Molecular Cancer (MOL CANCER)

Publications in this journal

• Article: Thymoquinone attenuates tumor growth in ApcMin mice by interference with Wnt-signaling.

  Michaela Lang, Melanie Borgmann, Georg Oberhuber, Rayko Evstatiev, Kristine Jimenez, Kyle W Dammann, Manuela Jambrich, Vineeta Khare, Christoph Campregher, Robin Ristl, Christoph Gasche
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  ABSTRACT: BACKGROUND: Patients with familial adenomatous polyposis (FAP) are at increased risk for the development of colorectal cancer. Surgery and chemoprevention are the most effective means to prevent cancer development. Thymoquinone (TQ) is considered the main compound of the volatile Nigella sativa seed oil and has been reported to possess anticarcinogenic properties. In this study we evaluated the chemopreventive properties of TQ in a mouse model of FAP. METHODS: APCMin mice were fed with chow containing 37.5 mg/kg or 375 mg/kg TQ for 12 weeks. H&E stained intestine tissue sections were assessed for tumor number, localization, size, and grade. Immunohistochemistry for beta-catenin, c-myc, Ki-67 and TUNEL-staining was performed to investigate TQ's effect on major colorectal cancer pathways. TQ's impact on GSK-3beta and beta-catenin were studied in RKO cells. RESULTS: 375 mg/kg but not 37.5 mg/kg TQ decreased the number of large polyps in the small intestine of APCMin mice. TQ induced apoptosis in the neoplastic tissue but not in the normal mucosa. Furthermore, upon TQ treatment, beta-catenin was retained at the membrane and c-myc decreased in the nucleus, which was associated with a reduced cell proliferation in the villi. In vitro, TQ activated GSK-3beta, which induced membranous localization of beta-catenin and reduced nuclear c-myc expression. CONCLUSIONS: In summary, TQ interferes with polyp progression in ApcMin mice through induction of tumor-cell specific apoptosis and by modulating Wnt signaling through activation of GSK-3beta. Nigella sativa oil (or TQ) might be useful as nutritional supplement to complement surgery and chemoprevention in FAP.
  Molecular Cancer 05/2013; 12(1):41.
• Article: Expression and sub-cellular localization of an epigenetic regulator, co-activator arginine methyltransferase 1 (CARM1), is associated with specific breast cancer subtypes and ethnicity.

  Melissa B Davis, Xinyu Liu, Shiyao Wang, Jaxk Reeves, Andrey Khramtsov, Dezheng Huo, Olufunmilayo I Olopade
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  ABSTRACT: BACKGROUND: Co-Activator Arginine Methyltransferase 1(CARM1) is an Estrogen Receptor (ER) cofactor that remodels chromatin for gene regulation via methylation of Histone3. We investigated CARM1 levels and localization across breast cancer tumors in a cohort of patients of either European or African ancestry. METHODS: We analyzed CARM1 levels using tissue microarrays with over 800 histological samples from 549 female cancer patients from the US and Nigeria, Africa. We assessed associations between CARM1 expression localized to the nucleus and cytoplasm for 11 distinct variables, including; ER status, Progesterone Receptor status, molecular subtypes, ethnicity, HER2+ status, other clinical variables and survival. RESULTS: We found that levels of cytoplasmic CARM1 are distinct among tumor sub-types and increased levels are associated with ER-negative (ER-) status. Higher nuclear CARM1 levels are associated with HER2 receptor status. EGFR expression also correlates with localization of CARM1 into the cytoplasm. This suggests there are distinct functions of CARM1 among molecular tumor types. Our data reveals a basal-like subtype association with CARM1, possibly due to expression of Epidermal Growth Factor Receptor (EGFR). Lastly, increased cytoplasmic CARM1, relative to nuclear levels, appear to be associated with self-identified African ethnicity and this result is being further investigated using quantified genetic ancestry measures. CONCLUSIONS: Although it is known to be an ER cofactor in breast cancer, CARM1 expression levels are independent of ER. CARM1 has distinct functions among molecular subtypes, as is indicative of its sub-cellular localization and it may function in subtype etiology. These sub-cellular localization patterns, indicate a novel role beyond its ER cofactor function in breast cancer. Differential localization among ethnic groups may be due to ancestry-specific polymorphisms which alter the gene product.
  Molecular Cancer 05/2013; 12(1):40.
• Article: 6-Formylindolo (3,2-b)carbazole (FICZ) enhances retinoic acid (RA)-induced differentiation of HL-60 myeloblastic leukemia cells.

  Rodica P Bunaciu, Andrew Yen
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  ABSTRACT: BACKGROUND: The aryl hydrocarbon receptor (AhR) ligand 6-Formylindolo(3,2-b)carbazole (FICZ) has received increasing attention since its identification as an endogenous AhR ligand and a photoproduct of tryptophan. FICZ and its metabolites have been detected in human fluids. We recently reported that AhR promotes retinoic acid (RA)-induced granulocytic differentiation of HL-60 myeloblastic leukemia cells by restricting the nuclear abundance of the stem cell associated transcription factor Oct4. The standard clinical management of acute promyelocytic leukemia (APL) is differentiation induction therapy using RA. But RA is not effective for other myeloid leukemias, making the mechanism of RA-induced differentiation observed in a non-APL myeloid leukemia of interest. To our knowledge, this is the first study regarding the influence of FICZ on RA-induced differentiation in any type of leukemic blasts. METHODS: Using flow cytometry and Western blotting assays, we determined the effects of FICZ on RA-induced differentiation of HL-60 human leukemia cells. All experiments were performed in triplicate. The groups RA and FICZ + RA were compared using the Paired-Samples T-Test. Western blot figures present the typical blots. RESULTS: We demonstrate that FICZ enhances RA-induced differentiation, assessed by the expression of the membrane differentiation marker CD11b; cell cycle arrest; and the functional differentiation marker, inducible-oxidative metabolism. FICZ causes changes in signaling events that are known to drive differentiation, and notably augments the RA-induced sustained activation of the RAF/MEK/ERK axis of the mitogen-activated protein kinase (MAPK) cascade. FICZ also augments expression of the known MAPK signaling regulatory molecules c-Cbl, VAV1, pY458 p85 PI3K, Src-family kinases (SFKs), and IRF-1, a transcription factor associated with this putative signalsome that promotes RA-induced differentiation. Moreover, FICZ in combination with RA also increases expression of AhR and even more so of both Cyp1A2 and p47phox, which are known to be transcriptionally regulated by AhR. pY1021 PDGFRbeta, a marker associated with retinoic acid syndrome was also increased. CONCLUSIONS: Our data suggest that FICZ modulates intracellular signaling pathways and enhances RA-induced differentiation.
  Molecular Cancer 05/2013; 12(1):39.
• Article: Expression of HPV16 E5 down-modulates the TGFbeta signaling pathway.

  Deborah French, Francesca Belleudi, Maria Vittoria Mauro, Francesca Mazzetta, Salvatore Raffa, Vincenza Fabiano, Antonio Frega, Maria Rosaria Torrisi
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  ABSTRACT: BACKGROUND: Infection with high-risk human papillomavirus (HR-HPV) genotypes, mainly HPV16 and HPV18, is a major risk factor for cervical cancer and responsible for its progression. While the transforming role of the HPV E6 and E7 proteins is more characterized, the molecular mechanisms of the oncogenic activity of the E5 product are still only partially understood, but appear to involve deregulation of growth factor receptor expression. Since the signaling of the transforming growth factor beta (TGFbeta) is known to play crucial roles in the epithelial carcinogenesis, aim of this study was to investigate if HPV16 E5 would modulate the TGF-BRII expression and TGFbeta/Smad signaling. FINDINGS: The HPV16 E5 mRNA expression pattern was variable in low-grade squamous intraepithelial lesions (LSIL), while homogeneously reduced in high-grade lesions (HSIL). Parallel analysis of TGFBRII mRNA showed that the receptor transcript levels were also variable in LSILs and inversely related to those of the viral protein. In vitro quantitation of the TGFBRII mRNA and protein in human keratinocytes expressing 16E5 in a dose-dependent and time-dependent manner showed a progressive down-modulation of the receptor. Phosphorylation of Smad2 and nuclear translocation of Smad4 were also decreased in E5-expressing cells stimulated with TGFbeta1. CONCLUSIONS: Taken together our results indicate that HPV16 E5 expression is able to attenuate the TGFbeta1/Smad signaling and propose that this loss of signal transduction, leading to destabilization of the epithelial homeostasis at very early stages of viral infection, may represent a crucial mechanism of promotion of the HPV-mediated cervical carcinogenesis.
  Molecular Cancer 05/2013; 12(1):38.
• Article: Gene expression profiling of chronic myeloid leukemia with variant t(9;22) reveals a different signature from cases with classic translocation.

  Francesco Albano, Antonella Zagaria, Luisa Anelli, Nicoletta Coccaro, Luciana Impera, Crescenzio Francesco Minervini, Angela Minervini, Antonella Russo Rossi, Giuseppina Tota, Paola Casieri, Giorgina Specchia
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  ABSTRACT: BACKGROUND: The t(9;22)(q34;q11) generating the BCR/ABL1 fusion gene represents the cytogenetic hallmark of chronic myeloid leukemia (CML). About 5--10 % of CML cases show variant translocations with the involvement of other chromosomes in addition to chromosomes 9 and 22. The molecular bases of biological differences between CML patients with classic and variant t(9;22) have never been clarified. FINDINGS: In this study, we performed gene expression microarray analysis to compare CML patients bearing variant rearrangements and those with classic t(9;22)(q34;q11). We identified 59 differentially expressed genes significantly associated with the two analyzed groups. The role of specific candidate genes such as TRIB1 (tribbles homolog 1), PTK2B (protein tyrosine kinase 2 beta), and C5AR1 (complement component 5a receptor 1) is discussed. CONCLUSIONS: Our results reveal that in CML cases with variant t(9;22) there is an enhancement of the MAPK pathway deregulation and show that kinases are a common target of molecular alterations in hematological disorders.
  Molecular Cancer 05/2013; 12(1):36.
• Article: Role of p38 and JNK MAPK signaling pathways and tumor suppressor p53 on induction of apoptosis in response to Ad-eIF5A1 in A549 lung cancer cells.

  Catherine A Taylor, Qifa Zheng, Zhongda Liu, John E Thompson
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  ABSTRACT: BACKGROUND: The eukaryotic translation initiation factor 5A1 (eIF5A1) is a highly conserved protein involved in many cellular processes including cell division, translation, apoptosis, and inflammation. Induction of apoptosis is the only function of eIF5A1 that is known to be independent of post-translational hypusine modification. In the present study, we investigated the involvement of mitogen- and stress-activated protein kinases during apoptosis of A549 lung cancer cells infected with adenovirus expressing eIF5A1 or a mutant of eIF5A1 that cannot be hypusinated (eIF5A1K50A). METHODS: Using adenoviral-mediated transfection of human A549 lung cancer cells to over-express eIF5A1 and eIF5A1K50A, the mechanism by which unhypusinated eIF5A1 induces apoptosis was investigated by Western blotting, flow cytometry, and use of MAPK and p53 inhibitors. RESULTS: Phosphorylation of ERK, p38 MAPK, and JNK was observed in response to adenovirus-mediated over-expression of eIF5A1 or eIF5A1K50A, along with phosphorylation and stabilization of the p53 tumor suppressor protein. Synthetic inhibitors of p38 and JNK kinase activity, but not inhibitors of ERK1/2 or p53 activity, significantly inhibited apoptosis induced by Ad-eIF5A1. Importantly, normal lung cells were more resistant to apoptosis induced by eIF5A1 and eIF5A1K50A than A549 lung cancer cells. CONCLUSIONS: Collectively these data indicate that p38 and JNK MAP kinase signaling are important for eIF5A1-induced cell death and that induction of apoptosis was not dependent on p53 activity.
  Molecular Cancer 05/2013; 12(1):35.
• Article: Extranuclear ERalpha is associated with regression of T47D PKCalpha-overexpressing, tamoxifen-resistant breast cancer.

  Bethany Perez White, Mary Ellen Molloy, Huiping Zhao, Yiyun Zhang, Debra A Tonetti
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  ABSTRACT: BACKGROUND: Prior to the introduction of tamoxifen, high dose estradiol was used to treat breast cancer patients with similar efficacy as tamoxifen, albeit with some undesirable side effects. There is renewed interest to utilize estradiol to treat endocrine resistant breast cancers, especially since findings from several preclinical models and clinical trials indicate that estradiol may be a rational second-line therapy in patients exhibiting resistance to tamoxifen and/or aromatase inhibitors. We and others reported that breast cancer patients bearing protein kinase C alpha (PKCalpha)- expressing tumors exhibit endocrine resistance and tumor aggressiveness. Our T47D:A18/PKCalpha preclinical model is tamoxifen resistant, hormone independent, yet is inhibited by 17beta-estradiol (E2) in vivo. We previously reported that E2-induced T47D:A18/PKCalpha tumor regression requires extranuclear ERalpha and interaction with the extracellular matrix. METHODS: T47D:A18/PKCalpha cells were grown in vitro using two-dimensional (2D) cell culture, three dimensional (3D) Matrigel and in vivo by establishing xenografts in athymic mice. Immunofluoresence confocal microscopy and co-localization were applied to determine estrogen receptor alpha (ERalpha) subcellular localization. Co-immunoprecipitation and western blot were used to examine interaction of ERalpha with caveolin-1. RESULTS: We report that although T47D:A18/PKCalpha cells are cross-resistant to raloxifene in cell culture and in Matrigel, raloxifene induces regression of tamoxifen-resistant tumors. ERalpha rapidly translocates to extranuclear sites during T47D:A18/PKCalpha tumor regression in response to both raloxifene and E2, whereas ERalpha is primarily localized in the nucleus in proliferating tumors. E2 treatment induced complete tumor regression whereas cessation of raloxifene treatment resulted in tumor regrowth accompanied by re-localization of ERalpha to the nucleus. T47D:A18/neo tumors that do not overexpress PKCalpha maintain ERalpha in the nucleus during tamoxifen-mediated regression. An association between ERalpha and caveolin-1 increases in tumors regressing in response to E2. CONCLUSIONS: Extranuclear ERalpha plays a role in the regression of PKCalpha overexpressing tamoxifen-resistant tumors. These studies underline the unique role of extranuclear ERalpha in E2- and raloxifene-induced tumor regression that may have implications for treatment of endocrine-resistant PKCalpha expressing tumors encountered in the clinic.
  Molecular Cancer 05/2013; 12(1):34.
• Article: Phospholipid Scramblase 1, an interferon-regulated gene located at 3q23, is regulated by SnoN/SkiL in ovarian cancer cells.

  Karthik M Kodigepalli, Pavana Anur, Paul Spellman, Peter J Sims, Meera Nanjundan
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  ABSTRACT: BACKGROUND: Treatment of advanced stage ovarian cancer continues to be challenging due to acquired drug resistance and lack of early stage biomarkers. Genes identified to be aberrantly expressed at the 3q26.2 locus (i.e. SnoN/SkiL) have been implicated in ovarian cancer pathophysiology. We have previously shown that SnoN expression is increased in advanced stage ovarian cancers and alters cellular response to arsenic trioxide (As2O3). FINDINGS: We now demonstrate increased DNA copy number levels (TCGA data) of phospholipid scramblase (PLSCR1, located at 3q23) whose transcript expression in ovarian cell lines is highly correlated with SnoN mRNA. Interestingly, SnoN can modulate PLSCR1 mRNA levels in the absence/presence of interferon (IFN-2alpha). Both IFN-2alpha and As2O3 treatment can modulate PLSCR1 mRNA levels in ovarian carcinoma cells. However, SnoN siRNA does not lead to altered PLSCR1 protein implicating other events needed to modulate its protein levels. In addition, we report that PLSCR1 can modulate aspects of the As2O3 cellular response. CONCLUSIONS: Our findings warrant further investigation into the role of PLSCR1 in ovarian cancer development and chemoresistance.
  Molecular Cancer 04/2013; 12(1):32.
• Article: Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na+/K+-ATPase.

  Laetitia Moreno Banuls, Adriana Katz, Walter Miklos, Alessio Cimmino, Daniel M Tal, Elena Ainbinder, Martin Zehl, Ernst Urban, Antonio Evidente, Brigitte Kopp, Walter Berger, Olivier Feron, Steven Karlish, Robert Kiss
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  ABSTRACT: BACKGROUND: Surface-expressed Na+/K+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC50 in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human alpha1beta1 NaK. METHODS: The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, digitoxin, gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human alpha1beta1, alpha2beta1 and alpha3beta1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides. RESULTS: Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the alpha2beta1 and alpha3beta1 than for the alpha1beta1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for alpha1beta1 than for the alpha2beta1 and alpha3beta1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation. CONCLUSIONS: Altogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the alpha2beta1 and alpha3beta1 than for the alpha1beta1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth.
  Molecular Cancer 04/2013; 12(1):33.
• Article: Epidermal growth factor receptor variant type III markedly accelerates angiogenesis and tumor growth via inducing c-myc mediated angiopoietin-like 4 expression in malignant glioma.

  Yasufumi Katanasaka, Yasuo Kodera, Yuka Kitamura, Tatsuya Morimoto, Tomohide Tamura, Fumiaki Koizumi
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  ABSTRACT: BACKGROUND: Expression of the constitutively activated mutant EGFR variant III (EGFRvIII), the most common mutation in glioblastoma multiforme (GBMs), has been clinically correlated with tumor proliferation, invasion, and angiogenesis. In this study, we examined the role of EGFRvIII on the tumor microenvironment, especially on angiogenesis. METHODS: To study the role of EGFRvIII in tumor angiogenesis, we prepared LN229 glioblastoma transfected with enhanced green fluorescent protein (EGFP), wild-type EGFR, or EGFRvIII (LN229-WT or -vIII), and examined tumor growth and microvessel density in the tumors. Additionally, the potential angiogenic factors were identified by real-time PCR analysis, and the functions in LN229-vIII cells were examined. RESULTS: LN229-vIII cells showed more aggressive tumor growth and higher vascularity as compared to LN229-WT cells in vivo, although there was no significant difference in the cell growth rates in vitro. We next investigated the expression of 60 angiogenesis-related factors to clarify the mechanisms underlying the difference in vascularity between tumor xenografts of LN229-vIII and LN229-WT. We found that the mRNA and protein expressions of angiopoietin-like 4 (Angptl4), a secreted protein involved in angiogenesis and metabolism regulation, were significantly induced by EGFRvIII overexpression, both in vitro and in vivo. Constitutive knockdown of Angptl4 in LN229-vIII using shRNA significantly decreased the microvessel density in the tumor xenografts and suppressed tumor growth. To clarify the regulatory mechanisms of Angptl4 by EGFRvIII, we analyzed the signaling pathways and transcription factors by pharmacological inhibition and RNA interference. U0126, an ERK signal inhibitor dramatically suppressed Angptl4 expression. The transcription factor c-Myc, which is regulated by ERK, was activated in the LN229-vIII cells and knockdown of c-Myc using siRNA also attenuated Angptl4 expression in the LN229-vIII cells. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed increased recruitment of c-Myc to the promoter region of Angptl4 in the LN229-vIII cells. CONCLUSIONS: In summary, we demonstrated that EGFRvIII induces Angptl4 expression through the ERK/c-Myc pathway and promotes tumor angiogenesis in malignant gliomas.
  Molecular Cancer 04/2013; 12(1):31.
• Article: MicroRNA-32 (miR-32) regulates phosphatase and tensin homologue (PTEN) expression and promotes growth, migration, and invasion in colorectal carcinoma cells.

  Weiyun Wu, Jingfang Yang, Xiao Feng, Hao Wang, Shicai Ye, Pengchun Yang, Wenkai Tan, Guoli Wei, Yu Zhou
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  ABSTRACT: BACKGROUND: Colorectal carcinoma (CRC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs, miRs) play important roles in carcinogenesis. MiR-32 has been shown to be upregulated in CRC. In this study, we identified the potential effects of miR-32 on some important biological properties of CRC cells, and clarified the regulation of PTEN by miR-32. METHODS: The effect of miR-32 on PTEN expression was assessed in CRC cell lines with miR-32 mimics/inhibitor to increase/decrease miR-32 expression. Furthermore, the roles of miR-32 in regulating CRC cells biological properties were analyzed with miR-32 mimics/inhibitor-transfected cells. The 3[prime]-untranslated region (3[prime]-UTR) of PTEN combined with miR-32 was verified by dual-luciferase reporter assay. RESULTS: Gain-of-function and loss-of-function studies showed that overexpression of miR-32 promoted SW480 cell proliferation, migration, and invasion, reduced apoptosis, and resulted in downregulation of PTEN at a posttranscriptional level. However, miR-32 knock-down inhibited these processes in HCT-116 cells and enhanced the expression of PTEN protein. In addition, we further identified PTEN as the functional downstream target of miR-32 by directly targeting the 3[prime]-UTR of PTEN. CONCLUSIONS: Our results demonstrated that miR-32 was involved in tumorigenesis of CRC at least in part by suppression of PTEN.
  Molecular Cancer 04/2013; 12(1):30.
• Article: Replication stress induces specific enrichment of RECQ1 at common fragile sites FRA3B and FRA16D.

  Xing Lu, Swetha Parvathaneni, Toshifumi Hara, Ashish Lal, Sudha Sharma
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  ABSTRACT: BACKGROUND: Stalled replication forks at common fragile sites are a major cause of genomic instability. RecQ helicases, a highly conserved family of DNA-unwinding enzymes, are believed to ease 'roadblocks' that pose challenge to replication fork progression. Among the five known RecQ homologs in humans, functions of RECQ1, the most abundant of all, are poorly understood. We previously determined that RECQ1 helicase preferentially binds and unwinds substrates that mimic DNA replication/repair intermediates, and interacts with proteins involved in DNA replication restart mechanisms. METHOD: We have utilized chromatin immunoprecipitation followed by quantitative real-time PCR to investigate chromatin interactions of RECQ1 at defined genetic loci in the presence or absence of replication stress. We have also tested the sensitivity of RECQ1-depleted cells to aphidicolin induced replication stress. RESULTS: RECQ1 binds to the origins of replication in unperturbed cells. We now show that conditions of replication stress induce increased accumulation of RECQ1 at the lamin B2 origin in HeLa cells. Consistent with a role in promoting fork recovery or repair, RECQ1 is specifically enriched at two major fragile sites FRA3B and FRA16D where replication forks have stalled following aphidicolin treatment. RECQ1-depletion results in attenuated checkpoint activation in response to replication stress, increased sensitivity to aphidicolin and chromosomal instability. CONCLUSIONS: Given a recent biochemical observation that RECQ1 catalyzes strand exchange on stalled replication fork structures in vitro, our results indicate that RECQ1 facilitates repair of stalled or collapsed replication forks and preserves genome integrity. Our findings provide the first evidence of a crucial role for RECQ1 at naturally occurring fork stalling sites and implicate RECQ1 in mechanisms underlying common fragile site instability in cancer.
  Molecular Cancer 04/2013; 12(1):29.
• Article: The impact of Cysteine-Rich Intestinal Protein 1 (CRIP1) in human breast cancer.

  Natalie Ludyga, Sonja Englert, Kerstin Pflieger, Sandra Rauser, Herbert Braselmann, Axel Walch, Gert Auer, Heinz Höfler, Michaela Aubele
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  ABSTRACT: BACKGROUND: CRIP1 (cysteine-rich intestinal protein 1) has been found in several tumor types, its prognostic impact and its role in cellular processes, particularly in breast cancer, are still unclear. METHODS: To elucidate the prognostic impact of CRIP1, we analyzed tissues from 113 primary invasive ductal breast carcinomas using immunohistochemistry. For the functional characterization of CRIP1, its endogenous expression was transiently downregulated in T47D and BT474 breast cancer cells and the effects analyzed by immunoblotting, WST-1 proliferation assay and invasion assay. RESULTS: We found a significant correlation between CRIP1 and HER2 (human epidermal growth factor receptor 2) expression levels (p = 0.016) in tumor tissues. In Kaplan Meier analyses, CRIP1 expression was significantly associated with the distant metastases-free survival of patients, revealing a better prognosis for high CRIP1 expression (p = 0.039). Moreover, in multivariate survival analyses, the expression of CRIP1 was an independent negative prognostic factor, along with the positive prognosticators nodal status and tumor size (p = 0.029). CRIP1 knockdown in the T47D and BT474 breast cancer cell lines led to the increased phosphorylation of MAPK and Akt, to the reduced phosphorylation of cdc2, and to a significantly elevated cell proliferation in vitro (p < 0.001). These results indicate that reduced CRIP1 levels may increase cell proliferation and activate cell growth. In addition, CRIP1 knockdown increased cell invasion in vitro. CONCLUSIONS: Because the lack of CRIP1 expression in breast cancer tissue is significantly associated with a worse prognosis for patients and low endogenous CRIP1 levels in vitro increased the malignant potential of breast cancer cells, we hypothesize that CRIP1 may act as a tumor suppressor in proliferation and invasion processes. Therefore, CRIP1 may be an independent prognostic marker with significant predictive power for use in breast cancer therapy.
  Molecular Cancer 04/2013; 12(1):28.
• Article: Targeting the Vav3 oncogene enhances docetaxel-induced apoptosis through the inhibition of androgen receptor phosphorylation in LNCaP prostate cancer cells under chronic hypoxia.

  Takeo Nomura, Mutsushi Yamasaki, Kenichi Hirai, Toru Inoue, Ryuta Sato, Keiko Matsuura, Masatsugu Moriyama, Fuminori Sato, Hiromitsu Mimata
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  ABSTRACT: BACKGROUND: The Vav family of Rho/Rac guanosine nucleotide exchange factors comprises three members in mammalian cells. Vav3 enhances androgen receptor (AR) activity during progression to androgen independence in prostate cancer. We examined Vav3 small interfering RNA (siRNA) effects on cell proliferation and apoptosis in docetaxel-treated LNCaP cells under chronic hypoxia (LNCaPH). METHODS: We examined individual and combined effects of Vav3 siRNA (si-Vav3) and docetaxel on cell growth and apoptosis under chronic hypoxia by cell proliferation, flow cytometric, DNA fragmentation, and immunoblot analyses. To clarify the molecular basis of si-Vav3- and docetaxel-induced apoptosis, we analyzed alterations in phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulate kinase (ERK), c-jun N-terminal kinase (JNK), and AR pathways using kinase inhibitors in LNCaPH cells. The effects of si-Vav3/atelocollagen complex alone or in combination with docetaxel were assessed on xenografts in nude mice by tumor growth delay. RESULTS: Vav3 overexpression was observed in LNCaPH compared with the expression under normoxia. Interrupting Vav3 signaling using siRNA enhanced docetaxel-induced cell growth suppression compared with that induced by docetaxel alone by inhibition of Akt and ERK phosphorylation, resulting in AR phosphorylation inhibition. In addition to increased B-cell lymphoma 2 (Bcl-2) phosphorylation through JNK signaling in response to docetaxel, si-Vav3 enhanced docetaxel-induced apoptosis, as characterized by the accumulation of sub-G1 phase cells and DNA fragmentation, through Bcl-xL/Bcl-2-associated death promoter (Bad) dephosphorylation, resulting in increased caspase-9, caspase-3, and cleaved poly(ADP-ribose) polymerase activation. Xenograft tumor growth was slightly inhibited by si-Vav3/atelocollagen complex injection and combined use of si-Vav3/atelocollagen complex and docetaxel produced a greater effect than docetaxel alone. CONCLUSIONS: Interrupting Vav3 signaling enhances docetaxel-induced apoptosis in LNCaP cells under chronic hypoxia by inhibiting the PI3K/Akt, ERK, and AR signaling pathways. Therapy targeting Vav3 in combination with docetaxel may have practical implications for managing castration-resistant prostate cancer.
  Molecular Cancer 04/2013; 12(1):27.
• Article: IL-6 expression predicts treatment response and outcome in squamous cell carcinoma of the esophagus.

  Miao-Fen Chen, Ping-Tsung Chen, Ming Shian Lu, Paul Yang Lin, Wen-Cheng Chen, Kuan-Der Lee
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  ABSTRACT: BACKGROUND: The identification of potential tumor markers can improve therapeutic planning and patient management. The aim of this study was to highlight the significance of IL-6 in esophageal squamous cell carcinoma (SCC). METHODS: We retrospectively analyzed the clinical outcomes of 173 patients with esophageal SCC, and examined the correlation between IL-6 levels and clinical outcomes in esophageal cancer patients. Furthermore, the human esophageal SCC cell line CE81T was selected for cellular and animal experiments to investigate changes in tumor behavior and treatment response after manipulation of IL-6 expression. RESULTS: In clinical outcome analysis, positive IL-6 staining and poor treatment response was significantly associated with shorter survival. Furthermore, the frequency of IL-6 immunoreactivity was significantly higher in esophageal cancer specimens than in non-malignant epithelium, and this staining was positively linked to the development of distant metastasis (p = 0.0003) and lower treatment response rates (p = 0.0001).By ELISA analysis, IL-6 serum levels were significantly elevated in patients developing disease failure.When IL-6 expression was inhibited, aggressive tumor behavior and radiation resistance could be overcome in vitro and in vivo. The underlying changes included increased cell death, less epithelial-mesenchymal transition and attenuated STAT3 activation. IL-6 inhibition was also associated with attenuated angiogenesis in tumor-bearing mice. CONCLUSIONS: IL-6 was significantly associated with poor prognosis in patients with esophageal cancer. Targeting this cytokine could be a promising strategy for treatment of esophageal cancer, as evidenced by inhibition of aggressive tumor behavior and treatment resistance.
  Molecular Cancer 04/2013; 12(1):26.
• Article: 1H-NMR based metabonomic profiling of human esophageal cancer tissue.

  Liang Wang, Jie Chen, Longqi Chen, Pengchi Deng, Qian Bu, Pu Xiang, Manli Li, Wenjie Lu, Youzhi Xu, Hongjun Lin, Tianming Wu, Huijuan Wang, Jing Hu, Xiaoni Shao, Xiaobo Cen, Ying-Lan Zhao
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  ABSTRACT: BACKGROUND: The biomarker identification of human esophageal cancer is critical for its early diagnosis and therapeutic approaches that will significantly improve patient survival. Specially, those that involves in progression of disease would be helpful to mechanism research. METHODS: In the present study, we investigated the distinguishing metabolites in human esophageal cancer tissues (n = 89) and normal esophageal mucosae (n = 26) using a 1H nuclear magnetic resonance (1H-NMR) based assay, which is a highly sensitive and non-destructive method for biomarker identification in biological systems. Principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and orthogonal partial least-squares-discriminant anlaysis (OPLS-DA) were applied to analyse 1H-NMR profiling data to identify potential biomarkers. RESULTS: The constructed OPLS-DA model achieved an excellent separation of the esophageal cancer tissues and normal mucosae. Excellent separation was obtained between the different stages of esophageal cancer tissues (stage II = 28; stage III = 45 and stage IV = 16) and normal mucosae. A total of 45 metabolites were identified, and 12 of them were closely correlated with the stage of esophageal cancer. The downregulation of glucose, AMP and NAD, upregulation of formate indicated the large energy requirement due to accelerated cell proliferation in esophageal cancer. The increases in acetate, short-chain fatty acid and GABA in esophageal cancer tissue revealed the activation of fatty acids metabolism, which could satisfy the need for cellular membrane formation. Other modified metabolites were involved in choline metabolic pathway, including creatinine, creatine, DMG, DMA and TMA. These 12 metabolites, which are involved in energy, fatty acids and choline metabolism, may be associated with the progression of human esophageal cancer. CONCLUSION: Our findings firstly identify the distinguishing metabolites in different stages of esophageal cancer tissues, indicating the attribution of metabolites disturbance to the progression of esophageal cancer. The potential biomarkers provide a promising molecular diagnostic approach for clinical diagnosis of human esophageal cancer and a new direction for the mechanism study.
  Molecular Cancer 04/2013; 12(1):25.
• Article: Short-term single treatment of chemotherapy results in the enrichment of ovarian cancer stem cell-like cells leading to an increased tumor burden.

  Khalid Abubaker, Ardian Latifi, Rod Luwor, Simon Nazaretian, Hongjian Zhu, Michael A Quinn, Erik W Thompson, Jock K Findlay, Nuzhat Ahmed
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  ABSTRACT: Over 80% of women diagnosed with advanced-stage ovarian cancer die as a result of disease recurrence due to failure of chemotherapy treatment. In this study, using two distinct ovarian cancer cell lines (epithelial OVCA 433 and mesenchymal HEY) we demonstrate enrichment in a population of cells with high expression of CSC markers at the protein and mRNA levels in response to cisplatin, paclitaxel and the combination of both. We also demonstrate a significant enhancement in the sphere forming abilities of ovarian cancer cells in response to chemotherapy drugs. The results of these in vitro findings are supported by in vivo mouse xenograft models in which intraperitoneal transplantation of cisplatin or paclitaxel-treated residual HEY cells generated significantly higher tumor burden compared to control untreated cells. Both the treated and untreated cells infiltrated the organs of the abdominal cavity. In addition, immunohistochemical studies on mouse tumors injected with cisplatin or paclitaxel treated residual cells displayed higher staining for the proliferative antigen Ki67, oncogeneic CA125, epithelial E-cadherin as well as cancer stem cell markers such as Oct4 and CD117, compared to mice injected with control untreated cells. These results suggest that a short-term single treatment of chemotherapy leaves residual cells that are enriched in CSC-like traits, resulting in an increased metastatic potential. The novel findings in this study are important in understanding the early molecular mechanisms by which chemoresistance and subsequent relapse may be triggered after the first line of chemotherapy treatment.
  Molecular Cancer 03/2013; 12(1):24.
• Article: MicroRNA-497 increases apoptosis in MYCN amplified neuroblastoma cells by targeting the key cell cycle regulator WEE1.

  Laura Creevey, Jacqueline Ryan, Harry Harvey, Isabella M Bray, Maria Meehan, Adnan R Khan, Raymond L Stallings
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  ABSTRACT: BACKGROUND: Neuroblastoma is responsible for 15% of all childhood cancer deaths. Despite advances in treatment and disease management, the overall 5-year survival rates remain poor in high-risk disease (25-40%). MiR-497 was previously identified by our laboratory as a member of a miRNA expression signature, predictive of neuroblastoma patient survival and has been reported as a tumor suppressor in a variety of other cancers. WEE1, a tyrosine kinase regulator of the cell cycle and predicted target of miR-497, has emerged as an oncogene in several cancer types and therefore represents an attractive potential target for novel therapy approaches in high-risk neuroblastoma. Our aim was to investigate the potential tumor suppressive role of miR-497 in high-risk neuroblastoma. METHODS: Expression levels of miR-497 and WEE1 in tissues and cells were determined using RT-PCR . The effect of miR-497 and siWEE1 on cell viability was evaluated using MTS assays, apoptosis levels were determined using FACS analysis of Annexin V/PI stained cells, and target protein expression was determined using western blot. Luciferase reporter plasmids were constructed to confirm direct targeting. Results were reported as mean+/-S.E.M and differences were tested for significance using 2-tailed Students t-test. RESULTS: We determined that miR-497 expression was significantly lower in high-risk MYCN amplified (MNA) tumors and that low miR-497 expression was associated with worse EFS and OS in our cohort. Over-expression of miR-497 reduced cell viability and increased apoptosis in MNA cells. We identified WEE1 as a novel target for miR-497 in neuroblastoma. Furthermore, our analysis showed that high WEE1 levels are significantly associated with poor EFS and OS in neuroblastoma and that siRNA knockdown of WEE1 in MNA cell lines results in significant levels of apoptosis, supporting an oncogenic role of WEE1 in neuroblastoma. Cisplatin (CDDP) treatment of both miR-497 over-expressing cells and WEE1 inhibited cells, resulted in a significant increase in apoptosis in MNA cells, describing a synergistic effect and therefore a potential therapeutic for high-risk neuroblastoma. CONCLUSION: Our study's results are consistent with miR-497 being a candidate tumor suppressor in neuroblastoma, through the direct targeting of WEE1. These findings re-enforce the proposal of WEE1 as a therapeutic target in neuroblastoma.
  Molecular Cancer 03/2013; 12(1):23.