Parental HCT116 Cells Research Articles

Prediction of novel target genes and pathways involved in irinotecan-resistant colorectal cancer.

BackgroundAcquired drug resistance to the chemotherapeutic drug irinotecan (the active metabolite of which is SN-38) is one of the significant obstacles in the treatment of advanced colorectal cancer (CRC). The molecular mechanism or targets mediating irinotecan resistance are still unclear. It is urgent to find the irinotecan response biomarkers to improve CRC patients’ therapy.MethodsGenetic Omnibus Database GSE42387 which contained the gene expression profiles of parental and irinotecan-resistant HCT-116 cell lines was used. Differentially expressed genes (DEGs) between parental and irinotecan-resistant cells, protein-protein interactions (PPIs), gene ontologies (GOs) and pathway analysis were performed to identify the overall biological changes. The most common DEGs in the PPIs, GOs and pathways were identified and were validated clinically by their ability to predict overall survival and disease free survival. The gene-gene expression correlation and gene-resistance correlation was also evaluated in CRC patients using The Cancer Genomic Atlas data (TCGA).ResultsThe 135 DEGs were identified of which 36 were upregulated and 99 were down regulated. After mapping the PPI networks, the GOs and the pathways, nine genes (GNAS, PRKACB, MECOM, PLA2G4C, BMP6, BDNF, DLG4, FGF2 and FGF9) were found to be commonly enriched. Signal transduction was the most significant GO and MAPK pathway was the most significant pathway. The five genes (FGF2, FGF9, PRKACB, MECOM and PLA2G4C) in the MAPK pathway were all contained in the signal transduction and the levels of those genes were upregulated. The FGF2, FGF9 and MECOM expression were highly associated with CRC patients’ survival rate but not PRKACB and PLA2G4C. In addition, FGF9 was also associated with irinotecan resistance and poor disease free survival. FGF2, FGF9 and PRKACB were positively correlated with each other while MECOM correlated positively with FGF9 and PLA2G4C, and correlated negatively with FGF2 and PRKACB after doing gene-gene expression correlation.ConclusionTargeting the MAPK signal transduction pathway through the targeting of the FGF2, FGF9, MECOM, PLA2G4C and PRKACB might increase tumor responsiveness to irinotecan treatment.

Abstract 856: Notch-1 promotes a mesenchymal phenotype in colon cancer cells via Notch-3 and Smad-3 activation

Abstract A role for cancer initiating cells (CIC) in tumor growth, drug resistance, relapse, and metastasis via epithelial to mesenchymal transition (EMT) has been supported by experimental evidence. It is well-documented that Notch-1 receptor signaling participates in stem cell maintenance and lineage commitment in various organs, including the colon, and can regulate EMT. The function of Notch signaling in colorectal cancer (CRC) is not clear. We have reported that Notch-1 activation in the human colon tumor cell line HCT-116 resulted in upregulation of Jagged-1, Smad-3, CD44, and Slug proteins. These changes were accompanied by acquisition of a mesenchymal cell phenotype that included increased anchorage independent growth and migratory activity. Experiments with a γ-secretase inhibitor (DAPT) and soluble Jagged1-Fc protein demonstrated that Notch-1 signaling activates CD44 and Slug via other Notch receptors. In subsequent experiments, we observed an increase in Notch-3 expression in the presence of activated Notch-1. Herein, we tested the hypothesis that the stem cell-like mesenchymal phenotype induced by Notch-1 signaling in colon cancer cells is mediated by the Notch-3 receptor and Smad-3. The human colon tumor cell line HCT-116 was transduced with constitutively active Notch-1, and a Notch-3 shRNA to produce a Notch-3 null cell line with activated Notch-1 (ICN1-shN3 cells). ICN1-shN3 cells exhibited a 2.5-fold lower plating efficiency than the HCT-116/ICN1 cell line (p<0.01) that expresses constitutively active Notch-1 in the presence of Notch-3. In addition, Notch-3 null cells with constitutively active Notch-1 (ICN1-shN3 cells) were slower by 29% in completing wound healing as compared with the HCT-116/ICN1 cell line (p<0.05). Consistent with the lower plating efficiency, a 37% decrease in colosphere formation was observed with ICN1-shN3 cells as compared with the HCT-116/ICN1 cell line (p<0.001). These data indicated that loss of Notch-3 abrogated the Notch-1 induced mesenchymal cell phenotype. Western blot analysis demonstrated that the HCT-116/ICN1 cells with intact Notch-3 exhibited increased levels of the EMT associated proteins Smad-3, CD44 and Slug as compared to the parental HCT-116 cell line. Expression of CD44 and Slug in Notch-3 receptor null ICN1-shN3 cells was highly reduced compared with HCT-116/ICN1 cells and was similar to that detected in the parental HCT-116 cells. Treatment of HCT-116/ICN1 cells with a Smad-3 inhibitor resulted in significant reduction of the CD44 and Slug proteins. Meanwhile, Notch-3 receptor null ICN1-shN3 cells were also more sensitive to chemotherapy treatment than the HCT-116/ICN1 cell line (p<0.05). Collectively, our data provide evidence for a novel pathway in which Notch-1 signaling in colon tumor cells promotes a mesenchymal phenotype via activation of Jagged-1, Notch-3 and Smad-3, followed by expression of CD44 and Slug. Citation Format: George Sigounas, Fred E. Bertrand, Douglas A. Weidner, Kaitlyn E. Vinson, Alexander G. Clark, Azeem Khan. Notch-1 promotes a mesenchymal phenotype in colon cancer cells via Notch-3 and Smad-3 activation [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 856. doi:10.1158/1538-7445.AM2017-856

Abstract 3254: Identification of potent anti-tumor agents from the LOPAC library using a unified 2D/3D cell culture screening approach

Abstract Two-dimensional (2D) cultures of immortalized cancer cells have played a significant role cancer drug discovery and development process for decades. However, the limitations imposed by the long-term cultures of cancer cell lines are becoming increasingly apparent only recently. Therefore, there is a general shift towards three-dimensional (3D) cell cultures, which effectively mimic tumor complexities, for lead optimization and to accelerate translation research. 3D cell cultures not only model the in vivo architecture of tumors, but 3D-derived cells also retain numerous signatures of cancer stem cells compared to parental cells in 2D. In this study, using a unified 2D and 3D cell culture screening approach, we screened the LOPAC library from Sigma-Aldrich as a proof-of-concept study. The drug screening was performed using parental colorectal cancer HCT116 cells as a model cell line. 3D-derived cultures were generated from multicellular spheroids of HCT116 cells. Additionally, utilizing lightsheet fluorescence microscopy (LSFM), we studied the tissue penetration of drugs in spheroids of HCT116 and cells expressing hypoxia-inducible factor-1α (HIF-1α). We identified cc. 200 compounds from the LOPAC library that showed increased activity in 3D-derived cultures. A dose–response relationship study of top 20% compounds further confirmed the preferential activity of these drugs in 3D than 2D cultures. Next, studies in spheroid cultures resulted in the identification of five lead compounds with potent spheroid-size reducing effects. Three of these compounds were kinase inhibitors, one HIF-1α inhibitor, and one interestingly, a monoamine oxidase inhibitor. LSFM studies in HCT116-HIF-1α spheroids showed that two kinase inhibitors and the HIF-1α inhibitors significantly altered spheroid hypoxia, and presumably spheroid growth. We believe that this proof-of-concept study will further highlight the advantages of a combined 2D/3D screening approach for drug discovery and development, and consequently enable downstream testing at reduced cost. Citation Format: Viswanath Das, Tomáš Fürst, Soňa Gurská, Petr Džubák, Marián Hajdúch. Identification of potent anti-tumor agents from the LOPAC library using a unified 2D/3D cell culture screening approach [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 3254. doi:10.1158/1538-7445.AM2017-3254

Loss of CUL4A expression is underlying cisplatin hypersensitivity in colorectal carcinoma cells with acquired trabectedin resistance

Background:Colorectal carcinoma (CRC) is the third most common cancer worldwide. Platinum-based anticancer compounds still constitute one mainstay of systemic CRC treatment despite limitations due to adverse effects and resistance development. Trabectedin has shown promising antitumor effects in CRC, however, again resistance development may occur. In this study, we aimed to develop strategies to circumvent or even exploit acquired trabectedin resistance in novel CRC treatment regimens.Methods:Human HCT116 CRC cells were selected for acquired trabectedin resistance in vitro and characterised by cell biological as well as bioinformatic approaches. In vivo xenograft experiments were conducted.Results:Selection of HCT116 cells for trabectedin resistance resulted in p53-independent hypersensitivity of the selected subline against cisplatin. Bioinformatic analyses of mRNA microarray data suggested deregulation of nucleotide excision repair and particularly loss of the ubiquitin ligase CUL4A in trabectedin-selected cells. Indeed, transient knockdown of CUL4A sensitised parental HCT116 cells towards cisplatin. Trabectedin selected but not parental HCT116 xenografts were significantly responsive towards cisplatin treatment.Conclusions:Trabectedin selection-mediated CUL4A loss generates an Achilles heel in CRC cancer cells enabling effective cisplatin treatment. Hence, inclusion of trabectedin in cisplatin-containing cancer treatment regimens might cause profound synergism based on reciprocal resistance prevention.

Downregulation of caveolin-1 increases the sensitivity of drug-resistant colorectal cancer HCT116 cells to 5-fluorouracil.

Colorectal cancer is the third most common type of cancer in men and women. Chemotherapy is an important treatment strategy for patients with terminal stage cancer. However, the development of drug resistance hampers the effectiveness of chemotherapy. Therefore, an effective therapeutic approach to target chemoresistance-associated cellular molecules is required. In the present study, drug-resistant human colorectal cancer HCT116 cells were developed by treating HCT116 cells with increasing concentrations of 5-fluorouracil (5-FU). The present study indicated that the drug-resistance cells (DRC) were resistant to 5-FU compared with parental HCT116 cells by detecting cell survival using an MTT assay. Additionally, the expression of the chemoresistance-associated protein caveolin-1 (Cav-1) was assessed by reverse transcription-quantitative polymerase chain reaction and western blotting. The results revealed that the Cav-1 expression level was significantly higher in DRC compared with that in the parental HCT116 cells. Next, Cav-1 was silenced by small interfering RNA (siRNA) or was inhibited with its specific inhibitor methyl β-cyclodextrin (MCD). MTT assay demonstrated that Cav-1 siRNA and MCD resensitized DRC to 5-FU. These data reveal that Cav-1 was involved in the development of resistance, suggesting that Cav-1 is a potential target for the treatment of colorectal cancer chemoresistance. In addition, 5-FU combined with Cav-1 siRNA or its specific inhibitor may increase the effectiveness of the treatment strategy.

Downregulation of YEATS4 by miR-218 sensitizes colorectal cancer cells to L-OHP-induced cell apoptosis by inhibiting cytoprotective autophagy.

Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide. Deregulation of microRNAs (miRNAs) has been reported to participate in CRC progression. In the present study, we observed downregulation of miR-218 and upregulation of YEATS domain containing4 (YEATS4) in CRC tissues and in multidrug-resistant HCT-116/L-OHP cells compared with these levels in normal tissues and parental HCT-116 cells, respectively. The results indicated that miR-218 overexpression significantly decreased the IC50 value of oxaliplatin (L-OHP) in the HCT-116/L-OHP cells, and suppression of miR-218 significantly enhanced the IC50 of L-OHP in the HCT-116 cells. Flow cytometric analysis showed that miR-218 overexpression alone promoted cell apoptosis in the HCT-116/L-OHP cells, which was further enhanced in response to L-OHP, and miR-218 inhibition decreased cell apoptosis in the HCT-116 cells following treatment with L-OHP. Western blot analysis indicated that, compared with the small increase observed in HCT-116 cells, the relative LC3II level in HCT-116/L-OHP cells after lysosome inhibition via chloroquine (CQ) was markedly upregulated following L-OHP treatment, suggesting induction of autophagy. Exposure of HCT-116/L-OHP cells to L-OHP after control mimic transfection increased autophagic flux, as reflected by increased LC3II levels, while miR-218 overexpression partly reversed L-OHP-mediated LC3II accumulation. Additionally, both miR-218 overexpression and CQ treatment promoted L-OHP-induced HCT-116/L-OHP cell apoptosis. Molecularly, our results confirmed that miR-218 directly targets the YEATS4 gene and inhibits YEATS4 expression. Furthermore, YEATS4 overexpression without the 3'-untranslated region (3'-UTR) restored miR-218-inhibited YEATS4 and LC3II expression, and abolished miR-218-stimulated cell viability loss and cell apoptosis increase in response to L-OHP. In conclusion, miR-218 sensitized HCT-116/L-OHP cells to L-OHP-induced cell apoptosis via inhibition of cytoprotective autophagy by targeting YEATS4 expression.

Abstract 1141: The H1047R point mutation in p110 alpha of PI3K down-regulates INPP4B expression in human colon cancer HCT116 cells

Abstract INTRODUCTION: Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and Inositol polyphosphate-4-phosphatase, type II (INPP4B) are two important enzymes maintaining homeostasis of phosphoinositides in cells. Class IA PI3K produces Phosphatidylinositol-3,4,5-bisphosphate (PIP3) by phosphorylation of Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at D3 position, while INPP4B dephosphorylate Phosphatidylinositol-4,5-bisphosphate (PI(3,4)P2) at the D4 position to generate Phosphatidylinositol 3-phosphate (PI(3)P). All of these phospholipids are important second messengers. PI3K activates its downstream substrate Protein Kinase B (Akt) by PIP3. INPP4B, however, blocks Akt activities in epithelial cells through the turnover of PI(3,4)P2. PI3K/Akt pathway is involved in cell adhesion, cytoskeletal rearrangement, invadopodia formation and cell motility. Gain of function mutations in the catalytic subunit p110α of PI3K occur in up to 1/3 of human colorectal cancers (CRC). The kinase domain mutation H1047R is the most frequent cancer-specific mutation in p110α. Our previous report showed that this mutation in p110α results in an increase in cellular levels of PIP3 and in turn an enhancement of Akt activity. Since the H1047R mutation and INPP4B have an opposite effect on Akt activities, the interesting question is if there is crosstalk between the two or if H1047R mutation can affect cellular INPP4B expression. We hypothesize that H1047R mutated p110α down-regulates INPP4B and therefore results in increased Akt activity. METHODS: HCT116 cells engineered to contain either the H1047R mutant (MUT) or wild type (WT) PI3KCA allele were used in this study. Endogenous protein expression level of INPP4B was detected by immune-blotting analysis in parental, WT and MUT HCT116 cells. To further investigate the effect on INPP4B of the H1047R mutation, we conducted RNA-sequencing for profiling, quantifying RNA transcripts in WT and MUT HCT116 cells, and compared RNA transcript level of INPP4B between the two isogenic cell lines of HCT116 cells. RESULTS: 1) Endogenous expression level of INPP4B protein in HCT116WT cells was over 10-times higher than in HCT116MUT cells. 2) INPP4B RNA was down regulated by the H1047R mutation in p110α (PIK3CA) of Class 1A PI3K. CONCLUSION: Our results suggested that hyperactivity of Akt in H1047R mutation bearing cells may be also related to low level of cellular INPP4B, and that the H1047R mutated p110α may increase cell migration and cancer metastasis through down regulation of tumor suppressor gene INPP4B. Although further studies are needed to reveal the mechanism of H1047R mutated PI3K mediated INPP4B-regulation, INPP4B expression level may be a potential maker to predict PI3K mutation induced CRC metastasis. Citation Format: Guanghua Wan, Scott A. Ness, Ashwani Rajput. The H1047R point mutation in p110 alpha of PI3K down-regulates INPP4B expression in human colon cancer HCT116 cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1141.

Abstract 3013: Identification of PHF5A as a common cellular target of splicing-modulating chemical probes

Abstract Recent discoveries that splicing factors such SF3B1, U2AF1, SRSF2 are frequently mutated in multiple hematological malignancies including chronic lymphocytic leukaemia and myelodysplastic syndromes have generated interest in therapeutic approaches to target the splicesome dependency in cancer cells bearing mutations in splicing factors. Previously, several structurally unrelated natural compounds including pladienolide, herboxidiene, and FR901464 have been shown to exert potent anti-proliferative effects in cancer cells grown in vitro. Further optimization has led to the discovery of natural product analogs (e.g. E7107) with anti-tumor efficacy in vivo in xenograft models. Target identification has revealed the SF3B complex of the splicesome as the common action site for these compounds. Recent work has demonstrated biological and genetic evidence that single amino acid substitution (R1074H) in SF3B1 completely abolished the anti-proliferative effect of pladienolide derivative E7107, suggesting that SF3B1 is the direct binding partner for pladienolides. However, the same SF3B1 R1074H mutation does not provide equal level of protections for cells treated with herboxidiene derivatives, indicating differential mechanism of action for these two classes of splicing modulators. To identify targets for herboxidiene-like compounds, we have generated resistant HCT116 clones upon continuous administration of herboxidiene derivative H3B-37045 in vitro. Whole exome sequencing from 6 resistant clones revealed a common Y36C mutation in SF3B subunit component PHF5A (SF3B14b). Over-expression of PHF5A Y36C but not the wild-type form in parental HCT116 cells confirmed the protective effect of this mutation to H3B-37045. Surprisingly, PHF5A Y36C expression also conferred resistance to the pladienolide derivative E7107, which indicates that, unlike the SF3B1 R1074H mutation, PHF5A resides within a common node of action site among different splicing modulators. RNA-seq, biochemical and structure homology-modeling analysis suggested that PHF5A Y36C mutation disrupted the action of splicing modulators through interfering with the compounds’ interaction with the SF3B complex. Detailed analysis of the function of the Y36C mutant and wild-type PHF5A in the SF3B complex is currently ongoing. Understanding the function of PHF5A in splicing and the molecular mechanism of Y36C mutation shall provide new insights of the biological role of splicesome, and guide the development of next generation splicesome inhibitors. Citation Format: Teng Teng, Xiaoling Puyang, Shouyong Peng, Jacob Feala, Betty Chan, Jennifer Tsai, Benjamin Caleb, Craig Karr, Eunice Park, Laura Corson, Yoshiharu Mizui, Peter Smith, Nicholas Larsen, Lihua Yu, Markus Warmuth, Ping Zhu, Agustin Chicas. Identification of PHF5A as a common cellular target of splicing-modulating chemical probes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3013.

Biofabrication of 3D Alginate-Based Hydrogel for Cancer Research: Comparison of Cell Spreading, Viability, and Adhesion Characteristics of Colorectal HCT116 Tumor Cells.

Hydrogels are an important class of biomaterials as they could mimic the extracellular matrix (ECM). Among the naturally occurring biopolymers, alginate and gelatin are extensively used for many biomedical applications. For developing biofabrication constructs as three-dimensional (3D) cell culture models, realistic imaging of cell spreading and proliferation inside the hydrogels represents a major challenge. Therefore, we aimed to establish a system that can mimic the structural architecture, composition, and biological functions of the ECM for cancer research approaches. For this, we compared the cell behavior of human colon cancer HCT116 cells in two biofabricated hydrogels as follows: pure alginate and cross-linked alginate-gelatin (ADA-GEL) matrixes. Our data indicate that cells from the ADA-GEL matrix showed highest proliferation and cellular networks through the material. Analyzing the mRNA expression of several integrins of cells cultured inside of the matrix, we showed that mRNA expression of integrin subunits differed based on the cell focal adhesion characteristics. Furthermore, we showed that recultured ADA-GEL immobilized cells do not differ from parental HCT116 cells regarding migration and proliferation capabilities. Comparing adhesion and other phenotypic characteristics of HCT116 tumor cells, we suggest that ADA-GEL hydrogel is a more suitable 3D system than pure alginate and seems to optimally mimic the physiological behavior of the tumor microenvironment. For the first time, we present a functional 3D hydrogel construct for colon cancer cells, which are supporting their physiological cell attachment, spreading, and viability. We strongly believe that it will be applicable as a suitable in vitro 3D tumor model to study different aspects of tumor cell behavior.

Data showing the circumvention of oxaliplatin resistance by vatalanib in colon cancer.

We have recently reported that vatalanib, an orally active small molecule multi-tyrosine kinase inhibitor (Hess-Stumpp et al., 2005 [1]), can sensitize multidrug resistant (MDR) colon cancer cells to chemotherapy under hypoxia by inhibiting two MDR transporters ABCB1 and ABCG2 (To et al., 2015 [2]). This data article describes the possible circumvention of resistance to specifically platinum (Pt)-based anticancer drugs by vatalanib via inhibition of two other efflux transporters ABCC2 and ATP7A. Data from the flow cytometric transporter efflux assay showed specific inhibition of ABCC2 activity by vatalanib in stable transfected cells and ABCC2-overexpressing oxaliplatin-resistant colon cancer cells HCT116/Oxa. We also performed the transporter ABCC2 ATPase assay and showed an increase in ATP hydrolysis by ABCC2 in the presence of vatalanib. ATP7A mRNA expression was also shown to be upregulated in HCT116/Oxa cells. Vatalanib was shown to suppress this upregulated ATP7A expression. Data from the cellular Pt accumulation assay showed a lower Pt accumulation in HCT116/Oxa cells than the parental sensitive HCT116 cells. Vatalanib was shown to increase cellular Pt accumulation in a concentration-dependent manner. Combination of oxaliplatin and vatalanib was shown to restore the suppressed apoptosis in HCT116/Oxa cells.

Suppression of lysyl-tRNA synthetase, KRS, causes incomplete epithelial-mesenchymal transition and ineffective cell‑extracellular matrix adhesion for migration.

The cell-adhesion properties of cancer cells can be targeted to block cancer metastasis. Although cytosolic lysyl-tRNA synthetase (KRS) functions in protein synthesis, KRS on the plasma membrane is involved in cancer metastasis. We hypothesized that KRS is involved in cell adhesion-related signal transduction for cellular migration. To test this hypothesis, colon cancer cells with modulated KRS protein levels were analyzed for cell-cell contact and cell-substrate adhesion properties and cellular behavior. Although KRS suppression decreased expression of cell-cell adhesion molecules, cells still formed colonies without being scattered, supporting an incomplete epithelial mesenchymal transition. Noteworthy, KRS-suppressed cells still exhibited focal adhesions on laminin, with Tyr397-phopshorylated focal adhesion kinase (FAK), but they lacked laminin-adhesion-mediated extracellular signal-regulated kinase (ERK) and paxillin activation. KRS, p67LR and integrin α6β1 were found to interact, presumably to activate ERK for paxillin expression and Tyr118 phosphorylation even without involvement of FAK, so that specific inhibition of ERK or KRS in parental HCT116 cells blocked cell-cell adhesion and cell-substrate properties for focal adhesion formation and signaling activity. Together, these results indicate that KRS can promote cell-cell and cell-ECM adhesion for migration.

MicroRNA-425-5p regulates chemoresistance in colorectal cancer cells via regulation of Programmed Cell Death 10.

Acquired chemoresistance represents a major obstacle in cancer treatment, the underlying mechanism of which is complex and not well understood. MiR-425-5p has been reported to be implicated tumorigenesis in a few cancer types. However, its role in regulating chemoresistance has not been investigated in colorectal cancer (CRC) cells. Microarray analysis was performed in isogenic chemosensitive and chemoresistant HCT116 cell lines to identify differentially expressed miRNAs. miRNA quantitative real-time PCR was used to detect miR-425-5p expression levels between drug resistant and parental cancer cells. MiR-425-5p mimic and inhibitor were transfected, followed by CellTiter-Glo(®) assay to examine drug sensitivity in these two cell lines. Western Blot and luciferase assay were performed to investigate the direct target of miR-425-5p. Xenograft mouse models were used to examine in vivo function of miR-425-5p. Our data showed that expression of miR-425-5p was significantly up-regulated in HCT116-R compared with parental HCT116 cells. Inhibition of miR-425-5p reversed chemoresistance in HCT116-R cells. Programmed cell death 10 (PDCD10) is the direct target of miR-425-5p which is required for the regulatory role of miR-425-5p in chemoresistance. MiR-425-5p inhibitor sensitized HCT116-R xenografts to chemo drugs in vivo. Our study demonstrated that miR-425-5p regulates chemoresistance of CRC cells by modulating PDCD10 expression level both in vitro and in vivo. MiR-425-5p may represent a new therapeutic target for the intervention of CRC.

Abstract 3608: Prostaglandin E-2 promotes resistance to oxaliplatin in colorectal cancer cells

Abstract Oxaliplatin, a platinum-based DNA crosslinking agent, has been used successfully as a first-line chemotherapeutic agent for the treatment of metastatic colorectal cancer (CRC). However, long-term treatment with oxaliplatin results in resistance in nearly all patients, limiting its therapeutic efficacy. Previous studies have shown that treatment of CRC cells with selective inhibitors of the enzyme Cyclooxygenase-2 (COX-2) leads to the development of drug resistance. One of the terminal products of the COX-2 pathway, prostaglandin E-2 (PGE2), is a pro-inflammatory lipid that has been implicated in colorectal tumorigenesis; however, it is unknown whether PGE2 plays a role in the development of drug resistance. In this study, we have begun to investigate the relationship between PGE2 and oxaliplatin resistance using the human CRC cell lines, HT29 and HCT116. Oxaliplatin-resistant HT29 cells (HT29-OXR) were generated by continuous exposure of parental HT29 cells to increasing concentrations of oxaliplatin over a period of 3 months. We have found that HT29-OXR cells produce significantly higher levels of PGE2 (3-fold increase, p<0.05) compared to the parental HT29 cell line. In addition, we have observed concomitantly elevated levels of the terminal PGE2 synthase, microsomal prostaglandin E synthase-1 (mPGES-1). Furthermore, we have demonstrated that the addition of exogenous PGE2 to both parental HT29 and HCT116 cells significantly (p<0.0001) decreased their sensitivity (IC50) to oxaliplatin, measured by the MTT cell viability assay. These findings suggest that PGE2 promotes resistance to oxaliplatin in CRC cells. FACS analysis showed that co-treatment of HT29 cells with exogenous PGE2 for 24 hours leaded to 27% reduction in oxaliplatin-induced Annexin V staining for early apoptosis, while exogenous PGE2 maintained G2/M arrest and simultaneously reduced the proportion of dead sub-G1 cells by approximately 80.4% in parental HCT116 cells. Our findings reveal a role for PGE2 in oxaliplatin resistance, possibly via regulation of apoptosis, and provide new data suggesting that targeting mPGES-1 may be a useful strategy for overcoming oxaliplatin resistance. Citation Format: Huakang Huang, Daniel W. Rosenberg. Prostaglandin E-2 promotes resistance to oxaliplatin in colorectal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3608. doi:10.1158/1538-7445.AM2015-3608

Abstract 2932: Role of death receptor 5 (DR5) in apoptosis induced by anticancer agents in colon cancer cells

Abstract The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, also called death receptor 5 (DR5; TRAIL receptor2), is a member of the death receptor family that shares a cysteine-rich extracellular domain and a cytoplasmic death domain. DR5 is located on cell surface, becomes activated upon binding to its ligand TRAIL or overexpression, and then signals apoptosis initiation through an FADD/caspase 8-dependant caspase activation cascade. A number of previous studies showed that DR5 expression is induced upon chemotherapy treatment. However, whether DR5 induction is necessary for apoptosis induction has remained unclear. To determine its functional role in chemotherapy-induced apoptosis, we used homologous recombination to knock out (KO) DR5 in HCT116 colon cancer cells, and then analyzed response of DR5 KO cells to a panel of anticancer agents. Compared to parental HCT116 cells, DR5 KO cells are less sensitive to TRAIL, the DNA damaging drug 5-FU, the non-steroidal anti-inflammatory drug (NSAID) sulindac, as well as several other classes of anticancer agents. We also investigated how these agents induce apoptosis through DR5. Together, our results indicate a critical role of DR5 in mediating therapeutic response of colorectal cancer cells. Citation Format: Jingshan Tong, Jian Yu, Lin Zhang. Role of death receptor 5 (DR5) in apoptosis induced by anticancer agents in colon cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2932. doi:10.1158/1538-7445.AM2015-2932

Tumor suppression by resistant maltodextrin, Fibersol-2

Resistant maltodextrin Fibersol-2 is a soluble and fermentable dietary fiber that is Generally Recognized As Safe (GRAS) in the United States. We tested whether Fibersol-2 contains anti-tumor activity. Human colorectal cancer cell line, HCT116, and its isogenic cells were treated with FIbersol-2. Tumor growth and tumorigenesis were studied in vitro and in vivo. Apoptotic pathway and generation of reactive oxygen species (ROS) were investigated. We discovered that Fibersol-2 significantly inhibits tumor growth of HCT116 cells by inducing apoptosis. Fibersol-2 strongly induces mitochondrial ROS and Bax-dependent cleavage of caspase 3 and 9, which is shown by isogenic HCT116 variants. Fibersol-2 induces phosphorylation of Akt, mTOR in parental HCT116 cells, but not in HCT116 deficient for Bax or p53. It prevents growth of tumor xenograft without any apparent signs of toxicity in vivo. These results identify Fibersol-2 as a mechanism-based dietary supplement agent that could prevent colorectal cancer development.

Phosphatidylinositol 3-Kinase Mediates the Ability of Retinol to Decrease Colorectal Cancer Cell Invasion

Previously, we showed that retinol (vitamin A) decreased both colorectal cancer cell invasion and phosphatidylinositol 3-kinase (PI3K) activity through a retinoic acid receptor–independent mechanism. Here, we determined if these phenomena were related by using parental HCT-116 cells that harbor 1 allele of wild-type PI3K and 1 allele of constitutively active (ca) PI3K and 2 mutant HCT-116 cell lines homozygous for caPI3K. In vitro, treatment of parental HCT-116 cells with 10 μM retinol reduced cell invasion whereas treatment of mutant HCT-116 cell lines with retinol did not. Treatment with 10 μM retinol also decreased the activity of matrixmetalloproteinase-9 and increased tissue inhibitor of matrixmetalloproteinase-I levels in parental, but not mutant, HCT-116 cells. Finally, parental or mutant cells were intrasplenically injected into athymic mice consuming diets with or without supplemental vitamin A. As expected, vitamin A supplementation tended (P = 0.18) to reduce the incidence of metastases in mice injected with the parental cell line and consuming the supplemented diet. In contrast, metastatic incidence was not affected (P = 1.00) by vitamin A supplementation in mice injected with mutant cells. These data indicate that the capacity of retinol to inhibit PI3K activity confers its ability to decrease colorectal cancer metastasis.

Abstract 1952: Notch-1 regulated stemness and EMT in colorectal cancer

Abstract Colorectal cancer (CRC) is the second leading cause of cancer death nationwide. In 2013, nearly 156,000 patients will be diagnosed with CRC. Surgical resection for early stage disease offers the best chance for cure; however, up to 30% of patients will relapse. Current models of CRC progression hold that tumor development is driven by a small subpopulation of cancer initiating (CIC) or cancer stem (CSC) cells that share many properties of stem cells. These cells have the capacity to drive tumor growth, are largely responsible for relapse and have been implicated in metastasis via epithelial to mesenchymal transition (EMT), similar to normal counterparts in organogenesis. Notch-1 is the major receptor for a highly conserved signaling pathway that is well-documented to control stem cell and lineage commitment in various organs, including the colon. The function of Notch signaling in colorectal cancer, however, is not well-defined. Based on preliminary data in which colon tumor samples from CRC patients expressed Notch-1 but surrounding benign tissue did not, we sought to test the hypothesis that Notch-1 signaling in colon cells promotes tumorigenesis by enhancing stem cell and EMT characteristics of tumor cells. The human colon tumor line HCT-116 was transduced with a retroviral construct that expresses constitutively active Notch-1 (ICN1). Hes-1 protein levels were increased in the activated Notch-1 transduced cells, indicating that the construct was functional in activating Notch-signaling. These HCT116/ICN1 cells exhibited higher plating efficiency than the parental cell line. The average colony size of the HCT116/ICN1 cells was about two-fold smaller than the parental cell line (p<0.001). In addition, cells with constitutively active Notch-1 (HCT116/ICN1) had a longer duplication time than parental HCT-116 cells (p<0.03). Consistent with longer duplication time, HCT116/ICN1 cells were two-fold slower in completing wound healing as compared with the parental HCT-116 cell line (p<0.001). Transwell migration assays revealed a two-fold increase in migration of HCT116/ICN1 cells as compared with the parental control (p<0.03). Western blot analysis demonstrated that the HCT116/ICN1 cells with activated Notch-1 expressed highly increased levels of the EMT associated proteins Smad3, CD44 and Slug. This was accompanied by a four-fold decreased expression of E-cadherin. Meanwhile, E-cadherin was not altered in HCT116 cells transduced with a Notch-1 shRNA lentiviral construct that inactivated Notch-1 signaling. Finally, levels of the numb protein, a Notch-1 antagonist, appeared to be significantly reduced (five-fold decrease) by constitutive Notch-1 signaling. Alternatively, inactivation of Notch-1 resulted in a four-fold increased expression of the numb protein. Collectively, our data point to the Notch-1 pathway as being a potential key regulator of EMT in colorectal tumors and as such, may be of therapeutic or prognostic utility. Citation Format: Alex Fender, Makenzie Nutter, Timothy Fitzgerald, Fred Bertrand, George Sigounas. Notch-1 regulated stemness and EMT in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1952. doi:10.1158/1538-7445.AM2014-1952

Epithelial-Mesenchymal Transition Associates with Maintenance of Stemness in Spheroid-Derived Stem-Like Colon Cancer Cells

Despite earlier studies demonstrating characteristics of colon cancer stem cells (CCSCs) and the role of epithelial-mesenchymal transition (EMT) in tumor development, it remains controversial as to the relationship between CCSCs and EMT. In this study, in order to present an insight into this relationship in colon cancer, we developed HCT116 and HT29 sphere models, which are known to be the cells enriching cancer stem cells. Compared to their parental counterparts, spheroid cells displayed lower homotypic/heterotypic adhesion but higher in vitro migratory/invasive capacity, as well as higher tumorigenic and metastatic potential in vivo. The spheroid cells also demonstrated down-regulated E-cadherin and up-regulated α-SMA and Vimentin expression, which is the typical phenotype of EMT. In order to explore whether this phenomenon is associated to activation of Wnt/β-catenin pathway, we detected several key signaling molecules. Compared with their parental cells, HCT116 and HT29 spheroid cells demonstrated down-regulated expression of GSK3β, but up-regulated expression of Slug and Snail. And also, the up-regulation of nucleus β-catenin in spheroid cells indicated that the free β-catenin transferred from cytoplasm to cell nucleus. Our findings indicate that spheroid cells have the characteristics of colon cancer stem cells, and EMT may account for their stemness and malignancy. And persistent activation of Wnt/β-catenin pathway may play an important role in the EMT of CCSCs.

Sphingosine kinase 1 plays a role in the upregulation of CD44 expression through extracellular signal-regulated kinase signaling in human colon cancer cells

Our previous study has shown that the activity and expression of sphingosine kinase (SPHK) regulated the sensitivity of human colon cancer cells to the chemotherapeutic oxaliplatin (L-OHP). In addition, the cancer stem cell marker CD44 increases cell resistance to anticancer drugs. Here, we use colon cancer cell lines to examine the relationship between SPHK1 activity and CD44 expression.CD44 expression was measured by western blotting and quantitative PCR in two human colon cancer cell lines: L-OHP-resistant RKO and L-OHP-sensitive HCT116. The regulation of CD44 by SPHK1 was examined by either blocking or overexpressing SPHK1 and by using an L-OHP-resistant HCT116 clone (HCT116-R).The levels of SPHK1, CD44, phosphorylated-Akt, and phosphorylated-extracellular signal-regulated kinase (ERK) were much higher in the RKO cells than in the HCT116 cells. The treatment of RKO cells with the SPHK inhibitor or SPHK1 silencing by RNA interference suppressed CD44 protein expression. SPHK1 and CD44 levels were much higher in HCT116-R cells compared with the parental HCT116 cells. Transfection of HCT116 cells with SPHK1 cDNA enhanced the expression of both CD44 and phosphorylated-ERK. The increase in the CD44 protein level was abolished by the inhibition of ERK phosphorylation. Treatment of RKO cells with the sphingosine-1-phosphate (S1P)2 receptor antagonist suppressed ERK phosphorylation and the expression of CD44 mRNA and protein. Exogenous stimulation with S1P increased ERK phosphorylation and CD44 protein expression in HCT116 cells, but treatment with an MEK inhibitor and S1P2 receptor antagonist blocked this effect.These findings indicate that SPHK1 and its product, S1P, contribute toward the regulation of CD44 protein expression through the ERK signaling pathway through S1P2 in human colon cancer cells.

Abstract 5519: In vivo fluorescence lifetime imaging differentiates the bound versus unbound status of a cell surface receptor targeted ligand.

Abstract In vivo fluorescence lifetime imaging allows for the isolation of probe-specific fluorescence lifetime from surrounding tissue auto-fluorescence. Fluorescence lifetime is not affected by agent concentration or depth, or image acquisition parameters. However, fluorescence lifetime can be affected by the in vivo tissue microenvironment, e.g. pH or hypoxia. In this work, fluorescence lifetime is used to characterize the presence and fate of a high-affinity peptidyl delta opioid receptor (δOR) ligand conjugated to the near-infrared fluorescent IRDye800CW (Li-Cor). HCT116 colorectal cancer cells engineered to over-express the δOR and parental non-expressing HCT116 cells were bilaterally xenografted subcutaneously into the flanks of athymic nude mice. At a series of time-points following tail vein injection of the ligand, in vivo fluorescence images images were acquired using the Optix MX3 scanner (Advanced Research Technologies). For 24 h, the ligand fluoresence lifetime was significantly different (p<0.05) in the δOR positive tumors relative to the negative tumors. At 48 hours, the fluorescence lifetime in the negative tumor decreased and was no longer different. We hypothesize that the early difference in fluorescence lifetime is attributed to the bound state of the ligand on receptor positive tumors relative to the unbound state in negative tumors. Over time, unbound ligand clears from the negative tumor leaving only a low concentration of ligand retained by non-specific interactions. In vitro studies demonstrated that the fluoresecence lifetime of this ligand is not altered within the biological pH range. However, the tumor microenvironment is complex and multiple factors may be influencing the observed differences. Future plans include studies of 3D cell cultures using varied microenvironmental conditions and studies on δOR endogenous cell lines. Citation Format: Yoli JeuneSmith, Allison S. Cohen, Narges K. Tafreshi, Josef Vagner, Robert Gillies, David Morse. In vivo fluorescence lifetime imaging differentiates the bound versus unbound status of a cell surface receptor targeted ligand. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5519. doi:10.1158/1538-7445.AM2013-5519