Colon Cancer Cell Line LS174T Research Articles

O-GlcNAcylation inhibition redirects the response of colon cancer cells to chemotherapy from senescence to apoptosis.

The potential use of pro-senescence therapies, known as TIS (Therapy-Induced Senescence), for the treatment of colorectal cancer (CRC) generated significant interest since they require lower doses compared to those required for inducing apoptosis. However, the senescent cell cycle-arrested cancer cells are long-lived, and studies have revealed escape mechanisms contributing to tumor recurrence. To deepen our understanding of the survival pathways used by senescent cancer cells, we delved into the potential involvement of the hexosamine biosynthetic pathway (HBP). HBP provides UDP-GlcNAc, the substrate for O-GlcNAc transferase (OGT), which catalyzes O-GlcNAcylation, a post-translational modification implicated in regulating numerous cellular functions and aberrantly elevated in CRC. In this study, we demonstrated, in the p53-proficient colon cancer cell lines HCT116 and LS174T, that TIS induced by low-dose SN38 or etoposide treatment was accompanied with a decrease of GFAT (the rate limiting enzyme of the HBP), OGT and O-GlcNAcase (OGA) expression correlated with a slight reduction in O-GlcNAcylation levels. Further decreasing this level of O-GlcNAcylation by knocking-down GFAT or OGT redirected the cellular response to subtoxic chemotherapy doses from senescence to apoptosis, in correlation with an enhancement of DNA damages. Pharmacological inhibition of OGT with OSMI-4 in HCT116 and LS174T cells and in a patient-derived colon tumoroid model supported these findings. Taken together, these results suggest that combing O-GlcNAcylation inhibitors to low doses of conventional chemotherapeutic drugs could potentially reduce treatment side effects while preserving efficacy. Furthermore, this approach may increase treatment specificity, as CRC cells exhibit higher O-GlcNAcylation levels compared to normal tissues.

ST3GalIV drives SLeX biosynthesis in gastrointestinal cancer cells and associates with cancer cell motility

Expression of sialyl Lewis X (SLeX) is a well-documented event during malignant transformation of cancer cells, and largely associates with their invasive and metastatic properties. Glycoproteins and glycolipids are the main carriers of SLeX, whose biosynthesis is known to be performed by different glycosyltransferases, namely by the family of β-galactoside-α2,3-sialyltransferases (ST3Gals). In this study, we sought to elucidate the role of ST3GalIV in the biosynthesis of SLeX and in malignant properties of gastrointestinal (GI) cancer cells. By immunofluorescent screening, we selected SLeX-positive GI cancer cell lines and silenced ST3GalIV expression via CRISPR/Cas9. Flow cytometry, immunofluorescence and western blot analysis showed that ST3GalIV KO efficiently impaired SLeX expression in most cancer cell lines, with the exception of the colon cancer cell line LS174T. The impact of ST3GalIV KO in the biosynthesis of SLeX isomer SLeA and non sialylated Lewis X and A were also evaluated and overall, ST3GalIV KO led to a decreased expression of SLeA and an increased expression in both LeX and LeA. In addition, the abrogation of SLeX on GI cancer cells led to a reduction in cell motility. Furthermore, ST3GalVI KO was performed in LS174T ST3GalIV KO cells, resulting in the complete abolishment of SLeX expression and consequent reduced motility capacity of those cells. Overall, these findings portray ST3GalIV as the main, but not the only, enzyme driving the biosynthesis of SLeX in GI cancer cells, with a functional impact on cancer cell motility.

Anti-proliferative Effects of Ocimum basilicum Leaf Aqueous Extract on Colon Cancer Cell Lines and the Expression of Apoptotic Genes

Background: Colon cancer is one of the most diagnosed cancers in the modern world. Chemotherapy is one of the most relevant treatments for colon cancer, but this method of treatment has severe and sometimes irreversible side effects. Therefore, alternative treatments with fewer side effects are highly considered. Natural and herbal medicine has been proven to be a great source for these alternative treatment methods. Objectives: This study aimed to investigate the anti-proliferative and cytotoxic effects of Ocimum basillicum (OB) leaf aqueous extract on human colon cancer cell lines LS174T and COLO205 and the expression of apoptotic genes. Methods: Methyl thiazol tetrazolium (MTT) assay was performed to assess the cytotoxic effects of our extract on the aforementioned cell lines. The changes in the expression of BAX and BAD were analyzed by qPCR following the treatment of cells with our extract. Results: The results of the MTT assay indicated that the extract had a cytotoxic effect and could reduce the population of live cancer cells, especially LS174T cells. The expression of BAX and BAD genes showed a significant increase in qPCR analysis in both cell lines, especially in the LS174T cells. Conclusions: Our results showed that the extract had cytotoxic, anti-proliferative, and pro-apoptotic characteristics on previously mentioned cell lines, especially on the LS 174T cells.

Mechanism of acquired 5FU resistance and strategy for overcoming 5FU resistance focusing on 5FU metabolism in colon cancer cell lines.

Fluorouracil (5FU) is converted to its active metabolite fluoro-deoxyuridine monophosphate (FdUMP) through the orotate phosphoribosyl transferase (OPRT)-ribonucleotide reductase (RR) pathway and thymidine phosphatase (TP)-thymidine kinase (TK) pathway and inhibits thymidylate synthase (TS), leading to inhibition of thymidine monophosphate (dTMP) synthesis through a de novo pathway. We investigated the mechanism of 5FU resistance and strategies to overcome it by focusing on 5FU metabolism. Colon cancer cell lines SW48 and LS174T and 5FU-resistant cell lines SW48/5FUR and LS174T/5FUR were used. FdUMP amount was measured by western blotting. The FdUMP synthetic pathway was investigated by combining TP inhibitor (tipiracil hydrochloride; TPI) or RR inhibitor (hydroxyurea; HU) with 5FU. Drug cytotoxicity was observed by crystal violet staining assay. FdUMP was synthesized through the OPRT-RR pathway in SW48 cells but was scarcely synthesized through either the OPRT-RR or TP-TK pathway in SW48/5FUR cells. FdUMP amount in SW48/5FUR cells was reduced by 87% vs. SW48 cells. Expression levels of OPRT and TP were lower in SW48/5FUR when compared with these levels in the SW48 cells, indicating decreased synthesis of FdUMP-led 5FU resistance. These results indicated that fluoro-deoxyuridine (FdU) rather than 5FU promotes FdUMP synthesis and overcomes 5FU resistance. Contrastingly, FdUMP was synthesized through the OPRT-RR and TP-TK pathways in LS174T cells but mainly through the TP-TK pathway in LS174T/5FUR cells. FdUMP amount was similar in LS174T/5FUR vs. the LS174T cells. OPRT and RR expression was lower and TK expression was higher in LS174T/5FUR vs. the LS174T cells, indicating that dTMP synthesis increased through the salvage pathway, thus leading to 5FU resistance. LS174T/5FUR cells also showed cross-resistance to FdU and TS inhibitor, suggesting that nucleoside analogs such as trifluoro-thymidine should be used to overcome 5FU resistance in these cells. 5FU metabolism and mechanisms of 5FU resistance are different in each cell line. Both synthesized FdUMP amount and FdUMP sensitivity should be considered in 5FU-resistant cells.

Humanized Anti–Tumor-Associated Glycoprotein–72 for Submillimeter Near-Infrared Detection of Colon Cancer in Metastatic Mouse Models

BackgroundTumor-associated glycoprotein (TAG)–72 is a pancarcinoma antigen that is overexpressed in greater than 80% of colorectal adenocarcinomas. CC49 is a TAG-72–specific antibody. The aim of the present study was to demonstrate selective imaging of colon tumors and metastases with the humanized TAG-72 antibody (anti-huCC49) conjugated to a near-infrared fluorophore in orthotopic mouse models. MethodsAnti-huCC49 was conjugated to near-infrared dye IR800CW. Mouse imaging was performed with the Pearl Trilogy Small Animal and FLARE Imaging Systems. Subcutaneous mouse models of colon cancer cell line LS174T were used to determine the optimal dose of administration and timing of imaging. Orthotopic mouse models of LS174T were established by surgical orthotopic implantation of LS174T tumors onto the serosa of the cecum. Peritoneal carcinomatosis models were established by injection of LS174T cells into the peritoneum of nude mice. Mice were administered anti-huCC49–IR800 via tail vein injection. Mice were euthanized 72 h later and imaged after laparotomy. ResultsSubcutaneous LS174T xenografts demonstrated optimal tumor detection 72 h after administration with 50 μg anti-huCC49-IR800CW. Tumors were visualized with fluorescence imaging with a mean tumor-to-liver ratio of 7.39 (standard deviation: 2.76). In the orthotopic model, metastases smaller than 1 mm were fluorescently visualized that were invisible with bright light. ConclusionsAnti-huCC49–IR800CW provides sensitive and specific imaging of colon cancer and metastases at a submillimeter resolution in metastatic nude mice models. This provides a promising near-infrared probe for the imaging of colon cancer and metastases for preoperative diagnosis and fluorescence-guided surgery.

Whole-transcriptome Analysis of Fully Viable Energy Efficient Glycolytic-null Cancer Cells Established by Double Genetic Knockout of Lactate Dehydrogenase A/B or Glucose-6-Phosphate Isomerase.

Nearly all mammalian tumors of diverse tissues are believed to be dependent on fermentative glycolysis, marked by elevated production of lactic acid and expression of glycolytic enzymes, most notably lactic acid dehydrogenase (LDH). Therefore, there has been significant interest in developing chemotherapy drugs that selectively target various isoforms of the LDH enzyme. However, considerable questions remain as to the consequences of biological ablation of LDH or upstream targeting of the glycolytic pathway. In this study, we explore the biochemical and whole transcriptomic effects of CRISPR-Cas9 gene knockout (KO) of lactate dehydrogenases A and B [LDHA/B double KO (DKO)] and glucose-6-phosphate isomerase (GPI KO) in the human colon cancer cell line LS174T, using Affymetrix 2.1 ST arrays. The metabolic biochemical profiles corroborate that relative to wild type (WT), LDHA/B DKO produced no lactic acid, (GPI KO) produced minimal lactic acid and both KOs displayed higher mitochondrial respiration, and minimal use of glucose with no loss of cell viability. These findings show a high biochemical energy efficiency as measured by ATP in glycolysis-null cells. Next, transcriptomic analysis conducted on 48,226 mRNA transcripts reflect 273 differentially expressed genes (DEGS) in the GPI KO clone set, 193 DEGS in the LDHA/B DKO clone set with 47 DEGs common to both KO clones. Glycolytic-null cells reflect up-regulation in gene transcripts typically associated with nutrient deprivation / fasting and possible use of fats for energy: thioredoxin interacting protein (TXNIP), mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), PPARγ coactivator 1α (PGC-1α), and acetyl-CoA acyltransferase 2 (ACAA2). Other changes in non-ergometric transcripts in both KOs show losses in "stemness", WNT signaling pathway, chemo/radiation resistance, retinoic acid synthesis, drug detoxification, androgen/estrogen activation, and extracellular matrix reprogramming genes. These findings demonstrate that: 1) The "Warburg effect" is dispensable, 2) loss of the LDHAB gene is not only inconsequential to viability but fosters greater mitochondrial energy, and 3) drugs that target LDHA/B are likely to be ineffective without a plausible combination second drug target.

A Purified Aspartic Protease from Akkermansia Muciniphila Plays an Important Role in Degrading Muc2.

Akkermansia muciniphila can produce various mucin-degrading proteins. However, the functional characteristics of these proteins and their role in mucin degradation are unclear. Of the predicted protein-coding genes, Amuc_1434, which encodes for a hypothetical protein, is the focus in this study. A recombinant enzyme Amuc_1434 containing the 6× His-tag produced in Escherichia coli (hereinafter termed Amuc_1434*) was isolated to homogeneity and biochemically characterised. Results showed that the enzyme can hydrolyse hemoglobin with an activity of 17.21 U/μg. The optimal pH and temperature for hemoglobin hydrolysis of Amuc_1434* were found to be around 8.0 and 40 °C, respectively. Amuc_1434* is identified as a member of the aspartic protease family through the action of inhibitor pepstatin A. Amuc_1434* promotes the adhesion of colon cancer cell line LS174T, which can highly express Muc2. Significantly Amuc_1434* can degrade Muc2 of colon cancer cells. Amuc_1434 is mainly located in the colon of BALB/c mice. These results suggest that the presence of Amuc_1434 from Akkermansia muciniphila may be correlated with the restoration of gut barrier function by decreasing mucus layer thickness.

Oxytocin Inhibition of Metastatic Colorectal Cancer by Suppressing the Expression of Fibroblast Activation Protein-α.

Oxytocin (OXT) and its receptor (OXTR) are present in the gastrointestinal system and are involved in gastrointestinal tumorigenesis. However, the effect of OXTR signaling on the development of colorectal cancer (CRC) and its underlying mechanisms remain unexplored. To address these issues, we first examined the expressions of OXT, OXTR, and several cancer-associated proteins using colon “tissue chips” from a spectrum of malignant progression of the colon, which included normal colon tissue, chronic colitis, colorectal adenoma, and colorectal adenocarcinoma (CAC). The results showed that the expressions of OXT and OXTR decreased gradually with the malignant progression of the disease. Stimulation of CAC tissues with OXT increased OXTR expression while down-regulated FAPα and CCL-2 protein expressions in a concentration- and time-dependent manner. Moreover, cell invasion experiment showed that OXT treatment reduced the invasion ability of colon cancer cells and blocking OXTR with atosiban blocked OXT-reduced invasion ability of human colon cancer cell lines Ls174T and SW480. The results indicate that OXT has the potential to inhibit CRC development via down-regulating the immunosuppressive proteins FAPα and CCL-2. When the OXTR signaling is weakened, colon tissues may transform to CRC. These findings also highlight the possibility of applying OXT to inhibit CRC development directly.

Regulation of Ketogenic Enzyme HMGCS2 by Wnt/β-catenin/PPARγ Pathway in Intestinal Cells.

The Wnt/β-catenin pathway plays a crucial role in development and renewal of the intestinal epithelium. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme in the synthesis of ketone body β-hydroxybutyrate (βHB), contributes to the regulation of intestinal cell differentiation. Here, we have shown that HMGCS2 is a novel target of Wnt/β-catenin/PPARγ signaling in intestinal epithelial cancer cell lines and normal intestinal organoids. Inhibition of the Wnt/β-catenin pathway resulted in increased protein and mRNA expression of HMGCS2 and βHB production in human colon cancer cell lines LS174T and Caco2. In addition, Wnt inhibition increased expression of PPARγ and its target genes, FABP2 and PLIN2, in these cells. Conversely, activation of Wnt/β-catenin signaling decreased protein and mRNA levels of HMGCS2, βHB production, and expression of PPARγ and its target genes in LS174T and Caco2 cells and mouse intestinal organoids. Moreover, inhibition of PPARγ reduced HMGCS2 expression and βHB production, while activation of PPARγ increased HMGCS2 expression and βHB synthesis. Furthermore, PPARγ bound the promoter of HMGCS2 and this binding was enhanced by β-catenin knockdown. Finally, we showed that HMGCS2 inhibited, while Wnt/β-catenin stimulated, glycolysis, which contributed to regulation of intestinal cell differentiation. Our results identified HMGCS2 as a downstream target of Wnt/β-catenin/PPARγ signaling in intestinal epithelial cells. Moreover, our findings suggest that Wnt/β-catenin/PPARγ signaling regulates intestinal cell differentiation, at least in part, through regulation of ketogenesis.

Abstract 3733: Development of photoimmunotherapy for colon cancer in nude mice models

Abstract Introduction: Photoimmunotherapy (PIT) uses tumor specific monoclonal antibodies conjugated to photosensitizer phthalocyanine dye IR700 to produce cytotoxicity. To date, there is no published data on the use of PIT in colorectal cancers, which could be useful as an adjunct to surgery to prevent local recurrence, or as a primary therapy. Methods: Humanized anti-CEA antibody (m5A) was conjugated to IR700CW dye (LI-COR). In vitro PIT was performed using a human colon cancer cell line LS174T. Cancer cells (2x103) were seeded on a 96-well plate and incubated for 24 hours. m5A-IR700CW containing media was added to the cells. Cells received either 2 minutes of PIT or no treatment. PIT was administered at 4 J/cm2, 8 J/cm2 or 16 J/cm2. Cell viability was measured on a microplate reader with CellTiter 96 Aqueous One Solution (Promega, Madison, WI). Subcutaneous implantation of LS174T was performed on the bilateral flanks of four male nude mice. Tail vein injection of 25 micrograms of m5A-IR700CW reconstituted in 100 microliters PBS was performed for each mouse. Animal imaging was performed with a LI-COR Pearl Trilogy imaging system. Right flank tumors of three mice were treated with a 690 nm laser at 150 mW/cm2 for 30 minutes, delivering a total of 270 J/cm2. A non-treated mouse served as the control. Images were obtained prior to PIT, immediately after PIT and 10 minutes, 20 minutes, 30 minutes and 24 hours after treatment. Maximum fluorescence intensity was measured at each time point. Tumor size was measured prior to treatment, 24 hours, 48 hours, 5 days and 10 days after treatment. Results: In vitro PIT was effective to kill the cancer cells in a dose response manner. In vivo PIT decreased tumor fluorescence intensity in a time-dependent manner. PIT arrested tumor growth in all mice, compared to the control, which demonstrated continuous tumor growth. Conclusion: PIT affects cancer cell death in vitro and arrests tumor growth in colon cancer models. Citation Format: Hannah Hollandsworth, Siamak Amirfakhri, Filemoni Filemoni, Sahar Razemjooie, Paul Yazaki, Robert Hoffman, Michael Bouvet. Development of photoimmunotherapy for colon cancer in nude mice models [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 3733.

Screen for modulators of atonal homolog 1 gene expression using notch pathway-relevant gene transcription based cellular assays.

Atonal homolog 1 (Atoh1) is a basic helix-loop-helix 9 (bHLH) transcription factor acting downstream of Notch and is required for the differentiation of sensory hair cells in the inner ear and the specification of secretory cells during the intestinal crypt cell regeneration. Motivated by the observations that the upregulation of Atoh1 gene expression, through genetic manipulation or pharmacological inhibition of Notch signaling (e.g. γ-secretase inhibitors, GSIs), induces ectopic hair cell growth in the cochlea of the inner ear and partially restores hearing after injuries in experimental models, we decided to identify small molecule modulators of the Notch-Atoh1 pathway, which could potentially regenerate hair cells. However, the lack of cellular models of the inner ear has precluded the screening and characterization of such modulators. Here we report using a colon cancer cell line LS-174T, which displays Notch inhibition-dependent Atoh1 expression as a surrogate cellular model to screen for inducers of Atoh1 expression. We designed an Atoh1 promoter-driven luciferase assay to screen a target-annotated library of ~6000 compounds. We further developed a medium throughput, real-time quantitative RT-PCR assay measuring the endogenous Atoh1 gene expression to confirm the hits and eliminate false positives from the reporter-based screen. This strategy allowed us to successfully recover GSIs of known chemotypes. This LS-174T cell-based assay directly measures Atoh1 gene expression induced through Notch-Hes1 inhibition, and therefore offers an opportunity to identify novel cellular modulators along the Notch-Atoh1 pathway.

Enhancement of B-cell translocation gene-2 inhibits proliferation and metastasis of colon cancer cells

To inhibit the proliferation and metastasis of colon cancer cells by increasing the expression level of B-cell translocation gene-2 (BTG2). Western blot assay was used to detect the expression level of BTG2 protein in the normal intestinal epithelial HIEC cells and three colon cancer cell lines SW620, HT-29 and LS174T. The expression of BTG2 protein in normal colonic epithelial tissue, colon adenoma and colon cancer tissue was detected by immunohistochemistry. The plasmid with BTG2 gene full-length sequence was transfected into colon cancer SW620 cells, and the expression of BTG2 protein was detected by Western blot. The cell growth curve was drawn by MTT test. The Ki-67-positive rate was calculated using immunofluorescence staining. The cell migration of colon cancer cells was detected by scratch test and Transwell double chamber culture system, and the pseudopodia growth of tumor cells was detected by Matrigel 3D culture system. Western blot results showed that BTG2 relative expression levels were 0.83 ± 0.12, 0.18 ± 0.04, 0.20 ± 0.05 and 0.36 ± 0.07 in normal human intestinal epithelial cells HIEC, and human colon cancer cell line SW620, HT-29 and LS174T, respectively. The results of immunohistochemistry showed that the positive expression of BTG2 protein in normal colorectal tissue, colorectal adenoma and colorectal carcinoma tissues were 82.5% (33/40), 77.5%(31/40) and 17.5% (7/40), respectively, with a significant difference between two groups (P < 0.05). Immunofluorescence results showed that the positive rate of Ki-67 in the control group, empty vector group and BTG2 transfection group was (76.2 ± 8.0)%, (81.4 ± 9.7)% and (50.1 ± 7.1)%, respectively, showing a significant difference between two groups (P < 0.05). The scratch test results showed that in the control group, empty vector group and BTG2 transfection group, the distance of SW620 cells between two sides was (79.27 ± 11.24) µm, (80.65 ± 12.17) µm and (124.77 ± 19.63) µm, respectively, with a significant difference between two groups (P < 0.05). Transwell results showed that in the control group, empty plasmid group and BTG2 transfection group, the SW620 cell migration rate was (78.5 ± 13.1)%, (73.2 ± 12.9)% and (47.4 ± 9.1)%, respectively, showing a significant difference between two groups (P < 0.05). The number of neurospheres of BTG2 transfection group was decreased SW620, which had poor ductility. BTG2 gene is involved in colon cancer cell proliferation and metastasis, and effectively restores the function of BTG2 protein. Therefore, it may be expected to become a new option in gene therapy for colon cancer.

Mesenchymal stem cell-mediated, tumor stroma-targeted radioiodine therapy of metastatic colon cancer using the sodium iodide symporter as theranostic gene.

The tumor-homing property of mesenchymal stem cells (MSCs) allows targeted delivery of therapeutic genes into the tumor microenvironment. The application of sodium iodide symporter (NIS) as a theranostic gene allows noninvasive imaging of MSC biodistribution and transgene expression before therapeutic radioiodine application. We have previously shown that linking therapeutic transgene expression to induction of the chemokine CCL5/RANTES allows a more focused expression within primary tumors, as the adoptively transferred MSC develop carcinoma-associated fibroblast-like characteristics. Although RANTES/CCL5-NIS targeting has shown efficacy in the treatment of primary tumors, it was not clear if it would also be effective in controlling the growth of metastatic disease. To expand the potential range of tumor targets, we investigated the biodistribution and tumor recruitment of MSCs transfected with NIS under control of the RANTES/CCL5 promoter (RANTES-NIS-MSC) in a colon cancer liver metastasis mouse model established by intrasplenic injection of the human colon cancer cell line LS174t. RANTES-NIS-MSCs were injected intravenously, followed by (123)I scintigraphy, (124)I PET imaging, and (131)I therapy. Results show robust MSC recruitment with RANTES/CCL5-promoter activation within the stroma of liver metastases as evidenced by tumor-selective iodide accumulation, immunohistochemistry, and real-time polymerase chain reaction. Therapeutic application of (131)I in RANTES-NIS-MSC-treated mice resulted in a significant delay in tumor growth and improved overall survival. This novel gene therapy approach opens the prospect of NIS-mediated radionuclide therapy of metastatic cancer after MSC-mediated gene delivery.

Abstract 4287: Novel method of liver tumor detection and characterization using ultrasound-induced biomarker release

Abstract Blood biomarkers can revolutionize early cancer detection by earlier detection of circulating tumor biomarkers. We have shown that cell membrane permeabilization by low frequency ultrasound can release biomarkers from tumor cells. We use this non-invasive method to increase the detectable blood biomarker level from orthotopic liver tumors in mice, allowing for tumor characterization. The ability to focus ultrasound enables localization of the biomarker-release site. This novel strategy could lead to the earlier characterization of unknown incidental lesions leading to earlier detection and localization of cancer. METHODS: The colon cancer cell line LS174T transfected with the firefly luciferase2 reporter gene was used to produce liver tumors in nude mice. This cell line produces the biomarkers carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA-19-9), which were detected using an enzyme-linked immunosorbant assay. The liver tumors were produced by either injecting 3 million cells in matrigel or transplantation of a piece of previously established subcutaneous tumor into the liver. The tumor growth was followed using bioluminescence and size was measured using ultrasound. The liver was exposed to low frequency (1MHz) ultrasound for 6 minutes, serum was collected and biomarkers measured. The increase in serum biomarker was determined by the difference in pre and post-ultrasound-treatment levels. Control samples were from tumor-bearing mice not ultrasound treated. Cells in culture were treated with the low frequency and biomarker release studied with an increase in time (0, 10, 30 min). RESULTS: Both the methods used to produce the liver tumors showed an 80% rate of tumor establishment within a week of implantation. The other 20% showed tumor growth at a slower rate. The transplantation of the previously established subcutaneous tumor piece showed a better control of the tumor size compared to the cells implanted. The tumors showed an exponential growth in three weeks (week-3 mean volume 0.815 cm3). The ultrasound treated tumors showed a significant increase in blood biomarkers released (CEA: p&amp;lt;0.007, n=10; CA19-9: p&amp;lt;0.002, n=6), when compared to controls. The cells in culture showed significant biomarker release (CEA and CA19-9 p&amp;lt;0.05) when treated with ultrasound. Previously studied cell death has been shown to be below 5 % under all conditions. CONCLUSIONS: Increase in biomarkers released from cells in culture and liver tumors was observed with ultrasound-treatment. This allows for characterization of tumors depending on the amplification of tumor specific blood biomarkers. This orthotopic liver model can be used to study tumor characterization as well as has implications in diagnostic monitoring of lesions and therapy. This novel method has a clear pathway for application of MR-guided focused ultrasound in characterization of incidental tumors, merging the fields of imaging and in vitro diagnostics. Citation Format: Aloma L. D'Souza, Xinrui Yan, Sanjiv S. Gambhir. Novel method of liver tumor detection and characterization using ultrasound-induced biomarker release. [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 4287. doi:10.1158/1538-7445.AM2014-4287

Clinicopathological and biological significance ofcriptooverexpression in human colon cancer

To assess the clinicopathological and biological significance of cripto in human colorectal cancer. Real-time reverse-transcription polymerase chain reaction (PCR) was used to examine cripto mRNA levels in primary colon cancer and normal colon tissues as well as normal and metastatic lymph nodes from colon cancers. Human colon cancer LS-174T cells were transfected with cripto small interfering RNA (siRNA), and mRNA and protein levels were evaluated using real-time PCR and western blot analysis, respectively. The growth of cancer cells was evaluated using the MTT assay and colony formation in soft agar. Invasion was examined using a Transwell assay, and the expressions of matrix metalloproteinase (MMP)-7 and MMP-9 were determined using western blot assay. Cripto was significantly overexpressed in primary colon cancer and metastatic lymph nodes. Silencing cripto gene expression with cripto siRNA resulted in a significant decrease in colony formation in soft agar in the colon cancer cell line LS-174T. Cripto siRNA treatment decreased the migration and invasion capabilities of the colon cancer cell line LS-174T in vitro. Furthermore, cripto siRNA treatment inhibited the expression of matrix MMP-7 and MMP-9. The results provide evidence that cripto siRNA could be an effective approach for the inhibition of cancer cell invasion and migration and thus has potential for use in devising novel preventive and therapeutic strategies for colon cancer metastasis.

302 Dynamic Regulation of Intestinal Crypt Base Columnar Stem Cells by Notch Signaling

Background/Aim: The intestinal epithelium is a rapidly renewed tissue, requiring continual replenishment by intestinal stem cells (ISCs) in order to sustain life. The crypt base columnar cell (CBC) is an actively dividing ISC population marked by the expression of Lgr5, Ascl2, and Olfm4. Our recent studies show that Notch signaling regulates epithelial cell homeostasis by directing differentiated cell fate and promoting stem cell maintenance. In this study we examined Notch regulation of CBCs by characterizing Olfm4 expression and function. Methods: Gamma-secretase inhibitors (GSIs) were used to block Notch signaling in C57BL/ 6 mice (30μmol/kg dibenzazepine; DBZ) or in the human colon cancer cell line LS174T (40μM DAPT). Tissue was analyzed at various time points for progenitor and differentiated marker expression and epithelial proliferation by histological staining and qRT-PCR. LS174T cells were used to investigate the mechanism of Notch regulation of Olfm4 expression. Genetically engineered Olfm4-deficient mouse intestine was studied to examine the function of this CBC marker for intestinal epithelial cell homeostasis. Results: Chronic Notch inhibition for 6 days resulted in marked secretory cell hyperplasia, decreased cellular proliferation, and a striking decrease in Olfm4 mRNA, showing that Notch is critical for many different aspects of cellular renewal. Surprisingly, acute block of Notch with a single dose of DBZ was sufficient to cause a rapid and transient loss of Olfm4, in addition to a delayed, yet sustained surge in secretory cells and cellular proliferation. This surge in proliferation was in stark contrast to the loss of proliferation observed with chronic DBZ treatment. The acute DBZ model revealed exquisite Olfm4 regulation: mice treated with one dose showed a significant decrease in Olfm4 mRNA within 12 hours, but recovery to baseline within 2 days. Similarly, LS174T colon cancer cells showed decreased Olfm4 mRNA 4 hours after DAPT administration. To test whether changes in Olfm4 expression may mediate Notch effects in the intestine we examinedOlfm4-/mice. This analysis showed normal proliferation, cell fate, and CBC marker gene expression patterns, demonstrating that Olfm4 is not required for CBC function. However, we observed expression of other Olfactomedin family members in intestine, suggesting functional redundancy.Conclusions:Olfm4 is dynamically regulated by Notch signaling, with rapid loss of transcripts observed upon acute Notch inhibition. Thus this CBC marker is a sensitive read out of Notch activity in the intestine. Olfm4-/mice did not exhibit an intestinal phenotype, although related family members may play a compensatory role, suggesting compound mouse mutants may be needed to understand the function of Notch regulation of Olfm4 in the CBC.

303 High Altitude Journeys and Flights are Associated With the Increased Risk of Flares in IBD Patients

Background/Aim: The intestinal epithelium is a rapidly renewed tissue, requiring continual replenishment by intestinal stem cells (ISCs) in order to sustain life. The crypt base columnar cell (CBC) is an actively dividing ISC population marked by the expression of Lgr5, Ascl2, and Olfm4. Our recent studies show that Notch signaling regulates epithelial cell homeostasis by directing differentiated cell fate and promoting stem cell maintenance. In this study we examined Notch regulation of CBCs by characterizing Olfm4 expression and function. Methods: Gamma-secretase inhibitors (GSIs) were used to block Notch signaling in C57BL/ 6 mice (30μmol/kg dibenzazepine; DBZ) or in the human colon cancer cell line LS174T (40μM DAPT). Tissue was analyzed at various time points for progenitor and differentiated marker expression and epithelial proliferation by histological staining and qRT-PCR. LS174T cells were used to investigate the mechanism of Notch regulation of Olfm4 expression. Genetically engineered Olfm4-deficient mouse intestine was studied to examine the function of this CBC marker for intestinal epithelial cell homeostasis. Results: Chronic Notch inhibition for 6 days resulted in marked secretory cell hyperplasia, decreased cellular proliferation, and a striking decrease in Olfm4 mRNA, showing that Notch is critical for many different aspects of cellular renewal. Surprisingly, acute block of Notch with a single dose of DBZ was sufficient to cause a rapid and transient loss of Olfm4, in addition to a delayed, yet sustained surge in secretory cells and cellular proliferation. This surge in proliferation was in stark contrast to the loss of proliferation observed with chronic DBZ treatment. The acute DBZ model revealed exquisite Olfm4 regulation: mice treated with one dose showed a significant decrease in Olfm4 mRNA within 12 hours, but recovery to baseline within 2 days. Similarly, LS174T colon cancer cells showed decreased Olfm4 mRNA 4 hours after DAPT administration. To test whether changes in Olfm4 expression may mediate Notch effects in the intestine we examinedOlfm4-/mice. This analysis showed normal proliferation, cell fate, and CBC marker gene expression patterns, demonstrating that Olfm4 is not required for CBC function. However, we observed expression of other Olfactomedin family members in intestine, suggesting functional redundancy.Conclusions:Olfm4 is dynamically regulated by Notch signaling, with rapid loss of transcripts observed upon acute Notch inhibition. Thus this CBC marker is a sensitive read out of Notch activity in the intestine. Olfm4-/mice did not exhibit an intestinal phenotype, although related family members may play a compensatory role, suggesting compound mouse mutants may be needed to understand the function of Notch regulation of Olfm4 in the CBC.

The histone deacetylase inhibitor trichostatin A induces cell cycle arrest and rapid upregulation of gadd45β in LS174T human colon cancer cells

Histone deacetylase (HDAC) inhibitors are considered as promising therapeutic agents against several malignant diseases because they inhibit cancer cell proliferation. The stress sensor genes of the growth arrest and DNA damage-inducible protein (gadd45) family exhibit disordered expression in several types of malignant diseases and are thus a novel target for cancer therapy. However, there have been only few investigations of whether HDAC inhibitors affect the expression of gadd45 genes. We examined the effects of a HDAC inhibitor, trichostatin A (TSA), on the time-dependent expression of gadd45 genes in the human colon cancer cell line LS174T. Addition of TSA to LS174T cells induced inhibition of cell proliferation by arresting the cell cycle. We found that TSA treatment of LS174T cells induced rapid upregulation of gadd45β mRNA expression within 15 min, reaching a peak level at 3 h. Although the time-dependent expression pattern of gadd45β mRNA was similar to that of gadd45β mRNA, the peak level of gadd45β was lower than that of gadd45β. TSA treatment also upregulated the mRNA level of p21Waf1/Cip1, a prolif- eration inhibitor, after 3 h, but downregulated the mRNA levels of cyclin D1, a proliferation inducer, after 3 h, and of c-Myc after 1 h. TSA treatment induced a certain level of apoptosis, but the mRNA level of p53, a potent apoptosis inducer, was down-regulated after 3 h. These results suggest that the up-regulation of p21Waf1/Cip1 and apoptosis was independent of p53 and that the early upregulation of gadd45β gene, which precedes the upregulation of p21Waf1/Cip1 and the downregulation of cyclin D1, are important in TSA-treated LS174T cells.

P5-04-03: Personalized Cancer Treatment Selection Using Computational Signaling Pathway Models.

Abstract Introduction: In clinical oncology, with breast cancer in the front line, - cancer genome sequencing is entering clinical diagnostics; - novel drugs target signaling pathways that drive tumor growth; - metastasized cancer is expected to change into a chronic disease, requiring therapy response monitoring and repeated personalized choice of targeted therapy. Hence, there is a need to clinically interpret available genomic data in order to diagnose which signaling pathway is driving tumor growth in a patient, including the causative mutation, such that appropriate targeted drugs can be chosen. Currently available pathway analysis tools and databases are not suitable for this. Companion diagnostics: To address the above need, we are developing computational models of signaling pathways, which can be used as companion diagnostic tools to translate genomic data into meaningful clinical information to enable selection of targeted drugs. The aim is to develop such models for the 10 to 20 most relevant oncogenic pathways, and use them to predict which one is most likely to drive tumor growth in an individual patient, including the probable underlying genomic defect. A model of the Wnt pathway: As a proof of concept, we have built a computational model of the Wnt pathway, which is active in colon adenoma and a main player in colon cancer, but also relevant in breast cancer. Our first generation model of the Wnt pathway covers its transcriptional program, for which we carefully selected a list of Wnt target genes. We have modeled this pathway by a Bayesian network, which interprets the expression levels of the target genes (from Affymetrix U133Plus2.0 arrays), and infers a probability that the Wnt pathway is active in a certain patient tumor sample. The parameters of the model are partly derived from literature and partly fitted on experimental data. Results: We first fitted a model on data from six pairs of Wnt knockdown experiments on colon cancer cell line LS174T, and tested it on a public set of 32 normal colon samples plus 32 colon adenomas from patients (GSE8671). This model perfectly predicted no Wnt activity in the normal samples and an active Wnt pathway in the adenomas. Next, we fitted the model on these 64 patient samples, and tested it on a set of 145 normal colon and colon cancer samples from patients (GSE20916). The model again predicted no Wnt activity in all 44 normal samples, and an active Wnt pathway in all cancer samples, except for four of the 36 samples from surgically removed colon carcinomas, which most likely are the most heterogeneous. Thirdly, we took the latter model, trained on colon samples, and tested it on three breast cancer data sets, one with 51 breast cancer cell lines (GSE12777) and two sets of breast cancer samples from patients (GSE12276, n = 204; GSE21653, n = 266). Also on this different tissue type, our model correctly predicted Wnt activity for the few breast cancer cell lines that are known to have an active Wnt pathway. For the two patient studies, a significantly higher number of samples of the basal subtype were predicted to have an active Wnt pathway compared to the other subtypes (Fisher's exact test: p = 0.021 for GSE12276; p = 2.7e-5 for GSE21653), in line with increasing evidence for Wnt activation in the basal subtype. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-04-03.

Abstract 4881: A novel method of tumor characterization by protein and microRNA biomarker release using ultrasound

Abstract Earlier cancer diagnosis and monitoring of therapy could be improved by the earlier detection of circulating biomarkers. Ultrasound at low frequencies is known to permeabilize cell membranes and has been used for delivery of molecules into the cell. We hypothesize and prove that this bioeffect of ultrasound also causes the release of both protein and nucleic acid biomarkers from cells in culture and mice. The ability to focus ultrasonic waves allows for the localization of the potential biomarker source in vivo. This novel strategy could lead to the earlier identification, characterization and localization of incidental lesions, cancer and other disease. Methods: The colon cancer cell line LS174T that produces protein biomarkers (carcinoembryonic antigen-CEA and cancer antigen 19-9 – CA19-9) and microRNAs (miRNA – miR -16 and miR-141) was exposed to low frequency ultrasound (1 MHz) in culture using varying intensities (0, 0.1, 0.3. 0.5, 0.7, 1.0 W/cm2) and time (0, 10, 30 min). Subcutaneous tumors of LS174T in mice were also exposed to ultrasound (2 W/cm2; 6 min), directly over the tumors or at a non-tumor bearing site. Samples were collected pre and post-ultrasound treatment and compared for changes in biomarker levels. Protein biomarkers were detected using an enzyme-linked immunosorbant assay and miRNAs were detected using quantitative reverse-transcription polymerase chain reaction. Cell death was studied using Trypan blue staining. Results: LS174T cells treated with 1 MHz ultrasound in culture (n=4) at a low intensity of 0.3 W/cm2 released both CEA and CA19-9 with an increase in time (0, 10, 30 min; p&amp;lt;0.05). Ultrasound treatment increased the abundance of cell-free miRNAs released into the media in a time and intensity dependent manner. This release was seen to increase relative to pre-treatment levels, from 15-fold (0.1 W/cm2) to &amp;gt;1000-fold (1.0 W/cm2) for miR-16 and 100-fold (0.1 W/cm2) to 500-fold (1 W/cm2) for miR-141, within 30 min of ultrasound treatment. Cell death was less than 5% across all conditions. Subcutaneous tumors (n=10) showed an increase release of protein biomarkers when treated with ultrasound at 2 W/cm2 (CEA p&amp;lt;0.04; CA19-9 p&amp;lt;0.002), when comparing pre- and post-ultrasound measurements. Controls treated with no ultrasound or on non-tumor bearing sites of tumor-bearing mice did not show any significant increase in the release of biomarkers. Conclusions: Increase in protein and miRNA biomarkers were observed when ultrasound was directly applied to cells. We have developed a simple non-invasive method to amplify and spatially localize the biomarker signal from tumors. This method has implications in diagnosis and monitoring of therapy and has a clear pathway into clinical applications uniting the fields of imaging and in vitro diagnostics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4881. doi:10.1158/1538-7445.AM2011-4881