bluuebunny/crossref_metadata_2025 huggingface dataset:
Antitumor and Anti-inflammatory Effects of Trabectedin on Human Myxoid Liposarcoma Cells
Authors: Germano Giovanni , Frapolli Roberta , Simone Matteo , Tavecchio Michele , Erba Eugenio , Pesce Samantha , Pasqualini Fabio , Grosso Federica , Sanfilippo Roberta , Casali Paolo G. , Gronchi Alessandro , Virdis Emanuela , Tarantino Eva , Pilotti Silvana , Greco Angela , Nebuloni Manuela , Galmarini Carlos Maria , Tercero Juan Carlos , Mantovani Alberto , D'Incalci Maurizio , Allavena Paola
Published: March 2010

Inflammatory mediators present in the tumor milieu may promote cancer progression and are considered promising targets of novel biological therapies. We previously reported that the marine antitumor agent trabectedin, approved in Europe in 2007 for soft tissue sarcomas and in 2009 for ovarian cancer, was able to downmodulate the production of selected cytokines/chemokines in immune cells. Patients with myxoid liposarcoma (MLS), a subtype characterized by the expression of the oncogenic transcript FUS-CHOP, are highly responsive to trabectedin. The drug had marked antiproliferative effects on MLS cell lines at low nanomolar concentrations. We tested the hypothesis that trabectedin could also affect the inflammatory mediators produced by cancer cells. Here, we show that MLS express several cytokines, chemokines, and growth factors (CCL2, CCL3, CCL5, CXCL8, CXCL12, MIF, VEGF, SPARC) and the inflammatory and matrix-binder protein pentraxin 3 (PTX3), which build up a prominent inflammatory environment. In vitro treatment with noncytotoxic concentrations of trabectedin selectively inhibited the production of CCL2, CXCL8, IL-6, VEGF, and PTX3 by MLS primary tumor cultures and/or cell lines. A xenograft mouse model of human MLS showed marked reduction of CCL2, CXCL8, CD68+ infiltrating macrophages, CD31+ tumor vessels, and partial decrease of PTX3 after trabectedin treatment. Similar findings were observed in a patient tumor sample excised after several cycles of therapy, indicating that the results observed in vitro might have in vivo relevance. In conclusion, trabectedin has dual effects in liposarcoma: in addition to direct growth inhibition, it affects the tumor microenvironment by reducing the production of key inflammatory mediators. Cancer Res; 70(6); 2235–44
A Functional Polymorphism in the NAD(P)H Oxidase Subunit CYBA Is Related to Gene Expression, Enzyme Activity, and Outcome in Non–Hodgkin Lymphoma
Authors: Hoffmann Marion , Schirmer Markus A. , Tzvetkov Mladen V. , Kreuz Markus , Ziepert Marita , Wojnowski Leszek , Kube Dieter , Pfreundschuh Michael , Trümper Lorenz , Loeffler Markus , Brockmöller Jürgen ,   in collaboration with the German Study Group for High-Grade Non-Hodgkin Lymphoma (DSHNHL)
Published: March 2010

NAD(P)H oxidase is a major endogenous source of reactive oxygen species (ROS). ROS may not only be involved in carcinogenesis but also in efficacy of chemotherapeutic agents like doxorubicin. By a comprehensive genotyping approach covering 48 genetic polymorphisms (single-nucleotide polymorphisms) in five subunits of phagocytic NAD(P)H oxidase, we asked whether they affect gene expression, enzymatic activity, and outcome of CHO(E)P chemotherapy. A highly consistent effect was observed for the CYBA 640A>G variant. In peripheral blood granulocytes of 125 healthy volunteers, the G allele of 640A>G was associated with lower NAD(P)H oxidase activity (P = 0.006). Moreover, the G allele was associated with lower mRNA and protein expression (both P = 0.02). Of clinical importance, the outcome of patients suffering from non-Hodgkin lymphoma and treated with CHO(E)P regimen was dependent on the CYBA 640A>G polymorphism. In an exploratory study (n = 401), carriers of 640GG had an event-free survival (EFS) risk ratio of 1.95 [95% confidence interval (95% CI), 1.31–2.90; P = 0.001] compared with 640AA. In a confirmatory set (n = 477), the risk ratios were 1.53 (1.04–2.25, P = 0.03). The complete set of 878 patients showed a relative risk of 1.72 (1.30–2.26) and 1.59 (1.14–2.21) for EFS and overall survival, respectively. Further molecular-biological experiments showed lower expression and reduced stability of transcripts with the G allele in lymphoblastoid cell lines. Transfection of allele-specific plasmids into HEK293 cells elicited lower activity for the G allele in a luciferase reporter gene construct. Thus, CYBA 640A>G was shown to be a functional polymorphism with possible consequences for patients receiving CHO(E)P chemotherapy and might have further implications for other ROS-mediated modalities. Cancer Res; 70(6); 2328–38
Constitutively Active Stat3 Enhances Neu-Mediated Migration and Metastasis in Mammary Tumors via Upregulation of Cten
Authors: Barbieri Isaia , Pensa Sara , Pannellini Tania , Quaglino Elena , Maritano Diego , Demaria Marco , Voster Alessandra , Turkson James , Cavallo Federica , Watson Christine J. , Provero Paolo , Musiani Piero , Poli Valeria
Published: March 2010

The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in tumors of different origin, but the molecular bases for STAT3 requirement are only partly understood. To evaluate the contribution of enhanced Stat3 activation in a controlled model system, we generated knock-in mice wherein a mutant constitutively active Stat3C allele replaces the endogenous wild-type allele. Stat3C could enhance the tumorigenic power of the rat Neu oncogene in mouse mammary tumor virus (MMTV)–Neu transgenic mice, triggering the production of earlier onset, more invasive mammary tumors. Tumor-derived cell lines displayed higher migration, invasion, and metastatic ability and showed disrupted distribution of cell-cell junction markers mediated by Stat3-dependent overexpression of the COOH terminal tensin-like (Cten) focal adhesion protein, which was also significantly upregulated in Stat3C mammary tumors. Importantly, the proinflammatory cytokine interleukin-6 could mediate Cten induction in MCF10 cells in an exquisitely Stat3-dependent way, showing that Cten upregulation is a feature of inflammation-activated Stat3. In light of the emerging pivotal role of Stat3 in connecting inflammation and cancer, our identification of Cten as a Stat3-dependent mediator of migration provides important new insights into the oncogenic role of Stat3, particularly in the breast. Cancer Res; 70(6); 2558–67
Therapeutic Antibodies to Human L1CAM: Functional Characterization and Application in a Mouse Model for Ovarian Carcinoma
Authors: Wolterink Silke , Moldenhauer Gerhard , Fogel Mina , Kiefel Helena , Pfeifer Marco , Lüttgau Sandra , Gouveia Ricardo , Costa Julia , Endell Jan , Moebius Ulrich , Altevogt Peter
Published: March 2010

Recent work has identified L1CAM (CD171) as a novel marker for human carcinoma progression. Functionally, L1CAM promotes tumor cell invasion and motility, augments tumor growth in nude mice, and facilitates experimental tumor metastasis. These functional features qualify L1 as an interesting target molecule for tumor therapy. Here, we generated a series of novel monoclonal antibodies (mAb) to the L1CAM ectodomain that were characterized by biochemical and functional means. All novel mAbs reacted specifically with L1CAM and not with the closely related molecule CHL1, whereas antibodies to the COOH terminal part of L1CAM (mAb2C2, mAb745H7, pcytL1) showed cross-reactivity. Among the novel mAbs, L1-9.3 was selected and its therapeutic potential was analyzed in various isotype variants in a model of SKOV3ip cells growing i.p. in CD1 nude mice. Only therapy with the IgG2a variant efficiently prolonged survival and reduced tumor burden. This was accompanied by an increased infiltration of F4/80-positive monocytic cells. Clodronate pretreatment of tumor-bearing animals led to the depletion of monocytes and abolished the therapeutic effect of L1-9.3/IgG2a. Expression profiling of tumor-derived mRNA revealed that L1-9.3/IgG2a therapy induced altered expression of cellular genes associated with apoptosis and tumor growth. Our results establish that anti-L1 mAb therapy acts via immunologic and nonimmunologic effector mechanism to block tumor growth. The novel antibodies to L1CAM could become helpful tools for the therapy of L1-positive human carcinomas. Cancer Res; 70(6); 2504–15
Critical Roles of Mucin 1 Glycosylation by Transactivated PolypeptideN-Acetylgalactosaminyltransferase 6 in Mammary Carcinogenesis
Authors: Park Jae-Hyun , Nishidate Toshihiko , Kijima Kyoko , Ohashi Takao , Takegawa Kaoru , Fujikane Tomoko , Hirata Koichi , Nakamura Yusuke , Katagiri Toyomasa
Published: March 2010

The structure of O-glycosylated proteins is altered in breast cancer cells, but the mechanisms of such an aberrant modification have been largely unknown. We here report critical roles of a novel druggable target, polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), which is upregulated in a great majority of breast cancers and encodes a glycosyltransferase responsible for initiating mucin-type O-glycosylation. Knockdown of GALNT6 by small interfering RNA significantly enhanced cell adhesion function and suppressed the growth of breast cancer cells. Western blot and immunostaining analyses indicated that wild-type GALNT6 protein could glycosylate and stabilize an oncoprotein mucin 1 (MUC1), which was upregulated with GALNT6 in breast cancer specimens. Furthermore, knockdown of GALNT6 or MUC1 led to similar morphologic changes of cancer cells accompanied by the increase of cell adhesion molecules β-catenin and E-cadherin. Our findings implied that overexpression of GALNT6 might contribute to mammary carcinogenesis through aberrant glycosylation and stabilization of MUC1 and that screening of GALNT6 inhibitors would be valuable for the development of novel therapeutic modalities against breast cancer. Cancer Res; 70(7); 2759–69
A Large Prospective Study of Meat Consumption and Colorectal Cancer Risk: An Investigation of Potential Mechanisms Underlying this Association
Authors: Cross Amanda J. , Ferrucci Leah M. , Risch Adam , Graubard Barry I. , Ward Mary H. , Park Yikyung , Hollenbeck Albert R. , Schatzkin Arthur , Sinha Rashmi
Published: March 2010

Although the relation between red and processed meat intake and colorectal cancer has been reported in several epidemiologic studies, very few investigated the potential mechanisms. This study examined multiple potential mechanisms in a large U.S. prospective cohort with a detailed questionnaire on meat type and meat cooking methods linked to databases for estimating intake of mutagens formed in meats cooked at high temperatures (heterocyclic amines, polycyclic aromatic hydrocarbons), heme iron, nitrate, and nitrite. During 7 years of follow-up, 2,719 colorectal cancer cases were ascertained from a cohort of 300,948 men and women. The hazard ratios (HR) and 95% confidence intervals (95% CI) comparing the fifth to the first quintile for both red (HR, 1.24; 95% CI, 1.09–1.42; Ptrend < 0.001) and processed meat (HR, 1.16; 95% CI, 1.01–1.32; Ptrend = 0.017) intakes indicated an elevated risk for colorectal cancer. The potential mechanisms for this relation include heme iron (HR, 1.13; 95% CI, 0.99–1.29; Ptrend = 0.022), nitrate from processed meats (HR, 1.16; 95% CI, 1.02–1.32; Ptrend = 0.001), and heterocyclic amine intake [HR, 1.19; 95% CI, 1.05–1.34; Ptrend < 0.001 for 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and HR, 1.17; 95% CI, 1.05–1.29; Ptrend <0.001 for 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx)]. In general, the elevated risks were higher for rectal cancer than for colon cancer, with the exception of MeIQx and DiMeIQx, which were only associated with colon cancer. In conclusion, we found a positive association for red and processed meat intake and colorectal cancer; heme iron, nitrate/nitrite, and heterocyclic amines from meat may explain these associations. Cancer Res; 70(6); 2406–14
Antineoplastic Mechanisms of Niclosamide in Acute Myelogenous Leukemia Stem Cells: Inactivation of the NF-κB Pathway and Generation of Reactive Oxygen Species
Authors: Jin Yanli , Lu Zhongzheng , Ding Ke , Li Juan , Du Xin , Chen Chun , Sun Xiaoyong , Wu Yongbin , Zhou Jing , Pan Jingxuan
Published: March 2010

NF-κB may be a potential therapeutic target for acute myelogenous leukemia (AML) because NF-κB activation is found in primitive human AML blast cells. In this report, we initially discovered that the potent antineoplastic effect of niclosamide, a Food and Drug Administration–approved antihelminthic agent, was through inhibition of the NF-κB pathway in AML cells. Niclosamide inhibited the transcription and DNA binding of NF-κB. It blocked tumor necrosis factor–induced IκBα phosphorylation, translocation of p65, and expression of NF-κB–regulated genes. Niclosamide inhibited the steps TAK1→IκB kinase (IKK) and IKK→IκBα. Niclosamide also increased the levels of reactive oxygen species (ROS) in AML cells. Quenching ROS by the glutathione precursor N-acetylcysteine attenuated niclosamide-induced apoptosis. Our results together suggest that niclosamide inhibited the NF-κB pathway and increased ROS levels to induce apoptosis in AML cells. On translational study of the efficacy of niclosamide against AML, niclosamide killed progenitor/stem cells from AML patients but spared those from normal bone marrow. Niclosamide was synergistic with the frontline chemotherapeutic agents cytarabine, etoposide, and daunorubicin. It potently inhibited the growth of AML cells in vitro and in nude mice. Our results support further investigation of niclosamide in clinical trials of AML patients. Cancer Res; 70(6); 2516–27
Annexin 1 Induced by Anti-Inflammatory Drugs Binds to NF-κB and Inhibits Its Activation: Anticancer Effects In vitro and In vivo
Authors: Zhang Zhiquan , Huang Liqun , Zhao Wenping , Rigas Basil
Published: March 2010

Annexin A1 (ANXA1), a mediator of the anti-inflammatory action of glucocorticoids, is important in cancer development and progression, whereas NF-κB regulates multiple cellular phenomena, some of them associated with inflammation and cancer. We showed that glucocorticoids and chemopreventive modified nonsteroidal anti-inflammatory drugs, such as nitric oxide–donating aspirin (NO-ASA) and phospho-aspirin, induced ANXA1 in cultured human colon and pancreatic cancer cells. ANXA1 associated with NF-κB and suppressed its transcriptional activity by preventing NF-κB binding to DNA. The induction of ANXA1 by glucocorticoids was proportional to their anti-inflammatory potency, as was the suppression of NF-κB activity, which was accompanied by enhanced apoptosis and inhibition of cell growth mediated by changes in NF-κB–dependent cell signaling. The proposed novel mechanism was operational in the intestinal mucosa of mice treated with dexamethasone or NO-ASA. ANXA1-based oligopeptides displayed the same effects as ANXA1 on NF-κB. One such tripeptide (Gln-Ala-Trp) administered to nude mice inhibited the growth of SW480 human colon cancer xenografts by 58% compared with control (P < 0.01). Our findings reveal that ANXA1 is an inducible endogenous inhibitor of NF-κB in human cancer cells and mice, provide a novel molecular mechanism for the action of anti-inflammatory agents, and suggest the possibility of mechanism-driven drug development. Cancer Res; 70(6); 2379–88
Role of Epidermal Growth Factor Receptor Degradation in Cisplatin-Induced Cytotoxicity in Head and Neck Cancer
Authors: Ahsan Aarif , Hiniker Susan M. , Ramanand Susmita G. , Nyati Shyam , Hegde Ashok , Helman Abigail , Menawat Radhika , Bhojani Mahaveer S. , Lawrence Theodore S. , Nyati Mukesh K.
Published: March 2010

Cisplatin and its analogues are the most commonly used agents in the treatment of head and neck squamous cell carcinoma. In this study, we investigated a possible role of epidermal growth factor (EGF) receptor (EGFR) phosphorylation and degradation in cisplatin-induced cytotoxicity. Cisplatin treatment led to an increase in initial EGFR phosphorylation at Y1045, the binding site of ubiquitin ligase, Casitas B-lineage lymphoma (c-Cbl), followed by ubiquitination in the relatively cisplatin-sensitive cell lines. However, cisplatin-resistant cell lines underwent minimal EGFR phosphorylation at the Y1045 site and minimal ubiquitination. We found that EGFR degradation in response to cisplatin was highly correlated with cytotoxicity in seven head and neck cancer cell lines. Pretreatment with EGF enhanced cisplatin-induced EGFR degradation and cytotoxicity, whereas erlotinib pretreatment blocked EGFR phosphorylation, degradation, and cisplatin-induced cytotoxicity. Expression of a mutant Y1045F EGFR, which is relatively resistant to c-Cbl–mediated degradation, in Chinese hamster ovary cells and the UMSCC11B human head and neck cancer cell line protected EGFR from cisplatin-induced degradation and enhanced cell survival compared with wild-type (WT) EGFR. Transfection of WT c-Cbl enhanced EGFR degradation and cisplatin-induced cytotoxicity compared with control vector. These results show that cisplatin-induced EGFR phosphorylation and subsequent ubiquitination and degradation is an important determinant of cisplatin sensitivity. Our findings suggest that treatment with an EGFR inhibitor before cisplatin would be antagonistic, as EGFR inhibition would protect EGFR from cisplatin-mediated phosphorylation and subsequent ubiquitination and degradation, which may explain the negative results of several recent clinical trials. Furthermore, they suggest that EGFR degradation is worth exploring as an early biomarker of response and as a target to improve outcome. Cancer Res; 70(7); 2862–9
PEA-15 Inhibits Tumorigenesis in an MDA-MB-468 Triple-Negative Breast Cancer Xenograft Model through Increased Cytoplasmic Localization of Activated Extracellular Signal-Regulated Kinase
Authors: Bartholomeusz Chandra , Gonzalez-Angulo Ana M. , Kazansky Anna , Krishnamurthy Savitri , Liu Ping , Yuan Linda X. H. , Yamasaki Fumiyuki , Liu Shuying , Hayashi Naoki , Zhang Dongwei , Esteva Francisco J. , Hortobagyi Gabriel N. , Ueno Naoto T.
Published: March 2010

Purpose: To determine the role of PEA-15 in breast cancer.

Experimental Design: A reverse-phase protein array was used to measure PEA-15 expression levels in 320 human breast cancers; these levels were correlated with clinical and tumor characteristics. PEA-15 was overexpressed by an adenovirus vector or by stably expressing PEA-15 in different breast cancer cell lines. The effects on breast cancer cell survival and on the downstream apoptotic signaling pathway were measured in terms of cell proliferation (trypan blue for cell viability, bromodeoxyuridine incorporation for DNA synthesis), anchorage-independent growth (soft agar colony formation), and apoptosis (fluorescence-activated cell sorter analysis). The preclinical efficacy of Ad.PEA-15 given intratumorally was evaluated in nude mice bearing tumors from s.c. implanted human MDA-MB-468 triple-negative breast cancer cells.

Results: In human breast cancers, low levels of PEA-15 expression correlated with high nuclear grade (P < 0.0001) and with negative hormone receptor status (P = 0.0004). Overexpression of PEA-15 in breast cancer cells resulted in growth inhibition, reduction in DNA synthesis, and onset of caspase-8–dependent apoptosis. In athymic nude mice bearing MDA-MB-468 xenografts, tumor volumes were significantly smaller in mice treated intratumorally with Ad.PEA-15 than in control mice (P < 0.0001). Tumors from mice treated with Ad.PEA-15 had increased levels of activated (phosphorylated) extracellular signal-regulated kinase and reduced levels of Ki-67 compared with tumors from nontreated or control-adenovirus–treated mice.

Conclusion: PEA-15 has therapeutic potential in breast cancer. Further preclinical and clinical exploration of PEA-15 as a druggable target is warranted. Clin Cancer Res; 16(6); 1802–11
Phase I Pharmacokinetic and Pharmacodynamic Study of the Oral MAPK/ERK Kinase Inhibitor PD-0325901 in Patients with Advanced Cancers
Authors: LoRusso Patricia M. , Krishnamurthi Smitha S. , Rinehart John J. , Nabell Lisle M. , Malburg Lisa , Chapman Paul B. , DePrimo Samuel E. , Bentivegna Steven , Wilner Keith D. , Tan Weiwei , Ricart Alejandro D.
Published: March 2010

Purpose: To determine tolerability, pharmacokinetics, and pharmacodynamics of PD-0325901, a highly potent, selective, oral mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/2 inhibitor in advanced cancer patients.

Experimental Design: Sixty-six patients received PD-0325901 at doses from 1 mg once daily to 30 mg twice daily (BID). Cycles were 28 days; three administration schedules were evaluated. Pharmacokinetic parameters were assessed and tumor biopsies were done to evaluate pharmacodynamics.

Results: Common adverse events were rash, diarrhea, fatigue, nausea, and visual disturbances including retinal vein occlusion (RVO; n = 3). Neurotoxicity was frequent in patients receiving ≥15 mg BID. The maximum tolerated dose, 15 mg BID continuously, was associated with late-onset RVO outside the dose-limiting toxicity window. An alternative dose and schedule, 10 mg BID 5 days on/2 days off, was therefore expanded; one RVO event occurred. Three of 48 evaluable patients with melanoma achieved confirmed partial responses; 10 had stable disease ≥4 months. PD-0325901 exposure was generally dose proportional. Doses ≥2 mg BID consistently caused ≥60% suppression of phosphorylated ERK in melanoma. Fifteen patients showed significant decreases (≥50%) in Ki-67.

Conclusions: PD-0325901 showed preliminary clinical activity. The maximum tolerated dose, based on first cycle dose-limiting toxicities, was 15 mg BID continuously. However, 10 and 15 mg BID continuous dosing and 10 mg BID 5 days on/2 days off schedules were associated with delayed development of RVO; thus, further enrollment to this trial was stopped. Intermittent dose scheduling between 2 and 10 mg BID should be explored to identify a recommended dose with long-term PD-0325901 use. Clin Cancer Res; 16(6); 1924–37
Approaches to Phase 1 Clinical Trial Design Focused on Safety, Efficiency, and Selected Patient Populations: A Report from the Clinical Trial Design Task Force of the National Cancer Institute Investigational Drug Steering Committee
Authors: Ivy S. Percy , Siu Lillian L. , Garrett-Mayer Elizabeth , Rubinstein Larry
Published: March 2010

The goals and objectives of phase 1 clinical trials are changing to include further evaluation of endpoints such as molecular targeted effects, in addition to dose-toxicity profile of the investigational agent. Because of these changes in focus, the National Cancer Institute and Investigational Drug Steering Committee's Task Force on Clinical Trial Design met to evaluate the most efficient ways to design and implement early clinical trials with novel therapeutics. Clinical approaches discussed included the conventional 3 + 3 cohort expansion phase 1 design, multi-institutional phase 1 studies, accelerated titration designs, continual reassessment methods, the study of specific target patient populations, and phase 0 studies. Each of these approaches uniquely contributes to some aspect of the phase 1 study, with all focused on dose and schedule determination, patient safety, and limited patient exposure to ineffective doses of investigational agent. The benefit of labor-intensive generation of preliminary biomarker evidence of target inhibition, as well as the value of molecular profiling of the study population, is considered. New drug development is expensive and the failure rate remains high. By identifying patient populations expected to respond to the study agent and tailoring the treatment with a novel drug, investigators will be one step closer to personalizing cancer treatment. The “fail early and fast” approach is acceptable if the appropriate patient population is evaluated in the phase 1 trial. The approaches outlined in this overview address the merits, advantages, disadvantages, and obstacles encountered during first in human studies. Clin Cancer Res; 16(6); 1726–36
Phase II, Randomized Trial to Compare Anastrozole Combined with Gefitinib or Placebo in Postmenopausal Women with Hormone Receptor–Positive Metastatic Breast Cancer
Authors: Cristofanilli Massimo , Valero Vicente , Mangalik Aroop , Royce Melanie , Rabinowitz Ian , Arena Francis P. , Kroener Joan F. , Curcio Elizabeth , Watkins Claire , Bacus Sarah , Cora Elsa M. , Anderson Elizabeth , Magill Patrick J.
Published: March 2010

Purpose: This phase II randomized trial evaluated the efficacy and tolerability of anastrozole combined with gefitinib or anastrozole with placebo in women with hormone receptor–positive metastatic breast cancer (MBC).

Experimental Design: Postmenopausal women with hormone receptor–positive measurable or evaluable MBC who had not received prior endocrine therapy for this disease stage or who developed metastatic disease during/after adjuvant tamoxifen were eligible. The primary response variable was progression-free survival (PFS) and secondary response variables included clinical benefit rate, objective response rate, overall survival, safety and tolerability, and pharmacokinetics. Tumor biomarker evaluation was an exploratory objective.

Results: Forty-three patients were randomized to anastrozole plus gefitinib and 50 patients were randomized to anastrozole plus placebo of a planned total of 174 patients (enrollment was prematurely discontinued due to slow recruitment). PFS for patients receiving the combination of anastrozole and gefitinib was longer than for patients receiving anastrozole plus placebo [hazard ratio (gefitinib/placebo), 0.55; 95% confidence interval, 0.32-0.94; median PFS, 14.7 versus 8.4 months]. The clinical benefit rate was 49% versus 34%, and the objective response rate was 2% versus 12% with anastrozole plus gefitinib and anastrozole plus placebo, respectively. No evidence of interaction between baseline biomarker levels and relative treatment effect was found. No unexpected adverse events were observed.

Conclusion: This small randomized study showed that anastrozole in combination with gefitinib is associated with a marked advantage in PFS compared with anastrozole plus placebo, and that the combination was tolerated in postmenopausal women with hormone receptor–positive MBC. Further investigation of epidermal growth factor receptor inhibition in combination with endocrine therapy may be warranted. Clin Cancer Res; 16(6); 1904–14
GermlineBRCAMutations Denote a Clinicopathologic Subset of Prostate Cancer
Authors: Gallagher David J. , Gaudet Mia M. , Pal Prodipto , Kirchhoff Tomas , Balistreri Lisa , Vora Kinjal , Bhatia Jasmine , Stadler Zsofia , Fine Samson W. , Reuter Victor , Zelefsky Michael , Morris Michael J. , Scher Howard I. , Klein Robert J. , Norton Larry , Eastham James A. , Scardino Peter T. , Robson Mark E. , Offit Kenneth
Published: March 2010

Purpose: Increased prostate cancer risk has been reported for BRCA mutation carriers, but BRCA-associated clinicopathologic features have not been clearly defined.

Experimental Design: We determined BRCA mutation prevalence in 832 Ashkenazi Jewish men diagnosed with localized prostate cancer between 1988 and 2007 and 454 Ashkenazi Jewish controls and compared clinical outcome measures among 26 BRCA mutation carriers and 806 noncarriers. Kruskal-Wallis tests were used to compare age of diagnosis and Gleason score, and logistic regression models were used to determine associations between carrier status, prostate cancer risk, and Gleason score. Hazard ratios (HR) for clinical end points were estimated using Cox proportional hazards models.

Results: BRCA2 mutations were associated with a 3-fold risk of prostate cancer [odds ratio, 3.18; 95% confidence interval (95% CI), 1.52-6.66; P = 0.002] and presented with more poorly differentiated (Gleason score ≥7) tumors (85% versus 57%; P = 0.0002) compared with non–BRCA-associated prostate cancer. BRCA1 mutations conferred no increased risk. After 7,254 person-years of follow-up, and adjusting for clinical stage, prostate-specific antigen, Gleason score, and treatment, BRCA2 and BRCA1 mutation carriers had a higher risk of recurrence [HR (95% CI), 2.41 (1.23-4.75) and 4.32 (1.31-13.62), respectively] and prostate cancer–specific death [HR (95% CI), 5.48 (2.03-14.79) and 5.16 (1.09-24.53), respectively] than noncarriers.

Conclusions: BRCA2 mutation carriers had an increased risk of prostate cancer and a higher histologic grade, and BRCA1 or BRCA2 mutations were associated with a more aggressive clinical course. These results may have implications for tailoring clinical management of this subset of hereditary prostate cancer. Clin Cancer Res; 16(7); 2115–21. ©2010 AACR.
The Oncogene DEK Promotes Leukemic Cell Survival and Is Downregulated by both Nutlin-3 and Chlorambucil in B-Chronic Lymphocytic Leukemic Cells
Authors: Secchiero Paola , Voltan Rebecca , di Iasio Maria Grazia , Melloni Elisabetta , Tiribelli Mario , Zauli Giorgio
Published: March 2010

Purpose: To characterize the role of the oncogene DEK in modulating the response to either Nutlin-3, a small-molecule inhibitor of the MDM2/p53 interaction, or chlorambucil in primary B-chronic lymphocytic leukemia (B-CLL) cells.

Experimental Design: DEK mRNA and protein levels were evaluated in primary B-CLL samples (n = 21), p53wild-type SKW6.4, p53mutated BJAB lymphoblastoid cell lines, and normal CD19+ B lymphocytes–treated Nutlin-3 or chlorambucil (10 μmol/L, each). Knocking down experiments with either p53 or DEK small interfering RNA (siRNA) were done to investigate the potential role of p53 in controlling the expression of DEK and the role of DEK in leukemic cell survival/apoptosis.

Results: Both Nutlin-3 and chlorambucil downregulated DEK in primary B-CLL samples (n = 21) and SKW6.4 but not in BJAB cells. Knocking down p53 attenuated the effect of Nutlin-3 on DEK expression, whereas knocking down DEK significantly increased both spontaneous and Nutlin-3–induced apoptosis. Conversely, counteracting DEK downmodulation by using p53 small interfering RNA reduced Nutlin-3–mediated apoptosis. On the other hand, Nutlin-3 potently induced p53 accumulation, but it did not affect DEK levels in normal CD19+ B lymphocytes.

Conclusions: These data show that the downregulation of DEK in response to either Nutlin-3 or chlorambucil represents an important molecular determinant in the cytotoxic response of leukemic cells, and suggest that strategies aimed to downregulate DEK might improve the therapeutic potential of these drugs. Clin Cancer Res; 16(6); 1824–33
Targeted Inhibition of Inducible Nitric Oxide Synthase Inhibits Growth of Human Melanoma In vivo and Synergizes with Chemotherapy
Authors: Sikora Andrew G. , Gelbard Alexander , Davies Michael A. , Sano Daisuke , Ekmekcioglu Suhendan , Kwon John , Hailemichael Yared , Jayaraman Padmini , Myers Jeffrey N. , Grimm Elizabeth A. , Overwijk Willem W.
Published: March 2010

Purpose: Aberrant expression of inflammatory molecules, such as inducible nitric oxide (NO) synthase (iNOS), has been linked to cancer, suggesting that their inhibition is a rational therapeutic approach. Whereas iNOS expression in melanoma and other cancers is associated with poor clinical prognosis, in vitro and in vivo studies suggest that iNOS and NO can have both protumor and antitumor effects. We tested the hypothesis that targeted iNOS inhibition would interfere with human melanoma growth and survival in vivo in a preclinical model.

Experimental Design: We used an immunodeficient non-obese diabetic/severe combined immunodeficient xenograft model to test the susceptibility of two different human melanoma lines to the orally-given iNOS-selective small molecule antagonist N6-(1-iminoethyl)-l-lysine-dihydrochloride (L-nil) with and without cytotoxic cisplatin chemotherapy.

Results: L-nil significantly inhibited melanoma growth and extended the survival of tumor-bearing mice. L-nil treatment decreased the density of CD31+ microvessels and increased the number of apoptotic cells in tumor xenografts. Proteomic analysis of melanoma xenografts with reverse-phase protein array identified alterations in the expression of multiple cell signaling and survival genes after L-nil treatment. The canonical antiapoptotic protein Bcl-2 was downregulated in vivo and in vitro after L-nil treatment, which was associated with increased susceptibility to cisplatin-mediated tumor death. Consistent with this observation, combination therapy with L-nil plus cisplatin in vivo was more effective than either drug alone, without increased toxicity.

Conclusions: These data support the hypothesis that iNOS and iNOS-derived NO support tumor growth in vivo and provide convincing preclinical validation of targeted iNOS inhibition as therapy for solid tumors. Clin Cancer Res; 16(6); 1834–44
The Design of Phase II Clinical Trials Testing Cancer Therapeutics: Consensus Recommendations from the Clinical Trial Design Task Force of the National Cancer Institute Investigational Drug Steering Committee
Authors: Seymour Lesley , Ivy S. Percy , Sargent Daniel , Spriggs David , Baker Laurence , Rubinstein Larry , Ratain Mark J. , Le Blanc Michael , Stewart David , Crowley John , Groshen Susan , Humphrey Jeffrey S. , West Pamela , Berry Donald
Published: March 2010

The optimal design of phase II studies continues to be the subject of vigorous debate, especially studies of newer molecularly targeted agents. The observations that many new therapeutics “fail” in definitive phase III studies, coupled with the numbers of new agents to be tested as well as the increasing costs and complexity of clinical trials, further emphasize the critical importance of robust and efficient phase II design. The Clinical Trial Design Task Force (CTD-TF) of the National Cancer Institute (NCI) Investigational Drug Steering Committee (IDSC) has published a series of discussion papers on phase II trial design in Clinical Cancer Research. The IDSC has developed formal recommendations about aspects of phase II trial design that are the subject of frequent debate, such as endpoints (response versus progression-free survival), randomization (single-arm designs versus randomization), inclusion of biomarkers, biomarker-based patient enrichment strategies, and statistical design (e.g., two-stage designs versus multiple-group adaptive designs). Although these recommendations in general encourage the use of progression-free survival as the primary endpoint, randomization, inclusion of biomarkers, and incorporation of newer designs, we acknowledge that objective response as an endpoint and single-arm designs remain relevant in certain situations. The design of any clinical trial should always be carefully evaluated and justified based on characteristic specific to the situation. Clin Cancer Res; 16(6); 1764–9
Activation of Wnt Signaling Pathway by Human Papillomavirus E6 and E7 Oncogenes in HPV16-Positive Oropharyngeal Squamous Carcinoma Cells
Authors: Rampias Theodore , Boutati Eleni , Pectasides Eirini , Sasaki Clarence , Kountourakis Panteleimon , Weinberger Paul , Psyrri Amanda
Published: March 2010

We sought to determine the role of human papillomavirus (HPV) E6 and E7 oncogenes in nuclear β-catenin accumulation, a hallmark of activated canonical Wnt signaling pathway. We used HPV16-positive oropharyngeal cancer cell lines 147T and 090, HPV-negative cell line 040T, and cervical cell lines SiHa (bearing integrated HPV16) and HeLa (bearing integrated HPV18) to measure the cytoplasmic and nuclear β-catenin levels and the β-catenin/Tcf transcriptional activity before and after E6/E7 gene silencing. Repression of HPV E6 and E7 genes induced a substantial reduction in nuclear β-catenin levels. Luciferase assay showed that transcriptional activation of Tcf promoter by β-catenin was lower after silencing. The protein levels of β-catenin are tightly regulated by the ubiquitin/proteasome system. We therefore performed expression analysis of regulators of β-catenin degradation and nuclear transport and showed that seven in absentia homologue (Siah-1) mRNA and protein levels were substantially upregulated after E6/E7 repression. Siah-1 protein promotes the degradation of β-catenin through the ubiquitin/proteasome system. To determine whether Siah-1 is important for the proteasomal degradation of β-catenin in HPV16-positive oropharyngeal cancer cells, we introduced a Siah-1 expression vector into 147T and 090 cells and found substantial reduction of endogenous β-catenin in these cells. Thus, E6 and E7 are involved in β-catenin nuclear accumulation and activation of Wnt signaling in HPV-induced cancers. In addition, we show the significance of the endogenous Siah-1–dependent ubiquitin/proteasome pathway for β-catenin degradation and its regulation by E6/E7 viral oncoproteins in HPV16-positive oropharyngeal cancer cells. Mol Cancer Res; 8(3); 433–43
The Bloom Syndrome Protein Limits the Lethality Associated with RAD51 Deficiency
Authors: Lahkim Bennani-Belhaj Kenza , Rouzeau Sébastien , Buhagiar-Labarchède Géraldine , Chabosseau Pauline , Onclercq-Delic Rosine , Bayart Emilie , Cordelières Fabrice , Couturier Jérôme , Amor-Guéret Mounira
Published: March 2010

Little is known about the functional interaction between the Bloom's syndrome protein (BLM) and the recombinase RAD51 within cells. Using RNA interference technology, we provide the first demonstration that RAD51 acts upstream from BLM to prevent anaphase bridge formation. RAD51 downregulation was associated with an increase in the frequency of BLM-positive anaphase bridges, but not of BLM-associated ultrafine bridges. Time-lapse live microscopy analysis of anaphase bridge cells revealed that BLM promoted cell survival in the absence of Rad51. Our results directly implicate BLM in limiting the lethality associated with RAD51 deficiency through the processing of anaphase bridges resulting from the RAD51 defect. These findings provide insight into the molecular basis of some cancers possibly associated with variants of the RAD51 gene family. Mol Cancer Res; 8(3); 385–94
Prediction of Discharge Destination after Neurological Rehabilitation in Stroke Patients
Authors: Frank Matthias , Conzelmann Martin , Engelter Stefan
Published: March 2010

Background/Aims: Returning home is one of the most important aims of stroke patients when admitted to rehabilitation. Methods: A single-center prospectively ascertained database study was conducted. Results: Among 1,332 eligible patients (median age = 76.5 years), 828 (62.2%) returned home. Multiple logistic regression revealed 5 independent predictors: independent sitting balance, higher motor and social-cognitive functional independence measure subscores (all on admission to rehabilitation), living with a partner and younger age. The area under the curve (AUC) of this model was 0.86 (95% confidence interval (CI) 0.84–0.88). When age was excluded from the model, the AUC remained virtually the same (AUC = 0.85, 95% CI = 0.83–0.87). Conclusion: The discharge destination could be predicted in a majority of patients with easily available parameters. Availability of a partner as well as parameters linked to severity of the neurological and functional deficit were both important. Age alone adds very little to the predictive power of these variables, therefore access to rehabilitation should not be denied on grounds of age.
Pulmonary Small Cell Carcinoma Metastatic to the Ovary: A Clinicopathologic Study of One Case with Emphasis on the Importance of P53 Analysis in Diagnosis
Authors: Garcia Virginia , Velasco Ana , Gatius Sonia , Gonzalez F. Javier , Matias-Guiu Xavier
Published: March 2010

Background: Small cell carcinoma of the lung (SCCL) is rarely associated with ovarian metastases. Only occasional cases have been reported so far. Since SCCL is increasing in women because of smoking, the frequency of ovarian metastases from SCCL may increase in the near future. Case: A 54-year-old woman with SCCL presented with a left ovarian mass, 4.8 cm in diameter. The microscopic appearance of the ovarian tumor was identical to that of the previous bronchoscopic biopsy. Molecular analysis of p53 demonstrated an identical point mutation (S215) in both tumor sites. Moreover, a p53 DNA polymorphism (P52R) was identified in normal tissue, but present in homozygosity in both tumor sites. Conclusion: In this article, we report a new example of SCCL metastatic to the ovary, and we discuss the main features for differential diagnosis with primary ovarian small cell carcinoma, pulmonary type, with emphasis on the value of p53 analysis.
Highly Conserved Asp-204 and Gly-776 Are Important for Activity of the Quinoprotein Glucose Dehydrogenase of Escherichia coli and for Mineral Phosphate Solubilization
Authors: Sashidhar Burla , Inampudi Krishna Kishore , Guruprasad Lalitha , Kondreddy Anil , Gopinath Kodetham , Podile Appa Rao
Published: March 2010

Gram-negative bacteria membrane-bound glucose dehydrogenase (m-GDH) has pyrroloquinoline quinone [PQQ (2,7,9,-tricarboxyl-1H-pyrrolo[2,3-f]quinoline-4,5-dione)] as its prosthetic group, transferring electrons to ubiquinone (UQ) in the membrane. Based on the sequence homology of the C-terminal catalytic domain (151–796 amino acid residues) we have modeled the 3D structure of Escherichia coli GDH. The geometrical parameters of the homology model structure, validated using the Ramachandran plot, revealed 95.8% of residues in the allowed regions and 2.2% of the residues in disallowed regions. From the model, we have identified five different amino acids that are specifically involved in maintaining the PQQ in the correct configuration along with a Ca2+ ion in the active site, and two amino acids on the surface of the protein that might be involved in UQ binding or transfer of electrons to the UQ. Site-directed mutants R201A, D204A, E217L, E217A, R266Q, R266E, E591L, E591Q, E591K, L712W, L712R, G776K, G776D and G776L lost their GDH activity, while E217Q and G776A retained their function similar to that of wild-type GDH, both in terms of specific activity and mineral phosphate solubilization. Our conclusions are consistent with those previously based on model GDH produced by a different method and using a different template X-ray structure.
Macrophage Response to Apoptotic Cells Varies with the Apoptotic Trigger and Is Not Altered by a Deficiency in LRP Expression
Authors: Kozmar Ana , Greenlee-Wacker Mallary C. , Bohlson Suzanne S.
Published: March 2010

Rapid engulfment of apoptotic cells in the absence of inflammation is required for maintenance of normal tissue homeostasis. The low-density lipoprotein receptor-related protein-1 (LRP/CD91) is a receptor mediating interactions between macrophages and apoptotic cells, but recent reports have challenged the requirement of this surface protein in this process. To explore the role of LRP in the recognition of apoptotic cells, target cells were generated with two distinct inducers of apoptotic cell death, etoposide and actinomycin-D. Jurkat T cells rendered apoptotic with etoposide exposed phosphatidylserine (PtdSer) and triggered engulfment by murine bone marrow-derived macrophages (BMDM), however they failed to suppress lipopolysaccharide-driven inflammatory cytokine secretion or, correspondingly, NFĸB-dependent or TNFα promoter-driven transcriptional activity in transfected RAW264.7 macrophages. In contrast, induction of apoptosis in either Jurkat cells or HeLa epithelial cells with actinomycin-D resulted in diminution of proinflammatory signaling from RAW264.7 cells and BMDM. Treatment of actinomycin-treated Jurkat cells with Q-VD-OPh, an irreversible inhibitor of caspase activity, blocked apoptosis, as assessed by the inhibition of PtdSer exposure; however, the cells maintained anti-inflammatory activity. Anti-inflammatory signaling mediated by actinomycin-treated cells was not affected by a macrophage-specific deletion in LRP. Moreover, the presence of LRP on macrophages did not alter the efficiency of engulfment of apoptotic cells in vitro or in vivo. These data demonstrate that the method of induction of apoptosis of target cells influences subsequent macrophage responsiveness, and that LRP is not required for engulfment of apoptotic cells regardless of the method of induction.
In Cystic Fibrosis Homozygotes and Heterozygotes, Neutrophil Apoptosis Is Delayed and Modulated by Diamide or Roscovitine: Evidence for an Innate Neutrophil Disturbance
Authors: Moriceau Sandra , Lenoir Gérard , Witko-Sarsat Véronique
Published: March 2010

Cystic fibrosis (CF) is a chronic inflammatory lung disease characterized by polymorphonuclear neutrophil (PMN)-dominated airway inflammation. Defective apoptosis might explain PMN persistence at these inflammation sites. We previously reported that in CF patients PMN underwent delayed apoptosis, which was not always related to their infectious state and independent of the type of CF transmembrane regulator (CFTR) mutation. To understand the role of infection and PMN apoptosis in CF, PMN apoptosis was investigated in CF parents who are obligate heterozygotes for the CFTR mutation but without chronic bacterial infection. They also demonstrated delayed PMN apoptosis compared with healthy controls, as assessed by annexin-V labeling and caspase-3 cleavage. Diamide, a direct thiol-oxidizing agent, potentiated PMN apoptosis in controls and CF patients, resulting in similar levels of constitutive and Fas-potentiated apoptosis. The cyclin-dependent kinase inhibitor roscovitine provided another approach to restore normal PMN apoptosis. However, the selective CFTR inhibitor CFTRInh172 did not affect PMN apoptosis in control subjects. Apparently, the dysregulation of CF PMN is not only a consequence of the chronic infectious state in CF children but might also be related to CF ‘intrinsic’ factors. Restoration of normal PMN apoptosis by cellular redox modulation or roscovitine opens new research avenues to decrease PMN-mediated inflammation in CF.
Homocysteine induces caspase activation by endoplasmic reticulum stress in platelets from type 2 diabetics and healthy donors
Authors: Zbidi Hanene , Redondo Pedro , López Jose , Bartegi Aghleb , Salido Gines , Rosado Juan
Published: March 2010

Diabetes mellitus is a disease characterised by hyperglycaemia and associated with several cardiovascular disorders, including angiopathy and platelet hyperactivity, which are major causes of morbidity and mortality in type 2 diabetes mellitus. In type 2 diabetic patients, homo-cysteine levels are significantly increased compared with healthy subjects. Hyperhomocysteinaemia is an independent risk factor for macro-and microangiopathy and mortality. The present study is aimed to investigate the effect of homocysteine on platelet apoptosis. Changes in cytosolic or intraluminal free Ca2+ concentration were determined by fluorimetry. Caspase activity and phosphorylation of the eukaryotic initiation factor 2α (eIF2α) were explored by Western blot. Our results indicate that homocysteine releases Ca2+ from agonist sensitive stores, enhances eIF2α phosphorylation at Ser51 and activates caspase-3 and -9 independently of extracellular Ca2+. Homocysteine induced activation of caspase-3 and -9 was abolished by salubrinal, an agent that prevents endoplasmic reticulum (ER) stress-induced apoptosis. Homo-cysteine-induced platelet effects were significantly greater in type 2 diabetics than in healthy subjects. These findings demonstrate that homocysteine induces ER stress-mediated apoptosis in human platelets, an event that is enhanced in type 2 diabetic patients, which might be involved in the pathogenesis of cardiovascular complications associated with type 2 diabetes mellitus.