Lines SW480 Research Articles

Abstract 1762: Verbascoside promotes apoptosis by regulating HIPK2-p53 signaling in human colorectal cancer

Abstract Background: We investigated the role of the HIPK2-p53 signaling pathway in tumorigenesis and resistance to the drug Verbascoside (VB) in colorectal cancer (CRC), using in vivo and in vitro experiments. Methods: Primary human CRC samples and normal intestinal tissues from patients were analyzed for HIPK2 expression by immunohistochemistry (IHC) and its expression was correlated against patients’ clinicopathological characteristics. Human CRC HCT-116 cells were implanted in BALB/c nude mice; mice with xenografted tumors were randomly administrated vehicle (control), 20, 40, or 80 mg/mL VB, or 1 mg/mL fluorouracil (5-FU). HIPK2, p53, Bax, and Bcl-2 expression in these tumors were determined by IHC. In vitro effects of VB on CRC cell proliferation and apoptosis were measured by CCK-8 assay and flow cytometry; HIPK2, p53, p-p53, Bax, and Bcl-2 were measured by western blot. Results: IHC analysis for 100 human CRC tumor samples and 20 normal intestinal tissues, showed HIPK2 expression to inversely correlate with Dukes stage and depth of invasion in CRC (P <0.05). In vivo, the inhibition rates of 20, 40, and 80 mg/mL VB on CRC xenograft tumor weight were 42.79%, 53.90%, and 60.99%, respectively, and were accompanied by increased expression of HIPK2, p53, and Bax, and decreased Bcl-2 expression in treated tumors. In vitro, VB significantly inhibited proliferation of CRC cell lines HCT-116, HT-29, LoVo, and SW620, in a time- and dose-dependent manner. The apoptosis rates of 25, 50, and 100 μM VB on HCT-116 cells were 10.83 ± 1.28, 11.25 ± 1.54, and 20.19 ± 2.87%, and on HT-29 cells were 18.92 ± 6.12, 21.57 ± 4.05, and 25.14 ± 6.73%, respectively. In summary, VB treatment significantly enhanced the protein expression of pro-apoptotic HIPK2, p53, p-p53, Bax, and decreased anti-apoptotic Bcl-2 expression in CRC cells. Conclusions: HIPK2 protein modulates the phosphorylation status of p53, and levels of Bax and Bcl-2 in CRC. We also found that VB effectively activated the HIPK2-p53 signaling pathway, resulting in increased CRC cells apoptosis. Note: This abstract was not presented at the meeting. Citation Format: Lihong Zhou, Yuanyuan Feng, Yongjie Jin, Xuan Liu, Hua Sui, Ni Chai, Xingzhu Chen, Ningning Liu, Yan Wang, Qi Li. Verbascoside promotes apoptosis by regulating HIPK2-p53 signaling in human colorectal 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 1762. doi:10.1158/1538-7445.AM2015-1762

Abstract 2544: Combination of anti-cancer small molecule tolfenamic acid and curcumin effectively inhibits colon cancer cell growth

Abstract Tolfenamic acid (TA) is small molecule and non-steroidal anti-inflammatory drug which is known to inhibit human cancer cells and mouse tumor growth in some cancer models. TA is known to target the transcription factor, specificity protein1 (Sp1), that mediates the expression of several genes associated with cancer, including survivin, a key member of the inhibitor of apoptosis proteins (IAP) family. Curcumin is an aromatic constituent of the plant curcuma longa (turmeric) which is extensively studied in some malignancies including breast, pancreatic and colon cancers. Even though curcumin shows a broad spectrum of anti-cancer activity in pre-clinical studies, its clinical application is greatly affected by its low bioavailability. Hence, several strategies to improve the therapeutic response of curcumin are being pursued, including the synthesis of curcumin analogs and using curcumin in combination with other small molecules or drugs to increase bioavailability. In this study, using a colon cancer model, the therapeutic efficacy of curcumin and a curcumin analog EF31 in combination with an anti-cancer small molecule TA was evaluated. Colon cancer cell lines HCT116, HT29, and SW620 were treated with TA, curcumin, or EF31 and cell viability was measured at 24, 48, and 72 hours post-treatment. All agents showed a steady and consistent decrease in cell viability, following a clear dose and time-dependent response. Combination of TA with curcumin or EF31 showed higher growth inhibition compared to single agent treatment. Analysis of apoptosis was carried out to determine the mode of growth inhibition, using flow cytometry (JC-1 staining) Western blot analysis (poly-ADP ribose polymerase [PARP] cleavage), and caspase3/7 activity. Results demonstrated a significant increase in the apoptotic fraction (JC-1 staining) following combination treatment, when compared to individual effect. This was accompanied by the activation of caspases (caspase 3/7) and the PARP cleavage. Western blot results also revealed that combination treatment of TA with curcumin or EF31 significantly decreased expression of Sp1, Nuclear Factor-kappa light chain enhancer of B cells (NF-kB), and survivin, and modulated the expression of proteins associated with the cell cycle. These pre-clinical results demonstrate that the anti-cancer small molecule, tolfenamic acid, may enhance the therapeutic efficacy of curcumin or curcumin analogs in colon cancer. Mechanistic studies to delineate the pathways involved in combination treatment are under investigation. Citation Format: Umesh T. Sankpal, Ganji Purnachandra Nagaraju, Myrna Hurtado, Sriharika R. Gottipolu, Bassel El-Rayes, Mamoru Shoji, Christopher G. Jordan, Riyaz Basha. Combination of anti-cancer small molecule tolfenamic acid and curcumin effectively inhibits colon cancer cell growth. [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 2544. doi:10.1158/1538-7445.AM2015-2544

Abstract 2557: Efficacy of combination chemotherapy using a novel oral chemotherapeutic agent, TAS-102, with oxaliplatin on human colorectal or gastric cancers and 5-FU-resistant gastric cancer xenografts

Abstract Background: TAS-102 is a novel oral nucleoside antitumor agent composed of trifluridine (FTD) and tipiracil hydrochloride (TPI) at a molar ratio 1:0.5. FTD is the active antitumor component of TAS-102 and its triphosphate form is incorporated into DNA in tumor cells. TPI is a potent inhibitor of thymidine phosphorylase, an enzyme that degrades FTD, allowing adequate plasma concentrations of orally administered FTD to be maintained and potentiating the antitumor activity of FTD. In a recent international multicenter randomized double blind Phase III study (RECOURSE), use of TAS-102 was shown to significantly improve overall survival, progression-free survival and had a favorable safety profile in comparison to the placebo in patients with metastatic colorectal cancer refractory to standard chemotherapies. In this study, we evaluated the antitumor effects of TAS-102 in combination with oxaliplatin on gastrointestinal tumor xenografts, including a 5-FU-resistant subline, in a nude mouse model. Method: The human colorectal cell lines HCT-116 and SW48 and gastric cancer cell lines SC-2, MKN74 and MKN74/5-FU, a 5-FU-resistant cell line derived from MKN74, were implanted into nude mice subcutaneously. TAS-102 (150 mg/kg/day) was orally administered twice daily from days 1 to 14 and oxaliplatin (7 or 13 mg/kg/day) was administered intravenously on days 1 and 8. Growth inhibitory activity was evaluated by assessing tumor volume and the growth delay period, estimated based on the period required to reach a tumor volume five times greater than the initial volume (RTV5). Results: TAS-102 and oxaliplatin monotherapy were effective on all the evaluated colorectal and gastric cancers, even for a 5-FU-resistant gastric cancer cell line. The tumor growth inhibitory activity and RTV5 of the group receiving TAS-102 with oxaliplatin were significantly superior to those receiving either monotherapy for all evaluated cancers (P<0.05). Tumor growth inhibition in the combination treatment group was more than 70%. Furthermore, the RTV values on day 29 were less than 5 for SC-2, MKN74 and MKN74/5FU xenografts. These results demonstrate that combination therapy with TAS-102 and oxaliplatin is significantly more effective than either monotherapy in colorectal and gastric cancer xenografts, including a 5-FU-resistant xenograft, when evaluated by tumor growth inhibition and growth delay period. This combination appeared to be well-tolerated, as drug-related death or a reduction in body weight of more than 20% was not observed. Conclusion: The present preclinical findings indicate that combination therapy of TAS-102 and oxaliplatin is a promising treatment option for colorectal or gastric cancer, not only for chemo-naïve tumors, but also for recurrent tumors after 5-FU-based chemotherapy. Citation Format: Mamoru Nukatsuka, Fumio Nakagawa, Kazuaki Matsuoka, Hiroshi Tsukihara, Teiji Takechi. Efficacy of combination chemotherapy using a novel oral chemotherapeutic agent, TAS-102, with oxaliplatin on human colorectal or gastric cancers and 5-FU-resistant gastric cancer xenografts. [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 2557. doi:10.1158/1538-7445.AM2015-2557

Sa1829 Phytochemicals Target Epigenetic Signaling to Block Cancer Stem Cell-Driven Colon Carcinogenesis

Background: Phytochemicals modulate key cellular signaling pathways and have proven anticancer effects. Alcea rosea (AR; Hollyhock) is an ornamental plant in the Malvaceae family with known anti-inflammatory properties. Its role as an anti-cancer agent however, has not yet been confirmed. The present investigation is focused on the mechanistic insight of AR seed extract as an anti-cancer agent for colon cancer. Methodology: Colon cancer cell lines HCT116 and SW480 and normal colon epithelial cells were used in the study. Cell proliferation, cell cycle and stemness were determined by hexoseaminidase, flow cytometry and spheroid assays, respectively. TOP-Flash reporter and WIF1 promoter activities were performed in HEK-293T cells. For in vivo effects, we used the HCT116-tumor xenograft model for colon tumorigenesis. Real Time PCR, Western blot and immunohistochemistry studies were performed for determining gene expression. Results: The AR seed extract inhibited proliferation and colony formation in a dose and time dependent manner in colon cancer cell lines. The anti-proliferative activity appeared to be mediated by apoptotic mechanisms, as cleavage of PARP and increased expression of Bax accompanied decreased levels of BCL-xl protein. In addition, AR seed extract arrested cells at Go/G1 phase of cell cycle and exhibited decreases in Cyclin D1. Treatment of cells with AR seed extract also reduced the number and size of colonospheres in a dose-dependent manner which coincided with decreases in the expression of ALDH1A1 and Dclk1, markers of CSCs (Cancer Stem Cells). During analyses of pathways critical for CSC maintenance and colon cancer progression, AR seed extract attenuated levels of β-catenin, Jagged1, Notch-ICD, Hes1 and EZH2. TOP-flash reporter activity, a measure of Wnt signaling, decreased significantly in response to treatment while overexpression of wild type but not mutant EZH2, reversed the inhibitory effects. Moreover,WIF1 (aWnt antagonist) promoter activity increased dramatically following treatment with AR seed extract which phenocopied increases in WIF1 reporter activity following EZH2 knockdown. To determine the effect of AR seed extract on tumor growth in vivo, athymic nude mice bearing tumor xenografts were administered AR seed extract intraperitoneally at 200 mg/Kg daily for 21 days. The AR seed extract significantly inhibited tumor xenograft growth. In addition, there were reduced levels of EZH2, b-catenin, CyclinD1 and Ki-67 along with attenuated levels of Dclk1 in the xenograft tissues. Finally, preliminary run through HPLC yielded three prominent peaks and further identification via NMR spectroscopy is underway to identify the active ingredient(s). Conclusion: These results clearly suggest that AR seed extracts can be an effective preventative/therapeutic agent for colon cancer.

Positron emission tomography imaging of human colon cancer xenografts in mice with [18F]fluorothymidine after TAS-102 treatment.

TAS-102 is an orally administered anticancer agent composed of α,α,α-trifluorothymidine (FTD) and thymidine phosphorylase inhibitor (TPI). This study assessed 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) uptake after TAS-102 administration. The human colorectal carcinoma cell lines HCT116, HT29, HCT8 and SW620 were exposed to FTD for 2h, further incubated for 0, 2 and 24h, and assayed for [(3)H]FLT uptake, nucleoside transport, thymidine kinase 1 (TK1) expression and TK1 activity. Static and 2-hdynamic [(18)F]FLT positron emission tomography (PET) was performed in mice bearing HT29 or SW620 tumours orally administered with vehicle or TAS-102. FTD decreased the viability of all cell lines, whereas increased [(3)H]FLT uptake (P<0.05). Increased nucleoside transport and/or TK1 expression were observed 24h after FTD, but not in 0-2h. Static [(18)F]FLT PET in mice bearing HT29 tumours showed accumulation of [(18)F]FLT in tumours 1h (day 1) after TAS-102. Two-hour dynamic PET in mice bearing SW620 tumours showed increased influx constant and volume of distribution of phosphorylated [(18)F]FLT on days 1 and 8 (P<0.05) after TAS-102 with decreased dephosphorylation on day 1 (P<0.001). Ex vivo studies showed that SW620 tumours after TAS-102 had higher TK1 expression than those with vehicle on days 8 and 15. TAS-102 administration induces an increase in [(18)F]FLT uptake. Mechanisms may involve decreased dephosphorylation of [(18)F]FLT phosphate early after TAS-102 administration. Increased TK1 expression and/or nucleoside transporter may be related to increased [(18)F]FLT uptake at a later time. [(18)F]FLT PET has a potential to assess the pharmacodynamics of TAS-102 in cancer patients.

LYTAK1, a novel TAK1 inhibitor, suppresses KRAS mutant colorectal cancer cell growth in vitro and in vivo.

KRAS mutation in colorectal cancer (CRC) activates transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) to promote tumor progression. In the current study, we explored the potential effect of LYTAK1, a novel TAK1 inhibitor, against KRAS mutant CRC cells in vitro and in vivo. We found that LYTAK1 dose-dependently inhibited KRAS mutant CRC cell (HT-29 and SW-620 lines) growth, and induced cell cycle G1-S arrest. Further, LYTAK1 activated apoptosis in HT-29 cells and SW-620 cells, and apoptosis inhibitors almost reversed LYTAK1-mediated growth inhibition. While in KRAS wild-type (WT) CRC cell lines (DLD-1 and HCT-116), LYTAK1 had almost no effect on cell growth, cell cycle progression, or cell apoptosis. In KRAS mutant HT-29 cells and SW-260 cells, LYTAK1 blocked TAK1 activation or phosphorylation at Thr-184/187. Activation of nuclear factor κB (NF-κB) in these cells, detected by phosphorylations of p65 and IκB kinase α (IKKα) as well as expression of NF-κB-regulated gene cyclin D1, was significantly inhibited by LYTAK1. Further, LYTAK1 treatment resulted in downregulation of β-catenin and Wnt response gene Axin 2, indicating Wnt inactivation. In vivo, oral LYTAK1 significantly inhibited HT-29 xenograft growth in nude mice. Together, these results show that LYTAK1 inhibits KRAS mutant CRC cell growth both in vitro and in vivo. LYTAK1 might be investigated as a novel agent against CRC with KRAS mutation.

Data supporting the identification of anti-metastatic drug and natural compound targets in isogenic colorectal cancer cells.

To investigate molecular therapeutic targets in cancer metastasis, comparative proteomic analysis was performed using the isogenic colorectal cancer cell lines SW480 and SW620. Two potential metastasis related molecular targets were identified: fatty acid synthase and histone H4. Subsequently, metastatic SW620 cells were treated with six anti-cancerous components and suppressive effects were observed in target protein expression. Through comprehensive proteomic analysis, three of the tested compounds, oxaliplatin, ginsenoside 20(S)-Rg3 and curcumin, were determined to have a suppressive effect on fatty acid synthase and histone H4 expression [1]. The current article contains one table exhibiting a list of proteins differentially expressed in metastatic SW620 cell lines compared to the primary SW480 cell line (Supplementary Table 1). Additionally, six tables demonstrate proteome changes in SW620 resulting from the treatment of three chemotherapeutics and three natural components (Supplementary Tables 1–7). The anti-metastatic components revealed by the current proteomic analysis represent promising chemotherapeutic candidates for the treatment of colorectal adenocarcinoma.

Expression of long non-coding RNA associated with radiotherapy-resistance in colorectal cancer cell lines with different radiosensitivity

To screen long non-coding RNA (lncRNA) associated with radiosensitivity in colorectal carcinoma cell lines. Colony formation assay was performed in colorectal cancer cell lines HT29, SW480, RKO, Lovo and HCT116 after irradiation with different radiation doses. Radiation sensitivity of these 5 cell lines was detected through survival fraction at 2 Gy (SF2 value). High-throughput lncRNA chip was used to screen lncRNA genes with expression differences more than 2 folds among SW480, RKO and Lovo. Further experiment on the expression differences of lncRNAs selected was conducted by realtime PCR. The radiosensitivity order of these 5 cell lines from low to high (SF2 value from high to low) was HT29 (0.83 ± 0.03), SW480 (0.69 ± 0.02), RKO(0.53 ± 0.02), Lovo (0.47 ± 0.05), HCT116 (0.32 ± 0.03) (P < 0.01). Five lncRNAs associated with radiation sensitivity were screened. Among them, expression levels of R05532, NR_015441, and NR_033374 were positively correlated with radiation resistance(all P < 0.01), and expression levels of the other 2 lncRNAs, NR_073156 and AA745020, were not correlated with radiation resistance of colorectal cancer cells (both P>0.05). lncRNA R05532, NR_015441 and NR_033374 may be used as the predictive marker of radiosensitivity of colorectal cancer cells. Higher expression of these genes shows radiation resistance.

Abstract 3946: Wnt/ß-catenin signaling mediates resistance of colorectal cancer cells to chemoradiotherapy

Abstract Background: Preoperative chemoradiotherapy represents the standard treatment for patients with rectal cancer. However, the clinical response of individual tumors to multimodal treatment is not uniform, and ranges from complete response to complete resistance. Therefore, the identification of novel therapeutic targets whose modification could be harnessed to sensitize a priori resistant tumors is exceedingly important. In this context, we have previously demonstrated that the Wnt transcription factor TCF4 was over-expressed in rectal cancers that were resistant to chemoradiotherapy. The aim of this study was to explore the functional relevance of TCF4 and Wnt/β-catenin signaling for mediating treatment resistance. Methods: Colorectal cancer cell lines SW837, SW480, and HT-29 were transfected with shRNAs targeting TCF4. After establishing stable single-cell clone (SCC) populations, selected clones were irradiated at 0, 1, 2, 4, 6 and 8 Gy of X-rays. To assess the putative Wnt/β-catenin dependency, β-catenin was inhibited in cancer cell lines using siRNAs, the tankyrase inhibitors XAV939 / JW55 and the porcupine inhibitor LGK 974. Furthermore Wnt/β-catenin signaling activity was stimulated in normal retinal pigment epithelial (RPE) cells through incubation with Wnt3a. Results: RNAi-mediated silencing of TCF4 led to a significant radiosensitization in SW837 and SW480, two cell lines with a high basal TCF4-activity, whereas no effect was observed in HT-29 a cell line with low activity. Inhibition of β-catenin similarly caused a pronounced sensitization of CRC cells to clinically relevant doses of X-rays. Finally, exogenous stimulation of canonical Wnt signaling with Wnt3a resulted in a significantly decreased sensitivity of RPE cells to radiotherapy, i.e. induced radioresistance. Conclusion: We have uncovered a novel role of Wnt/β-catenin signaling in mediating resistance to chemoradiotherapy. Moreover, these data suggest that targeting key components of this pathway may represent a potential therapeutic strategy to increase the proportion of patients that respond to chemoradiotherapy, with considerable clinical implications. Citation Format: Georg Emons, Melanie Spitzner, Sebastian Reineke, Janneke Möller, Emil Kendziorra, Tim Beissbarth, Margret Rave-Fraenk, Jochen Gaedcke, Thomas Ried, Michael Ghadimi, Marian Grade. Wnt/ß-catenin signaling mediates resistance of colorectal cancer cells to chemoradiotherapy. [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 3946. doi:10.1158/1538-7445.AM2014-3946

Abstract 2858: Angiopoietin-like protein 2 as a novel serum biomarker for diagnosis and prognosis in patients with colorectal cancer

Abstract Background: Inflammation plays a role in the carcinogenesis of sporadic colorectal cancer (CRC), and this can be supported by several evidences showing that effectiveness of nonsteroidal anti-inflammatory drugs as chemopreventive agents for CRC. Angiopoietin-like protein 2 (ANGPTL2) is known to act as a causative mediator of chronic inflammation and inflammatory carcinogenesis. However, the biological role and clinical significance of ANGPTL2 expression remains poorly understood in human cancer. We investigated the functional roles of ANGPTL2 in vitro and evaluated the clinical significance of ANGPTL2 expression in both primary tumor and matched serum in patients with CRC. Methods: ANGPTL2 gene knockdown by siRNA transfection was performed to evaluate ANGPTL2 function in CRC cancer cell line (sw480). Next, we examined ANGPTL2 expression in CRC tissues (n=194) to evaluate the association between its expression and clinicopathological findings by immunohistochemistry (IHC). Finally, we screened serum ANGPTL2 levels from 32 CRC (16 Stage I and 16 Stage IV) and 24 normal controls (NC), and validated its levels using 194 serum samples from CRC and 48 from NC to evaluate its utility as a biomarker by ELISA. Results: Knockdown of ANGPTL2 resulted in significantly inhibition of cell proliferation, migration, invasion, and resistance to anoikis in vitro. ANGPTL2 overexpressed in CRC tissues compared to normal colonic mucosa and high ANGPTL2 expression was associated with advanced T stage (p&amp;lt;0.01), lymphatic metastasis (p=0.04) and hepatic metastasis (p=0.04) in CRC patients. Next, we screened serum ANGPTL2 levels in CRC patients and NC, revealing that its levels in CRC patients were significantly higher than that in NC (p&amp;lt;0.01), and differentiated CRC patients from NC with high accuracy (AUC=0.837). Validation step also indicated that serum ANGPTL2 levels in CRC patients were significantly higher compared to NC (p&amp;lt;0.0001), had a promising AUC (0.885) with high sensitivity and specificity to distinguish CRC patients from NC. Serum ANGPTL2 levels also discriminated early CRC patients (stage I) from NC (AUC=0.79). High serum ANGPTL2 was significantly associated with tumor progression such as larger tumor size (p=0.03), undifferentiated adenocarcinoma (p=0.03), advanced T stage (p&amp;lt;0.01), peritoneal metastasis (p&amp;lt;0.01) in CRC. Kaplan-Meier curves revealed that high serum ANGPTL2 was significantly associated with poor disease free survival (p=0.01) and overall survival (p=0.03) in CRC. Intriguingly, ANGPTL2 levels in serum from CRC patients closely correlated with IHC scores of ANGPTL2 expression in matched CRC tissues (r=0.14, p=0.03). Conclusion: Our study first reports that overexpression of ANGPTL2 in CRC cells increased malignant potential and metastasis. Serum ANGPTL2, which might be derived from primary CRC tumor, was a novel biomarker for diagnosis and prognosis in patients with CRC. Citation Format: Takahito Kitajima, Yuji Toiyama, Tadanobu Shimura, Shozo Ide, Hiroki Imaoka, Satoru Kondo, Mikio Kawamura, Yoshinaga Okugawa, Aya Kawamoto, Junichiro Hiro, Susumu Saigusa, Masaki Ohi, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri, Sekido Yoshitaka, Masato Kusunoki. Angiopoietin-like protein 2 as a novel serum biomarker for diagnosis and prognosis in patients with 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 2858. doi:10.1158/1538-7445.AM2014-2858

Increased diacylglycerol kinase ζ expression in human metastatic colon cancer cells augments Rho GTPase activity and contributes to enhanced invasion

BackgroundUnraveling the signaling pathways responsible for the establishment of a metastatic phenotype in carcinoma cells is critically important for understanding the pathology of cancer. The acquisition of cell motility is a key property of metastatic tumor cells and is a prerequisite for invasion. Rho GTPases regulate actin cytoskeleton reorganization and the cellular responses required for cell motility and invasion. Diacylglycerol kinase ζ (DGKζ), an enzyme that phosphorylates diacylglycerol to yield phosphatidic acid, regulates the activity of the Rho GTPases Rac1 and RhoA. DGKζ mRNA is highly expressed in several different colon cancer cell lines, as well as in colon cancer tissue relative to normal colonic epithelium, and thus may contribute to the metastatic process.MethodsTo investigate potential roles of DGKζ in cancer metastasis, a cellular, isogenic model of human colorectal cancer metastatic transition was used. DGKζ protein levels, Rac1 and RhoA activity, and PAK phosphorylation were measured in the non-metastatic SW480 adenocarcinoma cell line and its highly metastatic variant, the SW620 line. The effect of DGKζ silencing on Rho GTPase activity and invasion through Matrigel-coated Transwell inserts was studied in SW620 cells. Invasiveness was also measured in PC-3 prostate cancer and MDA-MB-231 breast cancer cells depleted of DGKζ.ResultsDGKζ protein levels were elevated approximately 3-fold in SW620 cells compared to SW480 cells. There was a concomitant increase in active Rac1 in SW620 cells, as well as substantial increases in the expression and phosphorylation of the Rac1 effector PAK1. Similarly, RhoA activity and expression were increased in SW620 cells. Knockdown of DGKζ expression in SW620 cells by shRNA-mediated silencing significantly reduced Rac1 and RhoA activity and attenuated the invasiveness of SW620 cells in vitro. DGKζ silencing in highly metastatic MDA-MB-231 breast cancer cells and PC-3 prostate cancer cells also significantly attenuated their invasiveness.ConclusionElevated DGKζ expression contributes to increased Rho GTPase activation and the enhanced motility of metastatic cancer cells. These findings warrant further investigation of the clinical relevance of DGKζ upregulation in colon and other cancers. Interfering with DGKζ function could provide a means of inhibiting invasion and metastasis.

Subcutaneous preconditioning increases invasion and metastatic dissemination in colorectal cancer models

Mouse colorectal cancer (CRC) models generated by orthotopic microinjection of human CRC cell lines reproduce the pattern of lymphatic, haematological and transcoelomic spread but generate low metastatic efficiency. Our aim was to develop a new strategy that could increase the metastatic efficiency of these models. We used subcutaneous implantation of the human CRC cell lines HCT116 or SW48 prior to their orthotopic microinjection in the cecum of nude mice (SC+ORT). This subcutaneous preconditioning significantly enhanced metastatic dissemination. In the HCT116 model it increased the number and size of metastatic foci in lymph nodes, lung, liver and peritoneum, whereas, in the SW48 model, it induced a shift from non-metastatic to metastatic. In both models the number of apoptotic bodies in the primary tumour in the SC+ORT group was significantly reduced compared with that in the direct orthotopic injection (ORT) group. Moreover, in HCT116 tumours the number of keratin-positive tumour buddings and single epithelial cells increased at the invasion front in SC+ORT mice. In the SW48 tumour model, we observed a trend towards a higher number of tumour buds and single cells in the SC+ORT group but this did not reach statistical significance. At a molecular level, the enhanced metastatic efficiency observed in the HCT116 SC+ORT model was associated with an increase in AKT activation, VEGF-A overexpression and downregulation of β1 integrin in primary tumour tissue, whereas, in SW48 SC+ORT mice, the level of expression of these proteins remained unchanged. In summary, subcutaneous preconditioning increased the metastatic dissemination of both orthotopic CRC models by increasing tumour cell survival and invasion at the tumour invasion front. This approach could be useful to simultaneously study the mechanisms of metastases and to evaluate anti-metastatic drugs against CRC.

Role of tumor suppression gene RNASET2 in tumorigenesis and progression of pancreatic cancer

Objective To investigate the expression of RNASET2 in pancreatic cancer and its significance.Methods The expression levels of RNASET2 mRNA and in pancreatic cancer (32 cases),adjacent benign tissue (24 cases),3 human pancreatic cancer cell lines and benign ductal epithelial cell line were quantitated by using reverse transcriptase-polymerase chain reaction (RT-PCR).The protein expression of RNASET2 was evaluated by Western blotting in 8 fresh frozen tissues and immunohistochemistry (IHC) in 10 paraffin-embedded specimens.Results RNASET2 was down-regulated significantly in pancreatic cancer (6.516 ±3.033,P ＜0.05) and cell lines BxPC-3,PANC-1 and SW1990 (2.814 ±0.325,2.342 ±0.454 and 5.473 ±0.682,P ＜0.01) respectively.The protein expression of RNASET2 was obviously down-regulated in pancreatic cancer tissues and tumor cell lines.The group with perineural invasion showed lower expression of RNASET2 (5.021 ± 3.102,P ＜ 0.05).Conclusion The expression of RNA-SET2 was down-regulated in pancreatic ductal adenocarcinoma and tumor cell lines,and this gene might be correlated to tumorigenesis and perineural invasion of pancreatic cancer. Key words: Pancreatic cancer; RNASET2 gene ; Tumorigenesis; Perineural

Abstract C38: Small molecule targeting of the mRNA stability factor HuR impacts COX-2 expression in colon cancer

Abstract Background: Colorectal cancer (CRC) is the third most common cancer among American adults and a leading cause of cancer-related mortality. The initiation and progression of colorectal tumorigenesis is characterized by genetic alterations that promote expression of tumor-promoting genes such as COX-2. In non-transformed cells, the mRNAs of these genes are often targeted for rapid decay through AU-rich elements (ARE) present in their 3'UTR. However, ARE-mediated mRNA decay is compromised during colorectal tumorigenesis, allowing for pathogenic gene overexpression. Our prior work identified the RNA-binding protein HuR to be overexpressed during colon tumorigenesis. In this capacity, HuR promotes COX-2 expression by interfering with rapid mRNA degradation and is associated with poor clinical prognosis. In the present study, we examined the therapeutic potential of inhibiting HuR using the small molecule MS-444. Methods: HCT116, HCA-7, SW480, RKO, and HT-29 colon cancer cells along with non-transformed intestinal epithelial (RIE-1) and colonocytes (YAMC) were used. MS-444 was obtained from Novartis. Cell proliferation and apoptosis were assayed by MTT assay and flow cytometry. HuR trafficking was visualized by immunofluorescence. qPCR, western blot, and immunohistochemistry were performed to assess changes in gene expression. In vivo studies were performed to xenograft tumor models of colon cancer cells using IVIS-based imaging for COX-2. Results: Treatment of the CRC cells lines HCT116, HCA-7, SW480, RKO, and HT-29 with MS-444 resulted in growth inhibition with IC50 values ranging between 5.6-12.8 μM, and this observed growth inhibition resulted from increased cell apoptosis. Importantly, treatment of non-transformed intestinal and colonic epithelial cells with MS-444 exhibited IC50 values of 40.7μM and 28.1 μM, respectively, and were resistant to MS-444-induced apoptosis. The expression of HuR was not impacted by MS-444, whereas MS-444 altered HuR's presence in the cytoplasm. HuR performs its mRNA stabilization function when present in the cytoplasm, and CRC cells treated with MS-444 displayed nuclear HuR as a result inhibition of HuR cytoplasmic trafficking. Consistent with growth-inhibitory IC50 values, MS-444 inhibited COX-2 mRNA and protein expression in CRC cells. This inhibition required the COX-2 ARE-containing 3'UTR and occurred on a post-transcriptional level. In vivo, delivery of MS-444 resulted in xenograft CRC tumor growth inhibition and prevented HuR cytoplasmic localization. Furthermore, treatment of MS-444 inhibited tumor COX-2 overexpression. Conclusions: These findings indicate that small molecule-based inhibition of HuR results in selective CRC cell death in part by promoting COX-2 mRNA degradation, while also indicating that HuR is a necessary component for colorectal tumor cell survival. Citation Format: Fernando F. Blanco, Yuan Li, Nicole Meisner-Kober, Shrikant Anant, Dan A. Dixon. Small molecule targeting of the mRNA stability factor HuR impacts COX-2 expression in colon cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C38.

Abstract C36: miR-22 represses Wnt Signaling in human colon cancer cells

Abstract Background: Wnt signaling is an evolutionarily conserved pathway that plays an important role in processes like development, differentiation and metastasis. Aberrant activation of Wnt signaling has been implicated in various tumors, predominantly of breast and colon. MicroRNAs are short non-coding RNAs that are frequently deregulated in cancer. Expression of the microRNA miR-22 was reported to be significantly lower in colorectal cancer tissues than that in normal adjacent mucosa. Based on bioinformatics predictions, we identified several Wnt signaling intermediates as potential targets for miR-22. As Wnt signalling is constitutively active in colon cancer cell lines SW480 and SW620, we hypothesize that miR-22 might be a potential regulator of Wnt signaling in colon cancer. Methods: Wnt signaling was stimulated in HEK293 cells by treating them with 25mM Lithium Chloride (LiCl). Wnt signaling activation was assayed using TOPFlash/FOPFlash luciferase activity in HEK293, SW480 and its metastatic derivative SW620, after overexpressing miR-22 using a plasmid construct. CyclinD1 promoter luciferase assay, Western blotting and quantitative RT-PCR were used to study the expression of downstream targets of Wnt signaling. Direct targets of miR-22 were validated by performing 3'-UTR luciferase assays. Plasmid constructs were used to overexpress or knockdown c-Myc. Results: Overexpression of miR-22 resulted in decreased TOP-Flash luciferase activity in LiCl-treated HEK293 cells and the colon cancer cells SW480 and SW620. miR-22 repressed Wnt signaling target genes CyclinD1, c-Myc and TCF7L2, at both mRNA and protein levels. It also decreased the mesenchymal markers (Snail and Vimentin) at the protein level. Overexpression of miR-22 down-regulated BCL9L protein levels and also targeted the BCL9L 3' UTR, as demonstrated by luciferase reporter assay. In HEK293 cells, overexpression of c-MYC repressed miR-22 levels whereas knockdown of endogenous c-Myc by shRNA up-regulated miR-22 levels. Conclusions: miR-22 inhibits Wnt signaling and potentially exerts this effect by targeting the Wnt activator BCL9L. The repression of mesenchymal markers by miR-22 suggests a role for it in Epithelial to Mesenchymal Transition. Further, the Wnt target gene c-Myc regulates the expression of miR-22, suggesting the existence of a positive feedback loop involving miR-22/Wnt Signaling/c-Myc by which miR-22 may indirectly regulate its own expression. Our findings therefore demonstrate a novel role for miR-22 in the regulation of Wnt signaling in colon cancer cells. Citation Format: Chetlangia Neha, Srinivasa Rao Rao, Devarajan Karunagaran. miR-22 represses Wnt Signaling in human colon cancer cells. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C36.

Expression of ligands of DNAM-1 and NKG2D in colonic cancer

Objective To investigate the expression of ligands of DNAM-1 and NKG2D in the colonic cancer.Methods The colonic cancer tissue and adjacent normal colonic tissues were collected from 42 colonic cancer patients who were admitted to the Tangdu Hospital of Fourth Military Medical University from June 2010 to January 2011 were retrospectively analyzed.The expressions of CD155,CD112 and MICA/B in the colonic cancer tissues and the normal colonic tissues were detected by immunohistochemistry.The expressions of CD155,CD112 and MICA/B in the colonic cell line SWll6,SW480,SW620 and Colo205 in the Duke's A,B,C and D phases were detected by cell cytometry.The relationship of the expressions of the 3 ligands and the clinicopathological parameters was analyzed using the Mann-Whitney U test,chi-square test and Fisher exact probobility.Results Week expression of CD155 was found in the normal colonic tissues,while the expressions of CD112 and MICA/B were not found.In the colonic cancer tissues,the expressions of CD155,CD112 and MICA/B were 81.0％,52.4％ and 47.6％,which were significantly increased.The expressions of CD155,CD112 and MICA/B were not correlated with the gender,tumor differentiation,lymph node metastasis and Duke's staging (P ＞ 0.05).The overall expression rates of CD155,CD112 and MICA/B in the colonic cancer cell line SWll6,SW480,SW620 and Colo205 were 88.9％,67.4％ and 42.3％,respectively.The overall expression of CD155 was significantly higher than CD112 and MICA/B (F =23.17,P ＜ 0.05).Conclusion CD155,CD112 and MICA/B express in the colonic cancer tissues and colonic cancer cell line SW116,SW480,SW620 and Colo205,and the expression of CD155 is the highest. Key words: Colonic neoplasms ; Natural killer cells ; Receptor; Immunotherapy

Abstract 2584: Tandutinib inhibits the PI3 Kinase/Akt/mTOR signaling pathway to inhibit colon cancer growth.

Abstract Background: Despite therapeutic advances, colon cancer remains the second leading cause of death in the United States. The c-Kit receptor can activate distinct signaling pathways including phosphatidylinositol-3- kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR). Aberrant c-Kit activation protects cells from apoptosis and enhances invasion of colon carcinoma cells. Tandutinib is a novel quinazoline-based inhibitor of the type III receptor tyrosine kinases including c-Kit and PDGFR. The current study is designed to determine the effect of tandutinib on colon cancer growth and identified a mechanism of action. Method: Colon cancer cell lines HCT116, HT-29 and SW480 and normal colon epithelial cells were used in the study. Cell growth was measured by hexoseaminidase and clonogenicity assays. Apoptosis was determined by measuring caspase 3/7 activities. Cell cycle analysis was measured by Flow cytometry. Signaling proteins were quantified by western blot analysis. For in vivo effects, HCT116 xenografts were developed in the flanks of nude mice. Immunohistochemistry was performed for CD31 and Akt/mTOR signaling proteins. Results: Tandutinib treatment resulted in a dose and time dependent inhibition of proliferation and colony formation in all three cell lines but did not affect normal colonic epithelial cells. Treatment also induced colon cancer cells to undergo apoptosis and G0/G1 arrest. Apoptosis was further confirmed by increased levels of activated caspase 3 and Bax/Bcl2 ratio, coupled with a reduction in cyclin D1. There was also a downregulation of cyclooxygenase 2 (COX-2), vascular endothelial growth factor (VEGF) and interleukin-8 expression suggesting effects on cancer promoting genes. Moreover, tandutinib inhibited phosphorylation of c-Kit, Akt, mTOR, p70S6 Kinase and 4EBP1. Overexpression of constitutively active Akt1 overcame this inhibitory effect suggesting a critical role for this pathway. To determine the effect of tandutinib on tumor growth in vivo, nude mice harboring HCT116 tumor xenografts in their flanks were administered the compound intraperitoneally every day for 21 days. Tandutinib treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Microvessel density, based on CD31 staining was also significantly lower in the tumors following tandutinib treatment when compared to controls suggesting inhibition of angiogenesis. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression cancer promoting genes COX-2 and VEGF, and suppressed the activation of Akt/mTOR signaling proteins in the tandutinib-treated xenograft tissues. Conclusion: Together, these data suggest that tandutinib is a novel potent therapeutic agent that can target the Akt/mTOR/p70S6K signaling pathway to inhibit tumor growth and angiogenesis. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, David Standing, Parthasarathy Rangarajan. Tandutinib inhibits the PI3 Kinase/Akt/mTOR signaling pathway to inhibit colon cancer growth. [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 2584. doi:10.1158/1538-7445.AM2013-2584

Abstract 3743: Effect of folic acid supplementation on epigenetics and cancer stem cell populations in established colorectal cancer cell lines.

Abstract Mandated fortification of folic acid, a B vitamin involved in nucleotide synthesis and DNA CpG methylation, has led to a subsequent increase in blood folate concentration as well as a simultaneous increase in colorectal cancer incidence in North America. Recent findings suggest that a proportion of tumor cells, termed cancer stem cells (CSC), share properties with native stem cells and are vital in the development and perpetuation of tumor metastasis. Both epigenetic DNA modifications as well as the activation of established stem cell signaling pathways are responsible for the activation and maintenance of (CSC). CSC share properties with native stem cells and are vital in the development of primary lesions and the perpetuation of tumor metastasis. We hypothesize that folate, a CpG methylation altering agent, and extracellular microenvironment factors can influence the development and maintenance of CSCs. By isolating and characterizing the CSC fraction in established human colorectal cancer cell lines HCT116, Caco2, and SW480, LS174T and PC7, a subline of DLD-1, we have assessed the effect of folate on CSC signaling pathways and global DNA methylation. Cell lines were grown in suspension and supplemented with specialized stem cell media to promote the growth and expansion of the CSC fraction. Western blot analysis and immunofluorescence were used to characterize the protein expression and nuclear localization of proteins associated with established CSC maintenance pathways, respectively. Global DNA methylation status was assessed using an ELISA-based assay. HCT116 and SW480 show an increased ability to form colonospheres in culture compared to Caco2 and IEC18 (normal rat intestinal cell line). Cells grown in suspension show variable activation of stem cell renewal pathways compared to monolayer. Low folic acid levels led to reduced methylation status and colonosphere yield while high folic acid levels led to increased global methylation and an increased in colonosphere yield. Thus, altered folic acid exposure in vitro can influence the methylation status and CSC self-renewal ability of human colorectal cancer cells. The relationship between folic acid and cancer stem cell maintenance may be an important factor in the apparent duel modulatory effect of folic acid on colorectal cancer and warrants further investigation. Citation Format: Nathan Farias, Brenda Coomber. Effect of folic acid supplementation on epigenetics and cancer stem cell populations in established colorectal cancer cell lines. [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 3743. doi:10.1158/1538-7445.AM2013-3743

Abstract LB-183: Suppression of colon carcinogenesis by targeting Notch signaling.

Abstract Background and aims: Recent studies have shown that aberrant Notch signaling contributes to the pathogenesis of colorectal cancer (CRC). Notch signaling can be abrogated in cells by treatment with γ-secretase inhibitors (GSI) that suppress Notch signaling via inhibition of Notch cleavage. This effect is associated with suppression of cell proliferation in several human cancer cell lines. However, the potential therapeutic benefits of Notch pathway inhibitors, including GSIs, on colon carcinogenesis are still unresolved. In the present study, the effects of GSI treatment and its underlying mechanisms in colon carcinogenesis were investigated both in vitro and in vivo. We also evaluated the expression level of Krüppel-like factor 4 (KLF4), a downstream repression target of Notch signaling, in human polyps. Methods: Human colon cancer cell lines HCT116 (Wt and p21−/−) and SW480 were treated with 0-100 μM GSI, N-[N-3,5-Difluorophenacetyl]-L-alanyl-S-phenylglycine Methyl Ester (DAPM), for either 48 or 72 hours to evaluate the effects of DAPM on cell proliferation and protein expression by the MTT assay and Western blotting analysis, respectively. For the mouse study, male A/J mice were injected with azoxymethane (AOM, 10 mg/kg, bw) once a week for 6 weeks. Nine weeks after the last AOM injection, the presence of tumors in the distal colon was confirmed by colonoscopic imaging. Mice were then treated with 1 mg/kg bw of DAPM or vehicle control by ip injection every other day for a total of 4 weeks. Tumor frequency and size was determined 14 weeks after the last injection of AOM. In addition, human specimens were obtained from eighteen patients undergoing routine screening colonoscopy at the University of Connecticut Health Center and screened for the presence of KLF4 and p21 expression. Results: DAPM suppressed cell proliferation and induced the expression of KLF4 and p21 in human colon cancer cells. Interestingly, p21−/− HCT116 cells were largely resistant to the suppressive effects of DAPM on cell proliferation compared with the parental cells, indicating a key role for p21 activity in the response to DAPM. The frequency of both large (≥ 4 mm) and small (&amp;lt; 1 mm) colon tumors was significantly reduced by DAPM treatment (reduced by 60% and 33%, respectively) in AOM-treated mice. Furthermore, colon tumors in the DAPM-treated mice displayed induction of KLF4 and p21 expression accompanied by reduced Ki-67 staining compared to controls. Notably, in human colon tumor biopsies, KLF4 and p21 expression were present within serrated hyperplastic polyps, but the levels of both proteins were markedly reduced within tubular adenomas. Conclusion: Our results suggest that inhibition of Notch signaling by DAPM provides a potential chemopreventive strategy for patients with tubular adenomas, in part through activation of the KLF4-p21 axis. Citation Format: Shingo Miyamoto, Daniel W. Rosenberg. Suppression of colon carcinogenesis by targeting Notch signaling. [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 LB-183. doi:10.1158/1538-7445.AM2013-LB-183

Abstract 1588: Canonical Wnt signaling mediates resistance of colorectal cancer cells to radiation therapy.

Abstract Background: The clinical response of locally advanced rectal cancers to preoperative chemoradiotherapy is very heterogeneous. We previously identified TCF4, the key downstream effector of the Wnt/β-catenin signaling pathway, as over-expressed in tumors that were resistant to chemoradiotherapy. The aim of this study was to explore the potential functional relevance of Wnt/ß-catenin signaling for mediating treatment resistance. Methods: Using RNA interference, TCF4 and β-catenin were inhibited in the colorectal cancer cell lines SW837 and SW480. Subsequently, these cell lines were irradiated with varying doses of X-rays. In addition, normal retinal epithelial cells (RPE) were stimulated with Wnt-3a for 28 hours, and also irradiated. Wnt/β-catenin activity was assessed using the TOPFLASH/FOPFLASH reporter assay, and induction of Axin2 was confirmed by Western blotting. Results: Protein levels of both TCF4 and β-catenin were considerably reduced following RNAi, accompanied by a decreased TOP/FOP reporter activity. Importantly, silencing of both proteins led to a pronounced radiosensitization in SW837 and SW480 cells. Exogenous stimulation of canonical Wnt signaling in RPE cells using Wnt-3a resulted in an increased Axin2 expression and an increased TOP/FOP reporter activity. Importantly, however, this activation of canonical Wnt signaling was associated with significant increase in resistance to radiation therapy. Conclusion: TCF4 was found to be over-expressed in resistant rectal carcinomas, and its RNAi-mediated silencing caused a significant radiosensitization. To determine whether this effect was dependent on canonical Wnt signaling, we inhibited ß-catenin, another key factor of this pathway and binding partner of TCF4, and observed a similar sensitization to radiation therapy. Follow-up experiments show that external stimulation of canonical Wnt signaling confers radiation resistance in normal cells. These data suggest that targeting canonical Wnt signaling may represent a potential therapeutic strategy for sensitizing tumor cells to radiation. In vivo studies are ongoing to validate these results. Citation Format: Georg Emons, Janneke Möller, Melanie Spitzner, Emil Kendziorra, Jochen Gaedcke, Margret Rave-Fränk, Tobias Pukrop, Michael Ghadimi, Thomas Ried, Marian Grade. Canonical Wnt signaling mediates resistance of colorectal cancer cells to radiation therapy. [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 1588. doi:10.1158/1538-7445.AM2013-1588