COLO 320DM Research Articles

The 5-/12-lipoxygenase-BLT2 cascade induces elevated expression of c-Myc, thus mediating the proliferation and migration of KRAS mutant colorectal cancer cells

ABSTRACTColorectal cancer (CRC) is the most common malignant tumor and one of the leading causes of cancer-related death worldwide. Oncogene KRAS is a commonly mutated in CRC and plays crucial roles in the colorectal tumorigenesis. Emerging evidence suggests that increased expression levels of c-Myc are critical for KRAS-mediated CRC progression. However, it is unclear exactly how c-Myc expression is regulated in CRC. In the present study, we found that blockade of 5/12-lipoxygenase (5/12-LO) or leukotriene B4 receptor (BLT2) markedly reduced c-Myc expression in KRAS-mutant LOVO cells, while the control COLO 320DM cells were not affected. When the 5/12-LO products LTB4 and 12(S)-HETE, ligands of BLT2, were added to LOVO cells, c-Myc expression levels were restored, together suggesting that ‘5/12-LO-BLT2 cascade’ regulates c-Myc expression in KRAS-mutant CRC cells. We also observed that ‘5/12-LO-BLT2’-mediated c-Myc upregulation contributes to cell proliferation by regulating the expression of cyclin D1 in LOVO cells. Moreover, ‘5/12-LO-BLT2-cMyc’ cascade regulates the expression of EMT-related proteins in KRAS-mutant CRC cells, and depletion of both c-Myc and BLT2 using siRNA significantly enhanced the level of E-cadherin and reduced the level of Vimentin in LOVO cells. Taken together, our findings suggest that the BLT2-linked cascade promotes KRAS-mutant CRC cell proliferation and migration through the elevated expression of c-Myc. Thus, our results suggest that BLT2 appears to be an effective therapeutic target for KRAS-mutant CRCs.

Effective combination of biocompatible zinc oxide nanocrystals and high-energy shock waves for the treatment of colorectal cancer

BackgroundColorectal cancer (CRC) is the third most diagnosed tumor worldwide, with a very high mortality rate, second only to lung cancer. Current treatments, such as surgery, chemotherapy or radiotherapy, are not effective enough and show several limitations. Among the emerging strategies, nanomedicine offers very powerful tools in cancer treatment. Recently, the combination of nanoparticle antitumor effect with a triggering external stimulation was formulated to boost up the cytotoxic activity.ResultsIn this work, we show the synergistic effect of oleic acid-capped zinc oxide nanocrystals (ZnO NCs) and mechanical high-energy shock waves (SW) in the treatment for CRC cells, in vitro. We tested two different types of ZnO NCs synthetized in our laboratory, the basal undoped ZnO NCs and the iron-doped ones (Fe:ZnO NCs). The presence of the oleic acid capping and the further amino-propyl functionalization guarantee a high colloidal stability to both NCs, while the iron doping confers to Fe:ZnO NCs interesting magnetic properties useful for imaging applications in a clinical perspective. Thus, the iron-doped ZnO NCs are very attractive as potentially theranostic nanoparticles, allowing both stimuli-responsive therapy and magnetic resonance imaging.Importantly, two colon adenocarcinoma cell lines, the HT-29 and the Dukes’ type C Colo 320DM cells were tested, both showing a good bio-tolerance and internalization rates of NCs. With the aim of eradicating the CRC cells, the possible synergism between the undoped/iron-doped ZnO NCs and an external physical stimulus, i.e., high-energy SW, was then here investigated in vitro. We demonstrated that the combined treatment resulted in an augmentation of the antitumor activity, especially for Colo 320DM cells, when compared to controls. Moreover, a repeated and sequenced SW treatment (three times/day, 3SW) after ZnO NCs exposure resulted in a further increased mortality of CRC cells.ConclusionOur work proposes the combination of the cytotoxic activity of ZnO NCs with the SW external stimulation to obtain a booster of the antitumor activity, which warrants further investigation in vivo on CRC as well as on other tumors.Graphical

Abstract 5138: Cetuximab resistance induced by hepatocyte growth factor is overcome by MET inhibition in KRAS, NRAS, and BRAF wild-type colorectal cancer

Abstract Background: Recent evidence has highlighted the role of hepatocyte growth factor (HGF) as a putative biomarker to predict EGFR inhibitor resistance. This study investigated the impact of plasma HGF levels on EGFR inhibition and the therapeutic implications of MET inhibition in KRAS, NRAS, and BRAF (RAS/RAF) wild-type colorectal cancer (CRC). Methods: Clinical outcomes were analyzed according to the plasma HGF levels in patients with metastatic CRC (mCRC) receiving palliative first-line cetuximab or bevacizumab + FOLFIRI chemotherapy. Then, in vitro experiments were conducted to validate the clinical findings and to establish pre-clinical evidence of MET inhibition in the HGF-high population. Results: From March 2015 to September 2019, a total of 80 patients were enrolled. The median age was 63 (range, 24-85) years. Of these, 45 patients (56.2%) were treated with bevacizumab + FOLFIRI and 35 patients (43.8%) were treated with cetuximab + FOLFIRI. Among patients with evaluable disease (43 patients (95.6%) in the bevacizumab group and 34 patients (97.1%) in the cetuximab group), the objective response rate was 60.0% and 37.8%, respectively (p=0.162). The median serum HGF level was 374.75 (range, 9.43-30,644.44) pg/mL, and the optimal cut-off value of HGF levels was 12.70 pg/mL by the maximal chi-square method. During a median follow-up of 29.3 (range, 1.2-71.3) months, both progression-free survival (PFS) and overall survival (OS) were significantly shorter in the high HGF group compared with the low HGF group: median 11.8 (95% confidence interval [CI], 10.5-13.1) months vs. 24.7 (95% CI, 23.4-25.9) months for PFS (p=0.009), and median 21.1 (95% CI, 17.9-24.3) months vs. not reached for OS (p=0.018). The difference in PFS and OS was statistically significant in the cetuximab group (p=0.003 for PFS and p=0.035 for OS), but not in the bevacizumab group. In five RAS/RAF wild-type CRC cell lines (Caco-2, COLO 320DM, KM12C, SNU-C1, and SNU-C4), the addition of HGF activated ERK1/2 and AKT via MET phosphorylation. In the cytotoxicity assays, Caco-2 and SNU-C4 were relatively sensitive to cetuximab compared with the others and, in the presence of HGF, cetuximab sensitivity was significantly decreased in the two cells. Moreover, capmatinib, a MET inhibitor, abrogated the effect of HGF on common downstream signaling pathways of EGFR and MET and thus the cetuximab resistance was significantly overcome in vitro. Conclusions: Among patients with mCRC receiving cetuximab + FOLFIRI, those with high plasma HGF levels had significantly worse PFS and OS. HGF induced cetuximab resistance by AKT and ERK activation while capmatinib, a MET inhibitor, significantly increased the anti-tumor effects of cetuximab in the presence of HGF in vitro. Our data may provide evidence of future clinical trials of dual EGFR and MET targeting strategies in patients with HGF-high, RAS/RAF wild-type mCRC. Citation Format: Sang-A Kim, Hyejoo Park, Kui-Jin Kim, Ji-Won Kim, Ji Hea Sung, Milang Nam, Ju Hyun Lee, Eun Hee Jung, Koung Jin Suh, Ji Yun Lee, Se Hyun Kim, Jeong-Ok Lee, Jin Won Kim, Yu Jung Kim, Jee Hyun Kim, Soo-Mee Bang, Jong Seok Lee, Keun-Wook Lee. Cetuximab resistance induced by hepatocyte growth factor is overcome by MET inhibition in KRAS, NRAS, and BRAF wild-type colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5138.

Abstract 2651: The tankyrase inhibitor OM-153 demonstrates anti-tumor effect with a therapeutic index above 10

Abstract The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) poly-ADP-ribosylate target proteins, including AXIN proteins, to earmark them for degradation by the ubiquitin-proteasomal system. Hence, inhibition of TNKS1/2 can stabilize AXIN proteins, and consequently β-catenin degradosomes, resulting in inhibition of WNT/β-catenin signaling. Although several potent small-molecules have been developed to inhibit TNKS1/2 and oncogenic WNT/β-catenin signaling, also showing convincing anti-tumor effects in numerous mouse cancer models, there are currently no TNKS1/2 inhibitors available in the clinic. The development has mainly been disadvantaged by concerns over previous reports unfolding intestinal toxicity, apparently caused by on-target and WNT/β-catenin signaling pathway-specific side effects, and a deficient therapeutic index. Here we show that the novel, highly potent and selective1,2,4-triazole-based TNKS1/2 inhibitor OM-153, displaying improved ADME-properties and mouse pharmacokinetics, reduced WNT/β-catenin signaling and tumor progression in COLO 320DM colon cancer xenografts upon peroral (PO) administration of 0.33-10 mg/kg twice daily (BID). In addition, combined OM-153 and anti-PD-1 treatment conferred a synergistic anti-tumor effect in a B16-F10 mouse melanoma model. In a 28-day repeated dose mouse toxicity study, body weight loss, intestinal damage and tubular damage in the kidney was documented after PO BID administration of 100 mg/kg. In contrast, in mice treated PO BID using 10 mg/kg, the intestinal architecture was intact and no atypical histopathological changes were observed in other organs, while clinical biochemistry and haematological analyses did not identify any changes indicating considerable toxicity. The results provide a scaffold for using OM-153 as a potential anti-cancer treatment and additional preclinical and clinical evaluations. Citation Format: Shoshy Alam Brinch, Enya Amundsen-Isaksen, Sandra Espada, Aleksandra Aizenshtadt, Lone Holmen, Clara Hammarström, Merete Høyem, Hanne Scholz, Gunnveig Grødeland, Sven T. Sowa, Albert Galera-Prat, Lari Lehtiö, Ilonka A.T.M. Meerts, Ruben G. G. Leenders, Anita Wegert, Stefan Krauss, Jo Waaler. The tankyrase inhibitor OM-153 demonstrates anti-tumor effect with a therapeutic index above 10 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2651.

Evaluation of antitumor effects of VEGFR-2 inhibitor F16in a colorectal xenograft model.

Colorectal cancer(CRC) is the third most prevalent type of cancer in the United States. The treatment options for cancer include surgery, chemotherapy, radiation, and/or targeted therapy, which show significant improvement in overall survival. Among the various available treatments, antagonizing VEGF/VEGFR-2 pathways have shown effectiveness in limiting colorectal cancer growth and improving clinical outcomes. In this regard, we hypothesized that F16, a novel VEGFR-2 inhibitor, would control colorectal cancer growth by blocking the VEGFR-2 singling pathway in both in vitro and in vivo conditions. Therefore, the current study was aimed to analyze the efficacy of F16 on the growth of Colo 320DM cells under in vitro and in vivo conditions. Human RT2 profiler PCR array analysis results clearly showed that angiogenesis and anti-apoptosis-related gene expressions were significantly reduced in HUVEC cells after F16 (5μM) treatment. In addition, Western blot results revealed that F16 attenuated the downstream signaling of the VEGFR-2 pathway in HUVEC cells by up-regulating the p53 and p21 levels and down-regulating the p-AKT and p-FAK levels. Accordingly, F16 confirmed potent cytotoxic effects against the cell viability of Colo 320DM tumors, with an IC50 valueof 9.52 ± 1.49µM. Furthermore, treatment of mice implanted with Colo 320DM xenograft tumors showed a significant reduction in tumor growth and increases in survival rate compared to controls. Immunohistochemistry analysis of tumor tissues showeda reduction in CD31 levels also in F16 treated groups. These results justify further evaluation of F16 as a potential new therapeutic agent for treating colorectal cancers.

Development of a 1,2,4-Triazole-Based Lead Tankyrase Inhibitor: Part II.

Tankyrase 1 and 2 (TNKS1/2) catalyze post-translational modification by poly-ADP-ribosylation of a plethora of target proteins. In this function, TNKS1/2 also impact the WNT/β-catenin and Hippo signaling pathways that are involved in numerous human disease conditions including cancer. Targeting TNKS1/2 with small-molecule inhibitors shows promising potential to modulate the involved pathways, thereby potentiating disease intervention. Based on our 1,2,4-triazole-based lead compound 1 (OM-1700), further structure–activity relationship analyses of East-, South- and West-single-point alterations and hybrids identified compound 24 (OM-153). Compound 24 showed picomolar IC50 inhibition in a cellular (HEK293) WNT/β-catenin signaling reporter assay, no off-target liabilities, overall favorable absorption, distribution, metabolism, and excretion (ADME) properties, and an improved pharmacokinetic profile in mice. Moreover, treatment with compound 24 induced dose-dependent biomarker engagement and reduced cell growth in the colon cancer cell line COLO 320DM.

Wogonoside: Anti-human colon cancer activities and survey of HMG-CoA ‎reductase inhibition properties with molecular modeling

IntroductionThe biological activities and interactions of wogonoside in the presence of HMG-COA reductase were investigated using the molecular docking study as a versatile theoretical approach. Wogonoside showed a considerable binding affinity to the enzyme with a docking score of -7.582 kcal/mol.Material and methodsThe in vitro cytotoxic and anti- colon ‎ carcinoma effects of biologically synthesized Wogonoside ‎against GP5d, MDST8‎, HCA-46‎, HT115, LS174T, and COLO 320DM cancer cell lines were ‎assessed.ResultsThe results indicated that the compound makes hydrophobic contacts with essential residues of the catalytic domain of the enzyme. Therefore, wogonosid could be considered as a potential inhibitor for HMG-COA reductase. The IC50 of the Wogonoside were 105, 198, 173, 382, 71, and 183 µg/mL against GP5d, ‎MDST8‎, HCA-46‎, HT115, LS174T, and COLO 320DM cancer cell lines. The anti-colon‎ ‎carcinoma properties of the Wogonoside could significantly remove GP5d, MDST8‎, HCA-46‎, ‎HT115, LS174T, and COLO 320DM cancer cell lines in a time and concentration-dependent ‎manner by MTT assay. ‎ConclusionsIt appears that the anti-human colorectal carcinoma effect of recent nanoparticles is due to their antioxidant effects. We have obtained results for the HMG-CoA Reductase enzyme at the micromolar level. In our study, inhibition result of on HMG-CoA reductase showed lower values 28.70±4.73 micromolar.

Wnt Signaling and Cadherin Expressions in Different Staging of Colorectal Cancer as Biomarkers for Metastasis: Study of SW480, COLO 320DM, and HCT116 Cellines

BACKGROUND: Early metastases is still unresolved problem in cancer management, eventually in colorectal cancer (CRC). In addition, many markers are useful just only in the late stage of CRC. AIM: This study evaluates the differences in the expression intensity of nuclear β-catenin, cytoplasmic β-catenin, E-cadherin, and N-cadherin between CRC SW480 cell line as control group and COLO320DM and HCT116 cell lines as case groups. MATERIALS AND METHODS: This study applied experimental research design with the different test methods. Culture growing and subcultures manufacturing for the CRC cell line models were done initially and followed by the immunofluorescence method by administering antibodies on β-catenin, E-cadherin, and N-cadherin, and continued with staining process using fluorescein-5-isothiocyanate and 4’, 6-diamidino-2-phenylindole. Observations were done using an immunofluorescence microscope. Calculation of area density in each cell to perceive the expressions of cytoplasmic and nuclear β-catenin, E-cadherin, and N-cadherin was conducted using ImageJ software, resulted in mean fluorescence intensity. RESULTS: There are significant differences in the expressions of cytoplasmic β-catenin, nuclear β-catenin, E-cadherin, and N-cadherin among SW480, COLO320DM, and HCT116 cell lines (p < 0.05). Despite no significant differences in cytoplasmic and nuclear β-catenin expressions between SW480 and HCT116 cell lines, and in E-cadherin and N-cadherin expressions between COLO320DM and HCT116 cell lines (p > 0.05). SW480 cell line has a higher expression of nuclear β-catenin than the cytoplasm (p < 0.05). CONCLUSION: This study reveals differences in the expression of nucleic and cytoplasmic β-catenin, E-cadherin, and N-cadherin in three stages of CRC (Duke B, C, and D) refer to different activation invasion, migration, and metastatic processes. Furthermore, the high expression of nuclear β-catenin and N-cadherin in the early stage of CRC indicate there is a metastatic process in that stage, so nuclear β-catenin and cadherin can be considered as potential biomarkers in the early stage of this cancer.

New insights of RA-V cyclopeptide as an autophagy inhibitor in human COLO 320DM cancer cell lines

Colon cancer is the leading cause for the malignancy in the gastrointestinal tract. Autophagy is a self-degradation process of the unnecessary, injured and aged organelles and proteins in the cell, which is followed by recovering of degraded products. Apoptosis is a programmed cell death which is characterized by membrane blebbing, chromosome condensation and nuclear fragmentation. Apoptosis and autophagy can occur frequently in a cell, predominantly in a series preceding apoptosis through autophagy by the formation of autophagosomes. In current research, the impact of autophagy inhibition and apoptosis activation were found to be the targeted strategies to treat colon cancer. This study is focused on the apoptotic potential of RA-V, a natural cyclopeptide through the inhibition of protective autophagy in colon cancer cells. Growth inhibitory properties were observed in the RA-V treated (125 μM) colo 320DM cells using cell viability assay. RA-V induced apoptosis of colo 320DM cells at the maximum concentration of 125 μM, which was observed using DAPI and Annexin - PI staining methods. In this study we also examined the mechanistic role of RA-V (125 μM) incolo 320DM cells in the presence of Rapamycin (mTOR inhibitor) and chloroquine (autophagy inhibitor) using MDC and AO staining methods.

SIRT1 stabilizes extrachromosomal gene amplification and contributes to repeat-induced gene silencing

Sirtuin 1 (SIRT1) is a protein deacetylase that maintains genome stability by preventing the activation of latent replication origins. Amplified genes in cancer cells localize on either extrachromosomal double minutes (DMs) or the chromosomal homogeneously staining region. Previously, we found that a plasmid with a mammalian replication initiation region and a matrix attachment region spontaneously mimics gene amplification in cultured animal cells and efficiently generates DMs and/or an homogeneously staining region. Here, we addressed the possibility that SIRT1 might be involved in initiation region/matrix attachment region–mediated gene amplification using SIRT1-knockout human COLO 320DM cells. Consequently, we found that extrachromosomal amplification was infrequent in SIRT1-deficient cells, suggesting that DNA breakage caused by latent origin activation prevented the formation of stable extrachromosomal amplicons. Moreover, we serendipitously found that reporter gene expression from the amplified repeats, which is commonly silenced by repeat-induced gene silencing (RIGS) in SIRT1-proficient cells, was strikingly higher in SIRT1-deficient cells, especially in the culture treated with the histone deacetylase inhibitor butyrate. Compared with the SIRT1-proficient cells, the gene expression per copy was up to thousand-fold higher in the sorter-isolated highest 10% cells among the SIRT1-deficient cells. These observations suggest that SIRT1 depletion alleviates RIGS. Thus, SIRT1 may stabilize extrachromosomal amplicons and facilitate RIGS. This result could have implications in cancer malignancy and protein expression.

GREEN SYNTHESIS OF GOLD NANOPARTICLES OF ISOLATED CITRUS BIOFLAVONOID FROM ORANGE: CHARACTERIZATION AND IN VITRO CYTOTOXICITY AGAINST COLON CANCER CELL LINES COLO 320DM AND HT29

The aim of the present research was to perform green synthesis of gold nanoparticles of isolated citrus bioflavonoid from Citrus sinensis (orange) peel extract and to evaluate its anticancer potential. Methanolic extract of orange peel was obtained by Soxhlet extraction and citrus bioflavonoid was isolated by using column chromatography. Gold nanoparticles were developed by green synthesis method, wherein 1 % aqueous solution of isolated citrus bioflavonoid were mixed with 1% solution of HAuCl4 and incubated at ambient temperature for 4 to 5 hours and observed for the color change which indicated formation of nanoparticles. Obtained gold nanoparticles were evaluated by UV visible spectra, FTIR, SEM, XRD analysis and for antimicrobial potential against E coli, S.aureus and P. aeruginosa. Cytotoxicity study was carried out by using in vitro assays, namely MTT, SRB and Tryphan blue assay, against colon cancer cell line Colo 320 DM, and HT 29. results of SEM showed that nanosized particles in the range of 80-100nm were formed. Results of cytotoxicity studies revealed that CBFGNP exhibited 72.28% inhibition, against Colo320 DM whereas pure CBF showed 70.46% inhibition. Against HT 29, CBFGNP exhibited 69.79% inhibition, whereas for MTT assay and SRB assay, CBFGNP showed 80.15% and 58.29% inhibition, respectively. Moreover, CBFGNP exhibited 90.29% and 85% non viability against Colo320 DM and HT29. Based on the results, it can be concluded that gold nanoparticles of citrus bioflavonoid (CBFGNP) exhibits more cytotoxicity against Colo320 DM and HT29 as compared to pure citrus bioflavonoid when assessed by three different in vitro cytotoxicity assays.

Anti-proliferation and radiation-sensitizing effect of an anthocyanidin- rich extract from purple-shoot tea on colon cancer cells

Teas are known to have several health promoting benefits due to polyphenol contents. Two varieties of tea cyanins, Purple-Shoot Tea (not registered) and TTES No. 12, were selected, dehydrated or prepared to green tea to evaluate the anti-proliferative and radiation-sensitizing effects on colon cancer cells. The tea cyanins were extracted by using three drying treatments which are freeze-dried, hot-air dried and green tea manufacturing. The composition analysis showed that the extracts of purple-shoot teas contained more polyphenols, flavonoids, condensed tannins, anthocyanins and proanthocyanidins than TTES No. 12 under various tea processes. Lyophilized tea samples had better antioxidant capacities and reducing power than those of samples from hot-air dried and green tea manufacturing. Anti-proliferation results from HT-29, Colo 320DM and CT-26 cell lines showed that Purple-shoot tea had prominent inhibitory activities which might be correlated to its antioxidative properties. The cell cycle studies confirmed that Purple-shoot tea arrested colon cancer cell at G0/G1 phase. Tested tea samples had radiation-sensitizing effect on the CT-26 cell with 2Gy Co-60 irradiation. Higher anthocyanidin and anthocyanin contents in Purple-shoot tea may contribute to enhanced cytotoxicity and induction of apoptosis after irradiation. The radiation-sensitive effect of Purple-shoot tea on carcinoma cells suggested that nature compounds such as tea polyphenol can be utilized to reduce the dosage in radiotherapy in the future.

Preclinical Lead Optimization of a 1,2,4-Triazole Based Tankyrase Inhibitor.

Tankyrases 1 and 2 are central biotargets in the WNT/β-catenin signaling and Hippo signaling pathways. We have previously developed tankyrase inhibitors bearing a 1,2,4-triazole moiety and binding predominantly to the adenosine binding site of the tankyrase catalytic domain. Here we describe a systematic structure-guided lead optimization approach of these tankyrase inhibitors. The central 1,2,4-triazole template and trans-cyclobutyl linker of the lead compound 1 were left unchanged, while side-group East, West, and South moieties were altered by introducing different building blocks defined as point mutations. The systematic study provided a novel series of compounds reaching picomolar IC50 inhibition in WNT/β-catenin signaling cellular reporter assay. The novel optimized lead 13 resolves previous atropisomerism, solubility, and Caco-2 efflux liabilities. 13 shows a favorable ADME profile, including improved Caco-2 permeability and oral bioavailability in mice, and exhibits antiproliferative efficacy in the colon cancer cell line COLO 320DM in vitro.

Double-strand breakage in the extrachromosomal double minutes triggers their aggregation in the nucleus, micronucleation, and morphological transformation.

Gene amplification plays a pivotal role in malignant transformation. Amplified genes often reside on extrachromosomal double minutes (DMs). Low-dose hydroxyurea induces DM aggregation in the nucleus which, in turn, generates micronuclei composed of DMs. Low-dose hydroxyurea also induces random double-strand breakage throughout the nucleus. In the present study, we found that double-strand breakage in DMs is sufficient for induction of DM aggregation. Here, we used CRISPR/Cas9 to introduce specific breakages in both natural and artificially tagged DMs of human colorectal carcinoma COLO 320DM cells. Aggregation occurred in the S phase but not in the G1 phase within 4 hours after breakage, which suggested the possible involvement of homologous recombination in the aggregation of numerous DMs. Simultaneous detection of DMs and the phosphorylated histone H2AX revealed that the aggregation persisted after breakage repair. Thus, the aggregate generated cytoplasmic micronuclei at the next interphase. Our data also suggested that micronuclear entrapment eliminated the DMs or morphologically transformed them into giant DMs or homogeneously staining regions (HSRs). In this study, we obtained a model explaining the consequences of DMs after double-strand breakage in cancer cells. Because double-strand breakage is frequently involved in cancer therapy, the model suggests how it affects gene amplification.

Abstract 3447: APC truncating mutations in Middle Eastern population: Tankyrase inhibitor is an effective strategy to sensitize APC mutant CRC to 5-FU chemotherapy

Abstract Colorectal Cancer (CRC) is the leading cause of cancer related death and the most common cancer affecting Saudi males. Mutation of the adenomatous polyposis coli (APC) gene is considered as the initiating step of transformation in familial and sporadic CRC. The prevalence and prognostic role of APC mutation spectra are not well established in Middle Eastern CRC. Next generation sequencing of 412 CRC tumors was performed to determine mutations in APC. Association with protein expression and clinico-pathological correlation was performed. APC truncating mutation is found at a frequency of 58.2% (240/412) and is significantly associated with superior overall survival, whereas no difference in survival between short truncating and long truncating APC was noted. To test the hypothesis that APC mutations might influence 5- Fluorouracil (5-FU) therapy, which is the first line chemotherapy used in CRC, we compared CRC cell lines and showed that those expressing truncated APC exhibit limited response to 5-FU, unlike cells which were APC wild type. In COLO-320 DM and HCT-15, APC mutated CRC cells, we were able to increase sensitivity of APC truncated cells to 5-FU by inhibiting Tankyrase (TNKs) which are members of PARP family, using XAV939. In conclusion, the study identified the prognostic value of APC truncating mutation in Middle Eastern ethnicity. In vitro data further showed that APC mutation can be used as a molecular biomarker to predict response to 5-FU. Furthermore, Tankyrase inhibitor XAV939 selectively targeting APC-mutated CRC cells can be an effective strategy to overcome 5-FU resistance in these cells. Note: This abstract was not presented at the meeting. Citation Format: Sandeep K. Parvathareddy, Abdul K. Siraj, Rong Bu, Prateeshkumar Poyil, Tariq Masoodi, Divya Sasidharan Padmaja, Rica Concepcion, Rafia Begum, Maria A. Sabido, Roxanne Melosantos, Nasser Al-Sanea, Fouad Al-Dayel, Khawla S. Al-Kuraya. APC truncating mutations in Middle Eastern population: Tankyrase inhibitor is an effective strategy to sensitize APC mutant CRC to 5-FU chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3447.

Synergistic Effect of Combined Treatment with Longan Flower Extract and 5-Fluorouracil on Colorectal Cancer Cells

To investigate the influence of longan flower extract (LFE) on the sensitization of colorectal cancer (CRC) cells to 5-fluorouracil (5-FU) treatment, HT-29, Colo 320DM and SW480 cells were treated with LFE and 5-FU alone and in combination, and the cell viability was then assessed by trypan blue exclusion, the cell cycle by propidium iodide staining, the mitochondria membrane potential by rhodamine 123 staining, and the expression levels of associated genes by immunoblotting and quantitative real-time polymerase chain reaction. LFE and 5-FU synergistically inhibited cell proliferation of HT-29 and Colo 320DM cells. Combined treatment also elevated the level of loss of mitochondria membrane potential of these two CRC cells and arrested HT-29 cells in the S phase of the cell cycle, in association with down-regulation of cyclin A mRNA expression. LFE synergistically potentiated chemosensitivity to 5-FU in at least two CRC cell lines. The results indicated that LFE has potential as a novel agent for the sensitization of CRC cells to 5-FU.

Abstract 5873: BAY-3827, a selective inhibitor of AMPK for the evaluation of the role of AMPK in Myc-dependent tumors

Abstract The AMP-activated protein kinase (AMPK) is a sensor of the energy status in the cells, playing a key role in controlling their metabolism. For many years, AMPK was mainly perceived as a tumor suppressor in agreement with being a component of the LKB1 tumor suppressor cascade, which inhibits mTORC1. However, in the last few years, some studies suggested that AMPK might actually exert a pro-tumorigenic role in certain contexts. For instance, Liu and colleagues demonstrated that dysregulated MYC expression renders tumor cells sensitive to AMPK depletion (Liu et al, 2012. Nature). The authors showed that, due to their increased anabolism, MYC-dependent cells rely on AMPK to restore ATP levels and to prevent an energy crisis that results in apoptosis and cell death. Here we report the discovery of a new lead structure for the inhibition of AMPK by biochemical high throughput screening. The optimization of this lead structure towards potency and selectivity led to the probe compound BAY-3827 and the use of this tool compound to evaluate the therapeutic potential of AMPK inhibition in MYC-dependent tumors. To demonstrate a cellular effect of BAY-3827 an HRTF® assay (Homogeneous Time Resolved Fluorescence, cisbio) for phospho-Acetyl-CoA carboxylase (p-ACC, Ser79), a direct substrate of AMPK, was used. ACC phosphorylation was strongly inhibited by BAY-3827 in COLO 320DM and IMR-32 cells. However, despite its high potency, BAY-3827 failed to inhibit the proliferation of cells with dysregulated c-MYC or N-MYC. In conclusion, we have identified a potent and selective AMPK inhibitor. Despite demonstrated inhibition of AMPK kinase activity, BAY-3827 treatment did not translate into antiproliferative activity in MYC-dependent cells. While we could not confirm our initial hypothesis, one might speculate that inhibition of AMPK might be of therapeutic utility in other biological contexts. Therefore, the availability of potent and selective inhibitors, as described here, will contribute to further insight into the potential of AMPK inhibition as a therapeutic target in cancer. Citation Format: Clara Lemos, Volker K. Schulze, Benjamin Bader, Clara D. Christ, Hans Briem, Oliver Politz, Florian Prinz, Simon Holton, Tobias Heinrich, Julien Lefranc, Philip Lienau, Arne Scholz, Franz von Nussbaum, Carl Friedrich Nising, Dominik Mumberg, Marcus Bauser, Andrea Hägebarth. BAY-3827, a selective inhibitor of AMPK for the evaluation of the role of AMPK in Myc-dependent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5873.

Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells.

Plasmids with both a mammalian replication initiation region (IR) and a matrix attachment region (MAR) are spontaneously amplified in transfected cells, and generate extrachromosomal double minute (DM) or chromosomal homogeneously staining region (HSR). We previously isolated the shortest core IR (G5) required for gene amplification. In this study, we ligated the G5 DNA to create direct or inverted repeats, mixed the repeats with an expression plasmid, and transfected the mixture into human COLO 320DM or hamster CHO DG44 cells. Consequently, we found that the transfected sequence generated DMs or HSR where, surprisingly, the plasmid sequence was embedded within a long stretch of G5 sequences. The amplified structure from the direct G5 repeats was stable, whereas that from the inverted repeats was not. The amplification might be explained by the efficient replication/multimerization of the G5 repeat and recombination with the co-transfected plasmid in an extrachromosomal context. The product might then be integrated into a chromosome arm to generate a HSR. The expression from the plasmid within the long G5 array was much higher than that from a simple plasmid repeat. Because G5 is a core IR that favors gene expression, a long array of G5 provides an excellent environment for gene expression from the embedded plasmid.

Curcumin inhibits growth potential by G1 cell cycle arrest and induces apoptosis in p53-mutated COLO 320DM human colon adenocarcinoma cells

Curcumin, a natural polyphenolic compound and it is isolated from the rhizome of Curcuma longa, have been reported to possess anticancer effect against stage I and II colon cancer. However, the effect of curcumin on colon cancer at Dukes’ type C metastatic stage III remains still unclear. In the present study, we have investigated the anticancer effects of curcumin on p53 mutated COLO 320DM human colon adenocarcinoma cells derived from Dukes’ type C metastatic stage. The cellular viability and proliferation were assessed by trypan blue exclusion assay and MTT assay, respectively. The cytotoxicity effect was examined by lactate dehydrogenase (LDH) cytotoxicity assay. Apoptosis was analyzed by DNA fragmentation analysis, Hoechst and propidium iodide double fluorescent staining and confocal microscopy analysis. Cell cycle distribution was performed by flow cytometry analysis. Here we have observed that curcumin treatment significantly inhibited the cellular viability and proliferation potential of p53 mutated COLO 320DM cells in a dose- and time-dependent manner. In addition, curcumin treatment showed no cytotoxic effects to the COLO 320DM cells. DNA fragmentation analysis, Hoechst and propidium iodide double fluorescent staining and confocal microscopy analysis revealed that curcumin treatment induced apoptosis in COLO 320DM cells. Furthermore, curcumin caused cell cycle arrest at the G1 phase, decreased the cell population in the S phase and induced apoptosis in COLO 320DM colon adenocarcinoma cells. Together, these data suggest that curcumin exerts anticancer effects and induces apoptosis in p53 mutated COLO 320DM human colon adenocarcinoma cells derived from Dukes’ type C metastatic stage.

The Discovery and Characterization of K-756, a Novel Wnt/β-Catenin Pathway Inhibitor Targeting Tankyrase.

The Wnt/β-catenin pathway is a well-known oncogenic pathway. Its suppression has long been considered as an important challenge in treating cancer patients. Among colon cancer patients in particular, most patients carry an adenomatous polyposis coli (APC) mutation that leads to an aberration of Wnt/β-catenin pathway. To discover the small molecule inhibitors of the Wnt/β-catenin pathway, we conducted high-throughput screening in APC-mutant colon cancer DLD-1 cells using a transcriptional reporter assay, which identified a selective Wnt/β-catenin pathway inhibitor, K-756. K-756 stabilizes Axin and reduces active β-catenin, and inhibits the genes downstream of endogenous Wnt/β-catenin. We subsequently identified that K-756 is a tankyrase (TNKS) inhibitor. TNKS, which belongs to the PARP family, poly-ADP ribosylates Axin and promotes Axin degradation via the proteasome pathway. K-756 binds to the induced pocket of TNKS and inhibits its enzyme activity. Moreover, PARP family enzyme assays showed that K-756 is a selective TNKS inhibitor. K-756 inhibited the cell growth of APC-mutant colorectal cancer COLO 320DM and SW403 cells by inhibiting the Wnt/β-catenin pathway. An in vivo study showed that the oral administration of K-756 inhibited the Wnt/β-catenin pathway in colon cancer xenografts in mice. To further explore the therapeutic potential of K-756, we also evaluated the effects of K-756 in non-small cell lung cancer cells. Although a single treatment of K-756 did not induce antiproliferative activity, when K-756 was combined with an EGFR inhibitor (gefitinib), it showed a strong synergistic effect. Therefore, K-756, a novel selective TNKS inhibitor, could be a leading compound in the development of anticancer agents. Mol Cancer Ther; 15(7); 1525-34. ©2016 AACR.