Peter Burfeind

Defective thrombus formation in mice lacking coagulation factor XII

Blood coagulation is thought to be initiated by plasma protease factor VIIa in complex with the membrane protein tissue factor. In contrast, coagulation factor XII (FXII)–mediated fibrin formation is not believed to play an important role for coagulation in vivo. We used FXII-deficient mice to study the contributions of FXII to thrombus formation in vivo. Intravital fluorescence microscopy and blood flow measurements in three distinct arterial beds revealed a severe defect in the formation and stabilization of platelet-rich occlusive thrombi. Although FXII-deficient mice do not experience spontaneous or excessive injury-related bleeding, they are protected against collagen- and epinephrine-induced thromboembolism. Infusion of human FXII into FXII-null mice restored injury-induced thrombus formation. These unexpected findings change the long-standing concept that the FXII-induced intrinsic coagulation pathway is not important for clotting in vivo. The results establish FXII as essential for thrombus formation, and identify FXII as a novel target for antithrombotic therapy.

Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis

Formation of fibrin is critical for limiting blood loss at a site of blood vessel injury (hemostasis), but may also contribute to vascular thrombosis. Hereditary deficiency of factor XII (FXII), the protease that triggers the intrinsic pathway of coagulation in vitro, is not associated with spontaneous or excessive injury-related bleeding, indicating FXII is not required for hemostasis. We demonstrate that deficiency or inhibition of FXII protects mice from ischemic brain injury. After transient middle cerebral artery occlusion, the volume of infarcted brain in FXII-deficient and FXII inhibitor–treated mice was substantially less than in wild-type controls, without an increase in infarct-associated hemorrhage. Targeting FXII reduced fibrin formation in ischemic vessels, and reconstitution of FXII-deficient mice with human FXII restored fibrin deposition. Mice deficient in the FXII substrate factor XI were similarly protected from vessel-occluding fibrin formation, suggesting that FXII contributes to pathologic clotting through the intrinsic pathway. These data demonstrate that some processes involved in pathologic thrombus formation are distinct from those required for normal hemostasis. As FXII appears to be instrumental in pathologic fibrin formation but dispensable for hemostasis, FXII inhibition may offer a selective and safe strategy for preventing stroke and other thromboembolic diseases.

Targeted deletion of murine coagulation factor XII gene-a model for contact phase activation in vivo

To analyze the biological role of factor XII (FXII, Hageman Factor) in vivo, we generated mice deficient for FXII using a gene targeting approach on two distinct genetic backgrounds, i.e. mixed C57Bl/6J X 129X1/SvJ and inbred 129X1/SvJ. Homozygous FXII knockout (FXII(-)/(-)) mice showed no FXII plasma activity and had a markedly prolonged activated partial thromboplastin time (aPTT). In contrast, coagulation factors XI, VIII, IX, X,VII, V, II and fibrinogen did not differ between FXII(-/-) mice and their wild-type littermates. Heterozygous matings segregated according to the Mendelian inheritance indicating that FXII deficiency does not increase fetal loss. Furthermore, matings of FXII(-/-) males and FXII(-/-) females resulted in normal litter sizes demonstrating that total FXII deficiency in FXII(-/-) females does not affect pregnancy outcome. Also, gross and histological anatomy of FXII(-/-) mice was indistinguishable from that of their wild-type littermates on both genetic backgrounds. Thus it appears that deficiency of murine FXII does not cause thrombophilia or impaired fibrinolysis in vivo. These results indicate that FXII deficiency does not affect hemostasis in vivo and we anticipate that the FXII(-/-) mice will be helpful to elucidate the biological role(s) of FXII in health and disease.

Sequence and developmental expression of a mRNA encoding a putative protein of rat sperm outer dense fibers.

We have isolated a cDNA from rat testis homologous to the Drosophila melanogaster gene mst(3)gl-9 by screening a rat testis cDNA library with mst(3)gl-9 and by direct PCR amplification of upstream sequences out of the cDNA library. Homologous genes are also expressed in testes of different mammalian species. In rat testis, two different transcripts are found. Evidences are presented which suggest that these two transcripts are alternative splicing products. As proved by Northern blot analysis of testis RNA prepared from rats of different ages and by in situ hybridization to rat testis tissue sections, the mRNAs are first transcribed in early spermatids. The longest open reading frame of the cDNA encodes a polypeptide of 244 amino acids which contains 16.4% cysteine, 9.9% proline, and 5.7% glycine and closely resembles the sizes and amino acid compositions of two major polypeptides isolated from outer dense fibers of rat spermatozoa. The COOH-terminal end consists mostly of the tripeptide motif Cys-Gly-Pro, the main motif in D. melanogaster mst(3)gl-9. It is suggested that the isolated rat cDNA encodes a polypeptide which is a protein component of the outer dense fibers of spermatozoa.

Dual silencing of insulin-like growth factor-I receptor and epidermal growth factor receptor in colorectal cancer cells is associated with decreased proliferation and enhanced apoptosis

Overexpression and activation of tyrosine kinase receptors are common features of colorectal cancer. Using the human colorectal cancer cell lines DLD-1 and Caco-2, we evaluated the role of the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) and epidermal growth factor receptor (EGFR) in cellular functions of these cells. We used the small interfering RNA (siRNA) technology to specifically down-regulate IGF-IR and EGFR expression. Knockdown of IGF-IR and EGFR resulted in inhibition of cell proliferation of DLD-1 and Caco-2 cells. An increased rate of apoptosis was associated with siRNA-mediated silencing of IGF-IR and EGFR as assessed by activation of caspase-3/caspase-7. The combined knockdown of both EGFR and IGF-IR decreased cell proliferation and induced cell apoptosis more effectively than did silencing of either receptor alone. Comparable effects on cell proliferation and apoptosis were observed after single and combinational treatment of cells by the IGF-IR tyrosine kinase inhibitor NVP-AEW541 and/or the EGFR tyrosine kinase inhibitor erlotinib. Combined IGF-IR and EGFR silencing by either siRNAs or tyrosine kinase inhibitors diminished the phosphorylation of downstream signaling pathways AKT and extracellular signal–regulated kinase (ERK)-1/2 more effectively than did the single receptor knockdown. Single IGF-IR knockdown inhibited IGF-I–dependent phosphorylation of AKT but had no effect on IGF-I– or EGF-dependent phosphorylation of ERK1/2, indicating a role of EGFR in ligand-dependent ERK1/2 phosphorylation. The present data show that inhibition of the IGF-IR transduction cascade augments the antipoliferative and proapoptotic effects of EGFR inhibition in colorectal cancer cells. A clinical application of combination therapy targeting both EGFR and IGF-IR could be a promising therapeutic strategy.[Mol Cancer Ther 2009;8(4):821–33]

Blockade of the type I IGF receptor expression in human prostate cancer cells inhibits proliferation and invasion, up-regulates IGF binding protein-3, and suppresses MMP-2 expression

The type I insulin‐like growth factor receptor (IGF‐IR) is involved in tumour cell proliferation, invasion, and cancer cell survival. Several studies indicate that the IGF axis contributes to prostate cancer pathogenesis, but there is no consensus regarding the relative expression of the IGF‐IR in benign and malignant prostate epithelium. In this study, endogenous IGF‐IR gene expression was reduced in stably transfected PC‐3 cells by employing an antisense RNA strategy which resulted in significant suppression of both PC‐3 cell invasion and proliferation in vitro. Furthermore, it was demonstrated that a direct correlation exists between the inhibition of IGF‐IR gene expression and either up‐regulation of IGF binding protein (BP)‐3 or down‐regulation of matrix metalloproteinase (MMP)‐2 expression in androgen‐independent PC‐3 cells. Moreover, inhibition of IGF‐IR gene expression in transfected PC‐3 cells leads to an enhanced rate of spontaneous apoptosis. In addition, expression analyses by quantitative RT‐PCR on RNA from laser microdissected matched normal prostate and prostate tumour samples revealed that IGF‐IR gene expression was up‐regulated in nine of 12 prostate cancers, whereas IGFBP‐3 gene expression was down‐regulated in all 12 prostate carcinomas analysed. These results indicate an important role for IGF‐IR and IGFBP‐3 in the homeostasis of prostate carcinoma cells and provide a further basis for targeting IGF‐IR or IGFBP‐3 gene expression in order to improve understanding of the IGF‐IR‐activated signalling pathways and as a potential treatment for prostate cancer. Copyright

Bax Inhibitor-1 Is Overexpressed in Prostate Cancer and Its Specific Down-Regulation by RNA Interference Leads to Cell Death in Human Prostate Carcinoma Cells

To analyze differential gene expression of putative prostate tumor markers we compared the expression levels of more than 400 cancer-related genes using the cDNA array technique in a set of capsule-invasive prostate tumor and matched normal prostate tissue. The overexpression of Bax inhibitor-1 (BI-1) in prostate carcinoma and prostate cancer cell lines was confirmed by using Northern blot and Western blot analyses. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) on intact RNAs from 17 paired laser-captured microdissected epithelial tissue samples confirmed up-regulated BI-1 expression in 11 of 17 prostate tumors. In addition, it was demonstrated that BI-1 expression is down-regulated in stromal cells as compared to matched normal epithelial cells of the prostate. In situ hybridization experiments on prostate sections also revealed that BI-1 expression is mainly restricted to epithelial cells. Furthermore, quantitative RT-PCR on RNAs derived from five benign prostate hyperplasia (BPH) samples showed no significant difference in BI-1 expression as compared to normal epithelial prostate tissue. To determine the function of BI-1 in vitro, human PC-3, LNCaP, and DU-145 prostate carcinoma cells were transfected with small interfering double-strand RNA (siRNA) oligonucleotides against the BI-1 gene leading to a specific down-regulation of BI-1 expression. Furthermore, transfection of PC-3, LNCaP, and DU-145 cells with BI-1 sequence-specific siRNAs caused a significant increase in spontaneous apoptosis in all cell lines. Taken together, our results indicate that the human BI-1 gene contains the potential to serve as a prostate cancer expression marker and as a potential target for developing therapeutic strategies for prostate cancer.

The relevance of estrogen receptor-β expression to the antiproliferative effects observed with histone deacetylase inhibitors and phytoestrogens in prostate cancer treatment

In the prostate, estrogen receptor β (ERβ), the preferred receptor for phytoestrogens, has features of a tumor suppressor. To investigate the mechanisms underlying the beneficial effects on prostate cancer of histone deacetylase inhibitor valproic acid (VPA) and phytoestrogen tectorigenin, we analyzed the expression of ERβ after tectorigenin or VPA treatment. For further functional analysis, we knocked down ERβ expression by RNA interference. LNCaP prostate cancer cells were treated with 5 mmol/L VPA or 100 μmol/L tectorigenin and transfected with small interfering RNA (siRNA) against ERβ. Control transfections were done with luciferase (LUC) siRNA. Expression of ERβ was assessed by Western blot. mRNA expression was quantitated by real-time reverse transcription-PCR. Expression of ERβ mRNA and protein markedly increased after VPA or tectorigenin treatment. When ERβ was knocked down by siRNA, the expression of prostate-derived Ets factor, prostate-specific antigen, prostate cancer–specific indicator gene DD3PCA3, insulin-like growth factor-1 receptor, the catalytic subunit of the telomerase, and ERα was up-regulated and the tectorigenin effects were abrogated. ERβ levels were diminished in prostate cancer and loss of ERβ was associated with proliferation. Here, we show that siRNA-mediated knockdown of ERβ increases the expression of genes highly relevant to tumor cell proliferation. In addition, we show that one prominent result of treatment with VPA or tectorigenin is the up-regulation of ERβ resulting in antiproliferative effects. Thus, these drugs, by restoring the regulatory function of ERβ in tumor cells, could become useful in the intervention of prostate cancer. [Mol Cancer Ther 2007;6(10):2626–33]

Increased microtubule assembly rates influence chromosomal instability in colorectal cancer cells

Chromosomal instability (CIN) is defined as the perpetual missegregation of whole chromosomes during mitosis and represents a hallmark of human cancer. However, the mechanisms influencing CIN and its consequences on tumour growth are largely unknown. We identified an increase in microtubule plus-end assembly rates as a mechanism influencing CIN in colorectal cancer cells. This phenotype is induced by overexpression of the oncogene AURKA or by loss of the tumour suppressor gene CHK2, a genetic constitution found in 73% of human colorectal cancers. Increased microtubule assembly rates are associated with transient abnormalities in mitotic spindle geometry promoting the generation of lagging chromosomes and influencing CIN. Reconstitution of proper microtubule assembly rates by chemical or genetic means suppresses CIN and thereby, unexpectedly, accelerates tumour growth in vitro and in vivo. Thus, we identify a fundamental mechanism influencing CIN in cancer cells and reveal its adverse consequence on tumour growth.

A gene expression signature for chemoradiosensitivity of colorectal cancer cells.

PURPOSE The standard treatment of patients with locally advanced rectal cancers comprises preoperative 5-fluorouracil-based chemoradiotherapy followed by standardized surgery. However, tumor response to multimodal treatment has varied greatly, ranging from complete resistance to complete pathologic regression. The prediction of the response is, therefore, an important clinical need. METHODS AND MATERIALS To establish in vitro models for studying the molecular basis of this heterogeneous tumor response, we exposed 12 colorectal cancer cell lines to 3 μM of 5-fluorouracil and 2 Gy of radiation. The differences in treatment sensitivity were then correlated with the pretherapeutic gene expression profiles of these cell lines. RESULTS We observed a heterogeneous response, with surviving fractions ranging from 0.28 to 0.81, closely recapitulating clinical reality. Using a linear model analysis, we identified 4,796 features whose expression levels correlated significantly with the sensitivity to chemoradiotherapy (Q <.05), including many genes involved in the mitogen-activated protein kinase signaling pathway or cell cycle genes. These data have suggested a potential relevance of the insulin and Wnt signaling pathways for treatment response, and we identified STAT3, RASSF1, DOK3, and ERBB2 as potential therapeutic targets. The microarray measurements were independently validated for a subset of these genes using real-time polymerase chain reactions. CONCLUSION We are the first to report a gene expression signature for the in vitro chemoradiosensitivity of colorectal cancer cells. We anticipate that this analysis will unveil molecular biomarkers predictive of the response of rectal cancers to chemoradiotherapy and enable the identification of genes that could serve as targets to sensitize a priori resistant primary tumors.