The CaSm (LSm1) oncogene promotes transformation, chemoresistance and metastasis of pancreatic cancer cells.

The cancer-associated Sm-like (CaSm) oncogene is overexpressed in 87% of human pancreatic tumor samples and CaSm knockdown has demonstrated therapeutic efficacy in murine models of pancreatic cancer. Evidence indicates that CaSm modulates messenger RNA degradation; however, its target genes and the mechanisms by which CaSm promotes pancreatic cancer remain largely unknown. Here, we demonstrate that the CaSm overexpression alters several hallmarks of cancer-including transformation, proliferation, chemoresistance and metastasis. Doxycycline-induced CaSm expression enhanced proliferation and both anchorage-dependent and -independent growth of the human Panc-1 cells in vitro. CaSm induction decreased gemcitabine-induced cytotoxicity and altered the expression of apoptotic regulation genes, including Bad, E2F1 and Bcl-XL. CaSm-overexpressing Panc-1 cells were twofold more migratory and fourfold more invasive than the driver controls and demonstrated characteristics of epithelial-to-mesenchymal transition such as morphological changes and decreased E-cadherin expression. CaSm induction resulted in changes in RNA expression of metastasis-associated genes such as MMP1, SerpinB5, uPAR and Slug. Using a murine model of metastatic pancreatic cancer, injection of CaSm-induced Panc-1 cells resulted in a higher abundance of hepatic metastatic lesions. Overall, CaSm overexpression contributed to a more aggressive cancer phenotype in Panc-1 cells, further supporting the use of CaSm as a therapeutic target against pancreatic cancer.

Epithelial-to-mesenchymal transition and the cancer stem cell phenotype: insights from cancer biology with therapeutic implications for colorectal cancer.

Although mortality from colorectal cancer (CRC) is decreasing, CRC is still the second highest cause of cancer-related deaths in America. Chemotherapy and radiation therapy now have central roles in our strategies to fight cancer, although we continue to lack novel strategies overcoming therapeutic resistance. Molecular mechanisms of therapeutic resistance in CRC continue to be under intense investigation. In this review, we highlight the recent evidence linking epithelial-to-mesenchymal transition (EMT) with aggressive tumor biology as well as with the cancer stem cells (CSCs) across multiple organ systems including colon cancer. Furthermore, in the era of neo-adjuvant treatment, the clinical implications are concerning that our treatments may have the potential to induce more aggressive cancer cells through EMT, perhaps even generating CSCs more capable of metastasis and further resistant to treatment. This concern and potential reality highlights the critical need for further understanding the impact of clinical therapy on the pathobiology of cancer and further supports the need to therapeutically target the CSC. Besides serving as potential biomarkers for aggressive tumor biology and therapeutic resistance, EMT and CSC molecular pathways may highlight novel therapeutic targets as strategies for improving the response to conventional anti-neoplastic agents translating into improved oncologic outcomes.

Transferrin receptor targeting nanomedicine delivering wild-type p53 gene sensitizes pancreatic cancer to gemcitabine therapy.

To overcome gene therapy barriers such as low transfection efficiency and nonspecific delivery, liposomal nanoparticles targeted by a single-chain antibody fragment to the transferrin receptor (TfRscFv) delivering wild-type (wt) human p53 (SGT-53) were developed for tumor-specific targeting. We hypothesize that SGT-53 in combination with gemcitabine will demonstrate enhanced therapeutic benefit in an in vivo metastatic pancreatic cancer model. Intrasplenic injection of 1 × 10(6) Panc02 murine pancreatic cancer cells was used to generate in vivo hepatic metastatic tumors. Nanoparticle localization was assessed by tail vein injection of TfRscFv with fluorescently labeled oligonucleotides (6-carboxyfluorescein phosphoramidite (6FAM) ODN) imaged by Xenogen IVIS 200 scan. SGT-53 (equivalent to 30 µg of p53 intravenously) and gemcitabine (20 mg/kg intraperitoneally) alone and in combination were administered biweekly and compared with untreated mice. Survival was determined by blinded daily assessment of morbidity. Human wtp53 expression and transferrin levels in the tumors were assessed by western blot analysis. Tumor burden was quantified by liver weight. Xenogen imaging demonstrated tumor-specific uptake of TfRscFv-6FAM ODN. Exogenous human wtp53 protein was detected in the SGT-53-treated tumors compared with control. Compared with untreated mice with metastatic tumors demonstrating median survival of 20 days, SGT-53, gemcitabine and the combination demonstrated improved median survival of 29, 30 and 37 days, respectively. The combination treatment prolonged median survival when compared with single drug treatment and decreased tumor burden. The tumor targeting liposomal-based SGT-53 nanoparticle is capable of sensitizing pancreatic cancer to conventional chemotherapy in pancreatic cancer models. This approach has the potential to be translated into a new, more effective therapy for pancreatic cancer. Further optimization is ongoing, moving towards a Phase 1B/2 clinical trial.

Impact of Fli-1 transcription factor on autoantibody and lupus nephritis in NZM2410 mice.

The transcription factor Fli-1 is implicated in the pathogenesis of both murine and human lupus. Increased levels of Fli-1 mRNA were present in the peripheral blood lymphocytes from lupus patients; furthermore, transgenic overexpression of Fli-1 in normal mice resulted in the development of a lupus-like disease. Lupus nephritis is a major cause of death in both lupus patients as well as in animal models. In this study, we generated Fli-1 heterozygous knockout (Fli-1(+/)⁻ ) NZM2410 mice (of which the wild-type is a widely used lupus murine model) that expressed decreased levels of Fli-1 and investigated the impact of Fli-1 expression on lupus nephritis development and survival. Ninety-three per cent of the Fli-1(+/)⁻ NZM2410 mice survived to the age of 52 weeks compared to only 35% of wild-type NZM2410 mice. Autoantibodies, including anti-dsDNA and anti-glomerular basement antigen, in Fli-1(+/)⁻ NZM2410 mice were statistically significantly lower when compared to wild-type NZM2410 mice at the ages of 30 and 34 weeks. Total B cell and activated B cell populations in the spleens from Fli-1(+/)⁻ NZM2410 mice were decreased significantly compared to wild-type NZM2410 mice. Fli-1(+/)⁻ NZM2410 mice also had remarkably diminished proteinuria and decreased renal pathological scores when compared with wild-type NZM2410 mice. Expression of early growth response 1 (Egr-1) was decreased significantly in the kidneys from Fli-1(+/)⁻ NZM2410 mice when compared to wild-type littermates. Our data indicate that expression of Fli-1 plays an important role in lupus disease development in NZM2410 mice.

Inhibition of thromboxane synthase activity modulates bladder cancer cell responses to chemotherapeutic agents.

Recently, we reported prognostic significance of thromboxane synthase (TXAS) gene expression in invasive bladder cancer. The positive correlation between elevated TXAS expression and shorter patient survival supports a potential role for TXAS-regulated pathways in tumor metastases. In this study, using immunohistochemical analysis, we found an increased expression of TXAS protein in bladder cancer. Treatment of T24 and transitional cell carcinoma TCC-SUP bladder cancer cells with the TXAS inhibitors furegrelate or ozagrel induced an apoptotic effect measured as an increase in caspase-3 activation and cell death, and decreased survivin expression. Pharmacological inhibition of TXAS using the TXAS inhibitor furegrelate increased sensitivity to the chemotherapeutic agents cisplatin and paclitaxel. Molecular inhibition of TXAS expression by siRNA significantly decreased cell growth and migration. In concordance with the pharmacological data, siRNA-mediated reduction of TXAS expression increased sensitivity to cisplatin and paclitaxel in T24 and TCC-SUP cells. In summary, the data support a role for the thromboxane A(2) pathway in the pathogenesis of bladder cancer and the potential utility of modulation of this signaling pathway for cancer chemotherapy.

Molecular mechanisms of megakaryopoiesis.

One function of bone marrow megakaryocytes (MKs) is the controlled release of platelets into the circulation. Over the past few years, molecular mechanisms that contribute to MK development and differentiation have begun to be elucidated. This review provides a brief overview of megakaryopoiesis and platelet function, and the importance of selected hematopoietic transcription factors (including GATA-1, FOG, Fli-1, AML1, and NF-E2) and target genes in this biological process. In addition, a discussion of human diseases affecting megakaryopoiesis and mouse models of thrombocytopenia are presented with emphasis on how these systems have and will continue to provide further insights into mechanisms that control the biological functions of the megakaryocytic cell lineage. Ultimately, such knowledge may provide the basis for novel therapeutic approaches for modulation of platelet number and function.

Surgical delay in acute admissions on warfarin: are we doing enough?

Warfarin anti-coagulation can cause significant delay in acute surgical admissions. We reviewed fracture neck of femur patients operated over a period of 4 years in our unit. There was an average delay to surgery of 4.36 days in patients on warfarin as against an average delay of 1.78 days in patients not on warfarin (p < 0.001). The review was followed up with a questionnaire-based survey of consultant haematologists, and a general agreement towards a protocol-based use of vitamin K(1) was noted. The reasons for limited use of vitamin K(1) include the lack of studies and guidelines specifically addressing the pre-operative emergency admissions. We highlight a practical problem shared across different specialities and identify the areas for future studies.

Warfarin for non-rheumatic atrial fibrillation: five year experience in a district general hospital.

OBJECTIVES: To assess the long term efficacy of and risks associated with computer aided oral anticoagulation for non-rheumatic atrial fibrillation (NRAF) in a district hospital setting. DESIGN: Retrospective, age stratified, event driven clinical database analysis. SETTING: District general hospital. PARTICIPANTS: 739 patients receiving warfarin for NRAF between 1996 and 2001. Patients were selected from an anticoagulation database through appropriate filter settings. MAIN OUTCOME MEASURES: Anticoagulation control (international normalised ratio (INR)) and hospitalisations for bleeding complications, thromboembolic events, and stroke. RESULTS: Over 1484 patient-years, computer assisted anticoagulation was uncontrolled in 38.3% of patients (INR < 2.0 or > 3.0). No significant differences in INR control were observed with respect to patient age (< 65, 65-75, and > 75 years), although to achieve adequate control of anticoagulation, the frequency of testing increased significantly with age. Annual risks of bleeding complications, thromboembolism, and stroke were 0.76%, 0.35%, and 0.84%, respectively. No significant differences in these events were observed between the three age groups studied. Patients who had thromboembolic events and haemorrhagic complications were significantly more likely to have been under-anticoagulated (INR < 2.0) and over-anticoagulated (INR > 3.0), respectively, at the time of their clinical event. CONCLUSIONS: Computerised long term oral anticoagulation for NRAF in a community setting of elderly and diverse patients is safe and effective. Anticoagulation control, bleeding events, thromboembolic episodes, and stroke rates are directly comparable with those reported in major clinical trials. The authors therefore support the strategy of rate control with long term oral anticoagulation for NRAF in general clinical practice.

CaSm/gemcitabine chemo-gene therapy leads to prolonged survival in a murine model of pancreatic cancer.

BACKGROUND: CaSm, the cancer-associated Sm-like oncogene, is overexpressed in greater than 80% of pancreatic tumors. We previously reported that an adenovirus expressing antisense RNA to CaSm (Ad-alpha CaSm) can decrease pancreatic tumor growth in vivo but is not curative. In the current study we investigated the mechanism of Ad-alpha CaSm's antitumor effect to rationally approach combinatorial therapy for improved efficacy. METHODS: AsPC-1 and Panc-1 human pancreatic cancer cells were treated with Ad-alpha CaSm and examined by MTT assay for in vitro proliferation changes. Flow cytometry determined the effect of CaSm down-regulation on the cell cycle, and then cells treated with Ad-alpha CaSm in combination with cisplatin, etoposide, or gemcitabine chemotherapies were reexamined by MTT assay. SCID-Bg mice bearing subcutaneous AsPC-1 tumors were treated with Ad-alpha CaSm, gemcitabine, or the combination and monitored for tumor growth and survival. RESULTS: Treatment with Ad-alpha CaSm reduced the proliferation of AsPC-1 and Panc-1 cells (59% and 44%, respectively; P <.05). The cell cycle revealed a cytostatic block with decreased G(1) phase and increased DNA content in treated cells. The combination of Ad-alpha CaSm with gemcitabine significantly reduced in vitro proliferation (66% vs 39% and 48% for controls), decreased in vivo AsPC-1 tumor growth by 71% (n = 10), and extended survival time from 57 to 100 days. CONCLUSIONS: Down-regulation of CaSm reduces the growth of pancreatic cancer cells by altering the cell cycle in a cytostatic manner. The combination of Ad-alpha CaSm with gemcitabine is more effective than either agent used separately.

Defects of DNA mismatch repair in human prostate cancer.

Loss of mismatch repair (MMR) function leads to the accumulation of errors that normally occur during DNA replication, resulting in genetic instability. Germ-line mutations of MMR genes in the patients with hereditary nonpolyposis colorectal cancer lead to inactivation of MMR protein functions, and the defects of MMR are well correlated to the high rate of microsatellite instability in their tumors. Previous studies (T. Uchida, et al. Oncogene, 10: 1019-1022, 1995; S. Egawa, et al. Cancer RES:, 55: 2418-2421, 1995; J. M. Cunningham, et al. Cancer RES:, 56: 4475-4482, 1996; X. Gao, et al. Oncogene, 9: 2999-3003, 1994; H. Rohrbach, et al. Prostate, 40: 20-27, 1999) have shown that genetic instability (chromosomal and microsatellite instability) is detectable in human prostate cancer. To elucidate the role of MMR genes in the tumorigenesis of prostate cancer, we evaluated the expression of these genes in human cancer cell lines and in tumor specimens. Using Western blot analysis, we detected loss among MSH2, MLH1, PMS2, and PMS1 proteins in DU145, LNCaP, p69SV40T, M2182, and M12 cells. In addition, genomic instability in the prostate cell lines including DU145, PC3, LNCaP, p67SV40T, M2182, and M12 was detected by a microsatellite mutation assay. Significantly, immunohistochemical analysis of prostatic tissue revealed the reduction or absence of MMR protein expression in the epithelium of prostate tumor foci compared with normal adjacent prostate tissue. In contrast to hereditary nonpolyposis colorectal cancer, characterized by defects predominantly in MLH1 and MSH2, the samples we examined showed more tumor foci with loss of PMS1 and PMS2. PMS1, which is only expressed in the basal cells in normal glands, is conspicuously absent in most prostate cancer. From these results, we conclude that there are defects of MMR genes in human prostate cancer.

Fli-1 inhibits collagen type I production in dermal fibroblasts via an Sp1-dependent pathway.

Fibrosis is characterized by the excessive deposition of extracellular matrix (ECM), especially collagen. Because Ets factors are implicated in physiological and pathological ECM remodeling, the aim of this study was to investigate the role of Ets factors in collagen production. We demonstrate that the expression of collagenous proteins and collagen alpha2(I) (COL1A2) mRNA was inhibited following stable transfection of Fli-1 in dermal fibroblasts. Subsequent analysis of the COL1A2 promoter identified a critical Ets binding site that mediates Fli-1 inhibition. In contrast, Ets-1 stimulates COL1A2 promoter activity. In vitro binding assays demonstrate that both Fli-1 and Ets-1 form DNA-protein complexes with sequences present in COL1A2 promoter. Furthermore, Fli-1 binding to the COL1A2 is enhanced via Sp1-dependent interaction. Studies using Fli-1 dominant interference and DNA binding mutants indicate that Fli-1 inhibition is mediated by both direct (DNA binding) and indirect (via protein-protein interaction) mechanisms and that Sp1 is an important mediator of the Fli-1 function. Furthermore, experiments using the Gal4 system in the context of different cell types as well as experiments with the COL1A2 promoter in different cell lines demonstrate that the direction and magnitude of the effect of Fli-1 is promoter- and cell context-specific. We propose that Fli-1 inhibits COL1A2 promoter activity by competition with Ets-1. In addition, we postulate that another factor (co-repressor) may be required for maximal inhibition after recruitment to the Fli-1-Sp1 complex. We conclude that the ratio of Fli-1 to Ets-1 and the presence of co-regulatory proteins ultimately control ECM production in fibroblasts.

Suppression of the Ewing's sarcoma phenotype by FLI1/ERF repressor hybrids.

Fusion of the 5' half of the Ewing's sarcoma (ES) gene EWS with the DNA-binding domain of several transcription factors has been detected in many human tumors. The t(11;22)(q24;q12) chromosomal translocation is specifically linked to ES and primitive neuroectodermal tumors and results, in the majority of cases, in the fusion of the amino terminus of the EWS gene to the carboxyl-terminal DNA-binding domain of the FLI1 gene. The chimeric protein has been shown to be oncogenic, a potent transcriptional activator, and necessary for the maintenance of the Ewing's phenotype, making it an attractive target for gene therapy. In this study, we demonstrate that the ES transformed phenotype can be suppressed by chimeric transcriptional repressors containing the DNA-binding domain of FLI1 and the regulatory and repressor domain of ERF, a transcription suppressor and member of the ets gene family. The hybrid repressor is expressed at levels comparable with EWS/FLI1, does not affect EWS/FLI1 expression, and exhibits similar DNA-binding specificity but suppresses transcriptional activity. The FLI1/ERF repressor, like the wild-type ERF, is regulated by mitogen-activated protein kinase-dependent subcellular localization. Our data suggest that transformation by EWS/FLI1 may partially be due to activation of specific EWS/FLI1-regulated genes involved in cell proliferation.

The cancer-associated Sm-like oncogene: a novel target for the gene therapy of pancreatic cancer.

BACKGROUND: The prognosis for pancreatic cancer (PC) remains dismal, providing a clear need for the development of novel therapies. We have previously shown that the cancer-associated Sm-like (CaSm) oncogene is overexpressed in the great majority of pancreatic tumors and is required to maintain the transformed phenotype. The purpose of this study was to determine whether the application of CaSm antisense gene therapy would generate a significant antitumor effect against PC. METHODS: An adenoviral vector (Ad-alphaCaSm) expressing a 900-base pair antisense RNA to CaSm was created. The PC cell lines AsPC-1 and Capan-1 were infected with this vector and examined for changes in in vitro proliferation by using methyl thiazol tetrazolium and soft agar assays. SCID-Bg mice bearing subcutaneous AsPC-1 tumors were treated with Ad-alphaCaSm (1 x 10(9) plaque-forming units) as a single intratumor injection with tumor growth and survival monitored. RESULTS: AsPC-1 and Capan-1 cells showed decreased in vitro proliferation (93%, P =.0041, and 70%, P =. 0038, respectively) and anchorage independent growth (55%, P =.02, and 45%, P =.03, respectively) after treatment. Ad-alphaCaSm reduced in vivo AsPC-1 tumor growth by 40% (n = 10), extending median survival time from 35 to 60 days. CONCLUSIONS: Ad-alphaCaSm demonstrates a significant antitumor effect against pancreatic cancer both in vitro and in vivo. These results support the role of CaSm as a significant gene involved in the neoplastic transformation of pancreatic tumors. Thus CaSm represents a novel gene target in PC and holds potential as a new treatment approach either alone or in combination with existing therapies.

Hemorrhage, impaired hematopoiesis, and lethality in mouse embryos carrying a targeted disruption of the Fli1 transcription factor.

The Ets family of transcription factors have been suggested to function as key regulators of hematopoeisis. Here we describe aberrant hematopoeisis and hemorrhaging in mouse embryos homozygous for a targeted disruption in the Ets family member, Fli1. Mutant embryos are found to hemorrhage from the dorsal aorta to the lumen of the neural tube and ventricles of the brain (hematorrhachis) on embryonic day 11.0 (E11.0) and are dead by E12.5. Histological examinations and in situ hybridization reveal disorganization of columnar epithelium and the presence of hematomas within the neuroepithelium and disruption of the basement membrane lying between this and mesenchymal tissues, both of which express Fli1 at the time of hemorrhaging. Livers from mutant embryos contain few pronormoblasts and basophilic normoblasts and have drastically reduced numbers of colony forming cells. These defects occur with complete penetrance of phenotype regardless of the genetic background (inbred B6, hybrid 129/B6, or outbred CD1) or the targeted embryonic stem cell line used for the generation of knockout lines. Taken together, these results provide in vivo evidence for the role of Fli1 in the regulation of hematopoiesis and hemostasis.

The Ets1 proto-oncogene is upregulated by retinoic acid: characterization of a functional retinoic acid response element in the Ets1 promoter.

The v-ets oncoprotein and its progenitor Ets1 belong to a family of transcription factors that are related by an 85 amino acid conserved DNA binding domain, the ets domain. Ets1 plays important role(s) in control of cell proliferation, differentiation and apoptosis. Abnormal expression of Ets1 could lead to disruption of these processes and contribute to development of malignancy. Retinoic acid (RA) inhibits proliferation, induces differentiation and regulates apoptosis in many different cell types. Here, we demonstrate that RA treatment increases the expression of Ets1 mRNA, but not that of Ets2, Elk1 or Fli1 in MC3T3-E1 cells. Ets1 induction is detectable after 4 h, can be maintained for at least 14 days, and is inhibited by Actinomycin D, which suggests that RA regulation of Ets1 occurs at the transcriptional level. The promoter region of Ets1 contains four retinoic acid response element (RARE) half sites located at -94, -152, -1765 and -2252 from the translation start site. We show that RARbeta is expressed by MC3T3-E1 cells in the presence of RA and demonstrate that it binds to the -94 RARE half site. Furthermore, RA induces transcription of Ets1 promoter-reporter constructs containing this RARE half site.

EAP1/Daxx interacts with ETS1 and represses transcriptional activation of ETS1 target genes.

ETS1 is a member of the evolutionarily conserved family of ets genes, which are transcription factors that bind to unique DNA sequences, either alone or by association with other proteins. In this study, we have used the yeast two-hybrid system to identify an ETS1 interacting protein. The ETS1 N-terminal amino acid region was used as bait and an interaction was identified with the Daxx protein, referred to as EAP1 (ETS1 Associated Protein 1)/Daxx. This interactin has been shown to exist in yeast and in vitro. EAP1/Daxx and ETS1 are co-localized in the nucleus of mammalian cells. The region in EAP1/Daxx which specifically binds to ETS1 is located within its carboxy terminal 173 amino acid region. The ETS1 interaction region is located within its N-terminal 139 amino acids and is referred as the Daxx Interaction Domain (DID). The DID appears to be conserved in several other ets family members, as well as in other proteins known to interact with Daxx. The EAP1/Daxx interacts with both isoforms of ETS1, p51-ETS1 and p42-ETS1. Interaction of EAP1/Daxx with ETS1 causes the repression of transcriptional activation of the MMP1 and BCL2 genes. The interaction domains of both ETS1 and EAP1/Daxx are required for this repression and deletion of either domain abolishes this activity.