bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin II induces monocyte chemoattractant protein-1 expression via a nuclear factor-κB-dependent pathway in rat preadipocytes
Authors: Tsuchiya Kyoichiro , Yoshimoto Takanobu , Hirono Yuki , Tateno Toru , Sugiyama Toru , Hirata Yukio
Published: May 2006

Both monocyte chemoattractant protein-1 (MCP-1), a member of chemokine family, and angiotensinogen, a precursor of angiotensin (ANG) II, are produced by adipose tissue and increased in obese state. MCP-1 has been shown to decrease insulin-stimulated glucose uptake and several adipogenic genes expression in adipocytes in vitro, suggesting its pathophysiological significance in obesity. However, the pathophysiological interaction between MCP-1 and ANG II in adipose tissue remains unknown. The present study was undertaken to investigate the potential mechanisms by which ANG II affects MCP-1 gene expression in rat primary cultured preadipocytes and adipose tissue in vivo. ANG II significantly increased steady-state MCP-1 mRNA levels in a time- and dose-dependent manner. The ANG II-induced MCP-1 mRNA and protein expression was completely abolished by ANG II type 1 (AT1)-receptor antagonist (valsartan). An antioxidant/NF-κB inhibitor (pyrrolidine dithiocarbamate) and an inhibitor of 1κB-α phosphorylation (Bay 11-7085) also blocked ANG II-induced MCP-1 mRNA expression. ANG II induced translocation of NF-κB p65 subunit from cytoplasm to nucleus by immunocytochemical study. Luciferase assay using reporter constructs containing MCP-1 promoter region revealed that two NF-κB binding sites in its enhancer region were essential for the ANG II-induced promoter activities. Furthermore, basal mRNA and protein of MCP-1 during preadipocyte differentiation were significantly greater in preadipocytes than in differentiated adipocytes, whose effect was more pronounced in the presence of ANG II. Exogenous administration of ANG II to rats led to increased MCP-1 expression in epididymal, subcutaneous, and mesenteric adipose tissue. In conclusion, our present study demonstrates that ANG II increases MCP-1 gene expression via ANG II type 1 receptor-mediated and NF-κB-dependent pathway in rat preadipocytes as well as adipose MCP-1 expression in vivo. Thus the augmented MCP-1 expression by ANG II in preadipocytes may provide a new link between obesity and cardiovascular disease.
Adenoviral expression of NHERF-1 in NHERF-1 null mouse renal proximal tubule cells restores Npt2a regulation by low phosphate media and parathyroid hormone
Authors: Cunningham Rochelle , Steplock Deborah , E Xiaofei , Biswas Rajat S. , Wang Fengying , Shenolikar Shirish , Weinman Edward J.
Published: May 2006

Sodium-dependent phosphate transport in NHERF-1−/−proximal tubule cells does not increase when grown in a low phosphate media and is resistant to the normal inhibitory effects of parathyroid hormone (PTH). The current experiments employ adenovirus-mediated gene transfer in primary cultures of mouse proximal tubule cells from NHERF-1 null mice to explore the specific role of NHERF-1 on regulated Npt2a trafficking and sodium-dependent phosphate transport. NHERF-1 null cells have decreased sodium-dependent phosphate transport compared with wild-type cells. Infection of NHERF-1 null cells with adenovirus-GFP-NHERF-1 increased phosphate transport and plasma membrane abundance of Npt2a. Adenovirus-GFP-NHERF-1 infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP demonstrated increased phosphate transport and Npt2a abundance in the plasma membrane when grown in low phosphate (0.1 mM) compared with high phosphate media (1.9 mM). PTH inhibited phosphate transport and decreased Npt2a abundance in the plasma membrane of adenovirus-GFP-NHERF-1-infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP. Interestingly, phosphate transport is inhibited by activation of protein kinase A and protein kinase C in wild-type proximal tubule cells but not in NHERF-1−/−cells. Together, these results highlight the requirement for NHERF-1 for physiological control of Npt2a trafficking and suggest that the Npt2a/NHERF-1 complex represents a unique PTH-responsive pool of Npt2a in renal microvilli.
Age-dependent alterations in Ca2+homeostasis: role of TRPV5 and TRPV6
Authors: van Abel Monique , Huybers Sylvie , Hoenderop Joost G. J. , van der Kemp Annemiete W. C. M. , van Leeuwen Johannes P. T. M. , Bindels René J. M.
Published: May 2006

Aging is associated with alterations in Ca2+homeostasis, which predisposes elder people to hyperparathyroidism and osteoporosis. Intestinal Ca2+absorption decreases with aging and, in particular, active transport of Ca2+by the duodenum. In addition, there are age-related changes in renal Ca2+handling. To examine age-related changes in expression of the renal and intestinal epithelial Ca2+channels, control (TRPV5+/+) and TRPV5 knockout (TRPV5−/−) mice aged 10, 30, and 52 wk were studied. Aging of TRPV5+/+mice resulted in a tendency toward increased renal Ca2+excretion and significantly decreased intestinal Ca2+absorption, which was accompanied by reduced expression of TRPV5 and TRPV6, respectively, despite increased serum 1,25(OH)2D3levels. Similarly, in TRPV5−/−mice the existing renal Ca2+loss was more pronounced in elder animals, whereas the compensatory intestinal Ca2+absorption and TRPV6 expression declined with aging. In both mice strains, aging resulted in a resistance to 1,25(OH)2D3and diminished renal vitamin D receptor mRNA levels, whereas serum Ca2+levels remained constant. Furthermore, 52-wk-old TRPV5−/−mice showed severe hyperparathyroidism, whereas PTH levels in elder TRPV5+/+mice remained normal. In 52-wk-old TRPV5−/−mice, serum osteocalcin levels were increased in accordance with the elevated PTH levels, suggesting an increased bone turnover in these mice. In conclusion, downregulation of TRPV5 and TRPV6 is likely involved in the impaired Ca2+(re)absorption during aging. Moreover, TRPV5−/−mice likely develop age-related hyperparathyroidism and osteoporotic characteristics before TRPV5+/+mice, demonstrating the importance of the epithelial Ca2+channels in Ca2+homeostasis.
Activation of ERK1/2 pathway mediates oxidant-induced decreases in mitochondrial function in renal cells
Authors: Nowak Grażyna , Clifton Ginger L. , Godwin Malinda L. , Bakajsova Diana
Published: May 2006

Previously, we showed that oxidant exposure in renal proximal tubular cells (RPTC) induces mitochondrial dysfunction mediated by PKC-ε. This study examined the role of ERK1/2 in mitochondrial dysfunction induced by oxidant injury and whether PKC-ε mediates its effects on mitochondrial function through the Raf-MEK1/2-ERK1/2 pathway. Sublethal injury produced by tert-butylhydroperoxide (TBHP) resulted in three- to fivefold increase in phosphorylation of ERK1/2 and p38 but not JNK. This was followed by decreases in basal and uncoupled respirations (41%), state 3 respiration and ATP production coupled to complex I (46%), and complex I activity (42%). Oxidant exposure decreased aconitase activity 30% but not pyruvate, α-ketoglutarate, and malate dehydrogenase activities. Inhibition of ERK1/2 restored basal and state 3 respirations, ΔΨm, ATP production, and complex I activity but not aconitase activity. In contrast, activation of ERK1/2 by expression of constitutively active MEK1 suppressed basal, uncoupled, and state 3 respirations in noninjured RPTC to the levels observed in TBHP-injured RPTC. MEK1/2 inhibition did not change Akt or p38 phosphorylation, demonstrating that the protective effect of MEK1/2 inhibitor was not due to activation of Akt or inhibition of p38 pathway. Inhibition of PKC-ε did not block TBHP-induced ERK1/2 phosphorylation in whole RPTC or in mitochondria. We conclude that 1) oxidant-induced activation of ERK1/2 but not p38 or JNK reduces mitochondrial respiration and ATP production by decreasing complex I activity and substrate oxidation through complex I, 2) citric acid cycle dehydrogenases are not under control of the ERK1/2 pathway in oxidant-injured RPTC, 3) the protective effects of ERK1/2 inhibition are not due to activation of Akt, and 4) ERK1/2 and PKC-ε mediate oxidant-induced mitochondrial dysfunction through independent pathways.
Spontaneous activation of the NF-κB signaling pathway in isolated normal glomeruli
Authors: Hayakawa Kunihiro , Meng Yiman , Hiramatsu Nobuhiko , Kasai Ayumi , Yao Jian , Kitamura Masanori
Published: May 2006

In this report, we describe that NF-κB is spontaneously activated in isolated, normal glomeruli. Ex vivo incubation of isolated rat glomeruli triggered expression of a NF-κB-dependent gene, monocyte chemoattractant protein-1 (MCP-1), in parallel with downregulation of IκBα and IκBβ proteins and activation of the p65 NF-κB subunit. The induction of MCP-1 was also observed in mesangial cells coincubated with isolated glomeruli or exposed to media conditioned by isolated glomeruli (GCM), which was abrogated by inhibition of NF-κB. The activation of NF-κB by glomerulus-derived factors was confirmed using reporter mesangial cells that produce secreted alkaline phosphatase (SEAP) under the control of the κB enhancer element. When the reporter cells were adoptively transferred into normal glomeruli, expression of SEAP mRNA and activity of SEAP were also upregulated in the explanted glomeruli. The molecular weight of factors responsible for activation of NF-κB was >50 kDa, and TNF-α was identified as one of glomerulus-derived activators. To examine upstream events involved, we focused on MAP kinases that are spontaneously activated in explanted glomeruli. Selective suppression of ERK or p38 MAP kinase significantly attenuated activation of NF-κB in mesangial cells triggered by coculture with isolated glomeruli. Interestingly, the suppressive effects by MAP kinase inhibitors were not observed in mesangial cells treated with GCM. These data suggested that NF-κB was spontaneously activated in explanted glomeruli via autocrine/paracrine factors including TNF-α and that the production of NF-κB activators by glomeruli was, at least in part, through MAP kinase pathways.
Restoration of Long-Term Potentiation in Middle-Aged Hippocampus After Induction of Brain-Derived Neurotrophic Factor
Authors: Rex Christopher S. , Lauterborn Julie C. , Lin Ching-Yi , Kramár Eniko A. , Rogers Gary A. , Gall Christine M. , Lynch Gary
Published: May 2006

Restoration of neuronal viability and synaptic plasticity through increased trophic support is widely regarded as a potential therapy for the cognitive declines that characterize aging. Previous studies have shown that in the hippocampal CA1 basal dendritic field deficits in the stabilization of long-term potentiation (LTP) are evident by middle age. The present study tested whether increasing endogenous brain-derived neurotrophic factor (BDNF) could reverse this age-related change. We report here that in middle-aged (8- to 10-mo-old) rats, in vivo treatments with a positive AMPA-type glutamate receptor modulator both increase BDNF protein levels in the cortical telencephalon and restore stabilization of basal dendritic LTP as assessed in acute hippocampal slices 18 h after the last drug treatment. These effects were not attributed to enhanced synaptic transmission or to facilitation of burst responses used to induce LTP. Increasing extracellular levels of BDNF by exogenous application to slices of middle-aged rats was also sufficient to rescue the stabilization of basal dendritic LTP. Finally, otherwise stable LTP in ampakine-treated middle-aged rats can be eliminated by infusion of the extracellular BDNF scavenger TrkB-Fc. Together these results indicate that increases in endogenous BDNF signaling can offset deficits in the postinduction processes that stabilize LTP.
Role for Brm in Cell Growth Control
Authors: Coisy-Quivy Marjorie , Disson Olivier , Roure Virginie , Muchardt Christian , Blanchard Jean-Marie , Dantonel Jean-Christophe
Published: May 2006

Recently, we have shown implication of Brm, the catalytic subunit of the SWI/SNF chromatin remodeling complex, in repression of cyclin A expression in quiescent cells. Here, we have examined the fate of cells lacking Brm throughout the cycle. We find that despite elevated levels of cyclins A and E, these cells can respond to serum starvation, however, without reaching a canonical G0 phase as they continue to express high levels of c-Myc and have an abnormally large average size. The response to serum starvation can be correlated with increased levels of Rb proteins p130 and p107 as well as increased association of p27 with the cyclin-dependent kinases, possibly compensating for the higher levels of G1 cyclins by reducing their associated kinase activity. After serum stimulation, reentry into the cycle occurs normally, but the S phase is delayed and shorter. In addition, the M phase has an increased duration, and we observed frequent faulty chromosome segregation events in anaphase. Altogether, our data suggest that cells can partially overcome the absence of Brm by activating several compensatory mechanisms to control the cell cycle. However, they remain profoundly affected, unable to enter a canonical quiescent state, presenting a shorter S phase, and finally unable to perform correct chromosome segregation. (Cancer Res 2006; 66(10): 5069-76)
The Phosphoinositide 3-Kinase/Akt Pathway: A New Target in Human Renal Cell Carcinoma Therapy
Authors: Sourbier Carole , Lindner Véronique , Lang Hervé , Agouni Abdelali , Schordan Eric , Danilin Sabrina , Rothhut Sylvie , Jacqmin Didier , Helwig Jean-Jacques , Massfelder Thierry
Published: May 2006

Metastatic renal cell carcinoma is resistant to current therapies. The phosphoinositide 3-kinase (PI3K)/Akt signaling cascade induces cell growth, cell transformation, and neovascularization. We evaluated whether targeting this pathway could be of therapeutic value against human renal cell carcinoma. The activation of the PI3K/Akt pathway and its role in renal cell carcinoma progression was evaluated in vitro in seven human cell lines by Western blot, cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, terminal deoxyribonucleotide transferase–mediated nick-end labeling assays, and fluorescence-activated cell sorting analysis, using two PI3K inhibitors, LY294002 and wortmannin, as well as by transfection with various Akt constructs and through Akt knockdown by small interfering RNA (siRNA). In vivo nude mice bearing human renal cell carcinoma tumor xenografts were treated with LY294002 (75 mg/kg/wk, 4 weeks, i.p.). Tumor growth was measured and tumors were subjected to Western blot and immunohistochemical analysis. Akt was constitutively activated in all cell lines. Constitutive phosphorylation of glycogen synthase kinase-3 (GSK-3) was observed in all cell lines, whereas forkhead transcription factor and mammalian target of rapamycin, although expressed, were not constitutively phosphorylated. Exposure to LY294002 or wortmannin decreased Akt activation and GSK-3 phosphorylation and reduced cell growth by up to 70% through induction of cell apoptosis. These effects were confirmed by transfection experiments with Akt constructs or Akt siRNA. Importantly, LY294002 induced up to 50% tumor regression in mice through tumor cell apoptosis. Tumor neovascularization was significantly increased by LY294002 treatment. Blood chemistries showed no adverse effects of the treatment. Our results suggest an important role of PI3K/Akt inhibitors as a potentially useful treatment for patients with renal cell carcinoma. (Cancer Res 2006; 66(10): 5130-42)
Acetyl-CoA Carboxylase α Is Essential to Breast Cancer Cell Survival
Authors: Chajès Véronique , Cambot Marie , Moreau Karen , Lenoir Gilbert M. , Joulin Virginie
Published: May 2006

Activation of de novo fatty acid synthesis is a characteristic feature of cancer cells. We have recently described an interaction between acetyl-CoA carboxylase α (ACCα), a key enzyme in fatty acid synthesis, and BRCA1, which indicates a possible connection between lipid synthesis and genetic factors involved in susceptibility to breast and ovarian cancers. For this reason, we explored the role of ACCα in breast cancer cell survival using an RNA interference (RNAi) approach. We show that specific silencing of either the ACCα or the fatty acid synthase (FAS) genes in cancer cells results in a major decrease in palmitic acid synthesis. Depletion of the cellular pool of palmitic acid is associated with induction of apoptosis concomitant with the formation of reactive oxygen species (ROS) and mitochondrial impairment. Expression of a small interfering RNA (siRNA)–resistant form of ACCα mRNA prevented the effect of ACCα-RNAi but failed to prevent the effect of FAS gene silencing. Furthermore, supplementation of the culture medium with palmitate or with the antioxidant vitamin E resulted in the complete rescue of cells from both ACCα and FAS siRNA–induced apoptosis. Finally, human mammary epithelial cells are resistant to RNAi against either ACCα or FAS. These data confirm the importance of lipogenesis in cancer cell survival and indicate that this pathway represents a key target for antineoplastic therapy that, however, might require specific dietary recommendation for full efficacy. (Cancer Res 2006; 66(10): 5287-94)
Mirk/Dyrk1b Mediates Cell Survival in Rhabdomyosarcomas
Authors: Mercer Stephen E. , Ewton Daina Z. , Shah Sejal , Naqvi Asghar , Friedman Eileen
Published: May 2006

Rhabdomyosarcoma is the most common sarcoma in children and is difficult to treat if the primary tumor is nonresectable or if the disease presents with metastases. The function of the serine/threonine kinase Mirk was investigated in this cancer. Mirk has both growth arrest and survival functions in terminally differentiating skeletal myoblasts. Maintenance of Mirk growth arrest properties would cause down-regulation of Mirk in transformed myoblasts. Alternatively, Mirk expression would be retained if rhabdomyosarcoma cells used Mirk survival capability. Mirk expression was significant in 12 of 16 clinical cases of rhabdomyosarcoma. Mirk was detected in each rhabdomyosarcoma cell line examined. Mirk was a functional kinase in each of three rhabdomyosarcoma cell lines, where it proved to be more active than in C2C12 skeletal myoblasts. Mirk mediated survival of the majority of clonogenic rhabdomyosarcoma cells. Knockdown of Mirk by RNA interference reduced the fraction of RD and of Rh30 rhabdomyosarcoma cells capable of colony formation 3- to 4-fold in multiple experiments. Depletion of Mirk induced cell death by apoptosis, as shown by increased numbers of terminal deoxynucleotidyl transferase–mediated nick-end labeling–positive cells and by increased binding of Annexin V. Mirk is a stress-activated kinase that mediates expression of contractile proteins in differentiating myoblasts, but Mirk is not essential for muscle formation in the embryo. It is likely that Mirk also facilitates survival of satellite cell–derived rhabdomyoblasts in regenerating skeletal muscle and aids their differentiation. This survival function is maintained in rhabdomyosarcoma, where Mirk may be a novel therapeutic target. (Cancer Res 2006; 66(10): 5143-50)
Lymph Node Metastasis in Breast Cancer Xenografts Is Associated with Increased Regions of Extravascular Drain, Lymphatic Vessel Area, and Invasive Phenotype
Authors: Pathak Arvind P. , Artemov Dmitri , Neeman Michal , Bhujwalla Zaver M.
Published: May 2006

Interactions between the tumor stromal compartment and cancer cells play an important role in the spread of cancer. In this study, we have used noninvasive in vivo magnetic resonance imaging (MRI) of two human breast cancer models with significantly different invasiveness, to quantify and understand the role of interstitial fluid transport, lymphatic-convective drain, and vascularization in the regional spread of breast cancer to the axillary lymph nodes. Quantitative fluorescence microscopy was done to morphometrically characterize lymphatic vessels in these tumors. Significant differences in vascular and extravascular transport variables as well as in lymphatic vessel morphology were detected between the two breast cancer models, which also exhibited significant differences in lymph node and lung metastasis. These data are consistent with a role of lymphatic drain in lymph node metastasis and suggest that increased lymph node metastasis may occur due to a combination of increased invasiveness, and reduced extracellular matrix integrity allowing increased pathways of least resistance for the transport of extravascular fluid, as well as tumor cells. It is also possible that lymph node metastasis occurred via the cancer cell–bearing tumoral lymphatic vessels. The congestion of these tumoral lymphatics with cancer cells may have restricted the entry and transport of macromolecules. (Cancer Res 2006; 66(10): 5151-8)
DNA Methylation Changes after 5-Aza-2′-Deoxycytidine Therapy in Patients with Leukemia
Authors: Yang Allen S. , Doshi Ketan D. , Choi Sang-Woon , Mason Joel B. , Mannari Rajan K. , Gharybian Vazganush , Luna Rene , Rashid Asif , Shen Lanlan , Estecio Marcos R.H. , Kantarjian Hagop M. , Garcia-Manero Guillermo , Issa Jean-Pierre J.
Published: May 2006

5-Aza-2′-deoxycytidine (decitabine) is postulated to have clinical activity in myeloid leukemias via its ability to inhibit DNA methylation. To study this, we examined DNA methylation in patients with leukemia treated with decitabine. Five days after the treatment, total genomic 5-methylcytosine/cytosine decreased on average by 14% (from 4.3% to 3.7%), whereas methylation of repetitive DNA elements showed a mean decrease of 9% and 16% for Alu and long interspersed nucleotide elements, respectively. Methylation decreased linearly with increasing doses between 5 and 20 mg/m2/d (r = 0.88; P = 0.05) but showed a plateau above that. Hypomethylation correlated with response in patients with acute myelogenous leukemia treated with low doses (5-20 mg/m2/d), but patients with chronic myelogenous leukemia treated with high doses (100-180 mg/m2/d) showed no such correlation. Aberrant methylation of p15 (>10%) was found in 27% of patients, and 80% of these showed a decrease by at least one third, but this did not correlate with response. The imprinted gene H19 showed little change in methylation after decitabine. In conclusion, we show dose-dependent hypomethylation after decitabine at low doses. Increasing the dose, which has been shown previously to result in a reduced response rate, was not accompanied by further hypomethylation. (Cancer Res 2006; 66(10): 5495-503)
Silencing of Bidirectional Promoters by DNA Methylation in Tumorigenesis
Authors: Shu Jingmin , Jelinek Jaroslav , Chang Hao , Shen Lanlan , Qin Taichun , Chung Woonbok , Oki Yasuhiro , Issa Jean-Pierre J.
Published: May 2006

CpG island methylation within promoters is known to silence individual genes in cancer. The involvement of this process in silencing gene pairs controlled by bidirectional promoters is unclear. In a screen for hypermethylated CpG islands in cancer, bidirectional promoters constituted 25.2% of all identified promoters, which matches with the genomic representation of bidirectional promoters. From the screen, we selected three bidirectional gene pairs for detailed analysis, WNT9A/CD558500, CTDSPL/BC040563, and KCNK15/BF195580. Levels of mRNA of all three pairs of genes were inversely correlated with the degree of promoter methylation in multiple cancer cell lines. Hypomethylation of these promoters induced by 5-aza-2′-deoxycytidine treatment reactivated or enhanced gene expression bidirectionally. The bidirectional nature of the WNT9A/CD558500 promoter was confirmed by luciferase assays, and hypermethylation down-regulated expression of both genes in the pair. Methylation of WNT9A/CD558500 and CTDSPL/BC040563 promoters occurs frequently in primary colon cancers and acute lymphoid leukemias (ALL), respectively, and methylation was correlated with decreased gene expression in ALL patient samples. Our study shows that hypermethylation of bidirectional promoter-associated CpG island silences two genes simultaneously, a property that should be taken into account when studying the functional consequences of hypermethylation in cancer. (Cancer Res 2006; 66(10): 5077-84)
Evolution of the Androgen Receptor Pathway during Progression of Prostate Cancer
Authors: Hendriksen Peter J.M. , Dits Natasja F.J. , Kokame Koichi , Veldhoven Antoine , van Weerden Wytske M. , Bangma Chris H. , Trapman Jan , Jenster Guido
Published: May 2006

The present work focused on the potential involvement of selective adaptations of the androgen receptor pathway in the initiation and progression of prostate cancer. We defined the androgen receptor pathway by selecting 200 genes that were androgen responsive in prostate cancer cell lines and/or xenografts. This androgen receptor pathway gene signature was then used for profiling prostate cancer xenografts and patient-derived samples. Approximately half of the androgen receptor pathway genes were up-regulated in well-differentiated prostate cancer compared with normal prostate. Functionally distinct parts of the androgen receptor pathway were specifically down-regulated in high-grade cancers. Unexpectedly, metastases have down-regulated the vast majority of androgen receptor pathway genes. The significance of this progressive down-regulation of androgen receptor pathway genes was shown for a few androgen receptor–regulated genes. Lower mRNA expression of HERPUD1, STK39, DHCR24, and SOCS2 in primary prostate tumors was correlated with a higher incidence of metastases after radical prostatectomy. HERPUD1 mRNA expression predicted the occurrence of metastases almost perfectly. In vitro experiments showed that overexpression of the stress response gene HERPUD1 rapidly induces apoptosis. Based on the functions of the genes within the distinct subsets, we propose the following model. Enhanced androgen receptor activity is involved in the early stages of prostate cancer. In well-differentiated prostate cancer, the androgen receptor activates growth-promoting as well as growth-inhibiting and cell differentiation genes resulting in a low growth rate. The progression from low-grade to high-grade prostate carcinoma and metastases is mediated by a selective down-regulation of the androgen receptor target genes that inhibit proliferation, induce differentiation, or mediate apoptosis. (Cancer Res 2006; 66(10): 5012-20)
The S100A4 Metastasis Factor Regulates Cellular Motility via a Direct Interaction with Myosin-IIA
Authors: Li Zhong-Hua , Bresnick Anne R.
Published: May 2006

S100A4, a member of the Ca2+-dependent S100 family of proteins, is a metastasis factor that is thought to regulate the motility and invasiveness of cancer cells. Previously, we showed that S100A4 specifically binds to nonmuscle myosin-IIA and promotes the unassembled state. S100A4, thus, provides a connection between the actomyosin cytoskeleton and the regulation of cellular motility; however, the step or steps in the motility cycle that are affected by S100A4 expression have not been investigated. To examine how the biochemical properties of S100A4 affect cell motility, we determined the effect of S100A4 expression on protrusive behavior during chemoattractant-stimulated motility. Our studies show that S100A4 modulates cellular motility by affecting cell polarization, with S100A4 expressing cells displaying few side protrusions and extensive forward protrusions during chemotaxis compared with control cells. To establish a direct link between S100A4 and the regulation of myosin-IIA function, we prepared an antibody to the S100A4 binding site on the myosin-IIA heavy chain that has comparable effects on myosin-IIA assembly as S100A4. Microinjection experiments show that the antibody elicits the same effects on cell polarization as S100A4. Our studies show for the first time that S100A4 promotes directional motility via a direct interaction with myosin-IIA. These findings establish S100A4 as a critical regulator of myosin-II function and metastasis-associated motility. (Cancer Res 2006; 66(10): 5173-80)
Identification of Novel Target Genes by an Epigenetic Reactivation Screen of Renal Cancer
Authors: Ibanez de Caceres Inmaculada , Dulaimi Essel , Hoffman Amanda M. , Al-Saleem Tahseen , Uzzo Robert G. , Cairns Paul
Published: May 2006

Aberrant promoter hypermethylation is a common mechanism for inactivation of tumor suppressor genes in cancer cells. To generate a global profile of genes silenced by hypermethylation in renal cell cancer (RCC), we did an expression microarray-based analysis of genes reactivated in the 786-0, ACHN, HRC51, and HRC59 RCC lines after treatment with the demethylating drug 5-aza-2 deoxycytidine and histone deacetylation inhibiting drug trichostatin A. Between 111 to 170 genes were found to have at least 3-fold up-regulation of expression after treatment in each cell line. To establish the specificity of the screen for identification of genes, epigenetically silenced in cancer cells, we validated a subset of 12 up-regulated genes. Three genes (IGFBP1, IGFBP3, and COL1A1) showed promoter methylation in tumor DNA but were unmethylated in normal cell DNA. One gene (GDF15) was methylated in normal cells but more densely methylated in tumor cells. One gene (PLAU) showed cancer cell–specific methylation that did not correlate well with expression status. The remaining seven genes had unmethylated promoters, although at least one of these genes (TGM2) may be regulated by RASSF1A, which was methylated in the RCC lines. Thus, we were able to show that up-regulation of at least 6 of the 12 genes examined was due to epigenetic reactivation. The IGFBP1, IGFBP3, and COL1A1 gene promoter regions were found to be frequently methylated in primary renal cell tumors, and further study will provide insight into the biology of the disease and facilitate translational studies in renal cancer. (Cancer Res 2006; 66(10): 5021-8)
Cyclooxygenase-2–Dependent Regulation of E-Cadherin: Prostaglandin E2 Induces Transcriptional Repressors ZEB1 and Snail in Non–Small Cell Lung Cancer
Authors: Dohadwala Mariam , Yang Seok-Chul , Luo Jie , Sharma Sherven , Batra Raj K. , Huang Min , Lin Ying , Goodglick Lee , Krysan Kostyantyn , Fishbein Michael C. , Hong Longsheng , Lai Chi , Cameron Robert B. , Gemmill Robert M. , Drabkin Harry A. , Dubinett Steven M.
Published: May 2006

Elevated tumor cyclooxygenase-2 (COX-2) expression is associated with tumor invasion, metastasis, and poor prognosis in non–small cell lung cancer (NSCLC). Here, we report that COX-2-dependent pathways contribute to the modulation of E-cadherin expression in NSCLC. First, whereas genetically modified COX-2-sense (COX-2-S) NSCLC cells expressed low E-cadherin and showed diminished capacity for cellular aggregation, genetic or pharmacologic inhibition of tumor COX-2 led to increased E-cadherin expression and resulted in augmented homotypic cellular aggregation among NSCLC cells in vitro. An inverse relationship between COX-2 and E-cadherin was shown in situ by double immunohistochemical staining of human lung adenocarcinoma tissue sections. Second, treatment of NSCLC cells with exogenous prostaglandin E2 (PGE2) significantly decreased the expression of E-cadherin, whereas treatment of COX-2-S cells with celecoxib (1 μmol/L) led to increased E-cadherin expression. Third, the transcriptional suppressors of E-cadherin, ZEB1 and Snail, were up-regulated in COX-2-S cells or PGE2-treated NSCLC cells but decreased in COX-2-antisense cells. PGE2 exposure led to enhanced ZEB1 and Snail binding at the chromatin level as determined by chromatin immunoprecipitation assays. Small interfering RNA–mediated knockdown of ZEB1 or Snail interrupted the capacity of PGE2 to down-regulate E-cadherin. Fourth, an inverse relationship between E-cadherin and ZEB1 and a direct relationship between COX-2 and ZEB1 were shown by immunohistochemical staining of human lung adenocarcinoma tissue sections. These findings indicate that PGE2, in autocrine or paracrine fashion, modulates transcriptional repressors of E-cadherin and thereby regulates COX-2-dependent E-cadherin expression in NSCLC. Thus, blocking PGE2 production or activity may contribute to both prevention and treatment of NSCLC. (Cancer Res 2006; 66(10): 5338-45)
Potent In vivo Anti–Breast Cancer Activity of IN-2001, a Novel Inhibitor of Histone Deacetylase, in MMTV/c-Neu Mice
Authors: Joung Ki E. , Min Kyung N. , An Jin Y. , Kim Dae-Kee , Kong Gu , Sheen Y. Yhong
Published: May 2006

A novel synthetic inhibitor of histone deacetylase (HDAC), 3-(4-dimethylaminophenyl)-N-hydroxy-2-propenamide (IN-2001), was examined for its antitumor activity and for the underlying molecular mechanisms of any such activity. IN-2001 effectively inhibited cellular HDAC activity (IC50, 5.42 nmol/L) in MCF-7 human breast cancer cells. Based on the Western blot analysis, this HDAC inhibitory effect of IN-2001 was confirmed by an increase in histone H4 acetylation from the IN-2001-treated breast cancer cells. IN-2001 suppressed mammary tumor growth in MMTV/c-Neu transgenic mice and also showed higher apoptotic index and lower lymphatic invasion compared with controls. In human breast cancer cells (MCF-7, T47D, MDA-MB-231, and MDA-MB-468), IN-2001 induced cell cycle arrest at G2-M phase through up-regulation of p21WAF1 and p27KIP1 and eventually caused apoptosis. IN-2001-induced apoptosis was caspase dependent and seems mediated through an increase in Bax/Bcl-2 ratio. Taken together, our data indicate that this novel HDAC inhibitor is a promising therapeutic agent against human breast cancer. (Cancer es 2006; 66(10): 5394-402)
Therapeutic Effect of Imatinib in Gastrointestinal Stromal Tumors: AKT Signaling Dependent and Independent Mechanisms
Authors: Tarn Chi , Skorobogatko Yuliya V. , Taguchi Takahiro , Eisenberg Burton , von Mehren Margaret , Godwin Andrew K.
Published: May 2006

Most gastrointestinal stromal tumors (GISTs) possess a gain-of-function mutation in c-KIT. Imatinib mesylate, a small-molecule inhibitor against several receptor tyrosine kinases, including KIT, platelet-derived growth factor receptor-α, and BCR-ABL, has therapeutic benefit for GISTs both via KIT and via unknown mechanisms. Clinical evidence suggests that a potential therapeutic benefit of imatinib might result from decreased glucose uptake as measured by positron emission tomography using 18-fluoro-2-deoxy-d-glucose. We sought to determine the mechanism of and correlation to altered metabolism and cell survival in response to imatinib. Glucose uptake, cell viability, and apoptosis in GIST cells were measured following imatinib treatment. Lentivirus constructs were used to stably express constitutively active AKT1 or AKT2 in GIST cells to study the role of AKT signaling in metabolism and cell survival. Immunoblots and immunofluorescent staining were used to determine the levels of plasma membrane-bound glucose transporter Glut4. We show that oncogenic activation of KIT maximizes glucose uptake in an AKT-dependent manner. Imatinib treatment markedly reduces glucose uptake via decreased levels of plasma membrane-bound Glut4 and induces apoptosis or growth arrest by inhibiting KIT activity. Importantly, expression of constitutively active AKT1 or AKT2 does not rescue cells from the imatinib-mediated apoptosis although glucose uptake was not blocked, suggesting that the potential therapeutic effect of imatinib is independent of AKT activity and glucose deprivation. Overall, these findings contribute to a clearer understanding of the molecular mechanisms involved in the therapeutic benefit of imatinib in GIST and suggest that a drug-mediated decrease in tumor metabolism observed clinically may not entirely reflect therapeutic efficacy of treatment. (Cancer Res 2006; 66(10): 5477-86)
Controlled Activation of ErbB1/ErbB2 Heterodimers Promote Invasion of Three-Dimensional Organized Epithelia in an ErbB1-Dependent Manner: Implications for Progression of ErbB2-Overexpressing Tumors
Authors: Zhan Lixing , Xiang Bin , Muthuswamy Senthil K.
Published: May 2006

Receptor tyrosine kinases of the ErbB family are implicated in a number of cancers, including that of the breast. ErbB receptors are activated by ligand-induced formation of homodimers and heterodimers. Receptor heterodimerization is thought to play a critical role in breast cancers overexpressing multiple members of the ErbB family. Although coexpression of ErbB receptors is associated with poor patient prognosis, the mechanisms by which receptor heterodimerization regulates tumor progression are not clear, due in part to a lack of methods that allow controlled activation of specific receptor heterodimers in mammary epithelial cells. Here, we report an approach to activate ErbB1-ErbB2 heterodimers in a nontumorigenic breast epithelial cell line, MCF-10A, without interference from endogenous ErbB receptors. Using such a method, we show that whereas both ErbB2 homodimers and ErbB1-ErbB2 heterodimers were equally potent in activating the Ras/mitogen-activated protein kinase pathway, the heterodimers were more potent in activating the phosphoinositide 3′-kinase (PI3K) and phospholipase Cγ1 pathways than ErbB2 homodimers. We combined the dimerization system with a three-dimensional cell culture approach to show that whereas both ErbB2 homodimers and ErbB1-ErbB2 heterodimers induced disruption of three-dimensional acini-like structures, only heterodimers promoted invasion of cells through extracellular matrix. The ability of heterodimers to induce invasion required the ErbB1 kinase activity and required activation of PI3K, Ras/mitogen-activated protein kinase, and phospholipase Cγ1 signaling pathways. Thus, we have identified cell invasion as a heterodimer-specific biological outcome and suggest that coexpression of ErbB1 may critically regulate invasive progression of ErbB2-positive breast cancers. (Cancer Res 2006; 66(10): 5201-8)
Accelerated Tumor Formation in a Fatless Mouse with Type 2 Diabetes and Inflammation
Authors: Nunez Nomeli P. , Oh Won-Jun , Rozenberg Julian , Perella Chris , Anver Miriam , Barrett J. Carl , Perkins Susan N. , Berrigan David , Moitra Jaideep , Varticovski Lyuba , Hursting Stephen D. , Vinson Charles
Published: May 2006

Epidemiologic studies show a positive association between obesity and cancer risk. In addition to increased body adiposity and secretion of fat-derived hormones, obesity is also linked to insulin resistance, type 2 diabetes, and chronic inflammation. We used the fatless A-ZIP/F-1 transgenic mouse to dissociate the relative role of each of these underlying factors in the development of cancer. These mice are unique in that they do not have white fat but do develop type 2 diabetes. In two cancer models, the classic two-stage skin carcinogenesis protocol and the C3(1)/T-Ag transgenic mouse mammary tumor model, A-ZIP/F-1 mice displayed higher tumor incidence, tumor multiplicity, and decreased tumor latency than wild-type mice. We examined circulating levels of adipokines, growth factors, and cytokines. As expected, adipokines (i.e., leptin, adiponectin, and resistin) were undetectable or found at very low levels in the blood of fatless mice. However, insulin, insulin-like growth factor-I, growth hormone, vascular endothelial growth factor, and proinflammatory Th2 cytokines, such as interleukin (IL)-1β, IL-4, and IL-6, were elevated in A-ZIP/F-1 mice. Additionally, we examined multiple phosphorylated proteins (i.e., protein kinase B/Akt and ErbB2/HER-2 kinase) associated with cancer development. Results show that many of these phosphorylated proteins were activated specifically in the A-ZIP/F-1 skin but not in the wild-type skin. These findings suggest that adipokines are not required for the promotion of tumor development and thus contradict the epidemiologic data linking obesity to carcinogenesis. We postulate that insulin resistance and inflammation are responsible for the positive correlation with cancer observed in A-ZIP/F-1 mice. (Cancer Res 2006; 66(10): 5469-76)
Acid-Mediated Tumor Invasion: a Multidisciplinary Study
Authors: Gatenby Robert A. , Gawlinski Edward T. , Gmitro Arthur F. , Kaylor Brant , Gillies Robert J.
Published: May 2006

The acid-mediated tumor invasion hypothesis proposes altered glucose metabolism and increased glucose uptake, observed in the vast majority of clinical cancers by fluorodeoxyglucose-positron emission tomography, are critical for development of the invasive phenotype. In this model, increased acid production due to altered glucose metabolism serves as a key intermediate by producing H+ flow along concentration gradients into adjacent normal tissue. This chronic exposure of peritumoral normal tissue to an acidic microenvironment produces toxicity by: (a) normal cell death caused by the collapse of the transmembrane H+ gradient inducing necrosis or apoptosis and (b) extracellular matrix degradation through the release of cathepsin B and other proteolytic enzymes. Tumor cells evolve resistance to acid-induced toxicity during carcinogenesis, allowing them to survive and proliferate in low pH microenvironments. This permits them to invade the damaged adjacent normal tissue despite the acid gradients. Here, we describe theoretical and empirical evidence for acid-mediated invasion. In silico simulations using mathematical models provide testable predictions concerning the morphology and cellular and extracellular dynamics at the tumor-host interface. In vivo experiments confirm the presence of peritumoral acid gradients as well as cellular toxicity and extracellular matrix degradation in the normal tissue exposed to the acidic microenvironment. The acid-mediated tumor invasion model provides a simple mechanism linking altered glucose metabolism with the ability of tumor cells to form invasive cancers. (Cancer Res 2006; 66(10): 5216-23)
Bcl-w Promotes Gastric Cancer Cell Invasion by Inducing Matrix Metalloproteinase-2 Expression via Phosphoinositide 3-Kinase, Akt, and Sp1
Authors: Bae In Hwa , Park Myung-Jin , Yoon Sung Hwan , Kang Sung Wook , Lee Seung-Sook , Choi Kyung-Mi , Um Hong-Duck
Published: May 2006

Given a previous report that Bcl-w is expressed in gastric cancer cells, particularly in those of an infiltrative morphology, we investigated whether Bcl-w expression influences the invasiveness of gastric cancer cells. To accomplish this, Bcl-w was overexpressed in adherent types of gastric adenocarcinoma cell lines, and this was found to result in an increase in their migratory and invasive potentials. These effects were not induced when Bcl-2 was overexpressed in the same cell types. Consistently, Bcl-w, but not Bcl-2, overexpression increased matrix metalloproteinase-2 (MMP-2) expression, and synthetic or natural inhibitors of MMP-2 abolished Bcl-w–induced cell invasion. Bcl-w overexpression also activated phosphoinositide 3-kinase (PI3K), Akt, and Sp1, and the blocking effects of each of these components using pharmacologic inhibitors, dominant-negative mutants, or small interfering RNA abolished the ability of Bcl-w to induce MMP-2 and cell invasion. The inhibition of PI3K/Akt signaling also prevented Sp1 activation. Overall, our data suggest that Bcl-w, which was previously shown to enhance gastric cancer cell survivability, also promotes their invasiveness by inducing MMP-2 expression via the sequential actions of PI3K, Akt, and Sp1. (Cancer Res 2006; 66(10): 4991-5)
Preclinical Evaluation of a Potent Novel DNA-Dependent Protein Kinase Inhibitor NU7441
Authors: Zhao Yan , Thomas Huw D. , Batey Michael A. , Cowell Ian G. , Richardson Caroline J. , Griffin Roger J. , Calvert A. Hilary , Newell David R. , Smith Graeme C.M. , Curtin Nicola J.
Published: May 2006

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by ionizing radiation and topoisomerase II poisons, such as etoposide and doxorubicin. A major pathway for the repair of DSB is nonhomologous end joining, which requires DNA-dependent protein kinase (DNA-PK) activity. We investigated the therapeutic use of a potent, specific DNA-PK inhibitor (NU7441) in models of human cancer. We measured chemosensitization by NU7441 of topoisomerase II poisons and radiosensitization in cells deficient and proficient in DNA-PKCS (V3 and V3-YAC) and p53 wild type (LoVo) and p53 mutant (SW620) human colon cancer cell lines by clonogenic survival assay. Effects of NU7441 on DSB repair and cell cycle arrest were measured by γH2AX foci and flow cytometry. Tissue distribution of NU7441 and potentiation of etoposide activity were determined in mice bearing SW620 tumors. NU7441 increased the cytotoxicity of ionizing radiation and etoposide in SW620, LoVo, and V3-YAC cells but not in V3 cells, confirming that potentiation was due to DNA-PK inhibition. NU7441 substantially retarded the repair of ionizing radiation–induced and etoposide-induced DSB. NU7441 appreciably increased G2-M accumulation induced by ionizing radiation, etoposide, and doxorubicin in both SW620 and LoVo cells. In mice bearing SW620 xenografts, NU7441 concentrations in the tumor necessary for chemopotentiation in vitro were maintained for at least 4 hours at nontoxic doses. NU7441 increased etoposide-induced tumor growth delay 2-fold without exacerbating etoposide toxicity to unacceptable levels. In conclusion, NU7441 shows sufficient proof of principle through in vitro and in vivo chemosensitization and radiosensitization to justify further development of DNA-PK inhibitors for clinical use. (Cancer Res 2006; 66(10): 5354-62)
Processing of Tumor-Associated Antigen by the Proteasomes of Dendritic Cells ControlsIn vivoT-Cell Responses
Authors: Chapatte Laurence , Ayyoub Maha , Morel Sandra , Peitrequin Anne-Lise , Lévy Nicole , Servis Catherine , Van den Eynde Benoît J. , Valmori Danila , Lévy Frédéric
Published: May 2006

Dendritic cells are unique in their capacity to process antigens and prime naive CD8+ T cells. Contrary to most cells, which express the standard proteasomes, dendritic cells express immunoproteasomes constitutively. The melanoma-associated protein Melan-AMART1 contains an HLA-A2-restricted peptide that is poorly processed by melanoma cells expressing immunoproteasomes in vitro. Here, we show that the expression of Melan-A in dendritic cells fails to elicit T-cell responses in vitro and in vivo because it is not processed by the proteasomes of dendritic cells. In contrast, dendritic cells lacking immunoproteasomes induce strong anti-Melan-A T-cell responses in vitro and in vivo. These results suggest that the inefficient processing of self-antigens, such as Melan-A, by the immunoproteasomes of professional antigen-presenting cells prevents the induction of antitumor T-cell responses in vivo. (Cancer Res 2006; 66(10): 5461-8)
Earlier Onset of Tumoral Angiogenesis in Matrix Metalloproteinase-19–Deficient Mice
Authors: Jost Maud , Folgueras Alicia R. , Frérart Françoise , Pendas Alberto M. , Blacher Silvia , Houard Xavier , Berndt Sarah , Munaut Carine , Cataldo Didier , Alvarez Jesus , Melen-Lamalle Laurence , Foidart Jean-Michel , López-Otín Carlos , Noël Agnès
Published: May 2006

Among matrix metalloproteinases (MMP), MMP-19 displays unique structural features and tissue distribution. In contrast to most MMPs, MMP-19 is expressed in normal human epidermis and down-regulated during malignant transformation and dedifferentiation. The contribution of MMP-19 during tumor angiogenesis is presently unknown. In an attempt to give new insights into MMP-19 in vivo functions, angiogenic response of mutant mice lacking MMP-19 was analyzed after transplantation of murine malignant PDVA keratinocytes and after injection of Matrigel supplemented with basic fibroblast growth factor. In situ hybridization and immunohistochemical analysis revealed that MMP-19 is produced by host mesenchymal cells but not by endothelial capillary cells or CD11b-positive inflammatory cells. Based on a new computer-assisted method of quantification, we provide evidence that host MMP-19 deficiency was associated with an increased early angiogenic response. In addition, increased tumor invasion was observed in MMP-19−/− mice. We conclude that, in contrast to most MMPs that promote tumor progression, MMP-19 is a negative regulator of early steps of tumor angiogenesis and invasion. These data highlight the requirement to understand the individual functions of each MMP to improve anticancer strategies. (Cancer Res 2006; 66(10): 5234-41)