Insulin-like <em>growth</em> <em>factor</em> binding proteins (IGFBPs) have been shown to serve as carrier proteins for the insulin-like <em>growth</em> <em>factors</em> (IGFs) and to modulate their biologic effects. Since extracellular matrix (ECM) has been shown to be a reservoir for IGF-I and IGF-II, we examined the ECM of cultured human fetal <em>fibroblasts</em> and found that IGFBP-5 was incorporated intact into ECM, while mostly inert proteolytic fragments were found in the medium. In contrast, two other forms of IGFBP that are secreted by these cells were either present in ECM in minimal amounts (IGFBP-3) or not detected (IGFBP-4). Likewise, when purified IGFBPs were incubated with ECM, IGFBP-5 bound preferentially. IGFBP-5 was found to bind to types III and IV collagen, laminin, and fibronectin. Increasing salt concentrations inhibited the binding of IGFBP-5 to ECM and accelerated the release of IGFBP-5 from ECM, suggesting an ionic basis for this interaction. ECM-associated IGFBP-5 had a sevenfold decrease in affinity for IGF-I compared to IGFBP-5 in solution. Furthermore, when IGFBP-5 was present in cell culture substrata, it potentiated the <em>growth</em> stimulatory effects of IGF-I on <em>fibroblasts</em>. When IGFBP-5 was present only in the medium, it was degraded to a <em>22</em>-kD fragment and had no effect on IGF-I-stimulated <em>growth</em>. We conclude that IGFBP-5 is present in <em>fibroblast</em> ECM, where it is protected from degradation and can potentiate the biologic actions of IGF-I. These findings provide a molecular explanation for the association of the IGF's with the extracellular matrix, and suggest that the binding of the IGF's to matrix, via IGFBP-5, may be important in mediating the cellular <em>growth</em> response to these <em>growth</em> <em>factors</em>.

This study compares the relative effects of advancing male age on multiple genomic defects in human sperm [DNA fragmentation index (DFI), chromatin integrity, gene mutations, and numerical chromosomal abnormalities], characterizes the relationships among these defects and with semen quality, and estimates the incidence of susceptible individuals for a well characterized nonclinical nonsmoking group of 97 men (<em>22</em>-80 years). Adjusting for confounders, we found major associations between age and the frequencies of sperm with DFI and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 gene (FGFR3) mutations associated with achondroplasia (P < 0.01) with no evidence for age thresholds. However, we found no associations between age and the frequencies of sperm with immature chromatin, aneuploidies/diploidies, FGFR2 mutations (Apert syndrome), or sex ratio in this cohort. There were also no consistent correlations among genomic and semen-quality endpoints, except between DFI and sperm motility (r = -0.65, P < 0.001). These findings suggest there are multiple spermatogenic targets for genomically defective sperm with substantially variable susceptibilities to age. Our findings predict that as healthy males age, they have decreased pregnancy success with trends beginning in their early reproductive years, increased risk for producing offspring with achondroplasia mutations, and risk of fathering offspring with Apert syndrome that may vary across cohorts, but with no increased risk for fathering aneuploid offspring (Down, Klinefelter, Turner, triple X, and XYY syndromes) or triploid embryos. Our findings also suggest that the burden of genomic damage in sperm cannot be inferred from semen quality, and that a small fraction of men are at increased risk for transmitting multiple genetic and chromosomal defects.

<p><div><b>BACKGROUND</b></div>Alterations in the gene encoding <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (<i>FGFR</i>) are common in urothelial carcinoma and may be associated with lower sensitivity to immune interventions. Erdafitinib, a tyrosine kinase inhibitor of FGFR1-4, has shown antitumor activity in preclinical models and in a phase 1 study involving patients with <i>FGFR</i> alterations.</p><p><div><b>METHODS</b></div>In this open-label, phase 2 study, we enrolled patients who had locally advanced and unresectable or metastatic urothelial carcinoma with prespecified <i>FGFR</i> alterations. All the patients had a history of disease progression during or after at least one course of chemotherapy or within 12 months after neoadjuvant or adjuvant chemotherapy. Prior immunotherapy was allowed. We initially randomly assigned the patients to receive erdafitinib in either an intermittent or a continuous regimen in the dose-selection phase of the study. On the basis of an interim analysis, the starting dose was set at 8 mg per day in a continuous regimen (selected-regimen group), with provision for a pharmacodynamically guided dose escalation to 9 mg. The primary end point was the objective response rate. Key secondary end points included progression-free survival, duration of response, and overall survival.</p><AbstractText>A total of 99 patients in the selected-regimen group received a median of five cycles of erdafitinib. Of these patients, 43% had received at least two previous courses of treatment, 79% had visceral metastases, and 53% had a creatinine clearance of less than 60 ml per minute. The rate of confirmed response to erdafitinib therapy was 40% (3% with a complete response and 37% with a partial response). Among the <em>22</em> patients who had undergone previous immunotherapy, the confirmed response rate was 59%. The median duration of progression-free survival was 5.5 months, and the median duration of overall survival was 13.8 months. Treatment-related adverse events of grade 3 or higher, which were managed mainly by dose adjustments, were reported in 46% of the patients; 13% of the patients discontinued treatment because of adverse events. There were no treatment-related deaths.</AbstractText><p><div><b>CONCLUSIONS</b></div>The use of erdafitinib was associated with an objective tumor response in 40% of previously treated patients who had locally advanced and unresectable or metastatic urothelial carcinoma with <i>FGFR</i> alterations. Treatment-related grade 3 or higher adverse events were reported in nearly half the patients. (Funded by Janssen Research and Development; BLC2001 ClinicalTrials.gov number, NCT02365597.).</p>

Angiogenesis is essential for tumor <em>growth</em> and metastasis and depends on the production of angiogenic <em>factors</em> by tumor cells. Neuroblastoma (NB) is a common pediatric tumor of neural crest origin, which is biologically and clinically heterogeneous. Increased tumor vascular index correlates with poor outcome of NB. To determine which angiogenic <em>factors</em> contribute to NB angiogenesis and thereby support tumor progression, we examined the expression of eight angiogenic <em>factors</em> [vascular endothelial <em>growth</em> <em>factor</em> (VEGF), VEGF-B, VEGF-C, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, angiopoietin (Ang)-1, Ang-2, transforming <em>growth</em> <em>factor</em> alpha, and platelet-derived <em>growth</em> <em>factor</em> (PDGF)] by semiquantitative RT-PCR in 37 NB primary tumors and in <em>22</em> NB cell lines. We also analyzed the relationship between angiogenic <em>factor</em> expression and clinicopathological <em>factors</em> as well as patient survival. All eight angiogenic <em>factors</em> examined were expressed at various levels in NB cell lines and tumors, suggesting their involvement in NB angiogenesis. The expression levels of most angiogenic <em>factors</em> were correlated with each other, suggesting their synergy in regulating the angiogenic process. Significantly higher expression levels of VEGF, VEGF-B, VEGF-C, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, Ang-2, transforming <em>growth</em> <em>factor</em> alpha, and PDGF-A (P < 0.0001-0.026) were found in advanced-stage tumors (stages 3 and 4) compared with low-stage tumors (stages 1, 2, and 4S). Expression of PDGF-A was significantly associated with patient survival (P = 0.04). The redundancy in angiogenic <em>factor</em> expression suggests that inhibition of VEGF bioactivity alone might not be a sufficient approach for antiangiogenic therapy of human NB.

Bone morphogenetic proteins (BMPs) induce the differentiation of cells of the osteoblastic lineage and enhance the function of the osteoblast. <em>Growth</em> <em>factors</em> are regulated by binding proteins, but there is no information about binding proteins for BMPs in skeletal cells. Noggin specifically binds BMPs, but its expression by cells of the osteoblastic lineage has not been reported. We tested for the expression of noggin and its induction by BMP-2 in cultures of osteoblast-enriched cells from <em>22</em>-d-old fetal rat calvariae (Ob cells). BMP-2 caused a time- and dose-dependent increase in noggin mRNA and polypeptide levels, as determined by Northern and Western blot analyses. The effects of BMP-2 on noggin transcripts were dependent on protein, but independent of DNA synthesis. BMP-2 increased the rates of noggin transcription as determined by nuclear run-on assays. BMP-4, BMP-6, and TGF-beta1 increased noggin mRNA in Ob cells, but basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, platelet- derived <em>growth</em> <em>factor</em> BB, and IGF-I did not. Noggin decreased the stimulatory effects of BMPs on DNA and collagen synthesis and alkaline phosphatase activity in Ob cells. In conclusion, BMPs induce noggin transcription in Ob cells, a probable mechanism to limit BMP action in osteoblasts.

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 <em>22</em> 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 17 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.

BACKGROUND

Tumor-induced osteomalacia (TIO) is a paraneoplastic syndrome of hypophosphatemia, decreased renal phosphate reabsorption, normal or low serum 1,25-dihydryxyvitamin-D concentration, myopathy, and osteomalacia. Fibroblast growth factor 23 (FGF23) is a phosphaturic protein overexpressed in tumors that cause TIO and is, at least partly, responsible for the manifestations of TIO.

OBJECTIVE

The objective of this study was to determine the sensitivity of FGF23 measurements in TIO.

METHODS

FGF23 concentrations were measured on stored samples with three ELISAs.

METHODS

This study was conducted at subspecialty referral centers.

METHODS

Twenty-two patients with suspected TIO, 13 with confirmed tumors, were studied.

METHODS

There were no interventions in this study.

METHODS

FGF23 concentration was the main outcome measure of this study.

RESULTS

Elevated FGF23 concentrations were detected using the Immunotopics C-terminal assay in 16 of 22 TIO patients (for a sensitivity of 73%), the Immunotopics Intact assay in five of 22 patients (sensitivity, 23%), and the Kainos Intact assay in 19 of 22 patients (sensitivity, 86%). In the 13 patients with confirmed tumors, the sensitivity was higher with all assays: 92% for the Immunotopics C-terminal assay, 38% for the Immunotopics Intact assay, and 100% for the Kainos assay.

CONCLUSIONS

The Kainos Intact assay was the most sensitive, followed by the Immunotopics C-terminal assay. The findings of normal FGF23 concentrations in some patients with TIO may indicate that FGF 23 is not responsible for the hypophosphatemia in these patients or that FGF23 secretion by some tumors is partially responsive to serum phosphate. Normal FGF23 concentrations should be interpreted in relation to the serum phosphate and 1,25-dihydryxyvitamin-D concentrations.

Stem cells isolated from the fourth ventricle and spinal cord form neurospheres in vitro in response to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2)+heparin (H) or epidermal <em>growth</em> <em>factor</em> (EGF)+FGF2 together. To determine whether these <em>growth</em> <em>factor</em> conditions are sufficient to induce stem cells within the fourth ventricle and spinal cord to proliferate and expand their progeny in vivo, we infused EGF and FGF2, alone or together, with or without H, into the fourth ventricle for 6 days via osmotic minipumps. Animals were injected with bromodeoxyuridine (BrdU) on days 4, 5 and 6 of infusion in order to label cells proliferating in response to the <em>growth</em> <em>factors</em>. Infusions of EGF+FGF2+H into the fourth ventricle resulted in the largest proliferative effect, a 10.8-fold increase in the number of BrdU+ cells around the fourth ventricle, and a 33.5-fold increase in the number of BrdU+ cells around the central canal of the spinal cord, as compared to vehicle infused controls. The majority of the cells were nestin+ after 6 days of infusion. Seven weeks post-infusion, <em>22</em> and 30% of the number of BrdU+ cells induced to proliferate after 6 days of EGF+FGF2+H infusions were still detected around the fourth ventricle and central canal of the spinal cord, respectively. Analysis of the fates of the remaining cells showed that a small percentage of BrdU+ cells around the fourth ventricle and in the white matter of the spinal cord differentiated into astrocytes and oligodendrocytes. BrdU+ neurons were not found in the brainstem or in the grey matter of the cervical spinal cord 7 weeks post-infusion. These results show that endogenous stem cells and progenitors around the fourth ventricle and central canal of the spinal cord proliferate in response to exogenously applied <em>growth</em> <em>factors</em>, but unlike in the lateral ventricle where they generate some new neurons, they only produce new astrocytes and oligodendrocytes at 7 weeks post-infusion.

Vascular permeability <em>factor</em> (VPF), a tumor-secreted heparin-binding protein (Mr approximately 38,000), is responsible for increased vessel permeability and fluid accumulation associated with tumor <em>growth</em>. Vascular permeability <em>factor</em> also promotes the <em>growth</em> of human umbilical vein endothelial cells (EC) and bovine pulmonary ECs in vitro. It is shown for the first time that guinea pig VPF (half-maximal and maximal dose approximately 0.4 and <em>22</em> pmol/l (picomolar), respectively), as well as human VPF, are potent stimuli for human ECs resulting in [Ca2+]i increases (maximal three- to fourfold) and inositol triphosphate (IP3) formation. Unlike the maximal responses to thrombin and histamine, the [Ca2+]i response to a maximal VPF dose was preceded by a characteristic 10- to 15-second delay. Guinea pig VPF also selectively increased [Ca2+]i in cultured aortic and pulmonary artery ECs, but not aortic smooth muscle cells, human <em>fibroblasts</em>, or neutrophils. Affinity-purified rabbit antibody (raised to a synthetic peptide representing VPF N-terminal amino acids 1 to 24) adsorbed all vessel permeability-increasing activity, EC <em>growth</em>-promoting activity, and specifically all activity responsible for increasing EC [Ca2+]i. Similar to other mediators that increase [Ca2+]i in cultured ECs, VPF also induced a 200% increase in von Willebrand <em>factor</em> release. Together these data indicate that VPF acts directly on ECs and that rapid cellular events in its in vivo/in vitro actions are likely to involve phospholipase C activation, [Ca2+]i increase, and von Willebrand <em>factor</em> release.

Four isoforms of human <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2) result from alternative initiations of translation at three CUG start codons and one AUG start codon. Here we characterize a new 34-kDa FGF-2 isoform whose expression is initiated at a fifth initiation codon. This 34-kDa FGF-2 was identified in HeLa cells by using an N-terminal directed antibody. Its initiation codon was identified by site-directed mutagenesis as being a CUG codon located at 86 nucleotides (nt) from the FGF-2 mRNA 5' end. Both in vitro translation and COS-7 cell transfection using bicistronic RNAs demonstrated that the 34-kDa FGF-2 was exclusively expressed in a cap-dependent manner. This contrasted with the expression of the other FGF-2 isoforms of 18, <em>22</em>, <em>22</em>.5, and 24 kDa, which is controlled by an internal ribosome entry site (IRES). Strikingly, expression of the other FGF-2 isoforms became partly cap dependent in vitro in the presence of the 5,823-nt-long 3' untranslated region of FGF-2 mRNA. Thus, the FGF-2 mRNA can be translated both by cap-dependent and IRES-driven mechanisms, the balance between these two mechanisms modulating the ratio of the different FGF-2 isoforms. The function of the new FGF-2 was also investigated. We found that the 34-kDa FGF-2, in contrast to the other isoforms, permitted NIH 3T3 cell survival in low-serum conditions. A new arginine-rich nuclear localization sequence (NLS) in the N-terminal region of the 34-kDa FGF-2 was characterized and found to be similar to the NLS of human immunodeficiency virus type 1 Rev protein. These data suggest that the function of the 34-kDa FGF-2 is mediated by nuclear targets.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (Fgfs) are proteins with diverse functions in development, repair, and metabolism. The human Fgf gene family with <em>22</em> members can be classified into three groups, canonical, intracellular, and hormone-like Fgf genes. In contrast to canonical and intracellular Fgfs identified in invertebrates and vertebrates, hormone-like Fgfs, Fgf15/19, Fgf21, and Fgf23, are vertebrate-specific. The ancestral gene of hormone-like Fgfs was generated from the ancestral gene of canonical Fgfs by gene duplication early in vertebrate evolution. Later, Fgf15/19, Fgf21, and Fgf23 were generated from the ancestral gene by genome duplication events. Canonical Fgfs act as autocrine/paracrine <em>factors</em> in an Fgf receptor (Fgfr)-dependent manner. In contrast, hormone-like Fgfs act as endocrine <em>factors</em> in an Fgfr-dependent manner. Canonical Fgfs have a heparin-binding site necessary for the stable binding of Fgfrs and local signaling. In contrast, hormone-like Fgfs acquired endocrine functions by reducing their heparin-binding affinity during their evolution. Fgf15/19 and Fgf23 require βKlotho and αKlotho as co<em>factors</em>, respectively. However, Fgf21 might physiologically require neither. Hormone-like Fgfs play roles in metabolism at postnatal stages, although they also play roles in development at embryonic stages. Fgf15/19 regulates bile acid metabolism in the liver. Fgf21 regulates lipid metabolism in the white adipose tissue. Fgf23 regulates serum phosphate and active vitamin D levels. Fgf23 signaling disorders caused by hereditary diseases or tumors result in metabolic disorders. In addition, serum Fgf19 or Fgf21 levels are significantly increased by metabolic disorders. Hormone-like Fgfs are newly emerging and quite unique in their evolution and function.

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 (<em>22</em> with ecGVHD and 17 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.

The Glypican (GPC) family is a prototypical member of the cell-surface heparan sulfate proteoglycans (HSPGs). The HSPGs have been demonstrated to interact with <em>growth</em> <em>factors</em>, act as coreceptors and modulate <em>growth</em> <em>factor</em> activity. Here we show that based on oligonucleotide array analysis, GPC3 was upregulated in hepatocellular carcinoma (HCC). By northern blot analysis, GPC3 mRNA was found to be upregulated in 29 of 52 cases of HCC (55.7%). By Western blot analysis carried out with a monoclonal anti-GPC3 antibody we generated, the GPC3 protein was found to be overexpressed in 6 hepatoma cell lines, HepG2, Hep3B, HT17, HuH6, HuH7 and PLC/PRF/5, as well as <em>22</em> tumors (42.3%). To investigate the role of overexpressed GPC3 in liver cancer, we analyzed its effects on cell <em>growth</em> of hepatoblastoma-derived cells. Overexpression of GPC3 modulated cell proliferation by inhibiting <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) and bone morphogenetic protein 7 (BMP-7) activity. An interaction of GPC3 and FGF2 was revealed by co-immunoprecipitation, while GPC3 was found to inhibit BMP-7 signaling through the Smad pathway by reporter gene assay. The modulation of <em>growth</em> <em>factors</em> by GPC3 may help explain its role in liver carcinogenesis. In addition, the ability of HCC cells to express GPC3 at high levels may serve as a new tumor marker for HCC.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) have been implicated in numerous cellular processes, including proliferation, migration, differentiation, and survival. Whereas FGF-2, the prototypic ligand in a family of <em>22</em> members, activates all four tyrosine kinase FGF receptors (FGFR1-FGFR4), other members demonstrate a higher degree of selectivity. Oligodendrocytes (OLs), the myelin-producing cells of the CNS, are highly influenced by FGF-2 at all stages of their development. However, how other FGFs and their cognate receptors orchestrate the development of OLs is essentially undefined. Using a combination of specific FGF ligands and receptor blocking antibodies, we now show that FGF-8 and FGF-17 target OL progenitors, inhibiting their terminal differentiation via the activation of FGFR3, whereas FGF-9 specifically targets differentiated OLs, triggering increases in process <em>growth</em> via FGFR2 signaling; FGF-18 targets both OL progenitors and OLs via activation of both FGFR2 and FGFR3. These events are highly correlated with changes in FGF receptor expression from FGFR3 to FGFR2 as OL progenitors differentiate into mature OLs. In addition, we demonstrate that, although activation of FGFR1 by FGF-2 leads to proliferation of OL progenitors, it produces deleterious effects on differentiated OLs (i.e., aberrant reentry into cell cycle and down-regulation of myelin proteins with a loss of myelin membrane). These data suggest that ligand availability, coupled with changes in FGF receptor expression, yield a changing repertoire of ligand-receptor signaling complexes that contribute critically to the regulation of both normal OL development and potential OL/myelin pathogenesis.

The <em>growth</em> autonomy of human tumor cells is considered due to the endogenous production of <em>growth</em> <em>factors</em>. Transcriptional expression of candidates for autocrine stimulatory <em>factors</em> such as basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), acidic FGF, and transforming <em>growth</em> <em>factor</em> type beta were determined in human brain tumors. Basic FGF was expressed abundantly in 17 of 18 gliomas, 20 of <em>22</em> meninglomas, and 0 of 5 metastatic brain tumors. The level of mRNA expression of acidic FGF in gliomas was significant. In contrast, transforming <em>growth</em> <em>factor</em> type beta 1 was expressed in all the samples investigated. The mRNA for basic FGF and its peptide were localized in tumor cells in vivo by in situ hybridization and immunohistochemistry, showing that basic FGF is actually produced in tumor cells. Our results suggest that tumor-derived basic FGF is involved in the progression of gliomas and meningiomas in vivo, whereas acidic FGF is expressed in a tumor origin-specific manner, suggesting that acidic FGF works in tandem with basic FGF in glioma tumorigenesis.

The melanoma differentiation-associated gene 7 (mda-7), also called interleukin (IL)-24, suppresses the <em>growth</em> of some cancers in vitro and in vivo as a result of the ectopic expression of its protein. However, the function of the secreted form of the protein in cancer has not been previously studied. The purpose of this study was to determine the antiangiogenic function of a secreted form of the MDA-7/IL-24 protein (sMDA-7/IL-24). In vitro, sMDA-7/IL-24 inhibited both endothelial cell differentiation and migration of endothelial cells induced by vascular endothelial <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. The sMDA-7/IL-24-mediated inhibitory effect was 10-50 times more potent than endostatin, IFN-gamma, and IFN-inducible protein 10 in vitro. Furthermore, the inhibitory effect was not mediated by IFN or IFN-inducible protein 10. IL-<em>22</em> receptor mediated the antiangiogenic activity of sMDA-7/IL-24. Administration of a blocking antibody to IL-<em>22</em> receptor in conjunction with sMDA-7/IL-24 led to abrogation of inhibition of endothelial differentiation. sMDA-7/IL-24 inhibited vascular endothelial <em>growth</em> <em>factor</em>-induced angiogenesis as evidenced by reduced vascularization and hemoglobin content in in vivo Matrigel plug assays. In vivo, the <em>growth</em> of human lung tumor cells was significantly inhibited, and vascularization was reduced when the cells were mixed with 293 cells stably expressing sMDA-7/IL-24. Systemic administration of sMDA-7/IL-24 inhibited lung tumor <em>growth</em> in a mouse xenograft model. Associated with tumor <em>growth</em> inhibition was decreased tumor microvessel density and hemoglobin content, indicating the presence of antiangiogenic activity. These data demonstrate that sMDA-7/IL-24 is a novel and potent antiangiogenic effector and support the development of MDA-7/IL-24-based therapeutics.

To investigate the possibility that the epidermal <em>growth</em> <em>factor</em> (EGF) receptor functions as an oncogene product, we have determined the levels of EGF receptor protein and RNA in a variety of malignant and normal human cells, using a specific polyclonal antibody to the EGF receptor and a cDNA clone (plasmid pE7) that encodes the EGF receptor, respectively. Besides A431 epidermoid carcinoma cells, which are known to make large amounts of EGF receptor, cell lines from two ovarian cancers, two cervical cancers, and one kidney cancer were found to contain substantial amounts of receptor protein (11-<em>22</em>% of A431). Normal human <em>fibroblasts</em> (Detroit 551), a human lymphocyte line (IM-9), and a leukemic lymphocyte line (CEM) contained low or undetectable levels of EGF receptor. RNA blot analysis showed that among the human cell lines examined the levels of a 10- and a 5.6-kilobase species of pE7-specific RNA generally correlated with the amount of the EGF receptor protein. Genomic DNA blot analysis revealed that except for A431 none of these cell lines expressing high levels of EGF receptor protein possessed amplified receptor gene sequences. A431 cells are known to secrete a truncated form of the EGF receptor. An abundant 2.9-kilobase RNA is found only in A431 cells; it could encode the truncated form of the EGF receptor.

In the present study, we have investigated the effect of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) on retinal regeneration in the stage <em>22</em>-24 chick embryo. The neural retina was surgically removed in ovo leaving the retinal pigment epithelium (RPE) intact and then slow-release, plastic implants containing bFGF were inserted into the eye. Light microscopic examination of eyes 7 days later revealed that bFGF induced retinal regeneration in a dose-dependent manner. The absence of the RPE in these eyes and the reversed polarity of the regenerated neural retina is consistent with the hypothesis that this process occurs by transdifferentiation of the RPE. This represents the first time that a known molecule has been shown to induce retinal regeneration in vivo.

We have obtained the complete coding sequence of neurocan, a chondroitin sulfate proteoglycan of rat brain which is developmentally regulated with respect to its molecular size, concentration, carbohydrate composition, sulfation, and immunocytochemical localization. Two degenerate oligonucleotides, based on amino acid sequence data from the proteoglycan isolated from adult brain by immunoaffinity chromatography with the 1D1 monoclonal antibody, were used as sense and antisense primers in the polymerase chain reaction with a brain cDNA library as template to generate an unambiguous cDNA probe. A second probe for the N-terminal portion of the early postnatal form of the proteoglycan was obtained by reverse transcription/polymerase chain reaction. The composite sequence of overlapping cDNA clones is 5.2-kilobases (kb) long, including 1.3 kb of 3'-untranslated sequence and 76 base pairs of 5'-untranslated sequence. An open reading frame of 1257 amino acids encodes a protein with a molecular mass of 136 kDa containing 10 peptide sequences present in the adult and/or early postnatal brain proteoglycans. The deduced amino acid sequence revealed a <em>22</em>-amino acid signal peptide followed by an immunoglobulin domain, tandem repeats characteristic of the hyaluronic acid-binding region of aggregating proteoglycans, and an RGDS sequence. The C-terminal portion (amino acids 951-1215) has approximately 60% identity to regions in the C termini of the <em>fibroblast</em> and cartilage proteoglycans, versican and aggrecan, including two epidermal <em>growth</em> <em>factor</em>-like domains, a lectin-like domain, and a complement regulatory protein-like sequence. The central 595-amino acid portion of neurocan has no homology with other reported protein sequences. The proteoglycan contains six potential N-glycosylation sites and 25 potential threonine O-glycosylation sites. In the adult form of the proteoglycan (which represents the C-terminal half of neurocan) a single 32-kDa chondroitin 4-sulfate chain is linked at serin-944, whereas three additional potential chondroitin sulfate attachment sites (only two of which are utilized) are present in the larger proteoglycan species. A probe corresponding to a region of neurocan having no homology with versican or aggrecan hybridized with a single band at approximately 7.5 kb on Northern blots of mRNA from both 4-day and adult rat brain (but not with muscle, kidney, liver, or lung mRNA), indicating that the 1D1 proteoglycan of adult brain, containing a 68-kDa core protein, is generated by a developmentally regulated in vivo proteolytic processing of the 136-kDa species which is predominant in early postnatal brain.(ABSTRACT TRUNCATED AT 400 WORDS)

We tested the potential role of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) in the angiogenesis associated with experimental liver fibrogenesis induced by common bile duct ligation in Sprague-Dawley rats. In normal rats, VEGF and FGF-2 immunoreactivities were restricted to less than 3% of hepatocytes. One week after bile duct ligation, hypoxia was demonstrated by the immunodetection of pimonidazole adducts unevenly distributed throughout the lobule. After 2 weeks, hypoxia and VEGF expression were detected in >95% of hepatocytes and coexisted with an increase in periportal vascular endothelial cell proliferation, as ascertained by Ki67 immunolabeling. Subsequently, at 3 weeks the density of von Willebrand-labeled vascular section in fibrotic areas significantly increased. Semiquantitative reverse transcription polymerase chain reaction showed that VEGF(120) and VEGF(164) transcripts, that correspond to secreted isoforms, increased within 2 weeks, while VEGF(188) transcripts remained unchanged. FGF-2 mainly consisting of a <em>22</em>-kd isoform, according to Western blot, was identified by immunohistochemistry in 49% and 100% of hepatocytes at 3 and 7 weeks, respectively. Our data provide evidence that in biliary-type liver fibrogenesis, angiogenesis is stimulated primarily by VEGF in response to hepatocellular hypoxia while FGF-2 likely contributes to the maintenance of angiogenesis at later stages.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-1 mitogenic signal transduction is mediated in part by gene products that are specifically expressed in response to cell surface receptor binding and activation. We have used a targeted differential display method to identify FGF-1-inducible genes in murine NIH 3T3 <em>fibroblasts</em>. Here we report that one of these genes is predicted to encode a novel serine/threonine-specific protein kinase. This putative kinase has been named Fnk, for FGF-inducible kinase. The deduced Fnk amino acid sequence has 49, 36, 33, 32, and <em>22</em>% overall identity to mouse serum-inducible kinase (Snk), mouse polo-like kinase (Plk), Drosophila polo, Saccharomyces Cdc5, and mouse Snk/Plk-akin kinase (Sak), respectively. These proteins are all members of the polo subfamily of structurally related serine/threonine kinases. The Plk, polo, Cdc5, and Sak kinases are required for cell division. FGF-1 induction of Fnk mRNA expression is first detected at 30 min after mitogen addition, reflects transcriptional activation, and does not require de novo protein synthesis. FGF-2, platelet-derived <em>growth</em> <em>factor</em>-BB, calf serum, or phorbol myristate acetate treatment of quiescent cells also induces fnk gene expression. Fnk mRNA is expressed in vivo in a tissue-specific manner, with relatively high levels detected in newborn and adult mouse skin. These results indicate that Fnk may be a transiently expressed protein kinase involved in the early signaling events required for <em>growth</em> <em>factor</em>-stimulated cell cycle progression.

Interleukin-6 is a pleiotropic cytokine with a wide range of effects, including induction of B-cell and cytotoxic T-cell differentiation, and induction of acute phase reactant production by hepatocytes. Interleukin-6 also can act as an autocrine <em>growth</em> <em>factor</em> in malignancy. Various cell types produce interleukin-6, including T and B cells, monocytes, <em>fibroblasts</em>, and some solid tumor cells. In previous work we detected the production of substantial amounts of interleukin-6 by human ovarian cancer cells, including the ovarian cancer cell lines CAOV-3, OVCAR-3, and SKOV-3, and several primary ovarian tumor cultures. In this study we retrospectively examined 90 separate serum specimens for interleukin-6 in 36 patients with epithelial ovarian cancer. The mean serum interleukin-6 concentration of those ovarian cancer patients with macroscopic disease (n = 57) was 0.26 +/- 0.04 U/ml (mean +/- SEM). Healthy adult donors have interleukin-6 serum levels of 0.12 +/- 0.03 U/ml. Sixteen of 21 ovarian cancer patients with macroscopic disease (76%) had elevated (greater than 0.20 U/ml) levels of serum interleukin-6, with levels approaching 1 U/ml in some patients (p less than 0.01). Of those nine patients with bulky tumor (residual greater than 2 cm), eight (89%) had an elevated interleukin-6 level (mean, 0.31 +/- 0.05), while eight of 12 (66%) with minimal residual disease (less than 2 cm) had elevated levels. Only two of 15 (13%) patients who were in clinical remission and who had microscopic disease had elevated values. Of the 36 patients, <em>22</em> were CA 125 negative (less than 35 U/ml), and of these, four had elevated interleukin-6 levels. Of the 14 patients with an elevated CA 125 level, 12 (86%) had elevated interleukin-6 levels. In those 16 patients in whom serial levels of interleukin-6 were measured, rising levels were found over a 3 to 4 month interval in nine (56%); this correlated with tumor progression. Furthermore, the subsequent survival of patients was shown to correlate with the level of interleukin-6, such that patients whose levels were elevated greater than 0.20 U/ml interleukin-6 survived a mean of 12.5 months, compared with 27.2 months for patients with normal levels (p less than 0.001). These data support the concept that interleukin-6 may be a useful tumor marker in some patients with epithelial ovarian cancer, as it correlates with the tumor burden, clinical disease status, and survival.

With only a fraction of patients responding to cancer immunotherapy, a better understanding of the entire tumor microenvironment is needed. Using single-cell transcriptomics we chart the fibroblastic landscape during pancreatic ductal adenocarcinoma (PDAC) progression in animal models. We identify a population of carcinoma-associated <em>fibroblasts</em> (CAFs) programmed by transforming <em>growth</em> <em>factor</em> beta and expressing the leucine-rich repeat containing 15 (LRRC15) protein. These LRRC15+ CAFs surround tumor islets and are absent from normal pancreatic tissue. The presence of LRRC15+ CAFs in human patients was confirmed in >80,000 single-cells from <em>22</em> PDAC patients as well as immunohistochemistry on samples from 70 patients. Furthermore, immunotherapy clinical trials comprising over 600 patients across 6 cancer types revealed elevated levels of the LRRC15+ CAF signature correlated with poor response to anti-PD-L1 therapy. This work has important implications for targeting non-immune elements of the tumor microenvironment to boost responses of cancer patients to immune checkpoint blockade therapy.