bluuebunny/crossref_metadata_2025 huggingface dataset:
P-Glycoprotein Mediates Celecoxib-Induced Apoptosis in Multiple Drug-Resistant Cell Lines
Authors: Fantappiè Ornella , Solazzo Michela , Lasagna Nadia , Platini Francesca , Tessitore Luciana , Mazzanti Roberto
Published: May 2007

In several neoplastic diseases, including hepatocellular carcinoma, the expression of P-glycoprotein and cyclooxygenase-2 (COX-2) are often increased and involved in drug resistance and poor prognosis. P-glycoprotein, in addition to drug resistance, blocks cytochrome c release, preventing apoptosis in tumor cells. Because COX-2 induces P-glycoprotein expression, we evaluated the effect of celecoxib, a specific inhibitor of COX-2 activity, on P-glycoprotein–mediated resistance to apoptosis in cell lines expressing multidrug resistant (MDR) phenotype. Experiments were done using MDR-positive and parental cell lines at basal conditions and after exposure to 10 or 50 μmol/L celecoxib. We found that 10 μmol/L celecoxib reduced P-glycoprotein, Bcl-xL, and Bcl-2 expression, and induced translocation of Bax from cytosol to mitochondria and cytochrome c release into cytosol in MDR-positive hepatocellular carcinoma cells. This causes the activation of caspase-3 and increases the number of cells going into apoptosis. No effect was shown on parental drug-sensitive or on MDR-positive hepatocellular carcinoma cells after transfection with MDR1 small interfering RNA. Interestingly, although inhibiting COX-2 activity, 50 μmol/L celecoxib weakly increased the expression of COX-2 and P-glycoprotein and did not alter Bcl-xL and Bcl-2 expression. In conclusion, these results show that relatively low concentrations of celecoxib induce cell apoptosis in MDR cell lines. This effect is mediated by P-glycoprotein and suggests that the efficacy of celecoxib in the treatment of different types of cancer may depend on celecoxib concentration and P-glycoprotein expression. [Cancer Res 2007;67(10):4915–23]
The Farnesyltransferase Inhibitor R115777 Up-regulates the Expression of Death Receptor 5 and Enhances TRAIL-Induced Apoptosis in Human Lung Cancer Cells
Authors: Qiu Yuanzheng , Liu Xiangguo , Zou Wei , Yue Ping , Lonial Sagar , Khuri Fadlo R. , Sun Shi-Yong
Published: May 2007

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in transformed or malignant cells, thus exhibiting potential as a tumor-selective apoptosis-inducing cytokine for cancer treatment. Many studies have shown that the apoptosis-inducing activity of TRAIL can be enhanced by various cancer therapeutic agents. R115777 (tipifarnib) is the first farnesyltransferase inhibitor (FTI) that showed clinical activity in myeloid malignancies. In general, R115777, like other FTIs, exerts relatively weak effects on the induction of apoptosis in cancer cells with undefined mechanism(s). In the current study, we studied its effects on the growth of human lung cancer cells, including induction of apoptosis, and examined potential underlying mechanisms for these effects. We showed that R115777 induced apoptosis in human lung cancer cells, in addition to inducing G1 or G2-M arrest. Moreover, we found that R115777 up-regulated the expression of death receptor 5 (DR5), an important death receptor for TRAIL, and exhibited an augmented effect on the induction of apoptosis when combined with recombinant TRAIL. Blockage of DR5 induction by small interfering RNA (siRNA) abrogated the ability of R115777 to enhance TRAIL-induced apoptosis, indicating that R115777 augments TRAIL-induced apoptosis through up-regulation of DR5 expression. Thus, our findings show the efficacy of R115777 in human lung cancer cells and suggest that R115777 may be used clinically in combination with TRAIL for treatment of human lung cancer. [Cancer Res 2007;67(10):4973–80]
Inactivation of Glutathione Peroxidase Activity Contributes to UV-Induced Squamous Cell Carcinoma Formation
Authors: Walshe Jennifer , Serewko-Auret Magdalena M. , Teakle Ngari , Cameron Sarina , Minto Kelly , Smith Louise , Burcham Philip C. , Russell Terry , Strutton Geoffrey , Griffin Anthony , Chu Fong-Fong , Esworthy Stephen , Reeve Vivienne , Saunders Nicholas A.
Published: May 2007

Cutaneous squamous cell carcinomas (CSCC) are a common malignancy of keratinocytes that arise in sites of the skin exposed to excessive UV radiation. In the present study, we show that human SCC cell lines, preneoplastic solar keratoses (SK), and CSCC are associated with perturbations in glutathione peroxidase (GPX) activity and peroxide levels. Specifically, we found that two of three SKs and four of five CSCCs, in vivo, were associated with decreased GPX activity and all SKs and CSCCs were associated with an elevated peroxide burden. Given the association of decreased GPX activity with CSCC, we examined the basis for the GPX deficiency in the CSCCs. Our data indicated that GPX was inactivated by a post-translational mechanism and that GPX could be inactivated by increases in intracellular peroxide levels. We next tested whether the decreased peroxidase activity coupled with an elevated peroxidative burden might contribute to CSCC formation in vivo. This was tested in Gpx1−/− and Gpx2−/− mice exposed to solar-simulated UV radiation. These studies showed that Gpx2 deficiency predisposed mice to UV-induced CSCC formation. These results suggest that inactivation of GPX2 in human skin may be an early event in UV-induced SCC formation. [Cancer Res 2007;67(10):4751–8]
Rap1 Integrates Tissue Polarity, Lumen Formation, and Tumorigenic Potential in Human Breast Epithelial Cells
Authors: Itoh Masahiko , Nelson Celeste M. , Myers Connie A. , Bissell Mina J.
Published: May 2007

Maintenance of apico-basal polarity in normal breast epithelial acini requires a balance between cell proliferation, cell death, and proper cell-cell and cell-extracellular matrix signaling. Aberrations in any of these processes can disrupt tissue architecture and initiate tumor formation. Here, we show that the small GTPase Rap1 is a crucial element in organizing acinar structure and inducing lumen formation. Rap1 activity in malignant HMT-3522 T4-2 cells is appreciably higher than in S1 cells, their nonmalignant counterparts. Expression of dominant-negative Rap1 resulted in phenotypic reversion of T4-2 cells, led to the formation of acinar structures with correct polarity, and dramatically reduced tumor incidence despite the persistence of genomic abnormalities and baseline growth. The resulting acini contained prominent central lumina not observed when other reverting agents were used. Conversely, expression of dominant-active Rap1 in T4-2 cells inhibited phenotypic reversion and led to increased invasiveness and tumorigenicity. Thus, Rap1 acts as a central regulator of breast architecture, with normal levels of activation instructing polarity during acinar morphogenesis, and increased activation inducing tumor formation and progression to malignancy. [Cancer Res 2007;67(10):4759–66]
Carcinoembryonic Antigen Inhibits Anoikis in Colorectal Carcinoma Cells by Interfering with Trail-R2 (DR5) Signaling
Authors: Samara Raed N. , Laguinge Luciana M. , Jessup J. Milburn
Published: May 2007

Carcinoembryonic antigen (CEA) is a tumor marker that is associated with metastasis, poor response to chemotherapy of colorectal cancer (CRC), and anoikis, a form of apoptosis caused by cell detachment from matrix that is dependent on TRAIL-R2 (DR5) and caspase-8 activation in CRC. Although CEA is a homophilic binding protein that may provide survival signals through homotypical cell aggregation, we now report that CEA binds TRAIL-R2 (DR5) directly in two-hybrid assays to decrease anoikis through the extrinsic pathway. Deletion of the PELPK sequence (delPELPK) of CEA (delPELPK CEA) restores sensitivity to anoikis while it maintains its cell aggregation function. Wild-type (WT) CEA also increases experimental hepatic metastasis, whereas the delPELPK CEA does not. Thus, membrane CEA interacts with DR5 to inhibit anoikis and increase metastatic potential in CRC. [Cancer Res 2007;67(10):4774–82]
Scavenger Receptor-A–Targeted Leukocyte Depletion Inhibits Peritoneal Ovarian Tumor Progression
Authors: Bak S. Peter , Walters Julie Jo , Takeya Motohiro , Conejo-Garcia Jose R. , Berwin Brent L.
Published: May 2007

Immunosuppressive leukocytes are emerging as a critical factor in facilitating tumor progression. These leukocytes are converted by the tumor microenvironment to become tolerogenic, facilitate metastasis, and to aid in neovascularization. The predominant variety of suppressive leukocytes found in human and murine ovarian cancer are called vascular leukocytes (VLC), due to sharing functions and cell surface markers of both dendritic cells and endothelial cells. Using the ID8 murine model of ovarian cancer, the aim of this study was to test the efficacy of VLC elimination as an ovarian tumor therapy. We show that carrageenan-mediated depletion of peritoneal tumor-associated leukocytes inhibits ovarian tumor progression. We then identified scavenger receptor-A (SR-A) as a cell surface receptor that is robustly and specifically expressed within human and murine ovarian tumor ascites upon VLCs. Administration of anti–SR-A immunotoxin to mice challenged with peritoneal ID8 tumors eliminated tumor-associated VLCs and, importantly, substantially inhibited peritoneal tumor burden and ascites accumulation. Moreover, the toxin required targeting to SR-A because mice that received untargeted toxin did not exhibit inhibition of tumor progression. We conclude that SR-A constitutes a novel and specific target for efficacious immunotherapeutic treatment of peritoneal ovarian cancer. [Cancer Res 2007;67(10):4783–9]
Loss of Functional Fas Ligand Enhances Intestinal Tumorigenesis in the Min Mouse Model
Authors: Fingleton Barbara , Carter Kathy J. , Matrisian Lynn M.
Published: May 2007

Fas ligand (FasL/CD95L), a member of the tumor necrosis factor family, interacts with a specific receptor Fas, ultimately leading to cell death. Tumor expression of FasL has been proposed to aid in immune evasion through a “Fas counterattack” mechanism but has also been described as a proinflammatory factor. Here, we tested the role of FasL in a mouse model of spontaneous tumor development. We used the Min mouse in which multiple benign polyps develop in the intestine due to a mutation in the Apc tumor suppressor gene. Mutant mice deficient in functional FasL, termed gld/gld, were crossed to Min mice to generate tumor-prone animals lacking functional FasL. Comparison of FasL-deficient versus proficient Min mice revealed a significant increase in polyp number in the gld/gld mice. We next assessed immune cell infiltration into adenomas. There was no difference in the number of either lymphocytes or macrophages; however, the number of tumor-infiltrating neutrophils was 3-fold lower in the gld/gld specimens compared with controls. Neutrophil migration in vitro was stimulated by wild-type but not mutant FasL. In a nontumor-bearing colitis model in vivo, neutrophil recruitment to the intestine was also reduced in gld/gld mice. Although the Fas counterattack hypothesis suggests that the absence of FasL would result in increased immune-mediated tumor elimination, the opposite is true in the Min model with lack of functional FasL associated with reduced neutrophil influx and increased tumor development. Thus, the proinflammatory rather than counterattack role of tumor FasL is more relevant. [Cancer Res 2007;67(10):4800–4806]
Extralymphatic Tumors Prepare Draining Lymph Nodes to Invasion via a T-Cell Cross-Tolerance Process
Authors: Preynat-Seauve Olivier , Contassot Emmanuel , Schuler Prisca , Piguet Vincent , French Lars E. , Huard Bertrand
Published: May 2007

Metastases often develop in lymphoid organs. However, the immunologic mechanism allowing such invasion is not known because these organs are considered to be hostile to tumor cells. Here, we analyzed the interactions between tumor cells and CD8+ T cells in such lymphoid organs. Tumor cells implanted into lymph nodes were able to induce tumor-specific cytotoxic CD8+ T-cell responses, conducting to tumor rejection, in contrast to primary extralymphatic tumors rapidly anergizing T cells in draining lymph nodes (DLN) via a cross-presentation process. This abortive CD8+ T-cell response to extralymphatic tumor is independent of the subcellular localization of antigen in tumor cells and is consistent with a lack of CD4+ T-cell help. Notably, this anergy was potent enough to allow successful DLN implantation of tumor cells. Such distant cross-tolerization of tumor-specific CD8+ T cells may be a determinant permissive event leading to invasion of DLN. In this situation, metastatic tumor cells do not need to down-regulate their immunogenicity to spread. [Cancer Res 2007;67(10):5009–16]
Ras Transformation of RIE-1 Cells Activates Cap-Independent Translation of Ornithine Decarboxylase: Regulation by the Raf/MEK/ERK and Phosphatidylinositol 3-Kinase Pathways
Authors: Origanti Sofia , Shantz Lisa M.
Published: May 2007

Ornithine decarboxylase (ODC) is the first and generally rate-limiting enzyme in polyamine biosynthesis. Deregulation of ODC is critical for oncogenic growth, and ODC is a target of Ras. These experiments examine translational regulation of ODC in RIE-1 cells, comparing untransformed cells with those transformed by an activated Ras12V mutant. Analysis of the ODC 5′ untranslated region (5′UTR) revealed four splice variants with the presence or absence of two intronic sequences. All four 5′UTR species were found in both cell lines; however, variants containing intronic sequences were more abundant in Ras-transformed cells. All splice variants support internal ribosome entry site (IRES)–mediated translation, and IRES activity is markedly elevated in cells transformed by Ras. Inhibition of Ras effector targets indicated that the ODC IRES element is regulated by the phosphorylation status of the translation factor eIF4E. Dephosphorylation of eIF4E by inhibition of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) or the eIF4E kinase Mnk1/2 increases ODC IRES activity in both cell lines. When both the Raf/MEK/ERK and phosphatidylinositol 3-kinase/mammalian target of rapamycin pathways are inhibited in normal cells, ODC IRES activity is very low and cells arrest in G1. When these pathways are inhibited in Ras-transformed cells, cell cycle arrest does not occur and ODC IRES activity increases, helping to maintain high ODC activity. [Cancer Res 2007;67(10):4834–42]
Expression of Nicotinic Acetylcholine Receptor Subunit Genes in Non–Small-Cell Lung Cancer Reveals Differences between Smokers and Nonsmokers
Authors: Lam David Chi-leung , Girard Luc , Ramirez Ruben , Chau Wing-shun , Suen Wai-sing , Sheridan Shelley , Tin Vicky P.C. , Chung Lap-ping , Wong Maria P. , Shay Jerry W. , Gazdar Adi F. , Lam Wah-kit , Minna John D.
Published: May 2007

Nicotine and its derivatives, by binding to nicotinic acetylcholine receptors (nAChR) on bronchial epithelial cells, can regulate cellular proliferation and apoptosis via activating the Akt pathway. Delineation of nAChR subtypes in non–small-cell lung cancers (NSCLC) may provide information for prevention or therapeutic targeting. Expression of nAChR subunit genes in 66 resected primary NSCLCs, 7 histologically non-involved lung tissues, 13 NSCLC cell lines, and 6 human bronchial epithelial cell lines (HBEC) was analyzed with quantitative PCR and microarray analysis. Five nonmalignant HBECs were exposed to nicotine in vitro to study the variation of nAChR subunit gene expression with nicotine exposure and removal. NSCLCs from nonsmokers showed higher expression of nAChR α6 (P < 0.001) and β3 (P = 0.007) subunit genes than those from smokers, adjusted for gender. In addition, nAChR α4 (P < 0.001) and β4 (P = 0.029) subunit gene expression showed significant difference between NSCLCs and normal lung. Using Affymetrix GeneChip U133 Sets, 65 differentially expressed genes associated with NSCLC nonsmoking nAChR α6β3 phenotype were identified, which gave high sensitivity and specificity of prediction. nAChR α1, α5, and α7 showed significant reversible changes in expression levels in HBECs upon nicotine exposure. We conclude that between NSCLCs from smokers and nonsmokers, different nAChR subunit gene expression patterns were found, and a 65-gene expression signature was associated with nonsmoking nAChR α6β3 expression. Finally, nicotine exposure in HBECs resulted in reversible differences in nAChR subunit gene expression. These results further implicate nicotine in bronchial carcinogenesis and suggest targeting nAChRs for prevention and therapy in lung cancer. [Cancer Res 2007;67(10):4638–47]
Dominant-Negative but not Gain-of-Function Effects of a p53.R270H Mutation in Mouse Epithelium Tissue after DNA Damage
Authors: Wijnhoven Susan W.P. , Speksnijder Ewoud N. , Liu Xiaoling , Zwart Edwin , vanOostrom Conny Th. M. , Beems Rudolf B. , Hoogervorst Esther M. , Schaap Mirjam M. , Attardi Laura D. , Jacks Tyler , van Steeg Harry , Jonkers Jos , de Vries Annemieke
Published: May 2007

p53 alterations in human tumors often involve missense mutations that may confer dominant-negative or gain-of-function properties. Dominant-negative effects result in inactivation of wild-type p53 protein in heterozygous mutant cells and as such in a p53 null phenotype. Gain-of-function effects can directly promote tumor development or metastasis through antiapoptotic mechanisms or transcriptional activation of (onco)genes. Here, we show, using conditional mouse technology, that epithelium-specific heterozygous expression of mutant p53 (i.e., the p53.R270H mutation that is equivalent to the human hotspot R273H) results in an increased incidence of spontaneous and UVB-induced skin tumors. Expression of p53.R270H exerted dominant-negative effects on latency, multiplicity, and progression status of UVB-induced but not spontaneous tumors. Surprisingly, gain-of-function properties of p53.R270H were not detected in skin epithelium. Apparently, dominant-negative and gain-of-function effects of mutant p53 are highly tissue specific and become most manifest upon stabilization of p53 after DNA damage. [Cancer Res 2007;67(10):4648–56]
Immune Mechanisms in Non–Hodgkin Lymphoma: Joint Effects of the TNF G308A and IL10 T3575A Polymorphisms with Non–Hodgkin Lymphoma Risk Factors
Authors: Wang Sophia S. , Cozen Wendy , Cerhan James R. , Colt Joanne S. , Morton Lindsay M. , Engels Eric A. , Davis Scott , Severson Richard K. , Rothman Nathaniel , Chanock Stephen J. , Hartge Patricia
Published: May 2007

Two common single nucleotide polymorphisms in immunoregulatory genes (TNF G308A, rs1800629 and IL10 T3575A, rs1800890) have been recently reported as risk factors for non–Hodgkin lymphoma (NHL) in a large pooled analysis. We systematically investigated the effects of other established NHL risk factors in relation to the tumor necrosis factor (TNF) G308A or interleukin 10 (IL10) T3575A genotypes. We calculated odds ratios (OR) and 95% confidence intervals (95% CI) from 1,172 cases and 982 population-based controls in a U.S. multicenter study. We investigated NHL overall and two common subtypes [diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma]. NHL risks were increased among those with both an autoimmune condition and the TNF G308A GA/AA (ORNHL, 2.1; 95% CI, 1.0–4.2) or the IL10 T3575A TA/AA genotype (ORNHL, 1.6; 95% CI, 0.9–2.6) compared with individuals without an autoimmune condition and with the common TNF G308A GG or IL10 T3575A TT genotype, respectively; results were similar for DLBCL and follicular lymphoma. We found that elevated DLBCL risk associated with last-born status was more pronounced among those with TNF G308A GA/AA (ORDLBCL, 2.7; 95% CI, 1.1–6.4) or IL10 T3575A TA/AA (ORDLBCL, 2.9; 95% CI, 1.6–5.2). Similarly, elevated DLBCL risk associated with obesity (body mass index, ≥35 versus <25 kg/m2) was observed only among those with TNF G308A GA/AA (ORDLBCL, 2.5; 95% CI, 1.1–5.7) or IL10 T3575A TA/AA genotypes (ORDLBCL, 2.0; 95% CI, 1.1–3.5). These exploratory results require replication but provide evidence that autoimmune conditions, late birth order, and obesity act partly through a common inflammatory pathway, posing a greater risk to individuals with variant TNF and IL10 genotypes than those with wild-type alleles. [Cancer Res 2007;67(10):5042–54]
Survivin, a Member of the Inhibitor of Apoptosis Family, Is Induced by Photodynamic Therapy and Is a Target for Improving Treatment Response
Authors: Ferrario Angela , Rucker Natalie , Wong Sam , Luna Marian , Gomer Charles J.
Published: May 2007

We observed that photodynamic therapy (PDT) induces the expression and phosphorylation of the inhibitor of apoptosis (IAP) protein survivin in murine and human cancer cells and tumors. Survivin inhibits caspase-9, blocks apoptosis, and is associated with resistance to chemotherapy and radiation. Survivin is a client protein for the 90-kDa heat shock protein (Hsp-90), and the binding of survivin to Hsp-90 assists in the maturation, proper folding, assembly, and transport of this IAP protein. A derivative of the antibiotic geldanamycin, 17-allylamino-17-demethoxygeldanamycin (17-AAG), interferes with proper binding of client proteins, such as survivin, to Hsp-90 and leads to misfolding of client proteins, ubiquination, and proteasome degradation. We hypothesized that PDT efficacy may be reduced by treatment-mediated expression and phosphorylation of survivin, and therefore, targeting the survivin pathway could increase PDT responsiveness. To address this hypothesis, we examined cellular and molecular responses following exposure to PDT, 17-AAG, and the combination of PDT plus 17-AAG in human BT-474 breast cancer cells using Photofrin and NPe6 as photosensitizers. Cells treated with the combination of PDT and 17-AAG exhibited decreased expression of the Hsp-90 client proteins phosphorylated survivin, phosphorylated Akt, and Bcl-2. The decreased expression of these client proteins was accompanied by higher apoptotic indexes and increased cytotoxicity. To confirm a specific role for survivin in modulating PDT, we used a human melanoma cell line, YUSAC2/T34A-C4, stably transfected with an inducible dominant-negative survivin gene under the control of a tetracycline-regulated (tet-off) promoter. PDT treatment of melanoma cells expressing the dominant-negative survivin resulted in increased cleavage of the caspase substrate poly(ADP-ribose) polymerase, apoptosis, and cytotoxicity when compared with results following PDT of the same melanoma cell line expressing wild-type survivin. These results show for the first time that targeting survivin and possibly other Hsp-90 client proteins improves in vitro PDT responsiveness and suggest that manipulation of the antiapoptotic pathway maintained by survivin may enhance PDT-mediated cancer therapy. [Cancer Res 2007;67(10):4989–95]
EGFR-T790M Is a Rare Lung Cancer Susceptibility Allele with Enhanced Kinase Activity
Authors: Vikis Haris , Sato Mitsuo , James Michael , Wang Daolong , Wang Yian , Wang Min , Jia Dongmei , Liu Yan , Bailey-Wilson Joan E. , Amos Christopher I. , Pinney Susan M. , Petersen Gloria M. , de Andrade Mariza , Yang Ping , Wiest Jonathan S. , Fain Pamela R. , Schwartz Ann G. , Gazdar Adi , Gaba Colette , Rothschild Henry , Mandal Diptasri , Kupert Elena , Seminara Daniela , Viswanathan Avinash , Govindan Ramaswamy , Minna John , Anderson Marshall W. , You Ming
Published: May 2007

The use of tyrosine kinase inhibitors (TKI) has yielded great success in treatment of lung adenocarcinomas. However, patients who develop resistance to TKI treatment often acquire a somatic resistance mutation (T790M) located in the catalytic cleft of the epidermal growth factor receptor (EGFR) enzyme. Recently, a report describing EGFR-T790M as a germ-line mutation suggested that this mutation may be associated with inherited susceptibility to lung cancer. Contrary to previous reports, our analysis indicates that the T790M mutation confers increased Y992 and Y1068 phosphorylation levels. In a human bronchial epithelial cell line, overexpression of EGFR-T790M displayed a growth advantage over wild-type (WT) EGFR. We also screened 237 lung cancer family probands, in addition to 45 bronchoalveolar tumors, and found that none of them contained the EGFR-T790M mutation. Our observations show that EGFR-T790M provides a proliferative advantage with respect to WT EGFR and suggest that the enhanced kinase activity of this mutant is the basis for rare cases of inherited susceptibility to lung cancer. [Cancer Res 2007;67(10):4665–70]
The Antibody-Mediated Targeted Delivery of Interleukin-15 and GM-CSF to the Tumor Neovasculature Inhibits Tumor Growth and Metastasis
Authors: Kaspar Manuela , Trachsel Eveline , Neri Dario
Published: May 2007

Tumor-targeting immunocytokines represent a new class of anticancer pharmaceutical agents, which often display a superior therapeutic index compared with the corresponding unconjugated cytokines. In this article, we have studied the anticancer properties of interleukin-15 (IL-15) and granulocyte macrophage colony-stimulating factor (GM-CSF), fused to the human antibody fragment scFv(L19), specific to the EDB domain of fibronectin, a marker of angiogenesis. The immunocytokines L19-IL-15 and L19-GM-CSF were expressed in mammalian cells and purified to homogeneity, revealing no loss of cytokine activity in in vitro assays. Furthermore, the ability of the two immunocytokines to selectively localize to tumors in vivo was confirmed by biodistribution analysis with radioiodinated protein preparations. L19-IL-15 and L19-GM-CSF displayed a potent antitumor activity both in s.c. and in metastatic F9 and C51 murine models of cancer in immunocompetent mice. This therapeutic action was superior compared with IL-15–based and GM-CSF–based fusion proteins, containing antibodies of irrelevant specificity in the mouse, which were used as non–tumor-targeting controls. For both L19-IL-15 and L19-GM-CSF immunocytokines, CD8+ T cells seemed to mostly contribute to the therapeutic action as shown by in vivo cell depletion experiments. The results presented in this article are of clinical significance, considering the fact that the sequence of EDB is identical in mouse and man and that the tumor-targeting ability of the L19 antibody has been extensively shown in clinical trials in patients with cancer. [Cancer Res 2007;67(10):4940–8]
MMP-9 Short Interfering RNA Induced Senescence Resulting in Inhibition of Medulloblastoma Growth via p16INK4a and Mitogen-Activated Protein Kinase Pathway
Authors: Rao Jasti S. , Bhoopathi Praveen , Chetty Chandramu , Gujrati Meena , Lakka Sajani S.
Published: May 2007

The involvement of matrix metalloproteinases (MMP) has been suggested in cellular mechanisms leading to medulloblastoma, the most common malignant brain tumor in children. A significant association of the expression levels of MMP-9 with survival and M stage suggests that patients with medulloblastoma metastatic disease at diagnosis may benefit from the anti-MMP therapy. Here, we have evaluated the tumorigenicity of medulloblastoma cells after infection with an adenovirus containing a 21-bp short interfering RNA sequence of the human MMP-9 gene (Ad-MMP-9). Infection of Daoy medulloblastoma cells with Ad-MMP-9 reduced MMP-9 activity and protein levels compared with parental and Ad-SV controls. Ad-MMP-9 decreased the number of viable Daoy cells in a concentration-dependent manner. Fluorescence-activated cell sorting analysis indicated that Ad-MMP-9 infection caused a dose-dependent cell cycle arrest in the G0-G1 phase. Ad-MMP-9–induced cell cycle arrest seems to be mediated by the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway and the cell cycle inhibitor p16INK4a and is phenotypically indistinguishable from senescence. Ad-MMP-9 treatment inhibited medulloblastoma tumor growth in an intracranial model and was mediated by up-regulation of p16 expression. These studies validate the usefulness of targeting MMP-9 and provide a novel perspective in the treatment of medulloblastoma. [Cancer Res 2007;67(10):4956–64]
Synergistic Activation of the Tumor Suppressor, HLJ1, by the Transcription Factors YY1 and Activator Protein 1
Authors: Wang Chi-Chung , Tsai Meng-Feng , Dai Ting-Hao , Hong Tse-Ming , Chan Wing-Kai , Chen Jeremy J.W. , Yang Pan-Chyr
Published: May 2007

HLJ1 is a novel tumor and invasion suppressor that inhibits tumorigenesis and cancer metastasis. However, the mechanism of HLJ1 activation is currently unclear. Here, we identify an enhancer segment in the HLJ1 gene at −2,125 to −1,039 bp upstream of the transcription start site. A 50-bp element between −1,492 and −1,443 bp is the minimal enhancer segment, which includes the activator protein 1 (AP-1) site (−1,457 to −1,451 bp), an essential regulatory domain that binds the transcriptional factors FosB, JunB, and JunD. Chromatin immunoprecipitation assays confirm that these AP-1 family members bind to a specific site in the HLJ1 enhancer segment in vivo. Overexpression of either YY1 at promoter or AP-1 at enhancer results in a 3-fold increase in the transcriptional activity of HLJ1. We propose a novel mechanism whereby expression of the tumor suppressor, HLJ1, is up-regulated via enhancer AP-1 binding to promoter YY1 and the coactivator, p300, through DNA bending and multiprotein complex formation. The combined expression of AP-1 and YY1 enhances HLJ1 expression by more than five times and inhibits in vitro cancer cell invasion. Elucidation of the regulatory mechanism of HLJ1 expression may facilitate the development of personalized therapy by inhibiting cancer cell proliferation, angiogenesis, and metastasis. [Cancer Res 2007;67(10):4816–26]
UGT1A1 Polymorphism Can Predict Hematologic Toxicity in Patients Treated with Irinotecan
Authors: Côté Jean-François , Kirzin Sylvain , Kramar Andrew , Mosnier Jean-François , Diebold Marie-Danièle , Soubeyran Isabelle , Thirouard Anne-Sophie , Selves Janick , Laurent-Puig Pierre , Ychou Marc
Published: May 2007

Purpose: Irinotecan (CPT-11) is approved in metastatic colorectal cancer treatment and can cause severe toxicity. The main purpose of our study was to assess the role of different polymorphisms on the occurrence of hematologic toxicities and disease-free survival in high-risk stage III colon cancer patients receiving 5-fluorouracil (5FU) and CPT-11 adjuvant chemotherapy regimen in a prospective randomized trial.

Experimental Design: Four hundred patients were randomized in a phase III trial comparing LV5FU2 to LV5FU2 + CPT-11. DNA from 184 patients was extracted and genotyped to detect nucleotide polymorphism: 3435C>T for ABCB1, 6986A>G for CYP3A5, UGT1A1*28 and −3156G>A for UGT1A1.

Results: Genotype frequencies were similar in both treatment arms. In the test arm, no significant difference was observed in toxicity or disease-free survival for ABCB1 and CYP3A5 polymorphisms. UGT1A1*28 homozygous patients showed more frequent severe hematologic toxicity (50%) than UGT1A1*1 homozygous patients (16.2%), P = 0.06. Moreover, patients homozygous for the mutant allele of −3156G>A UGT1A1 polymorphism showed more frequent severe hematologic toxicity (50%) than patients homozygous for wild-type allele (12.5%), P = 0.01. This toxicity occurred significantly earlier in homozygous mutant than wild-type homozygous patients (P = 0.043). In a Cox model, the hazard ratio for severe hematologic toxicity is significantly higher for patients with the A/A compared with the G/G genotype [hazard ratio, 8.4; 95% confidence interval, 1.9–37.2; P = 0.005].

Conclusions: This study supports the clinical utility of identification of UGT1A1 promoter polymorphisms before LV5FU2 + CPT-11 treatment to predict early hematologic toxicity. The −3156G>A polymorphism seems to be a better predictor than the UGT1A1 (TA)6TAA>(TA)7TAA polymorphism.
Src Induces Urokinase Receptor Gene Expression and Invasion/Intravasation via Activator Protein-1/p-c-Jun in Colorectal Cancer
Authors: Leupold Jörg H. , Asangani Irfan , Maurer Gabriele D. , Lengyel Ernst , Post Stefan , Allgayer Heike
Published: May 2007

The urokinase receptor [urokinase plasminogen activator receptor (u-PAR)] promotes invasion and metastasis and is associated with poor patient survival. Recently, it was shown that Src induces u-PAR gene expression via Sp1 bound to the u-PAR promoter region −152/−135. However, u-PAR is regulated by diverse promoter motifs, among them being an essential activator protein-1 (AP-1) motif at −190/−171. Moreover, an in vivo relevance of Src-induced transcriptional regulators of u-PAR–mediated invasion, in particular intravasation, and a relevance in resected patient tumors have not sufficiently been shown. The present study was conducted (a) to investigate if, in particular, AP-1–related transcriptional mediators are required for Src-induced u-PAR–gene expression, (b) to show in vivo relevance of AP-1–mediated Src-induced u-PAR gene expression for invasion/intravasation and for resected tissues from colorectal cancer patients. Src stimulation of the u-PAR promoter deleted for AP-1 region −190/−171 was reduced as compared with the wild-type promoter in cultured colon cancer cells. In gelshifts/chromatin immunoprecipitation, Src-transfected SW480 cells showed an increase of phospho–c-Jun, in addition to JunD and Fra-1, bound to region −190/−171. Src-transfected cells showed a significant increase in c-Jun phosphorylated at Ser73 and also Ser63, which was paralleled by increased phospho–c-jun-NH2-kinase. Significant decreases of invasion/in vivo intravasation (chorionallantoic membrane model) were observed in Src-overexpressing cells treated with Src inhibitors, u-PAR–small interfering RNA, and dominant negative c-Jun (TAM67). In resected tissues of 20 colorectal cancer patients, a significant correlation between Src activity, AP-1 complexes bound to u-PAR region −190/−171, and advanced pN stage were observed. These data suggest that Src-induced u-PAR gene expression and invasion/intravasation in vivo is also mediated via AP-1 region −190/−171, especially bound with c-Jun phosphorylated at Ser73/63, and that this pathway is biologically relevant for colorectal cancer patients, suggesting therapeutic potential. (Mol Cancer Res 2007;5(5):485–96)
TAT-RasGAP317-326 Requires p53 and PUMA to Sensitize Tumor Cells to Genotoxins
Authors: Michod David , Widmann Christian
Published: May 2007

Although chemotherapy has revolutionized cancer treatment, the associated side effects induced by lack of specificity to tumor cells remain a challenging problem. We have previously shown that TAT-RasGAP317-326,a cell-permeable peptide derived from RasGAP, specifically sensitizes cancer cells to the action of genotoxins. The underlying mechanisms of this sensitization were not defined however. Here, we report that TAT-RasGAP317-326 requires p53, but not the Ras effectors Akt and extracellular signal-regulated kinase, to mediate its tumor sensitization abilities. The TAT-RasGAP317-326 peptide, although not modulating the transcriptional activity of p53 or its phosphorylation and acetylation status, nevertheless requires a functional p53 cellular status to increase the sensitivity of tumor cells to genotoxins. Genes regulated by p53 encode proapoptotic proteins, such as PUMA, and cell cycle control proteins, such as p21. The ability of TAT-RasGAP317-326 to sensitize cancer cells was found to require PUMA but not p21. TAT-RasGAP317-326 did not affect PUMA levels, however, but increased genotoxin-induced mitochondrial depolarization and caspase-3 activation. These results indicate that TAT-RasGAP317-326 sensitizes tumor cells by activating signals that intersect with the p53 pathway downstream of, or at the level of, proapoptotic p53 target gene products to increase the activation of the mitochondrial death pathway. (Mol Cancer Res 2007;16(1):497–507)
Down-Regulation of Sprouty2 in Non–Small Cell Lung Cancer Contributes to Tumor Malignancy via Extracellular Signal-Regulated Kinase Pathway-Dependent and -Independent Mechanisms
Authors: Sutterlüty Hedwig , Mayer Christoph-Erik , Setinek Ulrike , Attems Johannes , Ovtcharov Slav , Mikula Mario , Mikulits Wolfgang , Micksche Michael , Berger Walter
Published: May 2007

Sprouty (Spry) proteins function as inhibitors of receptor tyrosine kinase signaling mainly by interfering with the Ras/Raf/mitogen-activated protein kinase cascade, a pathway known to be frequently deregulated in human non–small cell lung cancer (NSCLC). In this study, we show a consistently lowered Spry2 expression in NSCLC when compared with the corresponding normal lung epithelium. Based on these findings, we investigated the influence of Spry2 expression on the malignant phenotype of NSCLC cells. Ectopic expression of Spry2 antagonized mitogen-activated protein kinase activity and inhibited cell migration in cell lines homozygous for K-Ras wild type, whereas in NSCLC cells expressing mutated K-Ras, Spry2 failed to diminish extracellular signal-regulated kinase (ERK) phosphorylation. Nonetheless, Spry2 significantly reduced cell proliferation in all investigated cell lines and blocked tumor formation in mice. Accordingly, a Spry2 mutant unable to inhibit ERK phosphorylation reduced cell proliferation significantly but less pronounced compared with the wild-type protein. Therefore, we conclude that Spry2 interferes with ERK phosphorylation and another yet unidentified pathway. Our results suggest that Spry2 plays a role as tumor suppressor in NSCLC by antagonizing receptor tyrosine kinase–induced signaling at different levels, indicating feasibility for the usage of Spry in targeted gene therapy of NSCLC. (Mol Cancer Res 2007;5(5):509–20)
Assessment of Methylation Events during Colorectal Tumor Progression by Absolute Quantitative Analysis of Methylated Alleles
Authors: de Maat Michiel F.G. , Umetani Naoyuki , Sunami Eiji , Turner Roderick R. , Hoon Dave S.B.
Published: May 2007

To date, the epigenetic events involved in the progression of colorectal cancer are not well described. To study, in detail, methylation during colorectal cancer development in high-risk adenomas, we developed an assay combining in situ (on-slide) sodium bisulfite modification (SBM) of paraffin-embedded archival tissue sections with absolute quantitative assessment of methylated alleles (AQAMA). We tested the performance of the assay to detect methylation level differences between paired pre-malignant and malignant colorectal cancer stages. AQAMA assays were used to measure methylation levels at MINT (methylated in tumor) loci MINT1, MINT2, MINT12, and MINT31. Assay performance was verified on cell line DNA and standard cDNA. On-slide SBM, allowing DNA methylation assessment of 1 to 2 mm2 of paraffin-embedded archival tissue, was employed. Methylation levels of adenomatous and cancerous components within a single tissue section in 72 colorectal cancer patients were analyzed. AQAMA was verified as accurately assessing CpG island methylation status in cell lines. The correlation between expected and measured cDNA methylation levels was high for all four MINT AQAMA assays (R ≥ 0.966, P < 0.001). Methylation levels at the four loci increased in 11% and decreased in 36% of specimens comparing paired adenoma and cancer tissues (P < 0.0001 by Kolmogorov-Smirnov test). Single-PCR AQAMA provided accurate methylation level measurement. Variable MINT locus methylation level changes occur during malignant progression of colorectal adenoma. Combining AQAMA with on-slide SBM provides a sensitive assay that allows detailed histology-oriented analysis of DNA methylation levels and may give new, accurate insights into understanding development of epigenetic aberrancies in colorectal cancer progression. (Mol Cancer Res 2007;5(5):461–71)
p75 Neurotrophin Receptor Inhibits Invasion and Metastasis of Gastric Cancer
Authors: Jin Haifeng , Pan Yanglin , He Lijie , Zhai Huihong , Li Xiaohua , Zhao Lina , Sun Li , Liu Jie , Hong Liu , Song Jiugang , Xie Huahong , Gao Juan , Han Shuang , Li Ying , Fan Daiming
Published: May 2007

The p75 neurotrophin receptor (p75NTR) is a focus for study at present. However, its function in gastric cancer was not elucidated. Here, we investigated its relation with metastasis of gastric cancer. By immunohistochemistry, we found that the positive rate of p75NTR expression in metastatic gastric cancer was 15.09% (16 of 106), which was lower compared with nonmetastatic gastric cancer (64.15%; 68 of 106). The average staining score in nonmetastatic gastric cancer was significantly higher than in metastatic gastric cancer (1.21 ± 0.35 versus 0.23 ± 0.18; P < 0.01). p75NTR protein level was also lowly expressed in the highly liver-metastatic gastric cancer cell line XGC9811-L compared with other gastric cancer cell lines by Western blotting. It could also significantly inhibit the in vitro adhesive, invasive, and migratory and in vivo metastatic abilities of gastric cancer cell lines SGC7901 and MKN45 by reducing urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9 proteins and by increasing tissue inhibitor of matrix metalloproteinase (TIMP)-1 protein. Further studies showed that p75NTR could suppress the nuclear factor-κB (NF-κB) signal. SN50, a specific inhibitor of NF-κB, which could inhibit in vitro invasive and migratory abilities of gastric cancer cells, reduced expression of uPA and MMP9 proteins and increased expression of TIMP1 protein. Taken together, p75NTR had the function of inhibiting the invasive and metastatic abilities of gastric cancer cells, which was mediated, at least partially, by down-regulation of uPA and MMP9 proteins and up-regulation of TIMP1 protein via the NF-κB signal transduction pathway. Our studies suggested that p75NTR may be used as a new potential therapeutic target in metastatic gastric cancer. (Mol Cancer Res 2007;5(5):423–30)
Silencing of Cyclooxygenase-2 Inhibits Metastasis and Delays Tumor Onset of Poorly Differentiated Metastatic Breast Cancer Cells
Authors: Stasinopoulos Ioannis , O'Brien David R. , Wildes Flonne , Glunde Kristine , Bhujwalla Zaver M.
Published: May 2007

Cyclooxygenases (COX) are rate-limiting enzymes involved in the conversion of PLA2-mobilized arachidonic acid into prostaglandins and thromboxanes. COX-2 is a key mediator of inflammation during both physiologic and pathologic responses to endogenous stimuli and infectious agents. Its overexpression has been detected in different cancers, including that of the breast. Using RNA interference, we have reduced the expression of COX-2 in the highly malignant breast cancer cell line MDA-MB-231 below detectable levels in response to interleukin-1β or 12-O-tetradecanoylphorbol-13-acetate treatment. Microarray analysis showed that COX-2 silencing resulted in the loss of mRNA expression of several oncogenic markers, such as matrix metalloproteinase-1, chemokine (C-X-C motif) receptor 4, and interleukin-11, which have been correlated with poor disease outcome, and in the up-regulation of antimetastatic transcripts, such as thrombospondin-1 and Epstein-Barr-Induced 3. Cells lacking COX-2 were less able to invade reconstituted extracellular matrix than parental cells in vitro. Consistent with these changes, loss of COX-2 resulted in the abolition or the significant delay of tumor onset when the cells were injected in the mammary fat pad of severe combined immunodeficient mice. Finally, silencing of COX-2 resulted in the inhibition of metastasis to the lungs of severe combined immunodeficient mice after intravenous injection. These data show that silencing of COX-2 abolishes the metastatic potential of MDA-MB-231 cells in vivo. (Mol Cancer Res 2007;5(5):435–42)
Apolipoprotein E Interrupts Interleukin-1β Signaling in Vascular Smooth Muscle Cells
Authors: Kawamura Akira , Baitsch Daniel , Telgmann Ralph , Feuerborn Renata , Weissen-Plenz Gabriele , Hagedorn Claudia , Saku Keijiro , Brand-Herrmann Stefan-Martin , von Eckardstein Arnold , Assmann Gerd , Nofer Jerzy-Roch
Published: May 2007

Objectives—

Apolipoprotein E (apoE) exerts antiatherogenic effects but precise mechanisms remain unclear. We here investigated the effect of apoE on intracellular signaling by interleukin-1β (IL-1β), a proinflammatory cytokine present in atherosclerotic lesions.

Methods and Results—

IL-1β-induced expression and activation of inducible nitric oxide synthase and cyclooxygenase-2 were inhibited by apoE in vascular smooth muscle cells (VSMCs). These inhibitory effects were linked to the suppression of both NF-κB and activating protein-1 (AP-1) transactivation, suggesting that the interruption of IL-1β signaling occurs upstream of transcription factors. Studies in VSMCs overexpressing IL-1β signaling intermediates revealed that NF-κB transactivation was inhibited by apoE in MyD88- and IRAK1- but not in TRAF6-transfected cells. Furthermore, apoE prevented IRAK1 phosphorylation and IRAK1-TRAF6 but not MyD88-IRAK1 complex formation. Inhibitory effects of apoE on IL-1β signaling were abolished after silencing LDL receptor-related protein-1 (LRP1) expression with siRNA. In addition, inhibitors of adenylyl cyclase and protein kinase A (PKA) restored IL-1β signaling in apoE-treated VSMCs, whereas apoE stimulated PKA activity. ApoE inhibited VSMC activation in response to IL-18 but not to tumor necrosis factor-α or polyinosinic:polycytidylic acid.

Conclusion—

ApoE targets IRAK-1 activation and thereby interrupts IL-1β and IL-18 signaling in VSMCs. This antiinflammatory effect represents a novel antiatherogenic activity of apoE.
Minimal Lipidation of Pre-β HDL by ABCA1 Results in Reduced Ability to Interact with ABCA1
Authors: Mulya Anny , Lee Ji-Young , Gebre Abraham K. , Thomas Michael J. , Colvin Perry L. , Parks John S.
Published: May 2007

Objectives—

The aim of this study was to determine the role of ATP binding cassette transporter A1 (ABCA1) on generation of different-sized nascent HDLs.

Methods and Results—

HEK293 cells stably-transfected with ABCA1 (HEK293-ABCA1) or non-transfected (control) cells were incubated with lipid free
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I-apoA-I for 24 hours. Incubation of apoA-I with HEK293-ABCA1 cells, but not control cells, led to the formation of heterogeneous-sized, pre-β migrating nascent HDL subpopulations (pre-β1 to -4) that varied in size (7.1 to 15.7 nm), lipid, and apoA-I content. Kinetic studies suggested that all subpopulations were formed simultaneously, with no evidence for a precursor-product relationship between smaller and larger-sized particles. When isolated nascent pre-β HDLs (pre-β1 to -4) were added back to HEK293-ABCA1 cells, their ability to bind to ABCA1 and efflux lipid was severely compromised. Heat-denaturation of pre-β1 HDL resulted in partial recovery of ABCA1 binding, suggesting that initial interaction of apoA-I with ABCA1 results in a constrained conformation of apoA-I that decreases subsequent binding.

Conclusions—

Interaction of apoA-I with ABCA1 results in the simultaneous generation of pre-β HDLs of discrete size and chemical composition. These nascent particles are poor substrates for subsequent lipidation by ABCA1 and presumably require additional non-ABCA1-mediated lipidation for further maturation.