bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of Autophagy Defect with a Malignant Phenotype and Poor Prognosis of Hepatocellular Carcinoma
Authors: Ding Zhen-Bin , Shi Ying-Hong , Zhou Jian , Qiu Shuang-Jian , Xu Yang , Dai Zhi , Shi Guo-Ming , Wang Xiao-Ying , Ke Ai-Wu , Wu Bin , Fan Jia
Published: November 2008

Hepatocellular carcinoma (HCC) is an aggressive cancer with a poor prognosis. The role of autophagy and the prognostic value of autophagic genes are largely unknown in HCC. Here, we showed decreased expression of autophagic genes and their corresponding autophagic activity and increased expression of the antiapoptotic gene Bcl-xL in HCC cell lines compared with a normal hepatic cell line. We also found decreased expression of the autophagic gene Beclin 1 in 44 HCC tissue samples compared with adjacent nontumor tissues. In addition, we found that the most aggressive malignant HCC cell lines and HCC tissues with recurrent disease displayed much lower autophagic levels, especially when Bcl-xL was overexpressed. Interestingly, in a tissue microarray study consisting of 300 HCC patients who underwent curative resection, the expression of Beclin 1 was only significantly correlated with disease-free survival (DFS; P < 0.0001) and overall survival (OS; P < 0.0001) in the Bcl-xL+ group. Multivariate and univariate analyses also revealed that Beclin 1 expression was an independent predictor for DFS and OS in Bcl-xL+ patients. In addition, we found a significant correlation between Beclin 1 expression and tumor differentiation in Bcl-xL+ but not in Bcl-xL− HCC patients. In conclusion, our data showed expression of autophagic genes and their corresponding autophagic activities were suppressed in HCC. The autophagy defects synergized with altered apoptotic activity might facilitate tumor malignant differentiation, which results in a more aggressive cancer cell phenotype and poor prognosis of HCC. [Cancer Res 2008;68(22):9167–75]
Hepatocyte Growth Factor Induces Gefitinib Resistance of Lung Adenocarcinoma with Epidermal Growth Factor Receptor–Activating Mutations
Authors: Yano Seiji , Wang Wei , Li Qi , Matsumoto Kunio , Sakurama Haruko , Nakamura Takahiro , Ogino Hirokazu , Kakiuchi Soji , Hanibuchi Masaki , Nishioka Yasuhiko , Uehara Hisanori , Mitsudomi Tetsuya , Yatabe Yasushi , Nakamura Toshikazu , Sone Saburo
Published: November 2008

Lung cancer with epidermal growth factor receptor (EGFR)–activating mutations responds favorably to the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. However, 25% to 30% of patients with EGFR-activating mutations show intrinsic resistance, and the responders invariably acquire resistance to gefitinib. Here, we showed that hepatocyte growth factor (HGF), a ligand of MET oncoprotein, induces gefitinib resistance of lung adenocarcinoma cells with EGFR-activating mutations by restoring the phosphatidylinositol 3-kinase/Akt signaling pathway via phosphorylation of MET, but not EGFR or ErbB3. Strong immunoreactivity for HGF in cancer cells was detected in lung adenocarcinoma patients harboring EGFR-activating mutations, but no T790M mutation or MET amplification, who showed intrinsic or acquired resistance to gefitinib. The findings indicate that HGF-mediated MET activation is a novel mechanism of gefitinib resistance in lung adenocarcinoma with EGFR-activating mutations. Therefore, inhibition of HGF-MET signaling may be a considerable strategy for more successful treatment with gefitinib. [Cancer Res 2008;68(22):9479–87]
“Active” Cancer Immunotherapy by Anti-Met Antibody Gene Transfer
Authors: Vigna Elisa , Pacchiana Giovanni , Mazzone Massimiliano , Chiriaco Cristina , Fontani Lara , Basilico Cristina , Pennacchietti Selma , Comoglio Paolo M.
Published: November 2008

Gene therapy provides a still poorly explored opportunity to treat cancer by “active” immunotherapy as it enables the transfer of genes encoding antibodies directed against specific oncogenic proteins. By a bidirectional lentiviral vector, we transferred the cDNA encoding the heavy and light chains of a monoclonal anti-Met antibody (DN-30) to epithelial cancer cells. In vitro, the transduced cells synthesized and secreted correctly assembled antibodies with the expected high affinity, inducing down-regulation of the Met receptor and strong inhibition of the invasive growth response. The inhibitory activity resulted (a) from the interference of the antibody with the Met receptor intracellular processing (“cell autonomous activity,” in cis) and (b) from the antibody-induced cleavage of Met expressed at the cell surface (“bystander effect,” in trans). The monoclonal antibody gene transferred into live animals by systemic administration or by local intratumor delivery resulted in substantial inhibition of tumor growth. These data provide proof of concept both for targeting the Met receptor and for a gene transfer–based immunotherapy strategy. [Cancer Res 2008;68(22):9176–83]
Identification of Alternative Splicing Markers for Breast Cancer
Authors: Venables Julian P. , Klinck Roscoe , Bramard Anne , Inkel Lyna , Dufresne-Martin Geneviève , Koh ChuShin , Gervais-Bird Julien , Lapointe Elvy , Froehlich Ulrike , Durand Mathieu , Gendron Daniel , Brosseau Jean-Philippe , Thibault Philippe , Lucier Jean-Francois , Tremblay Karine , Prinos Panagiotis , Wellinger Raymund J. , Chabot Benoit , Rancourt Claudine , Elela Sherif Abou
Published: November 2008

Breast cancer is the most common cause of cancer death among women under age 50 years, so it is imperative to identify molecular markers to improve diagnosis and prognosis of this disease. Here, we present a new approach for the identification of breast cancer markers that does not measure gene expression but instead uses the ratio of alternatively spliced mRNAs as its indicator. Using a high-throughput reverse transcription-PCR–based system for splicing annotation, we monitored the alternative splicing profiles of 600 cancer-associated genes in a panel of 21 normal and 26 cancerous breast tissues. We validated 41 alternative splicing events that significantly differed in breast tumors relative to normal breast tissues. Most cancer-specific changes in splicing that disrupt known protein domains support an increase in cell proliferation or survival consistent with a functional role for alternative splicing in cancer. In a blind screen, a classifier based on the 12 best cancer-associated splicing events correctly identified cancer tissues with 96% accuracy. Moreover, a subset of these alternative splicing events could order tissues according to histopathologic grade, and 5 markers were validated in a further blind set of 19 grade 1 and 19 grade 3 tumor samples. These results provide a simple alternative for the classification of normal and cancerous breast tumor tissues and underscore the putative role of alternative splicing in the biology of cancer. [Cancer Res 2008;68(22):9525–31]
Targeting HER2-Positive Breast Cancer with Trastuzumab-DM1, an Antibody–Cytotoxic Drug Conjugate
Authors: Lewis Phillips Gail D. , Li Guangmin , Dugger Debra L. , Crocker Lisa M. , Parsons Kathryn L. , Mai Elaine , Blättler Walter A. , Lambert John M. , Chari Ravi V.J. , Lutz Robert J. , Wong Wai Lee T. , Jacobson Frederic S. , Koeppen Hartmut , Schwall Ralph H. , Kenkare-Mitra Sara R. , Spencer Susan D. , Sliwkowski Mark X.
Published: November 2008

HER2 is a validated target in breast cancer therapy. Two drugs are currently approved for HER2-positive breast cancer: trastuzumab (Herceptin), introduced in 1998, and lapatinib (Tykerb), in 2007. Despite these advances, some patients progress through therapy and succumb to their disease. A variation on antibody-targeted therapy is utilization of antibodies to deliver cytotoxic agents specifically to antigen-expressing tumors. We determined in vitro and in vivo efficacy, pharmacokinetics, and toxicity of trastuzumab-maytansinoid (microtubule-depolymerizing agents) conjugates using disulfide and thioether linkers. Antiproliferative effects of trastuzumab-maytansinoid conjugates were evaluated on cultured normal and tumor cells. In vivo activity was determined in mouse breast cancer models, and toxicity was assessed in rats as measured by body weight loss. Surprisingly, trastuzumab linked to DM1 through a nonreducible thioether linkage (SMCC), displayed superior activity compared with unconjugated trastuzumab or trastuzumab linked to other maytansinoids through disulfide linkers. Serum concentrations of trastuzumab-MCC-DM1 remained elevated compared with other conjugates, and toxicity in rats was negligible compared with free DM1 or trastuzumab linked to DM1 through a reducible linker. Potent activity was observed on all HER2-overexpressing tumor cells, whereas nontransformed cells and tumor cell lines with normal HER2 expression were unaffected. In addition, trastuzumab-DM1 was active on HER2-overexpressing, trastuzumab-refractory tumors. In summary, trastuzumab-DM1 shows greater activity compared with nonconjugated trastuzumab while maintaining selectivity for HER2-overexpressing tumor cells. Because trastuzumab linked to DM1 through a nonreducible linker offers improved efficacy and pharmacokinetics and reduced toxicity over the reducible disulfide linkers evaluated, trastuzumab-MCC-DM1 was selected for clinical development. [Cancer Res 2008;68(22):9280–90]
A Prospective Study of Meat and Fat Intake in Relation to Small Intestinal Cancer
Authors: Cross Amanda J. , Leitzmann Michael F. , Subar Amy F. , Thompson Frances E. , Hollenbeck Albert R. , Schatzkin Arthur
Published: November 2008

Diets high in red and processed meats are associated with carcinogenesis of the large intestine, but no prospective study has examined meat and fat intake in relation to cancer of the small intestine. We prospectively investigated meat and fat intakes, estimated from a food frequency questionnaire, in relation to small intestinal cancer among half a million men and women enrolled in the NIH-AARP Diet and Health Study. We used Cox proportional hazards regression to estimate hazard ratios (HR) and 95% confidence intervals (95% CI). During up to 8 years of follow-up, 60 adenocarcinomas and 80 carcinoid tumors of the small intestine were diagnosed. Despite slightly elevated HRs for red meat, there were no clear associations for red or processed meat intake and either adenocarcinoma or carcinoid tumors of the small intestine. In contrast, we noted a markedly elevated risk for carcinoid tumors of the small intestine with saturated fat intake in both the categorical (highest versus lowest tertile: HR, 3.18; 95% CI, 1.62–6.25) and continuous data (HR, 3.72; 95% CI, 1.79–7.74 for each 10-g increase in intake per 1,000 kcal). Our findings suggest that the positive associations for meat intake reported in previous case-control studies may partly be explained by saturated fat intake.[Cancer Res 2008;68(22):9274–9]
Palomid 529, a Novel Small-Molecule Drug, Is a TORC1/TORC2 Inhibitor That Reduces Tumor Growth, Tumor Angiogenesis, and Vascular Permeability
Authors: Xue Qi , Hopkins Benjamin , Perruzzi Carole , Udayakumar Durga , Sherris David , Benjamin Laura E.
Published: November 2008

It has become clear that the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is central for promoting both tumor and tumor stroma and is therefore a major target for anticancer drug development. First- and second-generation rapalogs (prototypical mTOR inhibitors) have shown promise but, due to the complex nature of mTOR signaling, can result in counterproductive feedback signaling to potentiate upstream Akt signaling. We present a novel PI3K/Akt/mTOR inhibitor, Palomid 529 (P529), which inhibits the TORC1 and TORC2 complexes and shows both inhibition of Akt signaling and mTOR signaling similarly in tumor and vasculature. We show that P529 inhibits tumor growth, angiogenesis, and vascular permeability. It retains the beneficial aspects of tumor vascular normalization that rapamycin boasts. However, P529 has the additional benefit of blocking pAktS473 signaling consistent with blocking TORC2 in all cells and thus bypassing feedback loops that lead to increased Akt signaling in some tumor cells. [Cancer Res 2008;68(22):9551–7]
Ku80 Deletion Suppresses Spontaneous Tumors and Induces a p53-Mediated DNA Damage Response
Authors: Holcomb Valerie B. , Rodier Francis , Choi YongJun , Busuttil Rita A. , Vogel Hannes , Vijg Jan , Campisi Judith , Hasty Paul
Published: November 2008

Ku80 facilitates DNA repair and therefore should suppress cancer. However, ku80−/− mice exhibit reduced cancer, although they age prematurely and have a shortened life span. We tested the hypothesis that Ku80 deletion suppresses cancer by enhancing cellular tumor-suppressive responses to inefficiently repaired DNA damage. In support of this hypothesis, Ku80 deletion ameliorated tumor burden in APCMIN mice and increased a p53-mediated DNA damage response, DNA lesions, and chromosomal rearrangements. Thus, contrary to its assumed role as a caretaker tumor suppressor, Ku80 facilitates tumor growth most likely by dampening baseline cellular DNA damage responses. [Cancer Res 2008;68(22):9497–502]
Serotonin Metabolism Is Dysregulated in Cholangiocarcinoma, which Has Implications for Tumor Growth
Authors: Alpini Gianfranco , Invernizzi Pietro , Gaudio Eugenio , Venter Julie , Kopriva Shelley , Bernuzzi Francesca , Onori Paolo , Franchitto Antonio , Coufal Monique , Frampton Gabriel , Alvaro Domenico , Lee Sum P. , Marzioni Marco , Benedetti Antonio , DeMorrow Sharon
Published: November 2008

Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. Symptoms are usually evident after blockage of the bile duct by the tumor, and at this late stage, they are relatively resistant to chemotherapy and radiation therapy. Therefore, it is imperative that alternative treatment options are explored. We present novel data indicating that the metabolism of serotonin is dysregulated in cholangiocarcinoma cell lines, compared with normal cholangiocytes, and tissue and bile from cholangiocarcinoma patients. Specifically, there was an increased expression of tryptophan hydroxylase 1 and a suppression of monoamine oxidase A expression (enzymes responsible for the synthesis and degradation of serotonin, respectively) in cholangiocarcinoma. This resulted in an increased secretion of serotonin from cholangiocarcinoma and increased serotonin in the bile from cholangiocarcinoma patients. Increased local serotonin release may have implications on cholangiocarcinoma cell growth. Serotonin administration increased cholangiocarcinoma cell growth in vitro, whereas inhibition of serotonin synthesis decreases tumor cell growth both in vitro and in vivo. The data presented here represent the first evidence that serotonin metabolism is dysregulated in cholangiocarcinoma and that modulation of serotonin synthesis may represent an alternative target for the development of therapeutic strategies. [Cancer Res 2008;68(22):9184–93]
The Role of Parental and Grandparental Epigenetic Alterations in Familial Cancer Risk
Authors: Fleming Jessica L. , Huang Tim H-M. , Toland Amanda Ewart
Published: November 2008

Epigenetic alterations of the genome such as DNA promoter methylation and chromatin remodeling play an important role in tumorigenesis. These modifications take place throughout development with subsequent events occurring later in adulthood. Recent studies, however, suggest that some epigenetic alterations that influence cancer risk are inherited through the germline from parent to child and are observed in multiple generations. Epigenetic changes may be inherited as Mendelian, non-Mendelian, or environmentally induced traits. Here, we will discuss Mendelian, non-Mendelian, and environmentally induced patterns of multigenerational epigenetic alterations as well as some possible mechanisms for how these events may be occurring. [Cancer Res 2008;68(22):9116–21]
Genetic Predictors of MEK Dependence in Non–Small Cell Lung Cancer
Authors: Pratilas Christine A. , Hanrahan Aphrothiti J. , Halilovic Ensar , Persaud Yogindra , Soh Junichi , Chitale Dhananjay , Shigematsu Hisayuki , Yamamoto Hiromasa , Sawai Ayana , Janakiraman Manickam , Taylor Barry S. , Pao William , Toyooka Shinichi , Ladanyi Marc , Gazdar Adi , Rosen Neal , Solit David B.
Published: November 2008

Hyperactivated extracellular signal-regulated kinase (ERK) signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS, and upstream receptor tyrosine kinases. To characterize the mitogen-activated protein kinase/ERK kinase (MEK)/ERK dependence of lung cancers harboring BRAF kinase domain mutations, we screened a large panel of human lung cancer cell lines (n = 87) and tumors (n = 916) for BRAF mutations. We found that non–small cell lung cancers (NSCLC) cells with both V600E and non-V600E BRAF mutations were selectively sensitive to MEK inhibition compared with those harboring mutations in epidermal growth factor receptor (EGFR), KRAS, or ALK and ROS kinase fusions. Supporting its classification as a “driver” mutation in the cells in which it is expressed, MEK inhibition in V600EBRAF NSCLC cells led to substantial induction of apoptosis, comparable with that seen with EGFR kinase inhibition in EGFR mutant NSCLC models. Despite high basal ERK phosphorylation, EGFR mutant cells were uniformly resistant to MEK inhibition. Conversely, BRAF mutant cell lines were resistant to EGFR inhibition. These data, together with the nonoverlapping pattern of EGFR and BRAF mutations in human lung cancer, suggest that these lesions define distinct clinical entities whose treatment should be guided by prospective real-time genotyping. To facilitate such an effort, we developed a mass spectrometry-based genotyping method for the detection of hotspot mutations in BRAF, KRAS, and EGFR. Using this assay, we confirmed that BRAF mutations can be identified in a minority of NSCLC tumors and that patients whose tumors harbor BRAF mutations have a distinct clinical profile compared with those whose tumors harbor kinase domain mutations in EGFR. [Cancer Res 2008;68(22):9375–83]
PEA-15 Induces Autophagy in Human Ovarian Cancer Cells and Is Associated with Prolonged Overall Survival
Authors: Bartholomeusz Chandra , Rosen Daniel , Wei Caimiao , Kazansky Anna , Yamasaki Fumiyuki , Takahashi Takeshi , Itamochi Hiroaki , Kondo Seiji , Liu Jinsong , Ueno Naoto T.
Published: November 2008

Phospho-enriched protein in astrocytes (PEA-15) is a 15-kDa phosphoprotein that slows cell proliferation by binding to and sequestering extracellular signal–regulated kinase (ERK) in the cytoplasm, thereby inhibiting ERK-dependent transcription and proliferation. In previous studies of E1A human gene therapy for ovarian cancer, we discovered that PEA-15 induced the antitumor effect of E1A by sequestering activated ERK in the cytoplasm of cancer cells. Here, we investigated the role of PEA-15 in ovarian cancer tumorigenesis, the expression levels of PEA-15 in human ovarian cancer, and whether PEA-15 expression correlated with overall survival in women with ovarian cancer. We overexpressed PEA-15 in low-PEA-15-expressing cells and knocked down PEA-15 in high-PEA-15-expressing cells and analyzed the effects on proliferation, anchorage-independent growth, and cell cycle progression. We then assessed PEA-15 expression in an annotated tissue microarray of tumor samples from 395 women with primary epithelial ovarian cancer and tested whether PEA-15 expression was linked with overall survival. PEA-15 expression inhibited proliferation, and cell cycle analysis did not reveal apoptosis but did reveal autophagy, which was confirmed by an increase in LC3 cleavage. Inhibition of the ERK1/2 pathway decreased PEA-15–induced autophagy. These findings suggest that the antitumor activity of PEA-15 is mediated, in part, by the induction of autophagy involving activation of the ERK1/2 pathway. Multivariable analyses indicated that the women with high-PEA-15-expressing tumors survived longer than those with low-PEA-15-expressing tumors (hazard ratio, 1.973; P = 0.0167). Our findings indicate that PEA-15 expression is an important prognostic marker in ovarian cancer. [Cancer Res 2008;68(22):9302–10]
Targeting of the Bmi-1 Oncogene/Stem Cell Renewal Factor by MicroRNA-128 Inhibits Glioma Proliferation and Self-Renewal
Authors: Godlewski Jakub , Nowicki Michal O. , Bronisz Agnieszka , Williams Shanté , Otsuki Akihiro , Nuovo Gerard , RayChaudhury Abhik , Newton Herbert B. , Chiocca E. Antonio , Lawler Sean
Published: November 2008

MicroRNAs (miR) show characteristic expression signatures in various cancers and can profoundly affect cancer cell behavior. We carried out miR expression profiling of human glioblastoma specimens versus adjacent brain devoid of tumor. This revealed several significant alterations, including a pronounced reduction of miR-128 in tumor samples. miR-128 expression significantly reduced glioma cell proliferation in vitro and glioma xenograft growth in vivo. miR-128 caused a striking decrease in expression of the Bmi-1 oncogene, by direct regulation of the Bmi-1 mRNA 3′-untranslated region, through a single miR-128 binding site. In a panel of patient glioblastoma specimens, Bmi-1 expression was significantly up-regulated and miR-128 was down-regulated compared with normal brain. Bmi-1 functions in epigenetic silencing of certain genes through epigenetic chromatin modification. We found that miR-128 expression caused a decrease in histone methylation (H3K27me3) and Akt phosphorylation, and up-regulation of p21CIP1 levels, consistent with Bmi-1 down-regulation. Bmi-1 has also been shown to promote stem cell self-renewal; therefore, we investigated the effects of miR-128 overexpression in human glioma neurosphere cultures, possessing features of glioma “stem-like” cells. This showed that miR-128 specifically blocked glioma self-renewal consistent with Bmi-1 down-regulation. This is the first example of specific regulation by a miR of a neural stem cell self-renewal factor, implicating miRs that may normally regulate brain development as important biological and therapeutic targets against the “stem cell–like” characteristics of glioma. [Cancer Res 2008;68(22):9125–30]
Synergistic Proapoptotic Activity of Recombinant TRAIL Plus the Akt Inhibitor Perifosine in Acute Myelogenous Leukemia Cells
Authors: Tazzari Pier Luigi , Tabellini Giovanna , Ricci Francesca , Papa Veronica , Bortul Roberta , Chiarini Francesca , Evangelisti Camilla , Martinelli Giovanni , Bontadini Andrea , Cocco Lucio , McCubrey James A. , Martelli Alberto M.
Published: November 2008

To potentiate the response of acute myelogenous leukemia (AML) cells to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) cytotoxicity, we have examined the efficacy of a combination with perifosine, a novel phosphatidylinositol-3-kinase (PI3K)/Akt signaling inhibitor. The rationale for using such a combination is that perifosine was recently described to increase TRAIL-R2 receptor expression and decrease the cellular FLICE-inhibitory protein (cFLIP) in human lung cancer cell lines. Perifosine and TRAIL both induced cell death by apoptosis in the THP-1 AML cell line, which is characterized by constitutive PI3K/Akt activation, but lacks functional p53. Perifosine, at concentrations below IC50, dephosphorylated Akt and increased TRAIL-R2 levels, as shown by Western blot, reverse transcription-PCR, and flow cytometric analysis. Perifosine also decreased the long isoform of cFLIP (cFLIP-L) and the X-linked inhibitor of apoptosis protein (XIAP) expression. Perifosine and TRAIL synergized to activate caspase-8 and induce apoptosis, which was blocked by a caspase-8–selective inhibitor. Up-regulation of TRAIL-R2 expression was dependent on a protein kinase Cα/c-Jun-NH2-kinase 2/c-Jun signaling pathway activated by perifosine through reactive oxygen species production. Perifosine also synergized with TRAIL in primary AML cells displaying constitutive activation of the Akt pathway by inducing apoptosis, Akt dephosphorylation, TRAIL-R2 up-regulation, cFLIP-L and XIAP down-regulation, and c-Jun phosphorylation. The combined treatment negatively affected the clonogenic activity of CD34+ cells from patients with AML. In contrast, CD34+ cells from healthy donors were resistant to perifosine and TRAIL treatment. Our findings suggest that the combination of perifosine and TRAIL might offer a novel therapeutic strategy for AML. [Cancer Res 2008;68(22):9394–403]
Preferential Cytotoxicity of Bortezomib toward Hypoxic Tumor Cells via Overactivation of Endoplasmic Reticulum Stress Pathways
Authors: Fels Diane R. , Ye Jiangbin , Segan Andrew T. , Kridel Steven J. , Spiotto Michael , Olson Michael , Koong Albert C. , Koumenis Constantinos
Published: November 2008

Hypoxia is a dynamic feature of the tumor microenvironment that contributes to drug resistance and cancer progression. We previously showed that components of the unfolded protein response (UPR), elicited by endoplasmic reticulum (ER) stress, are also activated by hypoxia in vitro and in vivo animal and human patient tumors. Here, we report that ER stressors, such as thapsigargin or the clinically used proteasome inhibitor bortezomib, exhibit significantly higher cytotoxicity toward hypoxic compared with normoxic tumor cells, which is accompanied by enhanced activation of UPR effectors in vitro and UPR reporter activity in vivo. Treatment of cells with the translation inhibitor cycloheximide, which relieves ER load, ameliorated this enhanced cytotoxicity, indicating that the increased cytotoxicity is ER stress–dependent. The mode of cell death was cell type–dependent, because DLD1 colorectal carcinoma cells exhibited enhanced apoptosis, whereas HeLa cervical carcinoma cells activated autophagy, blocked apoptosis, and eventually led to necrosis. Pharmacologic or genetic ablation of autophagy increased the levels of apoptosis. These results show that hypoxic tumor cells, which are generally more resistant to genotoxic agents, are hypersensitive to proteasome inhibitors and suggest that combining bortezomib with therapies that target the normoxic fraction of human tumors can lead to more effective tumor control. [Cancer Res 2008;68(22):9323–30]
Humanized Radioiodinated Minibody For Imaging of Prostate Stem Cell Antigen–Expressing Tumors
Authors: Leyton Jeffrey V. , Olafsen Tove , Lepin Eric J. , Hahm Scott , Bauer Karl B. , Reiter Robert E. , Wu Anna M.
Published: November 2008

Purpose: Prostate stem cell antigen (PSCA) is a cell surface glycoprotein that is overexpressed in prostate cancer, including hormone refractory disease. Previous preclinical studies showed the intact anti-PSCA antibodies, 1G8 and hu1G8, localized specifically to PSCA-expressing xenografts. Optimal micro positron emission tomography (microPET) imaging using hu1G8, however, required a delay of 168 hours postinjection. In this study, the 2B3 minibody (an 80-kDa engineered antibody fragment) has been produced for rapid targeting and imaging.

Experimental Design: A gene encoding a PSCA-specific minibody, VL-linker-VH-hinge-huIgG1 CH3, was assembled. The minibody was expressed by secretion from mammalian cells and purified by cation exchange chromatography. Relative affinity and specificity were determined by competition ELISA and flow cytometry. Serial microPET imaging using a 124I-labeled minibody was conducted at 4 and 21 hours in mice bearing LAPC-9 AD, LAPC-9 AI, PC-3, and LNCaP-PSCA human prostate cancer xenografts. Tumor and tissue biodistribution was determined, and region of interest analysis of the images was conducted.

Results: Yields of 20 mg/L purified 2B3 minibody were obtained that showed specific binding to LNCaP-PSCA cells. Purified 2B3 minibody showed specific binding to LNCaP-PSCA cells with an apparent affinity of 46 nmol/L. Radioiodinated 2B3 minibody showed rapid nontarget tissue and blood clearance kinetics (t1/2β = 11.2 hours). MicroPET scanning using the 124I-2B3 minibody showed both androgen-dependent and -independent tumors as early as 4 hours and excellent high contrast images at 21 hours postinjection.

Conclusions: Imaging PSCA-positive prostate cancer is feasible using an intermediate size antibody fragment at 21 hours.
Molecular Imaging of Therapeutic Response to Epidermal Growth Factor Receptor Blockade in Colorectal Cancer
Authors: Manning H. Charles , Merchant Nipun B. , Foutch A. Coe , Virostko John M. , Wyatt Shelby K. , Shah Chirayu , McKinley Eliot T. , Xie Jingping , Mutic Nathan J. , Washington M. Kay , LaFleur Bonnie , Tantawy Mohammed Noor , Peterson Todd E. , Ansari M. Sib , Baldwin Ronald M. , Rothenberg Mace L. , Bornhop Darryl J. , Gore John C. , Coffey Robert J.
Published: November 2008

Purpose: To evaluate noninvasive molecular imaging methods as correlative biomarkers of therapeutic efficacy of cetuximab in human colorectal cancer cell line xenografts grown in athymic nude mice. The correlation between molecular imaging and immunohistochemical analysis to quantify epidermal growth factor (EGF) binding, apoptosis, and proliferation was evaluated in treated and untreated tumor-bearing cohorts.

Experimental Design: Optical imaging probes targeting EGF receptor (EGFR) expression (NIR800-EGF) and apoptosis (NIR700-Annexin V) were synthesized and evaluated in vitro and in vivo. Proliferation was assessed by 3′-[18F]fluoro-3′-deoxythymidine ([18F]FLT) positron emission tomography. Assessment of inhibition of EGFR signaling by cetuximab was accomplished by concomitant imaging of NIR800-EGF, NIR700-Annexin V, and [18F]FLT in cetuximab-sensitive (DiFi) and insensitive (HCT-116) human colorectal cancer cell line xenografts. Imaging results were validated by measurement of tumor size and immunohistochemical analysis of total and phosphorylated EGFR, caspase-3, and Ki-67 immediately following in vivo imaging.

Results: NIR800-EGF accumulation in tumors reflected relative EGFR expression and EGFR occupancy by cetuximab. NIR700-Annexin V accumulation correlated with cetuximab-induced apoptosis as assessed by immunohistochemical staining of caspase-3. No significant difference in tumor proliferation was noted between treated and untreated animals by [18F]FLT positron emission tomography or Ki-67 immunohistochemistry.

Conclusions: Molecular imaging can accurately assess EGF binding, proliferation, and apoptosis in human colorectal cancer xenografts. These imaging approaches may prove useful for serial, noninvasive monitoring of the biological effects of EGFR inhibition in preclinical studies. It is anticipated that these assays can be adapted for clinical use.
The N Staging System in Nasopharyngeal Carcinoma with Radiation Therapy Oncology Group Guidelines for Lymph Node Levels Based on Magnetic Resonance Imaging
Authors: Mao Yan-Ping , Liang Shao-Bo , Liu Li-Zhi , Chen Yong , Sun Ying , Tang Ling-Long , Tian Li , Lin Ai-Hua , Liu Meng-Zhong , Li Li , Ma Jun
Published: November 2008

Purpose: To evaluate the prognostic value of variables including nodal size, level, laterality, extranodal neoplastic spread (ENS), and necrosis in patients with nasopharyngeal carcinoma (NPC) and further explore the feasibility of an N-staging system using Radiation Therapy Oncology Group (RTOG) guidelines for lymph node levels based on magnetic resonance imaging (MRI).

Experimental Design: The MRI scans of 924 patients with histologically diagnosed nondisseminated NPC were reviewed retrospectively. The distribution of the tumors was mapped using RTOG guidelines and laterality. The multiplicity of each tumor was calculated, as well as the size and status of ENS and the necrosis of individual nodes.

Results: Nodal level, cervical lymph node laterality, and ENS were independent prognostic factors for disease failure and distant failure in multivariate analyses. There was no significant difference in the hazard ratios (HR) for distant failure between level II and retropharyngeal, level Ib, level V, or level III involvement, whereas patients with level IV and supraclavicular fossa involvement had a significant increase in HRs. The subsets that made up a given N stage group had similar HRs for distant failure. Both the HRs for disease failure and distant failure by the proposed N staging system between one stage and the next were statistically significant (P < 0.05). The survival curves of disease-free survival and distant metastasis-free survival for all subclassifications of N stage showed significant difference from the adjacent stage (P < 0.05). The overall distribution pattern of the proposed N staging was more equitable than that of the 6th American Joint Committee on Cancer N staging.

Conclusions: Nodal variables including level, cervical lymph node laterality, and ENS are independent prognostic factors for NPC. The proposed N staging system of NPC using RTOG guidelines based on MRI is highly predictive and may provide a more objective method for staging NPCs.
Polymorphisms and Clinical Outcome in Recurrent Ovarian Cancer Treated with Cyclophosphamide and Bevacizumab
Authors: Schultheis Anne M. , Lurje Georg , Rhodes Katrin E. , Zhang Wu , Yang Dongyun , Garcia Agustin A. , Morgan Robert , Gandara David , Scudder Sidney , Oza Amit , Hirte Hal , Fleming Gini , Roman Lynda , Lenz Heinz-Josef
Published: November 2008

Purpose: This study was designed to evaluate the associations between angiogenesis gene polymorphisms and clinical outcome in ovarian cancer patients treated with low-dose cyclophosphamide and bevacizumab.

Experimental Design: Seventy recurrent/metastatic epithelial ovarian cancer patients were enrolled in a phase II clinical trial. Genomic DNA was available from 53 blood samples. Polymorphisms were analyzed using the PCR-RFLP protocol. A 5′ end 33P γATP-labeled PCR protocol was used to analyze dinucleotide repeats.

Results: Patients genotyped A/A or A/T for the IL-8 T-251A gene polymorphism had a statistically significant lower response rate (19%; 0%) than those homozygous T/T (50%; P = 0.006, Fisher's exact test). Patients carrying a minimum one C allele (C/C; C/T) of the CXCR2 C+785T polymorphism showed a median progression-free survival (PFS) of 7.4 months compared with the PFS of 3.7 months for those homozygous T/T (P = 0.026, log-rank test). Patients with the VEGF C+936T polymorphism C/T genotype had a longer median PFS of 11.8 months, compared with those with the C/C and T/T genotype, which had median PFS of 5.5 months and 3.2 months, respectively (P = 0.061, log-rank test). Patients carrying both AM 3′end alleles <14 CA repeats had the shortest median PFS of 3.4 months; patients with at least one allele >14 repeats or both alleles >14 repeats showed a median PFS of 6.4 months and 7.2 months, respectively (P = 0.008, log-rank test).

Conclusion: Our data suggest that the IL-8 A-251T polymorphism may be a molecular predictor of response to bevacizumab-based chemotherapy. The CXCR2 C+785T, VEGF C+936T single nucleotide polymorphisms and the AM 3′ dinucleotide repeat polymorphisms may be molecular markers for PFS in ovarian cancer patients.
Identification of the Molecular Mechanisms for Dedifferentiation at the Invasion Front of Colorectal Cancer by a Gene Expression Analysis
Authors: Oku Yoshimasa , Shimoji Takashi , Takifuji Katsunari , Hotta Tsukasa , Yokoyama Shozo , Matsuda Kenji , Higashiguchi Takashi , Tominaga Toshiji , Nasu Toru , Tamura Koichi , Matsuura Masaaki , Miyata Satoshi , Kato Yo , Yamaue Hiroki , Miki Yoshio
Published: November 2008

Purpose: The aim of this study is to identify gene expression signatures that accompany dedifferentiation at the cancer invasion front in colorectal cancer.

Experimental Design: Two types of colorectal cancer were selected. Both types were well-differentiated adenocarcinomas at the superficial lesion. One type showed a dedifferentiated phenotype at the invasion front (type A, 13 samples); the other showed almost no dedifferentiated cancer cells at the invasion front (type B, 12 samples). Laser microdissection was combined with a cDNA microarray analysis to investigate the superficial lesions and the invasion front in colorectal cancers.

Results: Eighty-three genes were differentially expressed between types A and B in the superficial lesions, and the samples of superficial lesions were divided correctly into two clusters by these genes. Interestingly, the samples of the invasion front were also divided into the two same clusters by these genes. The text mining method selected 10 genes involved in potential mechanisms causing dedifferentiation of cancer cells at the invasion front. The potential mechanisms include the networks of transforming growth factor-β, Wnt, and Hedgehog signals. The expression levels of 10 genes were calculated by quantitative reverse transcription-PCR and 8 genes were confirmed to be significantly differentially expressed between two types (P < 0.05). The gene expression profiles of 8 genes divided 12 test cases into two clusters with one misclassification.

Conclusions: The molecular mechanisms constructed with 8 genes from three networks of transforming growth factor-β, Wnt, and Hedgehog signals were found to correlate with dedifferentiation at the invasion front of colorectal cancer.
Novel Jet-Injection Technology for Nonviral Intratumoral Gene Transfer in Patients with Melanoma and Breast Cancer
Authors: Walther Wolfgang , Siegel Robert , Kobelt Dennis , Knösel Thomas , Dietel Manfred , Bembenek Andreas , Aumann Jutta , Schleef Martin , Baier Ruth , Stein Ulrike , Schlag Peter M.
Published: November 2008

Purpose: This phase I clinical trial evaluated safety, feasibility, and efficiency of nonviral intratumoral jet-injection gene transfer in patients with skin metastases from melanoma and breast cancer.

Experimental Design: Seventeen patients were enrolled. The patients received five jet injections with a total dose of 0.05 mg β-galactosidase (LacZ)-expressing plasmid DNA (pCMVβ) into a single cutaneous lesion. Clinical and laboratory safety monitoring were done. Systemic plasmid clearance was monitored by quantitative real-time PCR of blood samples throughout the study. All lesions were resected after 2 to 6 days. Intratumoral plasmid DNA load, DNA distribution, and LacZ expression was analyzed by quantitative real-time PCR, quantitative reverse transcription-PCR, Western blot, immunohistochemistry, and 5-bromo-4-chloro-3-indolyl-β-d-galactoside staining.

Results: Jet injection of plasmid DNA was safely done in all patients. No serious side effects were observed. Thirty minutes after jet injection, peak plasmid DNA levels were detected in the blood followed by rapid decline and clearance. Plasmid DNA and LacZ mRNA and protein expression were detected in all treated lesions. Quantitative analysis revealed a correlation of plasmid DNA load and LacZ-mRNA expression confirmed by Western blot. Immunohistochemistry and 5-bromo-4-chloro-3-indolyl-β-d-galactoside staining showed LacZ-protein throughout the tumor. Transfected tumor areas were found close and distant to the jet-injection site with varying levels of DNA load and transgene expression.

Conclusion: Intratumoral jet injection of plasmid DNA led to efficient LacZ reporter gene expression in all patients. No side effects were experienced, supporting safety and applicability of this novel nonviral approach. A next step with a therapeutic gene product should determine antitumor efficacy of jet-injection gene transfer.
Relationship Between Tumor and Plasma Levels of hTERT mRNA in Patients with Colorectal Cancer: Implications for Monitoring of Neoplastic Disease
Authors: Terrin Liliana , Rampazzo Enrica , Pucciarelli Salvatore , Agostini Marco , Bertorelle Roberta , Esposito Giovanni , DelBianco Paola , Nitti Donato , De Rossi Anita
Published: November 2008

Purpose: Colorectal cancer (CRC) is one of the most common cancers in western countries. Identification of circulating markers for CRC would optimize early stage diagnosis and the monitoring for disease recurrence. Expression of telomerase reverse transcriptase (hTERT) is essential to the oncogenic process and might be used as a molecular marker of neoplastic disease.

Experimental Design: Eighty-five CRC samples (25 stage I, 15 stage II, 15 stage III, and 30 stage IV), the available corresponding noncancerous mucosa (n = 42), and plasma collected at the time of surgery (n = 49) were analyzed. Control plasma samples were obtained from 43 age-matched healthy subjects. All hTERT transcripts (hTERT-AT) and transcripts encoding the functional protein (hTERT-FL) were quantified by real-time PCR.

Results: hTERT-AT was found to correlate with hTERT-FL (r = 0.849; P < 0.0001) mRNA levels in tumors. Both hTERT mRNAs were significantly higher in tumors than in adjacent noncancerous mucosa and both significantly increased with tumor progression (P < 0.0001). In contrast to controls, all but two plasma samples from CRC patients were positive for hTERT mRNAs. Using the cutoff value of 180 copies hTERT-AT/mL, the sensitivity and specificity of the assay for CRC detection were 92% and 100%, respectively. Furthermore, hTERT-AT mRNA levels in plasma significantly correlated with hTERT-AT mRNA levels in tumors (r = 0.702, P < 0.0001).

Conclusions: These findings indicate that quantification of hTERT mRNA in plasma may be used as a marker for detection and monitoring of neoplastic colorectal disease.
Endosialin Protein Expression and Therapeutic Target Potential in Human Solid Tumors: Sarcoma versus Carcinoma
Authors: Rouleau Cecile , Curiel Maritza , Weber William , Smale Robert , Kurtzberg Leslie , Mascarello James , Berger Carol , Wallar Gina , Bagley Rebecca , Honma Nakayuki , Hasegawa Kazumasa , Ishida Isao , Kataoka Shiro , Thurberg Beth L. , Mehraein Khodadad , Horten Bruce , Miller Glenn , Teicher Beverly A.
Published: November 2008

Purpose: Endosialin/CD248/tumor endothelial marker 1 is expressed in stromal cells, endothelial cells, and pericytes in various tumors; however, few studies have focused on expression in malignant cells.

Experimental Design: We studied expression of endosialin in clinical specimens, cell culture, and animal models and designed an anti-endosialin therapeutic prototype.

Results: Fifty human tumor cell lines and 6 normal cell types in culture were assayed by reverse transcription-PCR and/or flow cytometry for endosialin. Cell surface protein was found on 7 sarcoma lines, 1 neuroblastoma, and 4 normal cell types in culture. A fully human anti-endosialin antibody bound to human A-673 Ewing's sarcoma cells and SK-N-AS neuroblastoma cells but not HT-1080 cells. Exposure of cells to an anti-human IgG conjugated to saporin resulted in growth inhibition only of endosialin-expressing cells. Endosialin expression was assessed by immunohistochemistry in 250 clinical specimens of human cancer including 20 cancer subtypes. Endosialin is frequently found in human cancers. Endosialin expression is mainly a perivascular feature in carcinomas, with some expression in stromal cells. In sarcomas, endosialin is expressed by malignant cells, perivascular cells, and stromal cells. Development and characterization of experimental models for studying endosialin biology in sarcomas and evaluating anti-endosialin therapies is presented.

Conclusions: Findings suggest that an anti-endosialin immunotoxin might be a promising therapeutic approach for endosialin-positive neoplasia, especially synovial sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, and osteosarcoma. Thus, a diagnostic/therapeutic targeted therapeutic approach to treatment of endosialin-expressing tumors may be possible.
Ubiquitin Proteasome System Stress Underlies Synergistic Killing of Ovarian Cancer Cells by Bortezomib and a Novel HDAC6 Inhibitor
Authors: Bazzaro Martina , Lin Zhenhua , Santillan Antonio , Lee Michael K. , Wang Mei-Cheng , Chan Kwun C. , Bristow Robert E. , Mazitschek Ralph , Bradner James , Roden Richard B.S.
Published: November 2008

Purpose: Elevated metabolic activity of ovarian cancer cells causes increased ubiquitin-proteasome-system (UPS) stress, resulting in their greater sensitivity to the toxic effects of proteasomal inhibition. The proteasomes and a potentially compensatory histone deacetylase 6 (HDAC6)-dependent lysosomal pathway mediate eukaryotic protein turnover. We hypothesized that up-regulation of the HDAC6-dependent lysosomal pathway occurs in response to UPS stress and proteasomal inhibition, and thus, ovarian cancer cell death can be triggered most effectively by coinhibition of both the proteasome- and HDAC6-dependent protein degradation pathways.

Experimental Design: To address this hypothesis, we examined HDAC6 expression patterns in normal and cancerous ovarian tissues and used a novel HDAC6-specific inhibitor, NK84, to address HDAC6 function in ovarian cancer.

Results: Abnormally high levels of HDAC6 are expressed by ovarian cancer cells in situ and in culture relative to benign epithelium and immortalized ovarian surface epithelium, respectively. Specific HDAC6 inhibition acts in synergy with the proteasome inhibitor Bortezomib (PS-341) to cause selective apoptotic cell death of ovarian cancer cells at doses that do not cause significant toxicity when used individually. Levels of UPS stress regulate the sensitivity of ovarian cancer cells to proteasome/HDAC6 inhibition. Pharmacologic inhibition of HDAC6 also reduces ovarian cancer cell spreading and migration consistent with its known function in regulating microtubule polymerization via deacetylation of α-tubulin.

Conclusion: Our results suggest the elevation of both the proteasomal and alternate HDAC6-dependent proteolytic pathways in ovarian cancer and the potential of combined inhibition of proteasome and HDAC6 as a therapy for ovarian cancer.
Nonclinical Antiangiogenesis and Antitumor Activities of Axitinib (AG-013736), an Oral, Potent, and Selective Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases 1, 2, 3
Authors: Hu-Lowe Dana D. , Zou Helen Y. , Grazzini Maren L. , Hallin Max E. , Wickman Grant R. , Amundson Karin , Chen Jeffrey H. , Rewolinski David A. , Yamazaki Shinji , Wu Ellen Y. , McTigue Michele A. , Murray Brion W. , Kania Robert S. , O'Connor Patrick , Shalinsky David R. , Bender Steve L.
Published: November 2008

Purpose: Axitinib (AG-013736) is a potent and selective inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases 1 to 3 that is in clinical development for the treatment of solid tumors. We provide a comprehensive description of its in vitro characteristics and activities, in vivo antiangiogenesis, and antitumor efficacy and translational pharmacology data.

Experimental Design: The potency, kinase selectivity, pharmacologic activity, and antitumor efficacy of axitinib were assessed in various nonclinical models.

Results: Axitinib inhibits cellular autophosphorylation of VEGF receptors (VEGFR) with picomolar IC50 values. Counterscreening across multiple kinase and protein panels shows it is selective for VEGFRs. Axitinib blocks VEGF-mediated endothelial cell survival, tube formation, and downstream signaling through endothelial nitric oxide synthase, Akt and extracellular signal-regulated kinase. Following twice daily oral administration, axitinib produces consistent and dose-dependent antitumor efficacy that is associated with blocking VEGFR-2 phosphorylation, vascular permeability, angiogenesis, and concomitant induction of tumor cell apoptosis. Axitinib in combination with chemotherapeutic or targeted agents enhances antitumor efficacy in many tumor models compared with single agent alone. Dose scheduling studies in a human pancreatic tumor xenograft model show that simultaneous administration of axitinib and gemcitabine without prolonged dose interruption or truncation of axitinib produces the greatest antitumor efficacy. The efficacious drug concentrations predicted in nonclinical studies are consistent with the range achieved in the clinic. Although axitinib inhibits platelet-derived growth factor receptors and KIT with nanomolar in vitro potencies, based on pharmacokinetic/pharmacodynamic analysis, axitinib acts primarily as a VEGFR tyrosine kinase inhibitor at the current clinical exposure.

Conclusions: The selectivity, potency for VEGFRs, and robust nonclinical activity may afford broad opportunities for axitinib to improve cancer therapy.