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Wang F.,Tongji University | Ma Y.-L.,Tongji University | Zhang P.,Tongji University | Shen T.-Y.,Tongji University | And 7 more authors.
Journal of Pathology | Year: 2013

Although recent studies indicate that DNA methylation contributes to the down-regulation of microRNAs (miRNAs) in colorectal cancer (CRC), this field remains largely unexplored. To identify methylation-silenced miRNAs and clarify their role in CRC, we performed a microarray analysis and screened for miRNAs that were induced in CRC cells by 5-aza-2′-deoxycytidine treatment or by the knockdown of DNA methyltransferases. The DNA methylation status of the candidate miRNA was analysed by bisulphite sequencing PCR and methylation-specific PCR. We found that miRNA-149 (miR-149) was epigenetically silenced in CRC and down-regulation of miR-149 was associated with hypermethylation of the neighbouring CpG island (CGI). Quantitative RT-PCR analysis demonstrated that the miR-149 level was markedly reduced in 51.6% of the CRC tissues compared with matched non-cancerous tissues. In addition, low expression of miR-149 was associated with a greater depth of invasion p = 0.012, lower 5-year survival rate p = 0.025, and was found to be an independent prognostic factor for overall survival p = 0.016 in a multivariate analysis. Moreover, transfection of miR-149 inhibited cell growth and invasion of CRC cells in vitro. We also identified mRNA for Specificity Protein 1 (SP1, Sp1), a potential oncogenic protein, as a target of miR-149. Our data suggest that, as a methylation-sensitive miRNA, miR-149 may play an important role as a tumour suppressor in CRC, which has prognostic and therapeutic implications. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Wang F.,Shanghai JiaoTong University | Yang Y.-Z.,Shanghai JiaoTong University | Shi C.-Z.,Shanghai JiaoTong University | Zhang P.,Shanghai JiaoTong University | And 4 more authors.
Annals of Surgical Oncology | Year: 2012

Purpose: To investigate whether ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1) expression is upregulated in colorectal cancer (CRC), whether UHRF1 promotes CRC cell growth and migration and the underlying molecular mechanism. Methods: UHRF1 protein expression was determined in 144 pairs of primary CRC and their corresponding adjacent nontumor tissues by immunohistochemistry with tissue microarrays. UHRF1 mRNA expression was assessed in 20 pairs of the above tissues and four colon cancer cell lines by quantitative reverse transcriptase-polymerase chain reaction. Associations of UHRF1 expression with demographic and clinicopathologic features were determined. Additionally, the effects of lentiviral-mediated RNA interference (RNAi) of UHRF1 on cell proliferation and migration, cell cycle and apoptosis, and the expression of p16ink4a and p21waf1/cip1 were investigated in CRC cell lines. Results: UHRF1 was overexpressed in CRC tissues and cell lines. UHRF1 protein expression levels correlated with the presence of lymph nodes (P = 0.005), distal metastasis (P = 0.030), poor Dukes staging (P = 0.001), and absence of p16ink4a expression (P = 0.002). RNAi of UHRF1 inhibited proliferation and migration, and induced apoptosis and cell cycle arrest at the G0/G1 phase. Furthermore, RNAi of UHRF1 enhanced the expression of p16ink4a, but not p21waf1/cip1, in CRC cells. Conclusions: UHRF1 expression is upregulated in CRC and is associated with the progression of CRC. Moreover, RNAi of UHRF1 decreases proliferation and migration but enhances apoptosis of CRC cells, with increased p16 ink4a expression. UHRF1 promotes CRC growth and metastasis, likely by repressing p16ink4a, and thus it may be used as a biomarker or even a therapeutic target for CRC. © 2012 Society of Surgical Oncology.


Ma Y.,University of Texas M. D. Anderson Cancer Center | Yang Y.,Tongji University | Wang F.,University of Texas M. D. Anderson Cancer Center | Moyer M.-P.,INCELL Corporation | And 8 more authors.
Gut | Year: 2015

Objective Long non-coding RNAs (lncRNAs) are emerging as key molecules in cancers, yet their potential molecular mechanisms are not well understood. The objective of this study is to examine the expression and functions of lncRNAs in the development of colorectal cancer (CRC). Methods LncRNA expression profiling of CRC, adenoma and normal colorectal tissues was performed to identify tumour-related lncRNAs involved in colorectal malignant transformation. Then, we used quantitative reverse transcription PCR assays to measure the tumour-related lncRNA and to assess its association with survival and response to adjuvant chemotherapy in 252 patients with CRC. The mechanisms of CCAL function and regulation in CRC were examined using molecular biological methods. Results We identified colorectal cancer-associated lncRNA (CCAL) as a key regulator of CRC progression. Patients whose tumours had high CCAL expression had a shorter overall survival and a worse response to adjuvant chemotherapy than patients whose tumours had low CCAL expression. CCAL promoted CRC progression by targeting activator protein 2α (AP-2α), which in turn activated Wnt/β-catenin pathway. CCAL induced multidrug resistance (MDR) through activating Wnt/β-catenin signalling by suppressing AP-2a and further upregulating MDR1/P-gp expression. In addition, we found that histone H3 methylation and deacetylases contributed to the upregulation of CCAL in CRC. Conclusions Our results suggest that CCAL is a crucial oncogenic regulator involved in CRC tumorigenesis and progression. © 2015 BMJ Publishing Group Ltd & British Society of Gastroenterology.


Rey O.,University of California at Los Angeles | Young S.H.,University of California at Los Angeles | Jacamo R.,University of Texas M. D. Anderson Cancer Center | Moyer M.P.,INCELL Corporation | Rozengurt E.,University of California at Los Angeles
Journal of Cellular Physiology | Year: 2010

The extracellular Ca 2+-sensing receptor (CaR) is increasingly implicated in the regulation of multiple cellular functions in the gastrointestinal tract, including secretion, proliferation and differentiation of intestinal epithelial cells. However, the signaling mechanisms involved remain poorly defined. Here we examined signaling pathways activated by the CaR, including Ca 2+ oscillations, in individual human colon epithelial cells. Single cell imaging of colon-derived cells expressing the CaR, including SW-480, HT-29, and NCM-460 cells, shows that stimulation of this receptor by addition of aromatic amino acids or by an elevation of the extracellular Ca 2+ concentration promoted striking intracellular Ca 2+ oscillations. The intracellular calcium oscillations in response to extracellular Ca 2+ were of sinusoidal pattern and mediated by the phospholipase C/diacylglycerol/inositol 1,4,5-trisphosphate pathway as revealed by a biosensor that detects the accumulation of diacylglycerol in the plasma membrane. The intracellular calcium oscillations in response to aromatic amino acids were of transient type, that is, Ca 2+ spikes that returned to baseline levels, and required an intact actin cytoskeleton, a functional Rho, Filamin A and the ion channel TRPC1. Further analysis showed that re-expression and stimulation of the CaR in human epithelial cells derived from normal colon and from colorectal adenocarcinoma inhibits their proliferation. This inhibition was associated with the activation of the signaling pathway that mediates the generation of sinusoidal, but not transient, intracellular Ca 2+ oscillations. Thus, these results indicate that the CaR can function in two signaling modes in human colonic epithelial cells offering a potential link between gastrointestinal responses and food/nutrients uptake and metabolism. © 2010 Wiley-Liss, Inc.


Zeng H.,U.S. Department of Agriculture | Trujillo O.N.,University of Arizona | Moyer M.P.,INCELL Corporation | Botnen J.H.,U.S. Department of Agriculture
Nutrition and Cancer | Year: 2011

Sulforaphane (SFN) is a naturally occurring chemopreventive agent; the induction of cell cycle arrest and apoptosis is a key mechanism by which SFN exerts its colon cancer prevention. However, little is known about the differential effects of SFN on colon cancer and normal cells. In this study, we demonstrated that SFN (15 μmol/L) exposure (72 h) inhibited cell proliferation by up to 95% in colon cancer cells (HCT116) and by 52% in normal colon mucosa-derived (NCM460) cells. Our data also showed that SFN exposure (5 and 10 μmol/L) led to the reduction of G1 phase cell distribution and an induction of apoptosis in HCT116 cells, but to a much lesser extent in NCM460 cells. Furthermore, the examination of mitogen-activated protein kinase (MAPK) signaling status revealed that SFN upregulated the phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) in NCM460 cells but not in HCT116 cells. In contrast, SFN enhanced the phosphorylation of stress-activated protein kinase (SAPK) and decreased cellular myelocytomatosis oncogene (c-Myc) expression in HCT116 cells but not NCM460 cells. Taken together, the activation of survival signaling in NCM460 cells and apoptotic signaling in HCT116 cells may play a critical role in SFN's stronger potential of inhibiting cell proliferation in colon cancer cells than in normal colon cells. Copyright © 2011, Taylor & Francis Group, LLC.


Zeng H.,U.S. Department of Agriculture | Briske-Anderson M.,U.S. Department of Agriculture | Wu M.,University of North Dakota | Moyer M.P.,INCELL Corporation
Nutrition and Cancer | Year: 2012

Methylselenol is hypothesized to be a critical selenium metabolite for anticancer action, and differential chemopreventive effects of methylselenol on cancerous and noncancerous cells may play an important role. In this study, the submicromolar concentrations of methylselenol were generated by incubating methionase with seleno-L methionine, and colon-cancer-derived HCT-116 cells and noncancerous colon NCM460 cells were exposed to methylselenol. Methylselenol exposure inhibited cell growth and led to an increase in G1 and G2 fractions with a concomitant drop in S-phase and an induction of apoptosis in HCT116, but to a much lesser extent in NCM460 colon cells. Similarly, the examination of mitogen-activated protein kinase (MAPK) and cellular myelocytomatosis oncogene (c-Myc) signaling status revealed that methylselenol inhibited the phosphorylation of extracellular-regulated kinase1/2 and p38 mitogen-activated protein kinase and the expression of c-Myc in HCT116 cells, but also to a lesser extent in NCM460 cells. The other finding is that methylselenol inhibits sarcoma kinase phosphorylation in HCT116 cells. In contrast, methylselenol upregulated the phosphorylation of sarcoma and focal adhesion kinase survival signals in the noncancerous NCM460 cells. Collectively, methylselenol's stronger potential of inhibiting cell proliferation/survival signals in the cancerous HCT116 cells when compared with that in noncancerous NCM460 cells may partly explain the potential of methylselenol's anticancer action. © 2012 Copyright Taylor and Francis Group, LLC.


Peng H.,Cedars Sinai Medical Center | Li T.W.H.,Cedars Sinai Medical Center | Yang H.,Cedars Sinai Medical Center | Yang H.,University of Southern California | And 4 more authors.
American Journal of Pathology | Year: 2015

Methionine adenosyltransferase 2B (MAT2B) encodes for variant proteins V1 and V2 that interact with GIT1 to increase ERK activity and growth in human liver and colon cancer cells. MAT2B or GIT1 overexpression activates MEK. This study explores the mechanism for MEK activation. We examined protein-protein interactions by co-immunoprecipitation and verified by confocal microscopy and pull-down assay using recombinant or in vitro translated proteins. Results were confirmed in an orthotopic liver cancer model. We found that MAT2B and GIT1-mediated MEK1/2 activation was not mediated by PAK1 or Src in HepG2 or RKO cells. Instead, MAT2B and GIT1 interact with B-Raf and c-Raf and enhance recruitment of Raf proteins to MEK1/2. MAT2B-GIT1 activates c-Raf, which is the key mediator for MEK/12 activation, because this still occurred in RKO cells that express constitutively active B-Raf mutant. The mechanism lies with the ability of MAT2B-GIT1 to activate Ras and promote B-Raf/c-Raf heterodimerization. Interestingly, MAT2B but not GIT1 can directly interact with Ras, which increases protein stability. Finally, increased Ras-Raf-MEK signaling occurred in phenotypically more aggressive liver cancers overexpressing MAT2B variants and GIT1. In conclusion, interaction between MAT2B and GIT1 serves as a scaffold and facilitates signaling in multiple steps of the Ras/Raf/MEK/ERK pathway, further emphasizing the importance of MAT2B/GIT1 interaction in cancer growth. © 2015 American Society for Investigative Pathology.


Norbury K.C.,An Acelity Company | Moyer M.P.,INCELL Corporation LLC
Mediators of Inflammation | Year: 2015

In a swine model of ischemia/reperfusion injury coupled with sepsis, we have previously shown attenuation of secondary organ injury and decreased mortality with negative pressure therapy (NPT). We hypothesized that NPT modulates the intestinal microenvironment by mediating the innate immune system. Sepsis was induced in 12 anesthetized female pigs. Group 1 (n = 6) was decompressed at 12 hrs after injury (T12) and treated with standard of care (SOC), and group 2 (n = 6) with NPT for up to T48. Immunoparalysis was evident as lymphocytopenia at T24 in both groups; however, survival was improved in the NPT group versus SOC (Odds ratio = 4.0). The SOC group showed significant reduction in lymphocyte numbers compared to NPT group by T48 (p < 0.05). The capacity of peritoneal fluid to stimulate a robust reactive oxygen species response in vitro was greater for the NPT group, peaking at T24 for both M1 (p = 0.0197) and M2 macrophages (p = 0.085). Plasma elicited little if any effect which was confirmed by microarray analysis. In this septic swine model NPT appeared to modulate the intestinal microenvironment, facilitating an early robust, yet transient, host defense mediated by M1 and M2 macrophages. NPT may help overcome immunoparalysis that occurs during inflammatory response to septic injury. © 2015 Kenneth C. Norbury and Mary Pat Moyer.


Sanchez-Lopez E.,University of California at San Diego | Zimmerman T.,CSIC - Biological Research Center | Gomez Del Pulgar T.,University Hospital Fundacion Jimenez Diaz | Moyer M.P.,INCELL Corporation | And 2 more authors.
Cell Death and Disease | Year: 2013

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction. © 2013 Macmillan Publishers Limited All rights reserved.


Zhang L.,University of Texas M. D. Anderson Cancer Center | Ren X.,University of Texas M. D. Anderson Cancer Center | Alt E.,University of Texas M. D. Anderson Cancer Center | Bai X.,University of Texas M. D. Anderson Cancer Center | And 6 more authors.
Nature | Year: 2010

Cancer chemoprevention uses natural, synthetic, or biological substances to reverse, suppress, or prevent either the initial phase of carcinogenesis or the progression of neoplastic cells to cancer. It holds promise for overcoming problems associated with the treatment of late-stage cancers. However, the broad application of chemoprevention is compromised at present by limited effectiveness and potential toxicity. To overcome these challenges, here we developed a new chemoprevention approach that specifically targets premalignant tumour cells for apoptosis. We show that a deficiency in the adenomatous polyposis coli (APC) gene and subsequent activation of Β-catenin lead to the repression of cellular caspase-8 inhibitor c-FLIP (also known as CFLAR) expression through activation of c-Myc, and that all-trans-retinyl acetate (RAc) independently upregulates tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptors and suppresses decoy receptors. Thus, the combination of TRAIL and RAc induces apoptosis in APC-deficient premalignant cells without affecting normal cells in vitro. In addition, we show that short-term and non-continuous TRAIL and RAc treatment induce apoptosis specifically in intestinal polyps, strongly inhibit tumour growth, and prolong survival in multiple intestinal neoplasms C57BL/6J-Apc Min/J (Apc Min) mice. With our approach, we further demonstrate that TRAIL and RAc induce significant cell death in human colon polyps, providing a potentially selective approach for colorectal cancer chemoprevention by targeting APC-deficient cells for apoptosis. © 2010 Macmillan Publishers Limited. All rights reserved.

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