bluuebunny/crossref_metadata_2025 huggingface dataset:
The Endothelin Receptor Blocker Bosentan Inhibits Doxorubicin-Induced Cardiomyopathy
Authors: Bien Sandra , Riad Alexander , Ritter Christoph A. , Gratz Matthias , Olshausen Florian , Westermann Dirk , Grube Markus , Krieg Thomas , Ciecholewski Sabine , Felix Stephan B. , Staudt Alexander , Schultheiss Heinz-Peter , Ewert Ralf , Völker Uwe , Tschöpe Carsten , Kroemer Heyo K.
Published: November 2007

Doxorubicin is a frequently used anticancer drug, but its therapeutic benefit is limited by acute and chronic cardiotoxicity, often leading to heart failure. The mechanisms underlying doxorubicin-induced cardiotoxicity remain unclear. It was previously shown in men that doxorubicin leads to increased endothelin-1 plasma levels. In addition, cardiac-specific overexpression of endothelin-1 in mice resulted in a cardiomyopathy resembling the phenotype following doxorubicin administration. We therefore hypothesized that endothelin-1 is involved in the pathogenesis of doxorubicin cardiotoxicity. In mice (C57Bl/10), we found that doxorubicin (20 mg/kg body weight, i.p.) impaired cardiac function with decreased ejection fraction, diminished cardiac output, and decreased end-systolic pressure points recorded by a microconductance catheter. This impaired function was accompanied by the up-regulation of endothelin-1 expression on mRNA and protein level. In vitro investigations confirmed the regulation of endothelin-1 by doxorubicin and indicated that the doxorubicin-mediated increase of endothelin-1 expression involves epidermal growth factor receptor signaling via the MEK1/2-ERK1/2 cascade, which was further confirmed by immunoblotting studies in the left ventricle of treated animals. Pretreatment of mice with the endothelin receptor antagonist bosentan (100 mg/kg body weight, p.o.) strikingly inhibited doxorubicin-induced cardiotoxicity with preserved indices of contractility. Moreover, bosentan pretreatment resulted in reduced tumor necrosis factor-α content, lipid peroxidation, and Bax expression, as well as increased GATA-4 expression. Thus, endothelin-1 plays a key role in mediating the cardiotoxic effects of doxorubicin and its inhibition may be of therapeutic benefit for patients receiving doxorubicin. [Cancer Res 2007;67(21):10428–35]
Identification of Human Aldehyde Dehydrogenase 1 Family Member A1 as a Novel CD8+ T-Cell–Defined Tumor Antigen in Squamous Cell Carcinoma of the Head and Neck
Authors: Visus Carmen , Ito Diasuke , Amoscato Andrew , Maciejewska-Franczak Malgorzata , Abdelsalem Ahmed , Dhir Rajiv , Shin Dong M. , Donnenberg Vera S. , Whiteside Theresa L. , DeLeo Albert B.
Published: November 2007

Few epitopes are available for vaccination therapy of patients with squamous cell carcinoma of the head and neck (SCCHN). Using a tumor-specific CTL, aldehyde dehydrogenase 1 family member A1 (ALDH1A1) was identified as a novel tumor antigen in SCCHN. Mass spectral analysis of peptides in tumor-derived lysates was used to determine that the CTL line recognized the HLA-A*0201 (HLA-A2) binding ALDH1A188-96 peptide. Expression of ALDH1A1 in established SCCHN cell lines, normal mucosa, and primary keratinocytes was studied by quantitative reverse transcription-PCR and immunostaining. Protein expression was further defined by immunoblot analysis, whereas ALDH1A1 activity was measured using ALDEFLUOR. ALDH1A188-96 peptide was identified as an HLA-A2–restricted, naturally presented, CD8+ T-cell–defined tumor peptide. ALDH1A188-96 peptide-specific CD8+ T cells recognized only HLA-A2+ SCCHN cell lines, which overexpressed ALDH1A1, as well as targets transfected with ALDH1A1 cDNA. Target recognition was blocked by anti-HLA class I and anti-HLA-A2 antibodies. SCCHN cell lines overexpressing ALDH1 had high enzymatic activity. ALDH1A1 protein was expressed in 12 of 17 SCCHN, and 30 of 40 dysplastic mucosa samples, but not in normal mucosa. ALDH1A1 expression levels in target cells correlated with their recognition by ALDH1A188-96 peptide-specific CD8+ T cells. Our findings identify ALDH1A1, a metabolic antigen, as a potential target for vaccination therapy in the cohort of SCCHN subjects with tumors overexpressing this protein. A smaller cohort of subjects with SCCHN, whose tumors express little to no ALDH1A1, and thus are deficient in conversion of retinal to retinoic acid, could benefit from chemoprevention therapy. [Cancer Res 2007;67(21):10538–45]
A Role for Endoglin as a Suppressor of Malignancy during Mouse Skin Carcinogenesis
Authors: Pérez-Gómez Eduardo , Villa-Morales María , Santos Javier , Fernández-Piqueras José , Gamallo Carlos , Dotor Javier , Bernabéu Carmelo , Quintanilla Miguel
Published: November 2007

Endoglin is a membrane glycoprotein that acts as a coreceptor for transforming growth factor-β. We and others have previously suggested a function of endoglin as a tumor suppressor in epithelial cancer. Here, we study the expression of endoglin during chemical mouse skin carcinogenesis. We find that shedding of membrane endoglin, allowing the secretion of a soluble endoglin form, is a late event associated with progression from squamous to spindle cell carcinomas. Knockdown of endoglin in transformed keratinocytes activates the Smad2/3 signaling pathway resulting in cell growth arrest, delayed tumor latencies, and a squamous to spindle phenotypic conversion. Forced expression of the long endoglin isoform in spindle carcinoma cells blocks transforming growth factor-β1 stimulation of Smad2/3 signaling and prevents tumor formation. In contrast, expression of the short endoglin isoform has no effect on spindle cell growth in vitro or in vivo. Our results show that endoglin behaves as a suppressor of malignancy during the late stages of carcinogenesis. Therefore, disruption of membrane endoglin emerges as a crucial event for progression to spindle cell carcinomas. [Cancer Res 2007;67(21):10268–77]
Association ofMatrix Metalloproteinase-8Gene Variation with Breast Cancer Prognosis
Authors: Decock Julie , Long Ji-Rong , Laxton Ross C. , Shu Xiao-Ou , Hodgkinson Conrad , Hendrickx Wouter , Pearce Eve G. , Gao Yu-Tang , Pereira Andresa C. , Paridaens Robert , Zheng Wei , Ye Shu
Published: November 2007

Animal and cell studies indicate an inhibitory effect of matrix metalloproteinase-8 (MMP8) on tumorigenesis and metastasis. We investigated whether MMP8 gene variation was associated with breast cancer metastasis and prognosis in humans. We first studied nine tagging single nucleotide polymorphisms (SNP) in the MMP8 gene in 140 clinically and pathologically well-characterized breast cancer patients. Four of the SNPs were found to be associated with lymph node metastasis, the most pronounced being a promoter SNP (rs11225395) with its minor allele (T) associating with reduced susceptibility to lymph node metastasis (P = 0.02). This SNP was further evaluated for association with cancer relapse and survival among a cohort of ∼1,100 breast cancer patients who had been followed for cancer recurrence and mortality for a median of 7.1 years. The T allele was associated with reduced cancer relapse and greater survival, particularly among patients with earlier stage cancer. Among patients of tumor-node-metastasis stage 0 to II, the adjusted hazard ratio of disease-free survival was 0.7 [95% confidence interval (95% CI), 0.5–0.9] for patients carrying T allele compared with those homozygous for the C allele (P = 0.02). In vitro experiments showed that the T allele had higher promoter activity than the C allele in breast cancer cells. Electrophoretic mobility shift assays showed binding of nuclear proteins to the DNA sequence at the SNP site of the T allele but not that of the C allele. The data suggest that MMP8 gene variation may influence breast cancer prognosis and support the notion that MMP8 has an inhibitory effect on cancer metastasis. [Cancer Res 2007;67(21):10214–21]
In vivoTargeting of Human Neutralizing Antibodies against CD55 and CD59 to Lymphoma Cells Increases the Antitumor Activity of Rituximab
Authors: Macor Paolo , Tripodo Claudio , Zorzet Sonia , Piovan Erich , Bossi Fleur , Marzari Roberto , Amadori Alberto , Tedesco Francesco
Published: November 2007

An in vivo model of human CD20+ B-lymphoma was established in severe combined immunodeficiency mice to test the ability of human neutralizing miniantibodies to CD55 and CD59 (MB55 and MB59) to enhance the therapeutic effect of rituximab. The miniantibodies contained single-chain fragment variables and the hinge-CH2-CH3 domains of human IgG1. LCL2 cells were selected for the in vivo study among six B-lymphoma cell lines for their high susceptibility to rituximab-dependent complement-mediated killing enhanced by MB55 and MB59. The cells injected i.p. primarily colonized the liver and spleen, leading to the death of the animals within 30 to 40 days. Thirty percent of mice receiving biotin-labeled rituximab (25 μg) i.p. on days 4 and 11 after cell injection survived to 120 days. Administration of biotin-labeled rituximab, followed by avidin (40 μg) and biotin-labeled MB55–MB59 (100 μg) at 4-h intervals after each injection resulted in the survival of 70% of mice. Surprisingly, 40% of mice survived after the sole injection of avidin and biotin-labeled MB55–MB59, an observation consistent with the in vitro data showing that the miniantibodies induced killing of ∼25% cells through antibody-dependent cell cytotoxicity. In conclusion, MB55 and MB59 targeted to tumor cells represent a valuable tool to enhance the therapeutic effect of rituximab and other complement-fixing antitumor antibodies. [Cancer Res 2007;67(21):10556–63]
Suppression of PTEN Expression Is Essential for Antiapoptosis and Cellular Transformation by Oncogenic Ras
Authors: Vasudevan Krishna Murthi , Burikhanov Ravshan , Goswami Anindya , Rangnekar Vivek M.
Published: November 2007

Ras is one of the most commonly mutated oncogenes in the array of human cancers. The mechanism by which Ras induces cellular transformation is, however, not fully elucidated. We present here evidence that oncogenic Ras suppresses the expression of the tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 (PTEN), and this action of oncogenic Ras is mediated by the Raf-mitogen-activated protein kinase/extracellular signal–regulated kinase (ERK) kinase (MEK)-ERK pathway via up-regulation of c-Jun. Jun+/+ cells undergo cellular transformation by oncogenic Ras, and restoration of wild-type PTEN, but not a phosphate-defective mutant of PTEN, induces apoptosis in these cells. Conversely, in Jun−/− cells, oncogenic Ras neither suppresses PTEN nor causes transformation, but rather it induces PTEN-dependent apoptosis. An apoptotic response to oncogenic Ras in Jun−/− cells can be prevented by suppressing PTEN expression. These findings imply that oncogenic Ras suppresses the apoptotic gene PTEN via the Raf-MEK-ERK-c-Jun pathway to induce antiapoptosis and cellular transformation. Together, our findings identify a novel molecular interface between the oncogenic and tumor suppressor pathways that regulates cellular transformation and survival. [Cancer Res 2007;67(21):10343–50]
A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma
Authors: Salvatore Giuliana , Nappi Tito Claudio , Salerno Paolo , Jiang Yuan , Garbi Corrado , Ugolini Clara , Miccoli Paolo , Basolo Fulvio , Castellone Maria Domenica , Cirafici Anna Maria , Melillo Rosa Marina , Fusco Alfredo , Bittner Michael L. , Santoro Massimo
Published: November 2007

Here, we show that the anaplastic thyroid carcinoma (ATC) features the up-regulation of a set of genes involved in the control of cell cycle progression and chromosome segregation. This phenotype differentiates ATC from normal tissue and from well-differentiated papillary thyroid carcinoma. Transcriptional promoters of the ATC up-regulated genes are characterized by a modular organization featuring binding sites for E2F and NF-Y transcription factors and cell cycle–dependent element (CDE)/cell cycle gene homology region (CHR) cis-regulatory elements. Two protein kinases involved in cell cycle regulation, namely, Polo-like kinase 1 (PLK1) and T cell tyrosine kinase (TTK), are part of the gene set that is up-regulated in ATC. Adoptive overexpression of p53, p21 (CIP1/WAF1), and E2F4 down-regulated transcription from the PLK1 and TTK promoters in ATC cells, suggesting that these genes might be under the negative control of tumor suppressors of the p53 and pRB families. ATC, but not normal thyroid, cells depended on PLK1 for survival. RNAi-mediated PLK1 knockdown caused cell cycle arrest associated with 4N DNA content and massive mitotic cell death. Thus, thyroid cell anaplastic transformation is accompanied by the overexpression of a cell proliferation/genetic instability-related gene cluster that includes PLK1 kinase, which is a potential molecular target for ATC treatment. [Cancer Res 2007;67(21):10148–57]
Androgen Induction of the Androgen Receptor Coactivator Four and a Half LIM Domain Protein-2: Evidence for a Role for Serum Response Factor in Prostate Cancer
Authors: Heemers Hannelore V. , Regan Kevin M. , Dehm Scott M. , Tindall Donald J.
Published: November 2007

Androgen receptor (AR) activity is critical for prostate cancer progression. Overexpression of several AR-associated coactivators has been shown to be essential for AR activation during disease progression. The stimuli and signaling pathways leading to overexpression of these coregulators, however, remain largely elusive. Here, we investigated whether androgen signaling, which demarcates critical transitions during prostate cancer disease progression, can affect coregulator expression. We found that expression of four and a half LIM domain protein-2 (FHL2), a key AR coactivator that is overexpressed in prostate cancer and associates with a poor prognosis, is induced strongly by androgens. Androgen induction of this coactivator established a feed-forward mechanism that robustly activated the AR. Stimulation of FHL2 after androgen exposure was time- and dose-dependent and relied on the presence of a functional AR. Androgen induction of FHL2 depended on active transcription of the FHL2 gene, mediated by action of serum response factor (SRF) on its proximal promoter. Loss of SRF, a transcription factor that preferentially regulates the expression of genes involved in mitogenic response and cytoskeletal organization, hampered prostate cancer cell proliferation. These results suggest a novel indirect mechanism of androgen action on FHL2 expression and provide evidence that SRF is an important determinant of AR action in prostate cancer cells. [Cancer Res 2007;67(21):10592–9]
A Novel Ovarian Xenografting Model to Characterize the Impact of Chemotherapy Agents on Human Primordial Follicle Reserve
Authors: Oktem Ozgur , Oktay Kutluk
Published: November 2007

Many chemotherapeutic agents, especially of the alkylating family, alter fertility in premenopausal females. However, it is not practically possible to quantify and characterize the impact of cancer drugs on ovarian reserve in a clinical setting. Thus, our specific aim was to develop a xenograft model to characterize the in vivo impact of chemotherapy agents on human ovary. Ovarian pieces from 24 weeks old abortuses were xenografted s.c. to severe combined immunodeficient mice (n = 52). Animals received either a single dose of 200 mg/kg of cyclophosphamide or the vehicle. Grafts were recovered from the control and treated mice 12 to 72 h after the cyclophosphamide injection and serially sectioned for primordial follicle counts. Apoptosis was assessed with terminal nucleotidyl transferase–mediated nick end labeling (TUNEL) assay. Platelet/endothelial cell adhesion molecule 1 (PECAM-1) staining, as well as intra-vital fluorescein-conjugated lectin and Evans blue labeling were done to assess microvasculature by confocal microscopy. Although there was 12% reduction in primordial follicle density by 12 h following treatment (P > 0.05), the follicle loss increased significantly at 24 h (53%, P < 0.01) and peaked at 48 h (93%, P < 0.0001). TUNEL staining peaked at 12 h, earlier than the diminishment in follicle numbers, and decreased thereafter. Xenograft vascularization pattern was similar to non-xenografted tissue, indicating appropriate in vivo drug delivery. The impact of cyclophosphamide on primordial follicle reserve in our human ovarian xenograft model is consistent with the clinical gonadotoxicity of this drug. Human ovarian xenografting is a promising model to characterize the gonadotoxic effects of current and emerging cancer drugs without a need for lengthy clinical studies. [Cancer Res 2007;67(21):10159–62]
Gene Expression Analysis of Preinvasive and Invasive Cervical Squamous Cell Carcinomas Identifies HOXC10 as a Key Mediator of Invasion
Authors: Zhai Yali , Kuick Rork , Nan Bin , Ota Ichiro , Weiss Stephen J. , Trimble Cornelia L. , Fearon Eric R. , Cho Kathleen R.
Published: November 2007

If left untreated, a subset of high-grade squamous intraepithelial lesions (HSIL) of the cervix will progress to invasive squamous cell carcinomas (SCC). To identify genes whose differential expression is linked to cervical cancer progression, we compared gene expression in microdissected squamous epithelial samples from 10 normal cervices, 7 HSILs, and 21 SCCs using high-density oligonucleotide microarrays. We identified 171 distinct genes at least 1.5-fold up-regulated (and P < 0.001) in the SCCs relative to HSILs and normal cervix samples. Differential expression of a subset of these genes was confirmed by quantitative reverse transcription-PCR and immunohistochemical staining of cervical tissue samples. One of the genes up-regulated during progression, HOXC10, was selected for functional studies aimed at assessing its role in mediating invasive behavior of neoplastic squamous epithelial cells. Elevated HOXC10 expression was associated with increased invasiveness of human papillomavirus–immortalized keratinocytes and cervical cancer–derived cell lines in both in vitro and in vivo assays. Cervical cancer cells with high endogenous levels of HOXC10 were less invasive after short hairpin RNA–mediated knockdown of HOXC10 expression. Our findings support a key role for the HOXC10 homeobox protein in cervical cancer progression. Other genes with differential expression in invasive SCC versus HSIL may contribute to tumor progression or may be useful as markers for cancer diagnosis or progression risk. [Cancer Res 2007;67(21):10163–72]
Cooperative Interactions between Androgen Receptor (AR) and Heat-Shock Protein 27 Facilitate AR Transcriptional Activity
Authors: Zoubeidi Amina , Zardan Anousheh , Beraldi Eliana , Fazli Ladan , Sowery Richard , Rennie Paul , Nelson Colleen , Gleave Martin
Published: November 2007

Androgen receptor (AR) transactivation is known to enhance prostate cancer cell survival. However, the precise effectors by which the prosurvival effects of androgen and AR drive prostate cancer progression are poorly defined. Here, we identify a novel feed-forward loop involving cooperative interactions between ligand-activated AR and heat-shock protein 27 (Hsp27) phospho-activation that enhance AR stability, shuttling, and transcriptional activity, thereby increasing prostate cancer cell survival. Androgen-bound AR induces rapid Hsp27 phosphorylation on Ser78 and Ser82 residues in an AR- and p38 kinase–dependent manner. After this androgen-induced, non-nuclear phospho-activation, Hsp27 displaces Hsp90 from a complex with AR to chaperone AR into the nucleus and interact with its response elements to enhance its genomic activity. Inhibition of Hsp27 phosphorylation, or knockdown using the antisense drug OGX-427, shifted the association of AR with Hsp90 to MDM2, increased proteasome-mediated AR degradation, decreased AR transcriptional activity, and increased prostate cancer LNCaP cell apoptotic rates. OGX-427 treatment of mice bearing LNCaP xenografts transfected with an androgen-regulated, probasin-luciferase reporter construct resulted in decreased bioluminescence and serum PSA levels as pharmacodynamic readouts of AR activity, as well as AR, Hsp27, and Hsp90 protein levels in LNCaP tumor tissue. These data identify novel nongenomic mechanisms involving androgen, AR, and Hsp27 activation that cooperatively interact to regulate the genomic activity of AR and justify further investigation of Hsp27 knockdown as an AR disrupting therapeutic strategy in prostate cancer. [Cancer Res 2007;67(21):10455–65]
Involvement of Sphingosine Kinase 2 in p53-Independent Induction of p21 by the Chemotherapeutic Drug Doxorubicin
Authors: Sankala Heidi M. , Hait Nitai C. , Paugh Steven W. , Shida Dai , Lépine Sandrine , Elmore Lynne W. , Dent Paul , Milstien Sheldon , Spiegel Sarah
Published: November 2007

Sphingosine-1-phosphate is a potent lipid mediator formed by phosphorylation of sphingosine, a metabolite of sphingolipids, catalyzed by two sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2. Expression of SphK2, which is enriched in the nucleus of MCF7 human breast cancer cells, increased expression of the cyclin-dependent kinase inhibitor p21 but had no effect on p53 or its phosphorylation. The anticancer drug doxorubicin is known to increase p21 via p53-dependent and p53-independent mechanisms. Down-regulation of endogenous SphK2 with small interfering RNA targeted to unique mRNA sequences decreased basal and doxorubicin-induced expression of p21 without affecting increased expression of p53. Down-regulation of SphK2 also decreased G2-M arrest and markedly enhanced apoptosis induced by doxorubicin. Moreover, siSphK2 reduced doxorubicin-induced p21 expression in p53-inactivated MCF7 cells. Likewise, in human wild-type p53- and p21-expressing HCT116 colon carcinoma cells, as well as in p53-null counterparts, down-regulation of SphK2 markedly reduced p21 induction by doxorubicin. Knockdown of SphK2 sensitized HCT116 cells to apoptosis induced by doxorubicin with concomitant cleavage of poly(ADP-ribose) polymerase. Collectively, our results show that endogenous SphK2 is important for p53-independent induction of p21 expression by doxorubicin and suggest that SphK2 may influence the balance between cytostasis and apoptosis of human cancer cells. [Cancer Res 2007;67(21):10466–74]
RasGRP1 Overexpression in the Epidermis of Transgenic Mice Contributes to Tumor Progression during Multistage Skin Carcinogenesis
Authors: Luke Courtney T. , Oki-Idouchi Carolyn E. , Cline J. Mark , Lorenzo Patricia S.
Published: November 2007

RasGRP1 is a guanine nucleotide exchange factor for Ras, activated in response to the second messenger diacylglycerol and its ultrapotent analogues, the phorbol esters. We have previously shown that RasGRP1 is expressed in mouse epidermal keratinocytes and that transgenic mice overexpressing RasGRP1 in the epidermis under the keratin 5 promoter (K5.RasGRP1) are prone to developing spontaneous papillomas and squamous cell carcinomas, suggesting a role for RasGRP1 in skin tumorigenesis. Here, we examined the response of the K5.RasGRP1 mice to multistage skin carcinogenesis, using 7,12-dimethylbenz(a)anthracene as carcinogen and 12-O-tetradecanoylphorbol-13-acetate (TPA) as tumor promoter. We found that whereas tumor multiplicity did not differ between transgenic and wild-type groups, the transgenic tumors were significantly larger than those observed in the wild-type mice (wild-type, 4.58 ± 0.25 mm; transgenic, 9.83 ± 1.05 mm). Histologic analysis further revealed that squamous cell carcinomas generated in the transgenic mice were less differentiated and more invasive than the wild-type tumors. Additionally, 30% of the transgenic mice developed tumors in the absence of initiation, suggesting that RasGRP1 overexpression could partially substitute for the initiation step induced by dimethylbenz(a)anthracene. In primary keratinocytes isolated from K5.RasGRP1 mice, TPA stimulation induced higher levels of Ras activation compared with the levels measured in the wild-type cells, indicating that constitutive overexpression of RasGRP1 in epidermal cells leads to elevated biochemical activation of endogenous Ras in response to TPA. The present data suggests that RasGRP1 participates in skin carcinogenesis via biochemical activation of endogenous wild-type Ras and predisposes to malignant progression in cooperation with Ras oncogenic signals. [Cancer Res 2007;67(21):10190–7]
Cell Growth Inhibition by Okadaic Acid Involves Gut-Enriched Kruppel-like Factor–Mediated Enhanced Expression of c-Myc
Authors: Zhang Liyue , Wali Anil , Ramana Chilakamarti V. , Rishi Arun K.
Published: November 2007

Human breast cancer (HBC) cell growth suppression by okadaic acid (OA) was previously found to involve elevated expression of oncogenes c-myc and c-fos and apoptosis. Since, c-Myc influences diverse pathways of cell growth, we hypothesized that elevated levels of c-Myc are involved in HBC growth suppression. Here, we investigated whether induction of c-Myc by OA or protein synthesis inhibitor cycloheximide contributed to HBC growth inhibition and the mechanisms involved. OA, cycloheximide, or the chemotherapeutic drug Taxol suppressed HBC cell growth. However, OA or cycloheximide treatments over 6 or 10 h, respectively, induced c-Myc expression. Depletion of c-Myc, on the other hand, resulted in enhanced HBC cell viabilities when exposed to OA or cycloheximide, but not by Taxol. OA induced c-myc transcription by targeting an 80-bp region from positions −11 to +70, relative to the P1 transcription start of mouse c-myc promoter. Gel mobility shift assays revealed binding of HBC cell nuclear proteins to the OA-responsive c-myc promoter fragment, whereas binding of one complex was elevated in the case of the OA-treated or cycloheximide-treated HBC cell nuclear extracts. Database search revealed presence of a consensus sequence for zinc finger protein gut-enriched Kruppel-like factor (GKLF) in OA-responsive region of the c-myc promoter. Mutation of GKLF consensus sequences abrogated OA responsiveness of the c-myc promoter, and OA treatments caused enhanced expression of GKLF in HBC cells. Thus, OA-dependent attenuation of HBC growth is accomplished, in part, by zinc finger transcription factor GKLF-mediated enhanced transcription of c-myc. [Cancer Res 2007;67(21):10198–206]
Telomerase-Specific T-Cell Immunity in Breast Cancer: Effect of Vaccination on Tumor Immunosurveillance
Authors: Domchek Susan M. , Recio Adri , Mick Rosemarie , Clark Carolyn E. , Carpenter Erica L. , Fox Kevin R. , DeMichele Angela , Schuchter Lynn M. , Leibowitz Michael S. , Wexler Michael H. , Vance Barbara A. , Beatty Gregory L. , Veloso Elizabeth , Feldman Michael D. , Vonderheide Robert H.
Published: November 2007

The human telomerase reverse transcriptase (hTERT) is nearly universally overexpressed in human cancer, contributes critically to oncogenesis, and is recognized by cytotoxic T cells that lyse tumors. CD8+ T cells specific for hTERT naturally occur in certain populations of cancer patients in remission, but it remains poorly understood whether such T cells could contribute to tumor immunosurveillance. To address this issue, we induced hTERT-specific T cells in vivo via peptide vaccination in 19 patients with metastatic breast cancer who otherwise had no measurable T-cell responses to hTERT at baseline. Tumor-infiltrating lymphocytes (TIL) were evident after, but not before vaccination, with 4% to 13% of postvaccine CD8+ TIL specific for the immunizing hTERT peptide. Induction of TIL manifested clinically with tumor site pain and pruritus and pathologically with alterations in the tumor microenvironment, featuring histiocytic accumulation and widespread tumor necrosis. hTERT-specific CD8+ T cells were also evident after vaccination in the peripheral blood of patients and exhibited effector functions in vitro including proliferation, IFN-γ production, and tumor lysis. An exploratory landmark analysis revealed that median overall survival was significantly longer in those patients who achieved an immune response to hTERT peptide compared with patients who did not. Immune response to a control cytomegalovirus peptide in the vaccine did not correlate with survival. These results suggest that hTERT-specific T cells could contribute to the immunosurveillance of breast cancer and suggest novel opportunities for both therapeutic and prophylactic vaccine strategies for cancer. [Cancer Res 2007;67(21):1–9]
Discovery of Endothelial to Mesenchymal Transition as a Source for Carcinoma-Associated Fibroblasts
Authors: Zeisberg Elisabeth M. , Potenta Scott , Xie Liang , Zeisberg Michael , Kalluri Raghu
Published: November 2007

Activated fibroblasts are associated with many different tumors. Myofibroblasts, activated fibroblasts, and perivascular mesenchymal cells such as pericytes play a role in cancer progression. Many studies suggest that myofibroblasts facilitate tumor growth and cancer progression. The source for myofibroblasts and other activated fibroblasts within the tumors is still debated. Although de novo activation of quiescent fibroblasts into α-smooth muscle actin (αSMA)–positive myofibroblasts is one likely source, epithelial to mesenchymal transition and bone marrow recruitment are also evolving as possible mechanisms for the emergence of a heterogeneous population of carcinoma-associated fibroblasts. Here, we show that transforming growth factor-β1 could induce proliferating endothelial cells to undergo a phenotypic conversion into fibroblast-like cells. Such endothelial to mesenchymal transition (EndMT) is associated with the emergence of mesenchymal marker fibroblast-specific protein-1 (FSP1) and down-regulation of CD31/PECAM. Additionally, we show EndMT in tumors using the B16F10 melanoma model and the Rip-Tag2 spontaneous pancreatic carcinoma model. Crossing Tie2-Cre mice with R26Rosa-lox-Stop-lox-LacZ mice allows for irreversible tagging of endothelial cells. We provide unequivocal evidence for EndMT at the invasive front of the tumors in these transgenic mice. Collectively, our results show that EndMT is a unique mechanism for the accumulation of carcinoma-associated fibroblasts and suggest that antiangiogenic treatment of tumors may have a direct effect in decreasing activated fibroblasts that likely facilitate cancer progression. [Cancer Res 2007;67(21):10123–8]
Microphthalmia-Associated Transcription Factor Gene Amplification in Metastatic Melanoma Is a Prognostic Marker for Patient Survival, But Not a Predictive Marker for Chemosensitivity and Chemotherapy Response
Authors: Ugurel Selma , Houben Roland , Schrama David , Voigt Heike , Zapatka Marc , Schadendorf Dirk , Bröcker Eva B. , Becker Jürgen C.
Published: November 2007

Purpose: The microphthalmia-associated transcription factor (MITF) is regarded as a key oncogene of the melanocytic lineage since it was detected by a genome-wide analysis to be strongly amplified in 15% to 20% of metastatic melanomas. MITF gene amplification was shown to be associated with a reduced survival in metastatic melanoma patients, and reduction of MITF activity was shown to sensitize melanoma cell lines to chemotherapeutics, suggesting the intratumoral MITF gene copy number as a predictive biomarker of response and survival after chemotherapy.

Patients and Methods: To validate this hypothesis, we investigated MITF gene amplification in tumor tissues obtained from 116 metastatic melanoma patients before an individualized sensitivity-directed chemotherapy using quantitative real-time PCR. MITF amplification rates were correlated with tumor chemosensitivity quantified by an ATP-based luminescence assay and with chemotherapy outcome in terms of response and survival.

Results: Of 116 tumor tissues, 104 were evaluable for MITF gene amplification. Strong amplification (≥4 copies per cell) was detected in 24 of 104 tissues (23%), whereas 62 of 104 tissues (60%) harbored >3 copies per cell. Strong MITF gene amplification was associated with a reduced disease-specific survival (P = 0.031). However, no correlation was found between MITF copy number and in vitro chemosensitivity or in vivo chemotherapy response.

Conclusion: Our findings suggest that strong amplifications of the melanoma oncogene MITF affects patient survival but does not influence tumor chemosensitivity and chemotherapy response. Thus, the MITF gene copy number seems a useful prognostic marker in metastatic melanoma but could not be confirmed as a predictive marker of chemosensitivity and chemotherapy response.
Gene Expression Profiling from Formalin-Fixed Paraffin-Embedded Tumors of Pediatric Glioblastoma
Authors: Haque Takrima , Faury Damien , Albrecht Steffen , Lopez-Aguilar Enrique , Hauser Péter , Garami Miklós , Hanzély Zoltán , Bognár László , Del Maestro Rolando F. , Atkinson Jeffrey , Nantel Andre , Jabado Nada
Published: November 2007

Purpose: Gene expression profiling has proved crucial for understanding the biology of cancer. In rare diseases, including pediatric glioblastoma (pGBM), the lack of readily available fresh frozen (FF) material limits the feasibility of this analysis, as well as its validation, on independent data sets, a step needed to ensure relevance, mandating the use of alternate RNA sources. To overcome the limitation of material number and to validate results we obtained on FF pGBM, we did microarray analysis on RNA extracted from formalin-fixed, paraffin-embedded archival samples from pGBM and control brains, wherein we had no control on the fixation process.

Experimental Design: RNA from 16 pGBM and 3 control brains was extracted and linearly amplified. Reverse transcription–PCR on housekeeping and formerly identified tumor-associated genes and microarray analysis were done on this RNA source. Results were validated by immunohistochemistry.

Results: Despite extensive RNA degradation, microarray analysis was possible on 16 of 19 samples and reproduced the pattern of results obtained on FF pGBM. Gene lists and ontology subgrouping were highly concordant in both sample types. Similar to the findings on FF samples, we were able to identify two subsets of pGBM based on their association/lack of association with evidence consistent with an active Ras pathway.

Conclusions: Archival formalin-fixed, paraffin-embedded tissues are an invaluable resource as they are the most widely available materials often accessible in conjunction with clinical and follow-up data. Gene expression profiling on this material is feasible and may represent a significant advance for understanding the biology of rare human diseases.
Targeting Poly(ADP-Ribose) Polymerase: A Two-Armed Strategy for Cancer Therapy
Authors: Plummer Elizabeth Ruth , Calvert Hilary
Published: November 2007

The DNA repair pathways are protective of the host genome in normal cells; however, in cancer cells, these pathways may be disrupted and predispose to tumorigenesis or their activity may overcome the potentially cytotoxic damage caused by anticancer agents and be a mechanism of resistance. Poly(ADP-ribose) polymerase inhibitors, which block base excision repair of single-strand breaks, have entered the clinic in the last few years. This article discusses the interactions between the pathways of single- and double-strand break repair, which explain the two clinical development strategies for this class of drugs.
Predictive Factors of Oxaliplatin Neurotoxicity: The Involvement of the Oxalate Outcome Pathway
Authors: Gamelin Laurence , Capitain Olivier , Morel Alain , Dumont Agnes , Traore Sory , Anne Le Bouil , Gilles Simard , Boisdron-Celle Michele , Gamelin Erick
Published: November 2007

Purpose: Oxaliplatin displays a frequent dose-limiting neurotoxicity due to its interference with neuron voltage-gated sodium channels through one of its metabolites, oxalate, a calcium chelator. Different clinical approaches failed in neurotoxicity prevention, except calcium-magnesium infusions. We characterized oxalate outcome following oxaliplatin administration and its interference with cations and amino acids. We then looked for genetic predictive factors of oxaliplatin-induced neurotoxicity.

Experimental Design: We first tested patients for cations and oxalate levels and did amino acid chromatograms in urine following oxaliplatin infusion. In the second stage, before treatment with FOLFOX regimen, we prospectively looked for variants in genes coding for the enzymes involved (a) in the oxalate metabolism, especially glyoxylate aminotransferase (AGXT), and (b) in the detoxification glutathione cycle, glutathione S-transferase π, and for genes coding for membrane efflux proteins (ABCC2).

Results: In the first 10 patients, urinary excretions of oxalate and cations increased significantly within hours following oxaliplatin infusion, accompanied by increased excretions of four amino acids (glycine, alanine, serine, and taurine) linked to oxalate metabolism. In a further 135 patients, a minor haplotype of AGXT was found significantly predictive of both acute and chronic neurotoxicity. Neither glutathione S-transferase π nor ABCC2 single nucleotide polymorphisms we looked for were linked to neurotoxicity.

Conclusion: These data confirm the involvement of oxalate in oxaliplatin neurotoxicity and support the future use of AGXT genotyping as a pretherapeutic screening test to predict individual susceptibility to neurotoxicity.
Immunization with Mimotopes Prevents Growth of Carcinoembryonic Antigen–Positive Tumors in BALB/c Mice
Authors: Brämswig Kira H. , Knittelfelder Regina , Gruber Silke , Untersmayr Eva , Riemer Angelika B. , Szalai Krisztina , Horvat Reinhard , Kammerer Robert , Zimmermann Wolfgang , Zielinski Christoph C. , Scheiner Otto , Jensen-Jarolim Erika
Published: November 2007

Purpose: The carcinoembryonic antigen (CEA) is a glycoprotein that is overexpressed in nearly 50% of all human and veterinarian tumors. At present, anti-CEA antibodies are being tested in clinical studies as passive immunotherapeutics. This study aims to establish an active immunotherapy for the poorly immunogenic CEA glycoprotein by generating antigen surrogates.

Experimental Design: We used the monoclonal anti-CEA antibody Col-1 and the biopanning method to generate peptide mimics (mimotopes) of the Col-1 epitope. The peptide showing the highest specificity and mimicry was synthesized as an octameric multiple antigenic mimotope (MAM). Subsequently, immunogenicity of the selected mimotope was examined in BALB/c mice. We assessed antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity mediated by the induced antibodies on CEA-expressing HT29 tumor cells. Furthermore, after immunization, the BALB/c mice were transplanted s.c. with Meth-A/CEA tumor cells.

Results: When BALB/c mice were immunized with this MAM, they generated a specific humoral immune response against CEA. The mimotope-induced polyclonal and poly-isotypic antibodies induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro. Furthermore, when MAM-immunized mice were transplanted s.c. with Meth-A/CEA cells expressing human CEA, a suppressed tumor growth was observed.

Conclusion: From our results, we can conclude that the Col-1 epitope of the glycoprotein CEA can be translated into an immunogenic peptide mimic. The mimotope-induced antibodies recognize CEA and do effectively inhibit growth of CEA-positive tumors. Based on these finding, we suggest that the generated mimotopes are candidates for active immunotherapy of CEA-expressing tumors.
Hypermethylation of Tachykinin-1 Is a Potential Biomarker in Human Esophageal Cancer
Authors: Jin Zhe , Olaru Alexandru , Yang Jian , Sato Fumiaki , Cheng Yulan , Kan Takatsugu , Mori Yuriko , Mantzur Carmit , Paun Bogdan , Hamilton James P. , Ito Tetsuo , Wang Suna , David Stefan , Agarwal Rachana , Beer David G. , Abraham John M. , Meltzer Stephen J.
Published: November 2007

Purpose: Our aim was to investigate whether and at what stage hypermethylation of the tachykinin-1 (TAC1) gene is associated with human esophageal neoplastic transformation.

Experimental Design: TAC1 promoter hypermethylation was examined by real-time methylation-specific PCR in 258 human esophageal specimens and 126 plasma samples from patients or tissues at various stages of neoplastic evolution.

Results: TAC1 hypermethylation in tissue samples showed highly discriminative receiver-operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) from normal esophagus (P < 0.0001). Both frequencies and normalized methylation values of TAC1 tissue methylation were significantly higher in Barrett's metaplasia (BE), dysplastic Barrett's esophagus, EAC, and ESCC than in normal esophagus (P < 0.01). The frequency of TAC1 hypermethylation increased dramatically and early during neoplastic progression, from 7.5% in normal esophagus to 55.6% in BE from patients with Barrett's metaplasia alone, 57.5% in dysplastic Barrett's esophagus, and 61.2% in EAC. There was a significant relationship between TAC1 hypermethylation and BE segment length, a known clinical risk factor for neoplastic progression. Twelve (50%) of 24 ESCC exhibited TAC1 hypermethylation. Overall patient survival correlated significantly with TAC1 methylation status in ESCC patients (mean survival, 22 versus 110 months; P = 0.0102, log-rank test), but not in EAC patients. Both mean normalized methylation values and frequency of TAC1 hypermethylation in plasma samples were significantly higher in EAC patients than in control subjects. Treatment of KYSE220 ESCC and BIC EAC cells with 5-aza-2′-deoxycytidine reduced TAC1 methylation and increased TAC1 mRNA expression.

Conclusions: TAC1 promoter hypermethylation is a common event in both major histologic types of human esophageal carcinoma, occurs early, correlates with other progression risk factors in esophageal adenocarcinogenesis, and is a tissue biomarker of a poor prognosis in ESCC. Circulating methylated TAC1 promoter DNA also offers potential as a biomarker for the diagnosis of EAC.
Repair Capacity for UV Light–Induced DNA Damage Associated with Risk of Nonmelanoma Skin Cancer and Tumor Progression
Authors: Wang Li-E , Li Chunying , Strom Sara S. , Goldberg Leonard H. , Brewster Abenaa , Guo Zhaozheng , Qiao Yawei , Clayman Gary L. , Lee J. Jack , El-Naggar Adel K. , Prieto Victor G. , Duvic Madeleine , Lippman Scott M. , Weber Randal S. , Kripke Margaret L. , Wei Qingyi
Published: November 2007

Purpose: To examine the role of suboptimal DNA repair capacity (DRC) for UV light–induced DNA damage in the development of nonmelanoma skin cancer (NMSC) and tumor progression.

Experimental Design: We conducted a hospital-based case-control study of 255 patients with newly diagnosed NMSC [146 with basal cell carcinoma (BCC) and 109 with squamous cell carcinoma (SCC)] and 333 cancer-free controls. We collected information on demographic variables and risk factors from questionnaires, tumor characteristics from medical records, and lymphocytic DRC phenotype by the host-cell reactivation assay. Multivariable logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (95% CI).

Results: Overall, there was a relative 16% reduction in DRC in NMSC patients compared with controls (P < 0.001 for BCC and for SCC, respectively). DRC below the controls' median value was associated with increased risk significantly for BCC (OR, 1.62; 95% CI, 1.07-2.45) but borderline for SCC (OR, 1.63; 95% CI, 0.95-2.79) after adjustment for age, sex, and other assay-related covariates. When the highest tertile of controls' DRC was used as the reference, the intermediate and low DRC were associated with a statistically significant trend for increasing risk for both BCC (Ptrend = 0.007) and SCC (Ptrend = 0.020). However, patients with aggressive or multiple SCC tended to have a higher DRC than those with nonaggressive or single SCC.

Conclusions: Reduced DRC is an independent risk factor for BCC and single or nonaggressive SCC but not for multiple primaries, local aggressiveness, or recurrence of NMSC.
A Contemporary Prognostic Nomogram for Men with Hormone-Refractory Metastatic Prostate Cancer: A TAX327 Study Analysis
Authors: Armstrong Andrew J. , Garrett-Mayer Elizabeth S. , Yang Yi-Chun Ou , de Wit Ronald , Tannock Ian F. , Eisenberger Mario
Published: November 2007

Purpose: To develop a prognostic model and nomogram using baseline clinical variables to predict death among men with metastatic hormone-refractory prostate cancer (HRPC).

Experimental Design: TAX327 was a clinical trial that randomized 1,006 men with metastatic HRPC to receive every three week or weekly docetaxel or mitoxantrone, each with prednisone. We developed a multivariate Cox model and nomogram to predict survival at 1, 2, and 5 years.

Results: Ten independent prognostic factors other than treatment group were identified in multivariate analysis: (a) presence of liver metastases [hazard ratio (HR), 1.66; P = 0.019], (b) number of metastatic sites (HR, 1.63 if ≥2 sites; P = 0.001), (c) clinically significant pain (HR, 1.48; P < 0.0001), (d) Karnofsky performance status (HR, 1.39 if ≤70; P = 0.016), (e) type of progression (HR, 1.37 for measurable disease progression and 1.29 for bone scan progression; P = 0.005 and 0.01, respectively), (f) pretreatment prostate-specific antigen (PSA) doubling time (HR, 1.19 if <55 days; P = 0.066), (g) PSA (HR, 1.17 per log rise; P < 0.0001), (h) tumor grade (HR, 1.18 for high grade; P = 0.069), (i) alkaline phosphatase (HR, 1.27 per log rise; P < 0.0001), and (j) hemoglobin (HR, 1.11 per unit decline; P = 0.004). A nomogram was developed based on this multivariate model and validated internally using bootstrap methods, with a concordance index of 0.69.

Conclusions: This multivariate model identified several new independent prognostic factors in men with metastatic HRPC, including PSA doubling time, and led to the successful development of a clinically applicable nomogram. External prospective validation may support the wider use of this prognostic baseline model for men with HRPC treated with chemotherapy.
Daily Physical-Rest Activities in Relation to Nutritional State, Metabolism, and Quality of Life in Cancer Patients with Progressive Cachexia
Authors: Fouladiun Marita , Körner Ulla , Gunnebo Lena , Sixt-Ammilon Petra , Bosaeus Ingvar , Lundholm Kent
Published: November 2007

Purpose: To evaluate daily physical-rest activities in cancer patients losing weight in relation to disease progression.

Experimental Design: Physical activity-rest rhythms were measured (ActiGraph, armband sensor from BodyMedia) in relation to body composition (dual-energy X-ray absorptiometry), energy metabolism, exercise capacity (walking test), and self-scored quality of life (SF-36, Hospital Anxiety and Depression Scale) in weight-losing outpatients with systemic cancer (71 ± 2 years, n = 53). Well-nourished, age-matched, and previously hospitalized non–cancer patients served as controls (74 ± 4 years, n = 8). Middle-aged healthy individuals were used as reference subjects (49 ± 5 years, n = 23).

Results: Quality of life was globally reduced in patients with cancer (P < 0.01), accompanied by significantly reduced spontaneous physical activity during both weekdays and weekends compared with reference subjects (P < 0.01). Spontaneous physical activity declined over time during follow-up in patients with cancer (P < 0.05). However, overall physical activity and the extent of sleep and bed-rest activities did not differ between patients with cancer and age-matched non–cancer patients. Spontaneous physical activity correlated weakly with maximum exercise capacity in univariate analysis (r = 0.41, P < 0.01). Multivariate analysis showed that spontaneous physical activity was related to weight loss, blood hemoglobin concentration, C-reactive protein, and to subjectively scored items of physical functioning and bodily pain (SF-36; P < 0.05-0.004). Anxiety and depression were not related to spontaneous physical activity. Patient survival was predicted only by weight loss and serum albumin levels (P < 0.01), although there was no such prediction for spontaneous physical activity.

Conclusions: Daily physical-rest activities represent variables which probably reflect complex mental physiologic and metabolic interactions. Thus, activity-rest monitoring provides a new dimension in the evaluation of medical and drug interventions during palliative treatment of patients with cancer.