Activity In Colorectal Cancer Cells Research Articles

Pre-clinical characterization of 4SC-202, a novel class I HDAC inhibitor, against colorectal cancer cells.

Histone deacetylase (HDAC) overactivity in colorectal cancer (CRC) promotes cancer progression. In the current study, we showed that 4SC-202, a novel class I HDAC inhibitor (HDACi), potently inhibited survival and proliferation of primary human colon cancer cells and established CRC lines (HT-29, HCT-116, HT-15, and DLD-1). Yet, the same 4SC-202 treatment was non-cytotoxic to colon epithelial cells where HDAC-1/-2 expressions were extremely low. 4SC-202 provoked apoptosis activation in CRC cells, while caspase inhibitors (z-VAD-CHO and z-DVED-CHO) significantly alleviated 4SC-202-exerted cytotoxicity in CRC cells. Meanwhile, 4SC-202 induced dramatic G2-M arrest in CRC cells. Further studies showed that AKT activation might be an important resistance factor of 4SC-202. 4SC-202-induced cytotoxicity was dramatically potentiated with serum starvation, AKT inhibition (by perifosine or MK-2206), or AKT1-shRNA knockdown in CRC cells. On the other hand, exogenous expression of constitutively active AKT1 (CA-AKT1) decreased the sensitivity by 4SC-202 in HT-29 cells. Notably, 4SC-202, at a low concentration, enhanced oxaliplatin-induced in vitro anti-CRC activity. In vivo, we showed that oral gavage of 4SC-202 inhibited HT-29 xenograft growth in nude mice, and when combined with oxaliplatin, its activity was further strengthened. Together, these pre-clinical results indicate that 4SC-202 may be further investigated as a valuable anti-CRC agent/chemo-adjuvant.

The Wnt Pathway Target Gene <i>CCND1</i> Changes Mitochondrial Localization and Decreases Mitochondrial Activity in Colorectal Cancer Cell Line SW480

Mutations leading to constitutive activation of the Wnt pathway and its target genes are frequently observed in cancer. The Wnt pathway promotes cell proliferation and increasing evidence supports its role also in cancer cell metabolism. This study aims to elucidate the role of the Wnt/β-catenin target gene CCND1 in these processes in colorectal cancer. We analyzed whether knock-down of CCND1 affects cell cycle progression and energy metabolism in a colorectal cancer cell line. Down-regulation of CCND1 led to retardation of the cell cycle. The proportion of cells in the G0 phase increased, while the amount of cells in the S- and G2/M phase decreased. Interestingly, knock-down of CCND1 changed the perinuclear localization of mitochondria into a homogeneous distribution within the cytosol. In addition CCND1 knock-down led to an increase of the intracellular ATP level indicating that cyclin D1 reduced mitochondrial activity. Our findings suggest that in addition to its role in cell cycle regulation, the Wnt target gene CCND1 regulates mitochondrial localization and inhibits mitochondrial activity in colorectal cancer cells.

Keratinocyte Growth Factor Regulation of Aryl Hydrocarbon Receptor Activation in Colorectal Cancer Cells.

Keratinocyte growth factor (KGF) stimulates normal growth, development and intestinal epithelial cell proliferation. Cyclin D1 promotes the cell cycle by inhibiting retinoblastoma protein (RB1). The activated aryl hydrocarbon receptor (AhR) has an important influence on the development of tumors through its interactions with the cell cycle. The aim of the present study was to explore a new role for AhR in KGF-induced colon cancer cell growth. Real-time PCR, western blot or immunofluorescence analysis were used to detect the expression of KGF, AhR, cyclin D1 and CYP1A1. Immunohistochemistry was used to observe the localization of AhR. MTT assay and flow cytometric analyses were performed to measure cell viability and the cell cycle. Real-time PCR analysis revealed that KGF, AhR, and CYP1A1 mRNAs were overexpressed in colorectal cancer tissues. Meanwhile, overexpression of AhR was primarily observed in epithelial cells. In in vitro assay, KGF promoted colon cancer cell growth, as well as up-regulated and activated AhR. At the same time, AhR-knockdown colon cancer cells were less responsive to KGF. Western blot analysis, real-time PCR, or immunofluorescence data indicated that cyclin D1 expression was up-regulated by KGF but this up-regulation was compromised when AhR was silenced, and the cell cycle was arrested in the G0/G1 stage in these cells. Our study suggests that KGF, AhR, and CYP1A1 are overexpressed in colorectal cancer tissues. Moreover, we reveal a new mechanism by which KGF promotes cell proliferation through the AhR-cyclin D1 pathway in colon cancer cells.

Molecular mechanisms underlying the antitumor activity of (E)-N-hydroxy-3-(1-(4-methoxyphenylsulfonyl)-1,2,3,4-tetrahydroquinolin-6-yl)acrylamide in human colorectal cancer cells in vitro and in vivo.

Upregulation of class I histone deacetylases (HDAC) correlates with poor prognosis in colorectal cancer (CRC) patients. Previous study revealed that (E)-N-hydroxy-3-(1-(4-methoxyphenylsulfonyl)-1,2,3,4-tetrahydroquinolin-6-yl)acrylamide (Compound 11) is a potent and selective class I HDAC inhibitor, exhibited significant anti-proliferative activity in various human cancer cell lines. In current study, we demonstrated that compound 11 exhibited significant anti-proliferative and cytotoxic activity in CRC cells. Notably, compound 11 was less potent than SAHA in inhibiting HDAC6 as evident from the lower expression of acetyl-α-tubulin, suggesting higher selectivity for class I HDACs. Mechanistically, compound 11 induced cell-cycle arrest at the G2/M phase, activated both intrinsic- and extrinsic-apoptotic pathways, altered the expression of Bcl-2 family proteins and exerted a potent inhibitory effect on survival signals (p-Akt, p-ERK) in CRC cells. Moreover, we provide evidence that compound 11 suppressed motility, decreased mesenchymal markers (N-cadherin and vimentin) and increased epithelial marker (E-cadherin) through down-regulation of Akt. The anti-tumor activity and underlying molecular mechanisms of compound 11 were further confirmed using the HCT116 xenograft model in vivo. Our findings provide evidence of the significant anti-tumor activity of compound 11 in a preclinical model, supporting its potential as a novel therapeutic agent for CRC.

Abstract 3254: Promotion of colon cancer invasion and metastasis via activation of Notch-Dab1-Abl-RhoGEF protein Trio

Abstract We have recently identified a metastasis suppressor gene for colorectal cancer (CRC); AES/Aes which encodes an endogenous inhibitor of Notch signaling. When Aes is knocked out in the adenomatous epithelium of intestinal polyposis mice, their tumors become malignant, showing marked submucosal invasion and intravasation. Here we show that one of the genes induced by Notch signaling in CRC is DAB1/Dab1. Genetic depletion of Dab1 suppresses cancer invasion and metastasis in the Notch signaling-activated mice. Dab1 is phosphorylated by Abl tyrosine kinase, which activates Abl reciprocally. Consistently, inhibition of Abl suppresses cancer invasion in mice. Furthermore, we show that one of the targets of Abl Tyr-kinase is Rac/Rho-GEF protein Trio, and that phosphorylation at its Tyr residue 2681 (pY2681) causes Rho activation in CRC cells. Its unphosphorylatable mutation Trio(Y2681F) reduces the RhoGEF activity, and inhibits invasion of CRC cells. Importantly, Trio(pY2681) correlates with significantly poorer prognosis of CRC patients after surgery. These results indicate that Trio(pY2681) is one of the downstream effects by Notch signaling activation in CRC, and can be a prognostic marker, helping determine the therapeutic modality of CRC patients. Citation Format: Mark Taketo. Promotion of colon cancer invasion and metastasis via activation of Notch-Dab1-Abl-RhoGEF protein Trio. [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 3254. doi:10.1158/1538-7445.AM2015-3254

Ergosterol peroxide from Chaga mushroom (Inonotus obliquus) exhibits anti-cancer activity by down-regulation of the β-catenin pathway in colorectal cancer

Aim of the studyIn this study, we examined the effect of different fractions and components of Chaga mushroom (Inonotus Obliquus) on viability and apoptosis of colon cancer cells. Among them, one component showed the most effective growth inhibition and was identified as ergosterol peroxide by NMR analysis. We investigated the anti-proliferative and apoptosis mechanisms of ergosterol peroxide associated with its anti-cancer activities in human colorectal cancer (CRC) cell lines and tested its anti-tumor effect on colitis-induced CRC developed by Azoxymethane (AOM)/Dextran sulfate sodium (DSS) in a mouse model. Materials and methodsWe used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, flow cytometry assays, Western blot analysis, colony formation assays, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and AOM/DSS mouse models to study the molecular mechanism of metastatic activities in CRC cells. ResultsErgosterol peroxide inhibited cell proliferation and also suppressed clonogenic colony formation in HCT116, HT-29, SW620 and DLD-1 CRC cell lines. The growth inhibition observed in these CRC cell lines was the result of apoptosis, which was confirmed by FACS analysis and Western blotting. Ergosterol peroxide inhibited the nuclear levels of β-catenin, which ultimately resulted in reduced transcription of c-Myc, cyclin D1, and CDK-8. Ergosterol peroxide administration showed a tendency to suppress tumor growth in the colon of AOM/DSS-treated mice, and quantification of the IHC staining showed a dramatic decrease in the Ki67-positive staining and an increase in the TUNEL staining of colonic epithelial cells in AOM/DSS-treated mice by ergosterol peroxide for both prevention and therapy. ConclusionOur data suggest that ergosterol peroxide suppresses the proliferation of CRC cell lines and effectively inhibits colitis-associated colon cancer in AOM/DSS-treated mice. Ergosterol peroxide down-regulated β-catenin signaling, which exerted anti-proliferative and pro-apoptotic activities in CRC cells. These properties of ergosterol peroxide advocate its use as a supplement in colon cancer chemoprevention.

ERK-dependent phosphorylation of HSF1 mediates chemotherapeutic resistance to benzimidazole carbamates in colorectal cancer cells.

Drugs containing the benzimidazole carbamate scaffold include anthelmintic and antifungal agents, and they are now also recognized as having potential applications in the treatment of colorectal and other cancers. These agents act by binding to β-tubulin, and in doing so they disrupt microtubules, arrest cell division, and promote apoptotic cell death in malignant cells. We have evaluated several commercially available benzimidazole carbamates for cytotoxic activity in colorectal cancer cells. In addition to cytotoxicity, we also observe activation of the transcription factor, heat shock factor-1 (HSF1). HSF1 is well known to mediate a cytoprotective response that promotes tumor cell survival and drug resistance. Here, we show that biochemical inhibition with the HSF1 inhibitor KRIBB11 or siRNA-based silencing of HSF1 results in a significant enhancement of drug potency, causing an approximately two-fold decrease in IC50 values of parbendazole and nocodazole. We also define a mechanism for drug-induced HSF1 activation, which results from a phosphorylation event at Ser326 that is dependent on the activation of the extracellular regulated protein kinase-1/2 (ERK-1/2) mitogen-activated protein kinase pathway. Inhibition of the upstream kinase MEK-1/2 with U0126 attenuates the phosphorylation of both ERK-1/2 and HSF1, and significantly enhances drug cytotoxicity. From these data we propose a unique model whereby the ERK-1/2-dependent activation of HSF1 promotes chemotherapeutic resistance to benzimidazole carbamates. Therefore, targeting the ERK-1/2 signaling cascade is a potential strategy for HSF1 inhibition and a means of enhancing the cytotoxicity of these agents.

A Protein Interaction between β-Catenin and Dnmt1 Regulates Wnt Signaling and DNA Methylation in Colorectal Cancer Cells.

Aberrant activation of the Wnt signaling pathway is an important step in the initiation and progression of tumor development in diverse cancers. The central effector of canonical Wnt signaling, β-catenin (CTNNB1), is a multifunctional protein, and has been extensively studied with respect to its roles in cell-cell adhesion and in regulation of Wnt-driven transcription. Here, a novel mass spectrometry-based proteomics technique in colorectal cancer cells expressing stabilized β-catenin, was used to identify a protein-protein interaction between β-catenin and DNA methyltransferase I (Dnmt1) protein, the primary regulator of DNA methylation patterns in mammalian cells. Dnmt1 and β-catenin strongly colocalized in the nuclei of colorectal cancer cells, and the interaction is mediated by the central domain of the Dnmt1 protein. Dnmt1 protein abundance is dependent upon the levels of β-catenin, and is increased in cells expressing stabilized mutant β-catenin. Conversely, the Dnmt1 regulates the levels of nuclear β-catenin and β-catenin/TCF-driven transcription. In addition, lysine-specific demethylase 1 (LSD1/KDM1A), a regulator of DNMT1 stability, was identified as a component of the Dnmt1-β-catenin protein complex and perturbation of the Dnmt1-β-catenin interaction altered DNA methylation. In summary, a functional protein-protein interaction was identified between two critically important oncoproteins, in turn revealing a link between Wnt signaling and downstream nuclear functions mediated by Dnmt1. Two critical oncoproteins, Dnmt1 and β-catenin, mutually regulate one each other's levels and activities in colorectal cancer cells.

Sa1993a Reduced Expression of Prostaglandin Transporter Promotes Angiogenesis in Gastric Cancer

Background & Aims: Colorectal cancer (CRC) cells (unlike normal colonic epithelial cells) secrete VEGF, which drives these cells proliferation and promotes tumor-related angiogenesis. The mechanism(s) of increased, aberrant VEGF expression in CRC cells is not known, but is likely related to transcriptional activation of VEGF gene.We hypothesized that: 1) upregulated importin-α in CRC cells (which facilitates nuclear transport of transcription factors: HIF1α and CREB, and is essential for VEGF gene promoter activation) is the underlying mechanism for increased VEGF expression in these cells and CRC cell proliferation, and, 2) inhibition and/or silencing of importin-α in CRC cells will inhibit VEGF gene activation and CRC cell proliferation. Ivermectin is a specific inhibitor of importin α nuclear transport (Biochem J. 2012: 1;443(3):851-6). We examined whether treatment of CRC cell lines with ivermectin could inhibit CRC cell growth. Methods: We used: (a) human CRC cell lines HCT116 & HT29 and (b) normal colonic epithelial cells NCM356 & NCM460. We treated cultured CRC cells with ivermectin (50 μM, 6 hr; specific inhibitor of importin-α nuclear transport), with importin α specific siRNA or control RNA (100nM, 48 hr). Add-back studies included treatment with exogenous VEGF (20ng/ml). Studies: 1) mRNA and protein expression of importin-α and VEGF by Real-Time RT-PCR, and/or Western blotting and immunostaining, respectively; 2) VEGF secretion into culture medium by ELISA; 3) translocation of importin to the nucleus in CRC vs. normal cells; and 4) cell proliferation by BrdU assay. Results: 1) CRC cell lines express high levels of VEGF and secrete large amounts of VEGF (up to 1530 pg/ml) into culture medium at 24 hours vs. normal epithelial cells, which only secrete minimal VEGF ( 40% (p< 0.05); 3) Selective inhibition of importin-α in CRC cells using ivermectin significantly reduced VEGF expression by ~3-fold (p<0.01) and proliferation of these cells by 2.4-fold (p<0.01). Treatment with exogenous VEGF partly reversed inhibited by ivermectin cell proliferation; 4) Importin-α silencing in CRC cells significantly reduced VEGF expression and proliferation of these cells by 2.2-fold and 2fold (both p<0.01), respectively; treatment with exogenous VEGF following importin-α silencing reversed inhibition of cell proliferation by 2-fold (p < 0.01). Conclusions: 1) VEGF gene activation in CRC cells requires functional importin-α; 2) importin-α mediates increased proliferation of CRC cells; downregulation of importin-α with specific siRNA or its inhibition with specific inhibitor ivermectin inhibits CRC cell proliferation; 3) These studies suggest a potential therapeutic role of ivermectin in inhibiting CRC cell proliferation.

Promotion of colorectal cancer invasion and metastasis through activation of NOTCH-DAB1-ABL-RHOGEF protein TRIO.

We have recently identified a metastasis suppressor gene for colorectal cancer: AES/Aes, which encodes an endogenous inhibitor of NOTCH signaling. When Aes is knocked out in the adenomatous epithelium of intestinal polyposis mice, their tumors become malignant, showing marked submucosal invasion and intravasation. Here, we show that one of the genes induced by NOTCH signaling in colorectal cancer is DAB1/Dab1. Genetic depletion of DAB1 suppresses cancer invasion and metastasis in the NOTCH signaling-activated mice. DAB1 is phosphorylated by ABL tyrosine kinase, which activates ABL reciprocally. Consistently, inhibition of ABL suppresses cancer invasion in mice. Furthermore, we show that one of the targets of ABL is the RAC/RHOGEF protein TRIO, and that phosphorylation at its Tyr residue 2681 (pY2681) causes RHO activation in colorectal cancer cells. Its unphosphorylatable mutation TRIO Y2681F reduces RHOGEF activity and inhibits invasion of colorectal cancer cells. Importantly, TRIO pY2681 correlates with significantly poorer prognosis of patients with colorectal cancer after surgery. These results indicate that TRIO pY2681 is one of the downstream effectors of NOTCH signaling activation in colorectal cancer, and can be a prognostic marker, helping to determine the therapeutic modality of patients with colorectal cancer.

The preclinical evaluation of the dual mTORC1/2 inhibitor INK-128 as a potential anti-colorectal cancer agent

The colorectal cancer is the leading contributor of cancer-related mortality. Mammalian target of rapamycin (mTOR), existing in 2 complexes (mTORC1/2), is frequently dysregulated and constitutively activated in colorectal cancers. It represents an important drug target. Here we found that INK-128, the novel ATP-competitive kinase inhibitor of mTOR, blocked both mTORC1 and mTORC2 activation in colorectal cancer cells (both primary and transformed cells). The immunoprecipitation results showed that the assembly of mTORC1 (mTOR-Raptor association) and mTORC2 (mTOR-Rictor-Sin1 association) was also disrupted by INK-128. INK-128 inhibited colorectal cancer cell growth and survival, and induced both apoptotic and non-apoptotic cancer cell death. Further, INK-128 showed no effect on Erk/MAPK activation, while MEK/Erk inhibition by MEK-162 enhanced INK-128-induced cytotoxicity in colorectal cancer cells. Meanwhile, INK-128 downregulated Fascin1 (FSCN1)/E-Cadherin expressions and inhibited HT-29 cell in vitro migration. In vivo, daily INK-128 oral administration inhibited HT-29 xenograft growth in mice, which was further enhanced by MEK-162 administration. Finally, we found that INK-128 sensitized 5-fluorouracil-(5-FU)-mediated anti-HT-29 activity in vivo and in vitro. Thus, our preclinical studies strongly suggest that INK-128 might be investigated for colorectal cancer treatment in clinical trials.

Matrine reduces the proliferation and invasion of colorectal cancer cells via reducing the activity of p38 signaling pathway.

Matrine has been used in anti-inflammatory and anti-cancer therapies for a long time. However, the anti-metastatic effect and related mechanism(s) in colorectal cancer (CRC) are still unclear. In this study, we investigated whether the administration of matrine could inhibit the proliferation, motility, and invasion of human CRC cells via regulating p38 signaling pathway. Results showed that matrine inhibited migration and invasion of CRC cells in vitro and in vivo. Additionally, after being treated with matrine for 24 h, the expression levels of matrix metalloproteinase-2 (MMP-2) and MMP-9 as well as proteinase activity in CRC cells were reduced in a dose-dependent manner. Moreover, matrine reduced the phosphorylation level of p38 obviously. Combined treatment with p38 inhibitor (SB203580) and matrine resulted in a synergistic reduction of invasion as well as MMP-2/-9 expression in CRC cells. It was also found that matrine inhibited the proliferation and metastasis of CRC tumor in vivo. In conclusion, p38 signaling pathway may involve in matrine's inhibitory effects on migration and invasion of CRC cells by reducing the expression of MMP-2/-9, suggesting that matrine may be a potential therapeutic agent for CRC.

A novel pyrido-thieno-pyrimidine derivative activates p53 through induction of phosphorylation and acetylation in colorectal cancer cells.

The tumor suppressor p53 plays a key role in regulation of the cell cycle, apoptosis and senescence in response to various stresses. We screened a library of 7920 chemical compounds for the p53 activator and identified N-[2-(dimethylamino)ethyl]-2,3-dimethyl-4-oxo-4H-pyrido[1,2-a]thieno[2,3-d]pyrimidine-9-carboxamide (PTP), which significantly increased p53-mediated reporter activity in colorectal cancer cells. PTP was found to induce p53 protein and activated transcription of downstream genes, such as p21 and PUMA, in HCT116 cells, leading to growth delay, G1-phase cell cycle arrest, cell senescence and cell death. Proximity ligation assay revealed that PTP weakened the interaction between p53 and murine double minute 2 (MDM2) in situ, thereby inhibiting MDM2-mediated p53 degradation. Although DNA damage has been known to promote phosphorylation of p53 and MDM2, thereby preventing their interaction and stabilizing p53, PTP did not cause DNA damage or activate any DNA damage response signaling. Instead, phosphorylation of p53 was mediated by Erk1/2 MAP kinase. In addition, PTP induced acetylation of p53 at Lys382 in a p300-dependent manner, but sirtuin (SIRT)1 and histone deacetylase (HDAC)1, a well-known p53-regulating deacetylase, were not involved. In the present study, the novel anticancer agent PTP was shown to cause the accumulation of p53 by inducing multiple post-translational modifications, as well as cell cycle arrest, senescence and cell death.

Novel role of Sarco/endoplasmic reticulum calcium ATPase 2 in development of colorectal cancer and its regulation by F36, a curcumin analog

Sarco/endoplasmic reticulum calcium ATPase (SERCA) enzymes play important roles in several signal transduction pathways that control proliferation, differentiation and apoptosis. Here, we reported that SERCA2 expression was positively correlated with tumor node metastasis (TNM) stages (n=75, P=0.0251) and grades (n=63, P=0.0146) of patients with colorectal cancer. The animal experiments demonstrated that SERCA2 expression was consistent with PCNA staining of intestinal tissues of male C57BL/6J-ApcMin/JNju mice. Besides, SERCA2 expression was also increased in undifferentiated HT-29 cells as compared with that in differentiated HT-29gal cells. Moreover, SERCA2 overexpression promoted proliferation and migration of SW480 cells via activating MAPK and AKT signaling pathways, while silence of SERCA2 inhibited the proliferation and migration of SW480 cells. In addition, we identified that a curcumin analog, F36, exhibited more potent inhibitory effect in colorectal cancer cells than curcumin through inhibiting SERCA2 expression. Taken together, our findings indicate that SERCA2 is involved in the malignant progress of colorectal cancer and maybe a therapeutic target for colorectal cancer treatment. Curcumin analog F36 shows enhanced anti-cancer activity in colorectal cancer cells by targeting SERCA2.

Abstract 5285: Protein phosphatases modulate kinase signaling mediated Wnt activation

Abstract Multiple growth factors, signaling through their respective kinase cascades, phosphorylate beta-catenin at specific Serine residues to cause its nuclear sequestration thereby fueling Wnt driven tumor cell proliferation. Our present work identifies a group of phosphatase genes that attenuate such growth factor mediated activation of Wnt signaling in colo-rectal cancer (CRC) cells. This proposed mechanism proposes an additional mode of regulation of Wnt activity in CRC cells. Using a lentiviral TCF-LEF reporter system, we isolated distinct SW480 (primary) and SW620 (metastatic) cell fractions based on their endogenous level of Wnt activity. Receptor Tyrosine Kinase (RTK) antibody arrays using cell lysates from these cell fractions identified that FGFR3, ALK and RET signaling pathways have a positive correlation with enhanced Wnt activity. Simultaneously qPCR arrays performed on these same cellular fractions identified a total of 10 phosphatase genes whose expression levels are significantly decreased with heightened Wnt activity, out of which 4 genes PHLPP, PHLPPL, PTPRK and PTPN6 are known to regulate the aforementioned RTK pathways. Of these phosphatase genes, PTPRK has been reported to undergo gene fusion events with RSPO3 (a non-canonical activator of Wnt) in CRC patients. Since the fusion gene product provides no additional Wnt activation that the wild-type RSPO3, we theorized that these fusion events occur either to allow CRC cells to utilize the enhanced promoter activity of the PTPRK gene promoter, or to nullify the Wnt attenuating properties of PTPRK. To test this hypothesis, we have presently cloned and identified PTPRK and RSPO3 gene promoter regions and wish to test their activity in distinct Wnt responsive cell fractions. Our work will allow a better understanding of how phosphatase genes may be involved in controlling the Wnt pathway and how their inactivation may contribute to CRC cell proliferation. Citation Format: Debasish Boral, Daotai Nie. Protein phosphatases modulate kinase signaling mediated Wnt activation. [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 5285. doi:10.1158/1538-7445.AM2014-5285

Abstract 2997: Vessel co-option in colorectal cancer liver metastases mediates resistance to VEGF-targeted therapy

Abstract The purpose of this study is to: (1) examine whether vessel co-option is associated with a lack of response to anti-angiogenic therapy in advanced colorectal cancer patients, (2) identify whether inhibition of pathways associated with vessel co-option sensitizes to anti-angiogenic therapy in vivo. Sprouting angiogenesis is the process whereby new blood vessels are induced to sprout from existing vessels. The growth of metastatic tumors is considered to require sprouting angiogenesis. This hypothesis has driven the development and clinical application of vascular endothelial growth factor (VEGF) targeted agents, such as bevacizumab. However, responses to such agents in cancer patients, including metastatic colorectal cancer (CRC), are variable. It is not currently clear why this is the case and we have no way of selecting patients suitable for such therapies. Importantly, it is now evident that tumors can also utilize a number of putative ‘VEGF-independent’ angiogenesis mechanisms, the existence of which may limit the efficacy of VEGF-targeted therapy. One such mechanism is ‘vessel co-option,’ whereby tumors engulf existing local blood vessels as they invade into surrounding host tissue. Published studies have reported that this mechanism occurs in ∼28-30% of CRC liver metastases. Here we present data from a retrospective study of patients with CRC liver metatases that were treated with bevacizumab. We demonstrate that, in this patient cohort, the presence of vessel co-option in liver metastases was strongly associated with lack of response to bevacizumab. We also show that a similar result is observed in a mouse model of colorectal cancer liver metastasis. Interestingly, when we disable actin nucleation activity in colorectal cancer cells, vessel co-option is compromised and sensitivity to VEGF-targeted therapy is restored in vivo. These data demonstrate a potential role for vessel co-option as a negative predictive biomarker for anti-angiogenic therapy and also have consequences for the development of novel therapies for targeting tumor angiogenesis. Citation Format: Sophia Frentzas, Victoria L. Thompson, Peter B. Vermeulen, Shane Foo, Gina Brown, David Cunningham, Andrew R. Reynolds. Vessel co-option in colorectal cancer liver metastases mediates resistance to VEGF-targeted therapy. [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 2997. doi:10.1158/1538-7445.AM2014-2997

Abstract 3146: Rac1 accumulates in the nucleus at the invasive front of colorectal cancer through promoting cell motility

Abstract [Background] Rac1 is one of the key regulators in a variety of physiological processes, including membrane trafficking, cell motility, and cancer invasiveness. Although evidence describing the function of the Rho family small GTPases in colorectal cancer has been recently accumulated, its mechanism in invasion and metastasis remains poorly explored. Here, we directly explore the interaction between Rac1 and β-catenin in colon cancer morphogenesis and metastasis. [Methods] Expressions of cell-cell junctional molecules, small GTPases and some tyrosine kinases such as IGF-1R and c-MET were evaluated by immunohistochemistry in the surgical specimens with human colon cancers. Ten colon cancer cell lines were assessed for Rac1 activation by pull-down assay. Invasion assay was performed to evaluate cells’ behavior. Cell adhesion associated proteins such as E-cadherin and β-catenin and actin cytoskeleton were evaluated with several kinase inhibitors by immunocytochemistry. [Results] First, we found that active Rac1 expression at the invasion front and distant liver metastatic site correlates with loss of E-cadherin and nuclear β-catenin accumulation in colon cancer patients. Next, rho-GTPase activation status was evaluated in various colon cancer cell lines with GST-fused Cdc42 and Rac interactive binding (CRIB) domain of PAK1 (PAK-CRIB). Expression level of active Rac1 correlates with cell invasiveness in colon cancer cell lines. The cell invasiveness was enhanced or inhibited by over expression of either constitutive active mutant of Rac (V12Rac) or dominant negative mutant of Rac (N17Rac) with Matrigel assay. The downstream signaling of Rac1-dependent pathway was inhibited by either dominant negative WAVE (WAVE-MT) or CA region of N-WASP (CA region). The finding that endogenous β-catenin and Rac1 directly were bound in nuclear fractions of cells was detected by co-immunoprecipitation assay. Rac1 inhibition by not only NSC23766, but also IGF-1R inhibitor restores the adhesion of colon cancer cells through inhibiting translocation of β-catenin to the nucleus. We confirmed that Rac1 inhibition induces cell-cell adhesion through restoring E-cadherin localization to the cell membrane and redistribution of β-catenin to the cytoplasm. [Conclusions] Taken together, our results raise the possibility that nuclear translocation of Rac1 and β-catenin at the invasive front could play a role in the invasive activity of colorectal cancer cells. Thus present study provides a concept for a new therapeutic strategy that targeting Rac1 molecul would be effective to suppress colon cancer invasive cell. Citation Format: Shinsuke Funakoshi, Hiromasa Takaishi, Ayano Niibe-Kabashima, Tomohiro Suzuki, Gen Sakai, Motoko Izumiya, Masayuki Adachi, Yasuo Hamamoto, Hajime Higuchi, Akinori Hashiguchi, Nobuhiro Tsukada, Takanori Kanai. Rac1 accumulates in the nucleus at the invasive front of colorectal cancer through promoting cell motility. [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 3146. doi:10.1158/1538-7445.AM2014-3146

IL-6R/STAT3/miR-34a feedback loop promotes EMT-mediated colorectal cancer invasion and metastasis

Members of the miR-34 family are induced by the tumor suppressor p53 and are known to inhibit epithelial-to-mesenchymal transition (EMT) and therefore presumably suppress the early phases of metastasis. Here, we determined that exposure of human colorectal cancer (CRC) cells to the cytokine IL-6 activates the oncogenic STAT3 transcription factor, which directly represses the MIR34A gene via a conserved STAT3-binding site in the first intron. Repression of MIR34A was required for IL-6-induced EMT and invasion. Furthermore, we identified the IL-6 receptor (IL-6R), which mediates IL-6-dependent STAT3 activation, as a conserved, direct miR-34a target. The resulting IL-6R/STAT3/miR-34a feedback loop was present in primary colorectal tumors as well as CRC, breast, and prostate cancer cell lines and associated with a mesenchymal phenotype. An active IL-6R/STAT3/miR-34a loop was necessary for EMT, invasion, and metastasis of CRC cell lines and was associated with nodal and distant metastasis in CRC patient samples. p53 activation in CRC cells interfered with IL-6-induced invasion and migration via miR-34a-dependent downregulation of IL6R expression. In Mir34a-deficient mice, colitis-associated intestinal tumors displayed upregulation of p-STAT3, IL-6R, and SNAIL and progressed to invasive carcinomas, which was not observed in WT animals. Collectively, our data indicate that p53-dependent expression of miR-34a suppresses tumor progression by inhibiting a IL-6R/STAT3/miR-34a feedback loop.

Mo1162 Inflammation Increases Notch1 Activity in Colorectal Cancer Cells and Is Counteracted by Eicosapentaenoic Acid-Free Fatty Acid

Mo1162 Inflammation Increases Notch1 Activity in Colorectal Cancer Cells and Is Counteracted by Eicosapentaenoic Acid-Free Fatty Acid

Yuja extract ameliorates DSS‐induced colitis and induces colon cancer cell death by reducing COX‐2 expressions

In this study, we first evaluated the inhibitory effect of 70% ethanol extract of Yuja (Citrus Junos Tanaka) on colitis and colorectal cancer cells. Yuja extract abolished the LPS‐induced secretions of nitric oxide, tumor necrosis factor‐alpha, and interleukin‐6 in RAW264.7 macrophages. We further investigated these anti‐inflammatory effects using experimental colitis models. Colitis was induced by 3% dextran sodium sulfate (DSS) in BALB/c mice. Oral administration of Yuja extract reduced the colon shortening induced by DSS. Cyclooxygenase‐2 (COX‐2) expression was also decreased in colon tissue after Yuja extract administration. Next we also evaluated whether Yuja extract exerts an anti‐tumor activity in colorectal cancer cells, because ulcerative colitis has been believed to be a risk factor of colorectal cancer. Yuja extract also accelerated cell death and apoptotic body appearance in HT‐29 colorectal cancer cells. This antitumor activity was accompanied by reducing iNOS and COX‐2 expressions. Taken together, Yuja extract may be useful for preventing colitis and colorectal cancer by reducing COX‐2 expressions.This work was supported by the Yuja Food Cluster Co., Ltd.