bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from MUC1, a New Hypoxia Inducible Factor Target Gene, Is an Actor in Clear Renal Cell Carcinoma Tumor Progression
Authors: Aubert Sébastien , Fauquette Valérie , Hémon Brigitte , Lepoivre Réjane , Briez Nicolas , Bernard David , Van Seuningen Isabelle , Leroy Xavier , Perrais Michaël
Published: March 2024

Abstract
The hypoxia inducible factor (HIF) signaling pathway is known as the main renal carcinogenetic pathway. MUC1, an O-glycoprotein membrane-bound mucin, is overexpressed in clear renal cell carcinomas (cRCC) with correlation to two major prognostic factors: tumor-node-metastasis stage and nuclear Fürhman grade. We questioned whether there is a direct link between the HIF pathway and MUC1 overexpression in renal tumors. Interestingly, we observed concomitant increase of HIF-1α and MUC1 in metastatic cRCC group versus nonmetastatic cRCC group. Using different renal cell models and small interfering RNA assays targeting either HIF-1α or YC-1, a HIF-1 pharmacologic inhibitor, we showed induction of MUC1 expression under hypoxia by a HIF-dependent mechanism. Chromatin immunoprecipitation assay showed a direct binding of HIF-1α at the MUC1 promoter. In addition, combined site-directed mutagenesis and gel shift assay allowed the identification of two functional putative hypoxia responsive elements at −1488/−1485 and at −1510/−1507 in the promoter. Using a rat kidney model of ischemia/reperfusion, we confirmed in vivo that clamping renal pedicle for 1 hour followed by 2 hours of reperfusion induced increased MUC1 expression. Furthermore, MUC1 knockdown induced significant reduction of invasive and migration properties of renal cancer cells under hypoxia. Altogether, these results show that MUC1 is directly regulated by HIF-1α and affects the invasive and migration properties of renal cancer cells. Thus, MUC1 could serve as a potential therapeutic target in cRCC. [Cancer Res 2009;69(14):5707–15]
Data from A Role for the Clock Gene Per1 in Prostate Cancer
Authors: Cao Qi , Gery Sigal , Dashti Azadeh , Yin Dong , Zhou Yan , Gu Jiang , Koeffler H. Phillip
Published: March 2024

Abstract
Circadian rhythms regulate diverse physiologic processes, including homeostatic functions of steroid hormones and their receptors. Perturbations of these rhythms are associated with pathogenic conditions, such as depression, diabetes, and cancer. Androgens play an important role in both normal development and carcinogenesis of the prostate. In the present study, we investigated a potential role for the core clock factor Per1 in the pathogenesis of prostate cancer. Serum-shocked synchronized prostate cancer cells displayed disrupted circadian rhythms compared with the normal prostate tissue. Using Oncomine to perform a meta-analysis of microarray expression studies, we found that Per1 is down-regulated in human prostate cancer samples compared with normal prostates. Reporter assays showed that Per1 inhibited transactivation of the androgen receptor (AR) both in 293T cells overexpressing the AR and in the prostate cancer cell line LNCaP. Forced expression of Per1 in LNCaP cells diminished the expression of known androgen-sensitive genes following stimulation with dihydrotestosterone. We showed that Per1 physically interacted with AR; in addition, we found that Per1 itself is regulated by androgens in prostate cancer cells. Overexpression of Per1 in prostate cancer cells resulted in significant growth inhibition and apoptosis. Our results support the emerging role of circadian genes as key players in malignant transformation. Further elucidating the connections between clock genes and the AR pathway could benefit the development of new therapeutic strategies for prostate cancer as well as provide insights into chronotherapy as a way to optimize current therapies. [Cancer Res 2009;69(19):7619–25]
Data from Defective Repair of Oxidative DNA Damage in Triple-Negative Breast Cancer Confers Sensitivity to Inhibition of Poly(ADP-Ribose) Polymerase
Authors: Alli Elizabeth , Sharma Vandana B. , Sunderesakumar Preethi , Ford James M.
Published: March 2024

Abstract
Subtypes of breast cancer that represent the two major types of epithelial cells in the breast (luminal and basal) carry distinct histopathologic profiles. Breast cancers of the basal-like subtype, which include the majority of hereditary breast cancers due to mutations in the breast cancer susceptibility gene 1 (BRCA1), frequently assume triple-negative status, i.e., they lack expression of estrogen receptor-α and progesterone receptor, and lack overexpression or amplification of the HER2/NEU oncogene. Defects in DNA damage response pathways result in genome instability and lead to carcinogenesis, but may also be exploited for therapeutic purposes. We analyzed repair of oxidative DNA damage by the base-excision repair (BER) pathway, which when aberrant leads to genomic instability and breast carcinogenesis, in cell lines that represent the different subtypes of breast cancer and in the presence of BRCA1 deficiency. We found that basal-like and BRCA1-mutated breast cancer cells were defective in BER of oxidative DNA damage, and that this defect conferred sensitivity to inhibition of poly(ADP-ribose) polymerase, a DNA repair enzyme. The defect may be attributed, at least in part, to a novel role for BRCA1 in the BER pathway. Overall, these data offer preventive, prognostic, and therapeutic usefulness. [Cancer Res 2009;69(8):3589–96]
Data from Expansion of Highly Cytotoxic Human Natural Killer Cells for Cancer Cell Therapy
Authors: Fujisaki Hiroyuki , Kakuda Harumi , Shimasaki Noriko , Imai Chihaya , Ma Jing , Lockey Timothy , Eldridge Paul , Leung Wing H. , Campana Dario
Published: March 2024

Abstract
Infusions of natural killer (NK) cells are an emerging tool for cancer immunotherapy. The development of clinically applicable methods to produce large numbers of fully functional NK cells is a critical step to maximize the potential of this approach. We determined the capacity of the leukemia cell line K562 modified to express a membrane-bound form of interleukin (IL)-15 and 41BB ligand (K562-mb15-41BBL) to generate human NK cells with enhanced cytotoxicity. Seven-day coculture with irradiated K562-mb15-41BBL induced a median 21.6-fold expansion of CD56+CD3- NK cells from peripheral blood (range, 5.1- to 86.6-fold; n = 50), which was considerably superior to that produced by stimulation with IL-2, IL-12, IL-15, and/or IL-21 and caused no proliferation of CD3+ lymphocytes. Similar expansions could also be obtained from the peripheral blood of patients with acute leukemia undergoing therapy (n = 11). Comparisons of the gene expression profiles of the expanded NK cells and their unstimulated or IL-2–stimulated counterparts showed marked differences. The expanded NK cells were significantly more potent than unstimulated or IL-2–stimulated NK cells against acute myeloid leukemia cells in vitro. They could be detected for >1 month when injected into immunodeficient mice and could eradicate leukemia in murine models of acute myeloid leukemia. We therefore adapted the K562-mb15-41BBL stimulation method to large-scale clinical-grade conditions, generating large numbers of highly cytotoxic NK cells. The results that we report here provide rationale and practical platform for clinical testing of expanded and activated NK cells for cell therapy of cancer. [Cancer Res 2009;69(9):4010–7]
Data from Association of LETM1 and MRPL36 Contributes to the Regulation of Mitochondrial ATP Production and Necrotic Cell Death
Authors: Piao Longzhen , Li Yuwen , Kim Soung Jung , Byun Hee Sun , Huang Song Mei , Hwang Soon-Kyung , Yang Keum-Jin , Park Kyeong Ah , Won Minho , Hong Janghee , Hur Gang Min , Seok Jeong Ho , Shong Minho , Cho Myung-Haing , Brazil Derek P. , Hemmings Brian A. , Park Jongsun
Published: March 2024

Abstract
Leucine zipper/EF hand–containing transmembrane-1 (LETM1) is a mitochondrial inner membrane protein that was first identified in Wolf-Hirschhorn syndrome, and was deleted in nearly all patients with the syndrome. LETM1 encodes for the human homologue of yeast Mdm38p, which is a mitochondria-shaping protein of unclear function. Here, we describe LETM1-mediated regulation of mitochondrial ATP production and biogenesis. We show that LETM1 overexpression can induce necrotic cell death in HeLa cells, in which LETM1 reduces mitochondrial biogenesis and ATP production. LETM1 acts as an anchor protein and associates with mitochondrial ribosome protein L36. Adenovirus-mediated overexpression of LETM1 reduced mitochondrial mass and expression of many mitochondrial proteins. LETM1-mediated inhibition of mitochondrial biogenesis enhanced glycolytic ATP supply and activated protein kinase B activity and cell survival signaling. The expression levels of LETM1 were significantly increased in multiple human cancer tissues compared with normals. These data suggest that LETM1 serves as an anchor protein for complex formation with the mitochondrial ribosome and regulates mitochondrial biogenesis. The increased expression of LETM1 in human cancer suggests that dysregulation of LETM1 is a key feature of tumorigenesis. [Cancer Res 2009;69(8):3397–404]
Data from Spleen Tyrosine Kinase Functions as a Tumor Suppressor in Melanoma Cells by Inducing Senescence-like Growth Arrest
Authors: Bailet Olivier , Fenouille Nina , Abbe Patricia , Robert Guillaume , Rocchi Stéphane , Gonthier Nadège , Denoyelle Christophe , Ticchioni Michel , Ortonne Jean-Paul , Ballotti Robert , Deckert Marcel , Tartare-Deckert Sophie
Published: March 2024

Abstract
Loss of tumor-suppressive pathways that control cellular senescence is a crucial step in malignant transformation. Spleen tyrosine kinase (Syk) is a cytoplasmic tyrosine kinase that has been recently implicated in tumor suppression of melanoma, a deadly skin cancer derived from pigment-producing melanocytes. However, the mechanism by which Syk suppresses melanoma growth remains unclear. Here, we report that reexpression of Syk in melanoma cells induces a p53-dependent expression of the cyclin-dependent kinase (cdk) inhibitor p21 and a senescence program. We first observed that Syk expression is lost in a subset of melanoma cell lines, primarily by DNA methylation–mediated gene silencing and restored after treatment with the demethylating agent 5-aza-2-deoxycytidine. We analyzed the significance of epigenetic inactivation of Syk and found that reintroduction of Syk in melanoma cells dramatically reduces clonogenic survival and three-dimensional tumor spheroid growth and invasion. Remarkably, melanoma cells reexpressing Syk display hallmarks of senescent cells, including reduction of proliferative activity and DNA synthesis, large and flattened morphology, senescence-associated β-galactosidase activity, and heterochromatic foci. This phenotype is accompanied by hypophosphorylated retinoblastoma protein (Rb) and accumulation of p21, which depends on functional p53. Our results highlight a new role for Syk tyrosine kinase in regulating cellular senescence and identify Syk-mediated senescence as a novel tumor suppressor pathway the inactivation of which may contribute to melanoma tumorigenicity. [Cancer Res 2009;69(7):2748–56]
Data from Inactivation of the Nijmegen Breakage Syndrome Gene Leads to Excess Centrosome Duplication via the ATR/BRCA1 Pathway
Authors: Shimada Mikio , Sagae Ruri , Kobayashi Junya , Habu Toshiyuki , Komatsu Kenshi
Published: March 2024

Abstract
Nijmegen breakage syndrome is characterized by genomic instability and a predisposition for lymphoma and solid tumors. Nijmegen breakage syndrome 1 (NBS1), the protein which is mutated in these patients, functions in association with BRCA1 and ATR as part of the cellular response to DNA double-strand breaks. We show here that NBS1 forms foci at the centrosomes via an interaction with γ-tubulin. Down-regulation of NBS1 by small interfering RNA induces supernumerary centrosomes, and this was confirmed with experiments using Nbs1 knockout mouse cells; the introduction of wild-type NBS1 (wt-NBS1) cDNA into these knockout mouse cells reduced the number of supernumerary centrosomes to normal levels. This phenotype in NBS1-deficient cells is caused by both centrosome duplication and impaired separation of centrioles, which have been observed in BRCA1-inhibited cells. In fact, supernumerary centrosomes were observed in Brca1 knockout mouse cells, and the frequency was not affected by NBS1 down-regulation, suggesting that NBS1 maintains centrosomes via a common pathway with BRCA1. This is consistent with findings that NBS1 physically interacts with BRCA1 at the centrosomes and is required for BRCA1-mediated ubiquitination of γ-tubulin. Moreover, the ubiquitination of γ-tubulin is compromised by either ATR depletion or an NBS1 mutation in the ATR interacting (FHA) domain, which is essential for ATR activation. These results suggest that, although centrosomes lack DNA, the NBS1/ATR/BRCA1 repair machinery affects centrosome behavior, and this might be a crucial role in the prevention of malignances. [Cancer Res 2009;69(5):1768–75]
Data from Concordant Gene Expression Signatures Predict Clinical Outcomes of Cancer Patients Undergoing Systemic Therapy
Authors: Williams Paul D. , Cheon Sooyoung , Havaleshko Dmytro M. , Jeong Hyeon , Cheng Feng , Theodorescu Dan , Lee Jae K.
Published: March 2024

Abstract
Conventional development of multivariate gene expression models (GEM) predicting therapeutic response of cancer patients is based on analysis of patients treated with specific regimens, which limits generalization to different or novel drug combinations. We overcome this limitation by developing GEMs based on in vitro drug sensitivities and microarray analyses of the NCI-60 cancer cell line panel. These GEMs were evaluated in blind fashion as predictors of tumor response and/or patient survival in seven independent cohorts of patients with breast (n = 275), bladder (n = 59), and ovarian (n= 143) cancer treated with multiagent chemotherapy, of which 233 patients were from prospectively enrolled clinical trials. In all studies, GEMs effectively stratified tumor response and patient survival independent of established clinical and pathologic tumor variables. In bladder cancer patients treated with neoadjuvant methotrexate, vinblastine, Adriamycin (doxorubicin), and cisplatin, the 3-year overall survival for those with favorable GEM scores was 81% versus 33% for those with less favorable scores (P = 0.002). GEMs for breast cancer patients treated with 5-fluorouracil, Adriamycin (doxorubicin), and cyclophosphamide and ovarian cancer patients treated with platinum-containing regimens also stratified patient survival [5-year overall survival 100% versus 74% (P = 0.05) and 3-year overall survival 68% versus 43% (P = 0.008), respectively]. Importantly, clinical prediction using our in vitro GEM was superior to that of conventionally derived GEMs. We show a facile yet effective approach to GEM derivation that identifies patients most likely to benefit from selected multiagent therapy. Use of such in vitro–based GEMs may provide a robust and generalizable approach to personalized cancer therapy. [Cancer Res 2009;69(21):8302–9]
Data from Tumor-Induced Suppression of CTL Expansion and Subjugation by gp96-Ig Vaccination
Authors: Schreiber Taylor H. , Deyev Vadim V. , Rosenblatt Joseph D. , Podack Eckhard R.
Published: March 2024

Abstract
Established tumors suppress antitumor immune responses and induce tolerance by incompletely characterized mechanisms, and this phenomenon is an important barrier to tumor immunotherapy. Single vaccination with tumor cells expressing gp96-Ig stimulates robust expansion of tumor-specific CTLs in tumor-naïve mice and this expansion is inhibited by established tumors. Interestingly, frequent vaccinations restore antitumor immune responses in the presence of established tumors. Syngeneic EG7 tumor-bearing mice have heterogeneous responses to frequent vaccination with EG7-gp96-Ig, with 32% complete responders and 68% partial responders. Comparison of responders to nonresponders revealed an inverse correlation between tumor-specific CTL expansion in the peripheral blood and tumor size. To identify immune cells and molecules associated with effective antitumor immune responses, reverse transcription-PCR arrays were performed using cells isolated from the vaccination site. ELISAs, cellular phenotyping, and tumor immunohistochemistry were also performed comparing vaccine responders to nonresponders. These data show that up-regulation of T-bet, RORγt, IFNγ, CCL8, CXCL9, and CXCL10 at the vaccination site are associated with vaccine-induced antitumor immunity. These data correlate with increased CTL expansion in the peripheral blood of responders, increased infiltration of responder tumors by CD8+ cells and interleukin-17+ cells, and decreased infiltration of responder tumors by CD11b+Gr-1+ cells and FoxP3+ cells. Furthermore, serum ELISAs revealed a significant elevation of transforming growth factor-β in nonresponders as compared with responders. Interestingly, CD8+ T cells isolated from responders and nonresponders have equivalent cytotoxic activity in vitro. Taken together, our data suggest that established tumors may escape immunosurveillance by preventing clonal expansion of tumor-specific CTL without inducing anergy. [Cancer Res 2009;69(5):2026–33]
Data from Identification of a Novel Pathway That Selectively Modulates Apoptosis of Breast Cancer Cells
Authors: Tinnikov Alexander A. , Yeung Kay T. , Das Sharmistha , Samuels Herbert H.
Published: March 2024

Abstract
Expression of the nuclear receptor interacting factor 3 (NRIF3) coregulator in a wide variety of breast cancer cells selectively leads to rapid caspase-2–dependent apoptotic cell death. A novel death domain (DD1) was mapped to a 30–amino acid region of NRIF3. Because the cytotoxicity of NRIF3 and DD1 seems to be cell type–specific, these studies suggest that breast cancer cells contain a novel “death switch” that can be specifically modulated by NRIF3 or DD1. Using an MCF-7 cell cDNA library in a yeast two-hybrid screen, we cloned a factor that mediates apoptosis by DD1 and refer to this factor as DD1-interacting factor-1 (DIF-1). DIF-1 is a transcriptional repressor that mediates its effect through SirT1, and this repression is attenuated by the binding of NRIF3/DD1. DIF-1 expression rescues breast cancer cells from NRIF3/DD1-induced apoptosis. Small interfering RNA (siRNA) knockdown of DIF-1 selectively leads to apoptosis of breast cancer cells, further suggesting that DIF-1 plays a key role in NRIF3/DD1-mediated apoptosis. A protein kinase A inhibitor (H89) also elicits apoptosis of breast cancer cells but not of the other cell types examined, and DIF-1 also protects these cells from H89-mediated apoptosis. In addition, H89 incubation results in a rapid increase in NRIF3 levels and siRNA knockdown of NRIF3 protects breast cancer cells from H89-mediated apoptosis. Our results indicate that DIF-1 plays a key role in breast cancer cell survival and further characterizing this pathway may provide important insights into developing novel therapies to selectively target breast cancer cells for apoptosis. [Cancer Res 2009;69(4):1375–82]
Data from Human Postmeiotic Segregation 2 Exhibits Biased Repair at Tetranucleotide Microsatellite Sequences
Authors: Shah Sandeep N. , Eckert Kristin A.
Published: March 2024

Abstract
The mismatch repair (MMR) system plays a major role in removing DNA polymerization errors, and loss of this pathway results in hereditary cancers characterized by microsatellite instability. We investigated microsatellite stability during DNA replication within human postmeiotic segregation 2 (hPMS2)–deficient and proficient human lymphoblastoid cell lines. Using a shuttle vector assay, we measured mutation rates at reporter cassettes containing defined mononucleotide, dinucleotide, and tetranucleotide microsatellite sequences. A mutator phenotype was observed in the hPMS2-deficient cell line. The mutation rate of vectors containing [G/C]10 or [GT/CA]10 alleles was elevated 20-fold to 40-fold in hPMS2-deficient cells, relative to an hPMS2-expressing cell line. We observed a 6-fold and 12-fold relative increase in mutation rate of [TTTC/AAAG]9 and [TTCC/AAGG]9 sequences, respectively, in hPMS2-deficient cells. Mutational specificity analyses suggested that repair by hPMS2 is biased. In the absence of hPMS2, a greater number of microsatellite expansion versus deletion mutations was observed, and expansion rates of the tetranucleotide alleles were similar. In the presence of hPMS2, we observed a 29-fold decrease in the [TTCC/AAGG]9 expansion rate but only a 6-fold decrease for the [TTTC/AAAG]9 allele. Our data indicate that hPMS2 is more protective of tetranucleotide expansions than deletions and that hPMS2 displays a sequence bias, wherein [TTCC/AAGG] sequences are stabilized to a greater extent than [TTTC/AAAG]. Our results allow for greater accuracy during identification of MMR defects by providing a mutational signature characteristic of hPMS2 defect. This study also provides clues to possible mechanisms of repair by hPMS2 in the context of the MMR system. [Cancer Res 2009;69(3):1143–9]
Data from Synergistic Activity of the Src Family Kinase Inhibitor Dasatinib and Oxaliplatin in Colon Carcinoma Cells Is Mediated by Oxidative Stress
Authors: Kopetz Scott , Lesslie Donald P. , Dallas Nikolas A. , Park Serk I. , Johnson Marjorie , Parikh Nila U. , Kim Michael P. , Abbruzzese James L. , Ellis Lee M. , Chandra Joya , Gallick Gary E.
Published: March 2024

Abstract
Chemotherapeutic regimens for the treatment of colorectal cancer generally include oxaliplatin, although inherent and acquired resistance is common. One potential mediator of oxaliplatin sensitivity is the nonreceptor protein tyrosine kinase, Src, the activity of which correlates with disease stage and patient survival. Therefore, we investigated the effects of Src inhibition using the tyrosine kinase inhibitor dasatinib on oxaliplatin sensitivity. We show that oxaliplatin acutely activates Src and that combination treatment with dasatinib is synergistic in a cell-line dependent manner, with the level of Src activation correlating with extent of synergy in a panel of six cell lines. Intracellular reactive oxygen species (ROS) are generated after oxaliplatin treatment, and ROS potently activates Src. Pretreatment with antioxidants inhibits oxaliplatin-induced Src activation. In oxaliplatin-resistant cell lines, Src activity is constitutively increased. In a mouse model of colorectal liver metastases, treatment with oxaliplatin also results in chronic Src activation. The combination of dasatinib and oxaliplatin results in significantly smaller tumors compared with single-agent treatment, corresponding with reduced proliferation and angiogenesis. Therefore, we conclude that oxaliplatin activates Src through a ROS-dependent mechanism. Src inhibition increases oxaliplatin activity both in vitro and in vivo. These results suggest that Src inhibitors combined with oxaliplatin may have efficacy in metastatic colon cancer and may provide the first indication of a molecular phenotype that might be susceptible to such combinations. [Cancer Res 2009;69(9):3842–9]
Data from The Novel Tubulin-Targeting Agent Pyrrolo-1,5-Benzoxazepine-15 Induces Apoptosis in Poor Prognostic Subgroups of Chronic Lymphocytic Leukemia
Authors: McElligott Anthony M. , Maginn Elaina N. , Greene Lisa M. , McGuckin Siobhan , Hayat Amjad , Browne Paul V. , Butini Stefania , Campiani Giuseppe , Catherwood Mark A. , Vandenberghe Elisabeth , Williams D. Clive , Zisterer Daniela M. , Lawler Mark
Published: March 2024

Abstract
Pyrrolo-1,5-benzoxazepine-15 (PBOX-15) is a novel microtubule depolymerization agent that induces cell cycle arrest and subsequent apoptosis in a number of cancer cell lines. Chronic lymphocytic leukemia (CLL) is characterized by clonal expansion of predominately nonproliferating mature B cells. Here, we present data suggesting PBOX-15 is a potential therapeutic agent for CLL. We show activity of PBOX-15 in samples taken from a cohort of CLL patients (n = 55) representing both high-risk and low-risk disease. PBOX-15 exhibited cytotoxicity in CLL cells (n = 19) in a dose-dependent manner, with mean IC50 of 0.55 μmol/L. PBOX-15 significantly induced apoptosis in CLL cells (n = 46) including cells with poor prognostic markers: unmutated IgVH genes, CD38 and zeta-associated protein 70 (ZAP-70) expression, and fludarabine-resistant cells with chromosomal deletions in 17p. In addition, PBOX-15 was more potent than fludarabine in inducing apoptosis in fludarabine-sensitive cells. Pharmacologic inhibition and small interfering RNA knockdown of caspase-8 significantly inhibited PBOX-15–induced apoptosis. Pharmacologic inhibition of c-jun NH2-terminal kinase inhibited PBOX-15–induced apoptosis in mutated IgVH and ZAP-70− CLL cells but not in unmutated IgVH and ZAP-70+ cells. PBOX-15 exhibited selective cytotoxicity in CLL cells compared with normal hematopoietic cells. Our data suggest that PBOX-15 represents a novel class of agents that are toxic toward both high-risk and low-risk CLL cells. The need for novel treatments is acute in CLL, especially for the subgroup of patients with poor clinical outcome and drug-resistant disease. This study identifies a novel agent with significant clinical potential. [Cancer Res 2009;69(21):8366–75]
Data from Structure-Function Approach Identifies a COOH-Terminal Domain That Mediates Heparanase Signaling
Authors: Fux Liat , Feibish Nir , Cohen-Kaplan Victoria , Gingis-Velitski Svetlana , Feld Sari , Geffen Chen , Vlodavsky Israel , Ilan Neta
Published: March 2024

Abstract
Heparanase is an endo-β-d-glucuronidase capable of cleaving heparan sulfate, activity that is strongly implicated in cellular invasion associated with tumor metastasis, angiogenesis, and inflammation. In addition, heparanase was noted to exert biological functions apparently independent of its enzymatic activity, enhancing the phosphorylation of selected protein kinases and inducing gene transcription. A predicted three-dimensional structure of constitutively active heparanase clearly delineates a TIM-barrel fold previously anticipated for the enzyme. Interestingly, the model also revealed the existence of a COOH-terminal domain (C-domain) that apparently is not an integral part of the TIM-barrel fold. We provide evidence that the C-domain is critical for heparanase enzymatic activity and secretion. Moreover, the C-domain was found to mediate nonenzymatic functions of heparanase, facilitating Akt phosphorylation, cell proliferation, and tumor xenograft progression. These findings support the notion that heparanase exerts enzymatic activity-independent functions, and identify, for the first time, a protein domain responsible for heparanase-mediated signaling. Inhibitors directed against the C-domain, combined with inhibitors of heparanase enzymatic activity, are expected to neutralize heparanase functions and to profoundly affect tumor growth, angiogenesis, and metastasis. [Cancer Res 2009;69(5):1758–67]
Data from Wnt/β-Catenin Signaling Regulates Cytokine-Induced Human Inducible Nitric Oxide Synthase Expression by Inhibiting Nuclear Factor-κB Activation in Cancer Cells
Authors: Du Qiang , Zhang Xinglu , Cardinal Jon , Cao Zongxian , Guo Zhong , Shao Lifang , Geller David A.
Published: March 2024

Abstract
The human inducible nitric oxide synthase (hiNOS) gene is regulated by nuclear factor κB (NF-κB) and has recently been shown to be a target of the Wnt/β-catenin pathway. In this study, we tested the hypothesis that Wnt/β-catenin signaling might regulate cytokine- or tumor necrosis factor α (TNFα)–induced hiNOS expression through interaction with NF-κB. A cytokine mixture of TNFα + interleukin (IL)-1β + IFNγ induced a 2- to 3-fold increase in hiNOS promoter activity in HCT116 and DLD1 colon cells, but produced a 2-fold decrease in SW480 colon cancer cells. A similar differential activity was seen in liver cancer cells (HepG2, Huh7, and Hep3B). Overexpression of β-catenin produced a dose-dependent decrease in NF-κB reporter activity and decreased cytokine mixture–induced hiNOS promoter activity. Gel shift for TNFα-induced hiNOS NF-κB activation showed decreased p50 binding and decreased NF-κB reporter activity in the β-catenin–mutant HAβ18 cells. Conversely, enhanced p50 binding and increased NF-κB reporter activity were seen in HAβ85 cells, which lack β-catenin signaling. Coimmunoprecipitation confirmed that β-catenin complexed with both p65 and p50 NF-κB proteins. NF-κB–dependent Traf1 protein expression also inversely correlated with the level of β-catenin. Furthermore, SW480 cells stably transformed with wild-type adenomatous polyposis coli showed decreased β-catenin protein and increased TNFα-induced p65 NF-κB binding as well as iNOS and Traf1 expression. Finally, β-catenin inversely correlated with iNOS and Fas expression in vivo in hepatocellular carcinoma tumor samples. Our in vitro and in vivo data show that β-catenin signaling inversely correlates with cytokine-induced hiNOS and other NF-κB–dependent gene expression. These findings underscore the complex role of Wnt/β-catenin, NF-κB, and iNOS signaling in the pathophysiology of inflammation-associated carcinogenesis. [Cancer Res 2009;69(9):3764–71]
Data from Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity
Authors: Nikolova Dessislava A. , Asangani Irfan A. , Nelson Laura D. , Hughes Dennis P.M. , Siwak Doris R. , Mills Gordon B. , Harms Andrea , Buchholz Erika , Pilz Lothar R. , Manegold Christian , Allgayer Heike
Published: March 2024

Abstract
Cetuximab, which blocks ligand binding to epidermal growth factor receptor (EGFR), is currently being studied as a novel treatment for non–small cell lung cancer (NSCLC). However, its mechanisms of action toward metastasis, and markers of drug sensitivity, have not been fully elucidated. This study was conducted to (a) determine the effect of Cetuximab on invasion and NSCLC-metastasis; (b) investigate urokinase-type plasminogen activator receptor (u-PAR), a major molecule promoting invasion and metastasis, as a target molecule; (c) delineate molecular mediators of Cetuximab-induced metastasis inhibition; and (d) identify biomarkers of drug sensitivity in NSCLC. Cetuximab treatment resulted in reduced growth and Matrigel invasion of H1395 and A549 NSCLC cell lines, in parallel with reduced u-PAR mRNA and protein. u-PAR down-regulation was brought about by suppressing the binding of JunD and c-Jun to u-PAR promoter motif −190/−171 in vivo, and an inhibition of MAP/ERK kinase signaling. Furthermore, Cetuximab inhibited NSCLC proliferation and metastasis to distant organs in vivo as indicated by the chicken embryo metastasis assay. Low E-cadherin and high u-PAR, but not EGFR, was associated with resistance to Cetuximab in seven NSCLC cell lines. Furthermore, siRNA knockdown of u-PAR led to a resensitization to Cetuximab. Moreover, low E-cadherin and high u-PAR was found in 63% of resected tumor tissues of NSCLC patients progressing under Cetuximab therapy. This is the first study to show u-PAR as a target and marker of sensitivity to Cetuximab, and to delineate novel mechanisms leading to metastasis suppression of NSCLC by Cetuximab. [Cancer Res 2009;69(6):2461–70]
Data from A Multiscale Mathematical Model for Oncolytic Virotherapy
Authors: Paiva Leticia R. , Binny Christopher , Ferreira Silvio C. , Martins Marcelo L.
Published: March 2024

Abstract
One of the most promising strategies to treat cancer is attacking it with viruses. Oncolytic viruses can kill tumor cells specifically or induce anticancer immune response. A multiscale model for virotherapy of cancer is investigated through simulations. It was found that, for intratumoral virus administration, a solid tumor can be completely eradicated or keep growing after a transient remission. Furthermore, the model reveals undamped oscillatory dynamics of tumor cells and virus populations, which demands new in vivo and in vitro quantitative experiments aiming to detect this oscillatory response. The conditions for which each one of the different tumor responses dominates, as well as the occurrence probabilities for the other nondominant therapeutic outcomes, were determined. From a clinical point of view, our findings indicate that a successful, single agent virotherapy requires a strong inhibition of the host immune response and the use of potent virus species with a high intratumoral mobility. Moreover, due to the discrete and stochastic nature of cells and their responses, an optimal range for viral cytotoxicity is predicted because the virotherapy fails if the oncolytic virus demands either a too short or a very large time to kill the tumor cell. This result suggests that the search for viruses able to destroy tumor cells very fast does not necessarily lead to a more effective control of tumor growth. [Cancer Res 2009;69(3):1205–11]
Data from Diaphanous-Related Formins Are Required for Invadopodia Formation and Invasion of Breast Tumor Cells
Authors: Lizárraga Floria , Poincloux Renaud , Romao Maryse , Montagnac Guillaume , Le Dez Gaëlle , Bonne Isabelle , Rigaill Guillem , Raposo Graça , Chavrier Philippe
Published: March 2024

Abstract
Proteolytic degradation of the extracellular matrix by metastatic tumor cells is initiated by the formation of invadopodia, i.e., actin-driven filopodia-like membrane protrusions endowed with matrix-degradative activity. A signaling cascade involving neural Wiskott-Aldrich syndrome protein and the Arp2/3 actin nucleating complex is involved in actin assembly at invadopodia. Yet, the mechanism of invadopodia formation is poorly understood. Based on their role as actin nucleators in cytoskeletal rearrangements, including filopodia formation, we examined the function of Diaphanous-related formins (DRF) in invadopodia formation and invasion by breast tumor cells. Using small interfering RNA silencing of protein expression in highly invasive MDA-MB-231 breast adenocarcinoma cells, we show that three members of the DRF family (DRF1–DRF3) are required for invadopodia formation and two-dimensional matrix proteolysis. We also report that invasion of a three-dimensional Matrigel matrix involves filopodia-like protrusions enriched for invadopodial proteins, including membrane type 1 matrix metalloproteinase, which depend on DRFs for their formation. These data identify DRFs as critical components of the invasive apparatus of tumor cells in two-dimensional and three-dimensional matrices and suggest that different types of actin nucleators cooperate during the formation of invadopodia. [Cancer Res 2009;69(7):2792–800]
Data from Circulating B-Cell Chronic Lymphocytic Leukemia Cells Display Impaired Migration to Lymph Nodes and Bone Marrow
Authors: Hartmann Tanja Nicole , Grabovsky Valentin , Wang Wei , Desch Petra , Rubenzer Gabriele , Wollner Stefan , Binsky Inbal , Vallon-Eberhard Alexandra , Sapoznikov Anita , Burger Meike , Shachar Idit , Haran Michal , Honczarenko Marek , Greil Richard , Alon Ronen
Published: March 2024

Abstract
Homing to secondary lymphoid organs and bone marrow (BM) is a central aspect of leukemic pathophysiology. We investigated the roles of the two major lymphocyte integrins LFA-1 and VLA-4 on B-cell chronic lymphocytic leukemia (CLL) cells in these processes. We found that the majority of CLL cells expressed significantly reduced LFA-1 due to low β2 integrin transcripts. VLA-4 expression was heterogenous but underwent rapid activation by the BM chemokine CXCL12. CLL cells failed to transmigrate across VCAM-1–expressing, ICAM-1–expressing, and CXCL12-expressing endothelium, whereas when LFA-1 expression was regained in subsets of CLL cells, these lymphocytes rapidly transmigrated the endothelium. Furthermore, when injected into tail veins of immunodeficient mice, normal B cells rapidly homed to lymph nodes (LN) in a LFA-1–dependent manner, whereas CLL cells did not. Nevertheless, only residual CLL subsets could reenter BM, whereas both normal and CLL cells homed to the mice spleen in an LFA-1–independent and VLA-4–independent manner. Our results suggest that CLL cells have a reduced capacity to adhere and transmigrate through multiple vascular endothelial beds and poorly home to lymphoid organs other than spleen. Integrin blocking could thus be an efficient strategy to prevent circulating CLL cells from reaching prosurvival niches in LNs and BM but not in spleen. [Cancer Res 2009;69(7):OF3121–30]
Data from The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells
Authors: Rao V. Ashutosh , Klein Sarah R. , Agama Keli K. , Toyoda Eriko , Adachi Noritaka , Pommier Yves , Shacter Emily B.
Published: March 2024

Abstract
Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chelator with selective anticancer activity. We investigated the mechanism whereby Dp44mT kills breast cancer cells, both as a single agent and in combination with doxorubicin. Dp44mT alone induced selective cell killing in the breast cancer cell line MDA-MB-231 when compared with healthy mammary epithelial cells (MCF-12A). It induces G1 cell cycle arrest and reduces cancer cell clonogenic growth at nanomolar concentrations. Dp44mT, but not the iron chelator desferal, induces DNA double-strand breaks quantified as S139 phosphorylated histone foci (γ-H2AX) and Comet tails induced in MDA-MB-231 cells. Doxorubicin-induced cytotoxicity and DNA damage were both enhanced significantly in the presence of low concentrations of Dp44mT. The chelator caused selective poisoning of DNA topoisomerase IIα (top2α) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2α knockout cells (top2α+/−) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2α+/+ or top2β−/− cells. Specificity for top2α was confirmed using top2α and top2β small interfering RNA knockdown in HeLa cells. The results show that Dp44mT is cytotoxic to breast cancer cells, at least in part, due to selective inhibition of top2α. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2α activity. [Cancer Res 2009;69(3):948–57]
Data from Heat-Induced Perturbations of DNA Damage Signaling Pathways are Modulated by Molecular Chaperones
Authors: Laszlo Andrei , Fleischer Ilona
Published: March 2024

Abstract
Heat is one of the most potent radiosensitizers known. Several randomized trials have shown that hyperthermia is a good adjuvant for radiotherapy at several different cancer sites. However, the mechanism(s) involved in the interaction of heat and radiation that lead to radiosensitization remain to be elucidated. In this report, we have determined that heat induces perturbations in some of the earliest events in the cellular response to DNA damage induced by ionizing radiation. We studied the effect of heat on the formation of complexes containing γ-H2AX/MDC1/53BP1 in heated-irradiated cells. We found that the formation of this complex was delayed in heated-irradiated cells, in a heat but not radiation dose–dependent manner. The length of the heat-induced delay of complex formation was attenuated in thermotolerant and heat radiosensitization–resistant cells. The length of the delay of γ-H2AX/MDC1/53BP1 complex formation correlated with the magnitude of heat radiosensitization and was modulated by the molecular chaperone Hsc70. Heat radiosensitization was attenuated in 53BP1-null cells, implying that the delay of the formation of the γ-H2AX/MDC1/53BP1 complex plays a role in heat radiosensitization. Heat also induced a delay of events in the DNA damage response that are downstream from 53BP1. Our results support the notion that heat-induced perturbations in the earliest events of the cellular response to ionizing radiation–induced DNA damage play a role in heat radiosensitization. [Cancer Res 2009;69(5):2042–9]
Data from Impaired Turnover of Prolactin Receptor Contributes to Transformation of Human Breast Cells
Authors: Plotnikov Alexandr , Varghese Bentley , Tran Thai H. , Liu Chengbao , Rui Hallgeir , Fuchs Serge Y.
Published: March 2024

Abstract
Signaling by polypeptide hormone prolactin (PRL) is mediated by its cognate receptor (PRLr). PRLr is commonly stabilized in human breast cancer due to decreased phosphorylation of residue Ser349, which when phosphorylated recruits the βTrcp E3 ubiquitin ligase and facilitates PRLr degradation. Here, we show that an impaired PRLr turnover results in an augmented PRL signaling and PRL-induced transcription. Human mammary epithelial cells harboring degradation-resistant PRLr display accelerated proliferation and increased invasive growth. Conversely, a decrease in PRLr levels achieved by either pharmacologic or genetic means in human breast cancer cells dramatically reduced transformation and tumorigenic properties of these cells. Consequences of alteration of PRLr turnover for homeostasis of mammary cells and development of breast cancers, as well as the utility of therapies that target PRLr function in these malignancies, are discussed. [Cancer Res 2009;69(7):3165–72]
Data from PIK3CA and PIK3CB Inhibition Produce Synthetic Lethality when Combined with Estrogen Deprivation in Estrogen Receptor–Positive Breast Cancer
Authors: Crowder Robert J. , Phommaly Chanpheng , Tao Yu , Hoog Jeremy , Luo Jingqin , Perou Charles M. , Parker Joel S. , Miller Melinda A. , Huntsman David G. , Lin Li , Snider Jacqueline , Davies Sherri R. , Olson John A. , Watson Mark A. , Saporita Anthony , Weber Jason D. , Ellis Matthew J.
Published: March 2024

Abstract
Several phosphoinositide 3-kinase (PI3K) catalytic subunit inhibitors are currently in clinical trial. We therefore sought to examine relationships between pharmacologic inhibition and somatic mutations in PI3K catalytic subunits in estrogen receptor (ER)–positive breast cancer, in which these mutations are particularly common. RNA interference (RNAi) was used to determine the effect of selective inhibition of PI3K catalytic subunits, p110α and p110β, in ER+ breast cancer cells harboring either mutation (PIK3CA) or gene amplification (PIK3CB). p110α RNAi inhibited growth and promoted apoptosis in all tested ER+ breast cancer cells under estrogen deprived-conditions, whereas p110β RNAi only affected cells harboring PIK3CB amplification. Moreover, dual p110α/p110β inhibition potentiated these effects. In addition, treatment with the clinical-grade PI3K catalytic subunit inhibitor BEZ235 also promoted apoptosis in ER+ breast cancer cells. Importantly, estradiol suppressed apoptosis induced by both gene knockdowns and BEZ235 treatment. Our results suggest that PI3K inhibitors should target both p110α and p110β catalytic subunits, whether wild-type or mutant, and be combined with endocrine therapy for maximal efficacy when treating ER+ breast cancer. [Cancer Res 2009;69(9):3955–62]
Data from Pharmacologic Abrogation of the Mitotic Spindle Checkpoint by an Indolocarbazole Discovered by Cellular Screening Efficiently Kills Cancer Cells
Authors: Stolz Ailine , Vogel Celia , Schneider Verena , Ertych Norman , Kienitz Anne , Yu Hongtao , Bastians Holger
Published: March 2024

Abstract
The mitotic spindle checkpoint represents a signal transduction pathway that prevents the onset of anaphase until all chromosomes are properly aligned on a metaphase plate. Partial inactivation of this checkpoint allows premature separation of sister chromatids and results in aneuploidy, which might contribute to tumorigenesis. Unlike other cell cycle checkpoints, the spindle checkpoint is essential for cell viability, giving rise to the idea that the spindle checkpoint itself might represent a valuable target for anticancer therapy. We used a cell-based screen and identified the indolocarbazole compound Gö6976 as a pharmacologic inhibitor of the spindle checkpoint. Gö6976 potently overrides a spindle checkpoint–mediated mitotic arrest by abrogating the phosphorylation and kinetochore localization of several spindle checkpoint proteins. We identified the Aurora-A and Aurora-B kinases, which have been previously implicated in proper mitotic progression and spindle checkpoint function, as targets for Gö6976. Accordingly, Gö6976 treatment causes severe mitotic abnormalities and chromosome alignment defects, which are not properly detected by the drug-inactivated spindle checkpoint. This results in an aberrant progression of mitosis, leading to apoptosis in various human cancer cell lines, including spindle checkpoint–compromised cancer cells. Thus, our work describes a novel and promising strategy for anticancer treatment that targets the mitotic spindle checkpoint. [Cancer Res 2009;69(9):3874–83]
Data from Suppressor of Cytokine Signaling 3 Promotes Bone Marrow Cells to Differentiate into CD8+ T Lymphocytes in Lung Tissue via Up-Regulating Notch1 Expression
Authors: Zhang Zhuohan , Zeng Bin , Zhang Zhiqian , Jiao Guohui , Li Haijie , Jing Zhizi , Ouyang Jiangbo , Yuan Xin , Chai Limin , Che Yongzhe , Zhang Yuan , Yang Rongcun
Published: March 2024

Abstract
Suppressor of cytokine signaling 3 (SOCS3) expression in bone marrow cells (BMC) was up-regulated upon exposure to interleukin 6, lipopolysaccharide, or tumor-associated factors. But, how the up-regulated SOCS3 affects differentiation of BMCs is incompletely characterized. Here, we showed that SOCS3 promoted BMCs to intently differentiate into CD8 T cells. Importantly, lung can be as one athymus tissue for the BMCs to differentiate into CD8+ T cells. Notch1 plays a critical role in the differentiation from SOCS3-transfected BMCs to CD8+ T cells. We conclude that the up-regulated SOCS3 in some pathologic conditions, such as tumor and inflammation, might promote BMCs to differentiate into CD8+ T lymphocytes in lung tissue via up-regulating Notch1 expression. This may represent a new mechanism against diseases such as tumor. [Cancer Res 2009;69(4):1578–86]
Data from In vivo Administration of Artificial Antigen-Presenting Cells Activates Low-Avidity T Cells for Treatment of Cancer
Authors: Ugel Stefano , Zoso Alessia , De Santo Carmela , Li Yu , Marigo Ilaria , Zanovello Paola , Scarselli Elisa , Cipriani Barbara , Oelke Mathias , Schneck Jonathan P. , Bronte Vincenzo
Published: March 2024

Abstract
The development of effective antitumor immune responses is normally constrained by low-avidity, tumor-specific CTLs that are unable to eradicate the tumor. Strategies to rescue antitumor activity of low-avidity melanoma-specific CTLs in vivo may improve immunotherapy efficacy. To boost the in vivo effectiveness of low-avidity CTLs, we immunized mice bearing lung melanoma metastases with artificial antigen-presenting cells (aAPC), made by covalently coupling pepMHC-Ig dimers and B7.1-Ig molecules to magnetic beads. aAPC treatment induced significant tumor reduction in a mouse telomerase antigen system, and complete tumor eradication in a mouse TRP-2 antigen system, when low-avidity CTLs specific for these antigens were adoptively transferred. In addition, in an in vivo treatment model of subcutaneous melanoma, aAPC injection also augmented the activity of adoptively transferred CTLs and significantly delayed tumor growth. In vivo tumor clearance due to aAPC administration correlated with in situ proliferation of the transferred CTL. In vitro studies showed that aAPC effectively stimulated cytokine release, enhanced CTL-mediated lysis, and TCR downregulation in low-avidity CTLs. Therefore, in vivo aAPC administration represents a potentially novel approach to improve cancer immunotherapy. [Cancer Res 2009;69(24):9376–84]