bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Array Comparative Genomic Hybridization Reveals Distinct DNA Copy Number Differences between Gastrointestinal Stromal Tumors and Leiomyosarcomas
Authors: Meza-Zepeda Leonardo A. , Kresse Stine H. , Barragan-Polania Ana H. , Bjerkehagen Bodil , Ohnstad Hege O. , Namløs Heidi M. , Wang Junbai , Kristiansen Bjørn E. , Myklebost Ola
Published: March 2023

Abstract
Leiomyosarcomas are spindle cell tumors showing smooth muscle differentiation. Until recently, most gastrointestinal stromal tumors (GIST) were also classified as smooth muscle tumors, but now GISTs are recognized as a separate entity, defined as spindle cell and/or epithelioid tumors localized in the gastrointestinal tract. Using microarray-based comparative genomic hybridization (array CGH), we have created a detailed map of DNA copy number changes for 7 GISTs and 12 leiomyosarcomas. Considerable gains and losses of chromosomal segments were observed in both tumor types. The most frequent aberration observed in GISTs was loss of chromosomes 14 and 22, with minimal recurrent regions in 14q11.2-q32.33 (71% of the tumors) and 22q12.2-q13.31 (100%). In leiomyosarcomas, frequent loss of chromosome 10 and 13q was observed, with minimal recurrent regions in 10q21.3 (75%) and 13q14.2-q14.3 (75%). Recurrent high-level amplification of 17p13.1-p11.2 was detected in leiomyosarcomas. Expression profiling using cDNA microarrays revealed four candidate genes in this region with high expression (AURKB, SREBF1, MFAP4, and FLJ10847). Altered expression of AURKB and SREBF1 has been observed previously in other malignancies. Hierarchical clustering of all samples separated GISTs and leiomyosarcomas into two distinct clusters. Statistical analysis identified six chromosomal regions, 1p36.11-p13.1, 9q21.11-9q34.3, 14q11.2-q23.2, 14q31.3-q32.33, 15q24.3-q26.3, and 22q11.21-q13.31, which were significantly different in copy number between GISTs and leiomyosarcomas. Our results show the potential of using array comparative genomic hybridization to classify histologically similar tumors such as GISTs and leiomyosarcomas. (Cancer Res 2006;66(18):8984-93)
Data from Contributions by MutL Homologues Mlh3 and Pms2 to DNA Mismatch Repair and Tumor Suppression in the Mouse
Authors: Chen Peng-Chieh , Dudley Sandra , Hagen Wayne , Dizon Diana , Paxton Leslie , Reichow Denise , Yoon Song-Ro , Yang Kan , Arnheim Norman , Liskay R. Michael , Lipkin Steven M.
Published: March 2023

Abstract
Germ line DNA mismatch repair mutations in MLH1 and MSH2 underlie the vast majority of hereditary non-polyposis colon cancer. Four mammalian homologues of Escherichia coli MutL heterodimerize to form three distinct complexes: MLH1/PMS2, MLH1/MLH3, and MLH1/PMS1. Although MLH1/PMS2 is generally thought to have the major MutL activity, the precise contributions of each MutL heterodimer to mismatch repair functions are poorly understood. Here, we show that Mlh3 contributes to mechanisms of tumor suppression in the mouse. Mlh3 deficiency alone causes microsatellite instability, impaired DNA-damage response, and increased gastrointestinal tumor susceptibility. Furthermore, Mlh3;Pms2 double-deficient mice have tumor susceptibility, shorter life span, microsatellite instability, and DNA-damage response phenotypes that are indistinguishable from Mlh1-deficient mice. Our data support previous results from budding yeast that show partial functional redundancy between MLH3 and PMS2 orthologues for mutation avoidance and show a role for Mlh3 in gastrointestinal and extragastrointestinal tumor suppression. The data also suggest a mechanistic basis for the more severe mismatch repair–related phenotypes and cancer susceptibility in Mlh1- versus Mlh3- or Pms2-deficient mice. Contributions by both MLH1/MLH3 and MLH1/PMS2 complexes to mechanisms of mismatch repair–mediated tumor suppression, therefore, provide an explanation why, among MutL homologues, only germ line mutations in MLH1 are common in hereditary non-polyposis colon cancer.
Data from Coexpression of NRASQ61R and BRAFV600E in Human Melanoma Cells Activates Senescence and Increases Susceptibility to Cell-Mediated Cytotoxicity
Authors: Petti Carlotta , Molla Alessandra , Vegetti Claudia , Ferrone Soldano , Anichini Andrea , Sensi Marialuisa
Published: March 2023

Abstract
Activating mutations in BRAF and NRAS oncogenes in human melanomas are mutually exclusive. This finding has suggested an epistatic relationship but is consistent even with synthetic lethality. To evaluate the latter possibility, a mutated NRASQ61R oncogene was expressed, under a constitutive or a doxycycline-regulated promoter, in a metastatic melanoma clone (clone 21) harboring an activated BRAFV600E oncogene. After the first 10 to 12 in vitro passages, the constitutive NRASQ61R transfectant displayed progressive accumulation in G0-G1 phase of the cell cycle and stained for the senescence-associated β-galactosidase activity (SA-β-Gal). Inducible expression of NRASQ61R, by the Tet-Off system, in clone 21 cells (21NRAS61ON) led to overactivation of the RAS/RAF/mitogen-activated protein kinase signaling pathway and, after the 10th in vitro passage, led to promotion of senescence. This was documented by reduced proliferation, flattened cell morphology, reduced growth in Matrigel, positive staining for SA-β-Gal, and expression of AMP-activated protein kinase and of the cell cycle inhibitor p21waf1/Cip1. These effects were detected neither in 21 cells with silenced NRASQ61R (21NRAS61OFF) nor in cells transfected with an inducible wild-type NRAS gene (21NRASWTON). In addition, when compared with parental 21 cells, or with 21NRAS61OFF, 21NRAS61ON and constitutive NRASQ61R transfectants cells showed increased susceptibility to cytotoxicity by both HLA class I antigen–restricted and nonspecific T cells and up-regulation of several MHC class I antigen processing machinery components. These results suggest a relationship of synthetic lethality between NRAS and BRAF oncogenes, leading to selection against “double-mutant” cells. (Cancer Res 2006; 66(13): 6503-11)
Data from Genetic Disruption of Cytosine DNA Methyltransferase Enzymes Induces Chromosomal Instability in Human Cancer Cells
Authors: Karpf Adam R. , Matsui Sei-ichi
Published: March 2023

Abstract
DNA methyltransferase 1 (DNMT1)–deficient mice are tumor-prone, and this has been proposed to result from the induction of genomic instability. To address whether loss of DNMT1, or the related protein DNMT3b, results in genomic instability in human cancer cells, we used a near-diploid human colorectal cancer cell line, HCT116, in which one or both DNMT genes were disrupted by homologous recombination. Array-based comparative genomic hybridization analyses indicated that double, but not single, DNMT knock-out cells display two specific alterations in regional DNA copy number, suggesting that DNMT deficiency and genomic DNA hypomethylation are not associated with widespread genomic amplifications or deletions in human cancer cells. However, spectral karyotype analyses revealed that DNMT-deficient HCT116 cells are highly unstable with respect to large-scale chromosomal alterations; furthermore, this effect is characterized by a high degree of individual cell heterogeneity. The induction of chromosomal alterations in DNMT-deficient cells was evidenced both by aneuploidy and by large increases in the number of novel chromosomal translocations. Studies of double knock-out cells indicated that the generation of chromosomal alterations is spontaneous and persistent in vitro, meeting the formal definition of genomic instability. In summary, we show that DNMT deficiency in human cancer cells results in constitutive genomic instability manifested by chromosomal translocations.
Data from Human Epididymis Protein 4 (HE4) Is a Secreted Glycoprotein that Is Overexpressed by Serous and Endometrioid Ovarian Carcinomas
Authors: Drapkin Ronny , von Horsten Hans Henning , Lin Yafang , Mok Samuel C. , Crum Christopher P. , Welch William R. , Hecht Jonathan L.
Published: March 2023

Abstract
Among the genes most commonly identified in gene expression profiles of epithelial ovarian carcinomas (EOC) is the gene for human epididymis protein 4 (HE4). To ascertain its clinical utility, we did a comprehensive assessment of HE4 protein expression in benign and malignant ovarian and nonovarian tissues by immunohistochemistry. In comparison with normal surface epithelium, which does not express HE4, we found that cortical inclusion cysts lined by metaplastic Mullerian epithelium abundantly express the protein. Its expression in tumors was restricted to certain histologic subtype: 93% of serous and 100% of endometrioid EOCs expressed HE4, whereas only 50% and 0% of clear cell carcinomas and mucinous tumors, respectively, were positive. Tissue microarrays revealed that the majority of nonovarian carcinomas do not express HE4, consistent with our observation that HE4 protein expression is highly restricted in normal tissue to the reproductive tracts and respiratory epithelium. HE4 is predicted to encode a secreted protein. Using reverse transcription-PCR, we identified ovarian cancer cell lines that endogenously overexpress HE4. Cultured medium from these cells revealed a secreted form of HE4 that is N-glycosylated. This observation is consistent with the recent report that HE4 circulates in the bloodstream of patients with EOC. Therefore, HE4 is a secreted glycoprotein that is overexpressed by serous and endometrioid EOCs. Its expression in cortical inclusion cysts suggests that formation of Mullerian epithelium is a prerequisite step in the development of some types of EOCs.
Data from Lymphatic Zip Codes in Premalignant Lesions and Tumors
Authors: Zhang Lianglin , Giraudo Enrico , Hoffman Jason A. , Hanahan Douglas , Ruoslahti Erkki
Published: March 2023

Abstract
Blood vessels in tumors are morphologically and functionally distinct from normal resting blood vessels. We probed lymphatic vessels in premalignant lesions and tumors by in vivo screening of phage-displayed peptide libraries, asking whether they too have distinctive signatures. The resulting peptides begin to define such signatures. One peptide identified the lymphatics in a human melanoma xenograft. Another recognized the lymphatics in prostate cancers but not in premalignant prostate lesions; this peptide similarly identifies human prostate cancer lymphatics. A third was selective for the lymphatics in the premalignant prostate lesions. A fourth identified the lymphatics in dysplasias and squamous carcinomas of the cervix and skin. None recognize lymphatics in normal tissues. Thus, tumor development is associated with organ- and stage-specific changes in lymphatics. Systemic treatment of mice with fusions of a lymphatic homing peptide and a proapoptotic motif reduced the number of tumor lymphatics in prostate tumor and melanoma, forecasting future lymphatic targeting agents for detection and therapeutic intervention. (Cancer Res 2006; 66(11): 5696-706)
Data from Lack of Correlation between p53-Dependent Transcriptional Activity and the Ability to Induce Apoptosis among 179 Mutant p53s
Authors: Kakudo Yuichi , Shibata Hiroyuki , Otsuka Kazunori , Kato Shunsuke , Ishioka Chikashi
Published: March 2023

Abstract
Tumor suppressor p53-dependent apoptosis is thought to be one of the most important tumor-suppressive functions in human tumorigenesis. However, whether the major mechanism underlying the p53-dependent apoptosis is transactivation dependent or independent remains unclear. Using 179 mutant p53s with diverse transcriptional activities for distinct p53-binding sequences in yeast, we evaluated both their sequence-specific transcriptional activities on six p53 target genes and their ability to induce apoptosis in Saos-2 cells. These mutant p53s also represented diversity in their ability to both transactivate target genes and induce apoptosis. We identified 17 mutant p53s with superior ability to induce apoptosis than wild-type p53 that tend to cluster at residues 121 or 290 to 292. There was no significant correlation between the two functional properties on any single target gene examined. Furthermore, the 17 mutant p53s were not classified in a specific cluster by hierarchical cluster analysis on their diverse transcriptional activities, indicating that these mutant p53s were not similar in the transcriptional activity of downstream genes. These results suggested that transactivation-dependent apoptosis does not always play a major role in p53-dependent apoptosis, indirectly supporting the importance role of the transactivation-independent mechanism.
Data from Tumor Cell–Derived and Macrophage-Derived Cathepsin B Promotes Progression and Lung Metastasis of Mammary Cancer
Authors: Vasiljeva Olga , Papazoglou Anna , Krüger Achim , Brodoefel Harald , Korovin Matvey , Deussing Jan , Augustin Nicole , Nielsen Boye S. , Almholt Kasper , Bogyo Matthew , Peters Christoph , Reinheckel Thomas
Published: March 2023

Abstract
Proteolysis in close vicinity of tumor cells is a hallmark of cancer invasion and metastasis. We show here that mouse mammary tumor virus–polyoma middle T antigen (PyMT) transgenic mice deficient for the cysteine protease cathepsin B (CTSB) exhibited a significantly delayed onset and reduced growth rate of mammary cancers compared with wild-type PyMT mice. Lung metastasis volumes were significantly reduced in PyMT;ctsb+/−, an effect that was not further enhanced in PyMT;ctsb−/− mice. Furthermore, lung colonization studies of PyMT cells with different CTSB genotypes injected into congenic wild-type mice and in vitro Matrigel invasion assays confirmed a specific role for tumor-derived CTSB in invasion and metastasis. Interestingly, cell surface labeling of cysteine cathepsins by the active site probe DCG-04 detected up-regulation of cathepsin X on PyMT;ctsb−/− cells. Treatment of cells with a neutralizing anti-cathepsin X antibody significantly reduced Matrigel invasion of PyMT;ctsb−/− cells but did not affect invasion of PyMT;ctsb+/+ or PyMT;ctsb+/− cells, indicating a compensatory function of cathepsin X in CTSB-deficient tumor cells. Finally, an adoptive transfer model, in which ctsb+/+, ctsb+/−, and ctsb−/− recipient mice were challenged with PyMT;ctsb+/+ cells, was used to address the role of stroma-derived CTSB in lung metastasis formation. Notably, ctsb−/− mice showed reduced number and volume of lung colonies, and infiltrating macrophages showed a strongly up-regulated expression of CTSB within metastatic cell populations. These results indicate that both cancer cell–derived and stroma cell–derived (i.e., macrophages) CTSB plays an important role in tumor progression and metastasis. (Cancer Res 2006; 66(10): 5242-50)
Data from Cancer Immunotherapy Based on Killing of Salmonella-Infected Tumor Cells
Authors: Avogadri Francesca , Martinoli Chiara , Petrovska Liljana , Chiodoni Claudia , Transidico Pietro , Bronte Vincenzo , Longhi Renato , Colombo Mario P. , Dougan Gordon , Rescigno Maria
Published: March 2023

Abstract
A major obstacle for the development of effective immunotherapy is the ability of tumors to escape the immune system. The possibility to kill tumor cells because they are recognized as infected rather than as malignant could help overcome immune escape mechanisms. Here we report a conceptually new approach of cancer immunotherapy based on in vivo infection of tumors and killing of infected tumor cells. Attenuated but still invasive, Salmonella typhimurium can be successfully exploited to invade melanoma cells that can present antigenic determinants of bacterial origin and become targets for anti-Salmonella–specific T cells. However, to fully appreciate the anticancer therapeutic properties of S. typhimurium, tumor-bearing mice need to be vaccinated against S. typhimurium before intratumoral Salmonella injection. Tumor infection when coupled to anti-Salmonella vaccination leads to 50% to 100% tumor-free mice with a better outcome on larger tumors. Invasive Salmonella also exert an indirect toxic effect on tumor cells through the recruitment of inflammatory cells and the cross-presentation of tumor antigens, which allow induction of tumor-specific immune response. This is effective in retarding the growth of untreated established distant tumors and in protecting the mice from subsequent tumor challenges.
Data from Ras-induced Modulation of CXCL10 and Its Receptor Splice Variant CXCR3-B in MDA-MB-435 and MCF-7 Cells: Relevance for the Development of Human Breast Cancer
Authors: Datta Dipak , Flaxenburg Jesse A. , Laxmanan Sreenivas , Geehan Christopher , Grimm Martin , Waaga-Gasser Ana Maria , Briscoe David M. , Pal Soumitro
Published: March 2023

Abstract
Interactions between chemokines and chemokine receptors have been proposed recently to be of importance in the development and progression of cancer. Human breast cancer cells express the chemokine CXCL10 (IP-10) and also its receptor CXCR3. In this study, we have investigated the role of Ras activation in the regulation of CXCL10 and its receptor splice variant CXCR3-B in two human breast cancer cell lines MDA-MB-435 and MCF-7. In cotransfection assays, using a full-length CXCL10 promoter-luciferase construct, we found that the activated form of Ras, Ha-Ras(12V), promoted CXCL10 transcriptional activation. Ras significantly increased CXCL10 mRNA and protein expression as observed by real-time PCR, fluorescence-activated cell sorting analysis, and ELISA. Selective inhibition of Ha-Ras by small interfering RNA (siRNA) decreased CXCL10 mRNA expression in a dose-dependent manner. Further, using effector domain mutants of Ras, we found that Ras-induced overexpression of CXCL10 is mediated primarily through the Raf and phosphatidylinositol 3-kinase signaling pathways. We also observed that the expression of the splice variant CXCR3-B, known to inhibit cell proliferation, was significantly down-regulated by Ras. Selective inhibition of CXCR3-B using siRNA resulted in an increase in CXCL10-mediated breast cancer cell proliferation through Gi proteins and likely involving CXCR3-A. Finally, we observed intense expression of CXCL10 and CXCR3 in association with human breast cancer in situ, indicating that these observations may be of pathophysiologic significance. Together, these results suggest that activation of Ras plays a critical role in modulating the expression of both CXCL10 and CXCR3-B, which may have important consequences in the development of breast tumors through cancer cell proliferation. (Cancer Res 2006; 66(19): 9509-18)
Data from Spontaneous Human Adult Stem Cell Transformation
Authors: Rubio Daniel , Garcia-Castro Javier , Martín María C. , de la Fuente Ricardo , Cigudosa Juan C. , Lloyd Alison C. , Bernad Antonio
Published: March 2023

Abstract
Human adult stem cells are being evaluated widely for various therapeutic approaches. Several recent clinical trials have reported their safety, showing them to be highly resistant to transformation. The clear similarities between stem cell and cancer stem cell genetic programs are nonetheless the basis of a recent proposal that some cancer stem cells could derive from human adult stem cells. Here we show that although they can be managed safely during the standard ex vivo expansion period (6-8 weeks), human mesenchymal stem cells can undergo spontaneous transformation following long-term in vitro culture (4-5 months). This is the first report of spontaneous transformation of human adult stem cells, supporting the hypothesis of cancer stem cell origin. Our findings indicate the importance of biosafety studies of mesenchymal stem cell biology to efficiently exploit their full clinical therapeutic potential.
Data from Immunologic and Clinical Responses after Vaccinations with Peptide-Pulsed Dendritic Cells in Metastatic Renal Cancer Patients
Authors: Wierecky Jan , Müller Martin R. , Wirths Stefan , Halder-Oehler Edith , Dörfel Daniela , Schmidt Susanne M. , Häntschel Maik , Brugger Wolfram , Schröder Stephen , Horger Marius S. , Kanz Lothar , Brossart Peter
Published: March 2023

Abstract
A phase I trial was conducted to evaluate the feasibility, safety, and efficacy of a dendritic cell–based vaccination in patients with metastatic renal cell carcinoma (RCC). Autologous mature dendritic cells derived from peripheral blood monocytes were pulsed with the HLA-A2-binding MUC1 peptides (M1.1 and M1.2). For the activation of CD4+ T-helper lymphocytes, dendritic cells were further incubated with the PAN-DR-binding peptide PADRE. Dendritic cell vaccinations were done s.c. every 2 weeks for four times and repeated monthly until tumor progression. After five dendritic cell injections, patients additionally received three injections weekly of low-dose interleukin-2 (1 million IE/m2). The induction of vaccine-induced T-cell responses was monitored using enzyme-linked immunospot and Cr release assays. Twenty patients were included. The treatment was well tolerated with no severe side effects. In six patients, regression of the metastatic sites was induced after vaccinations with three patients achieving an objective response (one complete response, two partial responses, two mixed responses, and one stable disease). Additional four patients were stable during the treatment for up to 14 months. MUC1 peptide–specific T-cell responses in vivo were detected in the peripheral blood mononuclear cells of the six patients with objective responses. Interestingly, in patients responding to the treatment, T-cell responses to antigens not used for vaccinations, such as adipophilin, telomerase, or oncofetal antigen, could be detected, indicating that epitope spreading might occur. This study shows that MUC1 peptide–pulsed dendritic cells can induce clinical and immunologic responses in patients with metastatic RCC. (Cancer Res 2006; 66(11): 5910-8)
Data from Genetic Profiling of Epithelial Cells Expressing E-Cadherin Repressors Reveals a Distinct Role for Snail, Slug, and E47 Factors in Epithelial-Mesenchymal Transition
Authors: Moreno-Bueno Gema , Cubillo Eva , Sarrió David , Peinado Héctor , Rodríguez-Pinilla Socorro María , Villa Sonia , Bolós Victoria , Jordá Mireia , Fabra Angels , Portillo Francisco , Palacios José , Cano Amparo
Published: March 2023

Abstract
The transcription factors Snail, Slug, and bHLH E47 have been recently described as direct repressors of E-cadherin and inducers of epithelial-mesenchymal transition (EMT) and invasion when overexpressed in epithelial cells. Although a role of those factors in tumor progression and invasion has been proposed, whether the different repressors play distinct or redundant roles in the tumorigenic process has not been established. To further investigate this important issue, we have analyzed the gene expression profiling of Madin-Darby canine kidney (MDCK) epithelial cells expressing the different repressors (MDCK-Snail, MDCK-Slug, and MDCK-E47 cells) versus control MDCK cells by cDNA microarrays. A total of 243 clones (228 genes and 15 expressed sequence tags) were found to be differentially expressed between either of the three MDCK-derived cell lines and control MDCK cells. Twenty two of the candidate genes were validated by Northern blot, Western blot, immunofluorescence, and promoter analyses in cell lines and by immunohistochemistry in xenografted tumors. Gene clustering analysis indicated that about a third of the 243 candidate genes were common to MDCK cells expressing Snail, Slug, or E47 factors, whereas the rest of the genes were regulated in only one or two cell types. Differentially regulated genes include those related to EMT (45 genes), transcriptional regulation (18 genes), cell proliferation and signaling (54 genes), apoptosis (12 genes), and angiogenesis (9 genes). These results indicate that Snail, Slug, and E47 transcription factors induce common and specific genetic programs, supporting a differential role of the factors in tumor progression and invasion. (Cancer Res 2006; 66(19): 9543-56)
Data from Insulin-Like Growth Factor Binding Protein-5 Is a Target of Matrix Metalloproteinase-7: Implications for Epithelial-Mesenchymal Signaling
Authors: Hemers Elaine , Duval Cedric , McCaig Catherine , Handley Mark , Dockray Graham J. , Varro Andrea
Published: March 2023

Abstract
Matrix metalloproteinase-7 (MMP-7) is localized to epithelial cells and is up-regulated in many cancers and in inflammation. We now report that MMP-7 targets a key mesenchymal cell type, the myofibroblast. Recombinant MMP-7 stimulated the proliferation and migration of human colonic myofibroblasts. These responses were partly attributable to activation of other MMPs, notably MMP-3 and MMP-8, and to stimulation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways. Using a proteomic approach, we identified insulin-like growth factor binding protein-5 (IGFBP-5) as a previously unsuspected target of MMP-7 produced by colonic myofibroblasts. We present evidence that the MMP-7 cleavage of IGFBP-5 liberates IGF-II that functions as an autocrine myofibroblast growth factor. Thus, MMP-7 may act as a signal from epithelial cells for local recruitment of myofibroblasts and stimulation of their proliferation. Similar effects of MMP-7 produced in epithelial tumors might account for the expansion of stroma through activation of myofibroblasts.
Data from Stem and Progenitor-Like Cells Contribute to the Aggressive Behavior of Human Epithelial Ovarian Cancer
Authors: Bapat Sharmila A. , Mali Avinash M. , Koppikar Chaitanyananda B. , Kurrey Nawneet K.
Published: March 2023

Abstract
The cellular mechanisms underlying the increasing aggressiveness associated with ovarian cancer progression are poorly understood. Coupled with a lack of identification of specific markers that could aid early diagnoses, the disease becomes a major cause of cancer-related mortality in women. Here we present direct evidence that the aggressiveness of human ovarian cancer may be a result of transformation and dysfunction of stem cells in the ovary. A single tumorigenic clone was isolated among a mixed population of cells derived from the ascites of a patient with advanced ovarian cancer. During the course of the study, yet another clone underwent spontaneous transformation in culture, providing a model of disease progression. Both the transformed clones possess stem cell–like characteristics and differentiate to grow in an anchorage-independent manner in vitro as spheroids, although further maturation and tissue-specific differentiation was arrested. Significantly, tumors established from these clones in animal models are similar to those in the human disease in their histopathology and cell architecture. Furthermore, the tumorigenic clones, even on serial transplantation continue to establish tumors, thereby confirming their identity as tumor stem cells. These findings suggest that: (a) stem cell transformation can be the underlying cause of ovarian cancer and (b) continuing stochastic events of stem and progenitor cell transformation define the increasing aggression that is characteristically associated with the disease.
Data from Proapoptotic Activity of Cell-Permeable Anti-Akt Single-Chain Antibodies
Authors: Shin Incheol , Edl Jeniffer , Biswas Swati , Lin P. Charles , Mernaugh Raymond , Arteaga Carlos L.
Published: March 2023

Abstract
We developed anti-Akt1 single-chain antibodies (scFv) by panning a mouse phage–displayed scFv recombinant antibody library. Recombinant scFv that bound glutathione S-transferase (GST)-Akt1 were screened for their ability to inhibit Akt activity in vitro in a kinase reaction containing human recombinant Akt1 and an Akt/serum glucocorticoid-inducible kinase (SGK) substrate. Michaelis-Menten analysis of kinase inhibition by a selected scFv was consistent with scFv-mediated competition with enzyme's substrate for the catalytic site of Akt. To generate a membrane-permeable version of the anti-Akt1 scFv, the scFv gene was subcloned into a GST expression vector carrying a membrane-translocating sequence (MTS) from Kaposi fibroblast growth factor. A purified GST–anti-Akt1–MTS fusion protein accumulated intracellularly in 293T, BT-474, and PyVmT cells in a dose- and time-dependent fashion. Intracellular accumulation correlated temporally with inhibition of p-Ser473 Akt and GSK-3α/β phosphorylation, suggesting that Ser473 is an Akt autophosphorylation site. Phosphorylated (activated) phosphoinositide-dependent kinase 1, mitogen-activated protein kinase, p38, and HER2 (erbB2) were not affected, supporting Akt kinase specificity for the inhibitory scFv. Exogenously expressed constitutively active Akt2 and Akt3 were also inhibited in vitro by the anti-Akt1 fusion protein. Furthermore, GST–anti-Akt1–MTS induced apoptosis in three cancer cell lines that express constitutively active Akt. Finally, systemic treatment with the anti-Akt scFv reduced tumor volume and neovascularization and increased apoptosis in PyVmT-expressing transgenic tumors implanted in mouse dorsal window chambers. Thus, GST–anti-Akt1–MTS is a novel cell-permeable inhibitor of Akt, which selectively inhibits Akt-mediated survival in intact cells both in vitro and in vivo.
Data from Epidermal Growth Factor Receptor Overexpression Results in Increased Tumor Cell Motility In vivo Coordinately with Enhanced Intravasation and Metastasis
Authors: Xue Chengsen , Wyckoff Jeffrey , Liang Fubo , Sidani Mazen , Violini Stefania , Tsai Kun-Lin , Zhang Zhong-Yin , Sahai Erik , Condeelis John , Segall Jeffrey E.
Published: March 2023

Abstract
Although overexpression of the epidermal growth factor receptor (EGFR; ErbB1) has been correlated with poor prognosis in breast and other cancers, clinical trials of ErbB1 inhibitors have shown limited efficacy in inhibiting tumor proliferation. To evaluate other possible roles of ErbB1 in tumor malignancy besides proliferation, we have developed a series of tools for analysis of intravasation. Overexpression of ErbB1 in MTLn3 mammary adenocarcinoma cells results in increased intravasation and lung metastasis from tumors formed by injection of cells in the mammary fat pad. However, increased ErbB1 expression has no effect on primary tumor growth and lung seeding efficiency of cells injected i.v. Chemotactic responses to low concentrations of EGF in vitro and cell motility in vivo in the primary tumor measured using intravital imaging are significantly increased by ErbB1 overexpression. The increased cell motility is restricted to ErbB1-overexpressing cells in tumors containing mixtures of cells expressing different ErbB1 levels, arguing for a cell-autonomous effect of increased ErbB1 expression rather than alteration of the tumor microenvironment. In summary, we propose that ErbB1 overexpression makes more significant contributions to intravasation than growth in some tumors and present a novel model for studying ErbB1 contributions to tumor metastasis via chemotaxis and intravasation. (Cancer Res 2006; 66(1): 192-7)
Data from Activation of Vav/Rho GTPase Signaling by CXCL12 Controls Membrane-Type Matrix Metalloproteinase–Dependent Melanoma Cell Invasion
Authors: Bartolomé Rubén A. , Molina-Ortiz Isabel , Samaniego Rafael , Sánchez-Mateos Paloma , Bustelo Xosé R. , Teixidó Joaquin
Published: March 2023

Abstract
Melanoma cells express the chemokine receptor CXCR4, which confers invasive signals on binding to its ligand CXCL12. We show here that knocking down membrane-type matrix metalloproteinase (MT1-MMP) expression translates into a blockade of invasion across reconstituted basement membranes and type I collagen gels in response to CXCL12, which is the result of lack of MMP-2 activation. Interference with MMP-2 expression further confirms its important role during this invasion. Vav proteins are guanine-nucleotide exchange factors for Rho GTPases that regulate actin dynamics and gene expression. We show that melanoma cells express Vav1 and Vav2, which are activated by CXCL12 involving Jak activity. Blocking Vav expression by RNA interference results in impaired activation of Rac and Rho by CXCL12 and in a remarkable inhibition of CXCL12-promoted invasion. Importantly, up-regulation of MT1-MMP expression by CXCL12, a mechanism contributing to melanoma cell invasion, is blocked by knocking down Vav expression or by inhibiting Jak. Together, these data indicate that activation of Jak/Vav/Rho GTPase pathway by CXCL12 is a key signaling event for MT1-MMP/MMP-2–dependent melanoma cell invasion. (Cancer Res 2006; 66(1): 248-58)
Data from CD24 Expression Causes the Acquisition of Multiple Cellular Properties Associated with Tumor Growth and Metastasis
Authors: Baumann Petra , Cremers Natascha , Kroese Frans , Orend Gertraud , Chiquet-Ehrismann Ruth , Uede Toshi , Yagita Hideo , Sleeman Jonathan P.
Published: March 2023

Abstract
The glycosylphosphatidylinositol-anchored membrane protein CD24 functions as an adhesion molecule for P-selectin and L1 and plays a role in B-cell development and neurogenesis. Over the last few years, a large body of literature has also implicated CD24 expression in tumorigenesis and progression. Here, we show that ectopic CD24 expression can be sufficient to promote tumor metastasis in experimental animals. By developing a doxycycline-inducible system for the expression of CD24 in breast cancer cells, we have also analyzed the cellular properties that CD24 expression influences. We found that CD24 expression increased tumor cell proliferation. Furthermore, in addition to promoting binding to P-selectin, CD24 expression also indirectly stimulated cell adhesion to fibronectin, collagens I and IV, and laminin through the activation of α3β1 and α4β1 integrin activity. Moreover, CD24 expression supported rapid cell spreading and strongly induced cell motility and invasion. CD24-induced proliferation and motility were integrin independent. Together, these observations implicate CD24 in the regulation of multiple cell properties of direct relevance to tumor growth and metastasis.
Data from Coordinated Functions of E-Cadherin and Transforming Growth Factor β Receptor II In vitro and In vivo
Authors: Andl Claudia D. , Fargnoli Brenton B. , Okawa Takaomi , Bowser Mark , Takaoka Munenori , Nakagawa Hiroshi , Klein-Szanto Andres , Hua Xianxin , Herlyn Meenhard , Rustgi Anil K.
Published: March 2023

Abstract
In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and loss of E-cadherin is a hallmark of tumor progression fostering cancer cell invasion and metastasis. To examine E-cadherin loss in squamous cell cancers, we used primary human esophageal epithelial cells (keratinocytes) as a platform and retrovirally transduced wild-type and dominant-negative forms of E-cadherin into these cells. We found decreased cell adhesion in the cells expressing dominant-negative E-cadherin, thereby resulting in enhanced migration and invasion. To analyze which molecular pathway(s) may modulate these changes, we conducted microarray analysis and found up-regulation of transforming growth factor β receptor II (TβRII) in the wild-type E-cadherin-overexpressing cells, which was confirmed by real-time PCR and Western blot analyses. To investigate the in vivo relevance of this finding, we analyzed tissue microarrays of paired esophageal squamous cell carcinomas and adjacent normal esophagus, and we could show a coordinated loss of E-cadherin and TβRII in ∼80% of tumors. To determine if there may be an E-cadherin-dependent regulation of TβRII, we show the physical interaction of E-cadherin with TβRII and that this is mediated through the extracellular domains of E-cadherin and TβRII, respectively. In addition, TβRI is recruited to this complex. When placed in the context of three-dimensional cell culture, which reflects the physiologic microenvironment, TβRII-mediated cell signaling is dependent upon intact E-cadherin function. Our results, which suggest that E-cadherin regulates TβRII function, have important implications for epithelial carcinogenesis characterized through the frequent occurrence of E-cadherin and TβRII loss. (Cancer Res 2006; 66(20): 9878-85)
Data from Functional Profiling: From Microarrays via Cell-Based Assays to Novel Tumor Relevant Modulators of the Cell Cycle
Authors: Arlt Dorit , Huber Wolfgang , Liebel Urban , Schmidt Christian , Majety Meher , Sauermann Mamatha , Rosenfelder Heiko , Bechtel Stephanie , Mehrle Alexander , Bannasch Detlev , Schupp Ingo , Seiler Markus , Simpson Jeremy C. , Hahne Florian , Moosmayer Petra , Ruschhaupt Markus , Guilleaume Birgit , Wellenreuther Ruth , Pepperkok Rainer , Sültmann Holger , Poustka Annemarie , Wiemann Stefan
Published: March 2023

Abstract
Cancer transcription microarray studies commonly deliver long lists of “candidate” genes that are putatively associated with the respective disease. For many of these genes, no functional information, even less their relevance in pathologic conditions, is established as they were identified in large-scale genomics approaches. Strategies and tools are thus needed to distinguish genes and proteins with mere tumor association from those causally related to cancer. Here, we describe a functional profiling approach, where we analyzed 103 previously uncharacterized genes in cancer relevant assays that probed their effects on DNA replication (cell proliferation). The genes had previously been identified as differentially expressed in genome-wide microarray studies of tumors. Using an automated high-throughput assay with single-cell resolution, we discovered seven activators and nine repressors of DNA replication. These were further characterized for effects on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling (G1-S transition) and anchorage-independent growth (tumorigenicity). One activator and one inhibitor protein of ERK1/2 activation and three repressors of anchorage-independent growth were identified. Data from tumor and functional profiling make these proteins novel prime candidates for further in-depth study of their roles in cancer development and progression. We have established a novel functional profiling strategy that links genomics to cell biology and showed its potential for discerning cancer relevant modulators of the cell cycle in the candidate lists from microarray studies.
Data from Novel 5′ Untranslated Region Variants of BCRP mRNA Are Differentially Expressed in Drug-Selected Cancer Cells and in Normal Human Tissues: Implications for Drug Resistance, Tissue-Specific Expression, and Alternative Promoter Usage
Authors: Nakanishi Takeo , Bailey-Dell Kimberly J. , Hassel Bret A. , Shiozawa Ken , Sullivan Daniel M. , Turner Joel , Ross Douglas D.
Published: March 2023

Abstract
To investigate transcriptional activation of the breast cancer resistance protein gene (BCRP/ABCG2), we examined the 5′ untranslated region of BCRP mRNA in cell lines with high BCRP transcriptional activity and in normal tissues. Human choriocarcinoma cells with high endogenous BCRP expression (JAR and BeWo) and human cancer cells (MCF-7 and Igrov1) and their BCRP-overexpressing, drug-selected, multidrug-resistant derivatives (MCF-7/AdrVp, Igrov1/MX3, and Igrov1/T8) were studied. Rapid amplification of 5′-cDNA ends-PCR (5′RACE-PCR) revealed at least three novel forms of the untranslated exon 1 (E1a, E1b, and E1c) that are spliced to a common exon 2, with differential expression of these splice variants in the drug-selected cell lines. Additionally, sequence analysis of the 5′RACE-PCR products revealed multiple transcriptional start sites for each variant, particularly in the drug-selected cells. The E1c isoform predominated in drug-selected MCF-7 cell lines and was translated more efficiently in MCF-7 cells than the E1a isoform. Varying patterns of expression of the exon 1 isoforms were observed in a variety of human tissues, suggesting that tissue-specific alternative promoters of BCRP exist. In summary, we find that BCRP overexpression in the drug-selected cells is accompanied by multiple transcriptional start sites and predominance of the more efficiently translated E1c isoform. The exon 1 variation we observe suggests that alternative promoters of the BCRP gene exist. (Cancer Res 2006; 66(10): 5007-11)
Data from Genotoxic Stress Induces Coordinately Regulated Alternative Splicing of the p53 Modulators MDM2 and MDM4
Authors: Chandler Dawn S. , Singh Ravi K. , Caldwell Lisa C. , Bitler Jaquelyn L. , Lozano Guillermina
Published: March 2023

Abstract
The tumor suppressor protein p53 is a transcription factor that induces G1 arrest of the cell cycle and/or apoptosis. The murine double-minute protein MDM2 and its homologue MDM4 (also known as MDMX) are critical regulators of p53. Altered transcripts of the human homologue of mdm2, MDM2, have been identified in human tumors, such as invasive carcinoma of the breast, lung carcinoma, and liposarcoma. MDM2 alternate forms act to negatively regulate the normal MDM2 gene product, thus activating p53. Although many reports have documented a plethora of tumor types characterized by MDM2 alternative transcripts, few have investigated the signals that might initiate alternative splicing. We have identified a novel role of these alternative MDM2 transcripts in the normal surveillance mechanism of the cell and in DNA damage response. We report that alternate forms of MDM2 are detected after UV irradiation. Furthermore, we show that mouse cells treated with UV are also characterized by alternative transcripts of mdm2, suggesting that this is an important and evolutionarily conserved mechanism for regulating the expression of MDM2/mdm2. An additional p53 regulator and mdm2 family member, MDM4, is likewise alternatively spliced following UV irradiation. By activating alternative splicing of both MDM2 and MDM4, yet another layer of p53 regulation is initiated by the cells in response to damage. A stepwise model for malignant conversion by which alternate forms of MDM2 and MDM4 place selective pressure on the cells to acquire additional alterations in the p53 pathway is herein proposed. (Cancer Res 2006; 66(19): 9502-8)
Data from Real-time Imaging of Lymphogenic Metastasis in Orthotopic Human Breast Cancer
Authors: Dadiani Maya , Kalchenko Vyacheslav , Yosepovich Ady , Margalit Raanan , Hassid Yaron , Degani Hadassa , Seger Dalia
Published: March 2023

Abstract
Metastatic spread to regional lymph nodes is one of the earliest events of tumor cell dissemination and presents a most significant prognostic factor for predicting survival of cancer patients. Real-time in vivo imaging of the spread of tumor cells through the lymphatic system can enhance our understanding of the metastatic process. Herein, we describe the use of in vivo fluorescence microscopy imaging to monitor the progression of lymph node metastasis as well as the course of spontaneous metastasis through the lymphatic system of orthotopic MDA-MB-231 human breast cancer tumors in severe combined immunodeficient mice. High-resolution noninvasive visualization of metastasizing cancer cells in the inguinal lymph nodes was achieved using cells expressing high levels of red fluorescent protein. Sequential imaging of these lymph nodes revealed the initial invasion of the tumor cells through the lymphatic system into the subcapsular sinuses followed by intrusion into the parenchyma of the nodes. FITC-dextran injected i.d. in the tumor area enabled simultaneous tracking of lymphatic vessels, labeled in green, and disseminated red cancer cells within these vessels. Fast snapshots of spontaneously metastasizing cells in the lymphatic vessels monitored the movement of a few tumor cells and the development of clumps clustered at lymphatic vessel junctions. Quantification of high interstitial fluid pressure (IFP) in the tumors and fast drainage rates of the FITC-dextran into the peritumoral lymphatic vessels suggested an IFP-induced intravasation into the lymphatic system. This work presents unprecedented live fluorescence images that may help to clarify the steps occurring in the course of spontaneous lymphogenic metastasis. (Cancer Res 2006; 66(16): 8037-41)
Data from Silencing of Peroxiredoxin 2 and Aberrant Methylation of 33 CpG Islands in Putative Promoter Regions in Human Malignant Melanomas
Authors: Furuta Junichi , Nobeyama Yoshimasa , Umebayashi Yoshihiro , Otsuka Fujio , Kikuchi Kanako , Ushijima Toshikazu
Published: March 2023

Abstract
Aberrant methylation of promoter CpG islands (CGI) is involved in silencing of tumor suppressor genes and is also a potential cancer biomarker. Here, to identify CGIs aberrantly methylated in human melanomas, we did a genome-wide search using methylation-sensitive representational difference analysis. CGIs in putative promoter regions of 34 genes (ABHD9, BARHL1, CLIC5, CNNM1, COL2A1, CPT1C, DDIT4L, DERL3, DHRS3, DPYS, EFEMP2, FAM62C, FAM78A, FLJ33790, GBX2, GPR10, GPRASP1, HOXA9, HOXD11, HOXD12, HOXD13, p14ARF, PAX6, PRDX2, PTPRG, RASD1, RAX, REC8L1, SLC27A3, TGFB2, TLX2, TMEM22, TMEM30B, and UNC5C) were found to be methylated in at least 1 of 13 melanoma cell lines but not in two cultured normal melanocytes. Among these genes, Peroxiredoxin 2 (PRDX2) was expressed in normal melanocytes, and its expression was lost in melanomas with methylation. The loss of expression was restored by treatment of melanomas with a demethylating agent 5-aza-2′-deoxycytidine. In surgical melanoma specimens, methylation of PRDX2 was detected in 3 of 36 (8%). Furthermore, immunohistochemical analysis of PRDX2 showed that disappearance of immunoreactivity tends to associate with its methylation. PRDX2 was recently reported to be a negative regulator of platelet-derived growth factor signaling, and its silencing was suggested to be involved in melanomas. On the other hand, 12 CGIs were methylated in ≥9 of the 13 melanoma cell lines and are considered as candidate melanoma biomarkers. (Cancer Res 2006; 66(12): 6080-6)
Data from Notch1-Induced Transformation of RKE-1 Cells Requires Up-regulation of Cyclin D1
Authors: Stahl Mark , Ge Changhui , Shi Shaolin , Pestell Richard G. , Stanley Pamela
Published: March 2023

Abstract
RKE-1 cells induced to overexpress activated Notch1 (RKE-ER-Nic) exhibit increased cyclin D1 transcripts and become transformed. However, the oncogenic pathway of Notch1-induced transformation is not known. Here, we use mutational analysis to functionally identify the sole region of the cyclin D1 promoter that responds to activated Notch1. The same region responds to activated Notch4 as well as to physiologic Notch ligand-induced Notch receptor signaling. The cyclin D1 gene was subsequently found to be a physiologic target of Notch signaling in Pofut1−/− mouse embryos defective in canonical Notch signaling and in embryos with an inactivating mutation in Notch1. To determine if Notch1-induced cyclin D1 expression in RKE-ER-Nic cells plays a direct role in transformation, cyclin D1 up-regulation was inhibited using a cyclin D1 antisense cDNA. We report here that transformation of RKE-ER-Nic cells is dependent on increased expression of cyclin D1 protein, which represents a new mechanism of Notch1-induced transformation. (Cancer Res 2006; 66(15): 7562-70)