Development Of Human Colon Cancer Research Articles

MicroRNA-128 inhibits the development of human colon cancer by targeting Rho family GTPase 3

MicroRNA-128 (miRNA-128) exhibits tumour regulatory role in several human cancers including the colon cancer. However, regulatory mechanics of miRNA-128 through Rho family GTPase-3 (RND3) targeting is elusive in colon cancer and was the objective of present study. The results showed that miRNA-128 has significant (P < 0.05) level of down-regulation in colon cancer. Over-expression of miRNA-128 significantly (P < 0.05) decreased the viability and colony forming ability of colon cancer cells through induction of apoptosis. The percentage of apoptosis was 6.01% and 37.02% for miR-NC and miR-128 mimics transfected HT-29 cancer cells, respectively. Induction of apoptosis was associated with up-regulation of Bax and down-regulation of Bcl-2. Rho family GTPase-3 (RND3) to be target of miRNA-128 and mediated the regulatory control of miRNA-128 in colon cancer. Finally, miR-128 was found to inhibit the growth of xenografted tumours in mice. Thus, miRNA-128 exerts tumour-suppressive role in human colon cancer and may serve in colon cancer management.

Mindin serves as a tumour suppressor gene during colon cancer progression through MAPK/ERK signalling pathway in mice.

Mindin is important in broad spectrum of immune responses. On the other hand, we previously reported that mindin attenuated human colon cancer development by blocking angiogenesis through Egr‐1–mediated regulation. However, the mice original mindin directly suppressed the syngenic colorectal cancer (CRC) growth in our recent study and we aimed to further define the role of mindin during CRC development in mice. We established the mouse syngeneic CRC CMT93 and CT26 WT cell lines with stable mindin knock‐down or overexpression. These cells were also subcutaneously injected into C57BL/6 and BALB/c mice as well as established a colitis‐associated colorectal cancer (CAC) mouse model treated with lentiviral‐based overexpression and knocked‐down of mindin. Furthermore, we generated mindin knockout mice using a CRISPR‐Cas9 system with CAC model. Our data showed that overexpression of mindin suppressed cell proliferation in both of CMT93 and CT26 WT colon cancer cell lines, while the silencing of mindin promoted in vitro cell proliferation via the ERK and c‐Fos pathways and cell cycle control. Moreover, the overexpression of mindin significantly suppressed in vivo tumour growth in both the subcutaneous transplantation and the AOM/DSS‐induced CAC models. Consistently, the silencing of mindin reversed these in vivo observations. Expectedly, the tumour growth was promoted in the CAC model on mindin‐deficient mice. Thus, mindin plays a direct tumour suppressive function during colon cancer progression and suggesting that mindin might be exploited as a therapeutic target for CRC.

Septin4 promotes cell death in human colon cancer cells by interacting with BAX.

Septin4 is a tumor suppressor protein that promotes cell programmed death in various cell types through specifically antagonizing XIAP (X linked inhibitor of apoptosis), little is known its other novel binding partner and role in colorectal cancer. In this study, we found that Septin4 significantly expressed lower in human colon cancer when compared to peri-tumor benign cells, and its low expression was significantly associated with worse prognostic outcomes. Furthermore, Septin4 participated in DOX-induced colon cancer cell death in vitro. Septin4-overexpressing colon cancer cells displayed augmented apoptotic cell death and ROS production. Additionally, Septin4-knockdown cells revealed a resistance of DOX-induced cell death and reduced ROS production. Importantly, we first identified that BAX is a novel Septin4 binding partner and the interaction is enhanced under DOX treatment. Finally, Septin4-knockdown promoted colon cells growth in vivo. These observations suggest that Septin4 as an essential molecule contribute to the occurrence and development of human colon cancer and provide new technical approaches for targeted treatment of this disease.

Rs187960998 polymorphism in miR-211 prevent development of human colon cancer by deregulation of 3&amp;rsquo;UTR in CHD5

BackgroundPrevious research indicated that overexpression of miRNA-211 could promote colorectal cancer cell growth by targeting tumor suppressive gene Chromodomain-helicase-DNA-binding protein 5 (CHD5) in human colon cancer (CC). Moreover, the function of the single-nucleotide polymorphism (SNP) located in the mature region of miR-211 has not been investigated. In this study, we found that SNP of rs187960998 in miR-211 was involved in the occurrence of CC by acting as a tumor suppressor by mal-regulation of its target gene CHD5.Materials and methodsThe genotype of total 685 CC patients was detected by real-time PCR, the proliferation of CC cell lines with different genotypes of miR-211 was determined by Cell Counting Kit-8, cell invasion evaluated by transwell and the activity of the CHD5 promoter in CC cell lines transfected with different miR-211 was determined by luciferase assay. The expression of CHD5 in CC patients was determined by the immunohistochemistry, and the relapse-free survival rate was analyzed by Kaplan–Meier analysis.ResultsC/T SNP of miR-211 could inhibit CC cell proliferation and invasion by upregulation of CHD5. And SNP in rs187960998 of miR-211 was associated with tumor size, metastasis and tumor differentiation in CC patients. Patients with CC genotype have significantly low CHD5 expression than the T-carrier, while no significant expression difference in miR-211 expression among different genotype subsets. Patients with CC genotype have significantly shorter postsurgery survival rate compared to the T-carrier.Conclusionrs187960998 in miR-211 was highly associated with a decreased risk of CC in the Chinese population by deregulating a tumor suppressive gene CHD5.

Identification of critical genes to predict recurrence and death in colon cancer: integrating gene expression and bioinformatics analysis

BackgroundThe purpose of this study was to screen the critical genes for future diagnosis and treatment of colon cancer by bioinformatics method.MethodsIn this study, we used bioinformatics approaches to identify gene alteration that contribute to colon cancer progression via analysis of TCGA RNA sequencing data and other publicly GEO microarray data. The Random forest survival model was used to screen gene sets related to the prognosis in DEGs. Gene ontology and KEGG pathway enrichment analysis were performed to determine the potential function of DEGs.ResultsWe identified versican (VCAN), a member of the aggrecan/versican proteoglycan family, as a key regulator in human colon cancer development and progression involved in cell adhesion, proliferation, migration and angiogenesis and plays a central role in tissue morphogenesis and maintenance. Interestingly, we found that VCAN is highly over-expressed in colon cancer and increased expression of VCAN was associated with the progression of colon cancer. High VCAN levels also predict shorter overall survival of colon cancer patients. Furthermore, in vitro assays of silencing VCAN inhibit HCT116 cell proliferation and invasion.ConclusionsThese data demonstrated VCAN were associated with tumorigenesis and may be as biomarker for identification of the pathological grade of colon cancer.

Rs35301225 polymorphism in miR-34a promotes development of human colon cancer by deregulation of 3\u2032UTR in E2F1 in Chinese population

BackgroundPrevious reports have revealed that down-regulation of miR-34a expression can promote colorectal cancer (CRC) cell growth by targeting cell cycle-related transcriptional factor E2F1. To date, the function of the single nucleotide polymorphism (SNP) located in the mature region of miR-34a has not been investigated.MethodsWe performed a case–control study including 685 CRC patients and 618 cancer-free controls. Genotyping, real-time PCR assay, cell transfection, and the dual luciferase reporter assay were used in our study. Cell proliferation and cell cycle analysis were measured in CRC cells including Hct-116 and SW480. The overall survival of different genotypes was also investigated.ResultsWe found that the rs35301225 polymorphism in miR-34a was involved in the occurrence of CRC by acting as a tumor suppressor by down-regulation of tumor-promoting gene E2F1. C/A SNP of miR-34a could promote CRC cell proliferation by up-regulation of E2F1. Also, C/A genotype can change the cell cycle by increasing the S phase percentage. Moreover, the SNP in rs35301225 of miR-34a was associated with tumor size and tumor differentiation, as well as metastasis in CRC patients; C/A SNP was related to the significantly enhanced expression of E2F1 and shorter survival in post-surgery CRC patients.Conclusionsrs35301225 in miR-34a was highly associated with a decreased risk of CRC in a Chinese population and might serve as a novel biomarker for colon cancer.

The Histone Acetyltransferase GCN5 Expression Is Elevated and Regulated by c-Myc and E2F1 Transcription Factors in Human Colon Cancer.

The histone acetyltransferase GCN5 has been suggested to be involved in promoting cancer cell growth. But its role in human colon cancer development remains unknown. Herein we discovered that GCN5 expression is significantly upregulated in human colon adenocarcinoma tissues. We further demonstrate that GCN5 is upregulated in human colon cancer at the mRNA level. Surprisingly, two transcription factors, the oncogenic c-Myc and the proapoptotic E2F1, are responsible for GCN5 mRNA transcription. Knockdown of c-Myc inhibited colon cancer cell proliferation largely through downregulating GCN5 transcription, which can be fully rescued by the ectopic GCN5 expression. In contrast, E2F1 expression induced human colon cancer cell death, and suppression of GCN5 expression in cells with E2F1 overexpression further facilitated cell apoptosis, suggesting that GCN5 expression is induced by E2F1 as a possible negative feedback in suppressing E2F1-mediated cell apoptosis. In addition, suppression of GCN5 with its specific inhibitor CPTH2 inhibited human colon cancer cell growth. Our studies reveal that GCN5 plays a positive role in human colon cancer development, and its suppression holds a great therapeutic potential in antitumor therapy.

Abstract 4591: Differential expression of tight junction polarity genes in human colon cancer

Abstract Tight junctions perform significant and multifactorial functions in cellular polarity maintenance and paracellular transport. A number of studies have found structural disruption of epithelial colon cells can significantly impact early tumorigenesis and progression. However, little is known about the direct contribution of polarity gene expression in human colon cancer development. We compared the expression of tight junction genes in colon cancer with normal control mucosa. Total RNA was extracted from fresh frozen tissue biopsies of six white male patients (N = 3 for early stage tumore, N = 3 for late stage tumor, and N = 2 for normal adjacent controls) and four unrelated normal colon mucosa from polyp-free individuals. We used the Qiagen RT2 Profiler PCR array to determine the expression of 84 genes in the tight junction pathway. Student's t test was used to compare the expression levels between different groups (control vs. early-stage tumor and late-stage tumor) using normalized gene expression data. Functional annotation clustering of statistically significant genes by group was conducted using DAVID. Significant (P&amp;lt;0.05) down regulation of claudin (CLDN5, CLDN6, CLDN7) expression was seen in both early and late-stage tumor compared with controls, all with fold change (FC)&amp;gt; 2; while CLDN1 (FC = 15) was only significantly upregulated (P = 0.0007) in early-stage tumor. Down regulation (FC&amp;gt;2) of polarity complex gene PARD6A (P&amp;lt;.001) and associated genes (JAM2, P&amp;lt;.001 and JAM3, P &amp;lt;.001) were present in both early and late-stage tumors along with associated down-regulation of tumor suppressor PTEN (P&amp;lt;.0001). We found differential expression of tight junction genes in normal versus colon tumor. Down regulation of polarity genes is likely to affect apico-basal polarity and promote random non-coordinated migration which can lead to disruption of epithelial structure and to carcinogenic tissue infiltration. Further larger studies are warranted to investigate the role of tight junction proteins in the progression of human colon tumor. Citation Format: Kathryn E. Royse, Liang Chen, Jocelyn Uriostegui, Michael Ittmann, David Y. Graham, Hashem El-Serag, Li Jiao. Differential expression of tight junction polarity genes in human colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4591. doi:10.1158/1538-7445.AM2015-4591

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KIT Signaling Promotes Growth of Colon Xenograft Tumors in Mice and Is Up-Regulated in a Subset of Human Colon Cancers

Receptor tyrosine kinase (RTK) inhibitors have advanced colon cancer treatment. We investigated the role of the RTK KIT in development of human colon cancer. An array of 137 patient-derived colon tumors and their associated xenografts were analyzed by immunohistochemistry to measure levels of KIT and its ligand KITLG. KIT and/or KITLG was stably knocked down by expression of small hairpin RNAs from lentiviral vectors in DLD1, HT29, LS174T, and COLO320 DM colon cancer cell lines, and in UM-COLON#8 and POP77 xenografts; cells transduced with only vector were used as controls. Cells were analyzed by real-time quantitative reverse transcription polymerase chain reaction, single-cell gene expression analysis, flow cytometry, and immunohistochemical, immunoblot, and functional assays. Xenograft tumors were grown from control and KIT-knockdown DLD1 and UM-COLON#8 cells in immunocompromised mice and compared. Some mice were given the RTK inhibitor imatinib after injection of cancer cells; tumor growth was measured based on bioluminescence. We assessed tumorigenicity using limiting dilution analysis. KIT and KITLG were expressed heterogeneously by a subset of human colon tumors. Knockdown of KIT decreased proliferation of colon cancer cell lines and growth of xenograft tumors in mice compared with control cells. KIT knockdown cells had increased expression of enterocyte markers, decreased expression of cycling genes, and, unexpectedly, increased expression of LGR5 associated genes. No activating mutations in KIT were detected in DLD1, POP77, or UM-COLON#8 cells. However, KITLG-knockdown DLD1 cells formed smaller xenograft tumors than control cells. Gene expression analysis of single CD44(+) cells indicated that KIT can promote growth via KITLG autocrine and/or paracrine signaling. Imatinib inhibited growth of KIT(+) colon cancer organoids in culture and growth of xenograft tumors in mice. Cancer cells with endogenous KIT expression were more tumorigenic in mice. KIT and KITLG are expressed by a subset of human colon tumors. KIT signaling promotes growth of colon cancer cells and organoids in culture and xenograft tumors in mice via its ligand, KITLG, in an autocrine or paracrine manner. Patients with KIT-expressing colon tumors can benefit from KIT RTK inhibitors.

MicroRNA-185 suppresses growth and invasion of colon cancer cells through inhibition of the hypoxia-inducible factor-2α pathway in vitro and in vivo

MicroRNAs (miRs) are small non‑coding RNAs with regulatory roles, which are involved in a broad spectrum of physiological and pathological processes, including cancer development and progression. However, the function of miR‑185 in the development of human colon cancer has not yet been investigated. In this study, the association between miR‑185 expression and the clinicopathological characteristics of patients with colon cancer was analyzed using quantitative polymerase chain reaction (qPCR). Using a gain‑of‑function approach, the effects of miR‑185 overexpression on the expression of hypoxia‑inducible factor‑2α (HIF‑2α), proliferating cell nuclear antigen (PCNA) and matrix metallopeptidase‑2 (MMP‑2) were investigated in SW620 colon cancer cells using qPCR and western blotting. Functional analysis of cellular proliferative activities, by MTT assay, and invasive potential, by Transwell assay, was conducted on SW620 cells expressing low levels of miR‑185. miR‑185 was found to be significantly downregulated in cancer tissues compared with adjacent non‑cancerous tissues, and was negatively correlated with lymph node metastasis of colon cancer (P<0.001). miR‑185 overexpression invitro impeded cellular proliferation and invasive potential with reduced expression of HIF‑2α, PCNA and MMP‑2 in SW620 cells transfected with an miR‑185 mimic. In addition, the tumor volumes in SW620 subcutaneous nude mouse models treated with miR‑185 were significantly smaller than those of the control group. In conclusion, these findings indicate that miR‑185 as a tumor suppressor may affect the development of colon cancer cells via inhibition of HIF‑2α signaling, suggesting that miR‑185 may serve as a potential therapeutic target in cancer treatment.

Esculetin Suppresses Proliferation of Human Colon Cancer Cells by Directly Targeting β-Catenin

The Wnt pathway is a promising therapeutic and preventive target in various human cancers. The transcriptional complex of β-catenin-T-cell factor (Tcf), a key mediator of canonical Wnt signaling, has been implicated in human colon cancer development. Current treatment of colon cancer depends on traditional cytotoxic agents with limited effects. Therefore, the identification of natural compounds that can disrupt the β-catenin-TcF complex to suppress cancer cell growth with fewer adverse side effects is needed. To identify compounds that inhibit the association between β-catenin and Tcf, we used computer docking to screen a natural compound library. Esculetin, also known as 6,7-dihydroxycoumarin, is a derivative of coumarin and was identified as a potential small-molecule inhibitor of the Wnt-β-catenin pathway. We then evaluated the effect of esculetin on the growth of various human colon cancer cell lines and its effect on Wnt-β-catenin signaling in cells and in an embryonic model. Esculetin disrupted the formation of the β-catenin-Tcf complex through direct binding with the Lys312, Gly307, Lys345, and Asn387 residues of β-catenin in colon cancer cells. In addition, esculetin effectively decreased viability and inhibited anchorage-independent growth of colon cancer cells. Esculetin potently antagonized the cellular effects of β-catenin-dependent activity, and in vivo treatment with esculetin suppressed tumor growth in a colon cancer xenograft mouse model. Our data indicate that the interaction between esculetin and β-catenin inhibits the formation of the β-catenin-Tcf complex, which could contribute to esculetin's positive therapeutic and preventive effects against colon carcinogenesis.

FXR silencing in human colon cancer by DNA methylation and KRAS signaling

Farnesoid X receptor (FXR) is a bile acid nuclear receptor described through mouse knockout studies as a tumor suppressor for the development of colon adenocarcinomas. This study investigates the regulation of FXR in the development of human colon cancer. We used immunohistochemistry of FXR in normal tissue (n = 238), polyps (n = 32), and adenocarcinomas, staged I-IV (n = 43, 39, 68, and 9), of the colon; RT-quantitative PCR, reverse-phase protein array, and Western blot analysis in 15 colon cancer cell lines; NR1H4 promoter methylation and mRNA expression in colon cancer samples from The Cancer Genome Atlas; DNA methyltransferase inhibition; methyl-DNA immunoprecipitation (MeDIP); bisulfite sequencing; and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) knockdown assessment to investigate FXR regulation in colon cancer development. Immunohistochemistry and quantitative RT-PCR revealed that expression and function of FXR was reduced in precancerous lesions and silenced in a majority of stage I-IV tumors. FXR expression negatively correlated with phosphatidylinositol-4, 5-bisphosphate 3 kinase signaling and the epithelial-to-mesenchymal transition. The NR1H4 promoter is methylated in ~12% colon cancer The Cancer Genome Atlas samples, and methylation patterns segregate with tumor subtypes. Inhibition of DNA methylation and KRAS silencing both increased FXR expression. FXR expression is decreased early in human colon cancer progression, and both DNA methylation and KRAS signaling may be contributing factors to FXR silencing. FXR potentially suppresses epithelial-to-mesenchymal transition and other oncogenic signaling cascades, and restoration of FXR activity, by blocking silencing mechanisms or increasing residual FXR activity, represents promising therapeutic options for the treatment of colon cancer.

TRF2 inhibits a cell-extrinsic pathway through which natural killer cells eliminate cancer cells

Dysfunctional telomeres suppress tumour progression by activating cell-intrinsic programs that lead to growth arrest. Increased levels of TRF2, a key factor in telomere protection, are observed in various human malignancies and contribute to oncogenesis. We demonstrate here that a high level of TRF2 in tumour cells decreased their ability to recruit and activate natural killer (NK) cells. Conversely, a reduced dose of TRF2 enabled tumour cells to be more easily eliminated by NK cells. Consistent with these results, a progressive upregulation of TRF2 correlated with decreased NK cell density during the early development of human colon cancer. By screening for TRF2-bound genes, we found that HS3ST4--a gene encoding for the heparan sulphate (glucosamine) 3-O-sulphotransferase 4--was regulated by TRF2 and inhibited the recruitment of NK cells in an epistatic relationship with TRF2. Overall, these results reveal a TRF2-dependent pathway that is tumour-cell extrinsic and regulates NK cell immunity.

Abstract 3561: Silencing of farnesoid X receptor in human colon cancer by epigenetic mechanisms is associated with cancer progression.

Abstract Background: Colon cancer is the third leading cause of cancer related deaths in the United States. Epidemiological studies suggest an increase in the intestinal bile-acid load resulting from the consumption of a high-fat diet is a significant risk factor for colon cancer. Bile acids are the endogenous ligands for the farnesoid X receptor (FXR), a ligand-activated transcription factor and member of the nuclear receptor superfamily, and high levels of bile acids can promote colon cancer development. FXR is essential for maintaining bile-acid homeostasis by regulating bile-acid synthesis and transport, preventing the accumulation of intestinal bile acid levels to cancer promoting levels. Previous studies have demonstrated that FXR knockout mice are more susceptible to the development of colon adenocarcinomas, indicating that FXR plays a suppressive role in colon tumor formation. This study investigates the role of FXR in the development of human colon cancer. Methods: Immunohistochemistry was used to label for FXR in normal human colon, colon polyps, and colon adenocarcinomas staged I-IV. SYBR green quantitative PCR and western blot analysis were used to measure expression of FXR and FXR target genes in normal human colon and colon cancers staged I-IV as well as colon cancer cell lines. Reverse phase protein array on colon cancer cell line lysates was used to correlate FXR expression with oncogenic signaling cascades. To test if FXR expression was suppressed by DNA methylation, colon cancer cell lines were treated with a DNA methyltransferase (DNMT) inhibitor and DNMT siRNA and FXR mRNA measured by real-time PCR. Immunoprecipitation with an antibody against 5-methylcytosine (MeDIP) analysis was done in human colon cancer cell lines to determine methylation of NR1H4 (gene encoding FXR) promoter. Results: IHC and qPCR analysis reveals that the expression and function of FXR is markedly reduced early in colon cancer progression, with suppression seen within precancerous lesions. Furthermore, FXR expression in colon cancer cell lines were negatively correlated to oncogenic PI3 kinase signaling cascades and associated with epithelial to mesenchymal transition (EMT). Results suggest DNA methylation as a mechanism of FXR silencing in colon cancer and confirms methylation of the FXR promoter. Conclusion: FXR deficiency in animals indicates FXR serves a tumor suppressive role. Our studies show that FXR is silenced in early in human colon cancer progression possibly by DNA methylation, which could be a cancer promoting event. The overall mechanism of FXR's anti-tumorigenic activity is not fully established but may be due to FXR's role in regulating EMT and bile acid homeostasis. Restoration and enhancement of FXR activity, by blocking DNA methylation or increasing baseline activity of FXR, represents a potential therapeutic option for the treatment of colon cancer. Citation Format: Ann M. Thomas, Le Zhan, Julie Izzo, Dipen Maru, Imad Shureiqi, Veera Baladandayuthapani, Han Liang, Grace L. Guo, Garth Powis. Silencing of farnesoid X receptor in human colon cancer by epigenetic mechanisms is associated with cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3561. doi:10.1158/1538-7445.AM2013-3561

IL-22 is related to development of human colon cancer by activation of STAT3

BackgroundIt has been previously reported that IL-22, one of the cytokines secreted by Th17 cells, demonstrates both a protective and inflammatory promotion effect in inflammatory bowel disease (IBD) through STAT3 signaling activation. We sought to investigate the role of IL-22 expression in colon cancer (CC).MethodsThe expression of IL-22 and related molecules were detected in human CC, the detail function and mechanism of IL-22 were investigated by in vivo and in vitro model.ResultsOur results demonstrated significant upregulation of IL-22 in human CC tumor infiltrated leukocytes (TILs) compared to peripheral lymphocytes. Moreover, our findings demonstrated that IL-22 expression was significantly higher in ulcerative colitis (UC) tissues versus normal colon tissues. Both IL-22 receptor α1 (IL-22RA1) and IL-23 were highly expressed in CC and UC tissues compared to normal controls. TILs exhibiting various IL-22 expression levels isolated from CC patients were demonstrated to enhance tumor growth and metastasis co-transplanted with Hct-116 cells underwent subcutaneous transplantation in mice model. Tumor growth and metastasis was promoted by STAT3 phosphorylation and upregulation of its downstream genes such as Bcl-xl, CyclinD1, and VEGF. In vitro studies confirmed the anti-apoptotic and pro-proliferation effect of IL-22 according to the BrdU cooperation assay and peroxide induced apoptosis analysis with or without the presence of IL-22.ConclusionIn this study we demonstrated that excessive IL-22 in the CC and UC microenvironment leads to tumor growth, inhibition of apoptosis, and promotion of metastasis depend on STAT3 activation.

Effect of lentivirus-mediated NOB1 gene silencing by RNA interference on proliferation and apoptosis of human colon cancer cells

To investigate the effect of NOB1 gene on proliferation and apoptosis of human colon cancer cell line RKO by RNA interference. Small interference RNA(siRNA) targeting NOB1 gene was cloned into lentivirus vector. Then the lentivirus particles expressing NOB1 short harpin RNA(shRNA) were infected into RKO cells. Real-time PCR and Western blot were performed to examine the expression of NOB1 in lentivirus infected cells. The Thermo Scientific Cellomics ArrayScan VTI HCS Reader was used to test the proliferation and colony-formation of RKO cells, and flow cytometry assay was performed to detect cell cycle and apoptosis. Xenograft tumor was established by injection of RKO cells into nude mice, then NOB1-shRNA was injected into the tumor and tumor volume was detected. Compared to negative controls, the expression levels of NOB1 mRNA and protein were both significantly down-regulated, the proliferation and colony-forming capacity of RKO cells were significantly inhibited, and cell apoptosis was increased after 3 days of NOB1-shRNA lentivirus infection(all P<0.05). The tumor volume was significantly smaller in NOB1-shRNA group than that in Scr-shRNA group[(405±102) mm(3) vs.(870±165) mm(3), P<0.05]. Silencing NOB1 gene by RNA interference may provide an inhibitive effect on human colon cancer development.

Abstract LB-296: Allikrein 14 induces epithelial-mesenchymal transition-like changes in colon cancer cells

Abstract Accumulating evidence indicates that serine proteinase family of the kallikrein-related-peptidases (KLK1-KLK15) are deregulated in cancer and their up-regulation is often associated with poor patient prognosis. Although primarily known as cancer biomarkers, recent evidence implicates KLKs in many cancer-related processes, including cell-growth, angiogenesis, invasion and metastasis. In addition, KLKs have emerged as signalling molecules by selectively targeting members of a unique subfamily of cell surface G-protein coupled receptors termed, proteinase-activated receptors (PARs). PARs are now considered important players in the development of human colon cancer. Indeed, we have previously demonstrated that in colonic tumours, upregulated PAR-2 and aberrantly expressed PAR-1 and PAR-4 control human colon cancer cell migration and proliferation. Recently, we showed that some members of the aberrantly expressed KLKs mediate these responses by signalling of through PARs. We hypothesized that KLK14 induced-cell migration and colonic tumour invasion are the consequence of epithelial-mesenchymal transition. We focused on KLK14 for which morphological changes were observed when colon cancer cells were treated with KLK14. We found that: 1) microscopic analysis showed redistribution of perijunctional F-actin in KLK14 treated HT29 cells, a colon cancer derived adenocarcinmoma; 2) HT29 cell treatment with KLK14 induced loss of E-cadherin-mediated cell-cell adhesion; 3) Biochemical studies showed down regulation of epithelial markers and upregulation of mesenchymal markers, the molecular mechanisms involved are under investigation. These results suggest for the first time that some KLKs are key mediators of induced ETM in human colonic tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-296. doi:1538-7445.AM2012-LB-296

Peroxisome proliferator‐activated receptor‐gamma ligand suppressed leptin‐mediated expression of interleukin‐8 and tumor angiogenesis in human colon cancer cells

Colorectal cancer is one of the leading causes of cancer death in western countries. Previous studies have demonstrated that overexpression of leptin is strongly associated with risk of human colon cancer. However, the effect of leptin on tumor angiogenesis during the development of human colon cancer has not been demonstrated well yet. The present study would investigate the role of peroxisome proliferators activated receptors (PPAR)‐gamma ligand in leptin–mediated expression of interleukin‐8 (IL‐8) and tumor angiogenesis in human colon cancer cells. Our results showed that leptin dose‐dependently (0, 0.0625, 0.25, 1 μM) induced the expression of IL‐8 in human colon cancer HT‐29 cells. Conditioned medium from leptin‐stimulated human colon cancer HT‐29 cells would also induce tubular formation in human umbilical vein endothelial cells (HUVEC). Treatment of Troglitazone (0.1 and 5μM), a specific PPAR‐ γ agonist, would suppress leptin‐mediated expression of IL‐8 in human colon cancer HT‐29 cells up to 9% and 38%, respectively. Furthermore, troglitazone–treated conditioned medium from cancer cells would suppress tubular formation in HUVEC. Take together; our results suggest that PPAR‐γ ligand could act as an important chemo‐preventive agent to suppress leptin‐mediated IL‐8 expression and tumor angiogenesis in human colon cancer.Grant Funding Source: National Science Council grant under agreement No. 97‐2320‐B‐039‐043‐MY3,

The cell adhesion nectin‐like molecules (Necl) 1 and 4 suppress the growth and tumorigenic ability of colon cancer cells

A key step in human colon cancer development includes the hyperactivation of Wnt/beta-catenin signaling and the induction of beta-catenin-TCF target genes that participate in colon cancer progression. Recent studies identified members of the immunoglobulin-like cell adhesion molecules (IgCAM) of the L1CAM family (L1 and Nr-CAM) as targets of beta-catenin-TCF signaling in colon cancer cells. L1 was detected at the invasive front of colon cancer tissue and confers metastasis when overexpressed in cells. In contrast to L1, we did not detect in colon cancer cells significant levels of another IgCAM family of molecules, the nectin-like (Necl) receptors Necl1 and Necl4, while Necl4 was previously found in the normal small intestine and colon tissues. We studied the properties of colon cancer cells in which Necl4 and Necl1 were expressed either alone, or in combination, and found that such cells display a wide range of properties associated with tumor suppression. Expression of both Necl1 and Necl4 was the most efficient in suppressing the tumorigenicity of colon cancer cells. This was associated with enhanced rates of apoptosis and change in several apoptosis-related markers. In contrast to its capacity to suppress tumorigenesis, Necl4 was unable to affect the highly malignant and metastatic capacities of colon cancer cells in which L1 was overexpressed. Our results suggest that various IgCAM receptor families play different roles in affecting the tumorigenic function of the same cells, and that Necl1 and Necl4 can fulfill a tumor suppressive role.