Tumor Formation In Nude Mice Research Articles

A novel 3p22.3 gene CMTM7 represses oncogenic EGFR signaling and inhibits cancer cell growth

Deletion of 3p12-22 is frequent in multiple cancer types, indicating the presence of critical tumor-suppressor genes (TSGs) at this region. We studied a novel candidate TSG, CMTM7, located at the 3p22.3 CMTM-gene cluster, for its tumor-suppressive functions and related mechanisms. The three CMTM genes, CMTM6, 7 and 8, are broadly expressed in human normal adult tissues and normal epithelial cell lines. Only CMTM7 is frequently silenced or downregulated in esophageal and nasopharyngeal cell lines, but uncommon in other carcinoma cell lines. Immunostaining of tissue microarrays for CMTM7 protein showed its downregulation or absence in esophageal, gastric, pancreatic, liver, lung and cervix tumor tissues. Promoter CpG methylation and loss of heterozygosity were both found contributing to CMTM7 downregulation. Ectopic expression of CMTM7 in carcinoma cells inhibits cell proliferation, motility and tumor formation in nude mice, but not in immortalized normal cells, suggesting a tumor inhibitory role of CMTM7. The tumor-suppressive function of CMTM7 is associated with its role in G1/S cell cycle arrest, through upregulating p27 and downregulating cyclin-dependent kinase 2 (CDK2) and 6 (CDK6). Moreover, CMTM7 could promote epidermal growth factor receptor (EGFR) internalization, and further suppress AKT signaling pathway. Thus, our findings suggest that CMTM7 is a novel 3p22 tumor suppressor regulating G1/S transition and EGFR/AKT signaling during tumor pathogenesis.

SND1 Affects Proliferation of Hepatocellular Carcinoma Cell Line SMMC‐7721 by Regulating IGFBP3 Expression

Staphylococcal nuclease domain containing 1 (SND1) is a ubiquitously expressed multifunctional protein involved in transcriptional regulation, RNA splicing and RNA metabolism. Ectopic expression of SND1 has been observed in various tumors including colon cancer, breast cancer, prostate cancer and hepatocellular carcinoma (HCC), indicating a positive role of SND1 in tumor initiation and progression. However, the exact role of SND1 in cancers has not been thoroughly investigated. In the present study, we investigated the role of SND1 in HCC. Immunohistochemistry analysis revealed that the expression level of SND1 was higher in HCC tissues than in adjacent nontumor tissues. Stable knock-down of SND1, performed on the HCC cell line SMMC-7721 using shRNA lentiviral expression system, led to reduced cell proliferation, clone formation and tumor formation in nude mice. The insulin-like growth factor (IGF) signaling pathway was frequently dysregulated in HCC, which could facilitate tumor progression. Screening of gene expression levels of the IGF pathway, using real-time PCR, revealed that a decrease in SND1 expression could increase the expression of IGF-binding protein 3 (IGFBP3), which can negatively regulate activation of the IGF pathway by restricting interactions between IGF and IGF receptors. Results from previous studies showed that the downregulation of IGFBP3 expression is a common feature in HCC, and the upregulation of IGFBP3 expression could suppress HCC cells proliferation. We further confirmed that stable knock-down of IGFBP3 could promote SMMC-7721 cells proliferation. Therefore, we concluded that SND1 could affect SMMC-7721 cells proliferation by regulating IGFBP3 expression and IGF signaling pathway.

ALLN hinders HCT116 tumor growth through Bax-dependent apoptosis

Continual high expression of cysteine proteases calpain I and II have been implicated in tumorigenicity; conversely, N-acetyl-leu-leunorleucinal (ALLN), which inhibits calpain I and II, should also influence tumor growth and carcinogenesis. To explore the role of ALLN against colon cancer and in promoting apoptosis, we used colon cancer HCT116 cell lines, p53 or Bax-deficient HCT116 cell lines. Cell viability and tumor growth decreased in a concentration-dependent manner when treated with 0–26μM ALLN. Treatment with ALLN induced apoptosis in HCT116 cell; however, flow cytometry showed that apoptosis significantly decreased in Bax-deficient HCT116 cell lines, but not in p53-deficient HCT116 cell lines. In addition, the ALLN-induced apoptosis response was through Bax translocation from cytosol to mitochondria. In this study we showed intraperitoneally injected ALLN to inhibit colon tumor formation in nude mice, and found ALLN to inhibit tumor growth in colon cancer cells, mainly through apoptosis that depends on translocation of Bax to a mitochondrial endogenous pathway; this implies a molecular mechanism for ALLN against human colon cancer. These results suggest that ALLN could become a novel agent for prevention of colon cancer.

Characterization of the oncogenic function of centromere protein F in hepatocellular carcinoma

Centromere protein F (CENPF) is an essential nuclear protein associated with the centromere-kinetochore complex and plays a critical role in chromosome segregation during mitosis. Up-regulation of CENPF expression has previously been detected in several solid tumors. In this study, we aim to study the expression and functional role of CENPF in hepatocellular carcinoma (HCC). We found CENPF was frequently overexpressed in HCC as compared with non-tumor tissue. Up-regulated CENPF expression in HCC was positively correlated with serum AFP, venous invasion, advanced differentiation stage and a shorter overall survival. Cox regression analysis found that overexpression of CENPF was an independent prognosis factor in HCC. Functional studies found that silencing CENPF could decrease the ability of the cells to proliferate, form colonies and induce tumor formation in nude mice. Silencing CENPF also resulted in the cell cycle arrest at G2/M checkpoint by down-regulating cell cycle proteins cdc2 and cyclin B1. Our data suggest that CENPF is frequently overexpressed in HCC and plays a critical role in driving HCC tumorigenesis.

The Pak4 protein kinase is required for oncogenic transformation of MDA-MB-231 breast cancer cells

The Pak4 protein kinase, normally expressed at low level in the mammary gland, is commonly overexpressed in breast cancer. Overexpression of Pak4 transforms mouse mammary epithelial cells in vitro and renders these cells tumorigenic in athymic mice in vivo. Here we show that Pak4 is also required for oncogenic transformation of the human breast cancer cell line MDA-MB-231. These high Pak4-expressing human breast cancer cells form highly disorganized three-dimensional (3D) structures in vitro and readily give rise to orthotopic xenograft tumors in nude mice. We have found that when Pak4 levels are reduced, MDA-MB-231 cells exhibit decreased proliferation and migration in vitro, as well as gross restoration of normal 3D mammary acinar organization, the latter in association with a strong induction of apoptosis. Similarly, Pak4 knockdown suppresses MDA-MB-231 breast xenograft tumor formation in nude mice in vivo. These results indicate that Pak4 has a key role in the oncogenic transformation of breast cells.

P21-activated kinase 5 plays essential roles in the proliferation and tumorigenicity of human hepatocellular carcinoma

To investigate the roles of P21-activated kinase 5 (PAK5) in proliferation and tumorigenicity of human hepatocellular carcinoma (HCC). HCC and matched paraneoplastictis tissue samples were obtained from 30 patients. Human HCC cell lines SMMC7721, HepG2, Hep3B, SK-HEP-1, Huh-7, and liver cell line HL-7702 were examined. The expression of PAK5 gene was studied using real-time qPCR and Western blotting. Cell proliferation was quantified with the MTT assay. Cell cycle was analyzed with flow cytometry. The tumorigenicity of Lv-shRNA-transfected HepG2 cells was evaluated in BALB/cA nude mice. The mRNA level of PAK5 was significantly higher in 25 out of 30 HCC samples compared to the matched paraneoplastic tissues. The HCC cell lines showed varying expression of PAK5 protein, and the highest level was found in the HepG2 cells. PAK5 gene silencing in HepG2 cells markedly reduced the cell proliferation and colony formation, and induced cell cycle arrest in the G1 phase. Furthermore, PAK5 gene silencing suppressed the tumor formation in nude mice, and significantly decreased the expression of HCC-related genes Cyclin D1 and beta-catenin. PAK5 may play essential roles in the initiation and progression of human HCC. Thus, it may be an effective therapeutic target or perhaps serve as a clinical diagnostic or prognostic marker in human HCC.

FGFR4 Role in Epithelial-Mesenchymal Transition and Its Therapeutic Value in Colorectal Cancer

Fibroblast growth factor receptor 4 (FGFR4) is vital in early development and tissue repair. FGFR4 expression levels are very restricted in adult tissues, except in several solid tumors including colorectal cancer, which showed overexpression of FGFR4. Here, FGFR4 mutation analysis discarded the presence of activating mutations, other than Arg388, in different colorectal cancer cell lines and tumoral samples. Stable shRNA FGFR4-silencing in SW480 and SW48 cell lines resulted in a significant decrease in cell proliferation, adhesion, cell migration and invasion. This decrease in the tumorigenic and invasive capabilities of colorectal cancer cells was accompanied by a decrease of Snail, Twist and TGFβ gene expression levels and an increase of E-cadherin, causing a reversion to a more epithelial phenotype, in three different cell lines. In addition, FGFR4-signaling activated the oncogenic SRC, ERK1/2 and AKT pathways in colon cancer cells and promoted an increase in cell survival. The relevance of FGFR4 in tumor growth was supported by two different strategies. Kinase inhibitors abrogated FGFR4-related cell growth and signaling pathways at the same extent than FGFR4-silenced cells. Specific FGFR4-targeting using antibodies provoked a similar reduction in cell growth. Moreover, FGFR4 knock-down cells displayed a reduced capacity for in vivo tumor formation and angiogenesis in nude mice. Collectively, our data support a crucial role for FGFR4 in tumorigenesis, invasion and survival in colorectal cancer. In addition, FGFR4 targeting demonstrated its applicability for colorectal cancer therapy.

Effects of manganese superoxide dismutase (MnSOD) expression on regulation of esophageal cancer cell growth and apoptosis in vitro and in nude mice

Manganese superoxide dismutase (MnSOD) catalyzes superoxide radical (O2 (-)) into hydrogen peroxide (H2O2), which is further catalyzed by the combined action of glutathione peroxidase (GPx) and catalase (CAT) into water and oxygen. MnSOD plays a role in cell protection from superoxide damage. This study aimed to investigate the effects of MnSOD on regulation of esophageal squamous cell carcinoma cell growth, apoptosis, and cell cycle distribution in vitro and tumor formation and growth in nude mouse xenografts. The data showed that differential levels of MnSOD expression had different effects on tumor cell proliferation, apoptosis, plating efficiency (PE), and cell cycle distribution in vitro and tumor formation and growth in nude mice. In particular, high levels of MnSOD expression promoted TE-1 cell growth and PE rate in vitro and in nude mice, whereas moderate MnSOD expression suppressed tumor cell growth and PE rate but induced more cell apoptosis. Thus, these data demonstrated the dual effects of MnSOD protein in esophageal squamous cell carcinoma and further study will confirm these current data.

Genomic analysis of diffuse pediatric low-grade gliomas identifies recurrent oncogenic truncating rearrangements in the transcription factorMYBL1

Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but, apart from BRAF kinase mutations or duplications in specific subclasses, few genetic driver events are known. Diffuse PLGGs comprise a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. We performed high-resolution copy-number analysis on 44 formalin-fixed, paraffin-embedded diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gain, was observed in 28% of diffuse astrocytoma grade IIs and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene v-myb avian myeloblastosis viral oncogene homolog (MYB) on 6q23.3. Whole-genome sequencing of a MYBL1-rearranged diffuse astrocytoma grade II demonstrated MYBL1 tandem duplication and few other events. Truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas.

Biological characteristics of CD133+ cells in nasopharyngeal carcinoma

Cancer stem cells are regarded as the cause of tumour formation and recurrence in nasopharyngeal carcinoma (NPC). However, ideal surface markers for stem cells in NPC remain unidentified. In the present study, we investigated the expression of CD133, Nanog and Sox2 in the nasopharyngeal carcinoma cell line CNE2 and primarily cultured NPC cells using immunofluorescence or flow cytometry. A cell population with a CD133+ phenotype was enriched using magnetic-activated cell sorting technology. We demonstrated that CD133+ cells exhibited a strong potential for self-renewal, proliferation and differentiation and a greater potential for in vivo tumour formation in nude mice compared to CD133− cells, although the percentage of CD133+ cells was small. However, the specific marker antigens Nanog and Sox2 were simultaneously expressed in normal cancer stem cells. Our results showed that CD133 can serve as a specific surface marker for nasopharyngeal cancer stem cells.

Abstract 3018: Identification of genes up-and down-regulated by dermcidin in G-361 malignant melanoma cell line.

Abstract Dermcidin (DCD) is a human gene mapped to chromosome 12q13 region, only identified in primates and humans, and normally expressed in the eccrine glands of skin and brain. Several studies have confirmed DCD-derived peptides contribute to innate and immune surveillance and in the oncogenic processes of breast, prostate and skin cancers, as revealed by its role as a growth factor and cell survival. We have further explored DCD function and its tumorigenic potential on skin melanocytes by specifically knocking down its expression in G-361 malignant melanoma cells via expressing constitutively short hairpin RNA against DCD mRNA. Biological and biochemical assays showed that the "knockdown" in the expression of DCD in two cell clones decreased significantly the in vitro growth in cell culture and tumor formation in nude mice. Similar results were obtained treating nude mice bearing G-361 melanoma xenografts with rabbit polyclonal antibodies against DCD protein (1 mg/Kg, i.p., once week, and one month). Here, we present a summary of a DNA microarray-based study that identified the genes that are up- and down-regulated in a G-361 pLKO control clone and a clone expressing constitutively short hairpin RNA against DCD mRNA. A total of 213 genes were differentially and repeatedly expressed in the G-361-DCD-RNAi clone IBC-I (>3-fold change and P < 0.005). Relative to pLKO vector control data profiling, 99 genes were up-regulated and 120 genes were down-regulated. Next, we validated various genes by real time-PCR. Systematic functional analysis of the differentially expressed genes using DAVID platform revealed significant enrichment for U1-U5 spliceosomal complex genes (SNORs) required for post-transcriptional processing of rRNA, mRNA and tRNA as well as small nucleolar RNA and non-coding RNAs (ncRNAs). Further studies are on way to prove the functional connections among ncRNAs in DCD-induced the development and progression of melanoma malignancy. Citation Format: Jose E. Belizario, Marcela Perez. Identification of genes up-and down-regulated by dermcidin in G-361 malignant melanoma cell line. [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 3018. doi:10.1158/1538-7445.AM2013-3018

Abstract 396: Osteopontin-c splicing isoform contributes to ovarian cancer progression.

Abstract Introduction: Ovarian carcinoma (OC) is one of the most aggressive gynecological diseases and generally diagnosed at advanced stages. Osteopontin (OPN) is overexpressed in OC and is involved in tumorigenesis and metastasis. Alternative splicing of OPN generates 3 isoforms, OPNa, OPNb, and OPNc. Objective: This study aims to characterize the expression profile and functional role of OPN splicing isoforms (OPN-SI) in OC. Methodology: The expression patterns of OPN-SI and a series of genes involved in cancer pathways were analyzed by real-time PCR. In vitro and in vivo functional assays were performed using the ovarian cancer cell line OvCar-3, stably overexpressing the three OPN-SI. Results: OPNc, but not OPNa and OPNb, was specifically expressed on OC samples. We then demonstrated that OPNc isoform has stimulatory effects on OvCar-3 cell proliferation, migration, invasion and anchorage independent cells growth, besides activating tumor formation in nude mice. We also found that these features are mainly mediated by PI3K/Akt signaling pathway. Moreover, OvCar-3 OPNc-overexpressing cells are able to modulate the expression of 34 genes involved in key cancer pathways. Notably, tumors formed by OPNc-overexpressing cells present high expression levels of typical angiogenic markers, such as VEGF-A, VEGFR-2 and CD34. Based on this observation, we also investigated the molecular mechanisms by which OPNc stimulates angiogenic processes. Our data showed that OPNc overexpression activates VEGF-A expression and secretion, also through the PI3K/Akt pathway. This splicing isoform is also able to activate the expression of c-Fos, c-Jun and phospho-c-Jun. OPNc role on activating the phosphorylation of c-Jun is mediated by integrin receptor, in an RGD-dependent manner. In addition, OPNc-conditioned medium is able to induce HUVEC endothelial cell proliferation, migration and adhesion. Conclusion: Thus, the specific targeting of OPNc and its regulated signaling network could be a putative novel strategy to inhibit key steps involved in OC progression and may represent a putative strategy for developing new therapeutic approaches using OPNc as interesting target for OC treatment strategies. Citation Format: Tatiana M. Tilli, Kivvi D. Mello, Vanessa Franco, Bruno Robbs, João Luiz Wanderley, Fabricio R A Silva, João P B Viola, Georg Weber, Vincent Castronovo, Akeila Bellahcène, Etel R P Gimba. Osteopontin-c splicing isoform contributes to ovarian 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 396. doi:10.1158/1538-7445.AM2013-396

Abstract 2054: GP88 (Progranulin) is a therapeutic and prognostic target for Non-Small Cell Lung Carcinoma.

Abstract Annually, about 220,000 patients are diagnosed with lung cancer in the US and 150,000 die of the disease. Advanced and recent therapeutic development in NSCLC addresses targets found in small percentages of patients, thereby emphasizing the need for continued discoveries of additional targets of lung cancer, in order to develop of novel therapies and diagnosis. We have investigated the role of the autocrine growth and survival factor GP88 (Progranulin) on the biology of Non-Small Cell Lung Carcinoma (NSCLC). Previously, GP88 had been shown to play a critical role in the proliferation and survival of breast cancer. Pathological studies with 600 cases of estrogen receptor positive invasive ductal carcinoma in correlation with clinical outcome data have also shown that GP88 tissue expression was associated with a 4-fold increased risk of recurrence and a 2.5-fold increase in mortality whereas inhibition of GP88 action by a neutralizing antibody resulted in inhibition of tumor formation of breast adenocarcinoma in vivo. In the present study, we examined the role of GP88 in NSCLC. Examination of GP88 protein expression by immunohistochemistry was carried out with formalin fixed paraffin embedded sections of lung cancer cases. Results showed that both human adenocarcinoma and squamous carcinoma stained positive for GP88 whereas normal lung tissues were negative. Pathological studies of tissue GP88 expression by IHC in 85 stage 1 and 2 NSCLC tumors in correlation with clinical outcomes demonstrated that high GP88 expression was associated with a 4-fold increase in the risk of recurrence (p=0.0004) indicating that GP88 is a prognostic factor for NSCLC. Based on these observations, expression and action of GP88 in NSCLC were examined using two representative human NSCLC cell lines A549 and H1299. Inhibition of GP88 expression by GP88 SiRNA resulted in an inhibition of cell migration and proliferation of these cells. Similarly, neutralization of GP88 action by treating the cells with AG1, an anti-human GP88 neutralizing monoclonal antibody expressed in recombinant form also inhibited cell proliferation and migration. Based on these results, studies were carried out to examine the effect of AG1 antibody on NSCLC tumor formation in nude mice. Preclinical studies show that AG1 treatment inhibits tumor formation by 30-50% when used as a single agent or in combination therapy with chemotherapeutic drugs used in the standard of care in NSCLC. These data to be presented here point out that GP88 is a novel therapeutic and prognostic target and provide new opportunities for targeted therapy with companion diagnostic for NSCLC. Citation Format: Ginette Serrero, Guanjun Xia, Jianping Dong, Binbin Yue, Amanda Schech, Douglas Hawkins, David Reisman, Pablo Bejarano, Jun Hayashi. GP88 (Progranulin) is a therapeutic and prognostic target for Non-Small Cell Lung Carcinoma. [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 2054. doi:10.1158/1538-7445.AM2013-2054

Abstract 5596: Linkage between the lipogenic pathway and the progression of breast carcinomas.

Abstract Fatty acid synthase (FASN) is a lipogenic enzyme responsible for the novo synthesis of fatty acids (FAs) from acetyl-CoA and malonyl-CoA in a NADPH dependent manner in cells. FAs are essential constituents of all biological membranes lipids and are important substrates for energy metabolism. While normal cells (except liver and adiposities) express low levels of FASN and have low to undetectable levels of FASN enzymatic activity, FASN expression and activity is observed in a vast majority of human cancers. Whether the increase in FASN is merely a consequence of disease progression or it is a “metabolic oncogene” that drives disease progression remains unclear. Here, we undoubtedly demonstrated that knockdown FASN results in inhibition of tumor formation and angiogenesis in athymic nude mice. Moreover, the histopathological features of the FASN knockdown remaining tissue in vivo appear globular, well differentiated with low nuclear grade compared to the control tumors, which appear invasive, undifferentiated, and of high nuclear grade. When tissues where stained for Ki-67, Vimentin and E-Cadherin the classical mesenchymal to epithelial transition (MET) was observed in the tissue derived from FASN knockdown tumors. Our studies establish a possible role for FASN as a “metabolic oncogene” in breast cancer. Given the data presented here, we propose that FASN is a perfect marker to develop personalized targeted therapy. Citation Format: Anatilde M. Gonzalez-Guerrico, Cheol Park-Hong, Celina Kleer, Ingrid Espinoza, Ruth Lupu. Linkage between the lipogenic pathway and the progression of breast carcinomas. [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 5596. doi:10.1158/1538-7445.AM2013-5596

Mutations in Critical Domains Confer the Human mTOR Gene Strong Tumorigenicity*

Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates cell growth, proliferation, and survival. mTOR is frequently activated in human cancers and is a commonly sought anticancer therapeutic target. However, whether the human mTOR gene itself is a proto-oncogene possessing tumorigenicity has not been firmly established. To answer this question, we mutated evolutionarily conserved amino acids, generated eight mutants in the HEAT repeats (M938T) and the FAT (W1456R and G1479N) and kinase (P2273S, V2284M, V2291I, T2294I, and E2288K) domains of mTOR, and studied their oncogenicity. On transient expression in HEK293T cells, these mTOR mutants displayed elevated protein kinase activities accompanied by activated mTOR/p70S6K signaling at varying levels, demonstrating the gain of function of the mTOR gene with these mutations. We selected P2273S and E2288K, the two most catalytically active mutants, to further examine their oncogenicity and tumorigenicity. Stable expression of the two mTOR mutants in NIH3T3 cells strongly activated mTOR/p70S6K signaling, induced cell transformation and invasion, and remarkably, caused rapid tumor formation and growth in athymic nude mice after subcutaneous inoculation of the transfected cells. This study confirms the oncogenic potential of mTOR suggested previously and demonstrates for the first time its tumorigenicity. Thus, beyond the pivotal position of mTOR to relay the oncogenic signals from the upstream phosphatidylinositol 3-kinase/Akt pathway in human cancer, mTOR is capable potentially of playing a direct role in human tumorigenesis if mutated. These results also further support the conclusion that mTOR is a major therapeutic target in human cancers.

Calmodulin protects Aurora B on the midbody to regulate the fidelity of cytokinesis

Aurora B kinase is an integral regulator of cytokinesis as it stabilizes the intercellular canal within the midbody to ensure proper chromosomal segregation during cell division. Here we identified an E3 ligase subunit, F box protein FBXL2, that by recognizing a calmodulin binding signature within Aurora B, ubiquitinates and removes the kinase from the midbody. Calmodulin, by competing with the F box protein for access to the calmodulin binding signature, protected Aurora B from FBXL2. Calmodulin co-localized with Aurora B on the midbody, preserved Aurora B levels in cells, and stabilized intercellular canals during delayed abscission. Genetic or pharmaceutical depletion of endogenous calmodulin significantly reduced Aurora B protein levels at the midbody resulting in tetraploidy and multi-spindle formation. The calmodulin inhibitor, calmidazolium, reduced Aurora B protein levels resulting in tetraploidy, mitotic arrest, and apoptosis of tumorigenic cells and profoundly inhibiting tumor formation in athymic nude mice. These observations indicate molecular interplay between Aurora B and calmodulin in telophase and suggest that calmodulin acts as a checkpoint sensor for chromosomal segregation errors during mitosis.

Enhancer of Zeste Homolog 2 Silences MicroRNA-218 in Human Pancreatic Ductal Adenocarcinoma Cells by Inducing Formation of Heterochromatin

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that is overexpressed by pancreatic ductal adenocarcinoma (PDAC) cells and increases their aggressiveness. We identified microRNAs (miRs) that are regulated by EZH2 and studied their functions in PDAC cells. We performed miR profile analysis of PDAC cells incubated with EZH2 inhibitor 3-deazaneplanocin A, and pancreatic ductal epithelial cells that overexpressed EZH2. Expression levels of miRs and the targets of miRs were analyzed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. We expressed different forms of EZH2 to analyze functional domains and used small interfering RNAs to reduce its level in PDAC cells. Expression of miR-218 was repressed by EZH2 in PDAC cells. Levels of miR-218 were significantly reduced in primary PDAC tumor samples compared with paired, adjacent nontumor tissue. Overexpression of miR-218 in SW1990 cells reduced their proliferation and tumor formation and metastasis in nude mice. Loss of miR-218 from SW1990 cells increased levels of UDP-glycosyltransferase 8 and miR-218 was found to bind to its 3'-UTR. Levels of UDP-glycosyltransferase protein and messenger RNA were associated with the metastatic potential of PDAC cell lines and progression of tumors in patients. EZH2 was found to silence miR-218 by binding to its promoter, promoting heterochromatin formation, and recruiting the DNAs methyltransferase 1, 3A, and 3B. EZH2 is up-regulated in PDAC samples from patients and silences miR-218. MicroRNA-218 prevents proliferation of PDAC cells in culture, and tumor growth and metastasis in nude mice. MicroRNA-218 reduces levels of UDP-glycosyltransferase, which is associated with the metastatic potential of PDAC tumors in mice and progression of human PDAC.

Integrated Metabolite and Gene Expression Profiles Identify Lipid Biomarkers Associated With Progression of Hepatocellular Carcinoma and Patient Outcomes

We combined gene expression and metabolic profiling analyses to identify factors associated with outcomes of patients with hepatocellular carcinoma (HCC). We compared metabolic and gene expression patterns between paired tumor and nontumor tissues from 30 patients with HCC, and validated the results using samples from 356 patients with HCC. A total of 469 metabolites were measured using liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. Metabolic and genomic data were integrated, and Kaplan-Meier and Cox proportional hazards analyses were used to associate specific patterns with patient outcomes. Associated factors were evaluated for their effects on cancer cells in vitro and tumor formation in nude mice. We identified 28 metabolites and 169 genes associated with aggressive HCC. Lipid metabolites of stearoyl-CoA-desaturase (SCD) activity were associated with aberrant palmitate signaling in aggressive HCC samples. Expression of gene products associated with these metabolites, including SCD, were associated independently with survival times and tumor recurrence in the test and validation sets. Combined expression of SCD and α-fetoprotein were associated with outcomes of patients with early-stage HCC. Levels of monounsaturated palmitic acid, the product of SCD activity, were increased in aggressive HCCs; monounsaturated palmitic acid increased migration and invasion of cultured HCC cells and colony formation by HCC cells. HCC cells that expressed small interfering RNA against SCD had decreased cell migration and colony formation in culture and reduced tumorigenicity in mice. By using a combination of gene expression and metabolic profile analysis, we identified a lipogenic network that involves SCD and palmitate signaling and was associated with HCC progression and patient outcomes. The microarray platform and data have been submitted to the Gene Expression Omnibus public database at NCBI following MIAME guidelines. Accession numbers: GPL4700 (platform), and GSE6857 (samples).

Critical Roles of Myc-ODC Axis in the Cellular Transformation Induced by Myeloproliferative Neoplasm-Associated JAK2 V617F Mutant

The acquired mutation (V617F) of Janus kinase 2 (JAK2) is observed in the majority of patients with myeloproliferative neoplasms (MPNs). In the screening of genes whose expression was induced by JAK2 (V617F), we found the significant induction of c-Myc mRNA expression mediated by STAT5 activation. Interestingly, GSK-3β was inactivated in transformed Ba/F3 cells by JAK2 (V617F), and this enhanced the protein expression of c-Myc. The enforced expression of c-Myc accelerated cell proliferation but failed to inhibit apoptotic cell death caused by growth factor deprivation; however, the inhibition of GSK-3β completely inhibited the apoptosis of cells expressing c-Myc. Strikingly, c-Myc T58A mutant exhibited higher proliferative activity in a growth-factor-independent manner; however, this mutant failed to induce apoptosis. In addition, knockdown of c-Myc significantly inhibited the proliferation of transformed cells by JAK2 (V617F), suggesting that c-Myc plays an important role in oncogenic activity of JAK2 (V617F). Furthermore, JAK2 (V617F) induced the expression of a target gene of c-Myc, ornithine decarboxylase (ODC), known as the rate-limiting enzyme in polyamine biosynthesis. An ODC inhibitor, difluoromethylornithine (DFMO), prevented the proliferation of transformed cells by JAK2 (V617F). Importantly, administration of DFMO effectively delayed tumor formation in nude mice inoculated with transformed cells by JAK2 (V617F), resulting in prolonged survival; therefore, ODC expression through c-Myc is a critical step for JAK2 (V617F)-induced transformation and DFMO could be used as effective therapy for MPNs.

HPV陽性および陰性の口腔扁平上皮がんに対するin vitro多段階発がんモデル

Oral squamous cell carcinomas (OSCCs) are considered to arise from human oral keratinocytes. DNAs of human papillomaviruses (HPVs), predominantly types 16 and 18, etiological agents of cervical cancers, have been detected in approximately 25% of OSCCs. In accordance with the established roles of E6 and E7 in inactivating p53 and pRb, respectively, mutations of p53 and inactivation of p16INK4a are frequently observed in HPV-negative OSCCs. In addition, other alterations such as overexpression of epidermal growth factor receptor (EGFR) are often observed in both HPV-positive and -negative OSCCs. However, the causal relation between accumulation of these abnormalities and multistep carcinogenesis is not fully understood. To elucidate underlying processes, we transduced either HPV16 E6/E7 or a mutant CDK4 (CDK4R24C), cyclin D1, and human telomerase reverse transcriptase (TERT) into primary human tongue keratinocytes (HTK), and obtained immortal cell populations, HTK-16E6E7 and HTK-K4DT. Additional transduction of oncogenic HR AS or EGFR together with MYC into HTK-16E6E7 and dominant negative p53 expressing HTK-K4DT resulted in anchorage-independent growth and subcutaneous tumor formation in nude mice. These results indicate that either HRAS mutation or activation of EGFR in cooperation with MYC overexpression play critical roles in transformation of HTKs on a background of inactivation of the pRB and p53 pathways and telomerase activation. This in vitro model system recapitulating the development of OSCCs should facilitate further studies of mechanisms of carcinogenesis in the oral cavity.