Abstract 1209: Par-4 as therapy for colon cancer

Background: Colon cancer is the third leading cause of cancer related deaths in the United States. Epidemiological studies suggest an increase in the intestinal bile-acid load resulting from the consumption of a high-fat diet is a significant risk factor for colon cancer. Bile acids are the endogenous ligands for the farnesoid X receptor (FXR), a ligand-activated transcription factor and member of the nuclear receptor superfamily, and high levels of bile acids can promote colon cancer development. FXR is essential for maintaining bile-acid homeostasis by regulating bile-acid synthesis and transport, preventing the accumulation of intestinal bile acid levels to cancer promoting levels. Previous studies have demonstrated that FXR knockout mice are more susceptible to the development of colon adenocarcinomas, indicating that FXR plays a suppressive role in colon tumor formation. This study investigates the role of FXR in the development of human colon cancer. Methods: Immunohistochemistry was used to label for FXR in normal human colon, colon polyps, and colon adenocarcinomas staged I-IV. SYBR green quantitative PCR and western blot analysis were used to measure expression of FXR and FXR target genes in normal human colon and colon cancers staged I-IV as well as colon cancer cell lines. Reverse phase protein array on colon cancer cell line lysates was used to correlate FXR expression with oncogenic signaling cascades. To test if FXR expression was suppressed by DNA methylation, colon cancer cell lines were treated with a DNA methyltransferase (DNMT) inhibitor and DNMT siRNA and FXR mRNA measured by real-time PCR. Immunoprecipitation with an antibody against 5-methylcytosine (MeDIP) analysis was done in human colon cancer cell lines to determine methylation of NR1H4 (gene encoding FXR) promoter. Results: IHC and qPCR analysis reveals that the expression and function of FXR is markedly reduced early in colon cancer progression, with suppression seen within precancerous lesions. Furthermore, FXR expression in colon cancer cell lines were negatively correlated to oncogenic PI3 kinase signaling cascades and associated with epithelial to mesenchymal transition (EMT). Results suggest DNA methylation as a mechanism of FXR silencing in colon cancer and confirms methylation of the FXR promoter. Conclusion: FXR deficiency in animals indicates FXR serves a tumor suppressive role. Our studies show that FXR is silenced in early in human colon cancer progression possibly by DNA methylation, which could be a cancer promoting event. The overall mechanism of FXR's anti-tumorigenic activity is not fully established but may be due to FXR's role in regulating EMT and bile acid homeostasis. Restoration and enhancement of FXR activity, by blocking DNA methylation or increasing baseline activity of FXR, represents a potential therapeutic option for the treatment of colon cancer. Citation Format: Ann M. Thomas, Le Zhan, Julie Izzo, Dipen Maru, Imad Shureiqi, Veera Baladandayuthapani, Han Liang, Grace L. Guo, Garth Powis. Silencing of farnesoid X receptor in human colon cancer by epigenetic mechanisms is associated with cancer progression. [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 3561. doi:10.1158/1538-7445.AM2013-3561

Despite recent advances in the treatment of metastatic colon cancer (CC), most patients are not curable. Furthermore, whenever patients have exhausted all available standard chemotherapy and biologic agent treatment, patients are left with a very poor performance status. Thus, additional therapy for CC needs to be both active against the most advanced forms of the disease and tolerable for patients. Resveratrol (RV) is a phytoalexin from the skin of grapes that has been shown to have inhibitory effects on the proliferation of human tumors. It can be administered to cancer patients as an oral naturopathic/nutritional supplement. The current study investigated the capacity of RV to modulate apoptotic and immunoregulatory gene expression in CC cells and explored the functional consequences of modulation on in vitro tumor cell proliferation. Gene expression was assessed with a real time PCR assay and in vitro proliferation with a standard MTS assay. The ATCC cell line, T84 as well as cell suspensions prepared from metastatic CC specimens involving the stomach (SM), ovary (OM), and extensive peritoneal carcinomatosis (PC) were tested. Inhibition of proliferation was observed with RV alone in concentrations up to 5 ug/mL and this was enhanced further when RV was combined with the immunologic death ligands, rTNF and rTRAIL. For the T84 cell line, levels of proliferation inhibition were 28.0%, 0.5%, 5.4%, 44.5% and 51.8% with RV, TNF, TRAIL, RV+TNF, and RV+TRAIL respectively. The corresponding values for surgical specimens were SM: 5.6%, 1.0%, 0.3%, 28.3%, and 52.4% respectively; OM: 26.2%, 4.6%, 6.1%, 57.4%, and 75.0% respectively; and PC: 46.9%, 17.1%, 59.6%, 61.8% and 96.7%, respectively. These effects of RV + immunologic death ligands were reflected by changes in expression of apoptotic and immunoregulatory genes. For example, in OM cells some of the genes whose expression was up-regulated by RV, TRAIL, and RV+TRAIL were FAS (4.33, 1.57, & 6.22-fold, respectively), DAPK (3.07, 1.16, 3.68), CARD6 (1.96, -1.09, 3.01), and TRADD (2.05, 1.29, 2.31). Consistently, there was increased gene expression in response to the combination of RV +TNF/TRAIL to levels greater than those elicited by any single agent for the following genes: TNFRSF9; CASP10; BCL2L1; BAK1; and BAX. The results of this study demonstrate that RV, a well tolerated oral naturopathic/nutritional supplement can inhibit the in vitro proliferation of human CC cells and collaborate with the immunologic death ligands TNF and TRAIL to inhibit proliferation further. These effects are correlated with changes in expression of apoptotic and immunoregulatory genes in CC cells. Taken together, the results suggest that RV could be combined with cytokine-based therapy or other forms of immunotherapy which elicit immunologic death ligands for the treatment of metastatic CC in patients. 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 4763. doi:10.1158/1538-7445.AM2011-4763

Tightly coordinated Hedgehog (HH) signaling is critical during normal embryonic development. Aberrant HH signaling is involved in driving cell proliferation in multiple human cancers. Classical HH signaling involves the interaction of the soluble HH ligands with their receptor, Patched (Ptch), thereby releasing a transmembrane protein Smoothened (Smo) from Ptch-mediated inhibition. Smo then activates the Gli family of transcription factors that regulate HH target genes. We have previously reported that HH signaling provides essential survival support to human colon cancer cells and inhibition of this signal induces DNA damage and extensive cell death. However the downstream targets that govern the regulation of cell survival by HH/Gli in cancer cells are not completely defined. This study demonstrates that the Gli proteins transcriptionally regulate the expression of human telomerase reverse transcriptase (hTERT) gene in human cancer cells. hTERT is a known regulator of telomere homeostasis, which determines the replicative potential and hence the life span of cells. Suppression of both Gli1 and Gli2 functions by exogenous expression of a C-terminus truncated Gli3 repressor mutant (Gli3R), or by GANT61, a pharmacological inhibitor of Gli1 and Gli2 activity, reduced hTERT protein expression over a period of 72 hr in human colon, prostate and brain cancer cell lines. Further, exogenously expressed Gli2 significantly increased hTERT protein expression in human colon cancer cell lines. Exposure to GANT61 also inhibited hTERT mRNA expression within 24 hr in human colon cancer cell lines. Insilico analysis of the hTERT promoter revealed 7 putative Gli binding sites suggesting a transcriptional mode of regulation of hTERT expression by Gli2. Chromatin immunoprecipitation with Gli2 antibody precipitated fragments of the hTERT promoter in human colon cancer cell lines, indicating a direct interaction between Gli2 and the hTERT promoter. The binding between Gli2 and hTERT promoter was significantly reduced upon exposure to GANT61. Further, overexpression of the wild type hTERT cDNA in cancer cells prevented the cytotoxic effects of blocking the HH signaling pathway with GANT61. These findings demonstrate hTERT to be a direct transcriptional target and a critical mediator of the HH/Gli signaling pathway, and identify a previously unknown role of the HH/Gli axis in regulating the replication potential of cancer cells. These findings are of significance in understanding important regulatory mechanisms that determine the role of HH/Gli signaling in cancer cell survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4699A. doi:1538-7445.AM2012-4699A

Enterococcus faecalis is a human colonic commensal that induces colon inflammation and cancer in several mouse models. We have shown that E. faecalis-infected macrophages produce 4-hydroxy-2-nonenal (4-HNE), a lipid peroxidation byproduct of ω-6 polyunsaturated fatty acids, and that this reactive aldehyde is genotoxic and mediates bystander effects (BSE). To further investigate mechanisms for 4-HNE-mediated BSE, we treated primary murine colonic epithelial cells (YAMC) with 4-HNE and studied Wnt/β-catenin signaling. Western blotting showed activation of β-catenin after 1 hr exposure of YAMC cells to 1 μM 4-HNE. Immunofluorescence staining confirmed nuclear translocation of activated β-catenin 5 hrs following treatment. These findings suggested that 4-HNE as an endogenous carcinogen that potentially contributes to colorectal carcinogenesis. Because glutathione S-transferase alpha 4 (Gsta4) is a detoxifying enzyme for this reactive aldehyde, we determined whether exposure of YAMC cells to 4-HNE increased the production of Gsta4. Indeed, Gsta4 was significantly increased at 24-72 hrs following 4-HNE treatment, suggesting upregulation of Gsta4 in response to 4-HNE-induced genotoxicity. In addition, we measured Gsta4 in 6 human colon cancer cell lines and compared these results to normal human colonic epithelial cells (FHC). Our findings showed that Gsta4 was strongly produced by all cancer cell lines compared to FHC. Finally, we colonized interleukin (IL)-10 knockout mice with E. faecalis for short- (2 weeks), intermediate- (3 months), and long-term (9 months) periods of time. Gsta4 levels in serum significantly increased for Il10−/− mice colonized with E. faecalis at 2 weeks compared to no change for colonized wildtype mice. Immunohistochemical staining for Gsta4 was apparent in tissue macrophages from colon biopsies for colonized mice, implying macrophage activation. Gsta4-positivity significantly increased in colon macrophages after 3 and 9 months of colonization in areas of increased inflammation. Of note, although Gsta4-positive staining was not seen in epithelial cells in areas of inflammation, intense staining was noted in neoplastic epithelial cells. These results suggest that activation of Gsta4 may be an early event in macrophage activation and associated with neoplasia. In summary, in the IL-10 knockout model, E. faecalis-infected macrophages produce 4-HNE and this lipid peroxidation breakdown product induces Wnt/β-catenin signaling and Gsta4 production. These findings implicate Gsta4 as a potential surrogate biomarker for colon cancer and should permit the development of new chemopreventive strategies. Citation Format: Xingmin Wang, Yonghong Yang, Mark Huycke, Department of Veterans Affairs Medical Center. Glutathione S-transferase alpha 4 is a potential biomarker for Enterococcus faecalis-induced inflammation and colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1344. doi:10.1158/1538-7445.AM2013-1344

Objective To investigate the effect of siRNA silencing cell cycle-related kinase (CCRK) gene on the growth and apoptosis of colon cancer in mice, and to provide a new idea and method for the treatment of colon cancer. Methods Fifty BALB / C (nu / nu) nude mice were inoculated with human colorectal carcinoma SW480 cells, in which 30 nude mice were successfully established for transplantation tumor model and were randomly divided into three groups, siRNA CCRK transfection of human SW480 colon cancer cell line (experimental group) and normal cultured SW480 cells (blank control group), and carrying irrelevant sequence fragment shRNA slow transfection of virus SW480 cells (negative control group) respectively.The nude mice were sacrificed and the tumor tissues were removed after 42 days.The tumor weight, CCRKmRNA expression, CCRK protein expression and apoptosis rate were compared between the groups. Results siRNA transfection group, tumor weight, CCRK mRNA and CCRK protein expression [(0.54±0.15)g, 1.14±0.24, 0.18±0.05] were significantly lower than those of the control group [(1.05±0.14)g, 2.41±0.42, 0.49±0.07] and empty vector of siRNA group [(1.07±0.12)g, 2.39±0.47, 0.47±0.09, F=21.18, 23.47, 90.03; P 0.05). Cells apoptosis rate in siRNA transfection group (21.23±1.12)% was significantly higher than that in normal control group (6.78±0.37)% and transfected with empty vector of siRNA group (7.25±0.32)% (P 0.05). Conclusions CCRK targeting siRNA expression vector inoculation with colon cancer can significantly reduce the growth of colon cancer and inhibit the expression of CCRK gene and protein in nude mice; CCRK gene silencing by siRNA can promote the colon cancer cells apoptosis; thus , CCRK gene can serve as a new potential molecular target for rectal cancer in gene therapy by siRNA. Key words: Colonic neoplasms; Cell cycle-related kinase gene; Gene silencing; Apoptosis

The aim of this study was to improve disease-free survival and overall survival in orthotopic nude mouse models of human colon cancer with fluorescence-guided surgery (FGS). Fluorescent orthotopic or carcinomatosis models were established in nude mice using human colon cancer cell lines HCT-116 and HT-29 expressing either green fluorescent protein (GFP) or red fluorescent protein (RFP). The tumors were later resected by bright light surgery (BLS) or FGS. In orthotopic and carcinomatosis models, pre- and post-operative images were obtained with the OV-100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan) to assess the extent of surgical resection. In the orthotopic model, whole body imaging of the mice was performed in the postoperative period to assess cancer recurrence and to follow subsequent tumor progression. The mice were sacrificed when they became premorbid and their abdomens were exposed for intravital and ex vivo imaging. Tumor burden was measured in mm2 using ImageJ v1.440. A greater extent of tumor resection in mice with carcinomatosis was achieved using FGS compared to BLS (99.9% vs. 76.9%, p = 0.006). Furthermore, all mice with localized disease had a complete surgical resection with FGS. In contrast, complete resection was achieved in only 56% of the mice undergoing BLS. (p=0.001) Fewer mice in the FGS group had evidence of tumor recurrence (33%) compared to mice in the BLS group (58%), lengthening disease-free survival from 9 weeks in the BLS group to 27 weeks in the FGS group. Overall survival of the mice also increased from 17 weeks in the BLS group to 29 weeks in the FGS group. The results of the present study demonstrate that improved surgical outcomes in the treatment of colon cancer can be achieved with FGS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 372. doi:1538-7445.AM2012-372

Gastrointestinal cancers, in particular colon and pancreatic cancer, account for almost 100,000 deaths each year in the United States, ranking third and fourth respectively, in the most common cause of cancer related deaths. Our lab has been focused on developing strategies for chemoprevention rather than treatment after diagnosis. For pancreatic cancer, the prognosis is usually poor with high mortality rates after diagnosis. Thus, the objective of this work was to study the effects of chemopreventive combination regimens of a non-steroidal anti-inflammatory drug (NSAID) naproxen (N), in combination with resveratrol (R, an antioxidant) and sulforaphane (S, a broccoli extract) on human colon (HT-29 and HCT-116) and pancreatic (MIA PaCa-2 and Panc-1) cancer cell lines. MTS cell proliferation assay was conducted on all cell lines. For the MTS assay, upon 75 % confluence, 100µL of 2.5X103 cells each of HT-29, HCT116 and MIA PaCa-2 and 4X10 3 cells Panc-1 cells were transferred into each well of 96-well plates. The NRS chemopreventive agents alone, or in combination, were added to the cells and incubated for 72 h. The plates were then incubated for 1-4 h at 37°C in a humidified, 5% CO2 atmosphere. Absorbance was recorded at 490 nm using an ELISA plate reader. For HT-29 and HCT 116 colon cancer cell lines, the combinations of naproxen (500µM/L) with resveratrol (50µM/L) and sulforaphane (5µM/L) showed reduction in cell viability of 56.74% and 52.9%, respectively. The IC50 values for NRS were 1.46 mM/L,104.54µM/L and 17.81µM/L respectively on HT-29 cell line. Similarly, for MIA PaCa-2 and Panc-1 pancreatic cancer cell lines, combinations of naproxen (500µM/L) with resveratrol (6µM/L) and sulforaphane (5µM/L) showed reduction in cell viability of 66.67% and 58.8% respectively. The IC50 values for NRS were 778µM/L, 74.8µM/L and 11.82µM/L respectively, on MIA Paca-2 cell line. When used individually, none of the chemopreventive agents at the above concentrations demonstrated any decrease in cell viability, however when combined together, a synergistic effect was observed. Our results demonstrate that NRS drug combinations at low concentrations exhibit significant decrease in cell proliferation in both human colon and pancreatic cancer cell lines. This data provides compelling evidence of the potential of these combination regimens for the chemoprevention of colon and pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1632. doi:1538-7445.AM2012-1632

Natural products from various plants, microorganisms, fungi etc. have provided considerable value to the pharmaceutical industry over the past half century. But there still remains a vast potential reservoir of possible useful therapeutics, yet to be identifed. Among them, lichens (symbiotic associations between a fungus and an alga and/or a cyanobacterium), which produce more than a 1000 secondary chemicals. The United States is home to a diverse assemblage of lichen species. Lichens play many biological roles; however, their full potential as a source of anticancer drugs has been largely unexplored. To examine the possible anticancer role of several lichen species, we assessed the cytotoxic activity of 17 species collected from various parts of the US. Secondary compounds of these lichens were extracted with acetone and screened against Burkitt's Lymphoma cells (Raji) and the human colon cancer cell line (HT-29). Based on the cytotoxcity of these lichen extracts we selected two species Tuckermannopsis ciliaris and Xanthoparmelia chlorochroa for further testing. IC50 values for these two species showed particular promise - 28.8 and 28.6 μg/ml for Raji cells and 60 and 40 μg/ml for HT-29 cells respectively. The viability and cell growth of both cell lines was significantly reduced by the extracts from these two lichens but these extracts did not affect the viability and growth of normal lymphocytes. We performed morphological detection of apoptosis using Acridine orange and Propidium Iodide. Extracts from both lichens were found to induce apoptosis in both cancer cell lines when treated with the concentrations equivalent to the IC50. Cell proliferation of Raji and HT-29 cells, as measured by 3H-thymidine incorporation, was significantly reduced in treated cells (P <0.05) over 24 hours as compared with controls. To further investigate apoptosis we measured DNA fragmentation using gel electrophoresis. Within 24 hours of treatment, there was partial DNA degradation. Using flow cytometry, it was discovered that extracts from both lichens arrested cell-cycle progression in S phase. Using RT-PCR, it was found that the p53 gene was up-regulated in the treated cells during G1 phase while the DNA repair enzyme Thymidine kinase 1 (TK1) was down regulated. In conclusion, extracts from T. ciliaris and X. chlorochroa were both cytotoxic and induced apoptosis in cancer cells but not in normal cells. Hence extracts from these two lichens could hold potential as a non-toxic anticancer therapy. However, more research is required to further elucidate the molecular mechanisms behind these preliminary findings. Citation Format: Gajendra Shrestha, Atif M. El-Naggar, Sean C. Derenthal, Michael R. Boswell, Evita Weagel, Larry L. St. Clair, Richard Robison, Kim L. O'Neill. Cytototoxicity and mode of action of extract from of two lichens, Tuckermannopsis ciliaria (Ach.) Gyelnik and Xanthoparmelia chlorochroa (Tuck.) Hale against Burkitt's Lymphoma (Raji) cells and the Human colon cancer (HT-29) cell line. [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 2250. doi:10.1158/1538-7445.AM2013-2250

Colon cancer is the second leading cause of cancer-related mortality worldwide, and the prognosis of advanced colon cancer has remained poor in recent years. Galectin-9 (Gal-9) is a tandem-repeat type galectin that has recently been shown to exert antiproliferative effects on various types of cancer cells. The present study aimed to assess the effects of Gal-9 on human colon and colorectal cancer cells in vitro and in vivo, as well as to evaluate the microRNAs (miRNAs/miRs) associated with the antitumor effects of Gal-9. We examined the ability of Gal-9 to inhibit cell proliferation via apoptosis, and the effects of Gal-9 on cell cycle-related molecules in various human colon and colorectal cancer cell lines. In addition, Gal-9-mediated changes in activated tyrosine kinase receptors and angiogenic molecules were assessed using protein array chips in colon and colorectal cancer cells. Moreover, miRNA array analysis was performed to examine Gal-9-induced miRNA expression profiles. We also elucidated if Gal-9 inhibited tumor growth in a murine in vivo model. We found that Gal-9 suppressed the cell proliferation of colon cancer cell lines in vitro and in vivo. Our data further revealed that Gal-9 increased caspase-cleaved keratin 18 levels in Gal-9-treated colon cancer cells. In addition, Gal-9 enhanced the phosphorylation of ALK, DDR1, and EphA10 proteins. Furthermore, the miRNA expression levels, such as miR-1246, miR-15b-5p, and miR-1237, were markedly altered by Gal-9 treatment in vitro and in vivo. In conclusion, Gal-9 suppresses the cell proliferation of human colon cancer by inducing apoptosis, and these findings suggest that Gal-9 can be a potential therapeutic target in the treatment of colon cancer.

We have previously reported inhibition of cell migration and proliferation induced by difluoromethylornithine (DMFO) and metformin (Met) combination in human melanoma and colon cancer cells. We further investigated the mechanism of cell death with the combination treatment in melanoma and colon cancer cells. The in vivo effect of the combination in melanoma was also determined. Human colon cancer (HCT 116, HT 29) and melanoma (MEL1861, SK-23) were treated with Met, DFMO, or combination. Western blotting was performed to determine the expression of AMP kinase, mTOR, p70S6K and 4E-BP1. Apoptosis was measured by Annexin V assay. Autophagy was determined by fluorescence microscopy staining for LC3A/B and Western blot analysis of LC3A/B and Beclin-1 expression. For in vivo evaluation, SK-23 cells were injected SQ into BALB/c nu mice. After tumor nodule was established, 4 groups of 6 mice : group 1 - IP injections of vehicle and normal drinking water, group 2 - 2% (w/v) DFMO in drinking water; group 3 - IP Met (250 mg/kg/day), group 4 - DFMO plus Met. Mice body weight and tumor volume were measured every 3 days. Tumor weight was measured on Day 20 at necropsy. For comparison between groups, the student's t test was used and p< 0.05 was considered to be significant. DFMO and Met induced apoptosis in colon cancer and melanoma cells in a dose- and time-dependent manner. Significant increase in apoptosis was noted in the combined treatment group compared with either one alone. Increased number of autolysosomes was observed in the combination group under fluorescence microscopy. Up-regulation of Beclin-1 and LC3A/B expression were also increased with combination treatment compared with either treatment alone. Increased expression of phosphor-AMPK, decreases expression of p70S6 and 4EBP1 were observed with the combination treatment. Both DFMO and Met alone have significant in vivo anti-proliferative effect on human melanoma cells compared with control. However, the anti-proliferative effect of the combination treatment was significantly better than either regimen alone (p< 0.001). Average SK-23 tumor weight was 100 mg for control group compared with 45 mg for group 2 (p<0.05), 35mg for group 3 (p< 0.05), and 25mg for the combination group (p< 0.001). Our findings suggest that combination of DFMO and Met induced cancer cell death via apoptosis and autophagy though activation of AMPK pathway and suppression of the Akt/mTOR signaling pathway. DFMO and Met combination can have significant anti-tumor effect in vivo. Citation Format: Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. Induction of autophagy and apoptosis with polyamine synthesis inhibition and metformin in human melanoma and colon cancer cells. [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 1418. doi:10.1158/1538-7445.AM2014-1418

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to migrate to tumor stroma as well as injured tissue. We reported that, in an orthotopic nude mice model of colon cancer, MSCs travel to tumor stroma and differentiate into carcinoma-associated fibroblasts (CAFs), and then, they promote growth and metastasis of colon cancer. We also found that CAFs express platelet-derived growth factor receptor-beta (PDGFR-β), at a high level and that imatinib therapy targeting PDGFR in CAFs combined with administration of a cytotoxic drug significantly inhibits growth and metastasis of human colon cancer. A novel oral multi-kinase inhibitor, regorafenib not only inhibits oncogenic receptor tyrosine kinases such as c-KIT, RET, and B-RAF, but also inhibits stromal cells which express VEGFR1-3, TIE2, PDGFR-β, and fibroblast growth factor receptor 1 (FGFR1). These cell surface receptor tyrosine kinases are known to be involved in tumor neovascularization, vessel stabilization, lymphatic vessel formation as well as activation of stroma. We examined whether the effect of MSCs on tumor growth and metastasis was inhibited by regorafenib. KM12SM human colon cancer cells alone or KM12SM cells mixed with MSCs in a 1:2 ratio were transplanted into the cecal wall of nude mice. Orthotopic transplantation of KM12SM cells + MSCs, in comparison to transplantation of KM12SM cells alone, resulted in tumors of greater weight and volume, and higher rate of lymphatic metastasis. Co-injection of MSCs with tumor cells promoted tumor growth and metastasis of colon cancer by enhancing angiogenesis, lymphangiogenesis, and tumor cell proliferation and by inhibiting tumor cell apoptosis. When CRC tumor + MSCs bearing animals were treated with regorafenib (by oral gavage once daily at 10 mg/kg for 35 days), primary tumor size or the number of lymph node metastases were significantly decreased compared to those treated with vehicle, and reached to the level with no significant difference from KM12SM tumor cells alone. Our data suggest that targeting tumor microenvironmental signaling pathways by regorafenib influences the interaction between bone marrow-derived MSCs and tumor cells and, hence, inhibits the progressive growth of colon cancer. Citation Format: Kei Shinagawa, Yasuhiko Kitadai, Ryo Yuge, Mieko Onoyama, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Treatment with regorafenib inhibits the tumor-promoting effect of bone marrow-derived mesenchymal stem cells in an orthotopic nude mice model of colon cancer. [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 5068. doi:10.1158/1538-7445.AM2015-5068

Ecotropic viral integration site-1 (EVI1) is an oncogenic transcription factor that is frequently implicated in hematological malignancies and in some solid tumors. Deregulated expression of EVI1 impairs several pathways including proliferation, differentiation, apoptosis and cell cycle through different mechanisms in different cell types. EVI1 was found to be overexpressed in 53% of human colorectal cancer samples, 100% of colon adenocarcinoma samples, 100% of human colon cancer cell lines and its presence might affect disease progression and sensitivity to chemotherapy. To investigate the stage specific expression pattern of EVI1 in colon cancer patient samples, we analyzed two of the publicly available microarray datasets (GEO database) of colon cancer patients and observed that although EVI1 is upregulated in the initial stage of the disease, the expression level decreases with the progression of the disease. In cancer cells loss of epithelial adhesion molecule E-cadherin is considered to be a fundamental event in EMT. To decipher whether EVI1 is involved in EMT, we checked five transcription factors (SLUG, SNAIL, TWIST, ZEB1 and ZEB2) which controls the expression of E-cadherin in cancer cells. Significant negative correlation was observed between the expression levels of EVI1 and all the above transcription factors in colon cancer patient samples. Recently it was shown by using ChIP sequencing that EVI1 target SLUG and SNAIL in SKOV3 cells. To understand whether EVI1 directly binds to both SLUG and SNAIL, we did an in silico analysis and searched for EVI1 binding site(s) in the functional promoter region of both SLUG and SNAIL. Although we did not observe any binding site in SNAIL promoter region; we observed two EVI1 binding sites in the SLUG promoter region. To validate the predicted target sites in vivo, we did a ChIP assay and found that EVI1 binds to one of the site and not both of them. To determine the transcriptional regulation, we cloned the promoter region of SLUG (-1100 bp) in pGL3 vector and did luciferase assay, which revealed that EVI1 significantly represses SLUG activity in EVI1 transfected cells. Analysis of gene expression databases corroborates the above finding and shows that EVI1 negatively regulates SLUG expression in each stage of the disease as well as in colon cancer cell lines. We have worked on the functional aspects of the binding in terms of EMT which will be presented in the poster. Citation Format: Kasturi B. Nayak, Soumen Chakraborty. EVI1 transcriptionally represses SLUG to regulate epithelial-mesenchymal transition (EMT) in human colon 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 465. doi:10.1158/1538-7445.AM2014-465

Serine is a non-essential amino acid that is generated by the sequential actions of phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT1) and phosphoserine phosphatase (PSPH). Increased serine biosynthesis occurs in several cancers and supports tumor growth. In addition to serine synthesis, exogenous serine is taken up by cells and can also fuel tumor growth. Interestingly, colon cancer cells increase expression of serine biosynthesis enzymes in the absence of exogenous serine, suggesting a compensatory adaptive response to reduced availability of serine. This study explored the relative contributions of exogenous and synthesized serine to colon cancer cell growth, metabolism and response to anti-cancer therapy. We found that PSAT1 expression was markedly increased in human colonic adenomas and colorectal cancer (CRC) compared to normal adjacent tissue. Additionally, high levels of PSAT1 in CRC were associated with reduced patient survival. Deletion of PSAT1 only modestly reduced colon cancer cell proliferation in vitro. Removal of serine from the medium strongly suppressed cell proliferation whereas the combination of removing exogenous serine and deleting PSAT1 caused even greater inhibition of cell proliferation. Metabolite profiling revealed altered nucleotide levels upon exogenous serine depletion, an effect that was enhanced when PSAT1 was deleted. Global gene expression profiling showed approximately 3,000 differentially expressed genes when only exogenous serine was removed, with an additional ~800 changes in PSAT1 knockout (KO) cells grown in serine deficient medium. A marked DNA damage response was induced selectively in PSAT1KO cells in serine deficient medium, which was prevented by the addition of formate. Xenografts derived from PSAT1 sufficient and PSAT1KO cells showed that neither PSAT1 deletion alone nor removal of dietary serine alone affected tumor growth, but the combination resulted in profound growth suppression. Additionally, treatment with 5-fluorouracil (5-FU) in the xenograft model induced a strong regression of established tumors derived from mice carrying PSAT1KO cells fed a serine deficient diet. Complementary in vitro studies showed enhanced sensitivity of PSAT1KO cells grown in serine deficient medium to 5-FU induced death, which was preceded by specific metabolite changes including markedly reduced levels of TMP and TTP. Taken together, our results suggest that both exogenous and endogenous sources of serine are important contributors to colon cancer cell proliferation and metabolism. Limiting exogenously available and synthesized serine may be an effective strategy to limit colon cancer growth and enhance the efficacy of treatment. Citation Format: David C. Montrose, Miguel Foronda, Suchandrima Saha, Erin M. McNally, Xi Kathy Zhou, Jan Krumsiek, Akanksha Verma, Olivier Elemento, Rhonda K. Yantiss, Qiuying Chen, Steven S. Gross, Lorenzo Galluzzi, Lukas E. Dow, Andrew J. Dannenberg. Exogenous and endogenous sources of serine contribute to colon cancer metabolism and growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3745.

In order to identify molecular markers prognostic of initiation and/or progression of human colon cancer (CC), a genome-wide analysis was performed and highlighted a micro-deletion at the 1p36.11-12 region in 23% (n = 115) and 47% (n = 59) of adenomas and carcinomas, respectively. This region contains the E2F2 and ID3 genes, which are known to regulate the cell cycle and cell differentiation, respectively. In addition, E2F2, is described as either oncogenic or tumour suppressor, depending on the tissue or cell type. The micro-deletion incidence depends on tumour stages (60% in early stages whereas only 34% in metastatic stages and further clinical analysis showed that patients with deleted E2F2 had a lower rate of recurrence and a better overall survival. Also, RT-QPCR evidenced that E2F2 and ID3 transcript expression levels decrease in human CC. Therefore, to assess the role of the deletion in tumour growth and metastatic activity, we evaluated the functional consequences of the loss of these genes both in vitro in human colon cancer cells that were transiently depleted of either E2F2 or ID3 and in vivo in immunodeficient mice. Both gene transcript expressions were down-regulated by transient siRNA transfection in the human epithelial CC cell line Caco-2/TC7. Consequences were evaluated by immunocytochemistry for proteins involved in the cell architecture and in cell-cell and cell-matrix junctions, and at the expression level by RT-QPCR and Western Blot analyses. Functional analyses were assessed for the migratory potential with the wound healing assay, for proliferation with the MTS assay, and for adhesion on substrates such as laminin, collagen I and fibronectin. The in vivo metastastic potential was evaluated with E2F2- and ID3-silenced cells that were intra-splenically implanted. E2F2 down-regulation and that of ID3 at a lesser extend, reduced proliferation and induced severe morphological modifications, associated with relocalization of structural members of adherens junctions (beta-catenin, APC), tight junctions (Claudin-1, ZO-1) and cytoskeleton (F-Actin, Cytokeratin-19). The integrins alpha5, alphaV, alpha2 and beta-1, were downregulated and the adhesion properties on laminin-111, but not on collagen I or fibronectin were lost. Moreover, inhibition of E2F2 and ID3 expression leads to a decreased migratory potential. The intra-spenic injection in immunodeficient mice of HT29 cells stably transduced to underexpress E2F2 and ID3, leads to lower / slower development of liver metastases. In conclusion, for a clinical aspect, the micro-deletion of the 1p36.11-12 region is a good prognosis for the event-free survival, and from a more basic and functional aspect, the loss of E2F2 and ID3 expression regulates tumor growth and dissemination through functions involving migration and adhesion. Citation Format: Dominique Guenot, Manon Voegelin, Eric Guérin, Céline Nicolet, Marie-Pierre Gaub. The role of the E2F2 and ID3 transcription factors in colon tumour growth and cellular dissemination. [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 1915. doi:10.1158/1538-7445.AM2013-1915

Background: Hyperthermic intraperitoneal chemotherapy (HIPEC) is a promising procedure for the treatment of peritoneal carcinomatosis (PC). Heat shock proteins (HSPs) and other proteins involved in cellular repair mechanisms seem to induce cytoprotective processes during HIPEC therapy. Therefore, the aim of this study was to analyze the effects of HIPEC-related conditions on tumor cell proliferation and the expression of HSPs in human colon cancer. Methods: Human colon cancer cell lines HT29, SW480 and SW620 were exposed to different temperatures (37°C, 41°C, and 43°C) as well as defined cytostatic agents (Oxaliplatin, Mitomycin C, and 5-Fluorouracil). After cellular regeneration (30 min, 24 h, 48 h and 72 h) RNA isolation and whole cell extraction was performed. Gene and protein expression analysis of HSP27, 70, 72 and 90 as well as PCNA, Ki-67, BCl-2 and BCl-Xl were carried out using RT-qPCR and Western blot. Additionally, MTS cell proliferation assays were performed 24 h, 48 h, 72 h and 96 h post treatment. Moreover, AnnexinV apoptosis assays were conducted. Results: All colon cancer cells exposed to hyperthermic conditions showed initially up-regulated HSP gene expression. Highest expression was found after exposure to 43°C. Combined cytostatic and hyperthermic treatment demonstrated additional increase in HSP27 expression and in other HSPs to a lesser degree. Tumor cells exposed to cytostatic agents showed overall higher HSP gene expression compared to cells without chemotoxic treatment. Similar effects were detected for the expression of the proliferation marker PCNA and anti-apoptotic protein BCl-Xl. Apoptosis assay demonstrated decreased numbers of apoptotic cells at 43°C compared to normothermia. Additionally, proliferation assays revealed reduced chemosensitivity in cells treated with hyperthermia. Conclusion: Desired effects of hyperthermia used in HIPEC therapy to achieve anti-proliferative and apoptosis inducing effects seem to be negatively influenced by cell stress mediated repair mechanisms in colon cancer. Our in vitro findings suggest analyzed HSPs to be significantly involved in this hyperthermia and chemotoxicity mediated cellular repair mechanisms. While initial increase in HSP expression can counteract cytotoxic effects during HIPEC therapy their prolonged expression may promote lasting resistance to cellular stress. Citation Format: Tanja Grimmig, Kerstin Kloos, Rebecca Thumm, Romana Moench, Christoph T. Germer, Ana Maria Waaga-Gasser, Martin Gasser. Cell stress during HIPEC causes heat shock protein induction and reduced chemosensitivity in human colon cancer. [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 2945.