bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Therapeutic Combination Using PD 0332991 and Bortezomib: Study in the 5T33MM Myeloma Model
Authors: Menu Eline , Garcia Josefina , Huang Xiangao , Di Liberto Maurizio , Toogood Peter L. , Chen Isan , Vanderkerken Karin , Chen-Kiang Selina
Published: July 2008

Multiple myeloma (MM) remains incurable partly because no effective cell cycle–based therapy has been available to both control tumor cell proliferation and synergize with cytotoxic killing. PD 0332991 is an orally active small molecule that potently and specifically inhibits Cdk4 and Cdk6. It has been shown to induce rapid G1 cell cycle arrest in primary human myeloma cells and suppress tumor growth in xenograft models. To improve therapeutic targeting of myeloma progression, we combined tumor suppression by PD 0332991 with cytotoxic killing by bortezomib, a proteasome inhibitor widely used in myeloma treatment, in the immunocompetent 5T33MM myeloma model. We show that 5T33MM tumor cells proliferate aggressively in vivo due to expression of cyclin D2, elevation of Cdk4, and impaired p27Kip1 expression, despite inhibition of Cdk4/6 by p18INK4c and the maintenance of a normal plasma cell transcription program. PD 0332991 potently inhibits Cdk4/6-specific phosphorylation of Rb and cell cycle progression through G1 in aggressively proliferating primary 5T33MM cells, in vivo and ex vivo. This leads to tumor suppression and a significant improvement in survival. Moreover, induction of G1 arrest by PD 0332991 sensitizes 5T33MM tumor cells to killing by bortezomib. Inhibition of Cdk4/6 by PD 0332991, therefore, effectively controls myeloma tumor expansion and sensitizes tumor cells to bortezomib killing in the presence of an intact immune system, thereby representing a novel and promising cell cycle–based combination therapy. [Cancer Res 2008;68(14):5519–23]
Identification of Hypoxia-Inducible Factor-1α as a Novel Target for miR-17-92 MicroRNA Cluster
Authors: Taguchi Ayumu , Yanagisawa Kiyoshi , Tanaka Masaharu , Cao Ke , Matsuyama Yasushi , Goto Hidemi , Takahashi Takashi
Published: July 2008

MicroRNAs (miRNAs) are a distinct class of small noncoding RNAs that posttranscriptionally repress expression of target genes through imperfect base pairing with the 3′ untranslated region. We previously reported amplification and overexpression of the miR-17-92 miRNA cluster at 13q31.3 in lung cancers, as well as growth inhibition by treatment with antisense oligonucleotides against miR-17-5p and miR-20a, constituents of miR-17-92, specifically in miR-17-92–overexpressing lung cancer cell lines. Although these findings clearly suggested important roles of miR-17-92 overexpression in lung cancers, only a few targets for the miR-17-92 cluster have been identified thus far. In this study, we identified hypoxia-inducible factor (HIF)-1α as a novel direct target for miR-17-92 through global expression profiling by mass spectrometric analysis using an isobaric tagging reagent, iTRAQ, combined with bioinformatic target prediction. This is the first report to describe negative regulation of HIF-1α by miRNA, which seemed to occur without disrupting the induction of HIF-1α for cellular adaptation to hypoxia. In addition, overexpression of c-myc led to down-regulation of HIF-1α and induction of miR-17-92, the latter of which was previously reported to be a transcriptional activation activity, suggesting that the induction of miR-17-92 may play a role at least in part in c-myc–mediated repression of HIF-1α. Together with previous reports on the functional negative regulation of c-myc by HIF-1α, our findings suggest the possible existence of an intricate and finely tuned circuit involving c-myc, miR-17-92, and HIF-1α that may play a role in cancer cell proliferation under normoxia in a cellular context–dependent manner. [Cancer Res 2008;68(14):5540–5]
Recruitment and Activation of Natural Killer CellsIn vitroby a Human Dendritic Cell Vaccine
Authors: Gustafsson Karin , Ingelsten Madeleine , Bergqvist Linda , Nyström Jenny , Andersson Bengt , Karlsson-Parra Alex
Published: July 2008

Recruitment of circulating natural killer (NK) cells into inflamed lymph nodes is known to provide a potent, IFN-γ–dependent boost for Th1-polarized immune responses in mouse models. Such NK cell recruitment into draining lymph nodes is induced by certain s.c. injected adjuvants, including mature vaccine dendritic cells (DC), and is mediated by a CXCR3-dependent pathway. Here, we show that monocyte-derived immature human DCs stimulated with polyinosinic acid:polycytidylic acid, IFN-α, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IFN-γ, α-type 1–polarized DC (αDC1), secrete profuse amounts of the CXCR3 ligand CXCL9/MIG and substantial amounts of CXCL10/IP-10 and CXCL11/I-TAC after withdrawal of maturation stimuli. In sharp contrast, no measurable production of these chemokines was found in DCs after maturation with the current gold standard maturation cocktail for human DC-based cancer vaccines consisting of TNF-α, IL-1β, IL-6, and prostaglandin-E2 (PGE2-DC). PGE2-DCs preferentially produced the Th2 and regulatory T-cell–attracting chemokines CCL17/TARC and CCL22/MDC, whereas only marginal levels of these chemokines were produced by αDC1s. Functional studies in vitro showed that supernatants from mature αDC1s actively recruited CD3−CD56+ NK cells and that adding anti-CXCL9/MIG antibodies to the αDC1 supernatant substantially reduced this recruitment. Finally, αDC1s were able to induce IFN-γ production when cocultured with resting autologous NK cells, but only if concurrent CD40 ligation was provided. These novel findings indicate that injected human αDC1-based vaccines have the potential to recruit and activate NK cells during their arrival to draining lymph nodes and that this feature may be of relevance for efficient priming of Th1 cells and CTLs. [Cancer Res 2008;68(14):5965–71]
Overexpression of Vascular Endothelial Growth Factor and the Development of Post-Transplantation Cancer
Authors: Basu Aninda , Contreras Alan G. , Datta Dipak , Flynn Evelyn , Zeng Liling , Cohen Herbert T. , Briscoe David M. , Pal Soumitro
Published: July 2008

Cancer is an increasing and major problem after solid organ transplantation. In part, the increased cancer risk is associated with the use of immunosuppressive agents, especially calcineurin inhibitors. We propose that the effect of calcineurin inhibitors on the expression of vascular endothelial growth factor (VEGF) leads to an angiogenic milieu that favors tumor growth. Here, we used 786-0 human renal cancer cells to investigate the effect of cyclosporine (CsA) on VEGF expression. Using a full-length VEGF promoter-luciferase construct, we found that CsA markedly induced VEGF transcriptional activation through the protein kinase C (PKC) signaling pathway, specifically involving PKCζ and PKCδ isoforms. Moreover, CsA promoted the association of PKCζ and PKCδ with the transcription factor Sp1 as observed by immunoprecipitation assays. Using promoter deletion constructs, we found that CsA-mediated VEGF transcription was primarily Sp1 dependent. Furthermore, CsA-induced and PKC-Sp1–mediated VEGF transcriptional activation was partially inhibited by von Hippel-Lindau protein. CsA also promoted the progression of human renal tumors in vivo, wherein VEGF is overexpressed. Finally, to evaluate the in vivo significance of CsA-induced VEGF overexpression in terms of post-transplantation tumor development, we injected CT26 murine carcinoma cells (known to form angiogenic tumors) into mice with fully MHC mismatched cardiac transplants. We observed that therapeutic doses of CsA increased tumor size and VEGF mRNA expression and also enhanced tumor angiogenesis. However, coadministration of a blocking anti-VEGF antibody inhibited this CsA-mediated tumor growth. Collectively, these findings define PKC-mediated VEGF transcriptional activation as a key component in the progression of CsA-induced post-transplantation cancer. [Cancer Res 2008;68(14):5689–98]
Modulation of CD59 Expression by Restrictive Silencer Factor–Derived Peptides in Cancer Immunotherapy for Neuroblastoma
Authors: Donev Rossen M. , Gray Lisa C. , Sivasankar Baalasubramanian , Hughes Timothy R. , van den Berg Carmen W. , Morgan B. Paul
Published: July 2008

Tumor cells escape clearance by complement by abundantly expressing CD59 and other membrane complement regulators. Existing strategies for blocking/knocking down these regulators can contribute to tumor immunoclearance in vitro; however, there are numerous difficulties restricting their use in vivo. Here, we report a new strategy for suppression of CD59 expression in neuroblastoma using peptides that target regulators of CD59 expression. We identified the neural-restrictive silencer factor (REST) as a target for modulation of CD59 expression in neuroblastoma. We next designed plasmids that encoded peptides comprising different DNA-binding domains of REST and transfected them into neuroblastoma cell lines. These peptides suppressed CD59 expression, sensitizing neuroblastoma to complement-mediated killing triggered by anti-GD2 therapeutic monoclonal antibody. These CD59-modulating peptides might be effective therapeutic adjuvants to therapeutic monoclonal antibodies used for treatment of neuroblastoma and other cancer types sharing the same mechanism for regulation of CD59 expression. [Cancer Res 2008;68(14):5979–87]
An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer
Authors: Narisawa-Saito Mako , Yoshimatsu Yuki , Ohno Shin-ichi , Yugawa Takashi , Egawa Nagayasu , Fujita Masatoshi , Hirohashi Setsuo , Kiyono Tohru
Published: July 2008

Human papillomaviruses (HPV) are believed to be the primary causal agents for development of cervical cancer, and deregulated expression of two viral oncogenes E6 and E7 in basal cells, mostly by integration, is considered to be a critical event for disease progression. However, lines of evidence suggest that, besides expression of E6 and E7 genes, additional host genetic alterations are required for cancer development. To directly test this hypothesis, we first transduced HPV16 E6 and E7 with or without hTERT into several lines of normal human cervical keratinocytes (HCK) from independent donors and then searched for additional alterations required for carcinogenesis. Oncogenic HrasG12V (Hras) provided marked tumor forming ability in nude mice and ErbB2 or c-Myc (Myc) endowed weaker but significant tumor forming ability. Combined transduction of Myc and Hras to HCKs expressing E6 and E7 resulted in the creation of highly potent tumor-initiating cells. These results show that only one or two genetic changes occurring after deregulated expression of high-risk HPV oncogenes might be sufficient for development of cervical cancer. [Cancer Res 2008;68(14):5699–705]
Temozolomide Preferentially Depletes Cancer Stem Cells in Glioblastoma
Authors: Beier Dagmar , Röhrl Stefanie , Pillai Deepu R. , Schwarz Stefanie , Kunz-Schughart Leoni A. , Leukel Petra , Proescholdt Martin , Brawanski Alexander , Bogdahn Ulrich , Trampe-Kieslich Ariane , Giebel Bernd , Wischhusen Jörg , Reifenberger Guido , Hau Peter , Beier Christoph P.
Published: July 2008

The prognosis of patients suffering from glioblastoma (GBM) is dismal despite multimodal therapy. Although chemotherapy with temozolomide may contain tumor growth for some months, invariable tumor recurrence suggests that cancer stem cells (CSC) maintaining these tumors persist. We have therefore investigated the effect of temozolomide on CD133+ and CD133− GBM CSC lines. Although differentiated tumor cells constituting the bulk of all tumor cells were resistant to the cytotoxic effects of the substance, temozolomide induced a dose- and time-dependent decline of the stem cell subpopulation. Incubation with sublethal concentrations of temozolomide for 2 days completely depleted clonogenic tumor cells in vitro and substantially reduced tumorigenicity in vivo. In O6-methylguanine-DNA-methyltransferase (MGMT)–expressing CSC lines, this effect occurred at 10-fold higher doses compared with MGMT-negative CSC lines. Thus, temozolomide concentrations that are reached in patients were only sufficient to completely eliminate CSC in vitro from MGMT-negative but not from MGMT-positive tumors. Accordingly, our data strongly suggest that optimized temozolomide-based chemotherapeutic protocols might substantially improve the elimination of GBM stem cells and consequently prolong the survival of patients. [Cancer Res 2008;68(14):5706–15]
Jagged-1 and Notch3 Juxtacrine Loop Regulates Ovarian Tumor Growth and Adhesion
Authors: Choi Jung-Hye , Park Joon T. , Davidson Ben , Morin Patrice J. , Shih Ie-Ming , Wang Tian-Li
Published: July 2008

Notch3 gene amplification and pathway activation have been reported in ovarian serous carcinoma. However, the primary Notch3 ligand that initiates signal transduction in ovarian cancer remains unclear. In this report, we identify Jagged-1 as the highest expressed Notch ligand in ovarian tumor cells as well as in peritoneal mesothelial cells that are in direct contact with disseminated ovarian cancer cells. Cell-cell adhesion and cellular proliferation were reduced in Notch3-expressing ovarian cancer cells that were cocultured with Jagged-1 knockdown mesothelial and tumor feeder cells. Interaction of Notch3-expressing ovarian cancer cells with Jagged-1–expressing feeder cells activated the promoter activity of candidate Notch3 target genes, and this activity was attenuated by Notch3 siRNA. Constitutive expression of the Notch3 intracellular domain significantly suppressed the Jagged-1 shRNA–mediated growth inhibitory effect. In Notch3-expressing ovarian cancer cells, Jagged-1–stimulating peptides enhanced cellular proliferation, which was suppressed by γ-secretase inhibitor and Notch3 siRNA. Taken together, our results show that Jagged-1 is the primary Notch3 ligand in ovarian carcinoma and Jagged-1/Notch3 interaction constitutes a juxtacrine loop promoting proliferation and dissemination of ovarian cancer cells within the intraperitoneal cavity. [Cancer Res 2008;68(14):5716–23]
PIK3IP1, a Negative Regulator of PI3K, Suppresses the Development of Hepatocellular Carcinoma
Authors: He Xin , Zhu Zhenqi , Johnson Carla , Stoops John , Eaker Amanda E. , Bowen William , DeFrances Marie C.
Published: July 2008

Phosphatidylinositol-3-kinase (PI3K) is a well-known regulator of cell division, motility, and survival in most cell types. Recently, we characterized a novel protein that we call PI3K Interacting Protein 1 (PIK3IP1), which binds to the p110 catalytic subunit of PI3K and reduces its activity in vitro. Little is known about the role of PIK3IP1 in normal and neoplastic growth in vivo. Proper liver function and development depend on intact PI3K signal transduction; when dysregulated, the PI3K pathway is linked to the development of liver cancer. To begin to dissect the contribution of PIK3IP1 to hepatic PI3K signaling in vivo and to liver tumorigenesis in particular, we formulated the following hypothesis: because PIK3IP1 down-regulates PI3K signaling and uncontrolled PI3K signaling is associated with liver cancer, then PIK3IP1-mediated down-regulation of the PI3K pathway should inhibit hepatocellular carcinoma (HCC) development. To test this idea, we generated transgenic mice overexpressing PIK3IP1 in hepatocytes in a mouse strain prone to develop HCC. Isolated PIK3IP1 transgenic mouse hepatocytes showed blunted PI3K signaling, DNA synthetic activity, motility, and survival compared with controls. In vivo, spontaneous liver tumorigenesis was significantly dampened in the transgenic animals. This was accompanied by decreased hepatic PI3K activity and reduced hepatocyte proliferation in the transgenics compared with controls. We also observed that human HCC expressed less PIK3IP1 protein than adjacent matched liver tissue. Our data show that PIK3IP1 is an important regulator of PI3K in vivo, and its dysregulation can contribute to liver carcinogenesis. [Cancer Res 2008;68(14):5591–8]
PAX8 Regulates Telomerase Reverse Transcriptase and Telomerase RNA Component in Glioma
Authors: Chen Yu-Jen , Campbell Hamish G. , Wiles Anna K. , Eccles Michael R. , Reddel Roger R. , Braithwaite Antony W. , Royds Janice A.
Published: July 2008

Paired box (PAX) developmental genes are frequently expressed in cancers and confer survival advantages on cancer cells. We have previously found that PAX genes are deregulated in glioma. We have now investigated the expression of PAX genes in glioma and their role in telomere maintenance. The mRNA level of PAX8 showed a positive correlation with telomerase activity in glioma biopsies (r2 = 0.75, P < 0.001) and in established glioma cell lines (r2 = 0.97, P = 0.0025). We found that PAX8 is able to coordinately transactivate the promoter for both the telomerase catalytic subunit (hTERT) and the telomerase RNA component (hTR) genes. By electrophoretic mobility shift assay, quantitative PCR, and a telomerase activity assay, we show that PAX8 binds directly to the hTERT and hTR promoters, up-regulating hTERT and hTR mRNA, as well as telomerase activity. Additionally, PAX8 small interfering RNA down-regulated hTERT and hTR. Collectively, these results show that PAX8 may have a role in telomerase regulation. [Cancer Res 2008;68(14):5724–32]
Novel MEK1 Mutation Identified by Mutational Analysis of Epidermal Growth Factor Receptor Signaling Pathway Genes in Lung Adenocarcinoma
Authors: Marks Jenifer L. , Gong Yixuan , Chitale Dhananjay , Golas Ben , McLellan Michael D. , Kasai Yumi , Ding Li , Mardis Elaine R. , Wilson Richard K. , Solit David , Levine Ross , Michel Kathrin , Thomas Roman K. , Rusch Valerie W. , Ladanyi Marc , Pao William
Published: July 2008

Genetic lesions affecting a number of kinases and other elements within the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the pathogenesis of human non–small-cell lung cancer (NSCLC). We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this pathway that could contribute to lung tumorigenesis. We have identified in 2 of 207 primary lung tumors a somatic activating mutation in exon 2 of MEK1 (i.e., mitogen-activated protein kinase kinase 1 or MAP2K1) that substitutes asparagine for lysine at amino acid 57 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal–regulated kinase (ERK)-1/2 in human 293T cells and to growth factor–independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells. We also screened 85 NSCLC cell lines for MEK1 exon 2 mutations; one line (NCI-H1437) harbors a Q56P substitution, a known transformation-competent allele of MEK1 originally identified in rat fibroblasts, and is sensitive to treatment with AZD6244. MEK1 mutants have not previously been reported in lung cancer and may provide a target for effective therapy in a small subset of patients with lung adenocarcinoma. [Cancer Res 2008;68(14):5524–8]
Identification of a Novel Subgroup of Melanomas with KIT/Cyclin-Dependent Kinase-4 Overexpression
Authors: Smalley Keiran S.M. , Contractor Rooha , Nguyen Thiennga K. , Xiao Min , Edwards Robin , Muthusamy Viswanathan , King Alastair J. , Flaherty Keith T. , Bosenberg Marcus , Herlyn Meenhard , Nathanson Katherine L.
Published: July 2008

Although many melanomas harbor either activating mutations in BRAF or NRAS, there remains a substantial, yet little known, group of tumors without either mutation. Here, we used a genomic strategy to define a novel group of melanoma cell lines with co-overexpression of cyclin-dependent kinase 4 (CDK4) and KIT. Although this subgroup lacked any known KIT mutations, they had high phospho-KIT receptor expression, indicating receptor activity. Quantitative PCR confirmed the existence of a similar KIT/CDK4 subgroup in human melanoma samples. Pharmacologic studies showed the KIT/CDK4-overexpressing subgroup to be resistant to BRAF inhibitors but sensitive to imatinib in both in vitro and in vivo melanoma models. Mechanistically, imatinib treatment led to increased apoptosis and G1 phase cell cycle arrest associated with the inhibition of phospho-ERK and increased expression of p27KIP. Other melanoma cell lines, which retained some KIT expression but lacked phospho-KIT, were not sensitive to imatinib, suggesting that KIT expression alone is not predictive of response. We suggest that co-overexpression of KIT/CDK4 is a potential mechanism of oncogenic transformation in some BRAF/NRAS wild-type melanomas. This group of melanomas may be a subpopulation for which imatinib or other KIT inhibitors may constitute optimal therapy. [Cancer Res 2008;68(14):5743–52]
Atypical Protein Kinase Cι Expression and Aurothiomalate Sensitivity in Human Lung Cancer Cells
Authors: Regala Roderick P. , Thompson E. Aubrey , Fields Alan P.
Published: July 2008

The antirheumatoid agent aurothiomalate (ATM) is a potent inhibitor of oncogenic PKCι. ATM inhibits non–small lung cancer (NSCLC) growth by binding PKCι and blocking activation of a PKCι-Par6-Rac1-Pak-Mek 1,2-Erk 1,2 signaling pathway. Here, we assessed the growth inhibitory activity of ATM in a panel of human cell lines representing major lung cancer subtypes. ATM inhibited anchorage-independent growth in all lines tested with IC50s ranging from ∼300 nmol/L to >100 μmol/L. ATM sensitivity correlates positively with expression of PKCι and Par6, but not with the PKCι binding protein p62, or the proposed targets of ATM in rheumatoid arthritis (RA), thioredoxin reductase 1 or 2. PKCι expression profiling revealed that a significant subset of primary NSCLC tumors express PKCι at or above the level associated with ATM sensitivity. ATM sensitivity is not associated with general sensitivity to the cytotoxic agents cis-platin, placitaxel, and gemcitabine. ATM inhibits tumorigenicity of both sensitive and insensitive lung cell tumors in vivo at plasma drug concentrations achieved in RA patients undergoing ATM therapy. ATM inhibits Mek/Erk signaling and decreases proliferative index without effecting tumor apoptosis or vascularization in vivo. We conclude that ATM exhibits potent antitumor activity against major lung cancer subtypes, particularly tumor cells that express high levels of the ATM target PKCι and Par6. Our results indicate that PKCι expression profiling will be useful in identifying lung cancer patients most likely to respond to ATM therapy in an ongoing clinical trial. [Cancer Res 2008;68(14):5888–95]
Macrophage Depletion Combined with Anticoagulant Therapy Increases Therapeutic Window of Systemic Treatment with Oncolytic Adenovirus
Authors: Shashkova Elena V. , Doronin Konstantin , Senac Julien S. , Barry Michael A.
Published: July 2008

Liver tropism of systemically delivered adenoviruses (Ad) represents a considerable challenge for their use as anticancer therapeutics. More than 90% of i.v. injected Ad is rapidly taken up by the liver leading to hepatotoxicity, reduced virus uptake by target tumor tissue, and diminished therapeutic efficacy. The lack of clinical activity of systemically given oncolytic Ad demands for better understanding and improvement of virus pharmacokinetics. We studied the effects of Ad “detargeting” from liver macrophages (Kupffer cells) and hepatocytes on toxicity and anticancer efficacy using a nonattenuated oncolytic Ad expressing enhanced green fluorescent protein-firefly luciferase fusion protein (Ad-EGFPLuc). Kupffer cell depletion before i.v. injection of Ad-EGFPLuc increased transgene expression in the liver 40.7-fold on day 3 after the injection indicating compensatory enhancement of hepatocyte transduction due to increased bioavailability of the virus. Pretreatment of mice with the anticoagulant drug warfarin to block blood factor–dependent binding of the virus to hepatocytes markedly reduced luciferase expression in the liver and mediated the corresponding decrease of hepatotoxicity in mice with intact and depleted liver macrophages. Combined depletion of Kupffer cells and pretreatment with warfarin before a single i.v. injection of Ad-EGFPLuc significantly reduced tumor growth and prolonged survival of nude mice bearing subcutaneous xenografts of aggressive human hepatocellular carcinoma. The improved antitumor activity correlated with enhanced transgene expression and virus spread in the tumors. These data suggest that detargeting oncolytic Ad from liver macrophages and hepatocytes is an effective strategy to increase the therapeutic window for therapy against disseminated tumor sites. [Cancer Res 2008;68(14):5896–904]
Monitoring Serial Changes in Circulating Human Breast Cancer Cells in Murine Xenograft Models
Authors: Eliane Jean-Pierre , Repollet Madeline , Luker Kathryn E. , Brown Martha , Rae James M. , Dontu Gabriela , Schott Anne F. , Wicha Max , Doyle Gerald V. , Hayes Daniel F. , Luker Gary D.
Published: July 2008

Circulating tumor cells (CTC) are emerging as a powerful prognostic and predictive biomarker in several types of cancer, including breast, colon, and prostate. Studies of CTC in metastasis and further development of CTC as a biomarker in cancer have been limited by the inability to repetitively monitor CTC in mouse models of cancer. We have validated a method to enumerate CTC in blood samples obtained from living mice using a modified version of an in vitro diagnostic system for quantifying CTC in patients. Different routes of blood collection were tested to identify a method to reproducibly recover CTC from tumor-bearing mice without interference from contaminating normal murine epithelial cells. CTC are present in blood samples from mice bearing orthotopic xenografts of several different breast cancer cell lines and primary breast cancer cells from patient biopsies. We also show that this technology can be used for serial monitoring of CTC in mouse xenograft models of human breast cancer. These results establish a new method for studying CTC in mouse models of epithelial cancer, providing the foundation for studies of molecular regulation of CTC in cancer and CTC as biomarker for therapeutic efficacy. [Cancer Res 2008;68(14):5529–32]
Defining a Chromatin Pattern that Characterizes DNA-Hypermethylated Genes in Colon Cancer Cells
Authors: McGarvey Kelly M. , Van Neste Leander , Cope Leslie , Ohm Joyce E. , Herman James G. , Van Criekinge Wim , Schuebel Kornel E. , Baylin Stephen B.
Published: July 2008

Epigenetic gene regulation is a key determinant of heritable gene expression patterns and is critical for normal cellular function. Dysregulation of epigenetic transcriptional control is a fundamental feature of cancer, particularly manifesting as increased promoter DNA methylation with associated aberrant gene silencing, which plays a significant role in tumor progression. We now globally map key chromatin parameters for genes with promoter CpG island DNA hypermethylation in colon cancer cells by combining microarray gene expression analyses with chromatin immunoprecipitation-on-chip technology. We first show that the silent state of such genes universally correlates with a broad distribution of a low but distinct level of the PcG-mediated histone modification, methylation of lysine 27 of histone 3 (H3K27me), and a very low level of the active mark H3K4me2. This chromatin pattern, and particularly H3K4me2 levels, crisply separates DNA-hypermethylated genes from those where histone deacetylation is responsible for transcriptional silencing. Moreover, the chromatin pattern can markedly enhance identification of truly silent and DNA-hypermethylated genes. We additionally find that when DNA-hypermethylated genes are demethylated and reexpressed, they adopt a bivalent chromatin pattern, which is associated with the poised gene expression state of a large group of embryonic stem cell genes and is characterized by an increase in levels of both the H3K27me3 and H3K4me2 marks. Our data have great relevance for the increasing interest in reexpression of DNA-hypermethylated genes for the treatment of cancer. [Cancer Res 2008;68(14):5753–9]
Class I PI3 Kinase Inhibition by the Pyridinylfuranopyrimidine Inhibitor PI-103 Enhances Tumor Radiosensitivity
Authors: Prevo Remko , Deutsch Eric , Sampson Oliver , Diplexcito Julie , Cengel Keith , Harper Jane , O'Neill Peter , McKenna W. Gillies , Patel Sonal , Bernhard Eric J.
Published: July 2008

Cell signaling initiated at the epidermal growth factor receptor (EGFR), RAS oncoproteins, or PI3K contributes to a common pathway that promotes tumor survival after radiation-induced DNA damage. Inhibition of signaling at the level of EGFR, RAS, and PI3K has been tested, but clinical applicability has been shown only at the level of the EGFR or by inhibiting RAS indirectly with prenyltransferase inhibitors. Inhibition of PI3K with LY294002 or wortmannin lacks specificity and has shown unacceptable toxicity in preclinical studies. We previously showed that inhibiting class I PI3K expression with siRNA resulted in enhanced radiation killing of tumor cells. Here, we tested the possibility of achieving specific tumor cell radiosensitization with a pharmacologic inhibitor of class I PI3K, the pyridinylfuranopyrimidine inhibitor PI-103. Our results show that inhibiting PI3K activity reduces phosphorylation of AKT at serine 473. Reduced survival is seen in cells with AKT activation and seems preferential for tumor cells over cells in which AKT activity is not elevated. Reduced survival is accompanied by persistence of DNA damage as evidenced by persistence of γH2AX and Rad 51 foci after irradiation in the presence of the inhibitor. Reduced survival does not result from cell cycle redistribution during the PI-103 treatment intervals tested, although combining PI-103 treatment with radiation enhances the G2-M delay observed after irradiation. These results indicate that pharmacologic inhibitors with enhanced specificity for class I PI3K may be of benefit when combined with radiotherapy. [Cancer Res 2008;68(14):5915–23]
Genomic Alterations Indicate Tumor Origin and Varied Metastatic Potential of Disseminated Cells from Prostate Cancer Patients
Authors: Holcomb Ilona N. , Grove Douglas I. , Kinnunen Martin , Friedman Cynthia L. , Gallaher Ian S. , Morgan Todd M. , Sather Cassandra L. , Delrow Jeffrey J. , Nelson Peter S. , Lange Paul H. , Ellis William J. , True Lawrence D. , Young Janet M. , Hsu Li , Trask Barbara J. , Vessella Robert L.
Published: July 2008

Disseminated epithelial cells can be isolated from the bone marrow of a far greater fraction of prostate-cancer patients than the fraction of patients who progress to metastatic disease. To provide a better understanding of these cells, we have characterized their genomic alterations. We first present an array comparative genomic hybridization method capable of detecting genomic changes in the small number of disseminated cells (10–20) that can typically be obtained from bone marrow aspirates of prostate-cancer patients. We show multiple regions of copy-number change, including alterations common in prostate cancer, such as 8p loss, 8q gain, and gain encompassing the androgen-receptor gene on Xq, in the disseminated cell pools from 11 metastatic patients. We found fewer and less striking genomic alterations in the 48 pools of disseminated cells from patients with organ-confined disease. However, we identify changes shared by these samples with their corresponding primary tumors and prostate-cancer alterations reported in the literature, evidence that these cells, like those in advanced disease, are disseminated tumor cells (DTC). We also show that DTCs from patients with advanced and localized disease share several abnormalities, including losses containing cell-adhesion genes and alterations reported to associate with progressive disease. These shared alterations might confer the capability to disseminate or establish secondary disease. Overall, the spectrum of genomic deviations is evidence for metastatic capacity in advanced-disease DTCs and for variation in that capacity in DTCs from localized disease. Our analysis lays the foundation for elucidation of the relationship between DTC genomic alterations and progressive prostate cancer. [Cancer Res 2008;68(14):5599–608]
New Insights into Tumor Microstructure Using Temporal Diffusion Spectroscopy
Authors: Colvin Daniel C. , Yankeelov Thomas E. , Does Mark D. , Yue Zoe , Quarles Chad , Gore John C.
Published: July 2008

Magnetic resonance images (MRI) that depict rates of water diffusion in tissues can be used to characterize the cellularity of tumors and are valuable in assessing their early response to treatment. Water diffusion rates are sensitive to the cellular and molecular content of tissues and are affected by local microstructural changes associated with tumor development. However, conventional maps of water diffusion reflect the integrated effects of restrictions to free diffusion at multiple scales up to a specific limiting spatial dimension, typically several micrometers. Such measurements cannot distinguish effects caused by structural variations at a smaller scale. Variations in diffusion rates then largely reflect variations in the density of cells, and no information is available about changes on a subcellular scale. We report here our experiences using a new approach based on Oscillating Gradient Spin-Echo (OGSE) MRI methods that can differentiate the influence on water diffusion of structural changes on scales much smaller than the diameter of a single cell. MRIs of glioblastomas in rat brain in vivo show an increased contrast and spatial heterogeneity when diffusion measurements are selectively sensitized to shorter distance scales. These results show the benefit of OGSE methods for revealing microscopic variations in tumors in vivo and confirm that diffusion measurements depend on factors other than cellularity. [Cancer Res 2008;68(14):5941–7]
Myricetin Suppresses UVB-Induced Skin Cancer by Targeting Fyn
Authors: Jung Sung Keun , Lee Ki Won , Byun Sanguine , Kang Nam Joo , Lim Sung Hwan , Heo Yong-Seok , Bode Ann M. , Bowden G. Tim , Lee Hyong Joo , Dong Zigang
Published: July 2008

Skin cancer is currently the most common type of human cancer in Americans. Myricetin, a naturally occurring phytochemical, has potent anticancer-promoting activity and contributes to the chemopreventive potential of several foods, including red wine. Here, we show that myricetin suppresses UVB-induced cyclooxygenase-2 (COX-2) expression in mouse skin epidermal JB6 P+ cells. The activation of activator protein-1 and nuclear factor-κB induced by UVB was dose-dependently inhibited by myricetin treatment. Western blot and kinase assay data revealed that myricetin inhibited Fyn kinase activity and subsequently attenuated UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays revealed that myricetin competitively bound with ATP to suppress Fyn kinase activity. Importantly, myricetin exerted similar inhibitory effects compared with 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a well-known pharmacologic inhibitor of Fyn. In vivo mouse skin data also revealed that myricetin inhibited Fyn kinase activity directly and subsequently attenuated UVB-induced COX-2 expression. Mouse skin tumorigenesis data clearly showed that pretreatment with myricetin significantly suppressed UVB-induced skin tumor incidence in a dose-dependent manner. Docking data suggest that myricetin is easily docked to the ATP-binding site of Fyn, which is located between the N and C lobes of the kinase domain. Overall, these results indicated that myricetin exerts potent chemopreventive activity mainly by targeting Fyn in skin carcinogenesis. [Cancer Res 2008;68(14):6021–9]
Hepatitis B Virus–Associated Multistep Hepatocarcinogenesis: A Stepwise Increase in Allelic Alterations
Authors: Lee Joyce Man-Fong , Wong Chun-Ming , Ng Irene Oi-Lin
Published: July 2008

Hepatocarcinogenesis is a multistep process, but systematic analysis using a genetic or molecular approach to accurately delineate the different stages of hepatocellular carcinoma (HCC) development is scarce. In this study, we used genome-wide allelotyping to systematically evaluate the allelic alterations in the multisteps of hepatitis B virus–associated hepatocarcinogenesis. The overall fractional allelic loss (FAL) indices of cirrhosis, dysplastic nodules (DN), and HCC were significantly different, with a clear stepwise increase (P < 0.001). Loss of heterozygosity (LOH) was uncommon in cirrhotic livers (n = 24; mean FAL index ± SD, 0.09 ± 0.09; median, 0.07). In contrast, LOH was common in our 74 HCC nodules, which were predominantly hepatitis B virus–associated (mean FAL index ± SD, 0.40 ± 0.23; median, 0.38). The 18 DNs had FAL index (mean ± SD, 0.27 ± 0.19; median, 0.20) in between that of cirrhosis and HCC. Importantly, high-grade DNs had FAL index significantly higher than that of low-grade DNs (P = 0.031) and close to that of HCC, indicating that high-grade DNs were genetically closer to HCC. However, there was no significant difference in FAL indices between primary HCCs and their corresponding intrahepatic metastases, but this absence of major allelic losses in this transformation to a metastatic phenotype does not exclude small-scale chromosomal losses or gene deletions. To conclude, hepatitis B virus–associated hepatocarcinogenesis is a multistep process accompanied by stepwise increase in allelic losses from cirrhosis and low- and high-grade DN to HCC. Such allelic losses contribute to promote tumor development and progression. [Cancer Res 2008;68(14):5988–96]
Refractoriness to Antivascular Endothelial Growth Factor Treatment: Role of Myeloid Cells
Authors: Shojaei Farbod , Ferrara Napoleone
Published: July 2008

CD11b+Gr1+ cells, which include neutrophils, macrophages, and myeloid-derived suppressor cells, have been shown to contribute to tumor angiogenesis. Recently, we found that accumulation of CD11b+Gr1+ in tumors renders them refractory to angiogenic blockade by vascular endothelial growth factor (VEGF) antibodies. This effect was traced to a pathway of CD11b+Gr1+–mediated angiogenesis that is, at least in part, driven by the secreted protein Bv8, which is up-regulated by the important myeloid growth factor granulocyte colony-stimulating factor (G-CSF). Thus, G-CSF may promote tumor angiogenesis through a Bv8-dependent pathway that bypasses VEGF and renders tumors refractory to anti-VEGF therapy. [Cancer Res 2008;68(14):5501–4]
Regulation of the Adenomatous Polyposis Coli Gene by the miR-135 Family in Colorectal Cancer
Authors: Nagel Remco , le Sage Carlos , Diosdado Begoña , van der Waal Maike , Oude Vrielink Joachim A.F. , Bolijn Anne , Meijer Gerrit A. , Agami Reuven
Published: July 2008

Inactivation of the adenomatous polyposis coli (APC) gene is a major initiating event in colorectal tumorigenesis. Most of the mutations in APC generate premature stop codons leading to truncated proteins that have lost β-catenin binding sites. APC-free β-catenin stimulates the Wnt signaling pathway, leading to active transcription of target genes. In the current study, we describe a novel mechanism for APC regulation. We show that miR-135a&b target the 3′ untranslated region of APC, suppress its expression, and induce downstream Wnt pathway activity. Interestingly, we find a considerable up-regulation of miR-135a&b in colorectal adenomas and carcinomas, which significantly correlated with low APC mRNA levels. This genetic interaction is also preserved in full-blown cancer cell lines expressing miR-135a&b, regardless of the mutational status of APC. Thus, our results uncover a miRNA-mediated mechanism for the control of APC expression and Wnt pathway activity, and suggest its contribution to colorectal cancer pathogenesis. [Cancer Res 2008;68(14):5795–802]
CTCFL/BORIS Is a Methylation-Independent DNA-Binding Protein That Preferentially Binds to the Paternal H19 Differentially Methylated Region
Authors: Nguyen Phuongmai , Cui Hengmi , Bisht Kheem S. , Sun Lunching , Patel Krish , Lee Richard S. , Kugoh Hiroyuki , Oshimura Mitsuo , Feinberg Andrew P. , Gius David
Published: July 2008

The CTCF paralog BORIS (brother of the regulator of imprinted sites) is an insulator DNA-binding protein thought to play a role in chromatin organization and gene expression. Under normal physiologic conditions, BORIS is predominantly expressed during embryonic male germ cell development; however, it is also expressed in tumors and tumor cell lines and, as such, has been classified as a cancer-germline or cancer-testis gene. It has been suggested that BORIS may be a pro-proliferative factor, whereas CTCF favors antiproliferation. BORIS and CTCF share similar zinc finger DNA-binding domains and seem to bind to identical target sequences. Thus, one critical question is the mechanism governing the DNA-binding specificity of these two proteins when both are present in tumor cells. Chromatin immunoprecipitation (ChIP) in HCT116 cells and their hypermethylated variant showed that BORIS binds to methylated DNA sequences, whereas CTCF binds to unmethylated DNA. Electromobility shift assays, using both whole-cell extracts and in vitro translated CTCF and BORIS protein, and methylation-specific ChIP PCR showed that BORIS is a methylation-independent DNA-binding protein. Finally, experiments in murine hybrid cells containing either the maternal or paternal human chromosome 11 showed that BORIS preferentially binds to the methylated paternal H19 differentially methylated region, suggesting a mechanism in which the affinity of CTCF for the unmethylated maternal allele directs the DNA binding of BORIS toward the paternal allele. [Cancer Res 2008;68(14):5546–51]
Presence of a TA Haplotype in the APC Gene Containing the Common 1822 Polymorphism and Colorectal Adenoma
Authors: Egan Jan B. , Jacobs Elizabeth T. , Martínez María Elena , Gerner Eugene W. , Jurutka Peter W. , Thompson Patricia A.
Published: July 2008

Acquired or inherited mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are causally linked to colorectal cancer. Given the significance of APC in colorectal cancer, we investigated the association between common single-nucleotide polymorphisms (SNP) in the APC gene and the odds of developing metachronous colorectal adenomas as a surrogate measure of colorectal cancer risk. Coding SNPs at codons 486, 1678, 1822, 1960, and 2502 were analyzed in a total of 1,399 subjects who participated in two randomized clinical trials for the prevention of colorectal adenomas. No association was found for any single SNP and the odds of metachronous adenoma. In contrast, a TA haplotype (codons 486 and 1822) was associated with a statistically significant 27% and 26% reduction in the odds of any and nonadvanced metachronous adenoma after adjustment for baseline adenoma characteristics [odds ratio (OR), 0.73; 95% confidence interval (95% CI), 0.59–0.91 and OR, 0.74; 95% CI, 0.57–0.94], respectively. No significant reduction in odds was observed for advanced metachronous lesions. Diplotype analysis revealed a strong gene dose effect with carriers of two alleles containing TT-AA (codons 486 and 1822, respectively) having an 89% lower odds for advanced metachronous adenomas (OR, 0.11; 95% CI, 0.01–0.80) when compared with the common CC-AA diplotype (codons 486 and 1822, respectively). Our findings support an important role for germ-line allele sequence in the APC gene and individual risk of metachronous adenomatous polyps. [Cancer Res 2008;68(14):6006–13]
PTEN Expression in Endometrial Biopsies as a Marker of Progression to Endometrial Carcinoma
Authors: Lacey James V. , Mutter George L. , Ronnett Brigitte M. , Ioffe Olga B. , Duggan Máire A. , Rush Brenda B. , Glass Andrew G. , Richesson Douglas A. , Chatterjee Nilanjan , Langholz Bryan , Sherman Mark E.
Published: July 2008

Inactivation of PTEN tumor suppressor gene is common in endometrial carcinoma and its precursor, atypical endometrial hyperplasia (EH). We compared PTEN expression via immunohistochemistry in endometrial biopsies diagnosed as EH in 138 cases, who were diagnosed with EH and then endometrial carcinoma at least 1 year later (median, 6 years), and 241 individually matched controls, who were diagnosed with EH but did not progress to carcinoma during equivalent follow-up. We assessed PTEN status (normal versus null) in index biopsies containing EH to estimate the relative risk (RR) of developing endometrial carcinoma up to 25 years later. Analysis of 115 cases and 193 controls with satisfactory assays revealed PTEN-null glands in index biopsies of 44% of cases and 49% of controls [P = 0.85; RR, 1.51; 95% confidence interval (CI), 0.73–3.13]. For predicting progression to carcinoma, PTEN-null status had low sensitivity (44%; 95% CI, 45–54%) and specificity (51%; 95% CI, 44–58%). Among 105 cases with PTEN results for both index biopsy and carcinoma, 16% had a PTEN-null index biopsy, 23% had PTEN-null carcinoma, and 26% had both a PTEN-null index biopsy and carcinoma. Loss of PTEN expression in endometrial biopsies was neither associated with nor a sensitive and specific marker of subsequent progression to endometrial carcinoma. [Cancer Res 2008;68(14):6014–20]
Coupling of Mutated Met Variants to DNA Repair via Abl and Rad51
Authors: Ganapathipillai Suganthini S. , Medová Michaela , Aebersold Daniel M. , Manley Paul W. , Berthou Sylvie , Streit Bruno , Blank-Liss Wieslawa , Greiner Richard H. , Rothen-Rutishauser Barbara , Zimmer Yitzhak
Published: July 2008

Abnormal activation of DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems is a compelling likelihood with significant implications in both cancer biology and treatment. Here, we show that due to a potential substrate switch, mutated variants of the receptor for hepatocyte growth factor Met, but not the wild-type form of the receptor, directly couple to the Abl tyrosine kinase and the Rad51 recombinase, two key signaling elements of homologous recombination–based DNA repair. Treatment of cells that express the mutated receptor variants with the Met inhibitor SU11274 leads, in a mutant-dependent manner, to a reduction of tyrosine phosphorylated levels of Abl and Rad51, impairs radiation-induced nuclear translocation of Rad51, and acts as a radiosensitizer together with the p53 inhibitor pifithrin-α by increasing cellular double-strand DNA break levels following exposure to ionizing radiation. Finally, we propose that in order to overcome a mutation-dependent resistance to SU11274, this aberrant molecular axis may alternatively be targeted with the Abl inhibitor, nilotinib. [Cancer Res 2008;68(14):5769–77]