Expression In Human Cancer Cells Research Articles

Pre-Defined Stem-Loop Structure Library for the Discovery of L-RNA Aptamers that Target RNA G-Quadruplexes.

L-RNA aptamers have been developed to target G-quadruplexes (G4s) and regulate G4-mediated gene expression. However, the aptamer selection process is laborious and challenging, and aptamer identification is subjected to high failure rate. By analyzing the previously reported G4-binding L-RNA aptamers, we found that the stem-loop (SL) structure is favored by G4 binding. Herein, we present a robust and effective G4-SLSELEX-Seq platform specifically for G4 targets by introducing a pre-defined stem-loop structure library during SELEX process. Using G4-SLSELEX-Seq, we rapidly identified an L-RNA aptamer, L-Apt1-12 for EBNA1 RNA G4 (rG4) in just three selection rounds. L-Apt1-12 maintained the stem-loop structure initially introduced, and possessed a unique G-triplex motif that is important for the strong binding affinity and specificity to EBNA1 rG4. Notably, L-Apt1-12 effectively downregulated endogenous EBNA1 protein expression in human cancer cells and showed selective toxicity towards EBV-positive cancer cells, highlighting its potential for targeted therapy against EBV-associated cancers. Furthermore, we demonstrate the robustness and generality of G4-SLSELEX-Seq by selecting L-RNA aptamers for another two G4 targets-APP rG4 and HCV-1a rG4, also obtaining high-affinity aptamers in three selection rounds. These findings demonstrated G4-SLSELEX-Seq can be a robust and efficient platform for the selection of L-RNA aptamers targeting rG4.

Pre‐Defined Stem‐Loop Structure Library for the Discovery of L‐RNA Aptamers that Target RNA G‐Quadruplexes

L‐RNA aptamers have been developed to target G‐quadruplexes (G4s) and regulate G4‐mediated gene expression. However, the aptamer selection process is laborious and challenging, and aptamer identification is subjected to high failure rate. By analyzing the previously reported G4‐binding L‐RNA aptamers, we found that the stem‐loop (SL) structure is favored by G4 binding. Herein, we present a robust and effective G4‐SLSELEX‐Seq platform specifically for G4 targets by introducing a pre‐defined stem‐loop structure library during SELEX process. Using G4‐SLSELEX‐Seq, we rapidly identified an L‐RNA aptamer, L‐Apt1‐12 for EBNA1 RNA G4 (rG4) in just three selection rounds. L‐Apt1‐12 maintained the stem‐loop structure initially introduced, and possessed a unique G‐triplex motif that is important for the strong binding affinity and specificity to EBNA1 rG4. Notably, L‐Apt1‐12 effectively downregulated endogenous EBNA1 protein expression in human cancer cells and showed selective toxicity towards EBV‐positive cancer cells, highlighting its potential for targeted therapy against EBV‐associated cancers. Furthermore, we demonstrate the robustness and generality of G4‐SLSELEX‐Seq by selecting L‐RNA aptamers for another two G4 targets‐APP rG4 and HCV‐1a rG4, also obtaining high‐affinity aptamers in three selection rounds. These findings demonstrated G4‐SLSELEX‐Seq can be a robust and efficient platform for the selection of L‐RNA aptamers targeting rG4.

Abstract 5393: Characterization of small ubiquitin-like modifier (SUMO) protein complex expression in human cancer cells to identify novel therapeutic targets

Abstract SUMOylation is a post-translational modification where a number of small ubiquitin-like modifier proteins (SUMO) are conjugated to lysine residues of the target proteins and contribute to various cellular functions essential for cancer cell survival and proliferation. The unique behaviors, phenotypic and molecular adaptations found in the naked-mole rat (NMR) suggest a high stability and effective functioning of the molecular machinery that counteracts damage accumulation in its genome. NMR can reach 32 years, shows no signs of ageing, and possesses a very efficient mechanism of resistance to cancer. This study aims to investigate the regulation of SUMOylation machinery between NMR tissues in comparison to human cancer cells, to find novel targets for cancer therapy. Gene expression of SUMO isoforms between NMR and humans, were investigated through qRT-PCR. The data suggests a significant low expression level for most SUMO machinery (except PIAS4) in NMR brain and intestine tissues compared to MCF-7, DLD-1, SH-SY5Y, and MCF-10A cells. Western blotting analysis also revealed high protein expression of PIAS4 in NMR compared to SENP1 and Ubc9. Also, SUMO machinery expression levels from NMR are recapitulated in cancer cells either silencing (hUBE2I: shRNA, hSENP1: shRNA) or overexpression (Myc/hPIAS4: Exp). Further, cytotoxicity assay was analyzed in relevance to Doxorubicin in MCF-7, 5-Fluorouracil in DLD-1 and Methotrexate in SH-SY5. The functional assays such as clonogenic and invasion assays were performed including apoptotic mechanisms (Bcl-2, and BID). In conclusion, the data revealed important differences in SUMOylation machinery between NMR and human cancer cells, which provides a future therapeutic path for cancer treatment. Citation Format: Mohammed Abdullah Mohammed Salih, Ritchie Williamson, Sriharsha Kantamneni. Characterization of small ubiquitin-like modifier (SUMO) protein complex expression in human cancer cells to identify novel therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5393.

Abstract B022: A gradient expression of integrin αvβ6 expression in human cancer cells associates with TGFβ mediated immune evasion

Abstract Our previous results demonstrate an integrin-mediated mechanism of immune evasion in colorectal cancer. Increased levels of latent TGFβ in the experimental tumor environment led to the upregulation of the immune checkpoint marker PD-L1 in HCT116WT cells, facilitating escape from T-cell killing. This effect was rescued in immune co-cultures by pretreating cancer cells with the broad-spectrum integrin inhibitor GLPG-0187. To further elucidate this system, we investigated the TGFβ-integrin axis. Integrin αvβ6 is a major activator of the inert latent TGFβ into the active, immunosuppressive form. With its high specificity within malignant tissue, inhibition of αvβ6 poses an attractive therapeutic target for disrupting TGFβ signaling while avoiding the toxicities of systemic blockade. To identify an endogenous in vitro model of integrin αvβ6-expressing cancer, computational analysis of publicly available mRNA expression data was performed to determine cancer types with high αvβ6 upregulation. This investigation revealed a strong presence of αvβ6 in pancreatic, bladder, head and neck, and cervical cancer tissues. Further in-silico work identified the head and neck squamous cell carcinoma cell lines CAL-27 and FaDu, as well as the pancreatic adenocarcinoma line Capan-2, as suitable models for in vitro study. Flow cytometry of these cell lines was performed to determine the surface expression of integrin αvβ6, revealing that protein expression correlated with the mRNA data from the previous computational studies. Capan-2 and CAL-27 exhibited high levels αvβ6 of expression, while FaDu expressed moderate levels, and HCT116WT exhibited low αvβ6 expression. These cell lines were pretreated with latent TGFβ, simulating sequestered latent TGFβ in the extracellular matrix, and subsequently co-cultured with TALL-104 T-cells. Quantitative fluorescence microscopy revealed that resistance to killing by T-cells correlated with αvβ6 expression across cell lines. These findings are consistent with the hypothesis that upregulated expression of integrin αvβ6 functions as an immunosuppressive resistance mechanism by increasing tumor microenvironment levels of active TGFβ. Analysis of mRNA sequencing data from the TCGA Pancreatic Adenocarcinoma Firehose database revealed that integrin αvβ6 is a strong marker of poor prognosis in pancreatic adenocarcinoma. Future co-culture experiments will investigate the dynamics of TGFβ activation in the tumor microenvironment in relation to αvβ6 expression. Further in vivo studies can facilitate translation of our findings to therapeutic clinical trials. Citation Format: William J. MacDonald, Praveen R. Srinivasan, Vida Tajiknia, Maximilian Pinho-Schwermann, Connor Purcell, Brooke Verschleiser, Wafik S. El-Deiry. A gradient expression of integrin αvβ6 expression in human cancer cells associates with TGFβ mediated immune evasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B022.

REV7 is involved in outcomes of platinum-based chemotherapy in pancreatic cancer by controlling the DNA damage response.

REV7 is a multifunctional protein implicated in various biological processes, including DNA damage response. REV7 expression in human cancer cells affects their sensitivity to DNA-damaging agents. In the present study, we investigated the significance of REV7 in pancreatic ductal adenocarcinoma (PDAC). REV7 expression was immunohistochemically examined in 92 resected PDAC specimens and 60 endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) specimens of unresectable PDAC treated with platinum-based chemotherapy, and its association with clinicopathologic features was analyzed. Although REV7 expression was not significantly associated with the progression of primary tumors (T-factor and Stage) in either resected or unresectable PDAC, decreased levels of REV7 expression in EUS-FNAB specimens of unresectable PDAC were significantly associated with better outcomes of platinum-based chemotherapy and a favorable prognosis. REV7-deficient PDAC cell lines showed suppressed cell growth and enhanced sensitivity to cisplatin invitro. Tumor-bearing mice generated using REV7-deficient PDAC cell lines also showed enhanced sensitivity to cisplatin invivo. RNA sequencing analysis using WT and REV7-deficient PDAC cell lines revealed that REV7 inactivation promoted the downregulation of genes involved in the DNA repair and the upregulation of genes involved in apoptosis. Our results indicate that decreased expression of REV7 is associated with better outcomes of platinum-based chemotherapy in PDAC by suppressing the DNA damage response. It is also suggested that REV7 is a useful biomarker for predicting the outcome of platinum-based chemotherapy and the prognosis of unresectable PDAC and is a potential target for PDAC treatment.

Tolfenamic acid negatively regulates YAP and TAZ expression in human cancer cells.

Several diseases are associated with improper regulation of the Hippo pathway, which plays an important role in cell proliferation and cancer metastasis. Overactivation of the YAP and TAZ proteins accelerates cell proliferation, invasion, and migration during tumorigenesis. Tolfenamic acid (TA) is a non-steroidal anti-inflammatory drug (NSAID) that exhibits activity against various types of cancer. In this study, we observed that TA decreased YAP and TAZ protein levels in cancer cells. TA increased the phosphorylation of YAP and TAZ, leading to the degradation of YAP and TAZ in the cytoplasm and nucleus. TA predominantly affected multiple phosphodegron sites in the YAP and TAZ and lowered 14-3-3β protein expression, causing YAP and TAZ to enter the ubiquitination pathway. Proteins that affect YAP and TAZ regulation, such as NAG-1 and several YAP/TAZ E3 ligases, were not involved in TA-mediated YAP/TAZ degradation. In summary, our results indicate that TA affects phosphodegron sites on YAP/TAZ, demonstrating a novel effect of TA in tumorigenesis.

A highly sensitive and robust electrochemical biosensor for microRNA detection based on PNA-DNA hetero-three-way junction formation and target-recycling catalytic hairpin assembly amplification

Robust and sensitive methods for the detection of microRNAs (miRNAs) are crucial in the clinical diagnosis of cancers. In this study, a novel electrochemical biosensor with high sensitivity for miRNA-21 detection is developed, which relies on the formation of a peptide nucleic acid (PNA)-DNA hetero-three-way junction (H3WJ) and target-recycling catalytic hairpin assembly (CHA) amplification. The electroneutral PNA probes are initially immobilized onto a gold electrode to construct the sensor. Upon introduction of miRNA-21, target-recycling CHA is initiated, resulting in abundant double-stranded CHA products. Subsequently, association between the PNA probes and these products leads to the formation of PNA-DNA H3WJs. Consequently, the electrode surface is densely populated with numerous electroactive Ferrocene (Fc) groups, resulting in a significantly amplified current response for highly sensitive detection of miRNA-21 at concentrations as low as 0.15 fM. This approach demonstrates remarkable specificity towards target miRNAs and can be utilized for quantitative monitoring of miRNA-21 expression in human cancer cells. More importantly, the sensor exhibits exceptional stability and shows a significant reduction in background noise during miRNA detection, making this method a highly promising sensing platform for monitoring various miRNA biomarkers to facilitate the diagnosis of diverse cancers.

Comprehensive Transcriptomic Analysis of Novel Class I HDAC Proteolysis Targeting Chimeras (PROTACs).

The class I histone deacetylase (HDAC) enzymes;HDAC1,2 and 3 form the catalytic engine of at least seven structurally distinct multiprotein complexes in cells. These molecular machines play a vital role in the regulation of chromatin accessibility and gene activity via the removal of acetyl moieties from lysine residues within histone tails. Their inhibition via small molecule inhibitors has beneficial effects in a number of disease types, including the clinical treatment of hematological cancers. We have previously reported a library of proteolysis targeting chimeras (PROTACs) incorporating a benzamide-based HDAC ligand (from CI-994), with an alkyl linker and ligand for the von Hippel-Lindau (VHL) E3 ubiquitin ligase that degrade HDAC1-3 at submicromolar concentrations. Here we report the addition of two novel PROTACs (JPS026 and JPS027), which utilize a ligand for the cellular inhibitor of apoptosis (IAP) family of E3 ligases. We found that both VHL (JPS004)- and IAP (JPS026)-based PROTACs degrade HDAC1-3 and induce histone acetylation to a similar degree. However, JPS026 is significantly more potent at inducing cell death in HCT116 cells than is JPS004. RNA sequencing analysis of PROTAC-treated HCT116 cells showed a distinct gene expression signature in which cell cycle and DNA replication machinery are repressed. Components of the mTORC1 and -2 complexes were also reduced, leading to an increase in FOXO3 and downstream target genes that regulate autophagy and apoptosis. In summary, a novel combination of HDAC and IAP ligands generates a PROTAC with a potent ability to stimulate apoptosis and differential gene expression in human cancer cells.

Indoleamine 2,3-Dioxygenase Activity-Induced Acceleration of Tumor Growth, and Protein Kinases-Related Novel Therapeutics Regimens.

Indoleamine 2,3-dioxygenase (IDO) is overexpressed in response to interferon-gamma (IFN-γ). IDO-mediated degradation of tryptophan (Trp) along the kynurenine (Kyn) pathway by immune cells is associated with the anti-microbial, and anti-tumor defense mechanisms. In contrast, IDO is constitutively expressed by various tumors and creates an immunosuppressive microenvironment around the tumor tissue both by depletion of the essential amino acid Trp and by formation of Kyn, which is immunosuppressive metabolite of Trp. IDO may activate its own expression in human cancer cells via an autocrine aryl hydrocarbon receptor (AhR)- interleukin 6 (IL-6)-signal transducer and activator of transcription 3 (STAT3) signaling loop. Although IDO is not a unique marker, in many clinical trials serum IDO activity is suggested to be an important parameter in the pathogenesis of cancer development and growth. Measuring IDO activity in serum seems to be an indicator of cancer growth rate, however, it is controversial whether this approach can be used as a reliable guide in cancer patients treated with IDO inhibitors. Thus, IDO immunostaining is strongly recommended for the identification of higher IDO producing tumors, and IDO inhibitors should be included in post-operative complementary therapy in IDO positive cancer cases only. Novel therapies that target the IDO pathway cover checkpoint protein kinases related combination regimens. Currently, multi-modal therapies combining IDO inhibitors and checkpoint kinase blockers in addition to T regulatory (Treg) cell-modifying treatments seem promising.

Licochalcone A inhibits EGFR signalling and translationally suppresses survivin expression in human cancer cells.

Dysfunction of epidermal growth factor receptor (EGFR) signalling plays a critical role in the oncogenesis of non–small‐cell lung cancer (NSCLC). Here, we reported the natural product, licochalcone A, exhibited a profound anti‐tumour efficacy through directly targeting EGFR signalling. Licochalcone A inhibited in vitro cell growth, colony formation and in vivo tumour growth of either wild‐type (WT) or activating mutation EGFR‐expressed NSCLC cells. Licochalcone A bound with L858R single‐site mutation, exon 19 deletion, L858R/T790M mutation and WT EGFR ex vivo, and impaired EGFR kinase activity both in vitro and in NSCLC cells. The in silico docking study further indicated that licochalcone A interacted with both WT and mutant EGFRs. Moreover, licochalcone A induced apoptosis and decreased survivin protein robustly in NSCLC cells. Mechanistically, we found that treatment with licochalcone A translationally suppressed survivin through inhibiting EGFR downstream kinases ERK1/2 and Akt. Depletion of the translation initiation complex by eIF4E knockdown effectively inhibited survivin expression. In contrast, knockdown of 4E‐BP1 showed the opposite effect and dramatically enhanced survivin protein level. Overall, our data indicate that targeting survivin might be an alternative strategy to sensitize EGFR‐targeted therapy.

Abstract B34: PI3K/p110β modulates tumor suppressor gene NR0B2 expression in human liver cancer cells

Abstract Background: NR0B2 (nuclear receptor subfamily 0 group B member 2), also called SHP (small heterodimer partner), is an orphan nuclear receptor with a unique feature of lacking a conventional zinc-finger DNA binding domain. It acts as a transcriptional repressor by binding to other nuclear receptors and regulates various metabolic pathways. NR0B2 gene expression was significantly reduced in many human malignancies, including liver cancers, and overexpression of NR0B2 protein suppresses liver cancer development, indicating it is a tumor suppressor. Previous reports showed that NR0B2 gene expression is upregulated by several transcription factors, including FXR; however, it is not clear if and how cellular signaling pathways are involved in transcription factors-regulated NR0B2 gene expression in human cancer cells. Phosphatidylinositol 3-kinases (PI3Ks) are a group of major cellular signaling molecules that regulate multiple cellular signal pathways and their genetic abnormalities are found in most of human cancers. In liver cancers, animal study with genetic ablation of PTEN, the negative PI3K regulator, resulted in 60% of tumor development in the liver, suggesting an important role of PI3K-PTEN pathway in liver cancers. Methods: To investigate if PI3K pathway is involved in the regulation of NR0B2 expression in human liver cancer HepG2 cells, multiple PI3K inhibitors were used to suppress PI3K activity. NR0B2 gene expression was assessed using real-time RT-PCR assay, Western blot and luciferase reporter driven by NR0B2 gene promoter (2200-LUC). Results: At the mRNA level, NR0B2 gene expression is significantly elevated after being treated by PI3K inhibitor LY294002, along with JNK inhibitor SP600125. In contrast, MAPK/ERK inhibitors PD98059 and U0126 drastically suppressed NR0B2 gene expression. With the NR0B2 promoter-driven luciferase reporter (2200-LUC), luciferase assay results revealed that MAPK/ERK kinase inhibitors U0126 and PD184161 abolished the basal and FXR agonist GW4064 -stimulated luciferase activities, in agreement with the results at the mRNA levels. Interestingly, PI3K inhibitor BKM120/LY294002 and PI3K/p110β inhibitor BL140 drastically enhanced the basal luciferase activity of 2200-LUC reporter but had no effect on GW4064-stimulated 2200-LUC activities. We also determined that GW4064-stimualted NR0B2 protein expression was abolished by U0126 and PD184161 but not by PI3K inhibitors. In contrast, PI3K inhibitors increased NR0B2 protein levels at a time- and concentration-dependent manner. Conclusion: These data suggest that NR0B2 gene expression is regulated by two major intracellular signal pathways, PI3K and MAPK. While MAPK/ERK signal pathway is involved in FXR-mediated NR0B2 expression, PI3K pathway suppresses NR0B2 expression that might be involved in liver cancer development or progression. Citation Format: Yanjie Tu, Jean Li, Lisa Zhang. PI3K/p110β modulates tumor suppressor gene NR0B2 expression in human liver cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B34.

Expression Analysis of Canine CMTM6 and CMTM4 as Potential Regulators of the PD-L1 Protein in Canine Cancers.

Cancer is one of the most significant causes of death in dogs. Antibody drugs targeting the PD-1/PD-L1 axis represent a promising immunotherapy for both human and canine cancers. However, the regulation mechanisms of PD-L1 expression in canine cancers require further investigation to better understand the resistance mechanisms to anti-PD-L1 therapy. Recent reports have shown that CMTM6 and CMTM4 are critical regulators of PD-L1 protein expression in human cancer cells. By preventing PD-L1 from lysosome-mediated degradation, CMTM6 maintains PD-L1 expression on the cell surface. However, the literature has not reported on CMTM6 and CMTM4 in dogs, and their functions are completely unknown. To reveal a regulation mechanism of PD-L1 in canine cancers, this study firstly identified the gene sequences of CMTM6 and CMTM4. Then, the expression analysis of these proteins was performed by immunohistochemistry. Furthermore, the functions of CMTM6 and CMTM4 in regulating PD-L1 expression were examined by gene knockdown of CMTM6 and CMTM4. Canine CMTM6 and CMTM4 displayed high amino acid sequence identities compared with those of humans and mice. An immunohistochemical analysis using cross-reactive antibodies revealed that canine malignant melanoma and osteosarcoma express CMTM6, CMTM4, and PD-L1 simultaneously. Gene knockdown of CMTM6 and CMTM4 with RNA interference significantly reduced the cell surface expression of PD-L1 in a canine cell line. These results suggest that CMTM6 and CMTM4 are regulators of PD-L1 expression in canine cancers and could serve as potential therapeutic targets to enhance antitumor immunity.

Protein-partners of selenoprotein SELM and the role of selenium compounds in regulation of its expression in human cancer cells

The search of potential partners of human SELM in lysates of two cancer cell lines HT-1080 (fibrosarcoma) and MCF-7 (breast adenocarcinoma) was carried out. Two cytoplasmic actin isoforms: cytoplasmic actin 1 (cytoskeleton b-actin) and cytoplasmic actin 2 (cytoskeletal g-actin) was identified as partners. In addition, the influence of two widely used antitumor selenium compounds (sodium selenite and methylseleninic acid) on the expression SELM in cancer cells was studied. According to the results obtained by real-time PCR and Western blotting, we was concluded that 1 µM and 10 µM sodium selenite was not affected on the expression SELM in fibrosarcoma cells, whereas in breast adenocarcinoma cells 1 µM sodium selenite slightly increased of expression and 10 µM resulted in a significant decrease (about 2 times). Methylseleninic acid in both cancer cell lines increased the expression of SELM gene, the most pronounced effect was observed when fibrosarcoma cells were treated with 10 µM MSC (increased expression of the hSelm gene by almost 4 times).

Protein Partners of Selenoprotein SELM and the Role of Selenium Compounds in Regulation of Its Expression in Human Cancer Cells.

The search for potential partners of human SELM in lysates of two cancer cell lines, HT-1080 (fibrosarcoma) and MCF-7 (breast adenocarcinoma), was carried out. Two cytoplasmic actin isoforms-cytoplasmic actin 1 (cytoskeleton β-actin) and cytoplasmic actin 2 (cytoskeletal γ-actin)-were identified as partners. In addition, the influence of two widely used antitumor selenium compounds (sodium selenite and methylseleninic acid) on the expression of SELM in cancer cells was studied. On the basis of the results obtained by real-time PCR and Western blotting, we concluded that 1 μM and 10 μM sodium selenite did not affect the expression of SELM in fibrosarcoma cells, whereas in breast adenocarcinoma cells 1 μM sodium selenite slightly increased expression and 10 μM sodium selenite significantly (approximately 2 times) decreased it. Methylseleninic acid in both cancer cell lines increased the SELM gene expression; the most pronounced effect was observed when fibrosarcoma cells were treated with 10 µM MSA (the expression of the hSelm gene increased almost 4 times).

Chitosan-based nanoparticles with damnacanthal suppress CRM1 expression.

Cancer is one of the leading causes of mortality worldwide. Phytochemicals may be promising anticancer agents given their various chemical structures and diverse biological activities. Damnacanthal (DAM) is a major bioactive component of Noni, which has been investigated previously as a cancer-preventive or chemotherapeutic agent. DAM has also been reported to exhibit anti-proliferative activity in several cancer types. In the present study, it was identified that DAM downregulates chromosome maintenance protein 1 (CRM1) expression in human cancer cells. The application of chitosan-based nanoparticles (NPs) with DAM also induced CRM1 downregulation, which suggests that chitosan-based NPs may be effective vehicles for delivery of phytochemicals such as DAM. It was also identified that DAM increased the levels of the tumor suppressor non-steroidal anti-inflammatory drugs-activated gene 1 in the nucleus, thereby leading to enhanced anticancer effects. The results of the present study indicate that DAM and its nanoformulation may be a candidate anticancer drug.

Fascin1 suppresses RIG-I–like receptor signaling and interferon-β production by associating with IκB kinase Ε (IKKΕ) in colon cancer

Fascin1 is an actin-bundling protein involved in cancer cell migration and has recently been shown also to have roles in virus-mediated immune cell responses. Because viral infection has been shown to activate immune cells and to induce interferon-β expression in human cancer cells, we evaluated the effects of fascin1 on virus-dependent signaling via the membrane- and actin-associated protein RIG-I (retinoic acid-inducible gene I) in colon cancer cells. We knocked down fascin1 expression with shRNA retrovirally transduced into a DLD-1 colon cancer and L929 fibroblast-like cell lines and used luciferase reporter assays and co-immunoprecipitation to identify fascin1 targets. We found that intracellular poly(I·C) transfection to mimic viral infection enhances the RIG-I/MDA5 (melanoma differentiation-associated gene 5)-mediated dimerization of interferon regulatory factor 3 (IRF-3). The transfection also significantly increased the expression levels of IRF-7, interferon-β, and interferon-inducible cytokine IP-10 in fascin1-deleted cells compared with controls while significantly suppressing cell growth, migration, and invasion. We also found that fascin1 constitutively interacts with IκB kinase ϵ (IKKϵ) in the RIG-I signaling pathway. In summary, we have identified fascin1 as a suppressor of the RIG-I signaling pathway associating with IκB kinase ϵ in DLD-1 colon cancer cells to suppress immune responses to viral infection.

5'-UTR and 3'-UTR Regulation of MICB Expression in Human Cancer Cells by Novel microRNAs.

The treatment of cancer through the induction of natural killer group 2, member D (NKG2D) ligands is of interest, but understanding of mechanisms controlling expression of individual ligand is limited. The major histocompatibility complex (MHC) class I chain related protein B (MICB) is a member of NKG2D ligands. We aimed to investigate the role of 3′-untranslated (3′-UTR) and 5′-untranslated regions (5′-UTR) in post-transcriptional regulation of MICB. Nine novel microRNAs (miRNAs) predicted to interact with 3′-UTR and 5′-UTR using TargetScan, RNAhybrid and miBridge were identified. Their regulation of 3′-UTR, 5′-UTR and both 3′- and 5′-UTR sequences of MICB were indicated by the reduction of luciferase activities of luciferase reporter constructs. Mutations of miRNA binding sites at 3′- and 5′-UTRs resulted in increased luciferase activities confirming the regulation of nine candidate miRNAs. In addition, overexpression of candidate miRNAs also down-regulated the expression of reporter constructs. Consequently, the overexpression and inhibition of candidate miRNAs lead to the decreased and increased. MICB protein expressions on the cells tested, respectively. This study has identified a new role of miRNAs in regulation of MICB expression via both 3′-UTR and 5′-UTR sequences applicable for cancer immunotherapy.

Abstract 5087: Radiation enhances the intracellular delivery of anti-MGMT morpholino oligonucleotides (AMONs) to enhance the cytotoxicity of chemo-radiation in cancer cells

Abstract Background: This study evaluates the use of a sub-lethal dose of ionizing radiation to guide and enhance the intracellular delivery of morpholino antisense nucleotides designed to block O6-methylguanine DNA methyltransferase (MGMT) to enhance the cytotoxicity of chemo-radiation therapy (CRT). MGMT is a DNA repair enzyme and its expression is a well-known mechanism for resistance to CRT in glioblastoma. Epigenetic silencing of this enzyme by promotor methylation is associated with better response and clinically relevant survival benefits after CRT. We designed anti-MGMT morpholino oligonucleotides (AMON) using the stable vivo-morphalinos backbone. Intracellular delivery of morpholinos, particularly in a manner that could be clinically feasible, continues to be a major challenge. We use sublethal ionizing radiation to enhance the delivery of AMON to transiently reduced MGMT protein expression. We previously demonstrated that sublethal ionizing radiation enhances the delivery of AMONs to reduce MGMT expression in human cancer cells (T98G glioma and H460 and A549 non-small cell lung cancer cell lines) in vitro as well as the targeted knockdown of MGMT with systemically administered AMONsin pre-irradiated tumors. We now test whether blocking MGMT will enhance the cytotoxicity of CRT, and investigate the mechanism by which radiation permits cellular uptake of AMONs. Methods: MGMT expressing, human T98G glioblastoma cells were used for all in-vitro studies. AMONs and non-specific fluorescent tagged control morpholinos were synthesized and purchased from Gene Tools, LLC (Philomath, OR, USA). High-resolution widefield microscopy with image deconvolution was performed to confirm the intracellular delivery. Cells were exposed to 1 Gy radiation; 24 hrs post radiation cells were treated with AMONs. Cells were then exposed to temozolomide (75 or 150 μg/mL) on day 3. Lysates were obtained on days 4 and 7. Expression of cellular MGMT protein, apoptosis markers, and key endocytosis proteins were characterized using immunoblotting. Results: Our results demonstrate that MGMT silencing using a single dose of AMONs delivered with sublethal dose of radiation increased apoptosis by 50% and reduces cell viability by 50% at one and two days after therapy. Further, we demonstrate an increase in caveolin 1, clathrin and dynamin expression after radiation exposure. Conclusions: Our results suggest that blocking AMONs can enhance the cytotoxicity of CRT. Dynamin mediated endocytosis appears to play a key role in this enhanced cellular update of antisense nucleotides after a sublethal dose of radiation. Since radiation is already part of standard of care cancer therapy, the use of radiation to guide the delivery of systemically administered anti-sense nucleotides is a novel strategy to target key tumor associated proteins for therapeutic benefit key. Citation Format: Prakash Ambady, Jeffrey Wu, Cymon Kersch, DreeAnna Morris, Michael Pagel, Leslie L. Muldoon, Edward Neuwelt. Radiation enhances the intracellular delivery of anti-MGMT morpholino oligonucleotides (AMONs) to enhance the cytotoxicity of chemo-radiation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5087. doi:10.1158/1538-7445.AM2017-5087

OVA12 promotes tumor growth by regulating p53 expression in human cancer cells.

Ovarian cancer-associated antigen 12 (OVA12) was first identified in an ovarian carcinoma complementary DNA (cDNA) expression library and has been shown to play an important role in tumor growth. Here, we found that overexpression of OVA12 accelerated tumor growth in different tumor cells, whereas OVA12 depletion was associated with the opposite effect. Moreover, knocking down OVA12 led to a significant increase in the protein levels of p53, and the overexpression of OVA12 significantly decreased endogenous p53 levels. In addition, OVA12 stimulated p53 polyubiquitination and degradation by the proteasome and promoted tumor growth at least partially through the p53 pathway. Taken together, these results indicate that OVA12 is a negative regulator of p53 and that inhibition of OVA12 expression might serve as a therapeutic target to restore tumor suppression.

Abstract B38: Radiation enhances intracellular delivery of anti-MGMT oligomers to reduce protein expression in vitro and in a xenograft model

Abstract High levels of DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) in glioblastoma tumor are associated with temozolomide resistance and poor prognosis. MGMT provides a promising therapeutic target to overcome chemo-resistance and improve overall survival in cancer patients. In this study, we investigate the enhanced delivery of anti-MGMT morpholino oligonucleotide (AMON) using a sub-therapeutic dose of radiation to reduce MGMT expression of human cancer cells (T98G glioma and H460 and A549 non-small cell lung carcinoma) in vitro and in vivo. Compared to standard transfection techniques, sub-therapeutic dose of radiation enhanced intracellular AMON delivery and transiently reduced MGMT protein expression at 3 d in vitro. The optimal radiation dosage was cancer cell type dependent and ranged from 1-12 Gy. In addition, AMON delivered using sub-therapeutic dose of radiation increased cytotoxicity of T98G cells in response to temozolomide compared to radiation and temozolomide alone in vitro. In a H460 subcutaneous xenograft tumor model, intravenous AMON administration reduced MGMT protein level by 50% in irradiated tumors but not in contralateral non-irradiated lesions within the same animal. Tumor MGMT protein downregulation by AMON did not alter DNA promoter methylation status. Our results demonstrate for the first time the use of radiation to enhance the intracellular delivery of antisense oligonucleotides to modulate MGMT protein expression in vitro and in vivo. We have previously shown that antisense nucleotides can be delivered to the brain parenchyma using osmotic blood brain barrier disruption. Therefore, we believe that cancer patients with MGMT overexpression, unmethylated DNA promoter and/or resistance to DNA alkylating agents may benefit from this novel antisense therapeutic technology to improve overall survival. Citation Format: Jeffrey Wu, Prakash Ambady, DreeAnna Morris, Michael Pagel, Randy Woltjer, Joshua Walker, Leslie Muldoon, Edward Neuwelt. Radiation enhances intracellular delivery of anti-MGMT oligomers to reduce protein expression in vitro and in a xenograft model [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B38.