bluuebunny/crossref_metadata_2025 huggingface dataset:
The Cyclin-Dependent Kinase Inhibitor Flavopiridol Potentiates the Effects of Topoisomerase I Poisons by Suppressing Rad51 Expression in a p53-Dependent Manner
Authors: Ambrosini Grazia , Seelman Sharon L. , Qin Li-Xuan , Schwartz Gary K.
Published: April 2008

The results of a phase I clinical trial of the topoisomerase I (Topo I) poison CPT-11 followed by the cyclin-dependent kinase inhibitor flavopiridol in patients with advanced solid tumors indicate that patients whose tumors were wild-type, but not mutant, for p53 obtained the most clinical benefit from this combination therapy. We elected to elucidate the mechanistic basis for this effect in isogenic-paired HCT116 colon cancer cells that were either wild-type (+/+) or null (−/−) for p53. With the combination therapy of SN-38 (the active metabolite of CPT-11) followed by flavopiridol, the induction of apoptosis was 5-fold greater in the p53+/+ cells compared with the p53−/− cells. This sequential treatment induced phosphorylation of p53 at Ser15, which interacted with Rad51, a DNA repair protein involved in homologous recombination. Rad51 bound to p53-Ser15 within the first 5 hours of combination therapy, and then was transcriptionally suppressed at 24 hours by flavopiridol only in p53+/+ cells. Microarray analysis also revealed suppression of Rad51 in a p53-dependent manner. Depletion of Rad51 by small interfering RNA (siRNA) sensitized both p53+/+ and p53−/− cells to SN-38–induced apoptosis with increase of γH2AX, a marker of DNA damage. Conversely, overexpression of Rad51 rescued p53+/+ cells from SN→F–induced apoptosis. Because flavopiridol inhibits Cdk9, we found that inhibition of Cdk9 by DRB or by siRNA could recapitulate the flavopiridol effects, with suppression of Rad51 and induction of apoptosis only in p53+/+ cells. In conclusion, after DNA damage by Topo I poisons, flavopiridol targets homologous recombination through a p53-dependent down-regulation of Rad51, resulting in enhancement of apoptosis. [Cancer Res 2008;68(7):2312–20]
Epithelial-to-Mesenchymal Transition and Integrin-Linked Kinase Mediate Sensitivity to Epidermal Growth Factor Receptor Inhibition in Human Hepatoma Cells
Authors: Fuchs Bryan C. , Fujii Tsutomu , Dorfman Jon D. , Goodwin Jonathan M. , Zhu Andrew X. , Lanuti Michael , Tanabe Kenneth K.
Published: April 2008

Hepatocellular carcinoma (HCC) is associated with a poor prognosis due to late diagnoses and a lack of effective treatment options. Epidermal growth factor receptor (EGFR)–targeted therapies have been effective in other cancers. However, erlotinib and cetuximab have shown only modest efficacy in clinical trials of HCC. We examined epithelial-to-mesenchymal transition (EMT) as a determinant of sensitivity of HCC to EGFR inhibitors. A panel of 12 human hepatoma cell lines were classified as epithelial or mesenchymal based on their expression of E-cadherin and vimentin. The resulting classification correlated with a previous microarray analysis of human hepatoma cell lines whereby the mesenchymal cell lines were shown to have increased expression of genes involved in metastasis and invasion. Sensitivity to erlotinib, gefitinib, and cetuximab was assessed and the epithelial cell lines were found to be significantly more susceptible to all three agents. Analysis of the EGFR pathway showed that EMT status was independent of EGFR expression or downstream extracellular signal–regulated kinase activation and only the epithelial cell lines expressed ErbB3. Interestingly, mesenchymal cells resistant to EGFR inhibitors had increased AKT and signal transducer and activator of transcription-3 activation through elevated expression of integrin-linked kinase (ILK). Mesenchymal cell lines were therefore experimentally transformed with kinase-inactive ILK (KI-ILK) with a resulting decrease in ILK activity and activation of AKT. KI-ILK transformants showed increased sensitivity to EGFR inhibitors both in vitro and in an in vivo xenograft model. These data suggest that EMT predicts HCC sensitivity to EGFR-targeted therapies and that ILK is a novel target to overcome HCC resistance to EGFR inhibition. [Cancer Res 2008;68(7):2391–9]
Pancreatic Stellate Cells: Partners in Crime with Pancreatic Cancer Cells
Authors: Vonlaufen Alain , Joshi Swapna , Qu Changfa , Phillips Phoebe A. , Xu Zhihong , Parker Nicole R. , Toi Cheryl S. , Pirola Romano C. , Wilson Jeremy S. , Goldstein David , Apte Minoti V.
Published: April 2008

Pancreatic stellate cells (PSC) produce the stromal reaction in pancreatic cancer, but their role in cancer progression is not fully elucidated. We examined the influence of PSCs on pancreatic cancer growth using (a) an orthotopic model of pancreatic cancer and (b) cultured human PSCs (hPSC) and human pancreatic cancer cell lines MiaPaCa-2 and Panc-1. Athymic mice received an intrapancreatic injection of saline, hPSCs, MiaPaCa-2 cells, or hPSCs + MiaPaCa-2. After 7 weeks, tumor size, metastases, and tumor histology were assessed. In vitro studies assessed the effect of cancer cell secretions on PSC migration and the effect of hPSC secretions on cancer cell proliferation, apoptosis, and migration. Possible mediators of the effects of hPSC secretions on cancer cell proliferation were examined using neutralizing antibodies. Compared with mice receiving MiaPaCa-2 cells alone, mice injected with hPSCs + MiaPaCa-2 exhibited (a) increased tumor size and regional and distant metastasis, (b) fibrotic bands (desmoplasia) containing activated PSCs within tumors, and (c) increased tumor cell numbers. In vitro studies showed that, in the presence of pancreatic cancer cells, PSC migration was significantly increased. Furthermore, hPSC secretions induced the proliferation and migration, but inhibited the apoptosis, of MiaPaCa-2 and Panc-1 cells. The proliferative effect of hPSC secretions on pancreatic cancer cells was inhibited in the presence of neutralizing antibody to platelet-derived growth factor. Our studies indicate a significant interaction between pancreatic cancer cells and stromal cells (PSCs) and imply that pancreatic cancer cells recruit stromal cells to establish an environment that promotes cancer progression. [Cancer Res 2008;68(7):2085–93]
Insulin-like Growth Factor-I–Dependent Up-regulation of ZEB1 Drives Epithelial-to-Mesenchymal Transition in Human Prostate Cancer Cells
Authors: Graham Tisheeka R. , Zhau Haiyen E. , Odero-Marah Valerie A. , Osunkoya Adeboye O. , Kimbro K. Sean , Tighiouart Mourad , Liu Tongrui , Simons Jonathan W. , O'Regan Ruth M.
Published: April 2008

The epithelial-to-mesenchymal transition (EMT) is crucial for the migration and invasion of many epithelial tumors, including prostate cancer. Although it is known that ZEB1 overexpression promotes EMT primarily through down-regulation of E-cadherin in a variety of cancers, the soluble ligands responsible for the activation of ZEB1 have yet to be identified. In the present study, we investigated the role of insulin-like growth factor-I (IGF-I) in the regulation of ZEB1 during EMT associated with prostate tumor cell migration. We found that ZEB1 is expressed in highly aggressive prostate cancer cells and that its expression correlates directly with Gleason grade in human prostate tumors (P < 0.001). IGF-I up-regulates ZEB1 expression in prostate cancer cells exhibiting an epithelial phenotype. In prostate cancer cells displaying a mesenchymal phenotype, ZEB1 inhibition reverses the suppression of E-cadherin protein and down-regulates the expression of the mesenchymal markers N-cadherin and fibronectin. Furthermore, ZEB1 blockade decreases migratory and invasive potential in ARCaPM compared with the control. These results identify ZEB1 as a key transcriptional regulator of EMT in prostate cancer and suggest that the aberrant expression of ZEB1 in prostate cancer cells occurs in part in response to IGF-I stimulation. [Cancer Res 2008;68(7):2479–88]
The Majority of Viral-Cellular Fusion Transcripts in Cervical Carcinomas Cotranscribe Cellular Sequences of Known or Predicted Genes
Authors: Kraus Irene , Driesch Corina , Vinokurova Svetlana , Hovig Eivind , Schneider Achim , von Knebel Doeberitz Magnus , Dürst Matthias
Published: April 2008

Integration of human papillomavirus (HPV) DNA into the host genome is a frequent event in cervical carcinogenesis and is reported to occur at randomly selected chromosomal sites. However, as the databases are being up-dated continuously, the knowledge based on sequenced viral integration sites also expands. In this study, viral-cellular fusion transcripts of a preselected group of 74 cervical carcinoma or cervical intraepithelial neoplasia grade 3 (CIN3) biopsies harboring integrated HPV16, HPV18, HPV31, HPV33, or HPV45 DNA were amplified by 3′-rapid amplification of cDNA ends PCR and sequenced. Consistent with previous reports, integration sites were found to be distributed throughout the genome. However, 23% (17 of 74) of the integration sites were located within the cytogenetic bands 4q13.3, 8q24.21, 13q22.1, and 17q21, in clusters ranging from 86 to 900 kb. Of note is that clusters 8q24.21 and 13q22.1 are within 1.5 Mbp of an adjacent fragile site whereas clusters 4q13.3 and 17q21 are >15 Mbp distant to any known fragile sites. It is tempting to speculate that as yet unknown fragile sites may be identified on the basis of HPV integration hotspots. No correlation between HPV type and specific integration loci was found. Of 74 fusion transcripts, 28 contained cellular sequences, which were homologous to known genes, and 40 samples contained sequences of predicted genes. In 33 fusion transcripts, both viral and cellular sequences were in sense orientation, indicating that the gene itself or upstream sequences were affected by integration. These data suggest that the influence of HPV integration on host gene expression may not be a rare effect and should encourage more detailed analyses. [Cancer Res 2008;68(7):2514–22]
Attenuation of Junctional Adhesion Molecule-A Is a Contributing Factor for Breast Cancer Cell Invasion
Authors: Naik Meghna U. , Naik Tejal U. , Suckow Arthur T. , Duncan Melinda K. , Naik Ulhas P.
Published: April 2008

The metastatic potential of cancer cells is directly attributed to their ability to invade through the extracellular matrix. The mechanisms regulating this cellular invasiveness are poorly understood. Here, we show that junctional adhesion molecule A (JAM-A), a tight junction protein, is a key negative regulator of cell migration and invasion. JAM-A is robustly expressed in normal human mammary epithelium, and its expression is down-regulated in metastatic breast cancer tumors. In breast cancer cell lines, an inverse relationship between JAM-A expression and the ability of these cells to migrate on a collagen matrix was observed, which correlates with the known ability of these cells to metastasize. The T47D and MCF-7 cells, which migrate least, are found to express high levels of JAM-A, whereas the more migratory MDA-MB-468 cells have lower levels of JAM-A on the cell surface. MDA-MB-231 cells, which are highly migratory, express the least amount of JAM-A. Overexpression of JAM-A in MDA-MB-231 cells inhibited both migration and invasion through collagen gels. Furthermore, knockdown of JAM-A using short interfering RNAs enhanced the invasiveness of MDA-MB-231 cells as well as T47D cells. The ability of JAM-A to attenuate cell invasion correlated with the formation of increased numbers of focal adhesions and the formation of functional tight junctions. These results show for the first time that an immunoglobulin superfamily cell adhesion protein expressed at tight junctions could serve as a key negative regulator of breast cancer cell invasion and possibly metastasis. Furthermore, loss of JAM-A could be used as a biomarker for aggressive breast cancer. [Cancer Res 2008;68(7):2194–203]
Identification of Novel Methylation Markers in Cervical Cancer Using Restriction Landmark Genomic Scanning
Authors: Wang Sophia S. , Smiraglia Dominic J. , Wu Yue-Zhong , Ghosh Srimoyee , Rader Janet S. , Cho Kathleen R. , Bonfiglio Thomas A. , Nayar Ritu , Plass Christoph , Sherman Mark E.
Published: April 2008

Aberrant methylation of CpG islands in gene promoters often represents an early clonal event in carcinogenesis. Accordingly, defining methylation profiles may be useful for developing marker panels for early detection or predicting the risk of cancer precursors. To identify specific genes frequently methylated in cervical cancer, we conducted methylation profiling of 20 primary human cervical cancers using NotI-based restriction landmark genomic scanning (RLGS). Of 2,172 RLGS fragments analyzed (average, 1,753 CpG islands per patient), 186 RLGS fragments were lost in at least one tumor and 40 were lost in three or more. Methylation was identified in 19 (95%) of 20 tumor samples compared with normal DNA. Bisulfite sequencing was conducted to confirm RLGS results. Of the confirmed markers frequently methylated, we developed Methylight assays for two corresponding genes, nucleolar protein 4 (NOL4), and lipoma HMGIC fusion partner–like protein 4 (LHFPL4), which were methylated in 85% and 55% of cancers, respectively. Using these assays, we further confirmed frequent CpG island methylation in the original cancers and in another independent series of 15 cervical cancers. We also showed methylation at a reduced frequency in a set of carefully reviewed cytology specimens demonstrating cells exfoliated from cancer precursor lesions. In summary, we identified, for the first time, NOL4 and LHFPL4 as novel methylation targets specific for cervical cancer. Inclusion of NOL4 and LHFPL4 in evaluating methylation panels for early detection, risk prediction, and etiologic research on cervical cancer is warranted. [Cancer Res 2008;68(7):2489–97]
Lkb1 Deficiency Causes Prostate Neoplasia in the Mouse
Authors: Pearson Helen B. , McCarthy Afshan , Collins Christopher M.P. , Ashworth Alan , Clarke Alan R.
Published: April 2008

Mutation of LKB1 is the key molecular event underlying Peutz-Jeghers syndrome, a dominantly inherited condition characterized by a predisposition to a range of malignancies, including those of the reproductive system. We report here the use of a Cre-LoxP strategy to directly address the role of Lkb1 in prostate neoplasia. Recombination of a LoxP-flanked Lkb1 allele within all four murine prostate lobes was mediated by spontaneous activation of a p450 CYP1A1-driven Cre recombinase transgene (termed AhCre). Homozygous mutation of Lkb1 in males expressing AhCre reduced longevity, with 100% manifesting atypical hyperplasia and 83% developing prostate intraepithelial neoplasia (PIN) of the anterior prostate within 2 to 4 months. We also observed focal hyperplasia of the dorsolateral and ventral lobes (61% and 56% incidence, respectively), bulbourethral gland cysts associated with atypical hyperplasia (100% incidence), hyperplasia of the urethra (39% incidence), and seminal vesicle squamous metaplasia (11% incidence). PIN foci overexpressed nuclear β-catenin, p-Gsk3β, and downstream Wnt targets. Immunohistochemical analysis of foci also showed a reduction in Pten activation and up-regulation of both p-PDK1 (an AMPK kinase) and phosphorylated Akt. Our data are therefore consistent with deregulation of Wnt and phosphoinositide 3-kinase/Akt signaling cascades after loss of Lkb1 function. For the first time, this model establishes a link between the tumor suppressor Lkb1 and prostate neoplasia, highlighting a tumor suppressive role within the mouse and raising the possibility of a similar association in the human. [Cancer Res 2008;68(7):2223–32]
Frequency of the CHEK2 1100delC Mutation among Women with Breast Cancer: An International Study
Authors: Zhang Shiyu , Phelan Catherine M. , Zhang Phil , Rousseau Francois , Ghadirian Parviz , Robidoux Andre , Foulkes William , Hamel Nancy , McCready David , Trudeau Maureen , Lynch Henry , Horsman Douglas , De Matsuda Maria Lourdes Leon , Aziz Zeba , Gomes Magda , Costa Mauricio Magalhaes , Liede Alexander , Poll Aletta , Sun Ping , Narod Steven A.
Published: April 2008

A founder allele in the CHEK2 gene (1100delC) has been associated with an elevated risk of breast cancer. This allele is responsible for the majority of CHEK2-associated breast cancers in women from northern European countries; however, within Europe, it seems to be rare in countries that are close to the Mediterranean. The frequency of the 1100delC allele has not been measured in non-White populations. We measured the frequency of the CHEK2 founder allele in 3,882 breast cancer patients and 8,609 controls from various countries. The allele was not seen among Asian patients (from Pakistan or the Philippines) and was present in 1 of 155 cases from Brazil. Among White women, the allele was present in 1.5% of 825 familial cases of breast cancer and in 0.7% of 1,106 patients with nonfamilial breast cancer. The allele was equally frequent in Jewish and non-Jewish patients. We estimate that the CHEK2 1100delC allele is associated with an odds ratio of 2.6 for breast cancer, which corresponds to a lifetime risk of ∼24% in Ontario. [Cancer Res 2008;68(7):2154–7]
Exploration of Tumor-Suppressive MicroRNAs Silenced by DNA Hypermethylation in Oral Cancer
Authors: Kozaki Ken-ichi , Imoto Issei , Mogi Seiki , Omura Ken , Inazawa Johji
Published: April 2008

In the last few years, microRNAs (miRNA) have started a revolution in molecular biology and emerged as key players in the carcinogenesis. They have been identified in various tumor types, showing that different sets of miRNAs are usually deregulated in different cancers. To identify the miRNA signature that was specific for oral squamous cell carcinoma (OSCC), we first examined expression profiles of 148 miRNAs in a panel of 18 OSCC cell lines and the immortalized oral keratinocyte line RT7 as a control. Compared with RT7, the expression of 54 miRNAs (36.5%) was frequently down-regulated in OSCC lines (<0.5-fold expression, ≥66.7% of 18 lines). Among these 54 miRNAs, we further analyzed four of these miRNAs (i.e., miR-34b, miR-137, miR-193a, and miR-203), located around CpG islands, to identify tumor-suppressive miRNAs silenced through aberrant DNA methylation. The expression of those four genes was restored by treatment with 5-aza-2′-deoxycytidine in OSCC cells lacking their expression. In addition, expression levels of the four miRNAs were inversely correlated with their DNA methylation status in the OSCC lines. In primary tumors of OSCC with paired normal oral mucosa, down-regulation of miRNA expression through tumor-specific hypermethylation was more frequently observed for miR-137 and miR-193a than for miR-34b and miR-203. Moreover, the ectopic transfection of miR-137 or miR-193a into OSCC lines lacking their expressions significantly reduced cell growth, with down-regulation of the translation of cyclin-dependent kinase 6 or E2F transcription factor 6, respectively. Taken together, our results clearly show that miR-137 and miR-193a are tumor suppressor miRNAs epigenetically silenced during oral carcinogenesis. [Cancer Res 2008;68(7):2094–105]
Mitochondrial Function and Nuclear Factor-κB–Mediated Signaling in Radiation-Induced Bystander Effects
Authors: Zhou Hongning , Ivanov Vladimir N. , Lien Yu-Chin , Davidson Mercy , Hei Tom K.
Published: April 2008

Although radiation-induced bystander effects have been well described over the past decade, the mechanisms of the signaling processes involved in the bystander phenomenon remain unclear. In the present study, using the Columbia University charged particle microbeam, we found that mitochondrial DNA–depleted human skin fibroblasts (ρo) showed a higher bystander mutagenic response in confluent monolayers when a fraction of the same population were irradiated with lethal doses compared with their parental mitochondrial–functional cells (ρ+). However, using mixed cultures of ρo and ρ+ cells and targeting only one population of cells with a lethal dose of α-particles, a decreased bystander mutagenesis was uniformly found in nonirradiated bystander cells of both cell types, indicating that signals from one cell type can modulate expression of bystander response in another cell type. In addition, we found that Bay 11-7082, a pharmacologic inhibitor of nuclear factor-κB (NF-κB) activation, and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a scavenger of nitric oxide (NO), significantly decreased the mutation frequency in both bystander ρo and ρ+ cells. Furthermore, we found that NF-κB activity and its dependent proteins, cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS), were lower in bystander ρo cells when compared with their ρ+ counterparts. Our results indicated that mitochondria play an important role in the regulation of radiation-induced bystander effects and that mitochondria-dependent NF-κB/iNOS/NO and NF-κB/COX-2/prostaglandin E2 signaling pathways are important to the process. [Cancer Res 2008;68(7):2233–40]
Two Distinct Molecular Mechanisms Underlying Cytarabine Resistance in Human Leukemic Cells
Authors: Cai Jie , Damaraju Vijaya L. , Groulx Normand , Mowles Delores , Peng Yunshan , Robins Morris J. , Cass Carol E. , Gros Philippe
Published: April 2008

To understand the mechanism of cellular resistance to the nucleoside analogue cytarabine (1-β-d-arabinofuranosylcytosine, AraC), two resistant derivatives of the human leukemic line CCRF-CEM were obtained by stepwise selection in different concentrations of AraC. CEM/4×AraC cells showed low AraC resistance, whereas CEM/20×AraC cells showed high resistance. Both cell lines showed similar patterns of cross-resistance to multiple cytotoxic nucleoside analogues, with the exception that CEM/20×AraC cells remained sensitive to 5-fluorouridine and 2-deoxy-5-fluorouridine. Both cell lines were sensitive to 5-fluorouracil and to a variety of natural product drugs. Although both CEM/4×AraC and CEM/20×AraC cells displayed reduced intracellular accumulation of [3H]AraC, only CEM/4×AraC cells showed reduced uptake of [3H]uridine, which was used to assess nucleoside transport activities. Genes encoding proteins known to be involved in nucleoside transport, efflux, and metabolism were analyzed for the presence of mutations in the two cell lines. In CEM/4×AraC cells, independent mutations were identified at each allele of human equilibrative nucleoside transporter 1 (hENT1; SLC29A1), one corresponding to a single-nucleotide change in exon 4, the other being a complex intronic mutation disrupting splicing of exon 13. In contrast to CEM/20×AraC cells, CEM/4×AraC cells did not bind the hENT1/SLC29A1 ligand nitrobenzylmercaptopurine ribonucleoside and lacked detectable hENT1/SLC29A1 protein. In CEM/20×AraC cells, independent intronic mutations impairing splicing of exons 2 and 3 were found at each allele of the deoxycytidine kinase gene. These studies point to at least two distinct mechanisms of AraC resistance in leukemic cells. [Cancer Res 2008;68(7):2349–57]
The Role of Amphiregulin in Exemestane-Resistant Breast Cancer Cells: Evidence of an Autocrine Loop
Authors: Wang Xin , Masri Selma , Phung Sheryl , Chen Shiuan
Published: April 2008

Exemestane-resistant breast cancer cell lines (i.e., ExeR), derived from MCF-7 cells expressing a high level of aromatase (MCF-7aro), were generated in our laboratory. The epidermal growth factor (EGF)–like protein amphiregulin (AREG) was highly expressed in ExeR cells based on cDNA microarray analysis. The high levels of AREG mRNA in ExeR cell lines were confirmed by real-time reverse transcription-PCR. The high levels of AREG protein in ExeR cell lysates and culture media were confirmed by Western blot analysis and ELISA, respectively. Furthermore, our Western blot analysis showed that whereas no AREG was detected in the DMSO control, overnight treatment of parental MCF-7aro cells with 1 μmol/L exemestane strongly induced the expression of AREG. This induction was totally blocked by 100 nmol/L of pure antiestrogen ICI 182,780, implying estrogen receptor (ER) dependence of exemestane-induced AREG expression. MCF-7aro cells were not able to proliferate in hormone-free medium, but were able to proliferate in conditioned medium from ExeR cells, similar to the treatment of recombinant human AREG. Small interference RNA targeting AREG inhibited ExeR proliferation, confirming that AREG is truly functioning as a growth factor of ExeR cells. The specific inhibitors to ER (ICI 182,780), EGF receptor (EGFR; AG1478), and mitogen-activated protein kinase (MAPK; U0126) all showed dose-dependent suppression of the proliferation of ExeR cells, indicating the involvement of the ER, EGFR, and MAPK pathways. Based on these findings, we propose a possible mechanism that underlies exemestane resistance: exemestane induces AREG in an ER-dependent manner. AREG then activates the EGFR pathway and leads to the activation of the MAPK pathway that drives cell proliferation. [Cancer Res 2008;68(7):2259–65]
The Nucleolin Targeting Aptamer AS1411 Destabilizes Bcl-2 Messenger RNA in Human Breast Cancer Cells
Authors: Soundararajan Sridharan , Chen Weiwei , Spicer Eleanor K. , Courtenay-Luck Nigel , Fernandes Daniel J.
Published: April 2008

We sought to determine whether nucleolin, a bcl-2 mRNA-binding protein, has a role in the regulation of bcl-2 mRNA stability in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 μmol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 μmol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the half-life of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 μmol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death. [Cancer Res 2008;68(7):2358–65]
Phosphatase and Tensin Homologue Deleted on Chromosome 10 Deficiency Accelerates Tumor Induction in a Mouse Model of ErbB-2 Mammary Tumorigenesis
Authors: Dourdin Nathalie , Schade Babette , Lesurf Robert , Hallett Michael , Munn Robert J. , Cardiff Robert D. , Muller William J.
Published: April 2008

Loss of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and amplification or elevated expression of ErbB-2 are both involved in human breast cancer. To directly test the importance of these genetic events in mammary tumorigenesis, we have assessed whether mammary-specific disruption of PTEN could cooperate with activation of ErbB-2. Transgenic mice expressing ErbB-2 under the transcriptional control of its endogenous promoter (ErbB-2KI) were interbred with mice carrying conditional PTEN alleles and an MMTV/Cre transgene. Loss of one or both PTEN alleles resulted in a dramatic acceleration of mammary tumor onset and an increased occurrence of lung metastases in the ErbB-2KI strain. Tumor progression in PTEN-deficient/ErbB-2KI strains was associated with elevated ErbB-2 protein levels, which were not due to ErbB-2 amplification or to a dramatic increase in ErbB-2 transcripts. Moreover, the PTEN-deficient/ErbB-2KI–derived mouse mammary tumors display striking morphologic heterogeneity in comparison with the homogeneous pathology of the ErbB-2KI parental strain. Therefore, inactivation of PTEN would not only have a dramatic effect on ErbB-2–induced mammary tumorigenesis but would also lead to the formation of mammary tumors that, in part, display pathologic and molecular features associated with the basal-like subtype of primary human breast cancer. [Cancer Res 2008;68(7):2122–31]
MUC4 Mucin Interacts with and Stabilizes the HER2 Oncoprotein in Human Pancreatic Cancer Cells
Authors: Chaturvedi Pallavi , Singh Ajay P. , Chakraborty Subhankar , Chauhan Subhash C. , Bafna Sangeeta , Meza Jane L. , Singh Pankaj K. , Hollingsworth Michael A. , Mehta Parmender P. , Batra Surinder K.
Published: April 2008

MUC4, a high–molecular weight transmembrane glycoprotein, is overexpressed in pancreatic cancer and is implicated in its pathogenesis. It is a heterodimeric protein containing a large extracellular, heavily glycosylated subunit, MUC4α, and a transmembrane growth factor–like subunit, MUC4β. In the present study, we have shown the interaction of human MUC4 with the receptor tyrosine kinase HER2 in pancreatic adenocarcinoma cells by reciprocal coimmunoprecipitation and cocapping studies. MUC4 colocalized with HER2 at the cell surface and in the cytoplasm. Silencing of MUC4 by transient or stable expression of MUC4-targeted short-interfering RNA led to the down-regulation of HER2 with a concomitant decrease in its phosphorylated form (pY1248-HER2). Further analyses revealed that the MUC4-knockdown–mediated decrease in HER2 expression occurred due to the drop in the stability of the receptor. In MUC4-knockdown pancreatic cancer cells, we also observed a reduced phosphorylation of the focal adhesion kinase and p42/44 mitogen-activated protein kinase, which are downstream effector proteins in HER2 signaling. Our findings add a new dimension to MUC4 function as a modulator of cell signaling and provide mechanistic evidence for its role in pancreatic cancer progression. [Cancer Res 2008;68(7):2065–70]
Mammalian Target of Rapamycin and S6 Kinase 1 Positively Regulate 6-thioguanine-Induced Autophagy
Authors: Zeng Xuehuo , Kinsella Timothy J.
Published: April 2008

DNA mismatch repair (MMR) ensures the fidelity of DNA replication and is required for activation of cell cycle arrest and apoptosis in response to certain classes of DNA damage. We recently reported that MMR is also implicated in initiation of an autophagic response after MMR processing of 6-thioguanine (6-TG). It is now generally believed that autophagy is negatively controlled by mammalian target of rapamycin (mTOR) activity. To determine whether mTOR is involved in 6-TG–induced autophagy, we used rapamycin, a potential anticancer agent, to inhibit mTOR activity. Surprisingly, we find that rapamycin cotreatment inhibits 6-TG–induced autophagy in MMR-proficient human colorectal cancer HCT116 (MLH1+) and HT29 cells as measured by LC3 immunoblotting, GFP-LC3 relocalization, and acridine orange staining. Consistently, short interfering RNA silencing of the 70-kDa ribosomal S6 kinase 1 (S6K1), the downstream effector of mTOR, markedly reduces 6-TG–induced autophagy. Furthermore, we show that inhibition of mTOR by rapamycin induces the activation of Akt as shown by increased Akt phosphorylation at Ser473 and the inhibition of 6-TG–induced apoptosis and cell death. Activated Akt is a well-known inhibitor of autophagy. In conclusion, our data indicate that mTOR-S6K1 positively regulates autophagy after MMR processing of 6-TG probably through its negative feedback inhibition of Akt. [Cancer Res 2008;68(7):2384–90]
Aspirin, NSAID, and Acetaminophen Use and the Risk of Endometrial Cancer
Authors: Viswanathan Akila N. , Feskanich Diane , Schernhammer Eva S. , Hankinson Susan E.
Published: April 2008

To date, no prospective studies have explored the relationship between the use of aspirin, other nonsteroidal anti-inflammatory medications (NSAID), and acetaminophen and endometrial adenocarcinoma. Of the 82,971 women enrolled in a prospective cohort study, 747 developed medical record–confirmed invasive endometrial cancer over a 24-year period. Use of aspirin was ascertained from 1980 to 2004, and for other NSAIDs and acetaminophen, from 1990 to 2004. Cox regression models calculated multivariate relative risks (MV RR), controlling for body mass index (BMI), postmenopausal hormone (PMH) use, and other endometrial cancer risk factors. Currency, duration, and quantity of aspirin were not associated with endometrial cancer risk overall [current use: MV RR, 1.03; 95% confidence interval (CI) 0.83–1.27; >10 years of use: MV RR, 1.01; 95% CI, 0.78–1.30; and cumulative average >7 tablets per week: (MV RR, 1.10; 95% CI, 0.84–1.44)]. However, stratified analyses showed that a lower risk of endometrial cancer among obese (BMI, ≥30 kg/m2) women was seen with current aspirin use (MV RR, 0.66; 95% CI, 0.46–0.95). The greatest risk reduction for current aspirin users was seen in postmenopausal obese women who had never used PMH (MV RR, 0.43; 95% CI, 0.26–0.73). The use of other NSAIDs or acetaminophen was not associated with endometrial cancer. Our data suggest that use of aspirin or other NSAIDs does not play an important role in endometrial cancer risk overall. However, risk was significantly lower for current aspirin users who were obese or who were postmenopausal and had never used PMHs; these subgroup findings require further confirmation. [Cancer Res 2008;68(7):2507–13]
Mutation Frequencies and Spectra in DNA Polymerase η–Deficient Mice
Authors: Busuttil Rita A. , Lin Qingcong , Stambrook Peter J. , Kucherlapati Raju , Vijg Jan
Published: April 2008

The low-fidelity polymerase η (polη) is required for bypass of UV-induced pyrimidine dimers inserting adenine nucleotides opposite these lesions. Mutations in the polη gene are responsible for the genetic defect in xeroderma pigmentosum variant patients. To study if the lack of polη significantly elevates spontaneous mutation frequency in various organs and tissues of the mouse, we crossed polη-deficient mice with transgenic mice harboring a chromosomally integrated lacZ-plasmid reporter construct. In cultured embryonic fibroblasts from the lacZ-polη−/− mice, 2.5 J/m2 UV irradiation induced ∼5-fold more mutations than in cells from lacZ control mice, in which an ∼3-fold increase in mutation frequency was found compared with the normal level. Whereas untreated cells harbored mainly 1-bp deletions, UV induced both transitions and transversions, with the latter type more highly represented in the polη-null cells than in the controls. No difference in mutation induction between the polη-null cells and the wild-type cells was observed after treatment with N-ethyl-N-nitrosourea. Having shown the validity of the lacZ model to accurately identify polη-associated mutagenesis, we then determined the mutant frequency at the lacZ locus in liver, spleen, and small intestine of 12-month-old animals. No differences were found between polη-null, heterozygous, or littermate control mice. We conclude that the polη defect is specific for UV damage and has no effect on in vivo mutagenesis in mice. [Cancer Res 2008;68(7):2081–4]
Mcl-1: A Gateway to TRAIL Sensitization
Authors: Kim Seok-Hyun , Ricci M. Stacey , El-Deiry Wafik S.
Published: April 2008

The proapoptotic cytokine tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is being evaluated presently as a selective anticancer agent, but its limited effects against cancer cell lines has raised some concerns about its ultimate clinical utility. Here, we review recent findings that cancer cell sensitivity to TRAIL is greatly increased when the Bcl-2 family protein Mcl-1 is down-regulated by the Raf/vascular endothelial growth factor kinase inhibitor sorafenib, a Food and Drug Administration–approved cancer drug. Using the TRAIL-sorafenib combination as a tactic to more effectively kill cancer cells may provide an effective tool to attack a variety of human cancers that are largely presently untreatable. [Cancer Res 2008;68(7):2062–4]
Mitogen- and Stress-Activated Kinase 1–Mediated Histone H3 Phosphorylation Is Crucial for Cell Transformation
Authors: Kim Hong-Gyum , Lee Ki Won , Cho Yong-Yeon , Kang Nam Joo , Oh Sang-Muk , Bode Ann M. , Dong Zigang
Published: April 2008

Mitogen- and stress-activated kinase 1 (MSK1) belongs to a family of dual protein kinases that are activated by either extracellular signal-regulated kinase or p38 mitogen-activated protein kinases in response to stress or mitogenic extracellular stimuli. The physiologic role of MSK1 in malignant transformation and cancer development is not well understood. Here, we report that MSK1 is involved in 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced or epidermal growth factor (EGF)–induced neoplastic transformation of JB6 Cl41 cells. H89, a potent inhibitor of MSK1, strongly suppressed TPA-induced or EGF-induced cell transformation. When cells overexpressing wild-type MSK1 were treated with TPA or EGF, colony formation increased substantially compared with untreated cells or cells that did not overexpress MSK1. In contrast, MSK1 COOH terminal or NH2 terminal dead dominant negative mutants dramatically suppressed cell transformation. Introduction of small interfering RNA-MSK1 into JB6 Cl41 cells resulted in suppressed TPA-induced or EGF-induced cell transformation. In addition, cell proliferation was inhibited in MSK1 knockdown cells compared with MSK1 wild-type cells. In wild-type MSK1-overexpressing cells, activator protein (AP-1) activation increased after TPA or EGF stimulation, whereas AP-1 activation decreased in both MSK1 dominant-negative mutants and in MSK1 knockdown cells. Moreover, TPA-induced or EGF-induced phosphorylation of histone H3 at Ser10 was increased in wild-type cells but the induced phosphorylation was abolished in MSK1 dominant-negative mutant or MSK1 knockdown cells. Thus, MSK1 is required for tumor promoter-induced cell transformation through its phosphorylation of histone H3 at Ser10 and AP-1 activation. [Cancer Res 2008;68(7):2538–47]
Early Metabolic Response Evaluation by Fluorine-18 Fluorodeoxyglucose Positron Emission Tomography Allows In vivo Testing of Chemosensitivity in Gastric Cancer: Long-term Results of a Prospective Study
Authors: Ott Katja , Herrmann Ken , Lordick Florian , Wieder Hinrich , Weber Wolfgang A. , Becker Karen , Buck Andreas K. , Dobritz Martin , Fink Ulrich , Ulm Kurt , Schuster Tibor , Schwaiger Markus , Siewert Jörg-Rüdiger , Krause Bernd J.
Published: April 2008

Purpose: We prospectively evaluated the predictive value of positron emission tomography using fluorine-18 fluorodeoxyglucose (FDG-PET) for in vivo testing of chemosensitivity in locally advanced gastric cancer using an a priori definition of metabolic response (a decrease of >35% of the standard uptake value). The goal of the study was the definition of biologically different groups of patients prior to or early during induction therapy, with special emphasis on FDG non–avid tumors.

Experimental Design: Based on our data, which was published in 2003, at least 36 patients with metabolic response or FDG non–avid tumors had to be recruited for an analysis of the group of FDG non–avid tumors with sufficient statistical power. Seventy-one patients (32 metabolic nonresponders, 17 metabolic responders, and 22 patients with FDG non–avid tumors) underwent FDG-PET at baseline. In FDG-avid tumors, FDG-PET was repeated 14 days after the initiation of chemotherapy.

Results: Metabolic responders (17 of 49) showed a high histopathologic response rate (69%) and a favorable prognosis (median survival not reached), whereas metabolic nonresponders (32 of 49) had a poor prognosis (median survival, 24.1 months) and showed a histopathologic response in 17%. The histopathologic response rate (24%) for FDG-PET non–avid patients showed no significant difference compared with FDG-avid nonresponders (P = 0.72). Survival of FDG non–avid patients was 36.7 months (not significantly different from FDG-avid nonresponders, 24.1 months, P = 0.46).

Conclusion: In locally advanced gastric cancer, three different metabolic groups exist. Response and survival was predicted by PET in FDG-avid tumors. Metabolic response assessment was not possible in FDG non–avid tumors; however, due to unfavorable outcome, therapy modification might also be considered in FDG non–avid tumors.
Integrated Genomic and Transcriptomic Analysis of Ductal Carcinoma In situ of the Breast
Authors: Vincent-Salomon Anne , Lucchesi Carlo , Gruel Nadège , Raynal Virginie , Pierron Gaëlle , Goudefroye Rémi , Reyal Fabien , Radvanyi François , Salmon Rémy , Thiery Jean-Paul , Sastre-Garau Xavier , Sigal-Zafrani Brigitte , Fourquet Alain , Delattre Olivier ,   for the breast cancer study group of the Institut Curie
Published: April 2008

Purpose: To gain insight into genomic and trancriptomic subtypes of ductal carcinomas in situ of the breast (DCIS).

Experimental Design: We did a combined phenotypic and genomic analysis of a series of 57 DCIS integrated with gene expression profile analysis for 26 of the 57 cases.

Results: Thirty-two DCIS exhibited a luminal phenotype; 21 were ERBB2 positive, and 4 were ERBB2/estrogen receptor (ER) negative with 1 harboring a bona fide basal-like phenotype. Based on a CGH analysis, genomic types were identified in this series of DCIS with the 1q gain/16q loss combination observed in 3 luminal DCIS, the mixed amplifier pattern including all ERBB2, 12 luminal and 2 ERBB2-/ER- DCIS, and the complex copy number alteration profile encompassing 14 luminal and 1 ERBB2-/ER- DCIS. Eight cases (8 of 57; 14%) presented a TP53 mutation, all being amplifiers. Unsupervised analysis of gene expression profiles of 26 of the 57 DCIS showed that luminal and ERBB2-amplified, ER-negative cases clustered separately. We further investigated the effect of high and low copy number changes on gene expression. Strikingly, amplicons but also low copy number changes especially on 1q, 8q, and 16q in DCIS regulated the expression of a subset of genes in a very similar way to that recently described in invasive ductal carcinomas.

Conclusions: These combined approaches show that the molecular heterogeneity of breast ductal carcinomas exists already in in situ lesions and further indicate that DCIS and invasive ductal carcinomas share genomic alterations with a similar effect on gene expression profile.
A Phase I Pharmacokinetic and Pharmacodynamic Study of TKI258, an Oral, Multitargeted Receptor Tyrosine Kinase Inhibitor in Patients with Advanced Solid Tumors
Authors: Sarker Debashis , Molife Rhoda , Evans T.R. Jeffrey , Hardie Maryon , Marriott Cheryl , Butzberger-Zimmerli Priska , Morrison Rosemary , Fox Judith A. , Heise Carla , Louie Sharianne , Aziz Natasha , Garzon Felix , Michelson Glenn , Judson Ian R. , Jadayel Dalal , Braendle Edgar , de Bono Johann S.
Published: April 2008

Purpose: To determine the maximum tolerated dose (MTD) dose-limiting toxicity, and pharmacokinetic and pharmacodynamic profile of TKI258 (formerly CHIR-258).

Experimental Design: A phase I dose escalating trial in patients with advanced solid tumors was performed. Treatment was initially as single daily doses on an intermittent 7-day on/7-day off schedule. Following a protocol amendment, a second schedule comprised, during cycle 1, 7-day on/7-day off treatment followed by 14 days of continuous daily dosing; subsequent cycles comprised 28 days of daily dosing. Pharmacokinetics and evaluation of phosphorylated extracellular signal-regulated kinase (ERK) in peripheral blood mononuclear cells were done during the first 28 days of each schedule.

Results: Thirty-five patients were treated in four intermittent (25-100 mg/d) and three continuous (100-175 mg/d) dosing cohorts. Observed drug-related toxicities were nausea and vomiting, fatigue, headache, anorexia, and diarrhea. Dose-limiting toxicities were grade 3 hypertension in one patient at 100 mg continuous dosing, grade 3 anorexia in a second patient at 175 mg, and grade 3 alkaline phosphatase elevation in a third patient at 175 mg. One patient had a partial response (melanoma) and two patients had stable disease >6 months. TKI258 pharmacokinetics were linear over the dose range of 25 to 175 mg. Five of 14 evaluable patients had modulation of phosphorylated ERK levels.

Conclusions: The MTD was defined as 125 mg/d. Evidence of antitumor activity in melanoma and gastrointestinal stromal tumors warrants further investigation, and other phase I studies are ongoing. Further pharmacodynamic evaluation is required in these studies to evaluate the biological effects of TKI258.
Gene Expression–Based Recurrence Prediction of Hepatitis B Virus–Related Human Hepatocellular Carcinoma
Authors: Woo Hyun Goo , Park Eun Sung , Cheon Jae Hee , Kim Ju Han , Lee Ju-Seog , Park Bum Joon , Kim Won , Park Su Cheol , Chung Young Jin , Kim Byeong Gwan , Yoon Jung-Hwan , Lee Hyo-Suk , Kim Chung Yong , Yi Nam-Joon , Suh Kyung-Suk , Lee Kuhn Uk , Chu In-Sun , Roskams Tania , Thorgeirsson Snorri S. , Kim Yoon Jun
Published: April 2008

Purpose: The poor prognosis of hepatocellular carcinoma (HCC) is, in part, due to the high rate of recurrence even after “curative resection” of tumors. Therefore, it is axiomatic that the development of an effective prognostic prediction model for HCC recurrence after surgery would, at minimum, help to identify in advance those who would most benefit from the treatment, and at best, provide new therapeutic strategies for patients with a high risk of early recurrence.

Experimental Design: For the prediction of the recurrence time in patients with HCC, gene expression profiles were generated in 65 HCC patients with hepatitis B infections.

Result: Recurrence-associated gene expression signatures successfully discriminated between patients at high-risk and low-risk of early recurrence (P = 1.9 × 10−6, log-rank test). To test the consistency and robustness of the recurrence signature, we validated its prognostic power in an independent HCC microarray data set. CD24 was identified as a putative biomarker for the prediction of early recurrence. Genetic network analysis suggested that SP1 and peroxisome proliferator–activated receptor-α might have regulatory roles for the early recurrence of HCC.

Conclusion: We have identified a gene expression signature that effectively predicted early recurrence of HCC independent of microarray platforms and cohorts, and provided novel biological insights into the mechanisms of tumor recurrence.
Population Pharmacokinetic-Pharmacodynamic Model of the Vascular-Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid in Cancer Patients
Authors: Li Jing , Jameson Michael B. , Baguley Bruce C. , Pili Roberto , Baker Sharyn D.
Published: April 2008

Purpose: To develop a population pharmacokinetic-pharmacodynamic (PK-PD) model that defines the dose-concentration-effect relationship of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), using plasma 5-hydroxyindole-3-acetic acid (5-HIAA) as a biomarker for the antivascular effect of DMXAA.

Experimental Design: The plasma DMXAA and 5-HIAA concentration data were obtained from 124 patients receiving DMXAA monotherapy as a 20-minute i.v. infusion weekly or every 3 weeks at doses of 6 to 4,900 mg/m2. The PK and PD data were analyzed by nonlinear mixed effects modeling with NONMEM version 5.

Results: DMXAA concentration-time profiles were well described by a three-compartment model with saturable elimination (Michaelis-Menten kinetics). Body surface area (BSA) and sex were significant covariates on the volume of distribution of the central compartment (V1) and the maximum elimination rate (Vm), respectively. Population estimates for Vm, Km (concentration at which half Vm is achieved), and V1 were 112[1 + 0.474(2 − sex)] μmol/L/h, 102 μmol/L, and 8.19(BSA/1.8)0.857 liters, respectively (sex in Vm is equal to 1 for males and equal to 2 for females). The effect of DMXAA on plasma 5-HIAA was described by the stimulatory Emax model, where population estimates for baseline, Emax, and EC50 were 46.3 μmol/L, 2.62-fold increase of the baseline value, and 631 μmol/L, respectively.

Conclusions: DMXAA plasma disposition is characterized by a saturable elimination process. BSA-guided dosing is important. The present PK-PD model, with 5-HIAA as a biomarker, supports the use of DMXAA doses of 1,000 to 2,000 mg/m2 in phase II studies, and provides an example of how PK-PD models can be used to aid in selection of drug doses for phase II evaluation.