bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from SYT-SSX1 and SYT-SSX2 Interfere with Repression of E-Cadherin by Snail and Slug: A Potential Mechanism for Aberrant Mesenchymal to Epithelial Transition in Human Synovial Sarcoma
Authors: Saito Tsuyoshi , Nagai Makoto , Ladanyi Marc
Published: March 2023

Abstract
Synovial sarcoma is a primitive mesenchymal neoplasm characterized in almost all cases by a t(X;18) fusing the SYT transcriptional coactivator gene with either SSX1 or SSX2, with the resulting fusion gene encoding an aberrant transcriptional regulator. A subset of synovial sarcoma, predominantly cases with the SYT-SSX1 fusion, shows foci of morphologic epithelial differentiation in the form of nests of glandular epithelium. The striking spontaneous mesenchymal to epithelial differentiation in this cancer is reminiscent of a developmental switch, but the only clue to its mechanistic basis has been the observation that most cases of synovial sarcoma with glandular epithelial differentiation (GED) contain SYT-SSX1 instead of SYT-SSX2. We report here that SYT-SSX1 and SYT-SSX2 interact preferentially with Snail or Slug, respectively, and prevent these transcriptional repressors from binding to the proximal E-cadherin promoter as shown by coimmunoprecipitation and chromatin immunoprecipitation. Luciferase reporter assays reveal that SYT-SSX1 and SYT-SSX2 can respectively overcome the Snail- or Slug-mediated repression of E-cadherin transcription. This provides a mechanism by which E-cadherin expression, a prerequisite of epithelial differentiation, is aberrantly derepressed in synovial sarcoma and may also explain the association of GED with the SYT-SSX1 fusion because it interferes with Snail, the stronger repressor of the E-cadherin promoter. Thus, our data provide a mechanistic basis for the observed heterogeneity in the acquisition of epithelial characteristics in synovial sarcoma and highlight the potential role of differential interactions with Snail or Slug in modulating this phenotypic transition. (Cancer Res 2006; 66(14): 6919-27)
Data from Identification of Novel p53 Target Genes in Ionizing Radiation Response
Authors: Jen Kuang-Yu , Cheung Vivian G.
Published: March 2023

Abstract
The tumor suppressor p53 plays an essential role in cellular adaptation to stress. In response to ionizing radiation, p53 regulates the transcription of genes in a diverse set of pathways including DNA repair, cell cycle arrest, and apoptosis. Previously, we identified by microarray analysis a set of genes that are transcriptionally activated or repressed in response to radiation exposure. In this study, we use computational methods and molecular techniques, including location analysis (ChIP-on-chip assay), to identify ionizing radiation–responsive genes that are directly regulated by p53. Among the 489 ionizing radiation–responsive genes examined, 38 genes were found to be p53 targets. Some of these genes are previously known to be directly regulated by p53 whereas others are novel p53 targets. We further showed that the novel p53 target genes are transcriptionally regulated by p53. The binding of p53 to promoters of target genes correlated with increased transcript levels of these genes in cells with functional p53. However, p53 binding and subsequent transcriptional activation of these target genes were significantly diminished in cells with mutant p53 and in cells from patients with ataxia telangiectasia, which have impaired p53 activation following ionizing radiation exposure. Identification and characterization of ionizing radiation–responsive p53 target genes extend our knowledge of the diverse role that p53 plays in the DNA damage response.
Data from Imatinib Mesylate Inhibits Leydig Cell Tumor Growth: Evidence for In vitro and In vivo Activity
Authors: Basciani Sabrina , Brama Marina , Mariani Stefania , De Luca Gabriele , Arizzi Mario , Vesci Loredana , Pisano Claudio , Dolci Susanna , Spera Giovanni , Gnessi Lucio
Published: March 2023

Abstract
Leydig cell tumors are usually benign tumors of the male gonad. However, if the tumor is malignant, no effective treatments are currently available. Leydig cell tumors express platelet-derived growth factor (PDGF), kit ligand and their respective receptors, PDGFR and c-kit. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the c-kit and PDGFR tyrosine kinases, on the growth of rodent Leydig tumor cell lines in vivo and in vitro, and examined, in human Leydig cell tumor samples, the expression of activated PDGFR and c-kit and the mutations in exons of the c-kit gene commonly associated with solid tumors. Imatinib caused concentration-dependent decreases in the viability of Leydig tumor cell lines, which coincided with apoptosis and inhibition of proliferation and ligand-stimulated phosphorylation of c-kit and PDGFRs. Mice bearing s.c. allografts of a Leydig tumor cell line treated with imatinib p.o., had an almost complete inhibition of tumor growth, less tumor cell proliferation, increased apoptosis, and a lesser amount of tumor-associated mean vessel density compared with controls. No drug-resistant tumors appeared during imatinib treatment but tumors regrew after drug withdrawal. Human Leydig cell tumors showed an intense expression of the phosphorylated form of c-kit and a less intense expression of phosphorylated PDGFRs. No activating mutations in common regions of mutation of the c-kit gene were found. Our studies suggest that Leydig cell tumors might be a potential target for imatinib therapy.
Data from Cytogenetic Instability in Ovarian Epithelial Cells from Women at Risk of Ovarian Cancer
Authors: Pejovic Tanja , Yates Jane E. , Liu Hong Y. , Hays Laura E. , Akkari Yassmine , Torimaru Yumi , Keeble Winifred , Rathbun R. Keaney , Rodgers William H. , Bale Allen E. , Ameziane Najim , Zwaan C. Michael , Errami Abdellatif , Thuillier Philippe , Cappuccini Fabio , Olson Susan B. , Cain Joanna M. , Bagby Grover C.
Published: March 2023

Abstract
Fanconi anemia is an inherited cancer predisposition disease characterized by cytogenetic and cellular hypersensitivity to cross-linking agents. Seeking evidence of Fanconi anemia protein dysfunction in women at risk of ovarian cancer, we screened ovarian surface epithelial cells from 25 primary cultures established from 22 patients using cross-linker hypersensitivity assays. Samples were obtained from (a) women at high risk for ovarian cancer with histologically normal ovaries, (b) ovarian cancer patients, and (c) a control group with no family history of breast or ovarian cancer. In chromosomal breakage assays, all control cells were mitomycin C (MMC) resistant, but eight samples (five of the six high-risk and three of the eight ovarian cancer) were hypersensitive. Lymphocytes from all eight patients were MMC resistant. Only one of the eight patients had a BRCA1 germ-line mutation and none had BRCA2 mutations, but FANCD2 was reduced in five of the eight. Ectopic expression of normal FANCD2 cDNA increased FANCD2 protein and induced MMC resistance in both hypersensitive lines tested. No FANCD2 coding region or promoter mutations were found, and there was no genomic loss or promoter methylation in any Fanconi anemia genes. Therefore, in high-risk women with no BRCA1 or BRCA2 mutations, tissue-restricted hypersensitivity to cross-linking agents is a frequent finding, and chromosomal breakage responses to MMC may be a sensitive screening strategy because cytogenetic instability identified in this way antedates the onset of carcinoma. Inherited mutations that result in tissue-specific FANCD2 gene suppression may represent a cause of familial ovarian cancer. (Cancer Res 2006; 66(18): 9017-25)
Data from Vascular Endothelial Growth Factor-A Promotes Peritumoral Lymphangiogenesis and Lymphatic Metastasis
Authors: Björndahl Meit A. , Cao Renhai , Burton Jeremy B. , Brakenhielm Ebba , Religa Piotr , Galter Dagmar , Wu Lily , Cao Yihai
Published: March 2023

Abstract
Metastases are commonly found in the lymphatic system. The molecular mechanism of lymphatic metastasis is, however, poorly understood. Here we report that vascular endothelial growth factor (VEGF)-A stimulated lymphangiogenesis in vivo and that overexpression of VEGF-A in murine T241 fibrosarcomas induced the growth of peritumoral lymphatic vessels, which occasionally penetrated into the tumor tissue. As a result of peritumoral lymphangiogenesis, metastases in lymph nodes of mice were detected. VEGF-A–overexpressing tumors contained high numbers of infiltrating inflammatory cells such as macrophages, which are known to express VEGF receptor (VEGFR)-1. It seemed that in the mouse cornea, VEGF-A stimulated lymphangiogenesis through a VEGF-C/-D/VEGFR-3–independent pathway as a VEGFR-3 antagonist selectively inhibited VEGF-C–induced, but not VEGF-A–induced, lymphangiogenesis. Our data show that VEGF-A contributes to lymphatic mestastasis. Thus, blockage of VEGF-A–induced lymphangiogenesis may provide a novel approach for prevention and treatment of lymphatic metastasis.
Data from Protein Kinase C α but not PKCζ Suppresses Intestinal Tumor Formation in ApcMin/+ Mice
Authors: Oster Henrik , Leitges Michael
Published: March 2023

Abstract
Members of the protein kinase C (PKC) family of serine/threonine kinases play key regulatory roles in numerous cellular processes, including differentiation and proliferation. Of the 11 mammalian PKC isoforms known, several have been implicated in tumor development and progression. However, in most cases, isotype specificity is poorly defined, and even contrary functions for a single PKC have been reported mostly because appropriate molecular and genetic tools were missing to specifically assess the contribution of single PKC isoforms in vivo. In this report, we therefore used PKC genetic targeting to study the role of PKCα and PKCζ in colorectal cancer. Both isoforms were found to be strongly down-regulated in intestinal tumors of ApcMin/+ mice. A deletion of PKCζ did not affect tumorigenesis in this animal model. In contrast, PKCα-deficient ApcMin/+ mice developed more aggressive tumors and died significantly earlier than their PKCα-proficient littermates. Even without an additional Apc mutation, PKCα knockout mice showed an elevated tendency to develop spontaneous intestinal tumors. Transcriptional profiling revealed a role for this kinase in regulating epidermal growth factor receptor (EGFR) signaling and proposed a synergistic mechanism for EGFR/activator protein and WNT/APC pathways in mediating intestinal tumor development. (Cancer Res 2006; 66(14): 6955-63)
Data from Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Mediated Proliferation of Tumor Cells with Receptor-Proximal Apoptosis Defects
Authors: Baader Eva , Toloczko Agnieszka , Fuchs Uta , Schmid Irene , Beltinger Christian , Ehrhardt Harald , Debatin Klaus-Michael , Jeremias Irmela
Published: March 2023

Abstract
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) might represent a future cytotoxic drug to treat cancer as it induces apoptosis in tumor cells without toxicity in animal trials. We recently described that in contrast to apoptosis, TRAIL mediates tumor cell survival and proliferation in certain tumor cells. Here we studied the effect of TRAIL on 18 cell lines and 53 primary leukemia cells and classified these tumor cells into four groups: TRAIL, anti-DR4 or anti-DR5 induced apoptosis in group A cells, whereas they had no effect on group 0 cells and mediated proliferation in group P cells. To our surprise, TRAIL induced simultaneous apoptosis and proliferation in group AP cells. More than 20% of all cells tested belonged to group P and showed TRAIL-mediated proliferation even in the presence of certain cytotoxic drugs but not inhibitors of nuclear factor-κB. Transfection with B-cell leukemia/lymphoma protein 2 transformed group A cells into group 0 cells, whereas transfection with Fas-associated polypeptide with death domain (FADD)–like interleukin-1-converting enzyme–inhibitory protein (FLIP) transformed them into group AP cells. Loss of caspase-8 or transfection of dominant-negative FADD transformed group A cells into group P cells. Taken together, our data suggest that proliferation is a frequent effect of TRAIL on tumor cells, which is related to receptor-proximal apoptosis defects at the level of the death-inducing signaling complex and should be prevented during antitumor therapy with TRAIL.
Data from Epigenomic Profiling Reveals Novel and Frequent Targets of Aberrant DNA Methylation-Mediated Silencing in Malignant Glioma
Authors: Kim Tae-You , Zhong Sheng , Fields C. Robert , Kim Jeong Hoon , Robertson Keith D.
Published: March 2023

Abstract
Malignant glioma is the most common central nervous system tumor of adults and is associated with a significant degree of morbidity and mortality. Gliomas are highly invasive and respond poorly to conventional treatments. Gliomas, like other tumor types, arise from a complex and poorly understood sequence of genetic and epigenetic alterations. Epigenetic alterations leading to gene silencing, in the form of aberrant CpG island promoter hypermethylation and histone deacetylation, have not been thoroughly investigated in brain tumors, and elucidating such changes is likely to enhance our understanding of their etiology and provide new treatment options. We used a combined approach of pharmacologic inhibition of DNA methylation and histone deacetylation, coupled with expression microarrays, to identify novel targets of epigenetic silencing in glioma cell lines. From this analysis, we identified >160 genes up-regulated by 5-aza-2′-deoxycytidine and trichostatin A treatment. Further characterization of 10 of these genes, including the putative metastasis suppressor CST6, the apoptosis-inducer BIK, and TSPYL5, whose function is unknown, revealed that they are frequent targets of epigenetic silencing in glioma cell lines and primary tumors and suppress glioma cell growth in culture. Furthermore, we show that other members of the TSPYL gene family are epigenetically silenced in gliomas and dissect the contribution of individual DNA methyltransferases to the aberrant promoter hypermethylation events. These studies, therefore, lay the foundation for a comprehensive understanding of the full extent of epigenetic changes in gliomas and how they may be exploited for therapeutic purposes. (Cancer Res 2006; 66(15): 7490-501)
Data from Loss-of-Function of Nkx3.1 Promotes Increased Oxidative Damage in Prostate Carcinogenesis
Authors: Ouyang Xuesong , DeWeese Theodore L. , Nelson William G. , Abate-Shen Cory
Published: March 2023

Abstract
Despite the significance of oxidative damage for carcinogenesis, the molecular mechanisms that lead to increased susceptibility of tissues to oxidative stress are not well-understood. We now report a link between loss of protection against oxidative damage and loss-of-function of Nkx3.1, a homeobox gene that is known to be required for prostatic epithelial differentiation and suppression of prostate cancer. Using gene expression profiling, we find that Nkx3.1 mutant mice display deregulated expression of several antioxidant and prooxidant enzymes, including glutathione peroxidase 2 and 3 (GPx2 and GPx3), peroxiredoxin 6 (Prdx6), and sulfyhydryl oxidase Q6 (Qscn6). Moreover, the formation of prostatic intraepithelial neoplasia in these mutant mice is associated with increased oxidative damage of DNA, as evident by increased levels of 8-hydroxy-2′-deoxyguanosine. We further show that progression to prostate adenocarcinoma, as occurs in compound mutant mice lacking Nkx3.1 as well as the Pten tumor suppressor, is correlated with a further deregulation of antioxidants, including superoxide dismutase enzymes, and more profound accumulations of oxidative damage to DNA and protein, the latter manifested by increased levels of 4-hydroxynonenal. We propose that the essential role of Nkx3.1 in maintaining the terminally differentiated state of the prostate epithelium provides protection against oxidative damage and, thereby, suppression of prostate cancer. Thus, our findings provide a molecular link between a gene whose inactivation is known to be involved in prostate carcinogenesis, namely Nkx3.1, and oxidative damage of the prostatic epithelium.
Data from Retinoblastoma Pathway Defects Show Differential Ability to Activate the Constitutive DNA Damage Response in Human Tumorigenesis
Authors: Tort Frederic , Bartkova Jirina , Sehested Maxwell , Ørntoft Torben , Lukas Jiri , Bartek Jiri
Published: March 2023

Abstract
Loss of G1-S control and aberrations of the p16Ink4a-cyclin D1/cyclin-dependent kinase (CDK) 4(6)-pRb-E2F-cyclin E/CDK2 pathway are common in human cancer. Previous studies showed that oncogene-induced aberrant proliferation, such as on cyclin E overexpression, causes DNA damage and checkpoint activation. Here, we show that, in a series of human colorectal adenomas, those with deregulation of cyclin D1 and/or p16Ink4a showed little evidence of constitutive DNA damage response (DDR), contrary to cyclin E-overexpressing higher-grade cases. These observations were consistent with diverse cell culture models with differential defects of retinoblastoma pathway components, as overexpression of cyclin D1 or lack of p16Ink4a, either alone or combined, did not elicit detectable DDR. In contrast, inactivation of pRb, the key component of the pathway, activated the DDR in cultured human or mouse cells, analogous to elevated cyclin E. These results highlight differential effect of diverse oncogenic events on driving the ‘cancer cell cycles’ and their ability to deregulate the replication-driving CDK2 kinase and to alarm the DDR as a potential anticancer barrier in accordance with their hierarchical positions along the retinoblastoma pathway. Our data provide new insights into oncogene-evoked DDR in human tumorigenesis, with potential implications for individualized management of tumors with elevated cyclin D1 versus cyclin E, due to their distinct clinical variables and biological behavior. (Cancer Res 2006; 66(21): 10258-63)
Data from TMPRSS2 Fusions with Oncogenic ETS Factors in Prostate Cancer Involve Unbalanced Genomic Rearrangements and Are Associated with HDAC1 and Epigenetic Reprogramming
Authors: Iljin Kristiina , Wolf Maija , Edgren Henrik , Gupta Santosh , Kilpinen Sami , Skotheim Rolf I. , Peltola Mari , Smit Frank , Verhaegh Gerald , Schalken Jack , Nees Matthias , Kallioniemi Olli
Published: March 2023

Abstract
Translocations fusing the strong androgen-responsive gene, TMPRSS2, with ERG or other oncogenic ETS factors may facilitate prostate cancer development. Here, we studied 18 advanced prostate cancers for ETS factor alterations, using reverse transcription-PCR and DNA and RNA array technologies, and identified putative ERG downstream gene targets from the microarray data of 410 prostate samples. Out of the 27 ETS factors, ERG was most frequently overexpressed. Seven cases showed TMPRSS2:ERG gene fusions, whereas the TMPRSS2:ETV4 fusion was seen in one case. In five out of six tumors with high ERG expression, array-CGH analysis revealed interstitial 2.8 Mb deletions between the TMPRSS2 and ERG loci, or smaller, unbalanced rearrangements. In silico analysis of the ERG gene coexpression patterns revealed an association with high expression of the histone deacetylase 1 gene, and low expression of its target genes. Furthermore, we observed increased expression of WNT-associated pathways and down-regulation of tumor necrosis factor and cell death pathways. In summary, our data indicate that the TMPRSS2:ERG translocation is common in advanced prostate cancer and occurs by virtue of unbalanced genomic rearrangements. Activation of ERG by fusion with TMPRSS2 may lead to epigenetic reprogramming, WNT signaling, and down-regulation of cell death pathways, implicating ERG in several hallmarks of cancer with potential therapeutic importance. (Cancer Res 2006; 66(21): 10242-6)
Data from Preparing the “Soil”: The Primary Tumor Induces Vasculature Reorganization in the Sentinel Lymph Node before the Arrival of Metastatic Cancer Cells
Authors: Qian Chao-Nan , Berghuis Bree , Tsarfaty Galia , Bruch MaryBeth , Kort Eric J. , Ditlev Jon , Tsarfaty Ilan , Hudson Eric , Jackson David G. , Petillo David , Chen Jindong , Resau James H. , Teh Bin Tean
Published: March 2023

Abstract
Sentinel lymph node (SLN) metastasis is the first step in the spreading of cancer in many malignancies. Tumor-reactive lymphadenopathy in SLNs has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain unexplored. Using animal models, we show here that, before the establishment of metastasis in the SLN, there are reorganizations of the lymphatic channels and the vasculature. The node becomes a functional blood vessel–enriched and lymph vessel/sinus-enriched organ before metastasis. The enlargement of the lymph sinuses is correlated with the primary tumor weight. The newly emerged functional blood vessels develop from high endothelial venules (HEV), in which the proliferation rate of the endothelial cells is also significantly increased. Similar alterations of the HEVs are also characterized in the axillary lymph nodes from human breast cancer patients without the evidence of metastasis. These findings support the hypothesis that modification of the microenvironment for a secondary tumor (i.e., vasculature reorganization in the SLN) can be initiated by a primary tumor before and independent of the physical presence of metastatic cancer cells. (Cancer Res 2006; 66(21): 10365-76)
Data from N-cadherin Gene Expression in Prostate Carcinoma Is Modulated by Integrin-Dependent Nuclear Translocation of Twist1
Authors: Alexander Nelson R. , Tran Nhan L. , Rekapally Harish , Summers Carol E. , Glackin Carlotta , Heimark Ronald L.
Published: March 2023

Abstract
The gain of N-cadherin expression in carcinomas has been shown to be important in the regulation of cell migration, invasion, and survival. Here, we show that N-cadherin mRNA expression in PC-3 prostate carcinoma cells is dependent on β1 integrin–mediated cell adhesion to fibronectin and the basic helix-loop-helix transcription factor Twist1. Depletion of Twist1 mRNA by small interfering RNA resulted in decreased expression of both Twist1 and N-cadherin and the inhibition of cell migration. Whereas Twist1 gene expression was independent of β1 integrin–mediated adhesion, Twist1 protein failed to accumulate in the nuclei of cells cultured in anchorage-independent conditions. The increased nuclear accumulation of Twist1 following cell attachment was suppressed by treatment with an inhibitor of Rho kinase or a β1 integrin neutralizing antibody. The effect of Twist1 on induction of N-cadherin mRNA required an E-box cis-element located within the first intron (+2,627) of the N-cadherin gene. These data raise the possibility that integrin-mediated adhesion to interstitial matrix proteins during metastasis differentially regulates the nuclear/cytoplasmic translocation and DNA binding of Twist1, activating N-cadherin transcription. (Cancer Res 2006; 66(7): 3365-9)
Data from Targeted Anti–Vascular Endothelial Growth Factor Receptor-2 Therapy Leads to Short-term and Long-term Impairment of Vascular Function and Increase in Tumor Hypoxia
Authors: Franco Marcela , Man Shan , Chen Limor , Emmenegger Urban , Shaked Yuval , Cheung Alison M. , Brown Allison S. , Hicklin Daniel J. , Foster F. Stuart , Kerbel Robert S.
Published: March 2023

Abstract
Because antiangiogenic therapies inhibit the growth of new tumor-associated blood vessels, as well as prune newly formed vasculature, they would be expected to reduce the supply of oxygen and thus increase tumor hypoxia. However, it is not clear if antiangiogenic treatments lead only to consistent and sustained increases in hypoxia, or transient decreases in tumor hypoxia along with periods of increased hypoxia. We undertook a detailed analysis of an orthotopically transplanted human breast carcinoma (MDA-MB-231) over a 3-week treatment period using DC101, an anti–vascular endothelial growth factor receptor 2 antibody. We observed consistent reductions in microvascular density, blood flow (measured by high-frequency micro-ultrasound), and perfusion. These effects resulted in an increase in the hypoxic tumor fraction, measured with an exogenous marker, pimonidazole, concurrent with an elevation in hypoxia-inducible factor-1α expression, an endogenous marker. The increase in tumor hypoxia was evident within 5 days and remained so throughout the entire course of treatment. Vascular perfusion and flow were impaired at days 2, 5, 7, 8, 14, and 21 after the first injection, but not at 4 hours. A modest increase in the vessel maturation index was detected after the 3-week treatment period, but this was not accompanied by an improvement in vascular function. These results suggest that sustained hypoxia and impairment of vascular function can be two consistent consequences of antiangiogenic drug treatment. The implications of the results are discussed, particularly with respect to how they relate to different theories for the counterintuitive chemosensitizing effects of antiangiogenic drugs, even when hypoxia is increased. (Cancer Res 2006; 66(7): 3639-48)
Data from Interaction of Deleted in Liver Cancer 1 with Tensin2 in Caveolae and Implications in Tumor Suppression
Authors: Yam Judy Wai Ping , Ko Frankie Chi Fat , Chan Chung-Yiu , Jin Dong-Yan , Ng Irene Oi-Lin
Published: March 2023

Abstract
Deleted in liver cancer 1 (DLC1) is a recently identified tumor suppressor gene frequently underexpressed in hepatocellular carcinoma (HCC). DLC1 encodes a Rho GTPase-activating protein domain that exhibits growth-suppressive activity in HCC cell lines. Our recent finding has revealed that inhibition of Rho-mediated actin stress fiber formation by DLC1 is associated with its growth inhibitory activity. In the present study, we identified tensin2 as the novel binding partner of DLC1. Tensin2 belongs to a new family of focal adhesion proteins that play key roles in cytoskeleton organization and signal transduction. Dysregulation of tensin proteins has previously been implicated in human cancers. Tensin2 is highly expressed in human liver. Introduction of tensin2 into HCC cell lines with low expression of tensin2 caused significant growth inhibition and induction of apoptosis. Tensin2 directly interacted with DLC1 in vitro and in vivo. Both proteins localized to punctate structures in the cytoplasm. Sequence analysis of DLC1 and tensin2 identified caveolin-1 binding motif in both proteins. In vivo immunoprecipitation study confirmed that both proteins indeed interacted with endogenous caveolin-1, which is the major structural component of caveolae. Our findings presented here suggest a new model for the action of DLC1 in hepatocytes, whereby DLC1-tensin2 complex interacts with Rho GTPases in caveolae to effect cytoskeletal reorganization. (Cancer Res 2006; 66(17): 8367-72)
Data from E-Cadherin Cell-Cell Adhesion in Ewing Tumor Cells Mediates Suppression of Anoikis through Activation of the ErbB4 Tyrosine Kinase
Authors: Kang Hyung-Gyoo , Jenabi Jasmine M. , Zhang Jingsong , Keshelava Nino , Shimada Hiroyuki , May William A. , Ng Tony , Reynolds C. Patrick , Triche Timothy J. , Sorensen Poul H.B.
Published: March 2023

Abstract
Ability to grow under anchorage-independent conditions is one of the major hallmarks of transformed cells. Key to this is the capacity of cells to suppress anoikis, or programmed cell death induced by detachment from the extracellular matrix. To model this phenomenon in vitro, we plated Ewing tumor cells under anchorage-independent conditions by transferring them to dishes coated with agar to prevent attachment to underlying plastic. This resulted in marked up-regulation of E-cadherin and rapid formation of multicellular spheroids in suspension. Addition of calcium chelators, antibodies to E-cadherin (but not to other cadherins or β1-integrin), or expression of dominant negative E-cadherin led to massive apoptosis of spheroid cultures whereas adherent cultures were unaffected. This correlated with reduced activation of the phosphatidylinositol 3-kinase-Akt pathway but not the Ras-extracellular signal–regulated kinase 1/2 cascade. Furthermore, spheroid cultures showed profound chemoresistance to multiple cytotoxic agents compared with adherent cultures, which could be reversed by α-E-cadherin antibodies or dominant negative E-cadherin. In a screen for potential downstream effectors of spheroid cell survival, we detected E-cadherin–dependent activation of the ErbB4 receptor tyrosine kinase but not of other ErbB family members. Reduction of ErbB4 levels by RNA interference blocked Akt activation and spheroid cell survival and restored chemosensitivity to Ewing sarcoma spheroids. Our results indicate that anchorage-independent Ewing sarcoma cells suppress anoikis through a pathway involving E-cadherin cell-cell adhesion, which leads to ErbB4 activation of the phosphatidylinositol 3-kinase-Akt pathway, and that this is associated with increased resistance of cells to cytotoxic agents. [Cancer Res 2007;67(7):3094–105]
Data from Identification of Biomarkers Modulated by the Rexinoid LGD1069 (Bexarotene) in Human Breast Cells Using Oligonucleotide Arrays
Authors: Kim Hee-Tae , Kong Gu , DeNardo David , Li Yuxin , Uray Ivan , Pal Sunita , Mohsin Syed , Hilsenbeck Susan G. , Bissonnette Reid , Lamph William W. , Johnson Karen , Brown Powel H.
Published: March 2023

Abstract
Retinoids have been found to be promising chemopreventive agents that play an important role in regulating cell growth, differentiation, and apoptosis. The action of retinoids is mediated by retinoid receptors (retinoic acid receptors and retinoid X receptors), which are nuclear transcription factors that, when bound to retinoids, regulate gene expression. LGD1069 is a highly selective RXR agonist that has reduced toxicity compared with retinoids. Our previous studies have shown that RXR-selective ligands (or “rexinoids”), including LGD1069, can inhibit the growth of normal and malignant breast cells and can suppress the development of breast cancer in transgenic mice. For the current study, we attempted to identify biomarkers of the chemopreventive effect of the RXR-selective retinoid LGD1069. In these experiments, we used Affymetrix microarrays to identify target genes that were modulated by LGD1069 in normal human breast cells. Affymetrix and dChip analysis identified more than 100 genes that were up-regulated or down-regulated by LGD1069 treatment. We then tested 16 of these genes in validation experiments using quantitative reverse transcription-PCR and Western blotting of independently prepared samples, and found that 15 of 16 genes were modulated in a similar manner in these validation experiments as in the microarray experiments. Genes found to be regulated include known retinoid-regulated genes, growth regulatory genes, transcription factors, and differentiation markers. We then showed that the expression of several of these rexinoid-regulated biomarkers is modulated in vivo in mammary glands from mice treated with LGD1069. These critical growth-regulating proteins will be promising targets of future agents for the prevention and treatment of breast cancer. (Cancer Res 2006; 66(24): 12009-18)
Data from Targeting Cancer Cells by Novel Engineered Modular Transporters
Authors: Gilyazova Dinara G. , Rosenkranz Andrey A. , Gulak Pavel V. , Lunin Vladimir G. , Sergienko Olga V. , Khramtsov Yuri V. , Timofeyev Kirill N. , Grin Mikhail A. , Mironov Andrey F. , Rubin Andrey B. , Georgiev Georgii P. , Sobolev Alexander S.
Published: March 2023

Abstract
A major problem in the treatment of cancer is the specific targeting of drugs to these abnormal cells. Ideally, such a drug should act over short distances to minimize damage to healthy cells and target subcellular compartments that have the highest sensitivity to the drug. We describe the novel approach of using modular recombinant transporters to target photosensitizers to the nucleus, where their action is most pronounced, of cancer cells overexpressing ErbB1 receptors. We have produced a new generation of the transporters consisting of (a) epidermal growth factor as the internalizable ligand module to ErbB1 receptors, (b) the optimized nuclear localization sequence of SV40 large T-antigen, (c) a translocation domain of diphtheria toxin as an endosomolytic module, and (d) the Escherichia coli hemoglobin-like protein HMP as a carrier module. The modules retained their functions within the transporter chimera: they showed high-affinity interactions with ErbB1 receptors and α/β-importin dimers and formed holes in lipid bilayers at endosomal pH. A photosensitizer conjugated with the transporter produced singlet oxygen and ·OH radicals similar to the free photosensitizer. Photosensitizers-transporter conjugates have >3,000 times greater efficacy than free photosensitizers for target cells and were not photocytotoxic at these concentrations for cells expressing a few ErbB1 receptors per cell, in contrast to free photosensitizers. The different modules of the transporters, which are highly expressed and easily purified to retain full activity of each of the modules, are interchangeable, meaning that they can be tailored for particular applications. (Cancer Res 2006; 66(21): 10534-40)
Data from hTERT Is Expressed in Cancer Cell Lines Despite Promoter DNA Methylation by Preservation of Unmethylated DNA and Active Chromatin around the Transcription Start Site
Authors: Zinn Rebekah L. , Pruitt Kevin , Eguchi Sayaka , Baylin Stephen B. , Herman James G.
Published: March 2023

Abstract
hTERT, which encodes the catalytic subunit of telomerase and is expressed in most immortalized and cancer cells, has been reported to have increased DNA methylation in its promoter region in many cancers. This pattern is inconsistent with observations that DNA methylation of promoter CpG islands is typically associated with gene silencing. Here, we provide a comprehensive analysis of promoter DNA methylation, chromatin patterns, and expression of hTERT in cancer and immortalized cells. Methylation-specific PCR and bisulfite sequencing of the hTERT promoter in breast, lung, and colon cancer cells show that all cancer cell lines retain alleles with little or no methylation around the transcription start site despite being densely methylated in a region 600 bp upstream of the transcription start site. By real-time reverse transcription-PCR, all cancer cell lines express hTERT. Chromatin immunoprecipitation (ChIP) analysis reveals that both active (acetyl-H3K9 and dimethyl-H3K4) and inactive (trimethyl-H3K9 and trimethyl-H3K27) chromatin marks are present across the hTERT promoter. However, using a novel approach combining methylation analysis of ChIP DNA, we show that active chromatin marks are associated with unmethylated DNA, whereas inactive marks of chromatin are associated with methylated DNA in the region around the transcription start site. These results show that DNA methylation patterns of the hTERT promoter (−150 to +150 around the transcription start) are consistent with the usual dynamics of gene expression in that the absence of methylation in this region and the association with active chromatin marks allow for the continued expression of hTERT. [Cancer Res 2007;67(1):194–201]
Data from Adrenal Hyperplasia and Adenomas Are Associated with Inhibition of Phosphodiesterase 11A in Carriers of PDE11A Sequence Variants That Are Frequent in the Population
Authors: Horvath Anelia , Giatzakis Christoforos , Robinson-White Audrey , Boikos Sosipatros , Levine Elizabeth , Griffin Kurt , Stein Erica , Kamvissi Virginia , Soni Payal , Bossis Ioannis , de Herder Wouter , Carney J. Aidan , Bertherat Jérôme , Gregersen Peter K. , Remmers Elaine F. , Stratakis Constantine A.
Published: March 2023

Abstract
Several types of adrenocortical tumors that lead to Cushing syndrome may be caused by aberrant cyclic AMP (cAMP) signaling. We recently identified patients with micronodular adrenocortical hyperplasia who were carriers of inactivating mutations in the 2q-located phosphodiesterase 11A (PDE11A) gene. We now studied the frequency of two missense substitutions, R804H and R867G, in conserved regions of the enzyme in several sets of normal controls, including 745 individuals enrolled in a longitudinal cohort study, the New York Cancer Project. In the latter, we also screened for the presence of the previously identified PDE11A nonsense mutations. R804H and R867G were frequent among patients with adrenocortical tumors; although statistical significance was not reached, these variants affected significantly enzymatic function in vitro with variable increases in cAMP and/or cyclic guanosine 3′,5′-monophosphate levels in HeLa and HEK293 cells. Adrenocortical tissues carrying the R804H mutation showed 2q allelic losses and higher cyclic nucleotide levels and cAMP-responsive element binding protein phosphorylation. We conclude that missense mutations of the PDE11A gene that affect enzymatic activity in vitro are present in the general population; protein-truncating PDE11A mutations may also contribute to a predisposition to other tumors, in addition to their association with adrenocortical hyperplasia. We speculate that PDE11A genetic defects may be associated with adrenal pathology in a wider than previously suspected clinical spectrum that includes asymptomatic individuals. (Cancer Res 2006; 66(24): 11571-5)
Data from Blockade of Hedgehog Signaling Inhibits Pancreatic Cancer Invasion and Metastases: A New Paradigm for Combination Therapy in Solid Cancers
Authors: Feldmann Georg , Dhara Surajit , Fendrich Volker , Bedja Djahida , Beaty Robert , Mullendore Michael , Karikari Collins , Alvarez Hector , Iacobuzio-Donahue Christine , Jimeno Antonio , Gabrielson Kathleen L. , Matsui William , Maitra Anirban
Published: March 2023

Abstract
In the context of pancreatic cancer, metastasis remains the most critical determinant of resectability, and hence survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in pancreatic cancer invasion and metastasis because this is likely to have profound clinical implications. In pancreatic cancer cell lines, Hh inhibition with cyclopamine resulted in down-regulation of snail and up-regulation of E-cadherin, consistent with inhibition of epithelial-to-mesenchymal transition, and was mirrored by a striking reduction of in vitro invasive capacity (P < 0.0001). Conversely, Gli1 overexpression in immortalized human pancreatic ductal epithelial cells led to a markedly invasive phenotype (P < 0.0001) and near total down-regulation of E-cadherin. In an orthotopic xenograft model, cyclopamine profoundly inhibited metastatic spread; only one of seven cyclopamine-treated mice developed pulmonary micrometastases versus seven of seven mice with multiple macrometastases in control animals. Combination of gemcitabine and cyclopamine completely abrogated metastases while also significantly reducing the size of “primary” tumors. Gli1 levels were up-regulated in tissue samples of metastatic human pancreatic cancer samples compared with matched primary tumors. Aldehyde dehydrogenase (ALDH) overexpression is characteristic for both hematopoietic progenitors and leukemic stem cells; cyclopamine preferentially reduced “ALDH-high” cells by ∼3-fold (P = 0.048). We confirm pharmacologic Hh pathway inhibition as a valid therapeutic strategy for pancreatic cancer and show for the first time its particular efficacy against metastatic spread. By targeting specific cellular subpopulations likely involved in tumor initiation at metastatic sites, Hh inhibitors may provide a new paradigm for therapy of disseminated malignancies, particularly when used in combination with conventional antimetabolites that reduce “bulk” tumor size. [Cancer Res 2007;67(5):2187–96]
Data from Activation of Insulin-like Growth Factor Signaling Induces Apoptotic Cell Death Under Prolonged Hypoxia by Enhancing Endoplasmic Reticulum Stress Response
Authors: Endo Hiroko , Murata Kohei , Mukai Mutsuko , Ishikawa Osamu , Inoue Masahiro
Published: March 2023

Abstract
Malignant cells in solid tumors survive under prolonged hypoxia and can be a source of resistance to current cancer therapies. Mammalian target of rapamycin (mTOR), one of the downstream molecules of the insulin-like growth factor (IGF) pathway, is a key regulator of translation, integrating multiple environmental and nutritional cues. The activity of mTOR is known to be suppressed under hypoxic conditions in cancer cells, whereas the contribution of this suppression to cell survival has not yet been clarified. We show that stimulating IGF signaling provoked caspase-dependent apoptosis under low oxygen tension in two cancer cell lines, COLO 320 and AsPC-1. In concurrence with increased levels of BAD phosphorylation, cell death was not accompanied by cytochrome c release from mitochondria. The cells were rescued from apoptosis when phosphatidylinositol 3-kinase (PI3K) or mTOR activity was inhibited, suggesting that these signals are critical in the observed cell death. IGFs and insulin enhanced the endoplasmic reticulum (ER) stress response as monitored by induction of the CCAAT/enhancer binding protein homologous protein (CHOP) proteins and the X box protein-1 splicing under hypoxic conditions, and this response was suppressed by inhibiting PI3K and mTOR activity. IGF-induced cell death under hypoxic conditions was prevented by treatment with cycloheximide, suggesting that de novo protein synthesis is required. Indeed, suppression of CHOP protein levels with small hairpin RNA reduced cell death. Taken together, the data suggest that stimulating IGF signaling under hypoxic conditions provokes apoptosis by enhancing the ER stress response. [Cancer Res 2007;67(17):8095–103]
Data from A Novel Transcriptional Inhibitor Induces Apoptosis in Tumor Cells and Exhibits Antiangiogenic Activity
Authors: Radhakrishnan Senthil K. , Gartel Andrei L.
Published: March 2023

Abstract
Using a high-throughput cell-based assay, we identified a nucleoside analogue 4-amino-6-hydrazino-7-β-d-ribofuranosyl-7H-pyrrolo(2,3-d)-pyrimidine-5-carboxamide (ARC), which has the properties of a general transcriptional inhibitor. Specifically, ARC inhibits the phosphorylation of RNA polymerase II by positive transcription elongation factor-b, leading to a block in transcriptional elongation. ARC was able to potently repress p53 targets p21 and hdm2 (human homologue of mdm2) protein levels, but dramatically increased p53 levels similar to other transcriptional inhibitors, including flavopiridol. This increase in p53 corresponded to the down-regulation of short-lived protein hdm2, which is a well-established negative regulator of p53. Remarkably, ARC induced potent apoptosis in human tumor and transformed, but not in normal cells, and possessed strong antiangiogenic activity in vitro. Although ARC promoted the accumulation of p53, ARC-induced apoptosis in tumor cells was p53-independent, suggesting that it may be useful for the treatment of tumors with functionally inactive p53. Furthermore, cell death induced by ARC had a strong correlation with down-regulation of the antiapoptotic gene survivin, which is often overexpressed in human tumors. Taken together, our data suggests that ARC may be an attractive candidate for anticancer drug development. (Cancer Res 2006; 66(6): 3264-70)
Data from Merlin/Neurofibromatosis Type 2 Suppresses Growth by Inhibiting the Activation of Ras and Rac
Authors: Morrison Helen , Sperka Tobias , Manent Jan , Giovannini Marco , Ponta Helmut , Herrlich Peter
Published: March 2023

Abstract
The small G-protein Ras is a tightly controlled regulator of cell fate. Prolonged or persistent arrest in the activated GTP-loaded state by mutation of Ras as in lung cancer or in a Ras–GTPase-activating protein as in neurofibromatosis type 1 promotes tumorigenesis. We now show that the tumor-suppressor protein merlin (mutated in neurofibromatosis type 2) also controls Ras activity. Systematic analysis of growth factor signaling located the step of merlin interference to the activation of Ras and Rac. Merlin independently uncouples both Ras and Rac from growth factor signals. In the case of Ras, merlin acts downstream of the receptor tyrosine kinase-growth factor receptor binding protein 2 (Grb2)-SOS complex. However, merlin does not bind either SOS or Ras, but it counteracts the ERM (ezrin, radixin, moesin)–dependent activation of Ras, which correlates with the formation of a complex comprising ERM proteins, Grb2, SOS, Ras, and filamentous actin. Because efficient signaling from Ras requires Rac-p21-activated kinase–dependent phosphorylations of Raf and mitogen-activated protein/extracellular signal-regulated kinase kinase, merlin can also inhibit signal transfer from dominantly active Ras mutants. We propose that the interference of merlin with Ras- and Rac-dependent signal transfer represents part of the tumor-suppressive action of merlin. [Cancer Res 2007;67(2):520–7]
Data from The Collagen Receptor Endo180 (CD280) Is Expressed on Basal-like Breast Tumor Cells and Promotes Tumor Growth In vivo
Authors: Wienke Dirk , Davies Gareth C. , Johnson Damian A. , Sturge Justin , Lambros Maryou B.K. , Savage Kay , Elsheikh Somaia Elbauomy , Green Andrew R. , Ellis Ian O. , Robertson David , Reis-Filho Jorge S. , Isacke Clare M.
Published: March 2023

Abstract
Tumor cell invasion into the surrounding stroma requires increased cell motility and extensive remodeling of the extracellular matrix. Endo180 (CD280, MRC2, urokinase-type plasminogen activator receptor-associated protein) is a recycling endocytic receptor that functions in both these cellular activities by promoting cell migration and uptake of collagens for intracellular degradation. In the normal breast, Endo180 is predominantly expressed by stromal fibroblasts. The contrary observation that Endo180 is expressed on epithelial tumor cell lines that display a high invasive capacity suggested that up-regulation of this receptor may be an associated and functional component in the acquisition of a more aggressive phenotype by tumor cells in vivo. Here, we show that high levels of Endo180 are found in a subset of basal-like breast cancers and that this expression is an independent prognostic marker for shorter disease-free survival. Two potential mechanisms for Endo180 up-regulation were uncovered. First, it was shown that Endo180 can be transcriptionally up-regulated in vitro following transforming growth factor-β treatment of breast cancer cells. Second, a proportion of Endo180+ tumors were shown to have Endo180 gene copy number gains and amplifications. To investigate the functional consequence of Endo180 up-regulation, MCF7 cells transfected with Endo180 were inoculated into immunocompromised mice. Expression of wild-type Endo180, but not an internalization-defective Endo180 mutant, resulted in enhanced tumor growth together with a reduction in tumor collagen content. Together, these data argue that elevated expression of this receptor in tumor cells could have important consequences in subsets of basal-like carcinomas for which there is a current lack of effective treatment. [Cancer Res 2007;67(21):10230–11]