Kirsi Narko

Cytoplasmic HuR Expression Is a Prognostic Factor in Invasive Ductal Breast Carcinoma

HuR is a ubiquitously expressed mRNA-binding protein. Intracellular localization of HuR is predominantly nuclear, but it shuttles between the nucleus and the cytoplasm. In the cytoplasm it can stabilize certain transcripts. Because nucleocytoplasmic translocation of HuR is necessary for its activity, it was hypothesized that cytoplasmic HuR expression in cancer cells could be a prognostic marker. To test the significance of HuR in carcinogenesis of the breast, we have investigated HuR expression in a mouse mammary gland tumor model and from 133 invasive ductal breast carcinoma specimens. HuR expression was elevated in the cyclooxygenase-2 transgene-induced mouse mammary tumors, and its expression was predominantly cytoplasmic in the tumor cells. In the human carcinoma samples, high cytoplasmic immunoreactivity for HuR was found in 29% (38 of 133) of the cases. Cytoplasmic HuR expression associated with high grade (P = 0.0050) and tumor size over 2 cm (P = 0.0082). Five-year distant disease-free survival rate was 42% [95% confidence interval (95% CI), 26-58] in cytoplasm-high category and 84% (95% CI, 76-91) in cytoplasm-negative or -low category (P < 0.0001), and high cytoplasmic expression of HuR was an independent prognostic factor in a Cox multivariate model (relative risk 2.07; 95% CI, 1.05-4.07). Moreover, high cytoplasmic HuR immunopositivity was significantly associated with poor outcome in the subgroup of node-negative breast cancer in a univariate analysis (P < 0.0007). Our results show that high cytoplasmic HuR expression is associated with a poor histologic differentiation, large tumor size, and poor survival in ductal breast carcinoma. Thus, HuR is the first mRNA stability protein of which expression associates with poor outcome in breast cancer.

Differential Hormonal Regulation of Vascular Endothelial Growth Factors VEGF, VEGF-B, and VEGF-C Messenger Ribonucleic Acid Levels in Cultured Human Granulosa-Luteal Cells1

The development of ovarian follicles and subsequent corpus luteum formation is accompanied by very active angiogenesis. Ovarian granulosa cells produce vascular endothelial growth factor (VEGF), which is a potent endothelial cell mitogen and an angiogenic agent. The complementary DNAs of two other factors structurally related to VEGF, namely VEGF-B and VEGF-C, were recently cloned, but little is known of their regulation in the ovary. We first studied the expression of the messenger RNAs (mRNAs) of the three VEGF isotypes in freshly isolated human granulosa-luteal (GL) cells obtained at oocyte retrieval for in vitro fertilization. The hormonal regulation of these mRNAs was subsequently studied in primary cultures of human GL cells. Analysis of cultured GL cell RNA by reverse transcription-PCR revealed that these cells express the alternatively spliced transcripts representing 121-, 145-, and 165-amino acid VEGF isoforms. Northern blot hybridization analyses indicated that transcripts of 4.5 and 3.7 kiloba...

Induction of cyclooxygenase-2 and prostaglandin F2alpha receptor expression by interleukin-1beta in cultured human granulosa-luteal cells.

Prostanoids are important regulators of ovarian function, especially during ovulation and luteolysis. Cyclooxygenase (Cox) is the rate-limiting enzyme in conversion of arachidonic acid to prostanoids. We have examined the expression and regulation of the inducible Cox isoform (Cox-2) and of the receptor for PGF2α (FP) in human granulosa cells obtained from women undergoing oocyte retrieval for in vitro fertilization. Freshly isolated granulosa cells express Cox-2 and FP receptor messenger RNAs (mRNAs). FP receptor mRNA is also expressed in cultured human granulosa-luteal (GL) cells, but Cox-2 transcripts are expressed only upon induction. Interleukin-1β (IL-1β) elevated Cox-2 mRNA steady state levels in a concentration-dependent manner, and kinetic studies showed that Cox-2 mRNA levels were already induced at the 2 h point and returned to the basal level after incubation for 24 h. The protein synthesis inhibitor, cycloheximide, induced Cox-2 mRNA expression and potentiated the effect of IL-1β. Degradation...

Effect of inflammatory cytokines on the expression of the vascular endothelial growth factor‐C

The development of the vascular system involves vasculogenesis and angiogenesis. In vasculogenesis the endothelium of blood vessels forms by in situ differentiation from precursor cells called angioblasts. During later embryogenesis and adult life the new blood vessels are formed mainly via angiogenesis, most commonly involving sprouting of capillaries from preexisting blood vessels (Hanahan & Folkman 1996). Angiogenesis is thus an important process in many physiological and pathological conditions such as female reproductive functions, wound repair, tumour growth and metastasis, and chronic inflammatory diseases (Hanahan & Folkman 1996). Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is an important angiogenic agent and the most specific known endothelial cell growth factor (Ferrara & Davis-Smyth 1997). VEGF also induces vascular permeability, regulates production of proteases and their inhibitors, and promotes endothelial cell differentiation, movement, and survival (Ferrara & Davis-Smyth 1997). Several VEGF isoforms are produced by alternative splicing of a single gene of which VEGF121, VEGF145 and VEGF165 are secreted soluble proteins and VEGF189 remains bound at the cell surface (Ferrara & Davis-Smyth 1997). VEGF homodimers bind and signal through tyrosine kinase receptors VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR) that are expressed by the endothelial cells. Recently Soker et al. (1998) reported binding of VEGF165 isoform also to neuropilin-1 which has been previously identified as a receptor for the collapsin/semaphorin family. Genetic disruption of VEGF and its receptors indicate that they are necessary for vasculogenesis and/or angiogenesis. Knock-out mice of VEGFR-1 have abnormal vascular organization (Fong et al. 1995) and VEGFR-2 deficient mice show complete inhibition of vascular development (Shalaby et al. 1995). In addition, heterozygous VEGF knock-out mice have impaired blood vessel formation (Carmeliet et al. 1996; Ferrara et al. 1996). VEGF-C (VEGF-related protein or VEGF-2) was initially isolated from conditioned media from PC-3 prostatic adenocarcinoma cells and cloned from PC-3 cell library (Joukov et al. 1996). The VEGF-C gene is 40 kb long and contains seven exons. Exons 3 and 4 are homologous with the VEGF gene, exons 5 and 7 encode cysteine-rich motifs, and exon 6 has motifs typical for the silk protein synthesized by the salivary gland of midge larvae. The 5′ untranslated region of the VEGF-C gene shows promoter activity in reporter gene assays and it contains putative binding sites for Sp-1, AP-2, and NF-κB transcription factors (Chilov et al. 1997). The human VEGF-C cDNA encodes a protein of 419 amino acids and the predicted molecular mass is 46.9 kD. VEGF-C is first synthesized as a preproprotein consisting N-terminal signal sequence, followed by N-terminal propeptide, the VEGF homology domain, and C-terminal propeptide. The major secreted VEGF-C form is a proteolytically cleaved homodimer. VEGF-C precursor protein has little activity, but the fully processed form binds and activates VEGFR-2 and VEGFR-3 (Flt-4) (Joukov et al. 1996). VEGF-C stimulates the migration of endothelial cells and increases vascular permeability (Joukov et al. 1996). However, unlike VEGF, it is relatively weak mitogen for blood vascular endothelial cells, but it stimulates proliferation of lymphatic endothelial cells (Joukov et al. 1997). VEGF-C mRNA is expressed at low levels in many tissues including lymph nodes, heart, placenta, skeletal muscle, ovary, and small intestine (Joukov et al. 1996) and it is a ligand for VEGFR-2 and VEGFR-3 (Joukov et al. 1996; Joukov et al. 1997). VEGFR-3 is expressed in most endothelial cells in early embryos, but later in development it becomes restricted to the venous compartment, and in adult tissues the expression of VEGFR-3 is restricted to the lymphatic endothelium (Kaipainen et al. 1995). Thus, VEGFR-3 is the first specific marker for the lymphatic endothelium and provides a new tool to investigate the lymphatic endothelial cell system, which has been less studied than the endothelial cells of blood vessels. Cardiovascular failure during embryonic development in VEGFR-3 knock out mice shows that VEGFR-3 has also a role in blood vessel formation (Dumont et al. 1998). The interaction of VEGF-C and lymphatic vessels is evident in mice overexpressing VEGF-C gene under transcriptional control of the human keratin 14 promoter that directs the expression of the transgene to the basal cells of stratified squamous epithelia. These mice develop hyperplastic lymphatic vessels in the skin that have overlapping endothelial junctions, anchoring filaments in the vessel wall, and a discontinuous and even partially absent basement membrane, all characteristics typical for lymphatic vessels (Jeltsch et al. 1997). The network of lymphatic vessels had similar mesh sizes in both normal and transgenic mice, but the diameter of the vessels was twice as large in transgenic animals. Overexpression of VEGF-C induced endothelial cell proliferation that lead to hyperplasia, but not to sprouting of lymphatic vessels or blood vessel angiogenesis. In addition, VEGF-C has been shown to induce lymphangiogenic response in avian chorioallantoic membrane assay (Oh et al. 1997). Other members of the VEGF-family include placenta growth factor (PlGF) and more recently discovered members of the VEGF family VEGF-B (VEGF-related factor), VEGF-D (c-fos-induced growth factor) and VEGF-E. PlGF shares a 56% identity at the amino acid level with the PDGF-like region of VEGF (Maglione et al. 1991). PlGF and VEGF can form heterodimers that bind VEGFR-2 and induce endothelial cell proliferation and migration (DiSalvo et al. 1995 and Cao et al. 1996). However, PlGF homodimers that only bind VEGFR-1 do not induce growth of endothelial cells (Park et al. 1994). VEGF-B binds to VEGFR-1 and regulates urokinase type plasminogen activator and plasminogen activator inhibitor 1 expression and activity in endothelial cells (Olofsson et al. 1996, 1998). It is expressed in most tissues and the expression is especially high in the heart and skeletal muscle. VEGF-D is related relatively closely to VEGF-C. Similarly to VEGF-C it binds to VEGFR-2 and VEGFR-3 and is an endothelial cell mitogen (Achen et al. 1998). VEGF-D is most abundantly expressed in the heart, the lung, skeletal muscle, colon, and small intestine VEGF-E is viral homologue of VEGF that binds to VEGFR-2 (Ogawa et al. 1998; Wise et al. 1999). Angiogenesis is an important component of chronic inflammatory diseases such as rheumatoid arthritis (RA) and psoriasis (Folkman 1995). Blood vessels maintain the chronic inflammatory state by transporting inflammatory cells to the site of inflammation and supplying nutrients and oxygen to the proliferating tissue. The synovium in RA is characterized by formation of highly vascularized synovial tissue that invades and destroys the cartilage and the bone. Levels of VEGF have been found to be high in the synovial fluid of RA patients (Koch et al. 1994) and VEGF mRNA and protein are expressed by synovial lining cells, magrophages, fibroblasts, and smooth muscle cells in highly vascularized areas in the RA synovial tissue (Fava et al. 1994). Tumour necrosis factor (TNF)-α and interleukin (IL)-1 are proinflammatory cytokines that have an important role in inflammatory conditions and they may account for the majority of magrophage-derived angiogenic activity in RA (Szekanecz et al. 1998). IL-1 and TNF-α stimulate expression of VEGF-C in human lung fibroblasts and in human umbilical vein endothelial cells (HUVEC) (Ristimaki et al. 1998). This cytokine-induced expression of VEGF-C may have a role in inflammation by controlling the composition and pressure of interstitial fluid and by facilitating lymphocyte trafficking. Similarly, the expression of VEGF has been shown to be stimulated by IL-1 and/or TNF-α in several cell types including human synovial fibroblasts (Ben-Av et al. 1995), rat aortic smooth muscle cells (Li et al. 1995), keratinocytes (Frank et al. 1995), and human lung fibroblasts (Ristimaki et al. 1998). In addition, IL-1 and TNF-α induce VEGFR-2 mRNA in HUVECs (Ristimaki et al. 1998; Giraudo et al. 1998). All this suggests that both production of VEGF and VEGF-C and responsiveness of these growth factors via modulation of VEGFR-2 expression is under tight control facilitated by proinflammatory cytokines. Further, the anti-inflammatory glucocorticoid dexamethasone inhibits IL-1-induced VEGF and VEGF-C mRNA expression (Ristimaki et al. 1998). In addition to cytokines, VEGF-C mRNA levels are increased after stimulation by platelet-derived growth factor, epidermal growth factor, and transforming growth factor-β (Enholm et al. 1997). Hypoxia, which is an important stimulus for angiogenesis and inducer of VEGF expression, does not induce VEGF-C expression (Ristimaki et al. 1998). Hypoxia induces VEGF expression by trascriptional activation via hypoxia-inducible factor-1 and by postranscriptional stabilization of the mRNA (Ikeda et al. 1995; Levy et al. 1995; Liu et al. 1995). Similarly, the mechanism of action of IL-1 on VEGF has been suggested to depend on both trascriptional and post-transcriptional regulation (Li et al. 1995). The rapid decay of the VEGF mRNA has been shown to be dependent on protein that binds to AU-rich instability motifs in 3′-untranslated region of VEGF mRNA (Levy et al. 1995) that are not present in the VEGF-C 3′-untranslated region. Indeed, expression of VEGF-C seems to be mainly regulated at the trascriptional level and not by stabilization of the mRNA (Enholm et al. 1997; Ristimaki et al. 1998). The upregulation of VEGF-C by proinflammatory cytokines may have an important role in inflammation by controlling composition and pressure of interstitial fluid and by facilitating lymphocyte trafficking.

Inhibition of cyclooxygenase-2 causes regression of gastric adenomas in trefoil factor 1 deficient mice.

Cyclooxygenase‐2 (Cox‐2) expression is a marker of reduced survival in gastric cancer patients, and inhibition of Cox‐2 suppresses gastrointestinal carcinogenesis in experimental animal models. To investigate the role of Cox‐2 in gastric carcinogenesis in vivo, we utilized trefoil factor 1 (Tff1) deficient mice, which model the neoplastic process of the stomach by developing gastric adenomas with full penetrance. These tumors express Cox‐2 protein and mRNA, and we have now investigated the effects of genetic deletion of the mouse Cox‐2 gene [also known as prostaglandin‐endoperoxide synthase 2 (Ptgs2)] and a Cox‐2 selective drug celecoxib. Our results show that genetic deletion of Cox‐2 in the Tff1 deleted background resulted in reduced adenoma size and ulceration with a chronic inflammatory reaction at the site of the adenoma. To characterize the effect of Cox‐2 inhibition in more detail, mice that had already developed an adenoma were fed with celecoxib for 8–14 weeks, which resulted in disruption of the adenoma that ranged from superficial erosion to deep ulcerated destruction accompanied with chronic inflammation. Importantly, mice fed with celecoxib for 16 weeks, followed by control food for 9 weeks, redeveloped a complete adenoma with no detectable inflammatory process. Finally, we determined the identity of the Cox‐2 expressing cells and found them to be fibroblasts. Our results show that inhibition of Cox‐2 is sufficient to reversibly disrupt gastric adenomas in mice.

Role of the Early Response Gene Cyclooxygenase (Cox)-2 in Angiogenesis

Cyclooxygenase (Cox)-2 is a recently-identified isoform of prostaglandin synthase, a rate-limiting enzyme in prostaglandin (PG) biosynthesis. PGs are short-lived mediators that regulate a variety of biological processes in the vasculature including angiogenesis. While PGE2 is a potent inducer of angiogenesis, the mechanisms involved are not well-understood. We have cloned the Cox-2 gene as a differentiation-induced early response gene from human umbilical vein endothelial cells (HUVEC). The expression of the Cox-2 gene is induced in a sustained manner by the inflammatory mediator interleukin-1 (IL-1) and is suppressed by the anti-inflammatory glucocorticoid dexamethasone (dex). Post-transcriptional regulation of Cox-2 mRNA stability is a major mechanism that regulates the expression of the Cox-2 gene. IL-1 stabilizes the Cox-2 transcript turn-over and thereby achieve a sustained induction of the Cox-2-dependent PG synthesis. In contrast to the Cox-1 enzyme, the expression of the Cox-2 enzyme is low in normal quiescent tissues; however, in acute inflammation, Cox-2 in induced transiently and is suppressed as the inflammatory response subsides. In chronic inflammatory diseases such as rheumatoid arthritis (RA), however, the Cox-2 gene is chronically expressed in an exaggerated manner. High levels of Cox-2 expression is correlated with the highly angiogenic state of the RA synovium. Because PGE2 is a major prostanoid produced and PGE2 also induces angiogenesis, the mechanisms of Cox-2–dependent angiogenesis was further investigated. While PGE2 does not directly induce angiogenic behavior in endothelial cells in vitro, PGE2-treated RA synovial fibroblasts expressed high levels of vascular endothelial cell growth factor (VEGF) mRNA. The induction appears to utilize the EP2 subtype of the PGE receptor which signals via the Gs/ cAMP/ protein kinase A pathway. These data suggest that Cox-2-derived PGE2 may be an indirect angiogenic factor in RA. In addition to RA, Cox-2 expression is also upregulated in the colorectal cancer. That Cox-2-derived PG may play a causative role in colorectal cancer development is suggested by epidemiological data which demonstrated that Cox inhibitors reduced the incidence of and death from colon cancer. It is not known how high-levels of Cox-2 expression contributes to the development of the malignant disease. However, the ability of Cox enzymes to activate mutagens as well as the property of prostanoids to induce cell proliferation, angiogenesis and immunosuppression may be involved. These data suggest that the dysregulated expression of the early response gene Cox-2 is an important component of many proliferative diseases and that induction of angiogenesis may be a critical component. Better understanding of the mechanisms involved in Cox-2 expression and function may yield novel therapeutic approaches for the control of proliferative and angiogenic diseases such as RA and colon cancer.