bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Dietary Genistein Inhibits Metastasis of Human Prostate Cancer in Mice
Authors: Lakshman Minalini , Xu Li , Ananthanarayanan Vijayalakshmi , Cooper Joshua , Takimoto Chris H. , Helenowski Irene , Pelling Jill C. , Bergan Raymond C.
Published: March 2023

Abstract
Dietary genistein has been linked to lower prostate cancer (PCa) mortality. Metastasis is the ultimate cause of death from PCa. Cell detachment and invasion represent early steps in the metastatic cascade. We had shown that genistein inhibits PCa cell detachment and cell invasion in vitro. Genistein-mediated inhibition of activation of focal adhesion kinase (FAK) and of the p38 mitogen-activated protein kinase (MAPK)–heat shock protein 27 (HSP27) pathway has been shown by us to regulate PCa cell detachment and invasion effects, respectively. To evaluate the antimetastatic potential of genistein, we developed an animal model suited to evaluating antimetastatic drug efficacy. Orthotopically implanted human PC3-M PCa cells formed lung micrometastasis by 4 weeks in >80% of inbred athymic mice. Feeding mice dietary genistein before implantation led to blood concentrations similar to those measured in genistein-consuming men. Genistein decreased metastases by 96%, induced nuclear morphometric changes in PC3-M cells indicative of increased adhesion (i.e., decreased detachment) but did not alter tumor growth. Genistein increased tumor levels of FAK, p38 MAPK, and HSP27 “promotility” proteins. However, the ratio of phosphorylated to total protein trended downward, indicating a failure to increase relative amounts of activated protein. This study describes a murine model of human PCa metastasis well suited for testing antimetastatic drugs. It shows for the first time that dietary concentrations of genistein can inhibit PCa cell metastasis. Increases in promotility proteins support the notion of cellular compensatory responses to antimotility effects induced by therapy. Studies of antimetastatic efficacy in man are warranted and are under way. [Cancer Res 2008;68(6):2024–32]
Data from EpCAM and α-Fetoprotein Expression Defines Novel Prognostic Subtypes of Hepatocellular Carcinoma
Authors: Yamashita Taro , Forgues Marshonna , Wang Wei , Kim Jin Woo , Ye Qinghai , Jia Huliang , Budhu Anuradha , Zanetti Krista A. , Chen Yidong , Qin Lun-Xiu , Tang Zhao-You , Wang Xin Wei
Published: March 2023

Abstract
The heterogeneous nature of hepatocellular carcinoma (HCC) and the lack of appropriate biomarkers have hampered patient prognosis and treatment stratification. Recently, we have identified that a hepatic stem cell marker, epithelial cell adhesion molecule (EpCAM), may serve as an early biomarker of HCC because its expression is highly elevated in premalignant hepatic tissues and in a subset of HCC. In this study, we aimed to identify novel HCC subtypes that resemble certain stages of liver lineages by searching for EpCAM-coexpressed genes. A unique signature of EpCAM-positive HCCs was identified by cDNA microarray analysis of 40 HCC cases and validated by oligonucleotide microarray analysis of 238 independent HCC cases, which was further confirmed by immunohistochemical analysis of an additional 101 HCC cases. EpCAM-positive HCC displayed a distinct molecular signature with features of hepatic progenitor cells including the presence of known stem/progenitor markers such as cytokeratin 19, c-Kit, EpCAM, and activated Wnt-β-catenin signaling, whereas EpCAM-negative HCC displayed genes with features of mature hepatocytes. Moreover, EpCAM-positive and EpCAM-negative HCC could be further subclassified into four groups with prognostic implication by determining the level of α-fetoprotein (AFP). These four subtypes displayed distinct gene expression patterns with features resembling certain stages of hepatic lineages. Taken together, we proposed an easy classification system defined by EpCAM and AFP to reveal HCC subtypes similar to hepatic cell maturation lineages, which may enable prognostic stratification and assessment of HCC patients with adjuvant therapy and provide new insights into the potential cellular origin of HCC and its activated molecular pathways. [Cancer Res 2008;68(5):1451–61]
Data from Characterization of TMPRSS2-ETS Gene Aberrations in Androgen-Independent Metastatic Prostate Cancer
Authors: Mehra Rohit , Tomlins Scott A. , Yu Jianjun , Cao Xuhong , Wang Lei , Menon Anjana , Rubin Mark A. , Pienta Kenneth J. , Shah Rajal B. , Chinnaiyan Arul M.
Published: March 2023

Abstract
Recurrent gene fusions between the androgen-regulated gene TMPRSS2 and the ETS transcription factor family members ERG, ETV1, and ETV4 have been identified as a critical event in prostate cancer development. In this study, we characterized the prevalence and diversity of these rearrangements in hormone-refractory metastatic prostate cancer. We used a fluorescence in situ hybridization (FISH) split probe strategy to comprehensively evaluate TMPRSS2-ETS aberrations across 97 nonosseous metastatic sites of prostate cancer from 30 rapid autopsies of men who died of androgen-independent disease. Tissue microarrays were constructed representing multiple metastatic sites from each patient, and split signal FISH probes for TMPRSS2, ERG, ETV1, and ETV4 were used to assess for TMPRSS2-ETS rearrangements. In patients exhibiting these aberrations, multiple sites from an individual case harbored the same gene fusion molecular subtype suggesting clonal expansion of disease. The most common prostate cancer gene fusion, TMPRSS2-ERG, can be generated by the mechanism of interstitial deletion (Edel) about 39% to 60% of the time in clinically localized disease. Interestingly, we observed that all of the androgen-independent metastatic prostate cancer sites harboring TMPRSS2-ERG were associated with Edel. These findings suggest that TMPRSS2-ERG with Edel is an aggressive and, in this study, uniformly lethal molecular subtype of prostate cancer associated with androgen-independent disease. [Cancer Res 2008;68(10):3584–90]
Data from LKB1 Is Necessary for Akt-Mediated Phosphorylation of Proapoptotic Proteins
Authors: Zhong Diansheng , Liu Xiuju , Khuri Fadlo R. , Sun Shi-Yong , Vertino Paula M. , Zhou Wei
Published: March 2023

Abstract
LKB1 plays the role of tumor suppressor, opposite to Akt, by negatively regulating mammalian target of rapamycin through the activation of AMP-activated protein kinase and TSC signaling. We have discovered a novel, potentially oncogenic role for LKB1 as a supporter of Akt-mediated phosphorylation of proapoptotic proteins. We found that Akt activation led to increased phosphorylation of FoxO3a at Thr32 in LKB1 wild-type cells but not in LKB1-null cells. Depletion of LKB1 in the cells with wild-type LKB1 resulted in attenuation of that phosphorylation of FoxO3a by activated Akt, whereas the restoration of LKB1 function in LKB1-null cells reestablished Akt-mediated FoxO3a phosphorylation. On expanding our analysis to other Akt targets, using isogenic LKB1 knockdown cell line pairs and a phospho-specific antibody microarray, we observed that there was a requirement for LKB1 in the phosphorylation of other Akt downstream targets, including Ask1 (Ser83), Bad (Ser136), FoxO1 (Ser319), FoxO4 (Ser197), and glycogen synthase kinase 3β (GSK3β; Ser9). Because the phosphorylation of these sites by Akt suppresses apoptosis, the requirement of LKB1 suggests that LKB1 may have an antiapoptotic role in tumor cells with constitutively active Akt. Indeed, we found that the suppression of LKB1 expression led to apoptosis in three cell lines in which Akt is constitutively active but not in two cell lines without Akt activation. This observation may explain the lack of LKB1 somatic mutations in brain, breast, and colon cancers, where Akt is frequently activated due to mutations in phosphatidylinositol 3-kinase, PTEN, or Akt itself. [Cancer Res 2008;68(18):7270–7]
Data from A Novel Mechanism of Late Gene Silencing Drives SV40 Transformation of Human Mesothelial Cells
Authors: Carbone Michele , Pannuti Antonio , Zhang Lei , Testa Joseph R. , Bocchetta Maurizio
Published: March 2023

Abstract
Suppression of the late gene expression, usually by integration of the viral DNA into the host genome, is a critical step in DNA tumor virus carcinogenesis. SV40 induces high rates of transformation in infected primary human mesothelial cells in tissue culture, leading to the formation of immortal cell lines (SV40-transformed human mesothelial cell lines, S-HML). The studies described here were designed to elucidate the unusual susceptibility of primary human mesothelial cells to SV40 carcinogenesis. We found that S-HML contained wild-type, mostly episomal SV40 DNA. In these cells, the early genes that code for the viral oncogenes are expressed; at the same time, the synthesis of the late genes, capsid proteins, is suppressed and S-HML are not lysed. Late gene suppression is achieved through the production of antisense RNA molecules. These antisense RNA molecules originate in the early region of the SV40 circular chromosome and proceed in antisense orientation into the late gene region, leading to the formation of highly unstable double-strand RNA, which is rapidly degraded. Our results reveal a novel biological mechanism responsible for the suppression of late viral gene products, an important step in viral carcinogenesis in humans. [Cancer Res 2008;68(22):9488–96]
Data from Negative Regulation of YAP by LATS1 Underscores Evolutionary Conservation of the Drosophila Hippo Pathway
Authors: Zhang Jianmin , Smolen Gromoslaw A. , Haber Daniel A.
Published: March 2023

Abstract
The Hippo pathway defines a novel signaling cascade regulating cell proliferation and survival in Drosophila, which involves the negative regulation of the transcriptional coactivator Yorkie by the kinases Hippo and Warts. We have recently shown that the human ortholog of Yorkie, YAP, maps to a minimal amplification locus in mouse and human cancers, and that it mediates dramatic transforming activity in MCF10A primary mammary epithelial cells. Here, we show that LATS proteins (mammalian orthologs of Warts) interact directly with YAP in mammalian cells and that ectopic expression of LATS1, but not LATS2, effectively suppresses the YAP phenotypes. Furthermore, shRNA-mediated knockdown of LATS1 phenocopies YAP overexpression. Because this effect can be suppressed by simultaneous YAP knockdown, it suggests that YAP is the primary target of LATS1 in mammalian cells. Expression profiling of genes induced by ectopic expression of YAP or by knockdown of LATS1 reveals a subset of potential Hippo pathway targets implicated in epithelial-to-mesenchymal transition, suggesting that this is a key feature of YAP signaling in mammalian cells. [Cancer Res 2008;68(8):2789–94]
Data from Transforming Growth Factor–β Regulates Mammary Carcinoma Cell Survival and Interaction with the Adjacent Microenvironment
Authors: Bierie Brian , Stover Daniel G. , Abel Ty W. , Chytil Anna , Gorska Agnieszka E. , Aakre Mary , Forrester Elizabeth , Yang Li , Wagner Kay-Uwe , Moses Harold L.
Published: March 2023

Abstract
Transforming growth factor (TGF)–β signaling has been associated with early tumor suppression and late tumor progression; however, many of the mechanisms that mediate these processes are not known. Using Cre/LoxP technology, with the whey acidic protein promoter driving transgenic expression of Cre recombinase (WAP-Cre), we have now ablated the type II TGF-β receptor (TβRII) expression specifically within mouse mammary alveolar progenitors. Transgenic expression of the polyoma virus middle T antigen, under control of the mouse mammary tumor virus enhancer/promoter, was used to produce mammary tumors in the absence or presence of Cre (TβRII(fl/fl);PY and TβRII(fl/fl);PY;WC, respectively). The loss of TGF-β signaling significantly decreased tumor latency and increased the rate of pulmonary metastasis. The loss of TGF-β signaling was significantly correlated with increased tumor size and enhanced carcinoma cell survival. In addition, we observed significant differences in stromal fibrovascular abundance and composition accompanied by increased recruitment of F4/80+ cell populations in TβRII(fl/fl);PY;WC mice when compared with TβRII(fl/fl);PY controls. The recruitment of F4/80+ cells correlated with increased expression of known inflammatory genes including Cxcl1, Cxcl5, and Ptgs2 (cyclooxygenase-2). Notably, we also identified an enriched K5+ dNp63+ cell population in primary TβRII(fl/fl);PY;WC tumors and corresponding pulmonary metastases, suggesting that loss of TGF-β signaling in this subset of carcinoma cells can contribute to metastasis. Together, our current results indicate that loss of TGF-β signaling in mammary alveolar progenitors may affect tumor initiation, progression, and metastasis through regulation of both intrinsic cell signaling and adjacent stromal-epithelial interactions in vivo. [Cancer Res 2008;68(6):1809–19]
Data from Blockade of Wnt Signaling Inhibits Angiogenesis and Tumor Growth in Hepatocellular Carcinoma
Authors: Hu Jie , Dong Aiwen , Fernandez-Ruiz Veronica , Shan Juanjuan , Kawa Milosz , Martínez-Ansó Eduardo , Prieto Jesus , Qian Cheng
Published: March 2023

Abstract
Aberrant activation of Wnt signaling plays an important role in hepatocarcinogenesis. In addition to direct effects on tumor cells, Wnt signaling might be involved in the organization of tumor microenvironment. In this study, we have explored whether Wnt signaling blockade by exogenous expression of Wnt antagonists could inhibit tumor angiogenesis and control tumor growth. Human Wnt inhibitory factor 1 (WIF1) and secreted frizzled-related protein 1 (sFRP1) were each fused with Fc fragment of human IgG1 to construct fusion proteins WIF1-Fc and sFRP1-Fc. The recombinant adenoviral vectors carrying WIF1-Fc and sFRP1-Fc driven by cytomegalovirus promoter were constructed. Ad-WIF1-Fc or Ad-sFRP1-Fc induced the expression and correct conformation of WIF1-Fc and sFRP1-Fc fusion proteins. These molecules caused down-regulation of E2F1, cyclin D1, and c-myc and promoted cell apoptosis in hepatocellular carcinoma cells. Treatment of established hepatocellular carcinoma tumors with Ad-WIF1-Fc and/or Ad-sFRP1-Fc resulted in significant inhibition of tumor growth and prolonged animal survival. The antineoplastic effect was associated with increased apoptosis of tumor cells, reduced microvessel density, and decreased expression of vascular endothelial growth factor and stromal cell–derived factor-1. Tube formation and migration of human microvascular endothelial cells and mouse endothelial progenitor cells (EPC) were significantly inhibited by both WIF1-Fc and sFRP1-Fc. In addition, these molecules blocked EPC differentiation and caused EPC apoptosis. Our data indicate that Wnt antagonists WIF1-Fc and sFRP1-Fc inhibit Wnt signaling and exert potent antitumor activity by increasing the apoptosis rate in tumor cells and by impairing tumor vascularization. [Cancer Res 2009;69(17):6951–59]
Data from Costimulation as a Platform for the Development of Vaccines: A Peptide-Based Vaccine Containing a Novel Form of 4-1BB Ligand Eradicates Established Tumors
Authors: Sharma Rajesh K. , Elpek Kutlu G. , Yolcu Esma S. , Schabowsky Rich-Henry , Zhao Hong , Bandura-Morgan Laura , Shirwan Haval
Published: March 2023

Abstract
Vaccines represent an attractive treatment modality for the management of cancer primarily because of their specificity and generation of immunologic memory important for controlling recurrences. However, the efficacy of therapeutic vaccines may require formulations that not only generate effective immune responses but also overcome immune evasion mechanisms employed by progressing tumor. Costimulatory molecules play critical roles in modulating innate, adaptive, and regulatory immunity and have potential to serve as effective immunomodulatory components of therapeutic vaccines. In this study, we tested the function of a novel soluble form of 4-1BB ligand (4-1BBL) costimulatory molecule in modulating innate, adaptive, and regulatory immunity and assessed its therapeutic efficacy in the HPV-16 E7-expressing TC-1 cervical cancer and survivin-expressing 3LL lung carcinoma mouse models. Vaccination with 4-1BBL activated dendritic cells and enhanced antigen uptake, generated CD8+ T-cell effector/memory responses, and endowed T effector cells refractory to suppression by CD4+CD25+FoxP3+ T regulatory cells. Immunization with 4-1BBL in combination with an E7 peptide or survivin protein resulted in eradication of TC-1 and 3LL tumors, respectively. 4-1BBL was more effective than TLR agonists LPS, MPL, and CpG and an agonistic 4-1BB antibody as a component of E7 peptide-based therapeutic vaccine for the generation of immune responses and eradication of TC-1 established tumors in the absence of detectable toxicity. Therapeutic efficacy was associated with reversal of tumor-mediated nonresponsiveness/anergy as well as establishment of long-term CD8+ T-cell memory. Potent pleiotropic immunomodulatory activities combined with lack of toxicity highlight the potential of 4-1BBL molecule as an effective component of therapeutic cancer vaccines. [Cancer Res 2009;69(10):4319–26]
Data from Direct Targeting of the Mucin 1 Oncoprotein Blocks Survival and Tumorigenicity of Human Breast Carcinoma Cells
Authors: Raina Deepak , Ahmad Rehan , Joshi Maya Datt , Yin Li , Wu Zekui , Kawano Takeshi , Vasir Baldev , Avigan David , Kharbanda Surender , Kufe Donald
Published: March 2023

Abstract
The mucin 1 (MUC1) oncoprotein is aberrantly overexpressed by ∼90% of human breast cancers. However, there are no effective agents that directly inhibit MUC1 and induce death of breast cancer cells. We have synthesized a MUC1 inhibitor (called GO-201) that binds to the MUC1 cytoplasmic domain and blocks the formation of MUC1 oligomers in cells. GO-201, and not an altered version, attenuates targeting of MUC1 to the nucleus of human breast cancer cells, disrupts redox balance, and activates the DNA damage response. GO-201 also arrests growth and induces necrotic death. By contrast, the MUC1 inhibitor has no effect on cells null for MUC1 expression or nonmalignant mammary epithelial cells. Administration of GO-201 to nude mice bearing human breast tumor xenografts was associated with loss of tumorigenicity and extensive necrosis, which results in prolonged regression of tumor growth. These findings show that targeting the MUC1 oncoprotein is effective in inducing death of human breast cancer cells in vitro and in tumor models. [Cancer Res 2009;69(12):5133–41]
Data from Mammary Epithelial-Specific Ablation of the Focal Adhesion Kinase Suppresses Mammary Tumorigenesis by Affecting Mammary Cancer Stem/Progenitor Cells
Authors: Luo Ming , Fan Huaping , Nagy Tamas , Wei Huijun , Wang Chenran , Liu Suling , Wicha Max S. , Guan Jun-Lin
Published: March 2023

Abstract
Focal adhesion kinase (FAK) has been implicated in the development of cancers, including those of the breast. Nevertheless, the molecular and cellular mechanisms by which FAK promotes mammary tumorigenesis in vivo are not well understood. Here, we show that targeted deletion of FAK in mouse mammary epithelium significantly suppresses mammary tumorigenesis in a well-characterized breast cancer model. Ablation of FAK leads to the depletion of a subset of bipotent cells in the tumor that express both luminal marker keratin 8/18 and basal marker keratin 5. Using mammary stem/progenitor markers, including aldehyde dehydrogenase, CD24, CD29, and CD61, we further revealed that ablation of FAK reduced the pool of cancer stem/progenitor cells in primary tumors of FAK-targeted mice and impaired their self-renewal and migration in vitro. Finally, through transplantation in NOD-SCID mice, we found that cancer stem/progenitor cells isolated from FAK-targeted mice have compromised tumorigenicity and impaired maintenance in vivo. Together, these results show a novel function of FAK in maintaining the mammary cancer stem/progenitor cell population and provide a novel mechanism by which FAK may promote breast cancer development and progression. [Cancer Res 2009;69(2):466–74]
Data from Fibulin-5 Suppresses Lung Cancer Invasion by Inhibiting Matrix Metalloproteinase-7 Expression
Authors: Yue Wen , Sun Quanhong , Landreneau Rodney , Wu Chuanyue , Siegfried Jill M. , Yu Jian , Zhang Lin
Published: March 2023

Abstract
The high mortality rate of lung cancer is largely due to the spread of disease to other organs. However, the molecular changes driving lung cancer invasion and metastasis remain unclear. In this study, we identified fibulin-5, a vascular ligand for integrin receptors, as a suppressor of lung cancer invasion and metastasis. Fibulin-5 was silenced by promoter hypermethylation in a majority of lung cancer cell lines and primary tumors. It inhibited lung cancer cell invasion and down-regulated matrix metalloproteinase-7 (MMP-7), which promoted lung cancer cell invasion. Knockdown of fibulin-5 was sufficient to stimulate cell invasion and MMP-7 expression. The expression levels of fibulin-5 and MMP-7 were inversely correlated in lung tumors. Suppression of MMP-7 expression by fibulin-5 was mediated by an integrin-binding RGD motif via the extracellular signal-regulated kinase (ERK) pathway. Furthermore, overexpression of fibulin-5 in H460 lung cancer cells inhibited metastasis in mice. Collectively, these results suggest that epigenetic silencing of fibulin-5 promotes lung cancer invasion and metastasis by activating MMP-7 expression through the ERK pathway. [Cancer Res 2009;69(15):6339–46]
Data from Angiopoietin-1 Overexpression Modulates Vascular Endothelium to Facilitate Tumor Cell Dissemination and Metastasis Establishment
Authors: Holopainen Tanja , Huang Huilian , Chen Caiping , Kim Kyung Eun , Zhang Luqing , Zhou Fei , Han Wencan , Li Chaojun , Yu Jun , Wu Jun , Koh Gou Young , Alitalo Kari , He Yulong
Published: March 2023

Abstract
The angiopoietin-1 (Ang1)/Tie2 signaling pathway is known to play an important role in the regulation of vascular maturation and maintenance of vessel integrity. In this study, we have investigated the effect of systemic Tie2 activation or inhibition on tumor growth and metastasis. We found that treatment with Ang1 delivered via an adenoviral vector promoted s.c. implanted tumor metastasis to the lungs. Ang1 treatment did not significantly increase vascular density in the tumors but induced enlargement of blood vessels in both the tumor and normal tissues, which increased tumor cell dissemination into the blood circulation. Ang1 also enhanced the formation of metastatic foci in the lungs when tumor cells were injected into the circulation via the tail vein. The effect of Ang1 on metastasis was validated by a simultaneous treatment with a soluble form of Tie2 (sTie2), which led to the suppression of Ang1-induced increase of tumor metastasis. Furthermore, using a highly metastatic tumor model, we confirmed that systemic treatment with sTie2 suppressed tumor metastasis to the lungs and lymph nodes, whereas tumor-associated angiogenesis and lymphangiogenesis were not significantly affected. This suggests that the Ang1/Tie2 signals contribute to tumor progression by increasing vascular entry and exit of tumor cells to facilitate tumor dissemination and establishment of metastases. [Cancer Res 2009;69(11):4656–64]
Data from Compartment-Specific Roles of ATP-Binding Cassette Transporters Define Differential Topotecan Distribution in Brain Parenchyma and Cerebrospinal Fluid
Authors: Shen Jun , Carcaboso Angel M. , Hubbard K. Elaine , Tagen Michael , Wynn Henry G. , Panetta John C. , Waters Christopher M. , Elmeliegy Mohamed A. , Stewart Clinton F.
Published: March 2023

Abstract
Topotecan is a substrate of the ATP-binding cassette transporters P-glycoprotein (P-gp/MDR1) and breast cancer resistance protein (BCRP). To define the role of these transporters in topotecan penetration into the ventricular cerebrospinal fluid (vCSF) and brain parenchymal extracellular fluid (ECF) compartments, we performed intracerebral microdialysis on transporter-deficient mice after an intravenous dose of topotecan (4 mg/kg). vCSF penetration of unbound topotecan lactone was measured as the ratio of vCSF-to-plasma area under the concentration-time curves. The mean ± SD ratios for wild-type, Mdr1a/b−/−, Bcrp1−/−, and Mdr1a/b−/−Bcrp1−/− mice were 3.07 ± 0.09, 2.57 ± 0.17, 1.63 ± 0.12, and 0.86 ± 0.05, respectively. In contrast, the ECF-to-plasma ratios for wild-type, Bcrp1−/−, and Mdr1a/b−/−Bcrp1−/− mice were 0.36 ± 0.06, 0.42 ± 0.06, and 0.88 ± 0.07. Topotecan lactone was below detectable limits in the ECF of Mdr1a/b−/− mice. When gefitinib (200 mg/kg) was preadministered to inhibit Bcrp1 and P-gp, the vCSF-to-plasma ratio decreased to 1.29 ± 0.09 in wild-type mice and increased to 1.13 ± 0.13 in Mdr1a/b−/−Bcrp1−/− mice, whereas the ECF-to-plasma ratio increased to 0.74 ± 0.14 in wild-type and 1.07 ± 0.03 in Mdr1a/b−/−Bcrp1−/− mice. Preferential active transport of topotecan lactone over topotecan carboxylate was shown in vivo by vCSF lactone-to-carboxylate area under the curve ratios for wild-type, Mdr1a/b−/−, Bcrp1−/−, and Mdr1a/b−/−Bcrp1−/− mice of 5.69 ± 0.83, 3.85 ± 0.64, 3.61 ± 0.46, and 0.78 ± 0.19, respectively. Our results suggest that Bcrp1 and P-gp transport topotecan into vCSF and out of brain parenchyma through the blood-brain barrier. These findings may help to improve pharmacologic strategies to treat brain tumors. [Cancer Res 2009;69(14):5885–92]
Data from Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth
Authors: Funa Nina S. , Kriz Vitezslav , Zang Guangxiang , Calounova Gabriela , Åkerblom Björn , Mares Jaroslav , Larsson Erik , Sun Ying , Betsholtz Christer , Welsh Michael
Published: March 2023

Abstract
Shb (Src homology 2 protein B) is an adapter protein downstream of the vascular endothelial growth factor receptor receptor-2 (VEGFR-2). Previous experiments have suggested a role for Shb in endothelial cell function. Recently, the Shb gene was inactivated and Shb null mice were obtained on a mixed genetic background, but not on C57Bl6 mice. The present study was performed to address endothelial function in the Shb knockout mouse and its relevance for tumor angiogenesis. Tumor growth was retarded in Shb mutant mice, and this correlated with decreased angiogenesis both in tumors and in Matrigel plugs. Shb null mice display an abnormal endothelial ultrastructure in liver sinusoids and heart capillaries with cytoplasmic extensions projecting toward the lumen. Shb null heart VE-cadherin staining was less distinct than that of control heart, exhibiting in the former case a wavy and punctuate pattern. Experiments on isolated endothelial cells suggest that these changes could partly reflect cytoskeletal abnormalities. Vascular permeability was increased in Shb null mice in heart, kidney, and skin, whereas VEGF-stimulated vascular permeability was reduced in Shb null mice. It is concluded that Shb plays an important role in maintaining a functional vasculature in adult mice, and that interference with Shb signaling may provide novel means to regulate tumor angiogenesis. [Cancer Res 2009;69(5):2141–8]
Data from Copy Number Alterations that Predict Metastatic Capability of Human Breast Cancer
Authors: Zhang Yi , Martens John W.M. , Yu Jack X. , Jiang John , Sieuwerts Anieta M. , Smid Marcel , Klijn Jan G.M. , Wang Yixin , Foekens John A.
Published: March 2023

Abstract
We have analyzed the DNA copy numbers for over 100,000 single-nucleotide polymorphism loci across the human genome in genomic DNA from 313 lymph node–negative primary breast tumors for which genome-wide gene expression data were also available. Combining these two data sets allowed us to identify the genomic loci and their mapped genes, having high correlation with distant metastasis. An estimation of the likely response based on published predictive signatures was performed in the identified prognostic subgroups defined by gene expression and DNA copy number data. In the training set of 200 patients, we constructed an 81-gene prognostic copy number signature (CNS) that identified a subgroup of patients with increased probability of distant metastasis in the independent validation set of 113 patients [hazard ratio (HR), 2.8; 95% confidence interval (95% CI), 1.4–5.6] and in an external data set of 116 patients (HR, 3.7; 95% CI, 1.3–10.6). These high-risk patients constituted a subset of the high-risk patients predicted by our previously established 76-gene gene expression signature (GES). This very poor prognostic group identified by CNS and GES was putatively more resistant to preoperative paclitaxel and 5-fluorouracil-doxorubicin-cyclophosphamide combination chemotherapy (P = 0.0048), particularly against the doxorubicin compound, while potentially benefiting from etoposide. Our study shows the feasibility of using copy number alterations to predict patient prognostic outcome. When combined with gene expression–based signatures for prognosis, the CNS refines risk classification and can help identify those breast cancer patients who have a significantly worse outlook in prognosis and a potential differential response to chemotherapeutic drugs. [Cancer Res 2009;69(9):3795–801]
Data from Tumor and Vascular Targeting of a Novel Oncolytic Measles Virus Retargeted against the Urokinase Receptor
Authors: Jing Yuqi , Tong Caili , Zhang Jin , Nakamura Takafumi , Iankov Ianko , Russell Stephen J. , Merchan Jaime R.
Published: March 2023

Abstract
Oncolytic measles virus (MV) induces cell fusion and cytotoxicity in a CD46-dependent manner. Development of fully retargeted oncolytic MVs would improve tumor selectivity. The urokinase-type plasminogen activator receptor (uPAR) is a tumor and stromal target overexpressed in multiple malignancies. MV-H glycoproteins fully retargeted to either human or murine uPAR were engineered and their fusogenic activity was determined. Recombinant human (MV-h-uPA) and murine (MV-m-uPA) uPAR–retargeted MVs expressing enhanced green fluorescent protein (eGFP) were rescued and characterized. Viral expression of chimeric MV-H was shown by reverse transcription-PCR and Western blot. In vitro viral replication was comparable to MV-GFP control. The receptor and species specificity of MV-uPAs was shown in human and murine cells with different levels of uPAR expression. Removal of the NH2-terminal fragment ligand from MV-uPA by factor X(a) treatment ablated the MV-uPA functional activity. Cytotoxicity was shown in uPAR-expressing human and murine cells. MV-h-uPA efficiently infected human endothelial cells and capillary tubes in vitro. I.v. administration of MV-h-uPA delayed tumor growth and prolonged survival in the MDA-MB-231 breast cancer xenograft model. Viral tumor targeting was confirmed by immunohistochemistry. MV-m-uPA transduced murine mammary tumors (4T1) in vivo after intratumor administration. MV-m-uPA targeted murine tumor vasculature after systemic administration, as shown by dual (CD31 and MV-N) staining of tumor capillaries in the MDA-MB-231 model. In conclusion, MV-uPA is a novel oncolytic MV associated with potent and specific antitumor effects and tumor vascular targeting. This is the first retargeted oncolytic MV able to replicate in murine cells and target tumor vasculature in a uPAR-dependent manner. [Cancer Res 2009;69(4):1459–68]
Data from EBV-Induced Human CD8+ NKT Cells Suppress Tumorigenesis by EBV-Associated Malignancies
Authors: Yuling He , Ruijing Xiao , Li Li , Xiang Ji , Rui Zhou , Yujuan Wang , Lijun Zhang , Chunxian Du , Xinti Tan , Wei Xiao , Lang Chen , Yanping Jiang , Tao Xiong , Mengjun Wu , Jie Xiong , Youxin Jin , Jinquan Tan
Published: March 2023

Abstract
The underlying mechanism of the protective and suppressive role of NKT cells in human tumor immunosurveillance remains to be fully elucidated. We show that the frequencies of CD8+ NKT cells in patients with EBV-associated Hodgkin's lymphoma or nasopharyngeal carcinoma are significantly lower than those in healthy EBV carriers. These CD8+ NKT cells in tumor patients are also functionally impaired. In human-thymus-severe combined immunodeficient (hu-thym-SCID) chimeras, EBV challenge efficiently promotes the generation of IFN-γ–biased CD8+ NKT cells. These cells are strongly cytotoxic, drive syngeneic T cells into a Th1 bias, and enhance T-cell cytotoxicity to EBV-associated tumor cells. Interleukin-4–biased CD4+ NKT cells are predominately generated in unchallenged chimeras. These cells are noncytotoxic, drive syngeneic T cells into a Th2 bias, and do not affect T-cell cytotoxicity. In humanized xenogeneic tumor-transplanted hu-thym-SCID chimeras, adoptive transfer with EBV-induced CD8+ NKT cells significantly suppresses tumorigenesis by EBV-associated malignancies. EBV-induced CD8+ NKT cells are necessary and sufficient to enhance the T-cell immunity to EBV-associated malignancies in the hu-thym-SCID chimeras. CD4+ NKT cells are synergetic with CD8+ NKT cells, leading to a more pronounced T-cell antitumor response in the chimeras cotransferred with CD4+ and CD8+ NKT cells. Thus, immune reconstitution with EBV-induced CD8+ NKT cells could be a useful strategy in management of EBV-associated malignancies. [Cancer Res 2009;69(20):7935–44]
Data from Transient Receptor Potential Type Vanilloid 1 Suppresses Skin Carcinogenesis
Authors: Bode Ann M. , Cho Yong-Yeon , Zheng Duo , Zhu Feng , Ericson Marna E. , Ma Wei-Ya , Yao Ke , Dong Zigang
Published: March 2023

Abstract
Blockade of the transient receptor potential channel vanilloid subfamily 1 (TRPV1) is suggested as a therapeutic approach to pain relief. However, TRPV1 is a widely expressed protein whose function might be critical in various nonneuronal physiologic conditions. The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is overexpressed in many human epithelial cancers and is a potential target for anticancer drugs. Here, we show that TRPV1 interacts with EGFR, leading to EGFR degradation. Notably, the absence of TRPV1 in mice results in a striking increase in skin carcinogenesis. The TRPV1 is the first membrane receptor shown to have a tumor-suppressing effect associated with the down-regulation of another membrane receptor. The data suggest that, although a great deal of interest has focused on TRPV1 as a target for pain relief, the chronic blockade of this pain receptor might increase the risk for cancer development. [Cancer Res 2009;69(3):905–13]
Data from PIK3CA and PIK3CB Inhibition Produce Synthetic Lethality when Combined with Estrogen Deprivation in Estrogen Receptor–Positive Breast Cancer
Authors: Crowder Robert J. , Phommaly Chanpheng , Tao Yu , Hoog Jeremy , Luo Jingqin , Perou Charles M. , Parker Joel S. , Miller Melinda A. , Huntsman David G. , Lin Li , Snider Jacqueline , Davies Sherri R. , Olson John A. , Watson Mark A. , Saporita Anthony , Weber Jason D. , Ellis Matthew J.
Published: March 2023

Abstract
Several phosphoinositide 3-kinase (PI3K) catalytic subunit inhibitors are currently in clinical trial. We therefore sought to examine relationships between pharmacologic inhibition and somatic mutations in PI3K catalytic subunits in estrogen receptor (ER)–positive breast cancer, in which these mutations are particularly common. RNA interference (RNAi) was used to determine the effect of selective inhibition of PI3K catalytic subunits, p110α and p110β, in ER+ breast cancer cells harboring either mutation (PIK3CA) or gene amplification (PIK3CB). p110α RNAi inhibited growth and promoted apoptosis in all tested ER+ breast cancer cells under estrogen deprived-conditions, whereas p110β RNAi only affected cells harboring PIK3CB amplification. Moreover, dual p110α/p110β inhibition potentiated these effects. In addition, treatment with the clinical-grade PI3K catalytic subunit inhibitor BEZ235 also promoted apoptosis in ER+ breast cancer cells. Importantly, estradiol suppressed apoptosis induced by both gene knockdowns and BEZ235 treatment. Our results suggest that PI3K inhibitors should target both p110α and p110β catalytic subunits, whether wild-type or mutant, and be combined with endocrine therapy for maximal efficacy when treating ER+ breast cancer. [Cancer Res 2009;69(9):3955–62]
Data from Interleukin-15 Enhances Proteasomal Degradation of Bid in Normal Lymphocytes: Implications for Large Granular Lymphocyte Leukemias
Authors: Hodge Deborah L. , Yang Jun , Buschman Matthew D. , Schaughency Paul M. , Dang Hong , Bere William , Yang Yili , Savan Ram , Subleski Jeff J. , Yin Xiao-Ming , Loughran Thomas P. , Young Howard A.
Published: March 2023

Abstract
Large granular lymphocyte (LGL) leukemia is a clonal proliferative disease of T and natural killer (NK) cells. Interleukin (IL)-15 is important for the development and progression of LGL leukemia and is a survival factor for normal NK and T memory cells. IL-15 alters expression of Bcl-2 family members, Bcl-2, Bcl-XL, Bim, Noxa, and Mcl-1; however, effects on Bid have not been shown. Using an adoptive transfer model, we show that NK cells from Bid-deficient mice survive longer than cells from wild-type control mice when transferred into IL-15-null mice. In normal human NK cells, IL-15 significantly reduces Bid accumulation. Decreases in Bid are not due to alterations in RNA accumulation but result from increased proteasomal degradation. IL-15 up-regulates the E3 ligase HDM2 and we find that HDM2 directly interacts with Bid. HDM2 suppression by short hairpin RNA increases Bid accumulation lending further support for HDM2 involvement in Bid degradation. In primary leukemic LGLs, Bid levels are low but are reversed with bortezomib treatment with subsequent increases in LGL apoptosis. Overall, these data provide a novel molecular mechanism for IL-15 control of Bid that potentially links this cytokine to leukemogenesis through targeted proteasome degradation of Bid and offers the possibility that proteasome inhibitors may aid in the treatment of LGL leukemia. [Cancer Res 2009;69(9):3986–94]
Data from Characterization of a Novel Mitogen-Activated Protein Kinase Kinase 1/2 Inhibitor with a Unique Mechanism of Action for Cancer Therapy
Authors: Daouti Sherif , Wang Huisheng , Li Wen-hui , Higgins Brian , Kolinsky Kenneth , Packman Kathryn , Specian Anthony , Kong Norman , Huby Nicholas , Wen Yang , Xiang Qing , Podlaski Frank J. , He Yang , Fotouhi Nader , Heimbrook David , Niu Huifeng
Published: March 2023

Abstract
The mitogen-activated protein kinase (MAPK) signal transduction pathway plays a central role in regulating tumor cell growth, survival, differentiation, and angiogenesis. The key components of the Ras/Raf/MEK/ERK signal module are frequently altered in human cancers. Targeting this pathway represents a promising anticancer strategy. Small molecule inhibitors targeting MEK1/2 have shown promise in the clinic; however, ultimate clinical proof-of-concept remains elusive. Here, we report a potent and highly selective non–ATP-competitive MEK1/2 inhibitor, RO4927350, with a novel chemical structure and unique mechanism of action. It selectively blocks the MAPK pathway signaling both in vitro and in vivo, which results in significant antitumor efficacy in a broad spectrum of tumor models. Compared with previously reported MEK inhibitors, RO4927350 inhibits not only ERK1/2 but also MEK1/2 phosphorylation. In cancer cells, high basal levels of phospho-MEK1/2 rather than phospho-ERK1/2 seem to correlate with greater sensitivity to RO4927350. Furthermore, RO4927350 prevents a feedback increase in MEK phosphorylation, which has been observed with other MEK inhibitors. We show that B-Raf rather than C-Raf plays a critical role in the feedback regulation. The unique MAPK signaling blockade mediated by RO4927350 in cancer may reduce the risk of developing drug resistance. Thus, RO4927350 represents a novel therapeutic modality in cancers with aberrant MAPK pathway activation. [Cancer Res 2009;69(5):1924–32
Data from Acyl-CoA Synthetase VL3 Knockdown Inhibits Human Glioma Cell Proliferation and Tumorigenicity
Authors: Pei Zhengtong , Sun Peng , Huang Ping , Lal Bachchu , Laterra John , Watkins Paul A.
Published: March 2023

Abstract
The contribution of lipid metabolic pathways to malignancy is poorly understood. Expression of the fatty acyl-CoA synthetase ACSVL3 was found to be markedly elevated in clinical malignant glioma specimens but nearly undetectable in normal glia. ACSVL3 levels correlated with the malignant behavior of human glioma cell lines and glioma cells propagated as xenografts. ACSVL3 expression was induced by the activation of oncogenic receptor tyrosine kinases (RTK) c-Met and epidermal growth factor receptor. Inhibiting c-Met activation with neutralizing anti–hepatocyte growth factor monoclonal antibodies reduced ACSVL3 expression concurrent with tumor growth inhibition in vivo. ACSVL3 expression knockdown using RNA interference, which decreased long-chain fatty acid activation, inhibited anchorage-dependent and anchorage-independent glioma cell growth by ∼70% and ∼90%, respectively. ACSVL3-depleted cells were less tumorigenic than control cells, and subcutaneous xenografts grew ∼60% slower than control tumors. Orthotopic xenografts produced by ACSVL3-depleted cells were 82% to 86% smaller than control xenografts. ACSVL3 knockdown disrupted Akt function as evidenced by RTK-induced transient decreases in total and phosphorylated Akt, as well as glycogen synthase kinase 3β, via a caspase-dependent mechanism. Expressing constitutively active myr-Akt rescued cells from the anchorage-dependent and anchorage-independent growth inhibitory effects of ACSVL3 depletion. These studies show that ACSVL3 maintains oncogenic properties of malignant glioma cells via a mechanism that involves, in part, the regulation of Akt function. [Cancer Res 2009;69(24):9175–82]
Data from Epidermal Growth Factor Receptor and PTEN Modulate Tissue Factor Expression in Glioblastoma through JunD/Activator Protein-1 Transcriptional Activity
Authors: Rong Yuan , Belozerov Vladimir E. , Tucker-Burden Carol , Chen Gang , Durden Donald L. , Olson Jeffrey J. , Van Meir Erwin G. , Mackman Nigel , Brat Daniel J.
Published: March 2023

Abstract
Hypoxia and necrosis are fundamental features of glioblastoma (GBM) and their emergence is critical for the rapid biological progression of this fatal tumor; yet, underlying mechanisms are poorly understood. We have suggested that vaso-occlusion following intravascular thrombosis could initiate or propagate hypoxia and necrosis in GBM. Tissue factor (TF), the main cellular initiator of coagulation, is overexpressed in GBMs and likely favors a thrombotic microenvironment. Epidermal growth factor receptor (EGFR) amplification and PTEN loss are two common genetic alterations seen in GBM but not in lower-grade astrocytomas that could be responsible for TF up-regulation. The most frequent EGFR mutation in GBM involves deletion of exons 2 to 7, resulting in the expression of a constitutively active receptor, EGFRvIII. Here, we show that overexpression of EGFR or EGFRvIII in human glioma cells causes increased basal TF expression and that stimulation of EGFR by its ligand, EGF, leads to a marked dose-dependent up-regulation of TF. In all cases, increased TF expression led to accelerated plasma coagulation in vitro. EGFR-mediated TF expression depended most strongly on activator protein-1 (AP-1) transcriptional activity and was associated with c-Jun NH2-terminal kinase (JNK) and JunD activation. Restoration of PTEN expression in PTEN-deficient GBM cells diminished EGFR-induced TF expression by inhibiting JunD/AP-1 transcriptional activity. PTEN mediated this effect by antagonizing phosphatidylinositol 3-kinase activity, which in turn attenuated both Akt and JNK activities. These mechanisms are likely at work in vivo, as EGFR expression was highly correlated with TF expression in human high-grade astrocytoma specimens. [Cancer Res 2009;69(6):2540–9]
Data from Attenuated Transforming Growth Factor β Signaling Promotes Nuclear Factor-κB Activation in Head and Neck Cancer
Authors: Cohen Jonah , Chen Zhong , Lu Shi-Long , Yang Xin Ping , Arun Pattatheyil , Ehsanian Reza , Brown Matthew S. , Lu Hai , Yan Bin , Diallo Oumou , Wang Xiao-Jing , Van Waes Carter
Published: March 2023

Abstract
Although constitutively activated nuclear factor-κB (NF-κB), attenuated transforming growth factor β (TGFβ) signaling, and TP53 mutations frequently occur in human cancers, how these pathways interact and together contribute to malignancy remains uncertain. Here, we found an association between overexpression of NF-κB–related genes, reduced expression of TGFβ receptor (TβR) subunits and downstream targets, and TP53 genotype in head and neck squamous cell carcinoma (HNSCC). In response to recombinant TGFβ1, both growth inhibition and TGFβ target gene modulation were attenuated or absent in a panel of human HNSCC lines. However, in HNSCC cells that retained residual TGFβ signaling, TGFβ1 inhibited both constitutive and tumor necrosis factor α–stimulated NF-κB activity. Furthermore, HNSCC lines overexpressing mutant (mt) TP53 and human tumor specimens with positive TP53 nuclear staining exhibited reduced TβRII and knocking down mtTP53 induced TβRII, increasing TGFβ downstream gene expression while inhibiting proinflammatory NF-κB target gene expression. Transfection of ectopic TβRII directly restored TGFβ signaling while inhibiting inhibitor κBα degradation and suppressing serine-536 phosphorylation of NF-κB p65 and NF-κB transcriptional activation, linking these alterations. Finally, experiments with TβRII conditional knockout mice show that abrogation of TGFβ signaling promotes the sustained induction of NF-κB and its proinflammatory target genes during HNSCC tumorigenesis and progression. Together, these findings elucidate a regulatory framework in which attenuated TGFβ signaling promotes NF-κB activation and squamous epithelial malignancy in the setting of altered TP53 status. [Cancer Res 2009;69(8):3415–24]
Data from Dynamic Gene Expression Analysis Links Melanocyte Growth Arrest with Nevogenesis
Authors: Yang Guang , Thieu Khanh , Tsai Kenneth Y. , Piris Adriano , Udayakumar Durga , Njauw Ching-Ni Jenny , Ramoni Marco F. , Tsao Hensin
Published: March 2023

Abstract
Like all primary cells in vitro, normal human melanocytes exhibit a physiologic decay in proliferative potential as it transitions to a growth-arrested state. The underlying transcriptional program(s) that regulate this phenotypic change is largely unknown. To identify molecular determinants of this process, we performed a Bayesian-based dynamic gene expression analysis on primary melanocytes undergoing proliferative arrest. This analysis revealed several related clusters whose expression behavior correlated with the melanocyte growth kinetics; we designated these clusters the melanocyte growth arrest program (MGAP). These MGAP genes were preferentially represented in benign melanocytic nevi over melanomas and selectively mapped to the hepatocyte fibrosis pathway. This transcriptional relationship between melanocyte growth stasis, nevus biology, and fibrogenic signaling was further validated in vivo by the demonstration of strong pericellular collagen deposition within benign nevi but not melanomas. Taken together, our study provides a novel view of fibroplasia in both melanocyte biology and nevogenesis. [Cancer Res 2009;69(23):9029–37]