bluuebunny/crossref_metadata_2025 huggingface dataset:
Tid1 Negatively Regulates the Migratory Potential of Cancer Cells by Inhibiting the Production of Interleukin-8
Authors: Kim Sung-Woo , Hayashi Masaaki , Lo Jeng-Fan , Fearns Colleen , Xiang Rong , Lazennec Gwendal , Yang Young , Lee Jiing-Dwan
Published: October 2005

Tid1 is the human homologue of the Drosophila tumor suppressor, Tid56. Reducing the expression of Tid1 in MDA-MB231 breast cancer cells enhanced their migration without affecting their survival or growth rate. From microarray screening, we discovered that after Tid1 depletion, the mRNA level of interleukin-8 (IL-8) was significantly increased in these cancer cells, which consequently increased secretion of IL-8 protein by 3.5-fold. The enhanced migration of these Tid1-knockdown cells was blocked by reducing the IL-8 expression or by adding an IL-8 neutralizing antibody to the culture medium, suggesting that enhancement of cell motility in these Tid1-deficient cells is dependent on the de novo synthesis of IL-8. Subsequently, we found that abrogating the nuclear factor κB binding site in the IL-8 promoter completely blocked the Tid1 depletion–induced IL-8 expression in the breast cancer cells. As increased IL-8 levels are known to promote tumor metastasis, we tested the effect of Tid1 knockdown on tumor metastasis and found that Tid1 depletion enhanced the metastasis of breast cancer cells in animals. Together, these results indicate that Tid1 negatively regulates the motility and metastasis of breast cancer cells, most likely through attenuation of nuclear factor κB activity on the promoter of the IL8 gene.
Role of the p38 Mitogen-Activated Protein Kinase Pathway in Cytokine-Mediated Hematopoietic Suppression in Myelodysplastic Syndromes
Authors: Katsoulidis Efstratios , Li Yongzhong , Yoon Patrick , Sassano Antonella , Altman Jessica , Kannan-Thulasiraman Padma , Balasubramanian Lakshmi , Parmar Simrit , Varga John , Tallman Martin S. , Verma Amit , Platanias Leonidas C.
Published: October 2005

The p38 mitogen-activated protein kinase (MAPK) pathway is activated by IFNs and other cytokines to mediate signals for important cellular functions, including transcriptional regulation and apoptosis. We examined the role of the p38 pathway in the generation of the effects of myelosuppressive cytokines on human hematopoiesis. Pharmacologic inhibition of p38 using BIX-01208 resulted in reversal of IFN-, tumor necrosis factor-α (TNF-α)–, and transforming growth factor-β (TGF-β)–mediated suppression of human erythroid (blast-forming unit-erythroid) and myeloid (granulocyte-macrophage colony-forming unit) colony formation, consistent with a key role for p38 in the generation of myelosuppressive signals by different cytokines. Similarly, the myelosuppressive effects of TNF-α and TGF-β were reversed by small interfering RNAs targeting p38α expression, further establishing the requirement of this kinase in the induction of myelosuppressive responses. As TNF overproduction has been implicated in the pathophysiology of bone marrow failure states, we determined whether pharmacologic inhibition of p38 reverses the hematopoietic defects seen in bone marrows from patients with myelodysplastic syndromes (MDS) and the anemia of chronic disease. Addition of pharmacologic inhibitors of p38 on such bone marrows resulted in increased numbers of erythroid and myeloid progenitors. Similarly, inhibition of the activity of the downstream effectors of p38, MAPK activated protein kinase-2, and mitogen and stress activated kinase 1 partially restored the hematopoietic defect seen in these bone marrows. Taken altogether, our data implicate the p38 MAPK in the pathophysiology of myelodysplasias and suggest that p38 pharmacologic inhibitors may have therapeutic applications in the treatment of MDS.
Haploinsufficiency of Atp2a2, Encoding the Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2 Ca2+ Pump, Predisposes Mice to Squamous Cell Tumors via a Novel Mode of Cancer Susceptibility
Authors: Prasad Vikram , Boivin Gregory P. , Miller Marian L. , Liu Lynne H. , Erwin Christopher R. , Warner Brad W. , Shull Gary E.
Published: October 2005

A null mutation in one copy of the Atp2a2 or ATP2A2 gene, encoding sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2), leads to squamous cell tumors in mice and to Darier disease in humans, a skin disorder that also involves keratinocytes. Here, we examined the time course and genetic mechanisms of tumor development in the mutant animals. Atp2a2+/− mice overexpressed keratins associated with keratinocyte hyperactivation in normal forestomachs as early as 2 months of age. By the age of 5 to 7 months, 22% of mutants had developed papillomas of the forestomach, and 89% of mutants older than 14 months had developed squamous cell papillomas and/or carcinomas, with a preponderance of the latter. Tumors occurred in regions that had keratinized epithelium and were subjected to repeated mechanical irritation. The genetic mechanism of tumorigenesis did not involve loss of heterozygosity, as tumor cells analyzed by laser capture microdissection contained the wild-type Atp2a2 allele. Furthermore, immunoblot and immunohistochemical analysis showed that tumor keratinocytes expressed the SERCA2 protein. Mutations were not observed in the ras proto-oncogenes; however, expression of wild-type ras was up-regulated, with particularly high levels of K-ras. Loss of the p53 tumor suppressor gene occurred in a single massive tumor, whereas other tumors had increased levels of p53 protein but no mutations in the p53 gene. These findings show that SERCA2 haploinsufficiency predisposes mice to tumor development via a novel mode of cancer susceptibility involving a global change in the tumorigenic potential of keratinized epithelium in Atp2a2+/− mice.
Bcr-Abl Expression Levels Determine the Rate of Development of Resistance to Imatinib Mesylate in Chronic Myeloid Leukemia
Authors: Barnes David J. , Palaiologou Danai , Panousopoulou Eleni , Schultheis Beate , Yong Agnes S.M. , Wong Alice , Pattacini Laura , Goldman John M. , Melo Junia V.
Published: October 2005

Chronic myeloid leukemia (CML) starts with the acquisition of a BCR-ABL fusion gene in a single hematopoietic stem cell, but the time to progression is unpredictable. Although the tyrosine kinase inhibitor imatinib mesylate is highly effective in the treatment of CML, its continuous administration is associated with development of resistance, particularly in advanced phase or blast crisis. We investigate here whether a feature of disease progression (i.e., elevated expression of Bcr-Abl in CD34+ progenitor cells from CML patients in blast crisis) has any bearing on the kinetics of resistance to imatinib. By studying cell lines that exogenously express Bcr-Abl over the range found from chronic phase to blast crisis of CML, we show that cells expressing high amounts of Bcr-Abl, as in blast crisis, are much less sensitive to imatinib and, more significantly, take a substantially shorter time for yielding a mutant subclone resistant to the inhibitor than cells with low expression levels, as in chronic phase. Our data suggest that the differential levels of the Bcr-Abl oncoprotein expressed by CD34+ CML cells may reflect the extent and duration of their response to imatinib; the relatively high levels of oncoprotein in advanced-phase disease may underlie the observed rapid development of resistance.
Inhibition of Rho-Kinase Affects Astrocytoma Morphology, Motility, and Invasion through Activation of Rac1
Authors: Salhia Bodour , Rutten Frederieke , Nakada Mitsutoshi , Beaudry Christian , Berens Michael , Kwan Allison , Rutka James T.
Published: October 2005

Malignant astrocytomas are highly invasive neoplasms infiltrating diffusely into regions of normal brain. Whereas the molecular and cellular mechanisms governing astrocytoma invasion remain poorly understood, evidence in other cell systems has implicated a role for the Rho-GTPases in cell motility and invasion. Here, we examine how the inhibition or activation of Rho-kinase (ROCK) affects astrocytoma morphology, motility, and invasion. ROCK was inhibited in astrocytoma cells by using 5 to 100 μmol/L of Y27632 or by expressing the dominant-negative ROCK mutant, RB/PH TT. ROCK activation was achieved by expressing a constitutively active mutant, CAT. ROCK inhibition led to morphologic and cytoskeletal alterations characterized by an increase in the number and length of cell processes, increased membrane ruffling, and collapse of actin stress fibers. Using two-dimensional radial migration and Boyden chamber assays, we show that astrocytoma migration and invasion were increased at least 2-fold by ROCK inhibition. On the contrary, ROCK activation significantly inhibited migration and invasion of astrocytoma cells. Furthermore, using a Rac-GTP pull-down assay, we show that Rac1 is activated as a consequence of ROCK inhibition. Finally, we show that treatment of astrocytoma cells with small interfering RNA duplexes specific for Rac1-reversed stellation, prevented membrane ruffling formation and abrogated the increased motility observed following treatment with Y27632. Our data show that Rac1 plays a major role in astrocytoma morphology, motility, and invasion. These findings warrant further investigation to determine precisely how the modulation of Rac1 and ROCK can be exploited to inhibit glioma invasion.
Topotecan-Triggered Degradation of Topoisomerase I Is p53-Dependent and Impacts Cell Survival
Authors: Tomicic Maja T. , Christmann Markus , Kaina Bernd
Published: October 2005

The anticancer drug topotecan belongs to the group of topoisomerase I (topo I) inhibitors. In the presence of topotecan, topo I cleaves the DNA but is unable to religate the single-strand break. This leads to stabilization of topo I-DNA–bound complexes and the accumulation of DNA strand breaks that may interfere with DNA replication. The molecular mechanism of controlling the repair of topo I-DNA covalent complexes and its impact on sensitivity of cells to topotecan is largely unknown. Here, we used mouse embryonic fibroblasts expressing wild-type p53 and deficient in p53, in order to elucidate the role of p53 in topotecan-induced cell death. We show that p53-deficient mouse embryonic fibroblasts are significantly more sensitive to topotecan than wild-type cells, displaying a higher frequency of topotecan-induced apoptosis and DNA strand breaks. Treatment of p53 wild-type cells with pifithrin-α, an inhibitor of the trans-activating activity of p53, caused reversal of the phenotype, making wild-type cells more sensitive to topotecan. Upon topotecan treatment, topo I was degraded in wild-type but not in p53-deficient cells. Topo I degradation was attenuated by the proteosomal inhibitor MG132. Similar data were obtained with human glioblastoma cells. U138 cells (p53 mutated) were significantly more sensitive to topotecan than U87 cells (p53 wild-type). Furthermore, U87 cells showed significant degradation of topo I upon topotecan treatment, whereas in U138 cells, this response was abrogated. Topo I degradation was again attenuated by pifithrin-α. The data suggests that p53 causes resistance of cells to topo I inhibitors due to stimulation of topotecan-triggered topo I degradation which may impact topotecan-based cancer therapy.
Effective Tumor Cell Death by σ-2 Receptor Ligand Siramesine Involves Lysosomal Leakage and Oxidative Stress
Authors: Ostenfeld Marie Stampe , Fehrenbacher Nicole , Høyer-Hansen Maria , Thomsen Christian , Farkas Thomas , Jäättelä Marja
Published: October 2005

Acquired resistance to classic caspase-mediated apoptosis is a common problem for the treatment of human cancer. Here, we show that siramesine, a novel σ-2 receptor ligand, effectively induces caspase-independent programmed cell death in immortalized and transformed cells of various origins. Siramesine-treated tumor cells displayed increased levels of reactive oxygen species, lysosomal membrane permeabilization, chromatin condensation, and shrinkage and detachment of cells. Lipid antioxidants (α-tocopherol and γ-tocopherol), but not other tested antioxidants (butylated hydroxyanisol or N-acetyl cysteine), effectively inhibited siramesine-induced morphologic changes and cell death. Cathepsin B inhibitors (CA-074-Me and R-2525) conferred similar, but less pronounced protection, whereas ectopic expression of antiapoptotic protein Bcl-2, lack of wild-type p53 as well as pharmacologic inhibitors of caspases (zVAD-fmk, DEVD-CHO, and LEHD-CHO), calpains (PD150606), and serine proteases (N-tosyl-l-phenylalanine chloromethyl ketone and pefabloc) failed to protect cells against siramesine-induced death. Importantly, transformation of murine embryonic fibroblasts with activated c-src or v-Ha-ras oncogenes greatly sensitized them to siramesine-induced cytotoxicity. Furthermore, p.o. administration of well-tolerated doses of siramesine had a significant antitumorigenic effect in orthotopic breast cancer and s.c. fibrosarcoma models in mice. These results present siramesine as a promising new drug for the treatment of tumors resistant to traditional therapies.
Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166/MEMD), a Novel Actor in Invasive Growth, Controls Matrix Metalloproteinase Activity
Authors: Lunter Pim C. , van Kilsdonk Jeroen W.J. , van Beek Hanneke , Cornelissen Ine M.H.A. , Bergers Mieke , Willems Peter H.G.M. , van Muijen Goos N.P. , Swart Guido W.M.
Published: October 2005

Activated leukocyte cell adhesion molecule (ALCAM/CD166/MEMD) could function as a cell surface sensor for cell density, controlling the transition between local cell proliferation and tissue invasion in melanoma progression. We have tested the hypothesis that progressive cell clustering controls the proteolytic cascade for activation of gelatinase A/matrix metalloproteinase-2 (MMP-2), which involves formation of an intermediate ternary complex of membrane type 1 MMP (MT1-MMP/MMP-14), tissue inhibitor of metalloproteinase-2 (TIMP-2), and pro–MMP-2 at the cell surface. Surprisingly, truncation of ALCAM severely impaired MMP-2 activation in a nude mouse xenograft model, in which we previously observed diminished primary tumor growth and enhanced melanoma metastasis. Comparative studies of two-dimensional monolayer and three-dimensional collagen-gel cultures revealed that extensive cell-to-cell contacts, wild-type ALCAM, and cell-to-matrix interactions were all indispensable for efficient conversion of pro–MMP-2 to its active form in metastatic melanoma cells. Truncated, dominant-negative ALCAM diminished MMP-2 activation via reduced transcript levels and decreased processing of MT1-MMP. Failure of the proteolytic cascade after selective ALCAM depletion by RNA interference was mainly due to incomplete MT1-MMP processing, which was otherwise promoted by extensive cell-to-cell contacts. These data attribute a novel signaling role to ALCAM in regulation of proteolysis and support its previously postulated sensor function in invasive growth.
Inhibition of Adhesion, Invasion, and Metastasis by Antibodies Targeting CEACAM6 (NCA-90) and CEACAM5 (Carcinoembryonic Antigen)
Authors: Blumenthal Rosalyn D. , Hansen Hans J. , Goldenberg David M.
Published: October 2005

CEACAM5 and CEACAM6 are overexpressed in many cancers and are associated with adhesion and invasion. The effects of three monoclonal antibodies targeting different epitopes on these antigens (NH2-terminal [MN-3] and A1B1 domains [MN-15] shared by CEACAM5 and CEACAM6 and the A3B3 domain [MN-14] restricted to CEACAM5) were evaluated in migration, invasion, and adhesion assays in vitro using a panel of human pancreatic, breast, and colonic cancer cell lines, and in the GW-39 human colonic micrometastasis model in vivo. MN-3 Fab′ and MN-15 Fab′ were both effective at inhibiting cell migration. MN-15 Fab′ treatment inhibited invasion, reducing cell penetration through an extracellular matrix (ECM). MN-3 Fab′ also decreased invasion but was less effective than MN-15 Fab′ in four of five cell lines. All three monoclonal antibody (mAb) Fabs decreased adhesion of tumor cells to endothelial cells by 49% to 58%. MN-15 Fab′ but not MN-3 or MN-14 Fabs induced a decrease in adhesion of three of six cell lines to the ECM protein, fibronectin, but adhesion to vitronectin, laminin, collagen-I, and collagen-IV was not affected. In vivo studies showed that treatment with MN-3 Fab′ or MN-15 Fab′ of mice implanted with GW-39 human colonic cancer cells increased their survival (P < 0.025 and P < 0.01, respectively). These studies show that antibody Fabs that target either CEACAM5 or CEACAM6 affect cell migration, cell invasion, and cell adhesion in vitro, and that MN-15 and MN-3 Fabs have antimetastatic effects in vivo, resulting in improved survival of mice with metastases. Thus, blocking the N and A1B1 domains of CEACAM5/CEACAM6 can impede the metastatic process.
Targeted Elimination of Prostate Cancer by Genetically Directed Human T Lymphocytes
Authors: Gade Terence P.F. , Hassen Waleed , Santos Elmer , Gunset Gertrude , Saudemont Aurore , Gong Michael C. , Brentjens Renier , Zhong Xiao-Song , Stephan Matthias , Stefanski Jolanta , Lyddane Clay , Osborne Joseph R. , Buchanan Ian M. , Hall Simon J. , Heston Warren D. , Rivière Isabelle , Larson Steven M. , Koutcher Jason A. , Sadelain Michel
Published: October 2005

The genetic transfer of antigen receptors is a powerful approach to rapidly generate tumor-specific T lymphocytes. Unlike the physiologic T-cell receptor, chimeric antigen receptors (CARs) encompass immunoglobulin variable regions or receptor ligands as their antigen recognition moiety, thus permitting T cells to recognize tumor antigens in the absence of human leukocyte antigen expression. CARs encompassing the CD3ζ chain as their activating domain induce T-cell proliferation in vitro, but limited survival. The requirements for genetically targeted T cells to function in vivo are less well understood. We have, therefore, established animal models to assess the therapeutic efficacy of human peripheral blood T lymphocytes targeted to prostate-specific membrane antigen (PSMA), an antigen expressed in prostate cancer cells and the neovasculature of various solid tumors. In vivo specificity and antitumor activity were assessed in mice bearing established prostate adenocarcinomas, using serum prostate-secreted antigen, magnetic resonance, computed tomography, and bioluminescence imaging to investigate the response to therapy. In three tumor models, orthotopic, s.c., and pulmonary, we show that PSMA-targeted T cells effectively eliminate prostate cancer. Tumor eradication was directly proportional to the in vivo effector-to-tumor cell ratio. Serial imaging further reveals that the T cells must survive for at least 1 week to induce durable remissions. The eradication of xenogeneic tumors in a murine environment shows that the adoptively transferred T cells do not absolutely require in vivo costimulation to function. These results thus provide a strong rationale for undertaking phase I clinical studies to assess PSMA-targeted T cells in patients with metastatic prostate cancer.
Src Inhibits Adriamycin-Induced Senescence and G2 Checkpoint Arrest by Blocking the Induction of p21waf1
Authors: Vigneron Arnaud , Roninson Igor B. , Gamelin Erick , Coqueret Olivier
Published: October 2005

DNA-damaging drugs stop tumor cell proliferation by inducing apoptosis, necrosis, or senescence. Cyclin-dependent kinase inhibitor p21waf1 is an important regulator of these responses, promoting senescence and preventing aberrant mitosis that leads to cell death. Because tumors expressing oncogenic tyrosine kinases are relatively resistant to DNA-damaging agents, the effects of Src on cellular responses to anticancer drug Adriamycin were investigated. Src expression increased drug survival in HT1080 fibrosarcoma cells, as measured by the colony formation assay, and strongly inhibited Adriamycin-induced senescence. Src also decreased the number of apoptotic cells while increasing the fraction of cells dying through necrosis. In addition, Src inhibited the G2 and G1 tetraploidy checkpoints of Adriamycin-treated cells, permitting these cells to proceed into mitosis and subsequently double their DNA content. Inhibition of senescence and G2-G1 checkpoints in Src-expressing cells was associated with the failure of these cells to up-regulate p21waf1 in response to Adriamycin. The failure of p21waf1 induction, despite increased expression of p53 and its binding to p21waf1 promoter, was mediated by the up-regulation of c-Myc, a negative regulator of p21waf1 transcription. Conversely, ectopic expression of p21waf1 inhibited Myc transcription in Src-expressing cells, an effect that was associated with the interaction of p21waf1 with the STAT3 transcription factor at the Myc promoter. These results reveal a complex effect of Src on cellular drug responses and provide an explanation for the effect of this oncogene on cellular drug resistance.
Sequence-Specific Knockdown of EWS-FLI1 by Targeted, Nonviral Delivery of Small Interfering RNA Inhibits Tumor Growth in a Murine Model of Metastatic Ewing's Sarcoma
Authors: Hu-Lieskovan Siwen , Heidel Jeremy D. , Bartlett Derek W. , Davis Mark E. , Triche Timothy J.
Published: October 2005

The development of effective, systemic therapies for metastatic cancer is highly desired. We show here that the systemic delivery of sequence-specific small interfering RNA (siRNA) against the EWS-FLI1 gene product by a targeted, nonviral delivery system dramatically inhibits tumor growth in a murine model of metastatic Ewing's sarcoma. The nonviral delivery system uses a cyclodextrin-containing polycation to bind and protect siRNA and transferrin as a targeting ligand for delivery to transferrin receptor–expressing tumor cells. Removal of the targeting ligand or the use of a control siRNA sequence eliminates the antitumor effects. Additionally, no abnormalities in interleukin-12 and IFN-α, liver and kidney function tests, complete blood counts, or pathology of major organs are observed from long-term, low-pressure, low-volume tail-vein administrations. These data provide strong evidence for the safety and efficacy of this targeted, nonviral siRNA delivery system.
Decreased Replication Ability of E1-Deleted Adenoviruses Correlates with Increased Brain Tumor Malignancy
Authors: Ghosh Subrata , Duigou Gregory J.
Published: October 2005

E1 region replacement adenoviruses are replication defective and are propagated in cells providing adenovirus E1A and E1B proteins. Although they are being developed for antitumor therapies, the proliferative behaviors of these viruses in normal brain tissues or in brain tumors are unknown. To address this, freshly cultured cells from normal human brain and common brain tumors (astrocytomas and meningiomas) were infected using wild-type species C adenoviruses and adenoviruses missing E1A (H5dl312) or E1A plus E1B (H5dl434). Viral DNA replication, late viral protein expression, and production of infectious progeny were characterized. Wild-type adenoviruses grew efficiently in normal brain and brain tumor cells. In comparison, E1-deleted adenovirus DNA replication was delayed and lower in cells derived from normal brain tissues, meningiomas, and low-grade astrocytomas. However, in contrast, E1-deleted adenovirus DNA replication did not occur or was extremely low in cells derived from malignancy grade III and IV astrocytic tumors. Because wild-type adenoviruses infected and replicated in all cells, the malignancy grade-based differential E1-deleted adenovirus DNA replication was not explained by differential virus uptake. Infectious H5dl312 and H5dl434 production correlated with viral DNA replication. Compared with a 5-day average for wild-type infections, advanced cytopathology was noted ∼4 weeks after H5dl312 or H5dl434 infection of meningioma, astrocytoma, and normal brain cells. Cytopathology was not observed after H5dl312 or H5dl434 infection of glioblastoma, anaplastic astrocytoma, and gliosarcoma cells. Because of this tumor grade–based differential growth, the E1-deleted adenoviruses may represent novel tools for studies of brain tumor malignancy.
E-Cadherin Regulates the Association between β-Catenin and Actinin-4
Authors: Hayashida Yasuharu , Honda Kazufumi , Idogawa Masashi , Ino Yoshinori , Ono Masaya , Tsuchida Akihiko , Aoki Tatsuya , Hirohashi Setsuo , Yamada Tesshi
Published: October 2005

The E-cadherin/catenin system acts as an invasion suppressor of epithelial malignancies. This invasion suppressive activity seems be mediated not only by the cell adhesive activity of E-cadherin but by other undetermined signaling pathways elicited by β-catenin. In fact, cancer cells that have infiltrated the stroma reduce the expression of E-cadherin and accumulate β-catenin. We attempted to identify the alternative partner proteins that make complexes with β-catenin in the absence of E-cadherin. An ∼100-kDa protein was constantly coimmunoprecipitated with β-catenin from SW480 colorectal cancer cells, which lack the expression of E-cadherin, and was identified as actinin-4 by mass spectrometry. Transfection of E-cadherin cDNA suppressed the association between β-catenin and actinin-4. Inhibition of E-cadherin by RNA interference transferred the β-catenin and actinin-4 proteins into the membrane protrusions of DLD-1 cells. Immunofluorescence histochemistry of clinical colorectal cancer specimens showed that the β-catenin and actinin-4 proteins were colocalized in colorectal cancer cells infiltrating the stroma. We reported previously that overexpression of actinin-4 induces cell motility and specifically promotes lymph node metastasis by colorectal cancer. The association between β-catenin and actinin-4 and its regulation by E-cadherin may represent a novel molecular link connecting cell adhesion and motility. Shutting down the signals mediating this association may be worth considering as a therapeutic approach to cancer invasion and metastasis.
Contributions by MutL Homologues Mlh3 and Pms2 to DNA Mismatch Repair and Tumor Suppression in the Mouse
Authors: Chen Peng-Chieh , Dudley Sandra , Hagen Wayne , Dizon Diana , Paxton Leslie , Reichow Denise , Yoon Song-Ro , Yang Kan , Arnheim Norman , Liskay R. Michael , Lipkin Steven M.
Published: October 2005

Germ line DNA mismatch repair mutations in MLH1 and MSH2 underlie the vast majority of hereditary non-polyposis colon cancer. Four mammalian homologues of Escherichia coli MutL heterodimerize to form three distinct complexes: MLH1/PMS2, MLH1/MLH3, and MLH1/PMS1. Although MLH1/PMS2 is generally thought to have the major MutL activity, the precise contributions of each MutL heterodimer to mismatch repair functions are poorly understood. Here, we show that Mlh3 contributes to mechanisms of tumor suppression in the mouse. Mlh3 deficiency alone causes microsatellite instability, impaired DNA-damage response, and increased gastrointestinal tumor susceptibility. Furthermore, Mlh3;Pms2 double-deficient mice have tumor susceptibility, shorter life span, microsatellite instability, and DNA-damage response phenotypes that are indistinguishable from Mlh1-deficient mice. Our data support previous results from budding yeast that show partial functional redundancy between MLH3 and PMS2 orthologues for mutation avoidance and show a role for Mlh3 in gastrointestinal and extragastrointestinal tumor suppression. The data also suggest a mechanistic basis for the more severe mismatch repair–related phenotypes and cancer susceptibility in Mlh1- versus Mlh3- or Pms2-deficient mice. Contributions by both MLH1/MLH3 and MLH1/PMS2 complexes to mechanisms of mismatch repair–mediated tumor suppression, therefore, provide an explanation why, among MutL homologues, only germ line mutations in MLH1 are common in hereditary non-polyposis colon cancer.
Increased Susceptibility of Vault Poly(ADP-Ribose) Polymerase–Deficient Mice to Carcinogen-Induced Tumorigenesis
Authors: Raval-Fernandes Sujna , Kickhoefer Valerie A. , Kitchen Christina , Rome Leonard H.
Published: October 2005

Vault poly(ADP-ribose) polymerase (VPARP) and telomerase-associated protein 1 (TEP1) are components of the vault ribonucleoprotein complex. Vaults have been implicated in multidrug resistance of human tumors and are thought to be involved in macromolecular assembly and/or transport. Previous studies showed that VPARP-deficient mice were viable, fertile, and did not display any vault-related or telomerase-related phenotype, whereas disruption of telomerase-associated protein 1 in mice led to reduced stability of the vault RNA and affected its stable association with vaults, although there were no telomerase-related changes. In this study, we evaluated the susceptibility of Vparp−/− and Tep1−/− mice to dimethylhydrazine-induced colon tumorigenesis and urethane-induced lung tumorigenesis. Mice received i.p. injections of either 1 g/kg body weight of urethane twice a week for 2 weeks or 20 mg/kg body weight of dimethylhydrazine once a week for 10 weeks and were analyzed after 10 and 60 weeks, respectively. The colon tumor incidence and multiplicity were significantly higher and colon tumor latency was significantly shorter in Vparp−/− mice compared with wild-type mice. Increased colon tumor incidence, multiplicity, and reduced tumor latency were also seen in Tep1−/− mice, however, these results were statistically not significant. Lung tumor multiplicities were increased in both Vparp−/− and Tep1−/− mice but were not significant. The increase in carcinogen-induced tumors in VPARP-deficient mice is the only phenotype observed to date, and suggests a possible role for VPARP, directly or indirectly, in chemically induced neoplasia.
The In vitro and In vivo Effects of JNJ-7706621: A Dual Inhibitor of Cyclin-Dependent Kinases and Aurora Kinases
Authors: Emanuel Stuart , Rugg Catherine A. , Gruninger Robert H. , Lin Ronghui , Fuentes-Pesquera Angel , Connolly Peter J. , Wetter Steven K. , Hollister Beth , Kruger Walter W. , Napier Cheryl , Jolliffe Linda , Middleton Steven A.
Published: October 2005

Modulation of aberrant cell cycle regulation is a potential therapeutic strategy applicable to a wide range of tumor types. JNJ-7706621 is a novel cell cycle inhibitor that showed potent inhibition of several cyclin-dependent kinases (CDK) and Aurora kinases and selectively blocked proliferation of tumor cells of various origins but was about 10-fold less effective at inhibiting normal human cell growth in vitro. In human cancer cells, treatment with JNJ-7706621 inhibited cell growth independent of p53, retinoblastoma, or P-glycoprotein status; activated apoptosis; and reduced colony formation. At low concentrations, JNJ-7706621 slowed the growth of cells and at higher concentrations induced cytotoxicity. Inhibition of CDK1 kinase activity, altered CDK1 phosphorylation status, and interference with downstream substrates such as retinoblastoma were also shown in human tumor cells following drug treatment. Flow cytometric analysis of DNA content showed that JNJ-7706621 delayed progression through G1 and arrested the cell cycle at the G2-M phase. Additional cellular effects due to inhibition of Aurora kinases included endoreduplication and inhibition of histone H3 phosphorylation. In a human tumor xenograft model, several intermittent dosing schedules were identified that produced significant antitumor activity. There was a direct correlation between total cumulative dose given and antitumor effect regardless of the dosing schedule. These results show the therapeutic potential of this novel cell cycle inhibitor and support clinical evaluation of JNJ-7706621.
The Transcription Factor Pokemon: A New Key Player in Cancer Pathogenesis
Authors: Maeda Takahiro , Hobbs Robin M. , Pandolfi Pier Paolo
Published: October 2005

Learning how critical cell regulatory pathways are controlled may lead to new opportunities for cancer treatment. We recently identified the transcription factor Pokemon as a central regulator of the important tumor suppressor ARF. Pokemon is overexpressed in multiple human cancers and cells lacking Pokemon are refractory to oncogenic transformation. These findings suggest that Pokemon may offer an effective new target for cancer therapeutics.
Functional Network Analysis Reveals Extended Gliomagenesis Pathway Maps and Three Novel MYC-Interacting Genes in Human Gliomas
Authors: Bredel Markus , Bredel Claudia , Juric Dejan , Harsh Griffith R. , Vogel Hannes , Recht Lawrence D. , Sikic Branimir I.
Published: October 2005

Gene expression profiling has proven useful in subclassification and outcome prognostication for human glial brain tumors. The analysis of biological significance of the hundreds or thousands of alterations in gene expression found in genomic profiling remains a major challenge. Moreover, it is increasingly evident that genes do not act as individual units but collaborate in overlapping networks, the deregulation of which is a hallmark of cancer. Thus, we have here applied refined network knowledge to the analysis of key functions and pathways associated with gliomagenesis in a set of 50 human gliomas of various histogenesis, using cDNA microarrays, inferential and descriptive statistics, and dynamic mapping of gene expression data into a functional annotation database. Highest-significance networks were assembled around the myc oncogene in gliomagenesis and around the integrin signaling pathway in the glioblastoma subtype, which is paradigmatic for its strong migratory and invasive behavior. Three novel MYC-interacting genes (UBE2C, EMP1, and FBXW7) with cancer-related functions were identified as network constituents differentially expressed in gliomas, as was CD151 as a new component of a network that mediates glioblastoma cell invasion. Complementary, unsupervised relevance network analysis showed a conserved self-organization of modules of interconnected genes with functions in cell cycle regulation in human gliomas. This approach has extended existing knowledge about the organizational pattern of gene expression in human gliomas and identified potential novel targets for future therapeutic development.
Stable RNA Interference–Mediated Suppression of Cyclophilin A Diminishes Non–Small-Cell Lung Tumor Growth In vivo
Authors: Howard Brandon A. , Furumai Ryohei , Campa Michael J. , Rabbani Zahid N. , Vujaskovic Zeljko , Wang Xiao-Fan , Patz Edward F.
Published: October 2005

Cyclophilin A (CypA) was recently reported to be overexpressed in non–small-cell lung cancer, and represents a potentially novel therapeutic target. To determine the role of CypA in oncogenesis, stable RNA interference (RNAi)–mediated knockdown of CypA was established in two non–small-cell lung cancer cell lines (ADLC-5M2 and LC-103H), and these cells were grown as xenografts in severe combined immunodeficient mice. Tumor cell proliferation, apoptosis, and angiogenesis were measured by Ki67, terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labeling, and CD31 immunohistochemistry, respectively. Tumor glucose metabolism was assessed by fluorodeoxyglucose positron emission tomography imaging. Knockdown of CypA correlated in vivo with slower growth, less fluorodeoxyglucose uptake, decreased proliferation, and a greater degree of apoptosis in the tumors. These results establish the relevance of CypA to tumor growth in vivo, specifically to proliferation and apoptosis. Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for lung cancer.
Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity
Authors: Kong Dehe , Park Eun Jung , Stephen Andrew G. , Calvani Maura , Cardellina John H. , Monks Anne , Fisher Robert J. , Shoemaker Robert H. , Melillo Giovanni
Published: October 2005

The identification of small molecules that inhibit the sequence-specific binding of transcription factors to DNA is an attractive approach for regulation of gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion, all of which are important for tumor progression and metastasis. To identify inhibitors of HIF-1 DNA-binding activity, we expressed truncated HIF-1α and HIF-1β proteins containing the basic-helix-loop-helix and PAS domains. Expressed recombinant HIF-1α and HIF-1β proteins induced a specific DNA-binding activity to a double-stranded oligonucleotide containing a canonical hypoxia-responsive element (HRE). One hundred twenty-eight compounds previously identified in a HIF-1–targeted cell-based high-throughput screen of the National Cancer Institute 140,000 small-molecule library were tested in a 96-well plate ELISA for inhibition of HIF-1 DNA-binding activity. One of the most potent compounds identified, echinomycin (NSC-13502), a small-molecule known to bind DNA in a sequence-specific fashion, was further investigated. Electrophoretic mobility shift assay experiments showed that NSC-13502 inhibited binding of HIF-1α and HIF-1β proteins to a HRE sequence but not binding of the corresponding proteins to activator protein-1 (AP-1) or nuclear factor-κB (NF-κB) consensus sequences. Interestingly, chromatin immunoprecipitation experiments showed that NSC-13502 specifically inhibited binding of HIF-1 to the HRE sequence contained in the vascular endothelial growth factor (VEGF) promoter but not binding of AP-1 or NF-κB to promoter regions of corresponding target genes. Accordingly, NSC-13502 inhibited hypoxic induction of luciferase in U251-HRE cells and VEGF mRNA expression in U251 cells. Our results indicate that it is possible to identify small molecules that inhibit HIF-1 DNA binding to endogenous promoters.
Targeted Virus Replication Plus Immunotherapy Eradicates Primary and Distant Pancreatic Tumors in Nude Mice
Authors: Sarkar Devanand , Su Zao-zhong , Vozhilla Nicolaq , Park Eun Sook , Randolph Aaron , Valerie Kristoffer , Fisher Paul B.
Published: October 2005

Pancreatic cancer is an aggressive neoplasm with no current viable, effective treatment options. In the majority of cases, at first diagnosis, pancreatic cancer has already become metastatic so that conventional treatment regimens provide minimal, if any, clinical benefit in prolonging life or ameliorating the negative prognosis of this disease. These harsh realities underscore the need for developing improved treatment paradigms for this cancer, with gene therapy and immunotherapy currently being evaluated as potential therapeutic options. We currently describe an adenovirus-based therapy for successfully managing pancreatic cancer, the cancer terminator virus (CTV), which is founded on targeted induction of viral replication from a cancer-specific progression elevated gene-3 (PEG-3) promoter (PEG-Prom) and immune modulation by IFN-γ. The PEG-Prom functions selectively in cancer cells of diverse lineages compared with their normal cellular counterparts. In the CTV, the PEG-Prom drives expression of the adenoviral early region 1A (E1A) gene, necessary for virus replication, with IFN-γ simultaneously being expressed from the E3 region. Infection of normal cells and pancreatic cancer cells with the CTV confirmed cancer cell–selective adenoviral replication, robust IFN-γ production coupled with virus replication, growth inhibition, and apoptosis induction. Infection of established pancreatic tumors in nude mice with the CTV promoted viral replication, IFN-γ production, and activation of antitumor immunity resulting in complete eradication of both primary and distant tumors, curing animals of disease. The CTV provides a novel reagent for treating pancreatic and other human cancers with potential for eliminating both primary tumors and metastatic disease.
Rho GTPase-Activating Protein Deleted in Liver Cancer Suppresses Cell Proliferation and Invasion in Hepatocellular Carcinoma
Authors: Wong Chun-Ming , Yam Judy Wai-Ping , Ching Yick-Pang , Yau Tai-On , Leung Thomas Ho-Yin , Jin Dong-Yan , Ng Irene Oi-Lin
Published: October 2005

Deleted in liver cancer (DLC1) is a candidate tumor suppressor gene recently isolated from human hepatocellular carcinoma. Structurally, DLC1 protein contains a conserved GTPase-activating protein for Rho family protein (RhoGAP) domain, which has been thought to regulate the activity of Rho family proteins. Previous studies indicated that DLC1 was frequently inactivated in cancer cells. In the present study, we aimed to characterize the tumor suppressor roles of DLC1 in hepatocellular carcinoma. We showed that DLC1 significantly inhibited cell proliferation, anchorage-independent growth, and in vivo tumorigenicity when stably expressed in hepatocellular carcinoma cells. Moreover, DLC1 expression greatly reduced the motility and invasiveness of hepatocellular carcinoma cells. With RhoGAP-deficient DLC1 mutant (DLC1-K714E), we showed that the RhoGAP activity was essential for DLC1-mediated tumor suppressor function. Furthermore, the 292– to 648–amino acid region and the steroidogenic acute regulatory related lipid transfer domain played an auxiliary role to RhoGAP and tumor suppressor function of DLC1. Taken together, our findings showed that DLC1 functions as a tumor suppressor in hepatocellular carcinoma and provide the first evidence to support the hypothesis that DLC1 suppresses cancer cell growth by negatively regulating the activity of Rho proteins.
Molecular Evidence for Increased Antitumor Activity of Gemcitabine by Genistein In vitro and In vivo Using an Orthotopic Model of Pancreatic Cancer
Authors: Banerjee Sanjeev , Zhang Yuxiang , Ali Shadan , Bhuiyan Mohammad , Wang Zhiwei , Chiao Paul J. , Philip Philip A. , Abbruzzese James , Sarkar Fazlul H.
Published: October 2005

Soy isoflavone genistein exhibits growth inhibitory activity against human pancreatic cancer cell lines. We previously reported the potential of genistein to augment chemotherapeutic response of pancreatic cancer cells in vitro. In the present study, we investigated whether genistein pretreatment could be used as a novel strategy for gemcitabine-induced killing in vitro and enhanced antitumor activity in vivo using an orthotopic tumor model. We conducted our studies using paired isogenic human pancreatic cancer cell line with differences in metastatic behavior (COLO 357 and L3.6pl). In vitro studies were done to measure growth inhibition and degree of apoptotic cell death induced by either genistein alone, gemcitabine alone, or genistein followed by gemcitabine. Our results show that pretreatment of cells with genistein for 24 hours followed by gemcitabine resulted in 60% to 80% growth inhibition compared with 25% to 30% when gemcitabine was used alone. The overall growth inhibition was directly correlated with apoptotic cell death irrespective of the metastatic potential of cells. Several genes that are known to inhibit apoptosis and contribute to chemoresistance such as nuclear factor-κB (NF-κB) and Akt were assessed to investigate the basis for the observed chemosensitizing effects of genistein. Genistein potentiated the gemcitabine-induced killing by down-regulation of NF-κB and Akt. In contrast, Akt and NF-κB were found to be up-regulated when pancreatic cancer cells were exposed to gemcitabine alone, suggesting the potential mechanism of acquired chemoresistance. In addition to in vitro results, we show here for the first time, that genistein in combination with gemcitabine is much more effective as an antitumor agent compared with either agent alone in our orthotopic tumor model. But most importantly, our data also showed that a specific target, such as NF-κB, was inactivated in genistein-treated animal tumors and that gemcitabine-induced activation of NF-κB was completely inhibited in animal tumors treated with genistein and gemcitabine. These results provide strong molecular in vivo evidence in support of our hypothesis that inactivation of NF-κB signaling pathway by genistein could also abrogate gemcitabine-induced activation of NF-κB resulting in the chemosensitization of pancreatic tumors to gemcitabine, which is likely to be an important and novel strategy for the treatment of pancreatic cancer.
Proficient Global Nucleotide Excision Repair in Human Keratinocytes but not in Fibroblasts Deficient in p53
Authors: Ferguson Bridget E. , Oh Dennis H.
Published: October 2005

The p53 tumor suppressor protein is important for many cellular responses to DNA damage in mammalian cells, but its role in regulating DNA repair in human keratinocytes is undefined. We compared the nucleotide excision repair (NER) response of human fibroblasts and keratinocytes deficient in p53. Fibroblasts expressing human papillomavirus 16 E6 oncoprotein had impaired repair of UV radiation–induced cyclobutane pyrimidine dimers in association with reduced levels of p53 and XPC, which is involved in DNA damage recognition. In contrast, keratinocytes expressing E6 alone or concurrently with the E7 oncoprotein, while possessing reduced levels of p53 but normal levels of XPC, continued to repair pyrimidine dimers as efficiently as control cells with normal p53 levels. Despite preservation of DNA repair, E6 and E6/E7 keratinocytes were hypersensitive to UV radiation. E6 fibroblasts exhibited markedly reduced basal and induced levels of mRNA encoding DDB2, another protein implicated in early events in global NER. In contrast, E6 or E6/E7 keratinocytes possessed basal DDB2 mRNA levels that were not significantly altered relative to control cells, although little induction occurred following UV radiation. Intact global NER was also confirmed in SCC25 cells possessing inactivating mutations in p53 as well as in cells treated with pifithrin-α, a chemical inhibitor of p53 that decreased sensitivity of cells to UV radiation. Collectively, these results indicate that human keratinocytes, unlike fibroblasts, do not require p53 to maintain basal global NER activity, but p53 may still be important in mediating inducible responses following DNA damage.
ATM and p21 Cooperate to Suppress Aneuploidy and Subsequent Tumor Development
Authors: Shen Kate C. , Heng Henry , Wang Yaolin , Lu Shu , Liu Guo , Deng Chu-Xia , Brooks S.C. , Wang Y. Alan
Published: October 2005

The DNA damage checkpoint protein kinase mutated in ataxia telangiectasia (ATM) is involved in sensing and transducing DNA damage signals by phosphorylating and activating downstream target proteins that are implicated in the regulation of cell cycle progression and DNA repair. Atm−/− cells are defective in cellular proliferation mediated by the Arf/p53/p21 pathway. In this report, we show that increased expression of p21 (also known as Waf1 or CDKN1a) in Atm−/− cells serves as a cellular defense mechanism to suppress further chromosomal instability (CIN) and tumor development because Atm−/−p21−/− mice are predisposed to carcinomas and sarcomas with intratumoral heterogeneity. It was found that Atm-deficient cells are defective in metaphase-anaphase transition leading to abnormal karyokinesis. Moreover, Atm−/−p21−/− primary embryonic fibroblasts exhibit increased CIN compared with either Atm−/− or p21−/− cells. The increased CIN is manifested at the cellular level by increased chromatid breaks and elevated aneuploid genome in Atm−/−p21−/− cells. Finally, we showed that the role of p21 in a CIN background induced by loss of Atm is to suppress numerical CIN but not structural CIN. Our data suggest that the development of aneuploidy precedes tumor formation and implicates p21 as a major tumor suppressor in a genome instability background.