This study evaluates the long-term outcome of farmer's lung (FL), adding high-resolution computed tomograms (HRCT) to previously reported procedures and verifying whether bronchoalveolar lavage (BAL) fluid markers or substrates of fibrosis (hyaluronic acid, Type III procollagen, fibronectin, and <em>fibroblast</em> <em>growth</em> <em>factors</em>) (FF) predict outcome. A total of 33 subjects with a history of FL dating back at least 6 yr were evaluated with pulmonary function tests, chest x-ray (CXR), and HRCT. All subjects had an initial evaluation, which included a BAL, 6 yr before the current study. Subjects were then either in acute FL (n = 19) or in clinical remission despite continued contact (n = 14). In the current study, pulmonary function tests revealed an obstructive profile in 13 subjects, restrictive changes in 1, an isolated decrease in lung diffusion capacity in 3, and normal values in 16. Chest radiographs (CXR) were normal in <em>22</em> subjects, abnormal with interstitial or reticulonodular changes in 6, and suggestive of emphysema in 5. HRCT revealed emphysema in 9 subjects; 3 had localized fibrotic changes, 2 a ground-glass pattern, and 19 were normal. There was a good correlation between the findings on pulmonary function tests and HRCT; however, CXR alone did not suggest the existence of emphysema in 4 subjects who had such findings on HRCT. No correlations were found between most outcome parameters and the level of the BAL FF measured 6 yr previously. We conclude that airflow obstruction with or without emphysema is an important long-term sequela of FL and that BAL FF do not predict outcome in this disease.

Wound healing is regulated by temporally and spatially restricted patterns of <em>growth</em> <em>factor</em> signaling, but there are few delivery vehicles capable of the "on-demand" release necessary for recapitulating these patterns. Recently we described a perfluorocarbon double emulsion that selectively releases a protein payload upon exposure to ultrasound through a process known as acoustic droplet vaporization (ADV). In this study, we describe a delivery system composed of fibrin hydrogels doped with <em>growth</em> <em>factor</em>-loaded double emulsion for applications in tissue regeneration. Release of immunoreactive basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) from the composites increased up to 5-fold following ADV and delayed release was achieved by delaying exposure to ultrasound. Releasates of ultrasound-treated materials significantly increased the proliferation of endothelial cells compared to sham controls, indicating that the released bFGF was bioactive. ADV also triggered changes in the ultrastructure and mechanical properties of the fibrin as bubble formation and consolidation of the fibrin in ultrasound-treated composites were accompanied by up to a <em>22</em>-fold increase in shear stiffness. ADV did not reduce the viability of cells suspended in composite scaffolds. These results demonstrate that an acoustic droplet-hydrogel composite could have broad utility in promoting wound healing through on-demand control of <em>growth</em> <em>factor</em> release and/or scaffold architecture.

Angiogenesis is one of the most important features of AIDS-associated Kaposi's sarcoma (AIDS-KS). Our studies suggested that spindle-shaped AIDS-KS cells from various AIDS-KS lesions play important roles in the development of KS lesions. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has been reported to be a predominant angiogenic <em>factor</em> expressed in AIDS-KS cells. However, our data from ELISA revealed the presence of the vascular endothelial <em>growth</em> <em>factor</em> (VEGF) molecule in large quantities in AIDS-KS cell-derived conditioned medium (AIDS-KS-CM) (12.1-21.4 ng/ml). In contrast, small amounts of bFGF were detected in AIDS-KS-CM (76-245 pg/ml). The combination of anti-VEGF and anti-bFGF IgGs completely inhibited endothelial cell <em>growth</em>-promoting activities in AIDS-KS-CM, while activities partially remained in the presence of anti-bFGF IgG or anti-VEGF IgG alone. VEGF and bFGF in AIDS-KS-CM were distinguished by heparin-affinity chromatography. Furthermore, the combination of VEGF and bFGF synergistically augmented the <em>growth</em> of endothelial cells. Both VEGF and bFGF revealed an angiogenic property that was inhibited by specific Abs, when applied to the rabbit cornea and chicken chorioallantoic membrane. On Western blots, anti-VEGF IgG gave two major bands of <em>22</em> and 24 kDa, similar to those of recombinant VEGF165. As detected on Northern blots, AIDS-KS cells expressed major 3.9-kb VEGF-specific mRNA. Thus, VEGF, in concert with bFGF, may play a crucial role in the angiogenesis of AIDS-KS lesions.

We have previously reported that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) can induce retinal regeneration in the stage <em>22</em>-24 chicken embryo. The present study was undertaken to identify the cellular source of the regenerate and to determine whether other <em>growth</em> <em>factors</em> also elicit regeneration in this animal model. Polymer implants containing bFGF were inserted into eyes of chicken embryos immediately after extirpation of the neural retina. The retinal pigment epithelium (RPE) was left intact. Evaluation by light microscopy revealed that in bFGF-treated eyes the new neural retina arose by transdifferentiation of the entire RPE layer. Differentiation of the new neural retina occurred in a sequence similar to that of normal development but proceeded in a reverse (vitread) direction. All retinal laminae had differentiated by Day 15. However, the regenerate displayed reversed polarity, with photoreceptors closest to the lens. The RPE, pecten, and optic nerve were absent. Focal areas of degeneration in the retinal regenerate became evident for the first time on Day 10. Retinal regeneration was also observed after treatment with higher doses of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>, but not with nerve <em>growth</em> <em>factor</em>-beta, transforming <em>growth</em> <em>factor</em>-beta 1, insulin, or insulin-like <em>growth</em> <em>factors</em> I or II. These results raise the possibility that FGFs may play a role in retinal differentiation during development.

We have developed an efficient, sensitive, and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry. Use of the MALDI Q-TOF enables selection of phosphopeptides and characterization by CID of the phosphopeptides performed on the same sample spot. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification before mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing <em>22</em> phosphorylation sites from low femtomolar amounts of a tryptic digest of beta-casein and alpha-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor kinase domain and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides.

Bone morphogenetic proteins (BMP) induce the differentiation of cells of the osteoblastic lineage and enhance the function of the osteoblast. <em>Growth</em> <em>factor</em> activity is regulated by binding proteins, and we previously showed that BMPs induce noggin, a glycoprotein that binds and blocks BMP action. Recently, additional BMP antagonists, such as gremlin, have been described, but there is no information about their expression or function in osteoblasts. We tested for the expression of gremlin and studied its induction by BMPs in cultures of osteoblast-enriched cells from <em>22</em>-day-old fetal rat calvariae (Ob cells). BMP-2 caused a time- and dose-dependent increase in gremlin messenger RNA and polypeptide levels, as determined by Northern and Western blot analyses. The effects of BMP-2 on gremlin transcripts were independent of new protein synthesis. BMP-2 increased the rate of gremlin transcription as determined by nuclear run-on assays. <em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 and platelet-derived <em>growth</em> <em>factor</em> BB also induced gremlin, but other hormones and <em>growth</em> <em>factors</em> had no effect. Gremlin prevented the stimulatory effects of BMP-2 on DNA, collagen, noncollagen protein synthesis, and alkaline phosphatase activity in Ob cells. In conclusion, BMPs induce gremlin transcription in Ob cells, a mechanism that probably limits BMP action in osteoblasts.

The post-translational methylation of the N-terminally extended or high molecular weight (HMW) forms of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) has been shown to affect the nuclear accumulation of the <em>growth</em> <em>factor</em>. In this study, we determined the extent and position of methyl groups in HMW FGF-2. Using mass spectrometry and amino acid sequence analysis, we have shown that the <em>22</em>- and <em>22</em>.5-kDa forms of HMW FGF-2 contain five dimethylated arginines located at positions -<em>22</em>, -24, -26, -36, and -38 using the methionine residue normally used to initiate the 18-kDa form as position 0. The 24-kDa form of HMW FGF-2 contains seven to eight dimethylated arginines located at positions -48, -50, and -52, in addition to positions -<em>22</em>, -24, -26, -36, and -38. In vitro methylation reactions demonstrate that the N-terminal extension of HMW FGF-2 acts as a specific substrate for yeast Hmt1p and human HRMT1L2 arginine methyltransferases. These findings indicate that HMW FGF-2, with the presence of five or more dimethylated Gly-Arg-Gly repeats, contains an RGG box-like domain, which may be important for protein-protein and/or protein-RNA interactions.

We have performed clinical, physiological, in vitro biochemical and genetic studies of a patient with severe insulin resistance associated with the phenotype of "pseudoacromegaly," defined as the presence of acromegaloid features in the absence of elevated levels of GH or insulin-like <em>growth</em> <em>factor</em>-I (IGF-I). Despite marked hyperinsulinemia, insulin and IGF-I binding to circulating blood cells and cultured skin <em>fibroblasts</em> was normal. Insulin and IGF-I-stimulated autophosphorylation of their respective receptors in cultured skin <em>fibroblasts</em> was also normal. However, neither insulin nor IGF-I were able to stimulate 2-deoxy D-glucose uptake by cultured skin <em>fibroblasts</em>. In contrast, the ability of insulin and IGF-I (or IGF-II) to stimulate amino acid uptake and thymidine incorporation into DNA was not impaired. This unique discordant signaling defect through both insulin and IGF-I receptors appeared not to be the consequence of altered expression or primary structure of the insulin receptor or the GLUT-4 glucose transporter, as assessed by several genetic and biochemical techniques. GLUT-4 expression in muscle was normal on Western blots, and SSCP screening of all 11 exons of the gene for nucleotide variation revealed no variations from normal. DNA sequencing and SSCP screening of exons 2-<em>22</em> of the insulin receptor gene revealed only one variation predicted to alter the amino acid sequence (Val985->>Met). No functional differences between Met985 and wild-type human insulin receptors were evident in studies performed with Chinese hamster ovary cell transfectants that overexpress either receptor. This data combined with our previously published epidemiological data concerning the frequency of the Met985 allele, indicate that this variant insulin receptor is not responsible for the insulin resistant glucose uptake or the clinical syndrome of pseudoacromegaly. We conclude that: 1) The molecular lesion responsible for the selective biochemical defect in this individual appears to involve a signaling intermediate required for insulin and IGF-I regulation of glucose transport, and/or an effector mechanism operative in this process. 2) Cells derived from this patient may be a valuable tool in the search for such molecular mechanisms. 3) The Met985 allele is a relatively common variant which has no demonstrable adverse consequences for insulin receptor function. 4) Pseudoacromegaly can be viewed as the expected result of hyperinsulinemia driving the unopposed mitogenic and anabolic actions of insulin.

Hepatitis C virus (HCV) induced liver disease is the leading indication for liver transplantation (LTx). Reinfection and accelerated development of fibrosis is a universal phenomenon following LTx. The molecular events that lead to fibrosis following HCV infection still remains poorly defined. In this study, we determined microRNA (miRNA) and mRNA expression profiles in livers from chronic HCV patients and normals using microarrays. Using Genego software and pathway finder we performed an interactive analysis to identify target genes that are modulated by miRNAs. <em>22</em> miRNAs were up regulated (>2 fold) and 35 miRNAs were down regulated (>2fold) compared to controls. Liver from HCV patients demonstrated increased expression of 306 genes (>3 fold) and reduced expression of 133 genes (>3 fold). Combinatorial analysis of the networks modulated by the miRNAs identified regulation of the phospholipase C pathway (miR200c, miR20b, and miR31through cellular proto-oncogene tyrosine-protein kinase Src (cSrc)), response to <em>growth</em> <em>factors</em> and hormones (miR141, miR107 and miR200c through peroxisome proliferator-activated receptor alpha and extracellular-signal-regulated kinases, and regulation of cellular proliferation (miR20b, miR10b, and miR141 through cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1 p21). Real time PCR (RT-PCR) validation of the miRNA in HCV infected livers demonstrated a 3.3 ±0.9 fold increase in miR200c. In vitro transfection of <em>fibroblasts</em> with miR200c resulted in a 2.2 fold reduction in expression of tyrosine-protein phosphatase non-receptor type 13 or FAS associated phosphatase 1 (FAP-1) and 2.3 fold increase in expression of cSrc. miR200c transfection resulted in significant increases in expression of collagen and <em>fibroblast</em> <em>growth</em> <em>factor</em> (2.8 and 3.4 fold, p<0.05). Therefore, we propose that HCV induced increased expression of miR200c can down modulate the expression of FAP1, a critical regulator of Src and MAP kinase pathway that play an important role in the production of fibrogenic <em>growth</em> <em>factors</em> and development of fibrosis.

A histopathology study of <em>22</em> pancreatic adenocarcinoma cases revealed that 13 of the patients presented with hyperplastic lesions (atypical and non-atypical hyperplasia, mucous cell hypertrophy, focal epithelial hyperplasia, and ductal papillary hyperplasia), and 9 exhibited fibrosis adjacent to the carcinoma. All lesions expressed high levels of epidermal <em>growth</em> <em>factor</em> receptor (EGF-R) (p<0.0001 and p=0.0008, respectively) as compared with normal ductal epithelium. Non-atypical and atypical hyperplastic lesions also had a higher proliferating cell nuclear antigen (PCNA) labeling index (p<0.001 and p=0.0008, respectively) than normal ductal epithelium. A gradient in PCNA+ nuclei was found in acinar cells adjacent to the tumors. In 16 cases with marked fibrosis, we observed a significant increase of PCNA+ nuclei in stromal <em>fibroblasts</em> (p=0.0041) and significant upregulation of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) mRNA expression in adjacent tumor cells (p=0.0213). These data suggest that the production of bFGF by pancreatic cancer cells induces ductal and stromal hyperplasia of the pancreas.

Although <em>growth</em> <em>factors</em> and anti-apoptotic peptides have been shown to be neuroprotective in stroke models, translation of these experimental findings to clinic is hampered by limited penetration of peptides to the brain. Here, we show that a large peptide like the basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and a small peptide inhibitor of caspase-3 (z-DEVD-FMK) can effectively be transported to the brain after systemic administration by incorporating these peptides to brain-targeted nanoparticles (NPs). Chitosan NPs were loaded with peptides and then functionalized by conjugating with antibodies directed against the transferrin receptor-1 on brain endothelia to induce receptor-mediated transcytosis across the blood-brain barrier (BBB). Pre-ischemic systemic administration of bFGF- or z-DEVD-FMK-loaded NPs significantly decreased the infarct volume after 2-hour middle cerebral artery occlusion and <em>22</em>-hour reperfusion in mice. Co-administration of bFGF- or z-DEVD-FMK-loaded NPs reduced the infarct volume further and provided a 3-hour therapeutic window. bFGF-loaded NPs were histologically detected in the brain parenchyma and also restored ischemia-induced Akt dephosphorylation. The neuroprotection was not observed when receptor-mediated transcytosis was inhibited with imatinib or when bFGF-loaded NPs were not conjugated with the targeting antibody, which enables them to cross the BBB. Nanoparticles targeted to brain are promising drug carriers to transport large as well as small BBB-impermeable therapeutics for neuroprotection against stroke.

White and brown adipose tissues (BATs), which store and burn lipids, respectively, play critical roles in energy homeostasis. <em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are signaling proteins with diverse functions in development, metabolism, and neural function. Among <em>22</em> FGFs, FGF1, FGF10, and FGF21 play roles as adipokines, adipocyte-secreted proteins, in the development and function of white and BATs. FGF1 is a critical transducer in white adipose tissue (WAT) remodeling. The peroxisome proliferator-activated receptor γ-FGF1 axis is critical for energy homeostasis. FGF10 is essential for embryonic white adipocyte development. FGF21 activates BAT in response to cold exposure. FGF21 also stimulates the accumulation of brown-like cells in WAT during cold exposure and is an upstream effector of adiponectin, which controls systemic energy metabolism. These findings provide new insights into the roles of FGF signaling in white and BATs and potential therapeutic strategies for metabolic disorders.

Spaceflight is known to diminish bone mass and reduce immune function, suggesting that repair of connective tissue might be impaired in a microgravity environment. Fisher 344 rats were used to test wound healing responses in the orbiting Space Shuttle Endeavour by preflight implantation of polyvinyl acetal sponge disks in which pellets were placed to release either platelet-derived <em>growth</em> <em>factor</em> (PDGF-BB), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), or placebo. Control groups on the ground included a matched environment group in similar housing modules and temperature control groups in cages at <em>22</em> degreesC and 28 degreesC. After 12 days of implantation and 10 days in orbit, the removed sponges were analyzed for histological and biochemical responses. <em>Growth</em> <em>factor</em> responses were histologically evident after release of PDGF-BB and bFGF in ground controls, whereas only immediate-release bFGF and delayed-release PDGF-BB showed significant responses in microgravity. Biochemical data confirmed that cellularity was increased by both <em>factors</em> in ground sponges; however, this response was significantly blunted in flight sponges (P<0.005, ANOVA), irrespective timing of <em>factor</em> release. Collagen content was 62% lower in sponges from animals with 10 days of microgravity exposure (P<0.01, ANOVA) and further reduced by bFGF. These data suggest that orbital exposure retards the capacity of wounds to heal and respond to exogenous stimuli.

MMP-8 (collagenase-2) is the most effective collagenase to initiate type I collagen degradation. Since initiation of lysis of the surrounding collagen matrix is an essential prerequisite for carcinoma cells to spread, this study investigated the expression of MMP-8 in squamous cell carcinoma (SCC) of the head and neck in vivo and in vitro. Most of the recently established head and neck carcinoma cell lines (<em>22</em>/25), corresponding tumour (5/7) and dermal (2/2) <em>fibroblasts</em>, commercial tongue carcinoma (HSC-3 and SCC-25), and transformed keratinocyte cell lines of the tongue (IHGK) and skin (HaCaT) expressed MMP-8 mRNA analysed by the PCR method. Western blotting revealed a latent 50 kD band in concentrated culture media of carcinoma cells and corresponding tumour and dermal <em>fibroblasts</em>. The expression of immunoreactive MMP-8 protein was reduced 30% by transforming <em>growth</em> <em>factor</em> beta-1 (TGF-beta1) at 1 ng/ml concentration and 60% at 10 ng/ml concentration, but up-regulated 2- and 2.5-fold after 10 nM and 100 nM phorbol 12-myristate 13 acetate (PMA), respectively. Immunohistological staining localized MMP-8 protein in a few malignant invading tumour cell islands, certain <em>fibroblasts</em>, polymorphonuclear neutrophils (PMNs), and plasma cells. In situ hybridization revealed a faint sporadic signal in carcinoma cells of all eight tissue sections analysed. It is concluded that tissue from head and neck carcinomas can express MMP-8 both in vivo and in vitro. Since the amount of MMP-8 in carcinoma and stromal cells is rather low, MMP-8 may have a potential role, with other collagenases, in the proteolysis of connective tissue associated with the spreading of invasive carcinoma.

Astroglial cells contribute to neuronal maintenance and function in the normal and diseased brain. Gap junctions formed predominantly by connexin43 (cx43) provide important pathways to coordinate astroglial responses. We have previously shown that <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2, which occurs ubiquitously in the CNS, downregulates gap junction communication in cortical and striatal, but not in mesencephalic astroglial cells in vitro (Reuss et al. Glia <em>22</em>:19-30, 1998). Other members of the FGF family expressed in the CNS include FGF-5 and FGF-9. We show that both FGF-5 and FGF-9, like FGF-2, downregulate astroglial gap junctions and functional coupling. However, their effects are strikingly different from different brain regions, with regard to astroglial cells. FGF-5 specifically affects mesencephalic astroglial cells without changing coupling of cortical and striatal astroglia, while FGF-9 reduces gap junctional coupling in astroglia from all three brain regions. Both cx43 mRNA and protein levels as well as functional coupling assessed by dye spreading are affected. To clarify whether brain region-specific effects of FGFs on astroglial coupling are due to differential expression of FGF receptors (FGFR), we monitored expression of the four known FGFR mRNAs in astroglial cultures by RT-PCR. Irrespective of their regional origin, astroglial cells express mRNAs for FGFR-2 and FGFR-3. In summary, our results provide evidence for an important role of FGF-2, -5, and -9 in a distinct, CNS region-specific regulation mechanism of astroglial gap junction communication. The molecular basis underlying the regionally distinct responsiveness of astrocytes to different FGFs may be sought beyond distinct FGFR expression.

Crotalinae snake venoms cause severe local myonecrosis and microvasculature failure at the bite site. We evaluated whether low-level laser therapy (LLLT) could accelerate angiogenesis and myoregeneration in male Swiss mice injected with Bothrops moojeni venom through immunohistochemistry of the vascular endothelial <em>growth</em> <em>factor</em> receptor-1 (VEGFR-1). Envenomed gastrocnemius was either unirradiated (V) or irradiated with HeNe (VHN, 632.8 nm) or GaAs (VGA, 904 nm, 10000 Hz). Animals sacrificed at 3 and 12 h were irradiated once (4 J cm(-2)), at 24 h (twice) and at 3, 7, 21 days (4, 8, <em>22</em> times, respectively). At 3 days, LLLT increased angiogenesis (80%:HeNe vs 40%:GaAs), decreased neutrophils and increased proliferation of regenerating cells. However, after 21 days, myoregeneration observed in the VHN group appeared delayed compared with the V group. As LLLT improved revascularization, the suggestive delay in myoregeneration could be a dose-response inhibitory effect caused by multiple irradiations in myogenesis. The immunodetection of VEGFR-1 in neutrophils, macrophages, satellite cells, <em>fibroblasts</em>, Schwann cells and skeletal and smooth muscle fibers (not seen in saline-controls) at only the acute stages of envenoming suggests a mediator role for VEGFR-1 in local alterations. This is the first time that VEGFR-1 expression, and its modulation by photostimulation, has been demonstrated in endothelial and nonendothelial cells of snake envenomed skeletal muscle.

BACKGROUND

Activating FGFR2 mutations are found in 10-16% of primary endometrial cancers and provide an opportunity for targeted therapy. We assessed the safety and activity of dovitinib, a potent tyrosine-kinase inhibitor of fibroblast growth factor receptors, VEGF receptors, PDGFR-β, and c-KIT, as second-line therapy both in patients with FGFR2-mutated (FGFR2(mut)) endometrial cancer and in those with FGFR2-non-mutated (FGFR2(non-mut)) endometrial cancer.

METHODS

In this phase 2, non-randomised, two-group, two-stage study, we enrolled adult women who had progressive disease after first-line chemotherapy for advanced or metastatic endometrial cancer from 46 clinical sites in seven countries. We grouped women according to FGFR2 mutation status and gave all women dovitinib (500 mg per day, orally, on a 5-days-on and 2-days-off schedule) until disease progression, unacceptable toxicity, death, or study discontinuation for any other reason. The primary endpoint was proportion of patients in each group who were progression-free at 18 weeks. For each group, the second stage of the trial (enrolment of 20 additional patients) could proceed if at least eight of the first 20 treated patients were progression free at 18 weeks. Activity was assessed in all enrolled patients and safety was assessed in all patients who received at least one dose of dovitinib. The completed study is registered with ClinicalTrials.gov, number NCT01379534.

RESULTS

Of 248 patients with FGFR2 prescreening results, 27 (11%) had FGFR2(mut) endometrial cancer. Between Feb 17, 2012, and Dec 13, 2013, we enrolled 22 patients in the FGFR2(mut) group and 31 patients in the FGFR2(non-mut) group. Seven (31·8%, 95% CI 13·9-54·9) patients in the FGFR2(mut) group and nine (29·0%, 14·2-48·0) in the FGFR2(non-mut) group were progression-free at 18 weeks. On the basis of predefined criteria, neither group continued to stage two: seven (35%) of the first 20 patients in the FGFR2(mut) group were progression free at 18 weeks, as were five (25%) of the first 20 in the FGFR2(mut) population. Rates of treatment-emergent adverse events were similar between groups and events were most frequently gastrointestinal. Overall, the most common grade 3 or 4 adverse events suspected to be related to the study drug were hypertension (nine patients; 17%) and diarrhoea (five; 9%). The most frequently reported serious adverse events suspected to be related to study drug were pulmonary embolism (four patients; 8%), vomiting (four; 8%), dehydration (three; 6%), and diarrhoea (three; 6%). Only one death was deemed to be treatment-related: one patient in the FGFR2(non-mut) group died from cardiac arrest with contributing reason of pulmonary embolism (grade 4, suspected to be study drug related) 4 days previously.

CONCLUSIONS

Second-line dovitinib in FGFR2(mut) and FGFR2(non-mut) advanced or metastatic endometrial cancer had single-agent activity, although it did not reach the prespecified study criteria. Observed treatment effects seemed independent of FGFR2 mutation status. These data should be considered exploratory and additional studies are needed.

BACKGROUND

Novartis Pharmaceuticals.

Cholesterol homeostasis is regulated by the liver X receptor (LXR) at the transcriptional level, but it remains unknown whether LXR can affect expression levels of intrahepatic lipolysis related gene. Recent evidence has demonstrated that <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) regulates hepatic lipolysis and fatty acid utilization. In the present study, we examined the role of LXR in FGF21 gene expression associated with regulation of cross-talk signals between cholesterol and triglyceride metabolism in the liver. An in vivo cholesterol feeding test revealed that intake of excess cholesterol increased cholesterol catabolism related gene expression as well as fatty-acid biosynthesis related gene expression. Moreover, the accumulated cholesterol suppressed FGF21 and hormone-sensitive lipase (HSL) gene expression. After 15-day cholesterol feeding, hepatic triglyceride concentrations were negatively correlated with expression levels of the FGF21 and HSL genes in the liver. An LXR agonist (TO-901317) repressed the FGF21 gene expression in mouse primary hepatocytes and HepG2 cells. A promoter deletion study and electrophoretic mobility shift assay revealed that the human FGF21 promoter has at least one LXR response element located from -37 to -<em>22</em> bp. In summary, LXR represses FGF21 gene expression at the transcription level and might suppress lipolysis and lipid utilization to protect the liver from excess accumulation of toxic cholesterol.

We examined the expression level of several genes that regulate different steps of metastasis in formalin-fixed, paraffin-embedded archival specimens of primary human colon carcinomas from patients with at least 5 years of follow-up. The expression of epidermal <em>growth</em> <em>factor</em> receptor, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, type IV collagenase, E-cadherin, and multidrug resistance (mdr-1) was examined by a colorimetric in situ mRNA hybridization technique concentrating on reactivity at the periphery of the neoplasms. The in situ hybridization technique revealed inter- and intratumor heterogeneity for expression of the metastasis-related genes. The expression of basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, collagenase type IV, epidermal <em>growth</em> <em>factor</em> receptor, and mdr-1 mRNA was higher in Dukes's stage D than in Dukes' stage B tumors. Among the <em>22</em> Dukes' stage B neoplasms, 5 specimens exhibited a high expression level of epidermal <em>growth</em> <em>factor</em> receptor, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and collagenase type IV. Clinical outcome data (5-year follow-up) revealed that all 5 patients with Dukes' stage B tumors developed distant metastasis (recurrent disease), whereas the other 17 patients with Dukes' stage B tumors expressing low levels of the metastasis-related genes were disease-free. Multivariate analysis identified high levels of expression of collagenase type IV and low levels of expression of E-cadherin as independent <em>factors</em> significantly associated with metastasis or recurrent disease. More specifically, metastatic or recurrent disease was associated with a high ratio >> 1.35) of expression of collagenase type IV to E-cadherin (specificity of 95%). Collectively, the data show that multiparametric in situ hybridization analysis for several metastasis-related genes may predict the metastatic potential, and hence the clinical outcome, of individual lymph-node-negative human colon cancers.

Over the last decade, much has been learned about the genetic changes that occur in human neoplasia and how they contribute to the neoplastic state. Oncogenes and tumor suppressor genes have been identified, and many powerful molecular genetic techniques have emerged. Brain tumors have been intensively studied as part of this process. Specific and recurring genetic alterations have been identified and are associated with specific tumor types. In astrocytomas, for example, losses of genetic material on chromosomes 10 and 17 and amplification of the epidermal <em>growth</em> <em>factor</em> receptor gene seem important in pathogenesis, with the loss of chromosome 10 and the amplification of epidermal <em>growth</em> <em>factor</em> receptor being strongly associated with glioblastoma multiforme. Meningiomas, on the other hand, have usually lost part or all of chromosome <em>22</em>. Brain tumors also express <em>growth</em> <em>factors</em> and <em>growth</em> <em>factor</em> receptors that may be important in promoting tumor <em>growth</em> and angiogenesis. These include epidermal <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-alpha, platelet-derived <em>growth</em> <em>factor</em>, the <em>fibroblast</em> <em>growth</em> <em>factors</em>, and vascular endothelial <em>growth</em> <em>factor</em>. In this article, we review the genetic aberrations that occur in the major types of brain tumors, including glial tumors, meningiomas, acoustic neuromas, medulloblastomas, primitive neuroectodermal tumors, and pituitary tumors. Wherever possible, clinical correlations have been made concerning the prognostic and therapeutic implications of specific aberrations. We also provide some background about the cytogenetic and molecular genetic techniques that have contributed to the description and understanding of these alterations and speculate as to some clinical and basic science issues that might be explored in the future.

We have investigated the epidermal <em>growth</em> <em>factor</em> (EGF)-stimulated tyrosine-specific protein kinase activity in quiescent cultures of diploid human <em>fibroblasts</em> that have a well characterized mitogenic response to EGF. We developed a method of permeabilizing cells with digitonin or other agents that permitted the rapid labeling of cellular proteins with exogenously added [gamma-32P]ATP while allowing only about 25% of marker cytosolic enzymes to escape from the cells. When phosphatases were inhibited with zinc and vanadate, EGF induced up to 8-fold stimulation of the incorporation of radioactivity from [gamma-32P]ATP into a 35-kDa band on sodium dodecyl sulfate gels. Alkali treatment of gels showed that EGF stimulated the phosphorylation of bands with apparent molecular masses of 170, 45, 35, 26, <em>22</em>, and 21 kDa. Phosphoamino acid analysis was performed on the 170- and 35-kDa bands and revealed that the EGF-stimulated phosphorylation was on tyrosyl residues. The 35-kDa band was resolved into four spots by two-dimensional gel electrophoresis. The most acidic form was the most prominent and it was precipitated by an antiserum against a 35-kDa protein from A-431 cells; heretofore, this protein has only been reported to be phosphorylated in an EGF-dependent manner by A-431 membranes in vitro (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). This antiserum also precipitated a 35-kDa phospho-protein from extracts of intact [32P]orthophosphate-labeled <em>fibroblasts</em> which was phosphorylated on tyrosine in an EGF-dependent manner. None of the forms of the 35-kDa phosphoproteins labeled in permeabilized cells were immunologically related to the 34-kDa protein that is a substrate for the tyrosyl kinase encoded by Rous sarcoma virus. Other mitogens (serum, insulin, platelet-derived <em>growth</em> <em>factor</em>, and thrombin) did not detectably stimulate phosphorylation in permeabilized cells.

OBJECTIVE

Fibroblast growth factor (FGF) signaling regulates tumor growth and vascularization and partly mediates antiangiogenic escape from VEGF receptor (VEGFR) inhibitors. Dovitinib (TKI258) is a tyrosine kinase inhibitor (TKI) that inhibits FGF receptor (FGFR), VEGFR, and platelet-derived growth factor receptor, which are known drivers of antiangiogenic escape, angiogenesis, and tumor growth in renal cell carcinoma (RCC).

METHODS

Patients with advanced or metastatic RCC were treated with oral dovitinib 500 mg/day (5-days-on/2-days-off schedule). The study population was enriched for patients previously treated with a VEGFR TKI and an mTOR inhibitor.

RESULTS

Of 67 patients enrolled, 55 patients (82.1%) were previously treated with ≥1 VEGFR TKI and ≥1 mTOR inhibitor (per-protocol efficacy set). The 8-week overall response rate and disease control rate in this population were 1.8% and 52.7%, respectively. Disease control rate during the entire study period was 56.4% (50.9% ≥4 months). Median progression-free survival and overall survival in the entire population were 3.7 and 11.8 months, respectively. Pharmacodynamic analyses demonstrated dovitinib-induced inhibition of VEGFR (as determined by increased levels of placental growth factor and decreased levels of soluble VEGFR2) and FGFR (as determined by increased FGF23 serum measures). The most frequently reported treatment-related adverse events of all grades included nausea (65.7%), diarrhea (62.7%), vomiting (61.2%), decreased appetite (47.8%), and fatigue (32.8%).

CONCLUSIONS

Dovitinib was shown to be an effective and tolerable therapy for patients with metastatic RCC who had progressed following treatment with VEGFR TKIs and mTOR inhibitors. Clin Cancer Res; 20(11); 3012-22. ©2014 AACR.

To study the regulation of human salivary-type gene expression we developed cell culture systems to support the <em>growth</em> and serial cultivation of salivary gland epithelial and <em>fibroblast</em>ic cell types. We have established <em>22</em> independent salivary gland epithelial cell strains from parotid or submandibular glands of human or macaque origin. Nineteen strains were derived from normal tissues and three from human parotid gland tumors. Both the normal and the tumor-derived salivary gland epithelial cells could be serially cultivated with the aid of a 3T3 <em>fibroblast</em> feeder layer in a mixture of Ham's F12 and Dulbecco's modified Eagle's media supplemented with fetal bovine serum, calcium, cholera toxin, hydrocortisone, insulin, and epidermal <em>growth</em> <em>factor</em>. Salivary gland epithelial cells cultured under these conditions conditioned to express the genes for at least two acinar-cell-specific markers at early passages. Amylase enzyme activity was detected in conditioned media from cultured rhesus parotid epithelial cells as late as Passage 5. Proline-rich-protein-specific RNAs were detected in primary cultures of both rhesus and human parotid epithelial cells. Neither amylase enzyme activity nor PRP-specific RNAs were detected in <em>fibroblasts</em> isolated from the same tissues. In addition, salivary gland epithelial cells cultured under our conditions retain the capacity to undergo dramatic morphologic changes in response to different substrata. The cultured salivary gland epithelial cells we have established will be important tools for the study of salivary gland differentiation and the tissue-specific regulation of salivary-type gene expression.

The Src homology 2 phosphotyrosyl phosphatase (SHP2) is a nonreceptor-type phosphatase that acts as a positive transducer of receptor Tyr kinase (RTK) signaling, particularly the Ras-REK and PI3K-Akt pathways. Recently, we have demonstrated that SHP2 is required for cell transformation induced by the constitutively active <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (K/E-FR3) (Oncogene, <em>22</em>, 6909-6918). In that study, we had detected a phosphotyrosyl protein of approximately 100 KDa (p100) in cells expressing dominant-negative SHP2 (R/E-SHP2), but its identity and relevance in SHP2-meditaed transformation was not known. Here, we report the identification of p100 as alpha-catenin, a vinculin-related protein involved in adherens junction-mediated intercellular adhesion. We show that alpha-catenin becomes Tyr phosphorylated in intercellular adhesion-dependent manner and this event is counteracted by SHP2. Substrate trapping in intact cells and immunocomplex phosphatse assays confirmed that alpha-catenin is in deed an SHP2 substrate. Tyr phosphorylation of alpha-catenin enhances its translocation to the plasma membrane and its interaction with beta-catenin, leading to enhanced actin polymerization and stabilization of adherens junction-mediated intercellular adhesion, a phenomenon commensurate with loss of the transformation phenotype. Site-directed mutagenesis studies also suggested that Tyr phosphorylation of alpha-catenin enhances its inhibitory role on cell transformation. Based on our previous work and the current report, we demonstrate that mediation of cell transformation by SHP2 is a complex process that involves modulation of the Ras-ERK and PI3K-Akt signaling pathways, intercellular adhesion, focal adhesion and actin cytoskeletal reorganization. To our knowledge, this is the first report showing regulation of alpha-catenin function by Tyr phosphorylation and its inhibitory effect on cell transformation.