL and Ox40L) via target modules.In summary, we developed a multifunctional and highly flexible modular system based on an anti-CD3 domain with lowest risk of side effects. In contrast to conventional anti-TAA-anti-CD3 bsAbs, the development of a novel modular system for different clinical indications is much easier and less time-consuming. Once the effector module containing the critical CD3 domain is optimized, the modular system can be flexibly applied to target any TAA simply by replacing the target module. Application of bispecific target modules or providing costimulatory signals via the modular system might prolong immune responses and further increase anti-tumor specificity and activity. Finally, the novel modular platform represents a valuable and promising tool for cancer immunotherapy. Disclosures:No relevant conflicts of interest to declare.

Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2

Although the Hsp90 chaperone family, comprised in humans of four paralogs, Hsp90α, Hsp90β, Grp94 and Trap-1, has important roles in malignancy, the contribution of each paralog to the cancer phenotype is poorly understood. This is in large part because reagents to study paralog-specific functions in cancer cells have been unavailable. Here we combine compound library screening with structural and computational analyses to identify purine-based chemical tools that are specific for Hsp90 paralogs. We show that Grp94 selectivity is due to the insertion of these compounds into a new allosteric pocket. We use these tools to demonstrate that cancer cells use individual Hsp90 paralogs to regulate a client protein in a tumor-specific manner and in response to proteome alterations. Finally, we provide new mechanistic evidence explaining why selective Grp94 inhibition is particularly efficacious in certain breast cancers.

Survivin-3B Potentiates Immune Escape in Cancer but Also Inhibits the Toxicity of Cancer Chemotherapy

Dysregulation in patterns of alternative RNA splicing in cancer cells is emerging as a significant factor in cancer pathophysiology. In this study, we investigated the little known alternative splice isoform survivin-3B (S-3B) that is overexpressed in a tumor-specific manner. Ectopic overexpression of S-3B drove tumorigenesis by facilitating immune escape in a manner associated with resistance to immune cell toxicity. This resistance was mediated by interaction of S-3B with procaspase-8, inhibiting death-inducing signaling complex formation in response to Fas/Fas ligand interaction. We found that S-3B overexpression also mediated resistance to cancer chemotherapy, in this case through interactions with procaspase-6. S-3B binding to procaspase-6 inhibited its activation despite mitochondrial depolarization and caspase-3 activation. When combined with chemotherapy, S-3B targeting in vivo elicited a nearly eradication of tumors. Mechanistic investigations identified a previously unrecognized 7-amino acid region as responsible for the procancerous properties of survivin proteins. Taken together, our results defined S-3B as an important functional actor in tumor formation and treatment resistance.

Abstract 1846: Potential of tumor-suppressive miR-596 targeting LGALS3BP as a therapeutic agent in oral cancer.

Abstract The incidence and mortality statistics for oral squamous cell carcinoma (OSCC) were 10th and 12th, respectively, in human cancers diagnosed worldwide in 2008. In this study, to identify novel tumor-suppressive microRNAs (TS-miRNAs) and their direct targets in OSCC, we performed methylation-based screening for 43 miRNAs encoded by 46 miRNA genes located within 500bp downstream of 40 CpG islands and genome-wide gene expression profiling in combination with a prediction database analysis, respectively, in 18 cell lines, resulting in the identification of a novel TS-miRNA miR-596 directly targeting LGALS3BP/Mac-2 BP/90K. DNA hypermethylation of CpG island located 5’-upstream of miR-596 gene was frequently observed in OSCC cell lines (100% of 18 cell lines) and primary OSCC cases (46.2% and 76.3% of 26 Japanese and 38 Thais primary cases) in a tumor-specific manner. The ectopic transfection of double-stranded RNA (dsRNA) mimicking miR-596 or specific siRNA for LGALS3BP significantly induced growth inhibition and apoptosis in cell lines lacking miR-596 expression or overexpressing LGALS3BP, respectively, in a manner associated with a suppression of ERK1/2 phosphorylation. Moreover, we also mention the effect of dsRNA mimicking miR-596 on the growth of an OSCC cell line in vivo. Our findings define a central role for miR-596 in OSCC and suggest the potential of miR-596 as an anti-cancer agent for miRNA replacement therapy in OSCC. Citation Format: Ken-ichi Kozaki, Hironori Endo, Tomoki Muramatsu, Mayuko Furuta, Narikazu Uzawa, Atiphan Pimkhaokham, Teruo Amagasa, Johji Inazawa. Potential of tumor-suppressive miR-596 targeting LGALS3BP as a therapeutic agent in oral cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1846. doi:10.1158/1538-7445.AM2013-1846

Abstract 4233: MeTA-array, a useful method to identify genes transcriptionally silenced by tumor-specific hypermethylation in cancer.

Abstract Aberrant DNA hypermethylation, the most well defined epigenetic changes in cancer, is associated with inappropriate gene silencing and this feature is utilized to search for tumor-specific DNA methylation biomarkers. Methyl-CpG binding domain (MBD) proteins (MBPs) can elicit the repressive potential of methylated DNA and play a major role in gene silencing mechanisms. Therefore, if the genes governed by MBPs are specifically reactivated, it should be possible to uncover them. We developed a method termed “methyl-CpG targeted transcriptional activation (MeTA)” that employs a fusion gene comprised of the MBD from MBD2 and the NFκB transcriptional activation domain. Microarray coupled with MeTA (MeTA-array) provides not only the information about methylated genes but also the one about transcriptional repression in a single experiment. We first applied either MeTA or a conventional DNA demethylating agent, 5-aza-cytidine (Aza-CR), to a human embryonic kidney cell line 293T and analyzed gene expression profiles by microarray; 138 and 202 genes that are upregulated 5-fold or more were identified by MeTA and Aza-CR, respectively. ChIP-Seq analysis indicated that MeTA-inducing protein containing MBD binds to more than 80% of genes selected by MeTA-array. We found associations between expressional restorations by MeTA, methylation status, and NFκB (AD)-MBD fusion protein bindings in CpG islands (CGIs) around the transcription start site of the genes. Importantly, MeTA can upregulate genes meeting the stringent criteria of CGIs defined by Takai and Jones (Proc Natl Acad Sci USA 99: 3740-3745, 2002) at the promoter region at higher frequency; 109 of 138 (79.0%) genes in MeTA vs. 121 of 202 (59.9%) genes in Aza-CR. We further applied MeTA-array to 12 pancreatic cancer cell lines along with HPDE (normal pancreatic ductal epithelial cell line) and identified 31 candidate tumor-specific hypermethylated genes; 26 of them have never been reported previously using the conventional DNA demethylating agents. Seven genes, IRX4, LHX6, NEFH, NEFL, NEFM, NPTX2 and TMEM204 were further examined their methylation statuses by MSP, and we found that 100% (21/21) of IRX4, 62% (13/21) of LHX6, 100% (21/21) of NEFH, 100% (21/21) of NEFL, 100% (21/21) of NEFM, 100% (21/21) of NPTX2 and 95% (20/21) of TMEM204 were methylated in our series of pancreatic cancer cell lines. Furthermore, 68% (15/22) of IRX4, 55% (12/22) of LHX6, 55% (12/22) of NEFH, 59% (23/22) of NEFL, 82% (18/22) of NEFM and 82% (18/22) of NPTX2 were also hypermethylated in primary pancreatic cancer specimens in a tumor-specific manner. Our results suggest that MeTA-array is a highly efficient method to identify methylation-mediated transcriptionally silenced genes in human cancer. Citation Format: Shinichi Fukushige, Akira Horii. MeTA-array, a useful method to identify genes transcriptionally silenced by tumor-specific hypermethylation in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4233. doi:10.1158/1538-7445.AM2013-4233

The Tumor-Suppressive miR-497-195 Cluster Targets Multiple Cell-Cycle Regulators in Hepatocellular Carcinoma

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression and commonly deregulated in carcinogenesis. To explore functionally crucial tumor-suppressive (TS)-miRNAs in hepatocellular carcinoma (HCC), we performed integrative function- and expression-based screenings of TS-miRNAs in six HCC cell lines. The screenings identified seven miRNAs, which showed growth-suppressive activities through the overexpression of each miRNA and were endogenously downregulated in HCC cell lines. Further expression analyses using a large panel of HCC cell lines and primary tumors demonstrated four miRNAs, miR-101, -195, -378 and -497, as candidate TS-miRNAs frequently silenced in HCCs. Among them, two clustered miRNAs miR-195 and miR-497 showed significant growth-suppressive activity with induction of G1 arrest. Comprehensive exploration of their targets using Argonute2-immunoprecipitation-deep-sequencing (Ago2-IP-seq) and genome-wide expression profiling after their overexpression followed by pathway analysis, revealed a significant enrichment of cell cycle regulators. Among the candidates, we successfully identified CCNE1, CDC25A, CCND3, CDK4, and BTRC as direct targets for miR-497 and miR-195. Moreover, target genes frequently upregulated in HCC in a tumor-specific manner, such as CDK6, CCNE1, CDC25A and CDK4, showed an inverse correlation in the expression of miR-195 and miR-497, and their targets. These results suggest the molecular pathway regulating cell cycle progression to be integrally altered by downregulation of miR-195 and miR-497 expression, leading to the aberrant cell proliferation in hepatocarcinogenesis.

Potential of tumor-suppressive miR-596 targeting LGALS3BP as a therapeutic agent in oral cancer

The incidence and mortality statistics for oral squamous cell carcinoma (OSCC) were 10th and 12th, respectively, in human cancers diagnosed worldwide in 2008. In this study, to identify novel tumor-suppressive microRNAs (TS-miRNAs) and their direct targets in OSCC, we performed methylation-based screening for 43 miRNAs encoded by 46 miRNA genes located within 500 bp downstream of 40 CpG islands and genome-wide gene expression profiling in combination with a prediction database analysis, respectively, in 18 cell lines, resulting in the identification of a novel TS-miRNA miR-596 directly targeting LGALS3BP/Mac-2 BP/90K. DNA hypermethylation of CpG island located 5'-upstream of miR-596 gene was frequently observed in OSCC cell lines (100% of 18 cell lines) and primary OSCC cases (46.2 and 76.3% of 26 Japanese and 38 Thais primary cases, respectively) in a tumor-specific manner. The ectopic transfection of double-stranded RNA (dsRNA) mimicking miR-596 or specific small interfering RNA for LGALS3BP significantly induced growth inhibition and apoptosis in cell lines lacking miR-596 expression or overexpressing LGALS3BP, respectively, in a manner associated with a suppression of ERK1/2 phosphorylation. Moreover, we also mention the effect of dsRNA mimicking miR-596 on the growth of an OSCC cell line in vivo. Our findings define a central role for miR-596 in OSCC and suggest the potential of miR-596 as an anticancer agent for miRNA replacement therapy in OSCC.

Progressive increase of glucose transporter-3 (GLUT-3) expression in estrogen-induced breast carcinogenesis

Increased glucose uptake and glycolysis are main metabolic characteristics of malignant cells. A family of glucose transporters (GLUTs) facilitates glucose movement across the plasma membranes in a tumor-specific manner. Glucose transporter-1 (GLUT-1), GLUT-3 and recently GLUT-12, have been previously shown in breast cancer cells and are found to be associated with poor prognosis. In addition, it has been shown that estrogen plays critical roles in GLUT regulation, however, the stage-specific GLUT regulation of mammary carcinogenesis is unclear. GLUT expression patterns were investigated in an in vitro-in vivo progressive, estrogen-induced, mammary carcinogenesis model which consisted of four cell lines, with same genetic background. In this model, different stages of tumor initiation and progression are represented, MCF-10F being the normal stage, E2 cells the transformed stage by estrogen, C5 cells, the invasive stage, and T4 cells the tumorigenic stage. In addition, loss of ductulogenesis and solid mass formation in collagen matrix and invasiveness of the cells were counted. Real time PCR showed that GLUT1 expression was downregulated in MCF10F after treatment with 17β-estradiol (E2), and in the invasive cell type (C5), but not in the tumor cells (T4), which had no changes compared to MCF10F. C5 and T4 cells showed the highest rate of GLUT-3 expression. These cells were also found to be associated with loss of ductulogenesis, solid mass formation and higher invasive capacity, whereas, GLUT-12 was downregulated in C5 and T4 cells. Estrogen-induced malignant transformation is associated with remarkable and progressive GLUT-3 expression, GLUT-1 re-expression at further stages, as well as GLUT-12 downregulation.

Abstract LB-367: Bioluminescent transposon screen identifies cancer cell specific promoters in Salmonella typhimurium

Abstract The ability of Salmonella Typhimurium to specifically localize to malignant tumors in vivo has been well documented and offers a powerful avenue for targeted anti-cancer therapies. S. Typhimurium, a common gastrointestinal pathogen, utilizes a complex array of type III secretion protein complexes, effector molecules and transcription factors during its normal pathophysiology. The expression of these genes is highly regulated in a spatiotemporal manner in response to the host environment. Akin to other host environments, such as the gastrointestinal tract and phagosomes, tumors have specific extracellular properties such as low pH and hypoxia. Identifying S. Typhimurium genes and their promoters that respond specifically to these tumor environmental properties has served as the basis for several lines of research with the hope of utilizing S. Typhimurium for anti-cancer based therapies. To identify such genes that are regulated in a tumor specific manner, we engineered a bioluminescent transposon reporter-trap to screen a S. Typhimurium library for genes specifically regulated by co-culture with malignant cells in vitro. Five previously undiscovered genes were identified (adiY, yohJ, STM1787, STM1791, and STM1793) and their promoter sequences were found to be specifically activated by the acidic microenvironment associated with cancer cells in vitro and tumors in vivo. Three of the genes (STM1787, STM1791, and STM1793) are controlled by a single promoter, which contains a recently discovered conserved tusp motif. We have uncovered that transcriptional regulation of this motif is more complex as it is sensitive to low pH in addition to hypoxia as was originally ascribed. The genes identified herein are highly expressed in an acidic pH tumor environment, but are not required for bacterial tumor targeting. Therefore, the promoters regulating these genes may be ideal candidates for utilization in therapeutic gene, pro-drug or toxin delivery studies. To address this inquiry, we utilized the cancer cell-activated STM1787 promoter sequence to regulate the expression of a toxic tumor transgene in a wild type strain of S. Typhimurium. Shiga-like toxin 2 (Stx2) is a holoenzyme produced by some strains of E.coli. It has been extensively studied for the potent translational inhibition characteristics of the A subunit in addition to the high tumor cell binding-specificity of the B subunit. Conditionally regulating the tumor-specific expression of Stx2 by STM1787's promoter resulted in striking cancer cell death in vitro, and shows promise in mouse xenografts in vivo. In conclusion, we have identified novel tumor-specific genes expressed by S. Typhimurium, identified the acidic pH-responsiveness of the tusp motif, and demonstrated that these promoters can be successfully engineered for targeted anti-cancer therapy using live S. Typhimurium. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-367. doi:1538-7445.AM2012-LB-367

Identification of epigenetically silenced genes in human pancreatic cancer by a novel method “microarray coupled with methyl-CpG targeted transcriptional activation” (MeTA-array)

Identification and characterization of epigenetically silenced genes is important for cancer research, because information from hypermethylated genes provides clues to understand roles of epigenetics in tumorigeneses and genes frequently methylated in a tumor-specific manner can be used as tumor markers. Here, we describe the identification of transcriptionally silenced hypermethylated genes in pancreatic cancer cells by using a novel method called “microarray coupled with methyl-CpG targeted transcriptional activation” (MeTA-array for short), which can effectively reactivate genes containing the stringent criteria of CpG islands at promoter regions. Three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2 and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE as a control, were examined with this method, and 19 genes were upregulated twofold or more in all the three cancer cell lines after MeTA; 16 of these 19 genes have not been detected previously when using a conventional DNA demethylating agent, 5-aza-2′-deoxycytidine. Among these 16 genes, CSMD2, SLC32A1, TMEM204 and TRH were further analyzed by methylation-specific PCR, and we found that 90% (19/21) of CSMD2, 100% (21/21) of SLC32A1, 95% (20/21) of TMEM204 and 100% (21/21) of TRH were methylated in our series of pancreatic cancer cell lines. Furthermore, CSMD2, SLC32A1 and TRH were also hypermethylated in primary pancreatic cancers in a tumor-specific manner. These results suggest that MeTA-array is a highly efficient method for identifying methylation-mediated transcriptionally silenced genes in human pancreatic cancer and that this method can be applied to other types of human cancer.

Association of mitochondrial DNA content in peripheral blood leukocyte with hepatitis B virus‐related hepatocellular carcinoma in a Chinese Han population

Increasing epidemiological evidence has indicated that inherited variations of mtDNA content could affect the genetic susceptibility of many malignancies in a tumor-specific manner. However, the association between mtDNA content and hepatocellular carcinoma (HCC) remains undetermined. In this study, mtDNA content of peripheral blood leukocytes was determined using quantitative real-time PCR in a case-control study consisting of 274 HCC cases, 126 non-cancer patient controls with chronic liver diseases (CLD), and 258 healthy controls. We found that HCC cases had a significant lower mtDNA content than CLD controls (median [range]: 0.77 [0.17-2.30] vs 0.84 [0.32-3.37]; P = 0.012) and healthy controls (0.77 [0.17-2.30] vs 0.84 [0.35-3.44]; P = 0.035). There was no difference in mtDNA content between CLD and healthy controls (0.84 [0.32-3.37] vs 0.84 [0.35-3.44]; P = 0.261). We further assessed the association between mtDNA content and HCC and found that, compared to individuals with high mtDNA content, those with low mtDNA content had a significantly increased risk of HCC when health controls (adjusted odds ratio [aOR] = 1.64, 95% confidence interval [CI] = 1.06-2.55), CLD controls (aOR = 1.57, 95% CI = 1.10-2.25) or combined controls (aOR = 1.55, 95% CI = 1.12-2.14) were used as reference. In addition, stratified analyses showed that the significant association was only evident in younger individuals, male individuals, ever-smokers, and never-drinkers. Collectively, our findings provided the first epidemiological evidence that mtDNA content in peripheral blood leukocytes is significantly associated with HCC, which warrants further validation in prospective studies.

Identification and functional characterization of glioma-specific promoters and their application in suicide gene therapy

Suicide gene therapy has been shown to be effective in inducing tumor regression. In this study, a human brain tumor-specific promoter was identified and used to develop transcriptionally targeted gene therapy. We searched for genes with brain tumor-specific expression. By insilico and reverse-transcription polymerase chain reaction screening, MAGE-A3 and SSX4 were found to be expressed in a tumor-specific manner. SSX4 gene promoter activity was high in human brain tumor cells but not in normal human astrocyte cells, whereas the MAGE-A3 promoter showed activity in both tumor and normal cells. A retrovirus vector carrying a suicide gene, the herpes simplex virus thymidine kinase gene controlled by the SSX4 promoter, was constructed to evaluate the efficacy of the promoter in tumor-specific gene therapy. Glioma and human telomerase catalytic subunit-immortalized fibroblast BJ-5ta cell lines transduced with retrovirus vectors were assayed for killing activity by ganciclovir. Glioma cell lines were effectively killed by ganciclovir in a concentration-dependent manner, whereas BJ-5ta cells were not. By contrast, MAGE-A3 promoter failed to induce cytotoxicity in a brain tumor-specific manner. In addition, mouse glioma RSV-M cells transduced with retrovirus vector also showed suppressed tumor formation activity in syngeneic mice in response to ganciclovir administration. Therefore, the SSX4 promoter is a candidate for brain tumor-specific gene therapy and supports the efficacy and safety of suicide gene therapy for malignant brain tumors.

Comparison of microwave and magnetic nanoparticle hyperthermia radiosensitization in murine breast tumors.

Hyperthermia has been shown to be an effective radiosensitizer. Its utility as a clinical modality has been limited by a minimally selective tumor sensitivity and the inability to be delivered in a tumor-specific manner. Recent in vivo studies (rodent and human) have shown that cancer cell-specific cytotoxicity can be effectively and safely delivered via iron oxide magnetic nanoparticles (mNP) and an appropriately matched noninvasive alternating magnetic field (AMF). To explore the tumor radiosensitization potential of mNP hyperthermia we used a syngeneic mouse breast cancer model, dextran-coated 110 nm hydrodynamic diameter mNP and a 169 kHz / 450 Oe (35.8 kA/m) AMF. Intradermally implanted (flank) tumors (150 ± 40 mm3) were treated by injection of 0.04 ml mNP (7.5 mg Fe) / cm3 into the tumor and an AMF (35.8 kA/m and 169 kHz) exposure necessary to achieve a CEM (cumulative equivalent minute) thermal dose of 60 (CEM 60). Tumors were treated with mNP hyperthermia (CEM 60), radiation alone (15 Gy, single dose) and in combination. Compared to the radiation and heat alone treatments, the combined treatment resulted in a greater than two-fold increase in tumor regrowth tripling time (tumor treatment efficacy). None of the treatments resulted in significant normal tissue toxicity or morbidity. Studies were also conducted to compare the radiosensitization effect of mNP hyperthermia with that of microwave-induced hyperthermia. The effects of incubation of nanoparticles within tumors (to allow nanoparticles to be endocytosed) before application of AMF and radiation were determined. This preliminary information suggests cancer cell specific hyperthermia (i.e. antibody-directed or anatomically-directed mNP) is capable of providing significantly greater radiosensitization / therapeutic ratio enhancement than other forms of hyperthermia delivery.