2-Methoxyestradiol (2-ME), an endogenous estrogen metabolite which disrupts microtubule function, has been shown to inhibit proliferating cells in vitro and suppress certain murine tumors in vivo. In vitro screening has determined that breast cancer cell lines are most sensitive to inhibition by 2-ME. Additionally, 2-ME has been shown to inhibit angiogenesis in vitro. We tested whether 2-ME suppresses cytokine-induced angiogenesis in vivo and inhibits <em>growth</em> of a human breast carcinoma in severe combined immunodeficient mice. A model of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF)-induced corneal neovascularization in C57BL/6 mice was used to evaluate the antiangiogenic effects of 2-ME and other microtubule inhibitors such as Taxol, vincristine, and colchicine. 2-ME (150 mg/kg p.o., n = 20) inhibited bFGF and VEGF-induced neovascularization by 39% and 54%, respectively. Taxol (6 mg/kg i.p., n = <em>17</em>) inhibited bFGF and VEGF-induced neovascularization by 45% and 37%, respectively. Vincristine (0.2 mg/kg i.p., n = 8) and colchicine (0.25 mg/kg i.p., n = 8) had no effect. Treatment with 2-ME (75 mg/kg p.o., n = 9) for 1 month suppressed the <em>growth</em> of a human breast carcinoma in mice by 60% without toxicity. Recognition of the antiangiogenic and antitumor properties of 2-ME and Taxol may be crucial in planning clinical applications to angiogenesis-dependent diseases.

Bleomycin-induced pulmonary fibrosis is characterized by inflammation, emergence of myo<em>fibroblasts</em>, and deposition of extracellular matrix. In an attempt to identify genes that may be involved in fibrosis, we used a 10,000 element (10 K) rat cDNA microarray to analyze the lung gene expression profiles in this model in the rat. Cluster analysis showed 628 genes were more than or equal to twofold up- or down-regulated, many of which were known to be involved in fibrosis. However, the most dramatic increase was observed with FIZZ1 (found in inflammatory zone; also known as RELM-alpha or resistin-like molecule-alpha), which was induced <em>17</em>-fold to approximately 25-fold at the peak of expression. In situ hybridization analysis revealed FIZZ1 expression to localize primarily to alveolar and airway epithelium, which was confirmed in vitro by analysis of isolated type II alveolar epithelial cells. However FIZZ1 expression was not detected in isolated lung <em>fibroblasts</em>. Co-culture of FIZZ1-expressing type II cells with <em>fibroblasts</em> stimulated alpha-smooth muscle actin and type I collagen expression independent of transforming <em>growth</em> <em>factor</em>-beta. Transfection of a FIZZ1-expressing plasmid into <em>fibroblasts</em> or treatment with glutathione S-transferase-FIZZ1 fusion protein stimulated alpha-smooth muscle actin and collagen I production. These results suggest a novel role for FIZZ1 in myofibroblast differentiation in pulmonary fibrosis.

Prostate cancer, the most prevalent cancer affecting men, frequently metastasizes to the axial skeleton where it produces osteoblastic lesions with <em>growth</em> rates often exceeding that of the primary tumor. To evaluate the role of tumor cell-host stromal interaction and stromal specific <em>growth</em> <em>factors</em> (GFs) in prostate cancer <em>growth</em> and progression, we coinoculated athymic mice with human prostate cancer cells (LNCaP) and various nontumorigenic <em>fibroblasts</em> s.c. LNCaP tumor formation was most consistently induced by human bone (MS) <em>fibroblasts</em> (62%), followed by embryonic rat urogenital sinus mesenchymal (rUGM) cells (31%) and Noble rat prostatic <em>fibroblasts</em> (<em>17</em>%), but not by NIH-3T3, normal rat kidney, or human lung CCD16 <em>fibroblasts</em>. Carcinomas formed preferentially in male hosts, demonstrating in vivo androgen sensitivity. The human prostate component of these tumors was confirmed with immunohistochemical staining for prostate-specific antigen (PSA), Northern analysis for PSA expression, and Southern analysis for human repetitive Alu sequences. Elevations in serum PSA paralleled the histomorphological and biochemical findings. LNCaP and <em>fibroblast</em> cell-conditioned media (CM) was used to determine whether autocrine and paracrine mitogenic pathways exist between LNCaP and <em>fibroblast</em> cells in vitro, and various defined GFs were tested to identify possible active <em>factors</em>. Mitogenic assays revealed a 200-300% bidirectional stimulation between LNCaP and bone or prostate <em>fibroblast</em>-derived CM. Lung, normal rat kidney, and 3T3 <em>fibroblast</em> CM were not mitogenic for LNCaP cells. Among the purified GFs tested basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was the most potent mitogen, stimulating LNCaP <em>growth</em> 180% in a concentration-dependent manner. Transforming <em>growth</em> <em>factor</em> alpha and epidermal <em>growth</em> <em>factor</em> were both minimally mitogenic. Coinoculation of LNCaP cells with a slowly absorbed matrix (Gelfoam) absorbed with bFGF or dialyzed and concentrated rUGM or MS CM was also capable of inducing LNCaP tumor formation in vivo. These observations illustrate that <em>fibroblasts</em> differentially modulate prostate cancer <em>growth</em> through the release of paracrine-mediated GFs, possibly including bFGF, and that tumor-stromal cell interactions play an important role in prostate cancer <em>growth</em> and progression.

<AbstractText><em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) 2 gene alterations are involved in the pathogenesis of cholangiocarcinoma. Pemigatinib is a selective, potent, oral inhibitor of FGFR1, 2, and 3. This study evaluated the safety and antitumour activity of pemigatinib in patients with previously treated, locally advanced or metastatic cholangiocarcinoma with and without FGFR2 fusions or rearrangements.</AbstractText><AbstractText>In this multicentre, open-label, single-arm, multicohort, phase 2 study (FIGHT-202), patients aged 18 years or older with disease progression following at least one previous treatment and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 recruited from 146 academic or community-based sites in the USA, Europe, the Middle East, and Asia were assigned to one of three cohorts: patients with FGFR2 fusions or rearrangements, patients with other FGF/FGFR alterations, or patients with no FGF/FGFR alterations. All enrolled patients received a starting dose of 13·5 mg oral pemigatinib once daily (21-day cycle; 2 weeks on, 1 week off) until disease progression, unacceptable toxicity, withdrawal of consent, or physician decision. The primary endpoint was the proportion of patients who achieved an objective response among those with FGFR2 fusions or rearrangements, assessed centrally in all patients who received at least one dose of pemigatinib. This study is registered with ClinicalTrials.gov, NCT02924376, and enrolment is completed.</AbstractText><AbstractText>Between Jan <em>17</em>, 20<em>17</em>, and March 22, 2019, 146 patients were enrolled: 107 with FGFR2 fusions or rearrangements, 20 with other FGF/FGFR alterations, 18 with no FGF/FGFR alterations, and one with an undetermined FGF/FGFR alteration. The median follow-up was <em>17</em>·8 months (IQR 11·6-21·3). 38 (35·5% [95% CI 26·5-45·4]) patients with FGFR2 fusions or rearrangements achieved an objective response (three complete responses and 35 partial responses). Overall, hyperphosphataemia was the most common all-grade adverse event irrespective of cause (88 [60%] of 146 patients). 93 (64%) patients had a grade 3 or worse adverse event (irrespective of cause); the most frequent were hypophosphataemia (18 [12%]), arthralgia (nine [6%]), stomatitis (eight [5%]), hyponatraemia (eight [5%]), abdominal pain (seven [5%]), and fatigue (seven [5%]). 65 (45%) patients had serious adverse events; the most frequent were abdominal pain (seven [5%]), pyrexia (seven [5%]), cholangitis (five [3%]), and pleural effusion (five [3%]). Overall, 71 (49%) patients died during the study, most frequently because of disease progression (61 [42%]); no deaths were deemed to be treatment related.</AbstractText><AbstractText>These data support the therapeutic potential of pemigatinib in previously treated patients with cholangiocarcinoma who have FGFR2 fusions or rearrangements.</AbstractText><AbstractText>Incyte Corporation.</AbstractText>

A dominant inhibitory mutation of Ha-ras which changes Ser-<em>17</em> to Asn-<em>17</em> in the gene product p21 [p21 (Asn-<em>17</em>)Ha-ras] has been used to investigate the role of ras in neuronal differentiation of PC12 cells. The <em>growth</em> of PC12 cells, in contrast to NIH 3T3 cells, was not inhibited by p21(Asn-<em>17</em>)Ha-ras expression. However, PC12 cells expressing the mutant Ha-ras protein showed a marked inhibition of morphological differentiation induced by nerve <em>growth</em> <em>factor</em> (NGF) or <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). These cells, however, were still able to respond with neurite out<em>growth</em> to dibutyryl cyclic AMP and 12-O-tetradecanoylphorbol-13-acetate (TPA). Induction of early-response genes (fos, jun, and zif268) by NGF and FGF but not by TPA was also inhibited by high levels of p21(Asn-<em>17</em>)Ha-ras. However, lower levels of p21(Asn-<em>17</em>) expression were sufficient to block neuronal differentiation without inhibiting induction of these early-response genes. Induction of the secondary-response genes SCG10 and transin by NGF, like morphological differentiation, was inhibited by low levels of p21(Asn-<em>17</em>) whether or not induction of early-response genes was blocked. Therefore, although inhibition of ras function can inhibit early-response gene induction, this is not required to block morphological differentiation or secondary-response gene expression. These results suggest that ras proteins are involved in at least two different pathways of signal transduction from the NGF receptor, which can be distinguished by differential sensitivity to p21(Asn-<em>17</em>)Ha-ras. In addition, ras and protein kinase C can apparently induce early-response gene expression by independent pathways in PC12 cells.

P450 oxidoreductase (POR) is the obligatory flavoprotein intermediate that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 enzymes. Although mouse Por gene ablation causes embryonic lethality, POR missense mutations cause disordered steroidogenesis, ambiguous genitalia, and Antley-Bixler syndrome (ABS), which has also been attributed to <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2) mutations. We sequenced the POR gene and FGFR2 exons 8 and 10 in 32 individuals with ABS and/or hormonal findings that suggested POR deficiency. POR and FGFR2 mutations segregated completely. Fifteen patients carried POR mutations on both alleles, 4 carried mutations on only one allele, 10 carried FGFR2 or FGFR3 mutations, and 3 patients carried no mutations. The 34 affected POR alleles included 10 with A287P (all from whites) and 7 with R457H (four Japanese, one African, two whites); <em>17</em> of the 34 alleles carried 16 "private" mutations, including 9 missense and 7 frameshift mutations. These 11 missense mutations, plus 10 others found in databases or reported elsewhere, were recreated by site-directed mutagenesis and were assessed by four assays: reduction of cytochrome c, oxidation of NADPH, support of <em>17</em>alpha-hydroxylase activity, and support of <em>17</em>,20 lyase using human P450c<em>17</em>. Assays that were based on cytochrome c, which is not a physiologic substrate for POR, correlated poorly with clinical phenotype, but assays that were based on POR's support of catalysis by P450c<em>17</em>--the enzyme most closely associated with the hormonal phenotype--provided an excellent genotype/phenotype correlation. Our large survey of patients with ABS shows that individuals with an ABS-like phenotype and normal steroidogenesis have FGFR mutations, whereas those with ambiguous genitalia and disordered steroidogenesis should be recognized as having a distinct new disease: POR deficiency.

We performed a high-throughput retroviral insertional mutagenesis screen in mouse mammary tumor virus (MMTV)-induced mammary tumors and identified 33 common insertion sites, of which <em>17</em> genes were previously not known to be associated with mammary cancer and 13 had not previously been linked to cancer in general. Although members of the Wnt and <em>fibroblast</em> <em>growth</em> <em>factors</em> (Fgf) families were frequently tagged, our exhaustive screening for MMTV insertion sites uncovered a new repertoire of candidate breast cancer oncogenes. We validated one of these genes, Rspo3, as an oncogene by overexpression in a p53-deficient mammary epithelial cell line. The human orthologs of the candidate oncogenes were frequently deregulated in human breast cancers and associated with several tumor parameters. Computational analysis of all MMTV-tagged genes uncovered specific gene families not previously associated with cancer and showed a significant overrepresentation of protein domains and signaling pathways mainly associated with development and <em>growth</em> <em>factor</em> signaling. Comparison of all tagged genes in MMTV and Moloney murine leukemia virus-induced malignancies showed that both viruses target mostly different genes that act predominantly in distinct pathways.

A completely serum-free assay method has been used to compare the mitogenic activities of polypeptide <em>growth</em> <em>factors</em> and estrogens with MCF-7 and T47D human breast cancer cells in culture. The lines were maintained in a viable, slowly dividing condition in Ham's F12 and Dulbecco's modified Eagle's medium (1:1) supplemented with sodium bicarbonate (2.2 g/liter), 15 mM 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid, human transferrin (10 micrograms/ml), and bovine serum albumin (200 micrograms/ml) (designated Tf/BSA). This medium allowed the assay of mitogenic activities as measured by multiple rounds of cell division and permitted comparisons of the biological potencies of <em>growth</em> <em>factors</em> within functional families as well as of dissimilar mitogens. Insulin-like <em>growth</em> <em>factor</em> I (IGF-I) was the most potent mitogen studied, showing ED50 values of 160 pg/ml and 1.7 ng/ml with the MCF-7 and T47D cells, respectively. Insulin-like <em>growth</em> <em>factor</em> II and insulin were less active, with ED50 values of 0.55 and 1.2 ng/ml with MCF-7 cells and 4.3 and 10 ng/ml with the T47D cell line, respectively. Mitogens sharing epidermal <em>growth</em> <em>factor</em>-like functional properties had ED50 values from 35 pg/ml to 2.5 ng/ml, while transforming <em>growth</em> <em>factor</em> type beta and platelet-derived <em>growth</em> <em>factor</em> had no detectable stimulatory effects. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> had ED50 values of 0.42 ng/ml and 3.7 ng/ml for the MCF-7 and T47D cells, respectively, while acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> was nearly inactive. In phenol red-free Tf/BSA, <em>17</em> beta-estradiol caused a 60% increase in MCF-7 cell numbers over controls in 8 days while having no effect on <em>growth</em> of the T47D cell line. From MCF-7 conditioned Tf/BSA medium, IGF-I was identified by biological activity, by radioimmunoassay (approximately equal to 2 pg/ml) and by estimation of molecular weight (8,000) under dissociating conditions. The concentration of IGF-I was not affected by <em>17</em> beta-estradiol treatment. The data indicate that induction of acid stable, low molecular weight autocrine <em>growth</em> <em>factors</em> involved more regulation than defined by estrogens alone. The minimal effects of <em>17</em> beta-estradiol in Tf/BSA opened several possibilities including the putative roles of other serum-borne hormones, <em>growth</em> <em>factors</em> and regulators in autocrine <em>growth</em> <em>factor</em> induction.

The insulin-like <em>growth</em> <em>factors</em> IGF-I and IGF-II circulate in blood bound to carrier proteins. The higher molecular mass IGF-binding protein complex (150 kDa) is composed of subunits, and one subunit that forms this complex is <em>growth</em> hormone dependent. In addition, many cell types and tissues secrete another form of IGF binding protein that is not <em>growth</em> hormone dependent. Both forms of the IGF binding protein are believed to inactivate the IGFs and to function as delivery systems to tissues. This conclusion was based on studies that determined the effects of impure preparations of these binding proteins or that examined the effect of these proteins only on the insulin-like actions of the IGFs. We report here that a pure preparation of the extracellular form of the IGF binding protein (purified from human amniotic fluid) markedly potentiated replication of several cell types in response to human IGF-I. Secondary cultures of human, mouse, and chicken embryo <em>fibroblasts</em> as well as porcine aortic smooth muscle cells showed marked enhancement of their DNA synthesis response (2.8- to 4.4-fold increases) to IGF-I in the presence of this protein. These responses were synergistic since the sum of the responses to either IGF-I or to the binding protein alone was between 8 and <em>17</em>% of the increase obtained in cultures exposed to both peptides. The binding protein not only potentiated the DNA synthesis response but also enhanced the increase in cell number in response to IGF-I. This stimulation is specific for <em>growth</em> <em>factors</em> that bind to the binding protein since incubation with insulin, which binds to the type I IGF receptor but not to the binding protein, did not result in potentiation of this response. We conclude that a form of IGF binding protein that is present in extracellular fluids and is secreted by many types of cells can markedly potentiate the cellular response to IGF-I.

OBJECTIVE

To determine the frequency and the prognostic value of fibroblast growth factor receptor 3 (FGFR3) mutations in patients with nonmuscle invasive bladder tumors according to tumor stage and grade.

METHODS

Seven hundred seventy-two patients with newly diagnosed bladder tumors were recruited. Tumors were reviewed by expert pathologists. Patients were prospectively followed-up (median, 62.6 months for disease-free patients) through review of hospital records and telephone interviews. The sequence of exons 7 and 10 of FGFR3 was analyzed by polymerase chain reaction and direct sequencing. We assessed the association of mutations with stage and grade. The predictive value of mutations for recurrence, progression, and mortality were assessed using Kaplan-Meier and Cox multivariable models.

RESULTS

Mutations were more common among low malignant potential neoplasms (LMPN; 77%) and TaG1/TaG2 tumors (61%/58%) than among TaG3 tumors (34%) and T1G3 tumors (17%). The S249C, Y375C, S248C, and G372C mutations accounted for 91.5% of all sequence changes. The A393E substitution was associated with LMPN (P < .001). The F386L polymorphism was more frequent among patients with low-grade tumors (odds ratio, 6.97; 95%CI, 1.40 to 47.06; P = .009). In the multivariable analysis of all superficial tumors, mutations were associated with increased risk of recurrence. However, in the stratified analyses only patients with TaG1 tumors had a significantly higher risk of recurrence (hazard ratio, 2.12; 95%CI, 1.28 to 3.53; P = .004).

CONCLUSIONS

The findings of this large study strongly support the notion that FGFR3 mutations characterize a subgroup of bladder cancers with good prognosis; patients with mutant TaG1 tumors have a higher risk of recurrence; and the F386L variant is selectively associated with low-grade tumors.

BACKGROUND

Angiogenesis is a critical event in wound healing, tumor growth, and the inflammatory vasculitides. Since women have a higher incidence of many vasculitic diseases, we examined the effects of female sex steroids, particularly estradiol, on human umbilical vein endothelial cell (HUVEC) behavior in vitro and on angiogenesis in vivo.

RESULTS

HUVECs were grown in estrogen-free medium before each assay. Exogenous 17 beta-estradiol (1 to 5 nmol/L) increased cell attachment to laminin, types I and IV collagen, and fibronectin, as well as to tissue culture plastic. After a confluent monolayer of cells was "wounded" by scraping, estradiol-treated (10(-8) mol/L) cells migrated into the wound three times faster than untreated cells. Cell proliferation on plastic and on laminin increased threefold to fivefold, respectively, in the presence of estradiol. Estradiol also enhanced the ability of HUVECs to organize into tubular networks when plated on a reconstituted basement membrane, Matrigel. Estradiol effects on both the "wounding" assay and tube formation were blocked by the specific estrogen receptor antagonist ICI 182,780. Ovariectomy markedly decreased in vivo vascularization of Matrigel plugs coinjected with basic fibroblast growth factor in mice. With estrogen replacement, angiogenesis was increased to the levels observed in nonovariectomized mice.

CONCLUSIONS

These studies demonstrate that, in vitro and in vivo, estradiol enhances endothelial cell activities important in neovascularization and suggest a promoting influence of estrogens on angiogenesis.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are pleiotrophic <em>growth</em> <em>factors</em> that control cell proliferation, migration, differentiation and embryonic patterning. During early zebrafish embryonic development, FGFs regulate dorsoventral patterning by controlling ventral bone morphogenetic protein (BMP) expression. FGFs function by binding and activating high-affinity tyrosine kinase receptors. FGF activity is negatively regulated by members of the Sprouty family, which antagonize Ras signalling induced by receptor tyrosine kinases. On the basis of similarities in their expression patterns during embryonic development, we have identified five genes that define a synexpression group -- fgf8, fgf3, sprouty2, sprouty4, as well as a novel gene, sef (similar expression to fgf genes). Sef encodes a conserved putative transmembrane protein that shares sequence similarities with the intracellular domain of the interleukin <em>17</em> receptor. Here we show that in zebrafish, Sef functions as a feedback-induced antagonist of Ras/Raf/MEK/MAPK-mediated FGF signalling.

We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-<em>17</em> to Asn-<em>17</em> in the gene product p21 [p21(Asn-<em>17</em>)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M<em>17</em>)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-<em>17</em>) via the mouse mammary tumor virus long terminal repeat and was <em>growth</em> inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M<em>17</em>) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal <em>growth</em> <em>factor</em> (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-<em>17</em>) expression, and stimulation by serum, <em>fibroblast</em> <em>growth</em> <em>factor</em>, and platelet-derived <em>growth</em> <em>factor</em> was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-<em>17</em>) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-<em>17</em> expression vector. In this assay, p21(Asn-<em>17</em>) inhibited chloramphenicol acetyltransferase expression induced by EGF and other <em>growth</em> <em>factors</em>. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-<em>17</em> expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.

The binding of polypeptide <em>growth</em> <em>factors</em> to their appropriate cell surface transmembrane receptors triggers numerous biochemical responses, including the transcriptional activation of specific genes. We have used a differential display approach to identify <em>fibroblast</em> <em>growth</em> <em>factor</em>-1-inducible genes in murine NIH 3T3 cells. Here, we report that the <em>fibroblast</em> <em>growth</em> <em>factor</em>-inducible-14 (Fn14) gene is a <em>growth</em> <em>factor</em>-regulated, immediate-early response gene expressed in a developmental stage- and adult tissue-specific manner in vivo. This gene, located on mouse chromosome <em>17</em>, is predicted to encode an 129-amino acid type Ia membrane protein with no significant sequence similarity to any known protein. We have used two experimental approaches, direct fluorescence microscopy and immunoprecipitation analysis of biotinylated cell surface proteins, to demonstrate that Fn14 is located on the plasma membrane. To examine the biological consequences of constitutive Fn14 expression, we isolated NIH 3T3 cell lines expressing variable levels of epitope-tagged Fn14 and analyzed their phenotypic properties in vitro. These experiments revealed that Fn14 expression decreased cellular adhesion to the extracellular matrix proteins fibronectin and vitronectin and also reduced serum-stimulated cell <em>growth</em> and migration. These results indicate that Fn14 is a novel plasma membrane-spanning molecule that may play a role in cell-matrix interactions.

OBJECTIVE

Pulmonary complications, including pulmonary fibrosis (PF) and pulmonary arterial hypertension (PAH), are the leading cause of mortality in patients with systemic sclerosis (SSc). The aim of this study was to compare the molecular fingerprint of lung tissue and matching primary fibroblasts from patients with SSc with that of lung tissue and fibroblasts from normal donors, patients with idiopathic pulmonary fibrosis (IPF), and patients with idiopathic pulmonary arterial hypertension (IPAH).

METHODS

Lung tissue samples were obtained from 33 patients with SSc who underwent lung transplantation. Tissues and cells from a subgroup of SSc patients with predominantly PF or PAH were compared to those from normal donors, patients with IPF, and patients with IPAH. Microarray data were analyzed using efficiency analysis for determination of the optimal data-processing methods. Real-time polymerase chain reaction and immunohistochemistry were used to confirm differential levels of messenger RNA and protein, respectively.

RESULTS

Consensus efficiency analysis identified 242 and 335 genes that were differentially expressed in lungs and primary fibroblasts, respectively. SSc-PF and IPF lungs shared enriched functional groups in genes implicated in fibrosis, insulin-like growth factor signaling, and caveolin-mediated endocytosis. Gene functional groups shared by SSc-PAH and IPAH lungs included those involved in antigen presentation, chemokine activity, and interleukin-17 signaling.

CONCLUSIONS

Using microarray analysis on carefully phenotyped SSc and comparator lung tissues, we demonstrated distinct molecular profiles in tissues and fibroblasts from patients with SSc-associated lung disease compared to idiopathic forms of lung disease. Unique molecular signatures were generated that are disease specific (SSc) and phenotype specific (PF versus PAH). These signatures provide new insights into the pathogenesis and potential therapeutic targets of SSc-related lung disease.

BACKGROUND

An unmet medical need exists for patients with metastatic renal cell carcinoma who have progressed on VEGF-targeted and mTOR-inhibitor therapies. Fibroblast growth factor (FGF) pathway activation has been proposed as a mechanism of escape from VEGF-targeted therapies. Dovitinib is an oral tyrosine-kinase inhibitor that inhibits VEGF and FGF receptors. We therefore compared dovitinib with sorafenib as third-line targeted therapies in patients with metastatic renal cell carcinoma.

METHODS

In this multicentre phase 3 study, patients with clear cell metastatic renal cell carcinoma who received one previous VEGF-targeted therapy and one previous mTOR inhibitor were randomly assigned through an interactive voice and web response system to receive open-label dovitinib (500 mg orally according to a 5-days-on and 2-days-off schedule) or sorafenib (400 mg orally twice daily) in a 1:1 ratio. Randomisation was stratified by risk group and region. The primary endpoint was progression-free survival (PFS) assessed by masked central review. Efficacy was assessed in all patients who were randomly assigned and safety was assessed in patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT01223027.

RESULTS

284 patients were randomly assigned to the dovitinib group and 286 to the sorafenib group. Median follow-up was 11·3 months (IQR 7·9-14·6). Median PFS was 3·7 months (95% CI 3·5-3·9) in the dovitinib group and 3·6 months (3·5-3·7) in the sorafenib group (hazard ratio 0·86, 95% CI 0·72-1·04; one-sided p=0·063). 280 patients in the dovitinib group and 284 in the sorafenib group received at least one dose of study drug. Common grade 3 or 4 adverse events included hypertriglyceridaemia (38 [14%]), fatigue (28 [10%]), hypertension (22 [8%]), and diarrhoea (20 [7%]) in the dovitinib group, and hypertension (47 [17%]), fatigue (24 [8%]), dyspnoea (21 [7%]), and palmar-plantar erythrodysaesthesia (18 [6%]) in the sorafenib group. The most common serious adverse event was dyspnoea (16 [6%] and 15 [5%] in the dovitinib and sorafenib groups, respectively).

CONCLUSIONS

Dovitinib showed activity, but this was no better than that of sorafenib in patients with renal cell carcinoma who had progressed on previous VEGF-targeted therapies and mTOR inhibitors. This trial provides reference outcome data for future studies of targeted inhibitors in the third-line setting.

BACKGROUND

Novartis Pharmaceuticals Corporation.

Squamous cell carcinomas (SCCs) of the head and neck are malignant tumors with high capacity to invade and metastasize. We have examined expression of the new collagenase, collagenase-3 (MMP-13), in SCCs of the head and neck. MMP-13 mRNAs were detected in 22 of 29 SCC cell lines: in 14 of 15 primary SCC cell lines and in 8 of 14 SCC cell lines from recurrent tumors or metastases. MMP-13 mRNAs were expressed by all 6 cell lines from highly invasive primary tumors and in all 4 cell lines from small aggressive tumors. Using in situ hybridization, MMP-13 mRNAs were detected in 15 of <em>17</em> SCC tumor samples. In most tumors, MMP-13 was expressed by tumor cells at the invading front of the tumors, but in a subset of SCCs, MMP-13 mRNA was also expressed by stromal <em>fibroblasts</em>. No MMP-13 expression was detected in intact skin or oral mucosa. MMP-13 mRNA levels in SCC cells were enhanced by transforming <em>growth</em> <em>factor</em>-beta, tumor necrosis <em>factor</em>-alpha, transforming <em>growth</em> <em>factor</em>-alpha, and keratinocyte <em>growth</em> <em>factor</em>. Specific expression of MMP-13 by SCC cells in vitro and in vivo strongly suggests a role for MMP-13 in the high invasion capacity of SCC cells.

In <em>fibroblast</em> cells, cAMP antagonizes <em>growth</em> <em>factor</em> activation of ERKs and cell <em>growth</em> via PKA and the small G protein Rap1. We demonstrate here that PKA's activation of Rap1 was mediated by the Rap1 guanine nucleotide exchange <em>factor</em> C3G, the adaptor Crk-L, the scaffold protein Cbl, and the tyrosine kinase Src. Src was required for cAMP activation of Rap1 and the inhibition of ERKs and cell <em>growth</em>. PKA activated Src both in vitro and in vivo by phosphorylating Src on serine <em>17</em> within its amino terminus. This phosphorylation was required for cAMP's activation of Src and Rap1, as well as cAMP's inhibition of ERKs and cell proliferation. This study identifies an antiproliferative role for Src in the physiological regulation of cell <em>growth</em> by cAMP.

We report that human breast cancer cells secrete a <em>growth</em> <em>factor</em> that is biologically and immunologically similar to platelet-derived <em>growth</em> <em>factor</em> (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or "competence" activity that is capable of inducing incorporation of [3H]thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. In addition, the conditioned medium contains an activity that competes with 125I-labeled PDGF for binding to PDGF receptors on normal human <em>fibroblasts</em>. The secretion of PDGF-like activity by the hormone-responsive cell line MCF-7 is stimulated by <em>17</em> beta-estradiol. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (95 degrees C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of approximately equal to 30 kDa on NaDodSO4/polyacrylamide gels. Immunoprecipitation with PDGF antiserum of proteins from metabolically labeled cell lysates and conditioned medium followed by analysis on nonreducing NaDodSO4/polyacrylamide gels identified proteins of 30 and 34 kDa. Upon reduction, the 30- and 34-kDa bands were converted to 15- and 16-kDa bands suggesting that the immunoprecipitated proteins were made up of two disulfide-linked polypeptides similar to PDGF. Hybridization studies with cDNA probes for the A chain of PDGF and the B chain of PDGF/SIS identified transcripts for both PDGF chains in the MCF-7 and MDA-MB-231 cells. The data summarized above provide conclusive evidence for the synthesis and hormonally regulated secretion of a PDGF-like mitogen by breast carcinoma cells. Production of a PDGF-like <em>growth</em> <em>factor</em> by breast cancer cell lines may be important in mediating paracrine stimulation of tumor <em>growth</em>.

The particular interest of IL-<em>17</em>, a homodimeric cytokine of about 32 kDa, is the strict requirement for an activation signal to induce its expression from a rather restricted set of cells, human memory T cells or mouse alpha beta TCR+CD4-CD8- thymocytes. In contrast with the tightly controlled expression pattern of this gene, the IL-<em>17</em> receptor, a novel cytokine receptor, is ubiquitously distributed but apparently more abundant in spleen and kidney. In addition to its capture by the T lymphotropic Herpesvirus Saimiri (HVS), this cytokine is inducing the secretion of IL-6, IL-8, PGE2, MCP-1 and G-CSF by adherent cells like <em>fibroblasts</em>, keratinocytes, epithelial and endothelial cells. IL-<em>17</em> is also able to induce ICAM-1 surface expression, proliferation of T cells, and <em>growth</em> and differentiation of CD34+ human progenitors into neutrophils when cocultured in presence of irradiated <em>fibroblasts</em>. In vitro, IL-<em>17</em> synergizes with other proinflammatory signals like TNF alpha for GM-CSF induction, and with CD40-ligand for IL-6, IL-8, RANTES and MCP-1 secretion from kidney epithelial cells. In vivo, injection of IL-<em>17</em> induces a neutrophilia, except in IL-6-KO mice. The involvement of IL-<em>17</em> in rejection of kidney graft has also been demonstrated. The role of this T cell secreted <em>factor</em> in various inflammatory processes is presently investigated.

Systemic sclerosis is a complex disease with widespread skin fibrosis and variable visceral organ involvement. Since transforming <em>growth</em> <em>factor</em>-beta (TGFbeta) has been implicated in driving fibrosis in systemic sclerosis, a mechanism-derived gene expression signature was used to assay TGFbeta-responsive gene expression in the skin of patients with systemic sclerosis (SSc). Primary dermal <em>fibroblasts</em> from patients with diffuse SSc (dSSc) and healthy controls were treated with TGFbeta, and the genome-wide gene expression was measured on DNA microarrays over a time course of 24 hours. Eight hundred and ninety-four probes representing 674 uniquely annotated genes were identified as TGFbeta responsive. Expression of the TGFbeta-responsive signature was examined in skin biopsies from <em>17</em> dSSc, seven limited SSc (lSSc), three morphea patients, and six healthy controls. The TGFbeta-responsive signature was expressed in 10 out of <em>17</em> dSSc skin biopsies, but was not found in lSSc, morphea, or healthy control biopsies. Expression of dSSC the TGFbeta-responsive signature stratifies patients into two major groups, one of which corresponds to the "diffuse-proliferation" intrinsic subset that showed higher modified Rodnan skin score and a higher likelihood of scleroderma lung disease. The TGFbeta-responsive signature is found in only a subset of dSSc patients who could be targeted by specific therapies.

OBJECTIVE

To investigate <em>factors</em> involved in inflammation of the prostate besides IL-15, we screened prostatic cells and tissues for IL-<em>17</em> and IL-<em>17</em> receptor expression.

METHODS

Normal prostate (n = 1), BPH (n = 19), and carcinoma (CaP, n = 12) specimens were screened for IL-<em>17</em>, IL-<em>17</em> receptor, CD45, IL-6, and IL-8 mRNA expression. The carcinoma cell lines DU145, PC3, LNCaP, and BPH-epithelial (EC), stromal cell (SC) preparations, and BPH-T-cell lines were analyzed for IL-<em>17</em> production by RT-PCR and ELISA. The effect of IL-<em>17</em> on IL-6, IL-8, TGF-beta1, and fibroblast growth factor (FGF-2) mRNA expression and/or release of SC was analyzed using real-time PCR and/or ELISA. Immunohistochemistry was used to localize both IL-<em>17</em> and IL-<em>17</em> receptor.

RESULTS

In the normal prostate, IL-<em>17</em> expression was very weak and restricted to lymphocytes. In 79% of BPH and 58% of CaP specimens, IL-<em>17</em> mRNA and protein expression was increased. IL-<em>17</em> mRNA expression could be shown for activated BPH-T-cells and to some extend for BPH-EC. Expression of IL-<em>17</em> receptor was ubiquitous. Release of IL-<em>17</em> was shown only for activated BPH-T-cells. IL-<em>17</em> stimulated expression of IL-6 (13-fold) and IL-8 (26-fold) by prostatic BPH-SC. In situ, however, the amount of IL-<em>17</em>mRNA in BPH-tissue did not correlate with the amount of IL-6 and IL-8 mRNA. In CaP tissue, significant correlation was found only between the amount of IL-6 and IL-8 mRNA.

CONCLUSIONS

Activated BPH-T-cells abundantly express IL-<em>17</em>. The increase of IL-<em>17</em> in BPH-tissues goes hand in hand with elevated levels of IL-15, a pro-inflammatory cytokine with T-cell growth factor properties. A clinical relevance of increased IL-<em>17</em> expression under pathological conditions is suggested by the demonstration of significant upregulation of IL-6 and IL-8 production of prostatic SC by IL-<em>17</em>.

Extensive chronic graft-versus-host disease (ecGVHD) is characterized by fibrosis similar to that of patients with systemic sclerosis (scleroderma). Since stimulatory autoantibodies against the platelet-derived <em>growth</em> <em>factor</em> (PDGF) receptor (PDGFR) have been found in patients with scleroderma and are responsible for the activation of skin <em>fibroblasts</em>, we tested the hypothesis that these autoantibodies are also present in patients affected by ecGVHD. Serum from 39 patients subjected to allogeneic stem cell transplantation for hematologic malignancies (22 with ecGVHD and <em>17</em> without cGVHD) and 20 healthy controls was assayed for the presence of stimulatory autoantibodies to the PDGFR by incubating purified IgG with mouse-embryo <em>fibroblasts</em> lacking PDGFR alpha or beta chains or with the same cells expressing PDGFR alpha. Stimulatory antibodies to the PDGFR were found selectively in all patients with ecGVHD but in none of the patients without cGVHD. Higher levels were detected in patients with generalized skin involvement and/or lung fibrosis. Antibodies recognized native PDGFR, induced tyrosine phosphorylation, accumulation of reactive oxygen species (ROS), and stimulated type 1 collagen gene expression through the Ha-Ras-ERK1/2-ROS signaling pathway. The biologic activity of these autoantibodies suggests a role in the development of fibrosis and argues for a common pathogenetic trait in ecGVDH and scleroderma phenotypes.

To study the early changes in the lower respiratory tract in persons exposed to periods of hyperoxia usually considered safe, we evaluated 14 normal subjects by bronchoalveolar lavage before and immediately after 16.7 +/- 1.1 hours of breathing more than 95 per cent oxygen. Hyperoxia caused a significant alveolar-capillary "leak" as detected by the presence of increased plasma albumin and transferrin in lavage fluid. These changes were reversible, as shown at repeat lavage in four subjects two weeks after oxygen administration. Hyperoxia for an average of <em>17</em> hours did not change the total number or type of lung inflammatory and immune effector cells recovered by lavage (P greater than 0.05, all comparisons). However, alveolar macrophages from subjects exposed to oxygen released increased amounts of fibronectin (P less than 0.05) and alveolar-macrophage--derived <em>growth</em> <em>factor</em> for <em>fibroblasts</em> (P less than 0.01)--mediators thought to modulate <em>fibroblast</em> recruitment and proliferation in the alveolar wall. Thus, although some of the effects of exposure to <em>17</em> hours of more than 95 per cent oxygen are reversible, hyperoxia for even this short period lowers the structural or functional barriers that normally prevent alveolar-capillary "leak" and induces processes that can culminate in fibrosis of the alveolar wall.