bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Epitope Enhancement of a PAX-FKHR Fusion Protein Breakpoint Epitope in Alveolar Rhabdomyosarcoma Cells Created by a Tumorigenic Chromosomal Translocation Inducing CTL Capable of Lysing Human Tumors
Authors: van den Broeke Leon T. , Pendleton C. David , Mackall Crystal , Helman Lee J. , Berzofsky Jay A.
Published: February 2006

Fusion proteins created by chromosomal translocations in tumors can create neoantigenic determinants at the breakpoint, which are unique to the tumor cells but shared by the vast majority of tumors of that histologic type. If the fusion protein is responsible for the malignant transformation, its expression cannot be lost by the tumor to escape immune responses against this tumor antigen. Here, we identify such a fusion protein breakpoint epitope in the PAX-FKHR fusion protein created by the t(2;13) translocation present in 80% of cases of alveolar rhabdomyosarcoma, a highly aggressive pediatric soft-tissue sarcoma. We use autologous dendritic cells pulsed with the RS10 breakpoint fusion peptide to raise a human CTL line from a normal healthy HLA-B7+ blood donor specific for this peptide. These CTLs are CD8+ (CD4−CD56−) and restricted by HLA-B7. These human peptide-specific CTL lyse human HLA-B7+ rhabdomyosarcoma tumor cells. Therefore, the fusion protein is endogenously processed to produce this natural epitope presented by HLA-B7 and thus this peptide is a bone fide human tumor antigen. We also define a substitution that increases the affinity for HLA-B7 without loss of antigenicity. This epitope-enhanced peptide may serve as a candidate cancer vaccine for HLA-B7+ patients with alveolar rhabdomyosarcoma. (Cancer Res 2006; 66(3): 1818-23)
Mutational and Epigenetic Evidence for Independent Pathways for Lung Adenocarcinomas Arising in Smokers and Never Smokers
Authors: Toyooka Shinichi , Tokumo Masaki , Shigematsu Hisayuki , Matsuo Keitaro , Asano Hiroaki , Tomii Kunitoshi , Ichihara Shuji , Suzuki Makoto , Aoe Motoi , Date Hiroshi , Gazdar Adi F. , Shimizu Nobuyoshi
Published: February 2006

Genetic and epigenetic alterations are considered to play important roles in lung cancer. Recent studies showed that EGFR and K-RAS mutations exhibited a mutually exclusive pattern in adenocarcinoma of the lung, suggesting the presence of two independent oncogenic pathways. However, it is unknown how epigenetic alterations were involved in lung carcinogenesis mediated by EGFR or K-RAS mutation. In this study, we examined the relationship between genetic and epigenetic alterations in 164 cases of lung adenocarcinoma. Somatic mutations were determined by direct sequence of EGFR exons 18 to 21 and K-RAS codons 12 and 13. Methylation status of p16INK4a, RASSF1A, APC, RARβ, and CDH13, frequently methylated in lung cancer, was determined by methylation-specific PCR and the degree of methylation was defined as the methylation index. Multivariate analysis adjusted for age, sex, and smoking dose showed that the probability of having EGFR mutation was significantly lower among those with p16INK4a and CDH13 methylation than in those without [p16INK4a: odds ratio (OR), 0.07; 95% confidence interval (95% CI), 0.02-0.33; CDH13: OR, 0.34; 95% CI, 0.15-0.77] and the methylation index was significantly lower in EGFR mutant cases than in wild type (OR, 0.70; 95% CI, 0.52-0.95). By contrast, K-RAS mutation was significantly higher in p16INK4a methylated cases than in unmethylated cases (OR, 4.93; 95% CI, 1.54-15.7) and the methylation index was higher in K-RAS mutant cases than in wild type with marginal significance (OR, 1.46; 95% CI, 0.95-2.25). Our results indicate the differences in the evolvement of epigenetic alterations between the EGFR- and K-RAS-mediated tumorigenesis and suggest the specific interaction of genetic and epigenetic changes in tumorigenesis of lung cancer. (Cancer Res 2006; 66(3): 1371-5)
Protein Chip Discovery of Secreted Proteins Regulated by the Phosphatidylinositol 3-Kinase Pathway in Ovarian Cancer Cell Lines
Authors: Moscova Michelle , Marsh Deborah J. , Baxter Robert C.
Published: February 2006

Ovarian cancer has the highest mortality among the gynecologic malignancies. The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated, leading to increased cell survival. This study aimed to identify secreted proteins regulated by the PI3K pathway in ovarian cancer cell lines. Surface-enhanced laser desorption-ionization time-of-flight mass spectrometry with cation-exchange protein-chips was used to analyze secreted proteins from five ovarian cancer cell lines (SKOV-3, PE01, OVCAR-3, OV167, and OV207). To activate the PI3K pathway, cells were treated with 50 ng/mL epidermal growth factor (EGF) with or without 10 μmol/L LY294002, a PI3K inhibitor. Proteins induced by EGF and inhibited by LY294002, in the m/z range 7,500 to 9,500, were purified chromatographically, identified by peptide mass fingerprinting and NH2-terminal sequencing, and confirmed by immunodepletion. Two immunologically related proteins, m/z ∼8,385 and 8,922, were identified as truncated and intact forms, respectively, of interleukin 8, a chemokine previously shown to be elevated in serum of ovarian cancer patients. Another protein, m/z 7,866, was identified as CXC chemokine ligand 1 (CXCL1) or GRO-α, a chemokine associated with melanoma formation and some epithelial cancers. EGF-stimulated CXCL1 levels were variably decreased by mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinase kinase and p38 MAPK inhibition in the five cell lines, but only LY294002 fully reversed the EGF effect in all cell lines. Immunoreactive CXCL1 levels in 160 conditioned media were highly correlated with corresponding peak intensities at m/z 7,866 by mass spectrometry, indicating the quantitative nature of these analyses. We conclude that proteomic analysis of cell models of human disease may facilitate the discovery of pathway-dependent proteins. (Cancer Res 2006; 66(3): 1376-83)
GATA5 Activation of the Progesterone Receptor Gene Promoter in Breast Cancer Cells Is Influenced by the +331G/A Polymorphism
Authors: Huggins Gordon S. , Wong Jason Y.Y. , Hankinson Susan E. , De Vivo Immaculata
Published: February 2006

Previously, a modest association was observed between the progesterone receptor +331G/A gene variant and breast cancer risk. Here, in a larger sample of breast cancer cases and controls (n = 1,322/n = 1,953) nested in the Nurses' Health Study cohort, we confirm a significant association (odds ratio, 1.41; 95% confidence interval, 1.10-1.79) and suggest a molecular model. The association of the +331G/A variant with breast cancer was particularly strong among obese women (body mass index > 30; odds ratio, 2.87; 95% confidence interval, 1.40-5.90). To help understand the molecular mechanism by which this variant may predispose women to breast cancer, we identified nearby transcription factor binding sites. This search predicted a binding site for the GATA family of transcriptional regulators adjacent to this hPR polymorphism. Importantly, we found GATA3, GATA4, and GATA6 are expressed in normal breast tissue and two breast cancer cell lines, whereas GATA5 is minimally expressed in normal mammary tissue and more strongly expressed in two breast cancer cell lines. This finding was relevant because GATA5 bound the site adjacent to the +331G/A polymorphism, and activated the hPR (−711 to +822)-luciferase reporter plasmid in breast cancer cells. Overexpression of GATA5 increased expression of the endogenous hPR transcript, and GATA5 more strongly activated an hPR promoter construct encoding the PR-B isoform. Finally, hPR promoter constructs including the +331A were more strongly activated by GATA5 than constructs including +331G. Our findings suggest that GATA5 interacts with the +331G/A polymorphism to stimulate hPR-B expression in mammary cells, which may contribute to breast cancer susceptibility. (Cancer Res 2006; 66(3): 1384-90)
Growth Properties of Colonic Tumor Cells Are a Function of the Intrinsic Mitochondrial Membrane Potential
Authors: Heerdt Barbara G. , Houston Michele A. , Augenlicht Leonard H.
Published: February 2006

Development of malignant transformation in the colonic mucosa includes disruption in the equilibrium between proliferation and apoptosis, decreased expression and deletions of the mitochondrial genome, alterations in mitochondrial enzymatic activity, and elevations in the mitochondrial membrane potential (Δψm). Focusing on the role of the Δψm in tumor development and progression, we generated novel isogenic colonic carcinoma cell lines that exhibit highly significant, stable differences in their intrinsic Δψm. Using these cell lines, we have recently shown that the intrinsic Δψm has a significant influence on steady state mitochondrial activity and the extent to which cells enter butyrate-mediated growth arrest and apoptotic cascades. Here, we report that the Δψm is also profoundly linked to important tumorigenic properties of the cells. Compared with cells with lower Δψm, cells with elevated intrinsic Δψm have an enhanced capacity to (a) respond to hypoxia by avoiding apoptosis and initiating angiogenesis, (b) escape anoikis and grow under anchorage-independent conditions, and (c) invade the basement membrane. Combined with our previous work, these data implicate the intrinsic Δψm of colonic carcinoma cells in determining the probability of tumor expansion and progression. (Cancer Res 2006; 66(3): 1591-6)
The Dispersal of Replication Proteins after Etoposide Treatment Requires the Cooperation of Nbs1 with the Ataxia Telangiectasia Rad3-Related/Chk1 Pathway
Authors: Rossi Rossella , Lidonnici Maria Rosa , Soza Samuela , Biamonti Giuseppe , Montecucco Alessandra
Published: February 2006

In mammalian cells, DNA replication takes place in functional subnuclear compartments, called replication factories, where replicative factors accumulate. The distribution pattern of replication factories is diagnostic of the different moments (early, mid, and late) of the S phase. This dynamic organization is affected by different agents that induce cell cycle checkpoint activation via DNA damage or stalling of replication forks. Here, we explore the cell response to etoposide, an anticancer drug belonging to the topoisomerase II poisons. Etoposide does not induce an immediate block of DNA synthesis and progressively affects the distribution of replication proteins in S phase. First, it triggers the formation of large nuclear foci that contain the single-strand DNA binding protein replication protein A (RPA), suggesting that lesions produced by the drug are processed into extended single-stranded regions. These RPA foci colocalize with DNA replicated at the beginning of the treatment. Etoposide also triggers the dispersal of replicative proteins, proliferating cell nuclear antigen and DNA ligase I, from replication factories. This event requires the activity of the ataxia telangiectasia Rad3-related (ATR) checkpoint kinase. By comparing the effect of the drug in cell lines defective in different DNA repair and checkpoint pathways, we show that, along with the downstream kinase Chk1, the Nbs1 protein, mutated in the Nijmegen breakage syndrome, is also relevant for this response and for ATR-dependent phosphorylation. Finally, our analysis evidences a critical role of Nbs1 in the etoposide-induced inhibition of DNA replication in early S phase. (Cancer Res 2006; 66(3): 1675-83)
Hepatocellular Neoplasms Induced by Low-Number Pancreatic Islet Transplants in Autoimmune Diabetic BB/Pfd Rats
Authors: Dombrowski Frank , Mathieu Chantal , Evert Matthias
Published: February 2006

It has been shown that combined high local hyperinsulinism and hyperglycemia after low-number islet transplantation into the livers of streptozotocin-diabetic rats lead to the development of hepatocellular neoplasms but a substantial cocarcinogenic effect of genotoxic streptozotocin could not be ruled out completely. Thus, we herein investigated this model in BB/Pfd rats (n = 805; nine experimental groups), which develop spontaneous autoimmune diabetes similar to human type 1 diabetes. After low-number islet transplantation (n = 450), the liver acini downstream of the islets show insulin-induced alterations: massive glycogen and/or fat accumulation, translocation of the insulin receptor, decrease in glucose-6-phosphatase activity, increase in expression of insulin-like growth factor (IGF)-I, IGF-II/mannose-6-phosphate receptor, insulin receptor substrate-1, Raf-1, and Mek-1, corresponding to clear cell preneoplastic foci of altered hepatocytes known from chemical hepatocarcinogenesis and identical to that in streptozotocin-diabetic Lewis rats. After 6 months, many altered liver acini progressed to other types of preneoplasias often accompanied by an overexpression of the glutathione-S transferase (placental form), IGF-I receptor, and transforming growth factor (TGF)-α. After 12 to 15 and 15 to 18 months, 52% and 100% of the animals showed one or multiple hepatocellular adenomas or hepatocellular carcinomas (HCCs), respectively. Conclusively, this study identifies combined hyperinsulinism and hyperglycemia as a carcinogenic mechanism for the development of HCCs in diabetic rats. Hepatocarcinogenesis is independent from additional genotoxic compounds (i.e., streptozotocin), but is primarily triggered by increased intracellular insulin signaling via pathways associated with cell growth and proliferation, such as the Ras-Raf-mitogen-activated protein kinase pathway and the IGF system, and secondarily involves other growth factors, such as TGF-α. (Cancer Res 2006; 66(3): 1833-43)
The High-Affinity IgG Receptor, FcγRI, Plays a Central Role in Antibody Therapy of Experimental Melanoma
Authors: Bevaart Lisette , Jansen Marco J.H. , van Vugt Martine J. , Verbeek J. Sjef , van de Winkel Jan G.J. , Leusen Jeanette H.W.
Published: February 2006

We examined the role of FcγR in antibody therapy of metastatic melanoma in wild-type and different FcγR knock-out mice. Treatment of B16F10-challenged wild-type mice with TA99 antibody specific for the gp75 tumor antigen resulted in a marked decrease in numbers of lung metastases. Treatment of individual FcγR knock-out mice revealed the high-affinity IgG receptor, FcγRI (CD64), to represent the central FcγR for TA99-induced antitumor effects. The potential of immune-modulating agents to further enhance the protective effect induced by monoclonal antibody (mAb) TA99 was examined in combination treatments consisting of mAb TA99 and a TLR-4 agonist, monophosphoryl lipid A (MPL). MPL did potently boost TA99 antibody-induced effects, and combination therapy was, again, found to be dependent on the presence of FcγRI. (Cancer Res 2006; 66(3): 1261-4)
Hypoxia-Inducible Factor-1α Promotes Nonhypoxia-Mediated Proliferation in Colon Cancer Cells and Xenografts
Authors: Dang Duyen T. , Chen Fang , Gardner Lawrence B. , Cummins Jordan M. , Rago Carlo , Bunz Fred , Kantsevoy Sergey V. , Dang Long H.
Published: February 2006

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that directly transactivates genes important for the growth and metabolism of solid tumors. HIF-1α is overexpressed in cancer, and its level of expression is correlated with patient mortality. Increased synthesis or stability of HIF-1α can be induced by hypoxia-dependent or hypoxia-independent factors. Thus, HIF-1α is expressed in both nonhypoxic and hypoxic cancer cells. The role of HIF-1α in nonhypoxia-mediated cancer cell proliferation remains speculative. We have disrupted HIF-1α by targeted homologous recombination in HCT116 and RKO human colon cancer cells. Loss of HIF-1α significantly reduced nonhypoxia-mediated cell proliferation in vitro and in vivo. Paradoxically, loss of HIF-1α expression did not grossly affect the hypoxic compartments within tumor xenografts in vivo, although HIF-1α promoted cell proliferation and survival under hypoxia in vitro. To further test the role of HIF-1α within tumor compartments, we generated cells with combined disruptions of both HIF-1α and vascular endothelial growth factor (VEGF). In all xenografts, disruption of VEGF led to marked expansion of the hypoxic compartments and growth delay. Nonetheless, the presence or absence of HIF-1α did not grossly affect these expanded hypoxic compartments. These data provide compelling evidence that, in a subset of colon cancers, (a) HIF-1α is a positive factor for nonhypoxia-mediated cell proliferation in vitro and in vivo and (b) HIF-1α is a positive factor for cell proliferation and survival under hypoxic conditions in vitro, but does not grossly contribute to the tumor hypoxic compartments in vivo. (Cancer Res 2006; 66(3): 1684-93)
P21-Activated Kinase 1 Regulation of Estrogen Receptor-α Activation Involves Serine 305 Activation Linked with Serine 118 Phosphorylation
Authors: Rayala Suresh K. , Talukder Amjad H. , Balasenthil Seetharaman , Tharakan Robbin , Barnes Christopher J. , Wang Rui-An , Aldaz Marcelo , Khan Sohaib , Kumar Rakesh
Published: February 2006

Here, we investigated the role of P21-activated kinase 1 (Pak1) signaling in the function of estrogen receptor-α (ER-α) as assessed by serine 305 (S305) activation and transactivation activity of ER. We found that Pak1 overexpression interfered with the antiestrogenic action of tamoxifen upon the ER transactivation function in hormone-sensitive cells. In addition, tamoxifen stimulation led to up-regulation of ER target genes in breast cancer cells with increased Pak1 expression. Tamoxifen also increased Pak1-ER interaction in tamoxifen-resistant but not in tamoxifen-sensitive cells. Results from the mutational studies discovered a role of ER-S305 phosphorylation in triggering a subsequent phosphorylation of serine 118 (S118), and these effects were further potentiated by tamoxifen treatment. We found that S305 activation-linked ER transactivation function requires a functional S118, and active Pak1 signaling is required for a sustaining S118 phosphorylation of the endogenous ER. All of these events were positively influenced by tamoxifen and thus may contribute toward the loss of antiestrogenic effect of tamoxifen. These findings suggest that Pak1 signaling-dependent activation of ER-S305 leads to an enhanced S118 phosphorylation presumably due to a conformational change, and such structural modifications may participate in the development of tamoxifen resistance. (Cancer Res 2006; 66(3): 1694-701)
mTOR Inhibition Induces Upstream Receptor Tyrosine Kinase Signaling and Activates Akt
Authors: O'Reilly Kathryn E. , Rojo Fredi , She Qing-Bai , Solit David , Mills Gordon B. , Smith Debra , Lane Heidi , Hofmann Francesco , Hicklin Daniel J. , Ludwig Dale L. , Baselga Jose , Rosen Neal
Published: February 2006

Stimulation of the insulin and insulin-like growth factor I (IGF-I) receptor activates the phosphoinositide-3-kinase/Akt/mTOR pathway causing pleiotropic cellular effects including an mTOR-dependent loss in insulin receptor substrate-1 expression leading to feedback down-regulation of signaling through the pathway. In model systems, tumors exhibiting mutational activation of phosphoinositide-3-kinase/Akt kinase, a common event in cancers, are hypersensitive to mTOR inhibitors, including rapamycin. Despite the activity in model systems, in patients, mTOR inhibitors exhibit more modest antitumor activity. We now show that mTOR inhibition induces insulin receptor substrate-1 expression and abrogates feedback inhibition of the pathway, resulting in Akt activation both in cancer cell lines and in patient tumors treated with the rapamycin derivative, RAD001. IGF-I receptor inhibition prevents rapamycin-induced Akt activation and sensitizes tumor cells to inhibition of mTOR. In contrast, IGF-I reverses the antiproliferative effects of rapamycin in serum-free medium. The data suggest that feedback down-regulation of receptor tyrosine kinase signaling is a frequent event in tumor cells with constitutive mTOR activation. Reversal of this feedback loop by rapamycin may attenuate its therapeutic effects, whereas combination therapy that ablates mTOR function and prevents Akt activation may have improved antitumor activity. (Cancer Res 2006; 66(3): 1500-8)
Counting Heads in the War against Cancer: Defining the Role of Annexin A5 Imaging in Cancer Treatment and Surveillance
Authors: Corsten Maarten F. , Hofstra Leonard , Narula Jagat , Reutelingsperger Chris P.M.
Published: February 2006

The unveiling of the heterogeneous nature of cell death modes has compromised the long-lived consensus that cancer treatment typically kills cancer cells through apoptosis. Moreover, it implies that measures of apoptosis may be misleading indicators of treatment efficacy. Simultaneously, it has become clear that phosphatidylserine exposition, traditionally considered a hallmark of apoptosis, is also associated with most other cell death programs, rendering phosphatidylserine an attractive target for overall cell death imaging. Annexin A5 binds with strong affinity to phosphatidylserine and hence offers an interesting opportunity for visualization of aggregate cell death, thus providing a fit benchmark for in vivo monitoring of anticancer treatment. This might be of significant value for pharmacologic therapy development as well as clinical monitoring of treatment success. (Cancer Res 2006; 66(3): 1255-60)
Activation of the Nuclear Factor κB Pathway by Astrocyte Elevated Gene-1: Implications for Tumor Progression and Metastasis
Authors: Emdad Luni , Sarkar Devanand , Su Zao-zhong , Randolph Aaron , Boukerche Habib , Valerie Kristoffer , Fisher Paul B.
Published: February 2006

Astrocyte elevated gene-1 (AEG-1) was initially identified as an HIV-1- and tumor necrosis factor α (TNF-α)–inducible transcript in primary human fetal astrocytes by a rapid subtraction hybridization approach. Interestingly, AEG-1 expression is elevated in subsets of breast cancer, glioblastoma multiforme and melanoma cells and AEG-1 cooperates with Ha-ras to promote transformation of immortalized melanocytes. Activation of the transcription factor nuclear factor κB (NF-κB), a TNF-α downstream signaling component, is associated with several human illnesses, including cancer, and NF-κB controls the expression of multiple genes involved in tumor progression and metastasis. We now document that AEG-1 is a significant positive regulator of NF-κB. Enhanced expression of AEG-1 via a replication-incompetent adenovirus (Ad.AEG-1) in HeLa cells markedly increased binding of the transcriptional activator p50/p65 complex of NF-κB. The NF-κB activation induced by AEG-1 corresponded with degradation of IκBα and nuclear translocation of p65 that resulted in the induction of NF-κB downstream genes. Infection with an adenovirus expressing the mt32IκBα superrepressor (Ad.IκBα-mt32), which prevents p65 nuclear translocation, inhibited AEG-1-induced enhanced agar cloning efficiency and increased matrigel invasion of HeLa cells. We also document that TNF-α treatment resulted in nuclear translocation of both AEG-1 and p65 wherein these two proteins physically interacted, suggesting a potential mechanism by which AEG-1 could activate NF-κB. Our findings suggest that activation of NF-κB by AEG-1 could represent a key molecular mechanism by which AEG-1 promotes anchorage-independent growth and invasion, two central features of the neoplastic phenotype. (Cancer Res 2006; 66(3): 1509-16)
Aberrant Activation of Notch Signaling in Human Breast Cancer
Authors: Stylianou Spyros , Clarke Rob B. , Brennan Keith
Published: February 2006

A role for Notch signaling in human breast cancer has been suggested by both the development of adenocarcinomas in the murine mammary gland following pathway activation and the loss of Numb expression, a negative regulator of the Notch pathway, in a large proportion of breast carcinomas. However, it is not clear currently whether Notch signaling is frequently activated in breast tumors, and how it causes cellular transformation. Here, we show accumulation of the intracellular domain of Notch1 and hence increased Notch signaling in a wide variety of human breast carcinomas. In addition, we show that increased RBP-Jκ–dependent Notch signaling is sufficient to transform normal breast epithelial cells and that the mechanism of transformation is most likely through the suppression of apoptosis. More significantly, we show that attenuation of Notch signaling reverts the transformed phenotype of human breast cancer cell lines, suggesting that inhibition of Notch signaling may be a therapeutic strategy for this disease. (Cancer Res 2006; 66(3): 1517-25)
Extracellular Signal–Regulated Kinase 1/2 Mitogen-Activated Protein Kinase Pathway Is Involved in Myostatin-Regulated Differentiation Repression
Authors: Yang Wei , Chen Yan , Zhang Yong , Wang Xueyan , Yang Ning , Zhu Dahai
Published: February 2006

The cytokines of transforming growth factor β (TGF-β) and its superfamily members are potent regulators of tumorigenesis and multiple cellular events. Myostatin is a member of TGF-β superfamily and plays a negative role in the control of cell proliferation and differentiation. We now show that myostatin rapidly activated the extracellular signal–regulated kinase 1/2 (Erk1/2) cascade in C2C12 myoblasts. A more remarkable Erk1/2 activation stimulated by myostatin was observed in differentiating cells than proliferating cells. The results also showed that Ras was the upstream regulator and participated in myostatin-induced Erk1/2 activation because the expression of a dominant-negative Ras prevented myostatin-mediated inhibition of Erk1/2 activation and proliferation. Importantly, the myostatin-suppressed myotube fusion and differentiation marker gene expression were attenuated by blockade of Erk1/2 mitogen-activated protein kinase (MAPK) pathway through pretreatment with MAPK/Erk kinase 1 (MEK1) inhibitor PD98059, indicating that myostatin-stimulated activation of Erk1/2 negatively regulates myogenic differentiation. Activin receptor type IIb (ActRIIb) was previously suggested as the only type II membrane receptor triggering myostatin signaling. In this study, by using synthesized small interfering RNAs and dominant-negative ActRIIb, we show that myostatin failed to stimulate Erk1/2 phosphorylation and could not inhibit myoblast differentiation in ActRIIb-knockdown C2C12 cells, indicating that ActRIIb was required for myostatin-stimulated differentiation suppression. Altogether, our findings in this report provide the first evidence to reveal functional role of the Erk1/2 MAPK pathway in myostatin action as a negative regulator of muscle cell growth. (Cancer Res 2006; 66(3): 1320-6)
β1 Integrin Inhibitory Antibody Induces Apoptosis of Breast Cancer Cells, Inhibits Growth, and Distinguishes Malignant from Normal Phenotype in Three Dimensional Cultures and In vivo
Authors: Park Catherine C. , Zhang Hui , Pallavicini Maria , Gray Joe W. , Baehner Frederick , Park Chong J. , Bissell Mina J.
Published: February 2006

Current therapeutic approaches to cancer are designed to target molecules that contribute to malignant behavior but leave normal tissues intact. β1 integrin is a candidate target well known for mediating cell-extracellular matrix (ECM) interactions that influence diverse cellular functions; its aberrant expression has been implicated in breast cancer progression and resistance to cytotoxic therapy. The addition of β1 integrin inhibitory agents to breast cancer cells at a single-cell stage in a laminin-rich ECM (three-dimensional lrECM) culture was shown to down-modulate β1 integrin signaling, resulting in malignant reversion. To investigate β1 integrin as a therapeutic target, we modified the three-dimensional lrECM protocol to approximate the clinical situation: before treatment, we allowed nonmalignant cells to form organized acinar structures and malignant cells to form tumor-like colonies. We then tested the ability of β1 integrin inhibitory antibody, AIIB2, to inhibit tumor cell growth in several breast cancer cell lines (T4-2, MDA-MB-231, BT474, SKBR3, and MCF-7) and one nonmalignant cell line (S-1). We show that β1 integrin inhibition resulted in a significant loss of cancer cells, associated with a decrease in proliferation and increase in apoptosis, and a global change in the composition of residual colonies. In contrast, nonmalignant cells that formed tissue-like structures remained resistant. Moreover, these cancer cell–specific antiproliferative and proapoptotic effects were confirmed in vivo with no discernible toxicity to animals. Our findings indicate that β1 integrin is a promising therapeutic target, and that the three-dimensional lrECM culture assay can be used to effectively distinguish malignant and normal tissue response to therapy. (Cancer Res 2006; 66(3): 1526-35)
Functional Coupling of p38-Induced Up-regulation of BiP and Activation of RNA-Dependent Protein Kinase–Like Endoplasmic Reticulum Kinase to Drug Resistance of Dormant Carcinoma Cells
Authors: Ranganathan Aparna C. , Zhang Lin , Adam Alejandro P. , Aguirre-Ghiso Julio A.
Published: February 2006

It has been proposed that occult, disseminated metastatic cells are refractory to chemotherapy due to lack of proliferation. We have shown that p38 activation induces dormancy of squamous carcinoma cells. We now show that p38 signaling in these cells activates a prosurvival mechanism via the up-regulation of the endoplasmic reticulum (ER) chaperone BiP and increased activation of the ER stress–activated eukaryotic translation initiator factor 2α kinase RNA-dependent protein kinase–like ER kinase (PERK) allowing dormant tumor cells to resist drug toxicity. RNA interference and dominant-negative expression studies revealed that both BiP and PERK signaling promote survival and drug resistance of dormant cells, and that BiP up-regulation prevents Bax activation. We propose that stress-dependent activation of p38 via BiP up-regulation and PERK activation protects dormant tumor cells from stress insults, such as chemotherapy. (Cancer Res 2006; 66(3): 1702-1711)
Delayed Onset of Igf2-Induced Mammary Tumors in Igf2r Transgenic Mice
Authors: Wise Thomas L. , Pravtcheva Dimitrina D.
Published: February 2006

The insulin-like growth factor-II (IGF-II) receptor (IGF2R) regulates the level or activity of numerous proteins, including factors that control growth and differentiation. Frequent loss or inactivation of this receptor in a diverse group of tumors indicates that it may act as a tumor suppressor, but it is not known which functions of this receptor are selected against in the tumors. Lysosomal targeting and degradation of the growth-promoting IGF-II has been proposed as a mechanism for the tumor suppressor effects of IGF2R. As a genetic test of this hypothesis in vivo, we have produced Igf2r transgenic mice that ubiquitously express the transgene and have crossed these mice with mice that develop mammary tumors as a consequence of Igf2 overexpression. Our findings indicate that the presence of the Igf2r transgene delays mammary tumor onset and decreases tumor multiplicity in Igf2 transgenic mice. These findings are relevant to human tumors and preneoplastic conditions accompanied by altered IGF2 expression. (Cancer Res 2006; 66(3): 1327-36)
Thiazolidinediones Inhibit Insulin-Like Growth Factor-I–Induced Activation of p70S6 Kinase and Suppress Insulin-Like Growth Factor-I Tumor-Promoting Activity
Authors: He Guobin , Sung You Me , DiGiovanni John , Fischer Susan M.
Published: February 2006

Thiazolidinediones are a novel class of antidiabetic drugs that improve insulin sensitivity in type 2 diabetic patients. Recently, these compounds have also been shown to suppress tumor development in several animal models. The molecular basis for their antitumor action, however, is largely unknown. We report here that oral administration of thiazolidinediones (rosiglitazone and troglitazone) remarkably inhibited insulin-like growth factor-I (IGF-I)–promoted skin tumor development by 73% in BK5.IGF-1 transgenic mice, although they were previously found to be ineffective in inhibiting UV- or chemically induced mouse skin tumorigenesis. The anti-IGF-I effect of troglitazone in mouse skin keratinocytes was due to, at least partially, inhibition of IGF-I–induced phosphorylation of p70S6 kinase (p70S6K) at Thr389, a site specifically phosphorylated by mammalian target of rapamycin (mTOR). Troglitazone did not directly inhibit mTOR kinase activity as shown by mTOR in vitro kinase assay but rapidly activated AMP-activated protein kinase (AMPK) through a yet undefined peroxisome proliferator-activated receptor γ–independent mechanism. Expression of a dominant-negative AMPK reversed the inhibitory effect of troglitazone on IGF-I–induced phosphorylation of p70S6K, suggesting that troglitazone inhibited IGF-I and p70S6K signaling through activation of AMPK. Collectively, these data suggest that thiazolidinediones specifically inhibit IGF-I tumor-promoting activity in mouse skin through activation of AMPK and subsequent inhibition of p70S6K. (Cancer Res 2006; 66(3): 1872-8)
Increased Levels of the FoxM1 Transcription Factor Accelerate Development and Progression of Prostate Carcinomas in both TRAMP and LADY Transgenic Mice
Authors: Kalin Tanya V. , Wang I-Ching , Ackerson Timothy J. , Major Michael L. , Detrisac Carol J. , Kalinichenko Vladimir V. , Lyubimov Alexander , Costa Robert H.
Published: February 2006

The proliferation-specific Forkhead Box M1 (FoxM1 or FoxM1b) transcription factor is overexpressed in a number of aggressive human carcinomas. Mouse hepatocytes deficient in FoxM1 fail to proliferate and are highly resistant to developing carcinogen-induced liver tumors. We previously developed a transgenic (TG) mouse line in which the ubiquitous Rosa26 promoter was used to drive expression of the human FoxM1b cDNA transgene in all mouse cell types. To investigate the role of FoxM1b in prostate cancer progression, we bred Rosa26-FoxM1b mice with both TRAMP and LADY TG mouse models of prostate cancer. We show that increased expression of FoxM1b accelerated development, proliferation, and growth of prostatic tumors in both TRAMP and LADY double TG mice. Furthermore, development of prostate carcinomas in TRAMP/Rosa26-FoxM1b double TG mice required high levels of FoxM1 protein to overcome sustained expression of the alternative reading frame tumor suppressor, a potent inhibitor of FoxM1 transcriptional activity. Depletion of FoxM1 levels in prostate cancer cell lines PC-3, LNCaP, or DU-145 by small interfering RNA transfection caused significant reduction in proliferation and anchorage-independent growth on soft agar. This phenotype was associated with increased nuclear levels of the cyclin-dependent kinase inhibitor protein p27Kip1 and diminished expression of S-phase promoting cyclin A2 and M-phase promoting cyclin B1 proteins. Finally, we show that elevated levels of FoxM1 protein correlate with high proliferation rates in human prostate adenocarcinomas. Our results suggest that the FoxM1 transcription factor regulates development and proliferation of prostate tumors, and that FoxM1 is a novel target for prostate cancer treatment. Cancer Res 2006; 66(3): 1712-20
Src Uses Cas to Suppress Fhl1 in Order to Promote Nonanchored Growth and Migration of Tumor Cells
Authors: Shen Yongquan , Jia Zhenyu , Nagele Robert G. , Ichikawa Hitoshi , Goldberg Gary S.
Published: February 2006

Anchorage independence and motility are hallmarks of tumor cell growth. Tumor cell growth and morphology can be normalized by contact with nontransformed cells. The Src tyrosine kinase phosphorylates specific sites on the focal adhesion adaptor protein Crk-associated substrate (Cas) to promote nonanchored cell growth and migration. We studied the effects of Src and Cas on the expression of >14,000 genes to identify molecular events that underlie these activities. Gene expression in tumor cells that were normalized by neighboring nontransformed cells was used as an additional filter to identify genes that control metastatic cell growth. This process enabled the identification of genes that play roles in anchorage-independent cell growth and migration. One candidate, four and a half LIM domains 1 (Fhl1), acts as a transcriptional regulator that can associate with cell junctions as well as with the nucleus. We show here that Src phosphorylates Cas to block Fhl1 expression. In addition, suppression of Fhl1 is required for Src to promote tumor cell growth. These data show that Fhl1 is a tumor suppressor gene that acts downstream of Src and Cas to specifically block anchorage-independent cell growth and migration. Moreover, Fhl1 was suppressed in tumors from several human tissues. Thus, identification of how Fhl1 controls fundamental aspects of tumor cell growth and metastasis may lead to the development of novel markers that can be used to diagnose human clinical specimens as well as open innovative avenues of investigations aimed at developing reagents that target cancer cells while minimizing damage to normal cells. (Cancer Res 2006; 66(3): 1543-52)
An Integrated Computational/Experimental Model of Tumor Invasion
Authors: Frieboes Hermann B. , Zheng Xiaoming , Sun Chung-Ho , Tromberg Bruce , Gatenby Robert , Cristini Vittorio
Published: February 2006

The intracellular and extracellular dynamics that govern tumor growth and invasiveness in vivo remain poorly understood. Cell genotype and phenotype, and nutrient, oxygen, and growth factor concentrations are key variables. In previous work, using a reaction-diffusion mathematical model based on variables that directly describe tumor cell cycle and biology, we formulated the hypothesis that tumor morphology is determined by the competition between heterogeneous cell proliferation caused by spatial diffusion gradients, e.g., of cell nutrients, driving shape instability and invasive tumor morphologies, and stabilizing mechanical forces, e.g., cell-to-cell and cell-to-matrix adhesion. To test this hypothesis, we here obtain variable-based statistics for input to the mathematical model from in vitro human and rat glioblastoma cultures. A linear stability analysis of the model predicts that glioma spheroid morphology is marginally stable. In agreement with this prediction, for a range of variable values, unbounded growth of the tumor mass and invasion of the environment are observed in vitro. The mechanism of invasion is recursive subspheroid component development at the tumor viable rim and separation from the parent spheroid. Results of computer simulations of the mathematical model closely resemble the morphologies and spatial arrangement of tumor cells from the in vitro model. We propose that tumor morphogenesis in vivo may be a function of marginally stable environmental conditions caused by spatial variations in cell nutrients, oxygen, and growth factors, and that controlling these conditions by decreasing spatial gradients could benefit treatment outcomes, whereas current treatment, and especially antiangiogenic therapy, may trigger spatial heterogeneity (e.g., local hypoxia), thus causing invasive instability. (Cancer Res 2006; 66(3): 1597-604)
Bone Marrow–Derived Stromal Cells Express Lineage-Related Messenger RNA Species
Authors: Direkze Natalie C. , Jeffery Rosemary , Hodivala-Dilke Kairbaan , Hunt Toby , Playford Raymond J. , Elia George , Poulsom Richard , Wright Nicholas A. , Alison Malcolm R.
Published: February 2006

Evidence has emerged that bone marrow cells have a greater degree of plasticity than previously thought. However, there has been a call to establish proof that these bone marrow–derived cells function appropriately in their new environment. We have already shown that the bone marrow contributes to myofibroblasts in multiple organs and that this is exacerbated by injury and occurs in a mouse tumor model. Here, we provide evidence that these cells are functioning appropriately by showing that bone marrow–derived myofibroblasts are expressing mRNA for the α1 chain of type I (pro)collagen using a new customized technique. This provides evidence that the bone marrow-tumor stroma axis is functionally relevant and may therefore subsequently be exploited to develop new strategies for anticancer therapy. (Cancer Res 2006; 66(3): 1265-9)
von Hippel-Lindau Tumor Suppressor Protein Regulates the Assembly of Intercellular Junctions in Renal Cancer Cells through Hypoxia-Inducible Factor–Independent Mechanisms
Authors: Calzada Maria J. , Esteban Miguel A. , Feijoo-Cuaresma Monica , Castellanos Maria C. , Naranjo-Suárez Salvador , Temes Elisa , Méndez Fernando , Yánez-Mo Maria , Ohh Michael , Landázuri Manuel O.
Published: February 2006

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is responsible for the development of renal cell cancers (RCC), pheochromocytomas, and tumors in other organs. The best known function of VHL protein (VHL) is to target the hypoxia-inducible factor (HIF) for proteasome degradation. VHL is also required for the establishment of an epithelial-like cell shape in otherwise fibroblastic-like RCC cell lines. However, the underlying mechanisms and whether this is linked to HIF remain undetermined. Because the breakage of intercellular junctions induces a fibroblastic-like phenotype in multiple cancer cell models, we hypothesized that VHL may be required for the assembly of intercellular junctions in RCC cells. Our experiments showed that VHL in RCC cell lines is necessary for the normal organization of adherens and tight intercellular junctions, the maintenance of cell polarity, and control of paracellular permeability. Additionally, 786-O cells reconstituted with wild-type VHL and with a constitutively active form of HIF-2α did not reproduce any of the phenotypic alterations of VHL-negative cells. In summary, we show that VHL inactivation in RCC cells disrupts intercellular junctions and cell shape through HIF-independent events, supporting the concept that VHL has additional functions beside its role in the regulation of HIF. (Cancer Res 2006; 66(3): 1553-60)
Ataxia Telangiectasia Mutated and Checkpoint Kinase 2 Regulate BRCA1 to Promote the Fidelity of DNA End-Joining
Authors: Wang Hui-Chun , Chou Wen-Cheng , Shieh Sheau-Yann , Shen Chen-Yang
Published: February 2006

Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are the two mechanisms responsible for repairing DNA double-strand breaks (DSBs) and act in either a collaborative or competitive manner in mammalian cells. DSB repaired by NHEJ may be more complicated than the simple joining of the ends of DSB, because, if nucleotides were lost, it would result in error-prone repair. This has led to the proposal that a subpathway of precise NHEJ exists that can repair DSBs with higher fidelity; this is supported by recent findings that the expression of the HR gene, BRCA1, is causally linked to in vitro and in vivo precise NHEJ activity. To further delineate this mechanism, the present study explored the connection between NHEJ and the cell-cycle checkpoint proteins, ataxia telangiectasia mutated (ATM) and checkpoint kinase 2 (Chk2), known to be involved in activating BRCA1, and tested the hypothesis that ATM and Chk2 promote precise end-joining by BRCA1. Support for this hypothesis came from the observations that (a) knockdown of ATM and Chk2 expression affected end-joining activity; (b) in BRCA1-defective cells, precise end-joining activity was not restored by a BRCA1 mutant lacking the site phosphorylated by Chk2 but was restored by wild-type BRCA1 or a mutant mimicking phosphorylation by Chk2; (c) Chk2 mutants lacking kinase activity or with a mutation at a site phosphorylated by ATM had a dominant negative effect on precise end-joining in BRCA1-expressing cells. These results suggest that the other two HR regulatory proteins, ATM and Chk2, act jointly to regulate the activity of BRCA1 in controlling the fidelity of DNA end-joining by precise NHEJ. (Cancer Res 2006; 66(3): 1391-400)
Checkpoint Kinase 2–Mediated Phosphorylation of BRCA1 Regulates the Fidelity of Nonhomologous End-Joining
Authors: Zhuang Jing , Zhang Junran , Willers Henning , Wang Hong , Chung Jay H. , van Gent Dik C. , Hallahan Dennis E. , Powell Simon N. , Xia Fen
Published: February 2006

The tumor suppressor gene BRCA1 maintains genomic integrity by protecting cells from the deleterious effects of DNA double-strand breaks (DSBs). Through its interactions with the checkpoint kinase 2 (Chk2) kinase and Rad51, BRCA1 promotes homologous recombination, which is typically an error-free repair process. In addition, accumulating evidence implicates BRCA1 in the regulation of nonhomologous end-joining (NHEJ), which may involve precise religation of the DSB ends if they are compatible (i.e., error-free repair) or sequence alteration upon rejoining (i.e., error-prone or mutagenic repair). However, the precise role of BRCA1 in regulating these different subtypes of NHEJ is not clear. We provide here the genetic and biochemical evidence to show that BRCA1 promotes error-free rejoining of DSBs in human breast carcinoma cells while suppressing microhomology-mediated error-prone end-joining and restricting sequence deletion at the break junction during repair. The repair spectrum in BRCA1-deficient cells was characterized by an increase in the formation of >2 kb deletions and in the usage of long microhomologies distal to the break site, compared with wild-type (WT) cells. This error-prone repair phenotype could also be revealed by disruption of the Chk2 phosphorylation site of BRCA1, or by expression of a dominant-negative kinase-dead Chk2 mutant in cells with WT BRCA1. We suggest that the differential control of NHEJ subprocesses by BRCA1, in concert with Chk2, reduces the mutagenic potential of NHEJ, thereby contributing to the prevention of familial breast cancers. (Cancer Res 2006; 66(3): 1401-8)
The Cyclin-Dependent Kinase 2 Inhibitor Down-regulates Interleukin-1β-Mediated Induction of Cyclooxygenase-2 Expression in Human Lung Carcinoma Cells
Authors: Mukhopadhyay Partha , Ali M. Aktar , Nandi Animesh , Carreon Peter , Choy Hak , Saha Debabrata
Published: February 2006

Overexpression of cyclooxygenase-2 (COX-2) is frequently observed in several human cancers, including lung, colon, and head and neck. Malignancies are also associated with the dysregulation of cell cycle events and concomitant elevated activity of cyclin-dependent kinases (CDK). CDK2 is a key cell cycle regulatory protein that controls the transition of cells from G1 to S phase. In this study, we furnish several lines of evidence that show a functional role for the CDK2 in interleukin-1β (IL-1β)–induced COX-2 expression in H358 human non–small cell lung carcinoma cell line by blocking CDK2 activity. First, we show that BMS-387032, a potent CDK2 inhibitor, blocks IL-1β-induced expression as well as steady-state mRNA levels of COX-2. Second, we show that small interfering RNA that abrogates CDK2 expression also blocks IL-1β-induced COX-2 expression. Third, results from in vitro kinase assays clearly show that IL-1β induces CDK2 activity in H358 cells and this activity is significantly inhibited by BMS-387032. Moreover, CDK2 inhibition blocks IL-1β-induced binding to the NF-IL6 element of the COX-2 promoter and inhibits transcription of the COX-2 gene. We also observed that BMS-387032 does not inhibit endogenous expression of COX-2 or prostaglandin synthesis in lung carcinoma cells. Finally, we provide evidence showing that IL-1β-induced signaling events, such as p38 mitogen-activated protein kinase, phosphorylated stress-activated protein kinase/c-Jun NH2-terminal kinase, phosphorylated AKT, and phosphorylated extracellular signal-regulated kinase 1/2, are not inhibited by CDK2 inhibitor. Taken together, the data suggest that CDK2 activity may play an important event in the IL-1β-induced COX-2 expression and prostaglandin E2 synthesis and might represent a novel target for BMS-387032. (Cancer Res 2006; 66(3): 1758-66)