bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Role for Extracellular Signal-Regulated Kinase 5 and Myocyte Enhancer Factor 2 in Medulloblastoma Cell Death
Authors: Sturla Lisa-Marie , Cowan Christopher W. , Guenther Lillian , Castellino Robert C. , Kim John Y.H. , Pomeroy Scott L.
Published: September 2005

Expression of the neurotrophin-3 receptor, tyrosine kinase C (TrkC), is associated with favorable prognosis in medulloblastoma patients. This may be due to increased tumor apoptosis induced by TrkC activation. Neurotrophin-3/TrkC–induced apoptosis is inhibited by the mitogen-activated protein (MAP) kinase (MAPK) pharmacologic antagonists SB203580 and PD98059. In addition to extracellular signal-regulated kinase (ERK)-1/2, PD98059 also inhibits the more recently identified neurotrophin-responsive MAPK, ERK5 (big MAPK 1). In the present study, we investigate the contribution of ERK5 and its target myocyte enhancer factor 2 (MEF2) to neurotrophin-3/TrkC–induced medulloblastoma cell death. Neurotrophin-3 not only enhanced ERK5 phosphorylation but also significantly enhanced the transcriptional activity of MEF2, a specific target of ERK5. Overexpression of both ERK5 and MEF2 induced a statistically significant increase in cell death of neurotrophin-3–responsive and nonresponsive medulloblastoma cell lines (Daoy-trkC and Daoy) and primary cultures of patched heterozygous mouse medulloblastomas. Only those cells expressing MAP/ERK kinase 5 (MEK5) plus ERK5 or MEF2 constructs underwent apoptosis, indicating that overexpression of either is sufficient to induce medulloblastoma cell death. Expression of a dominant-negative MEF2 or small interfering RNA for the ERK5 activator, MEK5, significantly inhibited neurotrophin-3–induced cell death. The dominant-negative MEF2 construct also blocked MEK5/ERK5-induced cell death, supporting a role for MEF2 downstream of ERK5. Coimmunoprecipitation studies revealed direct interaction of phosphorylated ERK5 with MEF2 in response to neurotrophin-3. Our investigation of the mechanism of neurotrophin-3/TrkC–induced apoptosis has identified a novel role for both MEK5/ERK5 and MEF2 in cell death, suggesting that these molecules can be exploited to induce apoptosis in both TrkC-expressing and nonexpressing medulloblastoma cells.
BLM Helicase Facilitates Mus81 Endonuclease Activity in Human Cells
Authors: Zhang Ran , Sengupta Sagar , Yang Qin , Linke Steven P. , Yanaihara Nozomu , Bradsher John , Blais Veronique , McGowan Clare H. , Harris Curtis C.
Published: September 2005

Bloom syndrome is a rare, autosomal recessive inherited disorder in humans. The product of the Bloom syndrome mutated gene, designated BLM, is a member of the RecQ helicase family. BLM has been proposed to function at the interface of replication and recombination, and to facilitate the repair of DNA damage. Here, we report in vivo physical interaction and colocalization of BLM and a DNA structure–specific endonuclease, Mus81, at sites of stalled replication forks outside the promyelocytic leukemia nuclear bodies during the S-phase arrest of the cell cycle. Amino acids 125 to 244 of Mus81 interact with the C-terminal region (amino acids 1,007-1,417) of BLM. Whereas Mus81 does not have any effect on the helicase activity of BLM, BLM can stimulate Mus81 endonuclease activity on the nicked Holliday junctions and 3′ flap. This stimulation is due to enhanced binding of Mus81 to the DNA substrates. These data suggest a new function of BLM in cooperating with Mus81 during processing and restoration of stalled replication forks.
NFBD1/Mdc1 Mediates ATR-Dependent DNA Damage Response
Authors: Peng Aimin , Chen Phang-Lang
Published: September 2005

Budding yeast Rad9 (scRad9) plays a central role in mediating Mec1-dependent phosphorylation by recruiting its downstream substrates. The human scRad9 orthologues 53BP1 and NFBD1 associate with ionizing radiation-induced foci (IRIF) at sites of DNA repair. RNAi-based gene silencing of 53BP1 or NFBD1 has shown impaired phosphorylation of SQ/TQ [ataxia-telangiectasia mutated/ATM and Rad3-related (ATM/ATR) substrates] at IRIF, intra-S, and G2-M checkpoints and has thereby revealed essential roles for 53BP1 and NFBD1 in the DNA damage signaling pathway. Whether 53BP1 and NFBD1 are required for activation of kinases and/or for recruitment of substrates at IRIF, however, is not clear. Here we show that both 53BP1 and NFBD1 are required for recruitment of ATR to DNA damage sites, as well as for ATR-dependent phosphorylation in response to DNA damage. NFBD1 is not required for ssDNA generation at DNA damage sites and is not recruited by replication protein A (RPA)–coated ssDNA. We therefore show that recruitment of NFBD1 and/or 53BP1, the factors downstream of H2AX, is independent of ssDNA generation and RPA coating, whereas both ssDNA and RPA coating play key roles in regulation of the ATR-dependent pathway. These novel findings help clarify where NFBD1 functions in DNA damage early responses.
Selective Apoptotic Killing of Malignant Hemopoietic Cells by Antibody-Targeted Delivery of an Amphipathic Peptide
Authors: Marks Alexandra J. , Cooper Margaret S. , Anderson Robert J. , Orchard Kim H. , Hale Geoffrey , North Janet M. , Ganeshaguru Kanagasabai , Steele Andrew J. , Mehta Atul B. , Lowdell Mark W. , Wickremasinghe R. Gitendra
Published: September 2005

The α-helical amphipathic peptide D-(KLAKLAK)2 is toxic to eukaryotic cells if internalized by a suitable targeting mechanism. We have targeted this peptide to malignant hemopoietic cells via conjugation to monoclonal antibodies, which recognize lineage-specific cell surface molecules. An anti-CD19/peptide conjugate efficiently killed 3/3 B lymphoid lines. However, an anti-CD33/peptide conjugate was cytotoxic to only one of three CD33-positive myeloid leukemia lines. The IC50 towards susceptible lines were in the low nanomolar range. Conjugates were highly selective and did not kill cells that did not express the appropriate cell surface cognate of the antibody moiety. Anti-CD19/peptide conjugates efficiently killed cells from patients with chronic lymphocytic leukemia but anti-CD33/peptide reagents were less effective against fresh acute myeloid leukemia cells. We therefore suggest that amphipathic peptides may be of value as targeted therapeutic agents for the treatment of a subset of hematologic malignancies.
One-Carbon Metabolism,MTHFRPolymorphisms, and Risk of Breast Cancer
Authors: Chen Jia , Gammon Marilie D. , Chan Wendy , Palomeque Caroline , Wetmur James G. , Kabat Geoffrey C. , Teitelbaum Susan L. , Britton Julie A. , Terry Mary Beth , Neugut Alfred I. , Santella Regina M.
Published: September 2005

Accumulating evidence from epidemiologic studies suggests that risk of breast cancer is reduced in relation to increased consumption of folate and related B vitamins. We investigated independent and joint effects of B vitamin intake as well as two polymorphisms of a key one-carbon metabolizing gene [i.e., methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C] on breast cancer risk. The study uses the resources of a population-based case-control study, which includes 1,481 cases and 1,518 controls. Significant inverse associations between B vitamin intake and breast cancer risk were observed among non-supplement users. The greatest reduction in breast cancer risk was observed among non-supplement users in the highest quintile of dietary folate intake [odds ratio (OR), 0.61; 95% confidence interval (95% CI), 0.41-0.93] as compared with non-supplement users in the lowest quintile of dietary folate intake (high-risk individuals). The MTHFR 677T variant allele was associated with increased risk of breast cancer (P, trend = 0.03) with a multivariate-adjusted OR of 1.37 (95% CI, 1.06-1.78) for the 677TT genotype. The 1298C variant allele was inversely associated with breast cancer risk (P, trend = 0.03), and was likely due to the linkage of this allele to the low-risk allele of 677C. The MTHFR-breast cancer associations were more prominent among women who did not use multivitamin supplements. Compared with 677CC individuals with high folate intake, elevation of breast cancer risk was most pronounced among 677TT women who consumed the lowest levels of dietary folate (OR, 1.83; 95% CI, 1.13-2.96) or total folate intake (OR, 1.71; 95% CI, 1.08-2.71). From a public heath perspective, it is important to identify risk factors, such as low B vitamin consumption, that may guide an effective prevention strategy against the disease.
BCR/ABL Promotes Dendritic Cell–Mediated Natural Killer Cell Activation
Authors: Terme Magali , Borg Christophe , Guilhot François , Masurier Carole , Flament Caroline , Wagner Erwin F. , Caillat-Zucman Sophie , Bernheim Alain , Turhan Ali G. , Caignard Anne , Zitvogel Laurence
Published: September 2005

BCR/ABL fusion gene, encoding a paradigmatic tyrosine kinase involved in chronic myelogenous leukemia (CML), can modulate the expression of genes involved in natural killer (NK) cell target recognition. Recent reports outline the role of allogeneic antileukemic NK effectors in the graft-versus-leukemia effect but the regulation of NK cell activation in the setting of graft-versus-leukemia effect remains unknown. Here we show that dendritic cells derived from monocytes of CML patients are selectively endowed with NK cell stimulatory capacity in vitro. We further show, using a gene transfer approach in mouse bone marrow progenitors, that ABL/ABL is necessary to promote dendritic cell–mediated NK cell activation. The dendritic cell/NK cell cross-talk in ABL/ABL-induced CML seems unique because JunB or IFN consensus sequence binding protein loss of functions, associated with other myeloproliferative disorders, do not promote dendritic cell–mediated NK cell activation. NK cell activation by leukemic dendritic cells involves NKG2D activating receptors and is blocked by imatinib mesylate. Indeed, ABL/ABL translocation enhances the expression levels of the NKG2D ligands on dendritic cells, which is counteracted by imatinib mesylate. Altogether, the clonal ABL/ABL dendritic cells display the unique and selective ability to activate NK cells and may participate in the NK cell control of CML. This study also highlights the deleterious role of imatinib mesylate at the dendritic cell level for NK cell activation.
Deletion, Methylation, and Expression of the NKX3.1 Suppressor Gene in Primary Human Prostate Cancer
Authors: Asatiani Ekatherine , Huang Wen-Xin , Wang Antai , Rodriguez Ortner Elizabeth , Cavalli Luciane R. , Haddad Bassem R. , Gelmann Edward P.
Published: September 2005

NKX3.1 is a prostate-specific homeoprotein and tumor suppressor that is affected by the loss of 8p21 in prostate cancer. In mice, Nkx3.1 haploinsufficiency results in prostatic dysplasia and complements cancer formation induced by loss of other suppressor genes. However, NKX3.1 expression can be immunohistochemically detected in most primary prostate cancers. We examined the relationship between suppressor gene haploinsufficiency, methylation, and quantitative NKX3.1 expression levels in primary prostate cancer. NKX3.1 gene copy number was assessed by microsatellite analysis, fluorescence in situ hybridization, and quantitative PCR. NKX3.1 gene methylation was determined in prostate cancer cell lines and we thereby identified potential CpG methylation sites for methylation-specific PCR analysis in tissues. We validated and then applied an internally controlled fluorescence immunomicroscopic assay for NKX3.1 protein expression in 48 primary prostate cancer specimens from radical prostatectomies. NKX3.1 loss of heterozygosity was found in 27 of 43 tissues tested. Classic CpG island methylation of the NKX3.1 gene was not found in either prostate cancer cell lines or tissues. However, in 33 of 40 samples tested, CpG sites at −921, −903, and −47 were methylated to a greater degree in malignant than in adjacent normal cells. In 43 of 48 samples, NKX3.1 protein expression was reduced from 0.34 to 0.90 compared with adjacent normal luminal epithelium (mean of all samples, 0.68; 95% confidence interval, 0.05). In 12 cases that also had high-grade prostatic intraepithelial neoplasia, NKX3.1 expression levels were similar in preinvasive and invasive cancer cells and significantly lower than adjacent normal cells. Even in the presence of allelic loss, NKX3.1 expression is reduced over a wide range in prostate cancer at the time of prostatectomy, suggesting that diverse factors influence expression. Samples with protein expression below the median level in cancer cells had both NKX3.1 deletion and selective CpG methylation.
Rapid Access of Antibodies to α5β1 Integrin Overexpressed on the Luminal Surface of Tumor Blood Vessels
Authors: Magnussen Anette , Kasman Ian M. , Norberg Scott , Baluk Peter , Murray Richard , McDonald Donald M.
Published: September 2005

Integrin α5β1 is overexpressed on endothelial cells of tumor vessels and is uniformly and rapidly accessible to antibodies in the bloodstream. Here, we determined whether antibodies rapidly gain access to integrin overexpressed on the abluminal (basolateral) surface of endothelial cells through vascular leakiness or whether the rapid accessibility results instead because the integrin is overexpressed on the luminal (apical) surface of endothelial cells due to loss of cell polarity. Using tumors in RIP-Tag2 transgenic mice as a model, we first compared the binding pattern of intravascular anti-α5β1 integrin antibody with the leakage pattern of nonspecific IgG. The distributions did not match: anti-α5β1 integrin antibody uniformly labeled the tumor vasculature, but IgG was located in patchy sites of leakage. We next injected an antibody to fibrinogen/fibrin, which resulted in patchy labeling of tumors that matched the leakage of IgG and the overall distribution of fibrin in tumors. Similarly, injected antibodies to the basement membrane protein fibronectin, a ligand of α5β1 integrin, or type IV collagen produced patchy sites of leakage instead of uniform labeling of vascular basement membrane. Differences in the kinetics of labeling, which for α5β1 integrin antibody was near maximal by 10 minutes but for the other antibodies gradually increased over 6 hours, indicated differences in accessibility of their respective targets. Isosurface rendering of confocal microscopic images was consistent with antibody binding to α5β1 integrin on the luminal surface of endothelial cells. Together, these findings indicate that the rapid accessibility of α5β1 integrin in RIP-Tag2 tumors results from overexpression of the integrin on the luminal surface of tumor vessels.
Association of Genetic Polymorphisms in the VEGF Gene with Breast Cancer Survival
Authors: Lu Hua , Shu Xiao-Ou , Cui Yong , Kataoka Nobuhiko , Wen Wanqing , Cai Qiuyin , Ruan Zhi-Xian , Gao Yu-Tang , Zheng Wei
Published: September 2005

The vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis and vascular permeability. VEGF overexpression has been associated with advanced stage and poor survival of several cancers. We evaluated the association of functional polymorphisms in the VEGF gene with breast cancer survival in a cohort of 1,193 breast cancer patients who were recruited as part of a population-based case-control study in Shanghai, China from 1996 to 1998 and followed for cancer recurrence and mortality between March 2000 and December 2002. Included in the study were three functional polymorphisms (C−460T, G+405C, and C+936T) in the VEGF gene. Carrying the −460C or +405G allele was associated with decreased overall survival. The age-adjusted hazard ratios (HR) were 1.5 [95% confidence interval (95% CI), 0.9-2.5] for −460CC genotype carriers and 1.6 (95% CI, 1.0-2.5) for +405GG genotype carriers compared with noncarriers. Further analyses showed that the −460T/+450C/+936C haplotype was related to increased survival (HR, 0.57; 95% CI, 0.4-0.9), whereas the −460C/+405G/+936T haplotype was associated with nonsignificantly decreased survival (HR, 2.1; 95% CI, −0.9 to 4.7). The C+936T polymorphism alone was not related to overall or disease-free survival. This study suggests that VEGF polymorphisms may be a significant genetic marker for breast cancer prognosis.
Decreased Androgen Receptor Levels and Receptor Function in Breast Cancer Contribute to the Failure of Response to Medroxyprogesterone Acetate
Authors: Buchanan Grant , Birrell Stephen N. , Peters Amelia A. , Bianco-Miotto Tina , Ramsay Katrina , Cops Elisa J. , Yang Miao , Harris Jonathan M. , Simila Henry A. , Moore Nicole L. , Bentel Jacqueline M. , Ricciardelli Carmella , Horsfall David J. , Butler Lisa M. , Tilley Wayne D.
Published: September 2005

Previously, we reported that androgen receptor (AR), but not estrogen receptor (ER) or progesterone receptor (PR), is predictive of response to the synthetic progestin, medroxyprogesterone acetate (MPA), in a cohort of 83 patients with metastatic breast cancer. To further investigate the role of AR in determining response to MPA in this cohort, we analyzed AR levels by immunohistochemistry with two discrete antisera directed at either the NH2 or the COOH termini of the receptor. Compared with tumors that responded to MPA (n = 31), there was a significant decrease in the intensity and extent of AR immunoreactivity with both AR antisera in tumors from nonresponders (n = 52). Whereas only a single AR immunostaining pattern was detected in responders to MPA, reflecting concordance of immunoreactivity with the two AR antisera, tumors from nonresponders exhibited four distinct AR immunostaining patterns: (a) concordance with the two antibodies (31%), (b) staining only with the COOH-terminal antibody (33%), (c) staining only with the NH2-terminal antibody (22%), or (d) no immunoreactivity with either NH2- or COOH-terminal antibody (14%). DNA sequencing and functional analysis identified inactivating missense gene mutations in the ligand-binding domain of the AR in tumors from two of nine nonresponders positive with the NH2-terminal AR antisera but negative for COOH-terminal immunoreactivity and lacking specific, high-affinity dihydrotestosterone binding in tumor cytosol fractions. Tumors with more AR than the median level (37 fmol/mg protein) had significantly lower levels of PR (30 fmol/mg protein) than tumors with low AR (PR; 127 fmol/mg protein) despite comparable levels of ER. Ligand-dependent activation of the AR in human T47D and MCF-7 breast cancer cells resulted in inhibition of estradiol-stimulated cell proliferation and a reduction in the capacity of the ER to induce expression of the PR. These effects could be reversed using a specific AR antisense oligonucleotide. Increasing the ratio of AR to ER resulted in a greater androgen-dependent inhibition of ER function. Collectively, these data suggest that reduced levels of AR or impaired AR function contribute to the failure of MPA therapy potentially due to abrogation of the inhibitory effect of AR on ER signaling.
Effects of the Mammalian Target of Rapamycin Inhibitor CCI-779 Used Alone or with Chemotherapy on Human Prostate Cancer Cells and Xenografts
Authors: Wu Licun , Birle Diana C. , Tannock Ian F.
Published: September 2005

Selective inhibition of repopulation of surviving tumor cells between courses of chemotherapy might improve the outcome of treatment. A potential target for inhibiting repopulation is the mammalian target of rapamycin pathway; PTEN-negative tumor cells are particularly sensitive to inhibition of this pathway. Here we study the rapamycin analogue CCI-779, alone or with chemotherapy, as an inhibitor of proliferation of the human prostate cancer cell lines PC-3 and DU145. The PTEN and phospho-Akt/PKB status and the effect of CCI-779 on phosphorylation of ribosomal protein S6 were evaluated by immunostaining and/or Western blotting. Expression of phospho-Akt/PKB in PTEN mutant PC-3 cells and xenografts was higher than in PTEN wild-type DU145 cells. Phosphorylation of S6 was inhibited by CCI-779 in both cell lines. Cultured cells were treated weekly with mitoxantrone or docetaxel for two cycles, and CCI-779 or vehicle was given between courses. Growth and clonogenic survival of both cell lines were inhibited in a dose-dependent manner by CCI-779, but there were minimal effects when CCI-779 was given between courses of chemotherapy. CCI-779 inhibited the growth of xenografts derived from both cell lines with greater effects against PC-3 than DU145 tumors. CCI-779 caused mild myelosuppression. The activity of mitoxantrone or docetaxel was limited, but CCI-779 given between courses of chemotherapy increased growth delay of PC-3 xenografts. Our results suggest that repopulation of PTEN-negative cancer cells between courses of chemotherapy might be inhibited by CCI-779.
Genetic Modeling of Human Rhabdomyosarcoma
Authors: Linardic Corinne M. , Downie Diane L. , Qualman Stephen , Bentley Rex C. , Counter Christopher M.
Published: September 2005

Rhabdomyosarcoma, a malignancy showing features of skeletal muscle differentiation, is the most common soft tissue sarcoma of childhood. The identification of distinct clinical presentation patterns, histologic tumor types, and risk groups suggests that rhabdomyosarcoma is a collection of highly related sarcomas rather than a single entity. In an effort to understand this seemingly heterogeneous malignancy, we constructed a genetically defined but malleable model of rhabdomyosarcoma by converting less differentiated human skeletal muscle cell precursors (SkMC) and committed human skeletal muscle myoblasts (HSMM) into their malignant counterparts by targeting pathways altered in rhabdomyosarcoma. Whereas the two cell types were both tumorigenic, SkMCs gave rise to highly heterogeneous tumors occasionally displaying features of rhabdomyosarcoma, whereas HSMMs formed rhabdomyosarcoma-like tumors with an embryonal morphology, capable of invasion and metastasis. Thus, despite introducing the same panel of genetic changes, altering the skeletal muscle cell of origin led to different tumor morphologies, suggesting that cell of origin may dictate rhabdomyosarcoma tumor histology. The ability to now genetically induce human rhabdomyosarcoma-like tumors provides a representative model to dissect the molecular mechanisms underlying this cancer.
Haploinsufficiency for Odc Modifies Mouse Skin Tumor Susceptibility
Authors: Guo Yongjun , Cleveland John L. , O'Brien Thomas G.
Published: September 2005

Numerous studies have linked overexpression of ornithine decarboxylase (Odc) gene with enhanced susceptibility to mouse skin tumorigenesis. However, there is little experimental evidence suggesting that modest reductions in Odc expression might reduce tumor susceptibility. To address this issue, here we report the use of the Odc+/− haploinsufficiency model, in which one copy of the murine Odc gene has been inactivated by a homologous recombination. Compared with Odc+/+ mice, Odc+/− mice exhibit reduced epidermal ODC enzyme activity and polyamine accumulation following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, following chronic TPA treatment, the characteristic hyperplastic response of the epidermis was diminished in Odc+/− mice. Finally, when subjected to a two-stage initiation-promotion protocol, substantially fewer skin papillomas developed in Odc+/− mice compared with wild-type littermates. These results support the concept that differences in tissue polyamine levels, resulting from either overexpression or reductions in ODC, are important modifiers of tumor susceptibility.
E-cadherin Suppression Accelerates Squamous Cell Carcinoma Progression in Three-Dimensional, Human Tissue Constructs
Authors: Margulis Alexander , Zhang Weitian , Alt-Holland Addy , Crawford Howard C. , Fusenig Norbert E. , Garlick Jonathan A.
Published: September 2005

We studied the link between loss of E-cadherin–mediated adhesion and acquisition of malignant properties in three-dimensional, human tissue constructs that mimicked the initial stages of squamous cell cancer progression. Suppression of E-cadherin expression in early-stage, skin-derived tumor cells (HaCaT-II-4) was induced by cytoplasmic sequestration of β-catenin upon stable expression of a dominant-negative E-cadherin fusion protein (H-2Kd-Ecad). In monolayer cultures, expression of H-2Kd-Ecad resulted in decreased levels of E-cadherin, redistribution of β-catenin to the cytoplasm, and complete loss of intercellular adhesion when compared with control II-4 cells. This was accompanied by a 7-fold decrease in β-catenin–mediated transcription and a 12-fold increase in cell migration. In three-dimensional constructs, E-cadherin–deficient tissues showed disruption of architecture, loss of adherens junctional proteins from cell contacts, and focal tumor cell invasion. Invasion was linked to activation of matrix metalloproteinase (MMP)–mediated degradation of basement membrane in H-2Kd-Ecad–expressing tissue constructs that was blocked by MMP inhibition (GM6001). Quantitative reverse transcription–PCR showed a 2.5-fold increase in MMP-2 and an 8-fold increase in MMP-9 in cells expressing the H-2Kd-Ecad fusion protein when compared with controls, and gel zymography showed increased MMP protein levels. Following surface transplantation of three-dimensional tissues, suppression of E-cadherin expression greatly accelerated tumorigenesis in vivo by inducing a switch to high-grade carcinomas that resulted in a 5-fold increase in tumor size after 4 weeks. Suppression of E-cadherin expression and loss of its function fundamentally modified squamous cell carcinoma progression by activating a highly invasive, aggressive tumor phenotype, whereas maintenance of E-cadherin prevented invasion in vitro and limited tumor progression in vivo.
Reduced FEZ1/LZTS1 Expression and Outcome Prediction in Lung Cancer
Authors: Nonaka Daisuke , Fabbri Alessandra , Roz Luca , Mariani Luigi , Vecchione Andrea , Moore G. William , Tavecchio Luca , Croce Carlo M. , Sozzi Gabriella
Published: September 2005

Chromosomal deletions are often observed in lung cancers suggesting that inactivation of tumor suppressor genes plays an important role in the development of this neoplasm. The region around chromosome 8p22 is a frequent and early target of these deletions and has therefore been investigated for the presence of candidate genes. The FEZ1/LZTS1 gene, located at 8p22, is inactivated in many cancers with 8p deletions, including prostate, esophageal, gastric, bladder, and breast cancer and the Fez1 protein has been shown to suppress growth of cancer cells and to regulate mitosis. To elucidate the role of FEZ1 in lung cancer, we have analyzed its expression by immunohistochemistry in 103 primary lung cancer specimens including 98 non–small cell lung cancers (57 adenocarcinomas, 32 squamous cell carcinomas, 7 large cell carcinomas, and 2 others) and five small cell carcinomas. Absence of Fez1 protein expression was observed in 27 cases (26%) and additional 43 cases (42%) showed strong reduction in immunoreactivity. There was a positive association between loss of FEZ1 expression and tumor grading (P = 0.0345) and a tendency toward a reduction in the mortality rate in subjects with strong FEZ1 expression. Overall, these data indicate an important role for FEZ1 in lung cancer and suggest the possibility that it may serve as a novel prognostic indicator.
p38 Regulates Cyclooxygenase-2 in Human Mammary Epithelial Cells and Is Activated in Premalignant Tissue
Authors: Gauthier Mona L. , Pickering Curtis R. , Miller Caroline J. , Fordyce Colleen A. , Chew Karen L. , Berman Hal K. , Tlsty Thea D.
Published: September 2005

The immediate-early gene, cyclooxygenase-2 (COX-2), is induced in a variety of inflammatory and neoplastic processes and is believed to play an important role in tumorigenesis. In this study, we identify an important upstream regulatory pathway of COX-2 expression in variant human mammary epithelial cells (vHMEC), which has been shown to exhibit phenotypes important for malignancy. We find that the stress-activated kinase, p38, is phosphorylated and activated in vHMEC compared with HMEC and is responsible for the expression of COX-2 in vHMEC as cells grow in culture. Furthermore in this capacity, p38 acts to stabilize the COX-2 transcript rather than activate COX-2 transcription. Inhibition of p38 kinase, using a chemical inhibitor, down-regulates COX-2 and decreases cell survival. Examination of archived tissue from women with ductal carcinoma in situ reveals epithelial cells that not only overexpress COX-2 but also have an abundance of activated phospho-p38 in the nucleus and cytoplasm, mirroring the expression observed in vitro. These epithelial cells are found within premalignant lesions as well as in fields of morphologically normal tissue that surround the lesions. In contrast, low phospho-p38 staining was observed in the majority of normal tissue obtained from reduction mammoplasty. These data help define the regulation of COX-2 expression in early carcinogenesis and provide alternative candidates for targeted prevention of COX-2-induced phenotypes and breast cancer.
Functional Proteomic Screen Identifies a Modulating Role for CD44 in Death Receptor–Mediated Apoptosis
Authors: Hauptschein Robert S. , Sloan Kevin E. , Torella Claudia , Moezzifard Roya , Giel-Moloney Maryann , Zehetmeier Carol , Unger Christine , Ilag Leodevico L. , Jay Daniel G.
Published: September 2005

Apoptotic evasion is a hallmark of cancer and its resistance to chemotherapeutic drugs. Identification of cellular proteins that mediate apoptotic programs is a critical step toward the development of therapeutics aimed at overcoming apoptosis resistance. We developed an innovative high-throughput screen to identify proteins that modulate Fas ligand–mediated apoptosis using fluorophore-assisted light inactivation (HTS-FALIpop). The FALI protein knockdown strategy was coupled to a caspase activity assay with the ability to detect both proapoptotic and antiapoptotic surface molecules expressed by HT-1080 human fibrosarcoma cells. FALI of the Fas receptor (Fas/CD95) using a fluorescein-conjugated anti-Fas antibody abrogated Fas ligand–mediated caspase activation. Ninety-six single-chain variable fragment antibodies (scFv), selected for binding to the surface of HT-1080 cells, were screened by HTS-FALIpop. Three of the scFvs caused decreases in caspase induction after FALI of their protein targets. One of the targets of these positive scFvs was identified as CD44 and was validated by performing FALI using a CD44-specific monoclonal antibody, which resulted in similar protection from Fas apoptosis. CD44-targeted FALI was antiapoptotic in multiple human cancer cell lines, including both Fas signaling type I and II cells, and was also protective against other ligands of the tumor necrosis factor death receptor family. FALI of CD44 inhibited formation and activation of the death-inducing signaling complex, suggesting that CD44 regulates Fas at the cell surface. This mechanism of death receptor regulation represents a novel means of apoptosis modulation that could be exploited by pharmacologic agents.
Centromere Protein H Is Up-regulated in Primary Human Colorectal Cancer and Its Overexpression Induces Aneuploidy
Authors: Tomonaga Takeshi , Matsushita Kazuyuki , Ishibashi Masumi , Nezu Masahiko , Shimada Hideaki , Ochiai Takenori , Yoda Kinya , Nomura Fumio
Published: September 2005

Chromosomal instability (CIN) has been recognized as a hallmark of human cancer and is caused by continuous chromosome missegregation during mitosis. Proper chromosome segregation requires a physical connection between spindle microtubules and centromeric DNA and this attachment occurs at proteinaceous structures called kinetochore. Several centromere proteins such as CENP-A and CENP-H are the fundamental components of the human active kinetochore, and inappropriate expression of the centromere proteins could be a major cause of CIN. We have previously shown that CENP-A was overexpressed in primary human colorectal cancer. In this study, we show that CENP-H was also up-regulated in all of 15 primary human colorectal cancer tissues as well as in CIN tumor cell lines. Surprisingly, transient transfection of CENP-H expression plasmid into the diploid cell line HCT116 remarkably induced aneupoidy. Moreover, CENP-H stable transfectant of mouse embryonic fibroblast/3T3 cell lines showed aberrant interphase micronuclei, characteristic of chromosome missegregation. In these CENP-H overexpressed cells, CENP-H completely disappeared from the centromere of mitotic chromosomes, which might be the cause of the chromosome segregation defect. These results suggest that the aberrant expression and localization of a kinetochore protein CENP-H plays an important role in the aneuploidy frequently observed in colorectal cancers.
Loss of RUNX3 Expression Significantly Affects the Clinical Outcome of Gastric Cancer Patients and Its Restoration Causes Drastic Suppression of Tumor Growth and Metastasis
Authors: Wei Daoyan , Gong Weida , Oh Sang C. , Li Qiang , Kim Won Dong , Wang Liwei , Le Xiangdong , Yao James , Wu Tsung T. , Huang Suyun , Xie Keping
Published: September 2005

Identification of precise prognostic marker and effective therapeutic target is pivotal in the treatment of gastric cancer. In the present study, we determined the level of RUNX3 expression in gastric cancer cells and gastric cancer specimens and the impact of its alteration on cancer biology and clinical outcome. There was a loss or substantial decrease of RUNX3 protein expression in 86 cases of gastric tumors as compared with that in normal gastric mucosa (P < 0.0001), which was significantly associated with inferior survival duration (P = 0.0005). In a Cox proportional hazards model, RUNX3 expression independently predicted better survival (P = 0.036). Moreover, various human gastric cancer cell lines also exhibited loss or drastic decrease of RUNX3 expression. Enforced restoration of RUNX3 expression led to down-regulation of cyclin D1 but to up-regulation of p27, caspase 3, 7, and 8 expression, cell cycle arrest, and apoptosis in vitro, and dramatic attenuation of tumor growth and abrogation of metastasis in animal models. Therefore, we offered both clinical and mechanistic evidence that RUNX3 was an independent prognostic factor and a potential therapeutic target for gastric cancer.
Overexpression of Cytochrome P450 1A1 and Its Novel Spliced Variant in Ovarian Cancer Cells: Alternative Subcellular Enzyme Compartmentation May Contribute to Carcinogenesis
Authors: Leung Yuet-Kin , Lau Kin-Mang , Mobley James , Jiang Zhong , Ho Shuk-Mei
Published: September 2005

Epithelial ovarian cancer derived from the human ovarian surface epithelium (HOSE) is the leading cause of death from gynecologic malignancies among American women. Metabolic activation of endogenous and exogenous chemicals by cytochrome P450 (CYP) class I enzymes has been implicated in its etiology. In this study, we showed overexpression of CYP1A1 mRNA, but not CYP1B1 transcripts, in ovarian cancer cell lines when compared with primary cultures or immortalized HOSE cell lines. Importantly, we identified a novel, enzymatically active, spliced variant of CYP1A1 (CYP1A1v) formed by excision of an 84-bp cryptic intron in exon 2. CYP1A1v is overexpressed in ovarian cancer cell lines and exhibits a unique subcellular distribution restricted to the nucleus and mitochondria, contrary to the endoplasmic reticulum localization of the wild-type enzyme. In concordance, total CYP1A1 activity, as measured by the ethoxyresorufin O-deethylase assay, was detected in mitochondrial, nuclear, and microsomal fractions of ovarian cancer cells but was notably absent in all subcellular fractions of HOSE cells. Immunocytochemistry studies in 30 clinical specimens revealed overexpression of CYP1A1 in various types of ovarian cancers compared with benign epithelia and frequent localization of the enzyme to cancer cell nuclei. Forced expression of CYP1A1wt or CYP1A1v in HOSE cells resulted in nuclear localization of the enzyme and acquisition of anchorage-independent growth, which was further exacerbated following exposure to benzo(a)pyrene or 17β-estradiol. Collectively, these data provided the first evidence that CYP1A1 overexpression and alternative splicing could contribute to ovarian cancer initiation and progression.
Up-Regulation of TWIST in Prostate Cancer and Its Implication as a Therapeutic Target
Authors: Kwok Wai Kei , Ling Ming-Tat , Lee Tak-Wing , Lau Tracy C.M. , Zhou Chun , Zhang Xiaomeng , Chua Chee Wai , Chan Kwok W. , Chan Franky L. , Glackin Carlotta , Wong Yong-Chuan , Wang Xianghong
Published: September 2005

Androgen-independent metastatic prostate cancer is the main obstacle in the treatment of this cancer. Unlike a majority of solid cancers, prostate cancer usually shows poor response to chemotherapeutic drugs. In this study, we have shown a potential novel target, TWIST, a highly conserved bHLH transcription factor, in the treatment of prostate cancer. Using malignant and nonmalignant prostate tissues, we found that TWIST expression was highly expressed in the majority (90%) of prostate cancer tissues but only in a small percentage (6.7%) of benign prostate hyperplasia. In addition, the TWIST expression levels were positively correlated with Gleason grading and metastasis, indicating its role in the development and progression of prostate cancer. Furthermore, down-regulation of TWIST through small interfering RNA in androgen-independent prostate cancer cell lines, DU145 and PC3, resulted in increased sensitivity to the anticancer drug taxol-induced cell death which was associated with decreased Bcl/Bax ratio, leading to activation of the apoptosis pathway. More importantly, inactivation of TWIST suppressed migration and invasion abilities of androgen-independent prostate cancer cells, which was correlated with induction of E-cadherin expression as well as morphologic and molecular changes associated with mesenchymal to epithelial transition. These results were further confirmed on the androgen-dependent LNCaP cells ectopically expressing the TWIST protein. Our results have identified TWIST as a critical regulator of prostate cancer cell growth and suggest a potential therapeutic approach to inhibit the growth and metastasis of androgen-independent prostate cancer through inactivation of the TWIST gene.
Hair Follicle–Derived Blood Vessels Vascularize Tumors in Skin and Are Inhibited by Doxorubicin
Authors: Amoh Yasuyuki , Li Lingna , Yang Meng , Jiang Ping , Moossa Abdool R. , Katsuoka Kensei , Hoffman Robert M.
Published: September 2005

We have recently shown that the neural-stem cell marker nestin is expressed in hair follicle stem cells and the blood vessel network interconnecting hair follicles in the skin of transgenic mice with nestin regulatory element–driven green fluorescent protein (ND-GFP). The hair follicles were shown to give rise to the nestin-expressing blood vessels in the skin. In the present study, we visualized tumor angiogenesis by dual-color fluorescence imaging in ND-GFP transgenic mice after transplantation of the murine melanoma cell line B16F10 expressing red fluorescent protein. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumor. Results of immunohistochemical staining showed that the blood vessel–specific antigen CD31 was expressed in ND-GFP–expressing nascent blood vessels. ND-GFP expression was diminished in the vessels with increased blood flow. Progressive angiogenesis during tumor growth was readily visualized during tumor growth by GFP expression. Doxorubicin inhibited the nascent tumor angiogenesis as well as tumor growth in the ND-GFP mice transplanted with B16F10-RFP. This model is useful for direct visualization of tumor angiogenesis and evaluation of angiogenic inhibitors.
The S100A7-c-Jun Activation Domain Binding Protein 1 Pathway Enhances Prosurvival Pathways in Breast Cancer
Authors: Emberley Ethan D. , Niu Yulian , Curtis Linda , Troup Sandra , Mandal Sanat K. , Myers Jeffery N. , Gibson Spencer B. , Murphy Leigh C. , Watson Peter H.
Published: September 2005

S100A7 is among the most highly expressed genes in preinvasive breast cancer, is a marker of poor survival when expressed in invasive disease, and promotes breast tumor progression in experimental models. To explore the mechanism of action, we examined the role of S100A7 in cell survival and found that overexpression of S100A7 in MDA-MB-231 cell lines promotes survival under conditions of anchorage-independent growth. This effect is paralleled by increased activity of nuclear factor-κB (3-fold) and phospho-Akt (4-fold), which are known to mediate prosurvival pathways. S100A7 and phospho-Akt are also correlated in breast tumors examined by immunohistochemistry (n = 142; P < 0.0001; r = 0.34). To explore the underlying mechanism, we examined the role of a putative c-Jun activation domain-binding protein 1 (Jab1)–binding domain within S100A7 using a panel of MDA-MB-231 breast cell lines stably transfected with either S100A7 or S100A7 mutated at the Jab1 domain. Structural analysis by three-dimensional protein modeling, immunoprecipitation, and yeast two-hybrid assay and functional analysis using transfected reporter gene and Western blot assays revealed that the in vitro effects of S100A7 on phospho-Akt and the nuclear factor-κB pathway are dependent on the Jab1-binding site and the interaction with Jab1. Enhanced epidermal growth factor receptor signaling was also found to correlate with the increased phospho-Akt. Furthermore, the Jab1-binding domain is also necessary for the enhanced tumorigenicity conferred by S100A7 expression in murine xenograft tumors in vivo. We conclude that the S100A7-Jab1 pathway acts to enhance survival under conditions of cellular stress, such as anoikis, which may promote progression of breast cancer.
Medullary Thyroid Carcinoma Arises in the Absence of Prolactin Signaling
Authors: Kedzia Cécile , Lacroix Ludovic , Ameur Nabahet , Ragot Thierry , Kelly Paul A. , Caillou Bernard , Binart Nadine
Published: September 2005

Prolactin, a pituitary hormone, exerts pleiotropic effects in various cells. These effects are mediated by a membrane receptor highly expressed in many tissues. To analyze prolactin effects on the thyroid gland, we first identified prolactin receptor (PRLR) mRNAs by in situ hybridization. To further evaluate the physiologic relevance of PRLR actions in the thyroid in vivo, we used PRLR knockout mice. Whereas the histologic structure of thyroid of PRLR-null mice was not disturbed, we show that T4 levels are lower in null animals (13.63 ± 2.98 versus 10.78 ± 2.25 pmol/L in null mice), confirming that prolactin participates in the control of thyroid metabolism. To further investigate thyroid effects in mice, we measured body temperature and thyroid-stimulating hormone in young and adult male and/or female PRLR-null mice and their normal siblings. Surprisingly, in null animals, we saw medullary thyroid carcinoma (MTC) arising from parafollicular C cells producing calcitonin. The incidence of these carcinomas attained 41% in PRLR-null mice, whereas this malignant tumor occurs sporadically or as a component of the familial cancer syndrome in humans. This finding suggests that PRLR-null mice could represent a valuable animal model for MTC, which could be compared with existing MTC models. These observations suggest a possible link between the appearance of this carcinoma and the absence of prolactin signaling.
Trastuzumab Therapy for Tamoxifen-Stimulated Endometrial Cancer
Authors: Osipo Clodia , Meeke Kathleen , Liu Hong , Cheng Dong , Lim Sherry , Weichel Alyssa , Jordan V. Craig
Published: September 2005

A novel in vivo model of tamoxifen-stimulated endometrial cancer was developed and the role of HER-2/neu investigated by using trastuzumab. Tamoxifen-stimulated tumors (ECC-1TAM) were growth stimulated by 17β-estradiol (E2), tamoxifen, or raloxifene. Trastuzumab inhibited growth of E2-stimulated ECC-1E2 tumors by 50% and tamoxifen-stimulated ECC-1TAM tumors by 100%. ECC-1 tumors expressed functional estrogen receptor α (ERα) as measured by induction of pS2 and c-myc mRNAs. E2 induced pS2 and c-myc mRNAs up to 40-fold in ECC-1E2 and ECC-1TAM. Tamoxifen induced pS2 and c-myc mRNAs up to 5-fold in ECC-1E2 tumors and up to 10-fold in ECC-TAM tumors. Trastuzumab blocked E2-induced pS2 mRNA (P < 0.01) in ECC-1E2 by 50% and tamoxifen-induced c-myc mRNA (P < 0.1) in ECC-1TAM tumors by 70%. Trastuzumab decreased phosphorylated and total HER-2/neu protein in ECC-1E2 and ECC-1TAM tumors. However, only phospho-ERK-1/2 and not phospho-Akt protein was decreased by trastuzumab in tamoxifen-treated ECC-1TAM tumors. The insulin-like growth factor (IGF-I) signaling pathway also activates extracellular signal-related kinase (ERK)-1/2 and could block the efficacy of trastuzumab in ECC-1E2 tumors. The results showed that IGF-I, IGF-IR mRNAs, and phospho-insulin receptor substrate-1 (IRS-1) protein were decreased in ECC-1TAM compared with ECC-1E2 tumors. The results show that trastuzumab is an effective therapy for both E2-stimulated and tamoxifen-stimulated endometrial cancer. The data suggest estrogenic activities of E2 and tamoxifen at ERα-regulated pS2 and c-myc genes are in part mediated by HER-2/neu. However, trastuzumab is a better growth inhibitor of ECC-1TAM tumors where there is diminished IGF-I signaling allowing for complete blockade of the downstream phospho-ERK-1/2 signal.
CY15, a Malignant Histiocytic Tumor That Is Phenotypically Similar to Immature Dendritic Cells
Authors: Kammertoens Thomas , Willebrand Ralf , Erdmann Bettina , Li Liangping , Li Yongping , Engels Boris , Uckert Wolfgang , Blankenstein Thomas
Published: September 2005

The origin and pathogenesis of histiocytic malignancies and the biology of the tumor cells are poorly understood. We have isolated a murine histiocytic tumor cell line (CY15) from a BALB/c IFNγ−/− mouse and characterized it in terms of phenotype and function. The morphology, as judged by electron microscopy, and the surface marker phenotype suggests that CY15 cells are similar to immature dendritic cells (CD11c low, MHC II low, CD11b+, B7.1+, B7.2+, and CD40+). The cells form tumors in BALB/c mice and metastasize to spleen, liver, lung, kidney, and to a lesser extend to lymph nodes and bone marrow, as judged by the growth of green fluorescent protein transfected tumor cells in mice. CY15 cells are capable of actively taking up antigen (FITC-ovalbumin) and can stimulate T lymphocytes in an allogenic mixed lymphocyte reaction but less effectively than their normal counterparts (immature dendritic cells). They respond to interleukin 4 (IL-4) with up-regulation of CD11c. If stimulated with IFNγ the cells up-regulate MHC II, CD40 B7.1, and B7.2. Lipopolysaccharide induces the cells to up-regulate B7.1 and B7.2 and to secrete tumor necrosis factor α and IL-12. Based on these data, CY15 is a dendritic cell–like tumor cell line and may serve as a transplantable tumor model for histiocytosis in humans.