bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Mist1-KrasG12D Knock-In Mice Develop Mixed Differentiation Metastatic Exocrine Pancreatic Carcinoma and Hepatocellular Carcinoma
Authors: Tuveson David A. , Zhu Liqin , Gopinathan Aarthi , Willis Nicholas A. , Kachatrian Leili , Grochow Rebecca , Pin Christopher L. , Mitin Natalia Y. , Taparowsky Elizabeth J. , Gimotty Phyllis A. , Hruban Ralph H. , Jacks Tyler , Konieczny Stephen F.
Published: March 2023

Abstract
Despite the prevalence of oncogenic Kras mutations in the earliest stages of pancreatic ductal adenocarcinoma, the cellular compartment in which oncogenic Kras initiates tumorigenesis remains unknown. To address this, we have gene targeted KrasG12D into the open reading frame of Mist1, a basic helix-loop-helix transcription factor that is expressed during pancreatic development and required for proper pancreatic acinar organization. Although the pancreata of Mist1KrasG12D/+ mutant mice predictably exhibited acinar metaplasia and dysplasia, the frequent death of these mice from invasive and metastatic pancreatic cancer with mixed histologic characteristics, including acinar, cystic, and ductal features, was unexpected and in contrast to previously described mutant mice that ectopically expressed the Kras oncogene in either acinar or ductal compartments. Interestingly, many of the mutant mice developed hepatocellular carcinoma, implicating Mist1KrasG12D/+ cells in both pancreatic and hepatic neoplasia. Concomitant Trp53+/− mutation cooperated with Mist1KrasG12D/+ to accelerate lethality and was associated with advanced histopathologic findings, including parenchymal liver metastasis. These findings suggest that Mist1-expressing cells represent a permissive compartment for transformation by oncogenic Kras in pancreatic tumorigenesis. (Cancer Res 2006; 66(1): 242-7)
Data from Involvement of the RASSF1A Tumor Suppressor Gene in Controlling Cell Migration
Authors: Dallol Ashraf , Agathanggelou Angelo , Tommasi Stella , Pfeifer Gerd P. , Maher Eamonn R. , Latif Farida
Published: March 2023

Abstract
We have previously shown that RASSF1A associates with the microtubules. This association alters the microtubule dynamics and seems essential for RASSF1A tumor suppressive function. Mutant variants of RASSF1A that do not associate fully with the microtubules have reduced ability to stabilize them and cause cell cycle arrest. Here we show that overexpression of RASSF1A diminished the ability of A549 non–small cell lung cancer cells to migrate either through a transwell filter or to close a wound. In addition, we employed gene knockdown as well as mouse embryonic fibroblasts (MEFs) from Rassf1a knockout mice to analyze RASSF1A function in controlling cell motility. A549 cells stably transfected with RASSF1A exhibited increased cell-cell adhesion and less refractive morphology compared with controls. Conversely, RASSF1A knockdown in HeLa caused loss of cell-cell adhesion and a more refractive morphology. RASSF1A-depleted HeLa cells as well as Rassf1a−/− MEFs displayed increased cell migration that could be partly phosphatidylinositol 3-kinase dependent. Time-lapse microscopy showed the RASSF1A-depleted cells are highly motile with fibroblast-like morphology and diminished cell-cell adhesion. Staining of the cytoskeleton in RASSF1A-depleted HeLa cells and MEFs show marked differences in terms of microtubules outgrowth and actin stress fibers formation. This observation was associated with increased activation of Rac1 in RASSF1A-knockdown cells and the Rassf1a−/− MEFs. In addition, expression of a dominant-negative variant of Rac1 in the RASSF1A-depleted HeLa cells reduced their ability to form lamellipodia and other protrusions. These findings represent a novel function for RASSF1A, which may help explain its tumor suppression ability independently of its effects on cell cycle and apoptosis.
Data from EZC-Prostate Models Offer High Sensitivity and Specificity for Noninvasive Imaging of Prostate Cancer Progression and Androgen Receptor Action
Authors: Seethammagari Mamatha R. , Xie Xiaoming , Greenberg Norman M. , Spencer David M.
Published: March 2023

Abstract
In vivo imaging advances have greatly expanded the use of animal cancer models. Herein, we describe two new models that permit prostate imaging ex vivo, in vivo, and in utero. Further, we show the use of these models for detecting small metastasis and testing reagents that modulate the androgen receptor (AR) axis. A luciferase reporter gene was directed to the prostate epithelium using three composite promoters called human kallikrein 2 (hK2)-E3/P, PSA-E2/P, and ARR2PB, derived from hK2, PSA, and rat probasin regulatory elements, to generate the EZC1, EZC2, and EZC3-prostate mice, respectively. EZC2 and EZC3-prostate display robust expression in the prostate with only minimal detectable expression in other organs, including testes and epididymis. Luciferase expression was detected as early as embryonic day 13 (E13) in the urogenital track. To image prostate cancer progression, lines of EZC mice were bred with prostate cancer models TRAMP and JOCK1, and imaged longitudinally. When crossed with prostate cancer models, EZC3 facilitated detection of metastatic lesions although total prostate luciferase expression was static or reduced due to weakening of AR-regulated promoters. Castration reduced luciferase expression by 90% and 97% in EZC2 and EZC3 mice, respectively, and use of GnRH antagonist also led to extensive inhibition of reporter activity. The EZC-prostate model permits prostate imaging in vivo and should be useful for imaging prostate development, growth, metastasis, and response to treatment noninvasively and longitudinally. These models also provide powerful new reagents for developing improved drugs that inhibit the AR axis. (Cancer Res 2006; 66(12): 6199-209)
Data from GRP78 as a Novel Predictor of Responsiveness to Chemotherapy in Breast Cancer
Authors: Lee Eunjung , Nichols Peter , Spicer Darcy , Groshen Susan , Yu Mimi C. , Lee Amy S.
Published: March 2023

Abstract
The discovery of predictive factors for chemoresistance is critical for improving adjuvant therapy for cancer patients. The 78-kDa glucose-regulated protein (GRP78), widely used as an indicator of the unfolded protein response (UPR), is induced in the tumor microenvironment. In vitro studies suggest that GRP78 confers chemoresistance to topoisomerase inhibitors, such as Adriamycin (doxorubicin). Here, we report on a retrospective cohort study of 127 stage II and III breast cancer patients who were treated with Adriamycin-based chemotherapy. Archival tumor specimens were available for analysis and the relationship of GRP78 expression level to “time to recurrence” (TTR), used as a surrogate marker for drug resistance, was examined. Our data show that 67% of the study subjects expressed high level of GRP78 in their tumors before the initiation of chemotherapy and suggest an association between GRP78 positivity and shorter TTR [hazard ratio (HR), 1.78; P = 0.16]. Interestingly, subgroup analysis reveals that the HR for the GRP78-positive group increased significantly among patients who did not receive further taxane treatment (HR, 3.00; P = 0.022) and among mastectomy patients (HR, 3.33; P = 0.027). The HR was even stronger among mastectomy patients who did not receive further taxane treatment (HR, 4.82; P = 0.010). The use of GRP78 as a predictor for chemoresponsiveness and the potential interaction of GRP78 and/or the UPR pathways with taxanes warrant larger studies. (Cancer Res 2006; 66(16): 7849-53)
Data from RNA Silencing of Checkpoint Regulators Sensitizes p53-Defective Prostate Cancer Cells to Chemotherapy while Sparing Normal Cells
Authors: Mukhopadhyay Utpal K. , Senderowicz Adrian M. , Ferbeyre Gerardo
Published: March 2023

Abstract
p53 is frequently mutated in patients with prostate cancer, especially in those with advanced disease. Therefore, the selective elimination of p53 mutant cells will likely have an impact in the treatment of prostate cancer. Because p53 has important roles in cell cycle checkpoints, it has been anticipated that modulation of checkpoint pathways should sensitize p53-defective cells to chemotherapy while sparing normal cells. To test this idea, we knocked down ataxia telangiectasia mutated (ATM) gene by RNA interference in prostate cancer cell lines and in normal human diploid fibroblasts IMR90. ATM knockdown in p53-defective PC3 prostate cancer cells accelerated their cell cycle transition, increased both E2F activity and proliferating cell nuclear antigen expression, and compromised cell cycle checkpoints, which are normally induced by DNA damage. Consequently, PC3 cells were sensitized to the killing effects of the DNA-damaging drug doxorubicin. Combining ATM knockdown with the Chk1 inhibitor UCN-01 further increased doxorubicin sensitivity in these cells. In contrast, the same strategy did not sensitize either IMR90 or LNCaP prostate cancer cells, both of which have normal p53. However, IMR90 and LNCaP cells became more sensitive to doxorubicin or doxorubicin plus UCN-01 when both p53 and ATM functions were suppressed. In addition, knockdown of the G2 checkpoint regulators ATR and Chk1 also sensitized PC3 cells to doxorubicin and increased the expression of the E2F target gene PCNA. Together, our data support the concept of selective elimination of p53 mutant cells by combining DNA damage with checkpoint inhibitors and suggest a novel mechanistic insight into how such treatment may selectively kill tumor cells.
Data from Inhibition of Mammalian Target of Rapamycin Reverses Alveolar Epithelial Neoplasia Induced by Oncogenic K-ras
Authors: Wislez Marie , Spencer M. Loreto , Izzo Julie G. , Juroske Denise M. , Balhara Kamna , Cody Dianna D. , Price Roger E. , Hittelman Walter N. , Wistuba Ignacio I. , Kurie Jonathan M.
Published: March 2023

Abstract
The serine/threonine kinase AKT and its downstream mediator mammalian target of rapamycin (mTOR) are activated in lung adenocarcinoma, and clinical trials are under way to test whether inhibition of mTOR is useful in treating lung cancer. Here, we report that mTOR inhibition blocked malignant progression in K-rasLA1 mice, which undergo somatic activation of the K-ras oncogene and display morphologic changes in alveolar epithelial cells that recapitulate those of precursors of human lung adenocarcinoma. Levels of phospho-S6Ser236/235, a downstream mediator of mTOR, increased with malignant progression (normal alveolar epithelial cells to adenocarcinoma) in K-rasLA1 mice and in patients with lung adenocarcinoma. Atypical alveolar hyperplasia, an early neoplastic change, was prominently associated with macrophages and expressed high levels of phospho-S6Ser236/235. mTOR inhibition in K-rasLA1 mice by treatment with the rapamycin analogue CCI-779 reduced the size and number of early epithelial neoplastic lesions (atypical alveolar hyperplasia and adenomas) and induced apoptosis of intraepithelial macrophages. LKR-13, a lung adenocarcinoma cell line derived from K-rasLA1 mice, was resistant to treatment with CCI-779 in vitro. However, LKR-13 cells grown as syngeneic tumors recruited macrophages, and those tumors regressed in response to treatment with CCI-779. Lastly, conditioned medium from primary cultures of alveolar macrophages stimulated the proliferation of LKR-13 cells. These findings provide evidence that the expansion of lung adenocarcinoma precursors induced by oncogenic K-ras requires mTOR-dependent signaling and that host factors derived from macrophages play a critical role in adenocarcinoma progression.
Data from Significance of Murine Retroviral Mutagenesis for Identification of Disease Genes in Human Acute Myeloid Leukemia
Authors: Erkeland Stefan J. , Verhaak Roel G.W. , Valk Peter J.M. , Delwel Ruud , Löwenberg Bob , Touw Ivo P.
Published: March 2023

Abstract
Retroviral insertion mutagenesis is considered a powerful tool to identify cancer genes in mice, but its significance for human cancer has remained elusive. Moreover, it has recently been debated whether common virus integrations are always a hallmark of tumor cells and contribute to the oncogenic process. Acute myeloid leukemia (AML) is a heterogeneous disease with a variable response to treatment. Recurrent cytogenetic defects and acquired mutations in regulatory genes are associated with AML subtypes and prognosis. Recently, gene expression profiling (GEP) has been applied to further risk stratify AML. Here, we show that mouse leukemia genes identified by retroviral insertion mutagenesis are more frequently differentially expressed in distinct subclasses of adult and pediatric AML than randomly selected genes or genes located more distantly from a virus integration site. The candidate proto-oncogenes showing discriminative expression in primary AML could be placed in regulatory networks mainly involved in signal transduction and transcriptional control. Our data support the validity of retroviral insertion mutagenesis in mice for human disease and indicate that combining these murine screens for potential proto-oncogenes with GEP in human AML may help to identify critical disease genes and novel pathogenetic networks in leukemia. (Cancer Res 2006; 66(2): 622-6)
Data from Identification of GATA3 as a Breast Cancer Prognostic Marker by Global Gene Expression Meta-analysis
Authors: Mehra Rohit , Varambally Sooryanarayana , Ding Lei , Shen Ronglai , Sabel Michael S. , Ghosh Debashis , Chinnaiyan Arul M. , Kleer Celina G.
Published: March 2023

Abstract
GATA binding protein 3 (GATA3) is a transcriptional activator highly expressed by the luminal epithelial cells in the breast. Here we did a meta-analysis of the available breast cancer cDNA data sets on a cohort of 305 patients and found that GATA3 was one of the top genes with low expression in invasive carcinomas with poor clinical outcome. To validate its prognostic utility, we did a tissue microarray analysis on a cohort of 139 consecutive invasive carcinomas (n = 417 tissue samples) immunostained with a monoclonal antibody against GATA3. Low GATA3 expression was associated with higher histologic grade (P < 0.001), positive nodes (P = 0.002), larger tumor size (P = 0.03), negative estrogen receptor and progesterone receptor (P < 0.001 for both), and HER2-neu overexpression (P = 0.03). Patients whose tumors expressed low GATA3 had significantly shorter overall and disease-free survival when compared with those whose tumors had high GATA3 levels. The hazard ratio of metastasis or recurrence according to the GATA3 status was 0.31 (95% confidence interval, 0.13-0.74; P = 0.009). Cox multivariate analysis showed that GATA3 had independent prognostic significance above and beyond conventional variables. Our data suggest that immunohistochemical analysis of GATA3 may be the basis for a new clinically applicable test to predict tumor recurrence early in the progression of breast cancer. (Cancer Res 2005; 65(24): 11259-64)
Data from Cancers as Wounds that Do Not Heal: Differences and Similarities between Renal Regeneration/Repair and Renal Cell Carcinoma
Authors: Riss Joseph , Khanna Chand , Koo Seongjoon , Chandramouli Gadisetti V.R. , Yang Howard H. , Hu Ying , Kleiner David E. , Rosenwald Andreas , Schaefer Carl F. , Ben-Sasson Shmuel A. , Yang Liming , Powell John , Kane David W. , Star Robert A. , Aprelikova Olga , Bauer Kristin , Vasselli James R. , Maranchie Jodi K. , Kohn Kurt W. , Buetow Ken H. , Linehan W. Marston , Weinstein John N. , Lee Maxwell P. , Klausner Richard D. , Barrett J. Carl
Published: March 2023

Abstract
Cancers have been described as wounds that do not heal, suggesting that the two share common features. By comparing microarray data from a model of renal regeneration and repair (RRR) with reported gene expression in renal cell carcinoma (RCC), we asked whether those two processes do, in fact, share molecular features and regulatory mechanisms. The majority (77%) of the genes expressed in RRR and RCC were concordantly regulated, whereas only 23% were discordant (i.e., changed in opposite directions). The orchestrated processes of regeneration, involving cell proliferation and immune response, were reflected in the concordant genes. The discordant gene signature revealed processes (e.g., morphogenesis and glycolysis) and pathways (e.g., hypoxia-inducible factor and insulin-like growth factor-I) that reflect the intrinsic pathologic nature of RCC. This is the first study that compares gene expression patterns in RCC and RRR. It does so, in particular, with relation to the hypothesis that RCC resembles the wound healing processes seen in RRR. However, careful attention to the genes that are regulated in the discordant direction provides new insights into the critical differences between renal carcinogenesis and wound healing. The observations reported here provide a conceptual framework for further efforts to understand the biology and to develop more effective diagnostic biomarkers and therapeutic strategies for renal tumors and renal ischemia. (Cancer Res 2006; 66(14): 7216-24)
Data from YC-1 Induces S Cell Cycle Arrest and Apoptosis by Activating Checkpoint Kinases
Authors: Yeo Eun-Jin , Ryu Ji-Hye , Chun Yang-Sook , Cho Young-Suk , Jang In-Jin , Cho HoSung , Kim Jinho , Kim Myung-Suk , Park Jong-Wan
Published: March 2023

Abstract
Hypoxia-inducible factor-1α (HIF-1α) seems central to tumor growth and progression because it up-regulates genes essential for angiogenesis and the hypoxic adaptation of cancer cells, which is why HIF-1α inhibition is viewed as a cancer therapy strategy. Paradoxically, HIF-1α also leads to cell cycle arrest or the apoptosis of cancer cells. Thus, the possibility cannot be ruled out that HIF-1α inhibitors unlock cell cycle arrest under hypoxic conditions and prevent cell death, which would limit the anticancer effect of HIF-1α inhibitors. Previously, we reported on the development of YC-1 as an anticancer agent that inhibits HIF-1α. In the present study, we evaluated the effects of YC-1 on hypoxia-induced cell cycle arrest and cell death. It was found that YC-1 does not reverse the antiproliferative effect of hypoxia, but rather that it induces S-phase arrest and apoptosis at therapeutic concentrations that inhibit HIF-1α and tumor growth; however, YC-1 did not stimulate cyclic guanosine 3′,5′-monophosphate production in this concentration range. It was also found that YC-1 activates the checkpoint kinase–mediated intra-S-phase checkpoint, independently of ataxia-telangiectasia mutated kinase or ataxia-telangiectasia mutated and Rad3-related kinase. These results imply that YC-1 does not promote the regrowth of hypoxic tumors because of its cell cycle arrest effect. Furthermore, YC-1 may induce the combined anticancer effects of HIF-1α inhibition and cell growth inhibition. (Cancer Res 2006; 66(12): 6345-52)
Data from MUC1 Oncoprotein Blocks Glycogen Synthase Kinase 3β–Mediated Phosphorylation and Degradation of β-Catenin
Authors: Huang Lei , Chen Dongshu , Liu Derek , Yin Li , Kharbanda Surender , Kufe Donald
Published: March 2023

Abstract
Dysregulation of β-catenin is of importance to the development of diverse human malignancies. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and associates with β-catenin. However, the functional significance of the MUC1-β-catenin interaction is not known. Here, we show that MUC1 increases β-catenin levels in the cytoplasm and nucleus of carcinoma cells. Previous studies have shown that glycogen synthase kinase 3β (GSK3β) phosphorylates β-catenin and thereby targets it for proteosomal degradation. Consistent with the up-regulation of β-catenin levels, our results show that MUC1 blocks GSK3β-mediated phosphorylation and degradation of β-catenin. To further define the interaction between MUC1 and β-catenin, we identified a serine-rich motif (SRM) in the MUC1 cytoplasmic domain that binds directly to β-catenin Armadillo repeats. Mutation of the SRM attenuated binding of MUC1 to β-catenin and MUC1-mediated inhibition of β-catenin degradation. Importantly, disruption of the MUC1-β-catenin interaction with the SRM mutant also attenuated MUC1-induced anchorage-dependent and -independent growth and delayed MUC1-mediated tumorigenicity. These findings indicate that MUC1 promotes transformation, at least in part, by blocking GSK3β-mediated phosphorylation and thereby degradation of β-catenin.
Data from Characterization of Endogenous Human Promyelocytic Leukemia Isoforms
Authors: Condemine Wilfried , Takahashi Yuki , Zhu Jun , Puvion-Dutilleul Francine , Guegan Sarah , Janin Anne , de Thé Hugues
Published: March 2023

Abstract
Promyelocytic leukemia (PML) has been implicated in a variety of functions, including control of TP53 function and modulation of cellular senescence. Sumolated PML is the organizer of mature PML bodies, recruiting a variety of proteins onto these nuclear domains. The PML gene is predicted to encode a variety of protein isoforms. Overexpression of only one of them, PML-IV, promotes senescence in human diploid fibroblasts, whereas PML-III was proposed to specifically interact with the centrosome. We show that all PML isoform proteins are expressed in cell lines or primary cells. Unexpectedly, we found that PML-III, PML-IV, and PML-V are quantitatively minor isoforms compared with PML-I/II and could not confirm the centrosomal targeting of PML-III. Stable expression of each isoform, in a pml-null background, yields distinct subcellular localization patterns, suggesting that, like in other RBCC/TRIM proteins, the COOH-terminal domains of PML are involved in interactions with specific cellular components. Only the isoform-specific sequences of PML-I and PML-V are highly conserved between man and mouse. That PML-I contains all conserved exons and is more abundantly expressed than PML-IV suggests that it is a critical contributor to PML function(s). (Cancer Res 2006; 66(12): 6192-8)
Data from Multiple Roles for the Receptor Tyrosine Kinase Axl in Tumor Formation
Authors: Holland Sacha J. , Powell Mark J. , Franci Christian , Chan Emily W. , Friera Annabelle M. , Atchison Robert E. , McLaughlin John , Swift Susan E. , Pali Erlina S. , Yam George , Wong Stephen , Lasaga Joe , Shen Mary R. , Yu Simon , Xu Weiduan , Hitoshi Yasumichi , Bogenberger Jakob , Nör Jacques E. , Payan Donald G. , Lorens James B.
Published: March 2023

Abstract
A focus of contemporary cancer therapeutic development is the targeting of both the transformed cell and the supporting cellular microenvironment. Cell migration is a fundamental cellular behavior required for the complex interplay between multiple cell types necessary for tumor development. We therefore developed a novel retroviral-based screening technology in primary human endothelial cells to discover genes that control cell migration. We identified the receptor tyrosine kinase Axl as a novel regulator of endothelial cell haptotactic migration towards the matrix factor vitronectin. Using small interfering RNA–mediated silencing and overexpression of wild-type or mutated receptor proteins, we show that Axl is a key regulator of multiple angiogenic behaviors including endothelial cell migration, proliferation, and tube formation in vitro. Moreover, using sustained, retrovirally delivered short hairpin RNA (shRNA) Axl knockdown, we show that Axl is necessary for in vivo angiogenesis in a mouse model. Furthermore, we show that Axl is also required for human breast carcinoma cells to form a tumor in vivo. These findings indicate that Axl regulates processes vital for both neovascularization and tumorigenesis. Disruption of Axl signaling using a small-molecule inhibitor will hence simultaneously affect both the tumor and stromal cell compartments and thus represents a unique approach for cancer therapeutic development.
Data from The Role of Base Excision Repair in the Sensitivity and Resistance to Temozolomide-Mediated Cell Death
Authors: Trivedi Ram N. , Almeida Karen H. , Fornsaglio Jamie L. , Schamus Sandra , Sobol Robert W.
Published: March 2023

Abstract
DNA-alkylating agents have a central role in the curative therapy of many human tumors; yet, resistance to these agents limits their effectiveness. The efficacy of the alkylating agent temozolomide has been attributed to the induction of O6-MeG, a DNA lesion repaired by the protein O6-methylguanine-DNA methyltransferase (MGMT). Resistance to temozolomide has been ascribed to elevated levels of MGMT and/or reduced mismatch repair. However, >80% of the DNA lesions induced by temozolomide are N-methylated bases that are recognized by DNA glycosylases and not by MGMT, and so resistance to temozolomide may also be due, in part, to robust base excision repair (BER). We used isogenic cells deficient in the BER enzymes DNA polymerase-β (pol-β) and alkyladenine DNA glycosylase (Aag) to determine the role of BER in the cytotoxic effect of temozolomide. Pol-β–deficient cells were significantly more susceptible to killing by temozolomide than wild-type or Aag-deficient cells, a hypersensitivity likely caused by accumulation of BER intermediates. RNA interference–mediated pol-β suppression was sufficient to increase temozolomide efficacy, whereas a deficiency in pol-ι or pol-λ did not increase temozolomide-mediated cytotoxicity. Overexpression of Aag (the initiating BER enzyme) triggered a further increase in temozolomide-induced cytotoxicity. Enhanced Aag expression, coupled with pol-β knockdown, increased temozolomide efficacy up to 4-fold. Furthermore, loss of pol-β coupled with temozolomide treatment triggered the phosphorylation of H2AX, indicating the activation of the DNA damage response pathway as a result of unrepaired lesions. Thus, the BER pathway is a major contributor to cellular resistance to temozolomide and its efficacy depends on specific BER gene expression and activity.
Data from Phosphorylation of Galectin-3 Contributes to Malignant Transformation of Human Epithelial Cells via Modulation of Unique Sets of Genes
Authors: Mazurek Nachman , Sun Yun Jie , Price Janet E. , Ramdas Latha , Schober Wendy , Nangia-Makker Pratima , Byrd James C. , Raz Avraham , Bresalier Robert S.
Published: March 2023

Abstract
Galectin-3 is a multifunctional β-galactoside-binding protein implicated in apoptosis, malignant transformation, and tumor progression. The mechanisms by which galectin-3 contributes to malignant progression are not fully understood. In this study, we found that the introduction of wild-type galectin-3 into nontumorigenic, galectin-3-null BT549 human breast epithelial cells conferred tumorigenicity and metastatic potential in nude mice, and that galectin-3 expressed by the cells was phosphorylated. In contrast, BT549 cells expressing galectin-3 incapable of being phosphorylated (Ser6→Glu Ser6→Ala) were nontumorigenic. A microarray analysis of 10,000 human genes, comparing BT549 transfectants expressing wild-type and those expressing phosphomutant galectin-3, identified 188 genes that were differentially expressed (>2.5-fold). Genes affected by introduction of wild-type phosphorylated but not phosphomutant galectin-3 included those involved in oxidative stress, a novel noncaspase lysosomal apoptotic pathway, cell cycle regulation, transcriptional activation, cytoskeleton remodeling, cell adhesion, and tumor invasion. The reliability of the microarray data was validated by real-time reverse transcription-PCR (RT-PCR) and by Western blot analysis, and clinical relevance was evaluated by real-time RT-PCR screening of a panel of matched pairs of breast tumors. Differentially regulated genes in breast cancers that are also predicted to be associated with phospho-galectin-3 in transformed BT549 cells include C-type lectin 2, insulin-like growth factor-binding protein 5, cathepsins L2, and cyclin D1. These data show the functional diversity of galectin-3 and suggest that phosphorylation of the protein is necessary for regulation (directly or indirectly) of unique sets of genes that play a role in malignant transformation.
Data from Effective Treatment of Tumors with Strong β-Catenin/T-Cell Factor Activity by Transcriptionally Targeted Oncolytic Herpes Simplex Virus Vector
Authors: Kuroda Toshihiko , Rabkin Samuel D. , Martuza Robert L.
Published: March 2023

Abstract
The Wnt/β-catenin/T-cell factor (Tcf) pathway is aberrantly up-regulated in the majority of colorectal cancers (CRC) and hepatoblastomas due to either an APC or β-catenin gene mutation. We constructed synthetic promoters, T and TE, which contain tandem repeats of a Tcf responsive element without and with the human 4F2 gene intronic enhancer, respectively. Although the T and TE promoters showed higher transcriptional activity than a control promoter in all CRC and hepatoblastoma cell lines tested, with low activities in most other tumor cell lines, the level of transcription varied considerably among the CRC and hepatoblastoma cell lines. In some CRC cell lines, the TE promoter displayed higher levels of transcription than even the human CMVIE promoter. In those CRC cells, the APC gene mutations were located within a small segment between the first and second 20-amino-acid repeats in the mutation cluster region of the APC protein. We created a transcriptionally targeted oncolytic herpes simplex virus vector (bM24-TE) in which replication is driven by the TE promoter. This vector efficiently and specifically replicated in and killed tumor cells with strong β-catenin/Tcf signaling. Intratumoral injection of bM24-TE significantly reduced the growth of highly β-catenin active SW480 CRC tumors and induced a complete response in half of them, whereas it had no effect on the growth of β-catenin-inactive A549 tumors. Our results suggest that a transcriptionally regulated oncolytic herpes vector targeting β-catenin/Tcf signal is very efficacious against CRC tumors carrying an APC gene mutation between the first and second 20-amino-acid repeats. (Cancer Res 2006; 66(20): 10127-35)
Data from The Syk Tyrosine Kinase Localizes to the Centrosomes and Negatively Affects Mitotic Progression
Authors: Zyss Déborah , Montcourrier Philippe , Vidal Benjamin , Anguille Christelle , Mérezègue Fabrice , Sahuquet Alain , Mangeat Paul H. , Coopman Peter J.
Published: March 2023

Abstract
We showed previously that the spleen tyrosine kinase Syk is expressed by mammary epithelial cells and that it suppresses malignant growth of breast cancer cells. The exact molecular mechanism of its tumor-suppressive activity remains, however, to be identified. Here, we show that Syk colocalizes and copurifies with the centrosomal component γ-tubulin and exhibits a catalytic activity within the centrosomes. Moreover, its centrosomal localization depends on its intact kinase activity. Centrosomal Syk expression is persistent in interphase but promptly drops during mitosis, obviously resulting from its ubiquitinylation and proteasomal degradation. Conversely, unrestrained exogenous expression of a fluorescently tagged Discosoma sp. red fluorescent protein (DsRed)-Syk chimera engenders abnormal cell division and cell death. Transient DsRed-Syk overexpression triggers an abrupt cell death lacking hallmarks of classic apoptosis but reminiscent of mitotic catastrophe. Surviving stable DsRed-Syk–transfected cells exhibit multipolar mitotic spindles and contain multiple abnormally sized nuclei and supernumerary centrosomes, revealing anomalous cell division. Taken together, these results show that Syk is a novel centrosomal kinase that negatively affects cell division. Its expression is strictly controlled in a spatiotemporal manner, and centrosomal Syk levels need to decline to allow customary progression of mitosis.
Data from Ephrin-B3 Ligand Promotes Glioma Invasion through Activation of Rac1
Authors: Nakada Mitsutoshi , Drake Kelsey L. , Nakada Satoko , Niska Jared A. , Berens Michael E.
Published: March 2023

Abstract
Eph receptor tyrosine kinases are involved in nervous system development. Eph ligands, termed ephrins, are transmembrane proteins that bind to Eph receptors, the mutual activation of which causes repulsive effects in reciprocally contacting cells. Previously, we showed that overexpression of EphB2 in glioma cells increases cell invasion. Here, expression profiles of ephrin-B family members were determined in four glioma cell lines and in invading glioblastoma cells collected by laser capture microdissection. Ephrin-B3 mRNA was up-regulated in migrating cells of four of four glioma cell lines (1.3- to 1.7-fold) and in invading tumor cells of eight of eight biopsy specimens (1.2- to 10.0-fold). Forced expression of ephrin-B3 in low expressor cell lines (U87, T98G) stimulated cell migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin-B3. In high expressor cell lines (U251, SNB19), ephrin-B3 colocalized with Rac1 to lamellipodia of motile wild-type cells. Cells transfected with ephrin-B3 small interfering RNA (siRNA) showed significant morphologic change and decreased invasion in vitro and ex vivo. Depletion of endogenous ephrin-B3 expression abrogated the increase of migration and invasion induced by EphB2/Fc, indicating increased invasion is dependent on ephrin-B3 activation. Furthermore, using a Rac1-GTP pull-down assay, we showed that ephrin-B3 is associated with Rac1 activation. Reduction of Rac1 by siRNA negated the increased invasion by addition of EphB2/Fc. In human glioma specimens, ephrin-B3 expression and phosphorylation correlated with increasing tumor grade. Immunohistochemistry revealed robust staining for phosphorylated ephrin-B and ephrin-B3 in invading glioblastoma cells. These data show that ephrin-B3 expression and signaling through Rac1 are critically important to glioma invasion. (Cancer Res 2006; 66(17): 8492-500)
Data from Synergistic Function of Smad4 and PTEN in Suppressing Forestomach Squamous Cell Carcinoma in the Mouse
Authors: Teng Yan , Sun An-Na , Pan Xiao-Chen , Yang Guan , Yang Lei-Lei , Wang Ming-Rong , Yang Xiao
Published: March 2023

Abstract
The genetic bases underlying esophageal tumorigenesis are poorly understood. Our previous studies have shown that coordinated deletion of the Smad4 and PTEN genes results in accelerated hair loss and skin tumor formation in mice. Herein, we exemplify that the concomitant inactivation of Smad4 and PTEN accelerates spontaneous forestomach carcinogenesis at complete penetrance during the first 2 months of age. All of the forestomach tumors were invasive squamous cell carcinomas (SCCs), which recapitulated the natural history and pathologic features of human esophageal SCCs. A small population of the SCC lesions was accompanied by adenocarcinomas at the adjacent submucosa region in the double mutant mice. The rapid progression of forestomach tumor formation in the Smad4 and PTEN double knockout mice corresponded to a dramatic increase in esophageal and forestomach epithelial proliferation. The decreased expression of p27, p21, and p16 together with the overexpression of cyclin D1 contributed cooperatively to the accelerated forestomach tumorigenesis in the double mutant mice. Our results point strongly to the crucial relevance of synergy between Smad4 and PTEN to suppress forestomach tumorigenesis through the cooperative induction of cell cycle inhibitors. (Cancer Res 2006; 66(14): 6972-81)
Data from A Novel Somatic Mouse Model to Survey Tumorigenic Potential Applied to the Hedgehog Pathway
Authors: Mao Junhao , Ligon Keith L. , Rakhlin Elena Y. , Thayer Sarah P. , Bronson Roderick T. , Rowitch David , McMahon Andrew P.
Published: March 2023

Abstract
We report a novel mouse model for the generation of sporadic tumors and show the efficiency of this approach by surveying Hedgehog (Hh)–related tumors. Up-regulation of the Hh pathway is achieved by conditionally regulated expression of an activated allele of Smoothened (R26-SmoM2) using either sporadic leakage or global postnatal induction of a ubiquitously expressed inducible Cre transgene (CAGGS-CreER). Following postnatal tamoxifen induction, CAGGS-CreER; R26-SmoM2 mice developed tumors with short latency and high penetrance. All mice exhibited rhabdomyosarcoma and basal cell carcinoma; 40% also developed medulloblastoma. In addition, mice showed a novel pancreatic lesion resembling low-grade mucinous cystic neoplasms in humans. In contrast, widespread activation of SmoM2 in the postnatal prostate epithelium results in no detectable morphologic outcome in 12-month-old mice. Comparison of gene expression profiles among diverse tumors identified several signature genes, including components of platelet-derived growth factor and insulin-like growth factor pathways, which may provide a common mechanistic link to the Hh-related malignancies. This experimental model provides a robust tool for exploring the process of Hh-dependent tumorigenesis and the treatment of such tumors. More generally, this approach provides a genetic platform for identifying tumorigenic potential in putative oncogenes and tumor suppressors and for more effective modeling of sporadic cancers in mice. (Cancer Res 2006; 66(20): 10171-7)
Data from Small-Molecule MDM2 Antagonists as a New Therapy Concept for Neuroblastoma
Authors: Van Maerken Tom , Speleman Frank , Vermeulen Joëlle , Lambertz Irina , De Clercq Sarah , De Smet Els , Yigit Nurten , Coppens Vicky , Philippé Jan , De Paepe Anne , Marine Jean-Christophe , Vandesompele Jo
Published: March 2023

Abstract
Circumvention of the p53 tumor suppressor barrier in neuroblastoma is rarely caused by TP53 mutation but might arise from inappropriately increased activity of its principal negative regulator MDM2. We show here that targeted disruption of the p53-MDM2 interaction by the small-molecule MDM2 antagonist nutlin-3 stabilizes p53 and selectively activates the p53 pathway in neuroblastoma cells with wild-type p53, resulting in a pronounced antiproliferative and cytotoxic effect through induction of G1 cell cycle arrest and apoptosis. A nutlin-3 response was observed regardless of MYCN amplification status. Remarkably, surviving SK-N-SH cells adopted a senescence-like phenotype, whereas CLB-GA and NGP cells underwent neuronal differentiation. p53 dependence of these alternative outcomes of nutlin-3 treatment was evidenced by abrogation of the effects when p53 was knocked down by lentiviral-mediated short hairpin RNA interference. The diversity of cellular responses reveals pleiotropic mechanisms of nutlins to disable neuroblastoma cells and exemplifies the feasibility of exploiting, by a single targeted intervention, the multiplicity of anticancer activities exerted by a key tumor suppressor as p53. The observed treatment effects without the need of imposing a genotoxic burden suggest that selective MDM2 antagonists might be beneficial for treatment of neuroblastoma patients with and without MYCN amplification. (Cancer Res 2006; 66(19): 9646-55)
Data from DNA Methylation Changes after 5-Aza-2′-Deoxycytidine Therapy in Patients with Leukemia
Authors: Yang Allen S. , Doshi Ketan D. , Choi Sang-Woon , Mason Joel B. , Mannari Rajan K. , Gharybian Vazganush , Luna Rene , Rashid Asif , Shen Lanlan , Estecio Marcos R.H. , Kantarjian Hagop M. , Garcia-Manero Guillermo , Issa Jean-Pierre J.
Published: March 2023

Abstract
5-Aza-2′-deoxycytidine (decitabine) is postulated to have clinical activity in myeloid leukemias via its ability to inhibit DNA methylation. To study this, we examined DNA methylation in patients with leukemia treated with decitabine. Five days after the treatment, total genomic 5-methylcytosine/cytosine decreased on average by 14% (from 4.3% to 3.7%), whereas methylation of repetitive DNA elements showed a mean decrease of 9% and 16% for Alu and long interspersed nucleotide elements, respectively. Methylation decreased linearly with increasing doses between 5 and 20 mg/m2/d (r = 0.88; P = 0.05) but showed a plateau above that. Hypomethylation correlated with response in patients with acute myelogenous leukemia treated with low doses (5-20 mg/m2/d), but patients with chronic myelogenous leukemia treated with high doses (100-180 mg/m2/d) showed no such correlation. Aberrant methylation of p15 (>10%) was found in 27% of patients, and 80% of these showed a decrease by at least one third, but this did not correlate with response. The imprinted gene H19 showed little change in methylation after decitabine. In conclusion, we show dose-dependent hypomethylation after decitabine at low doses. Increasing the dose, which has been shown previously to result in a reduced response rate, was not accompanied by further hypomethylation. (Cancer Res 2006; 66(10): 5495-503)
Data from Motexafin Gadolinium Disrupts Zinc Metabolism in Human Cancer Cell Lines
Authors: Magda Darren , Lecane Philip , Miller Richard A. , Lepp Cheryl , Miles Dale , Mesfin Mimi , Biaglow John E. , Ho Vincent V. , Chawannakul Danny , Nagpal Shailender , Karaman Mazen W. , Hacia Joseph G.
Published: March 2023

Abstract
To gain a better understanding of the mechanism of action of the metal cation–containing chemotherapeutic drug motexafin gadolinium (MGd), gene expression profiling analyses were conducted on plateau phase human lung cancer (A549) cell cultures treated with MGd. Drug treatment elicited a highly specific response that manifested in elevated levels of metallothionein isoform and zinc transporter 1 (ZnT1) transcripts. A549 cultures incubated with MGd in the presence of exogenous zinc acetate displayed synergistic increases in the levels of intracellular free zinc, metallothionein transcripts, inhibition of thioredoxin reductase activity, and cell death. Similar effects were observed in PC3 prostate cancer and Ramos B-cell lymphoma cell lines. Intracellular free zinc levels increased in response to treatment with MGd in the absence of exogenous zinc, indicating that MGd can mobilize bound intracellular zinc. These findings lead us to suggest that an important component of the anticancer activity of MGd is related to its ability to disrupt zinc metabolism and alter cellular availability of zinc. This class of compounds may provide insight into the development of novel cancer drugs targeting control of intracellular free zinc and the roles that zinc and other metal cations play in biochemical pathways relevant to cancer.
Data from A Mouse Model for the Molecular Characterization of Brca1-Associated Ovarian Carcinoma
Authors: Xing Deyin , Orsulic Sandra
Published: March 2023

Abstract
Little is known about the mechanisms that underlie Brca1-associated ovarian tumorigenesis, mainly due to the lack of an appropriate experimental model. We developed genetically defined primary mouse ovarian surface epithelial (OSE) cell lines in which the loss of functional Brca1 and p53 recapitulates the events that are thought to occur in early ovarian cancer development in patients with Brca1 mutations. This system allows for the introduction of additional oncogenes that are thought to cooperate with the loss of Brca1 and p53 to induce tumorigenesis. We showed that Myc is sufficient to induce transformation of ovarian cells that are deficient for both Brca1 and p53 but not sufficient for the transformation of cells that are deficient for either Brca1 or p53. The transformed Brca1-deficient OSE cells display an increased number of centrosomes, acquire complex chromosome aberrations, and lack Rad51 nuclear foci in the presence of DNA-damaging agents, such as mitomycin C and cisplatin. Immunocompetent mice injected with transformed OSE cells develop tumors that resemble human metastatic serous ovarian carcinoma, the most common type of ovarian cancer in women. Consistent with the reported platinum chemosensitivity in patients with Brca1-associated ovarian cancer, the Brca1-deficient OSE cells have increased sensitivity to the DNA-damaging agent cisplatin, whereas sensitivity to the microtubule poison paclitaxel is similar between Brca1 wild-type and Brca1-deficient cells. The Brca1 wild-type and Brca1-deficient mouse ovarian tumors and cell lines provide a new experimental system for the evaluation of therapies that target the Brca1 pathway. (Cancer Res 2006; 66(18): 8949-53)
Data from Identification of Novel Target Genes by an Epigenetic Reactivation Screen of Renal Cancer
Authors: Ibanez de Caceres Inmaculada , Dulaimi Essel , Hoffman Amanda M. , Al-Saleem Tahseen , Uzzo Robert G. , Cairns Paul
Published: March 2023

Abstract
Aberrant promoter hypermethylation is a common mechanism for inactivation of tumor suppressor genes in cancer cells. To generate a global profile of genes silenced by hypermethylation in renal cell cancer (RCC), we did an expression microarray-based analysis of genes reactivated in the 786-0, ACHN, HRC51, and HRC59 RCC lines after treatment with the demethylating drug 5-aza-2 deoxycytidine and histone deacetylation inhibiting drug trichostatin A. Between 111 to 170 genes were found to have at least 3-fold up-regulation of expression after treatment in each cell line. To establish the specificity of the screen for identification of genes, epigenetically silenced in cancer cells, we validated a subset of 12 up-regulated genes. Three genes (IGFBP1, IGFBP3, and COL1A1) showed promoter methylation in tumor DNA but were unmethylated in normal cell DNA. One gene (GDF15) was methylated in normal cells but more densely methylated in tumor cells. One gene (PLAU) showed cancer cell–specific methylation that did not correlate well with expression status. The remaining seven genes had unmethylated promoters, although at least one of these genes (TGM2) may be regulated by RASSF1A, which was methylated in the RCC lines. Thus, we were able to show that up-regulation of at least 6 of the 12 genes examined was due to epigenetic reactivation. The IGFBP1, IGFBP3, and COL1A1 gene promoter regions were found to be frequently methylated in primary renal cell tumors, and further study will provide insight into the biology of the disease and facilitate translational studies in renal cancer. (Cancer Res 2006; 66(10): 5021-8)
Data from Germ-Line Mutation of NKX3.1 Cosegregates with Hereditary Prostate Cancer and Alters the Homeodomain Structure and Function
Authors: Zheng S. Lilly , Ju Jeong-ho , Chang Bao-li , Ortner Elizabeth , Sun Jielin , Isaacs Sarah D. , Sun Jishang , Wiley Kathy E. , Liu Wennuan , Zemedkun Micheas , Walsh Patrick C. , Ferretti James , Gruschus James , Isaacs William B. , Gelmann Edward P. , Xu Jianfeng
Published: March 2023

Abstract
NKX3.1, a gene mapped to 8p21, is a member of the NK class of homeodomain proteins and is expressed primarily in the prostate. NKX3.1 exerts a growth-suppressive and differentiating effect on prostate epithelial cells. Because of its known functions and its location within a chromosomal region where evidence for prostate cancer linkage and somatic loss of heterozygosity is found, we hypothesize that sequence variants in the NKX3.1 gene increase prostate cancer risk. To address this, we first resequenced the NKX3.1 gene in 159 probands of hereditary prostate cancer families recruited at Johns Hopkins Hospital; each family has at least three first-degree relatives affected with prostate cancer. Twenty-one germ-line variants were identified in this analysis, including one previously described common nonsynonymous change (R52C), two novel rare nonsynonymous changes (A17T and T164A), and a novel common 18-bp deletion in the promoter. Overall, the germ-line variants were significantly linked to prostate cancer, with a peak heterogeneity logarithm of odds of 2.04 (P = 0.002) at the NKX3.1 gene. The rare nonsynonymous change, T164A, located in the homeobox domain of the gene, segregated with prostate cancer in a family with three affected brothers and one unaffected brother. Importantly, nuclear magnetic resonance solution structure analysis and circular dichroism studies showed this specific mutation to affect the stability of the homeodomain of the NKX3.1 protein and decreased binding to its cognate DNA recognition sequence. These results suggest that germ-line sequence variants in NKX3.1 may play a role in susceptibility to hereditary prostate cancer and underscore a role for NKX3.1 as a prostate cancer gatekeeper. (Cancer Res 2006; 66(1): 69-77)