bluuebunny/crossref_metadata_2025 huggingface dataset:
Afferent Responses During Experimentally Induced Semicircular Canalithiasis
Authors: Rajguru Suhrud M. , Rabbitt Richard D.
Published: January 2007

Benign paroxysmal positional vertigo (BPPV) is a common vestibular disorder that results in brief periods of vertigo and nystagmus, when the head is tipped relative to gravity. Symptoms are commonly attributed to the pathological presence of heavy calcium carbonate particles within the lumen of the semicircular canal(s)—a condition termed canalithiasis. In the present work, we induced canalithiasis in an animal model (oyster toadfish, Opsanus tau) by introducing heavy glass microbeads into the lumen of the lateral semicircular canal. Bead movement under the action of gravity and canal afferent nerve discharge were recorded in vivo. When the head was oriented nose-down, beads moved toward the nose and the lateral canal afferent discharge rate increased. Afferents that normally encoded angular velocity during oscillatory head rotations responded with tonic increases in the discharge rate during gravity-dependent bead movement. Other afferents, such as the units that rapidly adapt to a step increase in angular head velocity, responded with an initial increase in discharge rate followed by a period of adaptation. Afferent responses occurred in the complete absence of head movement and quantify the pathological inputs to the brain that arise from canalithiasis. The magnitude and time course of the responses reported here are sufficient to explain the symptoms of BPPV.
Physiologically Based Controller for Generating Overground Locomotion Using Functional Electrical Stimulation
Authors: Guevremont Lisa , Norton Jonathan A. , Mushahwar Vivian K.
Published: January 2007

The physiological control of stepping is governed both by signals descending from supraspinal systems and by circuitry residing within the lumbosacral spinal cord. The goal of this study was to evaluate the capacity of physiologically based controllers to restore functional overground locomotion after neurological damage, such as spinal cord injury when used in conjunction with functional electrical stimulation. For this purpose we implemented and tested two controllers: 1) an intrinsically timed system that generated a predetermined rhythmic output and 2) a sensory-based system that used feedback signals to make appropriate transitions between the unloaded (flexion) and loaded (extension) phases of the gait cycle. A third controller, a combination of the intrinsically timed and sensory-driven controllers, was implemented and two sessions were conducted to demonstrate the functional advantages of this approach. The controllers were tested in anesthetized cats, implanted with intramuscular electrodes in six major extensor and flexor muscles of the hindlimbs. The cats were partially supported on a sliding trolley that was propelled by the hindlimbs along a 2.5-m instrumented walkway. Ground reaction forces and limb positions were measured by force plates in the walkway and by accelerometers secured to the legs of the cat, respectively. The controllers were used to generate patterns of stimulation that would elicit alternating flexor (swing) and extensor (stance) movements in the hindlimbs. Using either the intrinsically timed or sensory-driven controllers, the cats were able to travel a distance of 2.5 m, taking five to 12 steps. Functional stepping sequences were more easily achieved using the intrinsically timed controller as the result of a lower sensitivity to the selection of initial stimulation parameters. However, unlike the sensory-driven controller, the intrinsically timed controller was unable to adjust to overcome walkway resistance and muscle fatigue. Neither system was consistently able to ensure load-bearing stepping. Therefore we propose the use of a “combined controller” that relies heavily on intrinsic timing but that can be reset based on sensory signals. A combined controller such as this one may provide the best solution for restoring robust overground locomotion after spinal cord injury.
Involvement of Persistent Na+ Current in Spike Initiation in Primary Sensory Neurons of the Rat Mesencephalic Trigeminal Nucleus
Authors: Kang Youngnam , Saito Mitsuru , Sato Hajime , Toyoda Hiroki , Maeda Yoshinobu , Hirai Toshihiro , Bae Yong-Chul
Published: January 2007

It was recently shown that the persistent Na+ current ( INaP) is generated in the proximal axon in response to somatic depolarization in neocortical pyramidal neurons, although the involvement of INaP in spike initiation is still unclear. Here we show a potential role of INaP in spike initiation of primary sensory neurons in the mesencephalic trigeminal nucleus (MTN) that display a backpropagation of the spike initiated in the stem axon toward the soma in response to soma depolarization. Riluzole (10 μM) and tetrodotoxin (TTX, 10 nM) caused an activation delay or a stepwise increase in the threshold for evoking soma spikes (S-spikes) without affecting the spike itself. Simultaneous patch-clamp recordings from the soma and axon hillock (AH) revealed that bath application of 50 nM TTX increased the delay in spike activation in response to soma depolarization, leaving the spike-backpropagation time from the AH to soma unchanged. This indicates that the increase in activation delay occurred in the stem axon. Furthermore, under a decreasing intracellular concentration gradient of QX-314 from the soma to AH created by QX-314–containing and QX-314–free patch pipettes, the amplitude and maximum rate of rise (MRR) of AH-spikes decreased with an increase in the activation delay following repetition of current-pulse injections, whereas S-spikes displayed decreases of considerably lesser degree in amplitude and MRR. This suggests that compared to S-spikes, AH-spikes more accurately reflect the attenuation of axonal spike by QX-314, consistent with the nature of spike backpropagation. These observations strongly suggest that low-voltage–activated INaP is involved in spike initiation in the stem axon of MTN neurons.
Epidermal Growth Factor Receptor Signaling Is Required for Microadenoma Formation in the Mouse Azoxymethane Model of Colonic Carcinogenesis
Authors: Fichera Alessandro , Little Nathaniel , Jagadeeswaran Sujatha , Dougherty Urszula , Sehdev Amikar , Mustafi Reba , Cerda Sonia , Yuan Weihua , Khare Sharad , Tretiakova Maria , Gong Can , Tallerico Michael , Cohen Greg , Joseph Loren , Hart John , Turner Jerrold R. , Bissonnette Marc
Published: January 2007

Colonic carcinogenesis involves the progressive dysregulation of homeostatic mechanisms that control growth. The epidermal growth factor (EGF) receptor (EGFR) regulates colonocyte growth and differentiation and is overexpressed in many human colon cancers. A requirement for EGFR in colonic premalignancy, however, has not been shown. In the current study, we used a specific EGFR antagonist, gefitinib, to investigate this role of the receptor in azoxymethane colonic premalignancy. The azoxymethane model shares many clinical, histologic, and molecular features of human colon cancer. Mice received azoxymethane i.p. (5 mg/kg/wk) or saline for 6 weeks. Animals were also gavaged with gefitinib (10 mg/kg body weight) or vehicle (DMSO) thrice weekly for 18 weeks, a dose schedule that inhibited normal receptor activation by exogenous EGF. Compared with control colonocytes [bromodeoxyuridine (BrdUrd), 2.2 ± 1.2%], azoxymethane significantly increased proliferation (BrdUrd, 12.6 ± 2.8%), whereas gefitinib inhibited this hyperproliferation (BrdUrd, 6.2 ± 4.0%; <0.005). Azoxymethane significantly induced pro-transforming growth factor-α (6.4 ± 1.3–fold) and increased phospho-(active) EGFR (5.9 ± 1.1–fold), phospho-(active) ErbB2 (2.3 ± 0.2–fold), and phospho-(active) extracellular signal-regulated kinase (3.3 ± 0.4–fold) in premalignant colonocytes. Gefitinib inhibited activations of these kinases by >75% (P < 0.05). Gefitinib also significantly reduced the number of large aberrant crypt foci and decreased the incidence of colonic microadenomas from 75% to 33% (P < 0.05). Gefitinib concomitantly decreased cell cycle–regulating cyclin D1 and prostanoid biosynthetic enzyme cyclooxygenase-2 in microadenomas, suggesting that these regulators are key targets of EGFR in colonic carcinogenesis. These results show for the first time that EGFR signaling is required for early stages of colonic carcinogenesis. Our findings suggest, moreover, that inhibitors of EGFR might be useful in chemopreventive strategies in individuals at increased risk for colonic malignancies. [Cancer Res 2007;67(2):827–35]
Nestin Is Expressed in the Basal/Myoepithelial Layer of the Mammary Gland and Is a Selective Marker of Basal Epithelial Breast Tumors
Authors: Li Hua , Cherukuri Pratima , Li Na , Cowling Victoria , Spinella Michael , Cole Michael , Godwin Andrew K. , Wells Wendy , DiRenzo James
Published: January 2007

Transcriptional profiling has identified five breast cancer subtypes, of which the basal epithelial is most aggressive and correlates with poor prognosis. These tumors display a high degree of cellular heterogeneity and lack established molecular targets, such as estrogen receptor-α, progesterone receptor, and Her2 overexpression, indicating a need for definitive diagnostic markers. We present evidence that nestin, a previously described marker of regenerative cells in diverse tissues, is expressed in the regenerative compartment of the normal human mammary gland. Colocalization studies indicate two distinct populations of mammary epithelia that express nestin: one expressing cytokeratin 14 (CK14) and ΔN-p63 and another expressing desmin. Immunohistochemical analysis indicates that ΔN-p63 and nestin are coordinately expressed during pregnancy in the murine mammary gland. In the embryonal carcinoma cell line NT2/D1, ectopic ΔN-p63-α disrupts retinoic acid–induced differentiation, thereby preserving expression of nestin; however, small interfering RNA–mediated ablation of nestin is insufficient to promote differentiation, indicating that whereas nestin may identify cells within the regenerative compartment of the mammary gland, it is insufficient to block differentiation and preserve replicative capacity. Immunohistochemical analysis of basal epithelial breast tumors, including those shown to carry BRCA1 mutations, indicates robust expression of nestin and CK14, punctate expression of p63, and low to undetectable levels of desmin expression. Nestin was not detected in other breast cancer subtypes, indicating selectivity for basal epithelial breast tumors. These studies identify nestin as a selective marker of the basal breast cancer phenotype, which displays features of mammary progenitors. [Cancer Res 2007;67(2):501–10]
Stromal-Epithelial Cell Interactions and Androgen Receptor-Coregulator Recruitment Is Altered in the Tissue Microenvironment of Prostate Cancer
Authors: Cano Patricia , Godoy Alejandro , Escamilla Rosalba , Dhir Rajiv , Onate Sergio A.
Published: January 2007

Tissue recombination experiments show that prostate mesenchyma directs prostate epithelial cell growth and development in an androgen-dependent manner, and that functional differentiation of prostate epithelium requires androgen-driven processes in both epithelia and stroma. The androgen induction of target genes in primary cultures of prostate stromal and epithelial cells was determined using an adenoviral expression system, which employed the MMTV-enhancer driven luciferase reporter as an androgen receptor (AR)–mediated transcription assay. These studies indicate that both cell types contain functional AR. Androgen induction of luciferase reporter activity is 3-fold in stromal cells compared with 10-fold in epithelial cells. AR-mediated transcription activity in stroma cells was enhanced by coculture with epithelial cells or epithelial cell–conditioned media. The elevated AR-mediated transcription activity in stromal cells that were exposed to epithelial factors correlated with increased recruitment of coactivators to the AR transcriptional complex. Epithelial cells facilitated interactions of AR with SRC-1 in an androgen-dependent manner. However, AR-mediated transcriptional activity in stromal cells isolated from prostate cancer was reduced compared with stromal cells isolated from benign prostate and continued to be reduced when cocultured with tumor-derived prostate epithelial cells. The occupancy of AR and coregulators on target genes showed that androgen-bound AR in prostate cancer stromal cells was associated with the corepressor silencing mediator for retinoid and thyroid hormone receptor. Thus, the ability of epithelial cells to modulate coregulator recruitment to the AR transcriptional complex on androgen-responsive genes seems altered in the stromal microenvironment of prostate cancer. [Cancer Res 2007;67(2):511–9]
Heat Shock Protein 70 Increases Tumorigenicity and Inhibits Apoptosis in Pancreatic Adenocarcinoma
Authors: Aghdassi Ali , Phillips Phoebe , Dudeja Vikas , Dhaulakhandi Dhara , Sharif Rifat , Dawra Rajinder , Lerch Markus M. , Saluja Ashok
Published: January 2007

Pancreatic carcinoma is a malignant disease that responds poorly to chemotherapy because of its resistance to apoptosis. Heat shock proteins (Hsp) are not only cytoprotective but also interfere with the apoptotic cascade. Here, we investigated the role of Hsp70 in regulating apoptosis in pancreatic cancer cells. Hsp70 expression was increased in pancreatic cancer cells compared with normal pancreatic ductal cells. This was confirmed by increased mRNA levels for Hsp70 in human pancreatic cancer tissue compared with neighboring normal tissue from the same patient. Depletion of Hsp70 by quercetin decreased cell viability and induced apoptosis in cancer cells but not in normal pancreatic ductal cells. To show that this is a specific effect of Hsp70 on apoptosis, levels of Hsp70 were knocked down by short interfering RNA treatment, which also induced apoptosis in cancer cells as indicated by Annexin V staining and caspase activation. Daily administration of quercetin to nude mice decreased tumor size as well as Hsp70 levels in tumor tissue. These findings indicate that Hsp70 plays an important role in apoptosis and that selective Hsp70 knockdown can be used to induce apoptosis in pancreatic cancer cells. [Cancer Res 2007;67(2):616–25]
SOX9 Is Expressed in Normal Prostate Basal Cells and Regulates Androgen Receptor Expression in Prostate Cancer Cells
Authors: Wang Hongyun , McKnight Nicole C. , Zhang Tao , Lu Michael L. , Balk Steven P. , Yuan Xin
Published: January 2007

SOX9 is a member of the SOX [Sry-related high-mobility group (HMG) box] family of HMG DNA-binding domain transcription factors and is required for the development and differentiation of multiple cell lineages. This report shows that basal epithelial cells express SOX9 in normal prostate, with no detectable expression in luminal epithelial cells. In contrast, SOX9 is expressed in primary prostate cancers in vivo, at a higher frequency in recurrent prostate cancer and in prostate cancer cell lines (LNCaP, CWR22, PC3, and DU145). SOX9 message and protein levels in prostate cancer cells were increased by treatment with glycogen synthase kinase 3β inhibitor (SB415286), and SOX9 was reduced when β-catenin was down-regulated by small interfering RNA (siRNA), indicating that SOX9 expression in prostate cancer is regulated by Wnt/β-catenin signaling. SOX9 bound specifically to androgen receptor (AR) DNA-binding domain glutathione S-transferase fusion proteins, and this interaction was dependent on a short peptide immediately COOH-terminal to the DNA-binding domain (the C-terminal extension), which is required for interactions between steroid hormone receptors and the architectural HMG proteins. Exogenous SOX9 expressed at high nonphysiologic levels decreased AR expression and activity; however, at lower levels, SOX9 increased AR protein expression. Significantly, down-regulation of SOX9 by siRNA in prostate cancer cells reduced endogenous AR protein levels, and cell growth indicating that SOX9 contributes to AR regulation and decreased cellular proliferation. These results indicate that SOX9 in prostate basal cells supports the development and maintenance of the luminal epithelium and that a subset of prostate cancer cells may escape basal cell requirements through SOX9 expression. [Cancer Res 2007;67(2):528–36]
Adenovirus-Mediated Expression of a Dominant Negative Ku70 Fragment Radiosensitizes Human Tumor Cells under Aerobic and Hypoxic Conditions
Authors: He Fuqiu , Li Ligeng , Kim Dooha , Wen Bixiu , Deng Xuelong , Gutin Philip H. , Ling Clifton C. , Li Gloria C.
Published: January 2007

Ku70 is one component of a protein complex, the Ku70/Ku80 heterodimer, which binds to DNA double-strand breaks and activates DNA-dependent protein kinase (DNA-PK), leading to DNA damage repair. Our previous work has confirmed that Ku70 is important for DNA damage repair in that Ku70 deficiency compromises the ability of cells to repair DNA double-strand breaks, increases the radiosensitivity of cells, and enhances radiation-induced apoptosis. Because of the radioresistance of some human cancers, particularly glioblastoma, we examined the use of a radio-gene therapy paradigm to sensitize cells to ionizing radiation. Based on the analysis of the structure-function of Ku70 and the crystal structure of Ku70/Ku80 heterodimer, we designed and identified a candidate dominant negative fragment involving an NH2-terminal deletion, and designated it as DNKu70. We generated this mutant construct, stably overexpressed it in Rat-1 cells, and showed that it has a dominant negative effect (i.e., DNKu70 overexpression results in decreased Ku-DNA end-binding activity, and increases radiosensitivity). We then constructed and generated recombinant replication-defective adenovirus, with DNKu70 controlled by the cytomegalovirus promoter, and infected human glioma U-87 MG cells and human colorectal tumor HCT-8 cells. We show that the infected cells significantly express DNKu70 and are greatly radiosensitized under both aerobic and hypoxic conditions. The functional ramification of DNKu70 was further shown in vivo: expression of DNKu70 inhibits radiation-induced DNA-PK catalytic subunit autophosphorylation and prolongs the persistence of γ-H2AX foci. If radiation-resistant tumor cells could be sensitized by down-regulating the cellular level/activity of Ku/DNA-PK, this approach could be evaluated as an adjuvant to radiation therapy. [Cancer Res 2007;67(2):634–42]
Bortezomib Inhibits Cell-Cell Adhesion and Cell Migration and Enhances Epidermal Growth Factor Receptor Inhibitor–Induced Cell Death in Squamous Cell Cancer
Authors: Lorch Jochen H. , Thomas Tarita O. , Schmoll Hans-Joachim
Published: January 2007

The lack of cell-cell adhesion and increased migration are key characteristics of cancer cells. The loss of expression of cell adhesion components and overexpression of components critical for cell migration, such as focal adhesion kinase (FAK), correlate with poor prognosis. Because alteration of protein turnover affects the expression levels and, in turn, may influence protein function, we investigated the effects of the proteasome inhibitor bortezomib on cell adhesion and migration in oral squamous cell cancer cell lines SCC68 and SCC15. Following treatment with bortezomib, protein levels of adherens junction components such as E-cadherin were unchanged. The desmosomal linker protein desmoplakin level was increased, whereas the protein level of the desmosomal cadherin, desmoglein 2, was diminished. Reduced desmoglein 2 levels correlated with the diminished strength of mechanical cell-cell adhesion. The protein level of the epidermal growth factor receptor (EGFR) increased after proteasome inhibition and EGFR inhibition with the EGFR-specific tyrosine kinase inhibitor PKI166 was able to restore cell-cell adhesion. Furthermore, we found that the combination of PKI166 with bortezomib enhanced the rate of cell death. Although the FAK protein level was unchanged following bortezomib treatment, recruitment of FAK phosphorylated at tyrosine residue 397 to the periphery of the cell was induced. Migration was reduced following treatment with bortezomib, which could potentially be explained by a prominent but disorganized actin fiber network revealed through immunofluorescence. Collectively, our results suggest that proteasome inhibition using bortezomib affects cell adhesion and cell migration profoundly and provides a rationale for its clinical use in conjunction with an EGFR inhibitor. [Cancer Res 2007;67(2):727–34]
Activation of Akt Predicts Poor Outcome in Neuroblastoma
Authors: Opel Daniela , Poremba Christopher , Simon Thorsten , Debatin Klaus-Michael , Fulda Simone
Published: January 2007

Whereas aberrant activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt pathway, a key survival cascade, has previously been linked to poor prognosis in several human malignancies, its prognostic effect in neuroblastoma has not yet been explored. We therefore investigated the phosphorylation status of Akt, S6 ribosomal protein as target of mammalian target of rapamycin, and extracellular signal–regulated kinase (ERK) in 116 primary neuroblastoma samples by tissue microarray and its correlation with established prognostic markers and survival outcome. Here, we provide for the first time evidence that phosphorylation of Akt at serine 473 (S473) and/or threonine 308 (T308), S6 ribosomal protein, and ERK frequently occurs in primary neuroblastoma. Importantly, we identified Akt activation as a novel prognostic indicator of decreased event-free or overall survival in neuroblastoma, whereas phosphorylation of S6 ribosomal protein or ERK had no prognostic effect. In addition, Akt activation correlated with variables of aggressive disease, including MYCN amplification, 1p36 aberrations, advanced disease stage, age at diagnosis, and unfavorable histology. Monitoring Akt at T308 or both phosphorylation sites improved the prognostic significance of Akt activation in neuroblastoma specimens compared with S473 phosphorylation. Parallel experiments in neuroblastoma cell lines revealed that activation of Akt by insulin-like growth factor (IGF)-I significantly inhibited tumor necrosis factor–related apoptosis-inducing ligand– or chemotherapy-induced apoptosis in a PI3K-dependent manner because the PI3K inhibitor LY294002 completely reversed the IGF-I–mediated protection of neuroblastoma cells from apoptosis. By showing that activation of Akt correlates with poor prognosis in primary neuroblastoma in vivo and with apoptosis resistance in vitro, our findings indicate that Akt presents a clinically relevant target in neuroblastoma that warrants further investigation. [Cancer Res 2007;67(2):735–45]
Drosophila split ends Homologue SHARP Functions as a Positive Regulator of Wnt/β-Catenin/T-Cell Factor Signaling in Neoplastic Transformation
Authors: Feng Ying , Bommer Guido T. , Zhai Yali , Akyol Aytekin , Hinoi Takao , Winer Ira , Lin Hua V. , Cadigan Ken M. , Cho Kathleen R. , Fearon Eric R.
Published: January 2007

Wnt ligands have pleiotropic and context-specific roles in embryogenesis and adult tissues. Among other effects, certain Wnts stabilize the β-catenin protein, leading to the ability of β-catenin to activate T-cell factor (TCF)-mediated transcription. Mutations resulting in constitutive β-catenin stabilization underlie development of several human cancers. Genetic studies in Drosophila highlighted the split ends (spen) gene as a positive regulator of Wnt-dependent signaling. We have assessed the role of SHARP, a human homologue of spen, in Wnt/β-catenin/TCF function in mammalian cells. We found that SHARP gene and protein expression is elevated in human colon and ovarian endometrioid adenocarcinomas and mouse colon adenomas and carcinomas carrying gene defects leading to β-catenin dysregulation. When ectopically expressed, the silencing mediator for retinoid and thyroid receptors/histone deacetylase 1-associated repressor protein (SHARP) protein potently enhanced β-catenin/TCF transcription of a model reporter gene and cellular target genes. Inhibition of endogenous SHARP function via RNA inhibitory (RNAi) approaches antagonized β-catenin/TCF-mediated activation of target genes. The effect of SHARP on β-catenin/TCF-regulated genes was mediated via a functional interaction between SHARP and TCF. β-Catenin–dependent neoplastic transformation of RK3E cells was enhanced by ectopic expression of SHARP, and RNAi-mediated inhibition of endogenous SHARP in colon cancer cells inhibited their transformed growth. In toto, our findings implicate SHARP as an important positive regulator of Wnt signaling in cancers with β-catenin dysregulation. [Cancer Res 2007;67(2):482–91]
Human Bone Marrow Activates the Akt Pathway in Metastatic Prostate Cells through Transactivation of the α-Platelet–Derived Growth Factor Receptor
Authors: Dolloff Nathan G. , Russell Mike R. , Loizos Nick , Fatatis Alessandro
Published: January 2007

The factors regulating the bone tropism of disseminated prostate cancer cells are still vaguely defined. We report that prostate cancer cells that metastasize to the skeleton respond to human bone marrow with a robust stimulation of the phosphatidylinositol 3-kinase/Akt pathway, whereas prostate cells that lack bone-metastatic potential respond negligibly. The majority of this Akt activation is dependent on α-platelet–derived growth factor receptor (α-PDGFR) signaling, which was shown using the small-molecule inhibitor of PDGFR signaling AG1296. Low concentrations of PDGF-AA and PDGF-BB found in bone marrow aspirates, which were detected by ELISA, do not account for the high levels of α-PDGFR signaling. Additionally, neutralizing PDGF binding using a α-PDGFR–specific antibody (IMC-3G3) failed to produce a significant inhibition of bone marrow–induced Akt activation. However, the inhibitory effect of IMC-3G3 rivaled that of AG1296 when incubation was done under conditions that stimulated α-PDGFR internalization. We conclude that α-PDGFR is activated by multiple soluble factors contained within human bone marrow, in addition to its natural ligands, and this transactivation is dependent on receptor localization to the plasma membrane. Therefore, α-PDGFR expression may provide select prostate phenotypes with a growth advantage within the bone microenvironment. [Cancer Res 2007;67(2):555–62]
Increased β1 Integrin Is Associated with Decreased Survival in Invasive Breast Cancer
Authors: Yao Evelyn S. , Zhang Hui , Chen Yunn-Yi , Lee Brian , Chew Karen , Moore Dan , Park Catherine
Published: January 2007

Aberrant microenvironments and loss of balance in cell-extracellular matrix signaling are associated with breast cancer invasion, metastasis, and resistance to therapy. We have recently shown that increased β1 integrin signaling is involved in malignant progression and that inhibitory antibody to β1 integrin leads to selective apoptosis and decreased proliferation in three-dimensional cultures and in xenograft models of breast cancer in vivo. To investigate the clinical importance of these findings, in the present study we examined the expression of β1 integrin and extracellular β1 integrin ligands fibronectin and laminin-1 in a cohort of 249 breast cancer patients who had a median follow-up of 8.4 years. Among the 149 scorable cases, the highest β1 integrin intensity score (3+ versus 0–2+) was associated with significantly decreased 10-year overall survival of 48% versus 71% (P < 0.03) and decreased disease-free survival of 50% versus 80% (P < 0.05). Importantly, high fibronectin expression was associated with decreased overall and disease-free survival on univariate analysis (P < 0.04) and β1 integrin intensity score was significantly correlated with fibronectin expression (Kendall's tau-b = 0.19; P = 0.03). In a multivariate Cox proportional hazards model, β1 integrin intensity score remained a significant independent predictor of overall survival [hazard ratio (HR), 1.69; 95% confidence interval (95% CI), 1.19–2.38; P < 0.003] and disease-free survival (HR, 1.87; 95% CI, 1.21–2.88; P < 0.005). These findings show that β1 integrin expression has potential prognostic value in invasive breast cancer and that coexpression of fibronectin may help identify patients with more aggressive tumors who may benefit from targeted therapy. [Cancer Res 2007;67(2):659–64]
A Novel Mechanism of Resistance to Epidermal Growth Factor Receptor Antagonism In vivo
Authors: Rajput Ashwani , Koterba Alan P. , Kreisberg Jeffrey I. , Foster Jason M. , Willson James K.V. , Brattain Michael G.
Published: January 2007

Epidermal growth factor receptor (EGFR) is widely expressed in a number of solid tumors including colorectal cancers. Overexpression of this receptor is one means by which a cell can achieve positive signals for survival and proliferation; another effective means is by constitutive activation of EGFR. We have elucidated the role of constitutive EGFR signaling in malignant progression by stably transfecting colon cancer cells with a human transforming growth factor-α cDNA (a ligand for EGFR) under repressible control by tetracycline. We show that constitutive expression of transforming growth factor-α and its subsequent constitutive activation of EGFR allows for cancer cell survival in response to environmental stress in vitro and in vivo as well. The reversal of constitutive EGFR activation results in the loss of downstream mitogen-activated protein kinase and Akt activation, and a reduction in xenograft size that is associated with decreased proliferation and increased apoptosis. We used CI-1033, a small molecule antagonist of EGFR, to dissect an activation pathway that shows the ability of ERBb2 to activate Akt, but not Erk in the face of EGFR antagonism. This novel escape mechanism is a possible explanation of why anti-EGFR therapies have shown disappointing results in clinical trials. [Cancer Res 2007;67(2):665–73]
O6-Methylguanine-DNA Methyltransferase Regulation by p53 in Astrocytic Cells
Authors: Blough Michael D. , Zlatescu Magdalena C. , Cairncross J. Gregory
Published: January 2007

Methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter (i.e., gene silencing) occurs in 40% to 50% of patients with glioblastoma and predicts benefit from temozolomide chemotherapy; when unmethylated, MGMT repairs DNA damage induced by temozolomide, contributing to chemoresistance. In this study, we tested the hypothesis that MGMT is regulated by p53 in astrocytic cells, the precursors of which may give rise to glioblastoma. p53 is of interest because, in addition to often being mutated in glioblastoma, inactivation sensitizes some astrocytoma cell lines to temozolomide. MGMT expression was examined in neonatal murine astrocytes and SF767 human astrocytic glioma cells following p53 inactivation by knockout (murine only) or RNAi methods. MGMT mRNA and protein were detected in murine wild-type p53 astrocytes. However, in knockout murine astrocytes and wild-type cells in which p53 was inhibited by RNAi, MGMT expression was reduced by >90%. This effect of p53 on MGMT expression was unrelated to MGMT promoter methylation—in both wild-type and p53-null astrocytes, the MGMT promoter was unmethylated. In wild-type astrocytes, the p53 protein localized to a regulatory region of the MGMT promoter. In SF767 human astrocytic glioma cells, transient knockdown of p53 led to the down-regulation of MGMT gene expression. In murine astrocytes and SF767 cells, p53 regulates MGMT expression without affecting promoter methylation; in astrocytes, this effect may be due to direct binding of p53 to the MGMT promoter. These results imply that the best use of temozolomide requires a thorough understanding of MGMT regulation. [Cancer Res 2007;67(2):580–4]
Systemic Efficacy with Oncolytic Virus Therapeutics: Clinical Proof-of-Concept and Future Directions
Authors: Liu Ta-Chiang , Kirn David
Published: January 2007

Oncolytic viruses that can destroy cancer cells have been engineered in a variety of ways with the aim of improving their selectivity and efficacy. Here, we review data from clinical investigations of these virotherapeutic agents, specifically those that have shown systemic efficacy: vaccinia, measles, mumps, viruses, Newcastle disease virus, and reovirus. Further directions for optimizing i.v. delivery and efficacy are discussed. [Cancer Res 2007;67(2):429–32]
Single Chemical Modifications of the C-1027 Enediyne Core, a Radiomimetic Antitumor Drug, Affect Both Drug Potency and the Role of Ataxia-Telangiectasia Mutated in Cellular Responses to DNA Double-Strand Breaks
Authors: Kennedy Daniel R. , Gawron Loretta S. , Ju Jianhua , Liu Wen , Shen Ben , Beerman Terry A.
Published: January 2007

The radiomimetic enediyne C-1027 induces almost exclusively DNA double-strand breaks (DSB) and is extremely cytotoxic. Unique among radiomimetics, ataxia-telangiectasia mutated (ATM) is dispensable for cellular responses to C-1027-induced DNA damage. This study explores the biological activity of three recently bioengineered C-1027 analogues: 7″-desmethyl-C-1027 (desmethyl), 20′-deschloro-C-1027 (deschloro), and 22′-deshydroxy-C-1027 (deshydroxy). Each compound maintains the characteristic ability of radiomimetics to cleave DNA in cell-free systems, varying in activity from 2-fold (deschloro) to 55-fold (desmethyl) less than C-1027. The induction of cellular DNA breaks based on pulsed field gel electrophoresis, comet analysis, and γH2AX activation was in the same rank order as cell-free DNA break induction, although the amount of breaks induced by desmethyl is greatly reduced compared with the other analogues. Despite the disparity in inducing DNA DSBs, all of the analogues produced G2-M cell cycle arrest and activated DNA DSB damage response proteins, such as p53-Ser15 and Chk2-Thr68, at concentrations in concordance with their ability to inhibit cell growth. Interestingly, of the three analogues, only the desmethyl-induced DNA damage response was similar to C-1027, as it did not cause hypersensitive cell growth inhibition in the absence of ATM nor require the kinase to phosphorylate p53 or Chk2. These findings show that simple modifications of the chromophore of C-1027 can result in varied induction of, and cellular response to, DNA DSBs. [Cancer Res 2007;67(2):773–81]
Nur77 Agonists Induce Proapoptotic Genes and Responses in Colon Cancer Cells through Nuclear Receptor–Dependent and Nuclear Receptor–Independent Pathways
Authors: Cho Sung Dae , Yoon Kyungsil , Chintharlapalli Sudhakar , Abdelrahim Maen , Lei Ping , Hamilton Stanley , Khan Shaheen , Ramaiah Shashi K. , Safe Stephen
Published: January 2007

Nerve growth factor–induced Bα (NGFI-Bα, Nur77) is an orphan nuclear receptor with no known endogenous ligands; however, recent studies on a series of methylene-substituted diindolylmethanes (C-DIM) have identified 1,1-bis(3′-indolyl)-1-(phenyl)methane (DIM-C-Ph) and 1,1-bis(3′-indolyl)-1-(p-anisyl)methane (DIM-C-pPhOCH3) as Nur77 agonists. Nur77 is expressed in several colon cancer cell lines (RKO, SW480, HCT-116, HT-29, and HCT-15), and we also observed by immunostaining that Nur77 was overexpressed in colon tumors compared with normal colon tissue. DIM-C-Ph and DIM-C-pPhOCH3 decreased survival and induced apoptosis in RKO colon cancer cells, and this was accompanied by induction of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) protein. The induction of apoptosis and TRAIL by DIM-C-pPhOCH3 was significantly inhibited by a small inhibitory RNA for Nur77 (iNur77); however, it was evident from RNA interference studies that DIM-C-pPhOCH3 also induced Nur77-independent apoptosis. Analysis of DIM-C-pPhOCH3–induced gene expression using microarrays identified several proapoptotic genes, and analysis by reverse transcription-PCR in the presence or absence of iNur77 showed that induction of programmed cell death gene 1 was Nur77 dependent, whereas induction of cystathionase and activating transcription factor 3 was Nur77 independent. DIM-C-pPhOCH3 (25 mg/kg/d) also inhibited tumor growth in athymic nude mice bearing RKO cell xenografts. These results show that Nur77-active C-DIM compounds represent a new class of anti–colon cancer drugs that act through receptor-dependent and receptor-independent pathways. [Cancer Res 2007;67(2):674–83]
Inhibition of Indoleamine 2,3-Dioxygenase in Dendritic Cells by Stereoisomers of 1-Methyl-Tryptophan Correlates with Antitumor Responses
Authors: Hou De-Yan , Muller Alexander J. , Sharma Madhav D. , DuHadaway James , Banerjee Tinku , Johnson Maribeth , Mellor Andrew L. , Prendergast George C. , Munn David H.
Published: January 2007

Indoleamine 2,3-dioxygenase (IDO) is an immunosuppressive enzyme that contributes to tolerance in a number of biological settings. In cancer, IDO activity may help promote acquired tolerance to tumor antigens. The IDO inhibitor 1-methyl-tryptophan is being developed for clinical trials. However, 1-methyl-tryptophan exists in two stereoisomers with potentially different biological properties, and it has been unclear which isomer might be preferable for initial development. In this study, we provide evidence that the d and l stereoisomers exhibit important cell type–specific variations in activity. The l isomer was the more potent inhibitor of IDO activity using the purified enzyme and in HeLa cell–based assays. However, the d isomer was significantly more effective in reversing the suppression of T cells created by IDO-expressing dendritic cells, using both human monocyte–derived dendritic cells and murine dendritic cells isolated directly from tumor-draining lymph nodes. In vivo, the d isomer was more efficacious as an anticancer agent in chemo-immunotherapy regimens using cyclophosphamide, paclitaxel, or gemcitabine, when tested in mouse models of transplantable melanoma and transplantable and autochthonous breast cancer. The d isomer of 1-methyl-tryptophan specifically targeted the IDO gene because the antitumor effect of d-1-methyl-tryptophan was completely lost in mice with a disruption of the IDO gene (IDO-knockout mice). Taken together, our findings support the suitability of d-1-methyl-tryptophan for human trials aiming to assess the utility of IDO inhibition to block host-mediated immunosuppression and enhance antitumor immunity in the setting of combined chemo-immunotherapy regimens. [Cancer Res 2007;67(2):792–801]
The Inflammatory Cytokine Tumor Necrosis Factor-α Generates an Autocrine Tumor-Promoting Network in Epithelial Ovarian Cancer Cells
Authors: Kulbe Hagen , Thompson Richard , Wilson Julia L. , Robinson Stephen , Hagemann Thorsten , Fatah Rewas , Gould David , Ayhan Ayse , Balkwill Frances
Published: January 2007

Constitutive expression of the inflammatory cytokine tumor necrosis factor-α (TNF-α) is characteristic of malignant ovarian surface epithelium. We investigated the hypothesis that this autocrine action of TNF-α generates and sustains a network of other mediators that promote peritoneal cancer growth and spread. When compared with two ovarian cancer cell lines that did not make TNF-α, constitutive production of TNF-α was associated with greater release of the chemokines CCL2 and CXCL12, the cytokines interleukin-6 (IL-6) and macrophage migration-inhibitory factor (MIF), and the angiogenic factor vascular endothelial growth factor (VEGF). TNF-α production was associated also with increased peritoneal dissemination when the ovarian cancer cells were xenografted. We next used RNA interference to generate stable knockdown of TNF-α in ovarian cancer cells. Production of CCL2, CXCL12, VEGF, IL-6, and MIF was decreased significantly in these cells compared with wild-type or mock-transfected cells, but in vitro growth rates were unaltered. Tumor growth and dissemination in vivo were significantly reduced when stable knockdown of TNF-α was achieved. Tumors derived from TNF-α knockdown cells were noninvasive and well circumscribed and showed high levels of apoptosis, even in the smallest deposits. This was reflected in reduced vascularization of TNF-α knockdown tumors. Furthermore, culture supernatants from such cells failed to stimulate endothelial cell growth in vitro. We conclude that autocrine production of TNF-α by ovarian cancer cells stimulates a constitutive network of other cytokines, angiogenic factors, and chemokines that may act in an autocrine/paracrine manner to promote colonization of the peritoneum and neovascularization of developing tumor deposits. [Cancer Res 2007;67(2):585–92]
Activation of Integrin-Linked Kinase Is a Critical Prosurvival Pathway Induced in Leukemic Cells by Bone Marrow–Derived Stromal Cells
Authors: Tabe Yoko , Jin Linhua , Tsutsumi-Ishii Yuko , Xu Yuanyuan , McQueen Teresa , Priebe Waldemar , Mills Gordon B. , Ohsaka Akimichi , Nagaoka Isao , Andreeff Michael , Konopleva Marina
Published: January 2007

Integrin-linked kinase (ILK) directly interacts with β integrins and phosphorylates Akt in a phosphatidylinositol 3-kinase (PI3K)–dependent manner. In this study, we examined the functional role of ILK activation in leukemic and bone marrow stromal cells on their direct contact. Coculture of leukemic NB4 cells with bone marrow–derived stromal mesenchymal stem cells (MSC) resulted in robust activation of multiple signaling pathways, including ILK/Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducers and activators of transcription 3 (STAT3), and Notch1/Hes. Blockade of PI3K or ILK signaling with pharmacologic inhibitors LY294002 or QLT0267 specifically inhibited stroma-induced phosphorylation of Akt and glycogen synthase kinase 3β, suppressed STAT3 and ERK1/2 activation, and decreased Notch1 and Hes1 expression in leukemic cells. This resulted in induction of apoptosis in both leukemic cell lines and in primary acute myelogenous leukemia samples that was not abrogated by MSC coculture. In turn, leukemic cells growing in direct contact with bone marrow stromal elements induce activation of Akt, ERK1/2, and STAT3 signaling in MSC, accompanied by significant increase in Hes1 and Bcl-2 proteins, which were all suppressed by QLT0267 and LY294002. In summary, our results indicate reciprocal activation of ILK/Akt in both leukemic and bone marrow stromal cells. We propose that ILK/Akt is a proximal signaling pathway critical for survival of leukemic cells within the bone marrow microenvironment. Hence, disruption of these interactions by ILK inhibitors represents a potential novel therapeutic strategy to eradicate leukemia in the bone marrow microenvironment by simultaneous targeting of both leukemic cells and activated bone marrow stromal cells. [Cancer Res 2007;67(2):684–94]
2-Methoxyestradiol Inhibits the Anaphase-Promoting Complex and Protein Translation in Human Breast Cancer Cells
Authors: Bhati Rajendra , Gökmen-Polar Yesim , Sledge George W. , Fan Cheng , Nakshatri Harikrishna , Ketelsen David , Borchers Christoph H. , Dial Michael J. , Patterson Cam , Klauber-DeMore Nancy
Published: January 2007

2-Methoxyestradiol (2ME2), an estradiol metabolite with antiproliferative and antiangiogenic activities, is in phase I/II clinical trials for breast cancer. 2ME2 inhibits microtubule polymerization and causes cells to arrest in G2-M. The purpose of this study was to further elucidate the molecular mechanism of 2ME2. MDA-MB-435 breast cancer cells were treated with 2ME2 (2 μmol/L) or vehicle alone. RNA was extracted and genomic profiling was done using 22k Agilent microarrays. Expression Analysis Systematic Explorer was used to determine enrichment of Gene Ontology categories. Protein isolates were subjected to Western blot analysis. Protein synthesis was measured with a [35S]methionine pulse assay. An MDA-MB-435 cell line with two β-tubulin mutations (2ME2R) was used to determine whether novel mechanisms were tubulin-dependent. Gene Ontology categories enriched include genes that regulate the mitotic spindle assembly checkpoint, apoptosis, and the cytosolic ribosome. The target of the mitotic spindle assembly checkpoint is the anaphase-promoting complex (APC). APC inhibition was confirmed by measuring protein levels of its targets securin and cyclin B1, which were increased in 2ME2-treated cells. Because gene expression in the cytosolic ribosome category was decreased, we evaluated whether 2ME2 decreases protein translation. This was confirmed with a pulse assay, which showed decreased isotope incorporation in 2ME2-treated cells, which was maintained in the tubulin-resistant 2ME2R cells. APC inhibition was not maintained in 2ME2R cells. 2ME2 induces tubulin-dependent cell cycle arrest through regulation of genes involved in the mitotic spindle assembly checkpoint, which results in inhibition of the APC and tubulin-independent inhibition of protein translation. [Cancer Res 2007;67(2):702–8]
Synergistic Tumor Suppression by Coexpression of FUS1 and p53 Is Associated with Down-regulation of Murine Double Minute-2 and Activation of the Apoptotic Protease-Activating Factor 1–Dependent Apoptotic Pathway in Human Non–Small Cell Lung Cancer Cells
Authors: Deng Wu-Guo , Kawashima Hiroyaki , Wu Guanglin , Jayachandran Gitanjali , Xu Kai , Minna John D. , Roth Jack A. , Ji Lin
Published: January 2007

FUS1 is a novel tumor suppressor gene identified in human chromosome 3p21.3 region. Loss of expression and deficiency of posttranslational modification of FUS1 protein have been found in a majority of human lung cancers. Restoration of wild-type FUS1 in 3p21.3-deficient human lung cancer cells exhibited a potent tumor suppression function in vitro and in vivo. In this study, we evaluated the combined effects of FUS1 and tumor suppressor p53 on antitumor activity and explored the molecular mechanisms of their mutual actions in human non–small cell lung cancer (NSCLC) cells. We found that coexpression of FUS1 and p53 by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate:cholesterol nanoparticle–mediated gene transfer significantly and synergistically inhibited NSCLC cell growth and induced apoptosis in vitro. We also found that a systemic treatment with a combination of FUS1 and p53 nanoparticles synergistically suppressed the development and growth of tumors in a human H322 lung cancer orthotopic mouse model. Furthermore, we showed that the observed synergistic tumor suppression by FUS1 and p53 concurred with the FUS1-mediated down-regulation of murine double minute-2 (MDM2) expression, the accumulation and stabilization of p53 protein, as well as the activation of the apoptotic protease-activating factor 1 (Apaf-1)–dependent apoptotic pathway in human NSCLC cells. Our results therefore provide new insights into the molecular mechanism of FUS1-mediated tumor suppression activity and imply that a molecular therapy combining two or more functionally synergistic tumor suppressors may constitute a novel and effective strategy for cancer treatment. [Cancer Res 2007;67(2):709–17]
Gene Expression Profiling of Childhood Adrenocortical Tumors
Authors: West Alina Nico , Neale Geoffrey A. , Pounds Stanley , Figueredo Bonald C. , Rodriguez Galindo Carlos , Pianovski Mara Albonei D. , Oliveira Filho Antonio G. , Malkin David , Lalli Enzo , Ribeiro Raul , Zambetti Gerard P.
Published: January 2007

Pediatric adrenocortical tumors (ACT) are rare and often fatal malignancies; little is known regarding their etiology and biology. To provide additional insight into the nature of ACT, we determined the gene expression profiles of 24 pediatric tumors (five adenomas, 18 carcinomas, and one undetermined) and seven normal adrenal glands. Distinct patterns of gene expression, validated by quantitative real-time PCR and Western blot analysis, were identified that distinguish normal adrenal cortex from tumor. Differences in gene expression were also identified between adrenocortical adenomas and carcinomas. In addition, pediatric adrenocortical carcinomas were found to share similar patterns of gene expression when compared with those published for adult ACT. This study represents the first microarray analysis of childhood ACT. Our findings lay the groundwork for establishing gene expression profiles that may aid in the diagnosis and prognosis of pediatric ACT, and in the identification of signaling pathways that contribute to this disease. [Cancer Res 2007;67(2):600–8]