bluuebunny/crossref_metadata_2025 huggingface dataset:
BCL6 Represses Smad Signaling in Transforming Growth Factor-β Resistance
Authors: Wang Degang , Long Jianyin , Dai Fangyan , Liang Min , Feng Xin-Hua , Lin Xia
Published: February 2008

Transforming growth factor-β (TGF-β) controls a wide spectrum of cellular processes. Deregulation of TGF-β signaling contributes to the pathogenesis of many diseases including cancer and autoimmune diseases. TGF-β signaling is generally mediated through intracellular signal transducers and transcription factors called Smads. Herein, we have identified the oncoprotein BCL6 as a transcriptional corepressor of tumor suppressor Smad4. BCL6 physically interacts with Smad3 and Smad4, disrupts the Smad-p300 interaction, and represses the transcriptional activity of Smad4. In accordance, B-cell lymphoma cells with a high expression level of BCL6 were found to be refractory to TGF-β antiproliferative response, whereas knockdown of BCL6 expression in B-cell lymphoma cells partially restores the TGF-β responses. This study provides strong evidence that overexpression of BCL6 contributes to TGF-β resistance in B-cell lymphoma. [Cancer Res 2008;68(3):783–9]
CCN3/Nephroblastoma Overexpressed Matricellular Protein Regulates Integrin Expression, Adhesion, and Dissemination in Melanoma
Authors: Vallacchi Viviana , Daniotti Maria , Ratti Francesca , Di Stasi Delia , Deho Paola , De Filippo Annamaria , Tragni Gabrina , Balsari Andrea , Carbone Antonino , Rivoltini Licia , Parmiani Giorgio , Lazar Noureddine , Perbal Bernard , Rodolfo Monica
Published: February 2008

CCN3/nephroblastoma overexpressed belongs to the CCN family of genes that encode secreted proteins associated with the extracellular matrix (ECM) and exert regulatory effects at the cellular level. Overexpression of CCN3 was shown in metastatic melanoma cells compared with cells of the primary tumor from the same patient. Analysis of short-term cultures from 50 primary and metastatic melanomas revealed a heterogeneous expression pattern of both the 46-kDa full-length cytoplasmic/secreted protein and the 32-kDa nuclear-truncated form. The different protein expression patterns were not associated with gene alterations or polymorphisms. Like the metastatic cells expressing high levels of the 46-kDa CCN3, cells transfected to overexpress CCN3 showed increased adhesion to ECM proteins, whereas inhibition of CCN3 expression by small interfering RNA decreased adhesion to laminin and vitronectin. CCN3 overexpression induced increased expression of laminin and vitronectin integrin receptors α7β1 and αvβ5 by increasing their mRNA production. Moreover, CCN3 secreted by melanoma cells acted as an adhesion matrix protein for melanoma cells themselves. Analysis of CCN3 protein expression with respect to melanoma progression detected the protein in all visceral metastases tested and in most nodal metastases from relapsing patients but in only a few nodal metastases from nonrelapsing patients and cutaneous metastases. Consistently, xenotransplantation in immunodeficient mice showed a higher metastatic potential of melanoma cells overexpressing CCN3. Together, these data indicate a role for CCN3 in melanoma cell interaction with the ECM by regulating integrin expression, resulting in altered cell adhesion and leading melanoma progression to aggressive disease. [Cancer Res 2008;68(3):715–23]
Modulation of Oncogenic Phenotype in Human Glioma Cells by Cytomegalovirus IE1–Mediated Mitogenicity
Authors: Cobbs Charles S. , Soroceanu Liliana , Denham Scott , Zhang Wenyue , Kraus Matthias H.
Published: February 2008

Recent evidence indicates that human cytomegalovirus (HCMV) infection occurs in a high percentage of human malignant gliomas in vivo, as the HCMV immediate early-1 (IE1) protein is detected in >90% of these tumors. The HCMV IE1 protein is essential for viral infection and has potent trans-activating and oncomodulatory properties. To investigate a potential role of HCMV in glioma biology, we stably expressed the HCMV IE1 gene product in immortalized and malignant human glial cells. Here we show that stable IE1 expression can differentially affect the growth of human glioblastoma cells, resulting in either growth proliferation or arrest. IE1 expression led to dysregulation of phosphatidylinositol 3-kinase/AKT activity, Rb phosphorylation, and expression of the p53 family of proteins. In U87 and U118 glioblastoma cells, IE1 induced cellular proliferation paralleled by reduction in steady-state expression level of Rb and p53 family proteins (including p53, p63, or p73) and simultaneous induction of the phosphatidylinositol 3-kinase/AKT signaling pathway. In contrast, IE1 expression in LN229 and U251 glioblastoma cells and immortalized human astrocytes was associated with increased expression of p53 family proteins, accompanied by growth arrest or lack of enhanced proliferation. Moreover, IE1 promoted cell cycle entry and DNA synthesis of human glioma cells on both stable expression in tumor-derived cell lines as well as transient expression in primary glioblastoma cells. These findings indicate that HCMV IE1 can significantly affect important oncogenic signaling pathways in glioblastoma cells. [Cancer Res 2008;68(3):724–30]
In vivo Switching of Human Melanoma Cells between Proliferative and Invasive States
Authors: Hoek Keith S. , Eichhoff Ossia M. , Schlegel Natalie C. , Döbbeling Udo , Kobert Nikita , Schaerer Leo , Hemmi Silvio , Dummer Reinhard
Published: February 2008

Metastatic melanoma represents a complex and heterogeneous disease for which there are no therapies to improve patient survival. Recent expression profiling of melanoma cell lines identified two transcription signatures, respectively, corresponding with proliferative and invasive cellular phenotypes. A model derived from these findings predicts that in vivo melanoma cells may switch between these states. Here, DNA microarray–characterized cell lines were subjected to in vitro characterization before s.c. injection into immunocompromised mice. Tumor growth rates were measured and postexcision samples were assessed by immunohistochemistry to identify invasive and proliferative signature cells. In vitro tests showed that proliferative signature melanoma cells are faster growing but less motile than invasive signature cells. In vivo proliferative signature cells initiated tumor growth in 14 ± 3 days postinjection. By comparison, invasive signature cells required a significantly longer (P < 0.001) period of 59 ± 11 days. Immunohistochemistry showed that regardless of the seed cell signature, tumors showed evidence for both proliferative and invasive cell types. Furthermore, proliferative signature cell types were detected most frequently in the peripheral margin of growing tumors. These data indicate that melanoma cells undergo transcriptional signature switching in vivo likely regulated by local microenvironmental conditions. Our findings challenge previous models of melanoma progression that evoke one-way changes in gene expression. We present a new model for melanoma progression that accounts for transcription signature plasticity and provides a more rational context for explaining observed melanoma biology. [Cancer Res 2008;68(3):650–6]
ABCG2 Expression and Side Population Abundance Regulated by a Transforming Growth Factor β–Directed Epithelial-Mesenchymal Transition
Authors: Yin Liqun , Castagnino Paola , Assoian Richard K.
Published: February 2008

We describe here the regulation of ABCG2 expression and side population (SP) abundance in MCF7 human breast cancer cells. The level of ABCG2 mRNA and protein were increased in purified MCF7 SP relative to non-SP cells, and incubation with an ABCG2-specific inhibitor or ABCG2 short interfering RNA eliminated the MCF7 SP. The purified MCF7 SP could generate a heterogeneous population containing both SP and non-SP cells in culture. In vivo tumorigenicity experiments showed that the purified MCF7 SP has an increased ability to colonize the mouse mammary gland. Importantly, the MCF7 SP was depleted by a transforming growth factor-β (TGFβ)–directed epithelial-mesenchymal transition (EMT), and this effect was associated with a strong down-regulation of ABCG2 gene expression, and an increased sensitivity to mitoxantrone. ABCG2 expression and SP abundance were restored upon the removal of transforming growth factor-β and reversion of the cells to an epithelial phenotype. Knock-down of E-cadherin also reduced SP abundance, but this effect was not accompanied by the loss of ABCG2 mRNA or protein. We conclude that ABCG2 expression in MCF7 cells is regulated during an EMT, and that the EMT effect reflects posttranslational regulation of ABCG2 function by E-cadherin as well as transcriptional repression of the ABCG2 gene. [Cancer Res 2008;68(3):800–7]
Modeling Genomic Diversity and Tumor Dependency in Malignant Melanoma
Authors: Lin William M. , Baker Alissa C. , Beroukhim Rameen , Winckler Wendy , Feng Whei , Marmion Jennifer M. , Laine Elisabeth , Greulich Heidi , Tseng Hsiuyi , Gates Casey , Hodi F. Stephen , Dranoff Glenn , Sellers William R. , Thomas Roman K. , Meyerson Matthew , Golub Todd R. , Dummer Reinhard , Herlyn Meenhard , Getz Gad , Garraway Levi A.
Published: February 2008

The classification of human tumors based on molecular criteria offers tremendous clinical potential; however, discerning critical and “druggable” effectors on a large scale will also require robust experimental models reflective of tumor genomic diversity. Here, we describe a comprehensive genomic analysis of 101 melanoma short-term cultures and cell lines. Using an analytic approach designed to enrich for putative “driver” events, we show that cultured melanoma cells encompass the spectrum of significant genomic alterations present in primary tumors. When annotated according to these lesions, melanomas cluster into subgroups suggestive of distinct oncogenic mechanisms. Integrating gene expression data suggests novel candidate effector genes linked to recurrent copy gains and losses, including both phosphatase and tensin homologue (PTEN)–dependent and PTEN-independent tumor suppressor mechanisms associated with chromosome 10 deletions. Finally, sample-matched pharmacologic data show that FGFR1 mutations and extracellular signal–regulated kinase (ERK) activation may modulate sensitivity to mitogen-activated protein kinase/ERK kinase inhibitors. Genetically defined cell culture collections therefore offer a rich framework for systematic functional studies in melanoma and other tumors. [Cancer Res 2008;68(3):664–73]
Tamoxifen Resistance in Breast Tumors Is Driven by Growth Factor Receptor Signaling with Repression of Classic Estrogen Receptor Genomic Function
Authors: Massarweh Suleiman , Osborne C. Kent , Creighton Chad J. , Qin Lanfang , Tsimelzon Anna , Huang Shixia , Weiss Heidi , Rimawi Mothaffar , Schiff Rachel
Published: February 2008

Not all breast cancers respond to tamoxifen, and many develop resistance despite initial benefit. We used an in vivo model of estrogen receptor (ER)–positive breast cancer (MCF-7 xenografts) to investigate mechanisms of this resistance and develop strategies to circumvent it. Epidermal growth factor receptor (EGFR) and HER2, which were barely detected in control estrogen-treated tumors, increased slightly with tamoxifen and were markedly increased when tumors became resistant. Gefitinib, which inhibits EGFR/HER2, improved the antitumor effect of tamoxifen and delayed acquired resistance, but had no effect on estrogen-stimulated growth. Phosphorylated levels of p42/44 and p38 mitogen-activated protein kinases (both downstream of EGFR/HER2) were increased in the tamoxifen-resistant tumors and were suppressed by gefitinib. There was no apparent increase in phosphorylated AKT (also downstream of EGFR/HER2) in resistant tumors, but it was nonetheless suppressed by gefitinib. Phosphorylated insulin-like growth factor-IR (IGF-IR), which can interact with both EGFR and membrane ER, was elevated in the tamoxifen-resistant tumors compared with the sensitive group. However, ER-regulated gene products, including total IGF-IR itself and progesterone receptor, remained suppressed even at the time of acquired resistance. Tamoxifen's antagonism of classic ER genomic function was retained in these resistant tumors and even in tumors that overexpress HER2 (MCF-7 HER2/18) and are de novo tamoxifen-resistant. In conclusion, EGFR/HER2 may mediate tamoxifen resistance in ER-positive breast cancer despite continued suppression of ER genomic function by tamoxifen. IGF-IR expression remains dependent on ER but is activated in the tamoxifen-resistant tumors. This study provides a rationale to combine HER inhibitors with tamoxifen in clinical studies, even in tumors that do not initially overexpress EGFR/HER2. [Cancer Res 2008;68(3):826–33]
Tamoxifen Stimulates the Growth of Cyclin D1–Overexpressing Breast Cancer Cells by Promoting the Activation of Signal Transducer and Activator of Transcription 3
Authors: Ishii Yuki , Waxman Samuel , Germain Doris
Published: February 2008

De novo or acquired resistance to tamoxifen is a major clinical challenge for the management of estrogen receptor (ER)–positive breast cancers. Although cyclin D1 overexpression is associated with a better outcome for breast cancer patients, its overexpression is also linked to tamoxifen resistance. We previously reported that the beneficial effect of cyclin D1 correlates with its ability to repress the antiapoptotic transcription factor signal transducer and activator of transcription 3 (STAT3). In contrast, molecular pathways linking overexpression of cyclin D1 to tamoxifen resistance have not been established. In the current study, the effect of tamoxifen on the growth of genetically matched high or low cyclin D1–expressing breast cancer cells was characterized and the interactions between cyclin D1, ER, and STAT3 in response to tamoxifen treatment were determined. We show that repression of STAT3 by cyclin D1 inhibits cell growth on Matrigel and in tumors in vivo; however, treatment with tamoxifen abolishes cyclin D1–mediated repression of STAT3 and growth suppression. We show that tamoxifen induces a redistribution of cyclin D1 from STAT3 to the ER, which results in the activation of both STAT3 and the ER. These results offer a molecular mechanism for the dual effect of cyclin D1 overexpression in breast cancer and support the notion that the level of cyclin D1 expression and activated STAT3 are important markers to predict response to tamoxifen treatment. [Cancer Res 2008;68(3):852–60]
Epitope Landscape in Breast and Colorectal Cancer
Authors: Segal Neil H. , Parsons D. Williams , Peggs Karl S. , Velculescu Victor , Kinzler Ken W. , Vogelstein Bert , Allison James P.
Published: February 2008

The finding that individual cancers contain many mutant genes not present in normal tissues has prompted considerable interest in the cancer epitope landscape. To further understand such effects, we applied in silico–based epitope prediction algorithms and high throughput post hoc analysis to identify candidate tumor antigens. Analysis of 1,152 peptides containing missense mutations previously identified in breast and colorectal cancer revealed that individual cancers accumulate on average ∼10 and ∼7 novel and unique HLA-A*0201 epitopes, respectively, including genes implicated in the neoplastic process. These data suggest that, with appropriate manipulation of the immune system, tumor cell destruction in situ may provide a polyvalent tumor vaccine without a requirement for knowledge of the targeted antigens. [Cancer Res 2008;68(3):889–92]
Raf and MEK Protein Kinases Are Direct Molecular Targets for the Chemopreventive Effect of Quercetin, a Major Flavonol in Red Wine
Authors: Lee Ki Won , Kang Nam Joo , Heo Yong-Seok , Rogozin Evgeny A. , Pugliese Angelo , Hwang Mun Kyung , Bowden G. Tim , Bode Ann M. , Lee Hyong Joo , Dong Zigang
Published: February 2008

Considerable attention has focused on the health-promoting effects of red wine and its nonflavonoid polyphenol compound resveratrol. However, the underlying molecular mechanisms and molecular target(s) of red wine or other potentially active ingredients in red wine remain unknown. Here, we report that red wine extract (RWE) or the red wine flavonoid quercetin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation of JB6 promotion-sensitive mouse skin epidermal (JB6 P+) cells. The activation of activator protein-1 and nuclear factor-κB induced by TPA was dose dependently inhibited by RWE or quercetin treatment. Western blot and kinase assay data revealed that RWE or quercetin inhibited mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) 1 and Raf1 kinase activities and subsequently attenuated TPA-induced phosphorylation of ERK/p90 ribosomal S6 kinase. Although either RWE or quercetin suppressed Raf1 kinase activity, they were more effective in inhibiting MEK1 activity. Importantly, quercetin exerted stronger inhibitory effects than PD098059, a well-known pharmacologic inhibitor of MEK. Resveratrol did not affect either MEK1 or Raf1 kinase activity. Pull-down assays revealed that RWE or quercetin (but not resveratrol) bound with either MEK1 or Raf1. RWE or quercetin also dose dependently suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are involved in the activation of MEK/ERK signaling. Docking data suggested that quercetin, but not resveratrol, formed a hydrogen bond with the backbone amide group of Ser212, which is the key interaction for stabilizing the inactive conformation of the activation loop of MEK1. [Cancer Res 2008;68(3):946–55]
The Human Trithorax Protein hASH2 Functions as an Oncoprotein
Authors: Lüscher-Firzlaff Juliane , Gawlista Isabella , Vervoorts Jörg , Kapelle Karsten , Braunschweig Till , Walsemann Gesa , Rodgarkia-Schamberger Chantal , Schuchlautz Henning , Dreschers Stephan , Kremmer Elisabeth , Lilischkis Richard , Cerni Christa , Wellmann Axel , Lüscher Bernhard
Published: February 2008

Regulation of chromatin is an important aspect of controlling promoter activity and gene expression. Posttranslational modifications of core histones allow proteins associated with gene transcription to access chromatin. Closely associated with promoters of actively transcribed genes, trimethylation of histone H3 at lysine 4 (H3K4me3) is a core histone mark set by several protein complexes. Some of these protein complexes contain the trithorax protein ASH2 combined with the MLL oncoproteins. We identified human ASH2 in a complex with the oncoprotein MYC. This finding, together with the observation that hASH2 interacts with MLL, led us to test whether hASH2 itself is involved in transformation. We observed that hASH2 cooperates with Ha-RAS to transform primary rat embryo fibroblasts (REF). Furthermore, transformation of REFs by MYC and Ha-RAS required the presence of rAsh2. In an animal model, the hASH2/Ha-RAS–transformed REFs formed rapidly growing tumors characteristic of fibrosarcomas that, compared with tumors derived from MYC/Ha-RAS transformed cells, were poorly differentiated. This finding suggests that ASH2 functions as an oncoprotein. Although hASH2 expression at the mRNA level was generally not deregulated, hASH2 protein expression was increased in most human tumors and tumor cell lines. In addition, knockdown of hASH2 inhibited tumor cell proliferation. Taken together, these observations define hASH2 as a novel oncoprotein. [Cancer Res 2008;68(3):749–58]
Self-Tolerance Does Not Restrict the CD4+ T-Helper Response against the p53 Tumor Antigen
Authors: Lauwen Marjolein M. , Zwaveling Sander , de Quartel Linda , Ferreira Mota S. Carmela , Grashorn Janine A.C. , Melief Cornelis J.M. , van der Burg Sjoerd H. , Offringa Rienk
Published: February 2008

Tumorigenesis is frequently associated with mutation and overexpression of p53, which makes it an attractive target antigen for T cell–mediated immunotherapy of cancer. However, the magnitude and breadth of the p53-specific T-cell repertoire may be restricted due to the ubiquitous expression of wild-type p53 in normal somatic tissues. In view of the importance of the CD4+ T-helper cell responses in effective antitumor immunity, we have analyzed and compared the p53-specific reactivity of this T cell subset in p53+/+ and p53−/− C57Bl/6 mice. This response was found to be directed against the same three immunodominant epitopes in both mouse types. Fine-specificity, magnitude, and avidity were not affected by self-tolerance. Immunization of p53−/− and p53+/+ mice with synthetic peptide vaccines comprising the identified epitopes induced equal levels of Th1 immunity. Our findings imply that the p53-specific CD4+ T-cell repertoire is not restricted by self-tolerance and is fully available for the targeting of cancer. [Cancer Res 2008;68(3):893–900]
Induction of EBV–Latent Membrane Protein 1–Specific MHC Class II–Restricted T-Cell Responses against Natural Killer Lymphoma Cells
Authors: Kobayashi Hiroya , Nagato Toshihiro , Takahara Miki , Sato Keisuke , Kimura Shoji , Aoki Naoko , Azumi Makoto , Tateno Masatoshi , Harabuchi Yasuaki , Celis Esteban
Published: February 2008

EBV-encoded latent membrane protein 1 (LMP1) has oncogenic potential and is expressed in many EBV-associated malignancies. Although LMP1 is regarded as a potential tumor-associated antigen for immunotherapy and several LMP1-specific MHC class I–restricted CTL epitopes have been reported, little is known regarding MHC class II–restricted CD4 helper T-lymphocyte (HTL) epitopes for LMP1. The goal of the present studies was to determine whether MHC class II–restricted CD4 T-cell responses could be induced against the LMP1 antigen and to evaluate the antitumor effect of these responses. We have combined the use of a predictive MHC class II binding peptide algorithm with in vitro vaccination of CD4 T cells using candidate peptides to identify naturally processed epitopes derived from LMP1 that elicit immune responses against EBV-expressing tumor cells. Peptide LMP1159-175 was effective in inducing HTL responses that were restricted by HLA-DR9, HLA-DR53, or HLA-DR15, indicating that this peptide behaves as a promiscuous T-cell epitope. Moreover, LMP1159-175–reactive HTL clones directly recognized EBV lymphoblastoid B cells, EBV-infected natural killer (NK)/T-lymphoma cells and naturally processed antigen in the form of LMP1+ tumor cell lysates presented by autologous dendritic cells. Because the newly identified epitope LMP1159-175 overlaps with an HLA-A2–restricted CTL epitope (LMP1159-167), this peptide might have the ability to induce simultaneous CTL and HTL responses against LMP1. Overall, our data should be relevant for the design and optimization of T-cell epitope–based immunotherapy against various EBV-associated malignancies, including NK/T cell lymphomas. [Cancer Res 2008;68(3):901–8]
A First-Generation Multiplex Biomarker Analysis of Urine for the Early Detection of Prostate Cancer
Authors: Laxman Bharathi , Morris David S. , Yu Jianjun , Siddiqui Javed , Cao Jie , Mehra Rohit , Lonigro Robert J. , Tsodikov Alex , Wei John T. , Tomlins Scott A. , Chinnaiyan Arul M.
Published: February 2008

Although prostate-specific antigen (PSA) serum level is currently the standard of care for prostate cancer screening in the United States, it lacks ideal specificity and additional biomarkers are needed to supplement or potentially replace serum PSA testing. Emerging evidence suggests that monitoring the noncoding RNA transcript PCA3 in urine may be useful in detecting prostate cancer in patients with elevated PSA levels. Here, we show that a multiplex panel of urine transcripts outperforms PCA3 transcript alone for the detection of prostate cancer. We measured the expression of seven putative prostate cancer biomarkers, including PCA3, in sedimented urine using quantitative PCR on a cohort of 234 patients presenting for biopsy or radical prostatectomy. By univariate analysis, we found that increased GOLPH2, SPINK1, and PCA3 transcript expression and TMPRSS2:ERG fusion status were significant predictors of prostate cancer. Multivariate regression analysis showed that a multiplexed model, including these biomarkers, outperformed serum PSA or PCA3 alone in detecting prostate cancer. The area under the receiver-operating characteristic curve was 0.758 for the multiplexed model versus 0.662 for PCA3 alone (P = 0.003). The sensitivity and specificity for the multiplexed model were 65.9% and 76.0%, respectively, and the positive and negative predictive values were 79.8% and 60.8%, respectively. Taken together, these results provide the framework for the development of highly optimized, multiplex urine biomarker tests for more accurate detection of prostate cancer. [Cancer Res 2008;68(3):645–9]
Loss of Lkb1 Provokes Highly Invasive Endometrial Adenocarcinomas
Authors: Contreras Cristina M. , Gurumurthy Sushma , Haynie J. Marshall , Shirley Lane J. , Akbay Esra A. , Wingo Shana N. , Schorge John O. , Broaddus Russell R. , Wong Kwok-Kin , Bardeesy Nabeel , Castrillon Diego H.
Published: February 2008

Mutations in the LKB1 tumor suppressor gene result in the Peutz-Jeghers syndrome, an autosomal dominant condition characterized by hamartomatous polyps of the gastrointestinal tract and a dramatically increased risk of epithelial malignancies at other sites, including the female reproductive tract. Here we show that female mice heterozygous for a null Lkb1 allele spontaneously develop highly invasive endometrial adenocarcinomas. To prove that these lesions were indeed due to Lkb1 inactivation, we introduced an adenoviral Cre vector into the uterine lumen of mice harboring a conditional allele of Lkb1. This endometrial-specific deletion of the Lkb1 gene provoked highly invasive and sometimes metastatic endometrial adenocarcinomas closely resembling those observed in Lkb1 heterozygotes. Tumors were extremely well differentiated and histopathologically distinctive and exhibited alterations in AMP-dependent kinase signaling. Although Lkb1 has been implicated in the establishment of cell polarity, and loss of polarity defines most endometrial cancers, Lkb1-driven endometrial cancers paradoxically exhibit (given their highly invasive phenotype) normal cell polarity and apical differentiation. In human endometrial cancers, Lkb1 expression was inversely correlated with tumor grade and stage, arguing that Lkb1 inactivation or down-regulation also contributes to endometrial cancer progression in women. This study shows that Lkb1 plays an important role in the malignant transformation of endometrium and that Lkb1 loss promotes a highly invasive phenotype. [Cancer Res 2008;68(3):759–66]
The EGFR-STAT3 Oncogenic Pathway Up-regulates the Eme1 Endonuclease to Reduce DNA Damage after Topoisomerase I Inhibition
Authors: Vigneron Arnaud , Gamelin Erick , Coqueret Olivier
Published: February 2008

The epidermal growth factor receptor (EGFR)-src-signal transducers and activators of transcription 3 (STAT3) oncogenic pathway plays a central role in tumorigenesis and is involved not only in cell transformation but also in tumor escape to genotoxic treatments. Despite its importance, the molecular mechanisms by which this signaling pathway induces resistance to DNA damage remain most of the time to be characterized. In this study, we show that the EGFR-src pathway is activated in response to topoisomerase I inhibition. After treatment, this signaling cascade induced the activation of STAT3 and the binding of the transcription factor to the promoter of the Eme1 gene. Eme1 is an endonuclease involved in the processing of DNA damage after topoisomerase I inhibition. These results suggest a model by which the STAT3-mediated activation of Eme1 prevents DNA damage and enhances cell survival in response to topoisomerase inhibition. This survival pathway was inhibited by a combined treatment with a src inhibitor, SKI, and with cetuximab, a monoclonal antibody directed against the EGFR that is widely used in the treatment of colorectal cancers. We therefore propose that the benefit of anti-EGFR therapy relies on an increase of DNA damage generated by topoisomerase I inhibition. [Cancer Res 2008;68(3):815–25]
Aggravated Endoplasmic Reticulum Stress as a Basis for Enhanced Glioblastoma Cell Killing by Bortezomib in Combination with Celecoxib or Its Non-Coxib Analogue, 2,5-Dimethyl-Celecoxib
Authors: Kardosh Adel , Golden Encouse B. , Pyrko Peter , Uddin Jasim , Hofman Florence M. , Chen Thomas C. , Louie Stan G. , Petasis Nicos A. , Schönthal Axel H.
Published: February 2008

The proteasome inhibitor bortezomib (Velcade) is known to trigger endoplasmic reticulum (ER) stress via the accumulation of obsolete and damaged proteins. The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib (Celebrex) causes ER stress through a different mechanism (i.e., by causing leakage of calcium from the ER into the cytosol). Each of these two mechanisms has been implicated in the anticancer effects of the respective drug. We therefore investigated whether the combination of these two drugs would lead to further increased ER stress and would enhance their antitumor efficacy. With the use of human glioblastoma cell lines, we show that this is indeed the case. When combined, bortezomib and celecoxib triggered elevated expression of the ER stress markers GRP78/BiP and CHOP/GADD153, caused activation of c-Jun NH2-terminal kinase and ER stress-associated caspase-4, and greatly increased apoptotic cell death. Small interfering RNA–mediated knockdown of the protective ER chaperone GRP78/BiP further sensitized the tumor cells to killing by the drug combination. The contribution of celecoxib was independent of the inhibition of COX-2 because a non-coxib analogue of this drug, 2,5-dimethyl-celecoxib (DMC), faithfully and more potently mimicked these combination effects in vitro and in vivo. Taken together, our results show that combining bortezomib with celecoxib or DMC very potently triggers the ER stress response and results in greatly increased glioblastoma cytotoxicity. We propose that this novel drug combination should receive further evaluation as a potentially effective anticancer therapy. [Cancer Res 2008;68(3):843–51]
Targeting the Eukaryotic Translation Initiation Factor 4E for Cancer Therapy
Authors: Graff Jeremy R. , Konicek Bruce W. , Carter Julia H. , Marcusson Eric G.
Published: February 2008

The eukaryotic translation initiation factor 4E (eIF4E) is frequently overexpressed in human cancers in relation to disease progression and drives cellular transformation, tumorigenesis, and metastatic progression in experimental models. Enhanced eIF4E function results from eIF4E overexpression and/or activation of the ras and phosphatidylinositol 3-kinase/AKT pathways and selectively increases the translation of key mRNAs involved in tumor growth, angiogenesis, and cell survival. Consequently, by simultaneously and selectively reducing the expression of numerous potent growth and survival factors critical for malignancy, targeting eIF4E for inhibition may provide an attractive therapy for many different tumor types. Recent work has now shown the plausibility of therapeutically targeting eIF4E and has resulted in the advance of the first eIF4E-specific therapy to clinical trials. These studies illustrate the increased susceptibility of tumor tissues to eIF4E inhibition and support the notion that the enhanced eIF4E function common to many tumor types may represent an Achilles' heel for cancer. [Cancer Res 2008;68(3):631–4]
Cancer-Associated Stromal Fibroblasts Promote Pancreatic Tumor Progression
Authors: Hwang Rosa F. , Moore Todd , Arumugam Thiruvengadam , Ramachandran Vijaya , Amos Keith D. , Rivera Armando , Ji Baoan , Evans Douglas B. , Logsdon Craig D.
Published: February 2008

Pancreatic adenocarcinoma is characterized by a dense background of tumor associated stroma originating from abundant pancreatic stellate cells. The aim of this study was to determine the effect of human pancreatic stellate cells (HPSC) on pancreatic tumor progression. HPSCs were isolated from resected pancreatic adenocarcinoma samples and immortalized with telomerase and SV40 large T antigen. Effects of HPSC conditioned medium (HPSC-CM) on in vitro proliferation, migration, invasion, soft-agar colony formation, and survival in the presence of gemcitabine or radiation therapy were measured in two pancreatic cancer cell lines. The effects of HPSCs on tumors were examined in an orthotopic murine model of pancreatic cancer by co-injecting them with cancer cells and analyzing growth and metastasis. HPSC-CM dose-dependently increased BxPC3 and Panc1 tumor cell proliferation, migration, invasion, and colony formation. Furthermore, gemcitabine and radiation therapy were less effective in tumor cells treated with HPSC-CM. HPSC-CM activated the mitogen-activated protein kinase and Akt pathways in tumor cells. Co-injection of tumor cells with HPSCs in an orthotopic model resulted in increased primary tumor incidence, size, and metastasis, which corresponded with the proportion of HPSCs. HPSCs produce soluble factors that stimulate signaling pathways related to proliferation and survival of pancreatic cancer cells, and the presence of HPSCs in tumors increases the growth and metastasis of these cells. These data indicate that stellate cells have an important role in supporting and promoting pancreatic cancer. Identification of HPSC-derived factors may lead to novel stroma-targeted therapies for pancreatic cancer. [Cancer Res 2008;68(3):918–26]
Use of a Combination of Approaches to Identify and Validate Relevant Tumor-Associated Antigens and Their Corresponding Autoantibodies in Ovarian Cancer Patients
Authors: Gagnon Audrey , Kim Jae-Hoon , Schorge John O. , Ye Bin , Liu Brian , Hasselblatt Kathleen , Welch William R. , Bandera Christina A. , Mok Samuel C.
Published: February 2008

Purpose: Novel biomarkers are urgently needed to increase the sensitivity of CA125 for the early detection of ovarian cancer. Indeed, it has been shown that as much as 20% of early-stage patients do not express significant levels of this biomarker. Therefore, the possibility of using autoantibodies directed against tumor-associated antigens as putative cancer markers is being more examined. Indeed, many autoantibodies have recently been shown to correlate with cancer patient prognosis or to be suitable for detection of the disease.

Experimental Design: In this study, we have used a new approach involving the use of proteomics, immunology, and ELISA methods to identify relevant autoantibodies in the plasma of ovarian cancer patients. To do so, we developed an innovative technique called two-dimensional differential gel electrophoresis analysis of immunoprecipitated tumor antigens.

Results: This strategy allowed us to successfully identify novel circulating autoantibodies directed against the S100A7 protein in the plasma of ovarian cancer patients. Further real-time reverse transcription-PCR and immunohistochemical studies confirmed that the S100A7 mRNA and protein were highly expressed in ovarian tumors but absent in normal and benign tissues. Moreover, a preliminary study involving 138 patients confirmed that the plasma levels of anti-S100A7 antibodies are significantly elevated in early- and late-stage ovarian cancer patients compared with healthy controls and with patients with benign gynecologic diseases.

Conclusions: This shows that our approach is a valuable tool to successfully identify autoantibodies and tumor-associated antigens in cancer patients and that future research assessing their putative clinical usefulness would be worthwhile.
Serum C-Reactive Protein as Independent Prognostic Variable in Patients with Ovarian Cancer
Authors: Hefler Lukas A. , Concin Nicole , Hofstetter Gerda , Marth Christian , Mustea Alexander , Sehouli Jalid , Zeillinger Robert , Leipold Heinz , Lass Harald , Grimm Christoph , Tempfer Clemens B. , Reinthaller Alexander
Published: February 2008

Purpose: To evaluate serum C-reactive protein (CRP) as prognostic variable in patients with epithelial ovarian cancer (EOC).

Experimental Design: In a multicenter study, preoperative serum CRP was evaluated in 623 patients with EOC. Results were correlated with clinical data.

Results: Mean (SD) preoperative serum CRP was 3.6 (4.8) mg/dL. Serum CRP was significantly associated with International Federation of Gynecologists and Obstetricians stage (P < 0.001) and postoperative residual tumor mass (P < 0.001) but not with histologic grade (P = 0.1) and type (P = 0.7), patients' age (Pearson's correlation coefficient = 0.05; P = 0.2), and serum CA 125 (Pearson's correlation coefficient = 0.02; P = 0.6). Patients with platinum-resistant EOC had significantly higher CRP serum levels compared with patients with platinum-sensitive EOC [6.0 (6.6) mg/dL versus 2.8 (3.8) mg/dL; P < 0.001]. Higher International Federation of Gynecologists and Obstetricians stage (P < 0.001), presence of postoperative residual tumor mass (P < 0.001), tumor grade (P = 0.001), serum CA 125 (P = 0.03), and serum CRP (P = 0.001) were independently associated with overall survival. Patients with serum CRP ≤1 mg/dL versus >1 mg/dL had an overall 5-year survival of 82% versus 58.5% (P < 0.001).

Conclusion: Serum CRP can be seen as a novel, widely available independent prognostic variable of ovarian cancer.
Association between Phosphatidylinositol 3-Kinase Regulatory Subunit p85α Met326Ile Genetic Polymorphism and Colon Cancer Risk
Authors: Li Li , Plummer Sarah J. , Thompson Cheryl L. , Tucker Thomas C. , Casey Graham
Published: February 2008

Purpose: The phosphatidylinositol 3-kinase signaling pathway is frequently activated in cancer. Emerging evidence supports the p85α regulatory subunit gene, PIK3R1, as a novel oncogene.

Experimental Design: We examined the association of a functional missense polymorphism (Met326Ile) of PIK3R1 with colon cancer risk in a population-based case-control study of 421 incident cases and 483 controls.

Results: In our base unconditional logistic regression model controlling for age, gender, and race, we observed a 47% increase in risk among those carrying one or two copies of the 326Ile variant (P = 0.01). Further adjustment for family history of colorectal cancer, body mass index, nonsteroidal anti-inflammatory drugs, smoking, alcohol consumption, and physical activity strengthened the association [odds ratio (OR), 1.73; 95% confidence interval (CI), 1.24-2.42, P = 0.001]. The association was more pronounced among those older than 64 years (OR, 2.10; 95% CI, 1.19-3.70, P = 0.01). Evaluation of the genotypes assuming an additive mode of inheritance showed a significant trend for gene-dose response, where compared with Met/Met, the OR estimates for Ile/Met and Ile/Ile were 1.68 (95% CI, 1.19-2.37) and 2.27 (95% CI, 0.98-5.29), respectively (P for trend = 0.001).

Conclusions: This study is the first to describe a significant association between a germ line functional variant in PIK3R1 and cancer, providing new evidence supporting a role for PIK3R1 in the development of colon cancer.
Tumor-Infiltrated Immune Response Correlates with Alterations in the Apoptotic and Cell Cycle Pathways in Hodgkin and Reed-Sternberg Cells
Authors: Álvaro Tomás , Lejeune Marylène , García Juan F. , Salvadó Ma. Teresa , López Carlos , Bosch Ramón , Jaén Joaquín , Escrivá Patricia , Pons Lluis E.
Published: February 2008

Purpose: To analyze tumor-microenvironment relationships in Hodgkin lymphoma (HL) as potential determinants in the decision-making process related to the alterations in cell cycle and apoptotic pathways of Hodgkin/Reed-Sternberg (H/RS) cells.

Experimental Design: Based on a cohort of 257 classic HL patients, we carried out a global descriptive correlational analysis and logistic regression study to identify tumor-infiltrated immune cell rate in HL that could be interconnected with genes involved in the regulation of apoptotic/proliferative pathways in H/RS cells.

Results: Our results reveal the existence of a connection between the reactive microenvironment and molecular changes in apoptotic/proliferative pathways in H/RS cells. A lesser incidence of infiltrated cytotoxic cells in the tumor (CD8+ T lymphocytes, CD57+ natural killer, and granzyme B+ cells) was associated with overexpression of antiapoptotic proteins (Bcl-XL, survivin, caspase-3, and nuclear factor-κB) in tumoral cells. Increased incidence of general infiltrated immune cells, such as CD4+ T lymphocytes, CD57+ natural killer cells, activated CTL, and dendritic cells, in the microenvironment of the tumor was associated with increased growth fraction of tumoral cells, including G1-S checkpoint (cyclin D and cyclin E) and tumor suppressor pathways (p16 and SKP2), and with the presence of EBV (signal transducers and activators of transcription 1 and 3 expression; STAT1/STAT3).

Conclusions: A lower level of cytotoxic cells correlated with an increase of antiapoptotic mechanisms in H/RS cells, whereas the global infiltrated immune population correlated with the growth fraction of the tumor. Our collective data suggest a causal relationship between infiltrated immune response and concurrent changes of the different proliferative checkpoints, tumor suppressor, and apoptotic pathways of H/RS cells in HL.
Development of a Multiplex RNA Urine Test for the Detection and Stratification of Transitional Cell Carcinoma of the Bladder
Authors: Holyoake Andrew , O'Sullivan Paul , Pollock Rob , Best Terry , Watanabe Jun , Kajita Yoichiro , Matsui Yoshiyuki , Ito Masaaki , Nishiyama Hiroyuki , Kerr Natalie , da Silva Tatley Fernanda , Cambridge Lisa , Toro Tumi , Ogawa Osamu , Guilford Parry
Published: February 2008

Purpose: New markers that enable the percentage of transitional cell carcinomas (TCC) of the bladder that are diagnosed before invasion of the bladder muscle layers to be increased would reduce the morbidity and mortality associated with this disease. The purpose of this study was to develop a simple, accurate urine test based on mRNA markers and simple gene signatures that (a) could detect TCC before muscle invasion while maintaining high specificity in patients with hematuria or urinary tract infections and (b) identify patients most likely to have grade 3 or stage ≥T1 disease.

Experimental Design: RNA markers with high overexpression in stage Ta tumors and/or T1 to T4 tumors but low expression in blood or inflammatory cells were characterized by quantitative reverse transcription-PCR using 2 mL of voided urine from 75 TCC patients and 77 control patients with other urological diseases.

Results: A combination of the RNAs CDC2, MDK, IGFBP5, and HOXA13 detected 48%, 90%, and 100% of stage Ta, T1, and >T1 TCCs, respectively, at a specificity of 85%. Detection of Ta tumors increased to 60% for primary (non-recurrent) Ta tumors and 76% for Ta tumors ≥1 cm in diameter. Test specificity was 80% for the 20 control patients with urinary tract infections. The combination of CDC2 and HOXA13 distinguished between grade 1 to 2 TCCs and grade 3 or stage ≥T1 TCCs with ∼80% specificity and sensitivity.

Conclusions: Simple gene expression signatures can be used as urine markers for the accurate detection and characterization of bladder cancer.
Genetic and Epigenetic Alterations in the von Hippel-Lindau Gene: the Influence on Renal Cancer Prognosis
Authors: Smits Kim M. , Schouten Leo J. , van Dijk Boukje A.C. , Hulsbergen-van de Kaa Christina A. , Wouters Kim A.D. , Oosterwijk Egbert , van Engeland Manon , van den Brandt Piet A.
Published: February 2008

Background: Inactivation of the von Hippel-Lindau (VHL) gene is considered as an early event in renal cancer tumorigenesis. The prognostic relevance of these changes, however, is not clear and previous results are contradictory. We have evaluated the influence of (epi)genetic alterations in VHL on cause-specific survival in clear-cell renal cell cancer (ccRCC) in a large, population-based group of cases.

Methods: One hundred and eighty-five cases of ccRCC, identified in the Netherlands Cohort Study on diet and cancer diagnosed in the period 1986 to 1997, were included in the analyses. Mortality information until December 2005, including causes of death, were obtained for all cases through linkage with the Central Bureau of Statistics. VHL mutations were determined with PCR single-strand conformational polymorphism and direct sequencing. VHL methylation was determined with methylation-specific PCR. Kaplan-Meier analyses and Cox proportional hazards models were used to assess associations between VHL alterations and cause-specific mortality.

Results: Median follow-up in our population was 6 years. The frequency of loss of function mutations and methylation, separately or combined, did not differ statistically significant between different cancer stages or between tumors with different sizes. We observed no influence of loss of function mutations or methylation of the VHL gene on cause-specific mortality (hazard ratio, 1.08; 95% confidence interval, 0.69-1.68, P = 0.735) as compared with patients with a wild-type or silent mutation in VHL.

Discussion: Our results indicate that (epi)genetic alterations in the VHL gene do not have prognostic value in ccRCC.
Endogenous Vascular Endothelial Growth Factor-C Expression Is Associated with Decreased Drug Responsiveness in Childhood Acute Myeloid Leukemia
Authors: de Jonge Hendrik J.M. , Weidenaar Alida C. , ter Elst Arja , Boezen H. Marike , Scherpen Frank J.G. , Bouma-ter Steege Jessica C.A. , Kaspers Gertjan J.L. , Goemans Bianca F. , Creutzig Ursula , Zimmermann Martin , Kamps Willem A. , de Bont Eveline S.J.M.
Published: February 2008

Purpose: We hypothesized that downstream effects of endogenous vascular endothelial growth factor (VEGF)/VEGF receptor signaling on acute myelogenous leukemia (AML) cell survival resulted in increased in vitro cellular drug resistance and a longer time to kill most leukemic cells in vivo upon drug exposure.

Experimental Design: In primary AML cells from pediatric patients, VEGFA and VEGFC mRNA expression and in vitro cellular resistance to nine cytotoxic drugs were studied. As in vivo equivalents for in vitro drug resistance, in vivo AML blast reduction upon drug exposure, measured as blast cell reduction on day 15 in the bone marrow and as time in days from diagnosis to complete remission (CR) were used.

Results: Increased endogenous VEGFC levels significantly correlated with increased in vitro resistance for six typical AML drugs in primary AML cells from pediatric patients. Patients with >5% blasts on day 15 showed a 12.9-fold increase in the median VEGFC level compared with patients with ≤5% blasts (P = 0.002). Time to reach CR was studied using linear regression analysis with VEGFC, age at diagnosis, sex, treatment protocol, FAB type, cytogenetic risk profile, and WBC counts as variables. There was a significant positive independent association between VEGFC levels and time to CR (b = 6.02, SE = 1.58, P ≤ 0.0001, n = 72).

Conclusions: These results suggest for the first time that higher endogenous VEGFC levels of AML cells are related to decreased in vitro and in vivo drug responsiveness.