bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Interplay of p53 and Mus81 in DNA Damage Responses and Cancer
Authors: Pamidi Ashwin , Cardoso Renato , Hakem Anne , Matysiak-Zablocki Elzbieta , Poonepalli Anuradha , Tamblyn Laura , Perez-Ordonez Bayardo , Hande M. Prakash , Sanchez Otto , Hakem Razqallah
Published: September 2007

Mus81 plays an integral role in the maintenance of genome stability and DNA repair in mammalian cells. Deficiency of Mus81 in human and mouse cells results in hypersensitivity to interstrand cross-linking (ICL) agents and elevated levels of genomic instability. Furthermore, Mus81-mutant mice are susceptible to spontaneous lymphomas. The role of cellular checkpoints in mediating the phenotypes observed in Mus81-deficient cells and mice is currently unknown. In this study, we have observed increased activation of p53 in Mus81−/− cells in response to ICL-induced DNA damage. In addition, p53 inactivation completely rescued the ICL hypersensitivity of Mus81−/− cells, signifying p53 is essential for the elimination of ICL-damaged cells in the absence of Mus81. Confirming that p53 acts as a critical checkpoint for the Mus81 repair pathway, a synergistic increase of spontaneous and ICL-induced genomic instability was observed in Mus81−/−p53−/− cells. To clarify the genetic interactions of Mus81 and p53 in tumor suppression, we monitored Mus81−/−p53−/− and control mice for the development of spontaneous tumors. Significantly, we show that loss of even a single allele of Mus81 drastically modifies the tumor spectrum of p53-mutant mice and increases their predisposition to developing sarcomas. Our results reveal a key role for p53 in mediating the response to spontaneous and ICL-induced DNA damage that occurs in the absence of Mus81. Furthermore, our data show that loss of Mus81, in addition to p53, is a key step in sarcoma development. [Cancer Res 2007;67(18):8527–35]
Inhibitors of the Proteasome Suppress Homologous DNA Recombination in Mammalian Cells
Authors: Murakawa Yasuhiro , Sonoda Eiichiro , Barber Louise J. , Zeng Weihua , Yokomori Kyoko , Kimura Hiroshi , Niimi Atsuko , Lehmann Alan , Zhao Guang Yu , Hochegger Helfrid , Boulton Simon J. , Takeda Shunichi
Published: September 2007

Proteasome inhibitors are novel antitumor agents against multiple myeloma and other malignancies. Despite the increasing clinical application, the molecular basis of their antitumor effect has been poorly understood due to the involvement of the ubiquitin-proteasome pathway in multiple cellular metabolisms. Here, we show that treatment of cells with proteasome inhibitors has no significant effect on nonhomologous end joining but suppresses homologous recombination (HR), which plays a key role in DNA double-strand break (DSB) repair. In this study, we treat human cells with proteasome inhibitors and show that the inhibition of the proteasome reduces the efficiency of HR-dependent repair of an artificial HR substrate. We further show that inhibition of the proteasome interferes with the activation of Rad51, a key factor for HR, although it does not affect the activation of ATM, γH2AX, or Mre11. These data show that the proteasome-mediated destruction is required for the promotion of HR at an early step. We suggest that the defect in HR-mediated DNA repair caused by proteasome inhibitors contributes to antitumor effect, as HR plays an essential role in cellular proliferation. Moreover, because HR plays key roles in the repair of DSBs caused by chemotherapeutic agents such as cisplatin and by radiotherapy, proteasome inhibitors may enhance the efficacy of these treatments through the suppression of HR-mediated DNA repair pathways. [Cancer Res 2007;67(18):8536–43]
Targeting Human Medulloblastoma: Oncolytic Virotherapy with Myxoma Virus Is Enhanced by Rapamycin
Authors: Lun Xue Qing , Zhou Hongyuan , Alain Tommy , Sun Beichen , Wang Limei , Barrett John W. , Stanford Marianne M. , McFadden Grant , Bell John , Senger Donna L. , Forsyth Peter A.
Published: September 2007

We have shown previously the oncolytic potential of myxoma virus in a murine xenograft model of human glioma. Here, we show that myxoma virus used alone or in combination with rapamycin is effective and safe when used in experimental models of medulloblastoma in vitro and in vivo. Nine of 10 medulloblastoma cell lines tested were susceptible to lethal myxoma virus infection, and pretreatment of cells with rapamycin increased the extent of in vitro oncolysis. Intratumoral injection of live myxoma virus when compared with control inactivated virus prolonged survival in D341 and Daoy orthotopic human medulloblastoma xenograft mouse models [D341 median survival: 21 versus 12.5 days; P = 0.0008; Daoy median survival: not reached (three of five mice apparently “cured” after 223 days) versus 75 days; P = 0.0021]. Rapamycin increased the extent of viral oncolysis, “curing” most Daoy tumor-bearing mice and reducing or eliminating spinal cord and ventricle metastases. Rapamycin enhanced tumor-specific myxoma virus replication in vivo and prolonged survival of D341 tumor-bearing mice (median survival of mice treated with live virus (LV) and rapamycin, versus LV alone, versus rapamycin alone, versus inactivated virus: 25 days versus 19, 13, and 11 days, respectively; P < 0.0001). Rapamycin increased the levels of constitutively activated Akt in Daoy and D341 cells, which may explain its ability to enhance myxoma virus oncolysis. These observations suggest that myxoma virus may be an effective oncolytic agent against medulloblastoma and that combination therapy with signaling inhibitors that modulate activity of the phosphatidylinositol 3-kinase/Akt pathway will further enhance the oncolytic potential of myxoma virus. [Cancer Res 2007;67(18):8818–27]
Progestins Reinitiate Cell Cycle Progression in Antiestrogen-Arrested Breast Cancer Cells through the B-Isoform of Progesterone Receptor
Authors: McGowan Eileen M. , Russell Amanda J. , Boonyaratanakornkit Viroj , Saunders Darren N. , Lehrbach Gillian M. , Sergio C. Marcelo , Musgrove Elizabeth A. , Edwards Dean P. , Sutherland Robert L.
Published: September 2007

Estrogen treatment of MCF-7 human breast cancer cells allows the reinitiation of synchronous cell cycle progression in antiestrogen-arrested cells. Here, we report that progestins also reinitiate cell cycle progression in this model. Using clonal cell lines derived from progesterone receptor (PR)–negative MCF-7M13 cells expressing wild-type or mutant forms of PRA and PRB, we show that this effect is mediated via PRB, not PRA. Cell cycle progression did not occur with a DNA-binding domain mutant of PRB but was unaffected by mutation in the NH2-terminal, SH3 domain interaction motif, which mediates rapid progestin activation of c-Src. Thus, the progestin-induced proliferative response in antiestrogen-inhibited cells is mediated primarily by the transcriptional activity of PRB. Analysis of selected cell cycle targets showed that progestin treatment induced levels of cyclin D1 expression and retinoblastoma protein (Rb) phosphorylation similar to those induced by estradiol. In contrast, progestin treatment resulted in only a 1.2-fold induction of c-Myc compared with a 10-fold induction by estradiol. These results support the conclusion that progestin, in a PRB-dependent manner, can overcome the growth-inhibitory effects of antiestrogens in estrogen receptor/PR-positive breast cancer cells by the induction of cyclin D1 expression. The mediation of this effect by PRB, but not PRA, further suggests a mechanism whereby abnormal regulation of the normal expression ratios of PR isoforms in breast cancer could lead to the attenuation of antiestrogen-mediated growth arrest. [Cancer Res 2007;67(18):8942–51]
Ionizing Radiation Predisposes Nonmalignant Human Mammary Epithelial Cells to Undergo Transforming Growth Factor β–Induced Epithelial to Mesenchymal Transition
Authors: Andarawewa Kumari L. , Erickson Anna C. , Chou William S. , Costes Sylvain V. , Gascard Philippe , Mott Joni D. , Bissell Mina J. , Barcellos-Hoff Mary Helen
Published: September 2007

Transforming growth factor β1 (TGFβ) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFβ activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFβ-mediated epithelial to mesenchymal transition (EMT). Nonmalignant HMEC (MCF10A, HMT3522 S1, and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFβ (0.4 ng/mL) or double treated. All double-treated (IR + TGFβ) HMEC underwent a morphologic shift from cuboidal to spindle shaped. This phenotype was accompanied by a decreased expression of epithelial markers E-cadherin, β-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Furthermore, double treatment increased cell motility, promoted invasion, and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFβ alone elicited EMT, although IR increased chronic TGFβ signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/mitogen-activated protein kinase (MAPK) signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFβ-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFβ, and treatment with U0126, a MAP/Erk kinase (MEK) inhibitor, blocked the EMT phenotype. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression. [Cancer Res 2007;67(18):8662–70]
Tobacco Smoke Induces Urokinase-Type Plasminogen Activator and Cell Invasiveness: Evidence for an Epidermal Growth Factor Receptor–Dependent Mechanism
Authors: Du Baoheng , Leung Helen , Khan K.M. Faisal , Miller Charles G. , Subbaramaiah Kotha , Falcone Domenick J. , Dannenberg Andrew J.
Published: September 2007

Multiple tobacco smoke–related premalignant and malignant lesions develop synchronously or metachronously in various organ sites, including the oral cavity. Both field cancerization and clonal migration seem to contribute to the occurrence of multiple tumors. Although the importance of endogenous factors (e.g., oncogenes) in regulating clonal migration is well established, little is known about the role of exogenous factors. Hence, the main objective of this study was to elucidate the mechanism by which tobacco smoke stimulated the migration of cells through extracellular matrix (ECM). Treatment of MSK-Leuk1 cells with a saline extract of tobacco smoke induced the migration of cells through ECM. Tobacco smoke induced the expression of urokinase-type plasminogen activator (uPA), resulting in plasmin-dependent degradation of ECM and increased cell migration. AG1478, a small-molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, a neutralizing antibody to EGFR, or an antibody to amphiregulin, an EGFR ligand, also blocked tobacco smoke–mediated induction of uPA and cell migration through ECM. PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase activity, caused similar inhibitory effects. Taken together, these results suggest that tobacco smoke activated the EGFR→extracellular signal-regulated kinase 1/2 MAPK pathway, causing induction of uPA. This led, in turn, to increased plasmin-dependent degradation of matrix proteins and enhanced cell migration through ECM. These data strongly suggest that chemicals in tobacco smoke can mimic the effects of oncogenes in regulating uPA-dependent cell invasion through ECM. These findings also strengthen the rationale for determining whether inhibitors of EGFR tyrosine kinase reduce the risk of tobacco smoke–related second primary tumors. [Cancer Res 2007;67(18):8966–72]
High SEPT9_v1 Expression in Human Breast Cancer Cells Is Associated with Oncogenic Phenotypes
Authors: Gonzalez Maria E. , Peterson Esther A. , Privette Lisa M. , Loffreda-Wren Janice L. , Kalikin Linda M. , Petty Elizabeth M.
Published: September 2007

Altered expression of the human septin gene, SEPT9, and its murine homologue, Sept9, has been implicated in neoplasia. However, their role(s) in oncogenesis remains poorly understood. We found amplification of SEPT9 in 67% of breast cancer cells (BCC) when compared with immortalized human mammary epithelial cells (IHMEC) as well as high levels of SEPT9 expression in the majority (61%) of the BCCs studied, unlike IHMECs. Expression profiling of variant SEPT9 transcripts and translated products revealed that high expression of the variant, SEPT9_v1, in contrast to other variants, was widespread in BCCs (55% of the BCCs) but not in IHMECs. High expression of SEPT9_v1 was also observed in primary breast cancer samples by immunohistochemical studies. We subsequently examined the phenotypic consequences of SEPT9_v1 expression in human breast cells. Retroviral expression of SEPT9_v1 in IHMEC cell culture models showed that SEPT9_v1 accelerated growth kinetics, stimulated cell motility, promoted invasion in Matrigel Transwell assays, increased genomic instability with the development of aneuploidy, and stimulated morphologic changes. Significant cytokinesis defects and disruption of tubulin microfilaments were also observed by immunofluorescence when SEPT9_v1 was ectopically expressed in IHMECs. Furthermore, SEPT9_v1 markedly enhanced neoplastic transformation in Hs578T cells, a BCC with no endogenous expression of the SEPT9_v1 isoform. Small interfering RNA–mediated and short hairpin RNA–mediated inhibition of SEPT9_v1 expression in two BCCs with high levels of endogenous SEPT9_v1 expression inhibited neoplastic growth properties of the cells. Taken together, our findings suggest that increased SEPT9_v1 expression contributes to the malignant pathogenesis of some breast tumors. [Cancer Res 2007;67(18):8554–11]
Identification of Tumorsphere- and Tumor-Initiating Cells in HER2/Neu-Induced Mammary Tumors
Authors: Liu Jeff C. , Deng Tao , Lehal Rajwinder S. , Kim Jinny , Zacksenhaus Eldad
Published: September 2007

A variety of human malignancies, including breast cancer, are thought to be organized in a hierarchy, whereby a relatively minor population of tumor initiating cells (TIC) is responsible for tumor growth and the vast majority of remaining cells is nontumorigenic. Analysis of TICs in model systems of breast cancer would offer uniform and accessible source of tumor cells and the power of mouse genetics to dissect these rare cells. The HER2/Neu proto-oncogene is overexpressed in an aggressive form of human breast cancer. Mouse mammary tumor virus (MMTV)-Neu transgenic mice develop mammary tumors that mimic human HER2 subtype breast cancer. Here, we report on the functional identification of mouse HER2/Neu TICs that can induce tumors after transplantation into the mammary gland of recipient mice. Secondary tumors formed after injecting MMTV-Neu TICs resemble primary tumors in the original transgenic mice and are organized in a hierarchy containing TICs as well as their nontumorigenic descendants. To study MMTV-Neu TICs in vitro, we grew tumorspheres under nonadherent culture conditions. Tumorsphere forming units (TFU) capable of producing tumorspheres retained tumorigenic potential and were indistinguishable by several criteria from TICs. Interestingly, MMTV-Neu TICs and TFUs were committed to the luminal cell fate when induced to differentiate in vitro. Our data define reproducible characteristics of the MMTV-Neu TIC and TFU, which help to explain marker expression profiles of HER2-positive breast cancer. In addition, the similarity between TICs and TFUs in this system provides a rationale for TFU-based screens to target tumor-initiating cells in HER2+ breast cancer. [Cancer Res 2007;67(18):8671–81]
Regulation of Phosphoglucose Isomerase/Autocrine Motility Factor Activities by the Poly(ADP-Ribose) Polymerase Family-14
Authors: Yanagawa Takashi , Funasaka Tatsuyoshi , Tsutsumi Soichi , Hu Huankai , Watanabe Hideomi , Raz Avraham
Published: September 2007

Phosphoglucose isomerase (PGI; EC 5.3.1.9) is a ubiquitous cytosolic enzyme essential for glycolysis and gluconeogenesis. PGI is a multifunctional dimeric protein that extracellularly acts as a cytokine [autocrine motility factor (AMF)] eliciting mitogenic, motogenic, and differentiation functions through binding to its cell surface receptor gp78/AMF receptor (AMFR). AMFR contains a seven-transmembrane domain with RING-H2 and leucine zipper motifs showing ubiquitin protein ligase (E3) activity and is exposed on the endoplasmic reticulum surface. Augmented expressions of both PGI/AMF and AMFR have been implicated in tumor progression and metastasis, and an intracellular binding partner of PGI/AMF is expected to regulate in part its diverse biological functions. Thus, we screened a cDNA library using a yeast two-hybrid system to search for interacting protein(s) and report on the finding of poly(ADP-ribose) polymerase-14 (PARP-14) to be a binding partner with PGI/AMF. PARP-14–PGI/AMF interaction was confirmed by coimmunoprecipitation and immunolocalization. We also report that PGI/AMF degradation is mainly regulated by the ubiquitin-lysosome system and RNA interference experiments revealed that PARP-14 inhibits PGI/AMF ubiquitination, thus contributing to its stabilization and secretion. This newly characterized PARP-14 protein should assist in understanding the regulation of PGI/AMF intracellular function(s) and may provide a new therapeutic target for inhibition of PGI/AMF inducing tumor cell migration and invasion during metastasis. [Cancer Res 2007;67(18):8682–9]
Deletion of the AU-Rich RNA Binding Protein Apobec-1 Reduces Intestinal Tumor Burden in Apcmin Mice
Authors: Blanc Valerie , Henderson Jeffrey O. , Newberry Rodney D. , Xie Yan , Cho Soo-Jin , Newberry Elizabeth P. , Kennedy Susan , Rubin Deborah C. , Wang Hanlin L. , Luo Jianyang , Davidson Nicholas O.
Published: September 2007

The RNA-specific cytidine deaminase apobec-1 is an AU-rich RNA binding protein that binds the 3′ untranslated region (UTR) of cyclooxygenase-2 (Cox-2) mRNA and stabilizes its turnover in vitro. Cox-2 overexpression accompanies intestinal adenoma formation in both humans and mice. Evidence from both genetic deletion studies as well as from pharmacologic inhibition has implicated Cox-2 in the development of intestinal adenomas in experimental animals and in adenomas and colorectal cancer in humans. Here, we show that small intestinal adenoma formation is dramatically reduced in compound Apcmin/+ apobec-1−/− mice when compared with the parental Apcmin/+ strain. This reduced tumor burden was found in association with increased small intestinal apoptosis and reduced proliferation in small intestinal crypt-villus units from compound Apcmin/+ apobec-1−/− mice. Intestinal adenomas from compound Apcmin/+ apobec-1−/− mice showed a <2-fold increase in Cox-2 mRNA abundance and reduced prostaglandin E2 content compared with adenomas from the parental Apcmin/+ strain. In addition, there was reduced expression in adenomas from compound Apcmin/+ apobec-1−/− mice of other mRNAs (including epidermal growth factor receptor, peroxisome proliferator–activated receptor δ, prostaglandin receptor EP4, and c-myc), each containing the apobec-1 consensus binding site within their 3′-UTR. Adenovirus-mediated apobec-1 introduction into HCA-7 (colorectal cancer) cells showed a dose-dependent increase in Cox-2 protein and stabilization of endogenous Cox-2 mRNA. These findings suggest that deletion of apobec-1, by modulating expression of AU-rich RNA targets, provides an important mechanism for attenuating a dominant genetic restriction point in intestinal adenoma formation. [Cancer Res 2007;67(18):8565–73]
Protein Kinase Cε Interacts with Signal Transducers and Activators of Transcription 3 (Stat3), Phosphorylates Stat3Ser727, and Regulates Its Constitutive Activation in Prostate Cancer
Authors: Aziz Moammir H. , Manoharan Herbert T. , Church Dawn R. , Dreckschmidt Nancy E. , Zhong Weixiong , Oberley Terry D. , Wilding George , Verma Ajit K.
Published: September 2007

Prostate cancer is the most common type of cancer in men and ranks second only to lung cancer in cancer-related deaths. The management of locally advanced prostate cancer is difficult because the cancer often becomes hormone insensitive and unresponsive to current chemotherapeutic agents. Knowledge about the regulatory molecules involved in the transformation to androgen-independent prostate cancer is essential for the rational design of agents to prevent and treat prostate cancer. Protein kinase Cε (PKCε), a member of the novel PKC subfamily, is linked to the development of androgen-independent prostate cancer. PKCε expression levels, as determined by immunohistochemistry of human prostate cancer tissue microarrays, correlated with the aggressiveness of prostate cancer. The mechanism by which PKCε mediates progression to prostate cancer remains elusive. We present here for the first time that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, including prostate cancer, interacts with PKCε. The interaction of PKCε with Stat3 was observed in human prostate cancer, human prostate cancer cell lines (LNCaP, DU145, PC3, and CW22rv1), and prostate cancer that developed in transgenic adenocarcinoma of mouse prostate mice. In reciprocal immunoprecipitation/blotting experiments, prostatic Stat3 coimmunoprecipitated with PKCε. Localization of PKCε with Stat3 was confirmed by double immunofluorescence staining. The interaction of PKCε with Stat3 was PKCε isoform specific. Inhibition of PKCε protein expression in DU145 cells using specific PKCε small interfering RNA (a) inhibited Stat3Ser727 phosphorylation, (b) decreased both Stat3 DNA-binding and transcriptional activity, and (c) decreased DU145 cell invasion. These results indicate that PKCε activation is essential for constitutive activation of Stat3 and prostate cancer progression. [Cancer Res 2007;67(18):8828–38]
Repression of Vascular Endothelial Growth Factor Expression by the Zinc Finger Transcription Factor ZNF24
Authors: Harper Jay , Yan Li , Loureiro Robyn M. , Wu Iinmin , Fang Jianmin , D'Amore Patricia A. , Moses Marsha A.
Published: September 2007

Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis. Although many positive regulators of VEGF have been identified, relatively little is known regarding the negative regulation of VEGF expression. We identified a zinc finger transcription factor, ZNF24, that may repress VEGF transcription. An inverse correlation between expression of VEGF and ZNF24 was observed in a series of independent studies. ZNF24 was up-regulated in angiogenic tumor nodules where VEGF expression is significantly decreased compared with preangiogenic nodules. In human breast carcinoma cells cultured under normoxic conditions, ZNF24 levels were significantly up-regulated whereas VEGF levels were low. In contrast, VEGF was significantly increased in hypoxic cells whereas ZNF24 was down-regulated. The same inverse correlation between ZNF24 and VEGF was also observed in 70% of matched cDNA pairs of normal and malignant tissues from human colon and breast biopsies. Overexpression of ZNF24 resulted in a significant down-regulation of VEGF, whereas silencing of ZNF24 with small interfering RNA led to increased VEGF expression. Cotransfection of ZNF24 and a VEGF promoter luciferase reporter construct in MDA-MB-231 cells resulted in a significant decrease in VEGF promoter activity. Taken together, these data suggest that ZNF24 is involved in negative regulation of VEGF and may represent a novel repressor of VEGF transcription. [Cancer Res 2007;67(18):8736–41]
Tie2-Expressing Monocytes and Tumor Angiogenesis: Regulation by Hypoxia and Angiopoietin-2
Authors: Lewis Claire E. , De Palma Michele , Naldini Luigi
Published: September 2007

Recent findings indicate that tumor-associated macrophages are important drivers of tumor angiogenesis. Here, we review the essential role played by Tie2-expressing monocytes (TEM) in this phenomenon. TEMs are present in human blood and tumors and their elimination in various tumor models suppresses tumor angiogenesis. A ligand for Tie2, angiopoietin-2 (Ang-2), is produced by angiogenic tumor vessels and is a chemoattractant for TEMs. Hypoxia up-regulates Tie2 expression on TEMs and, together with Ang-2, down-regulates their antitumor functions. Learning more about the regulation of TEMs by the tumor microenvironment may yield new strategies to ablate the tumor vasculature. [Cancer Res 2007;67(18):8429–32]
Relationship between B7-H4, Regulatory T Cells, and Patient Outcome in Human Ovarian Carcinoma
Authors: Kryczek Ilona , Wei Shuang , Zhu Gefeng , Myers Leann , Mottram Peter , Cheng Pui , Chen Lieping , Coukos George , Zou Weiping
Published: September 2007

B7-H4 is a recently identified B7 family member. We previously showed that ovarian tumor and associated macrophages expressed B7-H4; tumor B7-H4+ macrophages and CD4+CD25+FOXP3+ regulatory T cells (Treg cells) suppressed tumor-associated antigen–specific T-cell immunity. To determine the pathologic relationship between B7-H4, macrophages, and Treg cells in the tumor environment, in addition to Treg cell numbers, we quantified B7-H4 expression in the tumor and tumor-associated macrophages in 103 patients with ovarian carcinoma. We observed that the intensity of B7-H4 expression in macrophages was significantly correlated with Treg cell numbers in the tumor. Further, both Treg cells and macrophage B7-H4, but not tumor B7-H4, were negatively associated with patient outcome. Tumor Treg cells enabled macrophages to spontaneously produce interleukin (IL)-10 and IL-6. Tumor macrophages stimulated B7-H4 expression in an autocrine manner through IL-10 and IL-6. Our previous work showed that tumor-associated macrophages spontaneously produced chemokine CCL22 to mediate Treg cell trafficking into tumor, and Treg cells induced B7-H4 on antigen-presenting cells (APC) including macrophages. Altogether, our data support the concept that there is a mechanistic interaction between Treg cells and macrophage, and that Treg cells may convey the suppressive activity to APCs through B7-H4 induction in human ovarian cancer. [Cancer Res 2007;67(18):8901–05]
Nitric Oxide Radicals Choreograph a Radioadaptive Response
Authors: Matsumoto Hideki , Takahashi Akihisa , Ohnishi Takeo
Published: September 2007

The reduced biological effects of radiation exposure seen in cells after conditioning exposures to a low dose or at a low-dose rate (i.e., the acquisition of resistance against high-dose radiation) is called the “radioadaptive response” and many studies concerning this phenomenon have been reported since the 1980s. Radioadaptive responses have been observed using various end points, such as chromosomal aberrations, mutations, and clonogenic survival. However, the mechanisms of the radioadaptive response are not fully known. Here, we show that radiation-induced nitric oxide (NO) radicals contribute to the induction of radioresistance as determined by cell survival after a subsequent high-dose exposure. An accumulation of inducible NO synthase was produced, and the concentration of nitrite in the culture medium increased when cells were exposed to γ-rays at a low-dose rate or to X-rays for a low dose followed by an acute high-dose X-irradiation. In addition, the induction of radioresistance was not observed in the presence of an inhibitor of inducible NO synthase or a scavenger of NO radicals. Moreover, radioresistance was observed when cultures were treated with a NO radical–generating agent. These findings suggest that NO radicals are an initiator of the radioadaptive response. [Cancer Res 2007;67(18):8574–9]
MicroRNA Signatures in Human Ovarian Cancer
Authors: Iorio Marilena V. , Visone Rosa , Di Leva Gianpiero , Donati Valentina , Petrocca Fabio , Casalini Patrizia , Taccioli Cristian , Volinia Stefano , Liu Chang-Gong , Alder Hansjuerg , Calin George A. , Ménard Sylvie , Croce Carlo M.
Published: September 2007

Epithelial ovarian cancer (EOC) is the sixth most common cancer in women worldwide and, despite advances in detection and therapies, it still represents the most lethal gynecologic malignancy in the industrialized countries. Unfortunately, still relatively little is known about the molecular events that lead to the development of this highly aggressive disease. The relatively recent discovery of microRNAs (miRNA), a class of small noncoding RNAs targeting multiple mRNAs and triggering translation repression and/or RNA degradation, has revealed the existence of a new level of gene expression regulation. Multiple studies involving various types of human cancers proved that miRNAs have a causal role in tumorigenesis. Here we show that, in comparison to normal ovary, miRNAs are aberrantly expressed in human ovarian cancer. The overall miRNA expression could clearly separate normal versus cancer tissues. The most significantly overexpressed miRNAs were miR-200a, miR-141, miR-200c, and miR-200b, whereas miR-199a, miR-140, miR-145, and miR-125b1 were among the most down-modulated miRNAs. We could also identify miRNAs whose expression was correlated with specific ovarian cancer biopathologic features, such as histotype, lymphovascular and organ invasion, and involvement of ovarian surface. Moreover, the levels of miR-21, miR-203, and miR-205, up-modulated in ovarian carcinomas compared with normal tissues, were significantly increased after 5-aza-2′-deoxycytidine demethylating treatment of OVCAR3 cells, suggesting that the DNA hypomethylation could be the mechanism responsible for their overexpression. Our results indicate that miRNAs might play a role in the pathogenesis of human EOC and identify altered miRNA gene methylation as a possible epigenetic mechanism involved in their aberrant expression. [Cancer Res 2007;67(18):8699–707]
In Non-neoplastic Barrett's Epithelial Cells, Acid Exerts Early Antiproliferative Effects through Activation of the Chk2 Pathway
Authors: Zhang Hui-Ying , Zhang Xi , Hormi-Carver Kathy , Feagins Linda A. , Spechler Stuart J. , Souza Rhonda F.
Published: September 2007

Acid exerts pro-proliferative effects in Barrett's-associated esophageal adenocarcinoma cells. In non-neoplastic Barrett's epithelial (BAR-T) cells, in contrast, we have shown that acid exposure has antiproliferative effects. To explore our hypothesis that the acid-induced, antiproliferative effects are mediated by alterations in the proteins that regulate the G1-S cell cycle checkpoint, we exposed non-neoplastic Barrett's cells to acidic media (pH 4.0) and analyzed G1-S checkpoint proteins' expression, phosphorylation, and activity levels by Western blot. We studied acid effects on growth (by cell counts), proliferation (by flow cytometry and bromodeoxyuridine incorporation), cell viability (by trypan blue staining), and apoptosis (by annexin V staining), and we used caffeine and small interfering RNA to assess the effects of checkpoint kinase 2 (Chk2) inhibition on G1-S progression. Acid exposure significantly decreased cell numbers without affecting cell viability and with only a slight increase in apoptosis. Within 2 h of acid exposure, there was a delay in progression through the G1-S checkpoint that was associated with increased phosphorylation of Chk2, decreased levels of Cdc25A, and decreased activity of cyclin E–cyclin-dependent kinase 2; by 4 h, a continued delay at G1-S was associated with increased expression of p53 and p21. Caffeine and Chk2 siRNA abolished the acid-induced G1-S delay at 2 but not at 4 h. We conclude that acid exposure in non-neoplastic BAR-T cells causes early antiproliferative effects that are mediated by the activation of Chk2. Thus, we have elucidated a mechanism whereby acid can exert disparate effects on proliferation in neoplastic and non-neoplastic BAR-T cells. [Cancer Res 2007;67(18):8580–7]
SV40 Multiple Tissue Infection and Asbestos Exposure in a Hyperendemic Area for Malignant Mesothelioma
Authors: Comar Manola , Rizzardi Clara , de Zotti Renata , Melato Mauro , Bovenzi Massimo , Butel Janet S. , Campello Cesare
Published: September 2007

To assess the presence of SV40 in malignant mesothelioma tissue, 19 formalin-fixed paraffin-embedded pleural cancer samples of patients from a hyperendemic area of northeastern Italy were analyzed retrospectively. A total of 48 other tissues from the malignant mesothelioma subjects were investigated. The SV40 load was determined by real-time quantitative PCR. Exposure to asbestos was evaluated through a careful review of the occupational history of patients, supplemented by histology and isolation of asbestos bodies. Three of 19 (15.8%) malignant mesothelioma tissues harbored SV40 genomic signals. Two patients with SV40-positive malignant mesothelioma had viral sequences in another tissue. Overall, 3 of 18 (16.7%) normal liver tissues tested positive for SV40, as did 1 of 8 (12.5%) kidney tissues. SV40 viral loads were higher in malignant mesothelioma than in normal cells (P = 0.045). This survey shows that SV40 sustains infections in multiple tissues in malignant mesothelioma patients from a geographic area affected with asbestos-related mesothelioma. [Cancer Res 2007;67(18):8456–4]
TFAP2C Controls Hormone Response in Breast Cancer Cells through Multiple Pathways of Estrogen Signaling
Authors: Woodfield George W. , Horan Annamarie D. , Chen Yizhen , Weigel Ronald J.
Published: September 2007

Breast cancers expressing estrogen receptor-α (ERα) are associated with a favorable biology and are more likely to respond to hormonal therapy. In addition to ERα, other pathways of estrogen response have been identified including ERβ and GPR30, a membrane receptor for estrogen, and the key mechanisms regulating expression of ERs and hormone response remain controversial. Herein, we show that TFAP2C is the key regulator of hormone responsiveness in breast carcinoma cells through the control of multiple pathways of estrogen signaling. TFAP2C regulates the expression of ERα directly by binding to the ERα promoter and indirectly via regulation of FoxM1. In so doing, TFAP2C controls the expression of ERα target genes, including pS2, MYB, and RERG. Furthermore, TFAP2C controlled the expression of GPR30. In distinct contrast, TFAP2A, a related factor expressed in breast cancer, was not involved in estrogen-mediated pathways but regulated expression of genes controlling cell cycle arrest and apoptosis including p21CIP1 and IGFBP-3. Knockdown of TFAP2C abrogated the mitogenic response to estrogen exposure and decreased hormone-responsive tumor growth of breast cancer xenografts. We conclude that TFAP2C is a central control gene of hormone response and is a novel therapeutic target in the design of new drug treatments for breast cancer. [Cancer Res 2007;67(18):8439–43]
Clonal Evolution of Resistance to Imatinib in Patients with Metastatic Gastrointestinal Stromal Tumors
Authors: Desai Jayesh , Shankar Sridhar , Heinrich Michael C. , Fletcher Jonathan A. , Fletcher Christopher D. , Manola Judi , Morgan Jeffrey A. , Corless Christopher L. , George Suzanne , Tuncali Kemal , Silverman Stuart G. , Van den Abbeele Annick D. , van Sonnenberg Eric , Demetri George D.
Published: September 2007

Purpose: Resistance to imatinib mesylate is emerging as a clinical challenge in patients with metastatic gastrointestinal stromal tumors (GIST). Novel patterns of progression have been noted in a number of these patients. The objective of this study was to correlate molecular and radiologic patterns of imitinib-refractory disease with existing conventional criteria for disease progression.

Experimental Design: Patients with metastatic GIST treated with imatinib were followed with serial computed tomography/magnetic resonance imaging and [18F]fluoro-2-deoxy-d-glucose positron emission tomography. Where feasible, biopsies were done to document disease progression.

Results: A total of 89 patients were followed for a median of 43 months. Forty-eight patients developed progressive disease. A unique “resistant clonal nodule” pattern (defined as a new enhancing nodular focus enclosed within a preexisting tumor mass) was seen in 23 of 48 patients and was thought to represent emergence of clones resistant to imatinib. Nodules were demonstrable a median of 5 months (range, 0-13 months) before objective progression defined by tumor size criteria and were the first sign of progression in 18 of 23 patients. Median survival among patients whose first progression was nodular was 35.1 months, compared with 44.6 months for patients whose first progression met Southwest Oncology Group criteria (P = 0.31). Comparative tumor biopsies were done in 10 patients at baseline and from progressing nodules. Genotypic analyses of KIT and PDGFRA kinases were done, revealing new activating kinase mutations in 80% (8 of 10) of these patients.

Conclusion: The resistant clonal nodule is a unique pattern of disease progression seen in patients with GISTs after an initial response to imatinib and reflects the emergence of imatinib-resistant clones. Conventional tumor measurements (Southwest Oncology Group/Response Evaluation Criteria in Solid Tumors) do not detect this subtle finding. A new enhancing nodule growing within a preexisting tumor mass should be classified as a new lesion and be regarded, at least, as partial progression of GIST.
Preclinical Analysis of Tasidotin HCl in Ewing's Sarcoma, Rhabdomyosarcoma, Synovial Sarcoma, and Osteosarcoma
Authors: Garg Vaani , Zhang Wendong , Gidwani Pooja , Kim Mimi , Kolb E. Anders
Published: September 2007

Purpose: Dolastatins are a group of structurally unique peptides originally isolated from a sea hare, Dolabella auricularia, which seem to inhibit tubulin polymerization and mitosis. Tasidotin hydrochloride (tasidotin), a novel synthetic analogue of dolastatin 15, is evaluated in preclinical models of pediatric tumors.

Experimental Design: The cytotoxicity of tasidotin was evaluated in a panel of pediatric sarcoma cell lines in vitro and in vivo.

Results: The IC50 in Ewing's sarcoma, rhabdomyosarcoma, osteosarcoma, and synovial sarcoma lines ranged from 0.002 μ to 0.32 μmol/L. In the SK-ES1 and RH30 cell lines, tasidotin induced a G2-M arrest that persisted for 48 h after the drug was washed from the cells. In vitro, more than half the cells were in the early or late phase of apoptosis 48 h after treatment with tasidotin. In vivo, a significant increase in apoptotic nuclei was apparent in xenograft tumors harvested within 24 h after a 5-day course of tasidotin. In vivo response was determined in severe combined immunodeficient xenograft models of pediatric sarcomas implanted heterotopically. Significant antitumor activity was observed in all tumor lines tested. A complete response was observed in 2 synovial sarcoma lines, 1 osteosarcoma line, 1 rhabdomyosarcoma line, and 1 Ewing's sarcoma line. A partial response was observed in 1 rhabdomyosarcoma and 1 Ewing's sarcoma.

Conclusions: Tasidotin induces a G2-M block in treated cells ultimately resulting in apoptosis. Antitumor activity is confirmed in vivo in preclinical xenograft models of pediatric sarcomas.
Pathways Mediating the Expansion and Immunosuppressive Activity of Myeloid-Derived Suppressor Cells and Their Relevance to Cancer Therapy
Authors: Talmadge James E.
Published: September 2007

Cancer immunotherapy has focused on inducing and expanding CTLs and improving the immune recognition of weak antigenic determinants expressed by tumors. However, few positive clinical outcomes have been reported due, in part, to tumor-associated immunologic tolerance, supporting the need for an emphasis on overcoming immunosuppression. Systemic immunosuppression is associated with abnormal myelopoiesis secondary to tumor growth, myelosuppressive therapy, and growth factor administration and subsequent expansion/mobilization of bone marrow–derived immunosuppressive cells. These myeloid-derived suppressor cells (MDSC) reduce activated T-cell number and inhibit their function by multiple mechanisms, including depletion of l-arginine by arginase-1 (ARG1) production of nitric oxide, reactive oxygen species, and reactive nitrogen oxide species by inducible nitric oxide synthase. Increased numbers of MDSCs are associated with neoplastic, inflammatory, infectious, and graft-versus-host diseases where they restrain exuberant or novel T-cell responses. In this review, we discuss critical components of MDSC-mediated suppression of T-cell function, including cellular expansion and activation-induced secretion of immunosuppressive mediators. Both components of MDSC bioactivity are amenable to pharmacologic intervention as discussed herein. We also focus on the relationship between MDSCs, tumor growth, therapeutic responses, and the mechanisms of cellular expansion, activation, and immunosuppression.
Atorvastatin and Celecoxib Inhibit Prostate PC-3 Tumors in Immunodeficient Mice
Authors: Zheng Xi , Cui Xiao-Xing , Avila Gina E. , Huang Mou-Tuan , Liu Yue , Patel Jagruti , Kong Ah Ng Tony , Paulino Raphael , Shih Weichung Joe , Lin Yong , Rabson Arnold B. , Reddy Bandaru S. , Conney Allan H.
Published: September 2007

Purpose: To investigate the effects and mechanisms of atorvastatin and celecoxib administered individually or in combination on human prostate cancer PC-3 cells cultured in vitro or grown as xenograft tumors in immunodeficient mice.

Experimental Design: Human prostate cancer PC-3 cells in culture were treated with atorvastatin and celecoxib alone or in combination. Severe combined immunodeficient (SCID) mice were injected s.c. with PC-3 cells. The mice received daily i.p injections starting 2 days before tumor cell inoculation and continuing during the course of treatment with atorvastatin (10 μg/g body weight/d), celecoxib (10 μg/g/d), a combination of atorvastatin (10 μg/g/d) and celecoxib (10 μg/g/d), or a combination of atorvastatin (5 μg/g/d) and celecoxib (5 μg/g/d).

Results: Atorvastatin in combination with celecoxib had stronger effects on growth inhibition and apoptosis of PC-3 cells than either agent used individually. Atorvastatin and celecoxib in combination also had a stronger inhibitory effect on activation of nuclear factor-κB and extracellular signal-regulated kinase 1/2 in PC-3 cells than either agent alone. Treatment of SCID mice with combinations of atorvastatin and celecoxib more effectively inhibited the formation and growth of PC-3 tumors in the mice than either agent administered alone.

Conclusions: A combination of atorvastatin and celecoxib had a more potent inhibitory effect on the growth of PC-3 cells cultured in vitro or grown in SCID mice than either agent alone. A combination of atorvastatin and celecoxib may be an effective strategy for the prevention of prostate cancer.
Preoperative Plasma HER2 and Epidermal Growth Factor Receptor for Staging and Prognostication in Patients with Clinically Localized Prostate Cancer
Authors: Shariat Shahrokh F. , Bensalah Karim , Karam Jose A. , Roehrborn Claus G. , Gallina Andrea , Lotan Yair , Slawin Kevin M. , Karakiewicz Pierre I.
Published: September 2007

Purpose: Human epidermal growth factor receptor-2 (HER2) and epidermal growth factor receptor (EGFR) expression have been associated with disease progression in patients with prostate cancer. We tested the hypothesis that plasma levels of HER2 and/or EGFR are associated with prostate cancer stage and prognosis in patients with clinically localized disease.

Experimental Design: We measured preoperative plasma HER2 and EGFR levels using commercially available ELISAs on banked plasma from 227 patients treated with radical prostatectomy and bilateral lymphadenectomy for clinically localized prostate adenocarcinoma.

Results: Median preoperative plasma EGFR and HER2 levels were 31.4 ng/mL (interquartile range, 19.2 ng/mL) and 10.0 ng/mL (interquartile range, 2.7 ng/mL), respectively. HER2 was elevated in patients with seminal vesicle invasion (P = 0.033). In separate multivariate analyses that adjusted for the effects of standard preoperative predictors, lower EGFR, higher HER2, and higher HER2/EGFR ratio were associated with prostate-specific antigen (PSA) progression (P = 0.003, P < 0.001, and P < 0.001, respectively). In separate multivariate analyses that adjusted for the effects of standard postoperative predictors, lower EGFR and higher HER2/EGFR ratio were associated with PSA progression (P = 0.027 and P < 0.001, respectively). Among the patients who experienced PSA progression, HER2 was significantly higher (P = 0.023) and EGFR was lower (P = 0.04) in those with features of aggressive disease (i.e., development of metastasis, PSA doubling time <10 months, and/or failure to respond to local salvage radiation therapy).

Conclusion: Preoperative plasma HER2 and EGFR were associated with prostate cancer progression after radical prostatectomy. Plasma HER2 and EGFR may provide a tool for predicting long-term recurrence-free survival and early metastasis.
Targeting Methylguanine-DNA Methyltransferase in the Treatment of Neuroblastoma
Authors: Wagner Lars M. , McLendon Roger E. , Yoon K. Jin , Weiss Brian D. , Billups Catherine A. , Danks Mary K.
Published: September 2007

Purpose: The combination of temozolomide and irinotecan has preclinical schedule-dependent synergy against neuroblastoma but is not curative for relapsed high-risk patients. We hypothesized that the DNA repair protein methylguanine-DNA methyltransferase (MGMT) is an important resistance factor, and that inactivation of MGMT would sensitize neuroblastoma cells to these agents.

Experimental Design: MGMT protein expression was assessed in 74 primary neuroblastoma tumors. Growth inhibition assays were done to determine the IC50 and the extent of synergy observed with various concentrations of temozolomide, irinotecan, and the MGMT-inactivating agent O6-benzylguanine, using cultured syngeneic neuroblastoma cells with either low or high levels of MGMT expression. We then assessed efficacy in a mouse xenograft model of metastatic neuroblastoma.

Results: MGMT was expressed by all 74 tumors evaluated. Pretreatment of neuroblastoma cells with O6-benzylguanine reduced the IC50 of temozolomide by 10-fold regardless of level of MGMT expression, and pretreatment with BG followed by temozolomide + irinotecan further reduced the IC50 in cells with high MGMT expression another 10-fold, to well below clinically achievable concentrations. The combination index was 0.27 to 0.30 for all three drugs in both cell lines, indicating strong synergy. Survival at 100 days for mice with metastatic neuroblastoma was 56% with three-drug treatment, compared with untreated controls (0%, P < 0.001) or temozolomide + irinotecan (10%, P = 0.081).

Conclusions: MGMT is widely expressed in primary neuroblastoma tumors, and is a relevant therapeutic target. Both in vitro and in vivo studies suggest inactivation of MGMT with O6-benzylguanine may increase the activity of temozolomide and irinotecan against neuroblastoma.