bluuebunny/crossref_metadata_2025 huggingface dataset:
The Cytoplasmic Deacetylase HDAC6 Is Required for Efficient Oncogenic Tumorigenesis
Authors: Lee Yi-Shan , Lim Kian-Huat , Guo Xing , Kawaguchi Yoshiharu , Gao Yasheng , Barrientos Tomasa , Ordentlich Peter , Wang Xiao-Fan , Counter Christopher M. , Yao Tso-Pang
Published: September 2008

Histone deacetylase inhibitors (HDACI) are promising antitumor agents. Although transcriptional deregulation is thought to be the main mechanism underlying their therapeutic effects, the exact mechanism and targets by which HDACIs achieve their antitumor effects remain poorly understood. It is not known whether any of the HDAC members support robust tumor growth. In this report, we show that HDAC6, a cytoplasmic-localized and cytoskeleton-associated deacetylase, is required for efficient oncogenic transformation and tumor formation. We found that HDAC6 expression is induced upon oncogenic Ras transformation. Fibroblasts deficient in HDAC6 are more resistant to both oncogenic Ras and ErbB2-dependent transformation, indicating a critical role for HDAC6 in oncogene-induced transformation. Supporting this hypothesis, inactivation of HDAC6 in several cancer cell lines reduces anchorage-independent growth and the ability to form tumors in mice. The loss of anchorage-independent growth is associated with increased anoikis and defects in AKT and extracellular signal-regulated kinase activation upon loss of adhesion. Lastly, HDAC6-null mice are more resistant to chemical carcinogen-induced skin tumors. Our results provide the first experimental evidence that a specific HDAC member is required for efficient oncogenic transformation and indicate that HDAC6 is an important component underlying the antitumor effects of HDACIs. [Cancer Res 2008;68(18):7561–9]
Kaiso Contributes to DNA Methylation-Dependent Silencing of Tumor Suppressor Genes in Colon Cancer Cell Lines
Authors: Lopes Eloisi C. , Valls Ester , Figueroa Maria E. , Mazur Alexander , Meng Fan-Guo , Chiosis Gabriela , Laird Peter W. , Schreiber-Agus Nicole , Greally John M. , Prokhortchouk Egor , Melnick Ari
Published: September 2008

Aberrant CpG methylation of tumor suppressor gene regulatory elements is associated with transcriptional silencing and contributes to malignant transformation of different tissues. It is presumed that methylated DNA sequences recruit repressor machinery to actively shutdown gene expression. The Kaiso protein is a transcriptional repressor expressed in human and murine colorectal tumors that can bind to methylated clusters of CpG dinucleotides. We show here that Kaiso represses methylated tumor suppressor genes and can bind in a methylation-dependent manner to the CDKN2A in human colon cancer cell lines. The contribution of Kaiso to epigenetic silencing was underlined by the fact that Kaiso depletion induced tumor suppressor gene expression without affecting DNA methylation levels. As a consequence, colon cancer cells became susceptible to cell cycle arrest and cell death mediated by chemotherapy. The data suggest that Kaiso is a methylation-dependent “opportunistic” oncogene that silences tumor suppressor genes when they become hypermethylated. Because Kaiso inactivation sensitized colon cancer cell lines to chemotherapy, it is possible that therapeutic targeting of Kaiso could improve the efficacy of current treatment regimens. [Cancer Res 2008;68(18):7258–63]
Cytoglobin, the Newest Member of the Globin Family, Functions as a Tumor Suppressor Gene
Authors: Shivapurkar Narayan , Stastny Victor , Okumura Naoki , Girard Luc , Xie Yang , Prinsen Clemens , Thunnissen Frederik B. , Wistuba Ignacio I. , Czerniak Bogdan , Frenkel Eugene , Roth Jack A. , Liloglou Triantafillos , Xinarianos George , Field John K. , Minna John D. , Gazdar Adi F.
Published: September 2008

Cytoglobin (CYGB) is a recently discovered vertebrate globin distantly related to myoglobin with unknown function. CYGB is assigned to chromosomal region 17q25, which is frequently lost in multiple malignancies. Previous studies failed to detect evidence for mutations in the CYGB gene. Recent studies provided preliminary evidence for increased methylation of the gene in lung cancer. Our study was aimed at investigating the role of CYGB as a tumor suppressor gene. By nested methylation-specific DNA sequencing analysis of lung and breast cancer cell lines and bronchial and mammary epithelial cell lines, we identified that methylation of a 110-bp CpG-rich segment of the CYGB promoter was correlated with gene silencing. We specifically targeted this sequence and developed a quantitative methylation-specific PCR assay, suitable for high-throughput analysis. We showed that the tumor specificity of CYGB methylation in discriminating patients with and without lung cancer, using biopsies and sputum samples. We further showed the tumor specificity of this assay with multiple other epithelial and hematologic malignancies. To show tumor suppressor activity of CYGB, we performed the following: (a) RNA interference–mediated knockdown of CYGB gene on colony formation in a CYGB expression–positive lung cancer cell line, resulting in increased colony formation; (b) enforced gene expression in CYGB expression–negative lung and breast cancer cell lines, reducing colony formation; and (c) identification of potential proximate targets down-stream of the CYGB genes. Our data constitute the first direct functional evidence for CYGB, the newest member of the globin family, as a tumor suppressor gene. [Cancer Res 2008;68(18):7448–56]
Nordihydroguaiaretic Acid Inhibits an Activated Fibroblast Growth Factor Receptor 3 Mutant and Blocks Downstream Signaling in Multiple Myeloma Cells
Authors: Meyer April N. , McAndrew Christopher W. , Donoghue Daniel J.
Published: September 2008

Activating mutations within fibroblast growth factor receptor 3 (FGFR3), a receptor tyrosine kinase, are responsible for human skeletal dysplasias including achondroplasia and the neonatal lethal syndromes, Thanatophoric Dysplasia (TD) type I and II. Several of these same FGFR3 mutations have also been identified somatically in human cancers, including multiple myeloma, bladder carcinoma, and cervical cancer. Based on reports that strongly activated mutants of FGFR3 such as the TDII (K650E) mutant signal preferentially from within the secretory pathway, the inhibitory properties of nordihydroguaiartic acid (NDGA), which blocks protein transport through the Golgi, were investigated. NDGA was able to inhibit FGFR3 autophosphorylation both in vitro and in vivo. In addition, signaling molecules downstream of FGFR3 activation such as signal transducers and activators of transcription (STAT)1, STAT3, and mitogen-activated protein kinase (MAPK) were inhibited by NDGA treatment. Using HEK293 cells expressing activated FGFR3-TDII, together with several multiple myeloma cell lines expressing activated forms of FGFR3, NDGA generally resulted in a decrease in MAPK activation by 1 hour, and resulted in increased apoptosis over 24 hours. The effects of NDGA on activated FGFR3 derivatives targeted either to the plasma membrane or the cytoplasm were also examined. These results suggest that inhibitory small molecules such as NDGA that target a specific subcellular compartment may be beneficial in the inhibition of activated receptors such as FGFR3 that signal from the same compartment. [Cancer Res 2008;68(18):7362–70]
Interaction of Muc2 and Apc on Wnt Signaling and in Intestinal Tumorigenesis: Potential Role of Chronic Inflammation
Authors: Yang Kan , Popova Natalia V. , Yang Wan Cai , Lozonschi Ioanna , Tadesse Selam , Kent Scott , Bancroft Laura , Matise Ilze , Cormier Robert T. , Scherer Stefan J. , Edelmann Winfried , Lipkin Martin , Augenlicht Leonard , Velcich Anna
Published: September 2008

Somatic mutations of the adenomatous polyposis coli (APC) gene are initiating events in the majority of sporadic colon cancers. A common characteristic of such tumors is reduction in the number of goblet cells that produce the mucin MUC2, the principal component of intestinal mucus. Consistent with these observations, we showed that Muc2 deficiency results in the spontaneous development of tumors along the entire gastrointestinal tract, independently of deregulated Wnt signaling. To dissect the complex interaction between Muc2 and Apc in intestinal tumorigenesis and to elucidate the mechanisms of tumor formation in Muc2−/− mice, we crossed the Muc2−/− mouse with two mouse models, Apc1638N/+ and ApcMin/+, each of which carries an inactivated Apc allele. The introduction of mutant Muc2 into Apc1638N/+ and ApcMin/+ mice greatly increased transformation induced by the Apc mutation and significantly shifted tumor development toward the colon as a function of Muc2 gene dosage. Furthermore, we showed that in compound double mutant mice, deregulation of Wnt signaling was the dominant mechanism of tumor formation. The increased tumor burden in the distal colon of Muc2/Apc double mutant mice was similar to the phenotype observed in ApcMin/+ mice that are challenged to mount an inflammatory response, and consistent with this, gene expression profiles of epithelial cells from flat mucosa of Muc2-deficient mice suggested that Muc2 deficiency was associated with low levels of subclinical chronic inflammation. We hypothesize that Muc2−/− tumors develop through an inflammation-related pathway that is distinct from and can complement mechanisms of tumorigenesis in Apc+/− mice. [Cancer Res 2008;68(18):7313–22]
Multimodal Control of Cdc25A by Nitrosative Stress
Authors: Tomko Robert J. , Lazo John S.
Published: September 2008

Cdc25A propels cell cycle progression, is overexpressed in numerous human cancers, and possesses oncogenic and antiapoptotic activities. Reactive oxygen species, such as hydrogen peroxide, regulate Cdc25A, but the physiologic and pathologic effects of nitric oxide (•NO) and •NO-derived reactive species are not well defined. Herein, we report novel independent mechanisms governing Cdc25A in response to nitrosative insult. We observed direct and rapid inhibition of Cdc25A phosphatase activity after in vitro treatment with the low molecular mass cell-permeable S-nitrosothiol S-nitrosocysteine ethyl ester (SNCEE). In addition, treatment of cancer cells with SNCEE induced nitrosative stress and decreased Cdc25A protein levels in a time-dependent and concentration-dependent manner. Similarly, iNOS-derived •NO was sufficient to suppress Cdc25A expression, consistent with its role in mediating nitrosative stress. Whereas a decrease in Cdc25A half-life was not observed in response to SNCEE, we found the translational regulator eukaryotic initiation factor 2α (eIF2α) was hyperphosphorylated and total protein translation was decreased with kinetics consistent with Cdc25A loss. Inhibition of eIF2α decreased Cdc25A levels, supporting the hypothesis that SNCEE suppressed Cdc25A translation through inhibition of eIF2α. Nitrosative stress decreased the Cdc25A-bound fraction of apoptosis signal-regulating kinase-1 (ASK-1) and sensitized cells to apoptosis induced by the ASK-1–activating chemotherapeutic cis-diaminedichloroplatinum (II), suggesting that nitrosative stress–induced suppression of Cdc25A primed cells for ASK-1–dependent apoptosis. Together these data reveal novel •NO-dependent enzymatic and translational mechanisms controlling Cdc25A, and implicate Cdc25A as a mediator of •NO-dependent apoptotic signaling. [Cancer Res 2008;68(18):7457–65]
Quantitative Molecular Magnetic Resonance Imaging of Tumor Angiogenesis Using cNGR-Labeled Paramagnetic Quantum Dots
Authors: Oostendorp Marlies , Douma Kim , Hackeng Tilman M. , Dirksen Anouk , Post Mark J. , van Zandvoort Marc A.M.J. , Backes Walter H.
Published: September 2008

The objective of this study was to develop and apply cyclic Asn-Gly-Arg (cNGR)-labeled paramagnetic quantum dots (cNGR-pQDs) for the noninvasive assessment of tumor angiogenic activity using quantitative in vivo molecular magnetic resonance imaging (MRI). cNGR was previously shown to colocalize with CD13, an aminopeptidase that is highly overexpressed on angiogenic tumor endothelium. Because angiogenesis is important for tumor growth and metastatization, its in vivo detection and quantification may allow objective diagnosis of tumor status and evaluation of treatment response. I.v. injection of cNGR-pQDs in tumor-bearing mice resulted in increased quantitative contrast, comprising increased longitudinal relaxation rate and decreased proton visibility, in the tumor rim but not in tumor core or muscle tissue. This showed that cNGR-pQDs allow in vivo quantification and accurate localization of angiogenic activity. MRI results were validated using ex vivo two-photon laser scanning microscopy (TPLSM), which showed that cNGR-pQDs were primarily located on the surface of tumor endothelial cells and to a lesser extent in the vessel lumen. In contrast, unlabeled pQDs were not or only sparsely detected with both MRI and TPLSM, supporting a high specificity of cNGR-pQDs for angiogenic tumor vasculature. [Cancer Res 2008;68(18):7676–83]
CYR61 and αVβ5 Integrin Cooperate to Promote Invasion and Metastasis of Tumors Growing in Preirradiated Stroma
Authors: Monnier Yan , Farmer Pierre , Bieler Gregory , Imaizumi Natsuko , Sengstag Thierry , Alghisi Gian Carlo , Stehle Jean-Christophe , Ciarloni Laura , Andrejevic-Blant Snezana , Moeckli Raphael , Mirimanoff René-Olivier , Goodman Simon L. , Delorenzi Mauro , Rüegg Curzio
Published: September 2008

Radiotherapy is widely used to treat human cancer. Patients locally recurring after radiotherapy, however, have increased risk of metastatic progression and poor prognosis. The clinical management of postradiation recurrences remains an unresolved issue. Tumors growing in preirradiated tissues have an increased fraction of hypoxic cells and are more metastatic, a condition known as tumor bed effect. The transcription factor hypoxia inducible factor (HIF)-1 promotes invasion and metastasis of hypoxic tumors, but its role in the tumor bed effect has not been reported. Here, we show that tumor cells derived from SCCVII and HCT116 tumors growing in a preirradiated bed, or selected in vitro through repeated cycles of severe hypoxia, retain invasive and metastatic capacities when returned to normoxia. HIF activity, although facilitating metastatic spreading of tumors growing in a preirradiated bed, is not essential. Through gene expression profiling and gain- and loss-of-function experiments, we identified the matricellular protein CYR61 and αVβ5 integrin as proteins cooperating to mediate these effects. The anti-αV integrin monoclonal antibody 17E6 and the small molecular αVβ3/αVβ5 integrin inhibitor EMD121974 suppressed invasion and metastasis induced by CYR61 and attenuated metastasis of tumors growing within a preirradiated field. These results represent a conceptual advance to the understanding of the tumor bed effect and identify CYR61 and αVβ5 integrin as proteins that cooperate to mediate metastasis. They also identify αV integrin inhibition as a potential therapeutic approach for preventing metastasis in patients at risk for postradiation recurrences. [Cancer Res 2008;68(18):7323–31]
Epigenetic Silencing of Cell Adhesion Molecule 1 in Different Cancer Progenitor Cells of Transgenic c-Myc and c-Raf Mouse Lung Tumors
Authors: Reamon-Buettner Stella Marie , Borlak Juergen
Published: September 2008

Understanding molecular mechanisms underlying lung cancer is a prerequisite toward treatment. To enable mechanistic investigations into the epigenetic regulation of the tumor suppressor gene cell adhesion molecule 1 (Cadm1) in lung cancer progenitor cells, we developed 10 cell lines from single, spontaneously transformed lung tumor cells isolated from c-Myc and c-Raf double-transgenic mice. Specifically, we investigated Cadm1 promoter hypermethylation, which was significantly induced in transgenic transformed cells. Analysis of 69 CpGs displayed differential methylation pattern between and within progenitor cell lines, and the degree of methylation correlated well with transcriptional repression. Indeed, restoration of Cadm1 gene expression was achieved by treatment with the experimental demethylating drug 5-aza-2′-deoxycytidine. Furthermore, methylation of core CpGs in the binding sites of Sp1, Sp3, and zinc finger 5 along the promoter region of Cadm1 abrogated DNA-protein binding. Treatment with mithramycin A, an inhibitor of Sp1 or Sp3 binding, resulted in reduction of Cadm1 gene expression, therefore suggesting a potential role of Sp1/Sp3 in Cadm1 regulation. Identifying molecular rules for the epigenetic control of tumor suppressor genes enables mechanistic insights into lung cancer growth and opportunities for novel therapies. [Cancer Res 2008;68(18):7587–96]
Prevention of Both Direct and Cross-Priming of Antitumor CD8+ T-Cell Responses following Overproduction of Prostaglandin E2 by Tumor CellsIn vivo
Authors: Ahmadi Maryam , Emery David C. , Morgan David J.
Published: September 2008

Defects in antitumor immune responses have been associated with increased release of prostaglandin E2 (PGE2) as a result of overexpression of cyclooxygenase (COX)-2 by tumors. In this report, we examine the effects of PGE2 on antitumor CD8+ T-cell responses generated both by cross-presenting dendritic cells and by direct priming by tumor cells. Our data show that PGE2 inhibits dendritic cell maturation, resulting in the abortive activation of naive CD8+ T cells, and is dependent on interleukin-10 production by dendritic cells. Interaction of tumor cells with naïve CD8+ T cells in the presence of PGE2 in vitro results in the induction of CD8+ CD28− T cells, which fail to proliferate or exhibit effector function. In vivo, overexpression of COX-2 by tumor cells results in a decrease in number of tumor-infiltrating dendritic cells and confers the ability of tumor cells to metastasize to the tumor draining lymph nodes. [Cancer Res 2008;68(18):7520–9]
Functional Characterization of Filamin A Interacting Protein 1–Like, a Novel Candidate for Antivascular Cancer Therapy
Authors: Kwon Mijung , Hanna Engy , Lorang Dominique , He Mei , Quick John S. , Adem Asha , Stevenson Christina , Chung Joon-Yong , Hewitt Stephen M. , Zudaire Enrique , Esposito Dominic , Cuttitta Frank , Libutti Steven K.
Published: September 2008

Inhibiting angiogenesis has become a major therapeutic strategy for cancer treatment. To identify common intracellular mediators, we previously analyzed gene expression profiles of endothelial cells after treatment with angiogenesis inhibitors. Filamin A interacting protein 1-like (FILIP1L; previously known as down-regulated in ovarian cancer 1) was identified as one of the genes up-regulated in endothelial cells in response to these inhibitors. However, the expression and function of FILIP1L protein is uncharacterized. Here, we provide the first description of the expression and specific subcellular localization of FILIP1L protein in human tissue. Overexpression of FILIP1L resulted in inhibition of cell proliferation and migration and increased apoptosis. In addition, overexpression of FILIP1L truncation mutants showed differential antiproliferative activity. A COOH terminal truncation mutant (FILIP1LΔC103) was more potent than wild-type FILIP1L in mediating this activity. Targeted expression of FILIP1LΔC103 in tumor vasculature inhibited tumor growth in vivo. Overall, these findings suggest that the novel protein FILIP1L may be an important mediator of the effects of angiogenesis inhibitors and that FILIP1L has the potential to be an antivascular reagent for cancer therapy. [Cancer Res 2008;68(18):7332–41]
Regulation of Cyclin D1 RNA Stability by SNIP1
Authors: Bracken Cameron P. , Wall Steven J. , Barré Benjamin , Panov Kostya I. , Ajuh Paul M. , Perkins Neil D.
Published: September 2008

Cyclin D1 expression represents one of the key mitogen-regulated events during the G1 phase of the cell cycle, whereas Cyclin D1 overexpression is frequently associated with human malignancy. Here, we describe a novel mechanism regulating Cyclin D1 levels. We find that SNIP1, previously identified as a regulator of Cyclin D1 expression, does not, as previously thought, primarily function as a transcriptional coactivator for this gene. Rather, SNIP1 plays a critical role in cotranscriptional or posttranscriptional Cyclin D1 mRNA stability. Moreover, we show that the majority of nucleoplasmic SNIP1 is present within a previously undescribed complex containing SkIP, THRAP3, BCLAF1, and Pinin, all proteins with reported roles in RNA processing and transcriptional regulation. We find that this complex, which we have termed the SNIP1/SkIP–associated RNA-processing complex, is coordinately recruited to both the 3′ end of the Cyclin D1 gene and Cyclin D1 RNA. Significantly, SNIP1 is required for the further recruitment of the RNA processing factor U2AF65 to both the Cyclin D1 gene and RNA. This study shows a novel mechanism regulating Cyclin D1 expression and offers new insight into the role of SNIP1 and associated proteins as regulators of proliferation and cancer. [Cancer Res 2008;68(18):7621–8]
Development of Resistance to Targeted Therapies Transforms the Clinically Associated Molecular Profile Subtype of Breast Tumor Xenografts
Authors: Creighton Chad J. , Massarweh Suleiman , Huang Shixia , Tsimelzon Anna , Hilsenbeck Susan G. , Osborne C. Kent , Shou Jiang , Malorni Luca , Schiff Rachel
Published: September 2008

The effectiveness of therapies targeting specific pathways in breast cancer, such as the estrogen receptor or HER2, is limited because many tumors manifest resistance, either de novo or acquired, during the course of treatment. To investigate molecular mechanisms of resistance, we used two xenograft models of estrogen receptor–positive (ER+) breast cancer, one with and one without HER2 overexpression (MCF7/HER2-18 and MCF7 wt, respectively). Mice with established tumors were assigned to the following treatment groups: estrogen supplementation (E2), estrogen deprivation (ED), ED plus tamoxifen (Tam), all with or without the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (G). Another group received ED plus the antiestrogen fulvestrant (MCF7 wt only). Tumors with acquired or de novo resistance to these endocrine therapies were profiled for gene expression and compared with tumors in the E2 control group. One class of genes underexpressed in endocrine-resistant tumors (relative to E2-treated tumors) were estrogen inducible in vitro and associated with ER+ human breast cancers (luminal subtype). Another class of genes overexpressed in tumors with acquired resistance in both models represented transcriptional targets of HER2 signaling and was associated with ER−/HER2+ human cancers (ERBB2+ subtype). A third class of genes overexpressed in MCF7/HER2-18 tumors exhibiting de novo resistance to tamoxifen was associated with ER+ human cancers but not with estrogen-regulated genes. Thus, in response to various endocrine therapy regimens, these xenograft breast tumors shut down classic estrogen signaling and activate alternative pathways such as HER2 that contribute to treatment resistance. Over time, the molecular phenotype of breast cancer can change. [Cancer Res 2008;68(18):7493–501]
Identification of a Small Molecule with Synthetic Lethality for K-Ras and Protein Kinase C Iota
Authors: Guo Wei , Wu Shuhong , Liu Jinsong , Fang Bingliang
Published: September 2008

K-Ras mutations are frequently found in various cancers and are associated with resistance to treatment or poor prognosis. Similarly, poor outcomes have recently been observed in cancer patients with overexpression of protein kinase C iota (PKCι), an atypical protein kinase C that is activated by oncogenic Ras protein and is required for K-Ras–induced transformation and colonic carcinogenesis in vivo. Thus far, there is no effective agent for treatment of cancers with K-Ras mutations or PKCι overexpression. By synthetic lethality screening, we identified a small compound (designated oncrasin-1) that effectively kills various human lung cancer cells with K-Ras mutations at low or submicromolar concentrations. The cytotoxic effects correlated with apoptosis induction, as was evidenced by increase of apoptotic cells and activation of caspase-3 and caspase-8 upon the treatment of oncrasin-1 in sensitive cells. Treatment with oncrasin-1 also led to abnormal aggregation of PKCι in the nucleus of sensitive cells but not in resistant cells. Furthermore, oncrasin-1–induced apoptosis was blocked by siRNA of K-Ras or PKCι, suggesting that oncrasin-1 is targeted to a novel K-Ras/PKCι pathway. The in vivo administration of oncrasin-1 suppressed the growth of K-ras mutant human lung tumor xenografts by >70% and prolonged the survival of nude mice bearing these tumors, without causing detectable toxicity. Our results indicate that oncrasin-1 or its active analogues could be a novel class of anticancer agents, which effectively kill K-Ras mutant cancer cells. [Cancer Res 2008;68(18):7403–8]
CCN5/WISP-2 Expression in Breast Adenocarcinoma Is Associated with Less Frequent Progression of the Disease and Suppresses the Invasive Phenotypes of Tumor Cells
Authors: Banerjee Snigdha , Dhar Gopal , Haque Inamul , Kambhampati Suman , Mehta Smita , Sengupta Krishanu , Tawfik Ossama , Phillips Teresa A. , Banerjee Sushanta K.
Published: September 2008

Although previous in vitro studies predicted that CCN5/WISP-2 may act as an anti-invasive gene in breast cancer, the distribution pattern of CCN5 in breast cancer samples is conflicting. Thus, we systematically investigated the CCN5 expression profile in noninvasive and invasive breast tumor samples and its functional relevance in breast cancer progression. The studies showed that CCN5 expression is biphasic, such that in normal samples CCN5 expression is undetectable, whereas its expression is markedly increased in noninvasive breast lesions, including atypical ductal hyperplasia and ductal carcinoma in situ. Further, CCN5 mRNA and protein levels are significantly reduced as the cancer progresses from a noninvasive to invasive type. Additionally, we showed that CCN5 mRNA and protein level was almost undetectable in poorly differentiated cancers compared with the moderately or well-differentiated samples and its expression inversely correlated with lymph node positivity. The result was further supported by evaluating the RNA expression profile in microdissected sections using real-time PCR analysis. Therefore, our data suggest a protective function of CCN5 in noninvasive breast tumor cells. This hypothesis was further supported by our in vitro studies illuminating that CCN5 is a negative regulator of migration and invasion of breast cancer cells, and these events could be regulated by CCN5 through the modulation of the expression of genes essential for an invasive front. These include Snail-E-cadherin signaling and matrix metalloproteinase (MMP)-9 and MMP-2. Collectively, these studies suggest that the protective effect of CCN5 in breast cancer progression may have important therapeutic implications. [Cancer Res 2008;68(18):7606–12]
DNA Vaccination Controls Her-2+ Tumors that Are Refractory to Targeted Therapies
Authors: Whittington Paula J. , Piechocki Marie P. , Heng Henry H. , Jacob Jennifer B. , Jones Richard F. , Back Jessica B. , Wei Wei-Zen
Published: September 2008

Her-2/neu+ tumor cells refractory to antibody or receptor tyrosine kinase inhibitors are emerging in treated patients. To investigate if drug resistant tumors can be controlled by active vaccination, gefitinib and antibody sensitivity of four neu+ BALB/c mouse mammary tumor lines were compared. Significant differences in cell proliferation and Akt phosphorylation were observed. Treatment-induced drug resistance was associated with increased chromosomal aberrations as shown by spectral karyotyping analysis, suggesting changes beyond neu signaling pathways. When mice were immunized with pneuTM encoding the extracellular and transmembrane domains of neu, antibody and T-cell responses were induced, and both drug-sensitive and drug-resistant tumor cells were rejected. In T-cell–depleted mice, drug-sensitive tumors were still rejected by vaccination, but drug-refractory tumors survived in some mice, indicating their resistance to anti-neu antibodies. To further test if T cells alone can mediate tumor rejection, mice were immunized with pcytneu encoding full-length cytoplasmic neu that is rapidly degraded by the proteasome to activate CD8 T cells without inducing antibody response. All test tumors were rejected in pcytneu-immunized mice, regardless of their sensitivity to gefitinib or antibody. Therefore, cytotoxic T lymphocytes activated by the complete repertoire of neu epitopes were effective against all test tumors. These results warrant Her-2 vaccination whether tumor cells are sensitive or resistant to Her-2–targeted drugs or antibody therapy. [Cancer Res 2008;68(18):7502–11]
Enhancing Mammalian Target of Rapamycin (mTOR)–Targeted Cancer Therapy by Preventing mTOR/Raptor Inhibition-Initiated, mTOR/Rictor-Independent Akt Activation
Authors: Wang Xuerong , Yue Ping , Kim Young Ae , Fu Haian , Khuri Fadlo R. , Sun Shi-Yong
Published: September 2008

It has been shown that mammalian target of rapamycin (mTOR) inhibitors activate Akt while inhibiting mTOR signaling. However, the underlying mechanisms and the effect of the Akt activation on mTOR-targeted cancer therapy are unclear. The present work focused on addressing the role of mTOR/rictor in mTOR inhibitor-induced Akt activation and the effect of sustained Akt activation on mTOR-targeted cancer therapy. Thus, we have shown that mTOR inhibitors increase Akt phosphorylation through a mechanism independent of mTOR/rictor because the assembly of mTOR/rictor was inhibited by mTOR inhibitors and the silencing of rictor did not abrogate mTOR inhibitor-induced Akt activation. Moreover, Akt activation during mTOR inhibition is tightly associated with development of cell resistance to mTOR inhibitors. Accordingly, cotargeting mTOR and phosphatidylinositol 3-kinase/Akt signaling prevents mTOR inhibition-initiated Akt activation and enhances antitumor effects both in cell cultures and in animal xenograft models, suggesting an effective cancer therapeutic strategy. Collectively, we conclude that inhibition of the mTOR/raptor complex initiates Akt activation independent of mTOR/rictor. Consequently, the sustained Akt activation during mTOR inhibition will counteract the anticancer efficacy of the mTOR inhibitors. [Cancer Res 2008;68(18):7409–18]
Glucocorticoids Antagonize Estrogens by Glucocorticoid Receptor–Mediated Activation of Estrogen Sulfotransferase
Authors: Gong Haibiao , Jarzynka Michael J. , Cole Timothy J. , Lee Jung Hoon , Wada Taira , Zhang Bin , Gao Jie , Song Wen-Chao , DeFranco Donald B. , Cheng Shi-Yuan , Xie Wen
Published: September 2008

Glucocorticoids and estrogens are two classes of steroid hormones that have essential but distinct physiologic functions. Estrogens also represent a risk factor for breast cancer. It has been suggested that glucocorticoids can attenuate estrogen responses, but the mechanism by which glucocorticoids inhibit estrogenic activity is unknown. In this study, we show that activation of glucocorticoid receptor (GR) by dexamethasone (DEX) induced the expression and activity of estrogen sulfotransferase (SULT1E1 or EST), an enzyme important for the metabolic deactivation of estrogens, because sulfonated estrogens fail to activate the estrogen receptor. Treatment with DEX lowered circulating estrogens, compromised uterine estrogen responses, and inhibited estrogen-dependent breast cancer growth in vitro and in a xenograft model. We further showed that the mouse and human SULT1E1 genes are transcriptional targets of GR and deletion of Sult1e1/Est in mice abolished the DEX effect on estrogen responses. These findings have revealed a novel nuclear receptor–mediated and metabolism-based mechanism of estrogen deprivation, which may have implications in therapeutic development for breast cancers. Because glucocorticoids and estrogens are widely prescribed drugs, our results also urge caution in avoiding glucocorticoid-estrogen interactions in patients. [Cancer Res 2008;68(18):7386–93]
Systemic Targeting of CpG-ODN to the Tumor Microenvironment with Anti–neu-CpG Hybrid Molecule and T Regulatory Cell Depletion Induces Memory Responses in BALB-neuT Tolerant Mice
Authors: Sharma Sanjay , Dominguez Ana Lucia , Manrique Soraya Zorro , Cavallo Federica , Sakaguchi Shimon , Lustgarten Joseph
Published: September 2008

We have shown that neu transgenic mice are immunotolerant and that immunizations with dendritic cells (DC) pulsed with neu-derived antigens were not able to control tumor growth in these animals. We tested whether, by modulating the tumor microenvironment with Toll-like receptor ligands, it could be possible to induce the activation of antitumor responses in neu mice. Our results indicate that only intratumoral (i.t.) injections of CpG-ODN induce an antitumor response in neu mice. To target the CpG-ODN to the tumor site anywhere within the body, we chemically conjugated an anti–Her-2/neu monoclonal antibody (mAb) with CpG-ODN. The anti–neu-CpG hybrid molecule retained its ability to bind to Her-2/neu+ tumors, activate DCs, and induce antitumor responses. Our results indicated that injections of anti–neu-CpG induced the rejection of primary tumors in 100% of BALB/c mice and only in ∼30% of BALB-neuT mice. After challenging the BALB/c and BALB-neuT mice, we observed that BALB/c mice developed a protective memory response; in contrast, BALB-neuT mice succumbed to the challenge. After injections of anti–neu-CpG, T regulatory cells (T-reg) were drastically reduced at the tumor site, but a large number were still present in the lymphoid organs. When BALB-neuT mice were treated with anti–neu-CpG plus anti-GITR mAb, but not with anti-CD25 mAb, 100% of the BALB-neuT mice rejected the primary tumor and developed a protective memory response indicating the critical role of T-regs in regulating the repertoire against self antigens. Taken together, these results indicate that CpG-ODN–targeted therapy and depletion of T-regs optimally activate a primary response and generate a protective memory response against self-tumor antigens. [Cancer Res 2008;68(18):7530–40]
A Fluorescence In situ Hybridization Screen for E26 Transformation–Specific Aberrations: Identification of DDX5-ETV4 Fusion Protein in Prostate Cancer
Authors: Han Bo , Mehra Rohit , Dhanasekaran Saravana M. , Yu Jindan , Menon Anjana , Lonigro Robert J. , Wang Xiaosong , Gong Yusong , Wang Lei , Shankar Sunita , Laxman Bharathi , Shah Rajal B. , Varambally Sooryanarayana , Palanisamy Nallasivam , Tomlins Scott A. , Kumar-Sinha Chandan , Chinnaiyan Arul M.
Published: September 2008

Recurrent gene fusions involving E26 transformation–specific (ETS) transcription factors ERG, ETV1, ETV4, or ETV5 have been identified in 40% to 70% of prostate cancers. Here, we used a comprehensive fluorescence in situ hybridization (FISH) split probe strategy interrogating all 27 ETS family members and their five known 5′ fusion partners in a cohort of 110 clinically localized prostate cancer patients. Gene rearrangements were only identified in ETS genes that were previously implicated in prostate cancer gene fusions including ERG, ETV1, and ETV4 (43%, 5%, and 5%, respectively), suggesting that a substantial fraction of prostate cancers (estimated at 30–60%) cannot be attributed to an ETS gene fusion. Among the known 5′ gene fusion partners, TMPRSS2 was rearranged in 47% of cases followed by SLC45A3, HNRPA2B1, and C15ORF21 in 2%, 1%, and 1% of cases, respectively. Based on this comprehensive FISH screen, we have made four noteworthy observations. First, by screening the entire ETS transcription factor family for rearrangements, we found that a large fraction of prostate cancers (44%) cannot be ascribed to an ETS gene fusion, an observation which will stimulate research into identifying recurrent non-ETS aberrations in prostate cancers. Second, we identified SLC45A3 as a novel 5′ fusion partner of ERG; previously, TMPRSS2 was the only described 5′ partner of ERG. Third, we identified two prostate-specific, androgen-induced genes, FLJ35294 and CANT1, as 5′ partners to ETV1 and ETV4. Fourth, we identified a ubiquitously expressed, androgen-insensitive gene, DDX5, fused in frame with ETV4, leading to the expression of a DDX5-ETV4 fusion protein. [Cancer Res 2008;68(18):7629–37]
Gene Expression Profile and Angiogenic Markers Correlate with Response to Neoadjuvant Bevacizumab Followed by Bevacizumab plus Chemotherapy in Breast Cancer
Authors: Yang Sherry X. , Steinberg Seth M. , Nguyen Dat , Wu Thomas D. , Modrusan Zora , Swain Sandra M.
Published: September 2008

Purpose: To identify biomarkers and gene expression profile signatures to distinguish patients with partial response (PR) from those with stable disease (SD) and progressive disease (PD).

Experimental Design: Twenty patients with inflammatory breast cancer and one patient with locally advanced breast cancer received one cycle of bevacizumab followed by six cycles of bevacizumab plus docetaxel-doxorubicin before surgery. Baseline angiogenic/tumor markers were examined by immunohistochemistry and gene expression profiles were measured by Agilent Whole Human Genome arrays. All were assessed for clinical response.

Results: Fourteen patients (67%, 95% confidence interval, 43-85.4%) had PR, five had SD, and two had PD. Expression of CD31 and platelet-derived growth factor receptor-β (PDGFR-β) in the tumor vasculature by immunohistochemistry was significantly associated with response (PR versus SD/PD; CD31 median, 33.5 versus 13.2; P = 0.0004; PDGFR-β median, 5.9 versus 0.6; P = 0.01). Tumor VEGF-A showed a trend towards association with response (2.65 versus 0.25; P = 0.04). pVEGFR2(Y996), pVEGFR2(Y951), MVD, Ki67, apoptosis, grade, ER, HER-2/neu, and p53 were not associated with response. Twenty-six of 1,339 Gene Ontology (GO) classes at the gene transcriptional level were differentially expressed between patients with PR and SD/PD (P < 0.005). Representative significant GO classes include spindle (11 genes; P = 0.001), vascular endothelial growth factor receptor activity including PDGFR-β (5 genes; P = 0.002), and cell motility including CD31 (80 genes; P = 0.005).

Conclusions: Baseline CD31, PDGFR-β, and GO classes for vascular endothelial growth factor receptor activity and mitosis were significantly associated with response to bevacizumab followed by bevacizumab plus chemotherapy.
Impaired Tamoxifen Metabolism Reduces Survival in Familial Breast Cancer Patients
Authors: Newman William G. , Hadfield Kristen D. , Latif Ayshe , Roberts Stephen A. , Shenton Andrew , McHague Christopher , Lalloo Fiona , Howell Sacha , Evans D. Gareth
Published: September 2008

Purpose: Tamoxifen has been the mainstay adjuvant hormonal treatment for breast cancer for many years. Conversion of tamoxifen to its active metabolite, endoxifen, is reduced by low activity of the cytochrome P450 enzyme, CYP2D6. We examined the effect of reduced CYP2D6 activity on the response to tamoxifen in patients with familial early-onset breast cancer.

Experimental Design: We conducted a case note review and genotyping for the CYP2D6*3, CYP2D6*4, CYP2D6*5, and CYP2D6*41 alleles in 115 patients (47 BRCA1, 68 BRCA2) with familial breast cancer who had been treated with 20-mg tamoxifen following surgery.

Results: Eight (7%) individuals had genotypes consistent with poor metabolizer status, and 4 (3.5%) individuals took CYP2D6 inhibitor drugs concomitant with their tamoxifen and were also considered poor metabolizer. Time to tumor recurrence, disease-free survival, and overall survival were reduced in the patient group with poor metabolizer CYP2D6 activity. However, a significant effect was confined to patients with BRCA2 mutations with a worse overall survival (median survival, 7 versus 28 years; P = 0.008; adjusted hazard ratio, 9.7).

Conclusions: Poor metabolizer status for CYP2D6 predicts worse overall survival in patients with familial breast cancer. Therefore, CYP2D6 inhibitor drugs should not be prescribed concomitantly with tamoxifen. Prospective studies should be undertaken to establish the effect of CYP2D6 status on outcome in familial breast cancer patients treated with tamoxifen.
Monitoring Caspase-3 Activation with a Multimodality Imaging Sensor in Living Subjects
Authors: Ray Pritha , De Abhijit , Patel Manishkumar , Gambhir Sanjiv Sam
Published: September 2008

Purpose: Capsase-3 plays an important role in chemotherapy-induced apoptosis in many cancers. Herein, we applied a multimodality reporter vector to monitor caspase-3 activation indirectly in live cells and tumors of living animals undergoing apoptosis.

Experimental Design: A fusion protein (MTF) was constructed by combining three different reporter proteins, red fluorescent protein (mRFP1), firefly luciferase (FL), and HSV1-sr39 truncated thymidine kinase (TK), linked through a caspase-3 recognizable polypeptide linker. After cleavage by caspase-3, a significant gain in mRFP1, FL, and TK activity are observed by fluorescence-activated cell sorting and enzyme-based assays. A melanoma cell line (B16F10-mtf-hrl) stably expressing mtf (to measure caspase-3 activation) and hrl-IRES-gfp (to determine the decrease in a number of viable cells) vectors was generated to measure two independent molecular events upon treatment.

Results: Upon induction with 8 μmol/L staurosporine, the fusion protein showed a 2.8-fold increase in FL (P = 0.03), a 1.5-fold increase in TK (P = not significant), and a 2-fold increase in mRFP1 (P = 0.05) activity in 293T cells. Bioluminescence and micropositron emission tomography imaging of the apoptotic B16F10-mtf-hrl tumors showed a 2-fold higher FL activity (897 versus 416) and a 2-fold higher TK activity (10.3 versus 3.87) than control tumors when normalized with RL activity. Using a similar normalization approach, the time kinetics of caspase-3 activation by two protein kinase-C inhibitors was noninvasively monitored in living mice.

Conclusion: This multimodality caspase sensor vector could effectively and noninvasively monitor caspase-3 activation from single live cells to a multicellular tumor environment and, thus, would be a valuable tool for drug screening in preclinical models and future patient cell based therapy.
Randomized Study of Intravenous versus Subcutaneous Interleukin-2, and IFNα in Patients with Good Prognosis Metastatic Renal Cancer
Authors: Négrier Sylvie , Perol David , Ravaud Alain , Bay Jacques O. , Oudard Stéphane , Chabaud Sylvie , Fargeot Pierre , Delva Remy , Deplanque Gael , Gravis Gwenaelle , Escudier Bernard ,   for the French Immunotherapy Group
Published: September 2008

Purpose: Metastatic renal cancer patients with a single metastatic site are potentially amenable to interleukin 2 (IL-2) + IFN-α. A French immunotherapy intergroup multicenter trial assessed the potential benefit of i.v. over s.c. administration of IL-2 in this combination.

Experimental Design: Untreated patients with one metastatic site were randomized to continuous i.v. infusion (18 × 106 IU/m2/d; arm A) or twice daily s.c. injections (9 × 106 or 18 × 106 IU; arm B) of IL-2, associated with s.c. IFN-α (6 × 106 IU) 3 days per week in both arms. Tumor response was assessed (WHO criteria) at weeks 12 and 24 to 26. The primary end point was overall survival, with an expected 15% improvement at 4 years with i.v. IL-2. The planned sample size was 220 (80% power, 5% significance, one-sided test). Intent-to-treat analysis was done and survivals were compared using log-rank tests.

Results: From January 2000 to January 2005, 80 and 75 patients were randomized to arms A and B, respectively. Enrollment was stopped early because of low accrual; analysis was done at 42.5 months median follow-up. Patient characteristics were well balanced between groups. Response rates were 17.9% versus 21.3% in arms A and B. Progression-free survival rates were not significantly different. Overall survival difference was not significant: median 33 months (95% confidence interval, 27.0-40.2; P = 0.202).

Conclusions: In combination with IFN-α in selected, good prognosis metastatic renal cell carcinoma patients, i.v. IL-2 offers no significant advantage over s.c. IL-2 and induces higher toxicity. Although i.v. IL-2 induced longer responses, it seems unreasonable to continue recommending this regimen after the recent introduction of more effective therapies.
Predicting Clinical Outcome in Patients Diagnosed with Synchronous Ovarian and Endometrial Cancer
Authors: Ramus Susan J. , Elmasry Karim , Luo Zhiyuan , Gammerman Alex , Lu Karen , Ayhan Ayse , Singh Naveena , McCluggage W. Glenn , Jacobs Ian J. , Whittaker John C. , Gayther Simon A.
Published: September 2008

Purpose: Patients with synchronous ovarian and endometrial cancers may represent cases of a single primary tumor with metastasis (SPM) or dual primary tumors (DP). The diagnosis given will influence the patient's treatment and prognosis. Currently, a diagnosis of SPM or DP is made using histologic criteria, which are frequently unable to make a definitive diagnosis.

Experimental Design: In this study, we used genetic profiling to make a genetic diagnosis of SPM or DP in 90 patients with synchronous ovarian/endometrial cancers. We compared genetic diagnoses in these patients with the original histologic diagnoses and evaluated the clinical outcome in this series of patients based on their diagnoses.

Results: Combining genetic and histologic approaches, we were able make a diagnosis in 88 of 90 cases, whereas histology alone was able to make a diagnosis in only 64 cases. Patients diagnosed with SPM had a significantly worse survival than patients with DP (P = 0.002). Patients in which both tumors were of endometrioid histology survived longer than patients of other histologic subtypes (P = 0.025), and patients diagnosed with SPM had a worse survival if the mode of spread was from ovary to endometrium rather than from endometrium to ovary (P = 0.019).

Conclusions: Genetic analysis may represent a powerful tool for use in clinical practice for distinguishing between SPM and DP in patients with synchronous ovarian/endometrial cancer and predicting disease outcome. The data also suggest a hitherto uncharacterized level of heterogeneity in these cases, which, if accurately defined, could lead to improved treatment and survival.