Human Colon Cancer Tissues Research Articles

Oxaliplatin and Infliximab Combination Synergizes in Inducing Colon Cancer Regression.

BackgroundColon cancer is one of the most common malignant cancers and causes millions of deaths each year. There are still no effective treatments for colon cancer patients who are at advanced stage. Tumor necrosis factor-alpha (TNF-α) might be a good therapy target due to its widely-accepted roles in regulating multiple important biological processes, especially in promoting inflammation.Material/MethodsWe evaluated the expression of TNF-α in 108 human colon cancer tissue samples and 2 colon cancer cell lines (CT26 and HCT116), and analyzed its prognostic values. Further, we explored the roles and mechanism of anti-TNF-α treatment in combination with chemotherapy in vitro and in vivo.ResultsWe found that TNF-α was highly expressed in colon cancer cell lines. The survival analysis and Cox regression analysis indicated that high TNF-α was an independent adverse prognosticator of colon cancer. In addition, anti-TNF-α treatment enhanced the effects of chemotherapy in the xenograft mouse model through inducing ADCC and CDC effects.ConclusionsWe conclude that TNF-α is an independent adverse prognosticator of colon cancer, and anti-TNF-α might benefit colon cancer patients.

Gab3 is required for human colorectal cancer cell proliferation

Here, we focused on the potential function of Gab3, an uncommon Gab family protein, in human colorectal cancer (CRC) cells. We found that Gab3 was only expressed in human colon cancer tissues as well as in established (HCT-116 and HT-29 lines) and primary human CRC cells. It was however absent in normal human colon cancer tissues and in FHC colon epithelial cells. Knockdown of Gab3 by targeted-shRNAs inhibited proliferation of the CRC cells. Reversely, exogenous over-expression of Gab3 promoted CRC cell proliferation. At the signaling level, Gab3 co-precipitated with p85 and SHP2 in CRC cells, which was required for subsequent Akt and Erk activation. Gab3 shRNA knockdown inhibited Akt and Erk activation, yet Gab3 over-expression augmented it. In vivo, HCT-116 xenograft tumor growth in severe combined immune deficient (SCID) mice was suppressed following expressing Gab3 shRNAs. Meanwhile, Akt and Erk activation in Gab3 shRNA-expressing tumors was also largely inhibited. Together, our results suggest that Gab3 expression in CRC cells is important for Akt-Erk activation and cell proliferation.

P53 functional deficiency in human colon cancer cells promotes fibroblast-mediated angiogenesis and tumor growth.

Cancer-associated fibroblasts (CAFs) create a microenvironment that contributes to tumor growth; however, the mechanism by which fibroblasts are phenotypically altered to CAFs remains unclear. Loss or mutation of the tumor suppressor p53 plays a crucial role in cancer progression. Herein, we analyzed how the p53 status of cancer cells affects fibroblasts by investigating the in vivo and in vitro effects of loss of p53 function in cancer cells on phenotypic changes in fibroblasts and subsequent tumor progression in human colon cancer cell lines containing wild-type p53 and in cells with a p53 functional deficiency. The growth of p53-deficient tumors was significantly enhanced in the presence of fibroblasts compared with that of p53-wild-type tumors or p53-deficient tumors without fibroblasts. p53-deficient cancer cells produced reactive oxygen species, which activated fibroblasts to mediate angiogenesis by secreting vascular endothelial growth factor (VEGF) both in vivo and in vitro Activated fibroblasts significantly contributed to tumor progression. Deletion of fibroblast-derived VEGF or treatment with N-acetylcysteine suppressed the growth of p53-deficient xenograft tumors. The growth effect of blocking VEGF secreted from cancer cells was equivalent regardless of p53 functional status. Human colon cancer tissues also showed a significant positive correlation between p53 cancer cell staining activated fibroblasts and microvessel density. These results reveal that fibroblasts were altered by exposure to p53-deficient epithelial cancer cells and contributed to tumor progression by promoting neovascularization. Thus, p53 acts as a modulator of the tumor microenvironment.

MicroRNA-126 inhibits colon cancer cell proliferation and invasion by targeting the chemokine (C-X-C motif) receptor 4 and Ras homolog gene family, member A, signaling pathway.

MicroRNA-126 (miR-126) suppresses the migration, proliferation and invasion of colon cancer cells. However, the underlying mechanisms of miR-126 in colon cancer have not been fully elucidated. In this study, in vivo experiments revealed that miR-126 inhibits colon cancer growth and metastasis. Furthermore, miR-126 was down-regulated in human colon cancer tissue, and its expression was inversely correlated with TNM stage and metastasis of patients. Low level of miR-126 identified patients with poor prognosis. And we found that miR-126 expression was negatively correlated with the expression levels of chemokine (C-X-C motif) receptor 4 (CXCR4) and components of signaling pathway of Ras homolog gene family, member A (RhoA) in vitro and in vivo. Moreover, we verified that miR-126 negatively regulated CXCR4 and RhoA signaling in vitro. In addition, either in miR-126-overexpressing or in miR- 126-silenced colon cancer cells, the restoration of CXCR4 could significantly reverse the proliferation and invasion, as well as abolish the effects of miR-126 on RhoA signaling pathway. Collectively, these results demonstrated that miR-126 acts as a tumor suppressor by inactivating RhoA signaling via CXCR4 in colon cancer. And miR-126 may serve as a prognostic marker for monitoring and treating colon cancer.

Abstract 5166: Gene expression microarray analysis identifies association of genes in the integrin/fak signaling pathway with claudin-7 in human colon cancer tissue

Abstract The role and mechanism of the integrin/focal adhesion kinase (FAK)/ERK signaling pathway in the progression of many malignant tumor types, including colon cancer, have been widely reported. The claudin family of proteins is integral to the structure and function of tight junctions. The role of claudin-7 (Cldn-7, CLDN7) in the regulation of the integrin/FAK/ERK signaling pathway remains poorly understood. Therefore, we investigated differences in gene expression, primarily focusing on CLDN7 and integrin/FAK/ERK signaling pathway genes, in colon cancer and adjacent normal tissues. Quantitative real-time RT-PCR verified the results for mRNA expression, and immunohistochemistry verified the results for protein expression. In silico analysis was used to predict co-regulation between Cldn-7 and integrin/FAK/ERK signaling pathway members. Our results showed marked cancer stage-specific decreases in the protein expression of Cldn-7, Gelsolin, MAPK1 and MAPK3 in colon cancer samples. The observed changes in the expression of all proteins except Cldn-7 were in agreement with the changes in the corresponding mRNA levels. Cldn-7 was predicted not to directly regulate any genes in the integrin/FAK/ERK signaling pathway, but these factors may participate in a common network that regulates cancer progression, in which the MAPK pathway is the central node. Citation Format: Lu Kong, Lei Ding. Gene expression microarray analysis identifies association of genes in the integrin/fak signaling pathway with claudin-7 in human colon cancer tissue. [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 5166.

Abstract 4748: Targeting colon cancer stem cells: Novel marmelin analog THB suppresses DCLK1 and Notch Signaling

Abstract Background: Despite therapeutic advances, colon cancer remains the second leading cause of death in the United States. Previously, we have reported that the identification of a novel compound, HDNC or Marmelin from Aegle marmelos with potent anti-colon cancer activity. We have now developed a novel marmelin analogue THB and made it water soluble THB using β-cyclodextrin (THBCD). The current study is designed to determine whether THB affects stem cells and to identify a mechanism. Method: Colon cancer cell lines HCT116 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. Colosphere formation assay and FACS sorting were used for stem cells. For in vivo effects, we have performed HCT116 cells induced tumor xenografts. Immunohistochemistry was determined for stem cell markers and Notch signaling proteins. Results: THB treatment induced a significant dose-dependent inhibition of proliferation and colony formation of the two colon cancer cell lines, but not that of the normal cells. To demonstrate THB effects on stem cells, we performed colosphere assays. THB treatment significantly reduced the number and size of colospheres, suggesting effects on stem cells. In addition, colon stem cell marker proteins DCLK1, LGR5, and CD44 were also decreased. Further proof was obtained by flow cytometry analyses, where THB reduced the number of DCLK1+ cells. We next determined whether THB affects the Notch signaling pathway, a pathway that is important in maintaining CSC population. Notch receptor and its ligands are up-regulated in human colon cancer tissues. THB treatment significantly downregulated the expression of Notch1, its ligand Jagged1 and downstream target protein Hes1. Notch activation requires cleavage by the γ-secretase complex. THB treatment inhibits the expression of γ-secretase complex proteins Presenilin1, Nicastrin, APH1 and PEN2. Moreover, ectopic expression of the Notch Intracellular domain (NICD) rescued the cells from THB mediated growth suppression. These data demonstrate that THB mediated effects of colon cancer stem cells is in part through downregulating Notch1 activation. To determine the effect of THB on tumor growth in vivo, mice carrying HCT116 tumor xenografts were administered the compound intraperitoneally (5mg/kg bw) every day for 21 days. THB treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Western blot and immunohistochemistry analyses demonstrated significant inhibition of CSC marker proteins DCLK1, LGR5 and CD44 and also the Notch signaling proteins in the THB-treated xenograft tissues. Conclusion: Together, these data suggest that THB treatment suppresses colon cancer growth that targets stem cells by inhibiting Notch1 signaling pathway. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Prasad R. Dandawate, Ossama W. Tawfik, Roy A. Jensen, Scott J. Weir, Subhash B. Padhye, Shrikant Anant. Targeting colon cancer stem cells: Novel marmelin analog THB suppresses DCLK1 and Notch Signaling. [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 4748.

Claudin-7 indirectly regulates the integrin/FAK signaling pathway in human colon cancer tissue.

The claudin family of proteins is integral to the structure and function of tight junctions. The role of claudin-7 (Cldn-7, CLDN7) in regulating the integrin/focal adhesion kinase (FAK)/ERK signaling pathway remains poorly understood. Therefore, we investigated differences in gene expression, primarily focusing on CLDN7 and integrin/FAK/ERK signaling pathway genes, between colon cancer and adjacent normal tissues. Quantitative real-time reverse transcription-PCR and immunohistochemistry were utilized to verify the results of mRNA and protein expression, respectively. In silico analysis was used to predict co-regulation between Cldn-7 and integrin/FAK/ERK signaling pathway components, and the STRING database was used to analyze protein-protein interaction pairs among these proteins. Meta-analysis of expression microarrays in The Cancer Genome Atlas (TCGA) database was used to identify significant correlations between Cldn-7 and components of predicted genes in the integrin/FAK/ERK signaling pathway. Our results showed marked cancer stage-specific decreases in the protein expression of Cldn-7, Gelsolin, MAPK1 and MAPK3 in colon cancer samples, and the observed changes for all proteins except Cldn-7 were in agreement with changes in the corresponding mRNA levels. Cldn-7 might indirectly regulate MAPK3 via KRT8 due to KRT8 co-expression with MAPK3 or CLDN7. Our bioinformatics methods supported the hypothesis that Cldn-7 does not directly regulate any genes in the integrin/FAK/ERK signaling pathway. These factors may participate in a common network that regulates cancer progression in which the MAPK pathway serves as the central node.

Integrin α4 Induces Lymphangiogenesis and Metastasis via Upregulation of VEGF-C in Human Colon Cancer.

Vascular endothelial growth factor-C (VEGF-C) is a key regulator in lymphangiogenesis, and is overexpressed in various malignancies. Integrin α4β1, a new member of the VEGF-C/VEGF receptor pathway, was found to be overexpressed in melanoma tumors. However, little is known regarding the potential role of integrin α4β1 in lymphangiogenesis and other solid tumors. The aim of this study was to investigate the expression patterns of integrin α4 and VEGF-C in relation to lymphangiogenesis and clinicopathological parameters in human colon cancer. The expression of integrin α4, VEGF-C, and VEGFR-3 was assessed in 71 human colon cancer tissues and 30 paracancerous normal tissues by immunohistochemical staining. Lymphatic microvessel density (LMVD) was measured after D2-40-labeling, and the correlations among different factors were statistically analyzed. The expression of integrin α4, VEGF-C, VEGFR-3, and LMVD was higher in colon cancer tissues compared with the normal paracancerous colon tissues. There was a positive correlation between the expression of integrin α4 and VEGF-C. Integrin α4 and VEGF-C were significantly associated with the clinicopathological parameters (LMVD, Duke's stage, and lymph node metastasis). Kaplan-Meier analyses indicated that patients with high integrin α4 or VEGF-C expression had significantly shorter overall survival and tumor-free survival time. Multivariate analyses suggested that integrin α4 and VEGF-C may serve as independent prognostic factors for human colon cancer. Both integrin α4 and VEGF-C are involved in lymphangiogenesis and lymphatic metastasis. Our results demonstrated that integrin α4 is a novel prognostic indicator for human colon cancer. Anat Rec, 299:741-747, 2016. © 2016 Wiley Periodicals, Inc.

Upregulation of the Multidrug Resistance P‐glycoprotein ABCB1 by Transcription Factor Pituitary Homeobox 2 (Pitx2) in Human Colon and Kidney Cancers

BackgroundPituitary homeobox 2 (Pitx2) is a master regulator of organ asymmetry during development but is reactivated in neoplastic cells, contributing to cell proliferation, tumor cell invasion and cancer progression. Upregulation of the multidrug resistance P‐glycoprotein ABCB1 confers chemotherapeutic drug resistance to tumor cells, hampering effective treatment. We have previously shown that Pitx2 is upregulated in a multidrug resistant (MDR) kidney cancer cell line and serves as a positive transcriptional regulator of ABCB1 thereby increasing resistance to the anthracycline chemotherapeutic drug, doxorubicin (DOX) (Lee, W.‐K. et al. Int J Cancer 113, 556–67, 2013). The objective of this study was to determine if ABCB1 regulation by Pitx2 is also in engaged by other cancer types that exhibit intrinsic MDR.MethodsExpression of Pitx2 and ABCB1 was determined by immunofluorescence microscopy, immunoblotting and immunohistochemistry. Cell viability was assessed by MTT and clonogenic survival assays. Pitx2 transcriptional activity was measured by luciferase reporter gene assay. Isolation of nuclei was performed by Tenbroeck homogenization in hypoosmotic buffer and a discontinuous sucrose gradient.ResultsIn human colorectal adenocarcinoma Colo320DM cells, which exhibit MDR and harbor augmented ABCB1 expression, nuclear Pitx2 expression and Pitx2 reporter gene activity were significantly increased compared to less drug resistant Colo205 cells. The ABCB1 inhibitor PSC833 (0.1 μM) reversed drug resistance to 1 μM DOX after 24 h in Colo320DM cells. Further, knockdown of Pitx2 by RNAi in Colo320DM cells resulted in decreased ABCB1 protein by almost 3‐fold and diminished resistance to DOX resulting in attenuated cell survival. Moreover, the Pitx2 isoforms that are involved in regulation of ABCB1 in colon and kidney cancer cells were investigated. Both Pitx2A and Pitx2B were found to be present in the nuclear fraction of Colo320DM and MDR A498 kidney carcinoma cells. Analysis of human kidney and colon cancer tissue arrays by co‐immunostaining suggests a causal relationship between nuclear Pitx2 and ABCB1 expression.ConclusionPitx2 is a transcriptional regulator of ABCB1 in cancers of the colon and kidney suggesting a contributory role to the MDR phenotype.Support or Funding InformationFunded by an Internal Research Grant (UW/H) and ZBAF

RNA helicase YTHDC2 promotes cancer metastasis via the enhancement of the efficiency by which HIF-1α mRNA is translated

YTH domain containing 2 (YTHDC2) is a member of the DExD/H-box family of ATP-dependent RNA helicases. We previously found that YTHDC2 expression is up-regulated in several human cancer cells. In this study, we demonstrate novel roles for YTHDC2 in metastasis of colon tumor cells via translation-dependent pathway. Knockdown of YTHDC2 attenuated protein expression of metastasis-related genes, such as hypoxia-inducible factor-1alpha (HIF-1α), and inhibited metastasis of colon tumor cells in vitro and in vivo. To confirm that YTHDC2 promotes translation initiation by unwinding the 5′-untranslated region (5′UTR) of mRNA, we constructed a firefly luciferase reporter containing the 5′UTR of the HIF-1α mRNA and showed reduction in luciferase activity in YTHDC2-silenced cells. Furthermore, we examined expression levels of YTHDC2 by immunohistochemical staining in human colon cancer tissues from 72 patients and found a significantly positive correlation between YTHDC2 expression and the tumor stage, including metastasis. In conclusion, these results suggest that the RNA helicase YTHDC2 contributes to colon tumor metastasis by promoting translation of HIF-1α and that YTHDC2 is potentially a diagnostic marker and target gene for treating colon cancer patients.

NADPH Oxidase 1 Activity and ROS Generation Are Regulated by Grb2/Cbl-Mediated Proteasomal Degradation of NoxO1 in Colon Cancer Cells.

The generation of reactive oxygen species (ROS) is required for proper cell signaling, but must be tightly regulated to minimize deleterious oxidizing effects. Activation of the NADPH oxidases (Nox) triggers ROS production and, thus, regulatory mechanisms exist to properly control Nox activity. In this study, we report a novel mechanism in which Nox1 activity is regulated through the proteasomal degradation of Nox organizer 1 (NoxO1). We found that through the interaction between NoxO1 and growth receptor-bound protein 2 (Grb2), the Casitas B-lineage lymphoma (Cbl) E3 ligase was recruited, leading to decreased NoxO1 stability and a subsequent reduction in ROS generation upon epidermal growth factor (EGF) stimulation. Additionally, we show that EGF-mediated phosphorylation of NoxO1 induced its release from Grb2 and facilitated its association with Nox activator 1 (NoxA1) to stimulate ROS production. Consistently, overexpression of Grb2 resulted in decreased Nox1 activity, whereas knockdown of Grb2 led to increased Nox1 activity in response to EGF. CRISPR/Cas9-mediated NoxO1 knockout in human colon cancer cells abrogated anchorage-independent growth on soft agar and tumor-forming ability in athymic nude mice. Moreover, the expression and stability of NoxO1 were significantly increased in human colon cancer tissues compared with normal colon. Taken together, these results support a model whereby Nox1 activity and ROS generation are regulated by Grb2/Cbl-mediated proteolysis of NoxO1 in response to EGF, providing new insight into the processes by which excessive ROS production may promote oncogenic signaling to drive colorectal tumorigenesis.

PRC2 Epigenetically Silences Th1-Type Chemokines to Suppress Effector T-Cell Trafficking in Colon Cancer.

Infiltration of tumors with effector T cells is positively associated with therapeutic efficacy and patient survival. However, the mechanisms underlying effector T-cell trafficking to the tumor microenvironment remain poorly understood in patients with colon cancer. The polycomb repressive complex 2 (PRC2) is involved in cancer progression, but the regulation of tumor immunity by epigenetic mechanisms has yet to be investigated. In this study, we examined the relationship between the repressive PRC2 machinery and effector T-cell trafficking. We found that PRC2 components and demethylase JMJD3-mediated histone H3 lysine 27 trimethylation (H3K27me3) repress the expression and subsequent production of Th1-type chemokines CXCL9 and CXCL10, mediators of effector T-cell trafficking. Moreover, the expression levels of PRC2 components, including EZH2, SUZ12, and EED, were inversely associated with those of CD4, CD8, and Th1-type chemokines in human colon cancer tissue, and this expression pattern was significantly associated with patient survival. Collectively, our findings reveal that PRC2-mediated epigenetic silencing is not only a crucial oncogenic mechanism, but also a key circuit controlling tumor immunosuppression. Therefore, targeting epigenetic programs may have significant implications for improving the efficacy of current cancer immunotherapies relying on effective T-cell-mediated immunity at the tumor site.

Potential role of TRIM3 as a novel tumour suppressor in colorectal cancer (CRC) development

ABSTRACTObjective Colorectal cancer (CRC) is the third leading cause of cancer-related mortality in the United States. Recent cancer genome-sequencing efforts and complementary functional studies have led to the identification of a collection of candidate ‘driver’ genes involved in CRC tumorigenesis. Tripartite motif (TRIM3) is recently identified as a tumour suppressor in glioblastoma but this tumour-suppressive function has not been investigated in CRC. Material and methods In this study, we investigated the potential role of TRIM3 as a tumour suppressor in CRC development by manipulating the expression of TRIM3 in two authentic CRC cell lines, HCT116 and DLD1, followed by various functional assays, including cell proliferation, colony formation, scratch wound healing, soft agar, and invasion assays. Xenograft experiment was performed to examine in vivo tumour-suppressive properties of TRIM3. Results Small-interfering RNA (siRNA) mediated knockdown of TRIM3 conferred growth advantage in CRC cells. In contrast, overexpression of TRIM3 affected cell survival, cell migration, anchorage independent growth and invasive potential in CRC cells. In addition, TRIM3 was found to be down-regulated in human colon cancer tissues compared with matched normal colon tissues. Overexpression of TRIM3 significantly inhibited tumour growth in vivo using xenograft mouse models. Mechanistic investigation revealed that TRIM3 can regulate p53 protein level through its stabilisation. Conclusions TRIM3 functions as a tumour suppressor in CRC progression. This tumour-suppressive function is exerted partially through regulation of p53 protein. Therefore, this protein may represent a novel therapeutic target for prevention or intervention of CRC.

Electroporation-assisted penetration of zinc oxide nanoparticles in ex vivo normal and cancerous human colon tissue

In this study, we presented the research of the penetration of zinc oxide nanoparticles (ZnO NPs) (30 and 90 nm), and electroporation (EP) assisted penetration of the ZnO NPs in the human normal colon (NC) and adenomatous colon (AC) tissues studied with optical coherence tomography (OCT) and diffuse reflectance (DR) measurement. The results have shown that the attenuation coefficient of colon tissue after the application of 30 or 90 nm ZnO NPs alone decreased approximately by 28% and 14% for NC tissue, 35% and 22% for AC tissue, respectively; while the attenuation coefficient of colon tissue after combined application of 30 or 90 nm ZnO NPs/EP decreased approximately by 46% and 30% for NC tissue, and 53% and 42% for AC tissue, respectively. The results illustrate EP can significantly increase the penetration of ZnO NPs in the colon tissue, especially in AC tissue. Through the analysis of attenuation coefficient and reflectance intensity of the colon tissue, we find that the accumulation of the ZnO NPs in the colon tissue greatly influenced the tissue optical properties.

Butyrate inhibits pro-proliferative miR-92a by diminishing c-Myc-induced miR-17-92a cluster transcription in human colon cancer cells.

BackgroundCompromised colonic butyrate production resulting from low dietary fiber or altered gut microbiota may promote colon neoplasia. Previous reports indicate these actions are mediated in part by altered levels of miRNAs, including suppressed expression of the oncogenic miR-17-92a cluster. Here, we sought to identify the mechanisms underlying these effects of butyrate in colon cancer.MethodsmiR-92a levels were measured in archived human colon cancer and adjacent normal colon specimens by microarray and quantitative RT-PCR (qPCR). The effects of butyrate and other histone deacetylase inhibitors (suberoylanilide hydroxamic acid (SAHA) and valproic acid) on primary (pri-miR17-92a), precursor and mature miR-92a were analyzed in HCT-116 and HT-29 human colon cancer cells using qPCR. The effects of butyrate, SAHA and valproic acid on protein levels of c-Myc, Drosha and p57 were measured in HCT-116 cells using immunoblotting. Regulation of C13orf25 promoter activity by butyrate was analyzed by luciferase reporter assay using modified pGL3 constructs containing a wild-type or mutated c-Myc binding site. Expression of c-Myc was modulated using siRNA or adenovirus vectors. p57 mRNA and protein were measured before and after transfection with miR-92a-mimic molecules. Following butyrate treatment and miR-92a-mimic transfection, apoptosis was analyzed by TUNEL staining and caspase-3 immunoblotting.ResultsMicroarray, confirmed by qPCR, revealed a seven-fold increase in miR-92a levels in sporadic human colon cancer tissue compared to adjacent normal colon. Treating human colon cancer cells with butyrate reduced the levels of pri-miR17-92a, precursor and mature miR-92a, as well as c-Myc. SAHA and valproic acid had similar effects. Mutation of the c-Myc binding site diminished butyrate’s inhibitory effects on C13orf25 promoter activity. Silencing c-Myc expression reduced miR-92a levels. c-Myc over-expression neutralized butyrate-induced attenuation of pri-miR17-92a. Exogenous miR-92a inhibited butyrate-induced p57 expression and reversed the beneficial actions of butyrate on colon cancer cell proliferation and apoptosis.ConclusionsOur findings identify a novel cellular mechanism whereby butyrate inhibits miR-92a transcription by reducing c-Myc, thus augmenting p57 levels. These actions diminish colon cancer cell proliferation and stimulate apoptosis. This newly described regulation of oncogenic miRNA biogenesis expands our understanding of colon cancer cell biology and identifies novel therapeutic targets.

GSK3β and Gli3 play a role in activation of Hedgehog-Gli pathway in human colon cancer — Targeting GSK3β downregulates the signaling pathway and reduces cell proliferation

The role of Hedgehog-Gli (Hh-Gli) signaling in colon cancer tumorigenesis has not yet been completely elucidated. Here we provide strong evidence of Hh-Gli signaling involvement in survival of colon cancer cells, with the main trigger of activation being deregulated GSK3β.Our clinical data reveals high expression levels of GSK3β and Gli3 in human colon cancer tissue samples, with positive correlation between GSK3β expression and DUKES' stage. Further experiments on colon cancer cell lines have shown that a deregulated GSK3β upregulates Hh-Gli signaling and positively affects colon cancer cell survival. We show that inhibition of GSK3β with lithium chloride enhances Gli3 processing into its repressor form, consequently downregulating Hh-Gli signaling, reducing cell proliferation and inducing cell death. Analysis of the molecular mechanisms revealed that lithium chloride enhances Gli3–SuFu–GSK3β complex formation leading to more efficient Gli3 cleavage and Hh-Gli signaling downregulation. This work proposes that activation of the Hh-Gli signaling pathway in colon cancer cells occurs non-canonically via deregulated GSK3β. Gli3 seems to be the main pathway effector, highlighting the activator potential of this transcription factor, which is highly dependent on GSK3β function and fine tuning of the Gli3–SuFu–GSK3β platform.

Human Colon Tumors Express a Dominant-Negative Form of SIGIRR That Promotes Inflammation and Colitis-Associated Colon Cancer in Mice

Single immunoglobulin and toll-interleukin 1 receptor (SIGIRR), a negative regulator of the Toll-like and interleukin-1 receptor (IL-1R) signaling pathways, controls intestinal inflammation and suppresses colon tumorigenesis in mice. However, the importance of SIGIRR in human colorectal cancer development has not been determined. We investigated the role of SIGIRR in development of human colorectal cancer. We performed RNA sequence analyses of pairs of colon tumor and nontumor tissues, each collected from 68 patients. Immunoblot and immunofluorescence analyses were used to determine levels of SIGIRR protein in primary human colonic epithelial cells, tumor tissues, and colon cancer cell lines. We expressed SIGIRR and mutant forms of the protein in Vaco cell lines. We created and analyzed mice that expressed full-length (control) or a mutant form of Sigirr (encoding SIGIRR(N86/102S), which is not glycosylated) specifically in the intestinal epithelium. Some mice were given azoxymethane (AOM) and dextran sulfate sodium to induce colitis-associated cancer. Intestinal tissues were collected and analyzed by immunohistochemical and gene expression profile analyses. RNA sequence analyses revealed increased expression of a SIGIRR mRNA isoform, SIGIRR(ΔE8), in colorectalcancer tissues compared to paired nontumor tissues. SIGIRR(ΔE8) is not modified by complex glycans and is therefore retained in the cytoplasm-it cannot localize to the cell membrane or reduce IL1R signaling. SIGIRR(ΔE8) interacts with and has a dominant-negative effect on SIGIRR, reducing its glycosylation, localization to the cell surface, and function. Most SIGIRR detected in human colon cancer tissues was cytoplasmic, whereas in nontumor tissues it was found at the cell membrane. Mice that expressed SIGIRR(N86/102S) developed more inflammation and formed larger tumors after administration of azoxymethane and dextran sulfate sodium than control mice; colon tissues from these mutant mice expressed higher levels of the inflammatory cytokines IL-17A and IL-6 had activation of the transcription factors STAT3 and NFκB. SIGIRR(N86/102S) expressed in colons of mice did not localize to the epithelial cell surface. Levels of SIGIRR are lower in human colorectal tumors, compared with nontumor tissues; tumors contain the dominant-negative isoform SIGIRR(ΔE8). This mutant protein blocks localization of full-lengthSIGIRR to the surface of colon epithelial cells and itsability to downregulate IL1R signaling. Expression of SIGIRR(N86/102S) in the colonic epithelium of mice increases expression of inflammatory cytokines and formation and size of colitis-associated tumors.

Down-regulation of MicroRNA-381 promotes cell proliferation and invasion in colon cancer through up-regulation of LRH-1

The expression and roles of MicroRNA-381 (miR-381) has been explored in several types of human cancers. However, its biological functions in colon cancer remain largely unknown. Quantitative real-time PCR assays were used to detect the expression of miR-381 in human colon cancer tissues and adjacent normal tissues. miR-381 antisense oligos and mimics were introduced into SW480 and HCT116 cells. Bioinformatic prediction analysis was performed to identify the potential targets of miR-381. Protein expression analysis, luciferase assays and rescue assays were used to confirm the substrate of miR-381. We found that miR-381 was significantly down-regulated in human colon cancer tissues, compared with adjacent normal tissues. Introduction of miR-381 antisense oligos into SW480 and HCT116 cells promoted cell proliferation and invasion. Besides, inhibition of miR-381 could also support tumor growth in the nude mice. Additionally, bioinformatic prediction suggested that the nuclear receptor liver receptor homologue 1 (LRH-1) is a target gene of miR-381. Thus, our data suggested that down-regulation of miR-381 plays an important role in the colon cancer progression.

Abstract POSTER-BIOL-1302: Sucrose nonfermenting 1-related kinase (SNRK) expression and function in vitro in ovarian cancer cells

Abstract Purpose of study: Lipid metabolism and transport have recently been identified as potential targets for the treatment of cancers where adipocytes are a major component of the tumor microenvironment. Ovarian cancer metastases have a clear predilection for the omentum, which is primarily comprised of adipocytes. Studies have shown that human omental adipocytes promote migration and invasion of ovarian cancer cells and that adipocyte–ovarian cancer cell coculture promotes tumor growth and induces lipolysis in adipocytes and β-oxidation in cancer cells. SNRK is a serine/threonine kinase with sequence similarity to AMP-activated protein kinase (AMPK). AMPK proteins play a central role in cellular energy regulation. Active AMPK restricts cell proliferation, cell growth, and metabolic substrate synthesis, and increases ATP-generating processes, such as glucose and fatty acid oxidation. It has also been suggested that SNRK suppresses adipocyte inflammation via mTORC1 which is a pathway known to be aberrant in ovarian cancer. SNRK has been shown to reduce colon cancer cell proliferation and DNA synthesis and SNRK levels are decreased in human colon cancer tissue. Stable knockdown of SNRK increases in vitro colon cancer cell tumorigenicity indicating that SNRK is a regulator of cell proliferation. Because of the role SNRK appears to play in cell proliferation and metabolism and the link between SNRK and mTORC1 signaling, the goal of this study is to describe expression of SNRK in ovarian cancer cell lines and ovarian tissue from mice. Experimental Procedures: The ovarian cancer cell lines A2780 and SKOV3ip1 as well as a normal surface epithelial cell line IOSE, were grown in culture media and then immunostained for SNRK, as well as CC3 (apoptosis) and PH3 (proliferation). To assess the expression of SNRK in mice, ovarian tissue from wild type mice were harvested and immunostained for SNRK. Summary of data: Cell line immunostaining demonstrated differential expression of SNRK in both ovarian cancer cell lines and the benign ovarian epithelial cell line along with PH3 staining. There was no CC3 staining seen in any of the cell lines. The mouse ovarian tissue shows SNRK staining in the nuclei of follicular cells but no clear staining in the epithelial or stromal cells. Conclusions: Our results show the presence of SNRK in both benign and malignant epithelial ovarian cell lines as well as in benign mouse ovarian tissue. We plan to further explore SNRK expression levels in ovarian cancer by quantifying SNRK mRNA and protein levels in ovarian cancer cell lines as well as in human benign and malignant ovarian tissue. We also plan to knockdown SNRK expression in ovarian cancer cell lines to determine effects on cell proliferation, migration, and invasion as well as changes in proteins involved in proliferation and metabolism. Citation Format: Erin Bishop, Stephanie Cossette, Vakeel Padmanabhan, Ramani Ramchandran. Sucrose nonfermenting 1-related kinase (SNRK) expression and function in vitro in ovarian cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1302.

Abstract 4163: Targetting VPAC1 for intestinal cancer prevention

Abstract Vasoactive intestinal peptide (VIP) is a neurotransmitter that primarily functions as a vasodilator. VIP plays its role through binding to its receptors known as VIP/pituitary adenylate cyclase-activating peptide receptors (VPACs). In this study, we examined the expression of VPAC1 in human colon cancer tissues, analyzed the relationship between VPAC1 expression and cancer malignancy, and explored the possible mechanisms using immunohistochemistry and immunofluorescence double staining. The results showed that: 1) poorly-differentiated colon cancers have significantly higher VPAC1 expression than well-differentiated colon cancers do (p<0.01); 2) phospho-EGFR overexpression/activation in the cytoplasm of cancer cells is related to VPAC1 overexpression; 3) blood vessels surrounding colon cancer have significantly more VPAC1-positive than normal colon mucosa does; 4) Tumor-Associated Macrophages (TAMs) of colon cancer have a higher level of VPAC1 expression than macrophages in normal colon mucosa do. These data suggest that VPAC1 overexpression is associated with poorer differentiation of colon cancer, which is likely caused by subsequent EGFR activation in cancer cells. In addition, VPAC1 overexpression in both blood vessels and macrophages in tumors may also play an important role in the development of aggressive cancer. Note: This abstract was not presented at the meeting. Citation Format: Shaohua Liu, Yunjie Zeng, Nengtai Ouyang. Targetting VPAC1 for intestinal cancer prevention. [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 4163. doi:10.1158/1538-7445.AM2015-4163