bluuebunny/crossref_metadata_2025 huggingface dataset:
Breast Cancer in African-American Women: Differences in Tumor Biology from European-American Women
Authors: Amend Kandace , Hicks David , Ambrosone Christine B.
Published: September 2006

Disparities exist between African-American and European-American women in the incidence and nature of breast cancer. African-American women are more often diagnosed with breast cancer at an earlier age and with more aggressive disease, characterized by higher grade and negative estrogen and progesterone receptor status. Recent findings reveal specific gene expression patterns associated with the more aggressive breast cancers observed in African-American women. An overview of the current literature about racial differences in breast cancer prompts questions for future research to elucidate causes for the apparent disparities in tumor biology. (Cancer Res 2006; 66(17): 8327-30)
A Profile of Methyl-CpG Binding Domain Protein Occupancy of Hypermethylated Promoter CpG Islands of Tumor Suppressor Genes in Human Cancer
Authors: Lopez-Serra Lidia , Ballestar Esteban , Fraga Mario F. , Alaminos Miguel , Setien Fernando , Esteller Manel
Published: September 2006

Methyl-CpG binding domain (MBD) proteins have been shown to couple DNA methylation to transcriptional repression. This biological property suggests a role for MBD proteins in the silencing of tumor suppressor genes that are hypermethylated at their promoter CpG islands in cancer cells. Despite the demonstration of the presence of MBDs in the methylated promoter of several genes, we still ignore how general and specific is this association. Here, we investigate the profile of MBD occupancy in a large panel of tumor suppressor gene promoters and cancer cell lines. Our study shows that most hypermethylated promoters are occupied by MBD proteins, whereas unmethylated promoters are generally devoid of MBDs, with the exception of MBD1. Treatment of cancer cells with the demethylating agent 5-aza-2′-deoxycytidine results in CpG island hypomethylation, MBD release, and gene reexpression, reinforcing the notion that association of MBDs with methylated promoters is methylation-dependent. Whereas several promoters are highly specific in recruiting a particular set of MBDs, other promoters seem to be less exclusive. Our results indicate that MBDs have a great affinity in vivo for binding hypermethylated promoter CpG islands of tumor suppressor genes, with a specific profile of MBD occupancy that it is gene and tumor type specific. (Cancer Res 2006; 66(17): 8342-6)
Gadd45a Suppresses Ras-Driven Mammary Tumorigenesis by Activation of c-Jun NH2-Terminal Kinase and p38 Stress Signaling Resulting in Apoptosis and Senescence
Authors: Tront Jennifer S. , Hoffman Barbara , Liebermann Dan A.
Published: September 2006

The Gadd45 family of proteins is known to play a central role as cellular stress sensors that modulate the response of mammalian cells to stress inflicted by physiologic and environmental stressors. Gadd45a was shown to be a direct target to the p53 and BRCA1 tumor suppressor genes, whose loss of function is known to play a vital role in breast carcinogenesis; however, the role of Gadd45a in the suppression of breast cancer remains unclear. To address this issue, Gadd45a-deficient mice were crossed with breast cancer prone mouse mammary tumor virus–Ras mice to generate mice that express activated Ras and differ in their Gadd45a status. Using this mouse model, we show that the loss of Gadd45a accelerates Ras-driven mammary tumor formation, exhibiting increased growth rates and a more aggressive histologic phenotype. Moreover, it is shown that accelerated Ras-driven tumor formation in the absence of Gadd45a results in both a decrease in apoptosis, which is linked to a decrease in c-Jun NH2-terminal kinase (JNK) activation, and a decrease in Ras-induced senescence, which is correlated with a decrease in p38 kinase activation. Altogether, these results provide a novel model for the tumor-suppressive function of Gadd45a in the context of Ras-driven breast carcinogenesis, showing that Gadd45a elicits its function through activation of the stress-induced JNK and p38 kinases, which contribute to increase in apoptosis and Ras-induced senescence. (Cancer Res 2006; 66(17): 8448-54)
Expression of ΔDNMT3B Variants and Its Association with Promoter Methylation of p16 and RASSF1A in Primary Non–Small Cell Lung Cancer
Authors: Wang Jie , Walsh Garret , Liu Diane D. , Lee J. Jack , Mao Li
Published: September 2006

Despite the role of DNMT3B in de novo DNA methylation, a correlation between DNMT3B expression and promoter DNA methylation has not being established in tumors. We recently reported ΔDNMT3B, a subfamily of DNMT3B, with seven variants, as the predominant expression forms in non–small cell lung cancer (NSCLC). We hypothesized that expression of the ΔDNMT3B variants plays a role in promoter methylation formation during lung tumorigenesis. Expression of seven ΔDNMT3B variants was measured in 119 NSCLCs and the corresponding normal lungs using reverse transcription-PCR. The expression patterns of ΔDNMT3B variants were analyzed with the status of p16 and RASSF1A promoter methylation in the tumors as well as in patients' clinical variables, including outcomes. Expression of ΔDNMT3B variants was detected in 94 of 119 (80%) tumors but in only 22 (18%) of the corresponding normal lungs (P < 0.0001). ΔDNMT3B1, ΔDNMT3B2, and ΔDNMT3B4 were the most frequently detected transcripts in the tumors (62%, 76%, and 46%, respectively). The expression of ΔDNMT3B variants was associated with p16 and RASSF1A promoter methylation in the tumors, but the strongest association was between ΔDNMT3B4 and RASSF1A. Forty-two of 46 (91%) tumors with RASSF1A promoter methylation expressed ΔDNMT3B4 compared with only 13 of 73 (18%) tumors without the promoter methylation (P < 0.0001). Strong associations were also observed between expression of the variants in the tumors and in patients' clinical outcomes. Expression of ΔDNMT3B variants is common in NSCLC and may play an important role in the development of promoter methylation. (Cancer Res 2006; 66(17): 8361-6)
Aurora Kinase A Messenger RNA Overexpression Is Correlated with Tumor Progression and Shortened Survival in Head and Neck Squamous Cell Carcinoma
Authors: Reiter Rudolf , Gais Peter , Jütting Uta , Steuer-Vogt Miriam K. , Pickhard Anja , Bink Karin , Rauser Sandra , Lassmann Silke , Höfler Heinz , Werner Martin , Walch Axel
Published: September 2006

Purpose: Aurora kinase A (AURKA/STK15/BTAK) encodes a serine/threonine kinase associated with chromosomal distribution and its up-regulation induces chromosomal instability, thereby leading to aneuploidy and cell transformation in several types of cancer. In this study, we investigated the role of AURKA in head and neck squamous cell carcinoma (HNSCC).

Experimental Design: The mRNA expression levels of AURKA were compared in tumor tissues of 66 HNSCC patients with those in corresponding normal squamous epithelium by real-time quantitative reverse transcriptase-PCR. In addition, the association between AURKA mRNA and protein expression, centrosome abnormalities, and aneuploidy was studied in a subset of cases (n = 34). All molecular variables were correlated to histomorphologic findings and clinical follow-up data of the patients.

Results: AURKA mRNA up-regulation was significantly associated with tumor stage and the occurrence of regional lymph node, as well as distant metastasis (P < 0.0001 for all). Similarly, a correlation was found for protein expression and the occurrence of regional lymph node (P = 0.0183) and distant metastasis (P = 0.03). The mRNA was positively associated with protein expression (P = 0.003) and centrosome abnormalities (P = 0.03). Cox regression analysis revealed that AURKA mRNA up-regulation correlated with disease-free survival of the patients (P = 0.03) as well as shorter overall survival (P < 0.001).

Conclusions: We conclude that the up-regulation of AURKA mRNA may play a critical role in the tumor progression of HNSCC and provides useful information as a prognostic factor for HNSCC patients.
Microsatellite DNA Alterations of Gastrointestinal Stromal Tumors Are Predictive for Outcome
Authors: Schurr Paulus , Wolter Stefan , Kaifi Jussuf , Reichelt Uta , Kleinhans Helge , Wachowiak Robin , Yekebas Emre , Strate Tim , Kalinin Viacheslav , Simon Ronald , Sauter Guido , Schaefer Hansjoerg , Izbicki Jakob
Published: September 2006

Purpose: In gastrointestinal stromal tumors (GIST), loss of heterozygosity (LOH) on chromosome 22 and its presumptive biological function has been described. The prognostic value of these and other DNA regions for patient survival remains unclear.

Experimental Design: Sixty patients who underwent surgery at our institution between 1992 and 2003 for GIST were histopathologically reclassified by immunohistochemistry and the GIST consensus group criteria 2001. Twenty-one microsatellite loci on chromosomes 3, 9, 13, 17, 18, and 22 were screened for alterations in tumor and healthy DNA. Survival was calculated by Kaplan-Meier plots.

Results: Eleven (18.3%) of 60 patients showed metastases at presentation. Thirteen (21.7%) of 60 were high-risk GISTs. LOH was found in all tumors. Twenty-eight (46.7%) of 60 showed more than two LOH in 21 microsatellite marker sites. The frequency of single marker LOH varied from 1.7% to 28.3% among tumors. Frequent LOH was found on chromosomes 22 and 17. The correlation of LOH positivity and the consensus scoring was significant (P = 0.005, χ2 test). After a median observation time of 33.3 months (95% confidence interval, 23.9-42.6), overall survival was best for patients with tumors of very low, low, and intermediate risks with only 6 of 36 death events, whereas 14 of 24 high-risk and metastasized patients had died (P < 0.001, log-rank test). Likewise, LOH significantly predicted survival (P = 0.013) and the effect was particularly detrimental for LOH on chromosome 17 (P < 0.001).

Conclusions: LOH is a useful phenomenon for the prognosis of GIST. Rather than chromosome 22 markers, chromosome 17 markers independently predict survival.
Overexpression of Hypoxia-Inducible Factor 1α and p53 Is a Marker for an Unfavorable Prognosis in Gastric Cancer
Authors: Sumiyoshi Yasushi , Kakeji Yoshihiro , Egashira Akinori , Mizokami Ken , Orita Hiroyuki , Maehara Yoshihiko
Published: September 2006

Purpose: To investigate the effects of the expression of hypoxia-inducible factor 1α (HIF-1α) and p53 on the prognosis of human gastric cancer, the clinicopathologic characteristics of the tumors and the clinical outcome were analyzed.

Experimental Design: The expressions of HIF-1α and p53 proteins were studied by immunohistochemistry in 216 specimens of primary gastric cancer.

Results: HIF-1α(+)/p53(+) tumors more frequently showed an undifferentiated type, an infiltrative growth appearance, and an invasive lymphatic involvement compared with HIF-1α(−)/p53(−) tumors. HIF-1α(+)/p53(+) tumors also had more lymph node metastasis compared with HIF-1α(−)/p53(−) tumors. When stratified for HIF-1α and p53 positivity, the patients who were p53-negative and HIF-1α-negative had the most favorable prognosis, whereas patients who were p53-positive and HIF-1α-positive had the worst prognosis (P = 0.0018). Using a multivariate Cox regression analysis, the depth of invasion, lymph node metastasis, and HIF-1α positivity were all found to be independent prognostic factors in patients with gastric cancer.

Conclusion: Thus, HIF-1α is considered to be a useful independent prognostic factor in gastric cancer, and the combination of a HIF-1α protein overexpression with nonfunctional p53 tends to indicate a dismal prognosis.
Unique Tumor Antigens: Evidence for Immune Control of Genome Integrity and Immunogenic Targets for T Cell–Mediated Patient-Specific Immunotherapy
Authors: Sensi Marialuisa , Anichini Andrea
Published: September 2006

The molecular identification and characterization of antigenic epitopes recognized by T cells on human cancers has rapidly evolved since the cloning in 1991 of MAGEA1, the first gene reported to encode a CTL-defined human tumor antigen. In the expanding field of human tumor immunology, unique tumor antigens constitute a growing class of T cell–defined epitopes that exhibit strong immunogenicity. Some of these antigens, which often derive from mutation of genes that have relevant biological functions, are less susceptible to immunoselection and may be retained even in advanced tumors. Immunogenicity and constitutive expression of the unique tumor antigens provide a strong rationale for the design of novel, patient-tailored therapies that target such determinants. Here we discuss the immunologic relevance of unique tumor antigens in the light of the prospects for exploiting such epitopes as targets for patient-specific immune intervention strategies.
Rac Activation Is Associated with Hepatocellular Carcinoma Metastasis by Up-regulation of Vascular Endothelial Growth Factor Expression
Authors: Lee Terence K. , Poon Ronnie T.P. , Yuen Anthony P. , Man Kwan , Yang Zhen Fan , Guan Xin Yuan , Fan Sheung Tat
Published: September 2006

Purpose: Hepatocellular carcinoma (HCC) is associated with a propensity for vascular invasion and metastasis, which contribute to poor prognosis. Angiogenesis is a crucial process contributing to tumor growth and metastasis. Recently, Rac has been suggested to play a role in angiogenesis. However, the actual role of Rac in HCC angiogenesis remains unclear. Given that vascular endothelial growth factor (VEGF) is an important angiogenic factor in HCC, the purpose of this study was to evaluate the possible correlation between Rac activation and VEGF expression in HCC tumor samples, as well as the mechanism involved in Rac-induced HCC angiogenesis.

Experimental Design: We evaluated Rac and VEGF expression in the HCC tissue microarray of paired primary and metastatic HCC samples using immunohistochemical staining. The role of Rac-induced HCC angiogenesis was also evaluated in vitro in HCC cell lines.

Results: We first showed that activation of Rac was correlated with HCC metastasis (P < 0.001), and its expression was significantly correlated with VEGF expression by tissue microarray. Ectopic Rac-dominant active transfection in Hep3B cells increased VEGF secretion, which induced the morphologic change and proliferation of human umbilical vein endothelial cells, resulting in the promotion of angiogenesis. Rac induced the transcriptional activation of VEGF by direct interaction with hypoxia-inducible factor-1α (HIF-1α) expression. In hypoxic conditions, Rac promoted angiogenesis through an increase in HIF-1α stabilization.

Conclusion: This study shows that Rac is a novel angiogenic factor for HCC through the enhancement of HIF-1α protein stability, which provides a possible therapeutic target in the development of inhibitors of angiogenesis.
The Biological Role of the Low-Affinity p75 Neurotrophin Receptor in Esophageal Squamous Cell Carcinoma
Authors: Okumura Tomoyuki , Tsunoda Shigeru , Mori Yukiko , Ito Tetsuo , Kikuchi Keiji , Wang Timothy Cragin , Yasumoto Shigeru , Shimada Yutaka
Published: September 2006

In this study, we investigated the clinicopathologic significance of the low-affinity p75 neurotrophin receptor (p75NTR; which is expressed in the stem/progenitor cell fraction of normal esophageal epithelial cells) in 187 resected esophageal squamous cell carcinoma (ESCC) specimens and found that ∼50% of ESCC expressed p75NTR. Our investigation using ESCC cell lines showed that p75NTR was intensely expressed in the cells with high colony-forming capacity but they were sensitive to cell death on inhibition of p75NTR expression with transient transfection of small interfering RNA (siRNA). These findings suggest that p75NTR is necessary for survival and maintenance of ESCC tumors, providing us with a potential target for novel therapies.

Purpose: p75NTR is expressed in a stem/progenitor cell fraction of human normal esophageal epithelial cells. In this study, we investigated the expression and biological role of p75NTR in ESCC.

Experimental Design: The expression of p75NTR in 187 resected ESCC specimens was immunohistochemically investigated. The expression of p75NTR in 30 ESCC cell lines (KYSEs) was assessed by reverse transcription-PCR, immunocytochemistry, and flow cytometry. The p75NTR-bright and p75NTR-dim/negative cells were isolated from KYSE150 by magnetic beads and colony formation was investigated. The role of p75NTR in KYSEs was assessed by transient transfection of siRNA.

Results: p75NTR was expressed in 92 of 187 (49.2%) tumors. In well-differentiated tumors, positive staining was apparent in the first one to two layers from infiltrative margin of the tumors where most of the cells were actively proliferating. In moderately differentiated tumors, p75NTR was expressed in wider range from the margin of the tumors whereas p75NTR was diffusely distributed in poorly differentiated tumors. p75NTR was expressed in all examined KYSEs and the mean proportion of the p75NTR-bright fraction was 30.1%. The size of p75NTR-positive colonies was larger than that of p75NTR-negative colonies derived from KYSE150 (P < 0.0001). The purified p75NTR-bright cells formed p75NTR-positive large colonies more frequently than the p75NTR-dim/negative cells (P < 0.0001). Down-regulation of p75NTR expression by siRNA resulted in marked growth inhibition with induction of apoptosis.

Conclusions: Our findings suggest that p75NTR is necessary for survival and maintenance of ESCC tumors, providing us with a potential target for novel therapies.
Promoter Hypermethylation Profile of Kidney Cancer with New Proapoptotic p53 Target Genes and Clinical Implications
Authors: Christoph Frank , Weikert Steffen , Kempkensteffen Carsten , Krause Hans , Schostak Martin , Köllermann Jens , Miller Kurt , Schrader Mark
Published: September 2006

Purpose: Risk stratification of renal cell carcinoma is based on the histopathologic classification. Promoter hypermethylation as a mechanism of gene inactivation in renal cell carcinoma has been shown for only a small number of genes. We examined the usefulness of quantitative methylation analysis with a new set of p53 target genes for determining the clinical outcome and aggressiveness of the tumor disease.

Experimental Design: The genes selected were APAF-1, CASPASE-8, DAPK-1, IGFBP-3, and PML. The tissue samples analyzed were taken from tumor specimens obtained from 90 consecutive patients with clear cell renal carcinoma and from 20 normal kidney specimens. Quantitative methylation analysis of CpG sites in the promoter region was done by methylation-specific real-time PCR and the normalized index of methylation (NIM) was determined for each sample.

Results: Hypermethylation of the promoter region was common for APAF-1 (97%) and DAPK-1 (41%) but not for IGFBP-3 (3%), PML (3%), or CASP-8 (0%). The tumor patients had a median follow-up of 55 months. A correlation was found between the methylation level of APAF-1 and tumor size and nodal status, but not for tumor stage, grade, and age of patient. Kaplan-Meier analysis was able to identify patients with a higher risk of recurrence and tumor-related death by using APAF-1 (≥56% NIM) and DAPK-1 (≥10% NIM) methylation levels. In multivariate analysis, APAF-1 and DAPK-1 methylation levels were independent prognostic markers for metastatic disease and death from renal cell carcinoma.

Conclusions: Our findings indicate that promoter hypermethylation of APAF-1 and DAPK-1 is a marker of aggressive renal cell carcinoma and provides independent prognostic information on disease outcome.
Phase I and Pharmacokinetic Study of Tasidotin Hydrochloride (ILX651), a Third-Generation Dolastatin-15 Analogue, Administered Weekly for 3 Weeks Every 28 Days in Patients with Advanced Solid Tumors
Authors: Mita Alain C. , Hammond Lisa A. , Bonate Peter L. , Weiss Geoffrey , McCreery Heather , Syed Samira , Garrison Mitchell , Chu Quincy S.C. , DeBono Johann S. , Jones Christopher B. , Weitman Steve , Rowinsky Eric K.
Published: September 2006

Purpose: To determine the safety, tolerability, and pharmacokinetics and to seek preliminary evidence of anticancer activity of tasidotin (ILX651), a novel dolastatin analogue, when administered as a 30-minute i.v. infusion weekly for 3 weeks every 4 weeks.

Experimental Design: Thirty patients with advanced solid malignancies were treated with 82 courses at six dose levels ranging from 7.8 to 62.2 mg/m2 weekly, initially according to an accelerated dose-escalation scheme, which evolved into a Fibonacci scheme as a relevant degree of toxicity was observed. Plasma and urine were sampled to characterize the pharmacokinetic behavior of tasidotin.

Results: A high incidence of neutropenia complicated by fever (one patient), or precluding treatment on day 15 (three patients), was the principal toxicity of tasidotin, at doses above 46.8 mg/m2. At all dose levels, nonhematologic toxicities were generally mild to moderate and manageable. Grade 3 toxicities included diarrhea and vomiting (one patient each). Drug-induced neurosensory symptoms were mild and there was no evidence of cardiovascular toxicity, which has been previously associated with other dolastatins. Tasidotin pharmacokinetics were mildly nonlinear, whereas metabolite kinetics were linear. A patient with non–small cell lung carcinoma experienced a minor response, and a patient with hepatocellular carcinoma had stable disease lasting 11 months.

Conclusions: The recommended dose for phase II studies of tasidotin administered on this schedule is 46.8 mg/m2. The mild myelosuppression and manageable nonhematologic toxicities at the recommended dose, the evidence of antitumor activity, and the unique mechanistic aspects of tasidotin warrant further disease-directed evaluations on this and alternative schedules.
Aberrant Nuclear Accumulation of Glycogen Synthase Kinase-3β in Human Pancreatic Cancer: Association with Kinase Activity and Tumor Dedifferentiation
Authors: Ougolkov Andrei V. , Fernandez-Zapico Martin E. , Bilim Vladimir N. , Smyrk Thomas C. , Chari Suresh T. , Billadeau Daniel D.
Published: September 2006

Purpose: We have shown recently that glycogen synthase kinase-3 (GSK-3) β regulates nuclear factor-κB (NF-κB)–mediated pancreatic cancer cell survival and proliferation in vitro. Our objective was to determine the localization of GSK-3β in pancreatic cancer cells and assess the antitumor effect of GSK-3 inhibition in vivo to improve our understanding of the mechanism by which GSK-3β affects NF-κB activity in pancreatic cancer.

Experimental Design: Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the localization of GSK-3β in human pancreatic tumors. We studied the effect of GSK-3 inhibition on tumor growth, cancer cell proliferation, and survival in established CAPAN2 tumor xenografts using a tumor regrowth delay assay, Western blotting, bromodeoxyuridine incorporation, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling.

Results: We found nuclear accumulation of GSK-3β in pancreatic cancer cell lines and in 62 of 122 (51%) human pancreatic adenocarcinomas. GSK-3β nuclear accumulation is significantly correlated with human pancreatic cancer dedifferentiation. We have found that active GSK-3β can accumulate in the nucleus of pancreatic cancer cells and that inhibition of GSK-3 kinase activity represses its nuclear accumulation via proteasomal degradation within the nucleus. Lastly, we have found that inhibition of GSK-3 arrests pancreatic tumor growth in vivo and decreases NF-κB-mediated pancreatic cancer cell survival and proliferation in established tumor xenografts.

Conclusions: Our results show the antitumor effect of GSK-3 inhibition in vivo, identify GSK-3β nuclear accumulation as a hallmark of poorly differentiated pancreatic adenocarcinoma, and provide new insight into the mechanism by which GSK-3β regulates NF-κB activity in pancreatic cancer.
A Phase I Pharmacokinetic Study of HMN-214, a Novel Oral Stilbene Derivative with Polo-Like Kinase-1–Interacting Properties, in Patients with Advanced Solid Tumors
Authors: Garland Linda L. , Taylor Charles , Pilkington Deborah L. , Cohen Jan L. , Von Hoff Daniel D.
Published: September 2006

Purpose: HMN-214 is an oral prodrug of HMN-176, a stilbene derivative that interferes with the subcellular spatial location of polo-like kinase-1, a serine/threonine kinase that regulates critical mitotic events. We conducted a dose escalation study of HMN-214 in patients with advanced cancer to assess the safety profile and pharmacokinetics of HMN-214 and to establish the maximum tolerated dose.

Experimental Design: Thirty-three patients were enrolled onto four dosing cohorts of HMN-214 from 3 to 9.9 mg/m2/d using a continuous 21-day dosing schedule every 28 days, with pharmacokinetic sampling during cycle 1.

Results: A severe myalgia/bone pain syndrome and hyperglycemia were dose-limiting toxicities at 9.9 mg/m2/d. A dose reduction and separate enrollment by pretreatment status (lightly versus heavily pretreated) was undertaken, with one dose-limiting toxicity (grade 3 bone pain) at 8 mg/m2/d. The maximum tolerated dose was defined as 8 mg/m2/d for both treatment cohorts. Dose-proportional increases were observed in AUC but not Cmax. There was no accumulation of HMN-176, the metabolite of HMN-214, with repeated dosing. Seven of 29 patients had stable disease as best tumor response, including 6-month stable disease in a heavily pretreated breast cancer patient. A transient decline in carcinoembryonic antigen in a patient with colorectal cancer was noted.

Conclusions: The maximum tolerated dose and recommended phase II dose of HMN-214 when administered on this schedule was 8 mg/m2/d regardless of pretreatment status. Further development of HMN-214 will focus on patient populations for which high expression of polo-like kinase-1 is seen (i.e., prostate and pancreatic cancer patients).
Eliminating Established Tumor in nu/nu Nude Mice by a Tumor Necrosis Factor-α-Related Apoptosis-Inducing Ligand–Armed Oncolytic Adenovirus
Authors: Dong Fengqin , Wang Li , Davis John J. , Hu Wenxian , Zhang Lidong , Guo Wei , Teraishi Fuminori , Ji Lin , Fang Bingliang
Published: September 2006

Purpose: The tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) and oncolytic viruses have recently been investigated extensively for cancer therapy. However, preclinical and clinical studies have revealed that their clinical application is hampered by either weak anticancer activity or systemic toxicity. We examined whether the weaknesses of the two strategies can be overcome by integrating the TRAIL gene into an oncolytic vector.

Experimental Design: We constructed a TRAIL-expressing oncolytic adenovector designated as Ad/TRAIL-E1. The expression of both the TRAIL and viral E1A genes is under the control of a synthetic promoter consisting of sequences from the human telomerase reverse transcriptase promoter and a minimal cytomegalovirus early promoter. The transgene expression, apoptosis induction, viral replication, antitumor activity, and toxicity of Ad/TRAIL-E1 were determined in vitro and in vivo in comparison with control vectors.

Results: Ad/TRAIL-E1 elicited enhanced viral replication and/or stronger oncolytic effect in vitro in various human cancer cell lines than a TRAIL-expressing, replication-defective adenovector or an oncolytic adenovector–expressing green fluorescent protein. Intralesional administration of Ad/TRAIL-E1 eliminated all s.c. xenograft tumors established from a human non–small cell lung cancer cell line, H1299, on nu/nu nude mice, resulting in long-term, tumor-free survival. Furthermore, we found no treatment-related toxicity.

Conclusions: Viral replication and antitumor activity of oncolytic adenovirus can be enhanced by the TRAIL gene and Ad/TRAIL-E1 could become a potent therapeutic agent for cancer therapy.
Selective Profiling of Proteins in Lung Cancer Cells from Fine-Needle Aspirates by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry
Authors: Amann Joseph M. , Chaurand Pierre , Gonzalez Adriana , Mobley James A. , Massion Pierre P. , Carbone David P. , Caprioli Richard M.
Published: September 2006

Purpose: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to analyze tumor sections and can determine tumor type, nodal involvement, and survival, and shows promise in predicting therapeutic response. Our purpose was to develop a method compatible with MALDI-TOF MS that allows selective analysis of cancer cells in mixed clinical samples such as fine-needle aspirates.

Experimental Design: Lung cancer cell lines were cytocentrifuged onto metal-coated, transparent glass slides and used for optimization of fixation, staining, and RBC lysis protocols. Fine-needle aspirates from human tumors and mouse model tumors were used to provide fresh tissue samples for determining the feasibility of this method.

Results: The MALDI-TOF MS compatible fixation and staining techniques provided high-resolution cellular morphology, which allowed identification and selective spotting of tumor cells. The RBC lysis step efficiently removed contaminating RBC yielding spectra nearly free from hemoglobin peaks. Protein profiles of fine-needle aspirates were found highly reproducible and similar to the profiles of the tissue from which they were obtained. Using this method, we were able to differentiate between xenograft tumors derived from two different human cell lines, A549 and H460.

Conclusion: This procedure results in the production of high-quality, cancer cell–specific protein profiles. This highly reproducible technique could be applied to many other types of mixed clinical samples and has the potential to be very useful in the clinical diagnosis, classification, and, potentially, the individualized treatment of cancer patients.
Identification of Cripto-1 as a Novel Serologic Marker for Breast and Colon Cancer
Authors: Bianco Caterina , Strizzi Luigi , Mancino Mario , Rehman Aasia , Hamada Shin , Watanabe Kazuhide , De Luca Antonella , Jones Brenda , Balogh Gabriela , Russo Jose , Mailo Daniel , Palaia Raffaele , D'Aiuto Giuseppe , Botti Gerardo , Perrone Francesco , Salomon David S. , Normanno Nicola
Published: September 2006

Purpose: Human Cripto-1 (CR-1), a cell membrane glycosylphosphatidylinositol-anchored glycoprotein that can also be cleaved from the membrane, is expressed at high levels in several different types of human tumors. We evaluated whether CR-1 is present in the plasma of patients with breast and colon cancer, and if it can represent a new biomarker for these malignancies.

Experimental Design: We determined CR-1 plasma levels using a sandwich-type ELISA in 21 healthy volunteers, 54 patients with breast cancer, 33 patients with colon carcinoma, and 21 patients with benign breast lesions. Immunohistochemical analysis was also used to assess CR-1 expression in cancerous tissues.

Results: Very low levels of CR-1 (mean ± SD) were detected in the plasma of healthy volunteers (0.32 ± 0.19 ng/mL). A statistically significant increase in the levels of plasma CR-1 was found in patients with colon carcinoma (4.68 ± 3.5 ng/mL) and in patients with breast carcinoma (2.97 ± 1.48 ng/mL; P < 0.001). Although moderate levels of plasma CR-1 were found in women with benign lesions of the breast (1.7 ± 0.99 ng/mL), these levels were significantly lower than in patients with breast cancer (P < 0.001). Finally, immunohistochemical analysis and real-time reverse transcription-PCR confirmed strong positivity for CR-1 in colon and/or breast tumor tissues.

Conclusion: This study suggests that plasma CR-1 might represent a novel biomarker for the detection of breast and colon carcinomas.
Validity of Messenger RNA Expression Analyses of Human Saliva
Authors: Kumar Shalini V. , Hurteau Gregory J. , Spivack Simon D.
Published: September 2006

Purpose: The origins of expression microarray and reverse transcription-PCR (RT-PCR) signals in human saliva were evaluated.

Experimental Design: The “RNA” extracts from human saliva samples were treated with vehicle, DNase, or RNase. Two-step amplification and hybridization to Affymetrix 133A cDNA microarrays were then done. Confirmatory RT-PCR experiments used conventionally designed PCR primer pairs for the reference housekeeper transcripts encoding 36B4, β-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA sequences, which are known to be homologous to genomic DNA pseudogene sequences. Negative controls included the omission of reverse transcriptase (“no-RT”) to detect any DNA-derived signal. Finally, an RNA-specific RT-PCR strategy eliminated confounding signals from contaminating genomic DNA.

Results: Microarray experiments revealed that untreated, DNase-treated, and RNase-treated “RNA” extracts from saliva all yielded negligible overall signals. Specific microarray signals for 36B4, β-actin, and GAPDH were low, and were unaffected by RNase. Real-time quantitative RT-PCR reactions using conventional, non–RNA-specific primers on saliva samples yielded PCR products for 36B4, β-actin, and GAPDH; DNase-treated saliva samples did not yield a PCR product, and the “no-RT” and “+RT” conditions yielded similar amounts of PCR product. The RNA-specific RT-PCR strategy, across all conditions, yielded no PCR product from saliva.

Conclusions: The combination of (a) a minimal microarray signal, which was unaffected by RNase treatment, (b) the presence of a conventional RT-PCR housekeeper product in both RNase-treated and no-RT saliva samples, (c) the absence of a conventional RT-PCR housekeeper product in DNase-treated conditions, and (d) the absence of a RNA-specific RT-PCR product shows that any microarray or RT-PCR signal in the saliva must arise from genomic DNA, not RNA. Thus, saliva extracts do not support mRNA expression studies.
Liver Intestine-Cadherin (CDH17) Haplotype Is Associated with Increased Risk of Hepatocellular Carcinoma
Authors: Wang Xiao Qi , Luk John M. , Garcia-Barcelo Mercè , Miao Xiaoping , Leung Pauline P. , Ho David W. , Cheung Siu Tim , Lam Brian Y. , Cheung Cindy K. , Wong Ashley S. , Lau Silvana S. , So Man Ting , Yu Wan Ching , Cai Qi , Liu Karen S. , Hui Chee Kin , Lau George K. , Poon Ronnie T.P. , Wong John , Fan Sheung Tat
Published: September 2006

Purpose: Hepatocellular carcinoma (HCC), the most common form of liver cancer, is a leading cause of cancer death worldwide. We previously showed that aberrant mRNA splicing of the liver intestine-cadherin gene CDH17 in liver tissues was triggered by the specific constellation of two CDH17 single nucleotide polymorphisms (651T and IVS6+35G). CDH17 aberrant splicing was highly associated with tumor dissemination and shorter survival of HCC patients. Consequently, it is highly relevant to assess whether the presence of these single nucleotide polymorphisms in the general population represents a risk to the development of HCC.

Experimental Design: We conducted a case-control study including 164 HCC and 99 cirrhosis patients and 293 healthy controls. Genotyping was done by PCR and direct sequencing. Odds ratio (OR) and χ2 analysis were used to analyze genotypes and haplotypes.

Results: Genotypes 651TT [OR, 2.62; 95% confidence interval (95% CI), 1.34-5.03] and IVS6+35 GG (OR, 1.95; 95% CI, 1.04-3.62) were highly associated with HCC disease. The 651T (C>T) and IVS6+35G (A>G) alleles were also overrepresented in HCC patients and, in particular, the T-G haplotype was the most prevalent in HCC patients when compared with healthy controls (OR, 1.57; 95% CI, 1.167-2.109; P = 0.004), which was in agreement with the aberrant splicing observed in tumor tissues. There was no significant difference in genotype and allele frequencies between cirrhosis patients and controls.

Conclusion: The functional T-G haplotype of CDH17 (651 C>T and IVS6+35A>G) is a genetic susceptibility factor for the development of HCC in a Chinese population.
Preclinical Characterization of AEG35156/GEM 640, a Second-Generation Antisense Oligonucleotide Targeting X-Linked Inhibitor of Apoptosis
Authors: LaCasse Eric C. , Cherton-Horvat Gabriele G. , Hewitt Kimberley E. , Jerome Lori J. , Morris Stephen J. , Kandimalla Ekambar R. , Yu Dong , Wang Hui , Wang Wei , Zhang Ruiwen , Agrawal Sudhir , Gillard John W. , Durkin Jon P.
Published: September 2006

Purpose: Cancer cells can use X-linked inhibitor of apoptosis (XIAP) to evade apoptotic cues, including chemotherapy. The antitumor potential of AEG35156, a novel second-generation antisense oligonucleotide directed toward XIAP, was assessed in human cancer models when given as a single agent and in combination with clinically relevant chemotherapeutics.

Experimental Design: AEG35156 was characterized for its ability to cause dose-dependent reductions of XIAP mRNA and protein in vitro and in vivo, to sensitize cancer cell lines to death stimuli, and to exhibit antitumor activity in multiple human cancer xenograft models as a single agent or in combination with chemotherapy.

Results: AEG35156 reduced XIAP mRNA levels with an EC50 of 8 to 32 nmol/L and decreased XIAP protein levels by >80%. Loss of XIAP protein correlated with increased sensitization to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–mediated apoptosis in Panc-1 pancreatic carcinoma cells. AEG35156 exhibited potent antitumor activity relative to control oligonucleotides in three human cancer xenograft models (prostate, colon, and lung) and was capable of inducing complete tumor regression when combined with taxanes. Antitumor effects of AEG35156 correlated with suppression of tumor XIAP levels.

Conclusions: AEG35156 reduces XIAP levels and sensitizes tumors to chemotherapy. AEG35156 is presently under clinical assessment in multiple phase I trials in cancer patients as a single agent and in combination with docetaxel in solid tumors or cytarabine/idarubicin in leukemia.
Adenovirus-Mediated Calponin h1 Gene Therapy Directed against Peritoneal Dissemination of Ovarian Cancer: Bifunctional Therapeutic Effects on Peritoneal Cell Layer and Cancer Cells
Authors: Ogura Tomonori , Kobayashi Hiroaki , Ueoka Yousuke , Okugawa Kaoru , Kato Kiyoko , Hirakawa Toshio , Hashimoto Shigenari , Taniguchi Shun'ichiro , Wake Norio , Nakano Hitoo
Published: September 2006

Purpose: Calponin h1 (CNh1), one of the family of actin-binding proteins, stabilizes the filaments of actin and modulates various cellular biological phenotypes. Recent studies revealed the close correlation between the invasive tumor spread and the reduced expression of CNh1 and α-smooth muscle actin in the surrounding stromal cells. The purpose of this study is to evaluate the efficacy of i.p. CNh1 gene therapy against peritoneal dissemination of ovarian cancer.

Experimental Design: We used an adenoviral vector to induce the CNh1 gene into peritoneal cells and ovarian cancer cells as a means of enhancing or inducing the expression of α-smooth muscle actin as well as CNh1. The efficacy of gene transfer was examined by in vitro cell culture and in vivo animal experiments.

Results: The formation of longer and thicker actin fibers was observed in each transfected cell line, and the localization of these fibers coincided with that of externally transducted CNh1. With respect to changes in cell behavior, the CNh1-transfected peritoneal cells acquired an ability to resist ovarian cancer-induced shrinkage in cell shape; thus, cancer cell invasion through the monolayer of peritoneal cells was inhibited. In addition, CNh1-transfected ovarian cancer cells showed suppressed anchorage-independent growth and invasiveness, the latter of which accompanied impaired cell motility. The concomitant CNh1 transfection into both peritoneal cells and ovarian cancer cells produced an additive inhibitory effect with respect to cancer cell invasion through the peritoneal cell monolayer. By in vivo experiments designed to treat nude mice that had been i.p. inoculated with ovarian cancer cells, we found that the i.p. injected CNh1 adenovirus successfully blocked cancer-induced morphologic changes in peritoneal cell surface and significantly prolonged the survival time of tumor-bearing mice. Moreover, CNh1 adenovirus could successfully enhance the therapeutic effect of an anticancer drug without increase in side effects.

Conclusions: Thus, CNh1 gene therapy against peritoneal dissemination of ovarian cancer is bifunctionally effective (i.e., through inhibitory effects on the infected peritoneal cell layers that suppress cancer invasion and through direct antitumor effects against invasion and growth properties of cancer cells).
Effect of Raloxifene on the Incidence of Invasive Breast Cancer in Postmenopausal Women with Osteoporosis Categorized by Breast Cancer Risk
Authors: Lippman Marc E. , Cummings Steven R. , Disch Damon P. , Mershon John L. , Dowsett Sherie A. , Cauley Jane A. , Martino Silvana
Published: September 2006

Purpose: To assess the effect of raloxifene, indicated for osteoporosis treatment and prevention, on invasive breast cancer in subgroups of postmenopausal women defined by risk factors for breast cancer.

Experimental Design: Data from the 4-year Multiple Outcomes of Raloxifene Evaluation (MORE) trial (N = 7,705) and a follow-up study, the 4-year Continuing Outcomes Relevant to Evista (CORE) trial (N = 4,011), were analyzed. Prespecified subgroups were defined by age (≥65 versus <65 years), age at menopause (≥49 versus <49 years), body mass index (≥25 versus <25 kg/m2), family history of breast cancer (yes/no), serum estradiol level (5-10 versus <5, >10 versus <5 pmol/L), prior estrogen therapy (yes/no), and bone mass at MORE baseline, and 5-year predicted risk, assessed using the modified Gail model (≥1.67 versus <1.67%), at CORE baseline. Time-to-first invasive breast cancer was analyzed using Cox proportional hazards models.

Results: In the placebo group, older age, higher estradiol level, and a family history of breast cancer were associated with an increased breast cancer risk (P < 0.05). Raloxifene therapy was associated with a reduced breast cancer risk in both women at lower and those at higher breast cancer risk. Hazard ratio point estimates were 0.11 to 0.67, corresponding to a 33% to 89% reduction in breast cancer risk with raloxifene versus placebo. The therapy by family history interaction was significant (P = 0.04).

Conclusions: Raloxifene therapy was associated with a reduced risk of invasive breast cancer in postmenopausal women irrespective of the presence/absence of risk factors; its effect was greater in women with a family history of breast cancer.
Phase I/II Study of the Mammalian Target of Rapamycin Inhibitor Everolimus (RAD001) in Patients with Relapsed or Refractory Hematologic Malignancies
Authors: Yee Karen W.L. , Zeng Zhihong , Konopleva Marina , Verstovsek Srdan , Ravandi Farhad , Ferrajoli Alessandra , Thomas Deborah , Wierda William , Apostolidou Efrosyni , Albitar Maher , O'Brien Susan , Andreeff Michael , Giles Francis J.
Published: September 2006

Purpose: Everolimus (RAD001, Novartis), an oral derivative of rapamycin, inhibits the mammalian target of rapamycin (mTOR), which regulates many aspects of cell growth and division. A phase I/II study was done to determine safety and efficacy of everolimus in patients with relapsed or refractory hematologic malignancies.

Experimental Design: Two dose levels (5 and 10 mg orally once daily continuously) were evaluated in the phase I portion of this study to determine the maximum tolerated dose of everolimus to be used in the phase II study.

Results: Twenty-seven patients (9 acute myelogenous leukemia, 5 myelodysplastic syndrome, 6 B-chronic lymphocytic leukemia, 4 mantle cell lymphoma, 1 myelofibrosis, 1 natural killer cell/T-cell leukemia, and 1 T-cell prolymphocytic leukemia) received everolimus. No dose-limiting toxicities were observed. Grade 3 potentially drug-related toxicities included hyperglycemia (22%), hypophosphatemia (7%), fatigue (7%), anorexia (4%), and diarrhea (4%). One patient developed a cutaneous leukocytoclastic vasculitis requiring a skin graft. One patient with refractory anemia with excess blasts achieved a major platelet response of over 3-month duration. A second patient with refractory anemia with excess blasts showed a minor platelet response of 25-day duration. Phosphorylation of downstream targets of mTOR, eukaryotic initiation factor 4E-binding protein 1, and/or, p70 S6 kinase, was inhibited in six of nine patient samples, including those from the patient with a major platelet response.

Conclusions: Everolimus is well tolerated at a daily dose of 10 mg daily and may have activity in patients with myelodysplastic syndrome. Studies of everolimus in combination with therapeutic agents directed against other components of the phosphatidylinositol 3-kinase/Akt/mTOR pathway are warranted.
Signal Transducers and Activators of Transcription 3 Pathway Activation in Drug-Resistant Ovarian Cancer
Authors: Duan Zhenfeng , Foster Rosemary , Bell Debra A. , Mahoney Jennifer , Wolak Kathryn , Vaidya Ami , Hampel Constanze , Lee Hang , Seiden Michael V.
Published: September 2006

Purpose: One of the major obstacles in the treatment of ovarian cancer is the development of multidrug resistance. Recent evidence shows that high-grade ovarian cancer often shows activation of the signal transducers and activators of transcription 3 (Stat3) pathway with subsequent transcription of genes that support tumor growth and survival. Less studied is the role of the Stat3 pathway in acquired drug resistance. There is no information on Stat3 expression in chemotherapy naïve ovarian cancer as compared with tumors collected later in the natural history of the disease. To further clarify the significance of Stat3 activation in ovarian cancer, here we investigated the Stat3 expression and activation in ovarian cancer and ovarian cancer multidrug resistance cell lines.

Experimental Design: Western blotting, electrophoretic mobility shift assay, luciferase assays, ELISA assay, and real-time reverse transcription-PCR determined interleukin-6 and Stat3 pathway expression and activation in cell lines. Stat3 expression in ovarian cancer tissue microarray was evaluated by immunohistochemistry.

Results: Activated (phosphorylated) Stat3 is overexpressed in most paclitaxel-resistant ovarian cancer cells. Inhibition of Stat3 activation results in significant decreases in paclitaxel resistance and enhanced apoptosis. Drug-resistant recurrent tumors have significantly greater phosphorylated Stat3 (pStat3) expression as compared with matched primary tumors. Tumors with associated inflammatory cell infiltrates also have a higher proportion of cells staining intensely for nuclear phosphorylated Stat3 as compared with tumors without inflammatory infiltrates, consistent with paracrine activation of the Stat3 pathway by immune-mediated cytokines.

Conclusions: These data support the hypothesis that interruption of Stat3 signaling could reverse resistance to paclitaxel and perhaps other chemotherapy agents in human cancer.