bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacologic Inhibition of RAF→MEK→ERK Signaling Elicits Pancreatic Cancer Cell Cycle Arrest Through Induced Expression of p27Kip1
Authors: Gysin Stephan , Lee Sang-Hyun , Dean Nicholas M. , McMahon Martin
Published: September 2005

Expression of mutationally activated RAS is a feature common to the vast majority of human pancreatic adenocarcinomas. RAS elicits its effects through numerous signaling pathways including the RAF→mitogen-activated protein (MAP)/extracellular signal–regulated kinase (ERK) kinase [MEK]→ERK MAP kinase pathway. To assess the role of this pathway in regulating cell proliferation, we tested the effects of pharmacologic inhibition of MEK on human pancreatic cancer cell lines. In eight cell lines tested, MEK inhibition led to a cessation of cell proliferation accompanied by G0-G1 cell cycle arrest. Concomitant with cell cycle arrest, we observed induced expression of p27Kip1, inhibition of cyclin/cyclin-dependent kinase 2 (cdk2) activity, accumulation of hypophosphorylated pRb, and inhibition of E2F activity. Using both antisense and RNA interference techniques, we assessed the role of p27Kip1 in the observed effects of MEK inhibition on pancreatic cancer cell proliferation. Inhibition of p27Kip1 expression in Mia PaCa-2 cells restored the activity of cyclin/cdk2, phosphorylation of pRb, and E2F activity and partially relieved the effects of U0126 on pancreatic cancer cell cycle arrest. Consistent with the effects of p27Kip1 on cyclin/cdk2 activity, inhibition of CDK2 expression by RNA interference also led to G0-G1 cell cycle arrest. These data suggest that the expression of p27Kip1 is downstream of the RAF→MEK→ERK pathway and that the regulated expression of this protein plays an important role in promoting the proliferation of pancreatic cancer cells. Moreover, these data suggest that pharmacologic inhibition of the RAF→MEK→ERK signaling pathway alone might tend to have a cytostatic, as opposed to a cytotoxic, effect on pancreatic cancer cells.
Bone Marrow Mesenchymal Stem Cells Provide an Alternate Pathway of Osteoclast Activation and Bone Destruction by Cancer Cells
Authors: Sohara Yasuyoshi , Shimada Hiroyuki , Minkin Cedric , Erdreich-Epstein Anat , Nolta Jan A. , DeClerck Yves A.
Published: September 2005

The bone is the third most common site of cancer metastasis. To invade the bone, tumor cells produce osteoclast-activating factors that increase bone resorption by osteoclasts. Here we report that human neuroblastoma cells that form osteolytic lesions in vivo do not produce osteoclast-activating factors but rather stimulate osteoclast activity in the presence of human bone marrow mesenchymal stem cells. This alternative pathway of osteoclast activation involves a nonadhesive interaction between neuroblastoma cells and bone marrow mesenchymal stem cells. Stimulated bone marrow mesenchymal stem cells express markedly increased levels of interleukin-6, which is then responsible for osteoclast activation. This report describes a critical role of bone marrow mesenchymal stem cells in bone destruction in cancer.
Surrogate Tumor Antigen Vaccination Induces Tumor-Specific Immunity and the Rejection of Spontaneous Metastases
Authors: Lewis Jennifer D. , Shearer Michael H. , Kennedy Ronald C. , Bright Robert K.
Published: September 2005

The nonimmunogenic 4T1 murine mammary carcinoma model and a model surrogate tumor antigen (sTA) were employed to explore the possibility of inducing tumor-specific immunity through active immunization in the absence of defined tumor-associated antigens. Immunization of naive mice with protein-based sTA resulted in protection from s.c. challenge, with 4T1 modified to express the sTA (4T1.sTA), or from a sTA-expressing unrelated tumor cell line (mKSA). Immunization had no effect on parental 4T1 tumor growth or the formation of parental 4T1 spontaneous lung metastases. Mice that were sTA immunized and successfully rejected 4T1.sTA challenge also rejected a subsequent challenge in the contralateral flank with parental 4T1 and strikingly prevented the formation of spontaneous parental 4T1 lung metastases. The rejection of parental 4T1 seemed to be specific for and associated with unknown 4T1 tumor-associated antigens, because rejection of mKSA did not induce cross-protection against a challenge with parental 4T1. To evaluate the effect of this vaccine approach on established disease, mice were simultaneously challenged on day 0 with 4T1.sTA and parental 4T1 in contralateral flanks and then immunized on days 3, 10, 17, and 24 with sTA protein. Tumor growth and metastasis were delayed in four of five animals, and 20% (2 of 5) of the animals were tumor free at the completion of the experiment. Together, these data suggest that prior vaccination with a sTA followed by inoculation with poorly immunogenic tumor cells modified to express the sTA activates determinant spreading and the induction of systemic tumor immunity resulting in indigenous tumor rejection.
Linkage and Microarray Analyses of Susceptibility Genes in ACI/Seg Rats: A Model for Prostate Cancers in the Aged
Authors: Yamashita Satoshi , Suzuki Shugo , Nomoto Tomoko , Kondo Yasushi , Wakazono Kuniko , Tsujino Yoshimi , Sugimura Takashi , Shirai Tomoyuki , Homma Yukio , Ushijima Toshikazu
Published: September 2005

ACI/Seg (ACI) rats develop prostate cancers spontaneously with aging, similar to humans. Here, to identify genes involved in prostate cancer susceptibility, we did linkage analysis and oligonucleotide microarray analysis. Linkage analysis was done using 118 effective rats, and prostate cancer susceptibility 1 (Pcs1), whose ACI allele dominantly induced prostate cancers, was mapped on chromosome 19 [logarithm of odds (LOD) score of 5.0]. PC resistance 1 (Pcr1), whose ACI allele dominantly and paradoxically suppressed the size of prostate cancers, was mapped on chromosome 2 (LOD score of 5.0). When linkage analysis was done in 51 rats with single or no macroscopic testicular tumors, which had larger prostates and higher testosterone levels than those with bilateral testicular tumors, Pcs2 and Pcr2 were mapped on chromosomes 20 and 1, respectively. By oligonucleotide microarray analysis with 8,800 probe sets and confirmation by quantitative reverse transcription-PCR, only two genes within these four loci were found to be differentially expressed >1.8-fold. Membrane metalloendopeptidase (Mme), known to inhibit androgen-independent growth of prostate cancers, on Pcr1 was expressed 2.0- to 5.5-fold higher in the ACI prostate, in accordance with its paradoxical effect. Cdkn1a on Pcs2 was expressed 1.5- to 4.5-fold lower in the ACI prostate. Additionally, genes responsible for testicular tumors and unilateral renal agenesis were mapped on chromosomes 11 and 14, respectively. These results showed that prostate cancer susceptibility of ACI rats involves at least four loci, and suggested Mme and Cdkn1a as candidates for Pcr1 and Pcs2.
Dehydroepiandrosterone Can Inhibit the Proliferation of Myeloma Cells and the Interleukin-6 Production of Bone Marrow Mononuclear Cells from Patients with Myeloma
Authors: Liu Shangqin , Ishikawa Hideaki , Li Fu-Jun , Ma Zi , Otsuyama Ken-ichiro , Asaoku Hideki , Abroun Saeid , Zheng Xu , Tsuyama Naohiro , Obata Masanori , Kawano Michio M.
Published: September 2005

The serum levels of an adrenal sex hormone, dehydroepiandrosterone sulfate (DHEA-S), are significantly more decreased in human myelomas compared with the reduction brought by physiologic decline with age. In order to clarify the effect of DHEA on myeloma cells, we investigated whether DHEA and DHEA-S could inhibit interleukin-6 (IL-6) production of bone marrow mononuclear cells and the proliferation of myeloma cells from patients with myeloma. DHEA-S and DHEA suppressed IL-6 production from a bone marrow stromal cell line, KM-102, as well as in bone marrow mononuclear cells from patients with myeloma. Furthermore, DHEA inhibited in vitro growth of the U-266 cell line and primary myeloma cells from the patients, as well as the in vivo growth of U-266 cells implanted i.p. in severe combined immunodeficiency-hIL6 transgenic mice. DHEA up-regulated the expression of peroxisome proliferator–activated receptor (PPAR), PPAR β, but not PPARγ or PPARα, and the expression of IκBα gene in myeloma cells and bone marrow stromal cells, which could explain the suppressive effect of DHEA on IL-6 production through the down-regulation of NF-κB activity. Therefore, these data revealed that DHEA-S, as well as DHEA, had a direct effect on myeloma and bone marrow stromal cells to inhibit their proliferation and IL-6 production, respectively.
Down-regulation of CD9 in Human Ovarian Carcinoma Cell Might Contribute to Peritoneal Dissemination: Morphologic Alteration and Reduced Expression of β1 Integrin Subsets
Authors: Furuya Mitsuko , Kato Hiroaki , Nishimura Norihiro , Ishiwata Isamu , Ikeda Hitoshi , Ito Ryoko , Yoshiki Takashi , Ishikura Hiroshi
Published: September 2005

Peritoneal dissemination is one of the main causes of death in cancer patients. Pathophysiology of metastasis has been well investigated, but the mechanism of diffuse spread of tumor colonies in the peritoneal cavity is not fully understood. CD9 is a member of tetraspanin and its down-regulation is known to be involved in poor prognosis. To investigate the significance of the down-regulation of CD9, HTOA, an ovarian carcinoma cell line that highly expressed CD9, was transiently transfected with small interfering RNA (siRNA) against CD9, and CD9-negative cells (HTOACD9−) were purified. HTOACD9− showed altered adhesion patterns on Matrigel, collagen, fibronectin, and laminin compared with those of control siRNA–transfected HTOA (control-HTOA). Flow cytometry and fluorescence cytostainings revealed that the expression levels of integrins β1, α2, α3β1, α5, and α6 were lower in HTOACD9− than those of control-HTOA. HTOACD9− showed altered expression of junctional and cytoskeletal molecules. By time-lapse video microscopy, control-HTOA showed solid adhesion to extracellular matrix and formed cobblestone pattern, whereas HTOACD9− showed weaker adhesion and were distributed as diffuse spots. To examine whether the expression level of CD9 change during tumor dissemination, HTOA-P, a highly disseminative subclone of HTOA, was established. HTOA-P showed distinctive down-regulation of CD9 at mRNA and protein levels, and showed similar morphologic alteration as HTOACD9− did. These findings indicate that the down-regulation of CD9 may be an acquired event in the process of tumor dissemination. Down-regulated CD9 may attenuate the expression of several integrins and rearrange junctional and cytoskeletal molecules that might contribute to dissemination of ovarian carcinomas.
Breast Cancer Metastasis Suppressor 1 Inhibits Gene Expression by Targeting Nuclear Factor-κB Activity
Authors: Cicek Muzaffer , Fukuyama Ryuichi , Welch Danny R. , Sizemore Nywana , Casey Graham
Published: September 2005

Breast cancer metastasis suppressor 1 (BRMS1) functions as a metastasis suppressor gene in breast cancer and melanoma cell lines, but the mechanism of BRMS1 suppression remains unclear. We determined that BRMS1 expression was inversely correlated with that of urokinase-type plasminogen activator (uPA), a prometastatic gene that is regulated at least in part by nuclear factor-κB (NF-κB). To further investigate the role of NF-κB in BRMS1-regulated gene expression, we examined NF-κB binding activity and found an inverse correlation between BRMS1 expression and NF-κB binding activity in MDA-MB-231 breast cancer and C8161.9 melanoma cells stably expressing BRMS1. In contrast, BRMS1 expression had no effect on activation of the activator protein-1 transcription factor. Further, we showed that suppression of both constitutive and tumor necrosis factor-α–induced NF-κB activation by BRMS1 may be due to inhibition of IκBα phosphorylation and degradation. To examine the relationship between BRMS1 and uPA expression in primary breast tumors, we screened a breast cancer dot blot array of normalized cDNA from 50 breast tumors and corresponding normal breast tissues. There was a significant reduction in BRMS1 mRNA expression in breast tumors compared with matched normal breast tissues (paired t test, P < 0.0001) and a general inverse correlation with uPA gene expression (P < 0.01). These results suggest that at least one of the underlying mechanisms of BRMS1-dependent suppression of tumor metastasis includes inhibition of NF-κB activity and subsequent suppression of uPA expression in breast cancer and melanoma cells.
A Lack of DNA Mismatch Repair on an Athymic Murine Background Predisposes to Hematologic Malignancy
Authors: Campbell Marcia R. , Nation Patrick N. , Andrew Susan E.
Published: September 2005

Inheritance of a germline mutation in one of the DNA mismatch repair genes predisposes human individuals to hereditary nonpolyposis colorectal cancer, characterized by development of tumors predominantly in the colon, endometrium, and gastrointestinal tract. Mice heterozygous for a mismatch repair–null mutation generally do not have an increased risk of neoplasia. However, mice constitutively lacking mismatch repair are prone to tumor development from an early age, particularly thymic lymphomas. Mismatch repair–deficient mice crossed to Apc+/− mice develop an increased spontaneous intestinal tumor incidence, demonstrating that the tumor spectrum can be genetically influenced. Here, we bred Msh2- and Msh6-deficient mice to athymic nude mice, hypothesizing that a broader tumor spectrum may be observed if mice are able to survive longer without succumbing to thymic lymphomas. However, Msh2−/−;Foxn1nu/nu and Msh6−/−;Foxn1nu/nu mice developed primarily early-onset lymphoblastic lymphomas. Using B-cell–specific markers, we found these tumors to be predominately B-cell in origin. The development of hematologic malignancy in the mouse, even in the absence of a thymus, parallels the development of B- and T-cell lymphoma and leukemia in the few rare mismatch repair–null human patients that have been identified. The persistent development of hematologic malignancy both in the mouse and in human patients deficient in mismatch repair leads us to implicate mismatch repair as an important repair mechanism in normal B- and T-cell development. Thus, mismatch repair–deficient mice may prove to be a good model to study human hematologic malignancy.
In vitro Modeling of Human Pancreatic Duct Epithelial Cell Transformation Defines Gene Expression Changes Induced by K-ras Oncogenic Activation in Pancreatic Carcinogenesis
Authors: Qian Jiaying , Niu Jiangong , Li Ming , Chiao Paul J. , Tsao Ming-Sound
Published: September 2005

Genetic analysis of pancreatic ductal adenocarcinomas and their putative precursor lesions, pancreatic intraepithelial neoplasias (PanIN), has shown a multistep molecular paradigm for duct cell carcinogenesis. Mutational activation or inactivation of the K-ras, p16INK4A, Smad4, and p53 genes occur at progressive and high frequencies in these lesions. Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and is found early in the PanIN-carcinoma sequence, but its functional roles remain poorly understood. We show here that the expression of K-rasG12V oncogene in a near diploid HPV16-E6E7 gene immortalized human pancreatic duct epithelial cell line originally derived from normal pancreas induced the formation of carcinoma in 50% of severe combined immunodeficient mice implanted with these cells. A tumor cell line established from one of these tumors formed ductal cancer when implanted orthotopically. These cells also showed increased activation of the mitogen-activated protein kinase, AKT, and nuclear factor-κB pathways. Microarray expression profiling studies identified 584 genes whose expression seemed specifically up-regulated by the K-ras oncogene expression. Forty-two of these genes have been reported previously as differentially overexpressed in pancreatic cancer cell lines or primary tumors. Real-time PCR confirmed the overexpression of a large number of these genes. Immunohistochemistry done on tissue microarrays constructed from PanIN and pancreatic cancer samples showed laminin β3 overexpression starting in high-grade PanINs and occurring in >90% of pancreatic ductal carcinoma. The in vitro modeling of human pancreatic duct epithelial cell transformation may provide mechanistic insights on gene expression changes that occur during multistage pancreatic duct cell carcinogenesis.
Expression of Siva-1 Protein or Its Putative Amphipathic Helical Region Enhances Cisplatin-Induced Apoptosis in Breast Cancer Cells: Effect of Elevated Levels of BCL-2
Authors: Chu Fei , Barkinge John , Hawkins Sarah , Gudi Radhika , Salgia Ravi , Kanteti Prasad V.S.
Published: September 2005

cis-Diaminedichloroplatinum (II) (cisplatin) is routinely used to treat various types of cancers; however, a significant number develop resistance. One of the underlying factors that contribute to cisplatin resistance is the elevated level of BCL-2 and/or BCL-XL, which promotes cell survival. A potential method of overcoming such resistance is to use a potentiator that is capable of neutralizing the antiapoptotic effects of BCL-2/BCL-XL, such as Siva-1. We previously cloned the proapoptotic protein Siva-1 and showed a possible role for it in both extrinsic and intrinsic apoptosis. Using an adenovirus-based expression system, we now show that Siva-1 can synergize with cisplatin in inducing apoptosis in MCF7 and MDA-MB-231 breast cancer cells. In an anchorage-independent clonogenicity assay, MCF7/caspase-3 cells stably expressing Siva-1, but not the control cells, showed a dramatic decrease in the number of colonies formed on one-time cisplatin treatment. Further, we show that the unique putative amphipathic helical region (SAH) in Siva-1 (amino acid residues 36-55) is necessary and sufficient for the observed enhancement in cisplatin-induced apoptosis by Siva-1. Although cisplatin treatment results in significant elevation in the expression of Fas ligand and intracellular p21 levels, expression of Siva-1 has no additional benefit. Instead, the enhancement in apoptosis seems to be due to activation of intrinsic pathway that involves caspase-9 activation. Moreover, Siva-1 augments cisplatin-mediated cell death in MCF7 cells stably expressing BCL-2. We therefore propose that Siva-1 or its SAH region can be used as a potentiator of cisplatin-based chemotherapy.
Effect of Conditional Knockout of the Type II TGF-β Receptor Gene in Mammary Epithelia on Mammary Gland Development and Polyomavirus Middle T Antigen Induced Tumor Formation and Metastasis
Authors: Forrester Elizabeth , Chytil Anna , Bierie Brian , Aakre Mary , Gorska Agnieszka E. , Sharif-Afshar Ali-Reza , Muller William J. , Moses Harold L.
Published: September 2005

Transforming growth factor–β (TGF-β) isoforms are growth factors that function physiologically to regulate development, cellular proliferation, and immune responses. The role of TGF-β signaling in mammary tumorigenesis is complex, as TGF-β has been reported to function as both a tumor suppressor and tumor promoter. To elucidate the role of TGF-β signaling in mammary gland development, tumorigenesis, and metastasis, the gene encoding type II TGF-β receptor, Tgfbr2, was conditionally deleted in the mammary epithelium (Tgfbr2MGKO). Loss of Tgfbr2 in the mammary epithelium results in lobular-alveolar hyperplasia in the developing mammary gland and increased apoptosis. Tgfbr2MGKO mice were mated to the mouse mammary tumor virus-polyomavirus middle T antigen (PyVmT) transgenic mouse model of metastatic breast cancer. Loss of Tgfbr2 in the context of PyVmT expression results in a shortened median tumor latency and an increased formation of pulmonary metastases. Thus, our studies support a tumor-suppressive role for epithelial TGF-β signaling in mammary gland tumorigenesis and show that pulmonary metastases can occur and are even enhanced in the absence of TGF-β signaling in the carcinoma cells.
Loss of Heterozygosity Patterns Provide Fingerprints for Genetic Heterogeneity in Multistep Cancer Progression of Tobacco Smoke–Induced Non–Small Cell Lung Cancer
Authors: Pan Hongjie , Califano Joseph , Ponte Jose F. , Russo Andrea L. , Cheng Kuang-hung , Thiagalingam Arunthathi , Nemani Pratima , Sidransky David , Thiagalingam Sam
Published: September 2005

Dilution end point loss of heterozygosity (LOH) analysis, a novel approach for the analysis of LOH, was used to evaluate allelic losses with the use of 21 highly polymorphic microsatellite markers at nine chromosomal sites most frequently affected in smoking-related non–small cell lung cancers. Allelotyping was done for bronchial epithelial cells and matching blood samples from 23 former and current smokers and six nonsmokers as well as in 33 adenocarcinomas and 25 squamous cell carcinomas (SCC) and corresponding matching blood from smokers. Major conclusions from these studies are as follows: (a) LOH at chromosomal sites 8p, 9p, 11q, and 13q (P > 0.05, Fisher's exact test) are targeted at the early stages, whereas LOH at 1p, 5q, 17p, and 18q (P < 0.05, Fisher's exact test) occur at the later stages of non–small cell lung cancer progression; (b) LOH at 1p, 3p, 5q, 8p, 9p, 11q, 13q, 17p, and 18q occurs in over 45% of the tobacco smokers with SCC and adenocarcinoma; (c) compared with bronchial epithelial cells from smokers, there is a significantly higher degree of LOH at 1p, 5q, and 18q in adenocarcinoma and at 1p, 3p, and 17p in SCC (P < 0.05, Fisher's exact test). We propose that lung cancer progression induced by tobacco smoke occurs in a series of target gene inactivations/activations in defined modules of a global network. The gatekeeper module consists of multiple alternate target genes, which is inclusive of but not limited to genes localized to chromosomal loci 8p, 9p, 11q, and 13q.
ABCB5-Mediated Doxorubicin Transport and Chemoresistance in Human Malignant Melanoma
Authors: Frank Natasha Y. , Margaryan Armen , Huang Ying , Schatton Tobias , Waaga-Gasser Ana Maria , Gasser Martin , Sayegh Mohamed H. , Sadee Wolfgang , Frank Markus H.
Published: September 2005

Enhanced drug efflux mediated by ABCB1 P-glycoprotein and related ATP-binding cassette transporters is one of several mechanisms of multidrug resistance thought to impair chemotherapeutic success in human cancers. In malignant melanoma, its potential contribution to chemoresistance is uncertain. Here, we show that ABCB5, which functions as a determinant of membrane potential and regulator of cell fusion in physiologic skin progenitor cells, is expressed in clinical malignant melanoma tumors and preferentially marks a subset of hyperpolarized, CD133+ stem cell phenotype-expressing tumor cells in malignant melanoma cultures and clinical melanomas. We found that ABCB5 blockade significantly reversed resistance of G3361 melanoma cells to doxorubicin, an agent to which clinical melanomas have been found refractory, resulting in a 43% reduction in the LD50 from 4 to 2.3 μmol/L doxorubicin (P < 0.05). Our results identified ABCB5-mediated doxorubicin efflux transport as the underlying mechanism of resistance, because ABCB5 blockade significantly enhanced intracellular drug accumulation. Consistent with this novel ABCB5 function and mechanism in doxorubicin resistance, gene expression levels of the transporter across a panel of human cancer cell lines used by the National Cancer Institute for drug screening correlated significantly with tumor resistance to doxorubicin (r = 0.44; P = 0.016). Our results identify ABCB5 as a novel drug transporter and chemoresistance mediator in human malignant melanoma. Moreover, our findings show that ABCB5 is a novel molecular marker for a distinct subset of chemoresistant, stem cell phenotype-expressing tumor cells among melanoma bulk populations and indicate that these chemoresistant cells can be specifically targeted via ABCB5 to enhance cytotoxic efficacy.
Post-Transcriptional Regulation of the Androgen Receptor by Mammalian Target of Rapamycin
Authors: Cinar Bekir , De Benedetti Arrigo , Freeman Michael R.
Published: September 2005

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), an ErbB1 ligand and prostate stromal growth factor, is an antagonist of androgen receptor (AR) function. In the LNCaP prostate cancer model, HB-EGF reduced AR protein levels and AR transactivation without affecting AR mRNA level or protein turnover. The signal to attenuate AR was mediated by the mammalian target of rapamycin, as shown by genetic and pharmacologic methods, and was independent of ErbB2/HER-2, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase pathways. Additional evidence suggests that AR protein levels are highly sensitive to regulation by cap-dependent mRNA translation. These findings reveal a novel mechanism for regulation of AR by a classic growth factor system and indicate that a rapamycin-sensitive post-transcriptional pathway can attenuate or possibly bypass AR-mediated signaling.
Kringle 5 of Human Plasminogen Induces Apoptosis of Endothelial and Tumor Cells through Surface-Expressed Glucose-Regulated Protein 78
Authors: Davidson Don J. , Haskell Catherine , Majest Sandy , Kherzai Abdullah , Egan David A. , Walter Karl A. , Schneider Andrew , Gubbins Earl F. , Solomon Larry , Chen Zhebo , Lesniewski Rick , Henkin Jack
Published: September 2005

Kringle 5 (K5) of human plasminogen has been shown to inhibit angiogenesis by inducing the apoptosis of proliferating endothelial cells. Peptide regions around the lysine-binding pocket of K5 largely mediate these effects, particularly the peptide PRKLYDY, which we show to compete with K5 for the binding to endothelial cells. The cell surface binding site for K5 that mediates these effects has not been defined previously. Here, we report that glucose-regulated protein 78, exposed on cell surfaces of proliferating endothelial cells as well as on stressed tumor cells, plays a key role in the antiangiogenic and antitumor activity of K5. We also report that recombinant K5-induced apoptosis of stressed HT1080 fibrosarcoma cells involves enhanced activity of caspase-7, consistent with the disruption of glucose-regulated protein 78-procaspase-7 complexes. These results establish recombinant K5 as an inhibitor of a stress response pathway, which leads to both endothelial and tumor cell apoptosis.
The Homeobox Gene Gax Inhibits Angiogenesis through Inhibition of Nuclear Factor-κB–Dependent Endothelial Cell Gene Expression
Authors: Patel Sejal , Leal Alejandro D. , Gorski David H.
Published: September 2005

The growth and metastasis of tumors are heavily dependent on angiogenesis, but much of the transcriptional regulation of vascular endothelial cell gene expression responsible for angiogenesis remains to be elucidated. The homeobox gene Gax is expressed in vascular endothelial cells and inhibits proliferation and tube formation in vitro. We hypothesized that Gax is a negative transcriptional regulator of the endothelial cell angiogenic phenotype and studied its regulation and activity in vascular endothelial cells. Several proangiogenic factors caused a rapid down-regulation of Gax mRNA in human vascular endothelial cells, as did conditioned media from breast cancer cell lines. In addition, Gax expression using a replication-deficient adenoviral vector inhibited human umbilical vein endothelial cell migration toward proangiogenic factors in vitro and inhibited angiogenesis in vivo in Matrigel plugs. To identify putative downstream targets of Gax, we examined changes in global gene expression in endothelial cells due to Gax activity. Gax expression resulted in changes in global gene expression consistent with a quiescent, nonangiogenic phenotype, with increased expression of cyclin kinase inhibitors and decreased expression of genes implicated in endothelial cell activation and angiogenesis. Further analysis revealed that Gax down-regulated numerous nuclear factor-κB (NF-κB) target genes and decreased the binding of NF-κB to its target sequence in electrophoretic mobility shift assays. To our knowledge, Gax is the first homeobox gene described that inhibits NF-κB activity in vascular endothelial cells. Because NF-κB has been implicated in endothelial cell activation and angiogenesis, the down-regulation of NF-κB–dependent genes by Gax suggests one potential mechanism by which Gax inhibits the angiogenic phenotype.
Both Antiangiogenesis- and Angiogenesis-Independent Effects Are Responsible for Hepatocellular Carcinoma Growth Arrest by Tyrosine Kinase Inhibitor PTK787/ZK222584
Authors: Liu Yuqing , Poon Ronnie T. , Li Qinyu , Kok Tsz Wai , Lau Cecilia , Fan Sheung Tat
Published: September 2005

Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis of hepatocellular carcinoma. Inhibition of VEGF receptors could theoretically reduce angiogenesis and tumor growth in hepatocellular carcinoma, but this remains to be proven with an experimental study. This study examined the angiogenesis-dependent and angiogenesis-independent activities of PTK787/ZK222584 (PTK787), a tyrosine kinase inhibitor of VEGF receptors, in nude mice bearing human hepatocellular carcinoma xenografts. The in vitro effects of PTK787 on proliferation, apoptosis, and cell cycle distribution in human hepatocellular carcinoma cell lines were also studied. Oral administration of PTK787 resulted in a significant reduction in tumor volume and microvessel formation of hepatocellular carcinoma xenografts in nude mice. PTK787 inhibited tumor cell proliferation in a dose-dependent manner and also induced tumor cells to undergo apoptosis both in vivo and in vitro. The proapoptotic response was associated with down-regulation of Bcl-2 and Bcl-xL expression and induction of cleavage of caspase-3. In addition, PTK787 induced growth arrest in hepatocellular carcinoma cells, which was associated with G1 arrest and partial G2-M block. This effect correlated with an increase in p21WAF1/ CIP1 (p21) and p27KIP1 (p27) protein expression. In conclusion, this study showed that PTK787 is a potent inhibitor of tumor growth in hepatocellular carcinoma by both antiangiogenic effect and direct effects on tumor cell proliferation and apoptosis. Our data suggest that blockage of VEGF receptors may provide an effective therapeutic approach for human hepatocellular carcinoma.
An Identity Crisis for fps/fes: Oncogene or Tumor Suppressor?
Authors: Sangrar Waheed , Zirgnibl Ralph A. , Gao Yan , Muller William J. , Jia Zongchao , Greer Peter A.
Published: September 2005

Fps/Fes proteins were among the first members of the protein tyrosine kinase family to be characterized as dominant-acting oncoproteins. Addition of retroviral GAG sequences or other experimentally induced mutations activated the latent transforming potential of Fps/Fes. However, activating mutations in fps/fes had not been found in human tumors until recently, when mutational analysis of a panel of colorectal cancers identified four somatic mutations in sequences encoding the Fps/Fes kinase domain. Here, we report biochemical and theoretical structural analysis demonstrating that three of these mutations result in inactivation, not activation, of Fps/Fes, whereas the fourth mutation compromised in vivo activity. These results did not concur with a classic dominant-acting oncogenic role for fps/fes involving activating somatic mutations but instead raised the possibility that inactivating fps/fes mutations might promote tumor progression in vivo. Consistent with this, we observed that tumor onset in a mouse model of breast epithelial cancer occurred earlier in mice targeted with either null or kinase-inactivating fps/fes mutations. Furthermore, a fps/fes transgene restored normal tumor onset kinetics in targeted fps/fes null mice. These data suggest a novel and unexpected tumor suppressor role for Fps/Fes in epithelial cells.
Nuclear Factor-κB Induced by Doxorubicin Is Deficient in Phosphorylation and Acetylation and Represses Nuclear Factor-κB–Dependent Transcription in Cancer Cells
Authors: Ho Wai Chi , Dickson Kathleen M. , Barker Philip A.
Published: September 2005

The primary goal of chemotherapy is to cause cancer cell death. However, a side effect of many commonly used chemotherapeutic drugs is the activation of nuclear factor-κB (NF-κB), a potent inducer of antiapoptotic genes, which may blunt the therapeutic efficacy of these compounds. We have assessed the effect of doxorubicin, an anthracycline in widespread clinical use, on NF-κB activation and expression of antiapoptotic genes in breast cancer cells. We show that doxorubicin treatment activates NF-κB signaling and produces NF-κB complexes that are competent for NF-κB binding in vitro. Surprisingly, these NF-κB complexes suppress, rather than activate, constitutive- and cytokine-induced NF-κB–dependent transcription. We show that doxorubicin treatment produces RelA, which is deficient in phosphorylation and acetylation and which blocks NF-κB signaling in a histone deacetylase–independent manner, and we show that NF-κB activated by doxorubicin does not remain stably bound to κB elements in vivo. Together these data show that NF-κB signaling induced by doxorubicin reduces expression of NF-κB–dependent genes in cancer cells.
Epigenetic Up-regulation of C-C Chemokine Receptor 7 and C-X-C Chemokine Receptor 4 Expression in Melanoma Cells
Authors: Mori Takuji , Kim Joseph , Yamano Tomoki , Takeuchi Hiroya , Huang Sharon , Umetani Naoyuki , Koyanagi Kazuo , Hoon Dave S.B.
Published: September 2005

Histone deacetylation and DNA methylation establish epigenetic modifications, which through chromatin remodeling may result in gene silencing. We hypothesized that chemokine receptors C-C chemokine receptor 7 (CCR7) and C-X-C chemokine receptor 4 (CXCR4) on melanoma cells undergo epigenetic regulation. We investigated whether a histone deacetylase inhibitor and a demethylating agent influence CCR7 and CXCR4 expression on melanoma cells. Initially, microarray analysis was done to screen changes in chemokine receptor expression on melanoma cells after treatment with trichostatin A (TSA) and 5-Aza-2-deoxycytidine (5-Aza). CCR7 and CXCR4 mRNA expression were uniformly altered and selected for further investigation. Quantitative real-time reverse transcription-PCR assay, immunohistochemistry, and Western blot analysis were used to assess changes in mRNA and protein expression induced by TSA and 5-Aza in melanoma lines. Cell migration assays were conducted to assess the effects of altered CCR7 and CXCR4 expression on cell function. Treatment with TSA or 5-Aza increased gene expression of both CCR7 and CXCR4 in melanoma lines. TSA was the strongest enhancer. With combined treatment, CCR7 and CXCR4 mRNA expression was also up-regulated. Immunohistochemistry after combined treatment showed enhanced staining of both CCR7 and CXCR4 compared with control cells. Melanoma cell migration in TSA- and 5-Aza-treated cells was 7- and 2-fold higher than control cells for CCR7 and CXCR4, respectively. In summary, a histone deacetylase inhibitor and a demethylating agent up-regulated CCR7 and CXCR4 expression on melanoma cells. This increase in chemokine receptor expression correlated with functional activity. Most importantly, we have identified an epigenetic mechanism that may endogenously regulate chemokine receptor expression on melanoma cells.
SWI/SNF Chromatin-Remodeling Factors Induce Changes in DNA Methylation to Promote Transcriptional Activation
Authors: Banine Fatima , Bartlett Christopher , Gunawardena Ranjaka , Muchardt Christian , Yaniv Moshe , Knudsen Erik S. , Weissman Bernard E. , Sherman Larry S.
Published: September 2005

Brahma (Brm) and brahma-related gene-1 (Brg1) are mammalian homologues of SWI/SNF chromatin-remodeling factor subunits that can regulate both transcriptional activation and repression. Both Brg1 and Brm are mutated or deleted in numerous cancer cell lines, leading to the altered expression of genes that influence cell proliferation and metastasis. Here, we find that the promoters of two such genes, CD44 and E-cadherin, are hypermethylated in cells that have lost Brg1 or Brm. In two carcinoma cell lines that lack functional Brg1 and Brm, CD44 and E-cadherin expression are induced by the demethylating agent 5-aza-2′-deoxycytidine. Transfection with either Brg1 or Brm also induces CD44 and E-cadherin transcription and protein expression in these cells, as well as loss of methylation at sequences in the promoters of both genes. Chromatin immunoprecipitation assays show that Brg1 and Brm associate with these regions of the CD44 and E-cadherin promoters, suggesting that SWI/SNF protein complexes may directly influence the loss of DNA methylation. In vivo, Brm-deficient mice also show methylation and silencing of the CD44 promoter. Collectively, these data implicate loss of SWI/SNF-mediated transcriptional activation as a novel mechanism to increase DNA methylation in cancer cells and provide insight into the mechanisms underlying aberrant gene induction and repression during tumor progression.
Transitional Cell Hyperplasia and Carcinomas in Urinary Bladders of Transgenic Mice with Keratin 5 Promoter-Driven Cyclooxygenase-2 Overexpression
Authors: Klein Russell D. , Van Pelt Carolyn S. , Sabichi Anita L. , dela Cerda Jorge , Fischer Susan M. , Fürstenberger Gerhard , Müller-Decker Karin
Published: September 2005

The inducible form of cyclooxygenase (COX), COX-2, is up-regulated in many epithelial cancers and its prostaglandin products increase proliferation, enhance angiogenesis, and inhibit apoptosis in several tissues. Pharmacologic inhibition and genetic deletion studies showed a marked reduction of tumor development in colon and skin. COX-2 has also been strongly implicated in urinary bladder cancer primarily by studies with nonselective COX- and COX-2-selective inhibitors. We now show that forced expression of COX-2, under the control of a keratin 5 promoter, is sufficient to cause transitional cell hyperplasia (TCH) in 17% and 75% of the heterozygous and homozygous transgenic lines, respectively, in an age-dependent manner. TCH was strongly associated with inflammation, primarily nodules of B lymphocytes; some T cells and macrophage infiltration were also observed. Additionally, transitional cell carcinoma was observed in ∼10% of the K5.COX-2 transgenic mice; no TCH or transitional cell carcinoma was observed in wild-type bladders. Immunohistochemistry for vascular proliferation and vascular endothelial growth factor showed significant increases above that in wild-type urinary bladders. Our results suggest that overexpression of COX-2 is sufficient to cause hyperplasia and carcinomas in the urinary bladder. Therefore, inhibition of COX-2 should continue to be pursued as a potential chemopreventive and therapeutic strategy.
Effects of DNA Methylation on Galectin-3 Expression in Pituitary Tumors
Authors: Ruebel Katharina H. , Jin Long , Qian Xiang , Scheithauer Bernd W. , Kovacs Kalman , Nakamura Nobuki , Zhang Heyu , Raz Avraham , Lloyd Ricardo V.
Published: September 2005

Galectin-3 (Gal-3), a β-galactoside-binding protein is expressed in a specific cell–type manner in pituitary tumors. Here we questioned the mechanism of Gal-3 expression in pituitary tumors, by using methylation-specific PCR and DNA sequence analyses to analyze the methylation status of the promoter region of the LGALS3 gene. DNA analysis of a human pituitary tumor, breast carcinoma cell lines, and thyroid carcinoma cell lines showed that in cells expressing Gal-3 protein, the LGALS3 gene was unmethylated, whereas in Gal-3 null cells, the promoter of the LGALS3 gene was methylated. Treatment of cells with 30 μmol/L 5-aza-2′-deoxycytidine induced Gal-3 mRNA and protein expression. Among pituitary tumors, 30% (7/23), mainly in follicle-stimulating hormone/luteinizing hormone–producing (38%) and null cell (57%) adenomas, the promoter of the LGALS3 was found to be methylated and silenced, although prolactin- and adrenocorticotropic hormone–producing tumors, which were unmethylated, expressed the Gal-3 protein. These results show for the first time that Gal-3 expression is regulated in part by promoter methylation in pituitary as well as in other tumors. Because it is functionally involved in cancer progression and metastasis, Gal-3 may serve as a possible therapeutic target in the treatment of pituitary tumors.
Molecular Target-Based Treatment of Human Cancer: Summary of the 10th International Conference on Differentiation Therapy
Authors: Zelent Arthur , Petrie Kevin , Chen Zhu , Lotan Reuben , Lübbert Michael , Tallman Martin S. , Ohno Ryuzo , Degos Laurent , Waxman Samuel
Published: September 2005

The 10th International Conference on Differentiation Therapy was held between April 29 and May 3, 2004, in Shanghai, China. In the tradition of previous conferences from this series, which have been held biannually since the first meeting organized 20 years ago by Samuel Waxman and Giovanni Rossi in Sardinia, the organizers of the 10th International Conference on Differentiation Therapy aimed to gather basic and clinical cancer investigators in a setting of plenary sessions, workshops, and poster presentations to maximize the effective exchange of information and foster the establishment of collaborative interactions. Approximately 300 scientists attended the meeting with a mission to discuss targeted approaches to cancer treatment, which stem from our understanding of basic biological processes and the mechanisms of their deregulation during tumorigenesis.
Iterative Microarray and RNA Interference–Based Interrogation of the Src-Induced Invasive Phenotype
Authors: Irby Rosalyn B. , Malek Renae L. , Bloom Greg , Tsai Jennifer , Letwin Noah , Frank Bryan C. , Verratti Kathleen , Yeatman Timothy J. , Lee Norman H.
Published: September 2005

Src kinase has long been recognized as a factor in the progression of colorectal cancer and seems to play a specific role in the development of the metastatic phenotype. In spite of numerous studies conducted to elucidate the exact role of Src in cancer progression, downstream targets of Src remain poorly understood. Gene expression profiling has permitted the identification of large sets of genes that may be functionally interrelated but it is often unclear as to which molecular pathways they belong. Here we have developed an iterative approach to experimentally reconstruct a network of gene activity regulated by Src and contributing to the invasive phenotype. Our strategy uses a combination of phenotypic anchoring of gene expression profiles and loss-of-function screening by way of RNA-mediated interference. Using a panel of human colon cancer cell lines exhibiting differential Src-specific activity and invasivity, we identify the first two levels of gene transcription responsible for the invasive phenotype, where first-tier genes are controlled by Src activity and the second-tier genes are under the influence of the first tier. Specifically, perturbation of first-tier gene activity by either pharmacologic inhibition of Src or RNA-mediated interference–directed knockdown leads to a loss of invasivity and decline of second-tier gene activity. The targeting of first-tier genes may be bypassed altogether because knockdown of second-tier genes led to a similar loss of invasive potential. In this manner, numerous members of a “transcriptional cascade” pathway for metastatic activity have been identified and functionally validated.
Drastic Down-regulation of Krüppel-Like Factor 4 Expression Is Critical in Human Gastric Cancer Development and Progression
Authors: Wei Daoyan , Gong Weida , Kanai Masashi , Schlunk Christian , Wang Liwei , Yao James C. , Wu Tsung-Teh , Huang Suyun , Xie Keping
Published: September 2005

Krüppel-like factor 4 (KLF4) is highly expressed in epithelial tissues such as the gut and skin. However, the role of KLF4 in human gastric cancer development and progression is unknown. Here we show that KLF4 protein expression was decreased or lost in primary tumors and, in particular, lymph node metastases when compared with that in normal gastric mucosa. Moreover, loss of KLF4 expression in the primary tumors was significantly associated with poor survival, and also an independent prognostic marker in a multivariate analysis. Consistently, most human gastric cancer cell lines exhibited loss of or a substantial decrease in KLF4 expression at both RNA and protein levels. Enforced restoration of KLF4 expression resulted in marked cell growth inhibition in vitro and significantly attenuated tumor growth and total abrogation of metastasis in an orthotopic animal model of gastric cancer. Mechanism studies indicated that promoter hypermethylation and hemizygous deletion contributed to the down-regulation of KLF4 expression and the induction of apoptosis contributed to the antitumor activity of KLF4. Collectively, our data provide first clinical and casual evidence and potential mechanism that the alteration of KLF4 expression plays a critical role in gastric cancer development and progression.
Inactivation of the Mitogen-Activated Protein Kinase Pathway as a Potential Target-Based Therapy in Ovarian Serous Tumors with KRAS or BRAF Mutations
Authors: Pohl Gudrun , Ho Chung-Liang , Kurman Robert J. , Bristow Robert , Wang Tian-Li , Shih Ie-Ming
Published: September 2005

Activation of mitogen-activated protein kinase (MAPK) occurs in response to various growth stimulating signals and as a result of activating mutations of the upstream regulators, KRAS and BRAF, which can be found in many types of human cancer. To investigate the roles of MAPK activation in tumors harboring KRAS or BRAF mutations, we inactivated MAPK in ovarian tumor cells using CI-1040, a compound that selectively inhibits MAPK kinase, an upstream regulator of MAPK and thus prevents MAPK activation. Profound growth inhibition and apoptosis were observed in CI-1040-treated tumor cells with mutations in either KRAS or BRAF in comparison with the ovarian cancer cells containing wild-type sequences. Long serial analysis of gene expression identified several differentially expressed genes in CI-1040-treated MPSC1 cells harboring an activating mutation in BRAF (V599L). The most striking changes were down-regulation of cyclin D1, COBRA1, and transglutaminase-2 and up-regulation of tumor necrosis factor–related apoptosis-induced ligand, thrombospondin-1, optineurin, and palladin. These patterns of gene expression were validated in other CI-1040-treated tumor cells based on quantitative PCR. Constitutive expression of cyclin D1 partially reversed the growth inhibitory effect of CI-1040 in MPSC1 cells. Our findings indicate that an activated MAPK pathway is critical in tumor growth and survival of ovarian tumors with KRAS or BRAF mutations and suggest that the CI-1040 induced phenotypes depend on the mutational status of KRAS and BRAF in ovarian tumors.