bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression Profiling Identifies Altered Expression of Genes That Contribute to the Inhibition of Transforming Growth Factor-β Signaling in Ovarian Cancer
Authors: Sunde Jan S. , Donninger Howard , Wu Kongming , Johnson Michael E. , Pestell Richard G. , Rose G. Scott , Mok Samuel C. , Brady John , Bonome Tomas , Birrer Michael J.
Published: September 2006

Ovarian cancer is resistant to the antiproliferative effects of transforming growth factor-β (TGF-β); however, the mechanism of this resistance remains unclear. We used oligonucleotide arrays to profile 37 undissected, 68 microdissected advanced-stage, and 14 microdissected early-stage papillary serous cancers to identify signaling pathways involved in ovarian cancer. A total of seven genes involved in TGF-β signaling were identified that had altered expression >1.5-fold (P < 0.001) in the ovarian cancer specimens compared with normal ovarian surface epithelium. The expression of these genes was coordinately altered: genes that inhibit TGF-β signaling (DACH1, BMP7, and EVI1) were up-regulated in advanced-stage ovarian cancers and, conversely, genes that enhance TGF-β signaling (PCAF, TFE3, TGFBRII, and SMAD4) were down-regulated compared with the normal samples. The microarray data for DACH1 and EVI1 were validated using quantitative real-time PCR on 22 microdissected ovarian cancer specimens. The EVI1 gene locus was amplified in 43% of the tumors, and there was a significant correlation (P = 0.029) between gene copy number and EVI1 gene expression. No amplification at the DACH1 locus was found in any of the samples. DACH1 and EVI1 inhibited TGF-β signaling in immortalized normal ovarian epithelial cells, and a dominant-negative DACH1, DACH1-ΔDS, partially restored signaling in an ovarian cancer cell line resistant to TGF-β. These results suggest that altered expression of these genes is responsible for disrupted TGF-β signaling in ovarian cancer and they may be useful as new and novel therapeutic targets for ovarian cancer. (Cancer Res 2006; 66(17): 8404-12)
Silencing of the Metastasis Suppressor RECK by RAS Oncogene Is Mediated by DNA Methyltransferase 3b–Induced Promoter Methylation
Authors: Chang Hui-Chiu , Cho Chun-Yu , Hung Wen-Chun
Published: September 2006

RECK is a membrane-anchored glycoprotein that may negatively regulate matrix metalloproteinase activity to suppress tumor invasion and metastasis. Our previous study indicated that oncogenic RAS inhibited RECK expression via a histone deacetylation mechanism. In this study, we address whether DNA methyltransferases (DNMT) participate in the inhibition of RECK by RAS. Induction of Ha-RASVal12 oncogene increased DNMT3b, but not DNMT1 and DNMT3a, expression in 2-12 cells. In addition, induction of DNMT3b by RAS was through the extracellular signal-regulated kinase signaling pathway. Oncogenic RAS increased the binding of DNMT3b to the promoter of RECK gene and this binding induced promoter methylation, which could be reversed by 5′-azacytidine and DNMT3b small interfering RNA (siRNA). The MEK inhibitor U0126 also reversed RAS-induced DNMT3b binding and RECK promoter methylation. Treatment of 5′-azacytidine and DNMT3b siRNA restored RECK expression in 2-12 cells and potently suppressed RAS-stimulated cell invasion. In addition, the inhibitory effect of 5′-azacytidine on RAS-induced cell invasion was attenuated after knockdown of RECK by siRNA. Interestingly, human lung cancer cells harboring constitutively activated RAS exhibited lower RECK expression and higher promoter methylation of RECK gene. 5′-Azacytidine and DNMT3b siRNA restored RECK expression in these cells and effectively suppressed invasiveness. Collectively, our results suggest that RAS oncogene induces RECK gene silencing through DNMT3b-mediated promoter methylation, and DNMT inhibitors may be useful for the treatment of RAS-induced metastasis. (Cancer Res 2006; 66(17): 8413-20)
Farnesyltransferase Inhibitors Reverse Taxane Resistance
Authors: Marcus Adam I. , O'Brate Aurora M. , Buey Ruben M. , Zhou Jun , Thomas Shala , Khuri Fadlo R. , Andreu Jose Manuel , Díaz Fernando , Giannakakou Paraskevi
Published: September 2006

The combination of farnesyltransferase inhibitors (FTIs) and taxanes has been shown to result in potent antiproliferative and antimitotic synergy. Recent phase I and II clinical trials have shown that this combination shows clinical activity in taxane-refractory or taxane-resistant cancer patients. To understand the mechanism behind these clinical observations, we used a cancer cell model of paclitaxel resistance and showed that the FTI/taxane combination retains potent antiproliferative, antimitotic, and proapoptotic activity against the paclitaxel-resistant cells, at doses where each drug alone has little or no activity. To probe the mechanistic basis of these observations, paclitaxel activity was monitored in living cells using the fluorescently conjugated paclitaxel, Flutax-2. We observed that all FTIs tested increase the amount of microtubule-bound Flutax-2 and the number of microtubules labeled with Flutax-2 in both paclitaxel-resistant and paclitaxel-sensitive cells. Importantly, we observed a consequential increase in microtubule stability and tubulin acetylation with the combination of the two drugs, even in paclitaxel-resistant cells, confirming that the enhanced taxane binding in the presence of FTI affects microtubule function. Furthermore, this mechanism is dependent on the function of the tubulin deacetylase, HDAC6, because in cells overexpressing a catalytically inactive HDAC6, FTIs are incapable of enhancing Flutax-2–microtubule binding. Similar results were obtained by using an FTI devoid of farnesyltransferase inhibitory activity, indicating that functional inhibition of farnesyltransferase is also required. Overall, these studies provide a new insight into the functional relationship between HDAC6, farnesyltransferase, and microtubules, and support clinical data showing that the FTI/taxane combination is effective in taxane-refractory patients. (Cancer Res 2006; 66(17): 8838-46)
ARID3B Induces Malignant Transformation of Mouse Embryonic Fibroblasts and Is Strongly Associated with Malignant Neuroblastoma
Authors: Kobayashi Kenichiro , Era Takumi , Takebe Atsushi , Jakt Lars Martin , Nishikawa Shin-Ichi
Published: September 2006

ARID3B, a member of the AT-rich interaction domain (ARID) family of proteins, plays an essential role in the survival of neural crest during embryogenesis. Here, we report evidence that ARID3B is involved in the development of malignant neuroblastoma, a childhood tumor derived from neural crest. (a) ARID3B is expressed by all five cell lines derived from neuroblastoma tested by us. (b) Analysis of published DNA microarray data of fresh neuroblastoma tumors showed that ARID3B is expressed in 80% of stage IV tumors, whereas only in 9% of stage I-III+IVs tumors. (c) In vitro growth of several neuroblastoma cell lines is suppressed significantly by antisense as well as siRNA treatment. (d) An increase of the ARID3B expression level by transfection in the SY5Y neuroblastoma cell line enhances the malignancy in tumor growth assays in nu/nu mice. (e) ARID3B by itself can immortalize mouse embryonic fibroblasts (MEFs) in vitro and confers malignancy to MEF when transfected together with MYCN, the best characterized oncogene for neuroblastoma. Thus, ARID3B seems to play a key role in the malignant transformation of neuroblastoma and may serve not only as a marker of malignancy but also as a potential target for cancer therapy of stage IV neuroblastoma for which there is currently no effective treatment available. (Cancer Res 2006; 66(17): 8331-6)
Organic Cation Transporters Are Determinants of Oxaliplatin Cytotoxicity
Authors: Zhang Shuzhong , Lovejoy Katherine S. , Shima James E. , Lagpacan Leah L. , Shu Yan , Lapuk Anna , Chen Ying , Komori Takafumi , Gray Joe W. , Chen Xin , Lippard Stephen J. , Giacomini Kathleen M.
Published: September 2006

Although the platinum-based anticancer drugs cisplatin, carboplatin, and oxaliplatin have similar DNA-binding properties, only oxaliplatin is active against colorectal tumors. The mechanisms for this tumor specificity of platinum-based compounds are poorly understood but could be related to differences in uptake. This study shows that the human organic cation transporters (OCT) 1 and 2 (SLC22A1 and SLC22A2) markedly increase oxaliplatin, but not cisplatin or carboplatin, accumulation and cytotoxicity in transfected cells, indicating that oxaliplatin is an excellent substrate of these transporters. The cytotoxicity of oxaliplatin was greater than that of cisplatin in six colon cancer cell lines [mean ± SE of IC50 in the six cell lines, 3.9 ± 1.4 μmol/L (oxaliplatin) versus 11 ± 2.0 μmol/L (cisplatin)] but was reduced by an OCT inhibitor, cimetidine, to a level similar to, or even lower than that of, cisplatin (29 ± 11 μmol/L for oxaliplatin versus 19 ± 4.3 μmol/L for cisplatin). Structure-activity studies indicated that organic functionalities on nonleaving groups coordinated to platinum are critical for selective uptake by OCTs. These results indicate that OCT1 and OCT2 are major determinants of the anticancer activity of oxaliplatin and may contribute to its antitumor specificity. They also strongly suggest that expression of OCTs in tumors should be investigated as markers for selecting specific platinum-based therapies in individual patients. The development of new anticancer drugs, specifically targeted to OCTs, represents a novel strategy for targeted drug therapy. The results of the present structure-activity studies indicate specific tactics for realizing this goal. (Cancer Res 2006; 66(17): 8847-57)
Differential Antiproliferative Effects of Calcitriol on Tumor-Derived and Matrigel-Derived Endothelial Cells
Authors: Chung Ivy , Wong Michael K. , Flynn Geraldine , Yu Wei-dong , Johnson Candace S. , Trump Donald L.
Published: September 2006

The most active metabolite of vitamin D, calcitriol, is growth inhibitory for various tumor types in vitro and in vivo and inhibits the growth of endothelial cells freshly isolated from tumors [tumor-derived endothelial cells (TDEC)]. We compared the effects of calcitriol on Matrigel-derived endothelial cells (MDEC) and TDEC isolated from Matrigel plugs and squamous cell carcinoma tumors, respectively. TDEC and MDEC expressed vitamin D receptor (VDR) and responded to calcitriol by increasing VDR protein expression. Although no mutations were found in VDR from either cell type, Scatchard plot analysis revealed a higher ligand-binding affinity in TDEC (Kd, 0.26 nmol/L) than MDEC (Kd, 0.65 nmol/L). The VDR signaling axis in both cells was intact as shown using nuclear translocation and 24-hydroxylase promoter-luciferase reporter assays. However, unlike TDEC, MDEC were resistant to calcitriol-induced growth inhibition. Calcitriol (10 nmol/L) resulted in a 12.3% growth inhibition of MDEC compared with 47% in TDEC. In TDEC, calcitriol resulted in induction of G0/G1 arrest (10.75%) and reduction of S-phase cells (6.8%) with induction of p27 and down-regulation of p21 protein expression. Apoptotic effects, determined by Annexin V staining were also observed in calcitriol-treated TDEC (38.6%). Calcitriol caused reduced expression of p-Erk and p-Akt and an increase of poly(ADP-ribose) polymerase and caspase-3 cleavage in TDEC. By contrast, none of these effects on cell cycle or apoptosis were seen in calcitriol-treated MDEC. These results show that TDEC were more sensitive than MDEC to the antiproliferative effects of calcitriol despite apparently normal VDR content and structure of signaling axis in both cell types. (Cancer Res 2006; 66(17): 8565-73)
Functional Analysis of Alternative Isoforms of the Transcription Factor PAX3 in Melanocytes In vitro
Authors: Wang Qiuyu , Kumar Shant , Slevin Mark , Kumar Patricia
Published: September 2006

Transcription factor PAX3 has seven isoforms of which PAX3c has been studied extensively whereas the functions of the other isoforms are less well known. Here, we found that PAX3 isoforms in a stable transfection system have different biological functions in mouse melanocytes in vitro. PAX3a and PAX3b had negative effects on melanocyte proliferation but had no discernable effect on melanocyte growth in soft agar. PAX3a did not affect cell migration and apoptosis but PAX3b reduced migration and accelerated apoptosis. PAX3c and PAX3d promoted cell proliferation, migration, transformation, and survival. PAX3e reduced melanocyte growth; transformation and migration were unchanged and apoptosis was increased in vitro. PAX3g did not influence cell proliferation or apoptosis. Cells expressing PAX3g were able to grow in soft agar but migration was reduced. PAX3h increased cell proliferation, migration, survival, and transformation. These functional studies have advanced our understanding of the effects of PAX3 isoforms in melanocytes and their potential contribution in tumorigenesis. (Cancer Res 2006; 66(17): 8574-80)
Potential Role of Jun Activation Domain–Binding Protein 1 as a Negative Regulator of p27kip1 in Pancreatic Adenocarcinoma
Authors: Kouvaraki Maria A. , Korapati Anita L. , Rassidakis George Z. , Tian Ling , Zhang Qingxiu , Chiao Paul , Ho Linus , Evans Douglas B. , Claret François X.
Published: September 2006

Reduced expression of p27 has been associated with poor prognosis in most human cancers, including pancreatic adenocarcinoma. Jun activation domain–binding protein 1 (JAB1), an activator protein (AP-1) coactivator, previously implicated in p27 degradation, is overexpressed in various tumors and correlates with low p27 expression. We examined JAB1 and p27 in normal and neoplastic pancreatic tissues. Increased JAB1 expression was seen in pancreatic carcinoma samples but not in paired normal pancreatic tissues. Immunohistochemical analysis using tissue microarrays showed that JAB1 was overexpressed in all 32 (100%) pancreatic adenocarcinoma samples tested, predominantly nuclear in 23 (72%) samples and predominantly cytoplasmic in 9 (28%) tumors. When 10% was used as a cutoff for p27 positivity, p27 was expressed in 11 (34%) of tumors; however, p27 expression was localized in the nuclei of tumor cells in only 4 (13%) of the samples. Overexpression of the JAB1 in the pancreatic carcinoma cell lines Panc-1, Mia PaCa-2, and Panc-28 resulted in decreased p27 expression. Conversely, down-regulation of JAB1 by short interfering RNA substantially increased p27 expression and inhibited progression from G1 to S phase of the cell cycle. Interestingly, JAB1-mediated p27 degradation was not impaired when S-phase kinase-interacting protein 2 (Skp2), an F-box protein required for the ubiquitination and consequent degradation of p27, was silenced. Thus, JAB1 may have an Skp2-independent p27 degradation mechanism in pancreatic cancer cells. These findings suggest that JAB1 overexpression is involved in the pathogenesis of pancreatic cancer through JAB1-mediated p27 degradation and that control of JAB1 expression is a novel therapeutic target in patients with pancreatic adenocarcinomas. (Cancer Res 2006; 66(17): 8581-9)
DNA Damage Binding Protein Component DDB1 Participates in Nucleotide Excision Repair through DDB2 DNA-binding and Cullin 4A Ubiquitin Ligase Activity
Authors: Li Jinyou , Wang Qi-En , Zhu Qianzheng , El-Mahdy Mohamed A. , Wani Gulzar , Prætorius-Ibba Mette , Wani Altaf A.
Published: September 2006

Functional defect in DNA damage binding (DDB) activity has a direct relationship to decreased nucleotide excision repair (NER) and increased susceptibility to cancer. DDB forms a complex with cullin 4A (Cul4A), which is now known to ubiquitylate DDB2, XPC, and histone H2A. However, the exact role of DDB1 in NER is unclear. In this study, we show that DDB1 knockdown in human cells impaired their ability to efficiently repair UV-induced cyclobutane pyrimidine dimers (CPD) but not 6-4 photoproducts (6-4PP). Extensive nuclear protein fractionation and chromatin association analysis revealed that upon irradiation, DDB1 protein is translocated from a loosely bound to a tightly bound in vivo chromatin fraction and the DDB1 translocation required the participation of functional DDB2 protein. DDB1 knockdown also affected the translocation of Cul4A component to the tightly bound form in UV-damaged chromatin in vivo as well as its recruitment to the locally damaged nuclear foci in situ. However, DDB1 knockdown had no effect on DNA damage binding capacity of DDB2. The data indicated that DDB2 can bind to damaged DNA in vivo as a monomer, whereas Cul4A recruitment to damage sites depends on the fully assembled complex. Our data also showed that DDB1 is required for the UV-induced DDB2 ubiquitylation and degradation. In summary, the results suggest that (a) DDB1 is critical for efficient NER of CPD; (b) DDB1 acts in bridging DDB2 and ubiquitin ligase Cul4A; and (c) DDB1 aids in recruiting the ubiquitin ligase activity to the damaged sites for successful commencement of lesion processing by NER. (Cancer Res 2006; 66(17): 8590-7)
Vascular Endothelial Growth Factor Overexpression by Soft Tissue Sarcoma Cells: Implications for Tumor Growth, Metastasis, and Chemoresistance
Authors: Zhang Lianglin , Hannay Jonathan A.F. , Liu Juehui , Das Parimal , Zhan Maocheng , Nguyen Theresa , Hicklin Daniel J. , Yu Dihua , Pollock Raphael E. , Lev Dina
Published: September 2006

To better elucidate the role of vascular endothelial growth factor (VEGF)165 in soft tissue sarcoma (STS) growth, metastasis, and chemoresistance, we generated stably transfected human STS cell lines with VEGF165 to study the effect of VEGF165 on STS cells in vitro and the effect of culture medium from these cells on human umbilical vascular endothelial cells. Severe combined immunodeficient mice bearing xenografts of transfected cell lines were used to assess the effect of VEGF overexpression and the effect of VEGF receptor (VEGFR) 2 inhibition on STS growth, metastasis, and response to doxorubicin. VEGF165-transfected xenografts formed highly vascular tumors with shorter latency, accelerated growth, enhanced chemoresistance, and increased incidence of pulmonary metastases. Blockade of VEGFR2 signaling using DC101 anti-VEGFR2 monoclonal antibody enhanced doxorubicin chemoresponse; this combined biochemotherapy inhibited tumor growth and decreased pulmonary metastases without overt toxicity. Combined therapy reduced microvessel counts while increasing vessel maturation index. VEGF overexpression did not affect on the sarcoma cells per se; however, conditioned medium from VEGF transfectants caused increased endothelial cell proliferation, migration, and chemoresistance. Addition of DC101 induced endothelial cell sensitivity to doxorubicin and suppressed the activity of matrix metalloproteinases secreted by endothelial cells. We therefore conclude that VEGF is a critical determinant of STS growth and metastasis and that STS chemoresistance, in our model, is a process induced by the interplay between STS cells and tumor-associated endothelial cells. STS growth and metastasis can be interrupted by combined low-dose doxorubicin and anti-VEGFR2, a strategy that attacks STS-associated endothelial cells. In the future, such therapeutic approaches may be useful in treating STS before the development of clinically apparent metastases. (Cancer Res 2006; 66(17): 8770-7)
Mitogen-Activated Protein Kinase Phosphatase-1 Is Required for Cisplatin Resistance
Authors: Wang Zhaoqing , Xu Jing , Zhou Jun-Ying , Liu Yusen , Wu Gen Sheng
Published: September 2006

Mitogen-activated protein kinase (MAPK) phosphatase (MKP)-1 is a member of the MKP family that negatively regulates MAPK signaling. MKP-1 has been implicated in cell survival in response to stressful stimuli, including anticancer treatment, but its role in cisplatin resistance is not fully understood. Here, we show that cisplatin induces MKP-1 in several human cancer cell lines. Induction of MKP-1 by cisplatin was through the transcriptional mechanism regulated by extracellular signal-regulated kinase (ERK). Overexpression of MKP-1 rendered human lung cancer cells resistant to cisplatin. Conversely, down-regulation of MKP-1 by small interfering RNA silencing sensitized human lung cancer cells to cisplatin-induced cell death. Using primary mouse embryonic fibroblasts (MEF) from MKP-1 knockout mice, we show that induction of MKP-1 by cisplatin correlates with inactivation of c-Jun NH2-terminal kinase (JNK) but not ERK and p38. Furthermore, apoptosis induced by cisplatin was significant in MKP-1−/− MEFs, whereas such change was minimal in MKP-1+/+ MEFs. More importantly, cisplatin-induced cell death is inhibited by blocking JNK but not ERK and p38 activities. Collectively, our results establish a critical role of JNK in cisplatin-induced apoptosis and suggest that MKP-1 is required for cisplatin resistance. (Cancer Res 2006; 66(17): 8870-7)
The Generation and Regulation of Functional Diversity of Malignant Plasma Cells
Authors: Nadav Liat , Katz Ben-Zion , Baron Shoshana , Cohen Nir , Naparstek Elizabeth , Geiger Benjamin
Published: September 2006

Cellular diversity, which is a hallmark of malignancy, can be generated by both genetic and nongenetic mechanisms. We describe here variability in the adhesive and migratory behavior of malignant plasma cell populations, including multiple myeloma–derived lines and primary patient samples. Examination of the plasma cell lines ARH-77, CAG, and AKR revealed two distinct subpopulations of cells, one displaying highly adhesive properties (type A) and the other consisting of poorly adhesive, floating cells (type F). In the ARH-77 cell line, type A cells attach better to fibronectin and to human bone fragments and form paxillin-rich focal adhesions, whereas type F cells are highly motile and exert integrin-dependent bone marrow homing capacity in nonobese diabetic/severe combined immunodeficient mice. Flow cytometry indicated that type A cells express significantly higher levels of CD45 and CD56 and lower levels of CD138 compared with type F cells. Interestingly, culturing of either type A or type F cells under nonselective conditions resulted in the development of mixed cell population similar to the parental ARH-77 cells. Analysis of bone marrow aspirates of multiple myeloma patients revealed that spicules within the aspirates are enriched with type A–like cells. Nonadherent cells within the aspirate fluids express a marker profile similar to type F cells. This study indicates that multiple myeloma patients contain heterogeneous populations of malignant plasma cells that display distinct properties. Diverse subpopulations of malignant plasma cells may play distinct roles in the different biological and clinical manifestations of plasma cell dyscrasias, including bone dissemination and selective adhesion to bone marrow compartments. (Cancer Res 2006; 66(17): 8608-16)
Regulation of Transforming Growth Factor-β1–Induced Apoptosis and Epithelial-to-Mesenchymal Transition by Protein Kinase A and Signal Transducers and Activators of Transcription 3
Authors: Yang Yanan , Pan Xinchao , Lei Weiwei , Wang Jun , Shi Jian , Li Fuqiang , Song Jianguo
Published: September 2006

Apoptosis and epithelial-to-mesenchymal transdifferentiation or transition (EMT) are crucial for normal development and body homeostasis. The alterations of these events are closely related to some pathologic processes, such as tumor formation and metastasis, fibrotic diseases of liver and kidney, and abnormal development of embryos. The mechanism that underlies the simultaneously occurring apoptosis and EMT induced by transforming growth factor-β (TGF-β) has not been well studied. In this report, we investigated the potential mechanism that underlies TGF-β1–induced apoptosis and EMT. TGF-β1–induced apoptosis and EMT were associated with the activation of protein kinase A (PKA) and signal transducers and activators of transcription 3 (STAT3). Inhibition of PKA by specific PKA inhibitor H89 or by PKA inhibitor peptide blocked STAT3 activation and suppressed TGF-β1–induced apoptosis and EMT. Furthermore, overexpression of a phosphorylation-deficient form of STAT3, but not wild-type STAT3, produced an inhibitory effect on TGF-β1–induced apoptosis and EMT. The results indicate that PKA is an upstream regulator for TGF-β1–induced STAT3 activation and plays an important role in TGF-β1–mediated apoptosis and EMT. These studies provided a new insight into the signaling mechanism underlying the apoptosis and EMT, which could be of importance in understanding some related physiologic and pathologic processes. (Cancer Res 2006; 66(17): 8617-24)
The Isopeptidase USP2a Protects Human Prostate Cancer from Apoptosis
Authors: Priolo Carmen , Tang Dan , Brahamandan Mohan , Benassi Barbara , Sicinska Ewa , Ogino Shuji , Farsetti Antonella , Porrello Alessandro , Finn Stephen , Zimmermann Johann , Febbo Phillip , Loda Massimo
Published: September 2006

Deubiquitinating enzymes can prevent the destruction of protein substrates prior to proteasomal degradation. The ubiquitin-specific protease 2a (USP2a) deubiquitinates the antiapoptotic proteins Fatty Acid Synthase and Mdm2. Here, we show that when USP2a is overexpressed in nontransformed cells, it exhibits oncogenic behavior both in vitro and in vivo and prevents apoptosis induced by chemotherapeutic agents. Notably, USP2a silencing in several human cancer cell lines results in apoptosis. Gene set enrichment analysis, which focuses on groups of genes sharing biological function or regulatory pathways, was done on microarray expression data from human prostate cancers. The cell death–related gene set, as well as a selected cluster of validated p53 target genes, were significantly enriched in the low USP2a expression group of tumors. Conversely, genes implicated in fatty acid metabolism were significantly associated with tumors expressing high USP2a (44%). The expression profile analysis is consistent with the effects of USP2a on its known targets, i.e., Fatty Acid Synthase and Mdm2, defining a subset of prostate tumors resistant to apoptosis. USP2a thus represents a therapeutic target in prostate cancer. (Cancer Res 2006; 66(17): 8625-32)
Antiangiogenic and Antitumor Effects of Src Inhibition in Ovarian Carcinoma
Authors: Han Liz Y. , Landen Charles N. , Trevino Jose G. , Halder Jyotsnabaran , Lin Yvonne G. , Kamat Aparna A. , Kim Tae-Jin , Merritt William M. , Coleman Robert L. , Gershenson David M. , Shakespeare William C. , Wang Yihan , Sundaramoorth Raji , Metcalf Chester A. , Dalgarno David C. , Sawyer Tomi K. , Gallick Gary E. , Sood Anil K.
Published: September 2006

Src, a nonreceptor tyrosine kinase, is a key mediator for multiple signaling pathways that regulate critical cellular functions and is often aberrantly activated in a number of solid tumors, including ovarian carcinoma. The purpose of this study was to determine the role of activated Src inhibition on tumor growth in an orthotopic murine model of ovarian carcinoma. In vitro studies on HeyA8 and SKOV3ip1 cell lines revealed that Src inhibition by the Src-selective inhibitor, AP23846, occurred within 1 hour and responded in a dose-dependent manner. Furthermore, Src inhibition enhanced the cytotoxicity of docetaxel in both chemosensitive and chemoresistant ovarian cancer cell lines, HeyA8 and HeyA8-MDR, respectively. In vivo, Src inhibition by AP23994, an orally bioavailable analogue of AP23846, significantly decreased tumor burden in HeyA8 (P = 0.02), SKOV3ip1 (P = 0.01), as well as HeyA8-MDR (P < 0.03) relative to the untreated controls. However, the greatest effect on tumor reduction was observed in combination therapy with docetaxel (P < 0.001, P = 0.002, and P = 0.01, for the above models, respectively). Proliferating cell nuclear antigen staining showed that Src inhibition alone (P = 0.02) and in combination with docetaxel (P = 0.007) significantly reduced tumor proliferation. In addition, Src inhibition alone and in combination with docetaxel significantly down-regulated tumoral production of vascular endothelial growth factor and interleukin 8, whereas combination therapy decreased the microvessel density (P = 0.02) and significantly affected vascular permeability (P < 0.05). In summary, Src inhibition with AP23994 has potent antiangiogenic effects and significantly reduces tumor burden in preclinical ovarian cancer models. Thus, Src inhibition may be an attractive therapeutic approach for patients with ovarian carcinoma. (Cancer Res 2006; 66(17): 8633-9)
Smad3 Deficiency Promotes Tumorigenesis in the Distal Colon of ApcMin/+ Mice
Authors: Sodir Nicole M. , Chen Xuan , Park Ryan , Nickel Andrea E. , Conti Peter S. , Moats Rex , Bading James R. , Shibata Darryl , Laird Peter W.
Published: September 2006

Colorectal cancer, one of the most common human malignancies in the Western world, is often subdivided based on tumor location in either the distal or proximal colon. Several mouse models have been developed to study human colorectal cancer, but few display this clear distinction between the two colonic locations. By crossing ApcMin/+ and Smad3 mutant mice, we showed that combined activation of the Wnt pathway and attenuation of the transforming growth factor-β (TGF-β) pathway causes high multiplicity and rapid onset of invasive tumorigenesis almost exclusively in the distal colon, closely mimicking the familial adenomatous polyposis (FAP) disease and consisting with distinct colorectal cancer etiologies based on tumor location. Transcriptional profiling revealed higher expression of several TGF-β activators in the normal distal mucosa than in proximal mucosa, suggesting a stronger reliance on TGF-β-mediated growth control in the distal than in the proximal colon. ApcMin/+Smad3−/− mice provide an alternative model to ApcMin/+ mice to study FAP and distal sporadic colorectal cancer. This model will be useful in dissecting mechanistic and etiologic differences between proximal and distal colonic cancer, whereas the confinement of tumorigenesis to the distal colon offers unique advantages in monitoring tumor progression by in vivo imaging. (Cancer Res 2006; 66(17): 8430-8)
Prohibitin and Cofilin Are Intracellular Effectors of Transforming Growth Factor β Signaling in Human Prostate Cancer Cells
Authors: Zhu Beibei , Fukada Kei , Zhu Haining , Kyprianou Natasha
Published: September 2006

A proteomic analysis was pursued to identify new signaling effectors of transforming growth factor β1 (TGF-β1) that serve as potential intracellular effectors of its apoptotic action in human prostate cancer cells. The androgen-sensitive and TGF-β-responsive human prostate cancer cells, LNCaP TβRII, were used as in vitro model. In response to TGF-β, significant posttranslational changes in two proteins temporally preceded apoptotic cell death. TGF-β mediated the nuclear export of prohibitin, a protein involved in androgen-regulated prostate growth, to the cytosol in the LNCaP TβRII cells. Cofilin, a protein involved in actin depolymerization, cell motility, and apoptosis, was found to undergo mitochondrial translocation in response to TGF-β before cytochrome c release. Loss-of-function approaches (small interfering RNA) to silence prohibitin expression revealed a modest decrease in the apoptotic response to TGF-β and a significant suppression in TGF-β-induced cell migration. Silencing Smad4 showed that the cellular localization changes associated with prohibitin and cofilin action in response to TGF-β are independent of Smad4 intracellular signaling. (Cancer Res 2006; 66(17): 8640-7)
Enhanced Antitumor Activity of Murine-Human Hybrid T-Cell Receptor (TCR) in Human Lymphocytes Is Associated with Improved Pairing and TCR/CD3 Stability
Authors: Cohen Cyrille J. , Zhao Yangbing , Zheng Zhili , Rosenberg Steven A. , Morgan Richard A.
Published: September 2006

Little is known about the biology of murine T-cell receptors (TCR) expressed in human cells. We recently observed that a murine anti-human p53 TCR is highly functional when expressed in human lymphocytes. Herein, we compare human and mouse TCR function and expression to delineate the molecular basis for the apparent superior biological activity of murine receptors in human T lymphocytes. To this end, we created hybrid TCRs where we swapped the original constant regions with either human or mouse ones, respectively. We showed that murine or “murinized” receptors were overexpressed on the surface of human lymphocytes compared with their human/humanized counterparts and were able to mediate higher levels of cytokine secretion when cocultured with peptide-pulsed antigen-presenting cells. Preferential pairing of murine constant regions and improved CD3 stability seemed to be responsible for these observations. These enhanced biological properties translated into significantly greater antitumor response mediated by TCR with mouse constant regions. Furthermore, we were able to circumvent the natural low avidity of class I MHC TCR in CD4+ cells by introducing the murinized TCR into CD4+ lymphocytes, giving them the ability to recognize melanoma tumors. These findings have implications for human TCR gene transfer therapy and may provide new insights into the biology of the TCR/CD3 complex. (Cancer Res 2006; 66(17): 8878-86)
Overexpression of Tissue Transglutaminase Leads to Constitutive Activation of Nuclear Factor-κB in Cancer Cells: Delineation of a Novel Pathway
Authors: Mann Aman P. , Verma Amit , Sethi Gautam , Manavathi Bramanandam , Wang Huamin , Fok Jansina Y. , Kunnumakkara Ajaikumar B. , Kumar Rakesh , Aggarwal Bharat B. , Mehta Kapil
Published: September 2006

The transcription factor nuclear factor-κB (NF-κB) plays an important role in regulating cell growth, apoptosis, and metastatic functions. Constitutive activation of NF-κB has been observed in various cancers; however, molecular mechanisms resulting in such activation remain elusive. Based on our previous results showing that drug-resistant and metastatic cancer cells have high levels of tissue transglutaminase (TG2) expression and that this expression can confer chemoresistance to certain types of cancer cells, we hypothesized that TG2 contributes to constitutive activation of NF-κB. Numerous lines of evidence showed that overexpression of TG2 is linked with constitutive activation of NF-κB. Tumor cells with overexpression of TG2 exhibited increased levels of constitutively active NF-κB. Activation of TG2 led to activation of NF-κB; conversely, inhibition of TG2 activity inhibited activation of NF-κB. Similarly, ectopic expression of TG2 caused activation of NF-κB, and inhibition of expression of TG2 by small interfering RNA abolished the activation of NF-κB. Our results further indicated that constitutive NF-κB reporter activity in pancreatic cancer cells is not affected by dominant-negative IκBα. Additionally, coimmunoprecipitation and confocal microscopy showed that IκBα is physically associated with TG2. Lastly, immunohistochemical analysis of pancreatic ductal carcinoma samples obtained from 61 patients further supported a strong correlation between TG2 expression and NF-κB activation/overexpression (P = 0.0098, Fisher's exact test). We conclude that TG2 induces constitutive activation of NF-κB in tumor cells via a novel pathway that is most likely independent of IκBα kinase. Therefore, TG2 may be an attractive alternate target for inhibiting constitutive NF-κB activation and rendering cancer cells sensitive to anticancer therapies. (Cancer Res 2006; 66(17): 8788-95)
TMPRSS2:ERG Fusion-Associated Deletions Provide Insight into the Heterogeneity of Prostate Cancer
Authors: Perner Sven , Demichelis Francesca , Beroukhim Rameen , Schmidt Folke H. , Mosquera Juan-Miguel , Setlur Sunita , Tchinda Joelle , Tomlins Scott A. , Hofer Matthias D. , Pienta Kenneth G. , Kuefer Rainer , Vessella Robert , Sun Xiao-Wei , Meyerson Matthew , Lee Charles , Sellers William R. , Chinnaiyan Arul M. , Rubin Mark A.
Published: September 2006

Prostate cancer is a common and clinically heterogeneous disease with marked variability in progression. The recent identification of gene fusions of the 5′-untranslated region of TMPRSS2 (21q22.3) with the ETS transcription factor family members, either ERG (21q22.2), ETV1 (7p21.2), or ETV4 (17q21), suggests a mechanism for overexpression of the ETS genes in the majority of prostate cancers. In the current study using fluorescence in situ hybridization (FISH), we identified the TMPRSS2:ERG rearrangements in 49.2% of 118 primary prostate cancers and 41.2% of 18 hormone-naive lymph node metastases. The FISH assay detected intronic deletions between ERG and TMPRSS2 resulting in TMPRSS2:ERG fusion in 60.3% (35 of 58) of the primary TMPRSS2:ERG prostate cancers and 42.9% (3 of 7) of the TMPRSS2:ERG hormone-naive lymph node metastases. A significant association was observed between TMPRSS2:ERG rearranged tumors through deletions and higher tumor stage and the presence of metastatic disease involving pelvic lymph nodes. Using 100K oligonucleotide single nucleotide polymorphism arrays, a homogeneous deletion site between ERG and TMPRSS2 on chromosome 21q22.2-3 was identified with two distinct subclasses distinguished by the start point of the deletion at either 38.765 or 38.911 Mb. This study confirms that TMPRSS2:ERG is fused in approximately half of the prostate cancers through deletion of genomic DNA between ERG and TMPRSS2. The deletion as cause of TMPRSS2:ERG fusion is associated with clinical features for prostate cancer progression compared with tumors that lack the TMPRSS2:ERG rearrangement. (Cancer Res 2006; 66(17): 8337-41)
Type I Collagen Receptor (α2β1) Signaling Promotes the Growth of Human Prostate Cancer Cells within the Bone
Authors: Hall Christopher L. , Dai JinLu , van Golen Kenneth L. , Keller Evan T. , Long Michael W.
Published: September 2006

The most frequent site of prostate cancer metastasis is the bone. Adhesion to bone-specific factors may facilitate the selective metastasis of prostate cancer to the skeleton. Therefore, we tested whether prostate cancer bone metastasis is mediated by binding to type I collagen, the most abundant bone protein. We observed that only bone metastatic prostate cancer cells bound collagen I, whereas cells that form only visceral metastases failed to bind collagen. To confirm the relationship between collagen adhesion and bone metastatic potential, a collagen-binding variant of human LNCaP prostate cancer cells was derived through serial passage on type I collagen (LNCaPcol). Fluorescence-activated cell sorting analysis showed that LNCaPcol cells express increased levels of the integrin collagen I receptor α2β1 compared with LNCaP cells. Antibodies to the α2β1 complex inhibited LNCaPcol binding to collagen, confirming that integrins mediated the attachment. Correspondingly, LNCaPcol cells displayed enhanced chemotactic migration toward collagen I compared with LNCaP cells, an activity that could be blocked with α2β1 antibodies. To directly test the role of α2β1-dependent collagen binding in bone metastasis, LNCaP and LNCaPcol cells were injected into the tibia of nude mice. After 9 weeks, 7 of 13 (53%) mice injected with LNCaPcol developed bone tumors, whereas 0 of 8 mice injected with LNCaP cells had evidence of boney lesions. LNCaPcol cells were found to express increased levels of the metastasis-promoting RhoC GTPase compared with parental LNCaP. We conclude that collagen I attachment mediated by α2β1 initiates motility programs through RhoC and suggest a mechanism for prostate cancer metastasis to the bone. (Cancer Res 2006; 66(17): 8648-54)
Genome-wide Hypomethylation in Human Glioblastomas Associated with Specific Copy Number Alteration, Methylenetetrahydrofolate Reductase Allele Status, and Increased Proliferation
Authors: Cadieux Benoît , Ching Tsui-Ting , VandenBerg Scott R. , Costello Joseph F.
Published: September 2006

Genome-wide reduction in 5-methylcytosine is an epigenetic hallmark of human tumorigenesis. Experimentally induced hypomethylation in mice promotes genomic instability and is sufficient to initiate tumorigenesis. Here, we report that global hypomethylation is common in primary human glioblastomas [glioblastoma multiforme (GBM)] and can affect up to an estimated 10 million CpG dinucleotides per haploid tumor genome. Demethylation involves satellite 2 (Sat2) pericentromeric DNA at chromosomes 1 and 16, the subtelomeric repeat sequence D4Z4 at chromosomes 4q and 10q, and interspersed Alu elements. Severe hypomethylation of Sat2 sequences is associated with copy number alterations of the adjacent euchromatin, suggesting that hypomethylation may be one factor predisposing to specific genetic alterations commonly occurring in GBMs. An additional apparent consequence of global hypomethylation is reactivation of the cancer-testis antigen MAGEA1 via promoter demethylation, but only in GBMs and GBM cell lines exhibiting a 5-methylcytosine content below a threshold of ∼50%. Primary GBMs with significant hypomethylation tended to be heterozygous or homozygous for the low-functioning Val allele of the rate-limiting methyl group metabolism gene methylenetetrahydrofolate reductase (MTHFR), or had a deletion encompassing this gene at 1p36. Tumors with severe genomic hypomethylation also had an elevated proliferation index and deletion of the MTHFR gene. These data suggest a model whereby either excessive cell proliferation in the context of inadequate methyl donor production from MTHFR deficiency promotes genomic hypomethylation and further genomic instability, or that MTHFR deficiency–associated demethylation leads to increased proliferative activity in GBM. (Cancer Res 2006; 66(17): 8469-76)
Degradation of Tob1 Mediated by SCFSkp2-Dependent Ubiquitination
Authors: Hiramatsu Yoshihiro , Kitagawa Kyoko , Suzuki Toru , Uchida Chiharu , Hattori Takayuki , Kikuchi Hirotoshi , Oda Toshiaki , Hatakeyama Shigetsugu , Nakayama Keiichi I. , Yamamoto Tadashi , Konno Hiroyuki , Kitagawa Masatoshi
Published: September 2006

Tob1, a member of the Tob/BTG family, is involved in the control of G1-S progression by suppressing cyclin D1 expression and acts as a tumor suppressor gene. Tob1 was reported to have a quick turnover through the ubiquitin-proteasome pathway, but proteins involved in this process are still unknown. We showed that Skp2, a substrate-targeting subunit of the SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complex, was involved in ubiquitin-dependent degradation of Tob1. Skp2 interacted with Tob1 and facilitated ubiquitination of Tob1 in intact cells as well as in vitro. Skp2 mutants without the F-box or leucine rich repeat were not able to bind to Tob1 and did not enhance ubiquitination of Tob1. Tob1 was stabilized in both Skp2−/− mouse fibroblasts and Skp2 knockdown HeLa cells. Moreover, cyclin D1 expression was suppressed in Skp2 knockdown HeLa cells. These data suggest that Tob1 is a novel target for degradation by the SCF-Skp2 ubiquitin ligase. (Cancer Res 2006; 66(17): 8477-83)
N-myc Is an Essential Downstream Effector of Shh Signaling during both Normal and Neoplastic Cerebellar Growth
Authors: Hatton Beryl A. , Knoepfler Paul S. , Kenney Anna Marie , Rowitch David H. , de Alborán Ignacio Moreno , Olson James M. , Eisenman Robert N.
Published: September 2006

We examined the genetic requirements for the Myc family of oncogenes in normal Sonic hedgehog (Shh)–mediated cerebellar granule neuronal precursor (GNP) expansion and in Shh pathway–induced medulloblastoma formation. In GNP-enriched cultures derived from N-mycFl/Fl and c-mycFl/Fl mice, disruption of N-myc, but not c-myc, inhibited the proliferative response to Shh. Conditional deletion of c-myc revealed that, although it is necessary for the general regulation of brain growth, it is less important for cerebellar development and GNP expansion than N-myc. In vivo analysis of compound mutants carrying the conditional N-myc null and the activated Smoothened (ND2:SmoA1) alleles showed, that although granule cells expressing the ND2:SmoA1 transgene are present in the N-myc null cerebellum, no hyperproliferation or tumor formation was detected. Taken together, these findings provide in vivo evidence that N-myc acts downstream of Shh/Smo signaling during GNP proliferation and that N-myc is required for medulloblastoma genesis even in the presence of constitutively active signaling from the Shh pathway. (Cancer Res 2006; 66(17): 8655-61)
Male Germ Cell–Associated Kinase, a Male-Specific Kinase Regulated by Androgen, Is a Coactivator of Androgen Receptor in Prostate Cancer Cells
Authors: Ma Ai-Hong , Xia Liang , Desai Sonal J. , Boucher David L. , Guan Yi , Shih Hsiu-Ming , Shi Xu-Bao , deVere White Ralph W. , Chen Hong-Wu , Tepper Cliff G. , Kung Hsing-Jien
Published: September 2006

Androgen receptor (AR) is a ligand-induced transcriptional factor, which plays an important role in the normal development of prostate as well as in the progression of prostate cancer. Numerous coactivators, which associate with AR and function to remodel chromatin and recruit RNA polymerase II to enhance the transcriptional potential of AR, have been identified. Among these coactivators, few are protein kinases. In this study, we describe the characterization of a novel protein kinase, male germ cell–associated kinase (MAK), which serves as a coactivator of AR. We present evidence, which indicates that (a) MAK physically associates with AR (MAK and AR are found to be coprecipitated from cell extracts, colocalized in nucleus, and corecruited to prostate-specific antigen promoter in LNCaP as well as in transfected cells); (b) MAK is able to enhance AR transactivation potential in an androgen- and kinase-dependent manner in several prostate cancer cells and synergize with ACTR/steroid receptor coactivator-3 coactivator; (c) small hairpin RNA (shRNA) knocks down MAK expression resulting in the reduction of AR transactivation ability; (d) MAK-shRNA or kinase-dead mutant, when introduced into LNCaP cells, reduces the growth of the cells; and (e) microarray analysis of LNCaP cells carrying kinase-dead MAK mutant showed a significant impediment of AR signaling, indicating that endogenous MAK plays a general role in AR function in prostate cancer cells and likely to be a general coactivator of AR in prostate tissues. The highly restricted expression of this kinase makes it a potentially useful target for intervention of androgen independence. (Cancer Res 2006; 66(17): 8439-47)
p53 Is Preferentially Recruited to the Promoters of Growth Arrest Genes p21 and GADD45 during Replicative Senescence of Normal Human Fibroblasts
Authors: Jackson James G. , Pereira-Smith Olivia M.
Published: September 2006

Replicative senescence is the terminal growth arrest that most normal human cells enter into after a fixed number of divisions in vitro, limiting the proliferative potential of a cell and preventing genomic instability caused by critically short telomeres. Thus, senescence presents a tumor-suppressive mechanism and a barrier to tumor formation. However, senescent cells are inherently resistant to apoptosis and, as they accumulate in aging tissues, may contribute to organ dysfunction and promote tumor progression as part of the stromal environment. Replicative life span in normal human cells can be extended by inactivation of the tumor suppressor gene p53 or its direct target, the cyclin-dependent kinase inhibitor p21, suggesting a direct role for this pathway in senescence. However, p53 recruitment to promoters of target genes during replicative senescence has not been shown in live cells. In this study, we used chromatin immunoprecipitation to determine that p53 preferentially occupied the promoters of growth arrest genes p21 and GADD45 in senescent normal human diploid fibroblasts but not the promoters of other target genes that recruited p53 following doxorubicin-induced DNA damage, such as apoptosis regulators TNFRSF10b, TNFRSF6, and PUMA. This differential recruitment of p53 in senescent versus doxorubicin-treated fibroblasts was accompanied by differences in post-translational modification of p53. These data provide mechanisms for both the growth arrest mediated by p53 and the resistant nature of senescent cells to apoptosis despite p53 activity. (Cancer Res 2006; 66(17): 8356-60)