Proteomics identification of ITGB3 as a key regulator in reactive oxygen species-induced migration and invasion of colorectal cancer cells

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and second in females worldwide. Unfortunately 40-50% of patients already have metastatic disease at presentation when prognosis is poor with a 5-year survival of <10%. Reactive oxygen species (ROS) have been proposed to play a crucial role in tumor metastasis. We now show that higher levels of ROS accumulation are found in a colorectal cancer-derived metastatic cell line (SW620) compared with a cell line (SW480) derived from the primary lesion from the same patient. In addition, ROS accumulation can affect both the migratory and invasive capacity of SW480 and SW620 cells. To explore the molecular mechanism underlying ROS-induced migration and invasion in CRC, we have compared protein expression patterns between SW480 and SW620 cells using a two-dimensional electrophoresis-based proteomics strategy. A total of 63 altered proteins were identified from tandem MS analysis. Cluster analysis revealed dysregulated expression of multiple redox regulative or ROS responsive proteins, implicating their functional roles in colorectal cancer metastasis. Molecular and pathological validation demonstrated that altered expression of PGAM1, GRB2, DJ-1, ITGB3, SOD-1, and STMN1 was closely correlated with the metastatic potential of CRC. Functional studies showed that ROS markedly up-regulated expression of ITGB3, which in turn promoted an aggressive phenotype in SW480 cells, with concomitant up-regulated expression of STMN1. In contrast, knockdown of ITGB3 expression could mitigate the migratory and invasive potential of SW620 or H(2)O(2)-treated SW480 cells, accompanied by down-regulated expression of STMN1. The function of ITGB3 was dependent on the surface expression of integrin αvβ3 heterodimer. Furthermore, STMN1 expression and the PI3K-Akt-mTOR pathway were found to be involved in ROS-induced and ITGB3-mediated migration and invasion of colorectal cancer cells. Taken together, these studies suggest that ITGB3 plays an important role in ROS-induced migration and invasion in CRC.     

Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells

Silibinin, a flavonolignan isolated from the milk thistle plant (Silybum marianum), possesses anti-neoplastic properties. In vitro and in vivo studies have recently shown that silibinin inhibits the growth of colorectal cancer (CRC). The present study investigates the mechanisms of silibinin-induced cell death using an in vitro model of human colon cancer progression, consisting of primary tumor cells (SW480) and their derived metastatic cells (SW620) isolated from a metastasis of the same patient. Silibinin induced apoptotic cell death evidenced by DNA fragmentation and activation of caspase-3 in both cell lines. Silibinin enhanced the expression (protein and mRNA) of TNF-related apoptosis-inducing ligand (TRAIL) death receptors (DR4/DR5) at the cell surface in SW480 cells, and induced their expression in TRAIL-resistant SW620 cells normally not expressing DR4/DR5. Caspase-8 and -10 were activated demonstrating the involvement of the extrinsic apoptotic pathway in silibinin-treated SW480 and SW620 cells. The protein Bid was cleaved in SW480 cells indicating a cross-talk between extrinsic and intrinsic apoptotic pathway. We demonstrated that silibinin activated also the intrinsic apoptotic pathway in both cell lines, including the perturbation of the mitochondrial membrane potential, the release of cytochrome c into the cytosol and the activation of caspase-9. Simultaneously to apoptosis, silibinin triggered an autophagic response. The inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in silibinin-treated cells. Taken together, our data show that silibinin initiated in SW480 and SW620 cells an autophagic-mediated survival response overwhelmed by the activation of both the extrinsic and intrinsic apoptotic pathways.     

Anti-colorectal cancer activity of macrostemonoside A mediated by reactive oxygen species

Macrostemonoside A (MSS.A), an active steroidal saponin from Allium macrostemon Bung has been shown to possess anti-coagulation and anti-obesity effects. However, the functional role of MSS.A on tumor growth has not been elucidated. We found that MSS.A significantly inhibited human colorectal cancer cell growth in Caco2 and SW480 cells. Incubation of SW480 cells with MSS.A for 48 h resulted in cell cycle arrest. Moreover, MSS.A dose-dependently induced apoptosis in SW480 cells as shown by increased AnnexinV positively stained cell population, caspase activation, increased pro-apoptotic and reduced anti-apoptotic Bcl-2 family protein levels. Treatment of SW480 cells with MSS.A resulted in increased reactive oxygen species (ROS) generation. However, pre-incubation of SW480 cells with antioxidant N-acetylcysteine (NAC) attenuated the ROS generation and anti-colorectal cancer activities of MSS.A. Lastly, intra-peritoneal injections of MSS.A significantly inhibited tumor formation in BALB/c nude mice carcinogenesis xenograft model by reduced tumor volume and tumor weight when treated at dosages of 10, 50 or 100 mg/kg daily for 35 days compared with PBS control. Taken together, our results indicate that MSS.A suppressed colorectal cancer growth and induced cell apoptosis by inducing ROS production, and that MSS.A may have therapeutic relevance in the treatment of human colorectal cancer.     

Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model

It is known that cancer progresses by vertical gene transfer, but this paradigm ignores that DNA circulates in higher organisms and that it is biologically active upon its uptake by recipient cells. Here we confirm previous observations on the ability of cell-free DNA to induce in vitro cell transformation and tumorigenesis by treating NIH3T3 recipient murine cells with serum of colon cancer patients and supernatant of SW480 human cancer cells. Cell transformation and tumorigenesis of recipient cells did not occur if serum and supernatants were depleted of DNA. It is also demonstrated that horizontal cancer progression mediated by circulating DNA occurs via its uptake by recipient cells in an in vivo model where immunocompetent rats subjected to colon carcinogenesis with 1,2-dimethylhydrazine had increased rate of colonic tumors when injected in the dorsum with human SW480 colon carcinoma cells as a source of circulating oncogenic DNA, which could be offset by treating these animals with DNAse I and proteases. Though the contribution of biologically active molecules other than DNA for this phenomenon to occur cannot be ruled out, our results support the fact that cancer cells emit into the circulation biologically active DNA to foster tumor progression. Further exploration of the horizontal tumor progression phenomenon mediated by circulating DNA is clearly needed to determine whether its manipulation could have a role in cancer therapy.     

Matrine promotes apoptosis in SW480 colorectal cancer cells via elevating MIEF1-related mitochondrial division in a manner dependent on LATS2-Hippo pathway

Matrine, an alkaloid compound isolated from Sophora flavescens Ait, has been shown to exert cancer-killing actions in a variety of tumors; however, its anticancer mechanism in colorectal cancer (CRC) is not clear. The goal of our study was to characterize the anticancer effects and molecular mechanisms of matrine in SW480 CRC cells in vitro. Matrine treatment reduced mitochondrial metabolic function and ATP levels, repressed mitochondrial membrane potential, evoked mitochondrial reactive oxygen species accumulation, and promoted cyt-c-related mitochondrial apoptosis activation. In addition, we found that matrine treatment activated mitochondrial fission through upregulating mitochondrial elongation factor 1 (MIEF1); silencing of MIEF1 prevented matrine-mediated mitochondrial damage and reversed the decrease in SW480 cell viability. Moreover, matrine treatment affected MIEF1 expression via the large tumor suppressor-2 (LATS2)-Hippo axis, and LATS2 deficiency suppressed the anticancer actions exerted by matrine on SW480 cancer cells. In summary, we show for the first time that matrine inhibits SW480 cell survival by activating MIEF1-related mitochondrial division via the LATS2-Hippo pathway. These findings explain the anticancer mechanisms of matrine in CRC and also identify the LATS2-MIEF1 signaling pathway as an effective target for the treatment of CRC.     

Mediatory role of BLT2 in the proliferation of KRAS mutant colorectal cancer cells

Inflammatory lipid mediators play various roles in colorectal cancer progression through complex pathways. However, the mechanism by which lipoxygenase-derived inflammatory lipid mediators contribute to colorectal cancer progression remains elusive. In this study, we found that BLT2, a cell surface GPCR for leukotriene B₄ and 12‑hydroxyeicosatetraenoic acid, is highly upregulated in KRAS mutant LOVO and SW480 colorectal cancer cells and plays critical roles in mediating proliferation through activation of phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (Akt) and subsequent upregulation of cyclin D1. Exposure to BLT2 siRNA or LY255283, a specific BLT2 inhibitor, clearly suppressed the proliferation of KRAS mutant colorectal cancer cells and markedly increased cell cycle arrest by downregulating the PI3K/Akt-cyclin D1 cascade. Xenograft tumor formation by LOVO and SW480 cells in athymic mice was also substantially reduced by treatment with the BLT2 inhibitor in vivo. Together, our study demonstrates that BLT2 is necessary for the proliferation of LOVO and SW480 cells and thus may be a potential therapeutic target for the treatment of KRAS mutant colorectal cancer.     

Lupulone, a hop bitter acid, activates different death pathways involving apoptotic TRAIL-receptors, in human colon tumor cells and in their derived metastatic cells

Our study aimed to compare death signalling pathways triggered by lupulone in TRAIL-sensitive human colon cancer cells (SW480) and in their derived TRAIL-resistant metastatic cells (SW620). Lupulone (40 μg/ml) up-regulated expression of TRAIL DR4/DR5 death receptors at the cell surface of both cell lines, even in the absence of exogenous TRAIL ligand. Cell death induced by lupulone was inhibited in SW480 and SW620 cells exposed to blocking anti-DR4/DR5 antibodies. In SW480 cells, lupulone triggered cell death through a cross-talk between TRAIL-DR4/DR5 and the mitochondrial (intrinsic) pathways involving caspase-8 activation and Bid protein cleavage. As a consequence mitochondrial cytochrome c was released into the cytosol and activation of caspases-9 and -3 was observed. In the metastatic SW620 cells, lupulone restored the sensibility of these cells to TRAIL ligand and activated the extrinsic apoptotic pathway via DR4/DR5 death receptors and the involvement of the caspase-8/caspase-3 cascade. The demonstration that lupulone is able to activate TRAIL-death signalling pathways even in TRAIL resistant cancer cells highlights the potential of this natural compound for cancer prevention and therapy.     

Eps8 facilitates cellular growth and motility of colon cancer cells by increasing the expression and activity of focal adhesion kinase

In an attempt to study the role of Eps8 in human carcinogenesis, we observe that ectopic overexpression of Eps8 in SW480 cells (low Eps8 expression) increases cell proliferation. By contrast, expressing eps8 small interference RNA in SW620 and WiDr cells (high Eps8 expression) reduces their proliferation rate. Interestingly, attenuation of Eps8 decreases Src Pi-Tyr-416, Shc Pi-Tyr-317, and serum-induced FAK Pi-Tyr-397 and Pi-Tyr-861. Remarkably, by virtue of mammalian target of rapamycin/STAT3 Pi-Ser-727, Eps8 modulates FAK expression required for cell proliferation. Within 62% of colorectal tumor specimens examined, >2-fold enhancement of Eps8 as compared with their normal counterparts is observed, especially for those from the advanced stage. In agreement with the modulation of FAK by Eps8, the concomitant expression of these two proteins in tumor specimens is observed. Notably, Eps8 attenuation also impedes the motility of SW620 and WiDr cells, which can be rescued by ectopically expressed FAK. This finding discloses the indispensability of Eps8 and FAK in cell locomotion. These results provide a novel mechanism for Eps8-mediated FAK expression and activation in colon cancer cells.     

STAT3-siRNA对人结直肠癌细胞的干涉作用

目的:研究STAT3-siRNA对STAT3基因表达阳性的结直肠癌细胞凋亡的影响。方法:应用脂质体转染试剂将STAT3-siRNA表达盒(STAT3-siRNA expression cassettes,STAT3-SECs)体外转染至人结直肠癌SW480细胞及人成纤维细胞中,同时分别设立人成纤维对照组、SW480对照组、SW480错配链-SECs组和SW480空转染试剂组。于48h后收集细胞,先经荧光染色方法观察细胞表象变化,再通过流式细胞仪检测人结直肠癌SW480细胞凋亡情况,后分别提取细胞总RNA,用RT-PCR测定STAT3基因在mRNA水平的表达。结果:SW480STAT3-SECs组的细胞可见凋亡小体,出现明显的凋亡现象,而人成纤维对照组、人成纤维STAT3-SECs组、SW480对照组、SW480错配链-SECs组和SW480空转染试剂组未出现明显的凋亡现象。SW480STAT3-SECs组细胞的凋亡比率较SW480对照组、SW480错配链-SECs组和SW480空转染试剂组有明显的增高。RT-PCR所得数据经统计学处理得出:SW480STAT3-SECs组细胞的STAT3基因表达在mRNA水平上显著低于SW480对照组(P0.01);而人成纤维对照组与人成纤维STAT3-SECs组,SW480细胞对照组与SW480错配链-SECs组、SW480空转染试剂组之间无明显差异(P0.05)。结论:应用RNAi技术沉默STAT3基因可以降低人结直肠癌SW480细胞中STAT3的表达,诱导细胞的凋亡。     

Nogo-B (Reticulon-4B) functions as a negative regulator of the apoptotic pathway through the interaction with c-FLIP in colorectal cancer cells

Nogo-B is a member of the Nogo/Reticulon-4 family and has been reported to be an inducer of apoptosis in certain types of cancer cells. However, the role of Nogo-B in human cancer remains less understood. Here, we demonstrated the functions of Nogo-B in colorectal cancer cells. In clinical colorectal cancer specimens, Nogo-B was obviously overexpressed, as determined by immunohistochemistry; and Western blot analysis showed its expression level to be significantly up-regulated. Furthermore, knockdown of Nogo-B in two colorectal cancer cell lines, SW480 and DLD-1, by transfection with si-RNA (siR) resulted in significantly reduced cell viability and a dramatic increase in apoptosis with insistent overexpression of cleaved caspase-8 and cleaved PARP. The transfection with Nogo-B plasmid cancelled that apoptosis induced by siRNogoB in SW480 cells. Besides, combinatory treatment with siR-Nogo-B/staurosporine (STS) or siR-Nogo-B/Fas ligand (FasL) synergistically reduced cell viability and increased the expression of apoptotic signaling proteins in colorectal cancer cells. These results strongly support our contention that Nogo-B most likely played an oncogenic role in colorectal cancer cells, mainly by negatively regulating the extrinsic apoptotic pathway in them. Finally, we revealed that suppression of Nogo-B caused down-regulation of c-FLIP, known as a major anti-apoptotic protein, and activation of caspase-8 in the death receptor pathway. Interaction between Nogo-B and c-FLIP was shown by immunoprecipitation and immunofluorescence studies.In conclusion, Nogo-B was shown to play an important negative role in apoptotic signaling through its interaction with c-FLIP in colorectal cancer cells, and may thus become a novel therapeutic target for colorectal cancer.     

Protective effects of the ethanolic extract of Melia toosendan fruit against colon cancer

Colorectal cancer is one of the leading causes of death in the world. Plant-derived products have proven to be valuable sources for discovery and development of unique anticancer drugs. In this study, the inhibitory effects of ethanolic extract of Melia toosendan fruit (EMTF), a traditional medicine in the Chinese Pharmacopeia were evaluated in vitro and in vivo against colon cancer. Human colon cancer cells SW480 and murine colorectal adenocarcinoma cells CT26 were used to investigate cell proliferation. The results showed that EMTF inhibited cell proliferation of SW480 and CT26 by promoting apoptosis as indicated by nuclear chromatin condensation and DNA fragmentation. Through increasing mitochondrial membrane permeability and cytochrome c release from mitochondria, EMTF induced caspase-9 activity which further activated caspase-3 and poly(ADP-ribose) polymerase cleavage, leading the tumor cells to apoptosis. The in vivo results confirmed reduction of tumor volume and apoptotic effects and the side effects were not induced by EMTF. Therefore, EMTF may be an effective chemotherapeutic agent for colon cancer treatment.     

Knockdown of the inducible nitric oxide synthase (NOS2) splicing variant S3 promotes autophagic cell death from nitrosative stress in SW480 human colon cancer cells

Low levels of nitric oxide (NO) produced by constitutively expressed inducible NO synthase (NOS2) in tumor cells may be an important factor in their development. NOS2 expression is associated with high mortality rates for various cancers. Alternative splicing of NOS2 down-regulates its enzymatic activity, resulting in decreased intracellular NO concentrations. Specific probes to detect alternative splicing of NOS2 were used in two isogenic human colon cancer cell lines derived either from the primary tumor (SW480) or from a lymph node metastasis (SW620). Splicing variant of NOS2 S3, lacking exons 9, 10, and 11, was overexpressed in SW480 cells. NOS2 S3 was silenced in SW480 cells. Flow-cytometry analysis was used to estimate the intracellular NO levels and to analyze the cell cycle of the studied cell lines. Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine apoptosis and autophagy markers. SW480 and SW620 cells expressed NOS2 S3. Overexpression of the NOS2 S3 in SW480 cells downregulated intracellular NO levels. SW480 cells with knocked down NOS2 S3 (referred to as S3C9 cells) had higher intracellular levels of NO compared to the wild-type SW480 cells under serum restriction. Higher NO levels resulted in the loss of viability of S3C9 cells, which was associated with autophagy. Induction of autophagy by elevated intracellular NO levels in S3C9 cells under serum restriction, suggests that autophagy operates as a cytotoxic response to nitrosative stress. The expression of NOS2 S3 plays an important role in regulating intracellular NO production and maintaining viability in SW480 cells under serum restriction. These findings may prove significant in the design of NOS2/NO-based therapies for colon cancer.     

Expression profile analysis of colon cancer cells in response to sulindac or aspirin

Nonsteroidal anti-inflammatory drugs (NSAIDs) have a preventive effect against colorectal cancer. Although inhibition of cyclooxygenase-2 plays a crucial role in the suppression of tumors, precise mechanisms of their action remain to be disclosed. To identify genes involved in the growth-suppressive effect of NSAIDs, we utilized cDNA microarray containing 23,040 genes and analyzed time-dependent alteration of gene expression in response to sulindac or aspirin in NSAIDs-sensitive SW480 and SW948 colon-cancer cells as well as in relatively resistant SNU-C4 cells. Consequently we identified 112 genes with commonly altered expression by sulindac and 176 with commonly altered expression by aspirin in the three lines. Addition of sulindac and that of aspirin altered expression levels of 130 and 140 genes, respectively, in SW480 and SW948 cells but not in SNU-C4 cells. These data may lead to a better understanding of growth-suppressive effects on colonic epithelium, and may provide clues for identifying novel therapeutic and/or preventive molecular targets of colon cancer.     

Effects of silk sericin on the proliferation and apoptosis of colon cancer cells

Sericin is a silk protein woven from silkworm cocoons (Bombyx mori). In animal model, sericin has been reported to have anti-tumoral action against colon cancer. The mechanisms underlying the activity of sericin against cancer cells are not fully understood. The present study investigated the effects of sericin on human colorectal cancer SW480 cells compared to normal colonic mucosal FHC cells. Since the size of the sericin protein may be important for its activity, two ranges of molecular weight were tested. Sericin was found to decrease SW480 and FHC cell viability. The small sericin had higher anti-proliferative effects than that of the large sericin in both cell types. Increased apoptosis of SW480 cells is associated with increased caspase-3 activity and decreased Bcl-2 expression. The anti-proliferative effect of sericin was accompanied by cell cycle arrest at the S phase. Thus, sericin reduced SW480 cell viability by inducing cell apoptosis via caspase-3 activation and down-regulation of Bcl-2 expression. The present study provides scientific data that support the protective effect of silk sericin against cancer cells of the colon and suggests that this protein may have significant health benefits and could potentially be developed as a dietary supplement for colon cancer prevention.     

Proteomic profiling reveals key cancer progression modulators in shed microvesicles released from isogenic human primary and metastatic colorectal cancer cell lines

Extracellular vesicles comprise two main classes - exosomes and shed microvesicles (sMVs). Whilst much is known about exosome cargo content and functionality, sMVs are poorly understood. Here, we describe the large-scale purification of sMVs released from primary (SW480) and metastatic (SW620) human isogenic colorectal cancer (CRC) cell lines using a combination of differential ultracentrifugation and isopycnic iodixanol density centrifugation. The yield of SW480-sMVs and SW620-sMVs was 0.75 mg and 0.80 mg, respectively. Both SW480-/SW620-sMVs are heterogeneous in size (100–600 nm diameter) and exhibit identical buoyant densities (1.10 g/mL). In contrast to exosomes, sMVs are ALIX⁻, TSG101⁻, CD63⁻ and CD9⁻. Quantitative mass spectrometry identified 1295 and 1300 proteins in SW480-sMVs and SW620-sMVs, respectively. Gene Ontology enrichment analysis identified ‘cell adhesion’ (CDH1, OCLN, CTN families), ‘signalling pathway’ (KRAS, NRAS, MAPK1, MAP2K1), and ‘translation/RNA related’ processes (EIF, RPL, HNRNP families) in both sMV types. Strikingly, SW480- and SW620-sMVs exhibit distinct protein signatures - SW480-sMVs being enriched in ITGA/B, ANXA1, CLDN7, CD44 and EGFR/NOTCH signalling networks, while SW620-sMVs are enriched in PRKCA, MACC1, FGFR4 and MTOR/MARCKS signalling networks. Both SW480- and SW620-sMVs are taken up by NIH3T3 fibroblasts resulting in similar cell invasion capability. This study provides, for the first time, molecular insights into sMVs and CRC biology.     

NOP14 regulates the growth,migration, and invasion of colorectal cancer cells by modulating the NRIP1/GSK-3β/β-catenin signaling pathway

Colorectal cancer (CRC) is the third most common cancer diagnosed worldwide. Recently, nucleolar complex protein 14 (NOP14) has been discovered to play a critical role in cancer development and progression, but the mechanisms of action of NOP14 in colorectal cancer remain to be elucidated. In this study, we used collected colorectal cancer tissues and cultured colorectal cancer cell lines (SW480, HT29, HCT116, DLD1, Lovo), and measured the mRNA and protein expression levels of NOP14 in colorectal cancer cells using qPCR and Western blotting. GFP-NOP14 was constructed and siRNA fragments against NOP14 were synthesized to investigate the importance of NOP14 for the development of colorectal cells. Transwell migration assays were used to measure cell invasion and migration, CCK-8 kits were used to measure cell activity, and flow cytometry was applied to the observation of apoptosis. We found that both the mRNA and protein levels of NOP14 were significantly upregulated in CRC tissues and cell lines. Overexpression of GFP-NOP14 markedly promoted the growth, migration, and invasion of the CRC cells HT19 and SW480, while genetic knockdown of NOP14 inhibited these behaviors. Overexpression of NOP14 promoted the expression of NRIP1 and phosphorylated inactivation of GSK-3β, leading to the upregulation of β-catenin. Genetic knockdown of NOP14 had the opposite effect on NRIP1/GSK-3/β-catenin signals. NOP14 therefore appears to be overexpressed in clinical samples and cell lines of colorectal cancer, and promotes the proliferation, growth, and metastasis of colorectal cancer cells by modulating the NRIP1/GSK-3β/β-catenin signaling pathway.Key words: Colorectal cancer, NOP14, proliferation, migration, invasion     

A diterpenoid derivative 15-oxospiramilactone inhibits Wnt/β-catenin signaling and colon cancer cell tumorigenesis

The Wnt/β-catenin signaling pathway is a highly conserved pathway in organism evolution and regulates many biological processes. Aberrant activation of the Wnt/β-catenin signaling pathway is closely related to tumorigenesis. In order to identify potent small molecules to treat the over-activated Wnt signaling-mediated cancer, such as colon cancer, we established a mammalian cell line-based reporter gene screening system. The screen revealed a diterpenoid derivative, 15-oxospiramilactone (NC043) that inhibits Wnt3a or LiCl-stimulated Top-flash reporter activity in HEK293T cells and growth of colon cancer cells, SW480 and Caco-2. Treatment of SW480 cells with NC043 led to decreases in the mRNA and/or protein expression of Wnt target genes Axin2, Cyclin D1 and Survivin , as well as decreases in the protein levels of Cdc25c and Cdc2. NC043 did not affect the cytosol-nuclear distribution and protein level of soluble β-catenin, but decreased β-catenin/TCF4 association in SW480 cells. Moreover, NC043 inhibited anchorage-independent growth and xenograft tumorigenesis of SW480 cells. Collectively these results demonstrate that NC043 is a novel small molecule that inhibits canonical Wnt signaling downstream of β-catenin stability and may be a potential compound for treating colorectal cancer.