bluuebunny/crossref_metadata_2025 huggingface dataset:
TGFBI Deficiency Predisposes Mice to Spontaneous Tumor Development
Authors: Zhang Ye , Wen Gengyun , Shao Genze , Wang Cuidong , Lin Chyuansheng , Fang Hongbo , Balajee Adayabalam S. , Bhagat Govind , Hei Tom K. , Zhao Yongliang
Published: December 2008

Loss of TGFBI, a secreted protein induced by transforming growth factor-β, has been implicated in cell proliferation, tumor progression, and angiogenesis by in vitro studies. However, in vivo antitumor functions of TGFBI as well as the underlying molecular mechanism are not well understood. To these aims, we have generated a mouse model with disruption of TGFBI genomic locus. Mice lacking TGFBI show a retarded growth and are prone to spontaneous tumors and 7,12-dimethylbenz(a)anthracene–induced skin tumors. In relation to wild-type (WT) mouse embryonic fibroblasts (MEF), TGFBI−/− MEFs display increased frequencies of chromosomal aberration and micronuclei formation and exhibit an enhanced proliferation and early S-phase entry. Cyclin D1 is up-regulated in TGFBI−/− MEFs, which correlates with aberrant activation of transcription factor cyclic AMP–responsive element binding protein (CREB) identified by chromatin immunoprecipitation and luciferase reporter assays. TGFBI reconstitution in TGFBI−/− cells by either retroviral infection with WT TGFBI gene or supplement with recombinant mouse TGFBI protein in the culture medium leads to the suppression of CREB activation and cyclin D1 expression, and further inhibition of cell proliferation. Cyclin D1 up-regulation was also identified in most of the tumors arising from TGFBI−/− mice. Our studies provide the first evidence that TGFBI functions as a tumor suppressor in vivo. [Cancer Res 2009;69(1):37–44]
Identification of an Indispensable Role for Tyrosine Kinase 2 in CTL-Mediated Tumor Surveillance
Authors: Simma Olivia , Zebedin Eva , Neugebauer Nina , Schellack Carola , Pilz Andreas , Chang-Rodriguez Souyet , Lingnau Karen , Weisz Eva , Putz Eva Maria , Pickl Winfried F. , Felzmann Thomas , Müller Mathias , Decker Thomas , Sexl Veronika , Stoiber Dagmar
Published: December 2008

We showed previously that Tyk2−/− natural killer cells lack the ability to lyse leukemic cells. As a consequence, the animals are leukemia prone. Here, we show that the impaired tumor surveillance extends to T cells. Challenging Tyk2−/− mice with EL4 thymoma significantly decreased disease latency. The crucial role of Tyk2 for CTL function was further characterized using the ovalbumin-expressing EG7 cells. Tyk2−/− OT-1 mice developed EG7-induced tumors significantly faster compared with wild-type (wt) controls. In vivo assays confirmed the defect in CD8+ cytotoxicity on Tyk2 deficiency and clearly linked it to type I IFN signaling. An impaired CTL activity was only observed in IFNAR1−/− animals but not on IFNγ or IL12p35 deficiency. Accordingly, EG7-induced tumors grew faster in IFNAR1−/− and Tyk2−/− but not in IFNγ−/− or IL12p35−/− mice. Adoptive transfer experiments defined a key role of Tyk2 in CTL-mediated tumor surveillance. In contrast to wt OT-1 cells, Tyk2−/− OT-1 T cells were incapable of controlling EG7-induced tumor growth. [Cancer Res 2009;69(1):203–11]
NORE1B Is a Putative Tumor Suppressor in Hepatocarcinogenesis and May Act via RASSF1A
Authors: Macheiner Doris , Gauglhofer Christine , Rodgarkia-Dara Chantal , Grusch Michael , Brachner Andreas , Bichler Christoph , Kandioler Daniela , Sutterlüty Hedwig , Mikulits Wolfgang , Schulte-Hermann Rolf , Grasl-Kraupp Bettina
Published: December 2008

Recently, we found epigenetic silencing of the Ras effector genes NORE1B and/or RASSF1A in 97% of the hepatocellular carcinoma (HCC) investigated. This is strong evidence that the two genes are of major significance in hepatocarcinogenesis. Although RASSF1A serves as a tumor suppressor gene, the functions of NORE1B are largely unknown. Here, we studied the role of NORE1B for growth and transformation of cells. To understand the molecular mechanisms of action of the gene, we used the wild-type form and deletion mutants without the NH2 terminus and CENTRAL domain, the Ras association (RA) domain, or the COOH-terminal SARAH-domain. Intact RA and SARAH-domains were found to be necessary for NORE1B (a) to increase the G0-G1 fraction in hepatoma cells, (b) to suppress c-Myc/Ha-Ras–induced cell transformation, and (c) to interact closely with RASSF1A, as determined with fluorescence resonance energy transfer. In further studies, cell cycle delay by NORE1B was equally effective in hepatocyte cell lines with wild-type or mutant Ras suggesting that NORE1B does not interact with either Ras. In conclusion, NORE1B suppresses replication and transformation of cells as effectively as RASSF1A and thus is a putative tumor suppressor gene. NORE1B interacts physically with RASSF1A and functional loss of one of the interacting partners may lead to uncontrolled growth and transformation of hepatocytes. This may explain the frequent epigenetic silencing of NORE1B and/or RASSF1A in HCC. [Cancer Res 2009;69(1):235–42]
Distinct Roles for Mammalian Target of Rapamycin Complexes in the Fibroblast Response to Transforming Growth Factor-β
Authors: Rahimi Rod A. , Andrianifahanana Mahefatiana , Wilkes Mark C. , Edens Maryanne , Kottom Theodore J. , Blenis John , Leof Edward B.
Published: December 2008

Transforming growth factor-β (TGF-β) promotes a multitude of diverse biological processes, including growth arrest of epithelial cells and proliferation of fibroblasts. Although the TGF-β signaling pathways that promote inhibition of epithelial cell growth are well characterized, less is known about the mechanisms mediating the positive response to this growth factor. Given that TGF-β has been shown to promote fibrotic diseases and desmoplasia, identifying the fibroblast-specific TGF-β signaling pathways is critical. Here, we investigate the role of mammalian target of rapamycin (mTOR), a known effector of phosphatidylinositol 3-kinase (PI3K) and promoter of cell growth, in the fibroblast response to TGF-β. We show that TGF-β activates mTOR complex 1 (mTORC1) in fibroblasts but not epithelial cells via a PI3K-Akt-TSC2–dependent pathway. Rapamycin, the pharmacologic inhibitor of mTOR, prevents TGF-β–mediated anchorage-independent growth without affecting TGF-β transcriptional responses or extracellular matrix protein induction. In addition to mTORC1, we also examined the role of mTORC2 in TGF-β action. mTORC2 promotes TGF-β–induced morphologic transformation and is required for TGF-β–induced Akt S473 phosphorylation but not mTORC1 activation. Interestingly, both mTOR complexes are necessary for TGF-β–mediated growth in soft agar. These results define distinct and overlapping roles for mTORC1 and mTORC2 in the fibroblast response to TGF-β and suggest that inhibitors of mTOR signaling may be useful in treating fibrotic processes, such as desmoplasia. [Cancer Res 2009;69(1):84–93]
A New Model for Prediction of Drug Distribution in Tumor and Normal Tissues: Pharmacokinetics of Temozolomide in Glioma Patients
Authors: Rosso Lula , Brock Cathryn S. , Gallo James M. , Saleem Azeem , Price Patricia M. , Turkheimer Federico E. , Aboagye Eric O.
Published: December 2008

Difficulties in direct measurement of drug concentrations in human tissues have hampered the understanding of drug accumulation in tumors and normal tissues. We propose a new system analysis modeling approach to characterize drug distribution in tissues based on human positron emission tomography (PET) data. The PET system analysis method was applied to temozolomide, an important alkylating agent used in the treatment of brain tumors, as part of standard temozolomide treatment regimens in patients. The system analysis technique, embodied in the convolution integral, generated an impulse response function that, when convolved with temozolomide plasma concentration input functions, yielded predicted normal brain and brain tumor temozolomide concentration profiles for different temozolomide dosing regimens (75–200 mg/m2/d). Predicted peak concentrations of temozolomide ranged from 2.9 to 6.7 μg/mL in human glioma tumors and from 1.8 to 3.7 μg/mL in normal brain, with the total drug exposure, as indicated by the tissue/plasma area under the curve ratio, being about 1.3 in tumor compared with 0.9 in normal brain. The higher temozolomide exposures in brain tumor relative to normal brain were attributed to breakdown of the blood-brain barrier and possibly secondary to increased intratumoral angiogenesis. Overall, the method is considered a robust tool to analyze and predict tissue drug concentrations to help select the most rational dosing schedules. [Cancer Res 2009;69(1):120–7]
Pretargeted Radioimmunotherapy Using Anti-CD45 Monoclonal Antibodies to Deliver Radiation to Murine Hematolymphoid Tissues and Human Myeloid Leukemia
Authors: Pagel John M. , Matthews Dana C. , Kenoyer Aimee , Hamlin Donald K. , Wilbur Daniel S. , Fisher Darrell R. , Gopal Ajay K. , Lin Yukang , Saganic Laura , Appelbaum Frederick R. , Press Oliver W.
Published: December 2008

Radioimmunotherapy (RIT) for treatment of hematologic malignancies frequently fails because of disease recurrence. We therefore conducted pretargeted (P)RIT studies to augment the efficacy in mice of therapy using a pretargeted anti-human (h)CD45 antibody (Ab)-streptavidin (SA) conjugate followed by a biotinylated clearing agent and radiolabeled 1,4,7,10-tetraazacylodode cane N,N′,N″,N‴-tetraacetic (DOTA)-biotin. Tumor-to-blood ratios at 24 hours were 20:1 using pretargeted anti-hCD45 RIT and <1:1 with conventional RIT. In vivo imaging studies confirmed that the PRIT approach provided high-contrast tumor images with minimal blood-pool activity, whereas directly labeled anti-hCD45 Ab produced distinct tumor images but the blood pool retained a large amount of labeled Ab for a prolonged time. Therapy experiments showed that 90Y-DOTA-biotin significantly prolonged survival of mice treated with pretargeted anti-hCD45 Ab-SA compared with mice treated with conventional RIT using 90Y-labeled anti-hCD45 Ab at 200 μCi. Because human CD45 antigens are confined to xenograft tumor cells in this model, and all murine tissues are devoid of hCD45 and will not bind anti-hCD45 Ab, we also compared one-step and PRIT using an anti-murine (m)CD45 Ab where the target antigen is present on normal hematopoietic tissues. After 24 h, 27.3% ± 2.8% of the injected dose of activity was delivered per gram (% ID/g) of lymph node using 131I-A20-Ab compared with 40.0 ± 5.4% ID/g for pretargeted 111In-DOTA-biotin. These data suggest that pretargeted methods for delivering RIT may be superior to conventional RIT when targeting CD45 for the treatment of leukemia and may allow for the intensification of therapy, while minimizing toxicities. [Cancer Res 2009;69(1):185–92]
Consistent Up-regulation of Stat3 Independently of Jak2 Mutations in a New Murine Model of Essential Thrombocythemia
Authors: Senyuk Vitalyi , Rinaldi Ciro Roberto , Li Donglan , Cattaneo Francesca , Stojanovic Aleksandra , Pane Fabrizio , Du Xiaoping , Mahmud Nadim , Dickstein Jerome , Nucifora Giuseppina
Published: December 2008

Janus-activated kinase 2 (JAK2) mutations are common in myeloproliferative disorders; however, although they are detected in virtually all polycythemia vera patients, they are found in ∼50% of essential thrombocythemia (ET) patients, suggesting that converging pathways/abnormalities underlie the onset of ET. Recently, the chromosomal translocation 3;21, leading to the fusion gene AML1/MDS1/EVI1 (AME), was observed in an ET patient. After we forced the expression of AME in the bone marrow (BM) of C57BL/6J mice, all the reconstituted mice died of a disease with symptoms similar to ET with a latency of 8 to 16 months. Peripheral blood smears consistently showed an elevated number of dysplastic platelets with anisocytosis, degranulation, and giant size. Although the AME-positive mice did not harbor Jak2 mutations, the BM of most of them had significantly higher levels of activated Stat3 than the controls. With combined biochemical and biological assays we found that AME binds to the Stat3 promoter leading to its up-regulation. Signal transducers and activators of transcription 3 (STAT3) analysis of a small group of ET patients shows that in about half of the patients, there is STAT3 hyperactivation independently of JAK2 mutations, suggesting that the hyperactivation of STAT3 by JAK2 mutations or promoter activation may be a critical step in development of ET. [Cancer Res 2009;69(1):262–71]
Cryptotanshinone Inhibits Constitutive Signal Transducer and Activator of Transcription 3 Function through Blocking the Dimerization in DU145 Prostate Cancer Cells
Authors: Shin Dae-Seop , Kim Hye-Nan , Shin Ki Deok , Yoon Young Ju , Kim Seung-Jun , Han Dong Cho , Kwon Byoung-Mog
Published: December 2008

Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in most human solid tumors and is involved in the proliferation, angiogenesis, immune evasion, and antiapoptosis of cancer cells, researchers have focused on STAT3 as a target for cancer therapy. We screened for natural compounds that inhibit the activity of STAT3 using a dual-luciferase assay. Cryptotanshinone was identified as a potent STAT3 inhibitor. Cryptotanshinone rapidly inhibited STAT3 Tyr705 phosphorylation in DU145 prostate cancer cells and the growth of the cells through 96 hours of the treatment. Inhibition of STAT3 Tyr705 phosphorylation in DU145 cells decreased the expression of STAT3 downstream target proteins such as cyclin D1, survivin, and Bcl-xL. To investigate the cryptotanshinone inhibitory mechanism in DU145 cells, we analyzed proteins upstream of STAT3. Although phosphorylation of Janus-activated kinase (JAK) 2 was inhibited by 7 μmol/L cryptotanshinone at 24 hours, inhibition of STAT3 Tyr705 phosphorylation occurred within 30 minutes and the activity of the other proteins was not affected. These results suggest that inhibition of STAT3 phosphorylation is caused by a JAK2-independent mechanism, with suppression of JAK2 phosphorylation as a secondary effect of cryptotanshinone treatment. Continuing experiments revealed the possibility that cryptotanshinone might directly bind to STAT3 molecules. Cryptotanshinone was colocalized with STAT3 molecules in the cytoplasm and inhibited the formation of STAT3 dimers. Computational modeling showed that cryptotanshinone could bind to the SH2 domain of STAT3. These results suggest that cryptotanshinone is a potent anticancer agent targeting the activation STAT3 protein. It is the first report that cryptotanshinone has antitumor activity through the inhibition of STAT3. [Cancer Res 2009;69(1):193–202]
Paracrine Signaling by Platelet-Derived Growth Factor-CC Promotes Tumor Growth by Recruitment of Cancer-Associated Fibroblasts
Authors: Anderberg Charlotte , Li Hong , Fredriksson Linda , Andrae Johanna , Betsholtz Christer , Li Xuri , Eriksson Ulf , Pietras Kristian
Published: December 2008

Cancer results from the concerted performance of malignant cells and stromal cells. Cell types populating the microenvironment are enlisted by the tumor to secrete a host of growth-promoting cues, thus upholding tumor initiation and progression. Platelet-derived growth factors (PDGF) support the formation of a prominent tumor stromal compartment by as of yet unidentified molecular effectors. Whereas PDGF-CC induces fibroblast reactivity and fibrosis in a range of tissues, little is known about the function of PDGF-CC in shaping the tumor-stroma interplay. Herein, we present evidence for a paracrine signaling network involving PDGF-CC and PDGF receptor-α in malignant melanoma. Expression of PDGFC in a mouse model accelerated tumor growth through recruitment and activation of different subsets of cancer-associated fibroblasts. In seeking the molecular identity of the supporting factors provided by cancer-associated fibroblasts, we made use of antibody arrays and an in vivo coinjection model to identify osteopontin as the effector of the augmented tumor growth induced by PDGF-CC. In conclusion, we establish paracrine signaling by PDGF-CC as a potential drug target to reduce stromal support in malignant melanoma. [Cancer Res 2009;69(1):369–78]
Ligand-Independent Androgen Receptor Variants Derived from Splicing of Cryptic Exons Signify Hormone-Refractory Prostate Cancer
Authors: Hu Rong , Dunn Thomas A. , Wei Shuanzeng , Isharwal Sumit , Veltri Robert W. , Humphreys Elizabeth , Han Misop , Partin Alan W. , Vessella Robert L. , Isaacs William B. , Bova G. Steven , Luo Jun
Published: December 2008

Suppression of androgen production and function provides palliation but not cure in men with prostate cancer (PCa). Therapeutic failure and progression to hormone-refractory PCa (HRPC) are often accompanied by molecular alterations involving the androgen receptor (AR). In this study, we report novel forms of AR alteration that are prevalent in HRPC. Through in silico sequence analysis and subsequent experimental validation studies, we uncovered seven AR variant transcripts lacking the reading frames for the ligand-binding domain due to splicing of “intronic” cryptic exons to the upstream exons encoding the AR DNA-binding domain. We focused on the two most abundantly expressed variants, AR-V1 and AR-V7, for more detailed analysis. AR-V1 and AR-V7 mRNA showed an average 20-fold higher expression in HRPC (n = 25) when compared with hormone-naive PCa (n = 82; P < 0.0001). Among the hormone-naive PCa, higher expression of AR-V7 predicted biochemical recurrence following surgical treatment (P = 0.012). Polyclonal antibodies specific to AR-V7 detected the AR-V7 protein frequently in HRPC specimens but rarely in hormone-naive PCa specimens. AR-V7 was localized in the nuclei of cultured PCa cells under androgen-depleted conditions, and constitutively active in driving the expression of canonical androgen-responsive genes, as revealed by both AR reporter assays and expression microarray analysis. These results suggest a novel mechanism for the development of HRPC that warrants further investigation. In addition, as expression markers for lethal PCa, these novel AR variants may be explored as potential biomarkers and therapeutic targets for advanced PCa. [Cancer Res 2009;69(1):16–22]
PCPH/ENTPD5 Expression Confers to Prostate Cancer Cells Resistance against Cisplatin-Induced Apoptosis through Protein Kinase Cα–Mediated Bcl-2 Stabilization
Authors: Villar Joaquín , Quadri Humair S. , Song Insun , Tomita York , Tirado Oscar M. , Notario Vicente
Published: December 2008

Prostate cancer (PCa) frequently develops antiapoptotic mechanisms and acquires resistance to anticancer drugs. Therefore, identifying PCa drug resistance determinants should facilitate designing more effective chemotherapeutic regimens. Recently, we described that the PCPH protein becomes highly expressed in human prostatic intraepithelial neoplasia and in PCa, and that the functional interaction between PCPH and protein kinase Cδ (PKCδ) increases the invasiveness of human PCa. Here, we report that the functional interaction between PCPH and a different PKC isoform, PKCα, confers resistance against cisplatin-induced apoptosis to PCa cells. This interaction elicits a mechanism ultimately resulting in the posttranslational stabilization and subsequent elevated expression of Bcl-2. Stable knockdown of either PCPH, mt-PCPH, or PKCα in PCa cells decreased Ser70-phosphorylated Bcl-2 and total Bcl-2 protein, thereby increasing their cisplatin sensitivity. Conversely, forced expression of the PCPH protein or, in particular, of the mt-PCPH oncoprotein increased the levels of phosphorylated PKCα concurrently with those of Ser70-phosphorylated and total Bcl-2 protein, thus promoting cisplatin resistance. Consistently, Bcl-2 knockdown sensitized PCa cells to cisplatin treatment and, more importantly, reversed the cisplatin resistance of PCa cells expressing the mt-PCPH oncoprotein. Moreover, reexpression of Bcl-2 in PCPH/mt-PCPH knockdown PCa cells reversed the cisplatin sensitization caused by PCPH or mt-PCPH down-regulation. These findings identify PCPH and mt-PCPH as important participants in the chemotherapy response of PCa cells, establish a role for PCPH-PKCα-Bcl-2 functional interactions in the drug response process, and imply that targeting PCPH expression before, or simultaneously with, chemotherapy may improve the treatment outcome for PCa patients. [Cancer Res 2009;69(1):102–10]
Adenoviral E1A Function through Myc
Authors: Chakraborty Abhishek A. , Tansey William P.
Published: December 2008

The study of DNA tumor viruses has been invaluable in uncovering the cellular nodes and pathways that contribute to oncogenesis. Perhaps one of the best-studied oncoproteins encoded by a DNA tumor virus is adenovirus E1A, which modifies the function of key regulatory proteins such as retinoblastoma (Rb) and the chromatin remodeling protein p400. Although the interaction of E1A with Rb has long been known to target regulation of the E2F transcription factors, the downstream target of the E1A-p400 interaction has remained elusive. We have recently reported that a critical downstream link of the E1A-p400 nexus is the oncoprotein transcription factor c-Myc. Through its interaction with p400, E1A stabilizes Myc and promotes formation of Myc-p400 complexes on chromatin, leading to activation of Myc target genes. These findings point to an important role for p400 in Myc function and reveal that E1A drives oncogenesis by tapping into two important transcriptional networks: those of E2F and Myc. [Cancer Res 2009;69(1):6–9]
A Multifactorial Signature of DNA Sequence and Polycomb Binding Predicts Aberrant CpG Island Methylation
Authors: McCabe Michael T. , Lee Eva K. , Vertino Paula M.
Published: December 2008

Aberrant CpG island methylation is associated with transcriptional silencing of regulatory genes in human cancer. Although most CpG islands remain unmethylated, a subset accrues aberrant methylation in cancer via unknown mechanisms. Previously, we showed that CpG islands differ in their intrinsic propensity towards hypermethylation. We developed a classifier (PatMAn) based on the frequencies of seven DNA sequence patterns that discriminated methylation-prone (MP) and methylation-resistant (MR) CpG islands. Here, we report on the genome-wide application and direct testing of PatMAn in cancer. Although trained on data from a cell culture model of de novo methylation involving the overexpression of DNMT1, PatMAn accurately predicted CpG islands at increased risk of hypermethylation in cancer cell lines and primary tumors. Analysis of CpG islands predicted to be MP revealed a strong association with embryonic targets of polycomb-repressive complex 2 (PRC2), indicating that PatMAn predicts not only aberrant methylation, but also PRC2 binding. A second classifier (SUPER-PatMAn) that integrates the seven PatMAn DNA patterns with SUZ12 enriched regions as a marker of PRC2 occupancy showed improved performance (prediction accuracy, 81–88%). In addition to many non-PRC2 targets, SUPER-PatMAn identified a subset of PRC2 targets that were more likely to be hypermethylated in cancer. Genome-wide, CpG islands predicted to be MP were enriched in genes known to undergo hypermethylation in cancer, genes functioning in transcriptional regulation, and components of developmental pathways. These findings show that hypermethylation of certain gene loci is controlled in part by an underlying susceptibility influenced by both local sequence context and trans-acting factors. [Cancer Res 2009;69(1):282–91]
Sequence Variants at 22q13 Are Associated with Prostate Cancer Risk
Authors: Sun Jielin , Zheng Siqun Lilly , Wiklund Fredrik , Isaacs Sarah D. , Li Ge , Wiley Kathleen E. , Kim Seong-Tae , Zhu Yi , Zhang Zheng , Hsu Fang-Chi , Turner Aubrey R. , Stattin Pär , Liu Wennuan , Kim Jin Woo , Duggan David , Carpten John , Isaacs William , Grönberg Henrik , Xu Jianfeng , Chang Bao-Li
Published: December 2008

To search for genetic variants that are associated with prostate cancer risk in the genome, we combined the data from our genome-wide association study (GWAS) in a population-based case-control study in Sweden with publicly available GWAS data from the Cancer Genetic Markers of Susceptibility (CGEMS) study. We limited the cases to those with aggressive disease in an attempt to identify risk variants that are associated with this most clinically relevant form of the disease. Among the most likely candidate single nucleotide polymorphisms (SNP) identified from the two GWAS, we sequentially confirmed one SNP at 22q13 in two independent study populations: the remaining subjects in Cancer of the Prostate in Sweden and a hospital-based case-control study at Johns Hopkins Hospital. Association of aggressive prostate cancer with the SNP at 22q13 was also observed in the publicly available data of four additional study populations from the second stage of the CGEMS study. In all seven study populations examined, the frequency of allele “C” of rs9623117 at 22q13 was consistently higher in aggressive cases than in controls. The combined allelic test was highly significant, with P = 5.0 × 10−7. The odds ratio (OR) of allele C for aggressive prostate cancer was estimated to be 1.18 [95% confidence interval (95% CI), 1.11-1.26]. However, the SNP was also associated with nonaggressive prostate cancer, with an estimated OR of 1.11 (95% CI, 1.04-1.19; P = 0.004). The risk-associated variants are located within the genomic region of TNRC6B, a gene involved in miRNA-mediated mRNA degradation. Additional studies are warranted to further confirm the association. [Cancer Res 2009;69(1):10–5]
DNA Methylation Markers Predict Outcome in Node-Positive, Estrogen Receptor-Positive Breast Cancer with Adjuvant Anthracycline-Based Chemotherapy
Authors: Hartmann Oliver , Spyratos Frédérique , Harbeck Nadia , Dietrich Dimo , Fassbender Anne , Schmitt Manfred , Eppenberger-Castori Serenella , Vuaroqueaux Vincent , Lerebours Florence , Welzel Katrin , Maier Sabine , Plum Achim , Niemann Stephan , Foekens John A. , Lesche Ralf , Martens John W.M.
Published: December 2008

Purpose: We have shown that DNA methylation of the PITX2 gene predicts risk of distant recurrence in steroid hormone receptor-positive, node-negative breast cancer. Here, we present results from a multicenter study investigating whether PITX2 and other candidate DNA methylation markers predict outcome in node-positive, estrogen receptor-positive, HER-2-negative breast cancer patients who received adjuvant anthracycline-based chemotherapy.

Experimental Design: Using a microarray platform, we analyzed DNA methylation in regulatory regions of PITX2 and 60 additional candidate genes in 241 breast cancer specimens. Using Cox regression analysis, we assessed the predictive power of the individual marker/marker panel candidates. Clinical endpoints were time to distant metastasis, disease-free survival, and overall survival. A nested bootstrap/cross-validation strategy was applied to identify and validate marker panels.

Results: DNA methylation of PITX2 and 14 other genes was correlated with clinical outcome. In multivariate models, each methylation marker added significant information to established clinical factors. A four-marker panel including PITX2, BMP4, FGF4, and C20orf55 was identified that resulted in improvement of outcome prediction compared with PITX2 alone.

Conclusions: This study provides further evidence for the PITX2 biomarker, which has now been successfully confirmed to predict outcome among different breast cancer patient populations. We further identify new DNA methylation biomarkers, three of which can be combined into a panel with PITX2 to increase the outcome prediction performance in our anthracycline-treated primary breast cancer population. Our results show that a well-defined panel of DNA methylation markers enables outcome prediction in lymph node-positive, HER-2-negative breast cancer patients treated with anthracycline-based chemotherapy.
Cytoplasmic Sequestration of p27 via AKT Phosphorylation in Renal Cell Carcinoma
Authors: Kim Jinhee , Jonasch Eric , Alexander Angela , Short John D. , Cai Shengli , Wen Sijin , Tsavachidou Dimitra , Tamboli Pheroze , Czerniak Bogdan A. , Do Kim Anh , Wu Kevin J. , Marlow Laura A. , Wood Christopher G. , Copland John A. , Walker Cheryl Lyn
Published: December 2008

Purpose: p27 localization and expression has prognostic and predictive value in cancer. Little is known regarding expression patterns of p27 in renal cell carcinoma (RCC) or how p27 participates in disease progression or response to therapy.

Experimental Design: RCC-derived cell lines, primary tumors, and normal renal epithelial cells were analyzed for p27 expression, phosphorylation (T157 of the NLS), and subcellular localization. RCC-derived cell lines were treated with phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors and effects on p27 localization were assessed. The potential contribution of cytoplasmic p27 to resistance to apoptosis was also evaluated.

Results: p27 was elevated in tumors compared with matched controls, and cytoplasmic mislocalization of p27 was associated with increasing tumor grade. Cytoplasmic localization of p27 correlated with phosphorylation at T157, an AKT phosphorylation site in the p27 NLS. In RCC cell lines, activated PI3K/AKT signaling was accompanied by mislocalization of p27. AKT activation and phosphorylation of p27 was associated with resistance to apoptosis, and small interfering RNA knockdown of p27 or relocalization to the nucleus increased apoptosis in RCC cells. Treatment with the PI3K inhibitors LY294002 or wortmannin resulted in nuclear relocalization of p27, whereas mTOR inhibition by rapamycin did not.

Conclusions: In RCC, p27 is phosphorylated at T157 of the NLS, with increasing tumor grade associated with cytoplasmic p27. PI3K inhibition (which reduces AKT activity) reduces T157 phosphorylation and induces nuclear relocalization of p27, whereas mTOR inhibition does not. Clinical testing of these findings may provide a rational approach for use of mTOR and PI3K/AKT pathway inhibitors in patients with RCC.
Imaging Intratumoral Convection: Pressure-Dependent Enhancement in Chemotherapeutic Delivery to Solid Tumors
Authors: Gade Terence P.F. , Buchanan Ian M. , Motley Matthew W. , Mazaheri Yousef , Spees William M. , Koutcher Jason A.
Published: December 2008

Purpose: Low–molecular weight (LMW) chemotherapeutics are believed to reach tumors through diffusion across capillary beds as well as membrane transporters. Unexpectedly, the delivery of these agents seems to be augmented by reductions in tumor interstitial fluid pressure, an effect typically associated with high–molecular weight molecules that reach tumors principally through convection. We investigated the hypothesis that improved intratumoral convection can alter tumor metabolism and enhance the delivery of a LMW chemotherapeutic agent to solid tumors.

Experimental Design: For this purpose, we applied 31P/19F magnetic resonance spectroscopy (MRS) and magnetic resonance spectroscopic imaging (MRSI) to examine the influence of type I collagenase on tumor bioenergetics and the delivery of 5-fluorouracil (5FU) to HT29 human colorectal tumors grown s.c. in mice.

Results: Collagenase effected a 34% reduction in tumor interstitial fluid pressure with an attendant disintegration of intratumoral collagen. Neither mice-administered collagenase nor controls receiving PBS showed changes in 31phosphorus MRS–measured tumor bioenergetics; however, a time-dependent increase in the content of extracellular inorganic phosphate (Pie) was observed in tumors of collagenase-treated animals. 31Phosphorus MRSI showed that this increase underscored a more homogeneous distribution of Pie in tumors of experimental mice. 19Fluorine MRS showed that these changes were associated with a 50% increase in 5FU uptake in tumors of experimental versus control animals; however, this increase resulted in an increase in 5FU catabolites rather than fluoronucleotide intermediates that are required for subsequent cytotoxicity.

Conclusions: These data indicate that the modulation of convective flow within tumors can improve the delivery of (LMW) chemotherapeutics and show the potential role for noninvasive imaging of this process in vivo.
Intratumoral Immune Cell Infiltrates, FoxP3, and Indoleamine 2,3-Dioxygenase in Patients with Melanoma Undergoing CTLA4 Blockade
Authors: Ribas Antoni , Comin-Anduix Begoña , Economou James S. , Donahue Timothy R. , de la Rocha Pilar , Morris Lilah F. , Jalil Jason , Dissette Vivian B. , Shintaku Itsushi Peter , Glaspy John A. , Gomez-Navarro Jesus , Cochran Alistair J.
Published: December 2008

Purpose: CTL-associated antigen 4 (CTLA4)-blocking monoclonal antibodies induce long-term regression of metastatic melanoma in some patients, but the exact mechanism is unknown. In this study, biopsies of selected accessible tumor lesions from patients treated with tremelimumab were examined to further elucidate the mechanism of its antitumor activity.

Experimental Design: Fifteen tumor biopsies from 7 patients who had been treated with tremelimumab (CP-675,206) were collected. Samples were analyzed for melanoma markers, immune cell subset markers, the presence of the T regulatory-specific transcription factor FoxP3 and the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO).

Results: Clinically responding lesions had diffuse intratumoral infiltrates of CD8+ T cells that were markedly increased in cases where comparison with a baseline biopsy was available. Nonregressing lesions had sparse, patchy CD8+ intratumoral infiltrates. Patients with regressing lesions had an increased frequency of CD8+ cells with or without a concomitant increase in CD4+ cells. Two of 3 responding patients with paired samples showed a slight increase in the number of FoxP3+ cells in the postdosing biopsies. In patients with regressing lesions who had paired samples, the intensity of IDO staining in macrophages and/or melanoma cells showed no clear pattern of change postdosing.

Conclusions: Administration of tremelimumab was associated with massive intratumoral infiltrates of CD8+ CTLs in patients with regressing tumors but had varying effects on intratumoral infiltrates of CD4+ and FoxP3+ cells or intratumoral expression of IDO.
Population Pharmacokinetic/Pharmacodynamic Analyses of Pemetrexed and Neutropenia: Effect of Vitamin Supplementation and Differences between Japanese and Western Patients
Authors: Latz Jane E. , Schneck Karen Lee , Nakagawa Kazuhiko , Miller Mary Alice , Takimoto Chris H.
Published: December 2008

Purpose: The objectives of the analysis were to characterize the time course of neutropenia after pemetrexed administration using an established semimechanistic-physiologic model, characterize the relationship between pemetrexed exposure and neutropenia, and describe differences in neutropenic response by vitamin supplementation status and between Japanese and Western patients.

Experimental Design: An eight-compartment population pharmacokinetic/pharmacodynamic model was used to describe the absolute neutrophil count (ANC)-time profile (neutropenic response) following pemetrexed doses of 300 to 1,400 mg/m2 administered every 21 days. The analyses pooled data from 13 studies including 279 patients (161 supplemented with oral folic acid and intramuscular vitamin B12, and 118 unsupplemented; 248 Western and 31 Japanese) who received 857 treatment cycles.

Results: Vitamin supplementation status, ethnic origin, and drug exposure were the dominant predictors of neutropenic response. Vitamin supplementation diminishes neutropenic response to pemetrexed. Model-predicted ANC nadirs for the “typical” Western patient receiving 500 mg/m2 pemetrexed ± vitamin supplementation were 2.74 × 109/L and 1.70 × 109/L, respectively. Japanese patients had a less pronounced neutropenic response to pemetrexed relative to Western patients. The model-predicted ANC nadir for Japanese patients receiving 500 mg/m2 pemetrexed with vitamin supplementation was 2.66 × 109/L. Values for the 1,000 mg/m2 dose with vitamin supplementation were 1.91 × 109/L and 1.34 × 109/L for Japanese and Western patients, respectively. Increased albumin, decreased cystathionine, and decreased body surface area were also associated with increased neutropenic response.

Conclusions: The neutropenic response to higher pemetrexed doses administered with vitamin supplementation is tolerable. All other factors equal, Japanese patients have a lesser neutropenic response to pemetrexed relative to Western patients.
BMS-536924 Reverses IGF-IR-Induced Transformation of Mammary Epithelial Cells and Causes Growth Inhibition and Polarization of MCF7 Cells
Authors: Litzenburger Beate C. , Kim Hyun-Jung , Kuiatse Isere , Carboni Joan M. , Attar Ricardo M. , Gottardis Marco M. , Fairchild Craig R. , Lee Adrian V.
Published: December 2008

Purpose: This study aimed to test the ability of a new insulin-like growth factor receptor (IGF-IR) tyrosine kinase inhibitor, BMS-536924, to reverse the ability of constitutively active IGF-IR (CD8-IGF-IR) to transform MCF10A cells, and to examine the effect of the inhibitor on a range of human breast cancer cell lines.

Experimental Design: CD8-IGF-IR-MCF10A cells were grown in monolayer culture, three-dimensional (3D) culture, and as xenografts, and treated with BMS-536924. Proliferation, cell cycle, polarity, and apoptosis were measured. Twenty-three human breast cancer cell lines were treated in monolayer culture with BMS-536924, and cell viability was measured. MCF7, MDA-MB-231, and MDA-MB-435 were treated with BMS-536924 in monolayer and 3D culture, and proliferation, migration, polarity, and apoptosis were measured.

Results: Treatment of CD8-IGF-IR-MCF10A cells grown in 3D culture with BMS-536924 caused a blockade of proliferation, restoration of apical-basal polarity, and enhanced apoptosis, resulting in a partial phenotypic reversion to normal acini. In monolayer culture, BMS-536924 induced a dose-dependent inhibition of proliferation, with an accumulation of cells in G0/G1,, and completely blocked CD8-IGF-IR–induced migration, invasion, and anchorage-independent growth. CD8-IGF-IR-MCF10A xenografts treated with BMS-536924 (100 mg/kg/day) showed a 76% reduction in xenograft volume. In a series of 23 human breast cancer cell lines, BMS-536924 inhibited monolayer proliferation of 16 cell lines. Most strikingly, treatment of MCF7 cells grown in 3D culture with BMS-536924 caused blockade of proliferation, and resulted in the formation of hollow polarized lumen.

Conclusions: These results show that the new small molecule BMS-536924 is an effective inhibitor of IGF-IR, causing a reversion of an IGF-IR–mediated transformed phenotype.
Lenalidomide and Rituximab in Waldenstrom's Macroglobulinemia
Authors: Treon Steven P. , Soumerai Jacob D. , Branagan Andrew R. , Hunter Zachary R. , Patterson Christopher J. , Ioakimidis Leukothea , Chu Luis , Musto Paul , Baron Ari D. , Nunnink Johannes C. , Kash Joseph J. , Terjanian Terenig O. , Hyman Paul M. , Nawfel Elena L. , Sharon David J. , Munshi Nikhil C. , Anderson Kenneth C.
Published: December 2008

Purpose: Thalidomide and its more potent immunomodulatory derivative lenalidomide enhance rituximab-mediated antibody-dependent cell-mediated cytotoxicity. We therefore evaluated lenalidomide and rituximab in symptomatic Waldenstrom's macroglobulinemia (WM) patients naive to either agent.

Experimental Design: Intended therapy consisted of 48 weeks of lenalidomide (25 mg/d for 3 weeks and then 1 week off) along with rituximab (375 mg/m2/wk) dosed on weeks 2 to 5 and 13 to 16. Sixteen patients were enrolled, 12 of whom were previously untreated.

Results: Unexpectedly, we observed an acute decrease in hematocrit in 13 of 16 patients (median hematocrit decrease, 4.8%), which was attributable to lenalidomide patients and which led to cessation of further enrollment on this study. Lenalidomide-related anemia was observed even at doses as low as 5 mg/d and occurred in the absence of hemolysis or other cytopenias. The overall response and major response (<50% decrease in serum IgM) rates were 50% and 25%, respectively, on an intent-to-treat basis. With a median follow-up of 31.3 months, 4 of 8 responding patients have progressed with a median time to progression of 18.9 months.

Conclusion: Lenalidomide produces unexpected but clinically significant acute anemia in patients with WM. In comparison with our previous study with thalidomide and rituximab in an analogous patient population, the responses achieved in WM patients with lenalidomide and rituximab appear less favorable.
Phase II Study of Protracted Daily Temozolomide for Low-Grade Gliomas in Adults
Authors: Kesari Santosh , Schiff David , Drappatz Jan , LaFrankie Debra , Doherty Lisa , Macklin Eric A. , Muzikansky Alona , Santagata Sandro , Ligon Keith L. , Norden Andrew D. , Ciampa Abigail , Bradshaw Joanna , Levy Brenda , Radakovic Gospova , Ramakrishna Naren , Black Peter M. , Wen Patrick Y.
Published: December 2008

Purpose: Resistance to temozolomide chemotherapy is partly mediated by O6-methylguanine-DNA methlytransferase (MGMT). Protracted treatment with temozolomide potentially overcomes MGMT resistance and improves outcome. We conducted a phase II study of protracted daily temozolomide in adults with low-grade gliomas.

Experimental Design: Patients with newly diagnosed oligodendroglioma or oligoastrocytoma with a MIB-1 index of >5% or recurrent low-grade gliomas received temozolomide (75 mg/m2/day in 11-week cycles of 7 weeks on/4 weeks off). Treatment continued for a total of six cycles or until tumor progression or unacceptable toxicity. Primary end point was best overall response rate; secondary end points were progression-free survival, overall survival, and toxicity. We correlated response with MGMT promoter methylation and chromosome 1p/19q deletion status.

Results: Forty-four patients were treated (14 female, 30 male) with a median follow-up of 39.4 months. Median age was 43 years (range, 20-68 years) and median Karnofsky performance status was 90 (range, 70-100). The regimen was well tolerated. No patients had a complete response (0%), 9 had partial response (20%), 33 had stable disease (75%), and 2 had progressive disease (5%). A total of 21 patients eventually progressed with an overall median progression-free survival of 38 months. Patients with methylated MGMT promoter had a longer overall survival (P = 0.008). Deletion of either 1p or 19q chromosomes also predicted longer overall survival (hazard ratio, 0.17; 95% confidence interval, 0.03-0.93; log-rank P = 0.02).

Conclusions: A protracted course of daily temozolomide is a well-tolerated regimen and seems to produce effective tumor control. This compares favorably with historical data on the standard 5-day temozolomide regimen.
Advancing Bioluminescence Imaging Technology for the Evaluation of Anticancer Agents in the MDA-MB-435-HAL-Luc Mammary Fat Pad and Subrenal Capsule Tumor Models
Authors: Zhang Cathy , Yan Zhengming , Arango Maria E. , Painter Cory L. , Anderes Kenna
Published: December 2008

Purpose: Tumors grafted s.c. or under the mammary fat pad (MFP) rarely develop efficient metastasis. By applying bioluminescence imaging (BLI) technology, the MDA-MB-435-HAL-Luc subrenal capsule (SRC) model was compared with the MFP model for disease progression, metastatic potential, and response to therapy.

Experimental Design: The luciferase-expressing MDA-MB-435-HAL-Luc cell line was used in both MFP and SRC models. BLI technology allowed longitudinal assessment of disease progression and the therapeutic response to PD-0332991, Avastin, and docetaxel. Immunohistochemical analysis of Ki67 and CD31 staining in the primary tumors was compared in these models. Caliper measurement was used in the MFP model to validate the BLI quantification of primary tumors.

Results: The primary tumors in MDA-MB-435-HAL-Luc MFP and SRC models displayed comparable growth rates and vascularity. However, tumor-bearing mice in the SRC model developed lung metastases much earlier (4 weeks) than in the MFP model (>7 weeks), and the metastatic progression contributed significantly to the survival time. In the MFP model, BLI and caliper measurements were comparable for quantifying palpable tumors, but BLI offered an advantage for detecting the primary tumors that fell below a palpable threshold and for visualizing metastases. In the SRC model, BLI allowed longitudinal assessment of the antitumor and antimetastatic effects of PD-0332991, Avastin, and docetaxel, and the results correlated with the survival benefits of these agents.

Conclusions: The MDA-MB-435-HAL-Luc SRC model and the MFP model displayed differences in disease progression. BLI is an innovative approach for developing animal models and creates opportunities for improving preclinical evaluations of anticancer agents.
Phase I and Pharmacokinetic Study of Gimatecan Given Orally Once a Week for 3 of 4 Weeks in Patients with Advanced Solid Tumors
Authors: Zhu Andrew X. , Ready Neal , Clark Jeffrey W. , Safran Howard , Amato Antonino , Salem Nadia , Pace Silvia , He Xiaoying , Zvereva Nela , Lynch Thomas J. , Ryan David P. , Supko Jeffrey G.
Published: December 2008

Purpose: A phase I study was conducted to determine the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) of gimatecan, a lipophilic camptothecin analogue, administered orally once a week for 3 weeks.

Experimental Design: Adult patients with advanced solid tumors with good performance status and adequate hematologic, hepatic, and renal function were eligible for the study. The plasma pharmacokinetics of the drug was characterized during the initial 28-day cycle.

Results: A total of 33 patients were evaluated at 7 dose levels ranging from 0.27 to 3.20 mg/m2/wk. Anemia, fatigue, neutropenia, nausea, and vomiting were the principal toxicities. DLTs experienced by 3 of 7 patients in dose level 7 (3.20 mg/m2) were grade 2 hyperbilirubinemia and grade 3 to 4 fatigue. DLT (anorexia and nausea) occurred in only 1 of 11 patients evaluated at the MTD of 2.40 mg/m2. There were no objective responses, although disease stabilization was observed in 4 patients. Gimatecan has a very long apparent biological half-life (mean ± SD, 77 ± 37 h) and exists in plasma almost entirely as the pharmacologically active intact lactone form. At the MTD, mean peak concentrations of the drug in plasma ranged from 67 to 82 ng/mL for the 3 weekly doses and the mean concentration 7 days after dosing was 15 ± 18 ng/mL.

Conclusions: Administration of gimatecan orally once a week at doses that are well tolerated provides continuous exposure to potentially effective plasma concentrations of the biologically active form of the drug. This regimen deserves further evaluation to define its antitumor activity in specific tumor types either alone or in combination with other agents.
Tissue Specific Cytotoxicity of Colon Cancer Cells Mediated by Nanoparticle-delivered Suicide Gene In vitro and In vivo
Authors: Zhang Guiying , Liu Ting , Chen Yong-Heng , Chen Yuxiang , Xu Meihua , Peng Jie , Yu Shuyi , Yuan Jianwei , Zhang Xiuwu
Published: December 2008

Purpose: This study aimed to develop an efficient and safe strategy to introduce suicide genes into colon cancer cells.

Experimental Design: In this study, we fused an enhanced carcinoembryonic antigen promoter (CEA) to a suicide gene, cytosine deaminase (CD). This construct was delivered into colon cancer cells using calcium phosphate nanoparticles (CPNP). The cells were then treated with the prodrug 5-FC. The therapeutic effect was evaluated in vitro and in vivo.

Results: Our study showed that the CEA promoter–driven, CPNP-delivered suicide gene was only expressed in CEA-positive colon cancer cells, and resulted in significant cytotoxicity after administration of the prodrug 5-FC in vitro. Moreover, our in vivo study showed that CPNP-mediated CEA-CD delivery, together with 5-FC treatment, resulted in significant tumor growth delay in xenograft human colon carcinoma.

Conclusions: Our study indicates that the combination of CPNP and CEA-CD gene expression represents a novel approach for CEA-positive tumor gene therapy.