bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the Fanconi Anemia/BRCA Pathway and Recombination Repair in the Cellular Response to Solar Ultraviolet Light
Authors: Dunn Jessica , Potter Marisa , Rees Adam , Rünger Thomas M.
Published: December 2006

Recombination repair plays an important role in the processing of DNA double-strand breaks (DSB) and DNA cross-links, and has been suggested to be mediated by the activation of the Fanconi anemia (FA)/BRCA pathway. Unlike DNA damage generated by ionizing radiation or DNA cross-linking, UV light–induced DNA damage is not commonly thought to require recombination for processing, as UV light does not directly induce DSBs or DNA cross-links. To elucidate the role of recombination repair in the cellular response to UV, we studied the FA/BRCA pathway in primary skin cells exposed to solar–simulated light. UV-induced monoubiquitination of the FANCD2 protein and formation of FANCD2 nuclear foci confirmed the activation of the pathway by UV light. This was only observed when cells were irradiated during S phase and was not caused by directly UV-induced DSBs. UV-exposed cells did not exhibit FANCD2 nuclear foci once they entered mitosis or when growth-arrested. In addition, UV-induced nuclear foci of the recombination proteins, RAD51 and BRCA1, colocalized with FANCD2 foci. We suggest that in response to UV light, when nucleotide excision repair failed to repair, or when translesional DNA synthesis failed to bypass UV-induced DNA photoproducts, the FA/BRCA pathway mediates the recombination repair of replication forks stalled at DNA photoproducts as a third line of defense. (Cancer Res 2006; 66(23): 11140-7)
Topotecan Central Nervous System Penetration Is Altered by a Tyrosine Kinase Inhibitor
Authors: Zhuang Yanli , Fraga Charles H. , Hubbard K. Elaine , Hagedorn Nikolaus , Panetta John C. , Waters Christopher M. , Stewart Clinton F.
Published: December 2006

A potential strategy to increase the efficacy of topotecan to treat central nervous system (CNS) malignancies is modulation of the activity of ATP-binding cassette (ABC) transporters at the blood-brain and blood-cerebrospinal fluid barriers to enhance topotecan CNS penetration. This study focused on topotecan penetration into the brain extracellular fluid (ECF) and ventricular cerebrospinal fluid (CSF) in a mouse model and the effect of modulation of ABC transporters at the blood-brain and blood-cerebrospinal fluid barriers by a tyrosine kinase inhibitor (gefitinib). After 4 and 8 mg/kg topotecan i.v., the brain ECF to plasma AUC ratio of unbound topotecan lactone was 0.21 ± 0.04 and 0.61 ± 0.16, respectively; the ventricular CSF to plasma AUC ratio was 1.18 ± 0.10 and 1.30 ± 0.13, respectively. To study the effect of gefitinib on topotecan CNS penetration, 200 mg/kg gefitinib was administered orally 1 hour before 4 mg/kg topotecan i.v. The brain ECF to plasma AUC ratio of unbound topotecan lactone increased by 1.6-fold to 0.35 ± 0.04, which was significantly different from the ratio without gefitinib (P < 0.05). The ventricular CSF to plasma AUC ratio significantly decreased to 0.98 ± 0.05 (P < 0.05). Breast cancer resistance protein 1 (Bcrp1), an efficient topotecan transporter, was detected at the apical aspect of the choroid plexus in FVB mice. In conclusion, topotecan brain ECF penetration was lower compared with ventricular CSF penetration. Gefitinib increased topotecan brain ECF penetration but decreased the ventricular CSF penetration. These results are consistent with the possibility that expression of Bcrp1 and P-glycoprotein at the apical side of the choroid plexus facilitates an influx transport mechanism across the blood-cerebrospinal fluid barrier, resulting in high topotecan CSF penetration. (Cancer Res 2006; 66(23): 11305-13)
Hyporesponsiveness to Natural Killer T-Cell Ligand α-Galactosylceramide in Cancer-Bearing State Mediated by CD11b+ Gr-1+ Cells Producing Nitric Oxide
Authors: Yanagisawa Kazuhiko , Exley Mark A. , Jiang Xiaofeng , Ohkochi Nobuhiro , Taniguchi Masaru , Seino Ken-ichiro
Published: December 2006

CD1d-restricted natural killer T (NKT) cells are a potential therapeutic target for cancer, for which several clinical trials have already been reported. NKT cells are specifically activated by a synthetic glycolipid, α-galactosylceramide (α-GalCer). However, it is known that, in human cancer patients, NKT cells express a degree of hyporesponsiveness to α-GalCer. In this study, we have examined the mechanism by which hyporesponsiveness to α-GalCer can be induced. In cancer-bearing mice, α-GalCer-induced NKT cell expansion, cytokine production, cytotoxicity, and antimetastatic effect in vivo were all significantly impaired. In fact, α-GalCer could eliminate metastatic disease in naive animals but failed to protect cancer-bearing mice. CD11b+ Gr-1+ cells were particularly increased in cancer-bearing mice and were necessary and sufficient for the suppression of the α-GalCer response in a nitric oxide–mediated fashion. Administration of a retinoic acid to cancer-bearing mice reduced the population of CD11b+ Gr-1+ cells and effectively restored α-GalCer-induced protection. These results show a novel feature of NKT cell function in cancer. Furthermore, our data suggest a new strategy to enhance NKT cell-mediated anticancer immune responses by suppressing CD11b+ Gr-1+ cell functions. (Cancer Res 2006; 66(23): 11441-6)
Cbfβ Reduces Cbfβ-SMMHC–Associated Acute Myeloid Leukemia in Mice
Authors: Heilman Susan A. , Kuo Ya-Huei , Goudswaard Chantal S. , Valk Peter J. , Castilla Lucio H.
Published: December 2006

The gene encoding for core-binding factor β (CBFβ) is altered in acute myeloid leukemia samples with an inversion in chromosome 16, expressing the fusion protein CBFβ-SMMHC. Previous studies have shown that this oncoprotein interferes with hematopoietic differentiation and proliferation and participates in leukemia development. In this study, we provide evidence that Cbfβ modulates the oncogenic function of this fusion protein. We show that Cbfβ plays an important role in proliferation of hematopoietic progenitors expressing Cbfβ-SMMHC in vitro. In addition, Cbfβ-SMMHC–mediated leukemia development is accelerated in the absence of Cbfβ. These results indicate that the balance between Cbfβ and Cbfβ-SMMHC directly affects leukemia development, and suggest that CBF-specific therapeutic molecules should target CBFβ-SMMHC function while maintaining CBFβ activity. (Cancer Res 2006; 66(23): 11214-8)
Superior AntitumorIn vitroResponses of Allogeneic Matched Sibling Compared with Autologous Patient CD8+ T Cells
Authors: Kausche Sandra , Wehler Thomas , Schnürer Elke , Lennerz Volker , Brenner Walburgis , Melchior Sebastian , Gröne Mark , Nonn Marion , Strand Susanne , Meyer Ralf , Ranieri Elena , Huber Christoph , Falk Christine S. , Herr Wolfgang
Published: December 2006

Allogeneic cell therapy as a means to break immunotolerance to solid tumors is increasingly used for cancer treatment. To investigate cellular alloimmune responses in a human tumor model, primary cultures were established from renal cell carcinoma (RCC) tissues of 56 patients. In three patients with stable RCC line and human leukocyte antigen (HLA)-identical sibling donor available, allogeneic and autologous RCC reactivities were compared using mixed lymphocyte/tumor cell cultures (MLTC). Responding lymphocytes were exclusively CD8+ T cells, whereas CD4+ T cells or natural killer cells were never observed. Sibling MLTC populations showed higher proliferative and cytolytic antitumor responses compared with their autologous counterparts. The allo-MLTC responders originated from the CD8+ CD62L(high)+ peripheral blood subpopulation containing naive precursor and central memory T cells. Limiting dilution cloning failed to establish CTL clones from autologous MLTCs or tumor-infiltrating lymphocytes. In contrast, a broad panel of RCC-reactive CTL clones was expanded from each allogeneic MLTC. These sibling CTL clones either recognized exclusively the original RCC tumor line or cross-reacted with nonmalignant kidney cells of patient origin. A minority of CTL clones also recognized patient-derived hematopoietic cells or other allogeneic tumor targets. The MHC-restricting alleles for RCC-reactive sibling CTL clones included HLA-A2, HLA-A3, HLA-A11, HLA-A24, and HLA-B7. In one sibling donor-RCC pair, strongly proliferative CD3+CD16+CD57+ CTL clones with non-HLA-restricted antitumor reactivity were established. Our results show superior tumor-reactive CD8 responses of matched allogeneic compared with autologous T cells. These data encourage the generation of antitumor T-cell products from HLA-identical siblings and their potential use in adoptive immunotherapy of metastatic RCC patients. (Cancer Res 2006; 66(23): 11447-54)
HYAL1-v1, An Alternatively Spliced Variant of HYAL1 Hyaluronidase: A Negative Regulator of Bladder Cancer
Authors: Lokeshwar Vinata B. , Estrella Veronica , Lopez Luis , Kramer Mario , Gomez Pablo , Soloway Mark S. , Lokeshwar Bal L.
Published: December 2006

Tumor cells express HYAL1 hyaluronidase, which degrades hyaluronic acid. HYAL1 expression in bladder cancer cells promotes tumor growth, invasion, and angiogenesis. We previously described five alternatively spliced variants of HYAL1 that encode enzymatically inactive proteins. The HYAL1-v1 variant lacks a 30-amino acid sequence that is present in HYAL1. In this study, we examined whether HYAL1-v1 expression affects bladder cancer growth and invasion by stably transfecting HT1376 bladder cancer cells with a HYAL1-v1 cDNA construct. Although HYAL1-v1 transfectants expressed equivalent levels of enzymatically active HYAL1 protein when compared with vector transfectants, their conditioned medium had 4-fold less hyaluronidase activity due to a noncovalent complex formed between HYAL1 and HYAL1-v1 proteins. HYAL1-v1 transfectants grew 3- to 4-fold slower due to cell cycle arrest in the G2-M phase and increased apoptosis. In HYAL1-v1 transfectants, cyclin B1, cdc2/p34, and cdc25c levels were ≥2-fold lower than those in vector transfectants. The increased apoptosis in HYAL1-v1 transfectants was due to the extrinsic pathway involving Fas and Fas-associated death domain up-regulation, caspase-8 activation, and BID cleavage, leading to caspase-9 and caspase-3 activation and poly(ADP-ribose) polymerase cleavage. When implanted in athymic mice, HYAL1-v1-expressing tumors grew 3- to 4-fold slower and tumor weights at day 35 were 3- to 6-fold less than the vector tumors (P < 0.001). Whereas vector tumors were infiltrating and had high mitoses and microvessel density, HYAL1-v1 tumors were necrotic, infiltrated with neutrophils, and showed low mitoses and microvessel density. Therefore, HYAL-v1 expression may negatively regulate bladder tumor growth, infiltration, and angiogenesis. (Cancer Res 2006; 66(23): 11219-27)
Matrilysin 1 Influences Colon Carcinoma Cell Migration by Cleavage of the Laminin-5 β3 Chain
Authors: Remy Lionel , Trespeuch Cécile , Bachy Sophie , Scoazec Jean-Yves , Rousselle Patricia
Published: December 2006

Matrilysin 1 [matrix metalloproteinase 7 (MMP7)] is one of the most important metalloproteinases expressed in human tissues. This enzyme is generally not expressed by normal differentiated epithelial colon cells, but has been shown to be up-regulated in human colon adenomas and adenocarcinomas. Little is known about the role of MMP7 in cell invasion and its involvement in proteolytic processes. By searching the ligands of MMP7 in the colonic carcinoma cells HT29, we identified laminin-5/laminin-332 (LN5) as a specific target for MMP7 enzymatic activity. LN5, composed of α3, β3, and γ2 chains, is an important component of epithelial basement membranes where it induces firm adhesion and hemidesmosome formation. In this study, we show that LN5 and MMP7 are coexpressed in HT29 cells as well as in HT29 xenograft tumors and human colorectal adenocarcinomas. We provide evidence that human LN5 is a ligand for MMP7 and that a specific cleavage occurs in its β3 chain, giving rise to a carboxyl-terminal β3 chain fragment of 90 kDa. We have identified the MMP7 cleavage site at position Ala515-Ile516 in the β3 chain. Videomicroscopic analysis of HT29 cells plated on LN5 substrates reveals that the MMP7-processed LN5 significantly enhances cell motility. Moreover, the delayed migration of HT29 cells obtained after specific inhibition of MMP7 reinforces the hypothesis supporting its involvement in cell migration. Altogether, our results show that MMP7 is likely to play a crucial role in the regulation of carcinoma cell migration by targeting specific proteolytic processing of the LN5 β3 chain. (Cancer Res 2006; 66(23): 11228-37)
Epigenetic Silencing of Novel Tumor Suppressors in Malignant Melanoma
Authors: Muthusamy Viswanathan , Duraisamy Sekhar , Bradbury C. Matthew , Hobbs Cara , Curley David P. , Nelson Betsy , Bosenberg Marcus
Published: December 2006

Malignant melanoma is a common and frequently lethal disease. Current therapeutic interventions have little effect on survival, emphasizing the need for a better understanding of the genetic, epigenetic, and phenotypic changes in melanoma formation and progression. We identified 17 genes that were not previously known to be silenced by methylation in melanoma using a microarray-based screen following treatment of melanoma cell lines with the DNA methylation inhibitor 5-Aza-2′-deoxycytidine. Eight of these genes have not been previously shown to undergo DNA methylation in any form of cancer. Three of the genes, QPCT, CYP1B1, and LXN, are densely methylated in >95% of uncultured melanoma tumor samples. Reexpression of either of two of the silenced genes, HOXB13 and SYK, resulted in reduced colony formation in vitro and diminished tumor formation in vivo, indicating that these genes function as tumor suppressors in melanoma. (Cancer Res 2006; 66(23): 11187-93)
17β-Hydroxysteroid Dehydrogenase 14 Affects Estradiol Levels in Breast Cancer Cells and Is a Prognostic Marker in Estrogen Receptor–Positive Breast Cancer
Authors: Jansson Agneta K. , Gunnarsson Cecilia , Cohen Maja , Sivik Tove , Stål Olle
Published: December 2006

Estrogens have an important role in the progression of breast cancer. The 17β-hydroxysteroid dehydrogenase (17HSD) family has been identified to be of significance in hormone-dependent tissues. 17HSD1 and 17HSD2 are the main 17HSD enzymes involved in breast cancer investigated this far, but it is possible that other hormone-regulating enzymes have a similar role. 17HSD5 and 17HSD12 are associated with sex steroid metabolism, and 17HSD14 is a newly discovered enzyme that may be involved in the estrogen balance. The mRNA expression of 17HSD5, 17HSD12, and 17HSD14 were analyzed in 131 breast cancer specimens by semiquantitative real-time PCR. The results were compared with recurrence-free survival and breast cancer-specific survival of the patients. The breast cancer cell lines MCF7, SKBR3, and ZR75-1 were transiently transfected with 17HSD14 to investigate any possible effect on estradiol levels. We found that high 17HSD5 was related to significantly higher risk of late relapse in estrogen receptor (ER)–positive patients remaining recurrence-free later than 5 years after diagnosis (P = 0.02). No relation to 17HSD12 expression was found, indicating that 17HSD12 is of minor importance in breast cancer. Patients with ER-positive tumors with high expression levels of 17HSD14 showed a significantly better prognosis about recurrence-free survival (P = 0.008) as well as breast cancer-specific survival (P = 0.01), confirmed by multivariate analysis (P = 0.04). Transfection of 17HSD14 in the human breast cancer cells MCF7 and SKBR3 significantly decreased the levels of estradiol, presenting an effect of high expression levels of the enzyme. (Cancer Res 2006; 66(23): 11471-7)
Inhibition of Adenoma Progression to Adenocarcinoma in a 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone–Induced Lung Tumorigenesis Model in A/J Mice by Tea Polyphenols and Caffeine
Authors: Lu Gang , Liao Jie , Yang Guangyu , Reuhl Kenneth R. , Hao Xingpei , Yang Chung S.
Published: December 2006

The present study investigated the inhibitory effects of Polyphenon E [a standardized green tea polyphenol preparation containing 65% (−)-epigallocatechin-3-gallate] and caffeine on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)–induced lung tumor progression from adenoma to adenocarcinoma. Female A/J mice were treated with a single dose of NNK (103 mg/kg body weight, i.p.) and kept for 20 weeks for the mice to develop lung adenomas. The mice were then given a solution of 0.5% Polyphenon E or 0.044% caffeine as the sole source of drinking fluid until week 52. Both treatments significantly decreased the number of visible lung tumors. Histopathologic analysis indicated that Polyphenon E administration significantly reduced the incidence (by 52%) and multiplicity (by 63%) of lung adenocarcinoma. Caffeine also showed marginal inhibitory effects in incidence and multiplicity of adenocarcinoma (by 48% and 49%, respectively). Markers of cell proliferation, apoptosis, and related cell signaling were studied by immunohistochemistry, and the labeling index and staining intensity were quantified by the Image-Pro system. Polyphenon E and caffeine treatment inhibited cell proliferation (by 57% and 50%, respectively) in adenocarcinomas, enhanced apoptosis in adenocarcinomas (by 2.6- and 4-fold, respectively) and adenomas (both by 2.5-fold), and lowered levels of c-Jun and extracellular signal-regulated kinase (Erk) 1/2 phosphorylation. In the normal lung tissues, neither agent had a significant effect on cell proliferation or apoptosis. The results show that tea polyphenols (and perhaps caffeine) inhibit the progression of NNK-induced lung adenoma to adenocarcinoma. This effect is closely associated with decreased cell proliferation, enhanced apoptosis, and lowered levels of c-Jun and Erk1/2 phosphorylation. (Cancer Res 2006; 66(23): 11494-501)
A Small Interfering CD147-Targeting RNA Inhibited the Proliferation, Invasiveness, and Metastatic Activity of Malignant Melanoma
Authors: Chen Xiang , Lin Jing , Kanekura Takuro , Su Juan , Lin Wei , Xie Hongfu , Wu Yixi , Li Juan , Chen Mingliang , Chang Jing
Published: December 2006

CD147 plays a critical role in the invasive and metastatic activity of malignant melanoma cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases and vascular endothelial growth factor. We developed a system that blocks CD147 in the human malignant melanoma cell line, A375, using RNA interference. By transfecting melanoma cells with the small interfering RNA (siRNA) that targets human CD147, we were able to establish two stable clones in which CD147 expression was significantly down-regulated. This resulted in the decreased proliferation and invasion of A375 cells in vitro. CD147 siRNA also down-regulated the expression of vascular endothelial growth factor in these cells and reduced the migration of vascular endothelial cells. The reduction in the CD147 level suppressed the size of s.c. tumors and the microvessel density in an A375 s.c. nude mouse xenograft model. In addition, the in vivo metastatic potential of A375 cells transfected with CD147 siRNA was suppressed in a nude mouse model of pulmonary metastasis. (Cancer Res 2006; 66(23): 11323-30)
The PTEN/Akt Pathway Dictates the Direct αVβ3-Dependent Growth-Inhibitory Action of an Active Fragment of Tumstatin in Glioma Cells In vitro and In vivo
Authors: Kawaguchi Tomohiro , Yamashita Yoji , Kanamori Masayuki , Endersby Raelene , Bankiewicz Krystof S. , Baker Suzanne J. , Bergers Gabriele , Pieper Russell O.
Published: December 2006

The collagen type IV cleavage fragment tumstatin and its active subfragments bind to integrin αVβ3 and inhibit activation of focal adhesion kinase, phophoinositol-3 kinase, Akt, and mammalian target of rapamycin (mTOR) in what is thought to be an endothelial cell–specific manner. The resultant endothelial cell apoptosis accounts for the ability of tumstatin to function as an endogenous inhibitor of angiogenesis and an indirect suppressor of tumor growth. We hypothesized that the inability of tumstatin to directly suppress tumor cell growth might be the result of the constitutive activation of the Akt/mTOR pathway commonly seen in tumors. Consistent with this idea, several integrin αVβ3–expressing glioma cell lines with PTEN mutations and high levels of phospho-Akt (pAkt) were unaffected by exposure to an active fragment of tumstatin (T3), whereas αVβ3-expressing glioma cell lines with a functional PTEN/low levels of pAkt exhibited T3-induced growth suppression that could be bypassed by small interfering RNA–mediated suppression of PTEN, introduction of a constitutively expressed Akt, or introduction of the Akt and mTOR target eukaryotic translation initiation factor 4E. The direct tumor-suppressive actions of T3 were further shown in an αVβ3-deficient in vivo mouse model in which T3, while unable to alter the tumstatin-insensitive vasculature contributed by the αVβ3-deficient host, nonetheless suppressed the growth and proliferative index of i.c. implanted αVβ3-expressing PTEN-proficient glioma cells. These results show that tumstatin, previously considered to be only an endogenous inhibitor of angiogenesis, also directly inhibits the growth of tumors in a manner dependent on Akt/mTOR activation. (Cancer Res 2006; 66(23): 11331-40)
p21 Blocks Irradiation-Induced Apoptosis Downstream of Mitochondria by Inhibition of Cyclin-Dependent Kinase–Mediated Caspase-9 Activation
Authors: Sohn Dennis , Essmann Frank , Schulze-Osthoff Klaus , Jänicke Reiner U.
Published: December 2006

The role of the cyclin-dependent kinase (CDK) inhibitor p21 as a mediator of p53-induced growth arrest is well established. In addition, recent data provide strong evidence for new emerging functions of p21, including a role as a modulator of apoptosis. The mechanisms, however, by which p21 interferes with the death machinery, especially following ionizing radiation (IR), are largely unknown. Here, we report that IR induced caspase-9 and caspase-3 activation and subsequent apoptosis only in p21-deficient colon carcinoma cells, whereas similar treated wild-type cells were permanently arrested in the G2-M phase, correlating with the induction of cellular senescence. Interestingly, activation of the mitochondrial pathway, including caspase-2 processing, depolarization of the outer mitochondrial membrane, and cytochrome c release, was achieved by IR in both cell lines, indicating that p21 inhibits an event downstream of mitochondria but preceding caspase-9 activation. IR-induced p21 protein expression was restricted to the nucleus, and no evidence for a mitochondrial or cytoplasmic association was found. In addition, p21 did neither interact with caspase-3 or caspase-9, suggesting that these events are not required for the observed protection. Consistent with this assumption, we found that CDK inhibitors potently abrogated IR-induced caspase processing and activation without affecting mitochondrial events. In addition, in vitro caspase activation assays yielded higher caspase-3 activities in extracts of irradiated p21-deficient cells compared with extracts of similar treated wild-type cells. Thus, our results strongly indicate that p21 protects cells from IR-induced apoptosis by suppression of CDK activity that seems to be required for activation of the caspase cascade downstream of the mitochondria. (Cancer Res 2006; 66(23): 11254-62)
Hypoxia-Independent Overexpression of Hypoxia-Inducible Factor 1α as an Early Change in Mouse Hepatocarcinogenesis
Authors: Tanaka Hiroki , Yamamoto Masahiro , Hashimoto Norikazu , Miyakoshi Masaaki , Tamakawa Susumu , Yoshie Masumi , Tokusashi Yoshihiko , Yokoyama Kazunori , Yaginuma Yuji , Ogawa Katsuhiro
Published: December 2006

Hypoxia-inducible factor 1 (HIF-1) is involved in tumor progression/metastasis and activated in various cancers. Here we show that HIF-1α, which plays a major role in HIF-1 activation, is overexpressed in preneoplastic hepatocytic lesions from a very early stage during hepatocarcinogenesis in mice and man. Transcriptional targets of HIF-1, such as vascular endothelial growth factor, glut-1, c-met, and insulin-like growth factor II (IGF-II), were also overexpressed in mouse lesions. Oxygen tension within the lesions was not different from that of the normal hepatic tissues, indicating that HIF-1α expression was independent of hypoxia. On the other hand, Akt, the pathway of which can up-regulate HIF-1α expression, was activated in the mouse lesions, whereas HIF-1α was markedly down-regulated in the mouse hepatocellular carcinoma (HCC) cell lines after treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, indicating that HIF-1α expression is dependent on PI3K/Akt signaling. Conversely, HIF-1α knockdown by short interfering RNA in the HCC cell line resulted in decreased expression of activated Akt together with the HIF-1 target genes, indicating that Akt activation is reversely dependent on HIF-1 activation. Treating the HCC cells with IGF-II or epidermal growth factor (EGF) up-regulated both phospho-Akt and HIF-1α, whereas inhibition of IGF-II or EGF signaling down-regulated them both, suggesting that IGF-II and EGF can, at least in part, mediate the activation of Akt and HIF-1α. However, Akt was not activated by IGF-II or EGF in the HIF-1α knockdown cells, indicating that expression of the HIF-1 target genes is necessary for the Akt activation. These findings suggest that the reciprocal activation of PI3K/Akt signaling and HIF-1α may be important in the progression of hepatocarcinogenesis. (Cancer Res 2006; 66(23): 11263-70)
Prostate Cancer Cell ProliferationIn vitroIs Modulated by Antibodies against Glucose-Regulated Protein 78 Isolated from Patient Serum
Authors: Gonzalez-Gronow Mario , Cuchacovich Miguel , Llanos Carolina , Urzua Cristian , Gawdi Govind , Pizzo Salvatore V.
Published: December 2006

Circulating autoantibodies against the glucose-regulated protein of 78 kDa (GRP78) are present at high levels in prostate cancer patients and are a biomarker of aggressive tumor behavior. We purified the anti-GRP78 IgGs and examined their effect on 1-LN, PC-3, DU145, and LnCap human prostate cancer cells. We also evaluated its effects on the breast cancer MDA-MB231 and melanoma DM413 cell lines. The anti-GRP78 antibody binds only to cells expressing GRP78 on the surface, to a site also recognized by its physiologic agonist, activated α2-macroglobulin (α2M*). This antibody is completely specific for a peptide, including the primary amino acid sequence CNVKSDKSC, which contains a tertiary structural motif mimicking an epitope in GRP78. Tertiary structual analysis suggested the linear GRP78 primary amino acid sequence LIGRTWNDPSVQQDIKFL (Leu98-Leu115) as the putative binding site, containing the tertiary structual arrangement described above, which was confirmed experimentally. The anti-GRP78 antibodies from prostate cancer patients recognize almost exclusively this epitope. We produced animal antibodies against both these peptides, and they are able to mimic the effects of the human antibody. Our experiments also suggest this epitope as highly immunogenic, thereby explaining the specificity of the immune response against this epitope in GRP78, observed in humans. Using 1-LN cells as a model, we show that anti-GRP78 IgG purified from the sera of these patients mimics the proproliferative effects induced by α2M* via the common receptor, GRP78. Furthermore, increasing concentrations of human anti-GRP78 IgG show a dose-dependent protective effect on apoptosis induced by tumor necrosis factor α. (Cancer Res 2006; 66(23): 11424-31)
Targeted Degradation of the AML1/MDS1/EVI1 Oncoprotein by Arsenic Trioxide
Authors: Shackelford David , Kenific Candia , Blusztajn Agnieszka , Waxman Samuel , Ren Ruibao
Published: December 2006

Arsenic trioxide (ATO) has been found to be an effective treatment for acute promyelocytic leukemia patients and is being tested for treating other hematologic malignancies. We have previously shown that AML1/MDS1/EVI1 (AME), a fusion gene generated by a t(3;21)(q26;q22) translocation found in patients with chronic myelogenous leukemia during blast phase, myelodysplastic syndrome, or acute myelogenous leukemia (AML), impairs hematopoiesis and eventually induces an AML in mice. Both fusion partners of AME, AML1 and MDS1/EVI1, encode transcription factors and are also targets of a variety of genetic abnormalities in human hematologic malignancies. In addition, aberrant expression of ectopic viral integration site 1 (EVI1) has also been found in solid tumors, such as ovarian and colon cancers. In this study, we examined whether ATO could target AME and related oncoproteins. We found that ATO used at therapeutic levels degrades AME. The ATO treatment induces differentiation and apoptosis in AME leukemic cells in vitro as well as reduces tumor load and increases the survival of mice transplanted with these cells. We further found that ATO targets AME via both myelodysplastic syndrome 1 (MDS1) and EVI1 moieties and degrades EVI1 via the ubiquitin-proteasome pathway and MDS1 in a proteasome-independent manner. Our results suggest that ATO could be used as a part of targeted therapy for AME-, AML1/MDS1-, MDS1/EVI1-, and EVI1-positive human cancers. (Cancer Res 2006; 66(23): 11360-9)
Blastemal Expression of Type I Insulin-Like Growth Factor Receptor in Wilms' Tumors Is Driven by Increased Copy Number and Correlates with Relapse
Authors: Natrajan Rachael , Reis-Filho Jorge S. , Little Suzanne E. , Messahel Boo , Brundler Marie-Anne , Dome Jeffrey S. , Grundy Paul E. , Vujanic Gordan M. , Pritchard-Jones Kathy , Jones Chris
Published: December 2006

Most Wilms' tumors are of low stage, favorable histology, and have a high likelihood of cure with current multimodal therapy. Despite this, there remains a group of patients whose tumors recur for whom intensive salvage regimens result in survival of only 50%. Fitting a Cox proportional hazards model to microarray-based comparative genomic hybridization (aCGH) data on 68 Wilms' tumor samples, we identified a significant correlation between increased copy number at chromosome 15q26.3 insulin-like growth factor I receptor (IGFIR) and tumor relapse (adjusted P = 0.014). Wilms' tumors (13%) exhibited a low-level gain corresponding to three to four copies of the gene by aCGH analysis, 9 of 10 of which exhibited high IGFIR mRNA levels. Although IGFIR protein expression was restricted to the epithelial cells of fetal kidney and Wilms' tumors in most cases, 12% of tumors were also found to express IGFIR in the blastemal compartment. Blastemal IGFIR protein expression was associated with an increased copy number and a shorter relapse-free survival time (P = 0.027, log-rank test). In addition to the membrane localization, IGFIR was localized to the perinuclear region of the blastemal cells in 6% of Wilms' tumors. These data provide evidence that an increase in IGFIR gene copy number results in aberrant expression in the blastemal compartment of some Wilms' tumors and is associated with an adverse outcome in these patients. These findings suggest the possibility of use of targeted agents in the therapy of these children. (Cancer Res 2006; 66(23): 11148-55)
Adiponectin Modulates the Glycogen Synthase Kinase-3β/β-Catenin Signaling Pathway and Attenuates Mammary Tumorigenesis of MDA-MB-231 Cells in Nude Mice
Authors: Wang Yu , Lam Janice B. , Lam Karen S.L. , Liu Jing , Lam Michael C. , Hoo Ruby L.C. , Wu Donghai , Cooper Garth J.S. , Xu Aimin
Published: December 2006

Adiponectin is an adipokine that has pleiotropic beneficial roles in systemic insulin resistance and inflammation. Several recent clinical studies suggest that low serum levels of adiponectin are associated with increased risks of breast cancer. Here, we investigated the direct effects of adiponectin on breast cancer development in vitro and in vivo. Our results showed that adiponectin significantly attenuated the proliferations of two typical human breast cancer cells, MDA-MB-231 and T47D, in a cell type–specific manner. Further analysis revealed that adiponectin could induce apoptosis and arrest the cell cycle progression at G0-G1 phase in MDA-MB-231 cells. Prolonged treatment with adiponectin in this cell line blocked serum-induced phosphorylation of Akt and glycogen synthase kinase-3β (GSK-3β), suppressed intracellular accumulation of β-catenin and its nuclear activities, and consequently reduced expression of cyclin D1. Adiponectin-mediated suppression of cyclin D1 expression and attenuation of cell proliferation was abrogated by the GSK-3β inhibitor lithium chloride. These results suggest that the inhibitory role of adiponectin on MDA-MB-231 cell growth might be attributed to its suppressive effects on the GSK-3β/β-catenin signaling pathway. Furthermore, our in vivo study showed that both supplementation of recombinant adiponectin and adenovirus-mediated overexpression of this adipokine substantially reduced the mammary tumorigenesis of MDA-MB-231 cells in female nude mice. Taken together, these data support the role of adiponectin as a negative regulator of breast cancer development and also suggest that adiponectin might represent a novel therapeutic target for this disease. (Cancer Res 2006; 66(23): 11462-70)
Effects of Novel Retinoic Acid Metabolism Blocking Agent (VN/14-1) on Letrozole-Insensitive Breast Cancer Cells
Authors: Belosay Aashvini , Brodie Angela M.H. , Njar Vincent C.O.
Published: December 2006

Aromatase inhibitors are proving to be more effective than tamoxifen for postmenopausal estrogen receptor (ER)-positive breast cancer. However, the inevitable development of resistance to treatment is a concern. We investigated the effects of novel retinoic acid metabolism blocking agent, VN/14-1, in overcoming letrozole resistance in long-term letrozole cultured (LTLC) cells. Compared with MCF-7 cells stably transfected with aromatase (MCF-7Ca), LTLC cells were no longer sensitive to growth inhibition by aromatase inhibitors. The HER-2/phosphorylated mitogen-activated protein kinase (pMAPK) growth factor signaling pathways were activated, and ERα and coactivator amplified in breast cancer 1 (AIB1) were up-regulated ∼3-fold in LTLC cells. VN/14-1 inhibited aromatase activity and growth values of in MCF-7Ca cells with IC50 of 8.5 and 10.5 nmol/L, respectively. In human placental microsomes, aromatase activity was inhibited with IC50 of 8.0 pmol/L. The IC50 in LTLC cells was 0.83 nmol/L, similar to letrozole (IC50, 0.3 nmol/L) in MCF-7Ca cells. LTLC cells were 10-fold more sensitive to growth inhibition by VN/14-1 than MCF-7Ca cells. VN/14-1 treatment effectively down-regulated ERα, AIB1, pMAPK, HER-2, cyclin D1, cyclin-dependent kinase 4 (CDK4), and Bcl2 and up-regulated cytokeratins 8/18, Bad, and Bax. Tumor growth of LTLC cells in ovariectomized nude mice was independent of estrogens but was inhibited by VN/14-1 (20 mg/kg/d; P < 0.002). Decreases in ERα, cyclin D1, CDK4, and pMAPK and up-regulation of cytokeratins, Bad, and Bax with VN/14-1 in tumor samples may be responsible for the efficacy of this compound in inhibiting LTLC cell growth in vitro and in vivo. (Cancer Res 2006; 66(23): 11485-93)
Characterization of Murine JAK2V617F-Positive Myeloproliferative Disease
Authors: Bumm Thomas G.P. , Elsea Collin , Corbin Amie S. , Loriaux Marc , Sherbenou Daniel , Wood Lisa , Deininger Jutta , Silver Richard T. , Druker Brian J. , Deininger Michael W.N.
Published: December 2006

The JAK2V617F mutation is present in almost all patients with polycythemia vera (PV), large proportions of patients with essential thrombocythemia and idiopathic myelofibrosis, and less frequently in atypical myeloproliferative disorders (MPD). We show that transplantation of JAK2V617F-transduced bone marrow into BALB/c mice induces MPD reminiscent of human PV, characterized by erythrocytosis, granulocytosis, extramedullary hematopoiesis, and bone marrow fibrosis, but not thrombocytosis. Fluorescence-activated cell sorting of bone marrow and spleen showed proportional expansion of common myeloid progenitors, granulocyte-monocyte and megakaryocyte-erythrocyte progenitors. Megakaryocyte and late erythroid progenitors were dramatically increased, with only modest expansion of early erythroid progenitors. Erythropoietin (Epo) receptor expression was reduced on early, but normal on late erythroblasts. Serum levels of Epo and granulocyte colony-stimulating factor, but not granulocyte macrophage colony-stimulating factor, were reduced, whereas tumor necrosis factor-α was increased, possibly exerting a negative effect on JAK2V617F-negative hematopoiesis. These data suggest that erythrocytosis and granulocytosis in JAK2V617F mice are the net result of a complex interplay between cell intrinsic and extrinsic factors. There were no thromboembolic events and no animals succumbed to their disease, implicating additional factors in the manifestation of human disease. The disease was not transplantable and prolonged observation showed normalization of blood counts in most JAK2V617F mice, suggesting that the mutation may not confer self-renewal capacity. (Cancer Res 2006; 66(23): 11156-65)
Targeting the AIB1 Oncogene through Mammalian Target of Rapamycin Inhibition in the Mammary Gland
Authors: Torres-Arzayus Maria I. , Yuan Jing , DellaGatta Jamie L. , Lane Heidi , Kung Andrew L. , Brown Myles
Published: December 2006

Amplified in breast cancer 1 (AIB1), an estrogen receptor (ER) coactivator, is frequently amplified or overexpressed in human breast cancer. We previously developed a transgenic mouse model in which AIB1 can act as an oncogene, giving rise to a premalignant hyperplastic mammary phenotype as well as to a high incidence of mammary tumors that are primarily ER+. In this model, the AIB1 transgene is responsible for continued activation of the insulin-like growth factor-I receptor, suggesting a role for the activation of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway in the premalignant phenotype and tumor development. Here we show that treatment of AIB1 transgenic mice with the mTOR inhibitor RAD001 reverts the premalignant phenotype. Furthermore, treatment of cell lines derived from AIB1-dependent mammary tumors with RAD001 in culture leads to a G1 cell cycle arrest. Lastly, tumor growth after injection of ER+ AIB1 tumor cell lines into wild-type animals is inhibited by RAD001 treatment. In this ER+ model, inhibition of tumor growth by RAD001 was significantly better than inhibition by the antiestrogen 4-hydroxytamoxifen alone, whereas a combination of both RAD001 and 4-hydroxytamoxifen was most effective. Based on these results, we propose that the combination of mTOR inhibition and ER-targeted endocrine therapy may improve the outcome of the subset of ER+ breast cancers overexpressing AIB1. These studies provide preclinical support for the clinical development of RAD001 and suggest that AIB1 may be a predictive factor of RAD001 response. (Cancer Res 2006; 66(23): 11381-8)
Influence of Human CD8 on Antigen Recognition by T-Cell Receptor–Transduced Cells
Authors: Lyons Gretchen E. , Moore Tamson , Brasic Natasha , Li Mingli , Roszkowski Jeffrey J. , Nishimura Michael I.
Published: December 2006

The CD8 coreceptor on T cells has two functions. Namely, CD8 acts to stabilize the binding of the T-cell receptor (TCR) to the peptide-MHC complex while localizing p56lck (lck) to the TCR/CD3 complex to facilitate early signaling events. Although both functions may be critical for efficient activation of a CTL, little is known about how the structural versus signaling roles of CD8, together with the relative strength of the TCR, influences T-cell function. We have addressed these issues by introducing full-length and truncated versions of the CD8α and CD8β chains into CD8− Jurkat cell clones expressing cloned TCRs with known antigen specificity and relative affinities. Using a combination of antigen recognition and tetramer-binding assays, we find that the intracellular lck-binding domain of CD8 is critical for enhanced T-cell activation regardless of the relative strength of the TCR. In contrast, the extracellular domain of CD8 seems to be critical for TCRs with lower affinity but not those with higher affinity. Based on our results, we conclude that there are different requirements for CD8 to enhance T-cell function depending on the strength of its TCR. (Cancer Res 2006; 66(23): 11455-61)
Vascular Endothelial Growth Factor Tyrosine Kinase Inhibitor AZD2171 and Fractionated Radiotherapy in Mouse Models of Lung Cancer
Authors: Cao Carolyn , Albert Jeffrey M. , Geng Ling , Ivy Percy S. , Sandler Alan , Johnson David H. , Lu Bo
Published: December 2006

The vascular endothelial growth factor receptor (VEGFR) tyrosine kinases are being explored as targets for antiangiogenic cancer therapy. Radiotherapy also inhibits tumor growth and affects vasculature. We investigated the combination of the potent VEGFR tyrosine kinase inhibitor AZD2171 and ionizing radiation in cell culture and mouse models of lung cancer. We show that ionizing radiation induces expression of phosphorylated VEGFR-2 (Flk-1) in endothelial cells and that this phosphorylation is inhibited by AZD2171. Human umbilical vascular endothelial cells become more sensitive to radiation after treatment with AZD2171 as determined by clonogenic assay. Matrigel assay showed a decrease in in vitro endothelial tubule formation with AZD2171/radiation combination treatment. When similar combination was applied to the H460 lung cancer xenograft model in nude mice, loss of radiation-induced phosphorylated Flk-1 was observed in the combination treatment group, which also showed a large decrease in tumor vascular density by staining of the von Willebrand factor. H460 tumor growth delay was enhanced in the combination treatment group compared with the groups treated with AZD2171 or radiation alone. Additionally, after therapy, Ki67 index showed >4-fold reduction of tumor proliferation in the combination therapy group, which also showed increased intratumoral apoptotic index by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining. In conclusion, AZD2171 sensitizes lung tumor xenografts to radiation and inhibits angiogenesis both in vitro and in vivo. When used as a radiation enhancer, AZD2171 has the potential to improve tumor growth delay by inhibiting tumor proliferation and promoting apoptosis. Clinical trials are needed to determine the potential of this combination therapy in patients with locally advanced lung cancer. (Cancer Res 2006; 66(23): 11409-15)
Fhit Modulates the DNA Damage Checkpoint Response
Authors: Ishii Hideshi , Mimori Koshi , Inoue Hiroshi , Inageta Taeko , Ishikawa Kazuhiro , Semba Shuho , Druck Teresa , Trapasso Francesco , Tani Kenzaburo , Vecchione Andrea , Croce Carlo M. , Mori Masaki , Huebner Kay
Published: December 2006

In preneoplastic lesions, the DNA damage checkpoint is induced and loss of heterozygosity at the FRA3B/FHIT common chromosome fragile region precedes or is coincident with activation of the checkpoint response in these early stages. Introduction of exogenous Fhit into cells in vitro led to modulation of expression of checkpoint proteins Hus1 and Chk1 at mid-S checkpoint, a modulation that led to induction of apoptosis in esophageal cancer cells but not in noncancerous primary cultures. Mutation of the conserved Fhit tyrosine 114 resulted in failure of this function, confirming the importance of this residue. The results suggest that the DNA damage–susceptible FRA3B/FHIT chromosome fragile region, paradoxically, encodes a protein that is necessary for protecting cells from accumulation of DNA damage through its role in modulation of checkpoint proteins, and inactivation of Fhit contributes to accumulation of abnormal checkpoint phenotypes in cancer development. (Cancer Res 2006; 66(23): 11287-92)
Demonstration of a Genetic Therapeutic Index for Tumors Expressing Oncogenic BRAF by the Kinase Inhibitor SB-590885
Authors: King Alastair J. , Patrick Denis R. , Batorsky Roberta S. , Ho Maureen L. , Do Hieu T. , Zhang Shu Yun , Kumar Rakesh , Rusnak David W. , Takle Andrew K. , Wilson David M. , Hugger Erin , Wang Lifu , Karreth Florian , Lougheed Julie C. , Lee Jae , Chau David , Stout Thomas J. , May Earl W. , Rominger Cynthia M. , Schaber Michael D. , Luo Lusong , Lakdawala Ami S. , Adams Jerry L. , Contractor Rooja G. , Smalley Keiran S.M. , Herlyn Meenhard , Morrissey Michael M. , Tuveson David A. , Huang Pearl S.
Published: December 2006

Oncogenic BRAF alleles are both necessary and sufficient for cellular transformation, suggesting that chemical inhibition of the activated mutant protein kinase may reverse the tumor phenotype. Here, we report the characterization of SB-590885, a novel triarylimidazole that selectively inhibits Raf kinases with more potency towards B-Raf than c-Raf. Crystallographic analysis revealed that SB-590885 stabilizes the oncogenic B-Raf kinase domain in an active configuration, which is distinct from the previously reported mechanism of action of the multi-kinase inhibitor, BAY43-9006. Malignant cells expressing oncogenic B-Raf show selective inhibition of mitogen-activated protein kinase activation, proliferation, transformation, and tumorigenicity when exposed to SB-590885, whereas other cancer cell lines and normal cells display variable sensitivities or resistance to similar treatment. These studies support the validation of oncogenic B-Raf as a target for cancer therapy and provide the first evidence of a correlation between the expression of oncogenic BRAF alleles and a positive response to a selective B-Raf inhibitor. (Cancer Res 2006; 66(23): 11100-5)
Vaccination of Healthy Volunteers with Human Papillomavirus Type 16 L2E7E6 Fusion Protein Induces Serum Antibody that Neutralizes across Papillomavirus Species
Authors: Gambhira Ratish , Gravitt Patti E. , Bossis Ioannis , Stern Peter L. , Viscidi Raphael P. , Roden Richard B.S.
Published: December 2006

Oncogenic human papillomavirus (HPV) infection is a necessary cause of cervical cancer. Therefore, vaccination to prevent or eliminate HPV infection could reduce the incidence of cervical cancer. A fusion protein comprising HPV16 L2, E6, and E7 is a candidate combination preventive and therapeutic HPV vaccine. The L1- and L2-specific and neutralizing serum antibody titers and peripheral blood mononucleocyte antigen–specific proliferative responses generated by vaccination thrice at monthly intervals with HPV16 L2E7E6 were compared in two studies: a phase I randomized double-blind placebo controlled dose escalation trial in 40 healthy volunteers and a phase II trial of HPV16 L2E7E6 at the maximum dose in 29 women with high-grade anogenital intraepithelial neoplasia (AGIN). Vaccination of healthy volunteers induced L2-specific serum antibodies that were detected 1 month after the final vaccination (Pbinomial < 0.001). There was a significant trend to seroconversion for HPV16 and HPV18 neutralizing antibodies with increasing vaccine dose (P = 0.006 and P = 0.03, respectively). Seroconversion for HPV18 neutralizing antibodies showed a significant positive trend with increasing dose (P = 0.03) and was associated with seroconversion for HPV16 neutralizing antibodies (Pexact = 0.04). The antigen-specific proliferative response of vaccinated healthy volunteers also showed a significant trend with increasing vaccine dose (P = 0.04). However, AGIN patients responded less effectively to vaccination than healthy patients for induction of HPV16 L2–specific antibody (P < 0.001) and proliferative responses (P < 0.001). Vaccination of healthy volunteers thrice with 533-μg HPV16 L2E7E6 at monthly intervals induced L2-specific serum antibodies that neutralized across papillomavirus species. Responses in AGIN patients were infrequent. (Cancer Res 2006; 66(23): 11120-4)