bluuebunny/crossref_metadata_2025 huggingface dataset:
Down-regulation of Epidermal Growth Factor Receptor by Selective Expansion of a 5′-End Regulatory Dinucleotide Repeat in Colon Cancer with Microsatellite Instability
Authors: Baranovskaya Svetlana , Martin Yolanda , Alonso Sergio , Pisarchuk Ksenia L. , Falchetti Mario , Dai Yuichi , Khaldoyanidi Sophia , Krajewski Stan , Novikova Inna , Sidorenko Yuri S. , Perucho Manuel , Malkhosyan Sergei R.
Published: July 2009

Purpose: The epidermal growth factor receptor (EGFR) is overexpressed in several tumor types, and its expression is influenced by the length of a 5′-end microsatellite repeat (CA)n: the longer the repeat, the lower the expression. Dinucleotide repeats accumulate insertion/deletion types of mutations in tumors with microsatellite instability. We designed this study to estimate the occurrence of these mutations in EGFR(CA)n and their relevance in carcinogenesis of microsatellite instability–positive colon and gastric tumors.

Experimental Design: We analyzed the frequency of EGFR(CA)n mutations in vivo in 55 colorectal and 14 gastric microsatellite instability–positive cancers, and in vitro in single-cell clone cultures of microsatellite instability–positive colon tumor cell line LS174. Single-cell clone cultures with different repeat lengths were analyzed by fluorescent-activated cell sorter for EGFR cell-surface expression. A correlation analysis was done between EGFR(CA)n mutations and mutations in KRAS, BRAF, and p53.

Results: Unlike single-cell clone cultures, which exhibited higher rate of deletions compared with insertions, most of EGFR(CA)n mutations in colon and gastric tumors were insertions. Longer EGFR(CA)n correlated with lower EGFR cell-surface expression in single-cell clone cultures. In colon cancers, the elongation of the repeat was associated negatively with mutations in KRAS and BRAF, but not in p53.

Conclusions: The EGFR(CA)n elongation observed in tumors cannot be explained by an intrinsic property of this repeat favoring insertions versus deletions. Instead, a selection for repeat elongation occurs in microsatellite instability–positive tumors, leading to EGFR down-regulation. These findings suggest that in microsatellite instability–positive tumors current therapies targeting EGFR overexpression may have either no effect or an opposite to the expected effect.
The Influence of Prostate Volume on Prostate-Specific Antigen Performance: Implications for the Prostate Cancer Prevention Trial Outcomes
Authors: Elliott Christopher S. , Shinghal Rajesh , Presti Joseph C.
Published: July 2009

Purpose: Although showing a 25% reduction in the biopsy prevalence of cancer compared with placebo in the Prostate Cancer Prevention Trial, finasteride was associated with a higher prevalence of high-grade disease. This observation was driven by “for-cause” biopsies. We sought to understand how volume-dependent changes in prostate-specific antigen test performance characteristics may have contributed.

Experimental Design: A retrospective review was done on 1,304 men referred for initial biopsy with a prostate-specific antigen between 4 and 10 ng/mL or an abnormal digital rectal examination. Receiver-operator curves and positive predictive values were ascertained for prostate-specific antigen stratified by diagnosis and prostate volume.

Results: The performance of prostate-specific antigen changed for any and high-grade (Gleason, ≥3 + 4) cancer in a volume-specific manner. For any cancer, the area under the curve (AUC) decreased from 0.758 to 0.629 to 0.520 as prostate volume increased (<30, 30-50, >50 cm3, respectively). For high-grade cancer, a similar trend was shown (AUC, 0.712, 0.639, and 0.497, respectively). The positive predictive value of a prostate-specific antigen of ≥4 ng/mL was also affected by prostate volume. Trends for Gleason ≤ 6 decreased as prostate volume increased (positive predictive value for <30 cm3, 25.0%; positive predictive value for 30-50 cm3, 23.8%; and positive predictive value for >50 cm3, 17.3%). A more significant trend was seen for high-grade cancer (positive predictive value for <30 cm3, 39.0%; positive predictive value for 30-50 cm3, 22.3%; and positive predictive value for >50 cm3, 10.7%).

Conclusion: Decreases in prostate volume over time and the resultant change in prostate-specific antigen performance characteristics may have contributed a bias toward the detection of high-grade disease in the finasteride arm of the Prostate Cancer Prevention Trial.
15-Hydroxyprostaglandin Dehydrogenase Is Down-regulated in Gastric Cancer
Authors: Thiel Alexandra , Ganesan Aparna , Mrena Johanna , Junnila Siina , Nykänen Antti , Hemmes Annabrita , Tai Hsin-Hsiung , Monni Outi , Kokkola Arto , Haglund Caj , Petrova Tatiana V. , Ristimäki Ari
Published: July 2009

Purpose: We have investigated the expression and regulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in gastric cancer.

Experimental Design: Clinical gastric adenocarcinoma samples were analyzed by immunohistochemistry and quantitative real-time PCR for protein and mRNA expression of 15-PGDH and for methylation status of 15-PGDH promoter. The effects of interleukin-1β (IL-1β) and epigenetic mechanisms on 15-PGDH regulation were assessed in gastric cancer cell lines.

Results: In a gastric cancer cell line with a very low 15-PGDH expression (TMK-1), the 15-PGDH promoter was methylated and treatment with a demethylating agent 5-aza-2′-deoxycytidine restored 15-PGDH expression. In a cell line with a relatively high basal level of 15-PGDH (MKN-28), IL-1β repressed expression of 15-PGDH mRNA and protein. This effect of IL-1β was at least in part attributed to inhibition of 15-PGDH promoter activity. SiRNA-mediated knockdown of 15-PGDH resulted in strong increase of prostaglandin E2 production in MKN-28 cells and increased cell growth of these cells by 31% in anchorage-independent conditions. In clinical gastric adenocarcinoma specimens, 15-PGDH mRNA levels were 5-fold lower in gastric cancer samples when compared with paired nonneoplastic tissues (n = 26) and 15-PGDH protein was lost in 65% of gastric adenocarcinomas (n = 210).

Conclusions: 15-PGDH is down-regulated in gastric cancer, which could potentially lead to accelerated tumor progression. Importantly, our data indicate that a proinflammatory cytokine linked to gastric carcinogenesis, IL-1β, suppresses 15-PGDH expression at least partially by inhibiting promoter activity of the 15-PGDH gene.
Imaging Biomarkers Predict Response to Anti-HER2 (ErbB2) Therapy in Preclinical Models of Breast Cancer
Authors: Shah Chirayu , Miller Todd W. , Wyatt Shelby K. , McKinley Eliot T. , Olivares Maria Graciela , Sanchez Violeta , Nolting Donald D. , Buck Jason R. , Zhao Ping , Ansari M. Sib , Baldwin Ronald M. , Gore John C. , Schiff Rachel , Arteaga Carlos L. , Manning H. Charles
Published: July 2009

Purpose: To evaluate noninvasive imaging methods as predictive biomarkers of response to trastuzumab in mouse models of HER2-overexpressing breast cancer. The correlation between tumor regression and molecular imaging of apoptosis, glucose metabolism, and cellular proliferation was evaluated longitudinally in responding and nonresponding tumor-bearing cohorts.

Experimental Design: Mammary tumors from MMTV/HER2 transgenic female mice were transplanted into syngeneic female mice. BT474 human breast carcinoma cell line xenografts were grown in athymic nude mice. Tumor cell apoptosis (NIR700-Annexin V accumulation), glucose metabolism [2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography ([18F]FDG-PET)], and proliferation [3′-[18F]fluoro-3′-deoxythymidine-PET ([18F]FLT-PET)] were evaluated throughout a biweekly trastuzumab regimen. Imaging metrics were validated by direct measurement of tumor size and immunohistochemical analysis of cleaved caspase-3, phosphorylated AKT, and Ki67.

Results: NIR700-Annexin V accumulated significantly in trastuzumab-treated MMTV/HER2 and BT474 tumors that ultimately regressed but not in nonresponding or vehicle-treated tumors. Uptake of [18F]FDG was not affected by trastuzumab treatment in MMTV/HER2 or BT474 tumors. [18F]FLT-PET imaging predicted trastuzumab response in BT474 tumors but not in MMTV/HER2 tumors, which exhibited modest uptake of [18F]FLT. Close agreement was observed between imaging metrics and immunohistochemical analysis.

Conclusions: Molecular imaging of apoptosis accurately predicts trastuzumab-induced regression of HER2+ tumors and may warrant clinical exploration to predict early response to neoadjuvant trastuzumab. Trastuzumab does not seem to alter glucose metabolism substantially enough to afford [18F]FDG-PET significant predictive value in this setting. Although promising in one preclinical model, further studies are required to determine the overall value of [18F]FLT-PET as a biomarker of response to trastuzumab in HER2+ breast cancer.
A Phase I Clinical Trial of Darinaparsin in Patients with Refractory Solid Tumors
Authors: Tsimberidou Apostolia Maria , Camacho Luis H. , Verstovsek Srdan , Ng Chaan , Hong David S. , Uehara Cynthia K. , Gutierrez Catalina , Daring Shawn , Stevens Jan , Komarnitsky Philip B. , Schwartz Brian , Kurzrock Razelle
Published: July 2009

Purpose: Darinaparsin, an organic arsenic, targets essential cell survival pathways. We determined the dose-limiting toxicity (DLT) and maximum tolerated dose of darinaparsin in patients with advanced cancer.

Experimental Design: Patients with solid malignancies refractory to conventional therapies were treated with i.v. darinaparsin daily for 5 days every 4 weeks. The starting dose (78 mg/m2) escalated to 109, 153, 214, 300, 420, and 588 mg/m2. A conventional “3 + 3” design was used.

Results: Forty patients (median age, 61.5 years; median number of prior therapies, 5) received therapy; 106 cycles were given (median, 2; range, 1-12). Twenty patients reported no drug-related toxicities. No DLTs were reported at a dose of <420 mg/m2. At 588 mg/m2, two of four patients developed DLTs, including grade 3 altered mental status and ataxia. Of four patients treated at the de-escalated dose of 500 mg/m2, one developed similar toxicities. De-escalating the dose to 420 mg/m2 (n = 8) resulted in two neurologic DLTs. Further de-escalation to 300 mg/m2 (n = 3) resulted in no drug-related toxicities. Arsenic plasma levels peaked on treatment day 3, plateaued on day 5, and returned to baseline on day 7. Plasma levels varied within cohorts but increased with increasing doses. The median plasma arsenic half-life was 16.2 hours. Seven (17.5%) patients had stable disease for ≥4 months (median, 6; range, 4-11), including 4 of 17 with colorectal and 2 of 3 with renal cancer.

Conclusions: The recommended dose for phase II trials is 300 mg/m2 i.v. given daily for 5 days every 4 weeks.
EGFR/KRAS/BRAF Mutations in Primary Lung Adenocarcinomas and Corresponding Locoregional Lymph Node Metastases
Authors: Schmid Katharina , Oehl Natalie , Wrba Fritz , Pirker Robert , Pirker Christine , Filipits Martin
Published: July 2009

Purpose: The epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated with different frequencies in non–small cell lung cancer and mutations predict clinical response to EGFR inhibitors. The present study compared the mutational status of EGFR, KRAS, and BRAF in primary tumors with the one in corresponding lymph node metastases.

Experimental Design: Direct bidirectional sequencing of EGFR gene exons 18 to 21, KRAS gene codons 12/13 and 61 to 68, and BRAF exon 15 was done on 96 paired samples of primary lung adenocarcinomas and corresponding locoregional lymph node metastases. In addition, comparative genomic hybridization analyses in two pairs of corresponding primary and metastatic tumor samples with discordant EGFR mutation status were done.

Results: Mutations in EGFR, KRAS, and BRAF were observed in 7 (7%), 36 (38%), and 2 (2%) patients, respectively. Interestingly, KRAS mutations were observed in two patients with an EGFR mutation. Mutations in primary tumors and lymph node metastases were identical in 1 of 7 (14%) patients in case of EGFR and 11 of 36 (31%) patients in case of KRAS. One patient harbored different KRAS mutations in primary and corresponding metastatic tumors. Comparative genomic hybridization analysis revealed similar patterns of chromosomal changes, strongly supporting a common clonal origin of primary tumors and metastases.

Conclusions: The possibility of differences in the mutational status of EGFR, KRAS, BRAF between primary tumors and corresponding lymph node metastases should be considered whenever these mutations are used for the selection of patients for EGFR-directed tyrosine kinase inhibitor therapy.
Antiangiogenesis Targeting Tumor Microenvironment Synergizes Glucuronide Prodrug Antitumor Activity
Authors: Juan Ting-Yi , Roffler Steve R. , Hou Hsien-San , Huang Shih-Ming , Chen Kai-Chuan , Leu Yu-Lin , Prijovich Zeljko M. , Yu Cheng-Ping , Wu Chang-Chieh , Sun Guang-Huan , Cha Tai-Lung
Published: July 2009

Purpose: This study is aimed at investigating the in vivo antitumor activity of a novel cell-impermeable glucuronide prodrug, 9-aminocamptothecin glucuronide (9ACG), and elucidating the synergistically antitumor effects of antiangiogenesis therapy by targeting the tumor microenvironment.

Experimental Design: We analyzed the antitumor effects of 9ACG alone or combined with antiangiogenic monoclonal antibody DC101 on human tumor xenografts by measuring tumor growth and mouse survival in BALB/c nu/nu nude and NOD/SCID mice. The drug delivery, immune response, and angiogenesis status in treated tumors were assessed by high performance liquid chromatography, immunohistochemistry, and immunofluorescence assays.

Results: We developed a nontoxic and cell-impermeable glucuronide prodrug, 9ACG, which can only be activated by extracellular β-glucuronidase to become severely toxic. 9ACG possesses potent antitumor activity against human tumor xenografts in BALB/c nu/nu nude mice but not for tumors implanted in NOD/SCID mice deficient in macrophages and neutrophils, suggesting that these cells play an important role in activating 9ACG in the tumor microenvironment. Most importantly, antiangiogenic monoclonal antibody DC101 potentiated single-dose 9ACG antitumor activity and prolonged survival of mice bearing resistant human colon tumor xenografts by providing strong β-glucuronidase activity and prodrug delivery through enhancing inflammatory cell infiltration and normalizing tumor vessels in the tumor microenvironment. We also show that inflammatory cells (neutrophils) were highly infiltrated in advanced human colon cancer tissues compared with normal counterparts.

Conclusions: Our study provides in vivo evidence that 9ACG has potential for prodrug monotherapy or in combination with antiangiognesis treatment for tumors with infiltration of macrophage or neutrophil inflammatory cells.
Clinical Relevance of a Pharmacogenetic Approach Using Multiple Candidate Genes to Predict Response and Resistance to Imatinib Therapy in Chronic Myeloid Leukemia
Authors: Kim Dong Hwan (Dennis) , Sriharsha Lakshmi , Xu Wei , Kamel-Reid Suzanne , Liu Xiangdong , Siminovitch Katherine , Messner Hans A. , Lipton Jeffrey H.
Published: July 2009

Purpose: Imatinib resistance is major cause of imatinib mesylate (IM) treatment failure in chronic myeloid leukemia (CML) patients. Several cellular and genetic mechanisms of imatinib resistance have been proposed, including amplification and overexpression of the BCR/ABL gene, the tyrosine kinase domain point mutations, and MDR1 gene overexpression.

Experimental Design: We investigated the impact of 16 single nucleotide polymorphisms (SNP) in five genes potentially associated with pharmacogenetics of IM, namely ABCB1, multidrug resistance 1; ABCG2, breast-cancer resistance protein; CYP3A5, cytochrome P450-3A5; SLC22A1, human organic cation transporter 1; and AGP, α1-acid glycoprotein. The DNAs from peripheral blood samples in 229 patients were genotyped.

Results: The GG genotype in ABCG2 (rs2231137), AA genotype in CYP3A5 (rs776746), and advanced stage were significantly associated with poor response to IM especially for major or complete cytogenetic response, whereas the GG genotype at SLC22A1 (rs683369) and advanced stage correlated with high rate of loss of response or treatment failure to IM therapy.

Conclusions: We showed that the treatment outcomes of imatinib therapy could be predicted using a novel, multiple candidate gene approach based on the pharmacogenetics of IM.
SMAD4 Gene Mutations Are Associated with Poor Prognosis in Pancreatic Cancer
Authors: Blackford Amanda , Serrano Oscar K. , Wolfgang Christopher L. , Parmigiani Giovanni , Jones Siân , Zhang Xiaosong , Parsons D. Williams , Lin Jimmy Cheng-Ho , Leary Rebecca J. , Eshleman James R. , Goggins Michael , Jaffee Elizabeth M. , Iacobuzio-Donahue Christine A. , Maitra Anirban , Cameron John L. , Olino Kelly , Schulick Richard , Winter Jordan , Herman Joseph M. , Laheru Daniel , Klein Alison P. , Vogelstein Bert , Kinzler Kenneth W. , Velculescu Victor E. , Hruban Ralph H.
Published: July 2009

Purpose: Recently, the majority of protein coding genes were sequenced in a collection of pancreatic cancers, providing an unprecedented opportunity to identify genetic markers of prognosis for patients with adenocarcinoma of the pancreas.

Experimental Design: We previously sequenced more than 750 million base pairs of DNA from 23,219 transcripts in a series of 24 adenocarcinomas of the pancreas. In addition, 39 genes that were mutated in more than one of these 24 cancers were sequenced in a separate panel of 90 well-characterized adenocarcinomas of the pancreas. Of these 114 patients, 89 underwent pancreaticoduodenectomy, and the somatic mutations in these cancers were correlated with patient outcome.

Results: When adjusted for age, lymph node status, margin status, and tumor size, SMAD4 gene inactivation was significantly associated with shorter overall survival (hazard ratio, 1.92; 95% confidence interval, 1.20-3.05; P = 0.006). Patients with SMAD4 gene inactivation survived a median of 11.5 months, compared with 14.2 months for patients without SMAD4 inactivation. By contrast, mutations in CDKN2A or TP53 or the presence of multiple (≥4) mutations or homozygous deletions among the 39 most frequently mutated genes were not associated with survival.

Conclusions: SMAD4 gene inactivation is associated with poorer prognosis in patients with surgically resected adenocarcinoma of the pancreas.
Partially Matched Related Donor Transplantation Can Achieve Outcomes Comparable with Unrelated Donor Transplantation for Patients with Hematologic Malignancies
Authors: Xiao-Jun Huang , Lan-Ping Xu , Kai-Yan Liu , Dai-Hong Liu , Yu Wang , Huan Chen , Yu-Hong Chen , Wei Han , Jing-Zhi Wang , Yao Chen , Xiao-Hui Zhang , Hong-Xia Shi , Feng-Rong Wang , Fei-Fei Tang
Published: July 2009

Purpose: The study aimed to compare the outcomes of patients undergoing hematopoietic stem cell transplantation (HSCT) from partially matched related donors (PMRD) and unrelated donors (URD) for hematologic malignancies without the use of in vitro T cell depletion.

Experimental Design: HSCT was done on 297 consecutive patients from URDs (n = 78) and PMRDs (n = 219) during the same time period. Incidences of graft-versus-host disease (GVHD), relapse, nonrelapse mortality, overall survival, and leukemia-free survival between the PMRD and URD groups were compared.

Results: All patients achieved full engraftment. The cumct65ulative incidences of grades II to IV acute GVHD in the PMRD and URD cohorts were 47% [95% confidence interval (95% CI), 33-62%] versus 31% (CI, 20-42%; P = 0.033), with a relative risk of 1.72 (95% CI, 1.01-2.94; P = 0.046). The incidence of chronic GVHD did not differ significantly between the two cohorts (P = 0.17). The 2-year incidences of nonrelapse mortality and relapse were 20% (CI, 15-26%) versus 18% (CI, 10-27%), with P = 0.98, and 12% (CI, 8-16%) versus 18% (CI, 10-27%), with P = 0.12, for the PMRD versus the URD cohort, respectively. The 4-year overall survival and leukemia-free survival were 74% (CI, 67-80%) versus 74% (CI, 62-85%), with P = 0.98, and 67% (CI, 59-75%) versus 61% (CI, 47-74%), with P = 0.74, respectively.

Conclusions: Our comparisons show that every major end point, including relapse, nonrelapse mortality, overall survival, and leukemia-free survival, was comparable between the PMRD and the URD groups.
Revealing Tumor Immunity after Hematopoietic Stem Cell Transplantation
Authors: Wu Catherine J. , Ritz Jerome
Published: July 2009

Allogeneic hematopoietic stem cell transplantation is currently the most effective method for inducing tumor immunity. However, the diversity of target antigens recognized by donor T cells has not been established. New studies show that tumor-reactive T cells are directed against diverse tumor-specific targets as well as minor histocompatibility antigens.
Renal and liver tumors inTsc2+/− mice, a model of tuberous sclerosis complex, do not respond to treatment with atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor
Authors: Finlay Geraldine A. , Malhowski Amy J. , Polizzi Kristen , Malinowska-Kolodziej Izabela , Kwiatkowski David J.
Published: July 2009

Inactivating mutations of the tumor suppressor gene TSC2 are associated with tumorigenesis in tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis. Statins, as 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, have the potential to limit the growth of these tumors by limiting the isoprenylation of activated GTPases in Tsc2-null cells. We tested atorvastatin as a therapy for (a) ethylnitrosourea (ENU)-enhanced renal cystadenoma and (b) spontaneous liver hemangioma in 129Sv/Jae Tsc2+/− mice. ENU-treated Tsc2+/− mice were given atorvastatin chow (0.1%, w/w) for 1 or 3 months before sacrifice at 6 months; 129Sv/Jae Tsc2+/− mice were given atorvastatin chow (0.1%, w/w) for 6 months before sacrifice at 12 months. All treatment groups were compared with mice of identical genotype and strain background that were fed control chow. Pathologic analyses revealed a predominance of renal cystadenoma in ENU-treated and liver hemangioma in non–ENU-treated 129Sv/Jae Tsc2+/− mice. In both cohorts, serum cholesterol levels and levels of phosphorylated S6 and GTP-RhoA in healthy tissue were significantly (>50%) reduced in atorvastatin-treated mice as compared with controls. Following atorvastatin treatment, no significant reduction in tumor size, morphology, or phosphorylated S6 levels was observed for either ENU-associated renal cystadenoma or spontaneous liver hemangioma as compared with the untreated groups. In conclusion, although the marked reduction in cholesterol levels indicates that atorvastatin was effective as an 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, it did not inhibit the growth of tumors that develop in these Tsc2+/− models, suggesting that it is unlikely to have benefit as a single-agent therapy for TSC-associated tumors.[Mol Cancer Ther 2009;8(7):1799–807]
EB1089 inhibits the parathyroid hormone–related protein–enhanced bone metastasis and xenograft growth of human prostate cancer cells
Authors: Bhatia Vandanajay , Saini Manjit K. , Shen Xiaoli , Bi Lian X. , Qiu Suimin , Weigel Nancy L. , Falzon Miriam
Published: July 2009

Parathyroid hormone–related protein (PTHrP) plays a major role in prostate carcinoma progression and bone metastasis. Once prostate cancers become androgen-independent, treatment options become limited. Vitamin D analogues represent a potentially valuable class of agents in this clinical context. Using the prostate cancer cell line C4-2 as a model, we studied the effects of PTHrP and the noncalcemic vitamin D analogue EB1089 on markers of prostate cancer cell progression in vitro and in vivo. C4-2 is a second-generation androgen-independent LNCaP subline that metastasizes to the lymph nodes and bone when injected into nude mice and produces mixed lytic/blastic lesions, mimicking the in vivo situation. We report that PTHrP increases cell migration and invasion, and that a pathway via which EB1089 inhibits these processes is through down-regulation of PTHrP expression. PTHrP also increases anchorage-independent cell growth in vitro and xenograft growth in vivo; EB1089 reverses these effects. The in vivo PTHrP effects are accompanied by increased tumor cell proliferation and survival. Treatment with EB1089 reverses the proliferative but not the antiapoptotic effects of PTHrP. PTHrP also increases intratumor vessel density and vascular endothelial growth factor expression; EB1089 reverses these effects. Intracardially injected C4-2 cells produce predominantly osteoblastic lesions; PTHrP overexpression decreases the latency, increases the severity and alters the bone lesion profile to predominantly osteolytic. EB1089 largely reverses these PTHrP effects. A direct correlation between PTHrP immunoreactivity and increasing tumor grade is observed in human prostate cancer specimens. Thus, decreasing PTHrP production by treatment with vitamin D analogues may prove therapeutically beneficial for prostate cancer. [Mol Cancer Ther 2009;8(7):1787–98]
d,l-Sulforaphane causes transcriptional repression of androgen receptor in human prostate cancer cells
Authors: Kim Su-Hyeong , Singh Shivendra V.
Published: July 2009

d,l-Sulforaphane (SFN), a synthetic analogue of cruciferous vegetable–derived l-isomer, inhibits the growth of human prostate cancer cells in culture and in vivo and retards cancer development in a transgenic mouse model of prostate cancer. We now show that SFN treatment causes transcriptional repression of androgen receptor (AR) in LNCaP and C4-2 human prostate cancer cells at pharmacologic concentrations. Exposure of LNCaP and C4-2 cells to SFN resulted in a concentration-dependent and time-dependent decrease in protein levels of total AR as well as Ser210/213-phosphorylated AR. The SFN-mediated decline in AR protein level was accompanied by a decrease in intracellular as well as secreted levels of prostate-specific antigen, an AR-regulated gene product. The decrease in AR protein level resulting from SFN exposure was not reversed in the presence of the protein synthesis inhibitor cycloheximide. Reverse transcription-PCR analysis revealed a dose-dependent decrease in AR mRNA levels, indicating transcriptional repression of this ligand-activated transcription factor. The SFN treatment inhibited AR promoter activity as revealed by luciferase reporter assay. Synthetic androgen (R1881)–stimulated nuclear translocation of AR was markedly suppressed in the presence of SFN in both cell lines. The SFN treatment also inhibited R1881-stimulated proliferation of LNCaP cells. Naturally occurring thio analogues (iberverin, erucin, and berteroin), but not the sulfonyl analogues (cheirolin, erysolin, and alyssin sulfone), of SFN were also effective in reducing protein levels of AR in LNCaP cells. In conclusion, the present study shows for the first time that SFN treatment causes transcriptional repression of AR and inhibition of its nuclear localization in human prostate cancer cells. [Mol Cancer Ther 2009;8(7):1946–54]
Evaluation of current methods used to analyze the expression profiles of ATP-binding cassette transporters yields an improved drug-discovery database
Authors: Orina Josiah N. , Calcagno Anna Maria , Wu Chung-Pu , Varma Sudhir , Shih Joanna , Lin Min , Eichler Gabriel , Weinstein John N. , Pommier Yves , Ambudkar Suresh V. , Gottesman Michael M. , Gillet Jean-Pierre
Published: July 2009

The development of multidrug resistance (MDR) to chemotherapy remains a major challenge in the treatment of cancer. Resistance exists against every effective anticancer drug and can develop by multiple mechanisms. These mechanisms can act individually or synergistically, leading to MDR, in which the cell becomes resistant to a variety of structurally and mechanistically unrelated drugs in addition to the drug initially administered. Although extensive work has been done to characterize MDR mechanisms in vitro, the translation of this knowledge to the clinic has not been successful. Therefore, identifying genes and mechanisms critical to the development of MDR in vivo and establishing a reliable method for analyzing highly homologous genes from small amounts of tissue is fundamental to achieving any significant enhancement in our understanding of MDR mechanisms and could lead to treatments designed to circumvent it. In this study, we use a previously established database that allows the identification of lead compounds in the early stages of drug discovery that are not ATP-binding cassette (ABC) transporter substrates. We believe this can serve as a model for appraising the accuracy and sensitivity of current methods used to analyze the expression profiles of ABC transporters. We found two platforms to be superior methods for the analysis of expression profiles of highly homologous gene superfamilies. This study also led to an improved database by revealing previously unidentified substrates for ABCB1, ABCC1, and ABCG2, transporters that contribute to MDR. [Mol Cancer Ther 2009;8(7):2057–66]
Parathyroid Hormone-Related Protein Regulates Cell Survival Pathways via Integrin α6β4-Mediated Activation of Phosphatidylinositol 3-Kinase/Akt Signaling
Authors: Bhatia Vandanajay , Mula Ramanjaneya V. , Weigel Nancy L. , Falzon Miriam
Published: July 2009

Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissues and cancer cell lines. PTHrP enhances tumor cell growth and metastasis in vivo and up-regulates proinvasive integrin α6β4 expression in vitro. Hallmarks of malignant tumor cells include resistance to apoptosis and anchorage-independent cell growth. In this study, we used the human prostate cancer cell lines C4-2 and PC-3 as model systems to study the effects of PTHrP on these processes. We report that PTHrP protects these cells from doxorubicin-induced apoptosis and promotes anchorage-independent cell growth via an intracrine pathway. Conversely, autocrine/paracrine PTHrP action increases apoptosis in C4-2 cells and has no effect on apoptosis in PC-3 cells. The intracrine effects of PTHrP on apoptosis are mediated via activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. PTHrP also affects the phosphorylation state of Akt substrates implicated in apoptosis suppression, including glycogen synthase kinase-3 and Bad. The prosurvival effects of PTHrP are accompanied by increases in the ratio of antiapoptotic to proapoptotic members of the Bcl-2 family and in levels of c-myc. PTHrP also increases nuclear factor-κB activity via a PI3K-dependent pathway. Integrin α6β4 is known to activate PI3K. Here, we also show that knockdown of integrin α6β4 negates the PTHrP-mediated activation of the PI3K/Akt pathway. Taken together, these observations provide evidence of a link between PTHrP and the PI3K/Akt signaling pathway through integrin α6β4, resulting in the activation of survival pathways. Targeting PTHrP production in prostate cancer may thus prove therapeutically beneficial. (Mol Cancer Res 2009;7(7):1119–31)
PALB2 Functionally Connects the Breast Cancer Susceptibility Proteins BRCA1 and BRCA2
Authors: Zhang Fan , Fan Qiang , Ren Keqin , Andreassen Paul R.
Published: July 2009

BRCA1 and BRCA2 are prominently associated with inherited breast and ovarian cancer. The encoded proteins function in DNA damage responses, but no functional link between BRCA1 and BRCA2 has been established. We show here that PALB2 physically and functionally connects BRCA1 and BRCA2 into a DNA damage response network that also includes the RAD51 recombinase. PALB2 directly binds BRCA1, as determined with bacterially expressed fragments of each protein. Furthermore, PALB2 independently interacts with BRCA1 and BRCA2 through its NH2 and COOH termini, respectively. Critically, two point mutants (L21P and L24P) of the PALB2 coiled-coil domain or an NH2-terminal deletion (Δ1-70) disrupt its interaction with BRCA1. We have reconstituted PALB2-deficient cells with PALB2Δ1-70, PALB2-L21P, or PALB2-L24P, or with COOH-terminally truncated PALB2 that is deficient for interaction with BRCA2. Using extracts from these cells, we find that PALB2 mediates the physical interaction of BRCA2 with a COOH-terminal fragment of BRCA1. Analysis of the assembly of foci in these cells by BRCA1, PALB2, BRCA2, and RAD51 suggests that BRCA1 recruits PALB2, which in turn organizes BRCA2 and RAD51. Resistance to mitomycin C and the repair of DNA double-strand breaks by homologous recombination require the interaction of PALB2 with both BRCA1 and BRCA2. These results suggest that BRCA1 and BRCA2 cooperate in DNA damage responses in a PALB2-dependent manner, and have important implications for the genesis of breast/ovarian cancer and for chemotherapy with DNA interstrand cross-linking agents. (Mol Cancer Res 2009;7(7):1110–8)
Recombinant human C1-inhibitor prevents non-specific proteolysis by mutant pro-urokinase during optimal fibrinolysis
Authors: Pannell Ralph , Gurewich Victor
Published: July 2009

A single-site mutant of prouPA (M5) spared haemostatic fibrin during thrombolysis in dogs. Zymograms of plasma from these dogs showed an unusual inhibitor complex with C1-inhibitor (C1I). Purified C1I added to human plasma enhanced the fibrinspecificity of M5. In the present study, the effect of recombinant human C1I (recC1I) on high-dose M5 and tPA were compared using fluorescein-labeled standardised clots in a plasma milieu. The shortest time to complete clot lysis (maximum rate) was first determined. This was ∼65% per hour for both activators. By contrast, their top fibrin-specific lysis rate (<20% fibrinogen depletion) was less than half maximum (25–30% per hour). Adding recC1I (250–750 μg/ml) did not affect fibrinolysis, but prevented fibrinogenolysis and plasminogen depletion by M5, raising its fibrin-specific lysis rate to the maximum. With tPA, the recC1I modestly attenuated fibrinogenolysis, raising its fibrin-specific rate to about half the maximum. Consistent with this, the t½ inhibition by C1I was ∼90 min for tPA compared with ∼10 min for tcM5. The t½ of C1I for plasmin was ∼2 min. Zymograms of plasma after clot lysis indicated that recC1I prevented non-specific tcM5 generation from M5, as evidenced by suppression of tcM5:C1I complexes. In conclusion, recC1I raised the fibrin-specificity of M5 in plasma so that a maximum lysis rate could be achieved without fibrinogenolysis. The inhibition by C1I of nonspecific but not fibrin-dependent plasminogen activation could not be duplicated by other serpins. The findings provide a potential means to optimize both the efficacy and safety of thrombolysis.
Increased production of ADAMTS13 in hepatic stellate cells contributes to enhanced plasma ADAMTS13 activity in rat models of cholestasis and steatohepatitis
Authors: Watanabe Naoko , Kume Yukio , Satoh Yumiko , Kaneko Makoto , Takai Daiya , Tejima Kazuaki , Nagamine Masakazu , Mashima Hirosato , Tomiya Tomoaki , Noiri Eisei , Omata Masao , Matsumoto Masanori , Fujimura Yoshihiro , Yatomi Yutaka , Ikeda Hitoshi
Published: July 2009

Although hepatic stellate cells, endothelial cells, glomerular podocytes and plateles were reported to be a source of ADAMTS13, it is not clarified which source is involved in the regulation of plasma ADAMTS13 activity. It was demonstrated previously that selective hepatic stellate cell damage in rats caused decreased plasma ADAMTS13 activity.To further elucidate the potential contribution of hepatic stellate cells to the regulation of plasma ADAMTS13 activity, this study examined plasma ADAMTS13 activity when hepatic stellate cells proliferate during the process of liver fibrosis by employing rat models of liver fibrosis due to cholestasis, bile duct ligation, and steatohepatitis, a choline-deficient L-amino acid-defined-diet. ADAMTS13 expression was increased with co-localisation with smooth muscle α-actin, a marker of hepatic stellate cells, in bile duct-ligated livers up to four weeks, in which a close correlation between ADAMTS13 and smooth muscle α-actin mRNA expressions was determined. Plasma ADAMTS13 activity, measured by a sandwich ELISA involving a specific substrate to ADAMTS13, was increased in bile duct-ligated rats with a significant correlation with ADAMTS13 mRNA expression levels in the liver. Furthermore, ADAMTS13 mRNA expression was increased with enhanced mRNA expression in smooth muscle α-actin in the livers of rats fed a choline-deficient L-amino aciddefined-diet for 16 weeks, in which increased plasma ADAMTS13 activity was determined. Thus, increased plasma ADAMTS13 activity in cholestasis and steatohepatitis in rats may be due, at least in part, to enhanced ADAMTS13 production in the liver, suggesting a significant role of hepatic stellate cells in the regulation of plasma ADAMTS13 activity.
High-mobility group box 1 protein induces tissue factor expression in vascular endothelial cells via activation of NF-κB and Egr-1
Authors: Wang Haichao , Tang Yiting , Fan Zhang , Lv Ben , Xiao Xianzhong , Chen Fangping
Published: July 2009

High-mobility group box 1 protein (HMGB1), an abundant nuclear protein, was recently established as a proinflammatory mediator of experimental sepsis.Although extracellular HMGB1 has been found in atherosclerotic plaques, its potential role in the pathogenesis of atherothrombosis remains elusive. In the present study, we determined whether HMGB1 induces tissue factor (TF) expression in vascular endothelial cells (ECs) and macrophages. Our data showed that HMGB1 stimulated ECs to express TF (but not TF pathway inhibitor) mRNA and protein in a concentration and time-dependent manner. Blockade of cell surface receptors (including TLR4, TLR2, and RAGE) with specific neutralising antibodies partially reduced HMGB1-induced TF expression. Moreover, HMGB1 increased expression of Egr-1 and nuclear translocation of NF-κB (c-Rel/p65) in ECs. Taken together, our data suggest that HMGB1 induces TF expression in vascular endothelial cells via cell surface receptors (TLR4, TLR2, and RAGE), and through activation of transcription factors (NF-κB and Egr-1).
On the origin of (CD105+) circulating endothelial cells
Authors: Verhoef Cornelis , Gratama Jan , Sleijfer Stefan , Strijbos Michiel
Published: July 2009

Cells designated by the CellSearch™ assay as circulating endothelial cells (CEC) (CD146+/CD105+/CD45− nuclear cells) are thought to derive from damaged vasculature.As CD105 has been suggested to be expressed by endothelial cells from malignant vasculature particularly, it is currently unknown whether this assay is suitable to determine CECs in non-malignant diseases.Also,more insight is needed whether CECs as detected by this assay predominantly measures CECs or also endothelial progenitor cells (EPCs), which originate from the bone marrow and reflect angiogenesis rather than vascular damage. CEC counts were determined in nine patients treated with isolated limb perfusion with tumour necrosis factor (TNF) a and melphalan, and in 10 healthy donors. Given the severe vascular damage caused by venesection and cannulation of the main vessels, we expected a significant increase in CEC counts in case CEC were of vascular rather than of bone marrow origin.Additionally, this finding, as well as the presence of CD105+ CEC in the blood of healthy controls, would confirm that healthy endothelial cells express CD105. Numbers of CD146+/CD105+/CD45− nuclear CEC increased significantly after venesection and cannulation. After administration of TNF, a large fraction of non-intact, possibly apoptotic CEC appeared. This study shows that the Cell-Search™ assay detects CECs originating from damaged vasculature. Furthermore, CD105 expression is found on CEC from damaged normal vasculature rendering further exploration of the value of CEC determined by this assay worthwhile not only in malignant diseases but also in non malignant disorders characterised by vascular damage.
Two cases of congenital dysfibrinogenemia associated with thrombosis – Fibrinogen Praha III and Fibrinogen Plzeň
Authors: Reicheltová Zuzana , Malý Martin , Suttnar Jiří , Sobotková Alžbòta , Salaj Peter , Hirmerová Jana , Riedel Tomáš , Dyr Jan E. , Kotlín Roman
Published: July 2009

Congenital dysfibrinogenemia is a rare disease characterised by inherited abnormality in the fibrinogen molecule, resulting in functional defects. Two patients, a 26-year-old woman and a 61-year-old man, both with history of thrombotic events, had abnormal coagulation test results. DNA sequencing showed the heterozygous γY363N mutation (Fibrinogen Praha III) and the heterozygous Aα N106D mutation (Fibrinogen Plzeň), respectively. Fibrin polymerisation, after addition of either thrombin or reptilase, showed remarkably delayed polymerisation in both cases. Fibrinolysis experiments showed slower tPA initiated lysis of clots. SDS-PAGE did not show any difference between normal and Praha III and Plzeň fibrinogens. Both mutations had a significant effect on platelet aggregation. In the presence of either ADP or TRAP, both mutations caused the decrease of platelet aggregation. SEM revealed abnormal clot morphology, with a large number of free ends and narrower fibres of both fibrin Praha III and Plzeň. Praha III mutation was situated in the polymerisation pocket “a”. The replacement of the bulky aromatic side chain of tyrosine by the polar uncharged small side chain of asparagine may lead to a conformational change, possibly altering the conformation of the polymerisation pocket. The Plzeň mutation is situated in the coiled-coil connector and this replacement of polar uncharged asparagine residue by polar acidic aspartate changes the alpha-helical conformation of the coiled-coil connector;and may destabilise hydrogen bonds in its neighborhood. Although both mutations are situated in different regions of the molecule, both mutations have a very similar effect on fibrinogen functions and both are connected with thromboses.
The VEGF-induced transcriptional response comprises gene clusters at the crossroad of angiogenesis and inflammation
Authors: Schweighofer Bernhard , Testori Julia , Sturtzel Caterina , Sattler Susanne , Mayer Herbert , Wagner Oswald , Bilban Martin , Hofer Erhard
Published: July 2009

VEGF-A is the major trigger of vasculogenesis and physiologic angiogenesis. We have investigated to which extent the gene repertoire induced by VEGF-A in endothelial cells is distinct from that of other growth factors and inflammatory cytokines. Genes upregulated in human umbilical vein endothelial cells treated with VEGF, EGF or IL-1 were compared by microarray analysis and clusters characteristic for individual or combinations of inducers were defined. VEGF-A upregulated in comparison to EGF a five-fold larger gene repertoire, which surprisingly overlapped to 60% with the inflammatory repertoire of IL-1. As shown by real-time RT-PCR for selected genes, VEGFinduction was mostly mediated by VEGF receptor-2 and the capacity of VEGF-A to induce genes in common with IL-1 largely depended on activation of the calcineurin/NFAT pathway, since cyclosporin A inhibited this induction. Another angiogenic growth factor, bFGF, did not share a comparable induction of inflammatory genes, but partially induced a small group of genes in common with VEGF-A, which were not regulated by EGF. Thus, the data display that VEGF-A induces a distinct gene repertoire, which, contrasting with other growth factors such as EGF or bFGF, includes an inherent inflammatory component possibly contributing to the cross-regulation of angiogenesis and inflammation as further indicated by the VEGF-mediated induction of leukocyte adhesion. Furthermore, a small group of genes selectively induced by VEGF-A with potential importance for angiogenesis is defined.
Natural history of mesenteric venous thrombosis in patients treated with vitamin K antagonists
Authors: Ageno Walter , Witt Dan , Malato Alessandra , Clark Nathan , Garcia David , McCool Kathleen , Siragusa Sergio , Dyke Shannon , Crowther Mark , Dentali Francesco ,   for the WARPED consortium
Published: July 2009

summary

Knowledge on the natural history of mesenteric vein thrombosis (MVT) and of the efficacy and safety of long-term oral anticoagulant therapy (OAT) in this setting is based on small uncontrolled series of patients with a limited follow-up. It was the aim of the study to assess the natural history of MVT in a cohort of patients treated with OAT. The charts of all MVT patients currently attending or who have attended four anticoagulation clinics were reviewed. Information on risk factors, treatment, recurrence, major bleeding and mortality was collected. Seventyseven patients (mean age 49.2 years; 45 males) were included with a median follow-up of 36 months (range 2–204 months). Forty-six patients were treated with long-term OAT. Seven patients had venous thromboembolism (VTE) recurrence (5 splanchnic vein thromboses and two pulmonary emboli) for an incidence rate of 23.4 events /1,000 year patients. In two patients recurrentVTE occurred during OAT, for an incidence rate of 10.5 events /1,000 year patient. Five patients had VTE recurrence when OAT was suspened for an incidence rate of 45.9 events /1,000 year patient. Two patients (2.6%) had a major bleeding event. 97.3% of patients were alive at one year, and seven patients (9.1%) died during follow up. In conclusion, patients with MTV seem to have a low risk of recurrentVTE while receiving OAT. This risk appears increased after treatment is stopped.
Haplotype M2 in the annexin A5 (ANXA5) gene and the occurrence of obstetric complications
Authors: Tiscia Giovanni , Colaizzo Donatella , Chinni Elena , Pisanelli Daniela , Sciannamè Natale , Favuzzi Giovanni , Margaglione Maurizio , Grandone Elvira
Published: July 2009

Inherited or acquired thrombophilias have been largely explored as a cause of pregnancy complications. However, pathogenesis of obstetric complications, as fetal loss and pregnancyrelated hypertensive disorders is still partly unexplained. Recently, a common haplotype (M2) within the annexin A5 (ANXA5) gene has been described as a risk factor in recurrent fetal losses (RFL). It has been demonstrated to reduce the promoter activity of the ANXA5 promoter in luciferase reporter assays. Aim of this study was to investigate the prevalence of M2 haplotype in three different settings of women with previous obstetric complications: RFL, intra-uterine fetal death (IUFD) and pregnancy-related hypertension (gestational hypertension [GH] and pre-eclampsia [PE]). One hundred three patients with previous RFL, 54 with IUFD, 158 with hypertensive disease (67 GH, 91 PE) were investigated. As controls,195 women from the same ethnic background with uneventful pregnancies were enrolled. Logistic regression, correcting for age, gravidity and parity showed that the ANXA5 haplotype is significantly and independently associated with the occurrence of RFL (3.1; 95%CI: 1.1–9.5; p=0.047) and pregnancy-related hypertensive disorders (2.1; 95%CI: 1.2–3.5; p=0.008).The M2 haplotype might be a new and relevant risk factor for obstetric complications.